Marine Bioprocess Engineering

Marine Bioprocess Engineering

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edited by M.J. Bull (1st reprint 1983) edited by M.J. Bull edited by M.J. Bull edited by M.E. Bushell Microbial Polysaccharides, edited by M.E. Bushell Modem Applications of Traditional Biotechnologies, edited by M.E. Bushell Innovations in Biotechnology, edited by E.H. Houwink and R.R. van der Meer Statistical Aspects of the Microbiological Analysis of Foods, by B. Jarvis Moulds and Filamentous Fungi in Technical Microbiology, by O. Fassatiovfi Micro-organisms in the Production of Food, edited by M.R. Adams Biotechnology of Animo Acid Production, edited by K. Aida. I. Chibata, K. Nakayama, K. Takinama and H. Yamada Computers in Fermentation Technology, edited by M.E. Bushell Rapid Methods in Food Microbiology, edited by M.R. Adams and C.F.A. Hope Bioactive Metabolites from Microorganisms, edited by M.E. Bushell and U. Gr~ife Micromycetes in Foodstuffs and Feedstuffs, edited by Z. Jesenskfi .4spergillus: 50 years on, edited by S.D. Martinelli and J.R. Kinghorn Bioactive Secondary Metabolites of Microorganisms, edited V. Betina Techniques in Applied Microbiology, edited by B. Sikyta Biotransformations" Microbial Degradation of Health Risk Compounds, edited by V.P. Singh Microbial Pentose Utilization. Current Applications in Biotechnology, by A. Singh and P. Mishra Culture Media for Food Microbiology, edited by J.E.L. Corry, G.D.W. Curtis and R.M. Baird (second impression 1999) . .

Marine Bioprocess I: ng=neerlng

Proceedings of an International Symposium organized under auspices of The Working Party on Applied Biocatalysis of the Eurpean Federation of Biotechnology and The European Society for Marine Biotechnology, Noordwijkerhout, The Netherlands, November 8-11, 1998 "~. .

Edited by R. Osinga

Food and Bioprocess Engineering Group, WageningenAgricultural University, Wageningen, The Netherlands

J. Tramper

Food and Bioprocess Engineering group, WageningenAgricultural University, Wageningen, The Netherlands

J. G. Burgess

Department of Biological Sciences, Heriott-Watt University, Edinburgh, United Kingdom

R.H. Wijffels

Food and Bioprocess Engineering group, WageningenAgricultural University, Wageningen, The Netherlands

progress in industrial microbiology

1999

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Reprinted from Journal of Biotechnology, Vol. 70/1999

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Contents Cited in: Biological Abstracts; Biological & Agricultural Index; Cambridge Scientific Abstracts; Chemical Abstracts; Elsevier BlOBASE/Current Awareness in Biological Sciences; Current Contents (Agriculture, Biology & Environmental Sciences); EMBASE/Excerpta Medica; Index Medicus

Special Issue Marine Bioprocess Engineering

Editorial

1

Opening keynote

S.A. Pomponi (USA) The bioprocess-technological potential of the sea

Products

M.H.G. Munro, J.W. Blunt, E.J. Dumdei, S.J.H. Hickford, R.E. Lill, S. Li, C.N. Battershill and A.R. Duckworth (New Zealand) The discovery and development of marine compounds with pharmaceutical potential

15

J.G. Burgess, E.M. Jordan, M. Bregu, A. Mearns-Spragg and K.G. Boyd (UK) Microbial antagonism: a neglected avenue of natural products research

27

T. Matsunaga, H. Takeyama, T. Nakao and A. Yamazawa (Japan) Screening of marine microalgae for bioremediation of cadmium-polluted seawater

33

E.G. Vrieling, T.P.M. Beelen, R.A. van Santen and W.W.C. Gieskes (The Netherlands) Diatom silicon biomineralization as an inspirational source of new approaches to silica production

39

M. Turkiewicz, E. Gromek, H. Kalinowska and M. Zieliriska (Poland) Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

53

S. Kreitlow, S. Mundt and U. Lindequist (Germany) Cyanobacteria--a potential source of new biologically active substances

61

F. Sponga, L. Cavaletti, A. Lazzarini, A. Borghi, I. Ciciliato, D. Losi and F. Marinelfi (Italy) Biodiversity and potentials of marine-derived microorganisms

65

Z. CsSg6r, D. Melgar, K. Schmidt and C. Posten (Germany) Production and particle characterization of the frustules of Cyclotella cryptica in comparison with siliceous earth

71

G. Liebezeit, T.D. K(Jnnemann and G. Gad (Germany) Biotechnological potential of North Sea salt marsh plants--a review of traditional knowledge

77

A. Koulman, L.M.C. Pruijn, T.S.A. Sandstra, H.J. Woerdenbag and N. Pras (The Netherlands) The pharmaceutical exploration of cold water ascidians from the Netherlands: a possible source of new cytotoxic natural products

85

Energy J. Miyake, M. Miyake and Y. Asada (Japan) Biotechnological hydrogen production: research for efficient light energy conversion

89

i. Eroglu, K. Asian, U. GEmdEiz, M. YScel and L. TEIrker (Turkey) Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor

103

V. Sediroglu, i. Eroglu, M. YEicel, L. TElrker and U. GdndEIz (Turkey) The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production

115

D.O. Yi~it, U. GSnd~z, L. T~rker, M. YScel and ]. Ero~lu (Turkey) Identification of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O.U. 001 grown in the waste water of a sugar refinery

125

Cultivation of marine organisms B. Rinkevich (Israel) Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements

133

R. Osinga, P.B. de Beukelaer, E.M. Meijer, J. Tramper and R.H. Wijffels (The Netherlands) Growth of the sponge Pseudosuberites (aff.) andrewsi in a closed system

155

E. Armstrong, J.D. McKenzie and G.T. Goldsworthy (UK) Aquaculture of sponges on scallops for natural products research and antifouling

163

B.J.B. Wood, P.H.K. Grimson, J.B. German and M. Turner (UK, USA) Photoheterotrophy in the production of phytoplankton organisms

175

M.E. de Swaaf, T.C. de Rijk, G. Eggink and L. Sijtsma (The Netherlands) Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii

185

R.D. Bowles, A.E. Hunt, G.B. Bremer, M.G. Duchars and R.A. Eaton (UK) Long-chain n - 3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production

193

H. Helmholz, P. Etoundi and U. Lindequist (Germany) Cultivation of the marine basidiomycete Nia vibrissa (Moore & Meyers)

203

S. La Barre, S. Singer, E. Erard-Le Denn and M. Jozefowicz (France) Controlled cultivation of Alexandrium minutum and [33p] orthophosphate cell labeling towards surface adhesion tests

207

J.C. Ogbonna, S. Tomiyama and H. Tanaka (Japan) Production of c~-tocopherol by sequential heterotrophic-photoautotrophic cultivation of Euglena gracilis

213

K. Tsukahara, S. Sawayama, T. Yagishita and T. Ogi (Japan) Effect of Ca2 + channel blockers on glycerol levels in Dunaliella tertiolecta under hypoosmotic stress

223

P. Garcia-Jimenez, F.D. Marian, M. Rodrigo and R.R. Robaina (Spain) Sporulation and sterilization method for axenic culture of Gelidium canariensis

227

Design and scale-up of in vitro cultures E. Molina Grima, F.G.A. Fernandez, F. Garcia Camacho and Y. Chisti (Spain) Photobioreactors: light regime, mass transfer, and scaleup

231

A. S~nchez Mir6n, A. Contreras GOmez, F. Garcia Camacho, E. Molina Grima and Y. Chisti (Spain) Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae

249

M.M. Rebolloso Fuentes, J.L. Garcia Sanchez, J.M. Fernandez Sevilla, F.G. Aci~n Fernandez, J.A. S~nchez P~rez and E. Molina Grima (Spain) Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters

271

J.C. Ogbonna, T. Soejima and H. Tanaka (Japan) An integrated solar and artificial light system for internal illumination of photobioreactors

289

G. Chini Zittelli, F. Lavista, A. Bastianini, L. Rodolfi, M. Vincenzini and M.R. Tredici (Italy) Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors

299

M.A. Borowitzka (Australia) Commercial production of microaigae: ponds, tanks, tubes and fermenters

313

M. Janssen, T.C. Kuijpers, B. Veldhoen, M.B. Ternbach, J. Tramper, L.R. Mur and R.H. Wijffels (Netherlands) Specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana under medium duration light/dark cycles: 13-87 s

323

D. Baquerisse, S. Nouals, A. Isambert, P.F. dos Santos and G. Durand (France) Modelling of a continuous pilot photobioreactor for microalgae production

335

P.C. Wright, C. Stevenson, E. McEvoy and J.G. Burgess (UK) Opportunities for marine bioprocess intensification using novel bioreactor design: frequency of barotolerance in microorganisms obtained from surface waters

343

N. Zou and A. Richmond (Israel) Effect of light-path length in outdoor flat plate reactors on output rate of cell mass and of EPA in Nannochloropsis sp.

351

K. Kotzabasis, A. Hatziathanasiou, M.V. Bengoa-Ruigomez, M. Kentouri and P. Divanach (Greece, Spain) Methanol as alternative carbon source for quicker efficient production of the microalgae Chlorella minutissima: Role of the concentration and frequence of administration

357

Product recovery L.A.M. van der Wielen and L.K. Cabatingan (The Netherlands, Philippines) Fishing products from the sea--rational downstream processing of marine bioproducts

363

S. Hirano, T. Nakahira, M. Nakagawa and S.K. Kim (Japan, South Korea) The preparation and applications of functional fibres from crab shell chitin

373

A. Robles Medina, L. Esteban Cerd~n, A. Gim6nez Gim~nez, B. Camacho Paez, M.J. Iba~ez Gonzalez and E. Molina Grima (Spain) Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils

379

L. Vandanjon, P. Jaouen, N. Rossignol, F. Qu~m~neur and J.-M. Robert (France) Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen

393

Closing keynote O.R. Zaborsky (USA) Marine bioprocess engineering: the missing link to commercialization

403

Author Index Subject Index

409 413

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JOUItNAL

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Biotecbnology ELSEVIER

Journal of Biotechnology 70 (1999) 1-3

Editorial

This special issue contains full papers of both oral and poster presentations of the international symposium 'Marine Bioprocess Engineering' which was held in Noordwijkerhout, The Netherlands, 8-11 November 1998. The symposium focused on the bioprocessing of marine natural products. Biotechnology is the application-oriented integration of biodisciplines such as cell biology, biochemistry, microbiology, molecular biology and molecular genetics, with engineering. Applications of biotechnology are found in health care, environmental remediation and the production of energy, food and fine chemicals. Bioprocess engineering has been the key to success in the commercialization of biotechnology, especially with respect to biopharmaceuticals. In marine biotechnology, both new and existing biotechnological techniques are developed and applied to organisms from marine sources. For marine biotechnology, bioprocess engineering represents the link between discovery and commercialization. The world's oceans represent one of the largest untapped biological resources. Appropriately, the United Nations has designated 1998 as the 'Year of the Ocean' in recognition of the importance of oceans to the well-being of this planet and the need for proper use of their resources for sustainable development. The diversity of marine life points to a myriad of new bioproducts waiting to be discovered and developed commercially through engineering research and a systems approach that spans activities from the identification of new marine bioproducts through production,

separation, formulation and delivery. Numerous potential pharmaceuticals (e.g. bryostatin, halichondrin), valuable biopolymers (e.g. chitin), compounds that can be used in the anti-fouling industry (as an alternative to toxic chemicals like organotins), products for the food industry (polyunsaturated fatty acids) and products for the cosmetic industry (e.g. phycobiliproteins) have been found in and isolated from marine organisms. In addition, marine microorganisms can be used to produce energy (biohydrogen). In order to stimulate research that leads to commercial exploitation of these natural resources, marine organisms need to be cultivated in vitro for the rational and sustainable exploitation of the sea as a source of food and natural products. While the importance of marine biotechnology has been recognized, the engineering component has not been fully integrated. At the symposium 'Marine Bioprocess Engineering' we have begun to bridge the gap between the isolation of products from marine organisms in the laboratory and industrial applications by focusing on the bioproLess-engineering aspects. Reviews and recent developments in product discovery, bio-energy production, cultivation of marine organisms, scale up and product recovery were presented and discussed during five sessions (oral and poster presentations):

I. Products

The focus was on the screening of marine or-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 5 1 - 6

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Editorial

ganisms for useful products, including novel screening strategies. In situ biochemical functions as well as biomedical properties of the target compounds were also discussed.

2. Energy

There is an increasing interest in the biotechnological production of hydrogen as an energy source. Biological aspects (organisms, metabolic processes), technological aspects as well as economic aspects of biological hydrogen production were discussed.

3. Cultivation of marine organisms

This topic included techniques for cultivating bacteria, fungi, algae, thraustochytrids and invertebrates, invertebrate-cell cultures, physiological barriers for in vitro cultivation and methods to stimulate the production of the target compounds by the cultured organisms.

4. Design and scale-up of in vitro cultures

Presentations in this session were focused on the possibilities and drawbacks of marine bioreactor design. This includes different types of bioreactors (for instance: photobioreactors), hydrodynamics and mass transfer.

5. Product recovery

Downstream processing is for many natural products the most expensive process step. The recovery of marine natural products may differ from conventional recovery techniques. Presentations that considered these differences or that illustrated new and emerging recovery techniques were given. The manuscripts presented in this special issue give an extensive and timely overview of the current research and applications in marine bioprocess engineering. We hope the manuscripts will

stimulate application-oriented research in the field of marine biotechnology and that co-operations between biologists and engineers will result in an integrated research-approach. Researchers from biological disciplines should already implement in their research strategy questions that will raise when the processes are scaled up (think big!). Similarly, industry and researchers applying or studying cultivation and product recovery should discuss scale-up aspects in an early stage of the research with researchers from biological disciplines (scale-up by scaling down!). The Guest Editors hope that engineering aspects of marine biotechnology will receive further attention in the future. Exploration of new bioproducts from the ocean should be followed up by a sustainable exploitation of these valuable resources. The organization committee of the international symposium 'Marine Bioprocess Engineering' acknowledges with gratitude the following organizations: Applikon Dependable Instruments, (The Netherlands), for sponsoring the session 'design and scale-up' of in vitro cultures; Shell Nederland by, for sponsoring the session 'energy'; The Foundation for Biotechnology in The Netherlands and The National Committee for International Cooperation and Sustainable Development, The Netherlands, who supported the participation of several participants. Publication of this special issue and a spin-off book was possible thanks to a grant from the Directorate General Science, Research and Development of the European Commission (BIO4CT98-4814). In addition, the Guest Editors are indebted to the following people, who served as the editorial board for the special issue by reviewing the submitted manuscripts: A. Ballesteros (Madrid, Spain); M.A. Borowitzka (Murdoch, Australia); G.B. Bremer (Portsmouth, UK); C. Bucke (London, UK); L. Dijkhuizen (Groningen, The Netherlands); M. Jaspars (Aberdeen, UK); Y. LeGal (Concarneau, France); F. Marinelli (Gerenzano, Italy); T. Matsunaga (Tokyo, Japan); M. Meiners (Emden, Germany); J. Miyake (Tsukuba, Japan); E. Molina Grima (Almeria, Spain); A. Muller-Feuga (Plouzan6,

Editorial

France); M.H.G. Munro (Christchurch, New Zealand); L.R. Mur (Amsterdam, The Netherlands); P.D. Nichols (Hobart, Tasmania, Australia); S.A. Pomponi (Fort Pierce, USA); B. Rinkevich (Haifa, Israel); G.L. Rorrer (Corvallis, USA); M.R. Tredici (Florence, Italy); and O.R. Zaborsky (Honolulu, USA). With the help of so many, we have been able to turn the international symposium 'Marine Bio-

3

process Engineering' into a high quality event, ultimately resulting in this special issue of the Journal of Biotechnology. The Guest Editors, R. Osinga, Wageningen, The Netherlands J. Tramper, Wageningen, The Netherlands J.G. Burgess, Edinburgh, United Kingdom R.H. Wijffels, Wageningen, The Netherlands

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9

J O U R N A L

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Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 5-13

The bioprocess-technological potential of the sea Shirley A. Pomponi * Division of Biomedical Marine Research, Harbor Branch Oceanographic Institution Inc., Fort Pierce, FL, USA

Received 19 October 1998; received in revised form 30 November 1998; accepted 22 December 1998

Abstract

Marine bioprocess engineers face a unique challenge for the millennium: designing methods for the sustainable development of known marine resources, as well as inventing a new generation of tools and processes that will enable a greater understanding of the ocean and its resources and lead to the discovery of new bioproducts for the future. The identification and application of novel, marine-derived pharmaceuticals, cosmetics, nutritional supplements, enzymes, and pigments have already been realized. The current and potential market value of these marine bioproducts is substantial. Continued discovery and development of marine resources will depend on a number of factors: identification of new bioproducts, sustainable use of the product, optimization of production, and efficient product recovery. Successfully addressing these challenges will require the integration and collaboration of mutidisciplinary teams of oceanographers, biologists, chemists, and engineers. 9 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Aquaculture; Invertebrate cell culture; Marine bioproducts; Marine pharmaceuticals; Sustainable use

I. Introduction

The marine environment has proven to be a rich source of both biological and chemical diversity. The oceans contain nearly 300 000 described species, but it is estimated that this number is only a small percentage of the total number of species that have yet to be discovered and described (Winston, 1988; Malakoff, 1997). Marine microorganisms represent the greatest percentage of undescribed marine species (Colwell, 1997). Marine bacteria alone could constitute as much as 10% of the total living biomass carbon of the biosphere (Parkes et al., 1994). * Fax: + 1-561-461-2221. E-mail address: [email protected] (S.A. Pomponi)

F r o m a relatively small number of these species that have been studied to date, thousands of chemical compounds have been isolated (Ireland et al., 1993), yet only a small percentage of these compounds has been studied for their potential as useful products. The oceans represent a virtually untapped resource for discovery of even more novel chemicals with potential as pharmaceuticals, nutritional supplements, cosmetics, agrichemicals, molecular probes, enzymes and fine chemicals. Each of these classes of marine bioproducts has a potential multi-billion dollar market value (BioScience, 1996). There are several marine-derived products currently on the market (Table 1). This discussion will focus on the current status and future poten-

0168-1656/99/$ - see front matter 9 1999 Published by Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00053-X

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S.A. Pomponi / Journal of Biotechnology 70 (1999) 5-13

tial of bioprocess engineering related to the discovery, development, and sustainable use of marine-derived compounds with biomedical applications. Marine bioprocess engineers will be an integral part of the multidisciplinary approach to discovery and development of new marine-derived pharmaceuticals and molecular probes to study human disease processes. Novel tools and processes are required for identification of new products, sustainable use of the products, optimization of production, and efficient product recovery.

2. Identification of new products and development of novel screening technologies Marine organisms for drug discovery research have, to date, been collected using relatively routine methods, such as scuba diving, submersibles, dredging and trawling. Submersibles enable scientists to access unusual habitats, such as vent communities and deep sea benthic habitats (Fig. 1),

and some systems are equipped with specialized tools and chambers that allow samples to be maintained under ambient conditions, i.e. high pressure and low temperature. There is still a need for the development of versatile bioreactors that can be deployed and operated in extreme environments (e.g. hypersaline, vent, anoxic, and deep sea habitats). Such bioreactors could be used for collection, at-sea maintenance, and evaluation of both macroorganisms and microorganisms, so that their metabolites could be evaluated under physiological conditions that are as similar as possible to ambient conditions. Another approach to the identification of new products is the incorporation of miniaturized biosensors into both collecting tools and bioreactors for rapid, in situ analysis of wild and cultivated marine organisms for target molecules. A number of miniaturized biosensors and probes to study human disease processes are in development. Adaptation of these for in situ evaluation of marine-derived products would be an interest-

Table 1 Some examples of commercially available marine bioproducts Product

Application

Original source

Method of production

Ara-A

Antiviral drug

Marine sponge

Ara-C

Anticancer drug

Marine sponge

Microbial fermen- McConnell et al., tation of analog 1994 Chemical synthesis McConnell et al., of analog 1994 Cell culture Tachibana et al., 1981 Wild harvest of Glaser and sponge Jacobs, 1986 Recombinant Mattila et al., protein 1991 Cell culture ESPGAN, 1991

Molecular probe: phosDinoflagellate phatase inhibitor Molecular probe: phospholi- Marine sponge, Luffariella Manoalide variabilis pase A 2 inhibitor Deep sea hydrothermal Vent TM DNA polymerase Polymerase chain reaction vent bacterium enzyme Fatty acids used as additive Marine microalgae Formulaid | (Martek Biosciences, Columbia, MD) in infant formula nutritional supplement Bioluminescent jellyfish, Bioluminescent calcium Aequorin Aequora victoria indicator Bioluminescent jellyfish, Reporter gene Green Fluorescent Protein Aequora victoria (GFP) Conjugated antibodies used Red algae Phycoerythrin in ELISAs and flow cytometry Resilience | (Estre Lauder) 'Marine extract' additive in Caribbean gorgonian, Pseudop terogorgia skin creams

Okadaic acid

elisabethae

References

Recombinant protein Recombinant protein Cell culture

Badminton et al., 1995 Chalfie et al., 1994 Glazer, 1989

Wild harvest of gorgonian

Look et al., 1986

S.A. Pomponi / Journal of Biotechnology 70 (1999) 5-13

Fig. 1. The drug discovery research program at Harbor Branch Oceanographic Institution uses the Johnson-Sea-Link manned submersibles to conduct targeted, controlled collections of unique invertebrates from deep-water habitats such as rocky slopes and steep vertical walls that are not accessible by dredging and trawling. ( 9 Branch Oceanographic Institution, Inc.)

ing bioengineering challenge. One example is GeneChip | technology (Affymetrix, Santa Clara, CA), a rapidly developing field in which high-density oligonucleotide arrays on prefabricated solid chips are used to simultaneously analyze expression levels of thousands of genes (Fodor et al., 1991). The GeneChip probe arrays are manufactured using a photolithographic process similar to that used for making a computer microchip. A photo-protected glass substrate is selectively exposed by light passing through a photolithographic mask. The unmasked areas are photo-activated, the substrate is incubated with nucleosides, and chemical coupling occurs at the activated sites. This process of photo-activation and chemical coupling is repeated until the desired set of probes is synthesized. The GeneChip probe array can contain tens to hundreds of thousands of different oligonucleotide or cDNA probes, arranged in precise sequences and locations. A fluorescently labelled sample is analyzed by hybridization with the GeneChip probe array. The GeneChip probe array is then scanned by a laser, and the fluorescent pattern is analyzed for matches to known sequences. Although GeneChip probe arrays are currently used for diagnostics

7

and research primarily in mammalian systems, the technology could be adapted to identify new or known molecules from marine invertebrates, algae, and microorganisms for drug discovery research. The engineering challenge is to adapt the technology for rapid, in situ screening of marine organisms and their bioproducts. It would involve specialized tools for sampling the target organisms, preparing the samples for hybridization with the oligonucleotide arrays, and analyzing the arrays with modified scanners--all in situ. Potential applications are the identification of new or previously untested species, as well as analysis of gene expression that is specific to a particular disease or therapeutic area. The biological evaluation of marine-derived extracts and pure compounds has been based on assays developed for the 'high-throughput' screening of large libraries of synthetic compounds. They measure a number of end-points, such as activation or inhibition of enzymes or receptors involved in human disease processes, inhibition of growth of human pathogenic microorganisms, and toxicity against human cancer cells (Suffness et al., 1989; Ireland et al., 1993; McConnell et al., 1994; Munro et al., 1994). None of the assays used in major pharmaceutical drug discovery programs takes into account the role of marinederived compounds in nature, i.e. the in situ biochemical functions of both primary and secondary metabolites, and how those functions may be applied to the discovery of new drugs and probes to study human disease processes. Marine organisms as model systems offer the potential to understand and develop treatments for disease based on the normal physiological role of their secondary metabolites. For example, the mechanisms of action of Conus toxins are well-known (Hopkins et al., 1995; Shon et al., 1997), and are currently being applied to the development of new classes of drugs. Development of in situ biosensors would enhance our ability to probe the expression of secondary metabolites in response to various stimuli, lead to a better understanding of the role of the secondary metabolites in nature, and perhaps provide clues to the potential biomedical utility of these compounds.

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S.A. Pomponi /Journal of Biotechnology 70 (I 999) 5-13

3. Sustainable use of marine resources

With the enormous potential for discovery, development, and marketing of novel marine bioproducts comes the obligation to develop methods by which these products can be supplied in a way that will not disrupt the ecosystem or deplete the resource. Supply of most marine-derived compounds is a major limiting factor for further pharmaceutical development. Often, the metabolite occurs in trace amounts in the organism, and a steady source of supply from wild harvest cannot provide enough of the target compound for preclinical studies. In general, the natural abundance of the source organisms will not support production based on wild harvest. Some options for sustainable use of marine resources are chemical synthesis, controlled harvesting, aquaculture of the source organism, in vitro production through cell culture of the macroorganism or microorganism source, and transgenic production. Each of these options has its advantages and limitations, not all methods will be applicable to supply of every marine bioproduct, and most of the biological supply methods are still in development. The approach to be used will be based on a number of factors: 3.1. Complexity of the molecule: can it be synthesized using an industr&lly feasible process7 Synthetic processes have been published for many marine bioproducts in development as pharmaceuticals (e.g. Kageyama and Tamura, 1990; Corey et al., 1996; Harried et al., 1997). Unfortunately, most of these are multi-step processes that are not amenable to economic, industrial-scale synthesis. The cost of reagents, affordable on a small scale, may be prohibitively expensive to use for large-scale synthesis. In addition, many natural products are complex molecules with several stereocenters, making it necessary to control the stereochemistry of the chemical reactions. If the compound is an enantiomer, and both isomers are synthesized, the process may require separation of the isomers by chromatographic techniques which could be costly on a large scale.

Other sources of bulk supply are generally required for early-stage drug development, during which time the pharmaceutical company may also optimize chemical synthesis at the industrial scale. There is thus an opportunity and a market for supply of marine bioproducts from aquaculture, in vitro production, and transgenic production, not only for long-term applications, but also for early-stage validation of their utility. 3.2. Abundance of the organism in nature: what do we know about the impact of collections on the habitat or species populations7 Prior to large-scale wild harvest of an organism for recovery of a bioproduct, harvesting feasibility studies should be conducted. These should define factors such as the standing stock of the organism, its growth rate and the factors that affect growth, and the impact of harvesting and postharvesting recovery of the target organism. These data could then be used not only to assess the potential of supply from wild harvest, but also to develop models for aquaculture and/or in vitro production. Unfortunately, this is rarely done. One notable exception is the survey of the New Zealand sponge, Lissodendoryx sp., which produces the halichondrins, potent antitumor compounds. The sponge is restricted in occurrence to one locality off the Kaikoura Peninsula. Research conducted by Battershill et al. (1998) demonstrated that harvesting by dredging significantly reduced the standing stock of the sponge, but that the population may recover rapidly as a result of asexual propagation of sponge fragments dispersed as a result of the dredging. This study suggested that harvesting is only feasible for smaller quantities of the sponge. On the other hand, the bryozoan Bugula neritina, the source of the anticancer compound bryostatin 1 (Pettit et al., 1982), is a common fouling organism found throughout the world in both temperate and tropical habitats. The US National Cancer Institute collected more than 12 000 kg wet weight of this 'nuisance' organism from docks and pilings with apparently little impact on the populations. Moreover, this single bulk collection was sufficient to supply about 18 g

S.A. Pomponi / Journal of Biotechnology 70 (1999) 5-13

of bryostatin 1--enough to conduct all preclinical and clinical trials (Schaufelberger et al., 1991). Recent studies indicate, however, that there are actually two 'chemotypes' of Bugula neritina: samples occurring deeper than 9 m generally contain bryostatin 1, whereas samples collected from depths shallower than 9 m do not. In addition, only the Pacific samples have consistently yielded bryostatin 1 (Dominick Mendola, CalBioMarine Technologies, personal communication). This type of information is not only necessary in planning controlled harvesting, but is also critical for selection of genetically superior brood stock for aquaculture and in vitro production.

3.3. Source of the compound: is it microbially produced? A significant number of marine bioproducts with pharmaceutical potential have been identified from heterotrophic marine microorganisms isolated from coastal sediments (Fenical, 1993; Kobayashi and Ishibashi, 1993; Davidson, 1995). In addition, some marine bioproducts originally isolated from macroorganisms, such as sponges, have been subsequently discovered to be localized in microbial associates (e.g. Bewley et al., 1996). If these symbiotic microorganisms can be isolated and cultured, optimization of production in marine microbial bioreactors may lead to an industrially feasible supply option. The large-scale, photoautotrophic production of polyunsaturated fatty acids for use as nutritional supplements (e.g. Formulaid | Martek Biosciences) demonstrates the commercial feasibility of photoautotrophic microbial culture for bulk supply. Bulk supply of bioproducts from both photoautotrophic and heterotrophic microbial culture presents some bioengineering challenges that are addressed by a number of papers in this volume. If the source of the compound is the macroorganism itself, development of in vitro production methods could provide bulk supply of the compound. Research in progress in our laboratory on in vitro production of sponge metabolites has resulted in the establishment of primary sponge cell cultures that can be stimulated by lectins and

9

other growth-regulating compounds to divide and continue to produce bioactive compounds after doubling (Fig. 2) (Pomponi et al., 1997, 1998). The objective of our research is to establish cell lines of bioactive marine invertebrates that can be used as models to study in vitro production of bioactive metabolites and the factors which control expression of production. This could ultimately lead to in vitro production of marine bioproducts, including production of compounds that may only be produced by an intact invertebrate-microorganism symbiotic association. More importantly, an understanding of the cellular and molecular processes that control production of these metabolites could be used to enhance upstream processing/culture optimization and to stimulate production of 'unnatural' natural products--i.e, chemicals that the organism would not produce under normal conditions, but which may be more potent than the 'natural' product. As discussed by Rinkevich (this volume), there are a few obstacles that still need to be overcome before marine invertebrate in vitro production can become a viable option for bulk supply of bio225

e,-

._Q m c (.1 c

8 O

.G

100

r162 c

0.00

u aays

36 hours

"firm Fig. 2. Concentration of stevensine per cell in cultures of the sponge Teichaxinella morchella incubated in phytohemagglutinin (PHA), expressed as percent of control. One population doubling occurred within 36 h in PHA-stimulated cultures; control cultures did not divide. After 8 days, PHA-treated cultures showed an increase in stevensine concentration per cell (relative units) (From Pomponi et al. (1997)).

10

S.A. Pomponi Journal of Biotechnology 70 (1999) 5-13

Fig. 4. Typical grow-out plate with aquacultured Bugula neritina colonies attached, following retrieval from the undersea structure by divers after 5 months in the sea. (Photo courtesy of CalBioMarine Technologies, Carlsbad, CA, USA.)

Fig. 3. Launching of CalBioMarine's prototype in-sea structure for aquaculture of Bugula neritina to yield bryostatin 1. (Photo courtesy of CalBioMarine Technologies, Carlsbad, CA.

USA.) products, but our data indicate that in vitro production of bioactive metabolites by marine invertebrate cells is feasible.

3.4. In situ growth conditions: is aquaculture an option for deep water organisms? Both in-the-sea and land-based aquaculture methods have been developed by CalBioMarine Technologies (Carlsbad, CA) for the bryozoan, Bugula neritina (Figs. 3 and 4), and for Ecteinascidia turbinata (Figs. 5 and 6) the ascidian from which the antitumor compound, ecteinascidin 743, has been isolated (Rinehart et al., 1990; Wright et al., 1990). These are both common, shallow-water organisms for which reproduction and growth have been studied, but the factors controlling production of the compounds are not yet completely known. The New Zealand deepwater sponge, Lissodendoryx sp., is the source of the antitumor compounds, the halichondrins. The sponge occurs at 85-105 m, but has been cultured successfully from cuttings on lantern arrays in shallower wa-

ter, and has maintained production of the halichondrins (Battershill et al., 1998). Current efforts are directed toward modification of metabolite production by altering the microenvironment (Battershill, personal communication). This indicates that aquaculture of some deep water sponges is feasible, however, species from deeper water may have more critical growth requirements, such as high pressure and low temperature. Although in-the-sea aquaculture is a cost-effective method of production, it still does not afford the opportunity for over-expression of production of the compounds or for complete control of environmental parameters. Development of

Fig. 5. Underwater close-up of aquacultured colony of Ecteinascidia turbinata on poly-line. (Photo courtesy of CalBioMarine Technologies. Carlsbad, CA, USA.)

S.A. Pomponi / Journal of Biotechnology 70 (1999) 5-13

Fig. 6. Lab-settled colonies of Ecteinascidia turbinata on polylines in an indoor aquaculture tank, Keys Marine Lab, Long Key, FL, USA. (Photo courtesy of CalBioMarine Technologies, Carlsbad, CA, USA.)

closed-system bioreactors for the culture of both shallow water and deep water organisms is a particularly challenging opportunity for marine bioprocess engineers. Research in progress by Osinga et al. (1997) (and this volume) demonstrates the feasibility of this approach for shallowwater sponges. A combination of cell culture and aquaculture techniques may be applied to development of novel, closed bioreactor systems. For example, three-dimensional matrices may be used to establish seed cultures in vitro from dissociated cells of biomedically important species. Once established, the seeded matrices could be transferred into larger, closed bioreactors for optimization, scaleup, and product recovery.

3.5. Biosynthetic pathway" is genetic engineering realistic for the compound7 If the biosynthesis of the target compound is understood, it may be possible to identify, isolate, clone, and express in a heterologous host the genes responsible for production of the metabo-

11

lite. In many cases, of course, biosynthesis of the product is not known, or it is a multi-step process involving several enzymatic reactions. For these cases, transgenic production is not a trivial process. Matsunaga (1998) succeeded in transgenic production of eicosapentaenoic acid (EPA) by cloning an EPA gene cluster, isolated from Shewanella putrefaciens, into the marine cyanobacterium Synechococcus. Alternatively, chemoenzymatic synthesis, by which marine bioproducts are synthesized in cellfree, enzyme-based systems, offers a complementary technique to in vitro and transgenic production methods. Kerr et al. (1996a,b) have demonstrated the conversion of basic biosynthetic building blocks to the target molecule for a number of marine bioproducts, including bryostatin from Bugula neritina and pseudopterosins from the gorgonian Pseudopterogorgia americana (Figs. 7 and 8).

4. Optimization of production Perhaps the area in which marine bioprocess engineering has had the greatest impact--and still has the greatest opportunity for novel developments--is in the design and optimization of bioreactors for marine metabolite production. Since bioreactor design is addressed by several other authors in this volume, suffice it to say that Secosteroid Production using Enzymes

from

Local C o r a l .

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Punfied Enzymes

STEROID (e.g Cholesterol)

Enzyme Powder

SECOSTEROIO ANTI-INFLAMMATORY .AGENTS - efl~oent, inexpensive new production method

Fig. 7. The first step in chemoenzymatic synthesis of antiinflammatory secosteroid involves production of active enzyme precipitate (PAP) from the gorgonian Pseudopterogorgia americana. (Photo courtesy of Dr Russell Kerr, Florida Atlantic University, Boca Raton, FL, USA.)

S.A. Pomponi /Journal of Biotechnology 70 (1999) 5-13

12

Enzyme Column STEROID

-- (1 m g

r.holesterol 1100 mL buffer)

Im P A l )

9(11)-SECOSTEROID

(100%

yield cholesterol)

-~

Fig. 8. A steroid (e.g. cholesterol) is transformed into the anti-inflammatory secosteroid product by the active enzyme precipitate (PAP) immobilized and packed in a column. (Photo courtesy of Dr Russell Kerr, Florida Atlantic University, Boca Raton, FL, USA.)

a variety of bioreactor designs have been implemented, with varying degrees of success. The opportunity to produce new, bioactive structural analogs of known compounds via manipulation of culture conditions presents marine bioprocess engineers and their collaborators in marine natural products biochemistry, cellular and molecular biology with a unique challenge for new bioproduct discovery. Innovations in media development (chemical engineering), bioreactor design (bioprocess engineering), and transgenic production (molecular engineering), coupled with efficient downstream processing and product recovery, will be necessary to meet the needs of both discovery and bulk production of novel marine bioproducts. In summary, marine bioprocess engineers face a unique challenge for the millennium: designing methods for the sustainable development of known marine resources, as well as inventing a new generation of tools and processes that will enable a greater understanding of the ocean and its resources and lead to the discovery of new bioproducts for the future. Successfully addressing these challenges will require the integration and collaboration of multidisciplinary teams of marine biologists, pharmacologists, cell and molecular biologists, biochemists, and engineers.

References Badminton, M.N., Kendall, J.M., Sala-Newby, G., Campbell, A.K., 1995. Nucleoplasmin-targeted aequorin provides evidence for a nuclear calcium barrier. Exp. Cell Res. 216, 236-243. Battershill, C.N., Page, M.J., Duckworth, A.R., Miller, K.A., Bergquist, P.R., Blunt, J.W., Munro, M.H.G., Northcote, P.T., Newman, D.J.. Pomponi, S.A., 1998. Discovery and sustainable supply of marine natural products as drugs, industrial compounds and agrochemicals: chemical ecology, genetics, aquaculture and cell culture. In: Origin and Outlook: 5th International Sponge Symposium 1998, Book of Abstracts. Queensland Museum, Brisbane, Australia, p. 16. Bewley, C.A., Holland, N.D., Faulkner, D.J., 1996. Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia 52, 716-722. BioScience, 1996. Marine Biotechnology Special Issue, 46. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802-805. Colwell, R.R., 1997. Microbial biodiversity and biotechnology. In: Reaka-Kudla, M.E., Wilson, E. (Eds.), Biodiversity II: Understanding and Protecting Our Biological Resources. Joseph Henry Press, Washington, DC, pp. 279-287. Corey, E.J., Gin, D.Y, Kania, R.S., 1996. Enantioselective total synthesis of ecteinascidin 743. J. Am. Chem. Soc. 118, 9202-9203. Davidson, B.S., 1995. New dimensions in natural products research: cultured marine microorganisms. Curr. Opin. Biotechnol. 6, 284-291. ESPGAN Committee on Nutrition, 1991. Comment on the content and composition of lipids in infant formulas. Acta Paediatr. Scand. 80, 887-896. Fenical, W., 1993. Chemical studies of marine bacteria: developing a new resource. Chem. Rev. 93, 1673-1683. Fodor, S.P.A, Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T., Solas, D., 1991. Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-773. Glaser, K.B., Jacobs, R.J., 1986. Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2. Biochem. Pharmacol. 35, 449-453. Glazer, A.N., 1989. Light guides. Directional energy transfer in a photosynthetic antenna. J. Biol. Chem. 264, 1-4. Harried, G.Y., Strawn, M.A., Myles, D.C., 1997. The total synthesis of (-)-discodermolide: an application of the chelation-controlled alkylation reaction. J. Org. Chem. 62, 6098-6099. Hopkins, C., Grilley, M., Miller, C., Shon, K.J., Cruz, L.J., Gray, W.R., Dykert, J., Rivier, J., Yoshikami, D., Olivera, B.M., 1995. A new family of Conus peptides targeted to the nicotinic acetylcholine receptor. J. Biol. Chem. 38, 2236122367.

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Ireland, C.M., Copp, B.R., Foster, M.D., McDonald, L.A., Radisky, D.C., Swersey, J.C., 1993. Biomedical potential of marine natural products. In: Attaway, D.H., Zaborsky, O.R. (Eds.), Marine Biotechnology. Pharmaceutical and Bioactive Natural Products, vol. 1. Plenum Press, New York, pp. 1-43. Kageyama, M., Tamura, T., 1990. Synthesis of bryostatin 7. J. Am. Chem. Soc. 112, 7404-7408. Kerr, R.G., Lawry, J., Gush, K.A., 1996a. In vitro biosynthetic studies of the bryostatins, anticancer agents from the marine bryozoan Bugula neritina. Tetrahedron Lett. 37, 8305-8308. Kerr, R.G., Rodriguez, L., Kellman, J., 1996b. A chemoenzymatic synthesis of 9(ll)-secosteroids using an enzyme extract of the marine gorgonian Pseudopterogorgia americana. Tetrahedron Lett. 37, 8301-8304. Kobayashi, J., Ishibashi, M., 1993. Bioactive metabolites of symbiotic marine microorganisms. Chem. Rev. 93, 17531769. Look, S.A., Fenical, W., Jacobs, R.S., Clardy, J., 1986. The pseudopterosins: anti-inflammatory and analgesic natural products from the sea whip Pseudopterogorgia elisabethae. Proc. Natl. Acad. Sci. USA 83, 6238-6240. Malakoff, D., 1997. Extinction on the high seas. Science 277, 486-488. Matsunaga, T., 1998. Screening of marine microalgae for fine chemicals and bioremediation. In: Wijffels, R.H., et al. (Eds.), International Symposium Marine Bioprocess Engineering, Wageningen Agricultural University, Wageningen, The Netherlands, Book of Abstracts, p. 14. Mattila, P., Korpela, J., Tenkanen, T., Pitkanen, K., 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase - - an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967-4973. McConnell, O.J., Longley, R.E., Koehn, F.E., 1994. The discovery of marine natural products with therapeutic potential. In: Gullo, V.P. (Ed.), The Discovery of Natural Products with Therapeutic Potential. Butterworth-Heinemann, Boston, pp. 109-174. Munro, M.H.G., Blunt, J.W., Lake, R.J.U., Litaudon, M., Battershill, C.N., Page, M.J., 1994. From seabed to sickbed: what are the prospects? In: Van Soest, R.W.M., Van Kempen, T.M.G., Braekman, J.-C. (Eds.), Sponges in Time and Space, Proceedings of the 4th International Porifera Congress. A.A. Balkema, Rotterdam, pp. 473484. Osinga, R., Tramper, J., Wijffels, R.H., 1997. Cultivation of marine sponges. In: Marino, D., Willensky, D., Capone, D. (Eds.), 4th International Marine Biotechnology Conference, Abstracts. Stazione Zoologica 'Anton Dohrn', Naples, Italy, p. 210. Parkes, R.J., Cragg, B.A., Bale, S.J., Getliff, J.M., Goodman, K., Rochelle, P.A., Fry, J.C., Weightman, A.J., Harvey, S.M., 1994. Deep bacterial biosphere in Pacific Ocean sediments. Nature (London) 371,410-413.

13

Pettit, G.R., Herald, C.L, Doubek, D.L, Herald, D.L., 1982. Isolation and structure of bryostatin 1. J. Am. Chem. Soc. 104, 6846-6848. Pomponi, S.A., Willoughby, R., Kaighn, M.E., Wright, A.E., 1997. Development of techniques for in vitro production of bioactive natural products from marine sponges. In: Maramorosch, K., Mitsuhashi, J. (Eds.), Invertebrate Cell Culture: Novel Directions and Biotechnology Applications. Science Publishers, Inc, Enfield, New Hampshire, USA, pp. 231-237. Pomponi, S.A., Willoughby, R., Wright, A.E., Pecorella, C., Sennett, S.H., Lopez, J., Samples, G., 1998. In vitro production of marine-derived antitumor compounds. In: Le Gal, Y., Halvorson, H.O. (Eds.), New Developments in Marine Biotechnology. Plenum Press, New York, pp. 7376. Rinehart, K.L., Holt, T.G., Fregeau, N.L., Stroh, J.G., Keifer, P.A., Sun, F., Li, L.H., Martin, D.G., 1990. Ecteinascidins 729, 743, 745, 759A, 759B, and 770: potent antitumor agents from the Caribbean tunicate Ecteinascidia turbinata. J. Org. Chem. 55, 4512-4515. Schaufelberger, D.E., Koleck, M.P., Beutler, J.A., Vatakis, A.M., Alvarado, A.B., Andrews, P., Marzo, L.V., Muschik, G.M., Roach, J., Ross, J.T., Lebherz, W.B., Reeves, M.P., Eberwein, R.B., Rodgers, L.L., Testerman, R.P., Snader, K.M., Forenza, S., 1991. The large-scale isolation of bryostatin 1 from Bugula neritina following good manufacturing practices. J. Nat. Prod. 54, 12651270. Shon, K.J., Grilley, M., Jacobsen, R., Cartie, G.E., Hopkins, C., Gray, W.R., Watkins, M., Hillyard, D.R., Rivier, J., Torres, J., Yoshikami, D., Olivera, B.M., 1997. A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom. Biochemistry 31, 9581-9587. Suffness, M., Newman, D.J., Snader, K., 1989. Discovery and development of antineoplastic agents from natural sources. In: Scheuer, P.J. (Ed.), Bioorganic Marine Chemistry, Vol. 3. Springer-Verlag, New York, pp. 131-168. Tachibana, K., Scheuer, P.J., Tsukitani, Y., Kikuchi, H., Van Engen, D., Clardy, J., Gopichand, Y., Schmitz, F.J., 1981. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Halichondria. J. Am. Chem. Soc. 103, 2469-2471. Winston, J.E., 1988. The Systematists' Perspective. In: Fautin, D.G. (Ed.), Biomedical Importance of Marine Organisms. California Academy of Sciences, San Francisco, CA, pp. 1-6. Wright, A.E., Forleo, D.A., Gunawardana, G.P., Gunasekera, S.P., Koehn, F.E., McConnell, O.J., 1990. Antitumor tetrahydroisoquinoline alkaloids from the colonial ascidian Ecteinascidia turbinata. J. Org. Chem. 55, 4508-4512.

This Page Intentionally Left Blank

JOURNAl.

OF

Biotechno,logy ELSEVIER

Journal of Biotechnology 70 (1999) 15-25

The discovery and development of marine compounds with pharmaceutical potential Murray H.G. Munro a,, John W. Blunt a, Eric J. Dumdei a, Sarah J.H. Hickford a, Rachel E. Lill a, Shangxiao Li ", Christopher N. Battershill b, Alan R. Duckworth b a Department of Chemistry, University of Canterbuo,, PB 4800, Christchurch, New Zealand b National Institute of Water and Atmospheric Research, PO Box 14-901, Wellington, New Zealand

Received 12 October 1998; received in revised form 1 December 1998; accepted 22 December 1998

Abstract An assessment of the current status of marine anticancer compounds is presented along with a case study on the aquaculture of Lissodendoryx n. sp. 1, a sponge that produces the antimitotic agents halichondrin B and isohomohalichondrin B. The use of polymer therapeutics to enhance the properties of marine natural products is considered. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Biodiversity; MarinLit; Sponge aquaculture; Anticancer; Antimitotic; Polymer therapeutics

1. The discovery phase In contrast to work on terrestrial natural products the first serious work on studying marine natural products started just 50 years ago with the pioneering work of Bergman (e.g. Bergrnan and Feeney, 1951). While the difficulties of collecting marine samples cannot be underestimated, a large number of easily accessible marine samples are available simply by shore-wading. That the opportunity was not seriously grasped until the * Corresponding author. Tel.: + 64-3-364-2434; fax: + 643-364-2110. E-mail address: [email protected] (M.H.G. Munro)

1940s is possibly a commentary on the difficulties of isolation and purification of marine natural products with the limited techniques available at that time. However, since the 1940s the field has blossomed and matured. In 1997 there were 713 papers published on marine natural products. This is out of a total of 10 311 papers recorded in MarinLit, a database dedicated to the marine natural products literature (MarinLit, 1998). At the time of the mid-year release of the 1998 version of MarinLit, 484 new papers had been included. From the marine literature it is the Porifera that have been the most studied phylum followed closely by the Cnidaria, Chromophycota, Rhodophycota, Mollusca, Chordata and the Echinodermata (MarinLit, 1998).

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00052-8

16

M.H.G. Munro et al. , Journal o f Biotechnology 70 (1999) 1 5 - 2 5

8,246

Microorganisms

Absolute number screened in itafics

Marine Plants

6,540

Marine Animals

Terrestrial Plants

18,293

Terrestrial animals

434

1

0

Percentage

2

Data by courtesy of Dr Peter Murphy, AIMS

Fig. 1. Distribution of samples with significant cytotoxicity in the NCI's preclinical screen.

Over the years some distinct trends have emerged in the study of marine natural products. One has been the emphasis on the discovery of new bioactive natural products. Initial work by Bergman was undoubtedly curiosity-driven, but it was his discovery of the biologically-active, pharmaceutically important and novel arabino-nucleosides from the sponge Cryptotethya crypta that sparked interest in marine natural products and served to highlight the biomedical potential of the field (Bergman and Feeney, 1951). With advances in chromatographic techniques for dealing with polar compounds along with better analytical and structural elucidation technology an increasing proportion of the compounds isolated have shown cytotoxic properties (suggestive of potential antitumour compounds). In an early review (Munro et al., 1987) covering the marine literature up to early 1986, 185 bioactive compounds were reported. In 1993 a review (Schmitz, 1994) covering the next 5 years commented on an additional 400 compounds. A survey of MarinLit reveals that this trend has continued with some 46% of all cytotoxic compounds in the database having been reported since 1993.

As a source of bioactive compounds with pharmaceutical potential how well does the marine environment compare with the more traditional areas such as terrestrial microorganisms and plants? The best comparative data is that published by Garson based on statistical data from the US National Cancer Institute (NCI) screening programme provided by Dr Peter Murphy. This clearly indicated that marine invertebrates are a preferred source due to the much higher incidence of significant cytotoxic activity (Garson, 1994) (Fig. 1). If those screening data for marine animals are in turn examined on a phylum basis certain phyla (e.g. Porifera, Bryozoa, Chordata) have a higher incidence of bioactivity with the trend becoming very obvious as species with very significant bioactivity (ICs0 < 2 lag ml-~) are selected (Fig. 2). As the data in Fig. 1 suggest, the sampling of oceanic life-forms enhances the probability of discovering species from natural sources with potential anticancer activities. This can be rationalised as a sampling strategy which accesses the widest range of phyla. Greater than 70% of all recorded living species belong to the animal kingdom.

M.H.G. Munro et al.//Journal of Biotechnology 70 (1999) 15-25

17

16- i

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02

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,

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~

------.

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~ ,/

.,

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//~/'

Bioactivity

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I

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Fig. 2. Cytotoxicity by phylum.

While only 28% of the animals are aquatic these in fact represent > 90% of the animal phyla (May, 1988). When searching for bioactivity the total number of samples collected is important, but it is not as important as sampling across phyla. Put simplistically, the probability of finding a bioactive species (a 'hit') can be expressed as:

tion of marine organisms than from any other source and perhaps explains why marine samples offer greater opportunity for the discovery of unique compounds with pharmaceutical potential.

'Hits' ~ Samples x 'Biodiversity of Samples' x Assays

2. Current status of bioactive marine natural products

Clearly, the greater the number of samples assayed in the greatest number of possible assays will enhance the probability of finding useful compounds. With the advent over the last decade of high throughput screening (HTS) the number of assays that can be applied to any sample has gone up by between one and two orders of magnitude. However, the key term in the expression is 'biodiversity of samples'. This is a qualitative factor relating to the number of unique structural classes of compounds sampled (adapted from Devlin, 1997). By sampling across phyla the probability of finding unique classes of compounds is higher than by sampling many species within one phylum. The factor associated with 'biodiversity of samples' is probably higher for a random collec-

In spite of the advances in computer-assisted drug design, in molecular biology and gene therapy there is still a pressing need for new drugs to counteract drug-resistant pathogens like, for instance, the mycobacterium that causes tuberculosis, or multi-drug resistant cancers, or even disease states such as Alzheimers which is of pressing concern as the age demographics of the Western World change. What of value has emerged from the marine resource over the past 50 years that would justify the investment of time and money and optimism in this field? In the area of cancer there have been valuable discoveries. Likewise in the area of inflammatory diseases (Fenical, 1996), while probably the first marinebased drug that will be marketed is in the area of

18

M.H.G. Munro et al. Journal of Biotechnology 70 (1999) 15-25

intractable pain management (Olivera, 1997). However, for the purposes of this overview only the cancer leads will be commented on. Progress in the cancer area is summarised in Table 1. Of the four compounds now in clinical trials two are derived from tunicates. These are ecteinascidin 743 and aplidine (dehydrodidemnin B). The other compound, dolastatin 10, originated from a bryozoan. Didemnin B, another tunicatederived compound closely related to aplidine, has previously been in clinical trials and reached as far as phase 2 as had bryostatin 1 before being withdrawn in late 1998. At a preclinical phase are halichondrin B, a sponge metabolite, and kahalalide F from a mollusc followed by a range of compounds from a variety of sources, but all with potent in vivo activities against a range of cancer cell lines. From the mechanism of action of these marine metabo-

Table 1 Testing status of anticancer marine metabolites Status

Compound (origin/activity)

Clinical Phase III Phase II

Ecteinascidin 743 (tunicate, antimitotic)

Phase I

Aplidine (dehydrodidemnin B) (tunicate protein synthesis inhibitor) Dolastatin 10 (sea hare/antimitotic)

Pre-clinical

Halichondrin B (sponge/antimitotic) Kahalalide F (mollusc/-)

In vivo active

Aplyalronine (sea hare/actin) Thiocoralline (marine microorganism RNA inhibition) Isohomohalichondrin B (sponge/antimitotic) Discodermolide (sponge/antimitotic) Sarcodictyins (soft coral/antimitotic) Eleutherobins (soft coral/antimitotic) Spongistatins/Altohyrtins/Cinchyrolide (sponges/antimitotic) Lamellarin N (mollusc/tunicate/sponge antimitotic) Cryptophycins (blue green alga/antimitotic)

Table 2 Marine-derived antimitotic agents Type

Marine compound

Type I (colchicine site) Type II (GTP binding site)

Halichondrin B Isohomohalichondrin B Spongistatins/ Altohyrtins/ Cinchyrolide Cryptophycins Dolastatin 10

Type III (microtubule stabilisation)

Eleutherobin Sarcodictyins Discodermolide

Type IV (microtubule network disorganisation)

Ecteinascidin 743 Lamellarin N

lites it is very obvious that the marine environment has been an excellent source of antimitotic agents. Cancer chemotherapy exploits differences between normal and malignant cells. Ideally, total selectivity between the cell types is required, but has not been achieved. The high proliferation rate of cancer cells is one area that is targeted when looking for cytotoxic agents. Compounds that block mitosis (anti-mitotic agents) which occurs during cell proliferation have become some of the most important anticancer agents, e.g. taxol, vincristine. Antimitotic agents can be divided into four categories depending on which particular step in the microtubule polymerisation/depolymerisation steps that they inhibit (as indicated in Table 2) (Avila, 1997). Two of these antimitotic agents are already in clinical trials and many of the others listed are being actively investigated as potential anticancer agents. Undoubtedly, some will proceed further to clinical trials. There seems little doubt now that one, or more, of these potent marine natural products (or synthetic analogues derived from them) will emerge in the future as new, therapeutic agents in the fight against cancer.

19

M.H.G. Munro et al. ,Journal of Biotechnology 70 (1999) 15-25

Table 3 Potential supply sources of the marine-derived anticancer compounds Natural sources

Aquaculture/fermentation

Synthesis

Synthesis? a

Didemnins Ecteinascidins Halichondrins

Halichondrins Cryptophycins Thiocoralline

Ecteinascidins Cryptophycins Dolastatin 10 Lamellarins

Halichondrins Eleutherobins Sarcodictyins Discodermolide Aplyronines Spongistatins/ Altohyrtins/ Cinchyrolide

a

The synthesis has been achieved, but yields and number of steps not yet applicable to production.

3. The development phase Given the difficulty in synthesising many of the marine natural products, perhaps the most significant hindrance to their development as drugs, or industrial biocides, is their limited supply. The marine resource offers the biological diversity for sampling in the discovery-phase of new drug development. What is less attractive about marine macroorganisms is the lack of knowledge about obtaining either the organism in bulk, or sourcing the key compound by a routine method. There is no routine, easy source of material for scale-up such as seeding out plantations, or fermentation on a 50 000 L scale as applies to plant, or microbial products. No matter how attractive a biological profile a compound might possess, unless an adequate supply stream can be generated the compound will remain of novelty value only. For example, at the NCI if an adequate initial supply of the compound can be obtained an in vivo active compound can proceed as far as Decision Network IIA (DNIIA), an advanced point in that organisation's preclinical evaluation of anticancer compounds, but unless arrangements can then be made for the purchase or supply of the compound in bulk the compound will not proceed to the next step, DNIIB. Halichondrin B is a good example of a compound in that situation. When a marine natural product succeeds in the development phases, techniques for large-scale commercial supply need to be employed immediately in order to maxirnise the patent, or license investment. Realistically, the bulk supply of

bioactive compounds can only be achieved by harvesting from natural origins, by aquaculture/ fermentation, or by synthesis. Neither tissue culture nor genome transfer from the producing organism to an appropriate vector can be considered as viable supply options at least for the foreseeable future. All but one of the compounds listed in Table 1 have been synthesised (kahalide F), but one has to distinguish between an 'academic' synthesis and an 'industrial' synthesis. The former is where the goal is simply to synthesise the compound, preferably being the first to do so, and by a novel and elegant route. In the latter case the aim is to provide a viable, low-cost synthesis with as few steps as possible. Not all compounds lend themselves readily to synthesis on a large scale. This is usually due either to the complexity of the skeleton, the number of stereogenic centres, or a combination of both. Other compounds, like those of peptide origin, are more amenable to synthesis and compounds such as dolastatin 10 fall into this category. Supply from natural sources by careful harvesting works well for rapidly growing and abundant species. One example of this is the supply of the tunicate-based antimitotic ecteinascidin 743. In other cases where the source organism is rare, or grows only in extreme conditions, then aquaculture might be the only way of obtaining sufficient compound. When an assessment ~ is made of the potential supply source of the current antitumour compounds (Table 1) the following pattern emerges (Table 3). I A personal judgement specialist (MHGM and JWB) assisted by Dr Johnathan Morris, a special synthetic chemist.

20

M.H.G. Munro et al./Journal of Biotechnology 70 (1999) 15-25

Of the compounds in clinical trials, adequate supplies have been obtained from natural sources by harvesting with good husbandry practices, by aquaculture, or by total synthesis. Of the candidates at the pre-clinical phase it would be possible to obtain adequate supplies of halichondrin B by aquaculture methods. This is the first time that a Porifera secondary metabolite has become accessible by aquaculture. The same comment also applies to the production of ecteinascidins and bryostatins, metabolites from the phyla Chordata and Bryozoa, respectively.

4. Production of halichondrin B by aquaculture In work in New Zealand over the past 14 years ~ 6000 samples have been collected and assessed for biological potential. The samples have been mainly sponges and tunicates from sites ranging from Antarctica, through the sub-Antarctic islands, and around the coast line of New Zealand up to the Northern tip of the North Island. The most promising candidate discovered to date is the sponge Lissodendoryx n. sp. 1. The bioactive components in this sponge are a series of compounds belonging to the halichondrin B family, which have now been found in a total of four sponges (the others are Halichondria okadai (Hirata and Uemura, 1986), an Axinella sp. and Phakellia carteri (Pettit et al., 1991, 1993)). Halichondrin B is being assessed by the NCI, while isohomohalichondrin B, a related compound (Litaudon et al., 1994), will be developed independently in Europe by PharmaMar SA. Lissodendoryx n. sp. 1 is a rare, deep water species ( - 80 to - 1 0 0 m) found exclusively off the Kaikoura Peninsula (Page and Battershill, 1998). An extensive environmental survey, conducted using an ROV and a benthic camera, established that the 'sponge field' was only ~ 5 km 2 with the mean biomass and abundance of sponge estimated to be 69 _+ 21 g m - 2 with 1.1 + 0.1 individuals per m 2 over the sponge field. This survey gave an estimated total biomass of the Lissodendoryx sponge of just 289 _+ 90 t total (Dumdei et al., 1998; Page and Battershill, 1998) and established quite unambiguously that the

halichondrins could never be supplied on a commercial scale by collection from the wild. However, based on the results of the survey a permit was obtained and a collection of 1 t made, from which the halichondrins were isolated (310 mg of halichondrin B (NCI) and a comparable amount of isohomohalichondrin B) providing sufficient mass for the initial preclinical trials only. To establish a supply option for the halichondrins the New Zealand National Institute of Water and Atmospheric Research (NIWA), in collaboration with the University of Canterbury and the NCI, have carried out aquaculture feasibility trials at various scales on Lissodendoryx n. sp. 1. There were early indications that one mode of Lissodendoryx reproduction was by fragmentation and advantage was taken of this observation. It was established that small explants were capable of extremely rapid growth (up to 5000% within 1 month) given the correct conditions (Battershill and Page, 1996). Preliminary experiments also established that use of a scallop lantern was feasible as a support and an 'analysis of variance' model was adopted to allow examination of variability of growth and target metabolite production patterns in response to the following factors: season, location, site (within location), and depth. The experiments were run over a period of 18 months. It was quickly apparent that summer transplants were not successful and that while significant growth of the explants was observed at the deep sites this was short-lived, as all sponges succumbed to bryozoan overgrowth and pathogen attack. Mortality was over 95%. In contrast, the winter explants (April) at all sites were generally successful (mortality less than 15%), especially at the greater depths, with the same previously observed growth rates observed through to December, when a decline was observed as fouling again became excessive. The mortality rate of the sponges was high in summer, especially at shallower depths, and a critical temperature of 18~ has been identified above which the sponge will not survive (Dumdei et al., 1998). The next most important question after getting the sponges to grow and survive was whether or not the halichondrins were being produced under these conditions. Samples of the biomass were

M.H.G. Munro et al. /Journal of Biotechnology 70 (1999) I5-25

21

There was a need, however, to scale-up experimentation to a level that would simulate commercial production conditions. This was carried out in late 1997 using a variety of culture methods. The selection of culture methods was dictated by consideration of those approaches that could ultimately be most readily converted to a large-scale mechanised operation. Wild samples of Lissodendoryx n. sp. 1 were collected from the sponge field off Kaikoura and immediately transferred to aerated buckets and flown by light aircraft to Wellington before deployment. Explants (8 cm 3) were allocated at random into the following treatments: lantern, tray, bag or disc, all at 10 m depth. The lanterns used were commercially available scallop lanterns 1 m in diameter, with ten tiers supported by wire hoops and covered in a 1.5 cm nylon mesh. Tray culture represented a prototype sponge deployment system which essentially held explants in a vertically aligned nylon mesh sandwich. Bag treatments similarly represented a prototype for a continuous stocking type culture system and finally, discs held sponges in a clutch-type culture array where explants were suspended on ropes without any surrounding mesh. The explants were monitored for three months. Growth and mortality were measured in situ. Fig. 4 shows the changes in volume (directly proportional to weight in this species).

taken at regular intervals across all sites and extracted and examined by bioactivity assays and HPLC for the presence of the various halichondrins. From the bioactivity profiles it could be established that halichondrins were being produced and by HPLC analysis of five bulk samples from two sites at differing depths the production and profile of the halichondrin production was established. Wild samples of the sponge typically contain ~ 400 gg k g - 1 of halichondrin B, ~ 200 lag kg-1 of homohalichondrin B and ~ 900 lag kg-1 of isohomohalichondrin B. The overall halichondrin content of the cultured sponge was not as high as that of the wild sponge, and the production of the individual halichondrins was site dependent (see Fig. 3) (Dumdei et al., 1998). The relative production of halichondrin B in the cultured samples was generally higher than that found for homohalichondrin B and isohomohalichondrin B and, depending on the site, the figure for the total production of halichondrins per kg of sponge ranged from 30 to 60% of that found in the wild sponge at four of the sites surveyed. This is a significant rate of production, especially when rate of growth is taken into account. These initial experiments showed that the sponge could be grown successfully in small-scale trials and that the halichondrins continued to be biosynthesised, even after several years of culture. 1600

Isohomohalicl~onddn-B~ IIHomohalichondrinB

m

1400 t I

[] Halichondrin B

1200--~ 1000 -~ Halichondrin Levels (microg/kg sponge)

800 600 . 400 '

200~ I --

0i Wild

Beatrix

Sponge Shallow

l

Beatrix Mid

13

Beatrix Deep

L_.M

Akaroa Akaroa Shallow Deep

Site Fig. 3. Halichondrin levels in Lissodendorvx samples.

"

.

.

.

.

.

.

.

.

M.H.G. Munro et al. /Journal of Biotechnology 70 (I 999) 15-25

22

6o

i

5O

4O Volume (mL)

30

/

/t"

10 o

~

. . . . . .

4 .

0

.

.

~

"~,~-~~

~"

--4-- Lantern l Tray - Bag I .--o- -Disc i

-~~k.

-

-o-

-

-"

<>" " - "--- .

.

.

~

. ~..

.

26

~

~:~. .

43

I .

.

.

57

79

Days in Culture

Fig. 4. G r o w t h rate as a function of culture substrate.

Sponges cultured in trays, or on discs did not grow well. In both cases, fouling became a significant problem as the plastic component of the support structures appeared to promote settlement of fouling species, predominantly Bugula flabellata. The best initial growth rate was observed in lantern cultures, but after 26 days sponges were observed to lose weight and condition. It was apparent that the enclosed nature of the apparatus retarded growth after a certain size was obtained and that the lanterns also soon became severely fouled. The bag-type culture system proved to be the best both in terms of promoting fast growth, as well as maintaining growth. Explants quickly grew through the mesh and were soon able to grow uninhibited, using the overgrown bag for support. This culture system was also the most readily adaptable to a mechanised system and could be developed to a largescale stocking-type culture system, not unlike traditional mussel-seeding aquaculture. This suite of experiments was carried out in summer. This is usually a period through which the sponge does not grow in nature and hence represents a worst-case example of the potential of sponge aquaculture. None the less it was established that in less than 26 days sponge explants could quadruple their volume and weight. In

other experiments, carried out at the same time but at different localities, explants were observed growing to 120 cm 3 in less than 30 days, clearly indicating that there will be locations which are more suitable for growing sponges (Battershill, unpublished data). These results show that aquaculture of sponges is a viable and reliable option for creating extractable biomass. Very preliminary estimates of biomass production suggested that the annual production of 5 t of sponge per 100 m of longline is achievable (Dumdei et al., 1998). These estimates were based on use of scallop lanterns, good husbandry to reduce overgrowth, two harvests a year, mortality restricted to < 50% and growth of 8 cm 3 explants to ~ 300-400 g. The use of stocking-type systems as opposed to scallop lanterns will influence both the likely yields as well as the economics of production. The economics of production is likely to be very competitive as the mechanised technology is already available for seeding and harvest using stocking type systems. In addition, sponges may be readily grown with other species such as mussels in successful polyculture operations. If the halichondrins progress satisfactorily through preclinical trials over the next year, gram quantities of halichondrin B and isohomohali-

23

M.H.G. Munro et al./Journal of Biotechnology 70 (1999) 15-25

chondrin B will be required. This equates to tonnes of sponge which can only be supplied by aquaculture. Harvesting at that scale from the limited wild-stock is not possible. The supply of this quantity of the compounds will give the opportunity to estimate more accurately the actual production parameters and economics of the production of Lissodendoryx n. sp. 1 by aquaculture. Should either halichondrin B, or isohomohalichondrin B then proceed through clinical trials and become established as pharmaceuticals the estimated amount of each compound required annually is ~ 5 kg. This estimate is based on the potency of the halichondrins in in vivo animal trials and projected dose regimes and corresponds to the annual production of at least 5000 t of Lissodendoryx n. sp.

5. F u t u r e trends

The development of a successful pharmaceutical requires that attention also be paid to delivery of the drug as well as supply. The antiproliferative properties of today's anticancer compounds will never overcome solid tumours because of the sensitivity of the surrounding tissue to the fatal effects of exposure to the compounds. This limits the use of high concentrations. What is required are alternative approaches that facilitate the specific targeting of tumours. One approach, known as polymer therapeutics, is a rapidly growing multidisciplinary field requiring the combined talents of organic chemists, polymer chemists, pharmacologists and oncologists (Duncan, 1992). The concept behind polymer therapeutics is shown schematically in Fig. 5. The drug is attached via a biodegradable linker to a water soluble polymer. In other, optional approaches, specific targeting residues can also be added. Polymer therapeutics not only offers improved pharmacokinetic properties, but better targeting of tumour tissue and higher selectivity. The basis for this better targeting and selectivity operates by what is known as the 'enhanced permeability and retention' effect (EPR) which leads to higher concentrations of the anticancer agent within the tumour. In vivo trials have established the success

of this approach. Two such drugs currently undergoing phase I/II clinical trials are PK1 and PK2 where the anticancer drug doxorubicin has been attached via a tetrapeptide linker to a watersoluble hydroxypropyl-methacrylamide (HPMA) polymeric backbone. The tetrapeptide linker was designed to resist peptidase activity in the bloodstream, but be susceptible to lysomal enzymatic hydrolysis following the transfer by endocytosis to the interior of the target tumour cells (Duncan et al., 1996). Polymer therapeutics is an ideal approach to enhancing the value of marine toxins. Compounds like halichondrin B are already established by in vivo trials as effective agents which can be transferred intravenously to remote sites within the test animal and inhibit the growth of a range of human tumour types. Any modifications that can enhance pharmacokinetic properties, reduce required plasma concentrations and exhibit enhanced selectivity can only be considered advantageous. To this end we are working in collaboration with the NCI and the London School of Pharmacy on the development of a polymeric therapeutic based on the halichondrin skeleton. An amino derivative of the halichondrin skeleton has been synthesised and converted into a polymeric form comparable to PK1 and is currently undergoing in vitro testing against a range of human tumour cell lines. Polymer

Bond to be cleaved Backbone 1 < Biodegradable spacer DRUG

(

9 peptidyl spacer designed for enzymic cleavage.

< < <

(

~>

Spacer . . . . . .

'------!

.....i Targeting Residue

9access to target receptor

Fig. 5. Schematic showing basic features of a polymer therapeutic drug.

24

M.H.G. Munro et al. / Journal of Biotechnology 70 (1999) 15-25

Ultimately, this approach will be applied to other marine toxins such as the mycalamides (Perry et al., 1988), the discorhabdins (Perry et al., 1986), pateamine (Northcote et al., 1991), calyculinamides and swinholides (Dumdei et al., 1997) that we have isolated from New Zealand marine organisms. The interest in the swinholides and calyculinamides will centre on the attempt to develop these two toxins into in vivo active polymeric drugs. Both classes of compound have potent activities in vitro, but are inactive in vivo. The swinholides disrupt the formation of actin filaments while the calyculinamides are protein phosphatase 2A inhibitors. These biological profiles complement the antimitotic properties of our initial polymeric drug based on aminohalichondrin.

6. Conclusion The sea offers a rich source of biodiversity from which a series of potential drugs, particularly in the area of cancer chemotherapy, have already been discovered. That aspect of the drug development process is dominated by chemists, zoologists and biochemists. The development phase, where there is the pressing need for supply of these compounds, will need strong leadership from the marine biotechnologists as not all marine-based drugs will be able to be synthesised, or obtained by fermentation technology. There will be a need for new, innovative approaches to the aquaculture of many species from phyla that traditionally have not been subject to aquaculture. For those compounds that do become marketable drugs there will exist the challenge to grow the producing organisms commercially by aquaculture. The chemists will continue to find new leads. Can the biotechnologists produce the compounds economically? That is the challenge for the future.

References Avila, J., 1997. The use of microtubule poisons on tumor cells. Cancer J. 10, 6.

Battershill, C.N., Page, M.J., 1996. Sponge aquaculture for drug production. Aquac. Updates 16, 5-6. Bergman, W., Feeney, R.J., 1951. Nucleosides of sponges. J. Org. Chem. 16, 981-987. Devlin, J.P., 1997. Chemical diversity and genetic equity: synthetic and naturally derived compounds. In: Devlin, J.P. (Ed.), High Throughput Screening. Dekker, New York, pp. 3-48. Dumdei, E.J., Blunt, J.W., Munro, M.H.G., Pannell, L.K., 1997. Isolation of calyculins, calyculinamides, and swinholide H from the New Zealand deep-water marine sponge Lamellomorpha strongylata. J. Org. Chem. 62, 2636-2639. Dumdei, E.J., Blunt, J.W., Munro, M.H.G., Battershill, C.N., Page, M.J., 1998. The whys and whats of sponge chemistry: why chemists ~extract sponges and what problems does this cause? In: Watanabe, Y., Fusetani, N. (Eds.), Sponge Sciences. Multidisciplinary Perspectives. Springer, Tokyo, pp. 353-364. Duncan, R., 1992. Drug-polymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs 3, 175-210. Duncan, R., Dimitrijevic, S., Evagorou, E.G., 1996. The role of polymer conjugates in the diagnosis and treatment of cancer. STP Pharma Sci. 6, 237-263. Fenical, W., 1996. Marine biodiversity and the medicine cabinet. The status of new drugs from marine organisms. Oceanography 9, 23-27. Garson, M.J., 1994. The biosynthesis of sponge secondary metabolites: why it is important. In: van Soest, R.W.M., van Kempen, T.M.G., Braekman, J-C. (Eds.), Sponges in Time and Space. Balkema, Rotterdam, pp. 427-440. Hirata, Y., Uemura, D., 1986. Halichondrins-antitumor polyether macrolides from a marine sponge. Pure Appl. Chem. 58, 701-710. Litaudon, M., Hart, J.B., Blunt, J.W., Lake, R.J., Munro, M.H.G., 1994. Isohomohalichondrin B: a new antitumour polyether macrolide from the New Zealand deep-water sponge Lissodendoryx sp. Tetrahedron Lett. 35, 94359438. MarinLit, 1998. A marine literature database maintained by the Marine Chemistry Group, University of Canterbury, Christchurch, New Zealand. May, R.M., 1988. How many species are there on earth? Science 241, I441-1449. Munro, M.H.G., Luibrand, R.T., Blunt, J.W., 1987. The search for antiviral and anticancer compounds from marine organisms. In: Scheuer, P.J. (Ed.), Bioorganic Marine Chemistry, vol. 1. Verlag Chemie, Berlin, pp. 93176. Northcote, P.T., Blunt, J.W., Munro, M.H.G., 1991. Pateamine: a potent cytotoxin from the New Zealand marine sponge, Mycale sp. Tetrahedron Lett. 32, 64116414. Olivera, B.M., 1997. Conus venoms: a highly evolved neuropharmacological strategy. The 9th NAITO Conference on Bioactive Natural Products and Their Modes of Action. October 15-18. Shonan Village Center, Kanagawa, Japan, pp, 30-31.

M.H.G. Munro et al. / Journal of Biotechnology 70 (1999) 15-25

Page M.J., Battershill C.N., 1998. Ecology and impact of harvesting the deep water sponge Lissodendoryx n.sp.1 (Poecilosclerida; Myxillidae) on the continental shelf adjacent to the Kaikoura Peninsula, New Zealand. Mar. Ecol. Prog. Ser. (submitted). Perry, N.B., Blunt, J.W., McCombs, J.D., Munro, M.H.G., 1986. Discorhabdin C; a highly cytotoxic pigment from a sponge of the genus Latrunculia. J. Org. Chem. 51, 54765478. Perry, N.B., Blunt, J.W., Munro, M.H.G., Pannell, L.K., 1988. Mycalamide A, an antiviral compound from a New Zealand sponge of genus Mycale. J. Am. Chem. Soc. 110, 4850-4851. Pettit, G.R., Herald, C.L., Boyd, M.R., Leet, J.E., Dufresne, C., Doubek, D.L., Schmidt, J.M., Cerny, R.L.J., Hooper,

25

N.A., Rutzler, K.C., 1991. Antineoplastic agents. 219. Isolation and structure of the cell growth inhibitory constituents from the Western Pacific marine sponge Axinella sp. J. Med. Chem. 34, 3339-3340. Pettit, G.R., Tan, R., Gao, F., Williams, M.D., Doubek, D.L., Boyd, M.R., Schmidt, J.M., Chapius, J-C., Hamel, E., Bai, R., Hooper, J.N.A., Tackett, L.P., 1993. Isolation and structure of halistatin 1 from the Eastern Indian Ocean marine sponge Phakellia carteri. J. Org. Chem. 58, 25382541. Schmitz, F.J., 1994. Cytotoxic compounds from sponges and related microfauna. In: van Soest, R.W.M., van Kempen, T.M.G., Braekman, J-C. (Eds.), Sponges in Time and Space. Balkema, Rotterdam, pp. 485-496.

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JOURNAL

ii

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Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 27-32

Microbial antagonism" a neglected avenue of natural products research J. Grant Burgess *, Elizabeth M. Jordan, Migena Bregu, Andrew Mearns-Spragg, Kenneth G. Boyd Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK Received 19 November 1998; received in revised form 10 December 1998; accepted 22 December 1998

Abstract Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces. We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates. Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds. This is a much higher proportion than free living marine isolates or soil bacteria. In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals. Thus the number of strains able to produce antibiotics appears to be much higher than previously thought. Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed. 9 1999 Elsevier Science B.V. All rights reserved.

Keywords: Marine epibiotic bacteria; Microbial antagonism; Antibiotic

1. Introduction

For the past 50 years antibiotics have revolutionised medicine by providing cures for formerly life-threatening diseases. However, strains of bacteria have recently emerged that are virtually un* Corresponding author. Tel.: + 44-131-4513187; fax: + 44131-4513009. E-mail addresses: [email protected] (J.G. Burgess)

responsive to antibiotics. Such multidrug resistance, arising mainly through antibiotic misuse, is now recognised as a global health problem. The situation is exacerbated by the fact that no novel chemical class of antibiotics have been discovered for 20 years. A l t h o u g h m a n y pre-existing antibiotics have been modified to yield new derivatives, bacteria have the potential to m u t a t e k n o w n resistance mechanisms to c o m b a t these (Hancock, 1997; Knowles, 1997; Levy, 1998). Par-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00054-1

J.G. Burgess et al./Journal of Biotechnology 70 (1999) 27-32

28

ticular strains which are causing problems at the moment are the vancomycin-resistant enterococci (VRE) (Frieden et al., 1993; Leclerc, 1997) and methicillin resistant Staphylococcus aureus (MRSA). It is clear that new classes of antibiotics are urgently needed. Many marine free-living and sediment-inhabiting marine bacteria have been shown to produce secondary metabolites that display antibacterial properties (Burgess et al., 1991). The first antibiotic from a marine bacterium was identified and characterised in 1966 (Burkholder et al., 1966). In addition, bacteria in biofilms on the surfaces of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Lemos et al., 1986). Marine epiphytic bacteria, associated with nutrient-rich algal surfaces and invertebrates, have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors (Jensen and Fenical, 1994; Bernan et al., 1997). A number of surface-associated marine bacteria have also been found to produce antibiotics. Trischman et al. (1994) isolated a species of Streptomyces from the surface of a jellyfish, which produced two new bicyclic peptides (salinamides A and B). These compounds have novel backbones, and exhibit activity against an array of Gram positive organisms. Furthermore, a species of Bacillus isolated from a marine worm in Papua New Guinea produced a novel cyclic decapeptide antibiotic, loloatin B, which inhibited the growth of MRSA and VRE (Gerard et al., 1996). A new bacterium Pelagiobacter variabilis from a

seaweed Pocockiella variegata was found to produce new phenazine antibiotics, termed pelagiomicins which exhibited activity against Gram positive and Gram negative bacteria (Imamura et al., 1997). Recently, the marine bacterium AIteromonas rava sp. nov. was found to produce a new antibiotic thiomarinol (Shiozawa et al., 1993). Thus, marine epibiotic bacteria can provide novel antimicrobial compounds. However, in order to find more novel structures, new ways of screening for these compounds must be applied. We have noticed in our studies that certain strains of bacteria can be induced to produce antibiotics (Mearns-Spragg et al., 1997, 1998). They appear to be doing so in response to chemical signals received from potential competitor strains which elicit an antagonistic response. Such a response, which is inducible rather than constitutive, has been observed in other species of bacteria (Patterson and Bolis, 1997) but remains a little studied phenomenon. In this work we have screened marine epiphytic bacteria, isolated from the surfaces of seaweeds, starfish and nudibranchs for production of secondary metabolites with antimicrobial activities against multidrug resistant pathogens. We have also investigated the ability of live cells and various chemicals to elicit or enhance antibiotic production in these strains.

2. Materials and methods

2.1. Growth and culture conditions

Clinical isolates were grown on Columbia horse

Table 1 Zone(s) of inhibition against pathogenic bacteria, following antibiotic disc diffusion tests using extracts from epiphytic bacteria isolated from the brittle star Marine strain

BS-3A BS-4 BS-5A BS-8B

Test strain [zone of inhibition (mm)] EMRSA

VRE

Acinetobacter spp.

P. aeruginosa

7 9 0 8

0 0 7 0

0 0 0 0

0 0 0 0

J.G. Burgess et al./Journal of Biotechnology 70 (1999) 27-32

29

Table 2 Inhibition of pathogenic bacteria, following antibiotic disc diffusion, using epiphytic bacteria isolated from the common starfish Marine strain

SV-2 SV-6 SV-10 SV-11

Test strains [zone of inhibition (mm)] EMRSA

VRE

Acinetobacter spp.

P. aeruginosa

7 0 0 8

0 0 7 0

0 0 0 0

0 7 0 0

blood agar, marine strains were grown on the following media; 50% Marine agar (MA) made with Marine Broth 2216 (Difco, Detroit). Samples of various seaweed and invertebrate species were collected at low tide. For isolation of epiphytic bacteria, the sample was washed three times in sterile seawater to remove loosely attached bacteria. The sample was then placed in another 10 ml of sterile seawater and vortexed to remove more tightly bound epiphytes. Cell suspensions obtained in this way were used to inoculate agar plates of 50% MA. Plates were then incubated (25~ 5 days) and representative colonies were picked and isolated by successive restreaking.

2.2. Test strains The following test strains were used; EMRSA (Epidemic MRSA strain) VRE, Pseudomonas aeruginosa, Enterococcus faecium, A cinetobacter spp. All test strains were isolated from hospitalised patients and were a kind gift from Edinburgh University Medical Microbiology laboratories. Each of the strains were maintained on Columbia horse blood agar medium at 37~ and re-plated every 2 weeks.

2.3. Antibiotic assays All bacterial strains isolated were screened for production of antibacterial substances, using the disc diffusion method, and methods for induction of antibiotic production as previously described with Bacillus subtilis used as the test strain in order to test additional Gram positive species

(Mearns-Spragg et al., 1998). Out of all tested strains, five strains that showed good antimicrobial activity were selected for these induction studies.

3. Results

3.1. Antimicrobial activity of surface-associated strains The marine strains MH22, isolated from the seaweed Fucus vesiculosus, and FS-25, FS-28 and FS-30, isolated from the seaweed F. serratus, displayed good antagonistic activity against VRE and Enterococcus faecium. However, only the MH22 strain displayed antibacterial activity against VRE and P. aeruginosa, following the disc diffusion test, with 8 mm zones of inhibition. Of the strains of bacteria isolated from the surface of the seaweed Corallina officinalis, only one strain, MBCoral-6A, displayed antibiotic activity, with an 8 mm zone of inhibition against the pathogen MRSA. Strains isolated from the brittle star, Ophiothrix fragilis (Table 1), and the common starfish Asterias rubens (Table 2), displayed the greatest activity against an epidemic strain of methicillin resistant S. aureus (MRSA) and vancomycin resistant enterococcus (VRE). The largest zone of inhibition against MRSA (9 mm) was exhibited by the brittle star strain BS-4. The percentage of antibiotic producing strains from the seaweed, C. officinalis, the brittle star and the Common

J.G. Burgess et al./Journal of Biotechnology 70 (1999) 27-32

30

starfish, was 4.8, 17.4 and 26.7%, respectively (Table 3). In addition to the strains described here, an additional 400 strains were screened and the overall hit rate for antimicrobial activity was 35% (results not shown). Despite this high overall hit rate, isolates from the sea slug, Archidoris pseudoargus, did not show any antimicrobial activity (Table 3).

3.2. Character&ation of producer stra&s Strain MBCoral 6A grew as a yellow pigmented bacterium on 50% MA and was further characterised as a Gram positive coccus which formed chains. It is also catalase and oxidase positive. Strain BS-4 was a non-pigmented bacterium, forming white pinprick colonies on 50% MA. It was a Gram positive coccus, forming bunches, was catalase positive and oxidase negative. Strain BS-8B was also a non-pigmented bacterium identified as a Gram negative rod, which was oxidase and catalase negative.

3.3. Induction of antibiotic activity by culture supernatants. Marine epibiotic bacteria enhanced their production of antibiotics, when exposed to bacterial cell-free supernatants. Four out of five strains tested, showed enhanced antimicrobial activity against at least one of the pathogenic organisms, when cell-free supernatants were introduced in the growth medium (data not shown). The induced activity against B. subtilis of two marine strains (MH46, a Fucus epiphyte, and SSE20, an isolate from the surface of the sponge Halichondria sp.) is shown in Fig. 1. Antibacterial activity against B.

subtilis was enhanced in both strains. In the case of MH46, supernatants from the marine epiphytes MH3, MH2 and MH1 showed the greatest ability to elicit production of antimicrobial compounds. Supernatants from all three marine epiphytes also increased antibiotic production by strain SSE20. MH46 did not show any antimicrobial activity under control growth conditions, but the addition of bacterial cell-free supernatants induced the production of antibacterial compounds by these strains. SSE20 was active against B. subtilis under normal conditions, however this antimicrobial activity was significantly enhanced when exposed to cell-free supernatants from the inducer strains. SSE20 showed enhanced antimicrobial activity against B. subtilis when challenged with Bacillus cell-free supernatant in their growth medium.

4. Discussion

In this work we have shown that bacteria associated with starfish and seaweed have the ability to produce antimicrobial compounds against other marine bacteria and also against terrestrial pathogenic bacteria. We have also demonstrated that addition of culture supernatants can elicit antibiotic production in marine surface-associated bacteria. It had previously been concluded that antibiotic-producing marine bacteria were often pigmented although this conclusion was reached after mainly testing of pigmented colonies (Lemos et al., 1986). Therefore, as the selection of colonies for testing was not carried out randomly. In this study a random selection of strains were tested, and the most potent antibiotic-producers found to be non-pigmented.

Table 3 Numbers and percentages of the bacterial strains, isolated from different organisms, that display antibiotic activity Organism

No. of strains isolated

No. of antibiotic producersstrains isolated

% Antibiotic producing strains

A. pseudoargus (sea slug) C. officinalis (seaweed) O. fragilis (brittle star) A. rubens (common starfish)

19 21 23 15

0/19 1/21 4/23 4/15

0 4.8 17.4 26.7

J.G. Burgess et al./Journal of Biotechnology 70 (1999) 27-32

Strain r

,$=e

~-

= '~"

o

O

|- 9 ' = |

E

31

MH46

8.5 8 7.5

7

5 .s = 6.56 i5

[--'3

.....

,r---],l

I

i I,

,

cont E.coli B.sub P.aur MH3 MH2 MH1

Ind uce r stra ins

,.=~' o

" E ~

,_~

12

10 o

8+l

6 jl--l,

oot

Eozl

Bs.b

P.aLr M-B

M-12

M-I1

Irducer slraim

Fig. 1. Effect of cell-flee supernatants on antibiotic production by marine strains MH46 and SSE20 after 3 days of incubation with extracts from other species of bacteria. The test organism is B. subtilis.

The standard filter paper (Whatman, 6 mm) used for the antibiotic disc assays is composed of cellulose (i.e. 13(1-4) linked glucose monomers). The many free hydroxyl groups present on each of the glucose residues, renders the surface of the disc hydrophilic (Braithwaite and Smith, 1990). Thus, if the compounds produced by the marine bacteria were cationic, they would be expected to adsorb to the surface of the disc, and not diffuse into the medium. Consequently, a strain may produce antibiotic compounds that are polar in nature, but which are therefore not noticeably antibacterial by the paper disc diffusion method. Thus the use of different disc materials may allow additional compounds to be discovered. Echinoderms, such as the brittle star and common starfish, are able to produce antifouling substances that prevent adhesion of micro-organisms, and hence formation of biofilms (McKenzie and Grigolava, 1996). Sub-cuticular bacteria (SCB),

the bacteria living in the spaces between the cuticle and epidermis of Echinoderms, have also been isolated (McKenzie and Kelly, 1994). It is interesting to note in this work that some strains appear to be associated with the surfaces of echinoderms, despite the production of antifouling substances by their hosts. In addition to being resistant against these antifouling substances, these strains were shown to possess antimicrobial properties themselves. In conclusion, in addition to showing that echinoderm and seaweed associated bacteria are able to produce antimicrobial compounds, we have confirmed earlier observations that bacteria can elicit antimicrobial responses in different species in a way which we do not yet understand. It is interesting to speculate that this response represents a chemically induced defence response when the antibiotic producer strain is faced with a potential competing organism in the marine envi-

32

J.G. Burgess et al./Journal of Biotechnology 70 (1999) 27-32

r o n m e n t . C u r r e n t l y we are d e t e r m i n i n g the chemical s t r u c t u r e o f these a n t i m i c r o b i a l c o m p o u n d s .

Acknowledgements This w o r k was f u n d e d in p a r t f o r m a g r a n t

from the N a t u r a l E n v i r o n m e n t R e s e a r c h Council, CS was f u n d e d by a W e l l c o m e T r u s t V a c a t i o n Scholarship. T h e a u t h o r s also t h a n k the British C o u n c i l ( T o k y o ) for financial s u p p o r t . W e also t h a n k Professors Sebastian A m y e s a n d J o h n G o v a n o f the D e p a r t m e n t o f M e d i c a l M i c r o b i o l o g y , U n i v e r s i t y o f E d i n b u r g h , for p r o v i s i o n of clinical isolates, s u p p o r t a n d advice.

References Bernan, V.S., Greenstein, M., Maiese, W.M., 1997. Marine micro-organisms as a source of new natural products. Adv. Appl. Microbiol. 43, 57-89. Braithwaite, A., Smith, F.J., 1990. Chromatographic Methods. Chapman and Hall, London. Burkholder, P.R., Pfister, R.M., Leitz, F.P., 1966. Production of a pyrrole antibiotic by a marine bacterium. Appl. Microbiol. 14, 649-653. Burgess, J.G., Miyashita, H., Sudo, H., Matsunaga, T., 1991. Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores. FEMS Microbiol. Lett. 84, 301 - 306. Frieden, T.R., Munsiff, S.S., Low, D.E., Willey, B.M., Williams, G., Faur, Y., Eisner, W., Warren, S., Kreiswirth, B., 1993. Emergence of vancomycin-resistant enterococci in New York city. Lancet 342, 76-79. Gerard, J., Haden, P., Kelly, M.T., Andersen, R.J., 1996. Loloatin B, a cyclic decapeptide antibiotic produced in culture by a tropical marine bacterium. Tetrahedron Lett. 37, 7201-7204. Hancock, R.E.W., 1997. The role of fundamental research and biotechnology in finding solutions to the global problem of antibiotic resistance. Clin. Infect. Dis. 24 (Suppl 1), S 148S150.

Imamura, N., Nishijima, M., Takadera, T., Adachi, K., 1997. New anticancer antibiotics pelagiomicins, produced by a new marine bacterium Pelagiobcter variabilis. J. Antibiot. 50 (1), 8-12. Jensen, P.R., Fenical, W., 1994. Strategies for the discovery of secondary metabolites from marine bacteria: ecological perspectives. Annu. Rev. Microbiol. 48, 559-584. Knowles, D.J.C., 1997. New strategies for antibacterial drug design. Trends Microbiol. 5 (10), 379-383. Leclerc, R., 1997. Enterococci acquire new kinds of resistance. Clin. Infect. Dis. 24 (Suppl 1), 80-83. Lemos, M.L., Toranzo, A.E., Barja, L.J., 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microb. Ecol. 11, 149-163. Levy, S.B., 1998. The challenge of antibiotic resistance. Sci. Am. 278 (3), 32-39. McKenzie, J.D., Kelly, M.S., 1994. A comparative study of sub-cuticular bacteria in brittle stars. Mar. Biol. 120, 6580. McKenzie, J.D., Grigolava, I.V., 1996. The echinoderm surface and its role in preventing microfouling. Biofouling 10 (1-3), 261-272. Mearns-Spragg, A., Boyd, K.G., Hubble, M.O., Burgess, J.G., 1997. Antibiotics from surface-associated marine bacteria. Proceedings of the Fourth Underwater Science Symposium, Society for Underwater Technology, London, ISBN 0906940311, pp. 147-157. Mearns-Spragg, A., Bregu, M., Boyd, K.G., Burgess, J.G., 1998. Cross-species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Lett. Appl. Microbiol. 27, 142-146. Patterson, G.L., Bolis, C.M., 1997. Fungal cell-wall polysaccharides elicit an antifungal secondary metabolite (phytoalexin) in the cyanobacterium Scytonema oeellatum. J. Phycol. 33, 54-60. Shiozawa, H., Kagasaki, T., Kinoshita, T., Haruyama, H., 1993. Thiomarinol, a new hybrid antimicrobial antibiotic produced by a marine bacterium. J. Antibiot. 46, 18341841. Trischman, J.A., Tapiolas, D.M., Jensen, P.R., Dwight, R., Fenical, W., Mckee, T.C., Ireland, C.M., Stout, T.J., Clardy, J., 1994. Salinamide-A and salinamide-B-anti-inflammatory depsipeptides from a marine streptomycete. J. Am. Chem. Soc. 116, 757-758.

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 33-38

Screening of marine microalgae for bioremediation of cadmium-polluted seawater Tadashi Matsunaga *, Haruko Takeyama, Takashi Nakao, Akira Yamazawa Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan Received 21 October 1998; received in revised form 19 November 1998; accepted 22 December 1998

Abstract

Twenty four strains out of 191 marine microalgal strains exhibited cadmium (Cd) resistance. They were tested for their Cd removal ability in growth media containing 50 laM Cd. Six strains out of 19 green algae and one out of five cyanobacteria removed more than 10% of total Cd from the medium. The marine green alga Chlorella sp. NKG16014 showed the highest removal of Cd 48.7% of total. Cd removal by NKG 16014 was further quantitatively evaluated by measuring the amount of cell adsorption and intracellular accumulation. After 12 days incubation, 67% of the removed Cd was accumulated intracellularly and 25% of the Cd removed was adsorbed on the algal cell surface. The maximum Cd adsorption (qm~x) was estimated to be 37.0 mg Cd (g dry cells)- ~ using the Langmuir sorption model. The Cd removal by freeze-dried NKG16014 cells was also determined. Cd was more quickly adsorbed by dried cells than that by living cells, with a qmax of 91.0 mg Cd (g dry cells)- 1. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Bioremediation; Cadmium; Screening; Marine microalgae; Marine Chlorella; Adsorption

1. Introduction In terms of biomass, microalgae form the world's largest group of primary producers. They play an important role in the conversion of solar energy into chemical energy through their photosynthetic activities. Photosynthetic conversion can be expected to be an efficient and alternative

* Corresponding author. Tel.: + 81-42-388-7020; fax: + 8142-385-7713. E-mail address: [email protected] (T. Matsunaga)

process in several industrial fields. Unique algal strains have been screened from marine environments for fine chemicals. Furthermore, they have been considered to play a promising role in environmental protection. Several marine microalgal strains which produce useful metabolites have been described, for example, novel plant growth regulators which promote redifferentiation, germination and plantlet formation (Wake et al., 1992); tyrosinase inhibitors (Wachi et al., 1995a); sulfated polysaccharide showing anti-HIV activity (Sudo et al., 1995); novel antibiotics with light regulated activity (Miura et al., 1993a); a UV-A

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 5 5 - 3

34

T. Matsunaga et al./Journal of Biotechnology 70 (1999) 33-38

absorbing compound (Wachi et al., 1995b) and ?-linolenic acid (Miura et al., 1993b). Marine rnicroalgae have been utilized for bioremediation against CO2 (Takano et al., 1992,) and heavy metal removal. Increasing environmental pollution by heavy metals results from their increasing utilization in industrial processes (Nriagu and Pacyna, 1988). Heavy metal pollution of the marine environment has become an issue of major concern, because most of the heavy metals are transported into the marine environment and accumulate without decomposition. Cadmium (Cd) has been used in such processes as electroplating and alloy preparation and resulting soil and ground water contamination leads to accumulation in plants, marine plankton and invertebrates. Cd bioaccumulation has been studied in bacteria (Trevors et al., 1986; Volesky and Holan, 1995), cyanobacteria (Les and Walker, 1984; Inthorn et al., 1996), algae (Sakaguchi et al., 1979; Geisweid and Urbach, 1983; Holan et al., 1993) and fungi (Fourest and Roux, 1992; Fourest et al., 1994; Holan and Volesky, 1995). Several algae have been used for elucidating the mechanisms for Cd accumulation (Les and Walker, 1984; Holan et al., 1993; Wilde and Benemann, 1993). Cd uptake occurs in two stages: short-term uptake involves physical sorption on the cell surface; and long-term uptake involves intracellular accumulation (Khummongkol et al., 1982). Uptake kinetics have been reported for the green algae Chlorella vulgaris, Akistrodesmus braunii and Eremosphaera viridis and their Cd adsorption abilities were evaluated using Langmuir sorption isotherms (Geisweid and Urbach, 1983). A brown seaweed Ascophyllum nodosum was found to be readily capable of Cd sequestering and the maximum Cd removal by homogenized and dried A. nodosum was calculated to be 215 mg Cd per g dry weight (Holan et al., 1993). This higher Cd adsorption was attributed to A. nodosum polysaccharide. In this paper, we describe screening of marine microalgae for Cd removal. Cd uptake by several strains was characterized and the maximum adsorption of Cd by the living or non-living cells was determined.

2. Materials and methods

2.1. Screening of Cd resistant marine microalgae Axenic marine microalgae from the culture collection of Department of Biotechnology, Tokyo University of Agriculture and Technology (composed of green algal and cyanobacterial isolates) were used in this work. They were cultured in BGll medium (2% NaC1) (ATCC catalogue, medium no. 617), 1/10 • A medium (2% NaC1) (Kumazawa and Mitsui, 1981) or seawater-based Eppley's medium (Eppley et al., 1967) under the light intensity of 50 ~tE m - 2 s - ~ at room temperature. A total of 191 strains (104 marine cyanobacterial strains and 87 marine green algal strains) were tested for Cd resistance. Cd in Cd(NO3)2 was used for all experiments. Primary screening for Cd resistance was performed using various Cd concentrations (0, 0.2, 2, 20, 50, 100, 500, 1000 ~tM Cd) in microtitre 96-well plates. Cell growth under each Cd concentration was evaluated by eyes: a strain in a well showing coloration after 2 weeks cultivation was denoted as resistant. 2.2. Cd removal from the medium To determine strains showing highest capability of Cd removal, strains showing Cd resistance in primary screening were cultured in 5 ml medium and Cd removal was evaluated. Evaluation of Cd removal was carried out by measuring the change in Cd concentration in the supernatant of the culture medium (50 pM Cd) after 2 weeks cultivation. The concentration of Cd was measured by atomic absorption spectrophotometry (AA-6600, Shimadzu). The Cd removal rate (%) was calculated as follows: (amount of removed Cd)/ (amount of initial Cd)x 100. Cell growth, the amount of intracellular Cd and Cd on the cell surface were investigated in a strain showing highest capability of Cd removal. Cells grown in 100 ml medium to mid-logarithmic phase were harvested and then suspended at a cell concentration of 5 x 10 7 cells ml-~ in fresh media (100 ml) containing 50 pM Cd, then cultured for 2 weeks under the light intensity of 50 ~tE m - 2

T. Matsunaga et al./Journal of Biotechnology 70 (1999) 33-38

s-~ at room temperature with aeration. Samples of 1.5 ml were assayed for pH, cell concentration and amount of removed Cd. Samples were centrifuged (5500 x g ) and residual Cd in the medium was determined from the supernatant. The pellet was washed with 1.5 ml of 0.2 M phosphate buffer (pH 7.5) and recentrifuged (5500 x g). The precipitated Cd was determined from this supernatant. The pellet was further washed with 1.5 ml of 10 mM EDTA solution for desorption of Cd from the cell surface and centrifuged (5500 • g). Adsorbed Cd on the cell surface was determined from this supernatant. The amount of intracellular accumulation of Cd was determined by measuring the Cd content in the pellet resuspended in 1.5 ml of 1 N HNO3 using atomic absorption spectrophotometry.

2.3. Cd removal by freeze-dried cells

3. Results and discussion

3.1. Screening of marine microalgae with high Cd removal ability Primary screening of 191 marine microalgae in our culture collection for Cd resistance identified 19 strains of marine green algae which grew in the media containing 100 laM Cd and five marine cyanobacteria which grew in 50 ~tM Cd. These 24 strains were tested for their ability to remove Cd from a liquid medium containing 50 laM Cd. Table 1 shows that six strains out of 19 green algae (Chlorella strains were cultured in BGll medium with 2% NaC1) and one out of five Table 1 Screening of marine microalgae having high capability of cadmium removal a Strain

Cells were centrifuged (8000 • g), washed with distilled water and frozen at - 8 0 ~ The frozen samples were then lyophilized. Freeze-dried cells (11 mg, corresponding to approximately 1.4 • 108 cells) were suspended in 1.4 ml of media containing various Cd concentrations. Cd removal was measured as described in Section 2.2.

Oscillatoria Oscillatoria Oscillatoria Synechococcus Synechococcus

In order to determine the maximum Cd adsorption to the living or dried cells, the equilibrium isotherm of Cd adsorption was calculated using the Langmuir sorption model (Langmuir, 1918). The Cd adsorption (q) for the construction of sorption isotherms was determined as follows:

Cf/ q = (1/ qmaxb ) 4- ( Cf/ qmax)

(1)

(2)

Cd removal (%)

540009 540010 540028 550038 15031a

1.9 0.6 3.0 1.6 10.8

0004 0005 0010 0012 0019 0109 031501 031601 033101 040302 040401 040402 131601 132301 133001 1337 1415 16014 16026

0.8 11.0 5.4 2.4 12.6 22.8 1.6 0.7 5.6 9.7 3.4 6.1 3.3 19.4 5.0 1.1 12.1 48.7 8.9

Green algae

2.4. Evaluation of maximum adsorption and uptake amount of Cd

where q is the Cd adsorption (mg Cd g-1 dry cells), qmax is the maximum adsorption (mg Cd g-1 dry cells), Cf is the final concentration of Cd in the solution (mg 1-~), b (1 mg-1) is the Langmuir constant, ratio of the adsorption/desorption rates. For the fitting of experimental data, the Langmuir model was linearized as follows:

Number

Cyanobacteria

Chlorella

q = qmaxbCf/(1 4- bCf)

35

a

Cd removal (%)

= (Amount of removed Cd)/(Amount of ininital C d ) x 100 The initial concentration of Cd in the medium was 50 ~tM. The amount of Cd in the medium was measured after 2 weeks cultivation.

T. Matsunaga et al./Journal of Biotechnology 70 (1999) 33-38

36

3.0

10

600

9

~ 500

-

2.0

8 300

!,.o o

6

0

5 0

2

4

6

8

10

12

[4

Time (days)

i

,,, 0

0

2

4

6

8

10

12

14

Time (days)

Fig. 1. Cd removal by Chlorella sp. NKG16014 from the medium: (a), cell growth ( 9 and pH change (El), in the medium containing Cd (initial Cd, 597 ~tg 100 ml-~ medium); (b), the amount of Cd in supernatant (11), intraceUular accumulation ( 9 and adsorption (O).

cyanobacteria (Oscillatoria strains and Synechococcus sp. NKBG550038 were cultured in 1/10 A medium and Synechococcus sp. NKBG15031a was cultured in BGll medium with 2% NaC1) removed more than 10% Cd from the medium. The marine green alga Chlorella sp. NKG16014 showed the highest removal rate of Cd (48.7%).

3.2. Cd uptake by the marine Chlorella sp. NKG16014 As the growth of Chlorella sp. NKG16014 in seawater-based Eppley's medium was superior to that in BG11 medium, the following experiments were performed using Eppley's medium. Cd uptake by NKG16014 in 100 ml Eppley's medium containing 50 ~tM Cd was investigated. Fig. l(a) shows the growth of NKG16014 and pH change in the medium. The pH varied between 7.5 and 8.5 during cell growth, which is similar to the natural variation in seawater, thus indicating no significant precipitation of Cd by alkalinization. Cd precipitation was observed in the same medium at pH above 9 (data not shown). Fig. 1(b) indicates the time course of Cd removed from the medium, intracellular accumulation and adsorption by NKG16014 cells during 2 weeks cultivation. Adsorption to the cell surface was found to reach saturation after 2 days. Intracellular ac-

cumulation of Cd increased continuously. The physico-chemical adsorption of Cd by algal biomass was reported to reach saturation much faster (within 30 min) (Sakaguchi et al., 1979). The longer saturation time in living cultures may be caused by the increase in total cell surface area during rapid cell growth and effect of ions in the media such as Na+, Mg 2§ Mn 2§ which are known to effect on adsorption: 65% of total Cd was observed to be removed from the media after 12 days of cultivation; 67% of removed Cd was accumulated intracellularly; 25% was adsorbed on the cell surface; 4% was precipitated Cd; and remaining 4% was unknown.

3.3. The maximum Cd adsorption by the living marine Chlorella sp. NKG 16014 The Cd adsorption profiles of NKG16014 cells under various Cd concentrations were investigated in order to determine the maximum Cd adsorption on the cell surface. The adsorption was found to saturate in 2 days and intracellular accumulation was observed to increase slowly in 8 days in all experiments (data not shown). The data was analyzed using the Langmuir sorption model. Fig. 2 shows the equilibrium isotherm of Cd adsorption by NKG16014. The inset shows the linear regression line of the adsorption rate

T. Matsunaga et al./Journal of Biotechnology 70 (1999) 33-38

using Eq. (2). The maximum Cd adsorption (qmax) and the Langmuir constant (b) were estimated from this linear regression line to be 37.0 mg Cd g-~ dry cells and 1.5 x 10 -2, respectively. In contrast, the maximum intracellular accumulation saturated under low Cd concentration and was estimated to be 2.4 mg Cd g-~ dry cells. Maximum Cd adsorptions were reported for several cyanobacteria, including Tolypothrix tenuis (19.6 mg Cd g-1 dry cells) by Inthorn et al. (1996) and green algae such as: C. vulgaris (5 mg Cd g-1 dry cells) by Geisweid and Urbach (1983) and C. regularis (7.9 mg Cd g-1 dry cells) by Sakaguchi et al. (1979).

3.4. Cd adsorption by the dried marine Chlorella sp. NKG16014

0

1

37

2

3

4

5

6

T i m e (h)

Fig. 3. Cd removal by non-living cells (freeze-dried) of Chlorella sp. NKG16014 from the medium. Dried cells (11 mg) was suspended in the 1.4 ml medium containing Cd (initial Cd, 6.9 lag 1.4 ml-1 medium). 100

In order to determine the optimum incubation time for adsorption, the kinetics of Cd removal from the medium was investigated. Approximately 1.4 • l0 s dried cells were incubated in the medium containing Cd. Cd removal rates were found to saturate within 30 min (Fig. 3). Furthermore, since 95% of removed Cd from the medium was desorbed by washing with 10 mM EDTA, Cd

i"1

[]

75 60

~D

u9

i

50 []

~r

'

~'~

o

25 Cr(mg r 1)

0

3o

100 ,, 7s

C (rag [-1)

0

1000

i

1500

3000

4500

6000

Fig. 4. Adsorption of Cd by non-living cells (freeze-dried) of Chlorella sp. NKG16014 at various Cd concentration. The inset shows the linear regression line of the adsorption rate using the Langrnuir model.

'

0

i

C f (rag I"l)

[]

,m~ 20

i

2000

3000

4000

C f ( m g !"1)

Fig. 2. Adsorption of Cd by ChloreUa sp. NKG16014 at various Cd concentration. The inset shows the linear regression line of the adsorption rate using the Langmuir model.

removed from the medium was attributed to adsorption to the dried cells. The equilibrium isotherm and the linear regression line of the adsorption rate is shown in Fig. 4. The maximum Cd adsorption (qmax) and the Langmuir constant (b) were 91.0 mg Cd g-1 dry cells and 3.0 • 10-3, respectively. The dried cells were thus found to be superior to the living cells for Cd adsorption capacity and kinetics.

38

T. Matsunaga et al. Journal ~/" Biotechnology 70 (1999) 33-38

4. Conclusion Marine microalgae were screened for efficient capability of Cd removal with the aim of on site heavy metal removal from the marine environment. The maximum uptake of NKG16014 was found to be 39.4 mg Cd g-~ dry cells (including adsorption and intracellular accumulation), which is higher than that of other previously characterised microalgae. This higher uptake can be attributed to adsorption on the cell surface. Furthermore, a higher level and more rapid adsorption was obtained by using dried cells. These results indicate that NKG16014 can possibly be used for sequestering Cd from the marine environment. Further investigation of the effects of several factors on adsorption, such as ions in seawater and pH, will contribute to further optimization of the adsorption efficientcy of this rnicroalgal strain.

References Eppley, R.W., Holmes, R.W., Strickland, J.D.H., 1967. Sinking rates of marine photoplankton measured with a fluorometer. J. Exp. Mari. Biol. Ecol. 1, 191-208. Fourest, E., Roux, J.-C., 1992. Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH. Appl. Micobiol. Biotechnol. 37, 399-403. Fourest, E., Canal, C., Roux, J.-C., 1994. Improvement of heavy metal biosorption by mycelial dead biomasses (Rhizopus arrhizus, Mucor miehei and Penicillium chrysogenum): pH control and cationic activation. FEMS Microbiol. Rev. 14, 325-332. Geisweid, H.J., Urbach, W., 1983. Sorption of Cd by the green microalgae Chlorella vulgaris, Ankistrodesmus braunii and Eremosphaera viridis. Z. Pflanzenphysiol. 109, 127-141. Holan, Z.R., Volesky, B., Prasetyo, I., 1993. Biosorption of Cd by biomass of marine algae. Biotechnol. Bioeng. 41, 819-825. Holan, Z.R., Volesky, B., 1995. Accumulation of Cd, lead and nickel by fungal and wood biosorbents. Appl. Biochem. Biotechnol. 53, 133-146. Inthorn, D., Nagase, H., Isaji, Y., Hirata, K., Miyamoto, K., 1996. Removal of Cd from aqueous solution by the filamentous cyanobacterium Tolypothrix tenuis. J. Ferment. Bioeng. 82, 580-584. Khummongkol, D., Canterford, G.S., Fryer, C., 1982. Accumulation of heavy metals in unicellular algae. Biotechnol. Bioeng. 24, 2643-2660.

Kumazawa, S., Mitsui, A., 1981. Characterization and optimization of hydrogen photoproduction by a saltwater blue-green alga, Oscillatoria sp. Miami BG7. I. Enhancement through limiting the supply of nitrogen nutrients. Int. J. Hydrogen Energy. 6, 339-348. Langmuir, I., 1918. The adsorption of gases on plane surfaces of glass, mica and platium. J. Am. Chem. Soc. 40, 13611403. Les. A.. Walker, R.W., 1984. Toxicity and binding of copper, zinc and Cd by the blue-green alga, Chroococcus paris. Water, Air, Soil Pollut. 23, 129-139. Miura. Y., Sode, K.. Narasaki, Y., Matsunaga, T., 1993a. Light-induced antimicrobial activity of extracts from marine Chlorella. J. Mar. Biotechnol. 1, 143-146. Miura. Y., Sode, K., Nakamura, N., Matsunaga, N., Matsunaga, T., 1993b. Production of ~,-linolenic acid from the marine green alga Chlorella sp. NKG 042401. FEMS Microbiol. Lett. 107, 163-168. Nriagu. J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature 333, 134-139. Sakaguchi. T.. Tsuji, T., Nakajima, A., Horikoshi, T., 1979. Accumulation of Cd by green microalgae. Eur. J. Appl. Microbiol. Biotechnol. 8, 207-215. Sudo. H., Burgess, J.G., Takemasa, H., Nakamura, N., Matsunaga, T., 1995. Sulfated exopolysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia. Curr. Microbiol. 30, 219-222. Takano, H., Takeyama, H., Nakamura, N., Sode, K., Burgess, J.G., Manabe, E., Hirano, M., Matsunaga, T., 1992. CO2 removal by high density culture of a marine cyanobacterium Synechococcus sp. using an improved photobioreactor employing light-diffusing optical fibers. Appl. Biochem. Biotechnol. 34/35, 449-458. Trevors, J.T., Stratton, G.W., Gadd, G.M., 1986. Cd transport, resistance and toxicity in bacteria, algae and fungi. Can. J. Microbiol. 32, 447-464. Volesky, B., Holan, Z.R., 1995. Biosorption of heavy metals. Biotechnol. Prog. 11, 235-250. Wachi, Y., Burgess, J.G., Takahashi, J., Nakamura, N., Matsunaga, T., 1995a. Tyrosinase inhibition by the water-soluble fraction of marine microalgae. J. Mar. Biotechnol. 2, 210-213. Wachi, Y., Burgess, J.G., Iwamoto, K., Yamada, N., Nakamura, N., Matsunaga, T., 1995b. Effect of ultraviolet-A (UV-A) light on growth, photosynthetic activity and production of biopterin glucoside by the marine UV-A resistant cyanobacterium Oscillatoria sp. Biochim. Biophys. Acta. 1244, 165-168. Wake, H., Akasaka, A., Umetsu, H., Ozeki, Y., Shimomura, K., Matsunaga, T., 1992. Enhanced germination of artificial seeds by marine cyanobacterial extract. Appl. Microbiol. Biotechnol. 36, 684-688. Wilde, E.W., Benemann, J.R., 1993. Bioremoval of heavy metals by the use of microalgae. Biotech. Adv. 11, 781812.

Biotecbno,logy ELSEVIER

Journal of Biotechnology 70 (1999) 39-51

Diatom silicon biomineralization as an inspirational source of new approaches to silica production Engel G. Vrieling a,,, Theo P.M. Beelen b, Rutger A. van Santen b, Winfried W.C. Gieskes a

a

Department of Marine Biology, Center for Ecological and Evolutionary Studies, University of Groningen, Biological Center, PO Box 14, 9750 AA Haren, The Netherlands b Schuit Institute of Catalysis, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands

Received 8 October 1998; received in revised form 25 November 1998; accepted 22 December 1998

Abstract

The demand for new materials and products is still growing and the interest in naturally formed biopolymers and biominerals, such as chitin, calcium precipitates and silica is increasing. Photosynthesizing microalgae of the family Bacillariophyceae (diatoms) produce silica exoskeletons with a potential to be used in specific industrial or technological processes, they also are an excellent model in studies of silicon biomineralization. In contrast to geologically aged diatomaceous earth, the freshly prepared silica of cultured or harvested natural diatoms has been characterized insufficiently with respect to the properties (e.g. purity, specific surface area, porosity) required for technological and industrial application. In this contribution we summarize aspects of cellular processes that are involved in silicon biomineralization of diatoms and the current knowledge of the characterization of diatomaceous silica, following methods used for synthetically derived silica-based materials. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Biogenic silica; Biomineralization; Physisorption; Small angle X-ray scattering (SAXS); Silicon (bio)chemistry

1. Introduction

Although silica is by far the most abundant compound in the earth crust, only a minor part can be applied directly or after refining (sand, clay, several minerals). For most commercial applications such as filling materials, filter agents, * Corresponding author. Tel.: + 31-50-3632286; fax: + 3150-3632261. E-mail address: [email protected] (E.G. Vrieling)

catalyst supports, ion-exchange materials or abrasives, very specific porous structures or surface properties are required. Therefore this silica has to be synthesized from scratch in various industrial large-scale processes. Because of the still growing demand for new materials and new products, silicas or silica-based compounds are developed and improved continuously. Also related products like zeolites, a class of crystalline and microporous silica or silica-alumina (Jansen et al., 1991; van Bekkum et al., 1991) and nanosized

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S0168-1656(99)00056-5

40

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

mesoporous silicas such as the MCM-compounds (Corma, 1997) contribute to the rich choice of silica-based materials. They are at present available with a continuous spectrum of micro- and mesoporosity and with a broad range of surface activities. The scientific background of the often quite complicated silica chemistry is described in recent monographs (Brinker and Scherer 1990; Bergna 1994). In nature silicas can be found in higher terrestrial plants (e.g. grasses), but only the aquatic environment supplies structures that are of potential interest for industrial applications, namely in the format of unicellular organisms, Radiolaria and Diatoms. Both types of organisms produce siliceous exoskeletons that remain intact when cells die. Microalgae of the family Bacillariophyceae (or diatoms) produce exoskeletons with detailed species-specific morphological features (a gigantic variety of silicified networks, pores, tubes and ribbons), creating high surface area of a kind that is interesting with respect to new developments in synthesis of silica-based materials. The remnants of diatom blooms sink to the ocean floor where they end up as diatomaceous 'ooze' and reappear to the surface after millions of years of aging as 'diatomaceous earth' (known also as Kieselguhr) in geological deposits, the latter has been used commercially (Iler, 1979). Diatomaceous earth, however, is much less interesting for new applications, because aging reactions that have been going on over millions of years destroyed most of the attractive morphological features, while contaminating minerals are incorporated more often than not. Fresh diatomaceous silica (prepared from cultures or harvested from the field) has hardly been characterized by the physico-chemical techniques that are applied for synthetically derived silica. It is also of interest to understand the diatom exoskeleton morphogenesis, because that may provide insight in the biological (organic) and chemical (inorganic) processes that are involved in the silicification of the diatom cell walls (called frustules). Better knowledge of the natural silicification processes, e.g. the role of biopolymers that have recently been found in various biogenic silicas (Zaremba and Stucky, 1996), eventually may

result in biomimicking the way diatoms perform their silicification. In our search for new, natureinspired approaches to silica production it seemed appropriate to outline the current knowledge of silicon biomineralization by diatoms and to characterize the resulting silica products by techniques such as 29Si-NMR, physisorption and combined small and wide angle X-ray analysis (SAXS and

WAXS). 2. Aspects of diatom valve morphogenesis Diatoms are photosynthetic algae of which over 104 species are known in a taxonomy based on the variety in the morphology of the exoskeletons, the so-called frustules (Round et al., 1990). These frustules are composed of two valves that fit together like a petri-dish, connected to each other by one or more girdle bands (Fig. 1). The valve morphogenesis has been studied mainly by ultrastructural analysis and has been complemented by biochemical studies (Simpson and Volcani, 1981; Pickett-Heaps et al., 1990). Valve formation in diatoms proceeds basically as follows. Prior to cell division each of the two daughter cells within the mother cell develops a new hypovalve and girdle bands in a special cellular compartment, the silicon deposition vesicle (SDV). During growth and maturation of the valve, silica precursors are transported from the cytoplasm to the SDV by silicon transport vesicles (STVs) that fuse continuously with the expanding SDV. In the final stage valves and girdle bands are excreted, but only after the siliceous parts have been covered by a protective layer. This coating of organic matter (proteins and carbohydrates) avoids spontaneous dissolution in the aqueous environment (Simpson and Volcani, 198 l; Pickett-Heaps et al., 1990; van de Poll et al., 1999). The final macro-morphology of the siliceous parts, especially the so-called raphe, fibs, areola, processes and honeycomb-shaped chambers, most probably reflects the cellular, genetically driven molding activity to shape the SDV during silica polymerization. The fact that all these processes have a genetic basis may explain the enormous diversity in frustule morphology

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

(Schmid, 1986) and the intra-species variation in silica content of the valve (Conley et al., 1989).

3. Role of organic molecules in diatom valve formation

Except the uptake mechanism of silicate from the aqueous environment (Bhattacharyya and Volcani, 1980), little is known of other biochemical pathways that are involved in storing and transporting silica in diatom cells. Proteins, however, are important since expression and repression levels of some are regulated by the availability of silicon (Okita and Volcani, 1980; Hildebrand et al., 1993, 1997). Amino acid and sugar analysis of purified cell walls of diatoms revealed that carbohydrates and proteinaceous matter are associated with or even present inside the SiO2-matrix (Hecky et al., 1973; Swift and Wheeler, 1991, 1992). These organic molecules may act as nucleators and structure directing

41

molecules inside the SDV in a similar way as organic template molecules (such as tetramethylor tetrapropylamine) that are used in silica chemistry, they are indispensable in the preparation of zeolites (Zones et al., 1998). The amino acids serine (8.4-25.2 mol%) and glycine (9.27-24.5 mol%) dominate the proteinaceous fraction after dissolution of the SiO2-matrix (Table 1). The hydroxyl residues of serine (potential to form H-bridges) have been proposed to initiate silica polymerization in diatoms (Hecky et al., 1973; Swift and Wheeler, 1992; Harrison, 1996). Polymers and polypeptides are efficient nucleators in biomineralization in which a rigid 13sheet conformation seems to be essential (Addadi et al., 1991). Computational modeling in which it is assumed that two adjacent serine residues occur in a 13-sheet peptide conformation revealed that the spacing of the - O H groups of a siloxane ring matches (with a difference of only 0.02 ,~) that of the serine - O H groups (Lobel et al., 1996). Furthermore, the required energy (AH = - 28 kcal

13 -..?.

9 r

Epivalve

,:.;Zi....

~--Girdle bands---.-

Hypovalve ?~:iv

Fig. I. Overview of the cell wall (the frustule) of (A) a centric diatom: Odontella spp. and (C) a pennate species: Navicula salinarum. A schematic presentation of the two valves, the epi- (Ev) and hypovalve (Hv) and the connective girdle bands (Gb) is shown in (B) Bar = 5.0 ~tm.

N P

Table 1 The amino acid composition of the soluble fractions, obtained after dissolution of the solid silica matrix of diatoms and diatomaceous earth" Amino acid

Cyclotella cryptica

Cyclotella meneghiniana'

Cyclotella stelligera

Navicula pelliculosa b.c.d

Nitzschia brevirostris

Melosira granulata

Melosira nummuloidesb

Diatomaceous earthc

Ala

4.9 2.7

8.22 0.26

6.0 1.5

10.7 0.8

5.80 4.30

9.6 2.0

8.0 3.2

10.6 0.4

8.77 2.08

ASP

13.6

10.03

10.3

10.5

10.40

8.3

11.7

12.8

0.25

CYS

0.9 6.1

0.1 10.9

0.3 10.0

n.d. 15.44

0. I 14.5

0.2 6.4

0.4 9.1

9.86 10.50

19.5

13.1 0. I

9.27

11.4

11.3

12.3

18.46

1.5 3.1 5.0 0.1 1.3 1.2

0.02 n.d. 24.46 0.86 2.53 4.31 1.81 n.d. 2.23 4.43

5.3 10.4 1.8 0.9 4.5 6.6

2.04 5.77 9.00 5.93 n.d. 3.18 7.23

0.8 3.7 7.3 3.6 0.4 5.2 8.5

0.3 6. I 7.8 2.9 0.2 1.7 0.9

0.1 4.5 7.5 1.6 I .9 4.2 7.5

1.57 2.70 7.90 3.23 n.d. 3.02 5.66

23.5

20.31

9.9

8.79

8.4

22.8

12.7

12.83

10.3 1.4 3.1

5.03 0.79 4.35

6.4 2. I 6.8

4.36 2.20 5.31

7.4 I .3 7.5

8.3 2. I 7.3

5.7 2.6 6.0

5.03 I .83 5.39

Glx

GlY His Ile Leu

LYs Met Phe Pro Ser Thr

TYr Val a

21.o 1.1

0.1 3.6 5.4 2.5 0. I 1.2 6.5 25.2 7.0 0.6 4.1

Notes: Values were taken from Hecky et al. (1973). Swift and Wheeler (1992) and right column: Vrieling et al. (unpublished results).

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

kDa 158.0

43

4. Chemical processes in diatoms valve formation

66A

~..62 ~-"56

36.5 | .

1

2

.

3

.

.

.

4

.

.

5

Fig. 2. Overview of peptide profiles obtained by 10% SDSPAGE (silverstained) of soluble fractions after Na2CO3 dissolution of the SiO2-matrix (the organic casing was removed prior to dissolution) of various diatom samples. Lane 1: Molecular mass markers (mass indicated as kDa), lanes 2 to 5 represent samples of the species Coscinodiscus wailesii, Cylindrotheca fusiformis, Navicula pelliculosa and a natural population of harvested benthic diatoms. Note the apparent similarity in protein profile with respect to the origin of the samples.

mol-~) to deposit one siloxane ring allows fast and energy-inexpensive polymerization. To identify the proteinaceous source within the SiO2-matrix we have extracted peptides by Na2CO 3 dissolution. Preliminary characterization revealed that two, probably homologue, peptides (with an apparent molecular mass of approximately 56 and 62 kDa, respectively) are present in diatoms of different origin (arrows Fig. 2). The amino-acid profiles of these peptide fractions are comparable to those found by Hecky et al. (1973) and Swift and Wheeler (1992), see Table 1. Most probably these proteins are identical to those found in a proteinaceous fraction (mass range of 45-60 kDa) obtained after gel permeation purification (Swift and Wheeler, 1991, 1992). Further characterization of these SiO2-matrix proteins is in progress to determine: (1) the sequence of amino acids; (2) if these peptides really can initiate silica polymerization in diatoms; and (3) if they are promising for in vitro assembly studies and eventual biomimicking of diatomaceous silica synthesis.

From general silica chemistry it is known that temperature, the concentration of silicate, the availability of salts and cations (such as AP +, Ca 2 +, Fe 3+, K+, Na+) and the pH affect the synthesis of silica and zeolites (Iler, 1979; Jansen et al., 1991; Wijnen et al., 1993). For diatoms temperature effects are negligible, but the other parameters seem to be involved in the following reaction equilibria: monomersooligomers~(protein/silica) precursorsc~,aggregates,~frustule

(1)

Although it is still not clear where exactly or in what form (dissolved, particles, aggregates) silicate is present inside the cell, it is known that the cellular concentration may increase to values of 400-700 nM cell -~ (Robinson and Sullivan, 1987). The intra-cellular molarities, however, vary among species from less than 1 to over 20 mM (recalculated from the cellular silica content and biovolumina of over 77 species summarized in Conley et al. (1989)). The amount of 20 mM cellular silica is equivalent to 1 weight% dissolved SiO 2 and this is low to induce aggregation of silica particles in solvents (Iler, 1979). Gordon and Drum (1994) proposed that silica polymerization in the SDV of diatoms proceeds as a process of diffusion limitation, as known in silica chemistry (Iler, 1979), in which the concentration of silica precursors is the limiting factor. It is possible that inside the diatom cell concentration gradients are built that result in elevated local silica concentrations near the expanding edges of the SDV, consequently the equilibrium in Eq. (1) is driven to mass aggregation and therewith frustule completion. The presence of salts (usually referred to as 'the salinity' of the environment) is a very important factor, comparison of the silicon content of freshwater and saltwater species showed that freshwater species contain more silica cell-~ (Conley et al., 1989). Even within one species the silica content per cell varies at different salinities (Romero, 1994; Vrieling et al., 1999c). It is thought that increased silicification at lower salinities happens

44

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

to maintain the turgor-strength under a shifted osmotic balance (Paasche, 1973; Romero, 1994), but this explanation is questionable. Presence of salts affects the development of silica particles and the aggregation (networks) of those particles (Iler, 1979; Vrieling et al., 1999c). The addition of cations (e.g. aluminium, germanium) has been shown to give various responses on diatoms with respect to silicification. Germanium can compete with silicon and proved to be useful in uptake studies following silicon limitation, but high concentrations are toxic, germanium oxide is used to remove epiphytic diatoms from macroalgae (Lewin, 1966). Gensemer (1990) observed an increase of the cellular silica (biogenic silica) content when diatoms were enriched with aluminium, but in another study (Vfieling et al., 1999c) Al-enrichments did not alter the biogenic silica content, although it did enhance growth of the pennate species Navicula salinarum. Soluble A1 (present as AI(OH)3) reacts preferentially with Si(OH)4 and it can be assumed that Al-silica complexes are formed. A1 was found to be associated with, most probably, the outer part of diatom frustules (van Bennekom et al., 1989, 1991), but inside the SiO2matrix less than 0.2% is present (Vrieling et al., 1999c). The molecular orientation of Al and Si atoms inside the SiO2-matrix is unknown, but is is expected that microcrystalline phases as in zeolites do not occur, since diatomaceous silica is amorphous (Fig. 4A; Perry 1989; Vrieling et al., 1999a). The pH of the reaction solution is an important factor and influences the equilibrium of Eq. (1). It is believed that the silica precursors are transported to the SDV via acidic STVs, of which most probably the H +-pumps remain functional in the SDV membrane after fusion. Probe analysis, using the weak base 3-(2,4-dinitroanilino)-3'-aminoN-methylpropylamine (DAMP), revealed that D A M P was accumulated specifically in acidic compartments: the lumen of thylakoidal membranes (Fig. 3A), microbodies (Fig. 3B) and inside the SDV where daughter valves were synthesized (Fig. 3 B - D ; Vrieling et al., 1999b). Sequential sampling during valve formation showed that the SDV, in which new valves are formed, becomes increasingly more acidic (Fig. 3C,D) due to both

the increase in the number of functional H +pumps and the polymerization of silica itself (Iler,

NaAZeolim

o... C

2

,

,.

,

,

,

k

,

B

C

8'

I

...I

f

1.3

lls

,16

llr

1.8

Log O (nm "I)

Fig. 4. WAXS spectra of silica of cultured diatoms. (A) Representative WAXS spectra of a pure crystalline specimen of sodium A zeolite (Proctor and Gamble) and amorphous diatomaceous silica of the pennate species Nitzschia thermalis. (B) Examples of diatomaceous silica samples containing crystalline contaminants (arrows indicate position of peaks); top spectrum is a field sample of Odontella sinensis with remaining salt crystals and the bottom spectrum is of a benthic species Stauroneis constricta in which sand grains (required as a solid substrate) were present. Notes: Presence of crystalline contaminants clearly obscure the WAXS spectra of the amorphous character of diatomaceous silica. Methods: Combined small and WAXS analysis was performed at the CCLRC Synchrotron facility (Daresbury Laboratories, Warrington, UK), using a high brilliance collimated X-ray beam (Vrieling et al., 1999a). Measurements were made on samples of acid cleaned and chemically dried diatomaceous silica that was mounted between Kapton windows in a rotating sample cell. WAXS was recorded as a function of scattering angle by means of a curved INEL pressurized gas detector. Scattering spectra were presented as log scattering intensity (arbitrary unit) versus log Q (size measurement related to the scattering angle denoted as nm- 1) to determine presence of peaks and their spacing using the fully crystalline sample of sodium A zeolite as calibration specimen.

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

o

/

45

~

Fig. 3. Immunocytochemical localization, using indirect immuno-gold labeling, of the probe DAMP that accumulates in acidic cellular compartments. (A) The clearcut labeling of the acidic lumen of thylakoidal membranes (pH ~ 5), B) the labeling of a microbody (pH ~ 5-6) and the daughter hypovalves (arrows) and (C, D) the specific accumulation of the probe in the silicon deposition vesicle where the new hypovalves are formed; increasing acidity is shown by the increase in gold particles between 1 and 4 h after valve formation has been induced in silicon-synchronized cells. Abbreviations: Chl, chloroplast; dv, daughter valve; m, microbody; R, raphe; and V, vacuole. Bars = 5.0 and 1.0 lam for A and B-D, respectively. Note: Experimental details and results will be published elsewhere (Vrieling et al., 1999b).

1979; Vrieling et al., 1999b). Acidification of the SDV allows a thermo-dynamically driven fast aggregation (diatoms complete new hypovalves within less than 8 h) of silica particles to form the final deposited valve and girdle bands. Especially when local silica precursor concentrations are high the equilibrium of Eq. (1) is shifted to the fight. In an in vitro study using liposomes loaded with 0.1-0.2 M silicate ions (pH > 10.8), the polymerization was observed to occur within 15 min after shifting the pH ( < 6); the primary particles (6-7 nm) finally formed large aggregates ( > 7 0 nm) in 2 h (Perry et al., 1991). The advantage of the low pH of the SDV for diatoms is that the formed siliceous parts are protected from continuous dissolution that may destroy fragile or important details such as processes, ribs and spiculae (Vrieling et al., 1999b).

5. Characterization of diatomaceous silica

In comparison to chemically derived silicabased materials, biogenic silica has been studied quite poorly. In the past diatomaceous earth has been examined, but this appeared to be less interesting for industrial applications since its characteristics (e.g. surface area, porosity, contamination) limit its use as alternative for synthetically derived silica materials. Minor attention has been paid to cultured or freshly harvested silica of diatoms and this most probably reflects a lack of interest or even a prejudice of chemists, because of the assumed close relation to highly aged diatomaceous earth. For the classification of diatoms electron microscopy is very important and even at low magnifications morphological alterations can easily be followed (Vrieling et al., 1999c). For detailed

46

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

analysis of the silica, high-resolution microscopy may give insight into crystalline phases (not observed yet in biogenic silica) or the size and arrangement of silica particles or spheres in amorphous systems. By combining microscopy with analytical methods such as energy dispersion Xray or microprobe analysis, the elemental composition of the SiO2-matrix can be determined (van Bennekom et al., 1989; Swift and Wheeler, 1992; Vrieling et al., 1999c). This approach, however, is most often not sensitive enough for elements that are present at concentration < 0.2%. In order to determine the coordination of silicon molecules in biogenic silica and silica-based materials 29Si N M R can be applied (Perry, 1989; Wijnen et al., 1993; Burkett and Davis, 1994). N M R is very sensitive and alterations such as replacements o f - O H groups by - O - b r i d g e s can be identified. Furthermore, N M R can discern silica samples that seem to be similar by powder diffraction methods (Perry 1989). For solids (as diatomaceous silica is) the outer boundaries of the particles can be studied and as a consequence the tetrahedral coordination of silicon can be determined; the diatom Navicula pelliculosa contains silicon as Si(OSi-)4, Si(OSi-)3OH and Si(OSi-=-)2(OH)2, but there is no evidence yet that S i - O - C or S i - O - N bonds are present in their frustule silica. In situ 29Si N M R measurements during dissolution of diatomaceous silica have not been carried out, but such observations may answer questions related to the bonds between organic matter (the potential nucleators or structure directing agents) and silicon. To determine the surface area or pore size range of silicas or zeolites, sorption methods have often been applied. For diatomaceous silica a number of measurements has been performed (Table 2), showing that the specific surface area differs among and even within species. As mentioned, the silica diatoms is covered by an organic coating before excretion to the aquatic environment. Apparently the removal of this matter is quite difficult and no efficient and reproducible procedures have been designed yet. Furthermore, the effect of temperature used to dry the samples (to remove remaining water from capillary cavities) and the often small sample size (in the order

of milligrams rather than grams) affect the estimation of the representative specific surface area as m 2 per gram material (Beelen, Dokter, Vrieling, unpublished results). As a consequence of uncertainties the measured specific surface area of the silica of diatoms has not been compared properly to areas of synthetically derived silica-based materials. Diffraction methods are useful to determine crystallinity of silica samples. Powder X-ray diffraction (referred to as XRD) examines domains over l0 nm and peaks in the spectra indicate presence of crystalline phases. Based on powder diffraction, diatomaceous silica of different origin (cultures, earth) seems to be comparable and based on the absence of clearcut peaks the amorphous character was determined. However, infrared spectroscopical analysis of the same samples showed that the absorption patterns and ratios of absorption intensities are different, indicating that the silica is not exactly identical among species (Kamantani, 1971; Perry, 1989). For diatoms, crystalline phases have not been reported (see above), but it was suspected that at domains < l0 nm (below the limit of XRD) crystalline phases exist (Pickett-Heaps et al., 1990). Heat-treated diatomaceous earth indeed revealed crystalline phases (Hurd, 1983), but recent analysis by wide angle X-ray scattering (WAXS) showed that at domains between 0.07 and 0.5 nm no crystalline phases are present in silica from recent freshly cultured and collected diatoms (Fig. 4; Vrieling et al., 1999b). Small angle X-ray scattering (SAXS) discerns between matter (in our case silica) and the surrounding (water, air). SAXS allows accurate analysis of silica-based materials with respect to the particle/pore sizes (distributed over the silica solids or in solutions) and organization of particles/pores in clusters or cluster sizes (Dokter, 1994; Beelen, 1996; de Moor, 1998; Vrieling et al., 1999a). Using SAXS, silica of a selection of cultured and field-harvested diatoms has now been examined, showing that at over a size domain of 5.0-35.0 nm the slopes of the scattering spectra of all species and field samples were comparable (Fig. 5). From these data it could be concluded that the scatterers (the pores in the silica of diatoms), over the measured size range, have a quite

E.G. Vrieling et al. /Journal of Biotechnology 70 (1999) 39-51

consistent dimension and architecture (Vrieling et al., 1999a). Detailed electron microscopical exami-

47

nation and SAXS revealed that pore size-range was determined to vary between 30 and 500 .~

Table 2 Specific surface area (m 2 g-1) for silica of cultured diatoms, field diatoms and diatomaceous earth determined by sorption analysis a Species

Specific surface area (m 2 g-1)

Method to remove organic matter

Sorption method

References

Coscinodiscus asteromphalus Lauderia borealis

89

Nitric acid

N2

Lewin (1961)

75-269 b

9

Navicula pelliculosa Nitzschia sigma N. sigma

123

Low temperature ashing Nitric acid

N2

van Beusekom and van Bennekom, personal communicaton Lewin (1961)

10.5-25.0 c 9.3

N2 N2

This contribution This contribution

N. sigma

9.0

N2

This contribution

N. sigma

12.0

N2

This contribution

Odontella sinensis

49

EGME d

van Bennekom et al. (1991)

Odontella sinensis

44

N2

This contribution

Rhizoselenia hebetata R. tesselata

60

Nitric acid H202, KMnO4, oxalic acid H2SO 4, KMnO4, oxalic acid K2S20 8, KMnO4, oxalic acid Low temperature ashing Low temperature ashing Nitric acid

N2

Kamantani and Riley (1979)

EGME d

van Bennekom et al. (1991)

Thalassiosira descipiens T. eccentrica

258

N2

Kamantani and Riley (1979)

9

T. nordenskirldii

16--127b

T. punctigera

15-43 b

Dutch Wadden Sea 1997 e Dutch Wadden Sea 1998 Dutch Wadden Sea 1998 Dutch Wadden Sea 1998 Dutch Wadden Sea 1998 Diatomaceous earth

87

Low temperature ashing Low temperature ashing Low temperature ashing H202, H2504

N2

van Beusekom and van Bennekom, personal communicaton van Beusekom and van Bennekom, personal communicaton van Beusekom and van Bennekom, personal communicaton This contribution

17-165 r

Nitric acid

N2

This contribution

84

H202, KMnO4, oxalic acid H2SO4, KMnO4, oxalic acid K2S208, KMnO4, oxalic acid Nitric acid

N2

This contribution

N2

This contribution

N2

This contribution

N2

Iler (1979)

50

19

69 48-52 22

Low temperature ashing Nitric acid

9 9

a Notes: b Surface area depended on culture condition with respect to additions of aluminium. c Surface area depended on the applied sorption instrumentation. d The ethylene glycol monoethylether technique of Heilman et al. (1965). e Natural populations of benthic diatoms harvested from tidal flats.

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

48

Overlap region of mean .lopee of Itm ~ samples tested by SAXS (Mmr97)

I !

Wldden Sm 2 Wmdden See 1 T.wliBIbgl T. ptmctlgem

m~l

ysis indicates that silica of cultured diatoms has its own characteristic features that are not found in industrial silica-based materials, but the translation of these data to a potential use of this type of silica has yet to be made.

"--

!

,

! ,,,

[

S. oonsldda S. coslalum N. lhefnlllil N. sigma N. dolltwJum N. lallnMlall N.CfylNOCqd~l O. #inenm L. bomells C. gnmit

I

_,

__

6. Conclusion and perspectives |

.

! I

A. )mx.~a

1

-1.00 10

-0.75 1

i

i

-0.50

-

-0.25

0.00

Log Q (nm"1)

1

1

1

i

Diatom silicon biomineralization is related to silica chemistry when chemical processes that are involved in the formation of the silica are consid-

-

I

1

9

0.25 1

i-

1

65 60 55 50 45 40 35 30 25 20 15 10 5

~

A

d (nm)

~ Fig. 5. Overview of the results obtained by SAXS of 14 diatoms and two natural populations of benthic species (indicated as Wadden Sea 1 and 2). The marked area represent the overlapping size region (5 < d < 35 nm) of the scattering pores in the silica; the SAXS spectra (see also Fig. 6) in this region are comparable for all species. Methods: SAXS was performed as described in Fig. 4, but scattering was now recorded as a function of scattering angle, applying a camera length of 3.50 m, by means of a multi-wire gas filled quadrant detector. Scattering spectra were presented as log scattering intensity (arbitrary unit) versus log Q (size measurement related to the scattering angle denoted as n m - 1) to determine the slope(s) as indicator for the structure, dimension and spatial relation/distance of pores in the silica (Vrieling et al., 1999a). For SAXS analysis the Q-axis was calibrated using a specimen of wet rat tail collagen.

=

-3,54

4

c"

B < 10")

6

and this is comparable to mesoporous systems (pores > 10 A), but not to microporous systems (pores < 10 A). In comparison to chemically derived silicas the SAXS spectra of the biogenic silica of diatoms reveal a porous structure rather than fractal aggregates. This structure is closely related to the shape and dimensions of the pores in the frustule and it can be altered by inducing changes in the silica by varying the salinity (the salt concentration, see above) or adding aluminium (cations, see above). Both parameters have effect on the scattering behaviour (the slopes of the SAXS spectra; Fig. 6A) and the preferential distribution of pores over the silica (the curvature of the SAXS spectra; Fig. 6B). Thus, SAXS anal-

4

-0.4

-o.2

o:o

0.2

-1

Log O (nm) Fig. 6. Effects of the salinity of the culturing medium and enrichments with A1CI3 on the SAXS spectra of the pennate diatom Navicula salinarum. (A) By lowering the salinity from PSU 28 (top spectrum) to PSU 20 (bottom 2 spectra) the structure or geometric dimensions of the pores in the silica (5 < d < 50 nm) change, resulting in a decrease of the slopes in SAXS-spectra. (B) By adding aluminium (Al/Si-ratios increase in spectra from top to bottom) the distribution of the pores and their preferential spacing is affected, resulting in variation in the curvatures of the spectra. Note: SAXS analysis was performed as described in Fig. 5.

E.G. Vrieling et al./Journal of Biotechnology 70 (1999) 39-51

ered. We have argued that the pH, the presence of cations and salts and the concentration of silica precursors play an important role in biosilicification of diatoms, apparently natural silicon biomineralization is quite conform to the rules known in 'classical' silica chemistry. The low contamination of 'fresh' diatomaceous silica with metals (e.g. A1, Fe, Ti) indicates that diatoms produce a nearly pure SiO2-matrix that does not require exhaustive purification, contaminants of this kind are often present in large-scale synthetic silica products. Although diatomaceous silica has hardly ever been characterized as to physicochemical properties, there are now indications that species or species complexes represent unique forms with respect to both morphology and molecular structure. Direct applications of silica obtained from cultured diatoms as alternatives for synthetically derived silica-based materials are not foreseen yet, but it is obvious that nature provides us with a wealth of new structures, many of them of interest within the field of industrial silica. Moreover, these new morphologies are an inspiration in the quest for new structure-directed synthesis methods. To exploit these new possibilities, more effort should be put into the characterization of diatomaceous silica with emphasis on: intra-species variability; the yield of silica by mass culturing; product optimalization (via specified growth conditions using e.g. altered salinity or adding cations); and the biological processes involved in diatom silicification. According to Ozin et al. (1997) and Yang et al. (1997) structure directing agents can be used to develop tailor-made mesoporous silica materials. By characterizing the organic molecules that are identified in the SiO2-matrix of diatoms, the role of these molecules in silicon biomineralization by diatoms can be determined. Eventually, such molecules can be useful in chemical syntheses to develop silica that mimicks the characteristic one of diatoms. The development of tailor-made silicas, with the variety of pore sizes observed in diatoms, provides not only alternatives to existing silica or zeolites, but has the potential to be used in new applications as well.

49

Acknowledgements We thank P.-P.E.A. de Moor, F. Giimiisburun and E. van Oers (Eindhoven University of Technology), B.U. Komanschek (Synchrotron Radiation Source, Daresbury Laboratory, UK) and W.H. van de Poll and M. Keijmel (Department of Marine Biology) for their assistance during various experiments, the results of which are presented in this contribution. A.J. van Bennekom (Netherlands Institute of Sea Research, NL) and J.E.E. van Beusekom (Biologische Anstalt Helgoland, Germany) kindly supplied data on the specific surface area of some diatoms. E.G.V. was supported by the Netherlands Technology Foundation (STW; grant GBi55.3883) which is subsidized by the Netherlands Organisation for the Advancement of Pure Research (NWO).

References Addadi, L., Moradian-Oldak, J., Weiner, S., 1991. Macromolecule-crystal recognition in biomineralization. Studies using synthetic polycarboxylate analogs. In: Sikes, C.S., Wheeler, A.P. (Eds.), Surface Reactive Peptides and Polymers; Discovery and Commercialization, vol. 444. ACS Symposium Series, Dallas, TX, pp. 13-27. Beelen, T.P.M., 1996. Inorganic particle gels. Curr. Opin. Coll. Intersurf. Sci. 1, 718-725. Bergna, H.E., 1994. The colloid chemistry of silica. Adv. Chem. Ser. 234, 517-531. Bhattacharyya, P., Volcani, B.E., 1980. Sodium-dependent silicate transport in the apochlorotic marine diatom Nitzschia alba. Proc. Natl. Acad. Sci. 77, 6383-6390. Brinker, C.J., Scherer, G.W., 1990. Sol-Gel Science. Academic Press, New York. Burkett, S.L., Davis, M.E., 1994. Mechanism of structure direction in the synthesis of Si-ZSM-5: an investigation by intermolecular IH-29Si CP MAS NMR. J. Phys. Chem. 98, 4647-4653. Conley, D.J., Kilham, S.S., Theriot, E., 1989. Differences in silica content between marine and freshwater diatoms. Limn. Oceanogr. 34, 205-213. Corma, A., 1997. Preparation and catalytic properties of new mesoporous materials. Top. Catal. 4, 249-260. Dokter, W.H., 1994. Transformations in silica gels and zeolite precursors. Ph.D. Thesis. Eindhoven University of Technology, The Netherlands. Gensemer, R.W., 1990. Role of aluminium and growth rate on changes on cell size and silica content of silica-limited populations of Asterionella ralfsii var. americana (Bacillariophyceae). J. Phycol. 26, 250-258.

50

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Gordon, R., Drum, R.W., 1994. The chemical basis of diatom morphogenesis. Int. Rev. Cyt. 150, 243-372. Harrison, C.C., 1996. Evidence for intramineral macromolecules containing protein from plant silica. Phytochemistry 41, 37-42. Hecky, R.E., Mopper, K., Kilham, P., Degens, E.T., 1973. The amino acid and sugarcomposition of diatom cell-walls. Mar. Biol. 19, 323-331. Heilman, M.D., Carter, D.L., Gonzalez, C.L., 1965. The ethylene glycol monoethyl-ether (EGME) technique for determining soil-surface area. Soil Sci. 100, 409-413. Hildebrand, M., Higgins, D.R., Busser, K., Volcani, B.E., 1993. Silicon-responsive cDNA clones isolated from the marine diatom Cylindrotheca fusiformis. Gene 132, 213218. Hildebrand, M., Volcani, B.E., Gassmann, W., Schroeder, J.I., 1997. A gene family of silicon transporters. Nature 385, 688-689. Hurd, D.C., 1983. Physical and chemical properties of siliceous skeletons. In: Aston, S.R. (Ed.), Silicon Geochemistry and Biogeochemistry. Academic Press, London, pp. 187-244. Iler, R.K., 1979. The Chemistry of Silica. Wiley, New York. Jansen, J.C., Strcker, M., Karge, H.G., Weitkamp, J., 1991. Advanced zeolite science and applications. Studies in Surface Science and Catalysis, vol. 85. Elsevier, Amsterdam. Kamantani, A., 1971. Physical and chemical characteristics of biogenous silica. Mar. Biol. 8, 89-95. Kamantani, A., Riley, J.P., 1979. Rate of dissolution of diatom silica in seawater. Mar. Biol. 55, 29-35. Lewin, J.C., 1961. The dissolution of silica from diatom walls. Geochim. Cosmochim. Acta 21, 182-198. Lewin, J.C., 1966. Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phycologia 6, 1-12. Lobel, K.D., West, J.K., Hench, L.L., 1996. Computational model for protein-mediated biomineralization of the diatom frustule. Mar. Biol. 126, 353-360. de Moor, P-P.E.A., 1998. The mechanism of organic-mediated zeolite crystallization. Ph.D. Thesis. Eindhoven University of Technology, The Netherlands. Okita, T., Volcani, B.E., 1980. Role of silicon in diatom metabolism. X. Polypeptide labelling pattems during the cell cycle, silicate starvation and recovery in Cylindrotheca fusiformis. Exp. Cell Res. 125, 471-481. Ozin, G.A., Yang, H., Sokolov, I., Coombs, N., 1997. Shell mimetics. Adv. Mater. 9, 662-667. Paasche, E., 1973. Silicon and the ecology of marine plankton diatoms. 2. Silicate-uptake kinetics in five diatom species. Mar. Biol. 19, 262-269. Perry, C.C., 1989. Chemical studies of biogenic silica. In: Mann, S., Webb, J., Williams, R.J.P. (Eds.), Biomineralization: Chemical and Biochemical Perspectives. VCH, New York, pp. 223-256. Perry, C.C., Fraser, M.A., Hughes, N.P., 1991. Macromolecular assemblages in controlled biomineralization. In: Sikes, C.S., Wheeler, A.P. (Eds.), Surface Reactive Peptides and

Polymers; Discovery and Commercialization, vol. 444. ACS Symposium Series, Dallas, TX, pp. 316-339. Pickett-Heaps, J., S~hmid, A.M., Edgar, L.A., 1990. The cell biology of diatom valve formation. Progr. Phycol. Res. 7, 1-168. Robinson, D.H., Sullivan, C.W., 1987. How do diatoms make silicon biominerals. TIBS 12, 151-154. Romero, O., 1994. Morphological variation of Skeletonema costatum (Greville) Cleve (Bacillariophyceae) in Aysen fiord, Chile. Rev. Biol. Mar. 29, 1-21. Round, F.E., Crawford, R.M., Mann, D.G, 1990. The Diatioms. Biology and Morphology of the Genera. Cambridge University Press, Cambridge. Schmid, A.M.M., 1986. Wall morphogenesis in Coscinodiscus wailesii Gran et Angst. II. Cytoplasmic events of valve morphogenesis. In: Ricard, M. (Ed.) Proceedings of the Eight International Symposium on Living and Fossil Diatoms. O. Koeltz, Koenigstein, Germany, pp. 294-314. Swift, D.M., Wheeler, A.P., 1991. Some structural and functional properties of a possible organic matrix from the frustules of the freshwater diatom Cyclotella meneghiniana. In: Sikes, C.S., Wheeler, A.P. (Eds.), Surface Reactive Peptides and Polymers; Discovery and Commercialization, vol. 444. ACS Symposium Series, Dallas, TX, pp. 340353. Swift, D.M., Wheeler, A.P., 1992. Evidence of an organic matrix from diatom biosilica. J. Phycol. 28, 202-209. Simpson, T.L., Volcani, B.E., 1981. Silicon and siliceous structures in biological systems. Springer, Heidelberg. Van Bekkum, H., Flanigan, E.M., Jansen, J.C., 1991. Introduction to zeolite science and practice. Elsevier, Amsterdam. Van Bennekom, A.J., Buma, A.G.L., Nolting, R.F., 1991. Dissolved aluminium in the Weddel-Scotia Confluence and the effect of A1 on the dissolution kinetics of biogenic silica. Mar. Chem. 35, 423-434. Van Bennekom, A.J., Jansen, J.H.F., van der Gaast, S.J., van Iperen, J.M., Pieters, J., 1989. Aluminium-rich opal: and intermediate in the preservation of biogenic silica in the Zaire(Congo) deep-sea fan. Deep Sea Res. 36, 173-190. Van de Poll, W.H., Vrieling, E.G., Gieskes, W.W.C., 1999. Location and expression of frustulins in the pennate diatoms Cylindrotheca fusiformis, Navicula pelliculosa and Navicula salinarum. J. Phycol. (submitted for publication). Vrieling, E.G., Beelen, T.P.M., van Santen, R.A., Gieskes, W.W.C., 1999a. Combined small and wide angle X-ray scattering reveals nano-scaled uniformity of pore architecture in diatomaceous silica. J. Phycol. (submitted for publications). Vrieling, E.G., Gieskes, W.W.C., Beelen, T.P.M., 1999b. Silicon deposition in diatoms: control by the pH inside the silicon deposition vesicle. J. Phycol. 35 (in press). Vrieling, E.G., Poort, L., Beelen, T.P.M. and Gieskes, W.W.C., 1999c. Growth and silicon content of the diatoms Thalassiosira weissflogii and Navicula salinarum at different salinities and enrichments with aluminium. Eur. J. Phycol. (submitted for publication).

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51

Zaremba, C.M., Stucky, G.D., 1996. Biosilicates and biomimetic silicate synthesis. Curr. Opin. Solid State Mater. Sci. 1,425-429. Zones, S.I., Nakagawa, Y., Lee, G.S., Chen, C.Y., Yuen, L.T., 1998. Searching for new high silica zeolites through a synergy of organic templates and novel inorganic conditions. Micropor. Mesopor. Mater. 21, 199-211.

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JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 53-60

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium

Sphingomonas paucimobilis Marianna Turkiewicz *, Ewa Gromek, Halina Kalinowska, Maria Zielifiska Institute of Technical Biochemistry, Technical University of Lodz, 4/10 Stefanowskiego Street, Lodz 90-924, Poland Received 14 October 1998; received in revised form 26 November 1998; accepted 22 December 1998

Abstract An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10~ i.e. below the temperature optimum for the strain growth (15"C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20-30~ and pH 6.5-7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0~ the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at - 5 and - 10~ 37 and 30%, respectively. The metalloprotease was stable up to 30~ for 15 min and up to 20~ for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme. 9 1999 Elsevier Science B.V. All rights reserved.

Keywords: Psychrophilic enzymes; Metalloprotease; Antarctic marine bacteria

1. Introduction

Abbreviations: BSA, bovine serum albumin; BzTyrOEt, Nbenzoyl-tyrosine ethyl ester; CFU, colony forming unit; EDTA, ethylenediaminetetraacetic acid; Hb, haemoglobin; IAA, iodoacetic acid; PAGE, polyacrylamide gel electrophoresis; pCMB, p-chloromercuribezoate; PMSF, phenylmethylsulfonyl fluoride; Rm, relative mobility; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane. * Corresponding author. Tel.: + 48-42-6366618; fax: + 4842-6313402. E-mail address: [email protected] (M. Turkiewicz)

The overwhelming majority of commercially important enzymes are produced by mesophilic microorganisms and applied in conditions resembling their environment in vivo. New biocatalysts active in unusual conditions are looked for a m o n g extremophilic microorganisms including psychrophiles and psychrotrophs (Adams et al., 1995). They constitute the most abundant group of organisms, since temperatures below 5~ occur in about 80% of biosphere and even more than 90% of marine environment (Margesin and Schinner, 1994; Brenchley, 1996).

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S0168-1656(99)00057-7

54

M. Turkiewicz et al./ Journal of Biotechnology 70 (1999) 53-60

Cold-adapted enzymes secreted by these microorganisms, also called psychrophilic enzymes or psychrozymes, show high catalytic efficiency at low temperatures, especially at 0-20~ i.e. a temperature range at which mesozymes are usually inactive, as well as reduced thermostability resulting in denaturation at moderate temperatures (Margesin and Schinner, 1994; Feller et al., 1996). Cold enzymes are potentially useful for numerous biotechnological processes, especially those which require a supply of exogenous energy, are exposed to higher risk of microbial contamination or temperature instability of reactants or products (Margesin and Schinner, 1994; Marshall, 1997). Another profit resulting from studies on microbial psychrozymes is an enrichment of pure culture collection by strains existing in permanently cold environments, e.g. marine polar regions (DeLong, 1997). Information acquired in this way are important for development of our still very limited knowledge of biodiversity.

rpm. The strain growth was observed by determination of CFUs on solid growth medium (0.3% of beef extract, 0.5% of bactotryptone and 5.51% of bacto-marine agar 2216, Difco) or by absorbance reading at 660 nm. All other chemicals employed in the studies were purchased from standard sources and were at least reagent grade.

2. Materials and methods

2.3. Protein determination

2.1. Organ&m and cultivation

Protein was assayed according to Lowry et al. (1951) using bovine serum albumin (BSA) as a standard, or spectrophotometrically at 280 nm in fractions eluted from chromatography columns.

Gram-negative rods Sphingomonas paucimobilis, strain no. 116, from the collection of the Department of Antarctic Biology of Polish Academy of Sciences in Warsaw, were used for the studies. This strain was isolated from a stomach of Antarctic krill, Euphausia superba Dana by Stuart Donachie at the Arctowski Polish Antarctic Station. The strain classification was done using API ZONE strips (Api System, Biomerieux, France) at 15~ as well as microscopic observations and Gram staining (Donachie, 1995). Shaken cultures of the strain were performed at pH 8.0 in a liquid medium containing synthetic marine water (Instant Ocean, Aquarium System Inc., France), 0.2% of bactopeptone and 1% of casein digest (Difco). One hundred and twenty ml of the medium in a 500 ml flask was inoculated with a standard inoculum (4% v/v, 1.5 x 105 CFU (colony forming units) ml-1) and cultivated at 10~ in a shaker (INFORS-Switzerland) at 160

2.2. Protease assay Proteolytic activity was determined according to Anson (1938) using urea denatured Hb (pH 7.0, 30~ reaction time 15 min) or spectrophotometrically according to Hummel (1959) using Nbenzoyl-tyrosine ethyl ester (BzTyrOEt) in 50 mM tris(hydroxymethyl)aminomethane (Tris)-HC1 buffer pH 8.0 at 20~ (increase of absorbance at 254 nm). In both methods, the activity was expressed in lamol of product (L-tyrosine present in trichloroacetic acid (TCA)-soluble hydrolysis products or N-benzoyl-L-tyrosine, respectively) liberated from respective substrate for 1 min in standard conditions.

2.4. Electrophoresis Polyacyrlamide gel electrophoresis (PAGE) was run in nondenatured conditions, according to Laemmli (1970), using 7.5% gels (0.6 x 8 cm) in 0.1 M borate buffer pH 9.0 and current intensity 2.5 mA per gel. Gels were fixed and stained with Coomassie brillant blue R-250. Proteolytic activity was assayed in 2 mm slices of nonstained gels against denatured Hb (2% v/w, 30~ pH 7.0) as a substrate.

2.5. Protease purification Purification of the protease was carried out at 4~ and in presence of Ca 2 + ions (0.5 mM) as a stabilizing factor. The protein was precipitated

M. Turkiewicz et al./Journal of Biotechnology 70 (1999) 53-60

from the centrifuged (5000 x g, 30 rnin) culture medium with ammonium sulphate (80% of saturation, 1 h, 0~ dissolved in 10 mM sodium phosphate buffer pH 7.0 and desalted on Sephadex G-25 (0.9 x 20 cm) equilibrated with the same buffer. Five ml protein fractions (A28o above 0.8) were pooled and applied to a bacitracin-Sepharose 4B column (0.9 x 10 cm, the buffer as above), prepared according to Stepanov and Rudenskaya (1983). The column was prewashed with the starting buffer (flow rate 20 ml h - ~) and the bound proteins were eluted using this buffer enriched with 25% (v/v) of isopropanol and 1 M of NaC1. Active fractions were pooled, dialysed against the starting buffer and stored in small aliquots at - 40~

3. Results

3.1. Effect of temperature on growth and protease secretion S. paucimobilis 116 was grown at temperatures from 0 to 30~ The highest proteolytic activity in the culture medium was observed in a range from 5 to 10~ (about 0.25-0.26 units ml-1, Fig. 1). At 15~ which is optimal for the strain growth, as well as at 0~ the activity was twice lower. At 30~ the strain did not excrete extracellular proteases. Above 30~ its growth was not observed. At 10~ which was chosen as a standard

55

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,

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5

"~

,

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7

cultivationtime(days) proteolyticactivity

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Fig. 2. Relationship between the strain growth at 10~ and the proteolytic activity in the culture medium during 7 days of cultivation. Proteolytic activity was assayed in standard conditions using urea denatured Hb.

strain cultivation temperature, the protease synthesis occurred to run simultaneously to the growth and reached the maximum after 72 h, i.e. at a late logarithmic phase of bacterial growth (Fig. 2). Protein pattern of the third day supernatant of the culture medium shows Fig. 3. Two protein bands were active against denatured Hb. The proteolytic activity present in this supernatant was inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF) (34% of inhibition) and 1 mM EDTA (ethylenediaminetetraacetic acid) (38% of inhibition) which proves that S. paucimobilis 116 70 -

65,2

50

0,30 "~

40

0,25 30

.~ 0,20 ==

20

0,15

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,* 0,10

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.-1

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7

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25 28

31

34

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Fig. 1. Effect of S. paucimobilis 116 growth temperature on synthesis of extracellular proteases. Proteolytic activity was assayed in culture medium after 72 h of cultivation, in standard conditions, using urea denatured Hb.

Fig. 3. Electophoretic pattern of proteins and its relationship with proteolytic activity in the culture medium after 72 h of the strain growth.

M. Turkiewicz et al./Journal of Biotechnology 70 (1999) 53-60

56 A~so 0,40 ]-

(-

- 0,35

i

0,35 -i-

+ 0,30 -," - 0,25 .~

0,30 i 0,25

T 036

0,20

:~

o,15

0,15

0,10

0,10

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0,05 0,00 I

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......... 21 26 31 ~activity

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41

46

51 56 fraction number

(-) pooled fraction

Fig. 4. Elution profile of proteins (A280) and proteolytic activity in affinity chromatography of the strain proteases on bacitracin-Sepharose 4B. The arrow shows the start of elution with 25% (v/v) isopropanol.

S. paucimobil& 116 protease was most active at 25-30~ for 15 min, and at 15-20~ for 60 min, against urea denatured Hb (Fig. 6). At - 1 0 to 0~ the enzyme retained 30-47% of maximal activity. The apparent energy of activation for the hydrolysis of urea denatured Hb calculated from an Arrhenius plot was 24.3 kJ mol-~ The S. paucimobilis 116 metalloprotease is very thermolabile, since the complete stability was observed for 15 min up to 30~ and for 60 min up to 20~ (Fig. 7). At 45~ only 38% of the activity remained after 15 min, and after 60 min it was inactive. The metalloprotease digested Hb at pH from 5.0 to 10.5 with maximum at pH 6.5-7.0

produces extracellular serine proteases and metalloproteases, p-Chloromercuribezoate (p-CMB) and iodacetic acid (IAA) did not influence the activity.

, . ,i

3.2. Metalloprotease properties The highly purified metalloprotease was obtained using the procedure described in Section 2. The enzyme was isolated from 760 ml of the supernatant from 72 h S. paucimobilis 116 culture medium (Table 1). Specific activity of the pooled active fractions after affinity chromatography (Fig. 4) was about 20-fold higher in comparison to the starting supernatant, both against urea denatured Hb and BzTyrOEt. Although bacitracin-Sepharose 4B coupled all the proteolytic activity (Fig. 4), the fractions eluted from this column contained only 10% of it. The eluted proteolytic activity was in 92% inhibited by EDTA (final concentration 1 mM, enzyme-inhibitor incubation time 1 h) and not influenced by PMSF. It proves that only the metalloprotease was eluted from the column and the serine protease present in the starting material remained bound to bacitracin-Sepharose 4B. The yield of purification was low (3-4%, Table 1). PAGE of this highly purified enzymic preparation run in non-denatured conditions showed one strong protein band with relative mobility (Rm) 0.58 contaminated with trace amount of another protein with R m 0.83 (Fig. 5).

..

9

'

:ri!'

Fig. 5. PAGE of the highly purified metalloprotease from S. paucimobilis 116 (pooled active fractions after bacitracin-sepharose 4B column).

Table 1 Purification of a metalloprotease from S. paucimobilis 116 ~~

Purification step

Volume (ml)

Protein (mg)

Activity Specific (units mgof protein)

'

Yield ('5))

Purification (fold)

Hb

Hb

\

Total (units)

BzTyrOEt

BzTyrOEt

6 1:

4,

Culture supernatant Salted out protein Sephadex G-25 Bacitracin-Sepharose 4B

760 10

43 5.4

313.20 29.84 11.70 0.46

Hb

BzTyrOEt

Hb

BzTyrOEt

0.23 1.40 2.33 5.60'

0.04 0.15 0.28 0.72'

71.82 41.84 27.30 2.60

11.70 4.39 3.29 0.33

* The activity was inhibited by EDTA (in 92%) and not by PMSF.

a 100

59 38 3.6

100

-

-

38 28 3

6 10

4 8

24

19

58

M. Turkiewic: et al ..... Journal of Biotechnology 70 (1999) 53-60

100

100-

80 60 20 :

-10

-5

40

b

0

5

I0

15

20

25

30

35

40

45

20-

50

temperature (~

0

Fig. 6. Effect of temperature on the activity of the S. pauchnobilis 116 metalloprotease. The activity was assayed for two reaction times: (a) 15; and (b) 60 min, using urea denatured Hb pH 7.0.

(Fig. 8). Optimal pH in BzTyrOEt hydrolysis was 8.0 and at pH 10.5 the enzyme showed 30% of the maximal activity (Fig. 8). At 4~ the protease was stable at pH 6.0-8.0 for 60 min, and at pH 9.5 exhibited 91% of maximal activity (data not presented). The preferred substrate of the highly purified metalloprotease from S. paucimobilis 116 was denatured Hb (Table 2). Its activity against casein, fibrinogen and native Hb was 20-30% lower. BSA was digested to a small extent.

4. Discussion

A metalloprotease active against various proteins at neutral and slightly alkaline pH was isolated from extracellular proteases secreted by

80

a

5,0

6,0

7,0

8,0

9,0

10,0

11,0 pH

Fig. 8. Influence of pH on the activity of the S. paucimobilis 116 metalloprotease. The enzyme activity was assayed against urea denatured Hb (a) and BzTyrOEt (b).

S. paucimobilis strain 116, isolated from stomachs of Antarctic krill E. superba Dana. S. paucimobilis 116 synthesizes extracellular proteases with highest intensity at a temperature markedly lower (5-10~ than corresponding to its maximal growth rate (15~ similarly to many other coldadapted microorganisms, including Antarctic ones (Margesin and Schinner, 1994; Feller et al., 1996). It is presumed that intensification of enzyme biosynthesis at low temperatures is one of the main mechanisms of adaptation of these organisms to surrounding environment (Margesin and Schinner, 1994; Brenchley, 1996). Secretion of proteases by S. paucimobilis 116 starts already in the phase of adaptation and its maximum coincides with the end of logarithmic phase, falling on 72 h. This period of time is shorter than observed e.g. by Margesin and Schinner (1992a) for a metalloprotease from Alpine

~ Table 2 Activity of the S. paucimobilis 116 metalloprotease against selected proteins

60*

20,'

0

a

5

10

20

30

35

40

45

50

temperature (~

Fig. 7. Thermostability of the S. paucimobilis 116 metalloprotease. The enzyme was incubated at different temperatures (0-50~ for: (a) 15; and (b) 60 rain and the residual activity towards urea denatured Hb was determined in standard conditions (30~ pH 7.0).

Substrate

Activitya (units m1-1)

Hb denatured Hb native Casein Fibrinogen BSA

0.249 0.160 0.200 0.177 0.017

a Assay conditions: 30~ pH 7.0, 2% solutions of protein substrates, 15 min reaction.

M. Turkiewic: et al. /Journal of Biotechnolog3" 70 (1999) 53-60

strain of Pseudomonas fluorescens (96 h) or for subtilisin from Antarctic Bacillus TA 39 (150 h, Feller et al., 1996). Electrophoretic analysis and inhibitory tests proved that S. paucimobilis 116 produces at least two extracellular proteases, i.e. a metallo- and a serine enzyme. However we have managed to purify only the metalloenzyme. The serine protease coupled with bacitracin-Sepharose 4B but was immobile in a milieu of the eluent employed in our studies. According to Stepanov and Rudenskaya (1983) ligand from bacitracin-Sepharose possesses various amino acid residues and therefore binds not only serine proteases, but some other too which was revealed in model experiments on papain, subtilisin and B. subtilis metalloprotease. Kinetic properties such as very weak thermostability and low optimal temperature (15-30~ depending on reaction time) of the S. paucimobilis 116 metalloprotease point to its increased adaptation to low temperatures in comparison to many other enzymes, also originating from permanently cold, e.g. Antarctic habitats. Reported metalloproteases from other strains inhabiting cold environments usually exhibit higher optimal temperatures. Good examples are proteases from Alpine strain of P. fluorescens 114, most active at 40-45~ for 30 rain (Hamamoto et al., 1994) and from Xanthomonas maltophilia with maximal activity at 50~ for 15 rain (Margesin and Schinner, 1991). However, a protease isolated by K/irst et al. (1994) from an unidentified Antarctic bacterial strain was most active against proteins at 28~ Another important feature pointing to the psychrophilic character of the metalloprotease from S. paucimobilis 116 is its high activity at temperatures from 0 to 5~ (47-60% of maximal activity), resembling the strain physiological temperature range, as well as the ability to protein hydrolysis below 0~ (e.g. a t - 1 0 ~ the enzyme shows 30% of maximal activity against urea denatured Hb) which was not reported for other coldadapted proteases. For comparison, a metalloprotease from P. fluorescens 114 exhibited 20-30% of maximal activity at temperatures from 0 to 10~ (Hamarnoto et al., 1994), enzymes from Alpine strains showed 15-18% of maximal activ-

59

ity at 10~ (Margesin and Schinner, 1992b), the protease from X. maltophilia did not hydrolyse azocasein at 0~ and at 10~ retained only about 5% of maximal activity (Margesin and Schinner, 1991). The value of activation energy calculated for the S. paucimobilis 116 (24.3 kJ mol-1) is lower than obtained for enzymes from X. maltophilia (61.9 kJ tool-l, Margesin and Schinner, 1991), psychrophilic P. fluorescens strains (36.938.0 kJ mol-~, Margesin and Schinner, 1992a) or Antarctic psychrophile Bacillus TA41 (38.5 kJ mol- 1, Davail et al., 1994). In all the cases, values of activation energies of mesozymes tested under the same conditions were even higher (Margesin and Schinner, 1991, 1992a; Davail et al., 1994). The reduction of energy barrier displayed by the S. pauchnobilis 116 metalloprotease and correlated with its low temperature optimum and weak thermostability may result from more flexible conformation of the molecule in comparison to enzymes mentioned above. A potentially useful property of the S. paucimobilis 116 metalloprotease might be its relatively high activity towards native Hb, equal to about 70'/o of the activity against urea denatured Hb. Cold-adapted proteases may be applied in many fields, e.g. in production of detergents for washing at low temperatures, in tannery, in the food industry (haze removal from beer, bakery, cheese-making, production of fermented foods, meat tenderisation) or high protein waste degradation (Margesin and Schinner, 1994; Brenchley, 1996). Our studies demonstrate that proteases synthesized by marine Antarctic bacteria might become an interesting alternative to currently applied mesophilic enzymes.

References Adams. M.W.W.. Perler. F.B., Kelly, R.M., 1995. Extremozymes: expanding the limits of biocatalysis. Bio/' Technology 13, 662-668. Anson, M.L., 1938. Estimation of pepsin, trypsin papain and cathepsin with haemoglobin. J. Gen. Physiol. 22, 79-82. Brenchley, J.E.. 1996. Psychrophilic micro-organisms and their cold-active enzymes. J. Ind. Microbiol. 17, 432-437. Davail, S., Feller. G., Narinx, E., Gerday. C., 1994. Cold adaptation of proteins. J. Biol. Chem. 269, 17448-17453.

60

M. Turkiewicz et al./Journal of Biotechnology 70 (1999) 53-60

DeLong, E.F., 1997. Marine microbial diversity. Trends Biotechnol. 15, 203-207. Donachie, S.P., 1995. Ecophysiological description of marine bacteria from Admiralty Bay (Antarctica) and the digestive tracts of selected Euphausiidae, Philosophy Doctor Thesis, Polish Academy of Sciences, Warsaw. Feller, G., Narinx, E., Arpigny, J.L., Aittaleb, M., Baise, E., Genicot, S., Gerday, Ch., 1996. Enzymes from psychrophilic organisms. FEMS Microbiol. Rev. 18, 189-202. Hamamoto, T., Kaneda, M., Horikoshi, K., Kudo, T., 1994. Characterization of a protease from a psychrotroph Pseudomonas fluorescens 114. Appl. Environ. Microbiol. 60, 3878-3880. Hummel, B.C.W., 1959. A modified spectrophotometric method for determination of chymotrypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-1400. K/irst, U., Woehl, M., Czempinski, K., Schmid R.D., 1994. Characterization of extracellular hydrolases from marine psychrophilic bacteria. In: Seventh International Congress of Bacteriology and Applied Microbiology, Praha, Communicate MO122.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 165-275. Margesin, R., Schinner, F., 1991. Characterization of a metalloprotease from psychrophilic Xanthomonas maltophilia. FEMS Microbiol. Lett. 79, 257-262. Margesin, R., Schinner, F., 1992a. Production and properties of an extracellular metalloprotease from a psychrophilic Pseudomonas fluorescens. J. Biotechnol. 24, 207-210. Margesin, R., Schinner, F., 1992b. A comparison of extracellular proteases from three psychrotrophic strains of Pseudomonasfluorescens. J. Gen. Appl. Microbiol. 38, 209-225. Margesin, R., Schinner, F., 1994. Properties of cold-adapted micro-organisms and their potential role in biotechnology. J. Biotechnol. 33, 1-14. Marshall, C.J., 1997. Cold-adapted enzymes. Trends Biotechnol. 15, 359-363. Stepanov, V.M., Rudenskaya, G.N., 1983. Proteinase affinity chromatography on bacitracin-Sepharose. J. Appl. Biochem. 5. 420-428.

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 61-63

Cyanobacteria

a potential source of new biologically active substances

Susann Kreitlow *, Sabine Mundt, Ulrike Lindequist Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University Greifswald, F.-L.-Jahn-Str. 15a, D- 17487 Greifswald, Germany

Received 9 October 1998; received in revised form 2 December 1998; accepted 22 December 1998

Abstract

Hydrophilic and lipophilic extracts of twelve cyanobacterial strains, isolated from flesh and brackish water, and two waterblooms, collected during the summer from the Baltic Sea, were investigated for their antibiotic activities against seven microorganisms. No inhibitory effects were found against the three Gram-negative bacteria Escherichia coli, Proteus mirabilis and Serratia marcescens and the yeast Candida maltosa. Of all cyanobacterial samples, extracts from seven species inhibited the growth of at least one of the Gram-positive bacteria Micrococcusflavus, Staphylococcus aureus and Bacillus subtilis. M. flavus proved to be the most sensitive bacterium in the agar diffusion test system. In particular, the hexane and dichlormethane extracts showed antimicrobial effects. But only one water extract, prepared from material of a natural waterbloom, was found to be active. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyanobacteria; Blue-green algae; Antibiotic; Antifungal; Agar diffusion method

Cyanobacteria (blue-green algae) provide a potential source of biologically active secondary metabolites (Teuscher et al., 1992). Investigations over the last decades have identified compounds with for instance cytotoxic, antifungal, antibacterial or antiviral activity. Most of the isolated substances belong to groups of polyketides, amides, alkaloids and peptides (Borowitzka, 1995; Falch, 1996; Kleinkauf and von D6hren, 1997). * Corresponding author. Tel.: + 49-3834-864865; fax: + 493834-864802. E-mail address: [email protected] (S. Kreitlow)

The blue-green algae are among the oldest photoautotrophic organisms. Their cultivation without organic substrates can be an economical advantage over other microorganisms. An optimised production of relevant compounds under controlled culture conditions is conceivable (Kulik, 1995). In view of the growing resistance of bacteria to c o m m o n antibiotics the search for new antimicrobially active compounds has become increasingly important (Trias and Gordon, 1997). In our screening programme we tested approximately fifty extracts from twelve different cyanobacterial strains and two waterblooms against different bacteria and one yeast. The re-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S0168-1656(99)00058-9

62

S. Kreitlow et al. 'Journal of Biotechnology 70 (1999) 61-63

sults of our study show the ability of cyanobacteria to produce compounds with antimicrobial effects. Ten filamentous cyanobacterial strains: Anabaena solitaria AS, Anabaena cylindrica 1611, Anabaena variabilis 1 and Anabaena sp. 7120 (Nostoccaceae); Oscillatoria tenuis 03, Oscillatoria sp. 022, Oscillatoria rubescens O16, Oscillatoria prolifica 02, Pseudanabaena catenata 154 and Limnothrix sp. 051 (Oscillatoriaceae) and the unicellular form Synechocystis aquatilis 428 (Chroococcaceae) were isolated from fresh water. The filamentous strain Nodularia spumigena 280 (Nostoccaceae) originated from brackish water. The waterblooms were collected from different regions of the Baltic Sea during the summer of 1997 and consist mainly of Anabaena lemmermannii (Waterbloom 97-7) and Microcystis ichthyoblabe, A. lemmermannii and Nodularia sp. (Waterbloom 97-8). The strains were cultivated batchwise in 2-1 flasks either in BG11- or Z1/2-medium (Meffert, 1971; Rippka et al., 1979). The cultures, which were illuminated continuously with warm-white light under room temperature (20_+ 2~ were harvested in their exponential growth phase by centrifugation (4~ 4000 x g). The freeze-dried cyanobacterial material (about 1 g) was taken for exhaustive extraction. Both lipophilic and hydrophilic extracts were screened for antibiotic activity. In general, the extraction was carried out successively with hexane, dichlormethane (or in one case: ethylacetate), methanol and water. After evaporation in vacuum, the extracts were stored at - 2 0 ~ until use. The antibacterial and antifungal activities of all samples were tested against the Gram-negative bacteria Escherichia coli, Serratia marcescens and Proteus mirabilis, the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, Micrococcus flavus and the yeast Candida maltosa. The in vitro antimicrobial test utilised for the investigation is based on the diffusion method on agar plates (Collins et al., 1989). Therefore, nutrient agar was inoculated with a standardised quantity of a suspension of the respective organism. Sterilised paper disks (0 9 ram) containing the extract (2 mg) were transferred to the prepared agar

dishes. The antibiotic substance ampicillin was assayed for reference purposes. The inhibition zones (diameter minus diameter of the paper disk) were measured after 16 h incubation at 37~ (M. flavus at 22 _+ 2~ In order to increase the sensitivity of the test system a prediffusion for 4 h at 4~ was granted. Because of the small yields the dichlormethane extracts were not tested against all bacteria. None of the examined extracts has shown activity against Gram-negative bacteria. In addition, there were no inhibitory effects against the yeast C. maltosa. Among the water extracts only that from the natural waterbloom (97-8) was effective against S. aureus. The hexane extract from this sample gave the widest inhibition zones (3-4 ram) against B. subtilis. Two lipophilic extracts of the Limnothrix sp. 051 inhibited notably the growth of at least two of the tested Gram-positive bacteria. The dichlormethane extract of Oscillatoria sp. 022 showed the strongest activity against S. aureus and generally the largest inhibition zone (13 mm) as measured in our screening system. But no inhibitory effects of the other extracts of these species could be tested. M. flavus was the most sensitive bacterium against the methanol extract of O. rubescens 016. As reported in Table 1, which summarizes all extracts that showed activity against at least one of the test organisms, mainly hexane and dichlormethane extracts showed antimicrobial activities. The screening of the extracts was extended by testing their cytotoxicity against human fibroblast cells (Lindl and Bauer, 1994). In general, the lipophilic extracts showed stronger cytotoxic effects in concentrations of and above 100 ~tg m l (data not shown). However, there were no remarkable differences in cytotoxicity between extracts with antibiotic activities and extracts without antibiotic activities. In this early stage of the investigation, a differentiation between cytotoxic and antibiotic substances is not possible because the crude extracts contain a variety of different compounds. We were able to show that six out of twelve cyanobacterial strains cultivated under laboratory conditions produce substances with antibacterial activity. The ability of the blue-green algae to

S. Kreitlow et al./Journal of Biotechnology 70 (1999) 61-63

63

Table 1 Activity of the lipophilic and hydrophilic extracts of different cyanobacterial strains in a concentration of 2 mg paper d i s k - l a Zones of inhibition (in mm diameter) Cyanobacteria

Extract

S. aureus

B. subtilis

M. flavus

O. tenuis 03

Dichlormethane Methanol Methanol Hexane Hexane Dichlormethane Hexane Ethylacetate Water

+ + + + + -

+ + + + + + -

+ + + + + + + +

O. rubescens 016 Anabaena sp. 7120 A. solitaria AS Oscillatoria sp. 022 Limnothrix sp. 051 Waterbloom 97-8

+ + + +

+ + +

+

+ + +

+ + + + + + + + +

a--, No inhibitory effect; + , width < 1 mm; + + , width 1-8 mm; + + + , width > 8 mm.

produce antibiotic compounds could be an advantage for their survival in their natural environment (Teuscher et al., 1992). A large scale production to obtain sufficient cyanobacterial biomass is the precondition for the isolation and characterisation of the antibiotically and/or cytotoxically active compounds. Studies on this aspect are in progress.

Acknowledgements The authors thank Professor Kohl, Institute of Ecology, Humboldt-University, Berlin; Professor Pohl, Institute of Pharmacy, University Kiel; Dr Fulda, Botanical Institute, University Rostock and Dr Hfibel, Institute of Ecology, University Greifswald for supplying the cyanobacterial strains. The financial support by the government of Mecklenburg-Vorpommern is gratefully acknowledged.

References Borowitzka, M.A., 1995. Microalgae as sources of pharmaceu-

ticals and other biologically active compounds. J. Appl. Phycol. 7, 3-15. Collins, C.H., Lynes, P.M., Grange, J.M., 1989. Antimicrobial sensitivity and assay tests. In: Collins, C.H., Lynes, P.M. (Eds.), Collins and Lyne's microbial methods. Butterworth, London, pp. 155-168. Falch, B., 1996. Was steckt in Cyanobakterien? Pharmazie in unserer Zeit. 25, 311-321. Kleinkauf, H., von D6hren, H., 1997. Products of Secondary Metabolism. In: Rehm, H.J., Reed, G. (Eds.), Biotechnology, vol. 7. Weinheim, pp. 308-309. Kulik, M.M., 1995. The potential for using cyanobacteria (blue-green algae) and algae in the biological control of plant pathogenic bacteria and fungi. European J. Plant Path. 101, 585-599. Lindl, T., Bauer, J., 1994. Zell- und Gewebekultur. GustavFischer-Verlag, Stuttgart, p. 201. Meffert, M.E., 1971. Cultivation and growth of two planktonic Oscillatoria spec. Mitt. Internat. Verein. Limnol. 19, 189-205. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria taxonomy redefinition of the blue-green algae to conform to the bacteriological code. J. Gen. Microbiol. 111, 1-16. Teuscher, E., Lindequist, U., Mundt, S., 1992. Cyanobakterien, Quellen biogener Wirkstoffe. Pharm. Ztg. Wiss. 137, 57-69. Trias, J., Gordon, E.M., 1997. Innovative approaches to novel antibacterial drug discovery. Curr. Opin. Biotech. 8, 757762.

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Biotecbnology ELSEVIER

Journal of Biotechnology 70 (1999) 65-69

Biodiversity and potentials of marine-derived microorganisms Federica Sponga *, Linda Cavaletti, Ameriga Lazzarini, Angelo Borghi, Ismaela Ciciliato, D. Losi, Flavia Marinelli Biosearch Italia, Via R. Lepetit 34, 21040 Gerenzano (Va), Italy

Received 9 October 1998; received in revised form 19 November 1998; accepted 22 December 1998

Abstract

The marine environment is a prolific resource for the isolation of less exploited microorganisms. As a matter of fact, in the sea, untapped habitats exist with unique characteristics. In addition, the potential contribution of marine sources to the discovery of new bioactive molecules was recently recognized. Biosearch Italia possesses a collection of about 40000 microorganisms, isolated from different ecological niches. In the search of new bioactive entities, investigations were expanded to marine habitats including marine sediments and organisms. More then 800 microorganisms have been isolated. About half belong to fungal genera, the others being actinomycetes. The frequency of antibiotic activities produced by these marine strains has been determined. Initial data are encouraging: marine isolates produce antibiotic activities with frequencies comparable to terrestrial ones. These activities probably represent a mixture of novel metabolites and known products previously discovered from terrestrial isolates. Further investigations are ongoing to assess the novelty of these observed microbiological activities. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Biodiversity; Marine microorganisms; Antibiotic activities

1. Introduction

In the process of searching for useful natural products, it is generally agreed that a diverse and less exploited repertoire of microbes is essential to obtain a variety of novel metabolites. Biosearch Italia possesses a collection of more than 40000 microorganisms. In order to enlarge the collection with a novel array of microorgan* Corresponding author. Fax: + 39-2-96474238. E-mail address: [email protected] (F. Sponga)

isms, different ecological areas have been investigated as potential sources of microbial diversity. The marine environment, representing more than two thirds of our planet, is still under-explored and is considered a prolific resource for the isolation of less exploited microorganisms. Data based on 16S r R N A sequencing show that streptomycete strains of marine origin can be representative of peculiar species (Stackebrandt et al., 1991). As a matter of fact, the increasing impact of marine organisms on the discovery of new bioactive molecules has been recognized (Fenical and

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00059-0

66

F. Sponga et al. Journal o/" Biotechnology 70 (1999) 65-69

Jensen, 1994; Liberra and Linderquist, 1995: Bernan et al., 1997; Romero et al., 1997). For all these reasons, our isolation programs have been recently focused on marine sediments (Cuomo et al., 1995) and marine sponges (Hohnk and Ulken, 1979; Imamura et al., 1993; Abrell et al., 1994; Numata et al., 1997). We report here on the isolation of actinomycetes and filamentous fungi from these two sources. The capability of the isolates to produce antibiotic activities is compared with that of the previously observed from terrestrial strains. We have also evaluated the effect of the presence of sea water in the fermentation media on the antibiotic productivity by actinomycetes. 2. Materials and methods

2.1. Isolation from marine sediments A total of 38 marine sediments were collected in different areas (Mediterranean Sea, Red Sea, Atlantic Ocean), at different depths but not exceeding 20 m, and stored at 4~ until use. Soil dilution plate method and a modified soil plate method (Warcup, 1960) were used to isolate fungi. The isolates were cultivated in agar media into Petri dishes at 22~ under dark conditions. A number of media were tested to determine the optimal growth conditions. Petri dishes were inspected daily for up to 1 month for the development of colonies on the agar. The isolated fungi were identified to the level of genus by microscopic morphology. Nomenclature of fungi follows that listed in Ainsworth et al. (1973) and in Kohlmeyer et al. (1991). In the case of actinomycetes, 1 g of sediment was suspended in 10 ml of sterile sea water, vortexed for 1 min and decanted for 30 min. Supernatant (100 ill) was seeded on low and high nutrient agar isolation media, containing sea water. After 2-3 weeks of growth at 28~ single colonies were macro/microscopically observed for a preliminary classification at genus level based on morphology, picked up and transferred on oatmeal agar plates and allowed to grow for a further 2 weeks at 28~

2.2. Isolation from marine sponges Freshly withdrawn samples of sponge (30), belonging to ten different species, from Ligurian coastal areas at 3 - 4 m of depth were used. The sponges were collected and classified up to species by C. Cerrano, Institute of Zoologia, University of Genova (Italy). One sample from an unclassified alga was also included into the isolation program. To isolate filamentous fungi, fresh sponge material was rinsed in marine sterile water, soaked in ethyl alcohol for 5 min and then rinsed in sterile water for 5 rain. Small pieces of each sponge were deposited on two different agar media in Petri dishes: one rich medium and the same diluted 1:1 with marine water. Then the isolation followed the protocol as before. In the case of actinomycetes, sponges were rinsed in sterile sea water, cut into small pieces and treated as described for sediments. Moreover, the same pieces were also deposed directly onto a set of isolation plates. After 2 - 3 weeks of incubation at 28~ single colonies were microscopically observed for morphological classification, picked up, transferred onto oatmeal agar plates, and reincubated until growth.

2.3. Fermentation and sample preparation The fungal strains were inoculated in liquid media routinely used for the production of secondary metabolites in 500 ml Erlenmeyer flasks. Flasks were then incubated on a rotatory shaker at 180 rpm and 22~ for 4 days. Pure cultures of actinomycetes were used to seed two sets of flasks containing two liquid media: a rich medium routinely used for the production of secondary metabolites, and the same medium diluted 1:1 with marine water. Flasks were then incubated on a rotatory shaker at 200 rpm at 28~ for 5 days. Samples were prepared by resin absorption/elution of all fermented broth cultures, with the aim to eliminate impurities and enzymatic activities which could interfere with the biological assays.

F. Sponga et al. ,' Journal of Biotechnology 70 (1999) 65-69 2.4. Assay of antimicrobial activity

r Spongia virgultosa

The following test-strains were used: Enterococcus faecium D399, as a representative of Grampositive bacteria, Escherichia coli clinical isolate, as a Gram-negative representative, and Candida albicans clinical isolate, as a representative of yeasts. Microbial assays were performed in 96-well microtitre plates, in liquid medium inoculated 10 4 cfu m l - 1 with the opportune test-microorganism. Microbial growth was then detected at 620 nm after overnight incubation at 37~ in appropriate humidity conditions. A sample was defined 'active' when able to inhibit the growth of the testmicroorganism in a percentage higher than 80 with respect to controls.

Mieroor~misms mFUNOI r--tSTREPTO NRARE

r Ircinia r Acanthella acuta r Chlatrina cerebrum r Agelas oroides r Petrosia ficiformis r Ascandra falcata r Spongia officinalis r

reniformis r Axinella damicornis 0

3. Results

To evaluate the potential of marine sources for natural product discovery, different parameters were considered: abundance, variety of isolates per source, and their ability to produce diverse antibiotic activities. A total of 395 fungi were isolated either from sediments or from marine organisms (Table 1). The contribution of sediments was about seven strains per sample, while from sponges, nine different fungal strains were isolated per sample. When compared with the number of the isolates from soil samples, both marine sources (marine sediments and sponges) appear rich in fungal strains. Among these marine fungi, about 75% sporulated on the media currently used. These sporulating fungi could be assigned to the followTable 1 Number of sources and related isolates Source

Strain number

Type

N~

Fungi

Sediment Organism

38a 31

a

Rare

Stm

130

17

265

284

22 129

Only 18 were used to isolate fungi.

67

20

40 60 80 100 120 number of isolates

Fig. 1. Distribution of microbial population among sponge species. Value in brackets represents the number of individuals used. ing genera: Acremonium, Alternaria, Aspergillus,

Cephalosporium, Chaetomium, Cladosporium, Geotricum, Fusarium, Gliomastix, Humicola, Paecilomyces, Penicillium, Pestalotia, Phoma, Plectosphaerella, Scopulariopsis, Stachybotrys, Trichoderma. In the case of actinomycetes isolation, marine organisms yielded an average of ten morphologically different strains per single source: 31% being streptomycetes, and 69% belonging to rare genera mainly represented by micromonosporas, followed by nocardioforms and actinomaduras/microtetrasporas. Marine sediments yielded an average of one isolate per source: 50% streptomycetes and 50% rare actinos as shown in Table 1. Even among these rare actinos, Micromonospora was the more represented genus, in accordance with published studies (Jensen et al., 1991). It appeared of interest to investigate the distribution of the microbial population in the different species of sponges. The results are reported in Fig. 1.

68

F. Sponga et al. ,,'Journal of Biotechnology 70 (1999) 65-69

All strains were fermented in standard production liquid media and assayed for antimicrobial activity against three representative microorganisms: a G + , a G - and one yeast. The percentages of active strains are reported in Table 2. Marine actinomycetes have also been fermented and assayed for antimicrobial activity in the production medium diluted 1:1 with sea water. In most cases the antimicrobial activities were detected in both conditions, however four strains produced activities only when fermented in the presence of sea water.

4. Conclusion Most of the fungi isolated from these marine samples belong to genera usually considered soil inhabitants. Among the actinomycetes, a good variety of known genera was represented. A general conclusion is that marine organisms yielded a number of isolates per source that is comparable with that obtained from soils. On the other side, the number of isolates obtained from marine sediments is lower, particularly in the case of actinomycetes. Therefore, in terms of abundance of both types of microbial communities, sponges could be considered a more suitable source than sediments. The abundance of microorganisms isolated is different in the different species of sponges. Further investigation is needed to determine if the microbial population is specifically associated to each species. The frequency of antimicrobial activities displayed by marine fungi and actinomycetes is similar to that previously observed in terrestrial Table 2 Percentage of strains producing antimicrobial activities in our assays % Active strains

Fungi Rare Stm

Test-microorganisms E. faecium

E. coli

C. albicans

1.2 3.5 6

0.6 0 1.3

2.2 0.3 0.7

strains during the course of our screening programs. The ability shown by fungal isolates to germinate and produce secondary metabolites under the conditions generally used for the terrestrial ones make it difficult to define them as marine fungi. The presence of a few antibacterial activities expressed only when some actinomycete strains are incubated in the fermentation medium diluted with sea water, suggests that these isolates could be defined as 'adapted' to the marine environment. Structural characterization of the thus far discovered antibiotic activities, will provide additional information on the potential of marine environments as sources of novel metabolites.

Acknowledgements The authors wish to thank Maurizio Rogina for supplying marine sediments, and also the staff of both the Institute of Zoologia and the Institute of Biochimica of the University of Genova, Italy for supplying marine sponges and for their valuable support on the classification and evaluation of these.

References Abrell, L.M., Cheng, X.C., Crews, P., 1994. New nectriapyrones by salt water culture of a fungus separated from an Indo-Pacific sponge. Tetrahedron Lett. 35 (49), 9159-9160. Ainsworth, G.C., Sparrow, F.K., Sussmann, A.S., 1973. The Fungi, an Advanced Treatise, Academic, New York, San Francisco, London, vols. IVA and IVB. Bernan, V.S., Greenstein, M., Maiese, W.M., 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43, 57-90. Cuomo, V., Palomba, I., Perretti, A., Guerriero, A., D'Ambrosio, M., Pietra, F., 1995. Antimicrobial activities from marine fungi. J. Mar. Biotech. 2, 199-204. Fenical, W., Jensen, P.R., 1994. Strategies for the discovery of secondary metabolites from marine bacteria: ecological perspective. Annu. Rev. Microbiol. 48, 559-584. Hohnk, W., Ulken, A., 1979. Pilze aus marinen schwammen Veroff. Inst. Meeresforsch. Bremerh. 17, 199-204. Imamura, N., Nishijma, M., Adachi, K., Sano, H., 1993. Novel antimycin antibiotics, urauchimycins A and B, produced by marine actinomycete. J. Antibiot. 46 (2), 241246.

F. Sponga et al./Journal of Biotechnology 70 (1999) 65-69 Jensen, P.R., Dwight, R., Fenical, W., 1991. Distribution of Actinomycetes in near-shore tropical marine sediments. Appl. Environ. Microbiol. 57 (4), 1102-1108. Kohlenmeyer et al., 1991. Illustrated key to the filamentous higher marine fungi. Bot. Mar. 34, 1-61. Liberra, K., Linderquist, U., 1995. Marine fungi-a prolific resource of biologically active natural products? Pharmazie 50, 583- 588. Numata, A., Amagata, T., Minoura, K., Ito, T., 1997. Gymnnastatins, novel cytotoxic metabolites produced by a fungal strain from a sponge. Tetrahedron Lett. 32, 5675-5678.

69

R omero, F., Espliego, F., Perez Baz, J., Garcia de Quesada, T., Gravalos, D., De la Calle, F., Fernandez-Puentes, J.L., 1997. Thiocoraline, a new despsipeptide with antitumor activity produced by a marine Micromonospora. J. Antibiot. 50, 734-737. Stackebrandt, E., Witt, D., Kemmerling, C., Kroppenstedt, R., Liesack, W., 1991. Designation of streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes. Appl. Environ. Microbiol. 57, 1468-1477. Warcup, J.H., 1960. Methods for isolation and estimation of activity of fungi in soil. In: Ecology of Soil Fungi. Liverpool University Press, pp. 3-21.

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Biotecbn,o,logy ELSEVIER

Journal of Biotechnology 70 (1999) 71-75

Production and particle characterization of the frustules of Cyclotella cryptica in comparison with siliceous earth Zsuzsa Cs6g6r *, Dyna Melgar, Karsten Schmidt, Clemens Posten 1 Institut fiir Mechanische Verfahrenstechnik und Mechanik, Universitiit Karlsruhe, D-76128 Karlsruhe, Germany Received 14 October 1998; received in revised form 3 December 1998; accepted 22 December 1998

Abstract Diatoms are p h o t o a u t o t r o p h i c micro-organisms that use inorganic carbon sources and light in photosynthesis.

Diatom frustules were characterized in terms of particle techniques and compared with siliceous earth, i.e. depositions of diatoms that have wide technical applications. To obtain enough biomass for frustule characterization Cyclotella cryptica has been cultivated in a 15 1 photobioreactor under controlled conditions. Native diatom frustules are characterized by a 1.5-fold lower density and 80-fold higher specific surface than siliceous earth. Therefore, native diatom frustules provides a material with novel properties which might be interesting for special technical application. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Diatoms; Cultivation; Photobioreactor; Frustules; Siliceous earth

I. Introduction

Inorganic materials with a complex pattern on a microscopic scale are of increasing research interest (Mann and Ozin, 1996). Possible technical applications could be the design of new types of catalyst supports, specific filter aid or chromatographic separation material, or biomedical implants with macroporosity. Siliceous earth is an inorganic material widely used in industrial scale because of its high poros* Corresponding author. Tel.: + 49-721-6086171; fax: + 49721-693965. E-mail addresses: [email protected] (Z. Cs6g6r), [email protected] (C. Posten) Tel.: + 49-721-6082410; fax: + 49-721-693965.

ity (e.g. as filtering aid) and its high surface area (e.g. as HPLC column filling material). Siliceous earth is a fossil deposition of diatoms. Diatoms are eukaryotic microalgae. The cells are enclosed in amorphous silica structures (frustules). The cells may adhere through organic polymers forming chains of different length. The chloroplasts are of brownish color because of the pigment fucoxanthin. Although siliceous earth consists of diatom frustules, the amorphous native particles may differ in various respects. Properties such as volumetric density, particle size, surface area, porosity may have changed after deposition. Particularly transport behavior, catalytic activity, and separation efficiency are influenced strongly by particle

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Z. Cs6g6r et al./Journal of Biotechnology 70 (1999) 71-75

size and surface area. Thus, native diatom frustules may prove to be an interesting material for high quality application. The aim of the work presented in this paper was to cultivate diatoms, characterize their frustules from the point of view of particle techniques and to compare native diatom frustules with siliceous earth. Such a comparison has not been reported in the literature, yet.

2. Materials and methods

2.1. Microorgan&ms and culture conditions Cyclotella cryptica Reimann, Lewin and Guillard 1070 was obtained from the G6ttingen Algal Culture Collection (Schl6sser, 1994). The algae were originally isolated from the highly polluted river Werra in Germany. The strain is free from algal or bacterial contamination. Preoculation was performed in 300 ml Erlenmeyer flasks containing 100 ml medium under aseptic conditions. The flasks were inoculated with 2 ml pregrown medium, stirred at 100 rpm on a rotary shaker and illuminated with lamps providing a photon flux density of 15 ~tE m - 2 s-1 at the surface of the cultures. They were illuminated continuously or under a light/dark regime of 14/10 h, respectively.

The basis for this medium optimization were the flesh water and sea water media for diatom cultivation given in the literature (Stein, 1973; Schl6sser, 1994). According to the high salt pollution in the river where the species has been isolated from, the optimization lead to a brackish water medium. It was found that in Erlenmeyer flasks best growth could be obtained with continuous illumination in media containing medium salt concentration ( ~ 18 g 1-I NaC1). Based on these findings, the medium described below was applied to cultivate C. cryptica in a photobioreactor. The macro-element solution contained 17.95 g 1-~ NaC1; 4.90 g 1-~ MgC12 6 H20; 1.19 g l Na2HPO4 2 H20; 0.87 g 1-1 KNO3; 0.109 g 1-1 Ca(NO3)2 4 H20; 0.093 g 1-~ NazSiO3 9 H20; 0.013 g 1- ~ NaHCO3; 0.001 g 1- i KHzPO4. This medium was supplemented with 1% micro-element solution containing 1.21 g 1-~ Fe-citrate; 0.29 g 1-1 H3BO3; 0.277 g 1-~ MnSO4 4 H/O; 0.028 g 1- 1 Z n S O 4 7 H20; 0.0127 g 1-~ C o ( N O 3 ) 6 6 H20; 0.010 g 1-~ CuSO4 5 H20; 0.0063 g 1NazMoO4 2 H20 and 1 ml 1-~ vitamin solution containing 0.05 g 1-~ vitamin B~, and 0.0005 g 1-~ vitamin BI2. All chemicals used were of analytical quality. The pH was adjusted to 7.5. Media were sterilized at 121~ for 20 min, microelement solution was sterilized separately, vitamin solution was filtered (0.2 lam) and added to the medium at room temperature.

2.3. Cultivation in photobioreactor 2.2. Growth medium In order to cultivate enough cells for particle characterization a medium had to be found that allows good growth of the diatoms. Preferably it had to be a defined medium to obtain reproducible results. The medium given in the literature for the cultivation of the C. cryptica species used (Schl6sser, 1994) does not support growth to a high cell density. Moreover it is a complex medium containing 1% soil extract. Therefore, a medium optimization study had been carried out prior to these experiments in to find a defined medium that allows good growth (Z. Cs6g6r et al., unpublished results).

A 15 1 stirred tank reactor (all glass) was illuminated continuously from outside with halogen lamps to give a photon flux density of 92 gE m - 2 s - l at the inner surface of the glass reactor. Ten 1 sterile defined medium was inoculated with 400 ml medium containing 5 x 109 cells ml-1 C. cryptica pregrown in Erlenmeyer flasks. The medium was bubbled with sterile air/CO2 mixture (1% CO2) at 1 1 m i n - l pH, pO2, temperature, and the concentrations of 02 and CO2 in the exhaust gas were measured on-line, the pH was maintained at 7.5 during the cultivation. Cell number and optical density (see below) were determined daily.

z. Cs6g6r et al. / Journal of Biotechnology 70 (1999) 71- 75 2.4. Determination o f growth and growth rate

The optical density was determined at 430 nm in a spectrophotometer (Unicam UV2). The cell concentration was determined in a Neubauer chamber to obtain the growth rate/z according to Cx(t) = Cx,o e ~' t, with cx determining the cell concentration and Cx.o the cell concentration at the beginning of the cultivation. The cultivation data from the exponential phase of growth were taken into account for the growth rate determination. The optical density correlated well with the cell concentration (r2= 0.98). The dry weight was determined according to Zhu and Lee (1997). Cell concentration, optical density, and dry weight were analyzed in duplicate, mean values are shown. 2.5. Preparation o f the frustules

For the determination of the specific surface and density, C. cryptica cultured in the 15 1 bioreactor was harvested and washed in demin, water, and separated by centrifugation (5 rain in a centrifuge BGH Hermle ZK 630 at 4~ and 2000 • g). The cells were broken with the protease mixture Novozym FM 2.0 L: 0.1 mg protease per 7.5 mg algal suspension was applied at 55~ for 5 h, then washed three times as described above. Chlorophylls and other organic material were removed through acetone extraction until the preparation lost colour (no extinction in the visible range of light). Finally the frustules were dried by lyophilization a t - 3 0 ~ and 0.375 mbar (Fa. Christ alpha 1-4), mechanically loosened with a spoon and stored in an exsiccator. For scanning electron microscopy the same procedure was performed. Subsequently the dried frustules were sputtered with palladium and platinum and scanned in a scanning electron microscope (Hitachi S 4500). 2.6. Characterization o f the frustules and siliceous earth

The diatom frustules cultivated as described above were characterized in terms of particle techniques and compared to siliceous earth (Merck

73

1.10601). The data shown are mean values of one sample analyzed in duplicate. A gaspycnometer (Micromeritics NP1305) was used for the measurements with helium as measuring gas. A gaspycnometer accomplishes the measurement of skeletal volumes by observing the reduction of gas capacity in the sample chamber caused by the presence of a sample. Since the measuring gas penetrates even the smallest pores and surface irregularities, the volume obtained permits computation of the ultimate theoretical density of the solid comprising the sample if there are no enclosed pores. For the determination of the size of the particles a monochromatic laser light diffractiometer was used (Sympatec Helos H9236). Ultrasound was applied for 2 min to achieve good dispersion before the measurements (Bransonic B12, 150 W, 48 kHz). The specific surface was determined according to the Brunauer-Emmett-Teller method (BET) which considers the adsorption of gas to the surface of the sample. The adsorption isotherm can be calculated by measuring one point only and assuming the ordinate origin as the second (one-point BET) or by measuring several points and calculating the isotherm (multiple-point BET). The gas sorption analyzer Nova 1200 (Quantachrome Co.) was used with nitrogen as measuring gas. The specific surface was determined with both one-point and multiple-point BET.

3. Results and discussion 3. I. Cultivation in photobioreactor

The cultivation of C. cryptica in the bioreactor resulted in a good growth (see Fig. 1). The growth rate determined in the exponential growth phase between day 4 and 9 was 0.55 d-1, this is about twice the best growth rates determined in Erlenmeyer flasks (data not shown). The higher growth rate is due to the higher concentration of CO2 (1% added in the reactor vs. air) and the better illumination in the reactor compared to cultures grown in Erlenmeyer flasks (92 laE m - 2 S--1 and 15 laE

Z. Cs6g6r et al./ Journal of Biotechnology 70 (1999) 71-75

74

m - 2 S - 1 , respectively). After 10 days the culture did not grow exponentially any more because it became light limited. The cells were harvested at a concentration of 9.3 • 107 cells ml-1. The cells were cleaned as described above and the frustules were taken for particle characterization.

3.2. Characterization of the frustules C. cryptica is a unicellular centric diatom (Fig. 2). The cells do not adhere like many other diatoms thus allowing detailed analysis of the particles. The cell wall is composed of amorphous, hydrated silica and organic components (Will6n, 1991). The frustules obtained in the 15 1 cultivation scale were purified from the organic components and characterized in terms of particle techniques in comparison with siliceous earth. The particle size distribution of both are given in Fig. 3. C. cryptica frustules are much smaller in

size than siliceous earth. The frustules are not equal in size as can also be seen in the picture in Fig. 2. The relatively wide distribution of siliceous earth is due to the fact that it contains a variety of different species. The density of both native diatom particles and siliceous earth were analyzed in a gaspycnometer. The results are given in Table 1. C. cryptica is characterized by a 1.5-fold lower density in comparison with siliceous earth. The latter consists of many different diatom species with different amounts of hydrate: SiO2 may contribute between 10 and 70% in native frustules (Will6n, 1991). The discrepancy may also be due to the fact that the native frustules are of amorphous structure whereas in siliceous earth a compression to a crystalline structure has taken place. The specific surface of both was determined, the results are given in Table 1. The one-point BET and the multiple-point BET give very similar re-

1,40E+10

1,20E+10

E L

9 1,00E+10 JD

E C ~ 8.O0E+09 U c o

m 6,00E+09 L c

o u C

0 4,00E+09 U 0

r

2,00E+09

O,OOE+O0

0

1

2

3

4

Cultivation

5

6

7

8

9

10

time [days]

Fig. 1. Growth of C. cryptica in a stirred 15 1 photo-bioreactor, illuminated from outside with halogen lamps to give an average photon flux densitiy of 92 laE m - 2 s - ] , bubbled with a 1% CO2/air mixture. Points are measured cell concentrations, line gives exponential approximation of growth between days 3 and 9.

Z. CsOg6r et al./Journal of Biotechnology 70 (1999) 71-75

75

100

....

90 t 3

: -=-~r -

~~~'~A='

70

~ 60

,0a0

Y -=

4-

20 [ 0

_~

J

i

i

..A&

I i.-:

cyc,o,o, cryptica [ "" ~''siliceous

I

. . . . . .

1

10 100 particle size x [IJm]

1000

Fig. 3. Mass particle size distribution of C. cryptica and siliceous earth after 2 min dispersion with ultrasound. Fig. 2. C. cryptica, scanning electron microscope picture; the dried frustules were sputtered with palladium and platinum (bar length is 5 ~tm).

suits. The specific surface of C. cryptica frustules is much higher than that of siliceous earth (80 fold). This is both due to the smaller particle size (about 10 and 100 lam, respectively) and to the higher porosity of the native fluso tules. As the comparison shows, the native frustules of C. cryptica clearly exceed siliceous earth in respect to surface and porosity, properties that have led to the wide use of the latter in many areas. Furthermore they are more uniform in shape and size because of the controlled cultivation conditions of unialgal and axenic cultures. Although the cultivation of diatoms is expensive, it may turn out that the frustules are of advantage for special application.

Table 1 Characterization of C. cryptica and siliceous earth in terms of particle techniques

Density (g cm- 3) Mean particle size (lam) Spec. surface (m 2 g-Z) (one-point BET) Spec. surface (m 2 g-1) (multiple-point BET)

C. cryptica

Siliceous earth

1.46 + 0.01 I0.1 26.21 + 0.48

2.22 +__0.02 92.5 0.35 +0.003

27.70 + 0.49

0.35 ___0.004

References Mann, S., Ozin, G.A., 1996. Synthesis of inorganic materials with complex form. Nature 382, 313-318. Schl6sser, U.G., 1994. SAG--Sammlung yon Algenkulturen at the University of G6ttingen Catalogue of Strains 1994. Bot. Acta 107, 113-186. Stein, J.R., 1973. Handbook of phycological methods. Cambridge University Press. Will6n, E., 1991. Planctonic diatoms-an ecological review. Alg. Stud. 62, 69-106. Zhu, C.J., Lee, Y.K., 1997. Determination of biomass dry weight of marine microalgae. J. Appl. Phycol. 9, 189-194.

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IOUINAL

i|

oF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 77-84

Biotechnological potential of North Sea salt marsh plants a review of traditional knowledge Gerd Liebezeit a,, Thorsten D. Ktinnemann b, Gunnar Gad b a Forschungszentrum Terramare, Schleusenstrafle 1, D-26382 Wilhelmshaven, Germany b FB Biologic, Universitdt Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany Received 13 October 1998; received in revised form 1 December 1998; accepted 22 December 1998

Abstract

Extreme environments are characterised by wide variations in physical factors. Tidal flats and their adjacent salt marshes along the North Sea coast experience this type of variability on a daily, seasonal and annual basis. Plants and animals living in these extreme environments have to adapt to these, at times rapid, changes. This can be done by developing specific physiological responses which often encompass the synthesis of unusual chemicals. A number of salt marsh plants have traditionally been used for medical, nutritional or even industrial purposes. Here, examples are presented for these plants. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Extreme environments; Salt marsh plants; Semiterrestrial

1. Introduction Organisms thriving in extreme habitats have developed a number of mechanisms to cope with their physical environment. These may include a.o. the production of antifreeze compounds or heatshock proteins in fish and other organisms (Sicherl and Yang, 1995; Berger et al., 1996), specific enzyme systems capable of operating at temperatures as high as 113 ~ (Bl6chl et al., 1997) or at deep sea pressures (Huber et al., 1989; Gross and Jaenicke, 1994). Therefore, considerable biotechnological interest in these environCorresponding author. E-mail address: [email protected] (G. Liebezeit) *

ments has recently been expressed (Jensen, 1993; Pennisi, 1997; Gross, 1998). Extreme environments are usually defined as being extreme in one or several physical parameters such as temperature (e.g. hydrothermal vents, ice-covered regions), salinity (hypersaline lagoons, Dead Sea) or pressure (deep sea). However, in these systems the external variables remain relatively stable over time and are extreme only by human standards. On the other hand, marine systems with a high variability in temperature, salinity or radiation can also be found. These include a.o. salt marshes, tidal flats and mangrove ecosystems. Organisms thriving in saline environments have been found to produce a large number of sec-

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G. Liebezeit et al./Journal of Biotechnology 70 (1999) 77-84

ondary natural products that might be of potential interest for pharmacological or industrial applications (Hay, 1996). Marine neurotoxins, for example, may be used to study ion channels and receptors (Myers et al. 1993). Bryostatins, isolated from bryozooans, are currently being investigated as powerful anti-leukemia agents (Olson, 1996). Salt marshes and tidal fiats experience regular (e.g. tides) and irregular (e.g. storms) external influences. Temperatures may vary from below -20~ in severe winters to > 40~ in extreme summers, while the salt regime may change from freshwater conditions after heavy rains to hypersaline after prolonged evaporation periods (Reineck and Flemming, 1990). To be able to deal with these variations in environmental conditions organisms inhabiting these ecosystems have developed a number of adaptation mechanisms. These include mechanisms for a.o. salt excretion or storage, osmoregulation (i.e. control of cellular ion concentrations) or desiccation protection. In this context it should be mentioned that out of 16 mangrove species tested for antiviral activity, 10 were found to contain active compounds (Padamakumar and Ayyakkannu, 1994), while of the marine macroalgae investigated only 30% behaved similarly. The documented production of saponins in seeds (Glenn et al., 1991) or other toxic compounds by salt marsh plants (Cooper, 1984) can be regarded as a mechanism to avoid or at least to minimise predation pressure. This suggests that semiterrestrial plants may be a particularly wealthy source for unusual chemical compounds. Marine biotechnology largely depends on the availability of information on the natural products chemistry of marine organisms (Rouhi, 1995; Colwell, 1983) which can be obtained in various ways. One of these is the installation of screening programmes, whereby organisms of interest are systematically investigated for usually a restricted number of biological activities. The latter may encompass antibacterial, antiviral or other compounds of pharmacological interest. A second approach is to use traditional knowledge as assembled in e.g. homeopathy text books or passed on by folklore. In these cases, generally no details on the structures of molecules of interest

will be made available if they are known at all. Rather, these sources can be used to obtain information on the biological activities displayed by different plants or parts thereof. In this review we will concentrate on this second approach, taking a closer look at selected plants from North Sea salt marshes, chiefly glasswort (Salicornia spp.), sea lavender (Limonium vulgare), arrowgrass (Triglochin maritima), sea wormwood (Artemisia maritima) and sea plantain (Plantago maritima). Other plants which have also been used traditionally will be mentioned briefly.

2. Methods

The information provided here is based on an intense literature search including World Wide Web sources, notably wwwl. In addition, a number of older, out-of-print text books on botany have been used, especially Hegi (1909, 1926). Furthermore, for information on homeopathic usage of plants the publications of Anton (no publication date); Alsamer (1901), Flamm and Kr6ber (1936) as well as Beckmann and Beckmann (1997) have been consulted.

3. Presentation of data

Information on several salt marsh plants and their traditional usage is summarised in Table 1.

3.1. Salicornia europaea Salicornia europaea (glasswort) (Fig. 1), an annual plant of the family Chenopodiaceae, is the first pioneer plant of saltmarshes colonising the border between tidal fiats and the salt marsh. It is dependent on the presence of salt and accumulates large amounts of salts including sodium, potassium, iodide and bromide compounds during growth. This is the main reason why its ash has been used in glass making (hence its popular name) as the high salt content of the ash lowers the melting point of glass. As it also contains sodium carbonate, it was used traditionally in France for washing purposes.

G. Liebezeit et al./Journal of Biotechnology 70 (1999) 77-84

79

Table 1 Salt marsh plants with traditional usage Plant species

Trivial name

Compound(s)

Traditional usage

Salicornia sp. L. vulgare T. maritima

Glasswort Sea lavender Seaside arrowgrass Sea wormwood Sea plantain

Inorganic salts Triglochinin taxiphyllin a.o.

Ash for glass production and washing agents Antidiarrheticum Cabbage substitute

Prickly glasswort

Saponins

Scurvy grass

Ascorbic acid

A. maritima P. maritima Aster tripolium Salsola kali Cochlearia sp. Odontites rubra ssp. litoralis

Antihelminthicum; insecticide (moths, lice, fleas) Spinach substitute; soda (ancient Egypt) Vegetable (S Holland, China) Deadly to cows in large quantities; fresh plant juice as diureticum; soda (ancient Egypt) Scurvy treatment Cures toothache when cooked in wine

The plant is particularly conspicuous in autumn, when it displays a bright red discolouration. Although the pigment signature of S. europaea is relatively simple (Fig. 2), no attempts have been made so far to structurally characterise the compounds in question. HPLC analysis of aqueous acetone extracts indicates one or more carotenoid pigments to be possibly responsible for the red discolouration. These elute close to the column dead volume and are thus highly polar in nature. The precursor compounds eluting at 13, 15.5 and 23 min have so far not been identified. While the on line-absorption spectra (Fig. 2) indicate that these are carotenoids, a positive identification is not possible with these data alone. Carotenoids have biotechnological potential (Ausich, 1997). Incidentally, a yet unidentified pigment from Hasleria ostrearia, a benthic diatom, is employed in its native form for oyster colouring (Groth-Nard et al., 1994). Cooper and Johnson (1984) reported that among eight salt marsh plants tested S. europaea was the only one not showing limiting effects during growth in media containing up to 10 mM manganese sulphate. As this is far above the natural concentrations encountered in salt marsh soils this capability might point to the presence of a powerful complexing agent in Salicornia sp. In recent years, another Salicornia species, S. bigelovii, has received considerable interest as a cultivar in salt water irrigated agriculture (Glenn et al., 1991, 1998). Its seeds, besides containing

unidentified saponins, were found to be rich in proteins and lipid material, notably fatty acids. Weete et al. (1970) have suggested that the variability of the hydrocarbon and fatty acid composition of S. bigelovii is governed by external factors, i.e. differences in environmental ion concentrations.

Fig. 1. Salicornia europaea.

G. Liebezeit et al./Journal of Biotechnology 70 (1999) 77-84

80 0.25 0.16

Z w I-

0.20

oI= <

0 or) m
23' 15'

D

0.12 LIJ 0 z
SALICORNIA EUROPAEA

m

w

13'

0.15

23'

15'

0.10 - -

13'

0.08

I

I

'

!

4OO

5OO

WAVELE NGTH [nm]

,ID =J .J >. uJ Z uJ I-

o o 0.05

o m ,<

0.04 --"

0

&

GREEN 11nIl

0.00 0.00

,~

--

' 0

10

20

I

3O

I 40

I e - - lImB I |

RED '1 .... ' 50 [rain]

Fig. 2. H P L C chromatograms of S. europaea extracts. Upper: green summer plant, lower: red autumn plant.

3.2. Limonium vulgare L. vulgare (sea lavender) (Fig. 3) is a perennial plant which can reach an age of up to 40 years. It belongs to the family Plumbaginaceae which has derived its name from the fact that it, especially the roots, produces a compound which discolours paper and hands lead-grey. This yellow compound, plumbagin (5-hydroxy-2-methyl-l,4-naphthochinone) has been used in Africa for tribal tattooing. The plants, especially the roots, contain compounds that have been used to treat diarrhoea, dysentery and bleeding. It is ascribed astringent action. Several amines, including methylamine, diand trimethylamine as well as putrescine (derived from ornithine) have been reported to occur in L. vulgare (Smith, 1980). Osmolytic regulators in five Limonium species investigated are [3-alanine and glycine betaines as well as choline-O-sulphate (Hanson et al., 1991) while Cavalieri and Huang (1979) reported on the accumulation of proline in

L. carolinianum at salinities exceeding 0.25 M NaC1. 3.3. Trigloch& maritima T. maritima (seaside-arrowgrass) (Fig. 4) contains two cyanogenic glycosides, triglochinin and taxiphillin (Majak et al., 1980; Poulton, 1983) which can cause cyanide poisoning especially in ruminants (Cooper, 1984). Nevertheless, it has been traditionally used, before flowering, as a cabbage substitute in Lower Saxony. A blood cleaning action has been ascribed to this nourishment (Fig. 5). In contrast to other salt marsh plants such as P. maritima or Festuca rubra, T. maritima is usually not infected by vesizular arbuscular mycorrhizal fungi (Rozema et al., 1986), pointing to the presence of a chemical defence mechanism in this species. When the leaves are ground a chlorine-like odour is emitted. Whether this is the result of a

G. Liebezeit et al. / Journal of Biotechnology 70 (1999) 77-84

81

mechanism to get rid of excess chloride is unclear. If so, this might be an example of chloride oxidation by plants. T. bulbosa and T. striata produce proline as an organic osmoregulator although the major effect in osmoregulation appears to originate from balancing cellular sodium, potassium, calcium and magnesium concentrations (Naidoo, 1994).

3.4. Artemisia maritima and other Artemisia species

A. maritima (sea wormwood, family Compositae) (Fig. 6) is, as its popular name implies, even today used as an anti-helminticum. Santonica, an extract from the unexpanded flower heads, was used by the ancient Greeks to cure ascariasis (Bowman and Rand, 1980, p. 37.15). In addition, the whole dried plant has been used to prevent moth damage in garments and against lice and fleas.

Fig. 4. Triglochin maritima.

The active compound, santonin (Fig. 7) may reach a maximum content in the flower heads of 2.3-3.6%. Santonin may cause fatal poisonings; it has also effects (even in small doses) on vision and can cause headaches, nausea and vomiting. Besides santonin, ,4. maritima contains artemesin and cineol (Fig. 7). The latter compound can be isolated as a yellow volatile oil responsible for the odour of the plant. Heptadeca1,9(Z),16-trien-4,6-diyn-3,8-diol, which was isolated from ,4. borealis by Wang et al. (1990) together with another polyacetylene, proved ac~.. O-GLC N

OH (R-)-taxiphyllin

Fig. 3. Limonium vulgare.

O-GLC HO2C / ~- \ H02(~ ~N triglochinin

Fig. 5. Structures of taxiphyllin and triglochinin, two cyanogenic glycosides from T. maritima.

82

G. Liebezeit et al./'Journal of Biotechnology 70 (1999) 77-84

.

o

I

jon

Fig. 8. Thujon, the ~bt

..

v

, t~'"!~

ki~

~

Y ~

Fig. 6. Artemisia maritima,

monocyclicterpene of Artemisia

;~a

plantain; family Plantaginaceae) (Fig. 9) has already been employed by the ancient Egyptians for soda production. In northern Frisia it has been used as spinach substitute and has been ascribed blood cleaning properties. It is a preferred food for sheep and has been cultivated in Wales for this purpose. P. maritima may contain large amounts of sorbitol which is used by the plant as an osmoregulator (Ahmad et al., 1979). In addition it contains acidic polysaccharides (mucus). The plant is used

tive in a shrimp bioassay and showed antifungal and larvicidal activity. The oil of A. absinthum contains thujon as major compound (Fig. 8). The presence of related terpenoids in A. maritima suggests that a detailed investigation might reveal the presence of other such compounds. Mugwort (A. vulgaris), the most commonly member of the family Artemisia has a traditional usage in the treatment of menstruation problems. A comprehensive list of secondary chemicals in Artemisia species has been provided by www3.

o santonin

1,8-cineol

Fig. 7. Structure of santonin, the main antihelmintic principle of sea wormwood, and of 1,8-cineol.

Fig. 9. Plantago maritima.

G. Liebezeit et al./Journal of Biotechnology 70 (1999) 77-84

as a soothing expectorant and has more or less marked stomachic properties. There is no information on the active ingredients. Its terrestrial counterparts P. major and P. lanceolata, on the other hand, have been studied in greater detail. Both plants are a.o. used to treat fresh wounds and insect bites by applying ground fresh leaves. P. major contains aucubin, a iridoid monoterpene (www2), which among other effects causes smoke aversion in smokers.

3.6. Other plants Some other salt marsh plant species have been traditionally used as summarised in Table 1. Among these, Cochlearia officinalis is of interest as its salt marsh counterpart has been used to treat scurvy but also as a blood cleaning agent and to further digestion. The use of C. officinalis in scurvy treatment has already been described in the 17th century by Fournier (www4).

4. Conclusions

The traditional usage of salt marsh plants for soda production, as food (both for cattle and humans) and as drugs, suggests that a number of compounds might exist in this chemically poorly characterised group of plants which can be of potential interest to biotechnologists. This short review shows that information derived from nonchemical sources might prove helpful in the search for marine organisms to be investigated for biotechnological applications. Most salt marshes of the North Sea coast are protected nature reserves or, as in Germany, national parks. This prevents large scale sampling from natural resources. On the other hand, appropriate cultivation techniques can be developed as shown by the example of S. bigelovii mentioned above. For L. vulgare, unsuccessful attempts to cultivate the plant have been made already during the 17th century and later in 1913 (Jantzen, 1987). As the preferred location of sea lavender in nature is in the upper parts of the glasswort zone, seawater irrigated culture may be possible. On the other hand, as semiterrestrial organisms

83

the salt marsh plants described above should be amenable to cell culture techniques once compounds of interest have been identified and characterised.

References Ahmad, I., Larher, F., Stewart, G.R., 1979. Sorbitol a compatible osmotic solute in Plantago maritima. New Phytol. 82, 671-678. Alsamer, A., 1901. Gottessegen in der Pflanzenwelt. Verlag der St. Josef-Brtiderschaft, Klagenfurt. Anton, C., no publication date. Der Kr/iuterarzt. Verlag der Ernst'schen Buchhandlung, Leipzig. Ausich, R.L., 1997. Commercial opportunities for carotenoid production by biotechnology. Pure Appl. Chem. 69, 21692173. Beckmann, D., Beckmann, B., 1997. Das geheime Wissen der Kr/iuterhexen. Deutscher Taschenbuchverlag, Miinchen. Berger, F., Morellet, N., Menu, F., Potier, P., 1996. Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55. J. Bacteriol. 178, 2999- 3007. B16chl, E., Rachel, R., Burggraf, S., Hafenbradl, D., Jannasch, H.W., Stetter, K.O., 1997. Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113~ Extremophiles 1, 14-21. Bowman, W.C., Rand, M.J., 1980. Textbook of Pharmacology, 2nd edn. Blackwell, Oxford. Cavalieri, A.J., Huang, A.H.C., 1979. Evaluation of proline accumulation in the adaptation of diverse species of marsh halophytes to the saline environment. Am. J. Bot. 66, 307-312. Colwell, R.R., 1983. Biotechnology in the marine sciences. Science 222, 19-24. Cooper, A., 1984. A comparative study of the tolerance of salt marsh plants to manganese. Plant Soil 81, 47-59. Cooper, M.R., Johnson, A.W., 1984. Poisonous Plants in Britain and Their Effects on Animals and Man. Her Majesty's Stationery Office, London. Flamm, S., Kr6ber, L., 1936. Die Heilkraft der Pflanzen. Hippokrates-Verlag, Stuttgart. Glenn, E.P., O'Leary, J.W., Watson, C., Thompson, T.L., Kuehl, R.O., 1991. Salicornia bigelovii Torr.: an oilseed halophyte for seawater irrigation. Science 251, 1065-1067. Glenn, E.P., Brown, J.J., O'Leary, J.W. 1998. Irrigating crops with seawater, Sci. Am., August, 56-61. Groth-Nard, C., Robert, J.M., Gandemer, G., 1994. Lipid composition of the diatom Haslea ostrearia. 3rd Int. Mar. Biotechnol. Conf.: Program, Abstracts and List of Participants. Tromsoe, Norway, p. 54. Gross, M., 1998. Life on the Edge Amazing Creatures Thriving in Extreme Environments. Plenum, New York.

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Gross, M., Jaenicke, R., 1994. Proteins under pressure: the influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes. Eur. J. Biochem. 22 I, 617-630. Hanson, A.D., Rathinasabapathi, B., Chamberlin, B., Gage, D.A., 1991. Comparative physiological evidence that 13-alanine betaine and choline-O-sulphate act as compatible osmolytes in halophytic Limonium species. Plant Physiol. 97, 1199-1205. Hay, M.E., 1996. Marine chemical ecology: what's known and what's next? J. Exp. Mar. Biol. Ecol. 200, 103-134. Hegi, G., 1909. Illustrierte Flora von Mitteleuropa. Carl Hanser Verlag, Mfinchen, vol. 1. Hegi, G., 1926. Illustrierte Flora von Mitteleuropa. Mit besonderer Beriicksichtigung von Deutschland, Oesterreich und der Schweiz. J.F. Lehmanns-Verlag, M iinchen, vol. 6, 2. Huber, R., Kurr, M., Jannasch, H.W., Stetter, K.O., 1989. A novel group of abyssal methanogenic archaebacteria. Nature 342, 833-834. Jantzen, F., 1987. Pflanzen am Meer. Landbuch-Verlag, Hannover. Jensen, A., 1993. Present and future needs for algae and algal products. Hydrobiologia 260/261, 15-23. Majak, W., McDiarmid, R.E., Hall, J.W., van Ryswyk, A.L., 1980. Seasonal variation in the cyanide potential of arrowgrass (Triglochin maritima). Can. J. Plant Sci. 60, 12351241. Myers, R.A., Cruz, L.J., Rivier, J.E., Olivera, B.M., 1993. Conus peptides as chemical probes for receptors and ion channels. Chem. Rev. 93, 1923-1936. Naidoo, G., 1994. Growth, water and ion relationships in the coastal halophytes Triglochin bulbosa and T. striata. Environ. Exp. Bot. 34, 419-426. Olson, S.G., 1996. Curing cancer through aquaculture. Sea Technol. 37, 89-94. Padamakumar, R., Ayyakkannu, K., 1994. Antiviral activity of marine algae and mangroves. 3rd Int. Mar. Biotechnol.

Conf.: Program, Abstracts and List of Participants. Tromsoe, Norway, p. 98. Pennisi, E., 1997. Biotechnology in industry, extremophiles begin to make their mark. Science 276, 705-706. Poulton, J.E., 1983. Cyanogenic compounds in plants and their toxic effects. In: Handbook of Natural Toxins 1. Plant and Fungal Toxins, pp. 117-157. Reineck, H.E., Flemming, B.W., 1990. Salzgehalte der Restn/isse auf oder in der obersten Sedimentschicht und der Porenw/isser im Eu- und Supralitoral der Jadewatten in Relation zu denen des Jadewassers. Senckenberg. Marit. 21, 33-54. Rouhi, M., 1995. Supply issues complicate trek of chemicals from sea to market. Chem. Eng. News Nov. 20, 42-44. Rozema, J., Arp, W., van Diggelen, J., van Esbroek, M., Broekman, R., Punte, H., 1986. Occurrence and ecological significance of vesicular arbuscular mycorrhiza in the salt marsh environment. Acta Bot. Neerl. 35, 457-467. Sicherl, F., Yang, D.S.C., 1995. Ice-binding structure and mechanism of an antifreeze protein from winter flounder. Nature 375, 427-431. Smith, T.A., 1980. Plant amines. In: Pirson, A., Zimmerman, M.H. (Eds.), Encyclopedia of Plant Physiology, vol. 8, pp. 433-460. Wang, Y., Toyota, M., Krause, F., Hamburger, M., Hostettmann, K., 1990. Polyacetylenes from Artemisia borealis and their biological activities. Phytochemistry 29, 3101-3105. Weete, J.D., Rivers, W.G., Weber, D.J., 1970. Hydrocarbon and fatty acid distribution in the halophyte, Salicornia bigelovii. Phytochemistry 9, 2041-2045. www 1: Grieve, M., A modern Herbal. http:// www. botanical.com/botanical/mgrnh/. www 2: http://www.cigno.com/exam.html. www 3: http://chili.rt66.com/hrbmoore/Constituents/. www4: http://www.pc-78-120, udac. se:8001/www/Nautica/ Medicine/Fournier(1667).htm.

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 85-88

......

The pharmaceutical exploration of cold water ascidians from the Netherlands" a possible source of new cytotoxic natural products Albert Koulman *, Loes M.C. Pruijn, Th6r6se S.A. Sandstra, Herman J. Woerdenbag, Niesko Pras Department of Pharmaceutical Biology, University of Groningen, A. Deusinglaan 1, 9713 A V Groningen, The Netherlands

Received 13 October 1998; received in revised form 26 November 1998; accepted 22 December 1998

Abstract Six ascidian species from the Dutch North Sea coast were screened for cytotoxic activity. Freeze-dried biological material was extracted with solvents of different polarity followed by determination of the cytotoxicity. The most active extracts were further separated using different chromatographic techniques. The microculture tetrazolium (MTT) assay was used to determine the cytotoxicity against two human tumor cell lines: COLO32 o (a colon adenocarcinoma) and GLC4 (a small cell lung carcinoma). The two cell lines were selected for their different response towards known cytostatics. GLC 4 is sensitive and COLO32 o is resistant to most of the known cytostatics. Three of the species tested yielded interesting fractions. From the in colonial Didemnum lahillei we have isolated a compound which is more active against the COLO32 o cell line (IC5o:33 lag m l - l) than the GLC 4 (IC5o:49 lag m l - 1). The structure is currently elucidated. Another colonial species, Aplidium glabrum, yielded a very cytotoxic fraction, with an IC5o of 5 lag m l - 1 against the COLO32o cell line. From our results it can be concluded that the North Sea could be another interesting source of new compounds with pharmaceutical potential. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Cytotoxicity; Microculture tetrazolium assay; North Sea; Ascidian; Didemnum lahillei; Aplidium glabrum

1. Introduction The class of the Ascidiacea ( C h o r d a t a phylum, U r o c h o r d a t a s u b p h y l u m ) form a g r o u p of marine organisms which have yielded a large n u m b e r of different biologically active c o m p o u n d s . A very * Corresponding author. Tel.: + 31-50-3633363; fax: + 3150-3637570. E-mail address: [email protected] (A. Koulman)

well k n o w n example is the class of didemnin-like c o m p o u n d s originally isolated f r o m the ascidian Trididemnum solidum ( R i n e h a r t et al., 1988). Especially tropical, colonial benthic species have been discovered as a rich source of constituents with a p r o n o u n c e d pharmaceutical potential (Watters and Van de Brink, 1993). Ascidians are not only present in tropical waters. There are at least 20 different ascidian species k n o w n from the N o r t h Sea ( K n i g h t - J o n e s

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00062-0

86

A. Koulman et al. ,/Journal of Biotechnology 70 (1999) 85-88

and Ryland, 1995). Several species have become very abundant in two Dutch estuaries (Lake Grevelingen and Lake Oosterschelde, both SW Netherlands) after, respectively, enclosure and partial enclosure of these estuaries through the Deltaworks (Leewis et al., 1994). In some parts of the Estuary Oosterschelde, colonial species like Didemnum lahillei and Aplidium glabrum may cover more than 25% of the hard substrate. Also, other solitary living species are very abundant. The abundance of different species like Ciona intestinalis and A. glabrum varies each year (Nienhuis and Smaal, 1994). All of these species seem to suffer from hardly any predation. This indicates the presence of a kind of defense system possibly based on the biosynthesis of bioactive compounds. The microculture tetrazolium (MTT) assay was used for the screening of the different ascidian species for the presence of possible cytotoxic compounds. Two human tumor cell lines were used in the assay: a small lung cell carcinoma cell line (GLC4) and a colorectal cancer cell line (COLO320). The GLC 4 cell line is very sensitive to all classes of chemotherapeutics, while the COLO320 cell line is much more resistant against most known chemotherapeutics.

2. Material and methods

2.1. Collection All animal material was collected in September and October 1996 using scuba diving at depths between 3 and 6 m (see Table 1). After collection, the tunic was thoroughly cleaned of sand, mutt and overgrowing organisms (of the genera Bryop-

sis, Halichondria, Haliclona, Metridium, Diadumene, Obelia, Eudendrium, Electra, Bugula, Ascidiella, Ciona and Botryllus) and stored at -20~ A few specimens of every species were stored as a reference at our laboratory. The species were identified by the authors (according to Buizer (1983) and Knight-Jones and Ryland (1995)).

2.2. Extraction Material was weighed before and after freezedrying giving freshweight/dryweight ratio (FW/ DW). A portion of 10 g freeze-dried and pulverized material was extracted with 3 • 200 ml MeOH. The collected extracts were evaporated under reduced pressure (using a rotor vaporator) and the remaining residue was partitioned between 40% aqueous MeOH and CHCl3.

2.3. MTT-assay A modified assay was used (according to Koulman et al. (1996), after Scudiero et al. (1988)). The COLO32o cell line (doubling time: 13-15 h) was used at a density of 2500 cells per well and GLC4 (doubling time: 18-21 h) at 3750 cells per well. These cells were continuously incubated (96 h) with a series of diluted extracts. Stock solutions of the extracts were made in DMSO. The final concentration of DMSO during the incubation was 1% or lower, which has been proven not to effect the cell growth (Beekman et al., 1997). Concentration series were prepared by successive 1-fold dilution from the stock solution. From the results the IC5o value (the concentration of a test compound causing 50% growth inhibition) was calculated, which was used as a parameter for cytotoxicity. Table 1 Collection and occurrence of the species used Species

Collection date and side

15th September 1996 at Dreischor; Lake Grevelingen 15th September 1996 at Ascidiella aspersa Dreischor; Lake Grevelingen (Mfiller, 1776) 15th September 1996 at Styela clara (Alder and Dreischor; Lake Grevelingen Hancock, 1848) 1st October 1996 at WilhelminaM. manhattensis (De harbour; Eems-Dollard estuary Kay, 1843) A. glabrum (Verril, 1871) 15th September 1996 at Dreischor; Lake Grevelingen 24th September 1996 at Zijpe; D. lahillei (Hartmeyer, Oosterschelde estuary 1909) C. intestinalis (L., 1767)

A. Koulman et al./Journal of Biotechnology 70 (1999) 85-88

87

Table 2 D W / F W ratio and IC5o values a of extracts from six Dutch ascidian species Species

DW/FW b

MeOH extract

40% MeOH

CHCI 3

C. intestinalis

0.053

COLO32 GLC 4

> 1.25 > 1.25

ND r

ND ~

Ascidiella aspersa

0.074

COLO32o GLC 4

0.96 0.80

> 1.25 > 1.25

0.45 0.23

Styela clara

0.166

COLO320 GLC 4

0.89 0.25

> 1.25 > 1.25

0.46 0.33

M. manhattensis

0.063

COLO32o GLC4

0.88 0.74

> 1.25 > 1.25

0.08 0.02

A. glabrum

0.152

COLO32 o GLC 4

0.19 0.15

> 1.25 > 1.25

0.08 0.07

D. lahillei

0.161

COLO32 o GLC4

0.25 0.20

> 1.25 > 1.25

0.23 0.19

a IC5o, concentration (in lag ml -~) giving 50% growth inhibition of the COLO32 o or GLC 4 cell line in the MTT-assay. As a reference the cytostatic cisplatin (Aldrich, Milwaukee, USA) gave the following IC5o values; COLO32 o. 0.8 lag m l - l (2.7 laM); GLCa: 0.3 lag m l - l (1.0 laM). b DW/FW, ratio of dry weight and fresh weight. r no detectable effect from the extract in the MTT-assay.

2.4. Bioguided isolation of active compounds The species with most active extracts were chosen (M. manhattensis, A. glabrum and D. lahillei ) for further fractionation over a silica 60 column using different solvents. The fractions were analyzed qualitatively by thin layer chromatography (TLC). For TLC, silica 60-F-254 (Merck) plates were used, with CHC13:MeOH (9:1) as the eluents. The spots were detected under UV light (254 and 366 nm) followed by spraying with anisaldehyde. Fractions with an identical TLC pattern were combined and tested for their cytotoxicity. For further purification this procedure was repeated with flash chromatography using a prepacked column (Merck) at a low pressure (4 atm).

3. Results

3.1. Screening for cytotoxic compounds The crude extracts of all species tested showed a

moderate cytotoxic acticity (Table 2). In comparison to known cytostatic cisplatin, the most active extracts in Table 2 were at least 103 times less cytotoxic. The DW/FW (Table 2) ratio shows large differences in the water content of the species, indicating that the concentrations of bioactive compounds in living tissue are not only low, but also highly variable. Based on their low IC5o values (Table 2) only the CHC13 extracts of M. manhattensis, A. glabrum and D. lahillei were used for the bioguided isolation of cytotoxic compounds.

3.2. Isolation of bioactive compounds A. glabrum yielded a very active fraction (IC5o = 5 lag ml-~), but TLC demonstrated that amounts of compounds were too small for further purification. M. manhattensis also gave a very active crude extract (IC50 = 8 lag ml-~), but only fractions with a moderate cytotoxicity were obtained by column chromatography.

88

A. Koulman et al./Journal of Biotechnology 70 (1999) 85-88

D. lahillei yielded an active, almost pure compound. To obtain this compound in sufficient quantities, the isolation was performed with 60 g freeze-dried material and finally 2 mg of an active compound was isolated. The ICso of the compound for COLO320 (33 ~tg ml-1) was lower than for GLC 4 (49 ~tg ml-1), which indicates a specific activity. Spectroscopic analysis (Infra red (IR), proton and carbon nuclear magnetic resonance (1H-NMR and 13C-NMR), ion electron mass spectrometry (IEMS) and chemical ionization mass spectrometry (CIMS)) of the compound however, showed impurities to be present. ElMS gave the following spectrum M:412 (26), 398 (13), 397 (15), 396 (43), 384 (100), 109 (14), 107 (15), 105 (12). The unknown compound is presumably a C25 compound (sesterterpene) giving large stable ions in ELMS. New material of D. lahillei collected (on the 19th of October 1997 at the same place) for a large scale isolation did not contain the active compound.

4. Conclusion The screening of the different ascidian species for the presence of cytotoxic compounds yielded fractions with an interesting activity of A. glabrum and D. lahillei, proving that in addition to tropical species, cold water ascidians might also form a source for new cytotoxic compounds. In these ascidians, the active compounds were present at very low concentrations. Large scale isolation will be necessary to obtain sufficient quantities of pure compounds for structure elucidation and further pharmacological evaluation. The already mentioned unknown compound from D. lahillei is of special interest because of its more specific cytotoxicity. However, there are probably seasonal variations in the secondary metabolite composition. The generally moderate activity of the majority of the extracts is remarkable because none of the presented species seem to suffer from predation. In addition, there is no relation between the cytotoxicity and the FW/DW ratio, which implies that

large differences will occur in the concentrations of bioactive compounds in living tissue of the different species. It is therefore likely that other factors, besides bioactive compounds, might play an additional role in the defense mechanism of the species investigated.

Acknowledgements The authors would like to thank Jaap Begeman and Dr Karen E. Voskamp for their help with the collection of the animal material. Dr J.K. Herreman, W.H. Kruizinga and Dr A. Kiewiet are gratefully acknowledged for the NMR and MS spectra.

References Beekman, A.C., Woerdenbag, H.J., Van Uden, W., Pras, N., Konings, A.W.T., Wikstr6m, H.V., Schmidt, T.J., 1997. Structure-cytotoxicity relationships of some helanolidetype sesquiterpene lactones. J. Nat. Prod. 60, 252-257. Buizer, D.A.G., 1983. De Nederlandse zakpijpen (manteldieren) en mantelvisjes. Wet. Med. K.N.N.V. 158. Nienhuis, P.H., Smaal, A.C., 1994. The Oosterschelde estuary, a case-study of a changing ecosystem: an introduction. Hydrobiology 282-283, 1-14. Knight-Jones, E.W., Ryland, J.S., 1995. Acorn-worms and sea squirts (phyla Hemichordata and Urochordata). In: Hayward, P.J., Ryland, J.S. (Eds.), Handbook of the Marine Fauna of North-West Europe. Oxford University Press, Oxford. Koulman, A., Proksch, P., Ebel, R., et al., 1996. Cytotoxicity and mode of action of aeroplysinin-1 and a related dienone from the sponge Aplysina aerophoba. J. Nat. Prod. 59, 591-594. Leewis, R.A., Waardenburg, H.W., Van Der Tol, M.W.M., 1994. Biomass and standing stock on sublittoral hard substrates in the Oosterschelde estuary (SW Netherlands). Hydrobiology 282, 397-412. Rinehart, K.A., Kishore, V., Bible, K.C., Sakai, S., Sullins, D.W., Kai-Ming, L., 1988. Didemnins and tunichlorin: novel natural products from the marine tunicate Trididirnnurn solidum. J. Nat. Prod. 51, 1-21. Scudiero, D.A., Shoemaker, R.H., Paull, K.D., et al., 1988. Evaluation of soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48, 4827-4833. Watters, D.J., Van de Brink, A.U, 1993. Toxins from ascidians. Toxicon 31 (11), 1349-1372.

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 89-101

Biotechnological hydrogen production" research for efficient light energy conversion Jun Miyake a,b,, Masato Miyake b, Yasuo Asada b National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan b National Institute of Bioseience and Human Technology, Tsukuba, lbaraki 305-8566, Japan

Received 27 October 1998; received in revised form 3 December 1998; accepted 22 December 1998

Abstract

The study of biological hydrogen production by using photosynthetic bacteria and cyanobacteria is described based on a national R&D project in Japan. We describe here the subjects examined in the research for photosynthetic bacteria: analysis of the relationship between the penetration of light to photobioreactor and hydrogen production, genetic engineering of photosynthetic bacteria to control the pigment content for making the light penetration easy. Examples of bench-scale reactors are shown. Genetic manipulation of hydrogenase to enhance the protein expression is also studied. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen; Cyanobacteria; Photosynthetic bacteria; Renewable energy; Solar energy

I. Introduction

Ever aware of limitations to the amount of resources and hazardous effects to the environment of fossil energy, development of renewable energy is an urgent task. Solar energy is the most abundant amongst various renewable energy sources. Its radiation provides the biggest flow of energy on the earth, ~ 5.7 x 1024 J year-1. This is 10000 times more than the total energy consumed by human beings. However, the efficiency of converting solar energy into any of a number of possible renewable * Corresponding author. Tel." + 81-298-54-2558; fax: + 81298-54-2558. E-mail address: [email protected] (J. Miyake)

energy carriers is very limited, representing inefficient use of the available solar energy. In general, so-called renewable sources have a significant drawback in that we have to accumulate solar energy before supplying it to industrial applications. Collecting solar energy with photovoltaic cells or thermal solar collectors yields rather higher conversion efficiency, but because solar radiation at the surface of the Earth is very low power at 1 kW m -2, we need a huge area to collect a sufficient amount of energy. This defect prohibits a commercial application. Biological energy conversion system as photosynthesis might not to be so efficient in the solar energy utilization. However, it is a remarkable process in the viewpoint of energy accumulation. Plants and photosynthetic microorganisms grow

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00063-2

90

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

by themselves and collect energy. Such spontaneous energy accumulation system is not obtainable in man-made industrial technologies. There are many types of energy carriers as hydrogen, methane, alcohol, and all other biomass, which could be produced biologically from renewable energy sources. Among them, hydrogen is a powerful energy source that does not produce carbon dioxide (Fig. 1). In this article, we describe briefly an approach for creating a safe and efficient energy production system. Although in the laboratory rather high efficiency of light to hydrogen conversion has been recorded, especially by photosynthetic bacteria, we have to improve the organisms and the reactor system further to realize the potentially high activity under applicational conditions. Not only the biological improvements, but also the engineering aspects are studied here. We should like to describe the basis of hydrogen biological production, genetic engineering and the reactor design

and field tests.

1.1. Thermodynamic advantage of biological energy conversion (Miyake, 1998a) In the case of organic substances, as waste water they are dissolved and diluted in the water, i.e. in a high entropy state, it is impossible to obtain their combustion enthalpy unless extracting these substances. The diluted carbohydrates contained in the wastewater of a tofu factory have a combustion enthalpy of 150 kJ 1-~ Evaporation of 1 1 of water requires about 2200 kJ 1-~, yet the available energy of the substance is far smaller. It is very difficult to obtain energy from dilute solutions by such a mechanical way. On the other hand, bacteria can convert such organic material in a dilute solution. Using methanogenic bacteria, we can obtain methane with an energy content of 145 kJ 1-~ (combustion enthalpy). By using photosynthetic bacteria and

BIOLOGICAL PRODUCTION OF HYDROGEN BY ENVIRONMENTALLY ACCEPTABLE TECNOLOGIES

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r

Separationand Production of usefulby-products

Fig. 1. Concept of biological hydrogen production and utilization.

Physiological active materials

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

illuminating the solution, we can obtain hydrogen that contains 185 kJ 1-l of chemical energy. In this way the dilute energy can be extracted in a usable form. It is, in a sense, an entropy reducing process which could not be realized by mechanical or chemical systems. Because of this special capability, we believe that renewable energy sources could be efficiently recovered by using a biological method as discussed in this article.

1.2. Mechanisms of biological hydrogen production (Miyake, 1998b) In nature, only bacteria and algae have the capability of hydrogen production. Amongst these organisms, those currently selected for research are anaerobic bacteria, photosynthetic bacteria, cyanobacteria and green algae. Green algae and cyanobacteria directly decompose water to hydrogen and oxygen with light energy: light

H20 ~ H2 + 1/20 2 This is the process by which the earth obtained oxygen in the distant past. The reaction requires only water and sunlight and is very attractive from the viewpoint of environment protection. However, naturally born organisms that carry out this process showed rather low efficiency of hydrogen production due to the complicated reaction system which remains to overcome the large free energy ( + 242 kJ mol-1 hydrogen). For future applications, many improvements are required. Photosynthetic bacteria do not utilize water as the starting compound for hydrogen production but use organic acids. An equation below shows the production of hydrogen from lactate by photosynthetic bacteria. light

C3H60 3 + 3H20 ~ 6H 2 + 3CO 2 In this reaction, free energy is + 8.5 kJ mol-1 hydrogen. Compared to the algal hydrolysis, less light energy is required to produce hydrogen in the case of photosynthetic bacteria when starting from organic substances. There are various kinds of photosynthetic bacteria and many kinds of organic substrates such as fatty acids, sugars,

91

starches, cellulose, etc. that could be used as starting materials for hydrogen production. Photosynthetic bacteria can decompose such organic substances completely. Hydrogen production could be combined with organic wastes treatment (waste fluid from food factories, pulp factories, etc.). Mechanisms of converting light to hydrogen by photosynthetic bacteria and cyanobacteria are shown in Fig. 2. In the project, due to differences in the mechanism of the hydrogen production, different methods of genetic engineering are employed for photosynthetic bacteria and cyanobacteria.

2. Materials and methods

2. I. Cultivation techniques Screening in nature in the early stage of the project provided strains with high efficiency of hydrogen production. The strains used throughout this project are Rhodobacter sphaeroides RV (photosynthetic bacteria), Synechococcus sp. PCC7942 (cyanobacteria), and Clostridium butyricum (anaerobic bacteria). Screening of other strains was also done and some useful ones have been found. Synechococcus PCC7942 (a cyanobacterial host for genetic engineering) was grown at 30~ in liquid BG-11 medium (Rippka et al., 1979) with aeration, under continuous illumination provided by fluorescent lamps (14 W m-2). R. sphaeroides RV and its UV-mutant, P3, were grown in aSy medium (pH 6.8) containing 0.1% yeast extract, 10 mM ammonium sulfate, and 30 mM sodium succinate. The pH was adjusted with NaOH. The cells were cultivated with a 5% inoculure at 30~ for 24 h under anaerobic conditions with tungsten lamps (250 W m-2). The 50 ml of culture was transferred into a 200-ml bottle. Then the culture was filled with gL medium (pH 6.8) containing 50 mM sodium lactate and 10 mM sodium glutamate for hydrogen production. The culture was further incubated at 30~ for 18 h under anaerobic conditions with tungsten lamps (500 W m-2). Only the cultures evolving hydrogen at 10-15 ml h - l were used for experiments.

92

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

photosvstem

N2ase Electron pool

Plant type Photons Photons

PSI

PS II

f ' ~ ADP ATP 2H20 4H++O2

Bacterial type

N N,

4ADP

PSI . .

ADP

ATP

e

Organic ~ substrates

H§

ATP pool

Fig. 2. Mechanism of light to hydrogen conversion by phototrophic bacteria and cyanobacteria.

2.2. Measurement of hydrogen production Hydrogen evolution from cyanobacteria and photosynthetic bacteria was measured using a hydrogen electrode system (Biott, Tokyo). The reactor volume was 2.5 ml. The light illumination area was 1.77 cm 2. A halogen lamp (Bromo lamp delux, LPL, Tokyo) was used as the light source. The light intensity was determined by a radiometer (model 4090, SJI, Tokyo).

buffer (272 mM sucrose, 8 mM HEPES, pH 7.4). The cells were re-suspended in SH buffer at a concentration of 1 mg chlorophyll ml-~. Hydrogenase solutions of 50 ~tl (0.4 units) were quickly added into 350 lal cell suspensions. An electric pulse with a time-constant of 4.2 ms was immediately applied to the cell suspension (within 1 min) at room temperature (25-30~ under dim light conditions. The cells were washed with Tris buffer to remove extracellular protein and was used for studies.

2.3. Pseudotransformation techniques 2. 4. Genetic manipulation techniques Cells were harvested at 30~ by centrifugation at 10000 x g for 10 min and washed with SH

DNA cloning was carried out according to a

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101 laboratory manual (Sambrook et al., 1989). Genetic manipulation of cyanobacteria was carried out as described by Poter (1988). Genetic manipulation of photosynthetic bacteria was carried out as described by Simon et al. (1984). 2.5. Photobioreactor research The photobioreactor was composed of four equal compartments. Each compartment had a 0.5-cm light path and a 37.5-cm 2 irradiation area and was made of polyacryl resin of 0.5 cm thickness using transparent resin for the windows and black resin for the frame (Fig. 3). The reactor was immersed into a water bath to keep the temperature at 30~ The compartments were aligned along the light path with a 0.3-cm gap between compartments for the circulation of water to control the temperature. The light path was set normal to the parallel planes of the compartments to analyze hydrogen evolution at various depths in the reactor.

Hydrogen

93

A tungsten lamp of 150 W (color temperature of 2900 K) was used for the light source. Light was passed through 4 cm of water in the water bath and 0.6 cm of polyacryl resin of the water bath vessel before reaching the surface of the reactor. The intensity of the light at the surface of the reactor was adjusted to be either 360 or 720 W m - 2. Hydrogen gas was collected in glass syringes to measure the evolution rates as reported previously (Miyake and Kawamura, 1987). The wavelength-dependent light penetration into the reactor was measured by using a spectrophotometer (Hitachi, model 330, Tokyo) with an integrating sphere. Light energy was measured with a radiometer (YSI-Kettering, model 65A, USA). The emission light spectrum from the lamp was measured using a diode array spectrophotometer (Otsuka Electronic, model MCP-1100, Osaka). The cell density of the photosynthetic bacteria was measured with a spectrophotometer (Hitachi, model 110). The efficiency of the conversion of light energy to hydrogen was calculated using the equation below. Efficiency (%) = (combustion enthalpy of hydrogen) /(absorbed light energy)x 100

3. Results and discussion

3.1. Cyanobacteria i

I

Fig. 3. Photobioreactor with four compartments. The compartments were made with polyacryl resin of 0.5 cm thickness. Irradiation area was 37.5 c m 2 and the light path of each compartment was 0.5 cm. Spectra of light at the depth are illustrated.

3.1.1. Strategy for genetic breeding The mechanism of hydrogen evolution by cyanobacteria has been extensively studied for its potential use as a method of energy production from sunlight. The photosynthetic system produces the reducing power and ATP from light energy. They are transferred to the enzyme system for hydrogen production, hydrogenase or nitrogenase. However, the supply of reducing power and/or ATP for hydrogenase or nitrogenase is not always directly dependent on photosynthetic energy-pro-

J. Miyake et al. Journal of Biotechnology 70 (1999) 89-101

94

Hydrogenase system ,

_.

I compounds t_ I _.~n_._nl

~..

H2

-,

fermentation p a t h w a y

j /

~H2aseJ

7 H2

Bacterial hydrogenase system .o

-----, electronflow Nitrogenase system

H2

Fig. 4. Genetic strategy for hydrogen production by cyanobacteria. PS, photosystem, fd., ferredoxin; H2ase, hydrogenase; N2ase, nitrogenase.

duction but sometimes involves the decomposition of the carbohydrates that have been photosynthetically produced. The involvement of carbohydrate metabolism may be one of the factors which reduce the conversion efficiency of light to hydrogen. An alternative electron transporting pathway is the direct connection of the photosynthetic system to the hydrogen producing enzymes that is expected for efficient hydrogen production (Fig. 4).

3.1.2. Pseudotransformation system As the first step to see the effect of clostridial hydrogenase incorporated into cyanobacterial cells, we have directly introduced the enzyme protein into the cyanobacterial cells by electroporation. We have called the method 'pseudotransformation' (Miyake and Asada, 1997). The cells temporally showed clostridial hydrogenase and were designated as 'pseudotransformants', which retain clostridial hydrogenase without genetic manipulation. These cells were used to investigate in vivo coupling between the photosynthetic system and clostridial hydrogenase in cyanobacteria.

A considerable amount of the active hydrogenase was incorporated into the cyanobacterial cells after electroporation with clostridial hydrogenase at 9 kV cm-~ of electric field strength (Fig. 5). Incorporation of hydrogenase is dependent on strength of electric field, since low levels of hydrogenase activity were detected at low ( < 9 kV cm-1) electric field strengths. Clostridial hy-

>,.~ 4-J

12

~9o

10

~

8

~0 "0 m

4

~m Ib ,-

2 0 0 5 10 15 Electric field strength (kV cm 9 "1)

Fig. 5. Direct electroporation of clostridial hydrogenase protein into cyanobacterial cells.

J. Miyake et al. / Journal of Biotechnology 70 (I 999) 89-1 O1 (b)

(a)

(c)

t-

E c .~/o n -C E e- ~ 8 d

c

.2

-

c

~t-.- g

time 10min

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~ 1~,, ~ Uru

o

~

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d time

time

10min

10min

light

light

x x

DCMU

it.,

2

o8

e-

95

x

\ [X;MU H20 1 / 2 0 2

CO2 fixation

H201/202

2

2H +

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Q - clostridial hydrogenase

Fig. 6. Light-dependent hydrogen evolution by pseudotransformants. (a) Wild-type cells after electroporation without clostridial hydrogenase. (b) and (c) Pseudotransformants retaining clostridial hydrogenase.

drogenase inside the cells was detected by Western blot analysis (data not shown; see Miyake and Asada, 1997). Light-dependent H2 evolution by the pseudotransformants was measured without any artificial electron carriers. Wild-type cells produced no H2 (Fig. 6a). Induction of hydrogen evolution via light irradiation were observed by the pseudotransformation (91 ~tE m - 2 s - ] ) (Fig. 6b). Dichlorophenyldimethylurea (DCMU) inhibits electron transfer between PS II and PS I. The hydrogen evolution was inhibited by DCMU (10 nM) (Fig. 6c). These results strongly suggest that clostridial hydrogenase could couple with photosynthetic water splitting in vivo.

In order to get permanent incorporation of clostridial hydrogenase in the cyanobacterial cells, we have worked to develop a strong expression system for the non-nitrogen-fixing cyanobacterium, Synechococcus sp. PCC7942. A 262 bpDNA fragment, no. 9 (Miyake et al., 1996), showing strong promoter activity was cloned from Synechococcus PCC7942 by using a promoterprobe plasmid vector, pKE4, based on a promoter-less cat gene cartridge from pKK232-8 and a shuttle vector, pECAN8, which replicates in Escherichia coli and PCC7942. A sigma-70 type promoter (Fig. 7) similar to the other strong promoters was found in fragment no. 9. Tran-35

3.1.3. Expression of clostridial hydrogenase in cyanobacteria Genetic manipulation to improve the hydrogen evolution enzyme system has been studied as the second step. Clostridial hydrogenase I, the powerful enzyme for hydrogen production, was expressed in cyanobacteria. This enzyme is more efficient than nitrogenase which the bacteria usually uses for hydrogen production.

-10

+1

promoter origine

ql~ TAC_~ . . . . (17nt) . . . . TA GATT . . . . A

psbDI psbAIII psbAII

"]['I'GCGA . . . . (18nt) . . . . . TATA CT---G___T_T

No.9 promoter

"FrGACA

E col~ a 7~

" ~ G TAA . . . . (16nt) . . . . TAT C A T - - - - - A CACA . . . . (16nt) . . . . . TAT TAT . . . . A

TATAAT

Fig. 7. Alignment of sigma-70 type promoters in Synechococcus sp. PCC7942.

J. Miyake et al. / Journal of Biotechnology 70 (1999) 89-101

96

Ampicillin resistant gene

cat gene

s~ r

t

pKE4-9 8.50 Kb

I cyanobacterlal

i replication ori

l~;No.9 promoter

Fig. 8. Structure of a shuttle vector between E. coli and Synechococcus sp. PCC7942, pKE4-9.

scription of mRNA started 5-bases downstream of the -10 region of the promoter. The content of CAT protein expressed by the no. 9 promoter reached 20% of the soluble proteins in PCC7942 cells. The no. 9 promoter could be used to express a clostridial hydrogenase I gene in cyanobacteria. A plasmid, pKE4-9 (Fig. 8), which retained the no. 9 promoter, was used to express clostridial hydrogenase gene in Synechococcus PCC7942. The clostridial hydrogenase I gene was cloned from the downstream region of the no. 9 promoter of pKE4-9. E. coli and C. pasteurianum ribosomal binding sequences were designed upstream of the clostridial hydrogenase I gene to investigate the effect of ribosomal binding sequence variation. Clostridial hydrogenase I protein was expressed exclusively by using an E. coli ribosomal binding sequence but not by using clostridial sequence. These results suggest that alteration of ribosomal binding sequence is essential for efficient expression of clostridial hydrogenase I in cyanobacteria. Precise control of the gene expression and enzymatic activity is underway.

3.2. Photosynthetic bacter& 3.2. I. Strategy for genetic breeding A purple bacterium, R. sphaeroides RV is a strong hydrogen producer (Miyake and Kawamura, 1987) and is applied for photobioreactors (Tsygankov et al., 1994; Nakada et al., 1995). The energy conversion efficiency from light to hydrogen is approximately 7% under a solar simulator.

However, the efficiency varies under different light sources. It is believed that the hydrogen production by photosynthetic bacteria may depend on the spectral distribution, since the bacteria utilize the specific light wavelengths for photosynthesis. In the intracytoplasmic membrane of photosynthetic bacteria, two different light-harvesting (LH) complexes are involved in absorption of light quanta and transfer of excitation energy to the photosynthetic reaction center (RC) (Richter and Drews, 1991). R. sphaeroides shows the absorption peaks at 800, 850 and 875 nm, corresponding to the LH2-bacteriochlorophyll B800, B850 and LH 1-bacteriochlorophyll, respectively. An approach for the improvement of hydrogen production by photosynthetic bacteria is the control of photosynthetic protein expression to allow efficient absorption of light energy.

3.2.2. Rearrangement of light harvesting system A mutant, P3 strain, was obtained by UV-irradiation from R. sphaeroides RV. The amount of bacteriochlorophylls in LH1 were reduced to 30% and those of LH2 were enhanced to 140% in P3 compared to R. sphaeroides RV. The contribution of each LH complex to the hydrogen production was examined under monochromatic light (Fig. 9). At 850 and 800 nm (absorption maxima for LH2), the energy conversion efficiencies of P3 were 6.3 and 4%, respectively, whereas those of RV were 5.5 and 3.4%. The maximal production rates of P3 were also 1.4 and 1.5 times higher than those of R. sphaeroides RV. At 875 nm (absorption maximum for LH1), the energy conversion efficiency and the maximal hydrogen production rate of P3 were still high despite a decrease in the level of LH1. Genetic transformation of P3 with a plasmid carrying genes encoding LH1 pigment-binding proteins restored the wild-type absorbance (original LH1/ LH2 ratio) and hydrogen production characteristics of R. sphaeroides RV. We consider that the alteration of the LH1/ LH2 ratio induces the change of the energy pathway from LH pigments to the photosynthetic RC. The change in the amount of bacteriochlorophylls effected the light penetration profile in the photo-

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

~r-

0.8

~ -o E

0.6

" -5 ,-- E ~ E ~

0.4

97

I

0.2

0 t_

-r~'

0

I

0

9

I

o

I

,

I

,

I

20 40 60 80

,

I

,

0

I

-.

I

o

I

..-

I

9

20 40 60 80

0

I

I

20 40 60 80 100

Light intensity (W/m 2)

Fig. 9. Comparison of hydrogen evolution rates between R. sphaeroidesRV and the UV-mutant, P3, under different monochromatic light conditions. Black circle, R. sphaeroides RV; white circle, the mutant P3. bioreactor which also affected the hydrogen production. Optimization of pigment composition by genetic engineering would improve the hydrogen production by photosynthetic bacteria.

3.2.3. Promoter competition A method for the enhancement of the bacterial light-dependent hydrogen production is proposed by rearrangement of light harvesting systems. Using R. sphaeroides RV, variants have been obtained by the 'promoter competition method' (Fig. 10), thereby making sure of the effectiveness of this method of breeding improvement. Competitive inhibition of the expression of puf operon encoding LH1 and RC were investigated by using multicopy-plasmids, pRKM-LPPluc, which was constructed by insertion of the p u f promoter region (Nagamine et al., 1996) into a broad host vector, pRKM-415, as a control plasmid. R. sphaeroides RV retaining pRKM-LPPluc (RV-LPPluc) showed a weaker accumulation of bacteriochlorophyll than a transformant retaining pRKM-415 (RV-415) after transferring the cultures from 'aerobic dark' to 'anaerobic light' conditions. It was shown that after changing growth conditions the strain RV-LPPluc demonstrated a considerable lag-period before initiating photoheterotrophic growth in comparison with control strain RV-415. The luciferase activity of RV-LPPluc was increased and the maximum luc-activity was observed in the beginning of exponential growth. The existing additional copies of p u f promoter cloned into plasmid may lead to prolongation of

the lag-period prior to photoheterotrophic growth. More detailed investigations of this effect of promoter competition are necessary and continuous cultivation of the bacterium would be preferred.

3.2.4. Basis for the breeding: light penetration into the cell suspension R. sphaeroides RV shows characteristic absorption bands at around 400, 800 and 850 nm. The two major absorption bands at 800 and 850 nm are attributed to the absorption of bacteriochlorophylls. The absorption band around 400 nm is

puf promoter fragment

. ~ ~

chromosome

RNApolymerese

?Oo O j

pRKM-LPPItm

Fig. 10. Competitive inhibition of puf operon expression by using a plasmid, pRKM-LPPluc, which carries puf promoter sequence and a luciferase gene as a reporter gene.

98

J. Miyake et al. /Journal of Biotechnology 70 (1999) 89-101

/ / oe i/

0.9 >,

g6~ /

'

~

-i

.2

3 5 0 ~ 500

'+o+++%

0

20

++~,~+

Fig. 11. Spectra of light at various depths. Light spectrum and energy differ very much at various depths. Up to 20 mm. light energy is mostly adsorbed.

attributed to the absorption of bacteriochlorophylls and hemes (Soret band). Light of wavelength around the absorption maxima of the bacterium (at 800 and 850 nm) decreased rapidly with passage through the cell culture. The spectrum of the light was greatly altered in the deep part of the reactor where the light had maximum energy at 500-780 nm (Fig. 11). The portion of incident light energy absorbed in this first compartment was 69%. The second compartment absorbed 21%, the third 7.0% and the fourth 2.0%. The incident light energy only effectively reaches the first cell. Cells deep in the reactor are only poorly illuminated. Light reaching bacterial cells in the second or third compartments showed a much different spectrum to that of the incident light at the reactor surface. Energy of light of wavelength around the absorption maxima of the cells (800-850 nm) was mostly lost. Only light of wavelength in the region of 500-780 nm (mainly 600-80 nm) reached deep in the reactor. Hydrogen was produced from the energy of this light because the wavelength of which was outside the range of the absorption maxima of bacteriochlorophylls. However, light in the wavelength range was used for hydrogen production in the deep part of the reactor (Fig. 12).

Inhomogeneity of the light distribution in the reactor lowers the overall conversion efficiency. To enhance the total efficiency of light to hydrogen conversion in a photobioreactor, light energy should be equally distributed throughout the reactor. The spectrum of light in the reactor also affected hydrogen evolution. Light with 600-780nm wavelength reached the deep part of the reactor and contributed to the hydrogen evolution. The above conclusion leads us to control the level of pigments in the cells genetically. Cells with less pigment enables the following advantages: light penetration to the deep region of the reactor, moderate quantity of light energy is absorbed at the shallow region of the reactor. These may lead to the total efficiency of a reactor, though the improvement reduces the activity of individual cell. Fig. 13-1 shows a photobioreactor with light diffuser distributing light into the deep part of the reactor. It provided a high efficiency of light to hydrogen conversion (E1-Shishtawy et al., 1997). Solar light was collected and delivered to a reactor by using a fiber glass (Fig. 13-2). This type of reactor has defects in that the production cost is high and there is difficulty in cleaning. In application, a large-scale reactor is necessary as shown in Fig. 13-3. The engineering study on how to satisfy the above light distribution and the simple construction is yet to be examined.

m L

._o ---= u o > c

3

e-

t'13.}

.o_ 2 I.s~:

~t

o "o

1

"1"

1

2

Compartment

E!

3 number

Fig. 12. The relationship between the depth and hydrogen production efficiency. Efficiency at the low light (deep region of the reactor) is high but at high light is low.

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

T

2

/7,/3, ,J ,..) oa"d

. . . .

,o

I" I: I:

i:

i

o,,~ .~.

I

-o

I

- . I

ol

I

C

/

Fig. 13. Some examples of the reactors developed in RITE projects. (13-1) A reactor with wall-type light diffuser by Kashima Co. (13-2) A reactor illuminated intrinsic illuminating rod by Kubota. Co. Light is supplied from a solar collector (Himawari, La Fore Eng. Co.). (13-3) A large scale reactor (800 1:2 x 4 x 0.1 m) set on the sea surface by Ishikawajima Heavy Industry Co. Ltd.

3.3. Potential of biological hydrogen production The hydrogen production using the photosynthetic microorganisms is regarded as an environmentally acceptable (or friendly) technology. Wastes could be treated while producing hydro-

99

gen at the same time. Hydrogen gas is a clean fuel because it does not produce carbon dioxide. In principle, it dispenses with the use of fossil fuels and is useful in avoiding global warming. An attempt has been made to evaluate the 'environmental friendliness', with the carbon dioxide disposal ( L C C O 2 ) from fossil fuel used in all stages from the construction of the plant to its operation as the index. An important point of the application of solar energy is the efficiency of the conversion. It has been said that the plant photosynthesis is done with an energy conversion efficiency as low as 1%. In our study, R. sphaeroides showed a remarkable conversion ratio of up to 7%. It is comparable to the amorphous semiconductors especially taking into account the low energy cost of system production. Further improvement of the bacteria could provide higher efficiencies. One of the characteristic features of hydrogen production by use of photosynthetic bacteria is the ability to recover and concentrate the energy from high water content organic resources such as waste effluents and sludges from which energy cannot be recovered by combustion. Compared with methane fermentation, a similar energy recovering method, the energy recovery ratio from biomass is theoretically elevated by that increment corresponding to the light energy taken in by the reaction of converting organic acids into hydrogen. Furthermore, the use of the gas obtained thus as a fuel for fuel cells will testify to the superior total energy efficiency of hydrogen production by this system, for hydrogen has higher power generating efficiency than methane. Since hydrogen production from photosynthetic bacteria requires waste matters or effluents containing organic substances as the substrate material, the location of the plant should be in an area where such materials are available, and the plant scale will be regulated by the output of these materials. The requirements for achieving commercialization in the hydrogen production cost are, (1) a steep enhancement of the energy conversion efficiency of the photosynthetic bacteria, (2) a substantial reduction in the installation cost for facilities ranging from pretreatment plants to photosynthetic reactors, and (3) the payback expected

100

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-101

from the effluent treatment, the by-product excess microorganisms and so forth. A preliminary estimation has clearly pointed out cost reduction, although the calculation of hydrogen production cost has not yet been reliably made.

3.3.1. A research project for biological hydrogen production in Japan Research Institute of Innovative Technology for the Earth (RITE) launched a project of biological hydrogen production (Development of Environmentally Friendly Technology for the Production of Hydrogen) in 1991. The collaboration included six private industries, and a national institute join the project. The project consists of scientific and engineering research. Genetic improvement of bacteria to produce more hydrogen and design and operation of model reactors are simultaneously carried out in the project. For the attainment of this target, the following R&D activities are underway: 1. Search for and improve photosynthetic microorganisms which have a high hydrogen production capacity. 2. Large scale cultivation techniques for maximizing the efficiency of hydrogen production making use of microorganisms. 3. Techniques for effectively separating and refining the hydrogen formed. 4. Techniques for recovering useful materials including physiologically active materials, etc. 5. Construction of integrated systems for hydrogen production and technical evaluations.

4. Conclusion Use of fossil energy enhances the carbon dioxide concentration in the atmosphere leading to the global warming. New energy sources and the carder should be developed to prevent environmental problems. An energy source which is renewable and easily obtainable is desired. Solar radiation could be the most promising. It is huge in amount, but its energy density is low at the surface of the earth. As a cartier of solar energy, hydrogen is the most promising one because it does not cause air pollution as carbon dioxide

formation. The RITE biological hydrogen production project was established to develop methods suitable for this low density energy. Utilization of photosynthesis could provide a system to accumulate solar radiation. Photosynthetic microorganisms grow by themselves to cover a large area. The biological system does not require factory products. In the studies on the search for and breeding improvement of photosynthetic microorganisms, genetic manipulation methods have been established. R&D of mass cultivation techniques has been carried out to make an economically feasible system of hydrogen production coupled with wastewater treatment.

Acknowledgements This research was done in part as RITE biohydrogen project supported by NEDO/MITI.

References E1-Shishtawy, R.M.A., Kawasaki, S., Morimoto, M., 1997. Biological hydrogen production using noble light-induced and diffused photobioreactor. Biotechnol. Tech. (in press). Miyake, J., 1998a. The science of biohydrogen--background, research and potential. In: Zaborsky, O., Benemann, J.R., Miyake, J., Matsunaga, T., San Pietro, A. (Eds.), Biohydrogen, Plenum, New York, NY, in press. Miyake, J., 1998b. Biological solar energy conversion. In: Miyamoto, K. (Ed.), Renewable Biological Systems for Alternative Sustainable Energy Production. FAO Agricultural Services Bulletin 128, FAO, UN, pp. 7-17. Miyake, M., Asada, Y., 1997. Direct electroporation of clostridial hydrogenase into cyanobacterial cells. Biotechnol. Tech. 11, 787-790. Miyake, J., Kawamura, S., 1987. Efficiency of light energy conversion to hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides. Int. J. Hydrog. Energy 12, 147149. Miyake, M., Yamada, J., Aoyama, K., Uemura, I., Hoshino, T., Miyake, J., Asada, Y.Y., 1996. Strong expression of foreign protein in Synechococcus PCC7942. J. Mar. Biotechnol. 4, 61-63. Nagamine, Y., Kawasugi, T., Miyake, M., Asada, Y., Miyake, J., 1996. Characterization of photosynthetic bacterium Rhodobacter sphaeroides RV for hydrogen production. J. Mar. Biotechnol. 4, 34-37.

J. Miyake et al./Journal of Biotechnology 70 (1999) 89-10I

Nakada, E., Asada, Y., Arai, T., Miyake, J., 1995. Light penetration into cell suspensions of photosynthetic bacteria, relation to hydrogen production. J. Ferment. Bioeng. 80, 53-57. Poter, R.D., 1988. DNA transformation. Methods Enzymol. 167, 703-712. Richter, P., Drews, G., 1991. Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus. J. Bacteriol. 137, 5336-5345. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanie, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1-61.

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Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Simon, R., Priefer, U., Puhler, A., 1984. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784-791. Tsygankov, A.A., Hirata, Y., Miyake, M., Asada, Y., Miyake, J., 1994. Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction. J. Ferment. Bioeng. 77, 575-578.

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iiii

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 103-113

Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor [nci Eroglu a

a,, Kadir Asian, Ufuk Giindiiz b Meral Yticel b Lemi Tiirker c

Department of Chemical Engineer&g, Middle East Technical University, 06531 Ankara, Turkey b Department of Biology, Middle East Technical University, 06531 Ankara, Turkey c Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey

Received 13 October 1998; received in revised form 4 December 1998; accepted 22 December 1998

Abstract

The effect of L-malic acid and sodium glutamate, which serve as the carbon and nitrogen source, respectively, on hydrogen production by Rhodobacter sphaeroides O.U.001 has been investigated in a batch water jacketed glass column photobioreactor (PBR), which has an inner volume of 400 ml. The PBR was operated at different carbon to nitrogen ratios at 32~ with a tungsten lamp at a light intensity of 200 W m-2. Carbon to nitrogen ratio was found to be an important parameter for bio-hydrogen production. Moreover, hydrogen gas production was dependent on certain threshold concentrations of sodium glutamate. L-malic acid consumption was found to be first order with respect to L-malic acid concentration, whereas sodium glutamate consumption was found to be second order with respect to glutamate concentration. It was concluded that there is a close relationship between the hydrogen production rate and substrate consumption rates. A kinetic model is developed, which relates hydrogen gas production per amount of biomass, L-malic acid, and sodium glutamate concentrations. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen production; Photobioreactor; Rhodobacter sphaeroides; Instantaneous fractional yield; Hydrogen production factor; Dual substrate

1. Introduction

By using sunlight, some photosynthetic microorganisms produce hydrogen. Hydrogen is considered to be a promising, ideal, and renew-

* Corresponding author. Tel.: + 90-312-2102609; fax: + 90312-2101264. E-mail address: [email protected] (i. Eroglu)

able form of energy. Hydrogen is also used industrially in the synthesis of ammonia, in hydrogenation reactions, and in many other important applications. Bioproduction of hydrogen by photosynthetic bacteria from various substrates and from wastes has previously been investigated. Sasikala et al. (1991, 1992, 1995) produced hydrogen by Rhodobacter sphaeroides O.U.001. Recently, bioprocesses have been developed for hydrogen production by R. sphaeroides RV (Tsy-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: SO 168-1656(99)00064-4

104

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

gankov et al., 1993; Minami, 1997; Kitajima et al., 1998). At the Middle East Technical University in Turkey, three different bio-systems were designed for hydrogen production: (1) by R. sphaeroides O.U.001, (2) by coupled systems of Halobacterium halobium and E. toll, and (3) photoelectrochemical hydrogen production by H. halobium. In previous work (Arik et al., 1996), a 150 ml glass column photobioreactor (PBR) was constructed for the production of hydrogen by R. sphaeroides O.U.001 under anaerobic conditions and with a fixed light intensity. The optimum hydrogen production conditions were determined to be as follows: pH between 7.3 and 7.8, temperature 31-36~ light intensity 200 W m -2, and cell concentration 1.6-1.8 g 1-1 (dry weight). The maximum hydrogen production rate obtained was 0.047 1 1-1 h-1 gas produced per unit volume of culture with 99% purity. Then, a continuous PBR was scaled up to 400 ml (Eroglu et al., 1998). It was found that both the growth medium and the cell concentration have an influence on photosynthetic hydrogen production. The effect of parameters such as cell concentration and dual substrate concentrations (g-malic acid and sodium glutamate) on hydrogen production rate should be determined. If the relationship between the hydrogen production rate and the substrate concentrations can be expressed in terms of kinetic models, it might be possible to achieve prolonged continuous hydrogen production in a larger column PBR. To date, no reference exists in the literature about the modeling of large scale hydrogen production. To achieve such modeling, primarily kinetic models that relate the consumption of the carbon source, the nitrogen source and the cell growth and hydrogen production rates are required. Therefore, the objective of the present study is to find relationships between the consumption rates of the two substrates--u-malic acid as a carbon source and sodium glutamate as a nitrogen s o u r c e ~ o n cell growth and hydrogen gas production. This paper presents an initial quantitative approach to describe kinetics of hydrogen production by photoheterotrophic bacteria.

2. Materials and methods

R. sphaeroides O.U.001 (DSM 5648) was grown under anaerobic and sterile conditions in a minimal medium of Biebl and Pfenning (1981). The growth medium contained n-malic acid as the carbon source and sodium glutamate as the nitrogen source, and a vitamin solution (thiamin and niacin; 0.0005 g 1-1). Temperature was 32~ The growth medium was illuminated using a tungsten lamp at a light intensity of 200 W m -2. The initial pH of the growth medium was 7. Argon gas was used to create anaerobic conditions. The column PBR was made up of a glass cylinder that had an inner volume of 400 ml and was surrounded by a water jacket. At the top of the reactor, there was an inlet for the medium and outlets for the argon and for the hydrogen gas that was collected in a gas-measuring burette. Fresh medium was added from a reservoir that was placed above the PBR. Microorganisms were inoculated through the septum. At the bottom of the column PBR, there was an outlet for the culture and an inlet for argon gas. Experiments were carried out using media containing different initial amounts of the substrates n-malic acid and sodium glutamate. In the various experiments the initial concentrations of u-malic acid were 7.5, 15 or 30 mM, whereas the initial concentrations of sodium glutamate were 1, 2 or 10 mM. Samples were taken at 12 h time intervals while flushing the column with argon. Bacterial cell concentrations were measured as an increase in absorbance at 660 nm (Hitachi Spectrometer). The pH of the samples was also measured. In order to determine the consumption of the substrates, the samples were centrifuged and the supernatant was subjected to HPLC analysis (Shimadzu HPLC, BIORAD Aminex Ion Exclusion Column). The gas produced was analyzed by gas chromatography (Hewlett Packard 5890, Series II). We replicated each of the nine runs at least twice.

3. Results and discussion

In a set of nine replicated experiments, different combinations of initial substrate concentrations of

105

I. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

L-malic acid (CLMAo) and sodium glutamate (CNGo) were tested. The experiments showed that hydrogen production had started at some time after the inoculation of the bacteria. Different hydrogen production starting times (to) were obtained in different runs. Table 1 summarizes the total volume of hydrogen gas evolved (VT), gas production starting time (to), duration of the gas production, and the maximum cell concentration measured in these runs. Fig. 1 illustrates total hydrogen gas produced with respect to time in Runs 4 - 6 where initial L-malic acid concentration was 15 mM in each run. Initial sodium glutamate concentrations were varied and set at 1, 2 and l0 mM, respectively. High initial concentration of sodium glutamate (10 mM) in Run 6 led to a 4-fold decrease in hydrogen production relative to Run 4. Fig. 2 shows the growth curves of R. sphaeroides O.U.001 obtained for the same set of runs. The maximum cell concentration obtained in Run 6 was almost two times greater than that of Run 4 and Run 5. It was observed that excess sodium glutamate enhanced the cell growth but inhibited the hydrogen production. The pH varied between 7 and 7.6 in these runs. That is, during the course of these experiments, a slight decrease is observed in the pH of the medium during the

cell growth period, but once the hydrogen was produced, the pH increased. A similar comparison was made between the results obtained in Run 1, Run 4 and Run 7. In all these experiments the initial sodium glutamate concentration was set at 1 mM, and the initial L-malic acid concentrations were varied to 7.5, 15 and 30 mM, respectively. The total volume of hydrogen gas evolved, the duration of the hydrogen production, and the maximum cell concentration were increased, as the initial L-malic acid concentration was increased. This might indicate that high L-malic acid concentration enhanced both the cell growth and the hydrogen production at low sodium glutamate concentrations (Run 1 and Run 4). The pH varied between 7 and 7.8 during those runs. The consumption of the substrates during the runs must also be taken into account. It was observed that hydrogen production ceased when the L-malic acid had been totally consumed in all those runs having low initial sodium glutamate concentration (Runs 1, 2, 4, 5, 7 and 8) (Eroglu et al., 1998). However, if high sodium glutamate was present in the system (Runs 3, 6 and 9), both substrates were not utilized completely and they were left in the culture when hydrogen production stopped. It should be emphasized that cell concen-

g ,,,,,,,

200

0

--4~--.. 1 mM

100

'-"

2mM

. - ~ k ~ l O mM "

0

-----

----t

30

~--

;-

f

60

,,~ 90

f 120

~ 150

,I 180

t

210

240

Time (hrs)

Fig. 1. Total hydrogen production at a substrate concentration of 15 mM L-malic acid and three different sodium glutamate concentrations (1, 2 and l0 raM).

Table 1 The results of the hydrogen production experiments

h n I\

Run I 2 3 4 5 6 7 8 9

Initial L-malic acid concentration (mmol I - ' )

7.5 7.5 7.5 15 15

15 30 30 30

Initial sodium glutamate concentration (mrnol 1- ') I 2

Total volume of H, produced (ml)

I 2

203 1 I7 52 312 3 14 86 512 477

10

101

10

1 2 10

Gas production starting time I, (h) 40 40 40 50 40 40 70 50 90

Duration of gas production (h) 91 73 55 174 97 50 229 287 87

Maximum cell concentration (g I - ] ) 3.6 4.5 8.5 5.0 5.6 10

7.4 6.4 9.0

6 3

%

s

107

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113 12

- - e - - 1 mM

.J

~10

~2mM

3=

--~ir-- 10 rr~

~'8

"t3

~6 u C

uo 4 u2 o ~==='~'~ 0

i

~

t

i

50

100

150

200

Time(hr)

250

Fig. 2. Growth curves of bacteria at a substrate concentration of 15 mM L-malic acid and three different sodium glutamate concentrations (1, 2 and 10 mM). tration reached very high values in a short time in these runs. That might indicate the positive effect of high sodium glutamate concentration on cell growth, but would also indicate the inverse effect on hydrogen production.

4. Dual substrate consumption rates Integral method of analysis followed, to interpret the substrate consumption data and to find the consumption rate equations for L-malic acid and sodium glutamate. During this analysis, the volume of the PBR was assumed to be constant, since small amounts of samples (2 ml) were taken out. Significant variations of temperature and concentration were not expected in the reactor, since the reactor was small and argon gas bubbled during sampling. The cells were suspended in the column, but some of the cells--most probably the dead cells--sank to the bottom, which caused small fluctuations in cell concentration values. Various rate models m including Monod type of equations--have been tried using the Microsoft Excel 5 package program. The following rate equations gave the best fit: the first order consumption rate equation for L-malic acid (Fig. 3):

(1)

-- rLMA = --dCLMA/dt = k1CLMA and the second order consumption sodium glutamate (Fig. 4):

rate

for

- ryG = -- dCyG/dt = k 2 C 2 G

(2)

Table 2 summarizes the best fitting rate parameters of Eqs. (1) and (2), where n denotes the order of the rate equation, kl and k2 are the rate constants for the first order (h-1) and for the second order (1 mol-1 h - l ) rate equations, respectively. R 2 measures the dispersion of distribution from the mean. It varies between values 0 and 1, where 1 denotes the maximum agreement of the experimental data to calculated data. It should be noted that these fits were quite acceptable, considering the experimental difficulties and the scatter in the experimental data. No direct relationship was found between the rate constants and the concentration of the other substrate. Therefore, these rate equations were considered to be independent. The second order consumption rate of sodium glutamate may indicate that two molecules of glutamate are involved in the consumption reaction as would be the case if the enzyme had two binding sites for glutamate.

5. Cell growth without hydrogen production The growth curves obtained were examined in two phases. Phase 1 is the cell growth without hydrogen production, and Phase 2 is the hydrogen production. In Phase 1, between initial time and tc, substrates were mainly consumed by the

108

I. Eroglu et al. / Journal of Biotechnology 70 (1999) 103-113

perimental data. For convenience in evaluating cell concentration and substrate distribution, two yield terms are introduced: instantaneous fractional yield of cells, y, and overall fractional yield of cells, Y. Let y,V/LMA(I) be the ratio of the rate of new cells formed to the rate of consumption of L-malic acid (in terms of mass) at any time t. YX/LMA is called instantaneous fractional yield of cells with respect to L-malic acid. Similarly, the instantaneous fractional yield of cells with respect to sodium glutamate, y x / N G ( t ) , is the ratio of the rate of mass of new cells formed to the rate of consumption of sodium glutamate (in terms of mass). The L-malic acid consumption rate, rLMA, and sodium glutamate consumption rate, -rNG, are related to the growth rate, rG, by yield factors YX/LMA and YX/NG. From Eqs. (1) and (3) and similarly from Eqs. (2) and (3), the following relations are obtained:

growing cells. The growth rate of the bacteria, rG (the change in cell concentration relative to the change in time) is: rG = d X / d t

= mm](

(3)

where X is cell concentration (g 1-1), t is time (h), and m m is the specific growth rate (h-1). Eq. (3) should be valid for the exponential growth phase of the bacteria. In this phase, the cells adapt themselves to their new environment. After the adaptation period, cells should multiply rapidly, and cell mass and cell number density should increase exponentially with time. This is a period of balanced growth in which all components of the cell grow at the same rate. That is, the average composition of a single cell remains approximately constant during this phase of growth. During balanced growth, the specific growth rate determined from either cell number or cell mass would be the same. Since the nutrient concentrations are large in this phase, the growth rate is assumed not to be limited by the nutrient concentration, hence the exponential growth rate could be expressed as first order. Time required for the inoculation of the bacteria is neglected. A plot of In X versus time yields mm which is the slope of the best fitting line to represent the cell growth data obtained for each run. It was interesting to note that mr, varied between 0.022 and 0.217 h for different runs (Table 2). This result might indicate the significance of dual initial substrate concentrations on specific growth rate. In the present work, a new approach, the yield model, has been introduced to represent the ex2,50

l

1,50 1,00

(4)

yx/yG(t) = mmX/(keC2GMWNG)

(5)

The instantaneous fractional yields of cells with respect to each substrate depend on time. Therefore, they should be estimated by introducing the concentrations measured at that time. Table 3 lists the instantaneous yield factors estimated at to. Overall fractional yield which is the ratio of total mass of new cells formed until tc to total mass of L-malic acid consumed until tr is the mean of the instantaneous fractional yields between the initial time and t~ with respect to each substrate:

=oo gx I

y

-.- 2,00

yX/LMA(I) = m m X / ( k , C L M A M W L M A )

'~' 0,50 0,00

~-

0

~

t

20

"

'

?"

40

I

I

I '

I

60 80 timelhour)

'

1 ~

100

I w

120

140

Fig. 3. The first order consumption rate model for L-malic acid in the medium containing 7.5 mM L-malic acid and 1 mM sodium glutamate initially.

]. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

109

2000 1600 ~'

z

1200 y = 4,7357x + 951 RZ = 0,8825

800 400 0

20

40

60 80 time (hour)

100

120

140

Fig. 4. The second order consumption rate model for sodium glutamate in the medium containing 7.5 mM L-malic acid and 1 mM sodium glutamate initially. X(tc) - Xo

YX/LMA = ( C N ~ o -

CN~(to)MWLMA)

(6)

Similarly, the overall fractional yield of cells with respect to sodium glutamate is: X(tc) - ;Co

YX/N~ = ( CNGo - CN~(tr

(7)

where Xo is the initial cell concentration, which is neglected in the present work. Table 3 lists the overall fractional yield factors estimated in Phase 1. The instantaneous fractional yields estimated at tr were greater than the overall fractional yields. The yields for L-malic acid were much less than the yields for sodium glutamate. Those results indicated the importance of sodium glutamate for cell growth.

6. Hydrogen production Hydrogen production rate is defined in three different ways: 1. The average hydrogen production rate per culture volume, Rn2av, which is calculated by dividing total volume of gas produced by the volume of the reactor and by the duration of gas production, and has the unit of l 1-1 h-z. 2. The maximum hydrogen production rate per culture volume, RH, which is estimated from the total volume of~gas produced versus time data by simulation with a polynomial curve fitting and differentiation with respect to time. The plot of hydrogen production rate versus time gives a maximum at some time, tm. This is the time corresponding to the maximum

hydrogen production rate per unit volume of the culture, where the units are 1 1-1 h-1. 3. The maximum hydrogen production rate per biomass, R h2, is calculated by dividing the maximum hydrogen production rate, RH2, by cell concentration, X, measured at /m, where the unit is 1 g-~ h-~. The maximum hydrogen production rates were found to be quite close to the average hydrogen production rates (Table 3), which might indicate that the hydrogen production rate does not change too much during the gas's evolution. The rate of hydrogen production obtained in Run 5 was the highest compared to the other runs (0.01 1 1-~ h - t or 0.0024 1 g - ~ h - ~). These results are quite comparable with the rates obtained by Kitajima et al. (1998) who found hydrogen production rates as 0.042 1 1-~ h-~ or 0.0042 1 g-~ h-~ in plane type photosynthetic bioreactor. Minami (1997) reported 0.015 1 1-~ h-~ for R. s p h a e r o i d e s RV in 10 1 continuous PBR, and 0.011 1 1-~ h for 1.4 1 continuous PBR. The cells grow at a slower rate after tc during hydrogen production. The cell growth rate in Phase 2 is: !

ro = d X / d t = m m X

(8)

where m m is the specific growth rate during hydrogen production, m m values were obtained from the slope of the best fitting line of In X versus time, between tc < t < tm, and are listed in Table 2. m m values were found to be almost an order of magnitude less than the mm values for Phase 1. That could be attributed to the increase in the rate of death of the cells. In continuous experi-

110

]. Eroglu et al. / Journal of Biotechnology 70 (1999) 103- 113

ments the difference between m m and mm is very critical. If the dilution rate is not adjusted to mm, the cells will wash out. Instantaneous fractional yields in Phase 2 were estimated from Eqs. (4) and (5) by replacing m m with mm, which might also involve the consumption of the substrates due to the maintenance of the cells and hydrogen production.

YHJx = 3.17(X/CNG2) T M

(R2=0.74)

(b) The interactive model: hydrogen production factor depends on the yield of both of the substrates. That is:

Y n J x oc (Yx/NG)/(Yx/LMA)

(13)

where Y n J x is proportional to (CLMA/CNG2). The best fitting equation is:

Y n J x = 3.48(CLMA/CNG2) ~

7. Yield models In order to compare the results of different runs, a factor (the hydrogen production factor) is defined:

yH2/X = R'H2tm

(12)

(R 2 = 0.82)

(14)

It is concluded that hydrogen production is related to the ratio of the concentration of Lmalic acid to the square of the concentration of sodium glutamate.

(9)

yn2/x, by definition, has the unit volume of gas produced per dry weight of bacteria (1 g-~). Table 3 lists the instantaneous fractional yields and the hydrogen production factor estimated at t m . Two yield models are proposed for the estimation of the hydrogen production factor: (a) The non-interactive model: the hydrogen production factor depends on the yield of one of the substrates: either on the yield of L-malic acid or on the yield of sodium glutamate: yH2/X W.YX/LMA OCX~ CLMA

(1 O)

YH2/X OCYNG/X Ct?.X/ CNG2

(1 1)

The hydrogen production factor did not depend ( X / C L M A ) , hence, R 2 was very small. However, the hydrogen production factor depended on (X/ CNG:). The best fitting equation is: on

8. Hydrogen production models considering carbon to nitrogen ratio It was observed that the hydrogen production rate was affected by the ratio of substrate consumption rates, as explained in the previous section. Moreover, the ratio of carbon to nitrogen could be taken as a parameter instead of the substrate concentrations individually (Minami, 1997). That is to say, the hydrogen production rate is related with residence time, the carbon to nitrogen ratio, and cell concentration. Using these parameters in trial runs, it was found that the following models give the largest R 2 values:

yH2/X = 6.25[(C/N)X] ~

(R 2 = 0.56)

Y n J x = 22.6[(C4/N)X] ~

(15)

(R 2 = 0.75)

(16)

Table 2 Summary of the rate parameters Run

k 1 (h -l)

R2

k2 (1 mol -I h -l)

R2

mm (h-1)

mm (h-1)

1 2 3 4 5 6 7 8 9

0.0179 0.0316 0.0089 0.0109 0.0183 0.0141 0.0075 0.0096 0.0101

0.92 0.94 0.96 0.90 0.88 0.91 0.87 0.87 0.70

4.74 3.43 0.19 2.68 2.45 0.27 2.78 8.12 0.30

0.88 0.63 0.86 0.72 0.95 0.89 0.79 0.82 0.73

0.058 0.038 0.217 0.068 0.164 0.089 0.072 0.022 0.14

0.0066 0.0061 0.0085 0.0021 0.0014 0.0286 0.0034 0.0020 0.0055

c. Table 3 Yield factors and hydrogen production rates I

22 21 242 20 56 18 23 3 72

218 196 406 20 668 56 887 37 490

4.5 4.8 14.9 3.0 4.6 2.6

I44 24 64 41

1.1

I09

70 65 65 130 80 70 190 175 160

96

18

8.0 3.4 -~ ~

--

33 13 __

-

0.006 0.008 0.002 0.007 0.012 0.005 0.006 0.006 0.003

I

')

R112.1" (1

0.006 0.004 0.002 0.005 0.010 0.005 0.006 0.004 0.003

h-' 1

I)

R;IJ

h - ' g - I ) Yl12/X(LJ (1 g - ' )

0.00 19 0.0018 0.0003 0.0020 0.0024 0.0009 0.00 10 0.0010 0.0005

0.133 0.117 0.020 0.260 0. I92 0.063 0. I90 0.175 0.080

Y X / L M A (1,)

YXING

3.7 8.2 12.9

32.3 38.8 21.5 51.4 8.0 49.0 63.3 28.2 20.3

1.1

0.6 15.1 3.3 2.4 3.7

(Irn)

2 4 a

112

]. Eroglu et al./Journal of Biotechnology 70 (1999) I03-113

where C/N denotes the ratio of the total moles of carbon per the total moles of nitrogen present in the culture, whereas C4/N denotes the moles of carbon present in e-malic acid per the total moles of nitrogen present in the culture. It is interesting to note that hydrogen production depends on the moles of carbon present in L-malic acid more than on the total moles of carbon present in the medium.

9. Conclusions The following conclusions can be drawn from this study: 1. Both of the substrates (e-malic acid and sodium glutamate) are vital for hydrogen production. Moreover, hydrogen gas production is dependent on certain threshold concentrations of sodium glutamate. Interestingly, as the concentration of sodium glutamate reaches a certain upper limit, hydrogen production ceases. Therefore, the stress condition exerted by sodium glutamate to produce hydrogen is operative in a certain range of the concentration. 2. Hydrogen production starts after a lag period (40-80 h) and continues even after, the cell concentration has leveled out. The specific growth rate of bacteria in the hydrogen production phase is less than the specific growth rate of the bacteria in the exponential cell growth phase without hydrogen production. This finding contradicts the conclusions of previous researchers (Sasikala et al., 1992; Arik et al., 1996), who found that hydrogen was mainly produced in the exponential phase of growth in smaller sized PBRs. 3. The e-malic acid consumption rate was found to be first order with respect to the e-malic acid concentration, whereas the sodium glutamate consumption rate was found to be second order with respect to the sodium glutamate concentration. 4. There is a relationship between the cell concentration and the hydrogen production rate. However, the hydrogen production rate basically depends on the L-malic acid to sodium

glutamate ratio. The maximum hydrogen production rate is observed with the growth medium initially containing 15 mM L-malic acid and 2 mM sodium glutamate concentrations.

Acknowledgements This research has been supported by the Turkish Scientific Research Council (TUBITAK) Project number TBAG 1535, and the Middle East Technical University (METU) Research Fund, project number AFP-96-07-02-02.

Appendix A. Nomenclature

C C/N C4/N kl k2 mm t

mm

MW RG R RH 2

Rh2 RH2av R2 t tc tm

concentration (mol 1-1) ratio of the total moles of carbon per moles of nitrogen present in the culture ratio of the moles of carbon present in L-malic acid per moles of nitrogen in the culture first order reaction rate constant (h -1) second order reaction rate constant (1 mol-1 h-l) specific growth rate in Phase 1 (h-') specific growth rate in Phase 2 (h -1) Molecular weight (g mol -~) growth rate of the bacteria (g 1h -l) consumption rate (mol 1-~ h-l) maximum hydrogen production rate per culture (1 1-~ h -~) maximum hydrogen production rate per biomass (1 g-1 h-l) average hydrogen production rate per culture (1 1-~ h -~) goodness of fit time (h) hydrogen gas production starting time (h) residence time of maximum hydrogen production rate (h)

i. Eroglu et al./Journal of Biotechnology 70 (1999) 103-113

Vv X Xmax

total volume of hydrogen gas evolved (ml) cell dry weight concentration (g 1-1 ) maximum cell concentration (g 1-1)

yH2/X

hydrogen production factor (1 g-l)

y

instantaneous fractional yield of cells overall fractional yield of cells

Y

Subscripts H2 LMA NG o

X

hydrogen gas L-malic acid sodium glutamate initial cells

References

Arik, T., Gunduz, U., Yucel, M., Turker, L., Sediroglu, V., Eroglu, I., 1996. Photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001, Proceedings of the 11th World Hydrogen Energy Conference, Stuttgart, Germany, vol. 3, 2417-2424.

113

Biebl, H., Pfenning, N., 1981. Isolation of member of the family Rhodosprillacaea. In: The Prokaryotes. SpringerVerlag, New York, pp. 267-273. Eroglu, I., Asian, K., Gfindfiz, U., Yficel, M., Tfirker, L., 1998. Continuos hydrogen production by Rhodobacter sphaeroides O.U.001. In: Zaborsky, O.R. (Ed.), BioHydrogen. Plenum press, New York, pp. 143-149. Kitajima, Y., E1-Shishtalwy, R.M.A., Ueno, Y., Otsuka, S., Miyake, J., Morimoto, M., 1998. Analysis of compensation points of light using plain type photosynthetic bioreactor. In: Zaborsky, O.R. (Ed.), BioHydrogen. Plenum Press, New York, pp. 359-367. Minami, M., 1997. Biohydrogen production using sewage sludge by photosynthetic bacteria. Paper presented in BioHydrogen '97, the International Conference on Biological Hydrogen Poduction, Kona, Hawaii, USA. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1991. Environmental regulation for optimal biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 16, 597-601. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1992. Photoproduction of hydrogen from the waste water of a distillery by Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 17, 23-27. Sasikala, K., Ramana, C.H.V., Rao, P.R., 1995. Regulation of simultaneous hydrogen photoproduction during growth by pH and glutamate in Rhodobacter sphaeroides O.U.001. Int. J. Hydrogen Energy 20, 123-126. Tsygankov, A.A., Hirata, Y., Miyake, M., Asada, Y., Miyake, J., 1993. Hydrogen evolution photosynthetic bacterium Rhodobacter sphaeroides RV immobilized on porous glass. New Energy Systems and Conversions, Universal Academy Press, pp. 229- 233.

This Page Intentionally Left Blank

i

)OUkI~AL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 115-124

The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production Vedat Sediroglu a Inci Eroglu a Meral Yiicel b,* Lemi Tiirker r Ufuk Giindiiz a

b

Department of Chemical Engineering, Middle East Technical University (METU), 06531 Ankara, Turkey b Department of Biology, Middle East Technical University (METU), 06531 Ankara, Turkey c Department of Chemistry, Middle East Technical University (METU), 06531 Ankara, Turkey

Received 13 October 1998; received in revised form 11 December 1998; accepted 22 December 1998

Abstract Hydrogen gas can be produced electrochemically by leading a current through two electrodes immersed in a NaC1 solution. Bacteriorhodopsin (BR) a protein found in the purple membrane of Halobacterium halobium, is known to pump protons across the membrane upon illumination. In this study, the effect of BR on photoelectrochemical hydrogen production was investigated. A batch type bio-photoelectrochemical reactor was designed and constructed. The photoelectrochemical hydrogen production experiments were performed with free H. halobium packed cells or immobilised H. halobium cells. The cells were either immobilised in polyacrylamide gel (PAG) or on cellulose acetate membrane (CAM). Experiments were also performed with purple membrane fragments of H. halobium immobilised on cellulose acetate membrane. It was found that the presence of bacteriorhodopsin (BR) in the reactor enhances the hydrogen production rate upon illumination. Immobilisation increased the amount of hydrogen produced per mole of BR. Compared to control experiments without BR, the power requirement of the photoelectrochemical reactor per amount of hydrogen produced decreased fourfold when purple membrane fragments immobilised on CAM were used. The presence of BR regulates the pH of the system, increases the hydrogen production rate and causes light-induced proton dissociation, which lowers the electrical power requirement for the electrochemical conversion. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen production; Bacteriorhodopsin; Halobacterium halobium; Cellulose acetate membrane; Immobilisation; Polyacrylamide gel; Photoelectrochemical reactor

1. Introduction

* Corresponding author. Fax: + 90-312-2101289. E-mail address: [email protected] (M. Y/icel)

Hydrogen, being a renewable and a clean energy source, is an i m p o r t a n t fuel and basic raw material for chemical industries. Rapid advancement in biotechnology in recent years has drawn

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00065-6

116

V. Sediroglu et al./Journal of Biotechnology 70 (1999) 115-124

attention to technologies for the production of hydrogen with micro-organisms and solar energy, as well as its conventional production methods such as steam reforming of hydrocarbons, coal gasification, and electrolysis (Shreve, 1984). There are many studies on photobiological hydrogen production by photosynthetic bacteria, green algae, cyanobacteria, and cell free systems (Hall et al., 1995; Markov et al., 1995). However, technical problems currently limit the yield and duration of these systems, and therefore further development of a practical photobiological hydrogen producing system is necessary. Halobacterium halobium is a photosynthetic bacteria that can survive in 4 M NaC1 solutions (Oesterhelt and Stoeckenius, 1971). The cell membrane of H. halobium is subdivided into five sections; red, brown, white, yellow, and purple membranes (Oesterhelt and Stoeckenius, 1974). Although they have different functions in H. halobium, 80% of the cell membrane is composed of purple fragments, which contain a light transducing retinal protein called bacteriorhodopsin (BR). BR uses light energy to pump protons across the cell membrane. The retinal protein BR is very stable and may retain its activity for several years even in isolated form. This property has attracted the attention of researchers (Birge, 1990; Trissl, 1990). In recent studies, systems containing H. halobium or separated BR have been used for the photo production of hydrogen. Although protons are generated upon illumination in these micro-organisms, protons cannot be converted to molecular hydrogen because they normally lack hydrogenase enzymes. A proton reduction system should be coupled to H. halobium to enable the hydrogen production. This has been achieved by coupling H. halobium to a salt tolerant Escherichia coli (Khan and Bhatt, 1989; Patel and Madamwar, 1994; Kaya et al., 1996; Khan and Bhatt, 1997). H. halobium packed cells coupled to an electrochemical system was also found to be very promising and advantageous, since nutrients are not required (Khan and Bhatt, 1990, 1992; Sediroglu et al., 1996a). In these systems NaC1, which is very cheap and abundant, is used as electrolyte. This system is

considered to have an industrial potential, but it needs further improvement. The main objective of this study is to investigate and compare the different process conditions for using Halobacterium halobium for photoelectrochemical hydrogen production. Also, new immobilisation techniques were developed in order to improve the stability of the system. The factors studied are sodium chloride concentration, concentration of BR, and novel immobilisation techniques.

2. Materials and methods

2.1. Preparation of H. halobium packed cells and purple membrane fragments The materials used were all of reagent quality and were obtained from Merck (Germany). H. halobium S-9 strain was kindly provided by Professor Oesterhelt from the Max Planck Institute (Munich, Germany). H. halobium cells were grown as described by Yiicel et al. (1995). After 7 days of growth, the cells were collected by centrifugation at 6380 rpm for 20 min in a Sorvail centrifuge (GSA rotor). The precipitated cells were named as packed cells. These cells were no longer alive, but BR was active for a long period of time. In the experiments, these cells were resuspended in 4 M NaCI solution which prevents the osmotic rupture of the cells and to keep these cells in intact form. The purple membrane fragments were prepared according to the method as described in the literature (Yticel et al., 1995). 2.2. Measurement of photoactivity of H. halobium packed cells and PM fragments

The photoactivity of H. halobium packed cells and PM fragments were measured as pH versus time by using a combined pH electrode connected to a pH transmitter at 25~ as described by Yficel et al. (1995). Illumination was achieved by means of a 1000 W projector lamp located at a distance of 50 cm away from the vessel.

V. Sediroglu et al. / Journal of Biotechnology 70 (1999) 115-124

All the membranes were kept in 4 M NaC1 solution at 4~ in the dark for at least 24 h prior to the experiments. H. halobium packed cells were immobilised in polyacryamide gel (PAG) using the procedure originally developed by Eroglu et al. (1994) for the immobilisation of PM. The only difference was that instead of PM, H. halobium packed cells were added to gel solution. The concentration of BR changes with the amount of H. halobium packed cells added. The gel solution polymerised within the circular holes of the immobilisation assembly and nestled down in the niche by sticking to the periphery of the hole (Fig. 1). Polymerisation was completed in about 30 min. The gel constituted assembly were sunk into 4 M NaC1 solution to attain the uppermost swelling of the gel, and the assembly was kept in this solution in a refrigerator.

2.3. Immobilisation techniques

Cellulose acetate membranes (CAM) having a pore size and thickness of 0.2 gm and 0.15 mm, respectively, were placed into a novel immobilisation assembly (Fig. 1). The immobilisation assembly made of Plexiglas had nine circular sieved holes, each of which was 2.5 cm in diameter and 0.35 cm in depth. The holes in the sieves are each 1 mm in diameter. Phospholipid solution was prepared by dissolving egg-yolk phosphatidyl cholin (lecithin) in chloroform (10 g 1-1). According to the selected lecithin to BR ratio, the required amount of phospholipid solution was supplied to one side of the CAM and dried (Sediroglu et al., 1996b). Then, packed cells or purple membrane (PM) fragments with a known amount of BR were spread onto the lecithin impregnated cellulose acetate membrane surfaces.

[ i p n , T ]11

~ 9 ~ ~

~ 9 ~

IO01

12

~ 9176

9

''~176176176'.:

+

-

117

.

~ ~

~

"9

9

~176

9

x "~ "

"

~

1 ~

.

"'~176

i

T" 9

lo "~

...

4

5 13

2I

1

~

o

o

Fig. 1. Experimental setup: (1) light source; (2) magnetic stirrer; (3) water bath; (4) temperature probe; (5) pH electrode; (6) anode and cathode; (7) power supply; (8) analog digital converter; (9) pressure transducer; (10) immobilization assembly; (11) pH transmitter; (12) potentiometer; (13) black plate.

118

V. Sediroglu et al./Journal of Biotechnology 70 (1999) 115-124

2.4. Photoelectrochemical hydrogen production The experimental design and the bio-photoelectrochemical reactor (Bio-PEC) are shown in Fig. 1. The Bio-PEC was a 1 1 glass tank placed in a constant temperature water bath (made of glass) and stirred with a magnetic stirrer at 100 rpm (Sediroglu et al., 1996a). The upper part of the reactor had four openings: inlets for the electrode assembly, the combined pH electrode, the temperature probe, and a H2 gas outlet. The electrode assembly contained a platinum electrode as the cathode (surface area was 0.8 cm 2) and a silver electrode as the anode (surface area was 4.5 cm 2) located 1 cm away from each other. The electrodes were connected to a constant voltage power supplier. Voltage and current were recorded by a computer connected to an analogue digital converter. The combined pH electrode attached to a pH transducer and a temperature probe connected to a potentiometer were employed. The hydrogen gas produced was collected in a 1.4 1 closed vessel and a pressure transducer (Cole Parmer, J-7352 Series) connected to an analogue digital converter, which was employed to record the increase in pressure. In control experiments the evolved gas was collected in a gas measuring burette by reversible displacement of water. The gas was sampled with a gas-tight syringe (Alltech) and analysed by gas chromatography (Hewlett 5890 Packard Series II). Control experiments did not involve any BR. The voltage applied was 0.88 V. Fresh NaC1 solution was used and both Pt and Ag electrodes were regenerated before each experiment. The experiments were repeated at least three times. Experiments were performed at 25~ under anaerobic conditions. Illumination was achieved at a light intensity of 1000 W m - 2 . During the experiments voltage, current, pressure change and pH were recorded with respect to time. In the control experiments, NaC1 concentrations were changed from 0.1 to 3.5 M. The ranges of total BR used were 0.098-1.76 ~tmol for free H. halobium packed cells, 0.033-0.098 ~tmol for the packed cells immobilised on CAM and 0.0980.294 lamol for the packed cells immobilised on PAG. Whereas, BR used was 0.067 jamol for PM fragments immobilised on CAM.

8.0

6.0

/ar z'

-~--. E 4.0

. . . . .

~ . . ~ : ~ ~ ' ~

,. . . , . ,

0.0 -.--0

i_r a

"' 200

400 Time(rain)

8.74 V

600

.

800

b)

8.34 ~ 7.94 7.54 7.14 -0

~ ........... 200

400 Tlme(mln)

600

800

c)

4.00 2.00 E [

0.00 200

L1

400

-2.00

& -4.00 Tlme(mln)

Fig. 2. Effect of NaC1 concentration on photoelectrochemical hydrogen production. (a) H2 produced as volume versus time. (b) Change of pH versus time. (c) Change of current versus time. (~ Power ON-light ON; T power OFF-light OFF; ( 0 ) 0.1 M, (11) 1 M, (e) 2 M, (A) 3.5 M).

3. Results and discussion

3.1. Effect of NaCl concentration on the photoelectrochemical hydrogen production The effect of NaC1 concentration on the photoelectrochemical hydrogen production without BR was investigated by changing the NaCI concentration of the solution from 0.1 to 3.5 M in control experiments. As seen in Fig. 2a the rate of hydrogen production and the total amount of H2 produced were increased by increasing the NaC1 concentration of the solution. The following reac-

V. Sediroglu et al./'Journal of Biotechnology 70 (1999) 115-124

tions are expected to occur at the electrodes: At the cathode, 2H20 + 2e- ---,H 2 + 2 O H -

E ~ =

-

0.83

V

(1) At the anode, Ag + C1- ~ AgC1 + e -

E ~ = - 0.22 V

(2)

and/or 2Ag + 2 O H - ~ Ag20 + H 2 0 + 2eE ~ = - 0.34 V

(3)

In all experiments, however, hydrogen could not be produced below - 0 . 8 8 V. As it is known from the literature, the solubility of silver chloride in water is very small. However, its solubility increases with increasing NaC1 concentration. In addition to that alkali chlorides convert silver oxides into silver chloride (Mellor, 1963). Therefore, at high NaC1 concentrations, chloride ions covering the silver surface may dissolve silver oxide formed on the anode by the following reactions" Ag20 + 2C1- + H20 ~ 2AgC1 + 2 O H -

(4)

AgC1 + C1- *-~AgClz-

(5)

or

AgCI*-~ Ag + + C1 -

(6)

Meanwhile, the following photoreaction might help the regeneration of Ag electrode (Mellor, 1963). hv

2AgC1 + 2 O H - ~ H 2 0 2 + 2Ag + 2C1-

(7)

The surface analysis of silver electrodes by scanning electron microscopy showed the formation of Ag20 on the silver electrodes after all the photoelectrochemical hydrogen production experiments. The lower hydrogen production rate observed at low NaC1 concentrations was attributed to the accumulation of Ag20 on the silver electrode surface. Also the cessation of hydrogen production after 400 min might be attributed to Ag20 accumulation on the electrode. However, it should be emphasized that H 2 could not be produced by replacing the Ag electrode with a new one, unless the Pt electrode was also changed. All

119

these findings imply that a thin hydrogen layer adsorbed on the Pt electrode might cause a large resistance, thus stopping the hydrogen production. Fig. 2b illustrates the change of pH versus time. As H2 was produced, the pH of the solution increased because O H - ions were produced according to reaction (1). It is interesting to note that minimum pH increase was observed at 3.5 M NaC1 concentration, although the maximum H 2 production was achieved under this condition (even the initial pH of the solution was higher). This might be due to consumption of hydroxyl ions by Ag at the anode, as indicated in reaction (3). This might explain why the rate of pH increase was higher in low NaC1 concentration compared to that in high NaC1 concentration. In the light of the observations above, 3.5 M NaC1 solution has been selected for photoelectrochemical hydrogen production. Fig. 2c illustrates the change of current while the system was in operation. The current decreased during the first 200 min of H2 production and then remained almost constant. Current direction was reversed after power was turned off.

3.2. Effect of H. halobium on the photoelectrochemical hydrogen production The results of experiments carried out with free or immobilised H. halobium packed cells on CAM or the purple membrane immobilised on CAM were compared with the control experiment. As it is seen from Fig. 3a, the H2 production is enhanced by increasing the amount of BR. It seems that BR affects the pH of the solution as it is illustrated in Fig. 3b. The highest production of hydrogen was obtained with the immobilised system containing the highest amount of BR (0.098 lamol). The pH increase was the lowest in this system. Upon illumination BR pumps out protons and most probably this process is accompanied by deprotonation of BR (Yiicel et al., 1995). Consequently, BR has a buffering effect on the solution, thus enhancing the hydrogen production. BR was found to be more effective in the immobilised system than in the free system (which might be due to the synergetic behaviour of BR in the

V. Sediroglu et al./Journal of Biotechnology 70 (1999) 115-124

120

immobilised system). The immobilisation might have increased the effective utilisation of light energy. In immobilised systems the membranes are directly and continuously illuminated because they are kept in place in the reactor. Whereas the free Halobacterium halobium cells in the suspension may be exposed to a less amount of light flux due to improper orientation while stirring. Photoelectrochemical hydrogen production was even higher with PM fragments immobilised on CAM.

27 25

a)

~,23 ~'19 17 150

'

*

i

200

400

600

&

800

'nn~min)

8.6

8.4 25.0

:~ 8.2

23.0 ~

b)

a)

"8.0

21.0

7.8

19.0

7.6,.

-

~

17.0

8 0

15.0 0

200 K

400

600

800

0.8

Time(min)

0.6

8.60

~

8.40

'0.0 ~ -0.2

8.20

~ .0.4

8.00

.0.6

_ _ .

~

c)

0.4 0.2 ,

) ~

8 0

Q.

.0.8 7.80

A

Time(rain)

7.60 0

100

A 0.80 0.60 0.40

200

300

400

500

600

700

Time(rata)

........................................................................ c)

0.20

Fig. 4. Effect of immobilized H. halobium packed cells in PAG (a) on rate of hydrogen production, in terms of pressure (b) pH change with respect to time (c) change of current versus time (T Power ON-light ON; ], power OFF-light OFF); (O) control; (M) PAG-Control; (@) PAG (BR = 0.098 lamol); ( 9 PAG (BR = 0.196 lamol); ( 9 PAG (BR = 0.294 lamol).

0.00

.~ .0.20 -0.40

-0.60 .0.80

Fig. 3. Effect of immobilization of BR on CAM. (a) On the rate of hydrogen production, in terms of pressure. (b) pH change with respect to time. (c) Change of current versus time. (up arrow) Power ON-light ON; (down arrow) power OFFlight OFF; (solid line) control; H. halobium packed cells immobilized on CAM (rectangle) BR = 0.033 lamol, (square) BR = 0.067 l~mol, (circle) BR = 0.098 lamol; PM immobilized on CAM (diamond) BR =0.067 ~tmol; free H. halobium packed cells (triangle) BR = 0.098 lamol.

Hydrogen production with H. halobium packed cells immobilised in PAG, having different amounts of total BR (ranged from 0.098 to 0.294 lamol), were compared with two control experiments. The light induced deprotonation of BR immobilised in PAG can be clearly seen in Fig. 4b. Although gas was produced at the beginning of the runs, a slight drop in pH was still observed. After some time, however, the pH increased. It was observed that the rate of hydrogen production was almost the same within the first 50 min for flesh and aged membranes of both types.

V. Sediroglu et al./Journal of Biotechnology 70 (1999) 115-124

As the experiments proceeded, however, a slight decrease in the hydrogen production for the aged membranes compared to the fresh ones was observed, probably due to the loss of surface deprotonation capacity by stabilisation of biosynthetic membranes. Results are compared on the basis of the hydrogen production ratio (HPR), average and initial hydrogen production rate, activity and power requirements. The hydrogen production ratio is defined as the ratio of the difference between the total number of moles of H 2 produced in the presence and absence of BR within a certain period of time, divided by the number of moles of BR present in the system. As seen in Table 1, HPR obtained by using free H. halobium packed cells was lower than the immobilised systems containing the same amount of BR. The immobilised systems were apparently more effective for the utilisation of the light energy. The highest hydrogen production ratio was obtained with immobilised PM on CAM. Initial rates is defined as the number of moles of hydrogen produced per hr within the first 50 min, and average rate is defined as the number of moles of hydrogen produced per hour within 520 min. Activity is defined as the average rate per mg cell. As expected, the initial rate increased as the concentration of BR increased. However, for the free H. halobium packed cells an increase of approximately one order of magnitude in the concentration of BR resulted only in a slight increase of the initial rate. For the same BR concentration (0.098 gmol BR) the initial rate was considerably higher for the H. halobium packed cells immobilised on CAM compared to that of the free H. halobium packed cells. Immobilisation capacity of CAM was found to be limited between 0.033 and 0.098 gmol BR (Sediroglu et al., 1998). The maximal amount of packed cells immobilised in PAG system resulted in amounts of BR (0.294 gmol BR) that were three times higher than that of the CAM system. Higher BR amounts immobilised in PAG led to higher hydrogen production rates, as shown in Table 1. Since the rate of hydrogen production decreases with time, the average hydrogen production rate was smaller than the corresponding initial rate, as

121

given in Table 1. The change in the average rates with increasing BR amount showed a similar trend as the initial rates of the corresponding systems. For the same systems, the percentage change in the average values of the amount of BR was approximately the same as the percentage change shown in the initial values. The maximum activity obtained was almost 1.5 times higher than that obtained by Khan and Bhatt (1992). Power requirement per mole of hydrogen produced was calculated from the current and voltage data obtained during the experiments, on the basis of either the initial or the average rate of hydrogen production, as given in Table 1. Power requirements based on the average rate of hydrogen production was about three times that of the ones based on the initial rates. It was remarkably higher in the absence of BR and it decreased as the concentration of BR increased. It should be noted that the minimum power consumption was observed with PM fragments immobilised on CAM. The light induced protonation of BR had decreased the power consumption per mole of hydrogen produced from 4.5 to 1 W mol-~ h-1 when the control experiment was compared with the experiment carried out with PM fragments immobilised on CAM. The power requirement was decreased by 30% if PM was immobilised on CAM instead of packed cells containing the same amount of BR (BR = 0.067 gmol). This indicates the advantage of using PM instead of packed cells due to the following reasons; 1. packed cells contain all cellular components, however PM contain only lipids and BR. 2. the size of the packed cells are greater than PM. Therefore self orientation of PM on CAM might be much better than the packed cells, and utilisation of light energy might thus be better for PM fragments. 3. present experiments revealed that the immobilisation of PM fragments is easier than the immobilisation of packed cells on CAM. Although, long term stability of packed cells has not been reported yet, some experiments of ours implied that the stability of PM fragment is higher than the packed cells.

I

N

Table 1 Summary of the experimental results Conditions"

BR used (pmol)

HPR

Average rate (pmol h - I )

Initial rate (pmol h-I)

Activity (pmol h - ' mg-')

Power based on initial rate (w mol-' h-I)

Power based on average rate ( w mol-' h-I)

S

ia B

a

QQ

a

-

227.78

-

26.28

136.66

-

4.24

17.54

2

I .600 0.760 0.098

20 228 9840 1350

512.50 469.79 455.55

177.95 318.43 2324.18

59.13 54.20 52.56

324.58 281.87 256.25

0.016 0.029 0.190

1.94 2.16 2.18

9.96 I 1.45 8.65

3

0.098 0.067 0.033 0.067

I350 900 450 900

683.33 605.03 320.31 697.56

4648.46 5630.59 2803.93 701 1.64

78.84 69.81 36.95 80.48

427.08 375.83 196.45 504.87

0.315 0.417 0.438 0.588

1.35 1.35 2.78 1.01

6.45 6.15 10.92 4.47

529.30 495.40 410.00 230.62

0. I56 0.148 0.303

1.13

4.98

1.15 1.31 1.68

5.10 7.43 10.13

CON

-

BI B2 B3 CI c2 c3 C4' PI P2 P3 P4d

Cell wet weight Total H, proinitially usedh duced (pmol) (mg)

0.294 0. I96 0.098 -

4050 2700 I350 -

811.45 740.27 633.50 313.19

1985.27 2614.74 4140 -

93.62 85.41 73.09 36.13

-

a Where CON denotes control experiments, B denotes free H. holohiurn packed cells, C denotes immobilised packed cells on CAM and P denotes immobilised packed cells in PAG. Density of packed cell is I g ml- I . Denotes immobilised PM fragments on CAM. Denotes control experiments with PAG.

,

5% B

gn

20

6

2

2 +

-.\

2

h

s

V. Sediroglu et al./Journal of Biotechnology 70 (1999) 115-124

However, packed cells are cheaper than PM fragments since PM fragments require sophisticated purification techniques that might be quite expensive and time consuming. Based on overall evaluation of these factors, further investigation is required as to whether PM fragments might be more economical than using the packed cells or not.

123

for the photoelectrochemical hydrogen production. It should be also noted that the regeneration possibility of the system, the stability of the biological membranes, the increase in hydrogen production rate and decrease in power requirement make the photoelectrochemical hydrogen production a promising new system. However, further investigations are needed for the optimisation of this system for any practical application.

4. Conclusion Acknowledgements The minimum voltage required for the hydrogen production was 0.88 V in the bioelectrochemical reactor designed and constructed for the present work. The presence of BR, either in the form of H. h a l o b i u m packed cells or as PM fragments, enhances the hydrogen production rate upon illumination. Hydrogen production also increased as the BR concentration increased. BR does not lose its photoactivity on the membrane surface of CAM and PAG for at least 2 months. Drastic improvement of the hydrogen production rate in the immobilisation systems compared to free H. h a l o b i u m packed cells in NaC1 solution has been observed. Larger amounts of BR can be immobilised in PAG than on CAM. One of the most promising results of the present work is the low power requirement per mole of hydrogen produced. The minimum power requirement is obtained with the photoelectrochemical system containing PM fragments immobilised on CAM. This power requirement is about one fourth of the control experiment, which did not contain H. h a l o b i u m . In the present system, NaC1 solution, which is cheap and abundant, is used instead of expensive alkaline solutions. These findings considerably increase the future potential of photoelectrochemical hydrogen production. It should be emphasised that the presence of BR does not only regulate the pH of the system, thus causing increased hydrogen production rate, but also causes light-induced proton dissociation, which may lower the required electrical power for the electrochemical conversion. The system presented in this study can be exploited in a device which can utilise light energy and electrical energy

This research is supported by M E T U Research Fund, project number AFP 96-07-02-02 and by Turkish Scientific and Research Council, project number TBAG 15 35.

References Birge, R.R., 1990. Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin. Biochem. Biophys. Acta 1016, 293-327. Ero~lu, |., Aydemir, A., Tiirker, L., Yficel, M., 1994. Photoresponse of bacteriorhodopsin immobilised in polyacrylamide gel membrane. J. Membr. Sci. 86, 171-179. Hall, D.O., Markov, S.A., Watanabe, Y., Rao, K.K., 1995. The potential applications of cyanobacterial photosynthesis for clean technologies. Photosynth. Res. 46, 129-167. Kaya, B., Giindiiz, U., Ero~,lu, i., Tiirker, L., Yiicel, M., 1996. Hydrogen gas production using coupled enzyme systems from E. coli and H. halobium. In: Proceedings of l lth World Hydrogen Energy Conference, vol. 3. Stuttgart, Germany, pp. 2595-2600. Khan, M.M.T., Bhatt, J.P., 1989. Light dependent hydrogen production by H. halobium MMT22 coupled to E. coli. Int. J. Hydrog. Energy 14, 643-645. Khan, M.M.T., Bhatt, J.P., 1990. Photoelectrochemical studies on H. halobium or continuous production of hydrogen. Int. J. Hydrog. Energy 15, 477-480. Khan, M.M.T., Bhatt, J.P., 1992. Large scale photobiological solar hydrogen generation using H. halobium MMT22 and silicon cell. Int. J. Hydrog. Energy 17, 93-95. Khan, M.M.T., Bhatt, J.P., 1997. Photosensitized continuous production of hydrogen by Halobacterium halobium MMT22 coupled to Escherichia coli. Int. J. Hydrog. Energy 22, 995-997. Markov, S.A., Bazin, M.J., Hall, D.O., 1995. The potential of using cyanobacteria in photobioreactors for hydrogen production. Adv. Biochem. Eng. 52, 60-86. Mellor, J.W., 1963. Supplement to Mellor's Comprehensive Treatise on Inorganic and Theoretical Chemistry, vol. 2. Longrnans, London, p. 390.

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Oesterhelt, D., Stoeckenius, J.W., 1971. Rhodopsin-like protein from the purple membrane of H. halobium. Nature New Biol. 233, 149-154. Oesterhelt, D., Stoeckenius, J.W., 1974. Isolation of the cell membrane of H. Halobium and its fraction into red and purple membrane. Methods Enzymol. 31, 667-671. Patel, S., Madamwar, D., 1994. Photohydrogen production from a coupled system of H. halobium and P. valderianum. Int. J. Hydrog. Energy 19, 733-738. Sediroglu, V., Gfindfiz, U., Yficel, M., Tfirker, L., Erofglu, i., 1996a. Photoelectrochemical H 2 production by H. halobium. In: Proceedings of l lth World Hydrogen Energy Conference, vol. 3. Stuttgart, Germany, pp. 2589-2594. Sediroglu, V., Giindiiz, U., Yiicel, M., Tiirker, L., Er@,lu, i., 1996b. Modeling of long term photo response of bacteri-

orhodopsin immobilized on cellulose acetate membranes. J. Membr. Sci. 113, 65-71. Sediroglu, V., Yficel, M., Gfindfiz, U., Tfirker, L., Ero~lu, i., 1998. The effect of Halobacterium halobium on the photoelectrochemical hydrogen production. In: Zaborsky, O.R. (Ed.), BioHydrogen, Plenum Press, New York, pp. 295-304. Shreve, N.R., 1984. Chemical process industries, fifth ed. McGraw-Hill, New York, pp. 98. Trissl, H.W., 1990. Photoelectric measurements of purple membranes. Photochem. Photobiol. 51 (6), 793-818. Yficel, M., Zabut, B.M., Er@,lu, [., Tfirker, L., 1995. Kinetic analysis of light induced proton dissociation and association of bacteriorhodopsin in purple membrane fragments under continuous illumination. J. Membr. Sci. 104, 65-72.

i

JOURNAL

i

i

i

O F

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 125-131

Identification of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O.U. 001 grown in the waste water of a sugar refinery Deniz Ozgfir Yi~,it

a

Ufuk Gfindfiz b , Lemi Tiirker c,, Meral Yiicel inci Ero~,lu d

b

Department of Biotechnology, Middle East Technical University, 06531 Ankara, Turkey b Department of Biology, Middle East Technical University, 06531 Ankara, Turkey c Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey d Department of Chemical Engineering, Middle East Technical University, 06531 Ankara, Turkey a

Received 13 October 1998; received in revised form 4 December 1998; accepted 22 December 1998

Abstract Rhodobacter sphaeroides O.U. 001 is able to produce hydrogen anaerobically upon illumination. The cells were screened for the presence of valuable by-products such as poly-13-hydroxy (PHB) butyric acid aiming to improve the feasibility of the system. Also waste water from a sugar refinery was used for bacterial growth to further increase the feasibility. Under aerobic conditions the standard growth media containing L-malic acid and sodium glutamate in 7.5/10 and 15/2 molar ratios and a medium containing 30% waste water from sugar refinery were used. In this case the maximum concentration of PHB produced were approximately 0.2 g 1-~ in both of the standard media whereas it was 0.3 g I-~ in medium containing 30% waste water. By using the medium containing 30% waste water, PHB and hydrogen productions were determined under anaerobic conditions. The maximum concentration of PHB produced was around 0.5 g 1-~ and the amount of gas collected was 35 ml in 108 h. From these results it can be concluded that PHB can be collected during hydrogen production. The use of waste water from sugar refinery increased the yield. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Rhodobacter sphaeroides; Hydrogen; Poly-13-hydroxy butyric acid; Waste water

I. Introduction

There are limited reserves of fossil fuels on earth so there is a strict need for d e v e l o p m e n t of * Corresponding author. Fax: + 90-312-2101-280. E-mail address: [email protected] (L. Tiirker)

renewable and clean energy sources. Since solar energy seems to be the m o s t suitable starting point m u c h attention has been given to the design of systems capable of t r a n s f o r m i n g solar energy into high energy c o m p o u n d s such as hydrogen, which is a clean and highly efficient fuel that results in water upon burning without generating CO2.

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00066-8

126

D.O. Yi~it et al. ,'Journal of Biotechnology 70 (1999) 125-131

Photobiological hydrogen production attracts much attention because biological transformation systems are simple in operation, stable, low energy consuming and renewable. Feasibility of a photobiological hydrogen production system can be increased by the use of cheaper substrates for the microorganism such as the waste water and the collection and recycling of useful by-products other than hydrogen. Rhodobacter sphaeroides O.U. 001 is able to produce hydrogen anaerobically in a photobioreactor (Ank et al., 1996). It was shown that waste water from sugar refinery can partially replace the C source of bacterial growth medium (Yeti~ et al., 1998). It was reported that R. sphaeroides O.U. 001 cells contain valuable by-products (Yamagishi, 1995). Poly-[3-hydroxy butyric acid (PHB) is one of the by-products which is a biodegradable thermoplastic that can be synthesized during unfavorable growth conditions by a wide range of bacteria (Byrom, 1987; Page, 1989; Kim et al., 1994). PHB has industrial applications; it can be used to construct biodegradable carriers for long term dosage of herbicides and insecticides, packaging containers, bottles and bags (Anderson and Dawes, 1990). PHB has also very important medical applications; it can be used as a biodegradable carrier for long term dosage of drugs and can be used to construct surgical pins, sutures, staples, swabs and it can also be used for wound dressing (Abe and Doi, 1992; Sang, 1996). The aim of the study was to test different parameters to optimize the conditions of a hydrogen production system for maximum feasibility. R. sphaeroides O.U. 001 cells were therefore screened for PHB accumulation. Bacterial growth was determined under aerobic conditions in standard growth media (Biebl and Pfennig, 1981) containing L-malic acid and sodium glutamate in 7.5/10 and 15/2 molar ratios and concentrations of PHB accumulated at different phases of growth were determined. It was shown before that bacterial growth was optimum at the ratio of 7.5/10 and hydrogen production was maximum at 15/2 (Ero~lu et al., 1997) so PHB production was determined at these ratios. To reduce the cost of growth medium; a medium containing 30% waste

Table 1 Contents of standard growth medium having different L-malic acid to sodium glutamate ratios L-malic acid/sodium glutamate (raM m M - 1 ) NB content (g 1-1) KH2PO 4 MgSO4.7H20 L-Malic acid Sodium glutamate NaC1 CaCI2.2H20 Niacin Thiamin Trace element solution 7 Ferric-citrate solution

7.5/10 0.5 0.2 1.0 1.8 0.4 0.05 0.0005 0.0005 1 ml

15/2 0.5 0.2 2.0 0.36 0.4 0.05 0.0005 0.0005 1 ml

5 ml

5 ml

water from sugar refinery was tested. Bacteria and PHB concentrations were determined and it was seen that the use of waste water increased the yield of PHB. When the experiment was repeated under anaerobic conditions it was observed that PHB was accumulated in a hydrogen producing system which can utilize waste water from sugar refinery so that the cost of growth medium can be decreased and valuable by-products can be collected to increase the feasibility.

Table 2 Contents of growth medium including 30% waste water from sugar refinery N B content

g 1-l

KH2PO 4 MgSO4-7H20 L-Malic acid Sodium glutamate NaC1 CaC12.2H20 Niacin Thiamin Trace element solution 7 Ferric-citrate solution Pretreated waste water

0.5 0.2 0.54 0.8 0.4 0.05 0.0005 0.0005 1 ml 5 ml 300 ml

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D.O. Yi~it et al. /'Journal of Biotechnology 70 (1999) 125-131

1.8

1.6

1.6

1.4

1.4

- 1.2

E ~.2 t-. C:) 1 t,D (,C) ~ , 0.8 a

- 0.8 -0.6

O o.6 0.4

'--.~I,--OD (660 nm)

0.2

(a)

r"-

~Dry

0 20

40

60

TimeS~hr)

0,2 0,18 0,16

Wt (g/L)

0.4 0.2 0

loo

12o

140

160

0,19~.

~" o,14 ~ 0,12 rn

0,1

N.~ 0,08 0,06

0,04 0,02 (b) 0

~

v

,

0

9

20

,

40

i

,

60

80

,

100

',

120

Time (hr) Fig. 1. The growth curves and poly-13-hydroxy (PHB) production curves in standard growth medium containing L-malic acid and sodium glutamate having 7.5/10 and 15/2 molar ratios under aerobic conditions. (a) Bacterial growth curve based on both OD at 660 nm and dry weight in growth media having 7.5/10 k-malic acid and sodium glutamate molar ratio. (b) PHB production (g 1- 1) in growth media having 7.5/10 L-malic acid and sodium glutamate molar ratio. (c) Bacterial growth curve based on both OD at 660 nm and dry weight in growth media having 7.5/10 L-malic acid and sodium glutamate molar ratio. (d) PHB production (g 1-1) in growth media having 15/2 L-malic acid and sodium glutamate molar ratio.

2. Materials and methods R. sphaeroides O.U. 001 ( D S M 5648) was grown in 50 ml flasks and anaerobic jars both aerobically and under argon atmosphere (anaerobically) at 36~ under illumination at 200 W m - 2 . Both the standard growth media containing L-malic acid and sodium glutamate having 7.5/10 and 15/2 molar ratios and a medium containing 30% waste water were used. Waste water was

obtained from sugar refinery at Etimesgut, Ankara. It was pretreated by incubation at 95~ for 45 min followed by filtration through cheese cloth. Then it was centrifuged at 3000 rpm for 30 min and filtered through glass prefilters and 0.45 lam W h a t m a n n filter paper. Its p H was adjusted to 7.0 and sterilized by autoclaving. It was stored at - 20~ until it was added to the growth media. The c o m p o n e n t s of the growth media are shown in Tables 1 and 2. Initial p H of the growth media

128

D.O. Yi~it et al./Journal of Biotechnology 70 (1999) 125-131 1,2

1,8 1,6 1,4

E r

C) CO r E3

0

1,2

0,8

r

1

- 0,6

0,8 0,6

I"-

- 0,4

0,4 ---e--

0,2

(c)

OD (660 nm)

- 0,2

---II-- Dry Wt (g/L)

0

9

20

,

L

40

60

!

80

100

120

140

0 160

Time (hr)

...J O) ~,~ rn "r-9 Q. (d)

0,2 0,18 0,16 0,14 0,12 0,1

0,18 g/L

0,08 0,06 0,04 0,02 0

0

I

I

I

|

20

40

60

80

,,=

i

100

Time (hr) Fig. 1. (Continued) were adjusted to 6.8-7. Growth curves were constructed by OD and dry weight measurements. Concentration of bacterial PHB was determined by the method of Bowker (1981). After the given incubation period cells were collected by centrifugation, digested by sodium hypochlorite treatment, PHB was extracted by hot chloroform treatment and it was converted to crotonic acid by sulfuric acid addi-

tion. Their OD were measured using a spectrophotometer. Concentration and %(w/w) PHB was calculated using the standard curve calibrated.

3. Results and discussion The growth of R. sphaeroides O.U. 001 and

129

D.O. Yi~it et al./Journal of Biotechnology 70 (1999) 125-131 0,35 0,3 0,25 ._J

0,2 0,15

"!"

0,1

Q.

0,05 ,,

0

20

,

- ,

40

T

60

-

,

100

80

Time (hr) Fig. 2. Poly-13-hydroxy (PHB) production (g 1-1) in growth medium containing 30% waste water from sugar refinery under aerobic conditions. 0,6 0,50g/L

0,5 .._1

0,4

1:~ 0,3 nn "1" 0,2 v

13_

0,1 -~ 0

-

" t

20

-

t

t

40

60

"

Time (hr)

t

80

-..

~' 100

"

120

Fig. 3. Poly-13-hydroxy (PHB) production (g 1-1) in growth medium containing 30% waste water from sugar refinery under anaerobic conditions. 35 30 25 20

E ~ "1-

15

10 5

0 0

20

40

60

Time (hr)

80

I00

120

Fig. 4. Collected gas amount (ml) in growth medium containing 30% waste water from sugar refinery under anaerobic conditions.

130

D.O. Yi~it et al./Journal of Biotechnology 70 (1999) 125-131

Table 3 Poly-[3-hydroxy (PHB) accumulation in different media Growth medium

Time (h)

Max PHB concentration (g 1-1)

% PHB (w/w)

15/2 7.5/10 30% ww (aerobic) 30% ww (anaerobic)

96 96 84 96

0.2 0.2 0.3 0.5

19.8 16.8 52.4 70.4

PHB production in standard growth medium containing L-malic acid and sodium glutamate at two different molar ratios (7.5/10 and 15/2) under aerobic conditions are shown in Fig. 1. It is seen that the maximum amounts of PHB accumulated are0.19 g l - 1 (16.8%PHB w/w) a n d 0 . 1 8 g l - I (19.8% PHB w/w) for 7.5/10 and 15/2 molar ratios, respectively. In the growth medium, containing L-malic acid and sodium glutamate at molar ratio of 15/2, the percentage of PHB is higher compared to the growth medium having 7.5/10 molar ratio. In both media PHB production was observed in a stationary phase. It was reported that PHB is produced during the stationary phase of the growth as a storage material under stress conditions; to be used during starvation (Yamagishi, 1995). PHB production curve in growth medium containing 30% waste water under aerobic conditions is shown in Fig. 2. It is seen that the amount of PHB accumulated is 0.30 g 1-~ and 52.4% PHB (w/w). Growth medium containing 30% waste water has been adjusted to contain nearly the same C/N molar ratio as standard growth 4 3.5 3 2.5 O3

2 1.5 1 0.5 0

v

v

0

50

Time (hr)

~

T.

y

100

v

150

Fig. 5. Ratio of hydrogen to poly-J3-hydroxy (PHB) production in growth medium containing 30% waste water under anaerobic conditions

medium containing L-malic acid and sodium glutamate at 15/2 molar ratio; since waste water also contains carbon 1.46 g l-1 L-malic acid is saved. The maximum amount of PHB produced is higher when waste water is present in the growth media. The reason for this may be due to the stress caused during utilization of carbon from waste water. In addition waste water may also contain some components which may cause stimulation of PHB production. PHB production in growth medium containing 30% waste water under anaerobic conditions is shown in Fig. 3. It is seen that the maximum amount of PHB accumulated is 0.50 g 1-l which corresponds to 70.4% PHB (w/w). This shows that PHB is also produced under anaerobic conditions and the production is even higher than aerobic conditions. It was also reported that PHB is accumulated in large quantities inside the bacterial cellular material of photosynthetic bacteria when cultured under anaerobic conditions (Yamagishi, 1995). R. sphaeroides O.U. 001 produces hydrogen gas under anaerobic conditions. In Fig. 4 the amount of collected gas under anaerobic conditions at time intervals in growth medium containing 30% waste water is shown. It is seen that 34.8 ml gas was collected in 108 h. The hydrogen production rate is nearly 0.006 1 l - l h - l ; and yield of hydrogen is 0.46 1 g-~. When this hydrogen production rate is compared with the literature data it is seen that 0.047 1 1-~ h -~ hydrogen was produced under optimized conditions (Arlk et al., 1996). On the other hand 0.006 1 1-1 h-~ hydrogen was produced by the use of waste water (Sasikala et al., 1992) and 0.042 1 1-~ h - l hydrogen was produced when artificial waste was used (Kitjama et al., 1997). The ratio of hydrogen to PHB production in growth medium containing 30%

D.O. Yi~it et al./Journal of Biotechnology 70 (1999) 125-131

waste water under anaerobic conditions is shown in Fig. 5. From this figure, it is evident that PHB production competes with light dependent hydrogen production. The ratio suddenly decreases after 60th h which is the starting point of PHB production. The reason for this competition is that PHB production and hydrogen production use the same reducing power that results from the metabolism of organic acids (Yamagishi, 1995). PHB accumulation in different growth media is summarized in Table 3. When these results are compared with the literature data it is seen that Alcaligenes latus produces the highest amounts of polyhydroxy alkonates (88% w/w PHA) (Steinbtichel and Ffichtenbusch, 1998). R. sphaeroides produces 24% (w/w) PHB by using acetate as carbon source (Gross et al., 1992). The important point is that PHB can be collected from the hydrogen producing photobioreactor that utilizes waste water from sugar refinery to increase the feasibility of the total system. The cells and their growth medium would also be screened for other possible by-products such as carotenoids, bacteriochlorophyll and ubiquinone.

References Abe, H., Doi, Y., 1992. Controlled release of lastet, an anticancer drug, from poly(3-hydroxybutyrate) microspheres containing acylclycerols. Macromolec. Rep. A29, 229-235. Anderson, A.J., Dawes, E.A., 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial poly hydroxy alkanolates. Microbiol. Rev. 54, 450-472. Ank, T., Gtindiiz, U., Yticel, M., Tiirker, L., Sedirofglu, V., Erofglu, I., 1996. Photoproduction of hydrogen by Rhodobacter sphaeroides O.U.00. Proceedings of the 1l th World Hydrogen Energy Conference, Vol. 3, Stuttgart, Germany, pp. 2417-2424. Biebl, H., Pfennig N., 1981. Isolation of member of the family Rhodosprillacaea. The Prokaryotes. Springer-Verlag, New

131

York, pp. 267-273. Bowker, R.R., 1981. Manual of Methods for General Bacteriology. American Society for Microbiology, Washington DC. Byrom, D., 1987. Polymer synthesis by microorganisms; technology and economics. Trend Biotechnol. 5, 246-250. Ero~lu, i., Asian, K., Gtindiiz, U., Yficel, M., Ttirker, L., 1997. Continuous hydrogen production by Rhodobacter sphaeroides OU 001. Paper presented at Biohydrogen 97 Int. Conference on Biological Hydrogen Production, Kona Hawaii, USA, pp. 143-149. Gross, A.R., Ulmer, H.W., Lenz, R.W., Tshudy, J.D., Udon, C.P., Brandt, H., Fiiller, R.C., 1992. Biodeuteration of poly ([3-hydroxybutyrate). Int. J. Biol. Macromol. 14, 3340. Kim, B.C., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K., Woo, S.I., 1994. Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol. Bioeng. 43, 892-898. Kitjama, Y., E1-Shishtawy, R., Ueno, Y., Otsuka, S., Miyake, J., Moritomo, M., 1997. Analysis of compensation point of light using Plane-Type photosynthetic bioreactor. Proceedings of the Int. Conference on Biological Hydrogen Production, Kona, HI, USA, pp. 359-367. Page, W.J., 1989. Production of poly-13-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources. Appl. Microbiol. Biotechnol. 31, 329-333. Sang, Yup Lee, 1996. Bacterial Polyhydroxyalkanoates. Biotechnol. Bioeng. 49, 1-14. Sasikala, K., Ramana, C.H.V., Roa, P.R., 1992. Photoproduction of hydrogen from the waste water of a distillary by Rhodobacter sphaeroides OU 001. Int. J. Hydrogen Energy 17, 23-27. Steinbtichel, A., Fiichtenbusch, B., 1998. Bacterial and other biological systems for polyester production. TIBTECH 16, 419-426. Yamagishi, K., 1995. Interim Evaluation Report of Development of Environmentally Friendly Technology for the Production of Hydrogen. New Energy and Industrial Technology Development Organization, Japan. Yetis, M., Gtindiiz, U., Ero~lu, i., Yficel, M., Tiirker, L., 1998. Photoproduction of Hydrogen from waste water of a sugar refinery by Rhodobacter sphaeroides OU 001. Paper to be presented in Marine Bioprocess Engineering, Noordwijkerhout, The Netherlands.

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Bio ELSEVIER

ology

Journal of Biotechnology 70 (1999) 133-153

Cell cultures from marine invertebrates" obstacles, new approaches and recent improvements Baruch Rinkevich * National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel

Received 13 October 1998; received in revised form 19 November 1998; accepted 22 December 1998

Abstract

The establishment of cell lines from marine invertebrates has been encountered with obstacles. Contrary to insects and arachnids where the development of a variety of cell lines has become routine, there is no single established cell line from marine invertebrates. This review examines the activity in the field of marine invertebrate cell cultures within the last decade (1988-1998). During this period, attempts (90 peer reviewed studies in addition to many other abstracts, chapters in books, symposia presentations and reports) were limited to a few species within only six phyla (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, Urochordata: in addition to freshwater/terrestrial annelids and platyhelminths). These studies which are summarized here, on one hand indicated ubiquitous problems and on the other, unique characterizations to each phylum studied. Only one-third of the studies revealed cultures of 1 month or longer but most of these were long-term cultures found or suspiciously considered to be contaminated by other unicellular eukaryotic organisms, mainly by thraustochytrids. Three unique approaches/obstacles for marine invertebrate cell cultures (source of cell, cryopreservation and eukaryotic contaminants) are further discussed. The overall impact of recent improvements and developed protocols raises the suggestion for testing different, novel routes in the establishment of cell cultures from marine invertebrates. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Marine invertebrates; Cell lines; Platyhelminths

I. Introduction

Cells under in vitro conditions are used as exceptionally important tools in a variety of scientific disciplines including biological and medical sciences. In vitro applications may also be used as alternative tools for animal experimentation, for biotechnological applications and pathological in* Tel.: + 972-4-851-5202; fax: + 972-4-851-1911. E-mail address: [email protected] (B. Rinkevich)

vestigations. Invertebrates may be regarded as a major source for such applications. They comprise more than 95% of the animal species and, within them, marine invertebrates are thought to account for more than 30% of all animal species ( N a g a n u m a et al., 1994). The large group of marine invertebrates, which encompasses more than 20 different phyla, represent a rich source of cell and tissue types that significantly differ from one group to another. Many cell types from a variety of invertebrates possess extensive morpho-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00067-X

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B. Rinkevich /'Journal of Biotechnology 70 (1999) 133-153

genetic potentialities (multipotency, totipotency, including neoplasia; Rinkevich et al., 1994; Rosenfield et al., 1994; Rinkevich and Rabinowitz, 1997). This leads to high in vivo plasticity of shapes, structures, cell replacements, proliferation processes and cell lineage in different invertebrate taxa, sometimes differs significantly even between systematically related groups of organisms. Attempts to maintain and grow invertebrate cells in vitro were made quite early in the history of tissue culture, nearly 100 years ago (Gomot, 1971; Rannou, 1968, 1971). However, while there have been more than 200 cell lines established from insects and ticks (Mitsuhashi, 1989), all efforts to develop permanent and proliferative cell cultures from marine invertebrates have been unsuccessful (Rinkevich et al., 1994). Moreover, contrary to the considerable attention that has been given to the in vitro use of marine fish and plants, the literature points out to only limited activity directed towards marine invertebrate cell cultures (Rosenfield, 1993)--largely due to the fact that experimental failures are not suitable for publication in most scientific journals. Within recent years, there has been veritable activity of many scientists in marine invertebrate cell cultures/cell lines. This stems from the understanding that basically, all cells of different taxa within the kingdom Animalia are the same, having similar nutrient requirements, are controlled by the same developmental and physiologicalbiochemical pathways and are under the expression of identical genes. It is therefore plausible that techniques developed in one scientific discipline may be adapted (with some alterations) to other fields, so that the outcome will serve the new requirements. For example, mitogens that have important roles in cells derived from mammals have been found to alert similar activities in invertebrate cells in vitro (Peddie et al., 1995; Raftos and Cooper, 1996). Such approaches that allow creative novel designs for the development of cell cultures from marine invertebrates, gave new dimensions to this field, providing new results, ideas and improvements to this relatively stagnant field. The present communication aims in the summarizing of attempts for cell cultures from a

variety of marine invertebrates within the last l0 years (1988-1998). General trends in the results, common obstacles, the new approaches used and recent achievements are reviewed with an eye for formulating, if possible, a more ubiquitous framework for marine invertebrates cell culture technology.

2. Activity during the last decade During the last decade, attempts to develop cell cultures from marine invertebrates were limited to a few species within six phyla (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, Urochordata; a short account for freshwater/terrestrial annelids and platyhelminths is also included below due to the possible adaptation of their developed techniques for the related marine taxa as well) out of > 20 invertebrate phyla available (Fig. 1). Within each studied phylum, only a limited number of scientific groups and species were studied for cell cultures. For example, no polychaete cells were employed in the phylum Annelida, only few groups of decapods were studied in the crustaceans, no single marine species from the flat worms, no schyphozoans in the Cnidaria, cephalopods, polyplacophores and opistobranchs in the Mollusca, are only few for the remarkable outcome of low diversity in the attempts to use marine invertebrates for in vitro approaches. A total of 90 publications (Fig. 1, 1988-1998; excluding abstracts or manuscripts published in symposia or journals which were unavailable) are included in the detailed examination of the literature dealing with tissue cultures from marine invertebrates. Only 27 publications (30.0%) describe cases where cultures were maintained for more than 1 month in vitro. In four phyla (Cnidaria, Crustacea, Mollusca, Echinodermata) < 29.0% of the publications described long-term studies, whereas in the Porifera and Urochordata, longterm studies composed 36.4 and 45.5%, respectively, of all publications. In 18 of the long-term reports (refer to the section on unicellular eukaryotic contaminations), it was confirmed or suspected that the developed cultures were

B. Rinkevich / Journal of Biotechnology 70 (1999) 133-153

contaminated by other unrelated cells. There is no single valid marine invertebrate cell line developed from any of the studies published in the last 10 years of research. This outcome further reveals the problematic configuration of this scientific discipline: many attempts (the above 90 publications, Fig. 1, > 50 additional abstracts that were never published in peer reviewed journals; other unpublished studies that failed to mature to the form of a scientific communication) but little success. Below are short accounts for the development of in vitro studies in the above-mentioned invertebrate phyla:

135

organisms, it is plausible to predict that sponges will be one of the most amenable sources for invertebrate cell cultures. However, all attempts to develop long-term, continuous cell lines from sponges (including the studies published during the last decade: Klautau et al., 1993, 1994; Pomponi and Willoughby, 1994; Imsiecke et al., 1995; Ilan et al., 1996; Pomponi et al., 1997; Leys, 1997, 1998; Rinkevich et al., 1998) have not been as rewarding and the methodology for establishing cell lines is still in its preliminary stages. Even the establishment of long-term cell cultures from several marine sponges (Klautau et al., 1993, 1994) has been found to be erroneous. Cells that were isolated and maintained in culture for years were identified as protozoan cells (Custodio et al., 1995; and unpublished data). This resulted in a suggestion to cultivate sponge fragments in bioreactors rather than sponge cell cultures, for biotechnological production of sponge metabolites (Osinga et al., 1988). Although simply by their structure, sponges exhibit a variety of obstacles for the development of cell culture, two of them unique to this phylum. (1) Many species (especially within the hexactinellid sponges) are syncytial animals, consisting of a

2.1. Porifera (marine and freshwater sponges) Sponge cell culture has been proposed as a model system for the study of a variety of cellular aspects. During the last few years, it was also evident that in vitro cultures of marine sponge cells may address the need for the commercial supply of novel biologically active chemical compounds with high pharmacological potential. Since sponges are considered to be the most simple metazoan group, and are frequently placed at the base of the evolutionary tree for multicellular 40 TOTAL PUBLICATIONS r - - ] LONG TERM CULTURES Z

30

r,..) ,..1

~ o

20

0Z 10

PORIFERA

CNIDAPdA

CRUSTACEA

__L

MOLLUSCA

ECHINODERMATA

UROCHORDATA

PHYLUM

Fig. 1. Marine invertebrate cell cultures: Total number of publications (closed bars; sorted from ASFA, MEDLINE and BIOSIS) and total number of publications describing long-term cell cultures ( > 1 month) open bars; during the last decade (1988-1998). Six marine invertebrate phyla are analyzed. Abstracts, some chapters in symposia/internal reports and publications in journals that are unavailable, are not included.

136

B. Rinkevich ,' Journal of Biotechnology 70 (I 999) 133-153

single multinucleated giant cell, the trabecular syncytium that is connected via perforated plugged junctions to cellular components. Under in vitro conditions, a dissociated tissue is aggregated by fusing the membranes to form a giant multinucleatal syncytium (Leys, 1998). Therefore, the basic cytological organization of this group differs substantially from that of other multicellular organisms including other sponge groups, raising questions regarding the amenability for hexactinellid sponges for cell cultures. (2) Unlike other metazoans, there are no areas of a sponge from which sterile, primary cultures can be obtained (Pomponi and Willoughby, 1994). A further complication of this situation is the fact that many sponges host endosymbiotic intracellular microorganisms. The use of antibiotics for the production of axenic conditions may be harmful or may have inhibitive effects on sponge cells as well. Without antibiotics, cultures are contaminated within 1-3 days (Pomponi and Willoughby, 1994). While there is yet no finite or continuous cell lines from sponges, primary cultures have been obtained in vitro for months (25 weeks for adultderived cultures, Ilan et al., 1996; 41 weeks for embryo-derived primary cultures, Rinkevich et al., 1998). Recent studies have revealed the positive effects of a variety of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, lectins (wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA), phytohemagglutinin (PHA), concanavalin A (ConA)), exogenous cAMP, and other additives and substrates on cell division and behavior in primary cultures (Gaino and Magnino, 1994, 1996; Pomponi and Willoughby, 1994; Pomponi et al., 1997; Rinkevich et al., 1998). In the same manner, improved conditions for the shipment of living material (whole portions of adult sponges at 4~ Ilan et al., 1996; cryopreserved cells, Pomponi et al., 1997) as well as the use of 'preferable parts' (sponge fragments without the cortex layer, Ilan et al., 1996; embryos, Rinkevich et al., 1998) have progressed survivorship and cell divisions in primary cultures. The best dissociation protocols used are mechanical (Ilan et al., 1996: Rinkevich et al., 1998) and the combination of

mechanical/chemical dissociations (Pomponi and Willoughby, 1994; Pomponi et al., 1997). Several sponge cell culture media are used but it is evident that culture optimization is only in its primary stages. Improvement of culture conditions should be addressed by a number of experimentation procedures to evaluate and reveal better in vitro conditions specific to sponge cells.

2.2. Cnidaria (corals, sea anemones, hydrozoans) The Cnidaria, one of the most ancestral invertebrate phyla, is the lowest metazoan group possessing an organized body structure. The body plan of the cnidarians consists of only two cell layers, ectoderm and endoderm, with acellular intermediate gelatinous matrix, the mesoglea. The simplicity of cnidarians' structure and their relatively limited number of cell types provides an excellent opportunity for studying biological processes in vitro. Moreover, the symbiotic relationships between many cnidarians and unicellular algae (zooxanthellae, zoochlorella) exhibit an additional scientific challenge and several recent studies were engaged with these symbioses in vitro (i.e. Gates and Muscatine, 1992; Gates et al., 1992; Apte et al., 1996) and with dissociation protocols developed for cnidarian tissues (Gates and Muscatine, 1992: Gates et al., 1992; Greber et al., 1992; Frank et al., 1994; Weber 1995). However, in only one study (Frank et al., 1994), have long-term cell cultures been developed. In this study, continuous cell cultures from ten taxa of sedentary colonial marine cnidarians So'lophora pistillata, Porites lutea, Favia favus (Anthozoa, Madeporaria), Parerythropodium fulrum fidt'um, Dendronephthya hemprichi, Nephthya sp., Heteroxenia fitscescence (Anthozoa, Alcyonacea), Clathraria rubrinoides, Plexaura sp. (Anthozoa, Gorgonacea) and Millepora dichotoma (Hydrozoa) were established in vitro. Primary cultures of various cell types and sizes (5-20 ~tm) were obtained from colony fragments and/or planula larvae using three dissociation approaches: a mechanical approach, chemical approach and a novel approach, spontaneous dissociation (see below). Cells were cultured in a modified Leibowitz L15 medium, with 5-10%

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heat-inactivated fetal bovine serum, diluted in seawater. Cell proliferation was observed in primary cultures within 7-20 days following dissociation. Several cell cultures were developed, maintained for approximately 1 year, subcloned and cryopreserved. However, frozen cell materials, when thawed, were dominated by eukaryotic unicellular organisms from the phylum Labyrinthulomycota (unpublished; see below). These organisms took over the cultures competitively, excluding the original cell types. Additional efforts to establish secondary cell cultures from corals (unpubl.) were not as rewarding and in most cases, cells remained in an arrested form for up to 1 year with no signs of proliferation. One of the interesting points, not yet explored in the phylum Cnidaria, is the topic of cell-substrate interactions. In some of the few investigations, a species-specificity has been revealed. Jellyfish tissues of striated muscle and endoderm adhered and spread on mesoglea in a species-specific manner (Schmid and Bally, 1988). The importance of the mesoglea as the preferable substratum was also documented for primary culture of neurons from a hydrozoan jellyfish (Przysiezniak and Spencer, 1989). In addition, a recent study (Frank and Rinkevich, in press) revealed that dissociated cells from a variety of cnidarians (including anthozoans and hydrozoans) adhered, spread and penetrated into the jellyfish mesoglea cubes while cells from other organisms, such as tunicates, responded to the same mesoglea as an intact substrate (unpubl). On the other hand, one of the most critical factors in Cnidaria cell culture is the dissociation method employed. Dissociation protocols have a significant impact on the survivorship of cells, tissue culture conditions and prolonged cell cultures (Frank et al., 1994) and several studies during the last decade have tested this approach (Schmid and Bally, 1988; Gates and Muscatine, 1992; Gates et al., 1992; Greber et al., 1992; Frank et al., 1994; Weber, 1995; Apte et al., 1996).

2.3. Platyhelmintes and Annelida Earlier studies on annelid neuron cultures (especially in the leech genus Hirudo) have already

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been reviewed (Townsel and Thomas, 1987). Cell cultures from endoparasitic worms, with relation to human health, are also of great interest (reviewed in Toledo et al., 1997; Bayne, 1998). With regard to oligochaetes, while earthworms of the family Lumbricidae are of great interest in a variety of scientific disciplines including invertebrate immunology, environmental pollution and evolution, almost nothing has been added to our knowledge on annelid tissue cultures during the last three decades (following the reviews of Gomot, 1971; Rannou 1971). During the past 10 years, only a single report (Battaglia and Davoli, 1997) has studied in vitro conditions for Eisenia foetida cells. Earthworm cultures were maintained in vitro for > 1 year, possessing mainly three different cell types. One of the cell types, the non-adherent cells which are approximately 20 lam in diameter, appeared to be similar in morphology to the earthworm's coelomocytes. No cell proliferation has been recorded. Relatively longterm cultures of freshwater planarian neoblasts ( > 14 weeks) has also been recorded (Shiirmann and Peter, 1988).

2.4. Crustacea (crayfish, lobsters, shrimps) In vitro techniques in crustacean biology have become important and sometimes vital tools for the study of crustacean endocrinology and diseases of edible species. The in vitro study of endocrinology, especially in aspects of the endocrine regulation of molting (Brody and Chang, 1989; Chang, 1997), provides an applied tool for the characterization of this complex system. The intensive agricultural practices, which are associated with worldwide viral infections, represent an emerging problem that hampers culture of several commercially valuable species. Crustacean in vitro cell cultures that can support in vitro viruses and other parasitic replications are an needed urgently to study viral infections (Rosenthal and Diamant, 1990; Nadala et al., 1993; Hsu et al., 1995; Tong and Miao, 1996; Loh et al., 1997; Tapay et al., 1997). These are the two main reasons why cell cultures of edible crustaceans (also freshwater species) have gained new attention during the last decade (Brody and Chang, 1989; Chang and

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Brody, 1989; Chen and Kou, 1989; Chen et al., 1989; Cook et al., 1989; Itami et al., 1989; Hu, 1990; Ke et al., 1990; Rosenthal and Diamant, 1990; Ellender et al., 1992; Luedman and Lightner, 1992; Najafabadi et al., 1992; Loh, 1993; Nadala et al., 1993; Cancre et al., 1995; Ghosh et al., 1995; Hsu et al., 1995; Lu et al., 1995; Tapay et al., 1995, 1997; Frerichs, 1996; Tong and Miao, 1996; Toullec et al., 1996; Chang, 1997; Loh et al., 1997). As a result, much focus was placed on the establishment of cell lines derived from various tissues including epidermal origin (branchial walls, limb), hematopoietic and lymphoid tissues, gonads (ovary, testicular cells) nerve cells and hepatocytes. However, most studies concentrated on short culture periods and only few attempted to raise long-lasting cultures and cell lines ( < 3 weeks, Rosenthal and Diamant, 1990; 10 weeks to several months, Toullec et al., 1996; 3-11 months, Brody and Chang, 1989; > 90 passage, Hsu et al., 1995). Neither one of the above attempts has successfully matured into developing a cell line. The study on cell cultures from crustaceans is hampered by the fact that almost each laboratory has chosen a different species and other organs/ tissues for in vitro studies. It is very difficult to compare the studies and to draw a line of experimental steps for further consideration. For example, even the same laboratory may develop several dissociation protocols for different organs tested. While enzymatic dissociation was not beneficial for hepatopancreas (mechanical and explant spontaneous dissociation were superior), it was the preferable technical approach for epidermal tissues of penaeid shrimps (Toullec et al., 1996). In the same way, shrimp and Palaemon tissues responded differently to different media tested (Nadala et al., 1993; Cancre et al., 1995; Tong and Miao, 1996; Toullec et al., 1996) or to different growth factors (Cancre et al., 1995; Hsu et al., 1995). Cell culture optimization in crustacean studies is only in its primary steps and even within the same laboratories, long-term utilization and development of primary culture techniques was not the main topic for research, and therefore has not been advanced significantly (for example, comparison of methodology between Brody and Chang (1989) and Chang (1997)).

2.5. Mollusca (mussels, oysters, clams, snails) Mollusc cell culture is probably the most intensively studied group of marine invertebrates. Mollusc cell lines have been used in the study of neurobiology (Tamse et al., 1995; earlier studies reviewed in Townsel and Thomas, 1987), evaluation of xenobiotic effects on the cellular level and monitoring deleterious effects of pollution (Auffret and Oubella, 1997), the study of the immune system, especially of molluscs that are intermediate hosts of human parasites (Laursen et al., 1997; Davids and Yoshino, 1998) or those exhibiting a variety of tumors, including sarcomas and hematopoietic neoplasia (reviewed in Rosenfield, 1993; Rosenfield et al., 1994), the study for physiological/biochemical aspects of calcification in pearl oysters (Machii, 1988; Awaji, 1991, 1997; Samata et al., 1994), but above all, on a variety of aspects (biochemistry, mechanisms controlling development, metamorphosis, growth, pathogenesis, etc.) on molluscs which are of major commercial importance such as mussels, oysters, clams, snails, abalone (Ellis et al., 1985; Ellis and Bishop, 1989; Machii and Wada, 1989; Abbot, 1990, Kumazawa et al., 1990; Boulo et al., 1991; Holden and Patterson, 1991; Noel et al., 1991; Odintsova and Khomenko, 1991; Auzoux et al., 1993; Cornet, 1993, 1995; Odintsova et al., 1993, 1994; Wen et al., 1993; Domart-Coulon et al., 1994; Naganuma et al., 1994; Takeuchi et al., 1994, 1995a,b; Odintsova and Tsal, 1995; Renault et al., 1995; Kleinschuster et al., 1996; Lebel et al., 1996; Walker et al., 1996). Disease epidemics in edible molluscs have been of great interest and importance for more than three decades and the use of cell culture for studying mollusc pathogenesis has been of high priority (literature cited in Li et al., 1966; Brewster and Nicholson, 1979, Ellis et al., 1985). The great interest in cell culture from molluscs during the 1970s has revealed two major important outcomes: (1) oyster amebocytes were successfully maintained in vitro for long periods of up to 6 months (Brewster and Nicholson, 1979); (2) the most significant achievement, the development and characterization of the first and the only molluscan cell line from embryos of the freshwa-

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ter snail Biomphalaria glabrata (Hansen, 1976). It is therefore of great disappointment that not much has been achieved regarding the development of cell lines from marine molluscs and, on the other hand, that so few researchers used the available single cell line for their studies (for example: Yoshino and Laursen, 1995; Laursen et al., 1997). Studies of mantle tissue culture from the pearl oyster are similarly disappointing. Pearl oyster cell culture started almost 25 years ago (Machii, 1974) with tissue explants. In this culture system, secretion of an organic substance was followed in vitro. A year later, the same laboratory documented mitoses in cell cultures from mantle tissue. Even with the importance of this industry of pearl oysters, recent studies (Awaji, 1991, 1997; and literature therein) do not show any significant improvement as compared to the 1970s cell culture studies. It is therefore even more evident that outside insects and ticks, primary cell or organ culture maintenance has rarely given subcultures of any cell type and almost no finite or continuous cell lines (summarized in Rosenfield, 1993). During the last 10 years, an impressive variety of organs and cells from molluscs have been cultured, including epithelial cells from embryos, gills and mantles (Machii, 1988; Awaji, 1991, 1997; Auzoux et al., 1993; Odintsova et al., 1994; Samata et al., 1994; Takeuchi et al., 1994; Cornet, 1995), neurons (Berdan et al., 1990; Tamse et al., 1995, and literature therein), digestive glands (Odintsova et al., 1994), muscles including heart cells (Ellis and Bishop, 1989; Odintsova and Khomenko, 1991; Wen et al., 1993; Domart-Coulon et al., 1994; Naganuma et al., 1994; Odintsova et al., 1994; Odintsova and Tsal, 1995; Takeuchi et al., 1995a,b; Kleinschuster et al., 1996) and the hematopoietic systems (Kumazawa et al., 1990; Boulo et al., 1991; Noel et al., 1991; Lebel et al., 1996; Auffret and Oubella, 1997; Davids and Yoshino, 1998). The results, however, are not so rewarding. For example, when focusing on Perkinsus disease, which causes mass mortality in oyster and clam cultures, Auzoux et al. (1993) planned to establish a suitable cell culture system for clam's gill for the purpose of studying host-pathogen relationships

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and to screen in vitro anti-parasitic candidate agents. However, only relatively short (approximately 1 month) cultures have been established. The aim of culturing nudibranch neurons for the study of cellular physiology and behavior have revealed only short functional cultures. In this system, signs of cellular deterioration appeared as soon as days 10-12 (Tamse et al., 1995). Wen et al. (1993) clearly stated that a critical review of the efforts to culture adult tissues, especially heart cells from marine molluscs, resulted in difficulties which hampered further development. In the same manner, even neoplastic tissues from molluscs were not amenable for long, in vitro cultures (Noel et al., 1991; Rosenfield et al., 1994; Walker et al., 1996). Some of the studies that documented long-term cultivation for a variety of mollusc tissues for longer periods of times (Ellis and Bishop, 1989; Odintsova and Khomenko, 1991; Wen et al., 1993; Domart-Coulon et al., 1994; Odintsova et al., 1994; Takeuchi et al., 1995a,b) are also probably subject to contamination by thraustochytrid species (Ellis and Bishop, 1989; see below). It may therefore be concluded that despite great efforts to establish marine mollusc cell lines, this field is still in its preliminary stages. In several cases, however, the current state-ofthe-art regarding in vitro conditions was good enough for the specific questions asked. For example, hemocyte aggregation in the oyster Crassostrea gigas has been used as a sensitive bioassay for in vitro measurement of the effect of xenobiotics (Auffret and Oubella, 1997), and oyster cell cultures have been used to show that the American oyster is unable to synthesize sterols (Holden and Patterson, 1991). In vitro spreading and motility of circulating phagocytic cells (hemocytes) of snails was tested for the hypothesis that this behavior is mediated through RGD-binding integrin-like surface receptors (Davids and Yoshino, 1998) and mollusc cell cultures have been explored for synthesis of adhesive proteins (Abbot, 1990). In the same way, a functional study of burst respiratory activity has been successfully analyzed in vitro on scallop hemocytes as well as the analysis of interactions with protozoan and prokaryotic pathogens (Boulo et al., 1991). These in vitro model systems have applied the

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accumulated knowledge of in vitro maintenance of mollusc cells, providing first-hand evidence for the potential of in vitro cell cultures in a variety of scientific needs. General in vitro conditions have been recently tested for further improvements, including cellular cryopreservation (Odintsova and Tsal, 1995), media supplementation (Wen et al., 1993; DomartCoulon et al., 1994; Lebel et al., 1996), substrate of choice (Odintsova et al., 1994) and the effects of dissociation protocols (Awaji, 1991; Takeuchi et al., 1995a,b). Therefore, all facets of in vitro conditions are being studied in order to obtain greater knowledge of mollusc cell cultures. There are still difficulties common to primary molluscan cultures, such as tissue sterilization, cell proliferation, cellular purification/characterization and thraustochytrid contamination (part of this list is summarized in Awaji (1997)). The above studies embraced the four culture situations uniquely characteristic of a variety marine molluscs: giant neurons routinely used in neurobiology (Tamse et al., 1995), pearl formation (Awaji 1991, 1997 and literature therein; Samata et al., 1994), mollusc neoplasia, frequently detected during epidemiological surveys of bivalve mollusc stocks (Noel et al., 1991; Rosenfield et al., 1994, and literature therein) and the appearance of many snails as intermediate hosts for human parasites (such as the blood fluke Schistosoma mansoni; Davids and Yoshino, 1998). These situations are seldom referred to, or are not applicable to other invertebrate phyla. They provide experimental in vitro challenges uniquely applied to the phylum Mollusca. 2. 6. Echinodermata Primary cultures of echinoderm cells during the last 10 years concentrated on a variety of cellular differentiation aspects. Studies were performed on sea urchin cells (Benson et al., 1990; Odintsova et al., 1994; Ermak and Odintsova, 1996), primary starfish cultures (Kaneko et al., 1995, 1997) and cultures from sea cucumbers (Blinova et al., 1993; Odintsova et al., 1994). Due to the questions asked, most studies concentrated on embryonic cells (Benson et al., 1990; Odintsova et al., 1994;

Kaneko et al., 1995, 1997; Ermak and Odintsova, 1996) or germ line cells (Poccia, 1988) and for relatively short periods of a few days to up to 40 days. There are two characteristics of primary echinoderm cultures which were not recorded as important in most other invertebrate cell cultures. (1) The impact of substrates on cellular differentiation. For example, two different cell types (epithelial or mesenchymal) may be developed 1 week after initiation of cell cultures from sea urchin larvae at the gastrula stage (Ermak and Odintsova, 1996). Epithelial cells predominated wells coated with polylysine (with a high level of [3H]thymidine incorporation), while the addition of fibronectin or oncoprecipitin A (oncoA, a specific glycoprotein isolated from an ascidian) resuited in syncytia of different shapes and sizes and conglomerates typical of primary mesenchyme cells were formed. (2) As shown years ago (Bertheussen and Seljelid, 1978), a variety of cell types in echinoderms tend to form aggregates and/or monolayer syncytia. As a result of the limited number of studies aimed at the production of continuous cell culture from echinoderms, there are almost no data for the response of echinoderm cells in long-term conditions. Dissociation protocols, media, general conditions and organs to be used as a source for culture requirements to be defined in future experimentation. 2. 7. Urochordata (solitary, colonial tunicates) Tunicates, the most primitive group of the phylum Chordata, may reveal critical answers in the phylogeny of several biological phenomena such as immunology, developmental biology, genetics, cellular biology and more. Following that, the importance of urochordates as model organisms has already been established in a variety of disciplines, reflecting as well the field of cell culture. During the past 10 years, most studies have attempted to develop cell cultures from solitary and colonial tunicate blood cells (Raftos et al., 1990, 1991; Raftos and Cooper, 1991, 1996; Rinkevich and Rabinowitz, 1993; Sawada et al., 1994; Peddie et al., 1995; Rinkevich et al., 1996) mainly

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due to their importance in allorecognition and other immune responses. Few studies focused on epithelial cell cultures (Kawamura and Fujiwara, 1995; Rinkevich and Rabinowitz, 1997) or on embryo-derived cell cultures (Rinkevich and Rabinowitz, 1994). Some of the studies evaluated short-term experimental sets. However, relatively more frequent than in other phyla studied, longterm experiments ( > 2 months; Raftos et al., 1990; Rinkevich and Rabinowitz, 1993, 1994; Sawada et al., 1994; Kawamura and Fujiwara, 1995) have resulted in cell cultures from Botryllus schlosseri (Rinkevich and Rabinowitz, 1994) and Polyandrocarpa misakiensis (Kawamura and Fujiwara, 1995). At least in the B. schlosseri cultures, it has been evident that opportunistic organisms contaminated the cultures (thraustochytrids, see below). The in vitro development of long-term primary cultures from tunicate blood cells is faced with a significant challenge. Tunicate hemocytes have a rapid turnover and in vivo circulatory hemocytes survive for only several weeks (Raftos et al., 1990). Keeping this fact in mind, several studies used explant cultures of 'hematopoietic organs' (such as pharyngeal sinuses of solitary ascidians) as target sources for cell-line development (Raftos et al., 1990, 1991; Raftos and Cooper, 1991; Sawada et al., 1994). Where no hematopoiesis centers are known, blood cells were collected directly from blood vessels for in vitro approaches (Rinkevich and Rabinowitz, 1993; Peddie et al., 1995). In the first approach, resident hemocytes developed, multiplied, matured and migrated outside of the explants for more than 2 months, with clear indications that proliferation renewed the pool of hemocytes within the explant tissues (Raftos et al., 1990). Tunicate blood cells are one of the major cell types used in urochordate tissue cultures where several factors (also of mammalian systems) are found to influence cell proliferation and activation (reviewed in Peddie et al., 1995; Raftos and Cooper, 1996). Activities of tunicate defense-related hemocytes were found to be modulated by regulatory signals using mammalian cytokines and plant lectins. A detailed study on this phenomenon has resulted in the characterization of

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proteins (designated as tunlLl~ and tunlL1 ]3; reviewed in Raftos and Cooper, 1996). These proteins have a variety of cellular effects in vitro other than mitogenesis. They increase in vitro phagocytosis and opsonization, working as extremely potent materials (being active at concentrations of as little as 150 ng/ml) and they activate hemocyte chemotaxis and metabolism (activation of incorporated amino acids and glucose). The use of different dissociation protocols (Rinkevich and Rabinowitz, 1994), a variety of mitogens (Rinkevich and Rabinowitz, 1993; Peddie et al., 1995), osmolarity (Kawamura and Fujiwara, 1995) and different media (Raftos et al., 1990) may have significant effects on cell viability and cellular proliferation. In vitro conditions for tunicate cells are still far from optimal. One example is the capacity for proliferation during regeneration of isolate Botrylloides ampullae (Rinkevich et al., 1996). While any peripheral isolated fragment of blood vessel can regenerate in vivo (within filtered seawater) into whole newly formed zooids in less than 2 weeks, in vitro conditions lead to a complete failure of this unique regeneration from totipotent blood cells. This example reveals the potentiality of the system and the lack of amenability of currently developed protocols.

3. Marine invertebrates: unique approaches/obstacles The lack of significant progress of marine invertebrate cell cultures may be related to an inappropriate comparison of invertebrate cell culture requirements with the culture conditions of vertebrate cell lines or may be due to special or unique requirements needed for invertebrate cell cultures (Rinkevich et al., 1994; Bayne, 1998). The brief discussion above, dealing with the scientific activity during the last decade, further reveals unique characteristics of cell cultures for even different invertebrate phyla. The syncytial cells of hexactinellid sponges (Leys, 1998) and the cells from sponge species harboring endosymbiotic intracellular microorganisms (Pomponi and Willoughby, 1994) clearly require different in vitro conditions

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than, for example, mollusc neoplastic cells (Noel et al., 1991; Rosenfield et al., 1994) or urochordate blood cells (Rinkevich and Rabinowitz, 1993). The hypothesis that culture requirements for vertebrates and invertebrates are not comparable has been discussed in detail by Goodwin (1991), who advocated that invertebrate cell surface (lipidic and extracellular matrix) specificities differ significantly from those of the vertebrates. At the same time, significant genetic homologies were revealed between vertebrate and invertebrate bioactive peptides (Goodwin, 1991) a fact which indicates the opposite conclusion. On the other hand, it is clear that invertebrate cell cultures require unique approaches and bear obstacles not recorded in vertebrate cell cultures. Some of these points (aseptic conditions, media supplements and substrate cell types and classification; Rinkevich et al., 1994) and others (selection of species, selection of basic media and additives, the merit of feeder layers, and more; Bayne, 1998) were reviewed recently. A number of questions may be addressed when summarizing efforts of the last decade for invertebrate cell cultures. These questions include the investigation for unique approaches and obstacles which are common, or even ubiquitous, to only marine invertebrate cell cultures. In general, we do not even know why marine invertebrate cells never propagate in vitro. One of the striking examples is the survival budding processes in the colonial urochordate Botrylloides (Rinkevich et al., 1996). In this group of organisms, any separated minute fragment of peripheral blood vessels with 100-300 blood cells gives rise within a few weeks to a fully organized organism. However, all efforts to induce regeneration of isolated ampullae in vitro have failed (Rinkevich et al., 1996). Are blocks to propagation inadvertently introduced in procedure protocols or are they a natural part of in vivo versus in vitro conditions? There are several topics, however, that have emerged during the last decade, providing us with a new look, ubiquitous to marine invertebrate in vitro approaches.

3.1. Cell sources for better in vitro results

While all attempts to develop long-term, continuous cell cultures from marine invertebrates have not been rewarding and this field is still in its preliminary stages, there is one promising approach which has proven to be valid in several studies: the target organ/cell source for in vitro studies. Cells for in vitro studies were obtained from invertebrate epithelia (gills, mantle, pharynx, buds, outer epithelium), muscles (heart, a variety of other muscles), neurons, blood/hemolymph, internal organs (glands, hepatopancreas), gonads and others. Two of the most promising approaches are the amenability of larval/embryonic cells and the use of organ culture/spontaneous dissociation protocols. The use of larval and embryonic cells in vitro has been employed in a variety of freshwater and marine invertebrates, including sponges (Imsiecke et al., 1995; Rinkevich et al., 1998), crustaceans (Frerichs, 1996; Tong and Miao, 1996; Toullec et al., 1996), molluscs (Hansen, 1976; Brewster and Nicholson, 1979; Ellis et al., 1985; Ellis and Bishop, 1989; Machii and Wada, 1989; Odintsova and Khomenko, 1991; Odintsova et al., 1994; Naganuma et al., 1994; Odintsova and Tsal, 1995; Takeuchi et al., 1995a,b), cnidarians (Schmid, 1974; Frank et al., 1994), echinoderms (Benson et al., 1990; Odintsova et al., 1994; Kaneko et al., 1995; Ermak and Odintsova, 1996) and urochordates (Rinkevich and Rabinowitz, 1994; Kaneko et al., 1997). In the invertebrates, embryonic cell lines, which are restricted to insects (for example, Stiles et al., 1992) showed their amenability as an excellent tool for a variety of approaches and scientific questions. This source of cells is also less vulnerable to contamination from bacteria, yeast and mold (Frank et al., 1994; Rinkevich et al., 1998). This is of further importance because available tissue sources from marine invertebrates, particularly those that contain intracellular micro-organisms and crypto-organisms cannot be completely sterilized (Rinkevich et al., 1994). Moreover, when compared to adult primary cultures, embryonal cultures survived longer (Rinkevich and Rabinowitz, 1994; Rinkevich et al., 1998) and in many cases, consistently yielded

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cultures that were capable of at least limited growth in vitro (Ellis and Bishop, 1989). However, while cell lineages of gastropod embryos, for example, are well characterized (Refs. in Naganuma et al., 1994), this is not the case for other marine invertebrates such as sponges, cnidarians or even protochordates. This fact may provide an obstacle in characterization of cellular components developed in vitro from dissociated larvae/ embryos. On the other hand, larvae/embryos may provide large numbers of synchronously developing cell lineages and posses cell populations with high mitotic indices which are better candidates for primary and continuous cultures than any other organ or tissue. Organ cultures and spontaneously dissociated tissue fragments are two approaches which employ similar protocols for two different purposes: long-term cultivation of tissue fragments which consist of foci for cell proliferation that migrate from the explants into the medium during culture, and short-term cultivation of tissues that dissociate spontaneously (without the employment of any chemical or enzymatic treatment) and provide a wide scale of cell types for in vitro applications. Organ culture for sustained viability of proliferation of cells is mainly carried out on solitary tunicates where cultured pharyngeal fragment sites of hematopoiesis are used (Raftos et al., 1990; Sawada et al., 1994; Raftos and Cooper, 1996). In this system, pharyngeal tissues remained viable and proliferated. This maintained the pool of hemocytes within the explants and facilitated the migration of hemocytes from explants into the culture medium (Raftos et al., 1990). The same methodology has been employed for establishing primary epithelial cultures from tunicate buds (Kawamura and Fujiwara, 1995; Rinkevich and Rabinowitz, 1997). Cells spread out from the epithelial explant and proliferated (at least for a short period of 1-2 weeks) on the substrate. Spontaneous dissociation is based on inserting tissue fragments or whole larvae into tissue culture medium, where they are dissociated without employing any chemical/enzymatic or further mechanical treatment. This was successfully performed on earthworm and platyhelminth cultures (Battaglia and Davoli, 1997; Toledo et al., 1997),

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shrimp cell cultures (Nadala et al., 1993; Tong and Miao, 1996; Toullec et al., 1996), bivalves (Auzoux et al., 1993; Wen et al., 1993; Samata et al., 1994) and cnidarian larvae (Frank et al., 1994). In two cases (Frank et al., 1994; Toullec et al., 1996) cultures of cells from explants were superior to cultures obtained from dissociated tissues and provided viable cells, sometimes even more cell types. This methodology may be explored further to find additional improvements which will reduce stress to cells during tissue dissociation.

3.2. Cryopreservation Cryopreservation has been widely used for long-term conservation of a variety of vertebrate cells, cell lines and insect cell cultures (Hink, 1979). Cryopreserved cells may provide a yearround supply of various cell types with similar qualifications and the same source for long-term comparative experimental studies. The establishing of the cryopreservation methodology is essential for holding and developing cell cultures and cell lines (Saxena et al., 1995). The cryopreservation methodology has been tested in marine invertebrate cell and tissue cultures as of 1992, by employing studies on two potentially different sources: cells already in vitro conditions and tissues amenable for future dissociation. Ellender et al. (1992) tried to store shrimp hemocytes at - 7 0 ~ for 2 months (10% DMSO (dimethylsulfoxide) solution). Poor cellular recovery ( < 26%) was recorded after vial thawing. On the other hand, Odintsova and Tsal (1995) have shown that cryopreservation of primary cell cultures of bivalves is not only a feasible goal (cell viability percentages after thawing the frozen materials were almost the same as in freshly prepared primary cultures) but that the use of 5-10% DMSO is favorable over 10% glycerol as cryoprotectant. No difference in viability was recorded after thawing primary cnidarian cell cultures (Frank et al., 1994) and embryo derived tunicate cells (Rinkevich and Rabinowitz, 1994) frozen in either 10% glycerol or 20% DMSO. Both protocols yielded high rates of cell viability. In the same

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manner Pomponi et al. (1997), routinely used cryopreserved sponge archaeocytes (15% DMSO) as a major source for the purpose of establishing primary cultures, monitoring cultures for production of bioactive metabolites and for microtiter plate assays. The above experiments, carried out on representative species from five phyla (Porifera, Cnidaria, Arthropoda, Mollusca, Urochordata), clearly indicate the feasibility of cryopreservation technology for future demands of cell supplies. Two studies also examined the feasibility of cryopreserved whole embryos from marine invertebrates for future studies. This was performed on bivalve trochophores and veligers (Odintsova and Tsal, 1995), and on sponge parenchymella larva (Rinkevich et al., 1998). With bivalve larvae, the veligers showed high cell viability of > 90% (in either 10% DMSO or 10% glycerol; similar to the control, unfrozen cells), while only < 35% of the trochophore cells survived the freezing-thawing protocol. In the sponge system, immediately after dissociation, cell viability was similar in both treatments (70_+ 7 . 8 ~ n = 30 for thawed embryos; 77.0_+ 1 3 . 0 ~ n - 50 for freshly collected embryos; P > 0.05) as were cell yields and cell type distributions (Rinkevich et al., 1998). However, for the cryopreserved embryos, 196 out of 200 wells (98%) were contaminated by bacteria along a 39-day observational period (140 wells, 70% were septic within 1 week). In the controls, during a total of 122 out of 300 wells (40.7%; P < 0.05) were contaminated, mainly by bacteria and thraustochythrids (see below), but also by fungi, cyanobacteria and amoebae. Most cases of thraustochythrid contamination developed after 3 weeks and those with bacteria, within the first 2 weeks. The reviving protocol for cryopreserved sponge embryos, while not affecting initial cell viability, results in increased bacterial contamination compared to controls. Bacteria are likely to take advantage of the fragile conditions imposed on embryo cells during the freezing-thawing cycle. This approach, has the advantage of reproducibility, high rates of cell viability and a potentially continuous supply of embryos, and deserves, therefore, further consideration (Rinkevich et al., 1998).

3.3. Unicellular eukaryotic contamination The morphologies of invertebrate cells in vitro may differ from those in vivo (Rinkevich et al., 1994). The additional high plasticity of shapes and structures of cells from a specific animal (Gomot, 1971) and the insufficient information for the classification of cell types/lineages in most invertebrates add to the confusion of in vitro cell identification. This may result in the culturing of alien unicellular eukaryotic contaminants in vitro as 'genuine' cells originating from the animal under investigation. One such example was recorded during the attempts to establish long-term cell cultures from several sponge species (Klautau et al., 1993, 1994). Cells that were isolated and maintained in culture for long periods (years) were subsequently identified as the protozoan Neoparamoeba aestuarina (Custodio et al., 1995, and unpublished data). Many other types of amoebas were also recorded in primary cell cultures from Negombata (sponge) adults and embryo preparations (Blisko, 1998). A much more common type of in vitro contamination in many cultures of marine invertebrate cells is the appearance of thraustochytrids (Fig. 2a-d), common marine and freshwater heterotrophic protists, that feed as saprophores, as parasites or as bacterivores (Porter, 1990; Raghukumar, 1992). Although they are very common in coastal waters (up to 5.6 x 10 4 cells 1- 1; Naganuma et al., 1998) and on a variety of living marine organisms such as sponges, corals, hydroids, bivalves, octopus, squids, nudibranches, echinoids, diatoms, sea grass and more (Cousserans et al., 1974; Raghukumar, 1988; Porter, 1990; Raghukumar and Balasbramanian, 1991; Bower, 1995, and Refs. therein) their evolutionary relationships and taxonomy are still poorly understood (Porter, 1990; Cavalier-Smith et al., 1994). For example, they were characterized recently as neither protozoa nor fungi, but as heterotrophic heterokontchromists (Cavalier-Smith et al., 1994). The interest in thraustochytrids as possible major contaminants of invertebrate cultures was initiated by Ellis and co-worker's work on molluscs cell cultures (Ellis et al., 1985; Ellis and Bishop, 1989). These thraustochytrids were encountered in

B. Rinke~'ich .. Journal of Biotechnology 70 (1999) 133-153

145

Fig. 2. Unicelluar eukaryotic contaminations in marine invertebrate primary cultures: (a) thraustochytrids in primary sponge cell culture (Negombata sp.). Large and dividing lumps of small cells (arrowheads) are the typical appearance for these contaminants ( x 400); (b) phase contrast of thraustochytrids from Porites lutea (a hermatypic coral), characteristic by the network of connecting filipodia between the small size cells ( x 400); (c) possible thraustochytrid infection in a tunicate (Boto'llus schlosseri) primary epithelial cell culture. Epithelial cells and thraustochytrids are both characterized by extended filipodia ( x 400); (d) unspecified unicellular eukaryotic contaminant from tunicate cell culture, characterized by dividing cells in the periphery of the cell mass

( • 200). approximately 30% of oyster cultures and about 27% of clam cultures, and appeared in a variety of forms as rapidly dividing round cells, round cells with filopods forming a stellate pattern around the cells, cells connected by net-like ectoplasmic processes or, as spherical-to-ellipsoid cells. Recently, the appearance of a variety of thraustochytrid organisms has been confirmed in cell cultures from sponges (Ilan et al., 1996; Blisko, 1998), corals (Frank et al., 1994), oysters (Awaji, 1997) and tunicates (Rinkevich and Rabinowitz, 1993, 1994, 1997; Table l, Fig. 2a-d). Moreover, detailed examination of the literature on marine invertebrate cell cultures during the last decade, especially the studies that described highly prolif-

erating cultures (Table 1), reveal that thraustochytrid protists are very common in these cultures from sponges, cnidarians, crustaceans, molluscs, echinoderms and tunicates. Possibly, many reports on invertebrate cell cultures describe the development and maintenance of thraustochytrids rather than the original animal cultures, further revealing a poorer yield of cell cultures from marine invertebrates (Table 1). The above list of 21 confirmed and suspicious cases for thraustochytrid contamination in marine invertebrate cell cultures (Table 1) points to the importance of these contaminants as an obstacle in the development of cell lines from a variety of marine organisms. Thraustochytrids usually de-

B. Rinkevich /Journal of Biotechnology 70 (1999) 133-153

146

velop in cultures following other, more opportunistic parasitic forms. In sponges, (i.e. Fig. 3; Blisko, 1998) most of the thraustochytrids developed at days 19-39, while most bacteria, fungi and other protists appeared within the first 3 weeks. Similar results were obtained in colonial tunicate cell cultures. Records from 17 experiments where thraustochytrids were developed in primary Botryllus schlosseri cultures (from buds and blood cells) indicated that in most cases, they appeared > 12 days after culture initiation (up to 48 days; unpublished). There are several ways to identify thraus-

tochytrids in vitro; unfortunately, none of them is conclusive. One way of confirmation can be obtained by the use of electron microscopy (Ellis and Bishop, 1989; Ilan et al., 1996; Blisko, 1998). Contrary to cells originating from marine invertebrates, thraustochytrids have cell walls composed of overlapping plate elements. This material can, however, sometimes be very thin and not easily discernible even under an electron microscope. The mitochondrial cristae of thraustochytrids have a tubular profile, whereas mitochondria with lamellar cristae profiles are the usual animal pattern. A specialized structure unique to the thraus-

Table 1 Confirmed (C; as recorded by original authors) and suspicious (S) identifications (ID) of thraustochytrid protists contamination in marine invertebrate cell cultures during the last decade a Phylum

Reference

ID

Remarks

Porifera

Ilan et al. (1996) Blisko (1998)

C C

Cnidaria

Frank et al. (1994)

C

Crustacea

Itami et al. (1989)

S

Ke et al. (1990)

S

Hsu et al. (1995)

S-C

Toullec et al. (1996)

S

Mollusca

Ellis and Bishop (1989) Auzoux et al. (1993) Takeuchi et al. (1994) Lebel et al. (1996) Awaji (1997)

C S S S C

Echinoderrnata

Kaneko et al. (1995)

S-C

Ermak and Odintsova (1996)

S-C

Rinkevich and Rabinowitz (1993, 1994, 1997) Sawada et al. (1994)

C

Kawamura and Fujiwara (1995)

S-C

All proliferating cultures were identified as thraustochytrids. About 10% of all adult and embryo derived cultures were infected. Found in all ten soft and hard coral cultured species. All secondary cultures and proliferating cell lines were contaminated. Longer cultures (30-54 days) of shrimp lymphoid cells developed cell connections similar to thraustochytrids (their Table 1). Cultures of up to 5 m from shrimp hepatopancreas resemble thraustochytrid cultures by cell structures and by forming centers of multiplications (their Figs 1-4). Shrimp lymphoid tissue developed typical thraustochytrid cultures (their Figs. 6-8). Epidermal and hepatopancreas isolated cells emitted filopods typical to thraustochytrids. Developed in marine bivalve primary cultures. Suspicious thraustochytrid development (their Fig. 3 and text). Suspicious thraustochytrid development (their Fig. 3 and text). Suspicious thraustochytrid development (their Fig. 1). Thraustochytrid developed in pearl oyster mantle cultures (literature cited provided two more confirmed cases from 1988 to 1989 studies of same author). Suspicious thraustochytrid development (their Figs. 1-3 and text). Fine network of cell processes between cellular elements from starfish embryos. Dense network of filipodial processes between embryonic sea urchin cells. Thraustochytrids developed in primary cultures from blood ceils, embryos and epithelial cell of a colonial tunicate. An EM study or solitary tunicate hemocytes reveals cells with cell wall, typical to thraustochytrids (their Fig. 2b). Suspicious thraustochytrid development (their Fig. 4 and text).

Urodordata

S

a S-C refer to cases where the original figures in the published manuscripts depict thraustochytrids while authors were unaware of their presence.

B. Rinkevich /'Journal of Biotechnology 70 (1999) 133-153 a Total Bacteria Fungi Thraustochytrids --~ Others 20

0

,,,

7

~

.~_ E

51

N

~

14 21 28 Culture period (d)

35

42

o 50

40

3O

2O

8

lo

C)

"

0

7

14

21 28 Culture period (d)

J

35

42

Fig. 3. Contamination of primary sponge cultures (Negombata sp.) within the first 39 days of in vitro conditions. (a) Adult sponge cultures ( n - 1 9 0 wells); (b) embryo-derived cultures ( n - 300 wells). In both cases, thraustochytrids appeared in 9-10% of total wells following bacteria and fungi contaminations. In total, 90 wells (47.4%) of adult sponge cultures and 122 (40.5%) of embryos were contaminated during this period (Blisko, 1998).

tochytrids is the sagenogenetosome (Porter, 1990), usually diagnostic in electron microscopy. This structure can, however, be difficult to find in some thraustochytrids since there may only be one in a cell of up to 100 lam in diameter. Under light epifluorescence microscopy, the use of acriflavine hydrochloride, which stains the sulfated polysaccharide cell walls of these organisms, is highly recommended (Raghukumar and Schaumann, 1993). Another characteristic feature is the cytoplasmic extensions. In thraustochytrids, these extensions have no organellar inclusions and at the light microscope level often form anastomosing networks that rather resemble the web of an

147

orb-weaving spider. Under certain culture conditions, thraustochytrids may produce biflagellated zoospores. The zoospores are not produced in some genera of thraustochytrids, so they cannot be relied upon to identify contamination. In some cases, zoospore production is restricted to a short interval following cell feeding (Ellis, pers. commun.). The culture medium may also serve as an indicative parameter. Many thraustochytrids are capable of growth in very simple media. The basic requirements include pyridoxal phosphate, niacinamide, and vitamin B12 and a carbon and nitrogen source (Ellis, pers. commun.). Most animal cells would be unable to proliferate in this medium while thraustochytrids can. However, lack of growth cannot entirely ensure that the isolates are not thraustochytrids which are dependent on a specific carbon or nitrogen source. Many thraustochytrids, survive and grow on pollen grains (Raghukumar, 1992), a feature which is not characteristic to any animal cell types. Ellis and Bishop (1989) were also successful in screening bivalves and thraustochytrid cells by biochemical markers. Contrary to the thraustochytrids, in bivalves and a few other invertebrates, there is no L-lactate dehydrogenase; instead, they have opine or D-lactate dehydrogenases and this trait may serve as a useful marker in their cell cultures. Thraustochytrids may be distinguished from all animal cells by their typical 18S mRNA signatures (Cavalier-Smith et al., 1994). Additionally, many thraustochytrids may produce and release antibiotic products into the medium (Raghukumar, pers. commun.; Rinkevich and Rabinowitz, 1994, our unpublished results) or may feed directly on bacteria found in the medium (unpublished); but so might sponge cells. Since, usually one of the above criteria for the confirmation of thraustochytrid cells in culture is insufficient, it is highly suggested to combine several checks. Another important point for consideration is that thraustochytrid contaminations are sometimes inevitable. We (unpublished) found these cells circulating in the blood systems of Botryllus schlosseri, a colonial tunicate, and within/on embryos of corals (Frank et al., 1994), tunicates (Rinkevich and Rabinowitz, 1994) and sponges (Blisko, 1998).

148

B. Rinkevich /Journal of Biotechnology 70 (1999) 133-153

4. Perspectives During the middle 1970s, there were 70 cell lines available from different species of invertebrates, insects and ticks (Hink, 1979). More than 20 years thereafter, and despite the substantial, pent-up demand for such cell cultures from a variety of taxa, and for numerous applications, there are several hundred insect-arachnid cell lines but no single cell line from marine invertebrates. During the last decade, numerous studies followed previous efforts and failures for the development of marine invertebrate cell cultures (90 peer-reviewed publications, > 50 abstracts and many unpublished reports). This continued failure to establish cell lines from these invertebrate taxa may suggest that we still lack vital information regarding invertebrate cell physiology/biochemistry and biology. For example, during the development of the only single cell line from freshwater snails, an extreme sensitivity of the molluscan cells to changes in vertebrate serum supplementation was evident by changing serum lots or by the use of different serum sources (Hansen, 1976). One obvious conclusion may be that the lack of progress in this discipline is related to inappropriate comparisons of marine invertebrate cell culture requirements with the culture requirements of the vertebrate cells (Goodwin, 1991). There are probably many more reasons and any attempt to anticipate the whole range of unique obstacles is somewhat problematic. Approaches and protocols that already have been developed have successfully yielded primary sterile cultures from many marine invertebrates. Others yielded artifacts (such as the appearance of thraustochytrids), but the problems are encountered with possible solutions. Any cell which is derived from a multicellular organism represents on one hand, the most elementary expression of the organism's genetic trait, and on the other, establishes in vitro a new entity, of its 'own life', without being influenced by any organized structure, humoral or neural factors (Rinkevich et al., 1994). Therefore, in the vertebrates as well as in insects, primary culture methodologies have opened up new approaches for studying in vitro cellular and molecular

events. Some of the vertebrate growth factors may have positive influences on a variety of marine invertebrate primary cultures (Rinkevich and Rabinowitz, 1993, 1994; Lebel et al., 1996; Pomponi et al., 1997) but others do not. Some basic media used for vertebrate cell cultures showed positive influences on marine invertebrate primary cultures as well but the same media were detrimental in other cases (reviewed in Bayne, 1998). The overall conclusion from the variety of studies on freshwater and marine invertebrate primary cultures indicate, however, that we need to consider the production of secondary and cloned cell lines. The literature of the last decade clearly points out that the methodologies for holding short-term, viable primary cultures have been established for a variety of marine organisms. Efforts should therefore be concentrated on such approaches as trangenesis (Rosenfield et al., 1994), mutagenesis by irradiation/chemicals, cell hybridization (Diekmann-Schuppert et al., 1989), replacement of vertebrate sera with lipids and other factors (Goodwin, 1991), the use of feeder layers and more. Such approaches many successfully replace the continuous use of the same methodologies for in vitro conditions of primary marine invertebrate cultures which probably have a limited benefit maintaining viable, but non-proliferating cells.

Acknowledgements This is part of the research was carried out at the Minerva Center for Marine Invertebrate Immunology and Development Biology and was also supported by the Joint German Israeli project on Biotechnology.

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Journal of Biotechnology 70 (1999) 155-161

Growth of the sponge Pseudosuberites (aft.) andrewsi in a closed system Ronald Osinga *, Peter B. de Beukelaer, Ellen M. Meijer, Johannes Tramper, Ren6 H. Wijffels Wageningen University, Food and Bioprocess Engineering Group, PO Box 8129, 6700 EV, Wageningen, The Netherlands

Received 19 October 1998; received in revised form 3 December 1998; accepted 22 December 1998

Abstract

Explants of the Indo-Pacific sponge Pseudosuberites aft. andrewsi were fed with the microalgae Chlorella sorokiniana and Rhodomonas sp. It was microscopically observed that these algae were ingested and digested by the sponge cells, suggesting that they were consumed by the sponges. The algae were further used for two growth experiments with five explants of P. aff. andrewsi and four explants of P. andrewsi. Growth was measured as the increase in projected body area. The explants showed considerable growth (up to 730% in 54 days for P. aft. andrewsi and up to 680% in 22 days for P. andrewsi), which is much higher than previously reported growth rates for sponges. Growth started after a stationary phase of 5-20 days in which the projected body area did not increase. The growth of P. aff. andrewsi appeared to be linear and was inhibited at the end of the experiment. Two explants of P. andrewsi showed exponential growth instead of linear growth. Hence, no general statements about the growth kinetics of these sponges can be made at this time. However, the high growth rates found in this study suggest a promising future for cultivation of sponges in closed systems. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Sponges; In vivo cultivation; Pseudosuberites (aft.) andrewsi; Growth

1. Introduction

Sponges (Porifera) are primitive multicellular aquatic animals. They live attached to solid substrata, such as rocks, corals or shells, and feed upon small particles (microalgae, bacteria and dead organic material), which they filter out of the surrounding water. Most of the sponge species * Corresponding author. E-mail address: [email protected] (R. Osinga)

described so far live in marine environments. The most characteristic feature of the sponge body is the so-called aquiferous system, a network of channels and chambers through which water flows continuously. This water current is generated by flagellated cells (choanocytes) that line the walls of the chambers. The space between the channels and chambers is called the mesohyl: a gelatinous matrix, containing free-floating cells (archaeocytes) and skeletal material. The choanocytes not only generate a water current, but these cells also trap and ingest the food

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S0168-1656(99)00068-1

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particles. Some of the particles are transferred to the archaeocytes, whose role it is, among others, to generate metabolic energy. During the past few decades, marine sponges have been recognised as potential candidates for biotechnology, due to the numerous potentially interesting novel compounds that have been isolated from this group of animals (Garson, 1994; Munro et al., 1994). However, the methodology to produce large amounts of sponge biomass is still in its infancy. Some progress has been made with sponge aquaculture for drug production (Battershill and Page, 1996; Duckworth et al., 1997), but techniques to produce sponge biomass under completely controlled conditions in bioreactors are not available at present (Osinga et al., 1998). Perhaps the single most important problem to address when developing an in vivo cultivation system is to provide a suitable food regimen. In situ, sponges feed rather unselectively upon the complex mixture of particles that is available in natural seawater. Selecting those food particles that provide the sponge with all their metabolic requirements may be the key to a successful in vivo sponge culture. In our laboratory, the Indo-Pacific demosponge species Pseudosuberites andrewsi (Kirkpatrick) is used as a model species for the development of such an in vivo cultivation method. This species was maintained in controlled systems for more than a year, using two microalgal species (the freshwater chlorophycean Chlorella sorokiniana and the marine cryptophycean Rhodomonas sp.) as a food source (Osinga et al., 1999). These algae have also been used in previous studies to feed cultures of the temperate marine sponge Halichondria panicea (Barthel and Theede, 1986; Thomassen and Riisggtrd, 1995). It is the aim of the present study to verify if the chosen food particles are indeed ingested by the sponge cells. In addition, we will quantify the growth rates and describe the growth kinetics of explants of P. andrewsi and Pseudosuberites aff. andrewsi cultivated under controlled conditions in a bioreactor using C. sorokiniana and Rhodomonas sp. as the food source.

2. Materials and methods

2. I. Sponges Fresh material of P. andrewsi was obtained from Blijdorp Zoo (Rotterdam, The Netherlands), where it was growing in a large, shallow basin, in which a strong water current was generated to simulate an intertidal environment. More sponge material was obtained from Artis Zoological Garden, Amsterdam. Here, the sponges grow in the central filtration system of the tropical marine aquaria. This biofilter consists of small pebbles (grain size approximately 5 mm). The sponges grow on top and within the first 2 cm of this pebble layer. All the organic dirt from the aquaria (food remainders, organic wastes, bacteria) is deposited onto this filter, which is therefore a good locality for the sponges in terms of food availability. The sponge material obtained from Artis Zoo was classified as P. aff. andrewsi. The spicules of these sponges resembled the form of the spicules of P. andrewsi as described by Kirkpatrick (1900), but were considerably smaller ( ~ 200 ~m instead of 350 lam). In our laboratory, the sponges were held in 200 dm 3 air-lift bioreactors containing artificial seawater (using Instant Ocean Reef Crystals artificial sea salt) with a salinity of ~ 32%o. This water was replaced continuously ( D = 0 . 0 3 3 day-~). The temperature in the bioreactor varied between 25 and 29~ In order to provide the sponges with a source of silica, 0.25 mmole d m - 3 Na203Si-9H20 was added to the artificial seawater. Measurements of the silica concentration in the outflowing water showed that this addition was sufficient to cope with the sponges' demands. Non-axenic batch cultures of C. sorokiniana (average size ,-~ 3 ~m) and Rhodomonas sp. (average size ~ 6 ~m) were added as a food source for the sponges. Twice a week, 1 dm 3 of a culture of Chlorella sorokiniana was added, containing ~ 1 x 107 cells cm -3. In addition, 1 dm 3 of a culture of Rhodomonas sp., containing ~ 1 • 106 cells cm-3, was added weekly. The algae were cultured at a temperature varying between 17 and 20~ A light-dark cycle of 14 h light and 10 h darkness was applied. The growth media for the algae are

R. Osinga et al./Journal of Biotechnology 70 (1999) 155-161

given in Table 1. When the cultures were added to the sponges, the algae were usually near the end of their logarithmic growth phase.

2.2. Particle ingestion studies To study the ingestion of the supplied food particles by the sponges, small pieces of sponge tissue were incubated in 100 cm 3 aerated, stirred beaker glasses filled with artificial seawater. A few cm 3 culture of either C. sorokiniana or Rhodomonas sp. was added as a food source. Dissociated cells obtained from the incubated sponges were microscopically examined for the presence of algal material. This was done before feeding, 2 h after feeding and 24 h after feeding. Dissociated sponge cells were obtained from the sponges by putting the sponge tissue into a petri disc with Ca 2 + and Mg 2+ free Artificial Seawater (CMFS) and by cutting it into smaller pieces with Table 1 Growth media for the algae (a freshwater medium for C. sorokiniana and a seawater medium for Rhodomonas sp.) a Component

NaHCO 3 KNO 3 NaH2PO 4 Instant Ocean Reef Crystals artificial seasalt MgSOa'7H20 CaCI2"2H20 EDTANa2.2H20 FeC13 Na2B407" 10H20 ZnSO4"7H20 CuSO4"5H20 MnSO4"H20 Na2MoOa'2H20 NiSOa'6H20 NaVO 3 Thyamin-HCl Cyanocobalamin Biotin

Freshwater medium concentration 10.0 1.00 0.10

Seawater mediumconcentration 5.00 0.50 0.05 ~ 3 3 g dm -3

4.99 0.272 0.391 0.148 4.72 x 10 -2 3.13 x 10 -2 3.20 x 10 -2 3.59 • 10 -2 2.07 x 10 -2 2.85 • 10 -3 2.85 x 10 -3 5.93 • 10 -5 5.90 • 10 -6 1.64 • 10 -6

aThe freshwater medium was based on the A9 medium described by Lee and Pirt (1981). Concentrations are given in mM, unless indicated otherwise.

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a razor blade. C M F S is used to prevent reaggregation of the dissociated sponge cells. Ca and Mg ions play a crucial role in the reaggregation process (Miiller, 1982). The cell and tissue suspension was sieved through a 70 ~tm mesh to obtain a cell suspension. This cell suspension was centrifuged for 5 min at 600 • g at a temperature of 10~ The pellet was resuspended in a few cm 3 of C M F S and this suspension was examined under a light microscope.

2.3. Growth experiment An experiment to determine the growth rate of P. aff. andrewsi was performed with five explants (small cuttings of sponge colonies). These explants were prepared using razor-sharp knifes and were obtained from different sponge colonies. However, they probably all originate from a single individual, coincidentally introduced into the aquaria of the zoo. The pieces of sponge tissue were tied onto glass slides with nylon fishing-line. The explants were placed in temperature controlled, 1.58 dm 3 bioreactors, equipped with a sparger for air supply and a magnetic stirrer to keep the food particles in suspension. A second, repetitive experiment was done with four explants from P. andrewsi, which were all obtained from a single sponge colony. The sponges were fed with C. sorokiniana (twice a week, 50 cm 3) and Rhodomonas sp. (once a week, 50 cm 3) using material from the batch cultures that were described in the previous section. Temperature and salinity in the bioreactor was kept constant at 25~ and 34 %0, respectively. The first experiment was run for a period of 54 days, and the second experiment for a period of 22 days. To determine growth, the size of the explants was followed during the experiment. Explant sizes were measured as a two-dimensional projections of body area, which were determined from photographs. A similar method was used by Ayling (1983) to measure in situ growth and regeneration of sponges. The advantage of this method is that the sponges used in experiments do not have to be removed from the water. Exposure to air can cause serious damage to sponge tissue (Foss~ and

158

R. Osinga et al. Journal of Biotechnology 70 (1999) 155-161

Fig. 1. Explants of P. aft. andrewsiplaced on the rack designed for photography of the body area. The black dots indicate a marked distance. Nilsen, 1996), and may therefore affect the growth rate that is to be determined. We have evaluated the pros and cons of this method, and compared it with another, novel method to measure sponge biomass: determination of the underwater weight. The results of this comparative study will be described elsewhere (Osinga et al., 1999). Photographs of the explants were taken on days 0, 5, 8, 19, 33, 39, 47 and 54 in the first experiment (P. aff. andrewsi) and on days 1, 7, 10, 20 and 22 in the second experiment (P. andrewsi ). The explants (always kept underwater) were placed onto a rack on which black dots were painted that indicated a known distance (Fig. 1). Photographs were taken under a straight angle with a digital camera (Hewlett Packard PhotoSmart Model C5340A). The digital images were printed, the areas of the sponges were cut out with scissors and these cuttings were weighed. The weights of these cuttings were converted to areas by comparing them with the weight of a cutting of a known area. The thus obtained values were converted to real surface areas using the marked distances on the rack as a reference.

3. Results and discussion

3.1. Particle &gestion studies Usually, a change in colour of the outer appearance of the sponges was observed approximately 1 h after feeding. The colour changed from

yellow into green after feeding with the greencoloured C. sorok&iana, and from yellow into brown after feeding with the red-coloured Rhodomonas sp. This indicated that the food particles accumulated in the sponge body. The changes in colour were also observed regularly in the large bioreactor and may be used as an indication of the condition of the sponges. Sponges that change colour rapidly exhibit a strong pumping activity. The microscopic observations on dissociated cells showed that both Chlorella and Rhodomonas cells were taken up by the archaeocytes of P. andrewsi (Fig. 2B,C). Complete algal cells were detected in many sponge cells 2 h after feeding. Thus, ingestion by choanocytes and transfer to archaeocytes takes place within 2 h. This is comparable with the rate at which the freshwater algae Chlamydomonas re&hardtii was ingested and transferred by the freshwater sponge Spongilla lacustris (Imsiecke, 1994). After 24 h, some sponge cells still contained intact algae, but other cells only exhibited a green or red colour, indicating that the algae were being digested. It can therefore be concluded from the microscopic observations that P. andrewsi is able to feed upon C. sorokiniana and Rhodomonas sp. It remains to be studied further whether these algae are also the most suitable food source for this sponge species.

3.2. Growth experiment The obtained growth curves clearly show that the sponges P. aft. andrewsi (Fig. 3A) and P. andrewsi (Fig. 3B) are able to grow in a closed system. Unfortunately, in situ growth rates for P. andrewsi are not available. We compared our results with growth rates from other sponge species that were described in the literature (Table 2). In this table, the total growth of the explants in Fig. 3 during the whole experimental periods (54 and 22 days, respectively) is expressed as a percentage of the initial body size. The growth rates for P. (aft.) andrewsi obtained in our systems are much higher than the literature values in Table 2. The five explants of P. aff. andrewsi (Fig. 3A) all exhibited a similar growth pattern: a stationary

R. Osinga et al. ,'Journal of Biotechnology 70 (1999) 155-161

phase during the first 5-20 days, followed by a period in which the projected body area increased rather linearly. At the end of the experiment, the growth rate tended to decrease, sometimes even to zero. We have no explanation for the irregular pattern observed for explant 4. The peak at day 8 will not be further discussed. The explants of P. andrewsi in the second experiment (Fig. 3B) also showed a stationary phase of 7 or 10 days followed by a period of considerable growth. However, a somewhat different growth pattern was observed: explants 1 and 2 exhibited exponential growth, explant 3 did not grow at all, and explant 4 grew irregularly. A uniform description of the growth kinetics of these sponges cannot be deduced from these results. A specific growth rate expressed in percentage per day can only be calculated from the curves obtained for explants 1 and 2 in Fig. 3b, as only these data can be described by the function In X / X o =/ut (in which X is the amount of sponge biomass, X0 is the amount of sponge biomass at t = 0 and /~ is the growth rate in percentage per day). The calculated growth rates were 0.08 day-~ and 0.11 day-~ for explants 1 and 2, respectively. This is again higher than previously reported growth rates, which range from 0.01 to 0.058 day-~ (Thomassen and Riisg~.rd, 1995).

159

The stationary phase that was observed in both experiments could be a response of the sponge to the cutting procedure: the tissue has to rearrange into a functional sponge. By determination of the underwater weight, it was found that most explants even lost some weight during the stationary phase (Osinga et al., 1999), which indicates that the first days after the cutting procedure are indeed a period of adaptation. At the end of the experiment with P. aff. andrewsi, the growth rate decreased. There may be two explanations for this decrease. Sponges are typical modular organisms (Kaandorp 1991), i.e. they grow by repeated production of nearly identical multicellular structures (modules). In the species studied by Kaandorp (1991), these modules are determined by the skeletal structure: a new skeleton module is built upon the previous layer. Other examples of modular growth may be the formation of new channels and choanocyte chambers, or the formation of a new osculum. The modular growth may result in periods of growth and periods of tissue rearrangement. During the latter, the increase in sponge biomass will be lower, and this may have occurred at the end of the experiment. A second explanation for the decreased growth at the end of the experiment may be found in growth limitation. The food regimen was not

~ D

Fig. 2. Microscopic view of dissociated archaeocyte cells: (A) before feeding: (B) 2 h after feeding with C. sorokiniana; (C) 2 h after feeding with Rhodomonas sp.; (D) 24 h after feeding with Rhodomonas sp.

R. Osinga et al./Journal of Biotechnology 70 (1999) 155-161

160

with batch cultures will result in short periods of starvation, which may not be in favour of optimal growth. The fact that all explants stopped growing at the end of the experiment supports the suggestion of growth limitation. The growth of P. aff. andrewsi should therefore be further studied under conditions of continuous food supply, thus excluding the possibility of food limitation.

A: Pseudosuberites aft. andrewsi ~e~explantl --@--explant 2 -,W--explant 3 -"B---explant 4 --'0--explant 9 5

A 04

[ ] [ ]

< 6 E o v

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B: Pseudosuberites andrewsi ~e~- explant --4P--explant ; --B--explant : --B--explant,

).5 A

2

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1 0,5

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.

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P. aff. andrewsi was found to ingest and digest both C. sorokiniana and Rhodomonas sp., which confirms that these algae can be used as a food source for in vivo cultures of this sponge species. It was further shown that the growth rates of P. andrewsi and P. aff. andrewsi in a closed system are much higher than previously reported growth rates for sponges, indicating that sponge culture in closed systems has a promising future. However, growth rates and kinetics show a large variability. The present data still leave too many uncertainties to deduce a model that fits to the growth curves obtained for this species under the applied conditions. Experiments are under way in which we will study the growth of P. aft. andrewsi under conditions of constant food availability.

days

Fig. 3. (A) Growth of five explants of P. aff. Andrewsi, expressed as the increase in projected body area (cm 2) during a 54-day period. (B) Growth of four explants of P. andrewsi, expressed as the increase in projected body area (cm 2) during a 22-day period.

changed during the experiment, despite the fact that the sponges were growing. Besides, feeding

Acknowledgements We thank Max Janse (Artis Zoo, Amsterdam) and Michael Laterveer (Blijdorp Zoo, Rotterdam) for providing us with fresh sponge material and Dr Rob W.M. van Soest (University of Amster-

Table 2 Growth of P. (aft.) andrewsi, expressed as a percentage of the initial body weight (i.e. 100 means no growth), compared with some data from the literature Species

Growth

Culture period (days)

Reference

P. andrewsi P. aff. andrewsi Hippospongia lachne Raspaila agminata Spongia officinalis Spongia agaricina

70-680 500--730 160-320 125 230 300

22 54 365 262 600 600

This study This study Storr (1964) Duckworth et al. (1997) Verdenal and Vacelet (1990) Verdenal and Vacelet (1990)

R. Osinga et al. Journal of Biotechnology 70 (I999) 155-161

dam) for identifying the material. Emilie P.E. Theunissen is acknowledged for analytical support.

References Ayling, A.L., 1983. Growth and regeneration rates in thinly encrusting Demospongiae from temperate waters. Biol. Bull. 165, 343-352. Barthel, D., Theede, H., 1986. A new method for the culture of marine sponges and its application for experimental studies. Ophelia 25, 75-82. Battershill, C.N., Page, M., 1996. Sponge aquaculture for drug production. Aquaculture Update 1996, 5-6. Duckworth, A.R., Battershill, C.N., Bergquist, P.R., 1997. Infuence of explant procedures and environmental factors on culture success of three sponges. Aquaculture 156, 251-267. Fosse., S.A., Nilsen, A.J., 1996. Korallenriff-Aquarium, Band 5. Einzellige Organismen, Schw/imme, marine W~irmer und Weichtiere im Korallenriff und ftir das Korallenriff-Aquarium. Birgit Schmettkamp Verlag, Bornheim. Garson, M.J., 1994. The biosynthesis of sponge secondary metabolites: why is it important? In: van Soest, R.W.M., van Kempen, T.M.G., Braekman, J.C. (Eds.), Sponges in Time and Space. Balkema, Rotterdam, pp. 427-440. Imsiecke, G., 1994. Ingestion and digestion of Chlamydomonas reinhardtii (Volvocales) by the freshwater sponge Spongilla lacustris (Spongillidae). In: van Kempen, T.M.G., Braekman, J.C. (Eds.), Sponges in Time and Space. Balkerna, Rotterdam, pp. 371-376.

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Kaandorp. J.A.. 1991. Modelling growth forms of the sponge Haliclona oculata (Porifera; Demospongiae) using fractal techniques. Mar. Biol. 110, 203-215. Kirkpatrick, R., 1900. On the sponges of Christmas Island. Proc. Zool. Soc. London 12&13, 127-140. Lee, Y.-K., Pirt, S.J., 1981. Energetics of photosynthetic algal growth: influence of intermittent illumination in short (40 s) cycles. J. General Microbiol. 124, 43-52. Munro, M.H.G., Blunt, J.W., Lake, R.J., Litaudon, M., Battershill, C.N., Page, M.J., 1994. From seabed to sickbed: what are the prospects? In: van Soest, R.W.M., van Kempen, T.M.G., Braekman, J.C. (Eds.), Sponges in Time and Space. Balkema, Rotterdam, pp. 473-484. Mtiller, W.E.G., 1982. Cell membranes in sponges. Int. Rev. Cytol. 77, 129-181. Osinga, R., Tramper, J.. Wijffels, R.H., 1998. Cultivation of marine sponges for metabolite production: applications for biotechnology? Trends Biotechnol. 16, 130-134. Osinga, R., Redeker, D., De Beukelaer, P.B., Wijffels, R.H., 1999. Measurement of sponge growth by the projected body area and the underwater weight. Memoirs of the Queensland Museum. 44 (in press). Storr, J.F., 1964. Ecology of the Gulf of Mexico commercial sponges and its relation to the fishery. Special Scientific Report--Fisheries no. 466, United States Department of the Interior. Thomassen, S., Riisg~rd, H.U., 1995. Growth and energetics of the sponge Halichondria panicea. Mar. Ecol. Prog. Ser. 128, 239- 246. Verdenal, B., Vacelet, J., 1990. Sponge culture on vertical ropes in the Northwestern Mediterranean Sea. In: Rtitzler, K. (Ed.), New Perspectives in Sponge Biology. Smithsonian Institution Press, Washington, DC, pp. 416-424.

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JOURNAl

OIF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 163-174

Aquaculture of sponges on scallops for natural products research and antifouling Evelyn Armstrong a, j. Douglas McKenzie a , , Gordon T. Goldsworthy b Marine Biotechnology Group, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Scotland PA34 4AD, UK b Loch Fyne Seafarms Ltd., Tarbet Trading Estate, Tarbet, Loch Fyne, Argyll, Scotland PA29 6SX, UK

Received 12 October 1998; received in revised form 30 November 1998; accepted 22 December 1998

Abstract Suspended culture of scallops (Pectinidae: Bivalvia) is plagued by invertebrate fouling. Scallops covered in an orange sponge (Suberites ficus ssp. rubrus), however, do not have any other invertebrates fouling their shells and the sponge is easily removed. Sponges may also be valuable sources of bioactive compounds. Seven species of sponge were found to be associated with Chlamys opercularis but none were found associated with Pecten maximus. Standing crop of sponge on the scallop farm was just over one tonne. If all the scallops were covered by sponge this would rise to approximately 4.5 tonnes (from three million scallops). Primary cell cultures of (S.f. rubrus) were successfully established but no cell lines were achieved. A number of techniques were tried for establishing cells and pieces of sponge tissue onto scallop shells but none gave satisfactory results. Likewise mechanical methods for sticking small pieces of sponge to scallop shells were deemed impractical even where they resulted in subsequent sponge growth. Improving natural settlement of sponges onto the scallops was the only economically practical method for increasing sponge yield for C. opercularis, though other methods would need to be devised for P. maximus. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Adhesion; Cell culture; Chlamys opercularis; Fouling; Suberites ficus

I. Introduction Settlement of fouling invertebrates (Fig. 1) is a severe constraint on the practicality and economics of suspended culture of scallops (Pectinidae: Bivalvia). The price of a scallop is increased if the meat can be sold in its shell but this requires the * Corresponding author. Tel.: + 44-1631-567832; fax: + 441631-571150.

shells to be completely clean. Mechanical removal of fouling organisms such as mussels, tunicates and tube worms is not a cost-effective process so fouled scallops have to be expensively processed then sold at a lower price. Fouling (particularly by mussels) can also result in the death of scallops where it restricts the flow of water to the scallops in their culture bags. It was noted that scallops covered in an orange sponge (since identified as Suberites ficus ssp. rubrus) did not have any other

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S01 68-1656(99)00069-3

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E. Armstrong et al./ Journal of Biotechnology 70 (1999) 163-174

invertebrates fouling their shells (Figs. 2 and 3). This sponge can be easily cleaned off scallops using a water jet leaving a perfect shell. If sponge colonisation of the scallops could be made total on a farm of three million scallops, then this would result in a considerable increase in profitability for the farm (estimated at s additional profit per annum). While culture of the sponges is a highly attractive prospect in terms of the antifouling benefits alone, there is also the possibility of using the cultured sponges as a source of natural products initially for research or possibly for full scale commercial manufacture of any natural products of interest. Sessile invertebrate organisms must compete for their space on the substrate on which to live. To do this they often produce chemicals (secondary metabolites) to deter other settlers. Many potential antifouling chemicals have been

Fig. 2. Chlamys opercularis with sponge (S.f. rubrus) only on the right side of the valve. The left side which is free of sponge has been fouled by tube worms (probably a Pomatoceros species). Even this low amount of fouling would make the scallop useless to sell as a whole shell product.

found in invertebrate organisms (Fusetani et al., 1996). The sponges on scallop shells are probably also chemically defended given their efficacy in

Fig. 1. A badly fouled scallop line. The scallops are held within a thin plastic mesh bag. The scallops and the bag have been extensively fouled by mussels (Mytilus edulis), sea squirt (mostly Ciona intestinalis) and various tube worms. Arrow indicates a scallop (Chlamys opercularis) fouled by mussels and other encrusting organisms. Scale bar, approximately 10 cm.

Fig. 3. Chlamys opercularis with sponge (S.f. rubrus) growing over the whole valve. The sponge is obscuring the shell morphology. This cover completely deters fouling but is easily removed upon harvesting.

E. Armstrong et al. / Journal of Biotechnology 70 (I 999) 163-174

preventing fouling and their lack of any other obvious means of defence such as surface ornament. These compounds may also have medicinal uses: a wide range of cytotoxic and antimicrobial compounds have already been isolated from various marine organisms (e.g. Garson, 1994; Zilinskas et al., 1995). The ability to produce these metabolites on an industrial scale is vitally important if they are to be fully utilised. This factor is a major stumbling block in the production of drugs from natural sources (Osinga et al., 1998). As the compounds are often found in only extremely small amounts, large amounts of invertebrate tissue needs to be collected and processed. Because of both environmental concerns and economic limitations, it is unlikely that natural populations can provide sufficient tissue for extensive research or exploitation of interesting natural products. While it may be possible to synthesise some of the natural products or artificially produce analogues, many compounds are likely to be too costly to synthesise. This leaves culture are the only feasible method of producing sufficient quantities of these compounds. To produce suitably large quantities of natural products, sponges or their cells could be cultured in the laboratory and the compound of interest collected (Osinga et al., 1998). Alternatively the sponge could be produced by using aquaculture techniques more usually associated with food production. Sponges cultured on scallop shells would protect the scallops throughout their growth and then the sponge could be removed after harvesting the scallops and any economically interesting natural products extracted. The major advantages of this latter method are that large amounts of sponge could be collected without environmental damage and for little cost as it is a by-product of an existing aquaculture system. This paper reports the results of a 6-month study into the growth and distribution of sponges on cultured scallops and into methods to increase the yield of these sponges. Cell culture methodologies investigated for the dominant sponge, Suberites ficus rubrus, are described in detail.

165

2. Materials and methods

2.1. Identifications of species associated with commercial scallop species in Scotland Free-living and cultured scallops (Chlamys opercularis and Pecten maximus) were examined for the presence of sponge and identifications of the various species made. Sponges were identified by their spicules and general appearance. The spicules were released from the rest of the sponge tissue by digesting this tissue with bleach. After washing with distilled water the spicules were examined under a microscope.

2.2. Sponge standing crop on a scallop farm An assessment was made of the amount of sponge that is currently produced, and the potential production if sponge was attached to every scallop on the Loch Fyne Seafarms farm site at Barmore Island, Loch Fyne, Scotland. Sponges were collected from scallops on three different strings at three depths in the water column: 10, 14 and 18 m. These heights correspond to the depth of the scallops at the top, middle and bottom of the scallop strings. Scallops were examined from each string at each depth until ten scallops with sponge were found. The number of scallops without sponge were counted in order to calculate the percentage of scallops carrying sponge. The sponge was then scraped off the scallop shells and transported back to the Dunstaffnage Marine Laboratory where it was wet weighed. The sponge collected was a mixture of S. titus rubrus and S. ficus luridus (judging by the spicules and colour of the sponge). To compare the mass of sponge that was found on scallop shells at each height on each string, ANOVA with the all-pairwise multiple comparison Student- Newman- Keuls method was used.

2.3. Sponge cell culture As a prelude to developing novel methods of inoculating scallops with cells, methods of cell culture were investigated. S.f. rubrus was collected from Chlanl~'s opercularis from the scallop farm.

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E. Armstrong et al. Journal of Biotechnology 70 (1999) 163-174

A sterile scalpel was used to remove the sponge without exposing the tissue to air. However, the sponge was exposed to air briefly when the farmed scallops were raised from the water upon collection. The sponge tissue was rinsed a number of times in sterile filtered seawater ( < 0.2 ~tm) to remove any surface debris. It was then used directly or incubated for 24, 48 or 120 h at 4~ in sterile seawater with added gentamicin sulphate (0.5 mg ml-1 final concentration) or penicillin and streptomycin (100 units penicillin ml-1 and 0.1 mg streptomycin ml-1). Following incubation, the sponge was cut into smaller pieces, ca 5 mm 2, with a sterile scalpel and placed in fresh sterile seawater with antibiotic for 1 h at room temperature. Three different methods of tissue dissociation were attempted: chemical dissociation; mechanical dissociation and spontaneous dissociation. Chemical dissociation (after Pomponi and Willoughby, 1994) was done directly after the sponge was removed from the scallop shell. Sponge tissue was rinsed in several changes of calcium and magnesium free seawater in a petri dish, without exposure to air, and then cut into pieces as small as possible with a sterile scalpel. These small pieces were transferred, in a minimum of liquid, to 10 mM E D T A in calcium- and magnesium-free seawater for 25 min (10:1 C M F volume:sponge volume). During this time the sponge cells were shaken periodically. Then the sponge suspension was filtered through a 70 ~m cell strainer to remove any large clumps of undissociated cells and spicules. The cells were harvested by centrifugation at 300 x g for 10 min and washed 3 or 4 x in sterile filtered seawater. For mechanical dissociation (after Ilan et al., 1996), each sponge piece was placed in a microcentrifuge tube with 1 ml of sterile seawater with added gentamicin sulphate (0.5 mg ml-~ final conc.) or penicillin and streptomycin (100 units of penicillin and 0.1 rng streptomycin ml-~ final conc.). The tissue was then homogenised against the side of the tube with a sealed glass pasteur pipette. Once the majority of the cells was removed from the tissue (seen as the sponge piece becoming pale yellow in colour) the cells and seawater were removed from the tube and filtered

through a 70 ~tm cell strainer. The cells were harvested by centrifugation at 400 x g for 10 min and then washed 4 x in filtered seawater or filtered seawater with added antibiotics. On one occasion the outer layer of the sponge was removed before dissociation in an attempt to limit contamination problems, as suggested by Ilan et al. (1996). The removal of the cortex was only possible where the sponge tissue was thick. This was usually only the case in tissue freshly collected from the scallop farm. Sponge appeared to regress when it was brought to the aquarium conditions at the laboratory. For spontaneous dissociation (after Ilan et al., 1996) antibiotic-treated sponge tissue was cut into small pieces ca. 1-1.5 mm 2 and each piece placed in a well of a 96-multi-well plate with 200 ~tl of medium. After 24 and 48 h the plate was examined to see if sponge cells had emerged from the edges of the sponge tissue. Once the cells had emerged the sponge tissue was removed from the well. In addition to using this tissue, cells and tissue particles released into the seawater and antibiotic mix when the sponge was cut into smaller pieces was used as inoculum. After each dissociation the cultures were examined periodically and sub-cultures made as required. All cultures were incubated in the dark at 15~ 2.4. The following cell culture media were used 2. 4. I. Seawater and antibiotics Sterile filtered seawater (0.2 ~tm) was used as the base for the medium and then penicillin and streptomycin (100 units of penicillin and 0.1 mg streptomycin ml-~ final conc.) were added. In some cases heat-killed Escherichia coli were added as food. Heat-killed bacteria were prepared by harvesting the cells from an overnight nutrient broth culture and washing and resuspending them in sterile filtered phosphate-buffered saline (PBS). The bacterial suspension was then heated at 60~ for 2 h. After further washing the bacteria were stored at 4~ in fresh PBS. Seawater with gentamicin sulphate (0.5 mg ml-~) was also used as a medium but heat-killed E. coli were never added to this.

E. Armstrong et al. /Journal of Biotechnology 70 (1999) 163-174

2.4.2. Seawater, antibiotics and serum Sterile filtered seawater (0.2 lam) was used as the base for the medium with foetal bovine serum (5%) and penicillin (100 units m l - 1 final conc.) and streptomycin (0.1 mg m1-1 final conc.) added. 2.4.3. Seawater, serum, Leibovitz medium and antibiotics Sterile filtered seawater (0.2 lam) was used as the base with 5% serum and 5% Leibovitz medium added. Gentamicin sulphate was included at 0.5 mg ml-1 or penicillin and streptomycin at 100 units of penicillin and 0.1 mg streptomycin ml-1. Leibovitz medium was made up from powder (Sigma) with the following salts added to make the osmolarity as seawater (for 1 1): NaC1, 20.20 g; KC1, 0.54 g; CaCl2, 0.60 g; MgSOa.7H20 , 1.00 g; MgC12-6H20, 3.90 g. 2.4.4. Seawater, Leibovitz medium and antibiotics As for Section 2.4.3 except there was no serum added. 2.4.5. Seawater, Leibovitz medium, serum, antibiotics and antifungals As for Section 2.4.3 but with amphotericin added (2.5 ~tg ml-1). Mechanically dissociated cells from sponge tissue were used in attempts to separate different sponge cell types by density gradient centrifugation. Both Percoll and Ficoll were used to form the gradients. A Percoll discontinuous 60, 45, 30 and 15% gradient (Pomponi and Willoughby, 1994) was made up, mixing the Percoll with sterile filtered seawater. A total of 2 ml of each dilution was layered into a 15-ml centrifuge tube. Sponge cells, in 2 ml of seawater, were carefully placed on top of the Percoll. The gradient was then centrifuged at 400 x g for 20 rain to allow cell layers to form. In some cases, cells were stained with 0.001% methylene blue (final concentration) before centrifugation. Three different Ficoll-paque discontinuous gradients were set up: 11, 7, 5 and 2% (Uriz et al., 1996); 50, 40, 30 and 20%; and 45, 40, 35 and 30%. A total of 2 ml of each layer of the first gradient and 3 ml of each layer of the latter two gradients were set up in 15 ml cen-

167

trifuge tubes. A total of 2 ml of sponge cells was layered on top of the Ficoll and allowed to sediment for 3 h (Uriz et al., 1996). In addition, 2 ml of sponge cells were layered onto 100% Ficoll, which was centrifuged at 400 x g for 40 min or ca 11 000 x g for 35 min. Any cell layers were removed following centrifugation/sedimentation and examined before inoculation into medium in a petri dish. To improve cell attachment to the base of petri dishes following dissociation, a layer of collagen was put on the base of the dishes. A solution of collagen was made in acetic acid (0.1% collagen in 0.1 M acetic acid). This solution (0.5 ml) was spread over the base of a petri dish using a glass spreader and the excess allowed to evaporate. Medium was then added to the dish as normal. Cell cultures were examined periodically using an inverted microscope. Cultures which became contaminated by fungi or other cells, mainly protozoans such as ciliates, were discarded. The medium of chemically and mechanically dissociated cells was not changed for 2 weeks following inoculation after which the medium was changed as necessary. Cells were subcultured when they increased in number by washing them off the base of the culture vessel. The cells were transferred to a new petri dish containing fresh medium. Cells were identified as being sponge cells using differential interference contrast (DIC) light microscopy. Cultured and newly dissociated sponge cells were examined and compared with a compound microscope at x 1000 and an inverted microscope at x 400. In addition, cells were stained with acridine orange and examined using fluorescence microscopy with blue light. Cells were also stained with Syto 9 (Molecular Probes). This staining allowed the nuclei of the sponge cells to be seen more clearly.

2.5. Adhesion of sponge to scallops A number of methods were used to attempt attachment of sponge pieces. Ideally sponge would be attached to scallop shells in a rapid procedure where many shells could be processed at once. Therefore, some form of glue or adherent matrix was required; tissue glue was tried. This

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E. Armstrong et al. / Journal of Biotechnology 70 (1999) 163-174

was followed by attempts using the following natural adhesive mixtures:

2.5.1. Agar Agar was made up at 1.2% in seawater and empty scallop shells dipped in this once the agar was close to gelling. The shell was then dipped into seawater which contained small, thin pieces of sponge ca 2 mm 2. A thin sheen of agar gel formed over the surface which held a few pieces of sponge tissue.

2.5.2. Alginate A similar procedure was carried out for alginate (0.5 g in 50 ml of seawater, then dipped into 100 mM CaC12 in seawater). The scallop shells were placed in seawater tanks and examined periodically.

2.5.3. Phytogel This is another agar-like compound. It has been used previously in biofouling studies where it has been found to be capable of adhering to a surface for at least 6 weeks (Henrikson and Pawlik, 1995).

dry briefly on the scallop shells before the scallops were placed in seawater containing a homogenate of sponge. The scallops were left in this mixture for ca 90 min before they were removed and returned to their aquarium tank. Cultures of sponge were set up in sea water aquaria with flow-through sea water supplies. Sponge pieces were attached to monofilament line and suspended from a wire cage similar to that of Barthel and Theede (1986). Weights were required at the ends of the line so that the sponges were held below the water surface. Initially the sponge was tied onto the monofilament line but it was subsequently found that it was easier to thread the sponge onto the line. This also minimises the sponge's exposure time to air. The sponge pieces formed balls or cylinders around the monofilament line so the dimensions of the sponge decrease initially. The sponge was then measured at periodic intervals.

3. Results

3.1. Sponge species found associated with scallops 2.5.4. Agar and collagen Approximately 15 ml of molten agar were put in a petri dish and 2 ml of 0.1% 0.1 M acetic acid added and mixed in. Empty scallop shells were dipped into this mixture and then into seawater containing sponge pieces ca 1 x 1 mm. Collagen was also dropped directly onto scallop shells and then sponge laid on top. Other chemicals that may promote attachment of sponge such as collagen and poly-L-lysine were pipetted onto empty scallop shells and allowed to dry. The shells were then immersed in seawater which contained sponge tissue which had been homogenised giving large, small and microscopic pieces of tissue. The shells were left in this mixture for 24 h. The shells were then put in an aquarium tank supplied by a flow-through water system. A similar experiment was carried out with juvenile scallops because the sponge must be capable of surviving the movement of scallop. Scallops (4 x for each treatment) were covered with the following compounds: collagen; poly-L-lysine; gelatine and biofilm only. The compounds were allowed to

Sponges were never found on the King Scallop

Pecten maximus. Sponges were, however, frequently associated with the smaller Queen Scallop, Chlamys opercularis. The following species were found: Suberites ficus spp. rubrus: this was an orange encrusting sponge which appeared to be fairly robust. It was the most commonly found species. The sponge was found on scallops growing on the seabed and in scallop strings. S. ficus ssp. luridus: this sponge was very similar to S. rubrus in all except colour, S. luridus being yellow. Initially thought to be a single species, a study by Solr-Cava and Thorpe (1986) showed there to be three sub-species: S. ficus ssp. rubrus, S. ficus ssp. luridus and S. ficus ssp. pagurorum. To accurately separate these sub-species it is necessary to use molecular techniques. Mycale rotalis: this pink/purple encrusting sponge appeared to form patches on the surface although there was in fact sponge all over the

E. Armstrong et al./Journal of Biotechnology 70 (1999) 163-174

169

Table 1 Mean mass of sponge tissue on scallop shells at each height on three different scallop strings and the percentage of shells with sponge at each height on the three strings Depth (m)

10 14 18

Percentage of shells with sponge

Mean mass (g) String 1

String 2

String 3

String 1

String 2

String 3

0.172 0.063 0.169

0.120 0.063 0.373

0.133 0.145 0.119

16.5 27 16.2

31.9 11.8 20

23.3 25.6 46.2

scallop. There were small, orange (approx. 0.5 mm dia.), round structures in this sponge where it adhered to the scallop shell. It was assumed that these were gemmules, although they could have been developing larvae. This sponge was only found on scallops on the seabed, not on those found on strings. Mycale macilenta: this encrusting sponge was found in a number of different colours on scallops from different sites. It was orange to brownish on scallops from Loch Fyne Seafarms but yellow on scallops collected in Oban Bay. Hymeniacidon perleve: this yellow sponge forms thicker growth than the above three on the scallop shells, although it is still encrusting. The sponge is fairly elastic and forms mounds on the scallop surface. Spicules project from these areas. This sponge was not very common. An Anchinoe species: this was an orange/brown species that was thinly encrusting and contained a large number of holes. However, there were also channels connecting these holes indicating a leuconoid structure. A Myxilla species: this sponge was found on a scallop shell in a bag on a string at 18 m. It was a yellowish brown colour and reasonably thick on the surface when compared with the S. ficus species. The spicules indicated that this sponge was a Myxilla species, most likely M. incrustans.

3.1.1. Standing crop of sponges Table 1 shows the results of the survey of sponge biomass present on cultured Chlamys opercularis. This showed that there was significantly greater sponge on scallops found at 18 m than at 14 m. However, there were no differences between 18 and 10 m or between 14 and 10 m. In particu-

lar, there was more sponge on scallops at 18 m on string 2 than at 14 or 10 m and more sponge on scallops at 18 m on string 2 than on strings 1 and 3. When the percentage of scallop shells which had sponge was compared using the 2'2 test a significant difference was found in the numbers of scallops with sponge compared with those without sponge on string 2. When the data for all the strings was combined there were no significant differences. Overall the average mass of sponge per scallop was 0.15 g and the percentage of scallops with sponge was 24.3%. With an annual production of three million scallops this would mean that the farm was capable of producing 1093.5 kg of sponge. If sponge was attached to every shell it would be possible to produce 4500 kg of sponge. It was noticeable that the sponge collected from scallops held in pearl nets and from the sea bed was much thicker than the sponges on scallops in bags on strings

3.2. Cell culture Holding the S.f. rubrus tissue in seawater with antibiotics at 4~ greatly reduced contamination problems at later. Gentamicin sulphate (0.5 mg ml-~ final conc.) in the sterile filtered seawater was better than penicillin and streptomycin (100 units of penicillin and 0.1 mg streptomycin m l final conc.). The various dissociation methods gave mixed results. Chemical dissociation did not work particularly well. Lower numbers of cells were collected with this method compared with the others attempted. In addition the cells did not attach well and those that did attach did not appear to be sponge cells; contamination by protistan cells such as amoebae and flagellates was

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common. This may have been due to most of the sponge tissue used for chemical dissociation not being treated with antibiotics at 4~ before dissociation. Mechanical dissociation produced an abundance of cells. However, when the cells were inoculated into culture on some occasions there was a problem with the cells aggregating together again. In this state they tended not to stick to the base of the petri dish. Most of the cells that remained separated or in small number groups did adhere to the petri dish. Removing the cortex of the sponge before dissociation greatly reduced the contamination problems. Spontaneous dissociation was the most successful method of dissociation. When the tissue was examined in the plate wells, cells that had emerged from the tissue were obvious. When the sponge tissue was removed, the ceils appeared to remain healthy. However, when the cells were subcultured into a larger culture vessel the cells did not replicate sufficiently to fill these dishes. The survival of sponge cells after inoculation into different media is summarised in Table 2. When seawater with penicillin and streptomycin was used as the medium there was little evidence of growth by sponge cells. Amoeboid cells did grow and subculture of these cells was possible in this medium. These may have been amoebocyte cells from the sponge; however, they appeared very similar to the amoeba, N e o p a r a m o e b a sp. as noted by Custodio et al. (1995). Bacterial contamination was also a problem. The addition of

serum to this medium did not have a great effect on the cultures except to increase the growth of contaminating bacteria. The use of gentamicin sulphate as the antibiotic greatly decreased problems with contamination as did the incubation of sponge tissue with antibiotics prior to dissociation. The addition of Leibovitz to the medium enhanced cell survival. Seawater, serum, Leibovitz and gentamicin sulphate used with spontaneous dissociation producing the best results. Occasionally there were problems due to contamination with fungus so the addition of amphotericin to mechanically dissociated cells was attempted. However, this appeared to damage many of the sponge cells as few cells were found in the culture dish after 48 h incubation. The use of media without serum was also tried but there was little proliferation by cells that had been spontaneously dissociated while mechanically dissociated cells did not attach. These differing results may in part be due to the different dissociation methods used in conjunction with each medium. In general, cultures that were maintained or subcultured were put into seawater and gentamicin sulphate with Leibovitz and serum. This appeared to allow the best survival of sponge cells, despite occasional contamination problems. However, addition of serum and Leibovitz caused increased problems with bacterial contamination and contamination with thraustochytrids. Therefore it was decided to use only seawater and gentamicin sulphate as the growth

Table 2 Sponge cell survival in various media following cell dissociation by a number of methodsa Medium

Dissociation method Chemical

Seawater + penicillin + streptomycin Seawater+ penicillin+ streptomycin+ serum Seawater+ penicillin+ streptomycin+ serum + Leibovitz Seawater+ gentamicin sulphate+ serum + Leibovitz Seawater+ gentamicin sulphate+ Leibovitz Seawater + gentamicin sulphate+ serum + Leibovitz+ amphotericin Seawater + gentamicin sulphate

Mechanical

Spontaneous

-

n.d.

n.d.

+ n.d. n.d. n.d. n.d. +b

n.d. + +

n.d. + + +/n.d. +

+, cells survived and in some cases proliferated; - , cells did not survive, + / - results were mixed; n.d., no data. b When chemical and mechanical dissociation were combined.

a

E. Armstrong et al. /Journal of Biotechnology 70 (1999) 163-174

medium until the cultures were free of other contaminants when the other nutritional sources could be added. This medium proved best at controlling contamination and the cells appeared able to cope in this environment, adhering well to the petri dishes. However, after a few weeks of incubation many of the cells died off and in some cases protozoan cells begin to dominate the culture. The last attempt at cell culturing during this project appeared to be the most successful. Sponge was held in antibiotics in seawater for 4 days, mechanically dissociated and then inoculated into seawater and gentamicin sulphate. Cells were still abundant after a few days. They appeared to have been attracted to each other and were forming colonies. In most cases only a single cell band was formed in the Percoll density gradients. However, on one occasion two cell layers were formed in the Percoll density gradient. Flagellate-like cells were collected in the 30-15% interface while all the other cells were collected in the 45-30% layer. The cells in this layer could not be distinguished as any particular types. The cells were washed twice in sterile seawater before they were inoculated into seawater with Leibovitz, serum and streptomycin and penicillin. However, there was little attachment by the few remaining sponge cells after incubation for 1 week. When Ficoll was used as a density gradient at low concentrations (11, 7, 5 and 2%), all of the cells sedimented to the bottom of the tube within 3 h. At the higher concentrations all of the cells remained in the upper layers, i.e. the 20 and 30% layers. Therefore, the tubes were centrifuged at 400 x g for 40 min. After centrifugation all the cells were at the bottom of the tube and a precipitate had formed, also in the bottom layer. When 100% Ficoll was used for the density gradient the cells formed a single layer part way down the tube. When this layer was examined closely there were faint layers above and below the main cell layer. However, it was difficult to collect these thinner layers without the thicker cell layer. These results suggest that the use of a pre-formed continuous gradient may be a more advantageous way to collect the different cell groups. Mechanically dissociated cells were spun on a Ficoll concentrate gradient at ca

171

11 000 • g for 35 min, allowing the continuous gradient to form simultaneously with cell separation. The result was a thick layer with a faint layer above. The faint layer was not well formed. However, this may be due to the lack of cells. As a large number of cells formed the thick layer this indicates that most of the sponge cells are of the same density. The collagen layer on the bottom of petri dishes did not improve the attachment of sponge cells. It was noted, however, that attachment on multiwell plates was better than on the base of cell culture petri dishes.

3.3. Sponge adhesion Tissue glue caused exothermic polymerisation reactions which effectively 'cooked' smaller pieces of sponge. Larger pieces of sponge were used and attached at one end only. It was hoped that the undamaged piece of sponge would attach to the scallop shell but within the 2 weeks that the glue held there was no evidence of sponge attachment. After 9 days sponge was still attached to the scallop shell on agar coated shells but there was no evidence of sponge or alginate on the alginate coated shells. After a further 5 days, two pieces of sponge were still attached to the agar-coated shells but there was no evidence of growth. The agar subsequently came off the shells. Agar and collagen mixed was no more successful. The agar did not remain attached for any longer than in the previous experiment and the collagen did not cause any enhanced ability for the sponge to attach to the scallop shells. Phytagel did not remain attached to the scallop shells for more than a few days. In addition, sponge cells did not stick to the Phytagel surface. In the experiments using adhesion promoter factors (collagen and poly-L-lysine), large pieces of sponge initially attached to the scallop shells but then most gradually fell off the shells. However, some were still present 1 month after initial attachment. Forty days after initial attachment it was noted that there appeared to be very small colonies of sponge appearing on some of the scallops. In the experiments using juvenile scallops, after 2 days there were still some large pieces

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E. Armstrong et al. / Journal of Biotechnology 70 (1999) 163-174

of sponge left on the scallop shells, but by 7 days this sponge had been lost. The shells were examined again after another 22 days. This time there were a few small pieces of sponge on some shells. After a further 20 days the shells were again examined. Eight of the 16 shells had small sponge colonies on them. They were still very small. In both cases (empty and juvenile shells) these small sponge colonies could have arisen from tiny pieces of sponge or a few cells attaching to the scallop shells which gradually grew. However, there is also the possibility that sponge could have grown on the shells from larvae brought into the aquarium by the water flow or have been present on the scallop shells in small amounts before being moved from the farm (juveniles only). Later examination of the shells showed that some pink/ purple sponge grew on two juvenile scallops. This growth must be due to larvae entering the tanks through the water supply. Preliminary results with smaller pieces of sponge in suspended culture using a similar method to that of Fell et al. (1989) showed that the sponge took a considerable time to establish but then grew 1 or 2 ram. The sponge then appeared to stop growing.

4. Discussion

Approximately one-third of the Chlamys opercularis growing on the scallop farm had sponges growing on them. This represents a sizeable amount of readily available sponge which can be repeatedly collected without any environmental impact. C. opercularis acts as a substrate for a number of sponge species. This in contrast with the King Scallop Pecten maximus, where no species of sponge were ever found. The latter species normally lies with its upper valve covered in silt and sand so it is not surprising that sponges are absent from free-living specimens. Juvenile P. maximus being grown on the scallop farm are, however, of the same size and in the same environment as the C. opercularis. This suggests that sponge larvae are actively choosing to settle on C. opercularis and to avoid P. maximus. Such choice may be based on olfactory cues and would be an

interesting topic for further study in relation to finding ways to increase sponge larval settlement on scallops. This specificity also suggests that the relationship between the sponges and the scallops is not casual. There have been a number of studies previously which have highlighted the benefit to scallops of having sponge on their shells, mostly as an anti-predator defense (Bloom, 1975; Forester, 1979; Chernoff, 1987; Pitcher and Butler, 1987; Pond, 1992). Sponges have also been found to protect oysters (Feifarek, 1987). As many scallops are mobile they can take the sponge to areas of good water flow where there is abundant food and may also help them evade predators such as nudibranchs. Bioactivity screening studies are underway on the major species of sponge found on the farmed scallops but it is probable that they have some form of chemical defense and thus may contain interesting natural products. The immediate technical objectives of this project are to increase the numbers of scallops protected by coverings of sponge and to increase the amount of sponge available for natural products research. If sponges could be persuaded to grow on Pecten maximus then this would make suspended culture of this valuable species more feasible. At present fouling problems prevent suspended culture to marketable size for this and some other slow-growing scallop species or ones in areas of especially rapid fouling (e.g. New Zealand: Hayden and Woods, 1997). For natural products investigations and exploitation the challenge is to produce more sponge and, possibly, to increase the yield of secondary metabolites from the sponges. This is not necessarily the same objective as in producing 10% cover of the scallops. The current study has shown that none of the methods studied to inoculate scallop shells with sponge would be feasible alternatives to natural settlement. They either simply do not work or would involve too much labour to economically inoculate three million juvenile scallops. In contrast, larval settlement costs nothing and the postlarvae grow strongly and rapidly. Producing large volumes of multipotent sponge cells in culture and

E. Armstrong et al. / Journal of Biotechnology 70 (1999) 163-174

encapsulating viable populations in a sticky organic matrix which can then be applied to the scallop shells may be technically possible. It may be the only way to coat Pecten maximus with sponge. However, the state of the art in sponge cell culture is still somewhat removed from this, and in any case it is still not likely to compete economically with natural settlement. To increase the number of scallops coated with sponge, therefore, methods need to be devised to increase the larval supply to the juvenile scallops and further research should concentrate on the larval biology of S.f. rubrus. If the sponges could be induced to spawn while being held in a structure that also contained bags of scallop spat, then high larval densities would be achieved, leading to higher rates of settlement onto the scallop shells. If the sponges had no alternative substrate to settle on, it would be interesting to see if they would colonise P. maximus shells. Increasing the yield of sponges or their natural products may be easier to obtain. One way of increasing the output of sponge tissue would be to culture the scallops in lantern or pearl nets. S.f. rubrus generally grew in a very thin sheet when in suspended culture bags at high densities and with limited opportunities for movement. When in lantern nets or pearl nets the sponges grew more thickly and more closely resembled the thicknesses of growth found on free-living scallops. Whether these differences are due to the physical confinement or reduced feeding opportunities in the bags is not clear. A total of 225000 scallops would need to be cultured in pearl or lantern nets to achieve the same production of sponge as was being obtained from the string-suspended scallops. Preliminary studies also suggest that production of secondary metabolites in S.f. rubrus is highly seasonal (unpublished results) with peak production coinciding with spring settlement of fouling larvae. Post-harvest treatment of sponge tissue may also be a practical method for increasing the yield of secondary metabolites. The cell culture studies reported here show that S.f. rubrus appears no easier to culture than other sponge species (Klautau et al., 1994; Pomponi and Willoughby, 1994; Leys, 1997). Primary cell lines are easy to obtain but no progress beyond this

173

was made during the study. Cell culture offers the potential of producing large amounts of secondary metabolites under very controlled conditions. It may also be the only way of achieving sufficient yields to allow clinical trials of secondary metabolites from rare or deepwater species. In the short term at least, it is unlikely that cell culture will be competitive with natural products derived from sponges grown as part of existing aquaculture efforts. It would appear sensible, therefore, to thoroughly investigate the potential use of such species in natural products research. This means screening metabolites produced throughout the year and looking carefully for known metabolites of existing interest. Even if these species are producing known metabolites at lower yields than other sponge species from which the metabolites were first isolated, the ease with which they can be produced and the low environmental impact may mean that it is more effective to pursue aquaculture of these species than try to subvert the problems of, for instance, obtaining sufficient metabolites from deepwater species. If useful and interesting compounds are found in sponges associated with existing aquaculture species, then this will encourage further research into how to maximise yield from these species.

Acknowledgements This work was supported by grants from Highlands and Islands Enterprise and Highland Council and a European Union C R A F T Exploratory award to Biomar S.A. (MA-ST-6057).

References Barthel, D., Theede, H., 1986. A new method for the culture of marine sponges and its application for experimental studies. Ophelia 25 (2), 75-82. Bloom, S.A., 1975. The motile escape response of a sessile prey: a sponge-scallop mutualism. J. Exp. Mar. Biol. Ecol. 17, 311-321. Chernoff, H., 1987. Factors affecting mortality of the scallop Chlamys asperrima (Lamarck) and its epizooic sponges in South Australian waters. J. Exp. Mar. Biol. Ecol. 109, 155-171.

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Custodio, M.R., Imsiecke, G., Klautau, M., Rinkevich, B., Rogerson, A., Mfiller, W.E.G., 1995. Evolution of cell-adhesion systems-evidence for arg-gly-asp-mediated adhesion in the protozoan Neoparamoeba aestuarina. J. Eukaryotic Microbiol. 42, 721 - 724. Feifarek, B.P., 1987. Spines and epibionts as antipredator defenses in the thorny oyster Spondylus americanus Hermann. J. Exp. Mar. Biol. Ecol. 105, 39-56. Fell, P.E., Knight, P.-A., Rieders, W., 1989. Low-salinity tolerance of and salinity-induced dormancy in the estuarine sponge Microciona prolifera (Ellis et Solander) under longterm laboratory culture. J. Exp. Mar. Biol. Ecol. 133, 195-211. Forester, A.J., 1979. The association between the sponge Halichondria panicea (Pallas) and scallop Chlamys varia (L.): a commensal-protective mutualism. J. Exp. Mar. Biol. Ecol. 36, 1-10. Fusetani, N., Hiroto, H., Okino, T., Tomono, Y., Yoshimura, E., 1996. Antifouling activity of isocyanoterpenoids and related compounds isolated from a marine sponge and nudibranchs. J. Nat. Toxins 5 (2), 249-259. Garson, M., 1994. The biosynthesis of sponge secondary metabolites: why it is important. In: van Soest, R.W., van Kempen, T.M.G., Braekman, J.-C. (Eds.), Sponges in Space and Time. A.A. Balkema, Rotterdam, The Netherlands, pp. 427-440. Hayden, B., Woods, C., 1997. Scallops are particular about their culture conditions. Aquaculture Update (NIWA) 19, 4-5. Henrikson, A.A., Pawlik, J.R., 1995. A new antifouling assay method: results from field experiments using extracts of four marine organisms. J. Exp. Mar. Biol. Ecol. 194, 157-165. Ilan, M., Contini, H., Carmeli, S., Rinkevich, B., 1996. Progress towards cell cultures from a marine sponge that produces active compounds. J. Mar. Biotechnol. 4, 145149.

Klautau, M., Custodio, M.R., Borojevic, R., 1994. In vitro culture of primary cell lines from marine sponges. In: van Soest, R.W., van Kempen, T.M.G., Braekman, J.-C. (Eds.), Sponges in Space and Time. A.A. Balkema, Rotterdam, The Netherlands, pp. 401-406. Leys, S.P., 1997. Sponge cell culture: a comparative evaluation of adhesion to a native tissue extract and other culture substrates. Tissue Cell 29 (1), 77-87. Osinga, R., Tramper, J., Wijfteb, R.H., 1998. Cultivation of marine sponges for metabolite production: applications for biotechnology? Trends Biotechnol. 16, 130-134. Pitcher, C.R., Butler, A.J., 1987. Predation by asteroids, escape response, and morphometrics of scallops with epizoic sponges. J. Exp. Mar. Biol. Ecol. 112, 233-249. Pomponi, S.A., Willoughby, R., 1994. Sponge cell culture for production of bioactive metabolites. In: van Soest, R.W., van Kempen, T.M.G., Braekman, J.-C. (Eds.), Sponges in Space and Time. A.A. Balkema, Rotterdam, The Netherlands, pp. 395-400. Pond, D., 1992. Protective-commensal mutualism between the queen scallop Chlamys opercularis (Linnaeus) and the encrusting sponge Suberites. J. Molluscan Studies 58, 127134. Sol6-Cava. A.M., Thorpe, J.P., 1986. Genetic differentiation between morphotypes of the marine sponge Suberites ficus (Demospongiae: Hadromerida). Mar. Biol. 93, 247-253. Uriz, M.J., Becerro, M.A., Tur, J.M., Turon, X., 1996. Location of toxicity within the Mediterranean sponge Crambe crambe (Demospongiae: Poecilosclerida). Mar. Biol. 124, 583-590. Zilinskas, R.A., Colwell, R.R., Lipton, D.W., Hill, R.T., 1995. The Global Challenge of Marine Biotechnology: A report on the United States, Japan, Australia and Norway. Maryland Sea Grant Publication, College Park, MD.

i

i

IOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 175-183

Photoheterotrophy in the production of phytoplankton organisms B.J.B. W o o d a

a,*

P.H.K. Grimson a J.B. German

b,

M. Turner c

Department of Bioscience and Biotechnology, Royal College Building, University of Strathclyde, George Street, Glasgow, Scotland GI 1XW, UK b Department of Food Science and Technology, University of California at Davis, Davis, CA, USA c C.C.A.P., Scottish Association for Marine Sciences, Oban, Scotland, UK

Received 9 October 1998; received in revised form 30 November 1998; accepted 22 December 1998

Abstract Interest is growing in algae as sources of medicinal and other potentially useful compounds, as well as their use in fish rearing. We are interested in their production of polyunsaturated fatty acids (PUFA). Photoautotrophic growth gives the highest levels of unsaturation in the fatty acid pool, but biomass concentrations are low. Heterotrophy on sugars gives higher biomass but seems to give more saturation in the fatty acids. In freshwater algae acetate has proved to be a good carbon source for photoheterotrophic growth, giving a crop with reasonably high levels of PUFA. In addition it is possible to regulate acetic acid addition through the pH change as acetate is used up in a well-aerated system, so achieving high biomass yields in the presence of relatively low acetate concentration. When we attempted to extend this to marine algae (principally species used in fish farming), we found that acetic acid was ineffective or sometimes toxic to most species tested, even at high pH. However, glycerol stimulated growth in a number of the algae. We report on this stimulation, and on the fatty acid composition of the resulting algal crop, discuss the problems in regulating the addition of this metabolite to algal cultures, and speculate on applications in the production of other useful algal metabolites. We also show that some of these algae used in fish farming grow best when the salinity of the water is rather less than that found in standard sea water. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Acetic acid; Algae; Autotrophy; Fish farming; Glycerol; Lipid composition

1. Introduction Abbreviations: CCAP, Culture Collection of Algae and Protozoa, Oban, Scotland; UTEX, University of Texas algal collection strain number; FA, fatty acid; FAME, Fatty acid methyl ester; GLC, gas-liquid chromatography. * Corresponding author. Tel.: +44-141-548-2555; fax: + 44-141-553-1161. E-mail address: [email protected] (B.J.B. Wood)

When algae are grown with carbon dioxide as their sole carbon source, they depend upon light for all their energy, and for the production of biomass. Consequently the biomass levels (sometimes referred to as the 'standing crop', especially in studies on the phytoplankton levels in natural

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00070-X

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B.J.B. Wood et al. , Journal o f Biotechnology 70 (1999) 175-183

water bodies) are rather low when compared with those found in the more usual types of microbial bioprocess. These limitations are due to mutual shading, which limits light penetration into the culture; to rapid depletion of carbon dioxide; to the need to restrict both light input (to prevent overheating) and stirring (to limit physical damage to the algal cells). These considerations place constraints on what can be done with algae. In producing algae for fish farming, the earlier stages of algal production are carried out under strict asepsis, but the volumes of liquid which must be handled in the last stage(s) require that the cultivation is carried out in containers such as plastic bags, which make strict asepsis impossible. This compromise is satisfactory for fish farming, where the algae are being used for feeding the fish, often via intermediate grazing animals, such as rotifers. Even so, the liquid volumes restrict the amounts of algal biomass which can reasonably be produced. There is now an increasing awareness that algae are actual or potential sources of a wide range of interesting organic compounds. Our particular interests relate to the polyunsaturated fatty acids (PUFA) found in algae. Phytoplankton algae are thought to be a major source of the PUFA in fish oils. The nutritional and other applications of PUFA are very important, and, while some PUFA are also sourced from higher plants, some, such as eicosapentaenoic acid (EPA) are principally derived from fish oils. At present there is a particular interest in PUFA for supplementing 'formulas' used in infant feeding, and it is suggested that manufacturers of these preparations will need to look to algal sources of PUFA in the future. Thus there is a need to develop ways to grow plankton algae in quantity. Some, such as the freshwater members of the genus Chlorella, can grow on sugars (and other carbon sources) fully heterotrophically, that is to say aerobically in the dark, with the organic substrate acting as the source of both energy (through respiration) and carbon. In photoheterotrophic, or myxotrophic, growth, organisms use light as an energy source, and an organic compound such as a sugar as carbon source. This type of metabolism is proba-

bly significant where very high standing crops of algae are seen in places such as oxidation ponds for sewage treatment. Some undoubted algae, such as the diatom Nitzchia alba, have abandoned photosynthesis altogether, with loss of chlorophyll, while reports of algae with intact pigment systems being found in places where there is no significant light suggest that some algae can interchange between various nutrition modes as the conditions dictate. Maciasr and Eppley (1963) reported that Chlamydomonas mundana, isolated from a sewage treatment plant in the Mojave Desert, grew rapidly using acetate as carbon source in photoheterotrophic metabolism. Eke and Wood (1987) and other workers have shown that the media and conditions which Maciasr and Eppley developed for axenic culture of their alga will also support dense cultures of various freshwater species of Chlamydomonas, Chlorella, Scenedesmus and other algae. The increase in pH as acetate is used up can, in a system which is well aerated to remove carbon dioxide, be used to control further addition of acetic acid to the culture, so further increasing the biomass which can be attained, but without problems being created from excessive concentrations of salts of acetic acid, or from the toxicity of the free acid. Various studies of the effects which growth conditions have on the fatty acid composition of algae have shown that fully heterotrophic growth substantially reduces PUFA levels, and that even photoheterotrophic growth on substrates such as sugars produces some reduction in PUFA levels (Wood, 1988). Other sources cited in that same review indicate that photoheterotrophy on acetate can result in PUFA levels comparable with those found in the same organism when it is growing fully autotrophically. Initially we hoped to apply this type of work directly to marine phytoplankton species. However, in many cases acetate either did not stimulate algal growth, or even seemed to be toxic, despite the high pH of the marine salts medium being used. The significance of the last point lies in the suggestion that acetic acid toxicity results from the un-ionized molecule, which can penetrate the cell membrane and damage the cell

B.J.B. Wood et al. /Journal of Biotechnology 70 (1999) 175-183

interior by ionizing there. These were references in the literature to glycerol being used as a carbon source by various algae, and this compound is also noted for its role in osmotic regulation of the saline alga Dunaliella salina. The present report details some preliminary studies comparing marine algal growth, on acetate, glycerol and glucose, and on medium unsupplemented with organic carbon sources, and the fatty acid composition of the resulting biomass.

2. Materials and methods

2.1. Algae Algae used in these studies were purchased as pure cultures from the United Kingdom Culture Collection of Algae and Protozoa. They were maintained on Provasoli's marine algal medium (Sigma). Wherever possible stock cultures were kept as surface growth on this medium solidified with 1.5% agar, otherwise the cultures were maintained as liquid cultures in 50-ml portions of liquid medium in 250-ml conical flasks incubated at ambient temperature under constant illumination from an overhead bank of cool white fluorescent lights, with shaking, until a light but definite growth had developed. Once satisfactory growth had developed the stock cultures were stored in a refrigerator in the dark until required for subculture (at monthly intervals for cultures not currently being used in experiments).

2.2. Experimental conditions Inocula for experiments were prepared from stock cultures by inoculation into 50-ml portions of the basic Provasoli medium, then incubated as above until a definite growth had developed. Any infection of these cultures resulted in discoloured cultures, but was also checked for from time to time by plating onto nutrient agar plates, prepared both normally and in the presence of Provasoli's marine salts mixture. Media for studies on photoheterotrophic growth were prepared from the basic marine

177

medium supplemented with the appropriate level of organic compound before autoclaving. Nitrate (as the sodium salt) or ammonia (as the chloride) were similarly added. There are objections to autoclaving marine media, particularly after supplementations such as those used in the present experiments, but in practice no changes such as precipitate formation or colour development were observed, and algal growth was generally satisfactory, except as noted in the results, where the poor growth was a result of a specific supplementation. All experiments were conducted in 50-ml portions of the appropriate medium contained in 250-ml conical flasks plugged with foam plastic, sterilised by autoclaving for 15 min at 121~ then inoculated as soon as possible after cooling to ambient temperature. Inoculation was with 5-ml portions of a culture grown on the basic marine salts medium. Flasks were then incubated as described above. The shaker was set to operate at 90 cycles per minute. Light intensity was not completely uniform over the area of the shaker platforms, but was typically about 500 ~E m -2 s-~. Unfortunately there was no facility for cooling the incubator room, but in practice its temperature stayed around 25~ Equipment constraints meant that it was not practical to monitor the growth of organisms by a daily sampling routine. Consequently a given set of flasks, inoculated with the organism selected for that particular study, were incubated until the growth in those flasks where significant growth was developing had reached a reasonable level to give a harvest sufficient for subsequent lipid analysis, but before the organisms were showing changes in colour or other indications that the cultures were becoming senescent. All the flasks in a particular experiment were harvested at the same time, growth was estimated as described below, then the cell crops were immediately harvested at the centrifuge, lyophilised, and stored in closed containers in a freezer until required for lipid extraction. Typically incubation lasted for 7-10 days from inoculation to harvest, depending on the organism.

B.J.B. Wood et al. / Journal of Biotechnology 70 (1999) 175-183

178

2.3. Cell growth A comparative estimate of cell growth within a particular experiment, at the time selected for harvesting the set of cultures, was obtained by thoroughly mixing the contents of each flask (including dispersing any cell material growing on the flask wall), then measuring the optical density of the suspension at 750 nm, using a 1-cm pathlength cuvette. Attempts to obtain a more precise estimate by counting cell numbers in an haemocytometer were thwarted by the tendency of some algae to aggregate into small clumps.

2.4. Lipid analysis

on a fused-silica column. This polar phase is not ideal for FAME analyses, but gave reasonably satisfactory results in the present application. Programmed temperature operation was used, ramping at 1.5~ min-~ to a final 220~ Peaks were identified by comparison of retention times with a standard FAME mixture (Sigma FAME Mixture # 189-19); this is not satisfactory for identifying the fatty acids in a sample of unknown composition, but the FA composition of the algae being studied here is previously recorded (Wood, 1988) and the present study examines only changes in relative proportions of them.

3. Results and discussion

Lyophilised cell material was extracted with chloroform:methanol (2:1) mixture, cell debris removed by centrifugation and the liquid extract treated according to Folch et al. (1957). The chloroform extract was freed of solvent under vacuum and the lipid extract was converted to fatty acid methyl esters (FAME) by adding 1 ml of methanolic boron trifluoride reagent (Sigma), incubating for 30 min at 100~ in a closed tube under nitrogen, cooling and extracting the FAME into hexane. GLC was in a capillary system using Chrompack BP20 (TM) as the coating material

3.1. Cell growth Optical density measurements (means of measurements on samples from duplicate culture flasks) show marked differences between the various cultivars in their responses to different carbon sources (Table 1). The Nannochloropsis strains seemed to be particularly sensitive to acetate inhibition, and none of the organisms tested here was stimulated by it to the extent that some freshwater species are. It will be interesting to determine the

Table 1 G r o w t h of algae on various carbon and nitrogen sources a Carbon source

Nitrogen source

1

2

3

4

5

6

7

8

None

None Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate

0.36 0.19 0.20 0.44 1.02 -

0.30 0.30 0.25 0.31 0.26 -

0.85 0.09 0.10 1.03 0.93 -

0.31 1.03 0.73 0.52 0.67 1.28 1.08 0.85 1.10

1.45 1.56 1.11 1.24 1.42 1.69 1.49 1.62

0.62 0.81 0.35 0.52 0.86 0.96 0.73 0.89

1.29 1.25 1.53 0.09 0.09 1.39 1.44 1.23 1.64

1.04 1.01 0.68 0.17 1.32 1.25 1.02 1.18

Acetate Glycerol Glucose

a Readings are optical densities. Numbers at the top of columns identify organisms as follows: (1) Rhodomonas reticulata, preliminary experiment; (2) Chlorella C C A P strain 211/75, preliminary experiment; (3) 'Chlorella' strain UTEX2341; this is almost certainly a species of Nannochloropsis, preliminary experiment; (4) Rhodomonas reticulata, there were differences in colour of the cultures, depending on the conditions, with the cultures supplemented with either glycerol or glucose being a rust red, whereas the ones without organic carbon had a somewhat yellow appearance when viewed against the colour of the other cultures. Acetate-supplemented cultures were intermediate in colour; (5) Porphyridium purpureum; (6) Cyclotella cryptica CCAP1070/2; (7) Nannochloropsis strain CCAP879/5; (8) Nannochloropsis strain CCAP211/78, this organism was grown in half-strength sea water.

179

B.J.B. Wood et al. / Journal of Biotechnology 70 (1999) 175-183 1.20 m

1.00

~

~

m

/

.~ 0.80

m

/

o.60

0 o.40

/ /

0.20

q)

\ N

/ j

/

w~

0.00

\

S

D

/

~~,,,

.-

--x.\-.,,

q

,b

,

4= 1.6

3.3

s.~

8.5

~2

~5.5

~8.9

n4

~.o

~.4

~.o

~.3

T o t a l s a l t s g l "~

Fig. 1. Effect of salts concentration on growth of algae: (. . . . . . . ) Nannochloropsis CCAP211/78; (----) Cyclotella cryptica" ( - - - - ) ) Porphyridium purpureum" ( - - - - ) Chlamydomonas plethora'( ...... ) Chlorella UTEX2341.

Nannochloropsis CCAP879/5; (. . . . . . .

effect of acetate on other brackish water species. The marine alga Dunaliella salina was also sensitive to acetate (Eke and Wood, unpublished data). Stimulation by glycerol, although never very strong in this series of experiments, is evident in some cases. From looking at the flasks as the experiments progressed, an impression was formed that the stimulation by glycerol and glucose may have been more marked at the beginning of the growth cycle, with laggard cultures catching up as the experiment progressed, but this was a purely subjective observation which needs to be tested by properly designed experiments. Glycerol seemed to be utilised better than glucose by Nannochloropsis, Rhodomonas and Cyclotella. A useful attribute of acetic acid in work on freshwater species was the ability to use the increase in pH as it was metabolised and so removed from the medium, to control further addition of this nutrient. Doing something similar with glycerol would be attractive, as it would keep the actual concentration of the nutrient in the culture medium low, so possibly helping to ensure that light energy was used by the alga, and possibly predisposing it to a metabolism more like that of a fully autotrophic organism. Other studies (Wood, 1988) have shown that the extent of un-

saturation found in lipids is influenced by the concentration of sugars where these are present in media used for photoheterotrophy. The higher the sugar concentration, the more saturated the fatty acid profile. Clearly, however, pH is not available as a control here. If a glycerol-sensitive probe were available this might provide an answer, but on present knowledge the most reasonable suggestion is control through some kind of turbidostat. An examination into the effects of salinity on the growth of some organisms was undertaken in the expectation that Chlamydomonas plethora would grow best in the middle salinity range, as it did, but that the other species would grow best at the higher ranges of salinity. The latter expectation was confounded by the results obtained (Fig. 1); clearly these need confirmation, but seem to be reasonably internally consistent, showing that the algae being tested all grew best at salinities in the middle range of those tested. This could have significance for the production of algae for fish farming and other applications. Before using the lower salinities to produce algae for these applications there is a need to examine the nutritional value of algae grown at these intermediate salinities. If, as seems reasonable, the reduced salinity does not affect their composition significantly, these results suggest that productivity of algal

B.J.B. Wood et al./Journal of Biotechnology 70 (1999) 175-I83

180

cultures for fish farming could be improved by choosing the optimal salinity for growing these organisms. The data in Table 2 do, however, suggest that the proportion of the fatty acids represented by the most highly unsaturated ones is sensitive to salinity, with the highest salinity giving the best proportion of the putative 22:6

acid. This is in agreement with the results of Seto et al. (1984) who worked with an organism described as Chlorella minutissima, although this is more likely to have been a Nannochloropsis were it to be available for reassessment by more modern taxonomy.

Table 2 Effects of medium salt content on some fatty acids of the marine Chlorella strain CCAP 211/75 a

These are presented in Tables 2-6. They are too few in number and scope to permit drawing any very definitive conclusions, but some observations may be made. On the whole the fatty acid composition seems to be relatively indifferent to changes in the growth environment, (except the result in Table 2 which was noted above) which, if supported by more extensive analyses, is useful for those interested in growing algae, as it suggests that optimising conditions for biomass production (in terms of weight per unit volume) may not be too damaging in its effects on the quality of the biomass. It could even be that beneficial effects may be secured in some cases; for example Table 6 seems to suggest that nitrogen source is significant, with cells grown on nitrate giving higher levels of the acid tentatively identified as 20:6, than do otherwise comparable cultures supplied with ammonia. In some cases (e.g. Table 3 and Table 4, for growth on glucose) using nitrate as nitrogen source seems to affect the balance of the fatty acids in the palmitate (16 carbon atoms) group. Some data in Table 6 suggest possible effects of growth conditions on proportions of the more highly unsaturated acids, but the representation of currently unidentified acids in that set of experiments is such that it is difficult to produce reliable conclusions from it. Turning now to a somewhat more detailed consideration of the individual sets of fatty acid analyses. In Table 2, the most striking effect is on the proportions of palmitate (16:0) and the presumed 22:6 acid at the highest salt concentration examined here, with the former acid increasing in relative proportion as salinity increases, but then decreasing again at the highest salinity. Table 3 shows that the isolate UTEX 2341, which was inhibited by acetate but stimulated by glycerol (Table 1), showed decreases in 22:6 (tentative

Fatty acid

Total salts concentration (g 1 1.6

F A M E as % of total detected 16:0 20.5 16:2(?) 4.8 18:0 2.0 18:1 10.4 18:2 6.5 18:x(?) 18.0 22:6(?) 3.6

-i)

5.1

18.9

29.4

26.2 1.6 2.6 13.7 1.8 19.9 1.0

26.6 1.6 11.2 24.4 4.4

15.9 5.0 2.5 8.4 7.2 16.9 13.2

a Note: all identifications are very tentative, being based on a comparison of retention times with those of a single reference mixture, using a single type of chromatography column. Those marked with '(?)' are even more so, being based on interpolation, with the appropriate F A M E not being present in the reference mixture. Table 3 Effect of carbon and nitrogen sources on content of some fatty acids in 'Chlorella' (Nannochloropsis) strain UTEX 2341 a Fatty acid

Medium Basal ammonia

Glycerol + nitrate

F A M E as % of total detected 16:0 23.7 32.2 16:1 1.4 10.1 18:0 0.8 0.9 18:1 7.5 2.3 16:x(?) 4.2 20.6 18:2 1.9 (?) 18.0 7.5 20:2(?) 3.1 2.8 20:4 2.3 20:x(?) 1.1 22:6(?) 3.8 2.4 a See footnote to Table 2.

Glycerol + ammonia

29.1 30.7 1.4 6.3 1.2 2.8 1.1 0.8 3.7 5.3 -

3.2. Fatty acid profiles

B.J.B. Wood et al. / Journal of Biotechnology 70 (1999) 175-183

181

Table 4 Effect of carbon and nitrogen sources on content of some fatty acids of the diatom Cyclotella cryptica, strain CCAP 1070/2 a Fatty acid

Medium Basic + ammonia

F A M E as % of total detected 16:0 10.2 16.1 52.5 16:2(?) 4.0 16:x(?) 1.5 20:x(?) 8.4 24:x(?) -

Basic + nitrate

Glycerol + ammonia

Glucose + ammonia

Glucose + nitrate

16.0 46.9 5.1 1.7 8.6 3.4

10.3 26.8 1.7 27.5 5.1 1.6

14.4 25.3 1.3 31.5 3.3 1.5

15.6 49.5 4.4 1.5 8.8 3.2

a See footnote to Table 2. Table 5 Effect of carbon and nitrogen sources on content of some fatty acids of the green alga Nannochloropsis strain CCAP 879/5 a Fatty acid

Medium Basic

Basic+ammonia

F A M E as % of total detected 16:0 35.3 35.0 16:1 39.0 35.9 18:0 1.0 2.0 18:1 7.0 6.1 16:x(?) 0.9 18:2 1.2 1.9 18:x(?) t 20:4 1.9 1.0 20:x(?) 5.5 2.4

Glycerol+ammonia

Glycerol +nitrate

Glucose+ammonia

Glucose+ nitrate

34.2 29.1 1.4 4.2 9.8 1.7 1.3 1.6 4.2

34.2 22.2 1.1 3.5 15.2 1.7 2.0 1.5 3.5

33.3 23.4 1.7 3.8 14.2 2.0 1.6 1.0 2.1

31.4 28.8 1.2 3.9 8.6 1.9 t 2.3 7.2

a See footnote to Table 2.

identification) when grown in the presence of glycerol, but increases in the other compounds thought to be polyunsaturated acids in the 20-carbon group, and a striking increase in a possible polyunsaturated member of the palmitate family when grown on glycerol and nitrate. An unidentified FAME which chromatographed just after linoleate was an important component of the profile in the autotrophically grown cells, but was less significant in the cultures provided with glycerol. The diatom Cyclotella cryptica was somewhat inhibited by acetate, stimulated by glucose, more strongly stimulated by glycerol, and grew better on nitrate than on ammonia as nitrogen source irrespective of the carbon source being employed. Its fatty acid composition seemed to be affected principally in the degree of unsaturation

of the palmitate (C-16) group of acids, with the other FAME showing smaller variations with changes in carbon and nitrogen sources. The extent to which its growth was stimulated by supplementation by organic carbon was greater than had been anticipated, and demonstrates the need for more analyses of fatty acid profiles of this alga, which has a place in fish farming. The Nannochloropsis isolate CCAP 879/5, a representative of a genus widely used in fish farming, was very sensitive to acetate, with the cultures showing no significant growth and bleaching of the cells present in the inoculum, and did not seem to be very much affected either way in its growth by supplementation with glucose or glycerol, unlike the strain 211/78 which showed stimulation by glycerol (Table 1). Fatty acid profiles of strain

B.J.B. Wood et al./Journal of Biotechnology 70 (1999) 175-183

182

Table 6 Effect of carbon and nitrogen sources on content of some fatty acids of the green alga Chlamydomonas plethora C C A P 11/86A grown on half-strength sea water a Fatty acid

Medium Basic + ammonia

Basic+nitrate

F A M E as % of total detected 16:0 37.6 41.3 16:1 13.1 17.3 18:0 0.9 1.2 18:1 2.3 7.3 14.9 0.9 18:2 1.1 4.3 3.0 t 20:4 2.8 4.2 20:6(?) 3.8 15.0 O.D. 1.06 0.76

Glycerol + ammonia

Glycerol + nitrate

Glucose + ammonia

Glucose + nitrate

Acetate + ammonia

40.2 20.1 1.5 6.4 3.3 3.2 t 3.9 12.5 1.02

41.8 15.9 1.9 8.2 2.0 4.3 t 3.8 13.9 0.84

37.5 18.7 3.5 9.1 10.4 2.6 1.8 2.5 3.7 1.12

37.8 10.9 1.9 4.0 2.8 10.6 2.0 7.2 2.4 6.0 1.11

31.4 6.5 2.1 13.4 11.3 23.3 2.3 1.07

9

a See footnote to Table 2. Analyses are from cells grown at a salinity corresponding to half the strength of seawater. O.D., optical density as a measurement of relative growth.

879/5, however, showed differences in cells grown on different carbon sources, with the increases in the unidentified acid tentatively assigned to 16:x being particularly noticable (Table 5). This is somewhat similar to the results obtained with the diatom (Table 4) and with the UTEX strain 2341 (which is now thought to be a Nannochloropsis). On the other hand the acids thought to belong to the C-20 series appeared to be somewhat less sensitive to influence by the carbon and nitrogen sources which the cells were offered, as were acids of the stearate (C-18) series. The changes in acid 16:x were largely compensated for by countervailing changes in palmitoleate (16:1). Finally, the green Chlamydomonas plethora, originally isolated from brackish river water, showed much higher unsaturation of the C-20 series of fatty acids than do freshwater species of this genus (Wood, 1988) or the marine isolates examined by Chuecas and Riley (1969), and the addition of organic carbon sources to the medium seem to have had effects on the balance of fatty acids, even though effects on cell crop (as judged by the final optical density of the cultures) were small. Indeed, if the results in Table 6 are supported by more rigorous analyses, this alga might be a useful source of acids in this group. These results are so unexpected that

they must be treated with considerable caution pending a more exacting analysis backed up by methods such as argentation TLC, or GLC combined with mass spectroscopy. Overall the results to date serve mainly to underline the need for more rigorous analyses, which it is hoped to carry out in the near future. An additional matter which will be examined in future studies will be the distribution of the fatty acids of interest within the various lipid classes, which could have significance for producing enriched supplements of the essential fatty acids.

Acknowledgements Grateful thanks to Ms Lynn Curran for all her help with recalcitrant GLC equipment and performing the relevant analyses. Thanks also to Ms Elizabeth Clements for all the help with producing the Ms. The initial work which resulted in our undertaking the present study was carried out while B.J.B.W. was on Sabbatical leave at the University of California's Davis Campus; the Biochemical Society and the Carnagie Trust for the Universities of Scotland are thanked for their financial help toward this Sabbatical leave.

B.J.B. Wood et al. / Journal of Biotechnology 70 (1999) 175-183

References Chuecas, L., Riley, J.P., 1969. Component fatty acids of the total lipids of some marine phytoplankton. J. Mar. Biol. Assoc. UK 49, 97-1.1/5. Eke, L.O., Wood, B.J.B., 1987. Algal growth on acetic acid. Br. Phycol. J. 22, 314. Folch, J., Lees, M., Stanley Stone, G.M., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509.

183

Maciasr, F.M., Eppley, R.W., 1963. Development of EDTA media for the rapid growth of Chlamydomonas mundana. J. Protozool. 10, 242-246. Seto, A., Wang, H.L., Hesseltine, C.W., 1984. Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima. J. Am. Oil Chem. Soc. 61, 892-896. Wood, B.J.B., 1988. Lipids of algae and protozoa. In: Ratledge, C., Wilkinson, S.G. (Eds.), Microbial Lipids, vol. 1. Academic Press, London, pp. 807-868.

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JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 185-192

Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii Martin E. de Swaaf, Theo C. de Rijk, Gerrit Eggink, Lolke Sijtsma * Agrotechnological Research Institute (ATO-DLO), Bornsesteeg 59, Box 17, 6700 AA Wageningen, The Netherlands

Received 13 October 1998; received in revised form 16 November 1998; accepted 22 December 1998

Abstract

The heterotrophic micro alga Crypthecodinium cohnii was cultivated in media containing glucose, yeast extract and sea salt. Increasing amounts of yeast extract stimulated growth but influenced lipid accumulation negatively. Sea salt concentrations above half the average seawater salinity were required for good growth and lipid accumulation. C. cohnii was able to grow on a glucose concentration as high as 84.3 g 1-~, although concentrations above 25 g l decreased the growth rate. Comparison of growth at 27 and 30~ showed that the higher incubation temperature was more favourable for growth. However, lipid accumulation was higher at the lower incubation temperature. In a bioreactor the biomass concentration increased from 1.5 to 27.7 g 1-1 in 74 h. In the final 41 h of the process the lipid content of the biomass increased from 7.5 to 13.5%. In this period the percentage of docosahexaenoic acid of the lipid increased from 36.5 to 43.6%. The total amounts of lipid and docosahexaenoic acid after 91 h were 3.7 and 1.6 g 1-1, respectively. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Bioreactor; Crypthecodinium cohnii; Cultivation; Docosahexaenoic acid; Marine; Medium composition

I. Introduction

~-Linolenic acid (18:3), eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) are members of the omega-3 family of fatty acids. In recent years the interest in these longchain polyunsaturated fatty acids (PUFAs) has increased significantly due to their recognition as being beneficial for human health. They have been reported to play a role in prevention or treatment * Corresponding author. Tel.: + 31-317-475324; fax: + 31317-475347. E-mail address: [email protected] (L. Sijtsma)

of a variety of diseases such as arteriosclerosis (Dyerberg, 1986; Mehta et al., 1987), thrombosis (Urakaze et al., 1986), arthritis (Kremer et al., 1985) and several types of cancer (Braden and Carroll, 1986; Reddy and Maruyama, 1986). In addition, D H A is regarded to be essential for the proper visual and neurological development of infants (Nettleton, 1993). D H A is an important P U F A in human breast milk but is generally absent from infant formulas (Huisman, 1996). At present, several infant food manufacturers are interested in adding D H A into infant food products (Barclay et al., 1994).

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S01 68-1656(99)00071-1

186

M.E. de Swaaf et al./Journal of Biotechnology 70 (1999) 185-192

The traditional source of omega-3 fatty acids is fish oil. However, the use of fish oil as a food additive is limited due to problems associated with its typical fishy smell, unpleasant taste, and poor oxidative stability. Furthermore, the presence of EPA in fish oil is undesirable for application in infant food (Carlson, 1996). Therefore, alternative sources are of interest. The heterotrophic micro alga Crypthecodinium cohnii is an interesting source for DHA production (Kyle, 1996; Kyle et al., 1992) and for research on DHA biosynthesis (Beach et al., 1974; Henderson et al., 1988; Bell and Henderson, 1990; Henderson and Mackinlay, 1991) due to its unique fatty acid composition. C. cohnii can accumulate relatively high amounts of lipid ( > 20%) with 30-50% DHA of the fatty acids and no other polyunsaturated fatty acids present above 1% (Harrington and Holz, 1968; Beach and Holz, 1973). So far, a limited number of studies on growth of C. cohnii have been reported (Beach and Holz, 1973; Tuttle and Loeblich, 1975; Vazhappilly and Chen, 1998) and the media used are not applicable in large-scale cultivations as they are expensive and very laborious to prepare. In this report culture media were used which better meet the requirements of the bioprocess industry. The influence of medium components and culture conditions on biomass formation, lipid accumulation and DHA content during batch growth of C. cohnii was studied in shaken flask and bioreactor experiments. 2. Materials and methods

2.1. Chemicals Yeast extract was obtained from Oxoid. Structol sb2022 and structol j673 were obtained from Structol. Silicone SE-2 was obtained from Boom. Glucose-monohydrate was obtained from Merck. Sea salt, antifoam 204, c/s-4,7,10,13,16,19-docosahexaenoic acid methyl ester (99%), butylated hydroxy toluene (BHT) ( > 99%) and triheptadecanoic acid glycerate (17:0) (99%) were obtained from Sigma. Methyl docosanoate (22:0) ( > 99.5%) was obtained from Fluka.

2.2. Media, maintenance of cells and cultivation conditions All medium components were separately heat sterilised (12 ! ~ C. cohnii (ATCC 30772) cells were grown in standing cultures (50 ml in 250-ml shake flasks) on standard medium (9 g 1-~ glucose, 2 g 1yeast extract and 27.8 g 1-1 sea salt) at 25-27~ in the dark. The standing cultures were subcultured within 4 weeks for maintenance or used within 2 weeks as inoculum for precultures. Shaken flask cultivations (precultures and shaken flask experiments), containing 50 ml medium in 250-ml shake flasks, were carried out at 27~ pH 6.5, 100 rpm in a linear shaker. The cultures were inoculated with 10% (v/v) inoculum, unless otherwise indicated. Temperature effects on growth and lipid content were determined at 27 and 30~ in duplicate cultures. Precultures for shaken flasks experiments were grown on standard medium and precultures (100 ml in 500-ml shake flasks) for inoculation of batch cultivations in a bioreactor were grown on medium containing 27 g 1-1 glucose, 3.9 g 1yeast extract and 27.8 g 1-1 sea salt. The precultures were incubated for 3-4 days. In shaken flask experiments the cells were harvested after 50 h incubation, unless otherwise indicated. Batch cultivations were performed at 27~ in 14 laboratory bioreactors (Applikon). The dissolved oxygen tension was kept above 30% of air saturation by automatically increasing the stirrer speed (starting at 100 rpm) and flushing with 1.0 1 m i n - ' filter-sterilised air. The pH was kept constant at 6.5 +_ 0.1 by automatic addition of 2 M HC1. The foam was controlled by automatic addition of 5% (w/v) silicone SE-2. The medium contained 84.3 g 1- ~ glucose, 11.5 g 1- 1 yeast extract and 30.6 g 1 - ' sea salt. The inoculum was 20% (v/v), corresponding to 1.5 g 1-~ biomass. 2.3. Determination of biomass concentration and

optical density The biomass concentrations of culture samples (0.5-10 ml) were determined using cellulose ac-

M.E. de Swaaf et al. / Journal of Biotechnology 70 (1999) 185-192

etate filters (pore size 0.45 ~tm, Orange Scientific). After removal of the medium by filtration, the filters were washed with demineralised water and heated at 500 W in a microwave oven for 5 min (Samsung electronics, type M 9245). The optical density (OD) was measured at 470 nm (Pharmacia Biotech, Ultrospec 2000 UV/Visible Spectrophotometer).

2.4. Lipid analysis Culture samples for lipid analysis from shaken flask or bioreactor cultivations were harvested by centrifugation for 5 min at 1500 x g and washed once in demineralised water. Prior to and after freeze drying, the samples were stored at -20~ In order to determine the oil content of small amounts of biomass, the direct transmethylation by a modified procedure of Grayburn et al. (1992) was used. Freeze-dried cells (20-100 rag) were weighed accurately into a Pierce Reacti-Vial and 2 mg triheptadecanoic acid glycerate (17:0) was added as internal standard. Two ml of 1% H2SO4 in methanol were added, the vial was vortexed and heated to 80~ for 2 h. After cooling 2.0 ml chloroform and 1.0 ml water were added. The vial was vortexed and centrifuged at 1500 x g. The lower layer containing the fatty acid methyl esters (FAME) was stored at 4~ prior to gas chromatographic analysis. The amount of lipid was calculated from the total amount of fatty acids and the internal standard. Although this method is useful for determination of the lipid content in small samples, it appeared less useful for DHA analysis due to a relative large variability (Christie, 1973). This variability was not observed in the analysis of the other fatty acids present in C. cohnii. For larger amounts of biomass the oil was extracted from freeze-dried cells by a modified method of Bligh and Dyer (1959). Freeze-dried cells (100 mg or more) were weighed accurately into a 10-ml centrifuge tube. For extraction, 3 ml chloroform:methanol (2:1) containing 1.0 mg ml-1 methyl docosanoate (22:0) and 0.5 mg m l BHT was used and the tube was shaken gently overnight. After centrifugation at 1500 • g for 5 min, the supernatant containing the extracted oil was stored at 4~ until analysis.

187

Methyl esters of the fatty acids present in this Bligh-Dyer extract were prepared with trimethylsulphonium hydroxide according to Butte (1983). Fatty acid methyl esters were analysed on a Carlo-Erba GC. A 25 m • 0.25 mm i.d. (dr = 0.20 lain) Chrompack CP-58 CB column with helium (5.0 quality) as carrier gas. The column temperature was raised from 150 to 250~ at 10~ min-1, and kept at 250~ for 10 min. Samples of 1 ~tl were injected at a temperature of 275~ Detection was performed with a flame ionisation detector (FID) at a temperature of 280~ Percentage values of the lipid content of the biomass and the fatty acid composition have been expressed on a weight by weight basis. For identification purposes GC/MS was performed on a Carlo-Erba Mega GC linked to a QMD-1000 Mass Spectrometer by a direct interface, using the same column and temperature program. Positive ion electron-impact mass spectra (70 eV) were obtained at a source temperature of 200~ The scan range was 25-375 amu with a scan rate of 1 s-1. Peaks were identified by comparison of their retention times with authentic references and by comparison of the mass spectra with the spectra present in the Wiley/NIST Combined Library, 6th ed.

2.5. Glucose determination The glucose concentrations in the culture supernatants were determined with the GOD-PAP method (Trinder, 1969) (Boehringer Mannheim).

3. Results and discussion

3. I. Shaken flask cultivations 3.1.1. Agitation Growth of C. cohnii was improved significantly by enhancing the agitation speed from 50 to 100 rpm (Fig. 1), probably due to an enhanced oxygen supply. After 50 h growth, the culture grown at 100 rpm had reached a maximum OD of 4.7. This value was more than four times higher than the OD found for the culture grown at 50 rpm. The

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188

glucose was entirely depleted after 50 h growth at 100 rpm. This resulted in a decrease of the OD in the following period of incubation. Microscopic examination revealed no detrimental effects of agitation at 50 or 100 rpm, which is in contrast with the observations of Tuttle and Loeblich (1975) who found that rotary agitation above 40 rpm in flasks killed the C. cohnii cells. The importance of aeration conditions for the growth of C. cohnii was also indicated by Beach and Holz (1973) who showed that cells with an enhanced supply of O2, from shaken flask and bioreactor cultures multiplied more rapidly than cells grown with a restricted supply of oxygen.

3.1.2. Temperature The temperature optimum for C. cohnii is 27~ (Ishida, 1968). However, for large-scale cultivations a higher cultivation temperature is desirable, due to an improved cooling capacity at higher temperatures. Therefore, growth and lipid accumulation at incubation temperatures of 27 and 30~ were compared. At 27 and 30~ the optical densities of the cultures after 50 h growth were 4.7 and 5.8, respectively. Apparently, growth was more stimulated at the higher incubation temperature. Also, the lipid accumulation was clearly affected by the incubation temperature. When grown at 27~ the lipid content was 13.0% compared to 7.8% at 30~ In contrast, Beach and Holz (1973) found a higher lipid content in C. cohnii cells grown at 31.5~ (11%) than at 25~

0

I

0

....

50

I

100

150

time (h) Fig. 1. Growth curves of C. cohnii shaken at 50 (11) and 100 (71) rpm. Optical densities (OD) were measured at 470 nm.

(8.7%). These authors, however, harvested the cultures grown at 31.5 and 25~ at different cultivation times (after 4 and 2 days, respectively). This difference in time of harvest may strongly affect the lipid content. Not only the lipid content but also the percentage of DHA of the lipid was influenced by the incubation temperature. At 27~ the percentage of DHA was 35.9% compared to 40.4% at 30~

3.1.3. Carbon sources In order to determine suitable carbon sources, growth and lipid accumulation (after 50 h incubation) of C. cohnii grown on different carbon sources were studied. C. cohnii was able to grow on glucose and galactose. After 50 h incubation cultures grown on glucose and galactose had reached comparable OD values of 4.0 and 3.8, respectively. The lipid content of cells grown on glucose was 13.4%, whereas cells grown on galactose contained less, 11.4%. The DHA content of the lipids was similar (35.2% with glucose and 36.2% with galactose). No or marginal growth (less than one doubling) was observed when glycerol and sucrose were offered as carbon sources. 3.1.4. The influence of yeast extract, sea salt and glucose on growth and lipid accumulation For medium optimisation, the influence of variable amounts of glucose, yeast extract and sea salt on growth and total lipid content of C. cohnii was studied. C. cohnii was cultivated in a medium composed of 9 g 1-1 glucose, 27.8 g 1-~ sea salt and a range of yeast extract concentrations (0-10 g 1-1). With increasing yeast extract concentrations the resulting OD values after 50 h growth increased (Fig. 2a). The lipid content of the cells, however, decreased markedly at higher yeast extract concentrations. At a yeast extract concentration of 1 g 1-1, for example, an OD of 3.8 was reached, whereas the lipid content of the biomass was 20%. At a 5-g 1-~ yeast extract concentration, the OD increased 1.5 times to 5.8, but the lipid content was only 6%. These results are in agreement with the literature as in general lipid accumulation in micro-or-

M.E. de Swaaf et al./Journal of Biotechnology 70 (1999) 185-192 (a) 8

30 25

6

20 ~ O .,...

~4

15~ 10.~_ .,.

2 5 0

I

0

I

2

|

I

4 6 yeast extract (g I1)

j

8

10

(b) 8

30 25

O

0 4

e~ 10 ~.& 5 0

0

I

I

5

10

I

I

15 20 sea salt (g 11)

I

25

0

30

Fig. 2. Effect of different yeast extract and sea salt concentrations on O D (I-q) and the percentage of lipid of the biomass ( 9 after 50 h of incubation. (a) C. cohnii was grown on media containing 9 g 1- ~ glucose, 27.8 g 1- ~ sea salt and 0 - 1 0 g 1yeast extract. (b) C. cohnii was grown on media containing 9 g 1-~ glucose, 2 g 1-~ yeast extract and 2.8-27.8 g 1-~ sea salt. Optical densities (OD) were measured at 470 nm.

ganisms is stimulated by an excess of a carbon source and a limitation in one of the other nutrients, especially nitrogen (Leman, 1997). The effect of sea salt was studied in media containing 9 g 1-1 glucose, 2 g 1-~ yeast extract and sea salt concentrations, ranging from 2.8 to 27.8 g 1-1. Both growth and lipid accumulation were stimulated by increasing salinity. At 2.8 and 17.8 g 1 - 1 s e a salt the OD values after 50 h growth were 1.8 and 4.2 and the amounts of accumulated lipid were 6 and 16%, respectively (Fig. 2b). A higher salinity (27.8 g 1-~) as compared to 17.8 g 1-1 resulted in similar growth and lipid accumulation. Therefore, for optimal growth and lipid accumulation a minimal sea salt concen-

189

tration of 17.8 g 1-1 is required, which is about half of the average sea water salinity. The observation of growth inhibition at low salinity is in agreement with previous data. Inhibition of growth at low ( < 5 g/1 NaC1) and also high salinity ( > 50 g/1 NaC1) has been reported in shaken (Beach and Holz, 1973) and standing (Tuttle and Loeblich, 1975) cultivations of C. cohnii. For large-scale cultivation processes, the sea salt concentration should preferably be as low as possible in order to prevent corrosion problems. To study growth on high initial glucose concentrations, C. cohnii was incubated with 25, 50 or 75 g 1-~ glucose in the presence of 5 g 1-~ yeast extract and 17.8 g 1-1 sea salt. The highest OD values were reached with the highest glucose concentration (Fig. 3). The initial growth rate, however, decreased at concentrations above 25 g 1-~ glucose. Average doubling times between 16 and 40 h growth on 25, 50 and 75 g 1- ~ glucose were 10, 12.5 and 13.8 h, respectively. This inhibitory effect of high glucose concentrations on growth of C. cohnii has not been described before and is important for further development of batch and fed-batch cultivation modes for the production of DHA by C. cohnii.

3.1.5. The influence of different antifoaming agents on growth In order to study the influence of antifoaming agents on growth of C. cohnii four types of antifoam (antifoam 204, structol sb2022, structol j673 and silicone SE-2) were added to the standard medium at a concentration of 50 lag 1-~. In shaken flask cultivations only silicone SE-2 did not show detrimental effects on growth and lipid content. Therefore, this agent was selected as a suitable antifoam for high cell density cultivations. 3.2. Bioreactor cultivations Initial cultivations of C. cohnii in a 1-1 bioreactor showed that cells were able to grow at a stirrer speed as high as 800 rpm. Furthermore, the doubling time of C. cohnii, when grown on standard medium in a bioreactor, was 8.4 h. This doubling

M.E. de Swaaf et al./Journal of Biotechnology 70 (1999) 185-192

190 25

100

30

20

80

25 oo

20 ' ~

~0 60

15~ 10.~ ._=. 5

20

0

,=,

0

50

100 time (h)

150

200

Fig. 3. Effect of different glucose concentrations on growth of C. cohnii. Medium composition: 5 g 1-~ yeast extract, 17.8 g 1-1 sea salt and glucose, 25 (O), 50 ( 9 and 75 ( I ) g 1-~ Optical densities (OD) were measured at 470 nm.

time does not significantly differ from the one measured in shaken flask experiments (8.7 h) with standard medium. In order to study the possibility of high cell density cultivation of C. cohnii in a batch cultivation, and to follow the process of lipid accumulation and fatty acid composition during a growth cycle, a cultivation in a bioreactor was performed. Logarithmic growth was observed within the first 43 h of incubation. The average doubling time in the logarithmic growth phase was 13.3 h, which is similar to the doubling time of 13.8 h found for shaken flask experiments with 75 g 1glucose. After 43 h, the growth rate slowly decreased until growth completely ceased after about 74 h (Fig. 4). At this time the glucose was not entirely depleted. The maximal biomass concentration obtained was 27.7 g 1-l, which is the highest biomass concentration described so far for a batch cultivation of C. cohnii (Beach and Holz, 1973; Kyle et al., 1992). Although the biomass concentration did not further increase after 74 h growth, the glucose concentration continued to decrease, from 16.3 to 8.6 g 1-~ in the final 17 h. The lipid content increased from 7.5% at 50 h, to a maximum value of 13.5% at 91 h. This pattern of lipid accumulation fits the general theory that microbial lipid accumulation is a biphasic process (Leman, 1997). In the first phase rapid cell division occurs until a nutritional component be-

~

0

20

40

60 time (h)

80

0 100

Fig. 4. Growth and lipid content of C. cohnii grown in a 1-1 bioreactor. Glucose (in g 1- 1) (O), biomass (in g 1 - l ) ( I ) and the percentage of lipid of biomass ( 9

comes limiting. In the second phase, the growth rate decreases and lipids start to accumulate. DHA (22:6) was the major fatty acid in C. cohnii during the final 41 h of incubation (36.543.6%) (Table 1). Also abundant were the saturated fatty acids 16:0 (16.9-19.9%), 14:0 (16.6-18.6%), 12:0 (4.6-5.7%) and the monounsaturated fatty acid 18:1 (9.6-10.1%). The fatty acids 10:0, 16:1 and 18:0 were present in smaller amounts ( < 2.4%). Interestingly, the percentage of DHA steadily increased from 36.5 to 43.6% between 50 and 91 h. The percentages of other fatty acids remained at a constant level or decreased slightly. This pattern of DHA accumulation in the total lipid fraction was not observed in the study of Beach and Holz (1973). In their data the total fatty acid composition was not mentioned, but the fatty acid composition of both triglycerides and phosphatidylcholine (which accounted for 60% of the total lipid) were similar after 1, 2 and 4 days of growth. The maximal amounts of lipid and DHA (at 91 h) were 3.7 and 1.6 g 1-~, respectively. The average lipid and DHA productivities (at 74 h) were 46 and 19 mg 1-~ h - l , respectively. In order to produce DHA in a cost-effective process, research should especially focus on an increase of volumetric productivity. Rough cost estimations (Sijtsma et al., 1998) showed that in biocultivations with a relatively low productivity, the fixed costs are the most important cost factor

M.E. de Swaaf et al./Journal of Biotechnology 70 (1999) 185-192

191

Table 1 Major fatty acid composition of the total lipid content (%) of C. cohnii grown in a 1-1 bioreactor at 50, 67, 74 and 91 h growth Time (h)

10:0

12:0

14:0

16:0

16:1

18:0

18:1

22:6

50 67 74 91

1.5 1 1 0.8

5.7 5.7 5.4 4.6

16.8 18.6 17.6 16.6

19.9 18.5 17.0 16.9

0.5 1.3 1.3 1.3

2.3 1.6 1.5 1.6

10.1 9.9 9.6 9.6

36.5 38.3 41.1 43.6

of the total costs per unit of product. An increase in productivity would decrease the relative contribution of these fixed costs, and therefore significantly decrease the total product costs. The productivity can be increased by increasing the final biomass concentration, the lipid content of the biomass and the DHA content of the lipid or by decreasing the total process time. This study showed that at least two improvements of the production process of DHA by C. cohnii are possible. Firstly, from shaken flask cultivations it was concluded that C. cohnii cells can contain at least 20% lipid. Such percentages should also be possible in a bioreactor. Secondly, the total process time can be decreased. In a fed-batch cultivation for example, the glucose concentration can be kept below a critical value thereby preventing inhibition of growth.

Acknowledgements This work was financially supported by the European Community (FAIR CT 97-3146).

References Barclay, W.R., Meager, K.M., Abril, J.R., 1994. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. Appl. Phycol. 6, 123-129. Beach, D.H., Holz, G.G., 1973. Environmental influences on the docosahexaenoate content of the triacylglycerols and phosphatidylcholine of a heterotrophic, marine dinoflagellate, Crythecodinium cohnii. Biochim. Biophys. Acta 316, 56. Beach, D.H., Harrington, G.W., Gellerman, J.L., Schlenk, H., Holz, G.G., 1974. Biosynthesis of oleic acid and docosahexaenoic acid by a heterotrophic marine dinoflagellate Crythecodinium cohnii. Biochim. Biophys. Acta 369, 16-24.

Bell, M.V., Henderson, R.J., 1990. Molecular species composition of phosphatidylcholine from Crypthecodinium cohnii in relation to growth temperature. Lipids 25, 115-118. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911. Braden, L.M., Carroll, K.K., 1986. Dietary polyunsaturated fat in relation to mammary carcinogenesis in rats. Lipids 21,285-288. Butte, W., 1983. Rapid method for the determination of fatty acid profiles from fats and oils using trimethylsulphonium hydroxide for transesterification. J. Chromatogr. 261, 142145. Carlson, S.E., 1996. Arachidonic acid status of human infants: influence of gestational age at birth and diets with very long chain n-3 and n-6 fatty acids. J. Nutr. 126, 10921098. Christie, W.W., 1973. Lipid Analysis. Pergamon Press, Oxford, pp. 85-102. Dyerberg, J., 1986. Linolenate-derived polyunsaturated fatty acids and prevention of atherosclerosis. Nutr. Rev. 44, 125-134. Grayburn, W.S., Collins, G.B., Hildebrand, D.F., 1992. Fatty acid alteration by a 5-9 desaturase in transgenic tobacco tissue. Bio/Technology 10, 675-678. Harrington, G.W., Holz, G.G., 1968. The monoenoic and docosahexaenoic fatty acids of a heterotrophic dinoflagellate. Biochim. Biophys. Acta 164, 137-139. Henderson, R.J., Mackinlay, E.E., 1991. Polyunsaturated fatty acid metabolism in the marine dinoflagellate Crypthecodinium cohnii. Phytochemistry 30, 1781 - 1787. Henderson, R.J., Leftley, J.W., Sargent, J.R., 1988. Lipid composition and biosynthesis in the marine dinoflagellate Crypthecodinium cohnii. Phytochemistry 27, 1679-1683. Huisman, M., 1996. Effects of early infant nutrition and perinatal exposure to PCBs and dioxins on neurological development. Ph.D. Thesis. Rijksuniversiteit Groningen, Groningen, The Netherlands. Ishida, Y., 1968. Physiological studies on evolution of dimethyl sulfide from unicellular marine algae. Mem. Res. Inst. Food Sci. Kyoto Univ. 94, 48-82. Kremer, J.M., Bigauoette, J., Michalek, A.V., Timchalk, M.A., Lininger, L., Rynes, R.I., Huyck, C., Zieminski, J., Bartholomew, L.E., 1985. Effects of manipulation of dietary fatty acids on clinical manifestations of rheumatoid arthritis. Lancet 1, 184-187.

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Kyle, D.J., 1996. Production and use of a single cell oil which is highly enriched in docosahexaenoic acid. Lipid Technol. 8, 107-110. Kyle, D.J., Sicotte, V.J., Singer, J.J., Reeb. S.E., 1992. Bioproduction of docosahexaenoic acid (DHA) by microalgae. In: Kyle, D.J., Radledge, C. (Eds.), Industrial Applications of Single Cell Oils. American Oil Chemists" Society. Champaign, IL, pp. 287-300. Leman, J., 1997. Oleaginous microorganisms: an assessment of the potential. Adv. Appl. Microbiol. 43. 195-243. Mehta, J., Lopez, L.M., Wargovich, T., 1987. Eicosapentaenoic acid: its relevance in atherosclerosis and coronary artery disease. Am. J. Cardiol. 59, 155-159. Nettleton, J.A., 1993. Are n-3 fatty acids essential nutrients for fetal and infant development? J. Am. Dietetic Assoc. 93, 58-64. Reddy, B.S., Maruyama, H., 1986. Effect of dietary fish oil on azoxymethane-induced colon carcinogenesis in male F344 rats. Cancer Res. 46, 3367-3370. Sijtsma, L., Springer, J., Meesters, P.A.E.P., de Swaaf. M.E., Eggink, G., 1998. Recent advances in fatty acid synthesis

in oleaginous yeasts and microalgae. Recent Res. Dev. Microbiol. 2, 219-232. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with alternative oxygen acceptor. Ann. Clin. Biochem. 6, 24. Tuttle, R.C., Loeblich, A.R., 1975. An optimal growth medium for the dinoflagellate Crypthecodinium cohnii. Phycologia 14, 1-8. Urakaze, M., Hamazaki, T., Soda, Y., Miyamota, M., Ibuki, F., Yano, S., Kumagai, A., 1986. Infusion of emulsified trieicosapentaenoyl-glycerol into rabbits--the effects on platelet aggregation, polymorphonuclear leukocyte adhesion, and fatty acid composition in plasma and platelet phospholipids. Thromb. Res. 44, 673-682. Vazhappilly, R., Chen, F., 1998. Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth. J. Am. Oil Chem. Soc. 75, 393-397. Wiley/NIST Combined Library, 1994, 6th ed., Wiley-Interscience, New York.

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Bi,otecbnology ELSEVIER

Journal of Biotechnology 70 (1999) 193-202

Long-chain n - 3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids" screening of isolates and optimisation of docosahexaenoic acid production R.D. Bowles a,1 A.E. Hunt a G.B. Bremer a,, M.G. Duchars b R.A. Eaton a School of Biological Sciences, UniversiO, of Portsmouth, King Henry Building, Portsmouth P01 2DT, UK b Zeneea LifeScience Molecules, PO Box 2, Belasis Avenue, Billingham TS23 1YN, UK

Received 8 October 1998; received in revised form 26 November 1998; accepted 22 December 1998

Abstract Following an isolation programme for thraustochytrids (marine fungoid protists) from three different locations, 57 isolates were screened for biomass, oil and docosahexaenoic acid production (DHA). Although a common fatty acid profile for the thraustochytrid isolates emerged, there was considerable variation in the DHA content of the oil. In some isolates from a cold temperate environment, DHA represented almost 50% of the total fatty acids present. Although isolates from a sub-tropical environment produced higher levels of biomass, with up to 37% (w/w) oil, the DHA fraction of the fatty acids was low. Cool temperate isolates gave intermediate values. Studies to optimise biomass and DHA production by manipulation of growth medium composition were carried out on a tropical strain. Results indicated that medium with a high C:N ratio stimulated DHA production. The use of such media in bioreactor cultivations gave maximum biomass, lipid and DHA content of 14 g 1-1, 78 and 25% (w/w), respectively. Optimum DHA production was 2.17 g 1-~ after 107 h cultivation. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Docosahexaenoic acid; Eicosapentaenoic acid; Marine; N - 3 polyunsaturates; Protist; Thraustochytrid

1. Introduction Abbreviations: ASW, artificial seawater; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl ester; PUFA, polyunsaturated fatty acids; SCO, single cell oil. * Corresponding author. Tel.: + 44-1705-842018; fax: + 441705-842070. E-mail address: [email protected] (G.B. Bremer) Present address: Department of Biological and Earth Sciences, Liverpool John Moore's University, Byrom St., Liverpool L3 3AF, UK.

Increasing awareness and u n d e r s t a n d i n g o f the i m p o r t a n c e of the n - 3 polyunsaturates, eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid ( D H A , 22:6), in h u m a n development, health and disease ( D r a t z and Deese, 1986; Simopoulos, 1991) emphasises the need for sources o f these molecules, other than from fish oils (Yongmanitchai and W a r d , 1989). A range o f au-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00072-3

194

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

totrophic and heterotrophic microbes has been assessed for potential commercial sources of EPA and DHA by various workers including Barclay et al. (1994), Lewis et al. (1998) and Vazhappilly and Chen (1998), and the results of many studies in this area have been reviewed by Singh and Ward (1997) and Ratledge (1998). Particular attention has been given to the thraustochytrids, a group of fungoid protists generally considered obligately marine with a specific requirement for Na + ions (Siegenthaler et al., 1967). Recent work by Nakahara et al. (1996), Yaguchi et al. (1997), Yokochi et al. (1998) examined the production of DHA by a species of Schizochytrium and the ultrastructure and lipid production of a species of Thraustochytrium have been investigated by Weete et al. (1997). Thraustochytrids have attracted attention since their lipid contains proportionately large quantities of DHA; in some strains over 50% of the fatty acid content can consist of DHA (Yokochi et al., 1998). Assessment of EPA and DHA synthesis by thraustochytrids (Kendrick and Ratledge, 1992; Bajpai et al., 1991a,b; Li and Ward, 1994) indicated some potential, but both biomass and lipid yields were low. Two patents (Barclay, 1992, 1994) have however been filed, detailing the cultivation of thraustochytrid strains to produce lipids containing EPA and DHA, and indeed PUFA derived from these organisms are now available commercially in aquaculture products and chicken feed. The largest potential market for microbial oils containing DHA is perceived to be as an additive to infant formulae as an essential fatty acid for brain and retinal development (Horrobin, 1995). Ratledge (1998) considered that the presence of significant quantities of EPA in the thraustochytrid oils so far assessed, precluded its use for this purpose. Eicosapentaenoic acid is considered contra-indicatory in breast milk substitutes, but Ratledge suggested strain selection as a means of overcoming this difficulty. Previous studies on polyunsaturated fatty acid (PUFA) production have involved experimentation with only a fraction of the potential diversity of thraustochytrids which are ubiquitous microorganisms and common members of marine pi-

coplankton communities (Naganuma et al., 1998). This study has therefore focused on two aspects. A sampling programme to obtain thraustochytrid cultures from a range of habitats and ecological niches was carried out; the biomass production and PUFA profiles of these strains were assessed. Secondly, experimentation, including growth in bioreactors was undertaken to further optimise the production of both biomass and DHA.

2. Materials and methods

2.1. Isolation and screening programme 2.1. I. Isolation and maintenance of cultures Three distinct ecological and climatic environments were selected for the isolation programme: (1) cold temperate littoral (59-61 ~ (2) cool temperate littoral (50-51~ and (3) sub-tropical mangroves (25-27~ Thraustochytrids were isolated from water samples, sediment samples, mangrove leaf tissue, algae and marine angiosperms onto a medium comprising: yeast extract (1 g l-l), peptone (1 g 1-1) and agar (10 g 1-1 ) prepared in 70% seawater. A 2-ml solution of streptomycin and penicillin G (300 mg of each antibiotic per litre) was added to the isolation medium to prevent bacterial contamination during isolation. Methods for isolation were direct plating and baiting with ~,-irradiated (246 kGy) pine pollen as previously described by Gaertner (1968), Fell and Master (1973), Raghukumar et al. (1992) and Bremer (1995). The axenic cultures derived from the isolation programme were maintained on nutrient agar slopes overlaid with sterile autoclaved seawater. 2.1.2. Biomass production Isolates were grown in 250-ml Erlenmeyer flasks with 50 ml of cultivation medium comprising: yeast extract (2 g 1-~), peptone (2 g 1-1) and glucose (5 g 1-l) in 70% natural seawater. The flasks were inoculated with 1 ml of a zoospore suspension harvested from the seawater overlaying cultures on agar plates. Three replicate flasks of each isolate were incubated at 25~ on an orbital shaker at 150 rpm for 72 h. The biomass

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

from each flask was harvested separately by centrifugation, washed with phosphate-buffered saline and re-centrifuged. Each biomass pellet was lyophilised overnight and the dry weight determined.

2.2. Lipid extraction and fatty acid analysis 2.2.1. From biomass For the screening programme, biomass from the thraustochytrid isolates was analysed by taking 15-25 mg of dried cells, weighed in 5 rnl Reacti-vials. Lipids were extracted with 1 ml chloroform and esterified overnight in 2 ml BC13/ methanol (12%, w/w) reagent at 65~ Deionised water (1 ml) was added and the chloroform layer removed and dried under nitrogen. The residue was re-extracted in 1.5 ml hexane and 1 ml of deionised water was added. The solvent layer was removed and filtered through a 0.45-m filter with the syringe containing Na2SO 4. Fatty acid methyl esters (FAME) were analysed using gas chromatography (HP5890, series II GC with HP7673 injector and HP3365 Chemstation) on polar column (DB23 30 m length, 0.25 mm diameter) using flame ionisation detection with helium as the carrier gas (14.5 psi). FAMEs were identified and quantified using standard fatty acids. Nonadecanoic acid was used as an internal standard. Chloroform and hexane used for extraction contained 10 ppm butylated hydroxy-toluene. Extraction and determination of lipids from the optimisation studies was carried out using cells harvested from flask cultures. Cells were weighed (usually 2-3 g wet weight or 1 g dry weight) and lipids were extracted using chloroform:methanol (2:1) following the method of Bligh and Dyer (1959). 2.2.2. From culture liquids and bioreactor broths Total lipid extraction of the biomass from the bioreactor cultivation broths was based on the method of Christie (1989), which is in current commercial use. A known volume (minimum 100 ml) of medium was passed at a pressure of 800 bar, through a Soavi pressure homogeniser kept chilled on ice. The broth was then homogenised using an Ultra-turrax homogeniser together with

195

three times the volume of hexane. The resulting emulsion was centrifuged at 600 rpm at 4~ for 5 min. The upper layer of hexane was removed, filtered through Whatman IPS paper into a preweighed flask the method then followed that of Bligh and Dyer (1959). The fatty acids were esterified to their corresponding methyl esters and analysed using a modification of the method described above. Petroleum ether was used as the suspending solvent and heptadenoic acid was used as an internal standard. Peroxide values of the lipids extracted from cells of strain G13 were assessed using a method based on British Standard BS684, section 2.14. Of the three extraction methods used in this study, chloroform followed by re-extraction with hexane, chloroform:methanol and hexane, the first of these methods is being followed in current studies.

2.3. Optimisation and cultivation studies 2.3.1. Optimisation of biomass and DHA yield The organism selected for these studies was strain G13 from the University of Portsmouth culture collection, a thraustochytrid strain resembling Schizochytrium mangrovei Raghu Kumar isolated in the tropics (2~ from recently fallen leaves of the mangrove tree Sonneratia alba L. The effects of carbon (as glucose) and nitrogen (as yeast extract and peptone) on lipid and DHA synthesis by G13, were examined using batch cultures in 250-ml Erlenmeyer flasks. Inocula of the test strain were prepared from a dense suspension of zoospores derived from a culture grown in liquid medium in shaken flasks at 25~ for 12 h. Experimental media and conditions are detailed with the relevant results. 2.3.2. Cultivation Cultivation studies were carried out using B. Braun (Germany) bench-top bioreactors (2 1 capacity). Inocula were prepared as described for flask cultures and a total of 300 ml of inoculum was used to inoculate 1500 ml of the experimental medium in the bioreactor. Artificial seawater (Tropic Marin, Aquarientechnik, Wartenburg, Germany) was used throughout, except for the series of cultivations where 20 g 1-~ sodium sul-

196

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

phate was used as a substitute source of sodium. The media constituents and the conditions employed in the bioreactor for each separate investigation are detailed with the relevant results.

3. Results and discussion 3.1. Screen&g p r o g r a m m e

Biomass p r o d u c e d under standardised culture conditions by 57 t h r a u s t o c h y t r i d isolates obtained

from the three distinct climatic environments, is presented in Fig. l a. The 20 isolates f r o m a sub-tropical location p r o d u c e d biomass values ranging between 710 and 3140 mg 1- ~ dry weight after 72 h cultivation. These values were considerably greater than the biomass p r o d u c e d by thraustochytrid isolates f r o m both the cool temperate and cold t e m p e r a t e locations. Variability in biomass p r o d u c t i o n between individual isolates from each climatic e n v i r o n m e n t was a consistent feature.

F

i

Q

dt,,q,m~ tmpmo I~JIIJLU rluITIUmf

0 o

-r

<

30 20 10

1

2

Y..T__'--

3

Isolate number

cod I~xnm

Fig. 1. Biomass and DHA produced by 57 selected thraustochytrid isolates collected from cold temperate, cool temperate and sub-tropical sites. Isolates were grown at 25~ for 3 days at 150 rpm in a medium containing 5 g 1- 1 glucose, 2 g 1- 1 yeast extract and 2 g 1-~ peptone prepared in 70% seawater. Mean biomass values (a) were measured as mg 1-~. Mean DHA values (b) were measured as percent area of DHA in the fatty acid profile. The position of columns in (b) is identical to the biomass values for isolates in (a).

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

The percentage of DHA in the total fatty acids present in each of the isolates is shown in Fig. 1b. The sub-tropical isolates gave a range of 4-34% (mean 16.6%) of total fatty acids, the cool temperate isolates 2-39.6% (mean 13.7%) and the cold temperate isolates 17-47% (mean 35.9%). Wide differences in values were recorded, but overall the mean percentage of DHA in the oil was higher in thraustochytrid isolates from cold temperate locations. In general it would appear that the cold water isolates contained the largest amounts of PUFA, a feature that has been noted for other organisms from low-temperature environments (Erwin, 1973). Although baseline biomass values for these isolates were low (Fig. l a), they were grown at 25~ which is a temperature far higher than would be encountered in their natural habitat. Further exploratory cultivations will be carried out at temperatures below 25~ and these will include studies to compare the DHA content in the cells with different phases of growth. The oil and DHA content of the biomass from the 57 isolates was calculated and these results are presented in Fig. 2a-c. Although the range of values for total lipid in dried cells from the subtropical isolates was 1.8-37.3% (w/w), the maximum value (37.3%, w/w) was well in excess of that of the other sub-tropical isolates, where the range was 1.8-18.0% (w/w) (Fig. 2c). In contrast there was less total lipid extracted from the cold temperate isolates (2.0-7.1%, w/w) (Fig. 2a) and the cool temperate isolates (1.6-13.6%, w/w) (Fig. 2b). Fig. 2 a - c also depicts the DHA content of the dried thraustochytrid biomass (solid bar). The maximal values for DHA in dried cell biomass of isolates from the three environments ranged between 2.5% (w/w) for cool temperate isolates and 7.8% (w/w) for sub-tropical isolates. The mean values for DHA and EPA content of the dried cells of isolates from the three environments are summarised in Table 1. From these data, the isolate which had the highest oil content was identified from each geographical area. These isolates and their fatty acid profiles are summarised in Table 2. Docosahexaenoic acid constituted 45.7% of the fatty acid in the cold temperate isolate number 9 (SW4/4)

197

20 18 14 12

-o 8 ,g

6

~g 4 ~ 2 0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17

Isolate number

20 18 16 14 12 10 8 6 4 2 0 1 2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20

Isolate number

20 18 ~

. . . . .

.

.

.

c 37.3 !, II[

0~ 1 2

3 4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20

Isolate number

Fig. 2. Mean percentage total oil and D H A in dried cells of thraustochytrid isolates collected from (a) cold temperate, (b) cool temperate and (c) sub-tropical sites, grown at 25~ for 3 days at 150 rpm in a medium containing 5 g 1-~ glucose, 2 g 1-~ yeast extract and 2 g 1-~ peptone prepared in 70% seawater. ([Z) % w/w total oil; (11) % w/w DHA. Isolate numbers in (a), (b) and (c) correspond to isolate numbers in Fig. 1.

which was close to the maximum (47%) recorded for any isolate from this environment. The DHA from the other two isolates constituted significantly lower fractions of the fatty acid, 13.6% in the cool temperate isolate number 1 (HSED/14) and 23.8% in sub-tropical isolate number 2 (M6W/2), respectively. In addition, isolate HSED/14 contained large amounts of palmitic acid (38.8% of the total fatty acids present).

R.D. Bowles et al. /Journal of Biotechnology 70 (1999) 193-202

198

The results of the isolation and screening programme confirmed earlier findings (Ellenbogen et al., 1969; Findlay et al., 1986; Barclay, 1992; Weete et al., 1997) that thraustochytrids have characteristic fatty acid profiles with n - 3 PUFA as a significant component. Docosahexaenoic acid represented the major component of the PUFA content in most isolates, particularly those from the cold temperate environment. All of the thraustochytrids synthesised the n - 6 PUFA arachidonic acid in varying amounts, mainly as a minor component of the PUFA but in other organisms levels may be higher (Singh and Ward, 1997). Although the n - 3 PUFA EPA was present in the oil produced by all the isolates except two, the EPA content was generally low, varying from 0.2 to 0.6% (w/w) of the dried thraustochytrid cells.

3.2. Optimisation and cultivation studies 3.2. I. Optimisation studies When grown in liquid basal medium at 24~ in shaken flask cultures, strain G13 exhibited a

growth rate (max) of 0.2 h - ~ and a doubling time (td) of 3.4 h. The yields of biomass, the DHA content of that biomass and the yield of DHA achieved over a 4-day growth cycle are shown in Table 3. These data represent the baseline for subsequent experimentation to improve biomass and DHA yields. Raising biomass levels by medium manipulation is an obvious first step in optimising DHA yields. Previous studies reviewed by Singh and Ward (1997) and Ratledge (1998), using batch cultures in shake flasks and bioreactors, indicated biomass production was stimulated by increased glucose levels in the medium. Table 4 provides data on the effect of increasing glucose concentration while keeping organic nitrogen sources low. The results indicate that an increase in glucose concentration up to 10 g 1-~ leads to a linear increase in biomass production, suggesting a limiting factor other than glucose concentration beyond that point. The highest production of lipid (65-66%, w/w) was observed in concentrations of 20 and 30 g 1-~ of glucose. Although the DHA

Table 1 Mean biomass production, lipid, DHA and EPA content and percent area DHA in fatty acid profiles of selected isolates of thraustochytrids Isolate group

Biomass production (mg 1- l )

Lipid in biomass (%, w/w)

DHA in biomass (%, w/w)

EPA in biomass (%, w,'w)

% Area DHA in fatty acid profile

Cold temperate Cool temperate Sub-tropical

113-673 30-1587 710-3140

2.0-7.1 1.6-13.6 1.8-37.3

0.4-3.2 0.2-2.5 0.2-7.8

0.2-0.5 0.2-0.6 0.2-0.6

17-47 2-40 4-34

Table 2 Mean values for fatty acids in three thraustochytrid isolates measured as a percent of fatty acid profile a Fatty acid

Cold temperate isolate (SW4/4)

Cool temperate isolate (HSED/14)

Sub-tropical isolate (M6W/2)

13:0 14:0 16:0 20:4 20:5 22:6 Others b

3.05 1.60 19.28 3.30 8.19 45.73 18.85

1.51 7.59 38.88 4.67 4.36 13.6 29.39

1.28 8.43 17.71 16.04 1.48 23.79 31.27

a 13:0, tridecanoic acid; 14:0, myristic acid; 16:0, palmitic acid; 20:4, arachidonic acid; 20:5, eicosapentaenoic acid; 22:6, docosahexaenoic acid. b Other fatty acids included: C15:0, C16:1, C18:0, C18:1, C18:2, C20:2 and C22:4.

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202 Table 3 Mean biomass and D H A production of thraustochytrid strain G13 grown at 24~ 1 g 1-1 yeast extract, 1 g 1-I peptone, 1 g 1-1 glucose and 27 g 1-1 ASW

199

for 4 days at 120 rpm in a medium containing

Time (h)

Biomass (mg 1-1)

D H A in biomass (%, w/w)

DHA production (mg 1-1)

24 31 48 55 96

35 870 809 525 769

0.51 1.68 1.17 0.95 1.31

1.83 14.58 9.47 5.00 10.08

__+9128 ___29 + 99 + 18 + 30

+ + + + +

0.06 0.08 0.65 0.26 0.09

Table 4 Mean biomass production, total lipid and D H A content of thraustochytrid strain G13 grown in a medium adjusted with different glucose concentrations, 1 g 1-1 yeast extract, 1 g 1-1 peptone and 27 g 1-1 ASW at 24~ for 4 days at 120 rpm Glucose (g 1-1)

Dry biomass (g 1-1)

Lipid in biomass (%, w/w)

DHA in lipid (%, w/w)

D H A production (g l - l )

30 20 10 5 1 0

2.93 + 0.02 2.39 + 0.37 2.27 + 0.10 1.16 __+0.06 0.12 + 0.0 No growth

65.0 + 14.01 66.0 + 0.0 36.0 + 20.5 3.7 + 0.10 Insufficient biomass -

30.2 31.2 32.8 35.8 -

0.575 0.492 0.268 0.015 -

+ + + +

0.2 0.1 0.4 2.3

Table 5 Mean biomass production, lipid and D H A content of thraustochytrid strain G13 grown at 24~ for 4 days at 120 rpm in media containing different concentrations of glucose, yeast extract (YE) and peptone: all media contained 27 g 1-1 ASW Medium (g 1-1)

Peptone (g 1-1)

Dry biomass (g 1-1)

Lipid in biomass (0/0, W/W)

D H A in lipid (%, W/W)

D H A production (g 1--l)

40 40 20 20 40 40 20 20

20 20 20 20 0 0 0 0

8.93 5.87 7.48 5.83 7.02 5.91 7.45 5.22

5.60 3.90 11.23 7.27 6.13 5.03 6.15 7.00

18.8 38.5 18.7 29.4 18.9 29.0 24.7 26.0

0.094 0.088 0.157 0.124 0.081 0.086 0.113 0.095

glucose/40 glucose/20 glucose/40 glucose/20 glucose/40 glucose/20 glucose/40 glucose/20

YE YE YE YE YE YE YE YE

___0.25 + 0.45 + 0.89 ___0.45 + 0.13 + 0.38 + 0.55 + 0.27

content was highest at 35.8% in cultures grown with 5 g 1-' glucose, the highest yield of DHA (0.575 g 1-~) was achieved by cultures grown in 30 g 1-~ glucose. In an attempt to investigate the factor limiting biomass production, further steps to optimise biomass and lipid production involved increasing the yeast extract and peptone content of the medium and therefore reducing the C:N ratio. The data from this experiment are shown in Table 5. For all culture regimes, the concentration of

+ + + + + + + +

0.0 0.76 0.54 0.58 1.52 0.32 0.25 0.20

+ + + + + + + +

0.0 4.2 0.5 1.2 0.0 2.6 1.2 1.1

yeast extract was the determining factor for increasing biomass production. Although the medium containing 20 g 1-~ glucose, 40 g 1-1 yeast extract and 20 g 1-1 peptone supported high biomass and produced the highest lipid in the biomass (11.2%), only a relatively low proportion of the lipid (18.7%) was present as DHA. The highest percentage of DHA (38.5%) was found in the lipid from cultures grown in 40 g 1-1 glucose, 20 g 1-~ yeast extract and 20 g 1-l peptone, but the lipid content in biomass from this medium

200

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

was the lowest achieved (3.9%, w/w) in all of the media combinations tested. The values for D H A production in Tables 4 and 5 indicate the significance of C:N ratio in stimulating fatty acid production by thraustochytrids. This observation concurs with the work of Yokochi et al. (1998), who recorded an increase in total fatty acid content of the cells as the concentration of the nitrogen source introduced into the medium was reduced.

60

~

20

T

,

24

48

0 0

, .... 72

,,

,.=,

96

120

96

120

Incubation time (h)

80 ~,-.~ ~ ~, 70

3" .40~ ~

30

=~

20

~'-i

10

IIO

0-,. 0

24

48

72

Incubation time (h) Fig. 3. Biomass, lipid and DHA production and glucose utilisation by thraustochytrid strain G13 maintained in a 2-1 bioreactor: (a) for 118 h with medium composed of 40 g lglucose, 20 g 1-~ yeast extract and 27 g 1-s ASW; (b) for 107 h with medium composed of 40 g 1-1 glucose, 5 g 1-1 yeast extract and 20 g 1-~ sodium sulphate. A total of 30 ml Mazu added over 96 h; aeration, 300 ml min-1; agitation, 750 rpm; temperature, 24~ pH values ranged between (a) 6.1 and 7.6 and (b) 5.8 and 6.4.

3.2.2. Cultivation studies

The biomass, lipid and D H A produced by strain G13 over a 120-h period of growth in a bioreactor are presented in Fig. 3a. The growth curve depicted is typical of several achieved under the conditions documented, i.e. 40 g 1-~ glucose, 20 g 1- 1 yeast extract, 27 g 1-~ ASW, 30 ml Mazu antifoam added over 4 days, 300 rnl min-1 aeration and 750 rpm agitation. Maximum biomass production (ca 14 g 1-1) was reached at 41 h, close to the point of glucose exhaustion from the medium. This was also the point at which the lipid content of the biomass reached a maximum of 56% (w/w). The D H A content of the lipid remained stable at around 28% (w/w) from 63 to 120 h cultivation. There was a slight but insignificant rise from pH 6.5 throughout the growth period. The medium used in the cultivation study was modified further by substituting the ASW with 20 g 1-1 sodium sulphate as a source of sodium, and the concentration of yeast extract was reduced from 20 to 5 g 1-~, thus establishing a high carbon:nitrogen regime. A representative result from a bioreactor cultivation of strain G13 in this medium is shown in Fig. 3b. The culture produced a maximum biomass of 14 g 1-~ at 24 h around the point of glucose exhaustion from the medium. At 41 h the lipid content of the cells was maximal at 78% (w/w) of which the D H A content was 18% of the lipid. Thereafter, there was some increase in the D H A content of the lipid and a maximum D H A yield of 2.17 g 1-1 was achieved at 107 h. Recent investigations to optimise D H A production have recorded yields of 4 g 1-~ (Yokochi et al., 1998) and 13.3 g 1-1 (Yaguchi et al., 1997) from a strain of Schizochytrium sp. This clearly indicates the potential for further work with the thraustochytrid group in order to maximise even greater yields. The findings from the investigations described here are, firstly, that although thraustochytrids contain high levels of PUFA, there is wide diversity with regard to the synthesis of individual fatty acids. The possibility for 'tailor made' single cell oil (SCO) may exist. Thraustochytrids from warmer climates such as sub-tropical mangroves

R.D. Bowles et al./Journal of Biotechnology 70 (1999) 193-202

produced more biomass (at 25~ but this was not generally associated with high levels of DHA in the fatty acids present in the cells. In contrast, the cold water isolates were low biomass producers under the same culture conditions, but produced consistently higher levels of DHA in the total fatty acids. To ensure an optimum fatty acid profile, emphasis should be placed on strain selection when considering the use of thraustochytrids for SCO production for a specific application. Results indicate that the highest DHA production occurred at the time of glucose exhaustion and that high levels might be enhanced, or prolonged, by fed-batch cultivation. Thraustochytrid oil was found to be a free-flowing triacylglycerol with an acceptable peroxide value of less than 5 meq of peroxide oxygen kg-~ of oil, both features of advantage for commercial uses.

Ratledge (1998) considered that, despite improvements in the efficiency of photobioreactors, it is doubtful whether the growth of microalgae in bioreactors could be scaled up to satisfy even a modest demand for SCO rich in n - 3 PUFA, and suggested that heterotrophic microbes might be a more productive source. Our findings and those of other researchers highlight one group of marine microbes with a commercial future.

Acknowledgements R.D.B. and A.E.H. were supported by Industrial CASE studentships from the BBSRC in collaboration with Zeneca LifeScience Molecules, Billingham UK. Staff at Zeneca LifeScience Molecules are thanked for their assistance during the periods of experimentation and in particular, Dr D. Byrom and Dr A. Cornish for valuable discussions. Prof. J.W. Fell of the Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Florida and Prof. G. Daniel and Prof. T. Nilsson of the Dept. of Forest Products, Swedish University of Agricultural Sciences, University of Uppsala are thanked for providing laboratory facilities.

201

References Bajpai, P., Bajpai, B.P., Ward, O.P., 1991a. Production of docosahexaenoic acid by Thraustochytrium aureum. Appl. Microbiol. Biotechnol. 35, 706-710. Bajpai, P., Bajpai, B.P., Ward, O.P., 1991b. Optimisation of production of docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC 34304. J. Am. Oil Chem. Soc. 68, 509-514. Barclay, W.R., 1992. Process for the heterotrophic production of microbial oils with high concentrations of Omega-3 highly unsaturated fatty acids. US Patent no. 5,130,242. Barclay, W.R., 1994. Process for growing Thraustochytrium and Schizochytrium using non-chloride salts to produce a micro-floral biomass having omega-3 highly unsaturated fatty acids. US Patent no. 5,340,742. Barclay, W.R., Meager, K.M, Abril, J.R., 1994. Heterotrophic production of long chain omega-3 fatty acids utilising algae and algae-like microorganisms. J. Appl. Phycol. 6, 123. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Bremer, G.B., 1995. Lower marine fungi (Labyrinthulomycetes) and the decay of mangrove leaf litter. Hydrobiologia 295, 89-96. Christie, W.W., 1989. Gas Chromatography and Lipids. Oily Press, Dundee, Scotland. Dratz, E.A., Deese, A.J., 1986. The role of docosahexaenoic acid in biological membranes: examples from photoreceptors and model membrane bilayers. In: Simopoulos, A.P., Kifer, R.R., Martin, R.E. (Eds.), Health effects of polyunsaturated fatty acids in seafoods. Academic Press, Orlando, FL, pp. 319-330. Ellenbogen, B.B., Aaronson, S., Goldstein, S., Belsky, M., 1969. Polyunsaturated fatty acids of aquatic marine fungi: possible phylogenetic significance. Comp. Biochem. Physiol. 29, 805-811. Erwin, J., 1973. Comparative biochemistry of fatty acids in eukaryotic microorganisms. In: Erwin, J (Ed.), Lipids and Biomembranes of Eukaryotic Microorganisms. Academic Press, New York, pp. 41-143. Fell, J.W., Master, I.M., 1973. Fungi associated with the decay of mangrove (Rhizophora mangle L) leaves in South Florida. In: Stevenson, L.H., Colwell, R.R. (Eds.), Estuarine Microbial Ecology. University of S. Carolina Press, Columbia, SC, pp. 455-466. Findlay, R.H., Fell, J.W., Coleman, N.K., Vestal, J.R., 1986. Biochemical indicators of the role of fungi and thraustochytrids in mangrove detrital systems. In: Moss, S.T. (Ed.), The Biology of Marine Fungi. Cambridge University Press, Cambridge, pp. 91-103. Gaertner, A., 1968. Eine methode des quantitaven Nachweises Niederer, mit pollen koderbare pilze in Meereswasser und im sediment. Veroff. Inst. Meeresforsch. Bremerh. 3, 7592.

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Horrobin, D.F., 1995. Medical roles of metabolites of precursor EFA. INFORM 6, 428-435. Kendrick, A., Ratledge, C., 1992. Lipids of selected molds grown for the production of N-3 and N-6 polyunsaturated fatty acids. Lipids 27, 15-21. Lewis, T., Nichols, P.D., Hart, P.R., Nichols, D.S., McMeekin, T.A., 1998. Enrichment of rotifers Brachionus plicatilis with eicosapentaenoic acid and docosahexaenoic acid produced by bacteria. J. World Aquacult. Soc. 29 (3), 313-318. Li, Z.Y., Ward, O.P., 1994. Production of docosahexaenoic acid by Thraustochytrium roseum. J. Indust. Microbiol. 13, 233-241. Nakahara, T., Yakochi, T., Higashihara, S., Tanaki, T., Yaguchi, T., Honda, D., 1996. Production of docosahexaenoic and docosapentaenoic acids by Schizochytrium sp. isolated from Yap Island. J. Am. Oil. Chem. Soc. 73 (11), 1421-1426. Naganuma, T., Takasugi, H., Kimura, H., 1998. Abundance of thraustochytrids in coastal plankton. Mar. Ecol. Prog. Ser. 162, 105-110. Raghukumar, S., Raghukumar, C., Rajendran, A., 1992. Abundance of thraustochytrid fungi in the Arabian Sea. Estuar. Coast. Shelf Sci. 31, 351 - 358. Ratledge, C., 1998. Opportunities for marine microorganisms for the production of polyunsaturated fatty acids. In: Le Gal, Y., Muller-Feuga, A. (Eds.), Marine Microorganisms for Industry. Actes de colloque 21. IFREMER, Plouzane,

France, pp. 18-25. Siegenthaler, P.A., Belsky, M.M., Goldstein, S., 1967. Phosphate uptake in an obligately marine fungus: a specific requirement for sodium. Science 155, 93-94. Simopoulos, A.P., 1991. Omega-3 fatty acids in health and disease and growth and development. Am. J. Clin. Nutr. 54, 438-463. Singh, A., Ward, O.P., 1997. Microbial production of docosahexaenoic acid (DHA, C22:6). Adv. Appl. Microbiol. 45, 271-312. Vazhappilly, R., Chen, F., 1998. Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth. J. Am. Oil Chem. Soc. 75 (3), 393-397. Weete, J.D., Kim, H., Ghandhi, S.R., Wang, Y., Dute, R., 1997. Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185. Lipids 32 (8), 839-845. Yaguchi, T., Tanaka, S., Yokochi, T., Nakahara, T., Higashihara, T., 1997. Production of high yields of docosahexaenoic acid by Schizochytrium sp. Strain SR21. J. Am. Oil Chem. Soc. 74 (11), 1431-1434. Yokochi, T., Honda, D., Higashihara, T., Nakahara, T., 1998. Optimisation of docosahexaenoic acid production by Schi-ochytrium limacinum SR21. Appl. Microbiol. Biotechnol. 49 (1), 72-76. Yongmanitchai, W., Ward, O.P., 1989. N-3 fatty acids: alternative sources of production. Process Biochem. 117-125.

iuiii

JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 203-206

Cultivation of the marine basidiomycete Nia vibrissa (Moore & Meyers) H. Helmholz a,, p. Etoundi b, U. Lindequist b a GKSS Research Centre, Max Planck St., D-21502 Geesthacht, Germany b Institute of Pharmacy, Pharmaceutical Biology, Ernst-Moritz-Arndt University, F.-L.-Jahnstr. 17, D-17487 Greifswald, Germany

Received 9 October 1998; received in revised form 25 November 1998; accepted 22 December 1998

Abstract

An isolate of Nia vibrissa was fermented in liquid shake cultures and on agar. Suitable cultivation conditions are a prerequisite for the continuous synthesis of biological active metabolites. The growth response of N. vibrissa to four selected media and several environmental factors, such as salinity, pH and light, was studied. The amount of produced mycelia and the quantity and activity of the organic extracts were parameters for the optimal cultivation method. The ethanolic extracts of the mycelia of N. vibrissa grown in all the investigated nutrient media, showed an influence of the duration of cultivation on the biological activity. A synthetic medium with a pH of 7.5 was the preferred nutrient medium. The addition of wood and incubation under continuous light had no effect on growth but increased the activity of the ethanolic extract. The optimal agar medium salinity for colony growth was in the range between 5 and 25%0. At a salinity of 150%o growth was not observed. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Endotoxin; Marine fungi; Nia vibrissa

1. Introduction

Natural products research is turning to marine animals and plants. A lot of structurally and pharmacologically new and interesting substances have been, for instance, isolated from sponges, algae, molluscs and ascidians (KSnig and Wright, 1996; Pietra, 1997). Compared with these organisms, marine fungi are poorly investigated, but the general increased interest in marine and estuarine * Corresponding author. Tel.: + 49-4152-87-1933 fax: + 494152-87-1919. E-mail address: [email protected] (H. Helmholz)

habitats in the last decades has also led to an increase in studies on marine fungi (Liberra and Lindequist, 1995). Marine fungi are those that grow and sporulate in the oceans, in brackish waters and in estuarine habitats. Fungi are found in all marine environments from the high-water line down to the deep sea (Kohlmeyer and Kohlmeyer, 1979). The higher marine fungi occur as parasites on plants and animals, in symbiotic associations with algae and as saprobes on dead organic material like driftwood. They have developed unique metabolic and physiological capabilities that offer the potential for the production of biological active metabolites.

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00073-5

204

H. Helmholz et al. / Journal of Biotechnology 70 (1999) 203-206

Our investigations on the marine basidiomycete Nia vibrissa (Moore & Meyers) (Meyers and Moore, 1960) have led to the isolation of a mix of structurally similar glycolipids that may be of pharmaceutical interest. These glycolipids inhibit the binding of endotoxin LPS (lipopolysaccharide, a compound present in the cell membranes of Gram-negative bacteria) to a human endotoxin receptor (CD14). Suitable cultivation conditions are a prerequisite for the maximal production of mycelium and for the continuous synthesis of the biologically active glycolipids. In this paper, optimisation studies for the in vitro culture of Nia vibrissa are presented.

2. Materials and methods

The strain of N. vibrissa was kindly provided by Professor Dr H.P. Molitoris, Institute for Botany, University of Regensburg. The fungus was cultivated on an orbital shaker (120 rpm) in Erlenmeyer flasks in 100 ml of four different liquid nutrient media at room temperature for a maximum of 30 days. 2.1. Ingredients of the nutrient media 9 Hagem medium (Hag): 0.05% KHzPO4, 0.05% ammonium succinate, 0.05% MgSO4, 0.5% FeC13 solution (1%), 0.5% glucose, 0.5% malt extract; pH 7.5 9 synthetic medium (SM): 0.1% KH2PO4, 0.05% MgSO4"7H20, 3.0% glucose, 0.075% urea, vitamins and trace elements; pH 7.5 9 malt medium (malt): 3.0% malt extract; pH 7.5 9 sea water medium (SW): 0.1% malt extract, 0.05% pancreatic peptone, 0.01% yeast extract in artificial sea water; pH 7.5.

Hagem-agar medium with increasing concentrations of sodium chloride (pH 7.5) was used for a salt tolerance experiment. The colony growth was observed daily at concentrations of 0, 5, 10, 25, 50, 100 and 150%o. The diameter of ten colonies per each concentration was measured. In fermentations on synthetic medium (pH 7.5) variations in light regime were studied. Fermentations were performed under conditions of continuous light, darkness and a natural day and night rhythm. In some cultures under natural light regime, a panel of wood was added. The mycelium was removed from the medium by filtration and sampled for dry weight determination. The lyophilised mycelium was extracted with dichloromethane (5 h) and ethanol (24 h). The ethanolic extract was evaporated and distributed between ethylacetate and distilled water. The aqueous phase was evaporated to dryness and the biological activity of the residue was investigated. 2.3. Assessment of biological activity CD14-transfected CHO cells (Chinese Hamster ovarian cells) were kindly provided by Dr F. Stelter (Institute of Immunology, University of Greifswald) and cultivated in MEMa medium (Gibco, Paisley, UK) with 10% fetal calf serum (FCS) (Gibco), antibiotics and 20 ~tM methotrexate (Sigma) at 37~ in a humidified 5% CO2 atmosphere. The cells (6 x 106 m l - ' ) were incubated in phosphate buffer (0.1% NAN3) in the presence of FCS (20%), extract (100 ~g m l - ' ) and fluorescein isothiocyanate (FITC)-labelled LPS (Sigma) for 30 rain. The fluorescence was measured by flow cytometric analysis on a FACScan (Becton-Dickinson, Heidelberg, Germany). A complete inhibition was achieved by using polymyxin B (Sigma).

2.2. Varied environmental factors The strain of N. vibrissa was fermented in Hagem medium at pH 7.5 and 5.4. Although pH 7.5 more resembles the pH of seawater than pH 5.4, it has been shown that some marine fungi grow better in slightly acidic media (Curran, 1980).

3. Results and discussion

Nia vibrissa is a marine fungus that can be cultivated easily. There is a good growth under standard conditions compared with other marine and terrestrial fungi. The aim of these investiga-

H. Helmholz et al./Journal of Biotechnology 70 (1999) 203-206

tion was to increase the yield of mycelium combined with a high amount of ethanolic mycelium extract. Another focus was the biological activity of the ethanolic extract--the influence on the binding of LPS to an endotoxin receptor CD14. In Hagem medium, the yield of biomass and the biological activity of the ethanolic extract was increased at pH 7.5 compared to the results of the cultivation at pH 5.4 (Fig. 1). Therefore the strain was cultivated in malt, sea water and synthetic medium at the optimal pH of 7.5. After a 3-day cultivation, the yield of biomass was highest in the synthetic medium. A 3-day fermentation in the synthetic medium at pH 7.5 resulted in an increase of mycelium dry weight of about 19 times. It seems that glucose as the carbon source combined with a defined supply of vitamins and trace elements are required for the optimal growth of N. vibrissa. During the fermentation the yield of mycelium was decreased after the sixth day of cultivation. A reason might be the fast consumption of nutrients during the exponential phase of growth. In all fermentations, the biological activity of the ethanolic extracts of the mycelium was highest after the 12th day. Although fermentations in malt medium resulted in the highest biomass yield after 12 days (Fig. 1), the synthetic medium is to be preferred due to the much higher biological activity of the ethanolic extract (Fig. 2).

" 15 ._m .o E

9o

5

Hag 7,5

Hag 5,4

matt

SM

SM SM light SM wood darkness

SW

Fig. 1. Yield of mycelium dry weight on the 12th day of cultivation of N. vibrissa in different media with regard to dry weight of the inoculum.

205

.~ 60+ ...1

,

g .~_

~

20

i Hag 7,5

Hag 5,4

malt

SM

SM darkness

SM light

SM wood

Fig. 2. Inhibition of the binding of FITC-labeUed LPS on CD14 by the aqueous phase of the ethanolic extract of mycelium of N. vibrissa harvested on the 12th day of cultivation under different conditions (M + SD, n--6).

The influence of continuous light on N. vibrissa was investigated in synthetic medium. Twentyfour hour light and darkness showed no effect on the production of mycelia but increased the inhibition of the LPS binding to the endotoxin receptor CD14 (Figs. 1 and 2). The addition of wood panels had no significant influence on mycelial dry weight. However, an increased inhibition of the endotoxin-binding on CD14 to more than 95% could be achieved after a cultivation of 9 days in synthetic medium with addition of wood panels and an initial pH of 7.5. Another important factor for cultivation of marine fungi is the salinity of the nutrient broth. A main characteristic of the marine fungi is their higher salt tolerance compared with terrestrial fungi. Growth of N. vibrissa on petri dishes was observed. Hagem-agar with increasing salt concentrations at pH 7.5 was used. Nia vibrissa preferred salt concentrations of 5-25%o. There is absolutely no growth at a sodium chloride concentration of 150%o. Considering these results the cultivation of N. vibrissa in synthetic medium using a panel of wood could be recommended. The mycelium should be harvested between the ninth and the 12th days of fermentation because of the high amount of dry weight and the strong inhibition of the binding of FITC-labelled endotoxin on CD14 by the mycelium extract obtained during this period.

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Acknowledgements This work was supported by the Bundesministerium ffir Bildung, Wissenschaft, Forschung und Technologie (FK0310907).

References Curran, P., 1980. The effect of temperature, pH, light and dark on the growth of fungi from Irish coastal waters. Mycologia 72, 350-358.

Kohlmeyer, J., Kohlmeyer, E., 1979. Marine MycologywThe Higher Fungi. Academic Press, New York, San Francisco, London. K6nig, G., Wright, A., 1996. Marine natural products research: Current directions and future potential. Planta Med. 62, 193-211. Liberra, K., Lindequist, U., 1995. Marine fungi--a prolific resource of biologically active natural products? Pharma 50, 583- 588. Meyers, S., Moore, R., 1960. Thalassiomycetes II. A genera and species of deuteromycetes. Am. J. Bot. 47, 345-349. Pietra, F., 1997. Secondary metabolites from marine microorganisms: bacteria, protozoa, algae and fungi. Achievements and prospects. Nat. Prod. Rep. 14, 453-464.

JOURNAL

Bioteclinology ELSEVIER

Journal of Biotechnology 70 (1999) 207-212

Controlled cultivation of A lexandrium minutum and [33p] orthophosphate cell labeling towards surface adhesion tests St6phane La Barre

a,*

, St6phane Singer a, Evelyne Erard-Le Denn b Marcel Jozefowicz a

a Laboratoire de Recherche sur les Macromolkcules, CNRS URA 502. Universitk Paris-XIIl, Villetaneuse, France b DEL/EC/PD Centre de Brest, IFREMER, BP 70 29280 Plouzank, France

Received 22 October 1998; received in revised form 7 December 1998; accepted 22 December 1998

Abstract

We describe a protocol for preparing cultures of the PSP (paralytic shellfish poisoning) dinoflagellate Alexandrium minutum, towards subsequent studies of cell adhesion onto artificial substrates. First, phenotypic uniformity of the strain used and reproducibility of the standard growth profile are obtained by optimising key parameters. Batches of A. minutum at mid-exponential proliferation phase are radiolabeled with 33p-containing medium in order to later

quantify cell adhesion. A mortality corrective index is applied to the latter, using the vital fluorochrome acridine orange, i.e. dead cells show no nuclear incorporation under epifluorescence microscopy. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Alexandrium minutum; Cell numeration; Controlled proliferation: Radioactive labeling .

1. Introduction A l e x a n d r i u m m i n u t u m is a PSP dinoflagellate species (i.e. producer of the paralytic shellfish toxin saxitoxin and its derivatives), of importance in commercial bivalve farming in Europe (Lassus et al. 1994). We are in the process of studying specific interactions between A . m i n u t u m and artificial water-insoluble biomaterials (Jozefowicz and Jozefonvicz, 1997). The use of an elution column containing a solid phase of calibrated

* Corresponding author. Fax: + 33-1-42354841. address: [email protected] (s. La Barre) E-mail

binding affinity to a single species would be a novel alternative to existing techniques of specific cell sorting from natural plankton populations (Aguilera et al. 1996), which may simplify the time-consuming monitoring of populations of toxic microalgae in areas under surveillance (Shumway et al., 1995). In this note it is shown that we may obtain populations of A. m i n u t u m suitable for experimenting with artificial biomaterials, by a combination of simple and classical techniques. First, phenotypic uniformity may be achieved by using reproducible batches under well defined conditions, which is possible for most species of au-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00074-7

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S. La Barre et al. ' Journal o f Biotechnology 70 (1999) 207-212

totrophic dinoflagellates (Smayda, 1996), for which complete growth media have been especially formulated (Guillard, 1995). Second, an assay is required for measuring cell-substrate adhesion, a phenomenon which cannot be quantified directly by low-resolution microscopy on three-dimensional biomaterial substrata. Adhesion studies on bacteria and cultured vertebrate tissue generally use the detection of radioactivity levels from a suitable 'hot' ligand preincorporated into cell nuclei or chromosomes (Vaudaux et al., 1984). But since cell death results in rapid loss of radioactivity before evident signs of decay (S. La Barre, unpublished data), this method of measuring adhesion is only meaningful for cell populations shown to be 100% alive. Following radioactive feeding, we test aliquots of cells for viability prior to adhesion experiments, by visualizing cells capable of incorporating acridine orange (nuclei turn bright yellow) (Kasten, 1981).

2. Materials and methods

2.1. Algal culture

Cultures of clonal strain AM89BM of A. minutum from Morlaix, Britanny, were developed in 0.2 lam pre-filtered natural seawater enriched with 3 ml 1-~ Guillard's f/2 stock solution without Si and with traces of vitamins of the B complex (Guillard, 1995), followed by membrane sterilisation of the homogenised medium. Antibiotics were added as 1% per volume of a commercial (Life Technologies, Eragny, France) stock mixture of penicillin (5000 units m l - l ) and streptomycin (5000 units ml-~). Aliquots (300 ml) of growth medium were conditioned at 17~ in autoclavesterilised flasks. The cultures were inoculated by sterile transfer of a volume of inoculate calculated to contain 5 x 104 cells from a 15 to 20-day suspension of A. minutum dinophytes developed under similar conditions, and subsequent filling of flasks to 500 ml. The flasks were topped with porous silicone stoppers. Incubation conditions: automated 12"12 h light-dark regime (85 laE s-1 m -2 using cool white OSRAM 8w/20 neon tubes), with 17~ air temperature regulation.

2.2. Radioactive label

The following protocol was intended to show the rate of uptake of radio-element [33p] orthophosphate by the dinoflagellates and to establish the stability of the label, after eliminating unbound radioactivity from seawater medium. Single doses of 250 ul [33p] orthophosphate in HC1 (Amersham) were added to 100 ml aliquots of an 18-day-old culture of A. minutum. These aliquots were incubated 1, 2, 4 and 8 days, respectively, before determination of radioactivity. To eliminate excess radioactivity of supernatant, the aliquots were first reduced to 50 ml through a 5 lam porosity non-protein-absorbing filtration membrane (Durapore by Millipore). Then, 100-ml volumes of plain filtered seawater at 17~ were successively added and eluted ten times, with corresponding 200-1al aliquots saved in duplicate for radioactivity monitoring. Accordingly, duplicate 1 ml aliquots of washed cells were lysed in 1 M NaOH. All aliquots were placed in scintillation vials and homogenised in ten times their respective volumes of scintillation fluid (Wallac Optiphase Hisafe 2) prior to counts per minute readings of emitted beta particles in a beta counter (Wallac LKB 1214 Rackbeta). 2.3. Staining for cell viability

The following protocol was derived from Kasten (1981) to estimate the ratio of living versus dead dinoflagellate cells, especially after the radiolabeling procedure, and prior to experimentation with synthetic surfaces. Stock solution: 1 mg AO (acridine orange, Sigma-Aldrich) base in 2-ml HBSS buffer, itself prepared by mixing a 9.8-g dose of salt mixture (Sigma-Aldrich) and 0.35 g sodium bicarbonate in 1 1 distilled water, followed by pH 7.3 equilibration and membrane ultrafiltration. This stock solution was stored at 4~ A 20 ~tM working solution was prepared weekly by adding 1 volume of AO stock solution to 200 volumes of a 1:1 mixture of seawater and freshwater. Cell staining: 10-ml aliquots of cell suspension were gravity reduced to 2 ml on a 0.8-~tm cellulose nitrate filtration membrane, then completed back to 10 ml with 20 laM AO solution. After 10 rain,

S. La Barre et al. /Journal of Biotechnolog)' 70 (1999) 207-212

preparations were rinsed several times with seawater, then filtered cells were picked up from the membrane with 10% glycerol in seawater and counted on a Nageotte haemocytometer cell using a microscope (Leitz) equipped with a blue-green excitation filter (420-490 nm) and a 515 nm threshold filter (H3 Ploemopack, Leitz). Control staining: two 10 ml aliquots from same cell suspension (104 cells ml -~) were isolated; one aliquot (tube A) was heated above 60~ for several minutes to kill cells, and the other aliquot (tube B) was left at 17~ Both aliquots were stained according to the above procedure, and the ratio of living cells versus total population (viability index) was calculated.

3. Results and discussion

3.1. Algal culture Fig. l a depicts typical growth curve under the conditions described. The stationary phase is reached at around day 36, with the inflexion point of the exponential phase at 18-20 days after hardly any latency phase (Fig. l b). Division rate of 3.4 days at 17~ can be shortened to 2.2 days if cultured at 20~ but cell size becomes smaller and less homogenous on average (S. La Barre, personal observation). From day 14, the blooming culture consists of cells expressing a typical composition of cell surface receptors and membrane proteins (an important feature for performing adhesion studies), and as such, these cells are ready for metabolic radioactive labeling as described below. The population kinetics studies were initiated from well-selected cell lines in order to obtain homogeneous populations and, in turn, synchronous responses to some experimental variables (light, temperature, nutrient concentrations). According to earlier proliferation kinetics found for A. minutum (S. La Barre, unpublished results), reproducibility of this growth profile is achieved by maintaining exponential growth in 18-20 day cycles using vitamin-supplemented Guillard f/2 growth medium.

209

3.2. Radioactive label&g The uptake kinetics of [33p] orthophosphate by A. minutum are shown in Fig. 2. The concentration of incorporated radioactive nutrient increased linearly with time, i.e. no plateau was reached within the 8-day time course of the experiment. Adequate levels of incorporation, i.e. almost 10 counts per minute per cell (cpm cell-i) were obtained after 4 days incubation and after rinsing off unbound radioactivity. Radioactivity levels of the same population remained stable around 10 cpm cell-1 for another 3 days, which leaves more than enough time to complete the procedures related to adhesion experiments. Orthophosphate is one of the macroelements essential for growth, and is indeed a major component of Guillard's f/2 growth medium. The 33p nuclide is safe to handle (i.e. soft beta emissions) and has a short half-life of 25.3 days. It appeared to us to be the cheapest alternative to more expensive forms of cell labeling, using, e.g. cell-specific fluorescent probes, since our laboratory routinely uses radioisotopes and is suitably equipped for beta emission detection. Thymidine is often used as DNA-specific marker, but it is poorly incorporated in dinoflagellate DNA, where up to 85% of it is replaced by 5-hydroxymethyl uracil (Sigee, 1986), a fact which we verified experimentally (only 0.04 cpm cell -~ was detected using the protocol described above, against almost 10 cpm cell-~ for 33p), partly due to massive losses of radioactivity during supernatant replacement. 63Ni uptake is apparently rapid, its role as chromatin-stabilizing divalent cation making it a choice nuclear marker in autoradiographic readings (Sigee, 1982), but levels of incorporation are still too low for the purpose of monitoring cell adhesion using our method, and its long half-life (100 years) represents a safety problem when considering its fate as waste. 3.3. Stain&g for cell viability Over 85% of cells from tube B fluoresced bright green (some cytoplasmic components), yellow (nuclei) or intense orange (vacuoles), as living cells incorporate acridine orange; heat-killed cells

210

S. La Barre et al./Journal of Biotechnology 70 (1999) 207-212

pale green (empty thecae). Provided freshly stained preparations are examined, acridine or-

from tube A all autofluoresced uniformly faint red (whole cells), due to photosynthetic pigments, or

10

A.minutum growth kinetics

O v-

x m

E (D

t

v

days from 0

~

9

r

inoculation

,..

9

(a) 0

10

15

20

25

30

35

40

45

50

100000

0

ca

9

9

plateau

10000

exponential

1000 latence

semi-lo_o data days

(b)

100

1

0

,

4

"'

6

~,

,

10

,

12

,

14

,

18

.

20

.

24

26

.

28

.

32

,

34

38

42

46

,

48

Fig. 1. (a) Proliferation curve of a population of A. minutum cultivated under controlled temperature (17~ light (white, 85 ~tE s m - 2 ) and nutrient concentration (3 ml 1-~ of Guillards f/2 medium stock solution supplemented with B vitamins), inoculated with 5 x 104 cells and grown in a 500 ml vessel. Cells are marked with 33p at day 14. (b) Semi-log representation of the same curve, showing a short latency phase, the exponential growth phase and the plateau phase.

211

S. La Barre et al./Journal of Biotechnology 70 (1999) 207-212 12

~o

8

6

4

2

o

y 0

33P uptakeby A.minutum 2

4

6

8

days 10

12

Fig. 2. Rate of incorporation of [33p] orthophosphate (250 laCi in 100 ml medium) by A. minutum. ange at sublethal concentrations shows clear cut discrimination between dead and living cells, with excellent metachromatic photography representations (Fig. 3). We found that AO intake and hence brightness of stained organelles was enhanced when using mildly saline conditions (1.8%) rather than plain seawater (3.7% salinity), possibly by influencing fixation of stain on nucleic acids. AO usefulness for discriminating between dead and living cells was first reported by Strugger (1949) for bacteria, yeasts, paramecia and mammalian lyzosomal cells; its use has been extended to a wide variety of cell types (Kasten, 1981) including protists and it is now routinely used for the visualization of dinoflagellate nuclei, along with DAPI and fluorescent probes more suited to flow cytometry.

ity, e.g. cell receptor expression, which such experimentation requires. Under specific conditions we obtained reliable and reproducible kinetics, allowing the regular availability of cells at mid-exponential growth phase for radioactive labeling (and hence substrate interaction studies). [33p] Orthophosphate turned out to be an inexpensive, efficient and stable cell marker for subsequent cell

4. Conclusions A relatively simple and inexpensive protocol was devised in order to suitably prepare populations of A. m i n u t u m for observations of cell-substrate interactions (in progress). Homogenous populations represent the phenotypic homogene-

Fig. 3. Vital staining of cells of A. minutum using acridine orange. Living cells are brightly coloured (yellow to deep orange, top left), whereas dead cells appear pale (red, center), due to natural autofluorescence of photosynthetic pigments.

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S. La Barre et al. / Journal of Biotechnology 70 (1999) 207-212

adhesion measurements, provided live cells only were considered. Regarding this, acridine orange was found quite suitable as a vital fluorochrome for detection of living cells, using the staining procedure we developed. The overall methodology is currently used for cell adhesion experiments on synthetic substrates, but it may inspire ideas for other types of experimentation with living populations of microalgae.

References Aguilera, A., Gonzales-Gil, S., Keafer, B.A., Anderson, D.A., 1996. Immunomagnetic separation of cells of the toxic dinoflagellate Alexandrium fundyense from natural plankton samples. Mar. Ecol. Prog. Ser. 143, 255-269. Guillard, R.L., 1995. Culture methods. In: Hallegraeff, G.M.. Anderson, D.M., Cembella, A.D (Eds.), Manual on Harmful Marine Microalgae. IOC Manuals and Guides, no. 33. UNESCO, pp. 45-62. Jozefowicz, M., Jozefonvicz, J., 1997. Randomness and biospecificity: random copolymers are capable of biospecific molecular recognition in living systems. Biomaterials 18. 1633-1644.

Kasten, F.H., 1981. Methods for fluorescence microscopy. In: Clark, G. (Ed.), Staining Procedures, 4. Williams and Wilkins, Baltimore, pp. 39-104. Lassus, P., Ledoux, M., Bardouil, M., Bohec, M., Erard, E., 1994. Kinetics of Alexandrium minutum Halim toxin accumulation in mussels and clams. Nat. Toxins 2, 329-333. Shumway, S.E., van Egmont, H.P., Hurst, J.W., Bean, L.L., 1995. Management in shellfish resources. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D (Eds.), Manual on Harmful Marine Microalgae. IOC Manuals and Guides, no. 33. UNESCO, pp. 433-461. Sigee, D.C., 1982. Localized uptake of 63Ni into dinoflagellate chromosomes: an autoradiographic study. Protoplasma 110, 112-120. Sigee, D.C., 1986. The dinoflagellate chromosome. Adv. Bot. Res. 12, 206-264. Smayda, T.J, 1996. Dinoflagellate bloom cycles: what is the role of cellular growth rate and bacteria? In: Yasumoto, T., Oshima, T., Fukuyo, Y. (Eds.), Harmful and Toxic Algal Blooms. Proceedings of the Seventh International Conference on Toxic Phytoplankton, Sendai, Japan, 12-16 July 1995, IOC of UNESCO. UNESCO, pp. 331-334. Strugger, S., 1949. Fluoreszenzmikroskopie und Mikrobiologie. M.&H. Shaper, Hannover. Vaudaux, P., Waldvogel, F.A., Morgenthaler, J.J., Nydegger, U.E., I984. Adsorption of fibronectin onto polymethacrylate and promotion of Staphylococcus aureus adherence. Infect. Immun. 45, 768-774.

iii

IOU

--

RNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 213-221

Production of -tocopherol by sequential heterotrophic-photoautotrophic cultivation of Euglena

gracilis James C. Ogbonna *, Shota Tomiyama, Hideo Tanaka Institute of Applied Biochemistry, L)litersitv o/ Tsukuba, I-1-1 Tennodai, Tsukuba, Japan Received 6 October 1998; received in revised form 23 November 1998" accepted 22 December 1998

Abstract

Photoautotrophic cultivation of Euglena gracilis results in cells with high ~-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the ~-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of 7-tocopherol was therefore investigated for efficient 0t-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular ~-tocopherol content increased from 120 lag g-~ at the end of heterotrophic phase to more than 400 lag g-~ at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the a-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 lag g - 1. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for ~-tocopherol accumulation. In this way, it was possible to produce 7 g 1-1 cells containing about 1100 lag ~-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in 0t-tocopherol productivity of 100 lag 1-1 h -~ which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Euglena gracilis; a-Tocopherol" Photoautotrophic culture; Heterotrophic culture: Ethanol; Glucose; Corn steep liquor

1. Introduction * Corresponding author. Fax: + 81-298-534605. E-mail address:[email protected] (J.C. Og-

bonna)

Cultivation of photosynthetic cells for production of various useful metabolites has been under intensive investigation. Among them, antioxidants

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 7 7 - 2

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J.C. Ogbonna et al. Journal of Biotechnology 70 (1999) 213-221

such as tocopherol (vitamin E) are important because they can be used to prevent several diseases caused by oxidative damage. Tocopherol is also widely used as a food preservative in food processing industries. Currently, chemical synthesis and extraction from vegetable oils are the major sources of tocopherol. However, extracts from vegetable oils contain a mixture of ~-, [3(~{)- and 6-tocopherols and for pharmaceutical application there is a need to purify it to a more active ~-form. Among several microorganisms tested, Euglena was reported to accumulate the highest amount of tocopherol (Taketomi et al., 1983; Tani and Tsumura, 1989) and over 97% of the tocopherol produced by this strain is in the ~-form (Shigeoka et al., 1986). Thus there have been several reports on the production of ~-tocopherol by this strain (Tani and Osuka, 1989; Tani and Tsumura, 1989; Takeyama et al., 1997; Ogbonna and Tanaka, 1998; Ogbonna et al., 1998). Research on the production of useful metabolites by photosynthetic cells has concentrated on photoautotrophic cultures. However, due to light limitation, the cell concentrations attainable in photoautotrophic cultures are usually very low with resultant high cost of down stream processing. A lot of work has been done to improve the productivities of photoautotrophic cultures, ranging from optimization of open cultivation ponds (Laws et al., 1986; Ogbonna et al., 1995a; Ogbonna and Tanaka, 1996) to development of photobioreactors with high illumination surface to volume ratios. Examples include thin panels (Tredici et al., 1991), tubular (Richmond et al., 1993) and internally illuminated photobioreactors (Mori, 1985; Ogbonna et al., 1996; Ogbonna and Tanaka, 1997). However, the maximum cell concentrations reported for such reactors are still much lower than the values obtained in heterotrophic cultures (Ogbonna and M~irkl, 1993). Furthermore, the photobioreactors that give high cell concentrations are often either too complex or too costly for large scale production. Euglena can grow heterotrophically, using various organic substrates. Although very high cell concentrations can be obtained in heterotrophic cultures, the intracellular tocopherol contents of heterotrophically grown cells are usually much

lower than those obtained in photoautotrophic cultures (Takeyama et al., 1997; Ogbonna et al., 1998). However, by using ethanol as the organic carbon source and optimizing other culture conditions, a fed-batch heterotrophic culture can be used to obtain high Euglena cell concentration with relatively high tocopherol content (Ogbonna et al., 1998). In order to combine the advantages of heterotrophic (high cell concentrations) and photoautotrophic (high cellular contents of useful metabolites), we have proposed a sequential heterotrophic/autotrophic cultivation method as means of producing high concentrations of photosynthetic cells with high cellular contents of useful metabolites (Ogbonna et al., 1997). This method involves passing high concentrations of monoalgae biomass from a heterotrophic culture through a photobioreactor for accumulation of photosynthetic products. In this paper, application of this method for efficient production of ~-tocopherol was investigated.

2. Materials and methods

2. I. Microorganisms and media composition Euglena gracilis IAM E-6 (strain Z), obtained from the algal collection of the Institute of Applied Microbiology, University of Tokyo, Japan was used in this study. A modified Hutner medium was used as the basic medium for both the pre- and the main cultures. The composition of both the basic medium, the trace element and Fe-solutions were as described in our previous paper (Ogbonna et al., 1998) except that either glucose or ethanol was used as the organic carbon source. The medium was sterilized by autoclaving at 121 ~ for 15 min. Filter-sterilized vitamin B~2 and ethanol (when it was used as the organic carbon source) were added to the basic medium after autoclaving and cooling the medium to room temperature. 2.2. Pre-culture The stock culture was activated by inoculating 7.5 ml of the stock culture into 500 ml Elrenmeyer flask containing 150 ml of the basic medium and incubating on a rotary shaker. The flask was

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 213-221

wrapped with aluminum foil to avoid light penetration from outside. The rotation speed and cultivation temperature were 120 rpm and 25~ respectively.

215

When the organic carbon source concentration was reduced to zero, 0.5% corn steep liquor (CSL) was added and the reactor was illuminated by inserting four 4-W fluorescent lamps into the four housing glass tubes (Ogbonna et al., 1996). The light intensity at the surface of the glass tubes was 163 I.t mol m - 2 s-z, which is equivalent to a light supply coefficient of 0.374 kJ kg m -6 s - l (Ogbonna et al., 1995b). Air containing 5% CO2 was used for aeration (0.5 vvm) and the temperature was increased to 30~

2.3. Batch culture

The internally illuminated photobioreactor (Ogbonna et al., 1996; Ogbonna and Tanaka, 1997) containing 3.0 1 of the basic medium was inoculated with the pre-culture to give an initial cell concentration of 0.05 g 1-1. It was cultivated heterotrophically, using either glucose or ethanol as the organic carbon source, until the organic carbon source was completely consumed. During this heterotrophic phase, a mixture of ordinary air and nitrogen gas was used for aeration (0.5 vvrn) through the ring sparger. The agitation speed and dissolved oxygen concentration were controlled at their optimum values of 100 rpm and 0.81 pprn, respectively (Ogbonna et al., 1998). The dissolved oxygen concentration was controlled by varying the oxygen partial pressure in the aeration gas. The photobioreactor was not illuminated and aluminum foil was used to wrap the glass vessel. The cultivation was done at 25~ while the pH was controlled at 3.5.

2. 4. Continuous culture

A schematic diagram of the system used for continuous sequential heterotrophic/photoautotrophic cultivation is shown in Fig. 1. The heterotrophic phase is composed of a conventional 2.5 1 aerated stirred mini-jar fermentor (Iwashiya Bioscience Co Ltd, Saitama, Japan) with a working volume of 2.0 1 while the internally illuminated photobioreactor was used for the photoautotrophic phase. Both bioreactors were filled with the basal medium containing 8 g 1-1 ethanol and inoculated with the pre-culture to give an initial cell concentration of 0.05 g 1-1.

Exhaust gas Exhaust gas _

,, ~ . y Q

~--

Air

Culture broth

.... ~

.....~.)..-~----..:

sparger Culture broth "

Medium

4N HC! 4N NaOH

m

i

Mini.jar fermentor

Internally muminated photobioreactor

Fig. 1. A systematic diagram of the system used for continuous sequential heterotrophic/photoautotrophic cultivation of E. gracilis. Both pumps A and B were operated during the batch phase but during the continuous phase, only pump A was operated.

216

J.C. Ogbonna et al. Journal ~/ Biotechnology 70 (1999) 213-221

Both bioreactors were wrapped with aluminum foil and cultivated heterotrophically (the photobioreactor was not illuminated). The cultivation temperature, pH and aeration rate were as described for the batch heterotrophic culture. The agitation speeds for the jar fermentor and the photobioreactor were 150 rpm and 100 rpm, respectively. In order to ensure homogeneity in both bioreactors, pumps A and B (Fig. 1) were used to circulate the culture broth between the two bioreactors. When the ethanol concentration was reduced to zero, pump B was stopped and the photobioreactor was illuminated by four 4-W fluorescent lamps as described for the batch culture. Continuous sequential heterotrophic/photoautotrophic cultivation was started by continuously feeding a fresh medium containing 0.5% CSL into the mini-jar fermentor and feeding the effluent from the minijar fermentor into the photobioreactor. The feed rate was 41.7 ml h -1 which corresponds to a dilution rate of 0.021 h - 1 in the jar fermentor and a residence time in the photobioreactor of 72 h. This dilution rate is lower than the average specific growth rate (0.05 h - l ) observed during the batch phase. However, operating at a higher dilution rate would lead to shorter residence time in the photobioreactor and thus to lower ~-tocopherol content of the cells. The aeration gas during the photoautotrophic phase was 5% CO2 in air.

2.5. Analytical methods The dry cell concentration (Ogbonna and Tanaka, 1996); ethanol and glucose (Ogbonna et al., 1998) ~-tocopherol (Shigeoka et al.. 1986): chlorophyll and light intensity (Ogbonna et al., 1997) were determined as described in previous papers.

3. Results

3.1. Batch sequential heterotrophic /photoautotrophic cultivation, using glucose as the organic carbon source during the heterotrophic phase The result of batch sequential heterotrophic/

...,. "7_z 20

Heterotrophlcphase

'

[

~utotrophtc

plu~

500

40o.

=15 I

3oo~

~~10

200

i

10o 8

~ 9

' 00

20

40

__-- __--,-60 80 100 Cultivation lime (h)

,-120

, -0 140 16o

o

Fig. 2. Changes in glucose, cell and ~-tocopherol concentrations during batch sequential heterotrophic/photoautotrophic cultivation of E. gracilis, using glucose as the sole carbon source.

photoautotrophic cultivation of E. gracilis, using glucose as the organic carbon source during the heterotrophic phase is shown in Fig. 2. Heterotrophic cultivation for 72 h resulted in a cell concentration of 12 g 1-l but the ~-tocopherol content of the cells was only 120 gg g - ~cell. When glucose concentration in the medium was completely consumed, the condition was changed to the photoautotrophic condition. During this photoautotrophic phase, the cell concentration decreased slightly to about 9 g 1-1 but the 7-tocopherol content of the cells increased almost linearly to 400 gg g-1. However, the final a-tocopherol content was still low and there was a need for process optimization. The effects of light supply. nitrogen and carbon sources were therefore investigated.

3.2. Effect of light supply on ~-tocopherol accumulation during the photoautotrophic phase Since the effect of light intensity depends on cell concentration, light energy supplied per unit cell was used in this study. The effects of light energy supplied per unit cell on chlorophyll and ~-tocopherol contents of the cells are shown in Fig. 3. As the light supplied per unit cell was increased, the chlorophyll contents of the cells decreased but the 7-tocopherol content increased. Although the maximum 7-tocopherol content was obtained at a light energy supplied per unit cell of 1970 J g-1 s-1 (illuminating 2 g cell 1-~ in a 100 ml Roux bottle with a 2 cm light path at a light intensity of

J.C. Ogbonna et al. / Journal of Biotechnology 70 (I 999) 213- 221 800

"7 ,id

~ 600

ta

g

400 0

[] 200

II 0 85

299

560

1970

Light energy per Cell (J g-I s-l)

Fig. 3. Effects of light energy supplied per unit cell during the photoautotrophic phase on ~-tocopherol and chlorophyll contents of E. gracilis.

330 lamol m - : s-1), the volumetric ~-tocopherol concentration (1334 lag 1-1) was less than one third of the value (4095 lag 1-1) obtained at 560 J g-1 s-1 (light intensity = 330 lamol m - 2 s-1, cell concentration = 7 g 1-1). In the subsequent experiments, the cell concentration was therefore controlled at 7 g 1-1.

Fig. 4(A) shows the effect of addition of a nitrogen source (0.5%) to the culture during the photoautotrophic phase on ~-tocopherol accumulation. The cell concentration was 7 g 1-1 while isoo, B

750

450

15

-o

20

4~

6~

Illumination time (h)

o

20

4~

light intensity was 330 lamol m -2 s-1. Although addition of either sodium glutamate (data not shown), polypeptone or corn steep liquor (CSL) resulted in increased ~-tocopherol accumulation, the maximum value was obtained with CSL. We have already reported that the ~-tocopherol content of cells in heterotrophic culture was higher when ethanol was used as the organic carbon source (Ogbonna et al., 1998). The effect of second stage culture (photoautotrophic) on a-tocopherol content of cells cultivated heterotrophically on either ethanol or glucose is shown in Fig. 4(B). The cell concentration was adjusted to 7 g 1-1 and 0.5% CSL was added to the culture in a Roux bottle before illumination at a light intensity of 330 lamol m - 2 s-~. At the end of the heterotrophic phase, the ~-tocopherol contents of the cells were 114 and 416 lag g-1 for the cells cultivated on glucose and ethanol, respectively. During the 62 h of photoautotrophic culture, the ~-tocopherol content of the cells obtained from ethanol heterotrophic culture increased to 1200 lag g-1, which is 50% higher than that of cells cultivated in a glucose medium.

3.4. Batch sequential heterotrophic/photoautotrophic cultivation for high :~-tocopherol accumulation

3.3. Effect of nitrogen and carbon sources

--'-900

217

6~

80

Illumination time (It)

Fig. 4. Effects of nitrogen source addition during the photoautotrophic phase (A) and carbon source used for the heterotrophic phase (B) on ~-tocopherol accumulation during the photoautotrophic phase. In (A) the symbols are (O), corn steep liquor; (A), polypeptone and (11), no nitrogen source addition. In (B) the symbols are ( 9 ethanol and (O), glucose.

From the results obtained in a Roux bottle, it was concluded that high ~-tocopherol accumulation can be obtained by using ethanol as the organic carbon source for the heterotrophic phase, controlling the cell concentration at 7 g 1-1 and adding 0.5% CSL to the culture during the photoautotrophic phase. The results of batch cultivation under the above condition are shown in Fig. 5. After 56 h of heterotrophic culture, ethanol was completely consumed and photoautotrophic cultivation was started. During this phase, the cell concentration decreased gradually but the ~-tocopherol content of the cells increased to a very high value of 1700 lag g-1. This cz-tocopherol content is higher than the values reported for fed-batch heterotrophic cultivation on ethanol (Ogbonna et al., 1998), photoautotrophic cultures or fed-batch photoheterotrophic/photoautotrophic cultures (Takeyama et al., 1997) of this

218

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 213-221

--- ~alO

2~oo~

is00[

1200 ~

r

800~

9

i

w

I

o 0

20

40

60 Cultivation

80

100

120

9

14o

time (h)

Fig. 5. Changes in ethanol, cell and ~-tocopherol concentrations during batch sequential heterotrophic/photoautotrophic cultivation of E. gracilis, using ethanol as the sole carbon source.

strain. However, the productivity (62 ~tg l-~ h - !) is comparatively low due to the long cultivation time (time for heterotrophic phase + time for photoautotrophic phase). The use of continuous process for increased productivity was therefore investigated.

3.5. Continuous sequential heterotrophic/ photoautotrophic cultivation The results of continuous sequential heterotrophic/photoautotrophic cultivation are shown in Figs. 6 and 7. The continuous process was preceded by 50 h of heterotrophic batch cultivation using ethanol as the organic carbon source. From the start of the continuous process up to about 200 h of cultivation, the effluent cell concentration from the photoautotrophic phase was almost the same as that from the jar fermentor. However, during the later part of the culture, the cell con~,!

10~

6"~

0

~

400. ~

r~ .m O~ o

70

140

210 Cultivation

280

350

o

70

I

I

~

I

140 210 280 Cultivation time (it)

w

I

350

420

Fig. 7. Changes in a-tocopherol concentrations in the heterotrophic (O) and photoautotrophic ( 9 phases during continuous sequential heterotrophic/photoautotrophic cultivation of E. gracilis.

centrations in the effluents of the heterotrophic and photoautotrophic phases were 7 and 6 g 1-J, respectively. Ethanol concentration in the effluent of heterotrophic culture remained zero throughout the continuous process. This is important because if ethanol is passed over to the photobioreactor, the condition would be mixotrophic (photoheterotrophic) rather than photoautotrophic. This would decrease Qt-tocopherol accumulation since it is known that ~-tocopherol content of cells is higher in photoautotrophic than in photoheterotrophic cultures (Takeyama et al., 1997). The ~-tocopherol content of cells in the effluent of heterotrophic phase remained about 300 ~tg g-~ throughout the cultivation (Fig. 7). On the other hand, the ~-tocopherol content of the cells in the effluent of the photoautotrophic phase increased to more than 1200 ~tg g-~ and then started to decline gradually but remained above 1000 ~tg g - ~ throughout the process. This is lower than the value obtained in the batch process. However, using an average cell concentration of 6.5 g 1-~, ~-tocopherol content of 1100 ~tg g-1 and dilution rate (in the photobioreactor) of 0.014 h-1, the productivity was calculated to be 100 l.tg 1-~ h-1 which is higher than 62 l.tg 1-1 h-1 obtained in the batch culture.

420

time (h)

Fig. 6. Changes in ethanol (l l) and cell concentrations in the heterotrophic (O) and photoautotrophic ( 9 phases during continuous sequential heterotrophic/photoautotrophic cultivation of E. gracilis.

3.6. Comparison of ot-tocopherol productivities in various cultivation systems Table 1 shows a summary of 0~-tocopherol contents of the cells and productivities of various

219

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 213-221

High light intensity which favoured ~-tocopherol accumulation was inhibitory to chlorophyll synthesis. In photoautotrophic cultures, high light intensity cannot be used because it inhibits both chlorophyll synthesis and cell growth. However, light inhibition of cell growth is not a problem in sequential heterotrophic/photoautotrophic culture since cell growth is not necessary during the photoautotrophic phase. The reason for the inverse relationship between chlorophyll and ~-tocopherol contents under increasing light intensity is not clear. A possible explanation is that low chlorophyll content improves light penetration into the culture. The involvement of chlorophyll in a-tocopherol synthesis is not yet clear. Various studies in higher plants suggest that biosynthesis of a-tocopherol is closely related to chloroplasts whose differentiation is induced by light. It has also been reported that chloroplast envelope membranes are the only site of enzymatic prenylation in ~-tocopherol synthesis (Soll et al., 1980). Furthermore, Camara et al. (1982) noted that during a-tocopherol synthesis by Capsicum chloroplasts, the enzymes involved in the cyclization and methylation are both localized in the chloroplast membrane fraction. On the other hand, the occurrence of ~-tocopherol in the dark-adapted cells and the light-dependent increase of ~-tocopherol in bleached cells which lack chloroplasts (Shigeoka et al., 1986) suggest that in Euglena, chlorophyll may not be directly involved in ~-tocopherol biosynthesis. The rate of increase in a-tocopherol content of the cells during the photoautotrophic phase was about the same for the cells cultivated hetero-

systems. Although ~-tocopherol content of the cells is much lower in heterotrophic culture than that in the photoautotrophic culture, the productivity in the former is about twice as high due to better cell growth. When glucose was used as a carbon source for the sequential heterotrophic/ photoautotrophic batch cultivation, the ~-tocopherol content of the cells was almost four times higher than the value obtained in the batch heterotrophic culture but there was no significant increase in the productivity due to the longer cultivation time. However, by using ethanol for the heterotrophic phase and optimizing the photoautotrophic phase, both the ~-tocopherol content of the cells and the productivity increased about four and three times, respectively. A further 37% increase in the productivity was achieved by using a continuous culture, but the ~-tocopherol content of the cells was lower than the values obtained in the batch culture.

4. Discussion

The above results have shown that just by switching from the heterotrophic to the photoautotrophic condition (illumination of culture and aerating with 5% CO2 in air) the a-tocopherol content of the cells increased by almost four times in 72 h. Further increase in a-tocopherol accumulation was achieved by using ethanol for the heterotrophic phase as well as optimizing the light condition and adding a nitrogen source during the photoautotrophic phase.

Table 1 Comparison of a-tocopherol productivity of various cultivation systems System

Cellular 7-tocopherol content (lag g-cell-~)

Photoautotrophic Heterotrophic with glucose Batch sequential heterotrophic-autotrophic with glucose Batch sequential heterotrophic-autotrophic with ethanol + CSL Continuous sequential heterotrophic-autotrophic with ethanol + CSL

634.8 115 407.3 1700 1100

~t-Tocopherol productivity (lag 1-1 h -I) 10.5 21.7 22.4 61.9 100

220

J.C. Ogbonna et ai. Journal of Biotechnology 70 (1999) 213-221

trophically with ethanol and glucose. T h u s the c a r b o n source used for the h e t e r o t r o p h i c culture affects the overall 0c-tocopherol a c c u m u l a t i o n t h r o u g h its influence during the h e t e r o t r o p h i c phase but not in the subsequent p h o t o a u totrophic phase. A n a d v a n t a g e of using ethanol as the organic c a r b o n source is that it reduces the risk of c o n t a m i n a t i o n when strict sterile conditions are not m a i n t a i n e d during the cultivation. The ~-tocopherol productivity in the continuous process (100 ~tg 1-~ h - ~ ) is c o m p a r a b l e with the value o b t a i n e d in fed-batch culture with ethanol ( O g b o n n a et al., 1998) but a b o u t twice as high as that reported for two step p h o t o heterotrophic/photoautotrophic batch cultures ( T a k e y a m a et al., 1997). Relatively higher productivity (111 ~ 138 ~tg 1-~ h - ~ ) was obtained in a fed-batch two step p h o t o h e t e r o t r o p h i c phot o a u t o t r o p h i c culture ( T a k e y a m a et al., 1997). H o w e v e r , the later m e t h o d is complex (centrifugation of the p h o t o h e t e r o t r o p h i c culture and resuspension in a fresh medium for the p h o t o a u t o t r o p h i c cultivation) and difficult to apply for large scale production. The p h o t o a u totrophic phase is the rate limiting step and the productivity can be increased by using a p h o t o b i o r e a c t o r with higher light supply coefficient ( O g b o n n a et al., 1995b). Scale up potential of this process is high. We have already optimized heterotrophic cultivation of this strain in terms o f b o t h oxygen supply and h y d r o d y n a m i c stress in a mini-jar f e r m e n t o r ( O g b o n n a et al., 1998). Thus, a conventional stirred tank bioreactor can be used for large scale h e t e r o t r o p h i c culture. In order to reduce the cost o f p r o d u c t i o n , open cultivation p o n d s can be used for the p h o t o a u t o t r o p h i c phase. The risk o f c o n t a m i n a t i o n is low since high density pure culture from the heterotrophic phase is passed to the open pond.

References Camara, B., Bardat, F., Seye, A., D'Harlingue. A., Moneger. R., 1982. Terpenoid metabolism in plastids. Plant Physiol. 70, 1562-1563. Laws, E.A., Taguchi, S., Hirata, J., Pang, k., 1986. High algal

production rates achieved in a shallow outdoor fume. Biotechnol. Bioeng. 28, 191 - 197. Mori, K., 1985. Photoautotrophic bioreactor using visible solar rays condensed by fresnel lenses and transmitted through optical fibers. Biotechnol. Bioeng. Symp. 15, 331345. Ogbonna, J.C.. M~irkl, H., 1993. Nutrient-split feeding strategy for dialysis cultivation of E. coli. Biotechnol. Bioeng. 41, 1092-1100. Ogbonna, J.C., Yada. H., Tanaka, H., 1995a. Effect of cell movement by random mixing between the surface and bottom of photobioreactors on algal productivity. J. Ferment. Bioeng. 79, 152-157. Ogbonna. J.C., Yada, H., Tanaka, H., 1995b. Light supply coefficient -- a new engineering parameter for photobioreactor design. J. Ferment. Bioeng. 80, 369-376. Ogbonna. J.C.. Yada, H., Masui, H., Tanaka, H., 1996. A novel internally illuminated stirred tank photobioreactor for large-scale cultivation of photosynthetic cells. J. Ferment. Bioeng. 82, 61-67. Ogbonna. J.C.. Tanaka, H., 1996. Night biomass loss and changes in biochemical composition of cells during light/ dark cyclic culture of Chlorella pyrenoidosa. J. Ferment. Bioeng. 82. 558- 564. Ogbonna. J.C.. Tanaka. H., 1997. Industrial-size photobioreactors. Chemtech 27. 43-49. Ogbonna. J.C.. Masui. H.. Tanaka, H., 1997. Sequential heterotrophic autotrophic cultivation--an efficient method of producing Chlorella biomass for health food and animal feed. J. Appl. Phycol. 9, 359-366. Ogbonna. J.C.. Tanaka, H., I998. Cyclic autotrophic/heterotrophic cultivation of photosynthetic cells--a method of achieving continuous cell growth under light/dark cycles. Bioresearch Technol. 65, 65-72. Ogbonna. J.C.. Tomiyama, S., Tanaka, H., 1998. Heterotrophic cultivation of Euglena gracilis Z for efficient production of zc-tocopherol. J. Appl. Phycol. 10, 67-74. Richmond. A., Boussiba, S., Vonshak, V., Kopel, R., 1993. A ne~v tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol. 5, 327-332. Shigeoka, S.. Onishi. T.. Nakano, Y., Kitaoka, S., 1986. The contents and subcellular distribution of tocopherols in Euglena gracilis. Agric. Biol. Chem. 50, 1063-1065. Soll. J.. Kemmerling, M., Schultz, G., 1980. Tocopherol and plastoquinone synthesis in Spinach chloroplasts subfractions. Arch. Biochem. Biophys. 204, 544-550. Taketomi, H.. Soda. K., Katsui, G., 1983. Results of screening test in tocopherol in microbial realm. Vitamins (Japan) 57, 133-138. Takevama. H.. Kanamaru, A., Yoshino, Y., Kakuta, H., Kawamura, Y., Matsunaga, T., 1997. Production of antioxidant vitamins, [3-carotene, vitamin C, and vitamin E by two-step culture of Euglena gracilis Z. Biotechnol. Bioeng. 53. 185-190. Tani. Y.. Osuka, S.. 1989. ~-Tocopherol production by an analog-resistant strain of Euglena gracilis Z. Agric. Biol. Chem. 53, 2313- 2318.

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 213-221 Tani, Y., Tsumura, H., 1989. Screening' for tocopherol-producing microorganisms and ~-tocopherol production by Euglena gracilis Z. Agric. Biol. Chem. 53, 305-312.

221

Tredici, M.R.. Carlozzi, P., Chini Zittelli, G., Materassi, R., 1991. A vertical alveolar panel (VAP) for outdoor mass cultivation of microalgae and cyanobacteria. Bioresearch Technol. 38, 153-159.

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JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 223-225

Effect of C a 2 + channel blockers on glycerol levels in Dunaliella tertiolecta under hypoosmotic stress Kenichiro Tsukahara *, Shigeki Sawayama, Tatsuo Yagishita, Tomoko Ogi Biomass Division, National Institute for Resources and Environment, 16-30nogawa, Tsukuba, Ibaraki 305-8569, Japan

Received 8 October 1998; received in revised form 23 November 1998; accepted 22 December 1998

Abstract

The role of C a 2 + in glycerol dissimilation under hypoosmotic stress in the halotolerant alga Dunaliella tertiolecta was investigated using a pharmacological approach. A stretch-activated Ca 2 + channel blocker, GdC13, inhibited glycerol dissimilation under hypoosmotic stress. However, addition of voltage-dependent Ca 2 + channel blockers and inhibitors of mitochondrial and endoplasmic reticulum Ca 2 + channels did not affect the glycerol dissimilation under hypoosmotic stress. The results of the present study suggest that the influx of Ca 2 + from the extracellular space via the stretch-activated Ca 2 + channels localized in the plasma membrane is required for the transduction of osmotic signal of D. tertiolecta. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Ca2 + channel blocker; Dunaliella tertiolecta; Glycerol; Hypoosmotic stress

I. Introduction

Marine microalgal biomass is one of the most potentially energetic organic resources. It is renewable and reduces CO2 from the atmosphere. A marine unicellular green alga, Dunaliella tertiolecta has been actively studied because of the high levels of intracellular glycerol content. D. tertiolecta is a marine alga that can adapt to an extremely wide range of salinities (Ginzburg, 1987). Since Dunaliella lacks a rigid cell wall, it responds to osmotic changes by rapid alterations in cell volume; thus when exposed to a hypoos* Corresponding author. Fax: + 81-298-58-8158. E-mail address: [email protected] (K. Tsukahara)

motic stress the cells immediately swell. Later the cells resume their original volume by regulating the ion-transport machinery in the plasma membrane and by regulating the intracellular glycerol content, which is used as osmolyte and compatible solute (Maeda and Thompson, 1986). While several studies have been performed to examine the movement of ions as well as the enzymes involved in the altered carbohydrate metabolism caused by the reduced osmotic strength of the medium, little has been reported on the sensing mechanism and signal transduction cascade which allow Dunaliella to respond to changes in extracellular osmolarity. As changes in phosphoinositide metabolism have been observed after hypoosmotic stress (Einspahr et al., 1988), the processes

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00076-0

K. Tsukahara et al. /Journal of BiotechnoIogy 70 (1999) 223-225

224

that trigger the metabolic changes are probably initiated by Ca 2+-dependent signal transduction cascades. Ca 2+ has been shown to serve as a second messenger in signal transduction of environmental stimuli in plants (Bush, 1995). Environmental stimuli induce a rapid and transient increase in cytosolic Ca 2 + level, which is considered to be a primary event in the signal transduction. In order to obtain direct evidence for the involvement of Ca 2+ in the mechanism of osmotic signaling in Dunaliella, it is necessary to use pharmacological approaches elucidating the requirement for cytosolic Ca 2 + change under the osmotic stress. The aim of this paper is to study the role of Ca 2 + by using Ca 2 +channel blockers to elucidate the osmotic stress signal transduction pathway in D. tertiolecta. D. tertiolecta was grown photoautotrophically in medium containing MgC12"6H20 (1.5 g), MgSO4-VH20 (0.5 g), KC1 (0.2 g), CaCI2 (0.2 g), KNO3 (1.0 g), NaHCO3 (0.043 g), Tris (2.45 g), K2HPO4 (0.045 g), NaC1 (58.5 g), Fe-EDTA (3.64 mg), EDTA.2Na (1.89 mg), ZnSO4-7H20 (0.087 mg), H3BO3 (0.61 rag), COC12"H20 (0.015 rag), CuSO4"5H20 (0.06 mg), MnC12 (0.23 rag), (NH4)6MoTO24"4H20 (0.38 rag) in 1 1 distilled H20 (pH 8.0) at 25~ with constant aeration and shaking, under continuous light (68 la Era-2 s-1). The culture was harvested by centrifugation at 600 x g for 5 rain in the late exponential growth phase, 7 days after inoculation. The cell pellet was resuspended in fresh isoosmotic (1.0 M NaC1) growth medium in which a cell density was adjusted to approximately 106 cells ml-1 and incu-

bated for 12 h with gentle shaking under continuous light (68 la E m - 2 s-1). The cells were preincubated for 10 min with inhibitors before osmotic shock. To apply hypoosmotic stress (1.00.5 M NaC1) the cells were diluted with two volumes of the medium containing 0.25 M NaC1 and incubated for the indicated time in the continuous light (68 ~ E m - 2 s-1). In order to estimate glycerol content, the cell suspension was separated into cells and supernatant by centrifugation. Collected cells were resuspended in distilled water and heated at 100~ for 5 min to inactivate enzymes. The cell suspension was then sonicated at 0~ for 3 min and centrifuged to remove insoluble materials. The content of glycerol was determined using an enzymatic assay with glycerokinase (85 Units E ml-~), pyruvate kinase (600 Units ml-1) and lactate dehydrogenase (550 Units ml-1) in 50 mM glycylglycine buffer (NADH 0.99 mM, ATP 4.0 raM, phosphoenolpyruvate (PEP) 6.5 mM, MgSO4 8.3 raM, pH 7.4). The complete conversion of glycerol to lactate was followed by measuring the oxidation of NADH spectrophotometrically at 340 nm. When the external salinity dropped from 1.0 to 0.5 M, the decrease of glycerol content reached about 55% after 60 rain (Table 1), followed by a slow decline over the next 60 min. In an earlier paper, the D. viridis glycerol concentration has been shown to decrease by half within 1 h (Brown and Borowitzka, 1979), thereby allowing the swollen cells to resume their normal size. As the intracellular glycerol in D. tertiolecta is metabolized rather than leaked into the medium during

Table 1 Effect of Ca2+channel blockers on glycerol dissimilation under hypoosmotic stress (1.0---,0.5 M NaC1) in Dunaliella tertiolecta a Glycerol content per l06 cells (lag)

CCCP Ca 2 +channel blocker

No substance a

GdCI 3 Verapamil Ruthenium red

Before hypoosmotic stress

After hypoosmotic stress

10 jaM

58.3 + 2.6

53.9 + 1.5

5 mM 100 jaM 100 jaM

55.9 + 2.6 57.4 + 3.3 58.8 + 2.5

46.1 + 5.6 34.4 + 3.1 33.1 + 2.8

59.0 + 2.4

32.7 + 1.3

Glycerol content was measured 60 rain after hypoosmotic stress. Results are the mean + S.E. from five experiments.

K. Tsukahara et al. / Journal of Biotechnology 70 (1999) 223-225

culture dilution (Fujii and Hellebust, 1992), it is possible to measure intracellular glycerol dissimilation under hypoosmotic stress in D. tertiolecta. When the algae were incubated with the H + ionophore CCCP, glycerol dissimilation was almost completely inhibited (Table 1). Since immediate effects on D. salina cells by sudden changes in extracellular osmolarity include modulation of plasma membrane H +-ATPase (PM H + -ATPase) activity (Oren-Shamir et al., 1990), this result might be explained by the inhibition of PM H + -ATPase. As shown in Table 1, Gd 3 +, which is known to block stretch-type Ca 2 + channnels localized in the plasma membrane (Knight et al., 1992), inhibited the glycerol dissimilation to significant extents. In contrast, addition of verapamil, a voltage-dependent Ca 2 + channel blocker (Knight et al., 1992), and ruthenium red, a putative inhibitor of mitochondrial and endoplasmic reticulum Ca 2 + channnel (Knight et al., 1992), did not affect the glycerol dissimilation under hypoosmotic stress (Table 1). Because Gd 3 + blocked the stretch-activated Ca 2+ channels localized in the plasma membrane, our data indicate that the influx of Ca 2 + from the extracellular space via the stretchactivated Ca z + channels localized in the plasma membrane is required for the transduction of osmotic signal of D. tertiolecta. However, further

225

studies of signal transduction pathways are necessary to completely understand the role of Ca 2 + under hypoosmotic stress in Dunaliella.

References Brown, A.E., Borowitzka, L.J., 1979. Halotolerance of Dunaliella. In: Levandowsky, M., Hunter, S.H. (Eds.), Biochemistry and Physiology of Protozoa, vol. 1. Academic Press, New York, pp. 139-190. Bush, D.S., 1995. Calcium regulation in plant cells and its role in signaling. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 95-122. Einspahr, K.J., Peeler, T.C., Thompson, G.A. Jr., 1988. Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock. J. Biol. Chem. 263, 5775-5779. Fujii, S., Hellebust, J.A., 1992. Release of intracellular glycerol and pore formation in Dunaliella tertiolecta exposed to hypoosmotic stress. Can. J. Bot. 70, 1313-1318. Ginzburg, M., 1987. Dunaliella: a green alga adapted to salt. Adv. Bot. Res. 14, 93-180. Knight. M.R.. Smith, S.M., Trewavas, A.J., 1992. Wind-induced plant motion immediately increases cytosolic calcium. Proc. Natl. Acad. Sci. USA 89, 4967-4971. Maeda, M.. Thompson, G.A. Jr., 1986. On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock. J. Cell Biol. 102, 289-297. Oren-Shamir, M., Pick, U., Avron, M., 1990. Plasma membrane potential of the alga Dunaliella and its relation to osmoregulation. Plant Physiol. 93, 403-408.

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Journal of Biotechnology 70 (1999) 227-229

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Sporulation and sterilization method for axenic culture of Gelidium canariensis P. Garcia-Jimenez *, F.D. Marian, M. Rodrigo, R.R. Robaina Department of Biology, Faculty of Marine Sciences, University of Las Palrnas G.C., Box 550, Las Palmas, Gran Canaria, Spain

Received 21 October 1998; received in revised form 3 December 1998; accepted 22 December 1998

Abstract

A sporulation and sterilization procedure was used to establish axenic cultures of sporelings of Gelidium canariensis. Sporangial branchlets excised from the thallus were rinsed in distilled water twice and in 1% sodium hypochlorite (2 min). The branchlets were cultivated overnight in multiwell plates with 0.3 ml of autoclaved seawater to promote spore liberation in 90% of the cultivated branchlets. The branchlets were transferred to an antibiotic solution made of ampicillin, penicillin, rifampicin, nystatin (0.2 mg m l - ~ each) and 0.1 g m l - z of GeO2 in liquid PES for 45 days, during which clusters of spores (85-100 spores) were observed on the surface of the branchlet. After 55 days, they became axenic sporelings with the prostrate and erect system characteristic of Gelidium canariensis. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Axenic culture; Gelidium canariensis; Rhodophyta; Sporelings

I. Introduction

Heterotrophic culture may provide a cost-effective large-scale alternative method for cultivation of algae that utilize organic carbon sources (Chen, 1996). Axenic heterotrophic cell and tissue cultures from macroalgae can be established from precultured axenic carposporelings. For example, axenic heterotrophic cultures of the carragenophytic macroalga Grateloupia doryphora,

* Corresponding author. E-mail address: [email protected] (P. GarciaJimenez)

which produces secondary metabolites, sulfated polysaccharides and lipids, were successfully initiated from axenic spores (Garcia-Jimenez et al., 1996, 1998; Rodrigo and Robaina, 1997). Axenic cultures of Gelidium, another important genus of macroalgae, producing over 50% of the agar and agarose world market (Armisen and Galatas, 1987), have not been established yet, although laboratory and field cultures of its spores have already been reported (Rodriguez, 1996, and Refs. therein; Rojas et al., 1996). Here, a sporulation and sterilization method for producing axenic sporelings of Gelidium canariensis is described.

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P. Garcia-Jimenez et al. Journal of Biotechnology 70 (1999) 227-229

2. Materials and methods

Sporangial branchlets were excised from epiphyte-free thalli of G. canariensis collected at the North coast of Gran Canaria. Some branchlets were directly used to test the effect of the antibiotics according to the one-step antibiotic method (Saga and Sakai, 1982). As a result, we obtained an effective and non-inhibitory antibiotic solution mixture made of (final concentration in the culture medium): ampicillin (0.2 mg ml-1), penicillin (0.2 mg ml-1), rifampicin (0.2 mg ml-1) nystatin (0.2 mg m1-1) and germanium dioxide (0.1 g ml-1). To establish axenic cultures of sporelings we proceeded as follows: the branchlet was treated with 1% sodium hypochlorite for 2 rain and then incubated overnight in 0.3 ml of sterilised seawater in multiwell plates. The seawater evaporated partially (between 30 and 50%), thus promoting a hydric stress for sporulation. The sporulation and sterilization procedure went on by transferring the branchlets to petri dishes with 20 ml of PES (Provasoli-enriched seawater; Provasoli, 1968) medium and the antibiotic solution described above. Controls of axenity were performed in bacteriological Marine Agar (Difco) to check the presence of surface contaminants, namely bacte-

ria, fungi and other heterotrophic organisms. Thus, bacterial and fungal growth was not observed. Algal endophytes or epiphytes normally associated to Gelidium thalli were not recorded in the branchlets or sporelings.

3. Results and discussion

Sporulation was mainly observed when preceded by hydric stress. Ninety percent of all cultivated branchlets cultivated under hydric stress released spores, while in controls without hydric stress, only 40-50% of branchlets sporulated. The hydric stress overnight and rehydration of the branchlet in the medium with antibiotics seemed to push the spores out. The benefits of a hydric stress for sporulation have previously been reported in other algae (Edding et al., 1987). Clusters of axenic spores, attached to the branchlet in a mucous layer, were formed within 30-45 days after reculture (ca 85-100 spores per cluster. One or two clusters per branchlet (Fig. 1)). The development of the spores of G. canariensis was similar to that described in other Gelidiales (Macler and West, 1987; Fredriksen and Rueness, 1989). The first divisions were observed 3 or 4 days after, when the spores settled in the culture dishes and formed new cells (germ tube), that developed a pigmented cell which originated an erect axis. Within 55 days, the latter became an incipient prostrate system with small buds along the axis (Fig. 2). In conclusion, the sporulation induced by hydric stress and sterilization with an appropriate mixture of antibiotics made it possible to establish axenic cultures from spores of the agarophytic red seaweed Gelidium canariensis. It remains to investigate whether the spores will grow in media supplemented with organic carbon sources.

Acknowledgements Fig. 1. Part of a sporangial branchlet (b) of G. canariensis discharging tetraspores (small arrow). In detail, higher magnification of sporelings. Scale bar = 0.2 mm.

This work was financially supported by the Canarian Government grant PI1998/079.

P. Garcia-Jimenez et al. Journal of Biotechnology 70 (1999) 227-229

Fig. 2. Germinated and axenic sporelings showing elongation of a bud (b) after 45 days in Petri dish culture. Scale bar = 0.2 mm.

References Armisen, R., Galatas, F., 1987. Production, properties and uses of agar. In: McHugh, D.J. (Ed.), Production and Utilization of Products from Commercial Seaweed. FAO Fish Technical Papers. FAO, pp. 228-240. Chen, F., 1996. High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol. 14, 421-426. Edding, M., Le6n, C., Ambler, R., 1987. Growth of Gracilaria sp. in the laboratory. Hydrobiologia 151/152, 375-379. Fredriksen, S., Rueness, J., 1989. Culture studies of Gelidium

229

latifolium (Grev.) Born. et Thur. (Rhodophyta) from Norway. Growth and nitrogen storage in response to varying photon flux density, temperature and nitrogen availability. Bot. Mar. 32, 539-546. Garcia-Jimenez, P., Robaina, R.R., Luque, A., Tsekos, I., 1996. Glycerol-activated cellular and biosynthetic activity during growth and morphogenesis of carpospore seedlings of Grateloupia doryphora (Cryptonemiales, Rhodophyta). Phycologia 35, 261 - 269. Garcia-Jimenez, P., Rodrigo, M., Robaina, R.R., 1998. Influence of plant growth regulators, polyamines and glycerol interaction on growth and morphogenesis of carposporelings of Grateloupia cultured in vitro. J. Appl. Phycol. 10, 95-100. Macler, B.A., West, J.A., 1987. Life history and physiology of the red alga Gelidium coulteri, in unialgal culture. Aquaculture 61, 281 - 293. Provasoli. L., 1968. Media and prospects for the cultivation of marine algae. In: Watanabe, A., Hattori, A. (Eds.), Cultures and Collection of Algae. Japanese Society of Plant Physiologists, Tokyo. pp. 63-67. Rodrigo, M., Robaina, R.R., 1997. Stress tolerance of photosynthesis in sporellings of the red alga Grateloupia dorvphora compared to that of Stage III thalli. Mar. Biol. 128, 689-694. Rodriguez. D., 1996. Vegetative propagation by fragmentation of Gelidium sclerophyllum (Gelidiales, Rhodophyta). Hydrobiologia 326 327. 361-365. Rojas, H.R., Le6n, N.M., Rojas, O.R., I996. Practical and descriptive techniques for Gelidium rex (Gelidiales, Rhodophyta) culture. Hydrobiologia 326: 327, 367-370. Saga, N., Sakai, Y., 1982. A new method for pure culture of macroscopic algae, the one step selection method. Jpn. J. Phycol. 30, 40-43.

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Photobioreactors" light regime, mass transfer, and scaleup E. Molina Grima, F.G. Aci6n Fernfindez, F. Garcia Camacho, Yusuf Chisti * Department of Chemical Engineering, Unirersitv of Almeria, E-04071 Almeria, Spain

Received 7 September 1998; received in revised form 20 November 1998; accepted 22 December 1998

Abstract

Design and scaleup of tubular photobioreactors are discussed for outdoor culture of microalgae. Culture productivity is invariably controlled by availability of light, particularly as the scale of operation increases. Thus, light regime analysis is emphasized with details of a methodology for computation of the internal culture illumination levels in outdoor systems. Supply of carbon dioxide is discussed as another important feature of algal culture. Finally, potential scaleup approaches are outlined including promising novel concepts based on fundamentals of the unavoidable light-dark cycling of the culture. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Light regimen; Mass transfer; Microalgae; Photobioreactors; Scaleup

I. Introduction

Although the term 'photobioreactor' has been applied to open algal ponds and channels, it is best reserved for devices that allow monoseptic culture which is fully isolated from a potentially contaminating environment. This latter defining convention will be followed here. The available photobioreactor configurations are numerous (Lee, 1986; Tredici and Materassi, 1992; Borowitzka, 1996; Pulz and Scheinbenbogen, 1998), but most may be classified into one of two types: either tubular devices or fiat panels. These can be further categorized according to orientation of tubes or panels, the mechanism for circu* Corresponding author. Tel.: + 34-950-21-5032; fax: + 34950-21-5484. E-mail address: [email protected] (Y. Chisti)

lating the culture, the method used to provide light, the type of gas exchange system, the arrangement of the individual growth units, and the materials of construction employed. The developmental state of the photobioreactor technology has been reviewed comprehensively elsewhere (Lee, 1986; Borowitzka, 1996; Pulz and Scheinbenbogen, 1998); here the focus is on tubular photobioreactors (Fig. 1) which are amongst the most promising culture systems for potential large-scale production of microalgae-derived highvalue products. Some potential products are listed in Table 1. Design and scaleup methodologies for photobioreactors are poorly developed. Irrespective of the specific reactor configuration employed, several essential issues need addressing (Weissman et al., 1988): (i) effective and efficient provision of

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light; (ii) supply of carbon dioxide while minimizing losses; (iii) removal of photosynthetically generated oxygen that may inhibit metabolism or otherwise damage the culture if allowed to accumulate; and (iv) sensible scalability of the photobioreactor technology. Biomass productivity of a photobioreactor depends on close alignment of the culture environment to the needs of the selected algal strain. Some environmental factors, e.g. temperature and mineral nutrients supply, are relatively easily controlled, but others such as the supply of solar radiation are more difficult to regulate. Productivity is determined by the growth rate which, for fixed fluid-dynamics and temperature, is a function of the light profile within the reactor and the light regime to which the cells are subject. In dense microalgal cultures, light penetration is impeded by self-shading and light absorption (Rabe and Benoit, 1962; Frohlich et al., 1983: Erickson and Lee, 1986). These effects affect the radiation profile inside the culture. Consequently, within a photobioreactor exist zones of different levels of illumination. These zones may have different volumes. How long the cells reside in zones of different illumination is a function of the culture

fluid-dynamics (Philliphs and Myers, 1954; Terry, 1986; Grobbelaar, 1994; Grobbelaar et al., 1996). In addition to affecting light availability, fluid movement affects also the transport behavior, i.e. availability of carbon dioxide and other nutrients. In an optimal system where no other factors limit, the light availability determines the rate of photosynthesis and the productivity. However, excessive light can be harmful and is known to produce a photoinhibitory response (Bannister, 1979; Aiba, 1982). Here we address the essential aspects of providing light to outdoor photosynthetic cultures, supplying carbon dioxide as the principal carbon source, and scaling up of photobioreactors, with emphasis on the tubular types.

2. Light regime 2.1. How light affects productivity? Availability and intensity of light are the major factors controlling productivity of photosynthetic cultures (Lee and Low, 1992; Pulz and Scheinbenbogen, 1998). In continuous culture as typically practiced for microalgae, the biomass productivity

Fig. 1. Airlift-driven tubular photobioreactors. Two reactors are shown with the tubular loops immersed in a pond of cooling water. The two vertical columns at the far end are the airlift devices for circulating the culture through the horizontal loops. The 0.2-m 3 photobioreactor on the right was used in obtaining the data presented.

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247 Table 1 Potential high-value microorganisms a Product

products

from

Source organism b

Amphidinolides Amphidinium sp. and amphdinins Astaxanthin Haernatococcus pluvialis, Chlorella sp. [3-Carotene Dunaliella Docosahexaenoic acid "f-Linolenic acid

Isochrysis galbana

Other polyunsaturated fatty acids Fucoxanthin

Phaeodactylum tricornutum, Isochrysis galbana Phaeodactylum tricornutum Alexandrium hiranoi Oscillatoria agardhii

Goniodomins Oscillapeptin Phycobiliproteins Phycocyanin

Spirulina sp.

Red algae, cyanobacteria Spirulina platensis

photosynthetic

Current or potential use Antitumor agent Pigment Colorant, food supplement Essential fatty acid Essential fatty acid Health care, food supplement Antioxidant Antifungal agent Elastase inhibitor Colorants

els that express p in terms of the average irradiance raised to some power greater than unity better fit experimental observations (Fernfindez Sevilla, 1995; Pulz and Scheinbenbogen, 1998). Thus, using the previously developed (Molina Grima et al., 1994) Eq. (7) of Table 2 as a starting point, we established the equation (Molina Grima et al., 1996), ,t/max"I Ivb+ Tfoo) '~

(8)

Eq. (8) accounts for photoinhibition and the fact that the dependence of p on the average irradiance (Iav) varies with the incident irradiance level (Io). Eq. (8) was established with an outdoor culture of Phaeodactylum tricornutum UTEX 640 (Aci6n FernS.ndez et al., 1998); the specific values of the various growth parameters for that culture

Colorant Table 2 Models for light-dependent specific growth rate

Based on Yamaguchi (1997) and Benemann (1989). b Only representative examples are listed. a

Equation

(p) is a function of the cell concentration (C b) in the effluent and the dilution rate (D); thus,

p = DCb.

233

1.

12 = ~

~12maxI

(2)

,/'/max+ ~I

(1)

At steady state, the dilution rate equals the specific growth rate (/z) which is governed by the amount of light, the rate controlling factor. The dependence of /z on the average irradiance has been expressed variously as summarized in Table 2. Generally, p increases with increasing irradiance, reaching a maximum value, ,//max. Further increase in irradiance may actually inhibit growth-- a phenomenon known as photoinhibition. Although photoinhibition is well documented, it has often been disregarded. For example, Eqs. (2)-(4) and Eq. (7) in Table 2 do not take photoinhibition into account. In Table 2, only Eq. (5) and Eq. (6) consider the inhibitory effects of excessive light. Studies suggest that growth mod-

Reference Tamiya et al. (1953)

e,+.x) ,3, Van Oorschot (1955) z-~) 1 2 - -12max/e('~ /max ]'/maxI

(4) Steele (1977)

(5)

Bannister (1979)

(6)

Aiba (1982)

(Ki" + Ira)''

,]~rrlaxI 12=

12 K~+I+--

K,

6.

12-

PmaxI" I~,+I"

(7)

Molina Grima et al. (1994)

234

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

Table 3 Kinetic parameters (Eq. (8)) for an outdoor culture of P. tricornutum (Aci6n Femfindez et al., 1998) Parameter

Value a

flmax ( h - l)

0.063 94.3 3426 3.04 1.209 514.6

Ik (laE m -2 s - l ) Ki (laE m -2 s - l ) a b c

a The noted values apply over the full calendar year.

are noted in Table 3. Eq. (8) and others in Table 2 allow an estimation of the biomass productivity so long as an average irradiance value can be determined. This problem is addressed next. 2.2. What & average irradiance?

Even when the outdoor incident radiation level is constant, the irradiance within a culture varies as a function of position (Erickson and Lee, 1986). Cells nearer the light receiving surface experience a higher irradiance than cells elsewhere in the vessel (Frohlich et al., 1983). Cells closer to the light source shade those further away; hence, productivity varies with position and time (Tamiya et al., 1953; Bannister, 1979; Laws, 1980; Myers, 1980; Ree and Gotham, 1981). A mean value of irradiance may be defined as the volume average of the local irradiance values inside a culture. An average irradiance (I~) is the light level experienced by a single cell randomly moving inside the culture (Rabe and Benoit, 1962). In a cell-free system, average irradiance is independent of the state of mixing. When cells are distributed homogeneously, the average irradiance under given conditions is again the same for all cells; however, as discussed later, average irradiance is not a sufficient criterion of culture performance because it considers only the total length of the dark and the light periods, not the frequency of switch. Ignoring for the moment the dynamics of the cell, the average irradiance level (Ia~) inside culture depends on the following factors: the external irradiance (Io) on the surface of the reactor; the reactor geometry (Frohlich et al., 1983; Lee

and Low, 1991, 1992; Quiang et al., 1996; Aci6n Fernfindez et al., 1997); the concentration and morphology of cells; the level of cellular pigmentation; and the absorption characteristics of the pigment. An additional complicating factor is generally specific to outdoor culture: Outdoor cultures are subject to cyclic changes in irradiance levels. At least two cycles can be distinguished with substantially different cycling times: (i) a relatively long daily cycle; and (ii) a yet longer cycle based on the change of seasons during the year. A third cycle is due to fluid movement between zones of different illumination within a photobioreactor. Cycles (i) and (ii) affect only the incident radiation on the surface of a photobioreactor, but beyond that factor these cycles are unlikely to have any other impact on kinetics of the culture. The period of cycle (ii) is much longer than the residence time of the cells in a photobioreactor in continuous culture. The diurnal cycle means that a culture is light limited at dawn and dusk; however, during the midday peak light period, the culture may be photoinhibited. The peak light level may exceed 2000 laE m - 2 s - l , which is several times above the saturation irradiance. When the external irradiance level varies with time, an average irradiance is determined by time-averaging over short intervals. Current methods of estimating an 'average irradiance' level do not take into account the lightdark cycling associated with fluid motion in a bioreactor. In reality, light regime experienced by cells, i.e. the total cumulative illumination and the light-dark movement frequency, is what logically should affect biomass productivity. Identical average irradiance levels cannot necessarily mean identical productivities: if a culture requires a certain cumulative photon flux density level over a 6-h residence time, the same level could be provided over a shorter period without affecting the average light level over the residence time interval; however, this is bound to reduce productivity. Existing methods of estimating average illumination employ an approach consisting of the following: (i) estimation of the total photosynthetically active incident radiation at the surface of the photobioreactor; (ii) use of Beer-

E. Mo/ina Grima et al./Journal of Biotechnology 70 (1999) 231-247

Lambert law to determine the radiation level at any depth inside the culture; and (iii) some form of averaging of the radiation level inside the vessel. The various method vary in details of steps (i) and (iii). Step (ii) requires measured data on the attenuation of incident radiation with depth; the attenuation is a function of the concentration of the cells and the light absorption characteristics of the cellular pigments (Chrismadha and Borowitzka, 1994). Some of these steps are discussed later in this overview. 2.3. Quantifying average irradiance

To establish the average irradiance level inside a vessel, we must first determine the incident irradiance level as discussed below. 2.3.1. Incident radiation The radiation incident on the surface of a photobioreactor consists of direct sunlight, reflected radiation from the surroundings, and diffuse radi-

235

ation due to particulate matter in the atmosphere. The incident sunlight level is easily estimated (Liu and Jordan, 1960; Duffle and Beckman, 1980). The incident light level on an outdoor reactor is a function of time, the geographic location of the reactor, and environmental factors (Incropera and Thomas, 1978). Principles of solar power engineering provide methods for estimation of the incident photon flux anywhere on the surface of the Earth (Liu and Jordan, 1960; Duffle and Beckman, 1980) so long as the following are known: the day of year (N), the solar hour (h), and the geographic latitude (qS) of the photobioreactor. These variables determine the angle of incidence (0) of direct radiation on the photobioreactor's surface, i.e. the angle between the incident beam and the normal to the surface (Fig. 2). Two additional angles need to be known: the surface slope (fl), i.e. the angle between the photobioreactor's surface and the horizontal; and the surface azimuth angle (~,), i.e. the deviation of the projection on a horizontal plane of the normal

Zon~

t!Normalto horizontal

.... ~

--v~/~- ~ ,

Boam solar

~diation

N:rmalto slopin~,,~

~

i surface

ez---'--.~l

/

Diffuse solar

~i~on

Reflected solar radiation

(Diffuse re.ectance)

i

Fig. 2. The various angles relevant to estimation of solar radiation level incident on the flat surface of a photobioreactor with any general orientation relative to the land (see text for details). Adapted from Duffle and Beckman (1980).

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

236

to the surface from the local meridian, with zero due south, east negative, and west positive (Liu and Jordan, 1960; Duffle and Beckman, 1980). Under certain conditions, a culture vessel may have multiple values of fl and ?'. The total daily radiation (H), the daily diffuse radiation (Hal), and the daily direct radiation (HB) on a horizontal surface are all dependent on the level of the extraterrestrial radiation (Ho) obtained as (Liu and Jordan, 1960)

_

_

nilE: [0.409 + 0.5016"sin(cos 60)] 24 {

+ [0.6609 - 0.4767"(cos - 60)]cos co } ( cosco'cosco, ) (15) x \sin ~ss- cos'COScos ' where the angle co depends on the solar hour (Liu and Jordan, 1960): co = 15(12- h).

(16)

where ( is the universal solar constant (( = 1353 W m -2, Duffle and Beckman, 1980) and cos is given as (Liu and Jordan, 1960):

In Eq. (15) the factor E: is the photosynthetic efficiency of the solar radiation and it takes into account the fact that only a part of the total solar spectrum is photosynthetically active. Similarly, the photosynthetically active amount of the hourly diffuse radiation incident (ID) on a horizontal surface is obtained as a function of daily diffuse radiation (Ha) and the solar hour (Duffle and Beckman, 1980):

co~ = c o s - ~( -

ID--

Ho =

1 + 0.003"cos~ 365 ) ) 2ncos cos ~b'cos 6-sin cos + 360 "sin ~b.sin c5 , (9)

ngdEf( tan ~-tan

~b).

(10)

In Eq. (9) and Eq. (I0) ~b is the geographic latitude and 6 depends on the day of the year (Liu and Jordan, 1960) as follows, 9 {360(284 + N)) c5= 23.45"sln~, 3-6-5 "

(11)

Now, H, Ha, and HB are, obtained as noted by Liu and Jordan (1960); thus,

H=NHo,

(17)

Now, the photosynthetically active direct hourly radiation level (IB) is obtained as

IB=I--ID.

(18)

Eqs. (15)-(18) apply to a horizontal surface. When the surface is tilted, the 18 and ID values need to be corrected (Liu and Jordan, 1960); thus, cos 0 COS 0z'

IBt = IB.

(19)

and (12)

(1 + c o s fl) I D t = ID

Hd= (1.390 - 4.027N + 5.530N 2 - 3.108N3)H, (13) and

H~= H - Hd.

cos co'cos cos ) 24 \ s i n co,-- cos'COScos "

(14)

In these equations, N is the atmospheric clarity index which is a function of factors such as cloud cover and the amount of suspended matter in the atmosphere. The photosynthetically active amount of the total hourly radiation incident (I) on a horizontal surface is a function of H and the solar hour (Duffle and Beckman, 1980); thus,

2

(20) '

where IB, and 1D, are the equivalents of IB and ID for the surface tilted at angle fl relative to the horizontal. In Eq. (19) 0 is the angle of incidence which, according to Liu and Jordan (1960), is calculated as 0 = cos- l(sin ~ .sin 05"cos fl - sin ~-cos ~b.sin fl .cos ?, + cos 6.cos r

co

+ cos 6-cos q~-sin fl-cos ),'cos co + cos g-sin fl'sin ?,'sin co). The angle 0: (Eq. (19)) is estimated as:

(21)

E. Molina Grima et al .......Journal o f Biotechnolog)" 70 (1999) 2 3 1 - 2 4 7 0z -- COS - I(COS ~ "COS {~b"COS O.) "-it- s i n

,5 .sin q~). (22)

Solar beam

F

The level of radiation reflected from the ground (g) can be estimated using the equation (Liu and Jordan, 1960)

Ir+ (IB+ ID)p( 1 - c ~ fl) 2 '

237

j

Photobioreactor

surface /

(23)

b R

where p is the ground reflectivity. For the purpose of the work discussed here, a ground reflectivity (p) value of 0.5 was assumed (Garcia Camacho et al., 1999). For any three-dimensional photobioreactor, the total incident radiation is the sum of the direct, diffuse, and reflected, i.e. 18, + ID, + L; however, /~ may be ignored for a horizontal surface such as the top face of a flat plate. Once the photosynthetically active radiation incident on the surface of a photobioreactor at a given instant is established (Eqs. (19) and (20), and Eq. (23)), the next step is to determine the local radiation level as a function of position inside the culture. This is explained in the following section.

2.3.2. Local irradiance Even though the fluid in a photobioreactor may be well-mixed and with uniform optical properties, the illumination profile within is never uniform even when the culture depth is quite shallow. The irradiance at any point in the culture is a function of the total incident radiation at the surface of the culture, the optical properties of the culture, and the distance that the point is located from the surface. The mathematical relationship governing the local irradiance is the well-known Beer-Lambert law. For any photobioreactor, the distance (Pdirect) traveled by a direct ray from the tube's surface to a point within the culture may be determined from the position of the Sun, which establishes the point of incidence on the surface of the reactor, and the polar coordinates of the point (r;, ~0) in a cross-section of the tube. For a vertical tube as illustrated in Fig. 3, the distance (Pdirect) tO the point (r;, ~0) is calculated as (Garcia Camacho et al., 1999),

W !,,~narytangem surface

E

t(

sSolar._beam

N

Tube surf~

W

Fig. 3. Relationships among the various angles and distances associated with the path of direct radiation to any internal point (r,. ~o) inside the culture in a vertical tube. A plan view of the cross-section of the tube is shown in the lower part of the figure.

a;'cos co Pdirect --

R.sin e - r~ sin ~0 --

cos

-0"

,

cos

(24)

-0:

where the parameter a; is

a;

r; cos q~ - R'cos e R.sin e - r~ sin rp = sin ~ cos co "

(25)

The various lengths and angles relevant to Eq. (24) and Eq. (25) are shown in Fig. 3. The angle 0' in Eq. (24) is a function of the refractive indices of air and water; using Snell's law, O'z can be shown to be: 0'_ = sin-~(0.752.sin 0:).

(26)

Once, the distance to the point (Pdirect) is determined, Beer-Lambert law may be applied to obtain the local irradiance IB,(r,, ~0); hence,

Is,(r~, ~ ) =

IB, e x p ( -

KaCbPdirect),

(27)

E. Molina Grima et al. //Journal of Biotechnology 70 (1999) 231-247

238

difference was lower in the horizontal reactor. However, at noon, the irradiance distribution in the vertical vessel, unlike the horizontal tube, was fairly homogeneous, i.e. the local irradiance value did not depend on the angle 09. The reason for this phenomenon follows: around midday, the contribution of the dispersed irradiance to the total irradiance is large in comparison with the contribution of the direct irradiance in the vertical arrangement. Clearly, the level of irradiance is always higher in the horizontally placed tube irrespective of the solar hour. Once the local irradiance has been established, the average irradiance is calculated as explained next.

where K~ is the absorption coefficient and Ch is the concentration of biomass. Eq. (27) is written for the direct radiation; similar equations may be written for the disperse radiation and reflected radiation. The local value of ID, at location (r;, q~) is given as IDt(ri, qg) =

ID, e x p ( -

(28)

KaCbPdisperse),

where, based on trigonometric principles, the Pd~r~r~e can be shown to be Pdisperse

= w/(r, sin q9 - R sin e) 2 + (re cos 4o - R'cos e)z. (29) The computed local irradiance profiles in a horizontal and an equal-diameter vertical tube at solar hours 8 and 12 are shown in Fig. 4. As expected, in both cases, the local irradiance profiles change with the changing position of the Sun. In the morning (h = 8), in the vertical reactor the side facing the Sun had much higher direct local irradiance values than the opposite side. This

DIRECT RADIATION (SOLAR HOUR = a)

2.3.3. Average irradiance Once the local direct and local disperse irradiances--i.e. I~,(ri, q~) and ID,(h, q~), respectively-are estimated, the integration of the local values over the length and the radius of the tube yields the total average hourly irradiance inside the culture (Aci6n Fernfindez et al., 1997); thus,

DIRECT RADIATION (SOLAR HOUR = 12)

RADIAL DISTANCE = 0.02 m

S

~

s

i

? rl~

E

//

,7s

(/

it

. . . . . .

~

\

t...l~ .......... ii ............... , .

.'

\\

\

\

k\

\',

........ .I.....] .... I..... ~ L o ,

,

/ P~L/

=

,7 I

;1)-/;ool--!,<,,.r,-,s-,, '

'

'

Ii,' 4 ~ ~ " ~ ' ~ " ~ ' " ~ * ~ ~

I"-

~

""

I

r-

l

RADIAL DISTANCE=

i ~ , ~ 1

\

,

I'

/

VX 9•

,

..,..~ ./ ............ \'~

'

.

9 (RADIANS)

......... )1.. J.....,?1..... I. lo W

i l/~

\ 4 ~

5

5

N Fig. 4. Calculated radial irradiance profiles in cross-sections of vertical (dashed lines) and horizontal tubes (solid lines) at solar hours 8 and 12. Both tubes were 0.1 m in diameter; the assumed biomass concentration was 1.9 kg m - 3 of P. tricornutum with a pigment content of 2.21%.

E. Molina Grima et al. /Journal of Biotechnology 70 (1999) 231-247

Iav--~-~

IBt(ri, ~p)r dr dq~ ~0)r dr d~p de)}.

QI_ d[Cr] = (kLaL)co2([CO2]* --[COz])Sdx

(30)

The foregoing procedure disregards movement of cells among zones of different illumination. In effect, the above procedure assumes that the light exposure history of all cells is identical and frequency of light-dark movement is inconsequential. The first of these assumptions is generally valid in a given vessel. Furthermore, because the light-dark frequency is virtually constant in a given flow regime for a specific reactor, effects of the light-dark movement are masked in a given reactor; however, problems arise when comparing reactors of different scales. This aspect is examined in the section on scaleup.

3. Gas-liquid mass transfer: provision of carbon dioxide

Carbon dioxide is the usual carbon source for photosynthetic culture of microalgae. Carbon dioxide is typically supplied by continuous or intermittent injection of the gas at the beginning of a tubular solar receiver. As the carbon is consumed, oxygen is ultimately produced by photolysis of water. The generated oxygen is released into the culture fluid. The fluid in a tubular solar receiver is invariably in plug flow; hence, the concentration of carbon dioxide reflected in the culture pH changes (Livansky and Bartos, 1986) along the tube and so does the concentration of oxygen (Weissman et al., 1988). Gas-liquid mass transfer in such a system has been addressed (Camacho Rubio et al., 1999) following similar developments for open raceway culture (Livansky, 1982, 1990; Livansky and Bartos, 1986). For the liquid phase in plug flow, the changes in concentrations of dissolved oxygen and dissolved inorganic carbon along the loop are expressed as follows (Camacho Rubio et al., 1999):

QL d[O2] = (kLaL)%([O2]* --[O2])Sdx + Ro2(1 - eG)Sdx,

239

(31)

+ Rc%(1 - ea)Sdx,

(32)

where QL is the volumetric flow rate of liquid through the tube, kLat is the volumetric gas-liquid mass transfer coefficient, Sdx is the differential volume of the tube, and ea is the fractional gas holdup. The volumetric rates of generation of oxygen and consumption of carbon dioxide are represented as Ro2 and Rco 2, respectively. The liquid phase concentrations of oxygen, inorganic carbon, and carbon dioxide are represented as [O2], [CT], and [CO2], respectively. Note that the concentration values marked with asterisks are equilibrium concentrations, i.e. the maximum possible liquid-phase concentration of the component in contact with the gas phase of a given composition. In Eq. (31) and Eq. (32), the first term on the right-hand-side accounts for mass transfer to/from the gas phase and the second term accounts for generation or consumption. Depending on the situation, any of the terms on right-hand-side of Eq. (31) and Eq. (32) may be positive or negative. The mass balance considers the total inorganic carbon concentration [CT] and not just that of carbon dioxide. This is because CT includes the dissolved carbon dioxide, the carbonate (CO 2-) and the bicarbonate ( H C O 3 ) species (Livansky, 1990). The CT value is pH dependent as detailed later. As for the liquid phase, a component mass balance can be established also for the gas; hence,

dFo2 = - (kLaL)o2([02]* --[02])Sdx,

(33)

and dFco 2 = - (kLaL)c%([C02]* -- [C02])Sdx,

(34)

where F% and Fc% are the molar flow rates of the two components in the gas phase. Note that because of the changes in molar flow rates, the volumetric flow rate of the gas phase may change along the tube. This change is easily evaluated from the available equations of state for gases. This analysis assumes a constant molar flow rate for nitrogen and water within any section of the tube: nitrogen is neither consumed nor generated, and the gas phase is water saturated.

240

E. Molina Grima et al./'Journal of Biotechnology 70 (1999) 231-247

The equilibrium concentrations of the two gases in the liquid can be calculated using Henry's law; thus,

[02]*= =

Ho2Po 2

Ho2(Pr -

P~)

Fo2 FO2 + FCO2 -~- FN.~+ FH20' -

[Cv]=

K1

d[CT]

(35)

(

K,

K1

EGO2 Fo2 -~- Fco2 -~- FN2 -3t-FH20' (36)

where Ho2 and Hco 2 are the Henry's constants for oxygen and carbon dioxide. The other symbols are explained in the nomenclature. Eqs. (31)-(36), in combination with the known initial conditions and the dissociation equilibria for water and H2CO3, allow the determination of the CO2 and 02 axial profiles in the tubular loop. Similar methods have been applied to other zones of a photobioreactor, e.g. the riser, the downcomer, and the gas-liquid separating region of the airlift device that moves the fluid through the tubular solar loop (Camacho Rubio et al., 1999). As noted earlier, the total inorganic carbon concentration and the dissolved carbon dioxide in the culture are interrelated (Livansky, 1990). The dissolved carbon dioxide is in equilibrium with carbonate and bicarbonate species. These equilibria are pH dependent (Livansky and Bartos, 1986). The pH variation in culture is mainly due to consumption of carbon dioxide although variations due to consumption of other nutrients and/ or degradation of the excreted metabolites occur. Loss of dissolved carbon dioxide due to uptake into algal cells is partly compensated by regeneration from carbonates and bicarbonates (Livansky, 1990). Consequently, carbon dioxide uptake is accompanied by changes in pH. The total concentration of inorganic carbon is given by: [C021

q-

K,K2 )

1 + [H+] + [H +]2 d[CO2]

[CO2]* = Hco2Pco 2

[CT] =

[HCO3-] + [CO 2 -1.

(38)

where K~ and K2 are the first and second dissociation constants for H2CO3. Eq. (38)can be rewritten in a differential form as follows,

= = H c o 2 ( P T -- P~)

K1K2)[CO2],

I + [ H + ] + [ H +]2

(37)

Using Eq. (37) and the well-known dissociation equilibria for water and carbonate-bicarbonate system (Livansky, 1990), we obtain,

--

2K1K2~

[CO2] [H + 15 + [H + ]3] d[H + 1.

(39)

Taking into account the electroneutrality constraint, the well-known dissociation equilibria for water and H2CO 3, and assuming a constant concentration of all ions other than H +, O H - , carbonate, and bicarbonate in the culture, the following is easily derived (Camacho Rubio et al., 1999): d[H +]

Kl +2K1K2

:

K.. 1+~+

[H+]

[H +]2

d[CO2]. KI[COj 8K1KdCOj

]-------T[H + +

[H+] 3

(40)

Eq. (39) and Eq. (40) relate the three concentrations, [H + ], [CT] and [CO2]; hence, any two of those concentrations may be calculated if the third is known. Similar mass transfer models exist for open channel type algal culture systems (Livansky, 1982, 1990; Livansky and Bartos, 1986; Mfirkl and Mather, 1985). The noted model is potentially useful for limited scaleup; it does not consider possible inhibitory effects of accumulated oxygen on photosynthesis, i.e. Ro 2 and Rco 2 are assumed to be axially invariant and possible changes in cell concentration are disregarded. Furthermore, in a long tube, other parameters (e.g. gas holdup and kLaL) are likely to also vary. Camacho Rubio et al. (1999) used Eqs. (31)(40) for estimating the behavior of a culture system. Culture variables such as dissolved oxygen, carbon content in the fluid, the composition of the outlet gas, the carbon dioxide requirements, and the pH of the culture could be predicted. As

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247 350 ~J

O0

earlier; however, because the solar receiver tubes of the photobioreactor were located in a cooling pond that had a radiation concentrating effect (albedo = 2), the incident radiation level was appropriately corrected (Aci6n Fermindez et al., 1998). The efficiency factor Es (Eq. (15)) value used was 1.74 +_ 0.07 laE J - 1 as previously discussed (Aci6n Fern/mdez et al., 1998).

300 250 200

~ 150 ~ loo ~

5O t

I

t

4

8

12

"

241

t

i

t

16

20

24

Solar hour 0.215 ,.

.~ 0.210 9

9 n

9 1 4 9

=n

9 9

4 Scaleup

O.OOe ~

No reliable systematic scaleup method exists for photobioreactors. The mass transfer and the light regimen models outlined above provide a useful starting point as detailed elsewhere (Camacho Rubio et al., 1999). Predictive capability of the noted mass transfer model has been confirmed for a range of parameters (Camacho Rubio et al., 1999); however, as noted in the previous section, because of several restrictive assumptions, the model could not a priori predict the performance of a significantly larger reactor particularly if the tube diameter changed substantially. This point is important: other than 'scaleup' by multiplication of identical tubular modules, the only way to increase volume is by increasing length or/and diameter. Nevertheless, simulations based on the mass transfer model (Camacho Rubio et al., 1999) suggest that increasing tube length for a constant diameter will alter the culture pH at the tube exit, the dissolved oxygen content of the exiting fluid, and the carbon dioxide losses as shown in Fig. 6. The vertical bars around the simulated profiles in Fig. 6 indicate the maximum expected change when the air flow rate in the airlift pump is varied over 25-45 l m i n - l , the injection rate of carbon dioxide/air mixture for pH control varies over 70-110 l h - 1 , and the composition of the pH control gas mixture is changed to contain 0.4-1.0 mole fraction of carbon dioxide (Camacho Rubio et al., 1999). The predictions in Fig. 6 disregard any inhibition of photosynthesis due to accumulation of oxygen and any change in culture irradiance due to buildup of biomass. Unlike the predictions in Fig. 6, a plug flow reactor scaled up using established methods should have the same exit composition (oxygen

o.oo4_~

0.205 E

c7 0.2o0

0.008 ,=

" " " " "Y

~r"

0.OO20~

" " " "

0.195

0.000 4

8

12 Solar hour

16

20

24

160

8.0

140 120

0

0

0

0

o

&

0

0

o

7.8

0

"-" 100

74~ 0

40

72

~ o

I [ 70

4

8

12 Solar hour

16

20

24

Fig. 5. Predicted (solid lines) and measured values of the following variables: dissolved oxygen at the end of the tubular loop (top figure); oxygen and carbon dioxide mole fractions in the exit gas (middle figure); carbon dioxide losses in the exhaust gas and the culture pH (lower figure). All variables are shown as functions of solar hour. The predictions (continuous lines) are based on the mass transfer model described (Camacho Rubio et al., 1999). shown in Fig. 5, through any 24-h period, the model predictions agreed closely with the measurements; hence, demonstrating the predictive capability of the model. The data in Fig. 5 were obtained during an outdoor culture of P. t r i c o r n u turn (Camacho Rubio et al., 1999). An airliftdriven tubular photobioreactor was employed (Fig. 1) as detailed previously (Aci6n Fern~ndez et al., 1998). The average light level in the culture was established using the methodology outlined

242

E. Molina Grima et al. 1'Journal of Biotechnology 70 (1999) 231-247

increase. Performance of a reactor will change on scaleup--it will deteriorate unpredictably--unless the frequency of light-dark interchange of fluid is held constant upon scaleup. If the lightdark cycling time is allowed to increase with scale of operation, the reactor productivity will begin to decline as soon as the cycling time exceeds a maximum value, roughly the equivalent of the time interval between uptake of photons by the photosynthetic machinery when the light is available at saturation intensity or slightly higher. Data exists on the acceptable frequency of lightdark cycling for some algae and can be easily determined for others (Philliphs and Myers, 1954; Grobbelaar, 1994; Grobbelaar et al., 1996). In contrast, there is no information on the frequency of light-dark interchange of fluid in bioreactors, except in laminar flow when any radial interchange in a tube would be solely by diffusion or in effect no radial movement. Several approaches have been suggested for quantifying the light-dark cycling of fluid in a bioreactor. One approach relies on analogy with mass or heat transfer from the wall of a tube to the turbulently flowing fluid (Merchuk et al., 1998). In principle, available mass or heat transfer correlations could be used to determine a transfer coefficient. The latter could be used to determine a surface renewal time at the tube wall following the concepts of the classical surface renewal the-

content, carbon dioxide content, biomass concentration) as a smaller device: normally, in scaling up a plug flow reactor, the highest practicable dilution rate--i.e, the minimum residence time for a given exit biomass concentration--is established at a small scale. The same value of residence time applies to a longer tube of the same diameter as the smaller one; thus, the flow velocity in the longer pipe must be greater by a factor of length ratios of the larger-to-smaller tubes. Adhering to this criterion ensures that properties of the product stream leaving the longer tube match those established at the smaller scale. When this criterion is followed, all of the concentration profiles in the direction of flow will be identical at both scales. In any event, mass transfer of carbon dioxide and oxygen are not the principal factors limiting scalability. Supply of carbon dioxide is relatively easily managed irrespective of scale, whereas build-up of oxygen to inhibitory levels is a far lesser problem than adequacy of illumination. Unless the tubular reactor is scaled up by changing the length, any change in dimensions would imply a change in the relative volumes of the light and dark zones: under given conditions (external illumination, cell concentration, pigment content), the depth at which light intensity declines to a growth limiting level will not be affected, but the diameter of the dark zone will

~IE E ~-

1.1 1-

35

:

30

0.9

~

o 0.7__

8.0 pH of the culture To " ~ ~ ~ ~ " " " " ~ "

s. ~

O

.-""

iiDOOoggg~

7.9 L

.'J"

..-'t'"""

....~'"

..... 9"lD;ssolved oxyge

n O

7.8 |

o6=

#

iiilz

~ ~0

........ t " " "

I

25

~20

t5 0.5

t

Ca 0on,ossos

"1," C~

7.7

5 0.3

[_

0

25

t

50

t

75

{

100 Loop length, m

~

125

~

150

7.6 175

Fig. 6. Model predicted values of carbon dioxide loss, the culture pH, and the dissolved oxygen concentration at tube exit for various lengths of the tubular loop (Camacho Rubio et al., 1999).

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

ory of transport 9 This method would be valid only if the depth of the light zone was of the same order as the laminar boundary layer at the wall of the tube. The depth of the boundary layer is of the order of cellular dimensions; hence, this approach is entirely unrealistic except, possibly, in exceedingly dim light. Another similar approach relies on the velocity of the turbulent microeddies in the fluid. The eddy velocity u is easily determined using the wellknown Kolmogoroff's model of turbulence; hence, 1

U-'-

243

the maximum residence time of fluid in the dark zone should be < Oa. Therefore, the volumetric rate of fluid movement out of the dark zone is

QR =

dark zone volume ~r(dr- 2dL) 2 Oa = 40a

(45)

This QR value is on a unit tube length basis. Because all the fluid moving out of the dark zone must pass through the boundary between the light and dark zones, a fluid interchange velocity can be defined as

UR = Q---ER= ( d r - 2dz.). r~dk 40d '

(46)

~

kPL

thus,

where PL is the density of the fluid, p/~ is its viscosity, and 4' is the energy dissipation rate per unit mass calculated as,

- 2CfU3c. d

(42)

In Eq. (42) UL is the superficial liquid velocity, Cf is the Fanning friction factor, and d is the hydraulic diameter of the tube. Again, the calculated eddy velocity would be a measure of the lightdark cycle if the depth of the dark zone equaled the dimensions of the microeddies. The latter are quite small, usually less than 200 lam; thus, the approach is plainly unsound as a measure of the light-dark frequency in tubular photobioreactors. An alternative approach, not yet fully developed, follows. For any tube of diameter dr, the depth of the light zone dL can be calculated by applying Beer-Lambert law for known level of external incident illumination, the biomass content, the absorption coefficient, and the saturation irradiance value. Assuming a homogeneous external illumination, the depth of the dark zone in the tube becomes

Od= dk 4UR"

(47)

Increasing scale by increasing diameter does not change dL for otherwise fixed conditions; only dr, changes. To ensure identical performance at the two scales, the scaleup criterion becomes

a~ arge = (4 ) ma

1,

or

d~__~g~___~= 1,

(49)

where the subscripts S and L refer to small and large scales, respectively. If the scale factor is f, i.e. dkL =f'dks, then for identical performance, the large scale interchange velocity should be URL =fURs,

(50)

where f > 1. The radial flow velocities may be estimated from radial dispersion coefficients in turbulent flow; hence DR URoc~

(51)

dr"

dk = d r - 2dL.

(43)

Thus, the volume of the dark zone per unit tube length is Dark volume = i t ( d r - 2dL) 2 9 4

(44)

If 0a is the maximum acceptable duration of the dark period between successive light periods, then

Radial dispersion coefficients can be measured using suitably designed experiments. Correlations would need to be established for such dispersion coefficients as functions of the linear flow Reynolds number. In practice, radial flow may be enhanced by deploying static mixers inside a tube (Chisti, 1998). These mixers should be minimally intrusive

244

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

and should be confined to well within the dark core. One possibility is the use of a coaxially located rod with suitably spaced barbs or projections that have a somewhat flat profile and are suitably angled to direct the flow into the annular light zone. The projections should not extend into the light zone to prevent any loss of illumination in that zone. If for an algal species, the acceptable continuos dark time 0a is infinitesimally short, then scaleup without loss of productivity would be impossible by increasing the tube diameter. In theory, exceedingly high levels of turbulence and high radial velocities can be generated. In practice, an upper limit would be encountered at much lower than technically possible levels of turbulence in view of the known algal sensitivity to hydrodynamic shear and limited pressure tolerance of the typically employed transparent materials of construction. In addition, higher airlift devices would be necessary to generate higher flows through the tubes while compensating for the significant pressure drop due to static mixing elements (Chisti, 1989; Chisti et al., 1990).

6. Nomenclature a ai

b

CT r

D DR

( m 2 S-1)

dT

dks dL

5. Concluding remarks

Fco Of the many types of photobioreactors proposed for closed monoculture, tubular devices are amongst the more scaleable and suited to largescale production. Unlike the widely used open culture systems, the design of closed tubular photobioreactors is more complex. Irradiance levels in culture can be predicted as discussed. Similarly, carbon dioxide supply problems are relatively easily resolved. Difficulties arise with scaleup because relative volumes of light and dark zones change as the tube diameter increases; however, promising new leads in this area suggest that dependable scaleup based on fundamental principles should become feasible in the foreseeable future. At present the recommended method is to use a pipe diameter of no more than 0.1 m, and a continuous run length of about 80 m with a flow velocity of 0.3-0.5 m s -1. Multiple parallel run tubes originating and ending in common headers are apparently the best way to accommodate higher flows and volumes.

parameter in Eq. (8) path length parameter defined by Eq. (25) (m) parameter in Eq. (8) biomass concentration (kg m-3) Fanning friction factor total inorganic carbon in the liquid (tool m - 3) parameter in Eq. (8) dilution rate (s -1) radial dispersion coefficient

Fo FH20 FN f H

Hco

/40

Ho

diameter or hydraulic diameter (m) tube diameter (m) diameter of dark zone (m) diameter of dark zone at larger scale (m) diameter of dark zone at smaller scale (m) depth of light zone (m) photosynthetic efficiency of the solar radiation ( - 1.74 _+ 0.07 ~E J - ' ) carbon dioxide molar flow rate in the gas phase (mol s -1) oxygen molar flow rate in the gas phase (tool s-l) molar flow rate of water in the gas phase (tool s -l) nitrogen molar flow rate in the gas phase (tool s -l) scale factor total daily radiation on a horizontal surface (J m -2 day -1) daily direct radiation on a horizontal surface (J m -z day-l) Henry's law constant for carbon dioxide (mol m-3 a t m - l) daily diffuse radiation impinging on a horizontal surface (J m -2 day-1) daily global extraterrestrial solar radiation (J m -2 day-~) Henry's law constant for oxygen (mol m - 3 a t m - l)

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

h I

I.v

IBt(~, fO) IBt(ri, 09)

I,)

IDt IDt((-o) IDt(ri, (]9)

/max

Io Ir go K,

K~ K, Ks Kw (kLat)co 2

solar hour (h) hourly incident photosynthetic radiation on a horizontal surface (laE m-2 s-1) photosynthetically active hourly average irradiance inside culture (laE m-2 s-1) hourly PA direct irradiance on a horizontal surface (laE m-2 s - ' ) direct hourly PA irradiance on a vertical surface (~tE m-2 s-~) direct local hourly PA irradiance inside vertical column (~E m-2 s -1 ) hourly PA diffuse irradiance on horizontal surface (I~E m-2 s - ' ) hourly PA diffuse irradiance on inclined surface (~tE m-2 s - ' ) disperse hourly PA irradiance on vertical surface (~tE m - 2 S- I) local hourly disperse PA irradiance inside vertical column (~tE m-2 S-1) microalgal affinity for light (~tE m-2 S-1) saturation value of I (~tE m-2 S--I) solar PA irradiance impinging on the reactor's surface (IBt-+IDt) (~tE m - 2 S-1) hourly reflected PA irradiance on a surface (~tE m -2 s-l) absorption coefficient (m 2 g-l) first dissociation constant for H2CO 3 (mol m - 3) second dissociation constant for H2CO3 (tool m - 3) photoinhibition constant (I~E m-2 S-I) saturation constant (~tE m -2 S-1) dissociation constant for water (mol 2 m -6) volumetric gas-liquid mass transfer coefficient for carbon dioxide (s-1)

(kLaL)o2

245

volumetric gas-liquid mass transfer coefficient for oxygen

(S -1 ) L m N n PA p Pc%

Pdirect

tube length (m) exponent in Eq. (5) day of the year exponent in Eq. (6) photosynthetically active biomass productivity (kg m-3 s -I ) carbon dioxide partial pressure in the gas phase (atm) distance traveled by a direct incident ray from the tube's surface to any internal point (?'i, q))

(m) Pdisr~rse

Po2 PT P~,

QL QR R Rco 2 R02 r ri S UL UR

URL URs

u

distance traveled by disperse radiation from the tube's surface to any internal point (ri, ~0) (m) oxygen partial pressure in the gas phase (atm) total pressure in the system (arm) partial pressure of water vapor (atm) volumetric liquid flow rate (m3 s-') volumetric flow rate out of dark zone (m3s-') radius or hydraulic radius (m) carbon dioxide consumption rate (mol CO2 m - 3 s - 1 ) oxygen generation rate (mol 02 m -3 s -1) radial distance (m) distance in polar coordinates (m) cross-sectional area of the tube

(m2) superficial liquid velocity in the tube (m s-1) fluid interchange velocity (m s-l) fluid interchange velocity at larger scale (m s - ' ) fluid interchange velocity at smaller scale (m s -1) eddy velocity defined by Eq. (41) (m s-')

246

E. Molina Grima et al./Journal of Biotechnology 70 (1999) 231-247

distance in the direction of flow (m) liquid phase molar concentration of species in brackets (mol m - 3) liquid phase equilibrium concentration of species in brackets (mol m - 3)

x

[] []*

6.1. Greek symbols

c ~G

0 O' Od

O. !

Oz 12 /.1 L

Pmax

7E

P PL

O9 (_~0S

N

parameter in Eq. (2) surface tilt angle relative to the horizontal surface azimuth angle ( - 180~< 7 < 180~ declination of the angular position of the Sun at solar noon with respect to the plane of the equator, north positive ( - 23.45 ~ < fi < 23.45~ defined by Eq. (11) angle shown in Fig. 3 fractional gas holdup angle of incidence defined by Eq. (21) angle 0 modified by refraction in the culture maximum duration of dark period (s) zenith angle of the Sun, defined by Eq. (22) zenith angle of the Sun modified by refraction in the culture specific growth rate (s-~) viscosity of liquid (Pa s-l) maximum specific growth rate (s -1) energy dissipation rate per unit mass (W kg -~) pi ground reflectivity density of liquid (kg m - 3) geographic latitude angular position in polar coordinates angle corresponding to the solar hour, defined by Eq. (16) hour angle at sunrise, defined by Eq. (10) atmospheric clarity index estimated at 0.74 + 9% universal solar constant (( = 1353 W m -2)

Acknowledgements Some of the work described was supported by the Comisi6n Interministerial de Ciencia y Tecnologia, C.I.C.Y.T., Spain (Project Bio 95-0652) and the European Union (Project BRPR CT970537).

References Aci6n Fernfindez, F.G., Garcia Camacho, F., Sfinchez P6rez, J.A., Fernfindez Sevilla, J., Molina Grima, E., 1997. A model for light distribution and average solar irradiance inside outdoor tubular photobioreactors for microalgal mass culture. Biotechnol. Bioeng. 55, 701-704. Aci6n Fernfindez, F.G., Garcia Camacho, F., S~inchez P6rez, J.A., Fernfindez Sevilla, J., Molina Grima, E., 1998. Modelling of biomass productivity in tubular photobioreactors for microalgal cultures. Effects of dilution rate, tube diameter and solar irradiance. Biotechnol. Bioeng. 58, 605611. Aiba, S., 1982. Growth kinetics of photosynthetic microorganisms. Adv. Biochem. Eng. 23, 85-156. Bannister, T.T., 1979. Quantitative description of steady state, nutrient-saturated algal growth, including adaptation. Limnol. Oceanogr. 24 (1), 76-96. Benemann, J.R., 1989. The future of microalgal biotechnology. In: Cresswell, R.C., Rees, T.A.V., Shah, N. (Eds.), Algal and Cyanobacterial Biotechnology. Longman, London, pp. 317-337. Borowitzka, M.A., 1996. Closed algal photobioreactors: design considerations for large-scale systems. J. Mar. Biotechnol. 4, 185-191. Camacho Rubio, F., Aci6n Fern/mdez, F.G., S~inchez P6rez, J.A., Garcia Camacho, F., Molina Grima, E., 1999. Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture. Biotechnol. Bioeng. 62, 71-86. Chisti, Y., 1989. Airlift Bioreactors. Elsevier, London, p. 345. Chisti, Y., 1998. Pneumatically agitated bioreactors in industrial and environmental bioprocessing: hydrodynamics, hydraulics and transport phenomena. Appl. Mech. Rev. 51, 33-112. Chisti, Y., Kasper, M., Moo-Young, M., 1990. Mass transfer in external-loop airlift bioreactors using static mixers. Can. J. Chem. Eng. 68, 45-50. Chrismadha, T., Borowitzka, M.A., 1994. Effect of cell density and irradiance on growth proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular photobioreactor. J. Appl. Phycol. 6, 67-74. Duffle, J.A., Beckman, W.A., 1980. Solar Engineering of Thermal Processes. Wiley, New York, p. 762.

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Erickson, L.E., Lee, H.Y., 1986. Process analysis and design of algal growth systems. In: Barclay, W., McIntosh, R.P. (Eds.), Algal Biomass Technologies: An Interdisciplinary Perspective. Nova Hedwigia, Berlin, p. 197. Fernfindez Sevilla, J., 1995. Estudio del crecimiento simultaneamente fotolimitado y fotoinhibido de la microalga marina Isochrysis galbana. Productividad en ficidos grasos poliinsaturados n-3. Tesis doctoral. Universidad de Almeria, Spain. Frohlich, B.T., Webster, I.A., Ataai, M.M., Shuler, M.L., 1983. Photobioreactors: models for interaction of light intensity, reactor design and algal physiology. Biotechnol. Bioeng. Symp. 13, 331-350. Garcia Camacho, F., Contreras Gdmez, A., Aci6n Fern~indez, F.G., Molina Grima, E., 1999. Use of concentric tube air-lift photobioreactors for microalgal outdoor mass culture. Enzyme Microbial. Technol. 24, 164-172. Grobbelaar, J.U., 1994. Turbulence in algal mass cultures and the role of light/dark fluctuations. J. Appl. Phycol. 6. 331-335. Grobbelaar, J., Neddal, L., Tichy, V., 1996. Influence of high frequency light/dark fluctuations on photosynthetic characteristics of microalgae photo acclimated to different light intensities and implications for mass algal cultivation. J. Appl. Phycol. 8, 335-343. Incropera, F.P., Thomas, J.F., 1978. A model for solar radiation conversion to alga in shallow ponds. Solar Energy 2{). 157-165. Laws, E.A., 1980. Nutrient and light limited growth of Thalassiosirafluviatilis in continuous culture with implications for phytoplankton growth in the ocean. Limnol. Oceanogr. 25. 455-473. Lee, Y.K., 1986. Enclosed bioreactors for the mass cultivation of photosynthetic microorganisms: the future trend. Trends Biotechnol. 4, 186-189. Lee, Y.K., Low, C.S., 1991. Effects of photobioreactors inclination on the biomass productivity of an outdoor algal culture. Biotechnol. Bioeng. 38, 995-1000. Lee, Y.K., Low, C.S., 1992. Productivity of outdoor algal cultures in enclosed tubular photobioreactors. Biotechnol. Bioeng. 40, 1119-1122. Liu, B.Y.H., Jordan, R.C., 1960. The interrelationship and characteristic distribution of direct, diffuse and total solar radiation. Solar Energy 7, 53-65. Livansky, K., 1982. Effect of suspension temperature on mass transfer coefficient of carbon dioxide from algal suspension into air on a cultivation platform with baffles. Arch. Hydrobiol. Suppl. 63 (3), 363-367. Livansky, K., 1990. Losses of CO2 in outdoor mass cultures: determination of the mass transfer coefficient kl by means of measured pH course in NaHCO 3 solution. Algol. Stud. 58, 87-97. Livansky, K., Bartos, J., 1986. Relationship between pCO_, and pH in a medium for algal culture. Arch. Hydrobiol. Suppl. 73 (3), 425-431. Mfirkl, H., Mather, M., 1985. Mixing and aeration of shallow open ponds. Arc. Hydrobiol. Beih. 20, 85-93. Merchuk, J.C., Ronen, M., Giris, S., Arad, S., 1998. Light-

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dark cycles in the growth of the read microalgae Porphyridium sp. Biotechnol. Bioeng. 59, 705-713. Molina Grima, E., Garcia Camacho, F., Sfinchez P6rez, J.A., Fernfindez Sevilla, J.. Aci6n Fernfindez, F.G., Contreras Gomez. A.. 1994. A mathematical model of microalgal growth in light limited chemostat cultures. J. Chem. Technol. Biotechnol. 61, 167-173. Molina Grima. E.. Fernandez Sevilla, J.M., Sanchez Perez, J.A.. Garcia Camacho, F.. 1996. A study on simultaneous photolimitation and photoinhibition in dense microalgal cultures taking into account incident and averaged irradiances. J. Biotechnol. 45, 59-69. Myers. J.E.. 1980. On the algae: thoughts about physiology and measurement of efficiency. In: Falkowski, P.G. (Ed.), Primary Productivity in the Sea. Plenum Press, New York, pp. 1-16. Philliphs, J.N.. Myers. J.. 1954. Growth rate of Chlorella in flashing light. Plant Physiol. 29, 152-161. Pulz. O.. Scheinbenbogen. K., 1998. Photobioreactors: design and performance with respect to light energy input. Adv. Biochem. Eng. Biotechnol. 59, 123-152. Quiang. H.. Gutterman. H., Richmond, A., 1996. A flat inclined modular photobioreactor for outdoor mass cultivation of photo autotrophs. Biotechnol. Bioeng. 51, 5160. Rabe. A.E.. Benoit. A., 1962. Mean light intensity. A useful concept in correlating growth rates of dense cultures of microalgae. Biotechnol. Bioeng. 4. 337-390. Ree. G.Y.. Gotham. I.J.. 1981. The effect of environmental factors on phytoplankton growth: Light and interaction of light with nitrate limitation. Limnol. Oceanogr. 26, 649659. Steele, J.H.. 1977. In kapidus, L., Amundson, N.R. (Eds.), Microbial Kinetics and Dynamics in Chemical Reactor Theory. Prentice-Hall. Englewood Cliffs, NJ, pp. 405-483. Tamiva. H.. Hase. E.. Shibata, K., Mituya, A., Iwamura, T., Nihei. T.. Sasa, T., 1953. Kinetics of growth of Chlorella, with special reference to its dependence on quantity of available light and on temperature. In: Burlew, J.S. (Ed.), Algal Culture from Laboratory to Pilot Plant. Carnegie Institution of Washington, Washington, DC, pp. 204-232. Terry. K.L.. 1986. Photosynthesis in modulated light: Quantitative dependence of photosynthesis enhancement on flashing rate. Biotechnol. Bioeng. 28, 988-995. Tredici. M.R.. Materassi. R., 1992. From open ponds to vertical alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms. J. Appl. Phycol. 4, 221-231. Van Oorschot, J.L.P., 1955. Conversion of light energy in algal cultures. Med van Lund. Wang. 55, 225-277. Weissman. J.C., Goebel, R.P., Benemann, J.R., 1988. Photobioreactor design: mixing, carbon utilization, and oxygen accumulation. Biotechnol. Bioeng. 31, 336-344. Yamaguchi. K.. 1997. Recent advances in microalgal bioscience in Japan, with special reference to utilization of biomass and metabolites: a review. J. Appl. Phycol. 8, 487-502.

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i

JOURNAL

iiui

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 249-270

Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae Asterio Sfinchez Mir6n, Antonio Contreras G6mez, Francisco Garcia Camacho, Emilio Molina Grima, Yusuf Chisti * Department of Chemical Engineering, University of Almeria, E-04071 Almeria, Spain Received 27 October 1998; received in revised form 17 November 1998; accepted 22 December 1998

Abstract Engineering analyses combined with experimental observations in horizontal tubular photobioreactors and vertical bubble columns are used to demonstrate the potential of pneumatically mixed vertical devices for large-scale outdoor culture of photosynthetic microorganisms. Whereas the horizontal tubular systems have been extensively investigated, their scalability is limited. Horizontal tubular photobioreactors and vertical bubble column type units differ substantially in many ways, particularly with respect to the surface-to-volume ratio, the amount of gas in dispersion, the gas-liquid mass transfer characteristics, the nature of the fluid movement and the internal irradiance levels. As illustrated for eicosapentaenoic acid production from the microalga Phaeodactylum tricornutum, a realistic commercial process cannot rely on horizontal tubular photobioreactor technology. In bubble columns, presence of gas bubbles generally enhances internal irradiance when the Sun is low on the horizon. Near solar noon, the bubbles diminish the internal column irradiance relative to the ungassed state. The optimal dimensions of vertical column photobioreactors are about 0.2 m diameter and 4 m column height. Parallel east-west oriented rows of such columns located at 36.8~ latitude need an optimal inter-row spacing of about 3.5 m. In vertical columns the biomass productivity varies substantially during the year: the peak productivity during summer may be several times greater than in the winter. This seasonal variation occurs also in horizontal tubular units, but is much less pronounced. Under identical conditions, the volumetric biomass productivity in a bubble column is ~ 60% of that in a 0.06 m diameter horizontal tubular loop, but there is substantial scope for raising this value. 9 1999 Elsevier Science B.V. All rights reserved.

Keywords: Microalgae; Photobioreactors; Scale-up; Photosynthetic culture; Eicosapentaenoic acid; Phaeodactylum tricornutum

I. Introduction

* Corresponding author: Tel.: + 34-950-215566; fax: + 34950-215484. E-mail address: [email protected] (Y. Chisti) 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00079-6

P h o t o b i o r e a c t o r s for large-scale m o n o c u l t u r e of microalgae have conventionally been designed as devices with large s u r f a c e - t o - v o l u m e ratios. Various types of tubular p h o t o b i o r e a c t o r s are examples of this a p p r o a c h (Lee, 1986; Borowitzka,

250

A. Shnchez Mir6n et al./'Journal of Biotechnology 70 (1999) 249-270

1996). These reactors occupy vast land areas: they are expensive to build; difficult to maintain; and only somewhat scaleable. Tubular photobioreactors can usefully satisfy only medium level production demands. Attempted, large- scale production in horizontal tubular loops has failed quite spectacularly in one case (Fig. 1); hence, other reactor configurations are needed for the production of larger quantities of pharmacologically active compounds that certain microalgae can potentially produce. The areal productivity, i.e. productivity per unit land area, is low for conventional tubular photobioreactors and in large units as well as modular designs, sterile operation to the levels demanded in the pharmaceutical industry is difficult. Some low surface-to-volume, pneumatically agitated photobioreactors can potentially overcome these significant disadvantages. Examples of the latter type are bubble columns and airlift bioreactors. Large scale culture of microalgae in these systems has not been investigated as it has always been assumed that small surface-to-volume ratios of these devices would make them ineffective. This need not be so as reported in this work which deals with comparative outdoor evaluation of pilot scale bubble column photobioreactors with respect to performance in horizontal tubular loops. Data are reported on three aspects of comparative characterization: (a) gas-liquid hydrodynamics and mass transfer; (b) internal irradiance levels as functions of Sun's location relative to the photobioreactors; and (c) performance during culture of the microalga Phaeodactylum tricorntum. Also reported are the effects of hydrodynamics on survival behavior of algal cells.

2. I. Hydrodynamics

2. Comparison of performance

where PL is the density of the liquid, g is the gravitational acceleration and Uc is the superficial gas velocity in the column. Eqs. (1) and (2) were established by Chisti (1989) for tap water and 0.15 M aqueous sodium chloride, respectively. The equations apply to the bubble flow regime, or UG values less than about 0.05 m s-1 (PG/VL "~ 500 W m-3) The obvious decline in the rate of increase of gas holdup with increasing specific

The vertical and the horizontal tubular photobioreactors differ in several significant ways including differences in light regimens, gas-liquid hydrodynamics and mass transfer behavior. Some of these factors--e.g, hydrodynamics and light regimen--are interrelated. Their impact on culture performance is discussed below.

The hydrodynamics of flow in horizontal tubes and vertical columns are generally quite different. The necessarily gas sparged bubble columns and airlift reactors tend to have substantially greater gas holdups than do horizontal tubular solar receivers. The latter are virtually free of gas and any bubbles present are localized to a narrow zone along the upper portion of the tubes; moreover, the bubbles are relatively small. In contrast, there are many more and larger bubbles in vertical photobioreactors and the gas-liquid flow is much more chaotic than the highly directional flow in a small-diameter horizontal pipe. Differences in gas holdup and the bubble size affect light penetration, gas-liquid mass transfer, mixing and shear stress levels. 2. I.I. Gas holdup

Gas holdup measurements in a bubble column photobioreactor confirm that holdup increases with specific power input in accordance with previously published data (Chisti, 1989). Thus, the holdup data in tap water (Fig. 2) closely followed the equation e = 3 . 3 1 7 x 10- \ V L j

'

(1)

whereas data in sea water (Fig. 2) agreed with the correlation e = 7.958x10

-

5(P-~L)I249.

(2)

In these equations the power input due to aeration is calculated as

eo_ p~u~, v~

(3)

A. Shnchez Mir6n et al./'Journal of Biotechnolgoy 70 (1999) 249-270

251

Fig. 1. A commercial horizontal tubular bioreactor facility that failed to perform to expectations and was abandoned. This facility was located in Cartagena, Spain and it was owned by Photobioreactors Ltd.

A. Shnchez Mir6n et al./'Journal of Biotechnology 70 (1999) 249-270

252

used in microalgal culture in vertical photobioreactors because of the cell damaging potential of intense turbulence (Silva et al., 1987; Suzuki et al., 1995; Contreras et al., 1998; Chisti, 1999a). Data in Fig. 2 reveal that the flow transition occurs earlier in tap water, around a power input of 280

power input values above about 400 W m - 3 (Fig. 2b) is due to a well-known change in the flow regime from bubble to churn turbulent flow (Chisti, 1989). Equations for estimating gas holdup in the latter regime have been published (Chisti, 1989), but that regime is not likely to be 100

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Fig. 2. Variation of fractional gas holdup with specific power input in the bubble column" (a) tap water; and (b) sea water.

A. Sgmchez Mir6n et al./Journal of Biotechnolgoy 70 (1999) 249-270 Table 1 Composition of Almeria bay Mediterranean sea water (Contreras et al., 1998) Component

Amount (kg m -3)

Chloride Sulfate Bicarbonate Na + Mg 2+ Ca 2+ K+ Total dissolved solids Total organic carbon Ionic strength

20.812 2.866 0.168 10.552 1.362 0.519 0.413 36.608 Negligible 0.727

W m - 3 . The coalescence inhibiting effect of salts in sea water allows bubble flow to persist longer in that medium. Similar results have been reported earlier (Chisti, 1989). The data in Fig. 2 were obtained in an outdoor bubble column with a gas-free liquid height of 1.93 m. Either tap water or Mediterranean sea water was the liquid phase. The composition of the latter is noted in Table 1. Except during culture, a small amount of hypochlorite (50 ml corresponding to 35 g 1-1 free chlorine) was added to sea water to prevent microbial growth. Air was sparged through a

perforated pipe with 17 holes of 1 mm diameter, giving a gas flow area that was 0.05% of the total column cross-sectional area. The sparger head was shaped into a cross that was located centrally, 0.076 m above the base of the column. The internal diameter of the column was 0.19 m and it was made of 3.3 mm thick poly(methyl methacrylate), except for the lower 0.25 m section that was made of stainless steel. For the power input range of relevance here, Eqs. (1) and (2) are known to apply to bubble columns irrespective of the size of the sparger holes. Agreement of the data with Eqs. (1) and (2) suggests that changes in sparger characteristics are unlikely to affect the holdup in the two fluids.

2.1.2. Effects of holdup on irradiance The effect of gas holdup on the measured internal irradiance inside the column is shown in Figs. 3-6. The data in these figures were obtained on clear, cloudless days. The irradiance levels were measured using one spherical quantum s e n s o r - either a Biospherical QSL 100 (Biospherical Instruments Inc., San Diego, CA) unit or a LI-COR LI-196SA (Li-cor, Inc., Lincoln, N E ) d e v i c e - - l o cated always at the centerline of the column, 0.87 m below the static surface of the liquid. Data were

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TIME OF DAY (h) Fig. 3. Daily variation in internal irradiance in the bubble column in tap water. The gas holdup value was 0.101. The difference between the aerated and unaerated irradiance levels is also noted. The data are for 7 and 8 July, 1998.

A. SEmchez Mir6n et al. Journal o/ Biotechnology 70 (1999) 249-270

254

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Fig. 4. Daily variation in internal irradiance in the bubble c o l u m n in tap water. The gas h o l d u p value was 0.052. T h e difference between the aerated and u n a e r a t e d irradiance levels is also noted. T h e d a t a are for 25 June and 1 July, 1998.

acquired using a DaqBook/100-901 (IOtech, Inc., Cleveland, OH) data acquisition card connected to an IBM compatible 486 computer operating at 100 MHz. The trough in irradiance around 14 h (Figs. 3-6), or solar noon, occurs when the Sun is at its apex and most nearly aligned with the vertical axis of the bubble column (Fig. 7a). In this position, the direct sunlight flux on the column is at it's lowest, hence, the internal irradiance level is low. Also, around solar noon, because of a high angle of incidence, a greater amount of radiation is reflected off the surface of the column compared to when the angle of incidence is small (afternoon, morning). The irradiance peaks at 11:30 and 17:30 h in the absence of bubbling (Figs. 3-5) occur when the Sun is low on the horizon and most closely aligned with the normal to the bubble column's vertical axis (Fig. 7b). Typically, the internal irradiance levels at these times exceed those at solar noon. This general pattern is seen irrespective of whether the reactor is aerated or if it is gas-free. Of course, in a horizontally placed tubular loop the pattern of variation of irradiance with time will be different, the irradiance profile will have a single peak (Garcia Camacho et al., 1999) that will coincide with the solar noon.

As shown for tap water in Fig. 3, bubbling at a constant high aeration rate (UG = 0.051 m s - ] ) corresponding to a gas holdup of 0.101, may either enhance or reduce the level of internal irradiance depending on the position of the light source, the Sun. Irradiance is enhanced during periods when the Sun is most closely aligned with the normal to the vertical axis of the column (Fig. 7b). Around solar noon, when the Sun is best aligned with the vertical axis (Fig. 7a), presence of bubbles reduces internal irradiance. Depending on time, the difference between the aerated and unaerated irradiance levels may range from - 12 to + 35% (Fig. 3). The effect of aeration on internal irradiance diminishes as the gas flow rate is reduced (Fig. 4); however, even at a low superficial gas velocity of 0.0025 m s - l , corresponding to a fractional gas holdup of 0.008, or less than 1%, the irradiance level is affected by up to ~ 15% relative to gas-free operation in tap water (Fig. 5). Aeration at any level consistently shifts the irradiance maxima relative to the ungassed state (Figs. 3-6), the morning irradiance maximum occurs up to 1 h earlier than in the absence of bubbling. Similarly, the afternoon irradiance maximum occurs up to 1 h later than when no aeration is employed.

A. S(~nchez Mir6n et al. Journal of Biotechnolgo)' 70 (1999) 249-270

Irradiance enhancement and reduction by bubbles result from a combination of two factors: shading of culture by bubbles; and the phenomenon of total internal reflectance. In the bubble flow regime, the bubble shape is predominantly ellipsoidal (Fig. 8) with the major axis of the bubble roughly perpendicular to the vertical axis of the bubble column. At a given gas holdup and bubble size, the bubbles provide a greater shading effect when the light source is high on the horizon than when it is low (Fig. 8a). Furthermore, when the light source is high, i.e. closely aligned with the vertical axis of the column, the surface of the bubble reflects incident light poorly and much of the radiation penetrates into the gas phase. In contrast, when the light source is low on the horizon, it is more closely aligned with the major axis of the bubble (Fig. 8b) and the low approach angle of radiation relative to the gas-liquid interface causes total internal reflection of a large portion of the radiation (Fig. 8b). In effect, each bubble becomes a reflector that allows better dispersion of incident radiation within the liquid. This total internal reflectance more than compensates for the smaller shadows of sideways illumination, the shadow region behind a bubble is illuminated by reflectance from 3500

I

1

other nearby bubbles. Because of reflectance the total internal irradiance is greater relative to bubble-free operation when much of the incident radiatiol~ simply passes through the bubble column. Whether these effects will occur and to what extent, in optically less clear algal culture broths remains to be seen. Experiments are currently underway to establish this point. A comparison of Figs. 3-5 reveals that both the irradiance enhancement and reduction effects in tap water are stronger at the higher aeration rate when the gas holdup and the number of bubbles are greater. With more bubbles there are more reflectors in the fluid when the light source is low on the horizon. Similarly, the shadowing effect of many more bubbles is stronger when the Sun is at its apex. The data in Figs. 3 - 5 are for the bubble flow regime and the irradiance patterns could be different in the churn turbulent regime and also when the gas holdup is so high that clear patches of liquid allowing through penetration of light do not exist. Comparing Figs. 4 and 6 for tap and sea water, respectively, at identical aeration rates, the patterns of variation in irradiance are generally similar: however, the irradiance enhancement in sea water is somewhat greater than in tap water. In 1

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A. SEmchez Mirbn et al. Journal of Biotechnology 70 (1999) 249-270

256

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Fig. 6. Daily variation in internal irradiance in the bubble column in sea water. The gas holdup value was 0.065. The difference between the aerated and unaerated irradiance levels is also noted. The data are for 14 and 15 September, 1998.

contrast, the irradiance diminishing effect is virtually non-existent in sea water up to a gas velocity of 0.021 m s-1: the bubble size is slightly smaller than in tap water, the holdup is somewhat greater, and the sphericity of the bubbles is higher. These factors combine to reduce the shadowing effect of the bubbles during midday relative to that experienced in tap water. Quantitatively, the shadowing effect of bubbles can be shown to depend on bubble diameter and the gas holdup as follows: 8

Shadowing effect oc d-BB'

(4)

where e is the fractional gas holdup and dB is the mean bubble diameter. The above relationship originates from the fact that the light shadowing effect of bubbles depends on their number in a given volume (VD) of dispersion and on the mean diameter of bubbles, i.e. NB" d~ Shadowing effect o c t . v.

(5)

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vD'

(7)

relationship (4) is derived readily. Expression (4) does not consider bubble sphericity effects, however, with reference to Fig. 8a it is easily shown that at constant gas holdup an increase in sphericity should reduce the size of the shadow and hence the irradiance diminishing effect at midday. At much higher aeration rates than in Fig. 6, the situation in Fig. 9 is observed in sea water. Fig. 9 is for a highly turbulent system (PG/V~ .~ 500 W m-3) when the bubble size is quite small and the gas holdup is relatively high ( > 13%) so that the gas-liquid dispersion is cloudy (Fig. 10). Light penetration is now reduced quite a lot relative to the bubble-free case (Fig. 9). Note that in Fig. 9, the irradiance enhancement seen between 10 and 12 h is not repeated to the same extent in the afternoon around 16-18 h. This is because at commencement of aeration in the morning the fluid is not as cloudy as in the afternoon, the fine bubbles responsible for cloudiness accumulate with time and their concentration gradually increases to some steady state value from the time

A. S6nchez Mir6n et al./Journal of Biotechnolgoy 70 (I999) 249-270

of commencement of aeratior/. The size of these fine bubbles is such that even several hours after the gas flow is stopped, the water remains cloudy; however, overnight the bubbles rise and the water becomes clear. Note that the internal irradiance level is a function of the measurement position. The discussion here pertained to the lowest irradiance values, i.e. those at the deepest point (the centerline) from the principal illuminated surface or the walls of the vessel. 2.2. Light regimen 2.2.1. Effects of depth and fluid motion Light regimens in vertical bubble columns or airlift reactors and the horizontal tubular loops differ. Some of the factors responsible for the differences have already been discussed, including different orientations of the bioreactors and the presence of a large number of gas bubbles in pneumatically agitated vertical vessels. Another factor is the culture depth. Depth is typically

0

257

0.02-0.06 m in horizontal tubes, but much greater, e.g. 0.2 m or more, in vertical columns. At constant external illumination, small changes in culture depth are known to generally affect productivity of microalgae cultures (Kobayashi and Fujita, 1997), including those of P. tricornutum. Because of a lower depth and because the light intensity declines exponentially with depth according to Beer-Lambert law, the light level is always greater in the relatively thin horizontal tubes than in a larger diameter vertical column when the systems are compared at identical orientation of the light source relative to the reactors' surface. For otherwise fixed conditions, increase in diameter of tubes significantly reduces culture productivity (Kobayashi and Fujita, 1997). For example, Kobayashi and Fujita (1997) showed using Chlorella sp. cultures that growth rate and cell concentration were reduced with increasing diameter of thin vertical tubes over the range 0.016-0.050 m internal diameter. Although the volumetric productivity declined, the culture pro-

\ \

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(a)

(b)

Fig. 7. Schematic representation of the Sun's position with respect to the axes of the bubble column in the northern hemisphere: (a) at solar noon; and (b) in afternoon or early morning.

258

A. Sgmchez Mir6n et al. /Journal of Biotechnology 70 (1999) 249-270

(a)

(b) MINOR AXIS

MAJORAXIS

MINOR AXIS

~

MAJOR AXIS

BUBBLE

T

BUBBLE SMALLER PROJECTED SHADOW FOR SIDEWAYS ILLUMINATION TOTAL INTERNALREFLECTION

LARGER PROJECTED SHADOW FOR OVERHEAD ILLUMINATION

Fig. 8. Projected shadows of an ellipsoidal bubble illuminated from top or side (a). The shadow is much larger for overhead illumination compared to when sideways illumination occurs. (b) Total internal reflection at bubble-liquid interface when the incident radiation beam is closely aligned with the major axis of the bubble. The minor axis of the bubble is generally parallel to the vertical axis of the bubble column.

ductivity (kg m - 2 h-~) per unit bioreactor footprint area increased with increasing tube diameter (Kobayashi and Fujita, 1997) for a constant external level of illumination. The areal productivity more than doubled with increasing tube diameter over the noted range. Although culture depth is an important influence on production characteristics, consideration of depth alone is unsatisfactory; the nature and magnitude of fluid movement also need taking into account. In any photobioreactor, vertical or horizontal, the depth to which light penetrates depends on the external irradiance level, the biomass concentration and the absorption characteristics of the biomass. The culture volume adjacent to the illuminated walls is relatively better lit. At a certain depth, the light intensity falls to growth limiting levels and a 'dark zone' is encountered. Because short dark periods interspersed among sufficiently long and intense light periods do not affect the growth rate, the growth reducing potential of a dark zone is minimized by ensuring that elements of fluid do not reside in this zone

for long (Molina Grima et al., 1999). A dark zone must exist for at least part of the year even in a shallow photobioreactor tube that is optimally designed to make full use of the available light during summer noons. The depth of the dark zone is inevitably greater in large-diameter vertical columns than in narrow horizontal tubes; however, because of a highly chaotic flow regime and movement of bubbles, the radial movement of fluid that is necessary for improved light-dark cycling is substantially greater in bubble columns than in tubular loops. The latter have a highly directional flow in which the radial component is small compared to the axial component. Yet, the better radial flow in vertical aerated columns does not fully compensate for their larger diameter, hence, the volumetric productivity is reduced relative to narrow horizontal tubular photobioreactors. For best performance, bubble columns need to be operated at the highest feasible aeration rates consistent with the shear tolerance of the microalga; however, the aeration rate must not be so high as to produce a gas holdup level that

A. Sdnchez Mir6n et al./Journal of Biotechnolgoy 70 (I999) 249-270

prevents light transmission through the column. Ideally, the culture environment in the column should be manipulated to promote formation of relatively large bubbles (dB > 0.006 m) that rise rapidly. Radial flow into and out of the central dark core can be enhanced by ensuring that quite large spherical cap bubbles (dB ~ 0.02-0.04 m) rise through this zone intermittently (Fig. 11). These schemes are currently being experimented with. In summary, the low surface-to-volume photobioreactors can potentially approach the volumetric productivity of horizontal tubular loops, however, attaining this requires that: (i) the cumulative residence time of the cells in the light zone of the reactor should be comparable to that of the tubular device; and (ii) the frequency of light-dark transition cycle in the 'deeper' vessels should be comparable to that of tubular systems. These two objectives can be substantially achieved in bubble columns and airlift bioreactors by enhancing radial movement of cells. In airlift bioreactors, but not in batch bubble columns, radial flow may be enhanced by using static mixing elements.

259

2.2.2. Vertical photobioreactor emplacement The maximum number of the vertical column reactors that may be accommodated in a given area depends on the height of the column which, together with the position of the Sun, establishes the maximum extent of the column's shadow on the ground. The length of the shadow from the column's base is given by hc tan0e

L~ = ~ ,

(8)

where hc is the height of the column and Oi is the angle of incidence of the direct solar radiation. The angle of incidence--the inclination of the Sun from the normal to the vertical axis of the bubble columnmdepends on the geographic latitude ~b, the day of the year N, and the solar hour 1~; the angle of incidence is given as (Liu and Jordan, 1960): 0; = 90 ~ - cos- ~(cos~5 9cosq~ 9cosco + sine5 9sinq~) (9)

where 4~ is the geographic latitude. The angles co and c~ are related to the solar hour and the day of the year (Liu and Jordan, 1960), respectively, as follows:

4500

SEA W A T E R U G 0.051 m s -1

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--

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10 E

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3500

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,,

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2000

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Fig. 9. Daily variation in internal irradiance in the bubble column in sea water. The gas holdup value was 0.135. The difference between the aerated and unaerated irradiance levels is also noted. The data are for 8 and 9 September, 1998.

260

A. Sdnchez Mir6n et al. ~!Journal of Biotechnology 70 (1999) 249-270

Fig. 10. The bubble dispersion on the left allows through transmission of light whereas the cloudy dispersion on the right blocks light. These pictures were taken in sea water at aeration power inputs of 154 and 518 W m - 3 , respectively. The perforated pipe sparger hole diameter was 1 mm in both cases.

co =- 1 5 ( 1 2 - h),

(lo)

and 9 //360(284 + N ) t g = 23.45. sm~ 365 / "

(11)

ing of 0.35 m between centers of adjacent columns of 0.2 m diameter. Close spacing within east-west rows has no impact on illumination, but it improves efficiency of land use.

2.3. Shear effects The loci of the maximum extent of the shadow of a 2 m tall bubble column are plotted in Fig. 12 for representative days in winter, spring, and summer seasons at the geographic location of Almeria (36.8~ 2.9~ The maximum extent of the shadow in January is about 7.4 m, whereas the maximum extent in July is about 1.3 m. These distances are measured north-south between parallel east-west lines passing through the base of the vertical column and the tip of the column's shadow. Ideally, parallel east-west rows of bubble columns should be spaced by at least the maximum length of the shadow in winter. This would assure that the reactors are never mutually shaded, however, a more optimal setup would place the rows of reactors closer, about midway between the high extremes of the shadow length in the summer and the winter. Consequently, there will be no mutual shading in the summer but some shading would occur during the winter. In a single east-west row of columns the columns could be spaced quite close together; e.g. a spac-

Shear stress has been implicated as an important factor in culture of several microalgae (Silva et al., 1987; Suzuki et al., 1995; Contreras et al., 1998; Chisti, 1999a). A cell damaging hydrodynamic environment is easily attained in bubble columns and airlift reactors (Silva et al., 1987; Suzuki et al., 1995; Contreras et al., 1998; Chisti, 1998a), but damage to algal cells has never been documented within tubes of tubular photobioreactors. This may suggest that the damage is somehow linked with the presence of bubbles in pneumatically agitated devices, but extensive studies prove otherwise. With most microalgae, increasing aeration rate up to quite high values improves culture productivity (Silva et al., 1987; Contreras et al., 1998), but damage occurs when the turbulence is so intense that the fluid microeddy size approaches cellular dimension. Only in one case--that of the commercially important but unusually fragile marine alga Dunaliella--has damage been associated directly with the bubbles

A. Sdnchez Mir6n et al./Journal of Biotechnolgoy 70 (1999) 249-270

(Silva et al., 1987). Survival of Dunaliella in aerated systems is improved by supplementing the culture with viscosity enhancers such as carboxymethyl cellulose and agar (Silva et al., 1987), but this approach may not be applicable universally (Chisti, 1999a). In one study with D. tertiolecta, culture in a bubble column was quite successful, but when the bubble column was converted to an airlift device by inserting a vertical baffle, the productivity declined (Suzuki et al., 1995). Under conditions that were earlier identified as optimal, no growth was observed in the airlift reactor whereas good

LIGHT ZONE BUBBLE COLUMN DARK ZONE

SPHERICAL CAP BUBBLE

SPHEROIDAL BUBBLE

Fig. 11. Use of spherical cap bubbles in the central dark core to enhance radial light-dark interchange of fluid in a bubble column. The bubbles in the peripheral light zone are predominantly ellipsoidal with a diameter of about 0.006 m.

261

growth occurred in the bubble column. Microscopic examination showed significant disruption of the cells in the airlift device (Suzuki et al., 1995). This was associated with the hydrodynamic stresses generated as the culture flowed over the upper edge of the baffle into the downcomer (Suzuki et al., 1995). This effect could have been avoided, or at least minimized, by hydrodynamic smoothing of the upper and lower parts of the baffle to prevent flow separation (Chisti, 1998a). In the bubble column, the growth was sensitive to aeration rate: growth rate increased with increasing superficial gas velocity until a velocity of about 0.6 m min 1, or a specific power input of 98 W m-3. Further increase in aeration rate reduced growth, apparently because of hydrodynamic stresses in the fluid (Suzuki et al., 1995). Under non-growth conditions (no light), the specific death rate in the bubble column was shown to increase with superficial gas velocity for velocities exceeding 0.6 m min 1 (Suzuki et al., 1995). At a fixed aeration velocity (UG = 1 m min 1), the specific death rate decreased with increasing height of the culture fluid in the column (Suzuki et al., 1995), probably because the specific power input and, hence, the turbulence intensity declined (Chisti, 1998a). Similar behavior has been reported with animal cells in bubble columns (Emery et al., 1987; Tramper et ali,, 1987a,b)i Improved growth with increasing aeration up to a limit has been documented for several algae including Dunaliella (Silva et al., 1987); P. tricornutum (Contreras et al., 1998)i and others (Chisti, 1999a). This effect has been observed invariably under light limited conditions and it is best explained by improved light-dark cycling due to improved agitation. Indeed, the effect occurs even in the absence of gas when mec used to improve light-dark int( photobioreactors that contain In indoor batch cultures of P. out in a draft-tube sparged co~ nal-loop airlift photobioreact01 mination of 1200 ~tE m - 2 S the reactor, Contreras et al. ( the maximum specific g r o w t h increasing aeration velocity in increase occurred until a velo

A. Sdnchez Mirdn et al./Journal of Biotechnology 70 (1999) 249-270

262

NORTH

DISTANCE (m)

8 -

JANUARY 01

6 -

5- !

/

LOCI OF MAXIMUM EXTENT OF SHADOW

SHADOW AT SUNRISE

APRIL 01 JUNE 21

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0\

i

i

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5

10

15

DISTANCE (m) BUBBLE COLUMN

WEST

EAST

SOUTH

DAILY PATH OF SUN

IsuNl

Fig. 12. Locus of the shadow of a 2 m tall bubble column during the day. The loci are shown for three representative seasons for 8 h (January) or 10 h (April, June) days, centered at midday.

s - ] , corresponding to a specific power input of 270 W m -3 Whether improved growth was due to enhanced fluid interchange between the light and dark zones was unclear and other factors may have contributed. The cultures were clearly light limited whenever the biomass concentration exceeded about 1 kg m-3. Increase in aeration rate beyond 0.055 m s -~ substantially reduced the specific growth rate of P. tricornutum cultures (Contreras et al., 1998). At

the critical aeration rate value of 0.055 m s - ] , the calculated Kolmogoroff microeddy scale was 45 gm, or comparable to the dimensions of the algal cells (up to 35 lam long and 3 gm wide (Lewin et al., 1958)). Except for robust microorganisms, damage to cells has generally been observed when the dimensions of the isotropic turbulence microeddies approach those of the cells (Chisti, 1999a), however, the assumption of isotropic turbulence is almost never justifiable under typical

263

A. Sdnchez Mir6n et al./Journal of Biotechnolgoy 70 (1999) 249-270

operation of bubble columns and airlift systems (Chisti, 1998a). Why has cell damage not been observed in horizontal tubular photobioreactor pipes is readily explained. For a 200 m straight run smooth horizontal tube of 0.06 rn diameter, the culture velocity would have to exceed 1.14 rn s-1 for the microscale of turbulence to approach 45 g m - - t h e value that damaged the P. tricornutum cells in the vertical airlift device. This velocity value is easily estimated from the relationship between the Kolmogoroff microeddy length scale (f) and the specific energy dissipation rate (Chisti, 1999a):

__ (/AL~3/4E- 1/4.

(12)

k,P L f

The specific energy dissipation rate E in (Eq. (12)) is related with the pressured drop (AP), i.e. E=

ULAP

(13)

pL L '

where L is the length of the tube and UL is the culture velocity. The pressure drop in (Eq. (13)) is obtained using the expression: L A P = ZCf -~ pLU 2 .

(14)

In (Eq. (14)), the Fanning friction factor (C f) is obtained from the Blasius equation:

CF = 0.0792(P~ULd) -°2s ,

(15)

where d is the diameter of the tubing. For the above calculations, the measured viscosity of the algal culture was virtually the same as that of water. In practice, because of low pressure ratings of typically used transparent materials of construction, a culture velocity as high as 1.14 m s cannot be attained in long-run continuous tubings. Typically, the culture velocity in tubular loops does not exceed 0.5 m s-1, or less than half the damaging threshold value. In theory, high culture velocities should benefit productivity by increasing the allowable length of a continuous run tube before oxygen concentration builds up to high inhibitory levels. In reality, pumping becomes difficult as the tube length increases. The

cell damaging potential of pumps is another consideration that is especially relevant to tubular photobioreactor culture. Damage in pumps is treated elsewhere (Chisti, 1999a). 2.4. Gas- liquid mass transfer

Microalgae generate oxygen during photosynthesis, hence, dissolved oxygen levels equivalent to several times air saturation are easily reached in closed cultures. Oxygen concentrations above air saturation generally inhibit photosynthesis in microalgae (Aiba, 1982). In studies with Chlorella vulgaris, M/irkl and Mather (1985) noted that the rate of photosynthesis increased by 14% when there was almost no dissolved oxygen. Saturation of the medium with pure oxygen reduced the photosynthesis rate by 35%. Accumulation of oxygen to inhibitory levels is a major problem in horizontal tubular photobioreactors and this problem becomes more severe as the length of continuous run tubing increases. With Spirulina platensis under intense artificial illumination, oxygen production rates have been estimated at 0.35 0.5 g 1-1 h - 1 for radiation intensity levels of 15002600 gE m - 2 s-1 (Tredici et al., 1991). In one case, a tubular photobioreactor designed as a commercial production unit with several kilometer long tube runs (Fig. 1) failed to produce partly because of oxygen accumulation. This device was apparently the largest horizontal tube photobioreactor ever constructed and it's failure is clear evidence of limited scalability of this type of bioreactor. Existing functioning tubular photobioreactors are typically a mere fraction of the size of the device shown in Fig. 1. Oxygen removal into the exhaust gas-phase is substantially greater in pneumatically agitated vertical reactors than in horizontal tubes. For example, for a power input value of 24 W m - 3 the lowest value in Fig. 2a the overall volumetric gas-liquid mass transfer coefficient (kLaL) in the bubble column was estimated at 0.0037 s-1 using the equation

4(PG~ 0"86

kLaL=2.39 X 10- \ V L ]

"

(16)

264

A. Sfinchez Mir6n et al./Journal of Biotechnology 70 (1999) 249-270

Eq. (16) applies to air-water systems (Chisti, 1989). The estimated kLaL in the column was about 4-fold the estimated value for a horizontal tube with a reported 3% maximum gas holdup (Camacho Rubio et al., 1999). In view of these results, vertical tubular photobioreactors such as bubble columns should easily maintain a dissolved oxygen level only a little higher than the air saturation value. Consequently, vertical reactors will experience little oxygen inhibition. Indeed, in one case the dissolved oxygen level did not exceed the air saturation value during outdoor culture of P. tricornutumin a draft-tube airlift reactor (Contreras, 1996). Because of a reduced level of dissolved oxygen, the photooxidation-associated loss of biomass and product metabolite will be lower in vertical reactors. Photooxidation occurs especially when high levels of dissolved oxygen combine with an intensely irradiated environment.

2.5. Overall productivity: the case of EPA Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid that is potentially medically significant in treatment of certain cancers and heart disease (Rambjor et al., 1996; Simonsen et al., 1998). EPA is produced by several microalgae and some other microorganisms. EPA occurs also in fish oil. Current annual demand of EPA is approximately 125 tonnes in Japan alone and a world-wide demand has been estimated at 300 tonnes per annum (Corden, personal communication). A facility producing just 20 tonnes EPA per annum from microalgal sources would need to generate 24 tonnes of wet biomass daily, operating continuously for 95% of the calendar year. This figure assumes an EPA content of about 2% (w/w, dry weight basis) in P. tricorntum, an optimistic 80% recovery of the EPA and 85% moisture in the biomass. For this production capacity, an optimally operated horizontal tubular photobioreactor (0.06 m tube diameter) would have a volume of 2350 m 3. This volume is based on an experimentally observed dry biomass productivity of 2.02 kg m - 3 d - 1 in June, or an average annual productivity of 1.535 kg m -3 d 1. The latter value accounts for the experimentally documented 8% per month decline in productivity going from

mid summer to mid winter (Aci6n Fern/mdez et al., 1998). Such a reactor will occupy a surface area of about 14.1 ha. This estimation of surface area assumes an optimal distance of 0.11 m between adjacent parallel horizontal tube runs. In theory, this distance could be reduced by half although some loss of productivity would occur due to mutual shading. A bubble column tank farm for the same 20 tonne annual EPA production would have a total volume of 8220 m 3 based on experimentally measured dry biomass productivity of 0.64 kg m -3 d - 1 in June, or a mean annual productivity of 0.486 kg m - 3 d 1. Such a tank farm will occupy an area 16.3 ha. Each bubble column would be 0.2 m in diameter, 2.1 m tall, with a culture volume of about 0.06 m 3. East-west oriented rows, 995 m long, will have a spacing of 0.35 m between column centers, inter-row spacing will be 3.4 m measured between column centers. Because a 3.4 m inter-row spacing results in mutual shading of columns for part of the year, the mean annual volumetric productivity of biomass declines to 0.439 kg m - 3 d - 1 , or about 90% of the unshaded productivity. The land use efficiency may seem lower for the bubble column tank farm, however, the column height used was only 2.1 m and efficiency improves with increasing column height as shown in Fig. 13. Thus, for a more realistic column height of 4 m, the total volume needed to for 20 tonnes per annum EPA capacity will be about 10 120 m 3. The mean annual volumetric productivity of biomass will decline further to 0.356 kg m - 3 d - 1 because of increased mutual shading of the taller columns, but the area demand will reduce to ~ 40% of that with 2.1 m columns. Because of the larger volume per column, fewer columns will be needed. The effect of bubble column height on mean annual volumetric biomass and EPA productivities is shown in Fig. 14 for a facility with 46 columns arranged in four east west oriented rows with 3.4 m interrow spacing. The volumetric productivity declines with increasing column height because of increased duration of the mutually shaded period. The increased production with increasing column height (Fig. 13) occurs because the culture volume depends directly on height and more volume is

265

A. Sdnchez Mir6n et al./Journid of Biotechnolgoy 70 (1999) 249-270

accommodated on a given land surface when taller columns are employed. Because taller columns have reduced volumetric productivity as a result of longer periods of mutual shading, increasing height does not increase the annual production linearly (Fig. 13). Beyond a height of 5 m, the annual production from a given area is no longer sensitive to column height (Fig. 13) because the decline in volumetric productivity exactly balances the effect of increased volume. The biomass productivity numbers used in these estimations were obtained in a 0.2 m 3 horizontal tubular reactor that occupied an area of 12 m 2. The reactor was located on a reflective surface with an albedo of 2 and it produced P. tricorntum biomass. The culture was carried out in Mann and Myers (1968) medium (Table 2) formulated in sea water (Table 1) at 3-fold concentration than recommended by Mann and Myers (1968). The optimal dilution rate during the summer (June) was ~ 0.05 h -1, giving a biomass productivity of 2.02 kg m -3 d -1 with 4 kg m - 3 biomass in the effluent. The maximum a real productivity of the reactor was a mere 0.034 kg m - 2 d - 1 . 800

I

I

I

I

As noted earlier, the experimental productivity in a single 2 m tall column, 0.2 m in diameter, was 0.64 kg m - 3 d-1 in June. The maximum areal productivity of the single reactor was 0.093 kg m - 2 d - 1, accounting for the 1.3 m shadow length and 0.3 m effective width of the column. The culture medium was a modified Ukeles (1961) medium (Garcia Sfinchez, 1994) made in sea water (Table 1) at three times the components concentrations noted in Table 2. Differences in media had no effect on culture behavior. Comparing superficially, the mean annual volumetric productivity of the vertical column was only 30% of that of the horizontal tubular loop, however, it needs emphasizing that the horizontal device was located on a reflective surface that enhanced the total incident radiation on it's surface by a factor of two. If the vertical column is placed on a similarly reflective background its productivity will increase minimally by a factor of 1.8; hence, comparing on an equal basis, the volumetric productivity of the vertical unit is about 57% of that in the horizontal device.

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COLUMN HEIGHT (m) Fig. 13. Annual biomass and EPA production of a multiple bubble column facility as a function of column height. Column diameter was 0.2 m. Forty-fivecolumns were accommodated on a 13.9 x 5.9 m surface with 3.4 m center-to-center between east-west oriented rows.

A. Sdnchez Mir6n et al./Journal of Biotechnology 70 (1999) 249-270

266

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C O L U M N H E I G H T (m) Fig. 14. Effect of bubble c o l u m n height on m e a n a n n u a l volumetric productivity of biomass and EPA. Forty-five columns were placed on a 13.9 x 5.9 m surface with 3.4 m center-to-center between e a s t - w e s t oriented rows.

In any outdoor culture, the mean monthly productivity varies during the year because of cyclic changes in external irradiance: in horizontally oriented photobioreactors productivity peaks during summer when the Sun is highest on the horizon. Productivity is lower during winter. As noted earlier, the experimentally measured decline in productivity is about 8% per month going from mid summer to mid winter for horizontally placed tubes (Aci6n Fernfindez et al., 1998). Whether the same level of decline would occur in vertical tubes remains to be confirmed. Annual variation in productivity in a vertical photobioreactor may follow a different cyclic pattern than observed with horizontal devices. During summer when the Sun is highest on the horizon, a vertical bubble column should experience lower average irradiance t h a n in winter when the direct radiation from the Sun affects a greater part of the reactor's surface. This variation is similar to that seen in the gas-free state in Figs. 3 6, except that the cycle is annual. This annual cyclic pattern has important advantages for the vertical oriented photobioreactors. Higher irradiance levels in the winter should reduce any heating demand whereas

lower irradiance in the summer should reduce the need for cooling. Furthermore, the low surfaceto-volume ratio should help reduce heat losses during the night. The expected variation in biomass and EPA production for a bubble column farm and a horizontal tubular loop photobioreactor occupying the same land area (82 m 2) is shown in Fig. 15. The production rate values in Fig. 15 are for a system in which the adjacent horizontal tubes of 0.06 m diameter are spaced optimally at 0.11 m, whereas the bubble columns are spaced 0.3 m wall-to-wall within an east-west row and the inter-row spacing is 3.4 m. Again, the assumed mean annual culture biomass productivity is 0.438 kg m -3 d - 1 based on experimentally observed productivity of 0.64 kg m -3 d -1 in a single bubble column for cultures of P. tricornutum during June. For the tubular loop system, the assumed average annual productivity is 11535 kg m -3 d-1. The bubble column diameter used is 0.2 m and the column height is 2 m. This diameter is close to the maximum practicable in view of the light penetration needs.

A. Sdnchez Mir6n et al./Journal of Biotechnolgoy 70 (1999) 249-270

For a given land area and 0.2 m diameter columns, the annual biomass production increases with culture height as shown in Fig. 13 even though the volumetric productivity declines (Figl 14). The optimal column height is about 5 m (Fig. 13), but because of considerations such as wind speed and strength of optically transparent materials such as glass and thermoplastics, the maximum permissible height is 4 m. The effect of inter-row spacing of 2 m tall columns (0.2 m diameter) on annual production from a fixed land area of 82 m 2 is shown in Fig. 16. Clearly, to attain maximum yield while keeping the number of columns to a minimum implies an optimal inter-row spacing of between 2.5 and 3.5 m. Use of multiple vertical columns has other important advantages: (i) a more uniform and better controlled pH than could ever be possible in a plug flow tubular loop; (ii)improved culture homogeneity and, therefore, a consistent metabolic state of the cells; (iii) greater operational flexibility in view of the ability to easily vary the number of columns in production at a given time; (iv) ability to culture several different algae at the same time in separate units; and (v) substantially reduced Table 2 Composition of the media Component

C o n c e n t r a t i o n (kg m - 3 ) Mann and

Modified Ukeles'

M y e r s (1968)

medium

MgSO4 • 7H20 NaNO 3

1.20 1.00

1.00

CaC1 z

0.30

-

K2HPO 4

0.10

-

NaH2PO4 • 2H20 Na2EDTA

0.03

0.016 0.01

H3BO 3 FeSO 4 • 7H20

6 x 10- 3 2 x 10- 3

_

Fe(III) citrate

-

MnC12

1.4 x 1 0 - 3

MnC12 • 4 H 2 0 ZnSO 4 • 7H20 ZnC12

-

_

9.9 x 10 . 4

3.3 x 10 . 4 -

Co(NO3)2" 6H20

_ 4.9 x 1 0 - 3

1.36 x 1 0 - 4

7.0 x 10 - 6

267

need for pumping the culture as there would be no recirculation. Flow management among the individual column bioreactors as well as sampling can be easily automated (Chisti, 1998b). Similarly, rapid and automatic clean-in-place and chemical sterilization of individual photobioreactor units becomes feasible (Chisti and Moo-Young, 1994; Chisti, 1999b) while a part of the facility remains in production. Among factors that could significantly improve the EPA-from-microalgae process feasibility is improved yield. Raising EPA content of the biomass from the current 2% of dry weight to 3% under consistent outdoor production will reduce the production facility size by about 30%. Similarly, a 50% improvement in bubble column bioreactor productivity from the current 0.4860.729 kg m - 3 d-1 (no shading) will reduce the facility size further. Overall, with these two improvements, a 20 tonne per annum bubble column based EPA facility will require a volume of 3653 m 3, or less than 45% of the volume needed when the yield and illumination are not enhanced. Both the noted improvements are realislic. EPA content of biomass may be improved by genetic manipulation and selection of be~ter producing strains of P. tricornutum. Accompl!shing this will affect the bubble column and th~ tubular loop based facilities equally. Improved illumination and consequent biomass productivity enhancement is feasible only for the bubble column system as the tubular loop productivity numbers are already the maximum attainable in an outdoor system with the external irradiance level enhanced to twice the direct radiation from the Sun. The bubble column volumetric productivity can be further enhanced by employing artificial illumination at night, technology exists for plac!ng a low power vertical light source at the ax~s of the column. This kind of efficient internal illumination is not practicable in horizontal tubular loops. Of course, artificial illumination may add to capital cost and it will certainly add to the operating expense, but for sufficiently high-~alue products the improved productivity may more than compensate for the greater expense. i

,

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268

A. Sdnchez Mir6n et al./Journal of Biotechnology 70 (1999) 249-270 70

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MONTH Fig. 15. Mean monthly biomass and EPA production rates of a bubble column (0.2 m diameter, 2 m tall, 3.4 m center-to-center between east-west oriented rows) farm and a horizontal tubular loop photobioreactor (0.06 m tube diameter) occupying equal land areas (82 m2). The column farm accommodated 45 columns. 700 600 E c" ¢.(0 O)

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3. Conclusions

Horizontal tubular photobioreactors are generally believed to be the most practicable culture

system for fully contained large-scale monoculture of microalgae, nevertheless, as discussed here, detailed analyses reveal severe limitations of tubular photobioreactors. Unless the concentration of the

A. Sdnchez Mir6n et al./Journal of Biotechnolgoy 70 (1999) 249-270

length of the shadow from the column's base (m) length scale of microeddies (m) day of the year number of bubbles in dispersion pressure drop (Pa) power input due to aeration (W) superficial liquid velocity in the tube (m

desired microalgal metabolite in the biomass is unusually high and the market size for the product is exceedingly small, the use of horizontal tubular photobioreactors would be impossible in commercial production. Unlike horizontal tubular devices, vertical reactors such as bubble columns and airlift vessels appear to be the only ones that can be effectively used in large-scale culture of microalgae. As demonstrated here, overall, the vertical bubble columns perform better than the horizontal loops. Bubble columns are more scaleable and provide a relatively homogeneous culture environment. In addition, the low surface-to-volume bubble columns demand less cooling. Vertical columns experience less photoinhibition during periods of high light intensity; during low light seasons such as winter, the vertical reactors still receive substantial total radiation because they receive more reflected light than do horizontal tubes. So far, bubble columns and airlift bioreactors have been ignored for largescale culture of photosynthetic organisms, an obvious low surface-to-volume ratio of these devices relative to horizontal tubular loops has in the past been automatically perceived as a barrier to their use, but such perceptions have never been substantiated.

Greek symbols d declination the angular position of the Sun at solar noon with respect to the plane of the equator, north positive (-23.45 ° _< ~ <_ 23.45°), defined by Eq. (11) fractional gas holdup angle of incidence defined by Eq. (9) Oi viscosity of liquid (Pa s) gL density of liquid (kg m -3) PL geographic latitude O9 angle corresponding to the solar hour, defined by (Eq. (10)

4. Nomenclature

Acknowledgements

Ad

cross-sectional area of downcomer zone

Ar G

cross-sectional area of riser z o n e (m 2) Fanning friction factor diameter (m) mean bubble diameter (m) energy dissipation rate per unit mass (W kg -1) eicosapentaenoic acid gravitational acceleration (m s -z) height of the column (m) solar hour (h) overall volumetric gas-liquid mass transfer coefficient for oxygen

d

4 E EPA g hc h kLaL

(m2)

(s-') length of tubing (m)

LS

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269

s -1)

v~ v~ v~

volume of gas-liquid dispersion in bioreactor (m 3) volume of gas in liquid (m 3) volume of liquid in bioreactor (m 3)

This work was supported by the European Commission contract BRPR CT970537 and CICYT (BIO-98-0522), Spain.

References Aci6n Fern~.ndez, F.G., Garcia Camacho, F., S/mchez P6rez, J.A., Fern/mdez Sevilla, J.M., Molina Grima, E., 1998. Modelling of biomass productivity in tubular photobioreactors for microalgal cultures: Effects of dilution rate, tube diameter and solar irradiance. Biotechnol. Bioeng. 58, 605-616. Aiba, S., 1982. Growth kinetics of photosynthetic microorganisms. Adv. Biochem. Eng. 23, 85-156. Borowitzka, M.A., 1996. Closed algal photobioreactors: design considerations for large-scale systems. J. Mar. Biotechnol. 4, 185-191.

270

A. Sdnchez Mirdn et al./Journal of Biotechnology 70 (1999) 249-270

Camacho Rubio, F., Aci6n Fern/mdez, F.G., Sgmchez P6rez, J.A., Garcia Camacho, F., Molina Grima, E., 1999. Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture. Biotechnol. Bioeng. 62, 71-86. Chisti, Y., 1989. Airlift Bioreactors. Elsevier, New York, p. 355. Chisti, Y., 1998a. Pneumatically agitated bioreactors in industrial and environmental bioprocessing: Hydrodynamics, hydraulics and transport phenomena. Appl. Mech. Rev. 51, 33-112. Chisti, Y., 1998b. Biosafety. In: Subramanian, G. (Ed.), Bioseparations and Bioprocessing: A Handbook, vol. 2. Wiley-VCH, New York, pp. 379-415. Chisti, Y., 1999a. Shear sensitivity. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology. Wiley, New York, pp. 2379-2406. Chisti, Y., 1999b. Process hygiene: (e)modern systems of plant cleaning. In: Robinson, R., Batt, C., Patel, P. (Eds.), Encyclopedia of Food Microbiology, Academic Press, London (in press). Chisti, Y., Moo-Young, M., 1994. Clean-in-place systems for industrial bioreactors: design, validation and operation. J. Ind. Microbiol. 13, 201-207. Contreras, A., 1996. Caracterizaci6n de una columna de burbujeo con recirculaci6n interna. Aplicaci6n al cultivo de Phaeodactylum tricornutum. Ph.D. Thesis, University of Almeria, Almeria, Spain. Contreras, A., Garcia, F., Molina, E., Merchuk, J.C., 1998. Interaction between CO2-mass transfer, light availability and hydrodynamic stress in the growth of Phaeodactylum tricornutum in a concentric tube airlift photobioreactor. Biotechnol. Bioeng. 60, 318-325. Emery, A.N., Lavery, M., Williams, B., Handa, A., 1987. Large-scale hybridoma culture. In: Webb, C., Mavituna, F. (Eds.), Plant and Animal Cells: Process Possibilities. Ellis Horwood, Chichester, pp. 137-146. Garcia Camacho, F., Contreras G6mez, A., Aci~n Fern~indez, F.G., Fern/mdez Sevilla, J., Molina Grima, E., 1999. Use of concentric-tube airlift photobioreactors for microalgal outdoor mass cultures. Enzyme Microbial. Technol. 124, 164-172. Garcia S/mchez, J.L., 1994. Estudio multifactorial de nutrientes para el crecimiento de la microalga marina Isochrysis galbana. Evaluacion en un fotobiorrecator tubular externo. Ph.D. Thesis, University of Almeria, Spain. Kobayashi, K., Fujita, K., 1997. Tube diameter on tubular

photobioreactor for microalgal culture and its biomass productivity. J. Chem. Eng. Jpn. 30, 339-341. Lee, Y.K., 1986. Enclosed bioreactors for the mass cultivation of photosynthetic microorganisms: the future trend. Trends Biotechnol. 4, 186-189. Lewin, J.C., Lewin, R.A., Philpott, D.E., 1958. Observations on Phaeodactylum tricornutum. J. Gen. Microbiol. 18, 418-426. Liu, B.Y.H., Jordan, R.C., 1960. The interrelationship and characteristic distribution of direct, diffuse and total solar radiation. Solar Energy 7, 53-65. Mann, J.E., Myers, J., 1968. On pigments, growth and photosynthesis of Phaeodactylum tricornutum. J. Phycol. 4, 349355. MS.rkl, H., Mather, M., 1985. Mixing and aeration of shallow open ponds. Arch. Hydrobiol. Beih. 20, 85-93. Molina Grima, E., Aci~n Fernandez, F.G., Garcia Camacho, F., Chisti, Y., 1999. Photobioreactors: Light regime, mass transfer and scaleup. J. Biotechnol. 70, 233-249. Rambjor, G.S., Walen, A.I., Windsor, S.L., Harris, W.S., 1996. Eicosapentaenoic acid is primarily responsible for hpotriglyceridemic effect of fish oil in humans. Lipids 40 (Suppl. 5), 45-49. Silva, H.J., Cortifias, T., Ertola, R.J., 1987. Effect of hydrodynamic stress on Dunaliella growth. J. Chem. Technol. Biotechnol. 40, 41-49. Simonsen, S., van't Veer, P., Strain, J.J., et al., 1998. Adipose tissue omega-3 and omega-6 fatty acid content and breast cancer in the EURAMIC study. Am. J. Epidemiol. 147, 342-352. Suzuki, T., Matsuo, T., Ohtaguchi, K., Koide, K., 1995. Gas-sparged bioreactors for CO 2 fixation by Dunaliella tertiolecta. J. Chem. Technol. Biotechnol. 62, 351-358. Tramper, J., Joustra, D., Vlak, J.M., 1987a. Bioreactor design for growth of shear-sensitive insect cells. In: Webb, C., Mavituna, F. (Eds.), Plant and Animal Cells: Process Possibilities. Ellis Horwood, Chichester, pp. 125-136. Tramper, J., Smit, D., Straatman, J., Vlak, J.M., 1987b. Bubble column design for growth of fragile insect cells. Bioprocess. Eng. 2, 37-41. Tredici, M.R., Carlozzi, P., Zittelli, G.C., Materassi, R., 1991. A vertical alveolar panel (VAP) for outdoor mass cultivation of microalgae and cyanobacteria. Bioresour. Technol. 38, 153-159. Ukeles, R., 1961. The effect of temperature on the growth and survival of several marine algae species. Biol. Bull. 120, 255-264.

Bi0 ~ ELSEVIER

Journal of Biotechnology 70 (1999) 271-288

Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor" quantitative analysis of the daily cyclic variation of culture parameters M.M. Rebolloso Fuentes, J.L. Garcia Sfinchez, J.M. Fernfindez Sevilla, F.G. Aci6n Fernfindez, J.A. Sfinchez P6rez, E. Molina Grima * Departamento de Ingenieria Quimica, Universidad de Almeria, E-04071 Almeria, Spain Received 27 October 1998; received in revised form 23 November 1998; accepted 22 December 1998

Abstract

The present work reports on the daily cyclic variation of oxygen generation rates, carbon consumption rates, photosynthetic activities, growth rates and biochemical composition of the biomass in a pilot plant continuous outdoor culture of the microalgae Porphyridium cruentum. A linear relationship between the external irradiance and the average irradiance inside the culture was found. In addition, the oxygen generation and carbon consumption rates were found to be a function of the average irradiance inside the culture. A reduction in photosynthetic activity of the cells at noon and recovery in the afternoon was also observed. Therefore, the cells showed a short-term response of parameters such as oxygen generation rate as well as carbon consumption rate with external and average irradiance; a model of photosynthesis rate considering photoinhibition is proposed. This model is a useful tool for the operation and scaleup of tubular photobioreactors, and can be used for determining CO2 requirements of the system. The higher the photosynthesis rates, the lower the carbon losses, ranging from 25% at noon to 100% during the night. The growth rate showed a linear relationship with the daily mean average irradiance inside the culture with a long-term response. Likewise, a linear relationship among the oxygen generation rate and the growth rate was obtained. With respect to the biochemical composition of the biomass, the cells showed a long-term response of metabolic routes to mean daily culture conditions. During the illuminated period, energy was stored as carbohydrates and synthesis of proteins was low. During the night, the stored carbohydrates were consumed. The fatty acid dry weight (DW) content decreased during the daylight period, whereas the fatty acid profile, as total fatty acids, was a function of growth rate. A short-term variation of exopolysaccharides synthesis with solar irradiance was also observed, i.e. the higher the external irradiance the higher the excretion of exopolysaccharides as a protection against adverse culture conditions. 9 1999 Elsevier Science B.V. All rights reserved.

Keywords: Porphyridium cruentum; Tubular photobioreactor: Oxygen generation rate: Carbon dioxide consumption rate; Continuous culture; Elemental analysis

* Corresponding author. Tel.: + 34-950-215032; fax: + 34-950-215484. E-mail address: [email protected] (E. Molina Grima) 0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S01 68- 1656(99)00080-2

272

M.M. Rebolloso Fuentes et al. /'Journal of Biotechnology 70 (1999) 271-288

1. Introduction

Microalgae have traditionally been cultivated in open systems where the culture conditions are selected to favour algal growth while hindering the growth of potentially contaminating micro-organisms. Open systems permit little operation control and a certain level of contamination always occurs. Consequently, it is not possible to use open systems to grow algae requiring conditions that would allow proliferation of other potentially contaminating microbes. The production of high-value algal products from strains that cannot be maintained in open ponds requires closed systems. One of these strains is the microalga Porphyridium cruentum which has been referenced as a potential source of several highvalue products such as: arachidonic acid, an essential dietary constituent for man and precursor of a large group of C20 compounds (Nichols and Appleby, 1969; Ahem et al., 1983) polysaccharides containing about 10% half-sulphate esters (Ramus, 1972; Percival and Foyle, 1979; Arad et al., 1985), pigments such as phycocyanin and phycoerythrin (Arad, 1987), and antioxidants as superoxidedismutase (Gudin, 1989). Among the closed systems, the tubular photobioreactors, employing an airlift device for low-shear propulsion of culture fluid through a horizontal tubular loop that constitutes the solar receiver, have been repeatedly referenced to obtain the highest productivities (Gudin and Chaumont, 1983; Gudin and Therpenier, 1986; Lee, 1986; Tredici and Materassi, 1992; Richmond et al., 1993; Torzillo et al., 1993; Molina Grima et al., 1994a,b,c,d; Borowitzka, 1996). The temperature, nutrients and light availability at which the cells are exposed (Little, 1953; Richmond et at., 1993) determine the productivity of microalgal cultures. The temperature can be easily controlled by several system such as water spraying (Torzillo et al., 1991a,b), termostation by a stainless steel tube placed inside the reactor (Chini Zitelli et at., 1996) or by immersion of the solar receiver in a termostated water pool (Molina Grima et al,, 1994a, b). Mineral nutrients can be easily supplied to the system although the optimal level has not been studied until now and they are

usually supplied in excess to attain nutrient-saturated conditions. In the same way, the addition of inorganic carbon, usually supplied as pure CO2 by an on-demand injection either mixed in the airflow (Chini Zitelli et al., 1996) or directly in the culture (Molina Grima et al., 1994a,b), has not been optimised either. The light availability is determined by the solar irradiance, a function of the geographic location and the local climatic parameters (Duffle and Beckman, 1980), the reactor design (Alfano et al., 1986; Lee and Low, 1992, 1993) and the attenuation produced by the cells, which is a function of the biomass concentration and the extinction coefficient of the biomass (Evers, 1991; Sukenik et al., 1986; M olina Grima et al., 1994c). The influence of solar irradiance on the variation of culture conditions and the phenomena taking place in the culture has not been totally elucidated. Torzillo et al. (1991a,b) studied the influence of daily variations of temperature in the productivity of Spirulina platensis cultures developed in open and closed reactors, concluding that the higher biomass productivity was attained with the best temperature control. Guterman et al. (1990) obtained a macromodel for open pond algal mass production of S. platensis in which the dissolved oxygen, growth rate and pH of the culture were functions of the solar irradiance, obtaining expressions for these relationships based on theoretical approximations. Camacho Rubio et al. (1998) studied the daily variation of pH, dissolved oxygen and carbon losses in outdoor cultures of Porphyridium cruentum during the day giving attention to the mass transfer phenomena involved, but disregarding the interrelationship between the variables. Molina Grima et al. (1994b,d) described the daily variation of dissolved oxygen, biomass concentration and temperature of outdoor cultures of P. tricornutum and Isochrysis galbana, reporting a first approach to the phenomena taking place in such systems but not elucidating the relationships existing among the different variables. Qiang and Richmond (1996) studied the influence of biomass concentration on the daily growth rate of S. platensis cultures, showing that high biomass concentrations gave rise to low increases of growth

M.M. Rebolloso Fuentes et al. ,, Journal o f Biotechnology 70 (1999) 271-288

rate but also to low photoinhibition at noon. Photoinhibition phenomena, usually attributed to high irradiance levels, have been observed in S. platensis (Vonshak and Guy, 1992; Qiang and Richmond, 1996) and P. tricornutum (Aci6n Fern~mdez et al., 1998) outdoor cultures, resulting in a low yield of the system and even in inhibition of the nitrogen uptake in S. platensis cultures (Bocci et al., 1988). Daily variations of the biochemical composition derived from the daily variation of culture parameters have also been referenced. Several studies have shown that microalgae and cyanobacteria when grown under high light intensity synthesise mostly carbohydrates during the light periods that are consumed in protein synthesis in the dark (Bocci et al., 1988; Collos and Slawyk, 1980; Molina Grima et al., 1994a,b, 1995). This behaviour presume different nutrients uptake rates for different times in the daily cycle, thus the supply of nutrients should be changed during the course of the day. Moreover, the accumulation of lipids (Bocci et al., 1988) and variation of fatty acids content and profiles (Molina Grima et al., 1995) has been referenced. The higher the adequacy of culture conditions to the requirements of the strain, the higher the synthesis of proteins and structural lipids and the lower the neutral lipids storage (Molina Grima et al., 1995). These variations in culture conditions determine different biochemical composition along the day, which could be taken into account in order to harvest a biomass with the most adequate composition. The present work is aimed at studying the influence of the overall daily cyclic variations of several culture parameters and the phenomena taking place in P. cruentum outdoor cultures. The objective is to find the main variables determining the daily behaviour of the system, and the relationship between these variables in order to develop a model useful for the estimation of the culture parameters during the day. For this, the overall behaviour of a chemostat outdoor culture of P. cruentum in a tubular photobioreactor has been determined taking into account pH, temperature, dissolved oxygen, composition of the inlet and outlet gas, in addition to the elemental analy-

273

sis of the broth, supernatant and biomass, and the biochemical composition of the biomass.

2. Materials and methods

2.1. Micro-organism and culture conditions

The red microalga used was P. cruentum UTEX 161. The culture was carried out in continuous mode at a constant dilution rate of 0.049 h-~ for 10 h in the daylight period during March 1997. The culture medium was a modification of Hemerick's medium (1973) prepared from sterilised seawater. The pH of the culture was controlled at 7.6 by an on-demand automatic injection of carbon dioxide. The temperature was controlled at 20~ by thermostating the water pond in which the solar receiver was submerged. 2.2. The photobioreactor

The photobioreactor (Fig. 1) consisted of an airlift pump that drove the culture fluid through a horizontal tubular solar receiver (Camacho Rubio et al., 1998). The airlift section (riser, downcomer and degasser) had a height of 3.5 m. The solar receiver was made of transparent Plexiglas tubes (0.05 m internal diameter, 0.005 m wall thickness) joined into a loop configuration by Plexiglas joints to obtain a total horizontal length of 98.8 m. The solar receiver was submerged (0.05 m) in a shallow pool of water thermostated by a heat pump (Calorex 4000, Andrews, Sykes Ltd., Malcolm Essex, UK). The bottom and inside walls of the pool were painted in white to improve their reflectance. The surface area of the pool was 21.4 m 2. The total culture volume in the bioreactor was 0.220 m 3. A port located on the side of the degasser section was used to continuously harvest the culture. The temperature, dissolved oxygen and pH were measured by sensors located in the degassing zone. The data were read by a ML-4100 control unit (New Brunswick Scientific, New Brunswick, NY, USA) connected to a computer equipped with a data acquisition card for data logging. Air was continuously supplied at a flow rate of 0.0124 mol s-1 Carbon was added as

274

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

pure CO2 directly injected under pH requirement at a flow rate of 0.0014 mol s-1. The air supply point was located at the end of the loop (i.e. at the entrance of the riser) whereas the CO2 was injected at the entrance of the solar receiver. Both gas flows were continuously monitored.

2.3. Solar irradiance The instantaneous photon-flux density of the photosynthetically active radiation (PAR) inside the thermostatic pond and therefore the irradiance on the reactor surface, Iw, was measured on-line using a quantum scalar irradiance meter (LI-190 SA, Licor Instruments, Lincoln, NE, USA) connected to a data acquisition card. The average irradiance inside the culture, Iav, was cal-

culated by using a solar irradiance model for tubular photobioreactors previously proposed by Aci6n Ferndndez et al. (1997).

2.4. Absorption coefficient of the biomass The absorption coefficient of the biomass was determined using the method described by Aci6n Fern~.ndez et al. (1997). To ascertain the light attenuation at a given time of the day, culture samples were placed in a tubular device using the same type of tube as the external-loop with a sheath to house the quantum scalar irradiance meter sensor in the centre. Attenuation at the centre of the tube, A t, was calculated as, At= L n ( ~ )

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M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

with I0 and I being the irradiance measured at the tube centre when the biomass concentration is zero and not zero, respectively. The variation in attenuation with biomass concentration for each sample was obtained by consecutive dilution with seawater, obtaining a slope which is the product of absorption coefficient of the biomass, Ka, by the light path length, p. The light path length at a given time of the day was a function of sun position, and it was determined using the irradiance model proposed by Aci6n Fernfindez et al. (1997).

2.5. Oxygen generation rate, carbon consumption rate and C02 losses The mass flow rates of both the inlet air and carbon dioxide were measured by two mass flow meters (Model 824-13-0V1-PV1-V4, SIERRA Instruments, Monterey, CA, USA). A gas analyser (Servomex Analyser Series 1400) was used to monitor the compositions of inlet and exhaust gases. The mass flow meters and the gas analyser were connected to a data acquisition card (DaqBook/112 and DaqBoard/112, IOTECH Inc., Cleveland, OH, USA) and an IBM compatible 486 computer for monitoring. The photosynthesis rate in the entire volume of the culture in a given instant was calculated using the experimental measurement of the gas inlet and outlet compositions and an oxygen balance. The oxygen mass balance could be written as, 02 in + 02 generation = 0 2 out + 02 accumulation (2) Since the accumulation term resulted 100 times lower than the difference between the oxygen inlet and exhaust including at the first hours of light period when the slope of dissolved oxygen is the maximum, this term was rejected and the oxygen generation rate, Ro2 was calculated as,

R% = 02 out - 02 in (3) In the same way, the carbon dioxide consumption, Rc% was determined from the difference between the amount injected into the loop and the amount present in the exhaust gas; the accumulation term was again rejected as being negligible. Hence,

Rco

2 -- CO 2

in

-

CO 2

out

275

(4)

The losses of C O 2 w e r e expressed as the ratio of the net CO2 molar flow in the exhaust gas to the CO2 molar flow injected.

2. 6. Photosynthetic activity The measurement of photosynthetic activity of the cells was carried out by a method similar to that described by Vonshak et al. (1985) based on the determination of the oxygen generation rate under constant laboratory conditions. For this, samples of culture were diluted to adjust the optical density to 0.1 at 625 nm, put into a termostated (22~ vessel (1 1 capacity), and irradiated with 2000 laE m - 2 s-1 by a halogen lamp located 0.5 m from the vessel. Oxygen development was then measured every second during 30 min with a dissolved oxygen electrode (LisleMetrix 2200D/P91) connected to a data acquisition system. The photosynthetic activity of the cells was determined as the slope of the dissolved oxygen variation versus time.

2. 7. Analytical methods The biomass concentration was determined by dry weight (DW). Duplicate culture samples were centrifuged (1800 x g), washed with 0.5 M NaC1 and distilled water to remove non-biological material such as mineral salt precipitates, lyophilised for 3 days, and weighed. The elemental composition (C, H, N, S, O) of the biomass, broth and cell free culture medium was determined using an elemental analyser LECO CHNS-932 equipped with an additional furnace VT-900 for oxygen measurement. For the analysis of broth and cell free culture medium (supernatant), 5 lal samples were injected. For the analysis of lyophilised biomass, 2 mg were injected. The protein content of the biomass was estimated as 6.25 times the elemental nitrogen content. These results were checked by the Kjehldahl method to confirm their accuracy (Rebolloso Fuentes et al., 1998). The chlorophyll content was measured according to the Hansmann method

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

276 w 5000

140

~.~. 4000 e-

3000

I~

2oo0

._m

o A

&

o8~ O

r 120

13

o

A

08 a A

C ID

&o&& ~

&

,

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o 60

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0 r= n ww

o

0

4

20

0

i

8

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12 Hour aDey

16

20

24

0

T

~

|

r

i

l

4

8

12 Hour

16

20

24

2 &Day 3]

Fig. 2. Daily variation of irradiance on the water pond.

Fig. 3. Daily variation of dissolved oxygen within the culture.

(1973). Carotenoids determination was as described by Whyte (1987). The fatty acid profile of the biomass was determined by GC using a HP5938 gas chromatography analyser. Fresh centrifuged wet biomass was used for fatty acid analysis. Methylation was arried out by direct transesterification following a modification of the Lepage and Roy method (1984) proposed by Garcia S~.nchez et al. (1993). Nonadecanoic acid was used as an internal standard to quantify fatty acid content in biomass.

During the night, the dissolved oxygen decreases down to 60% saturation with air due to the consumption of oxygen by respiration (Fig. 3). On days 1 and 2, the dissolved oxygen reaches a maximum at 12:00 and then declines in spite of the high solar irradiance. On day 3, this decrease is not observed, the dissolved oxygen remaining high until the solar irradiance decreases, in this moment dissolved oxygen strongly decreasing (Fig. 3). This behaviour can be attributed to a photoinhibition effect on days 1 and 2 caused by the high external irradiance at noon (above 4000 ~E m - 2 S-1), being lower on day 3 (3000 laE m - 2 s-1) due to shadowing by clouds. In order to verify the existence of photoinhibition in outdoor conditions, the variation of photosynthetic activity of the cells during the day was determined (Fig. 4). The results show that the photosynthetic activity of the cells is the highest at early morning, 0.0040 mol O~ m - 3 s-1, whereas it is decreased to 0.0006 mol 02 m - 3 s-X at noon, when the

3. Results

The solar irradiance inside the water pond, Iw, presents a Gaussian-like variation during the day, from zero in the night to up 4000 gE m - 2 s - 1 in the central hours of the day (Fig. 2). Day to day differences observed were due to weather variations. Thus, different daylight mean external irradiance on the reactor surface, Iwm, of 2000, 1800 and 1600 l,tE m - 2 s-1 was measured for days 1 2 and 3, respectively. The relatively high solar irradiance values measured are due to the existence of albedo, e.g. the reflection of solar radiation in the walls and bottom of the pond. This effect has been referenced to increase the irradiance on the culture surface in tubular reactors compared to open systems (Aci6n Fern~indez et al., 1997). The dissolved oxygen in the culture increases with irradiance up to 120% saturation with air in the daylight period due to photosynthesis (Fig. 3).

0.0040 :~

0.0030 -

.~ E ~ _ o"~ 0.0o20 c0 >'E 0 ~ 0.oo10 -

',,,,

13.

0.0000

6

8

10

12

Hour

14

16

18

20

Fig. 4. Daily variation of photosynthetic activity of the cells.

277

M.M. Rebolloso Fuentes et al. j Journal o f Biotechnology 70 (1999) 271-288 21.6 .Co 21.2 20.8

0.60

140

0.50 c

120

0.40 ~

100

-

- 8.0 7.8 p.,

L

0.30 = --O E

~ 20.4

0.20 (~

~ 20.0

0.10

.

19.6 0.0

0.5

J t

.

. 1.0

.

.

0.00

.

1.5 2.0 "13me, days

o

2.5

~"

80

,.~

60

o

m r

3.5

3.0

7.6 9

.

t

i]

9

"6 7.4 :z:

"

7.2

20

. 0.0

J

Fig. 5. Daily variation of gas exhaust composition during the quasi steady state.

solar irradiance is the highest, and again an increase during the afternoon up to 0.0012 mol O2 m - 3 s - 1, when solar irradiance decreases (Fig. 4). The gas exhaust composition varied according to the variations in dissolved oxygen. The oxygen molar fraction of the exhaust gas varied around the oxygen molar fraction in the air, ranging from 21.4% in the daylight period to 19.8% during the night (Fig. 5). The carbon dioxide molar fraction of the exhaust gas was always higher than in the air, ranging from 0.35% in the central hours of the day to ~ 0.50% during the night (Fig. 5). The high values of carbon dioxide molar fraction in the gas exhaust compared to air are due to the injection of pure CO2 bubbles for pH control. Therefore, when the CO2 injection is released inside the reactor, the broth becomes locally saturated with pure CO2. Then, when the culture is aerated in the airlift system, the carbon dioxide is stripped to air. This happened in both the daylight period and night period because the pH control system was not switched off at night. Moreover, the carbon dioxide generated during the night by respiration also increases the carbon dioxide molar fraction in the exhaust gas. The daily variation of solar irradiance also influenced the pH of the culture and the carbon losses. Although the pH of the culture was controlled at 7.6_+0.3 by on-demand injection of pure CO2, a cyclic daily variation was observed (Fig. 6). The average pH of the culture in the daylight period was higher, 7.75 _+ 0.08, than during the night, 7.51 _+ 0.05. In the daylight period,

,

40

9 Oxygen ....~ - - O ~ x ) n dioxide I

8.2

.

0.5

. 1.0

.

.

.

7.0

1.5

2.0 Time, days

F i g ....Carbonlosses

2.5 ,r

3.0

3.5

PHI

Fig. 6. Daily variation of carbon losses and pH of the culture during the quasi steady state.

the pH was above the set point due to the carbon consumption by photosynthesis, whereas during the night, the pH was under the set point due to CO2 generation by respiration. Although the pH of the culture was controlled, the carbon losses were high, ~ 100% during the night, dawn and dusk, due to the low carbon consumption rate. In the central hours of the day, the carbon losses were lower, equal to 20%, due to the high carbon consumption rate (Fig. 6). On the daylight period, the biomass concentration increased indicating that the growth rate was higher than the imposed dilution rate ( D - 0.049 h-1) and the biomass was accumulated in the reactor (Fig. 7). During the night, a fraction of the accumulated biomass was consumed, thus the 112 110

~

Day 1, iJ=0.0107 h1 BL=9% Day 2 IJ=0.0049 h"1 BL=20%

~.~

1 08 o r

106-

r

..j 1 0 2 1 O0 0.98 0.96

8

10

12

14 Hour

16

18

20

i O Day 1 IEIDay 2 A Day 3 I

Fig. 7. Daily variation of biomass concentration, additional growth rat in the daylight period, ~so~, and biomass losses, LB, during the quasi steady state.

278

M.M. Rebolloso Fuentes et al. Journal of Biotechnology 70 (1999) 271-288

biomass concentration at first hours in the morning being lower. The 3-day average quasi steady state biomass concentration was 3.50 g 1-1, this resulted in an average biomass productivity of 1.76 g 1-ld-~. In front, the biomass night losses were ~ 14% of the biomass concentration at the end of daylight time. The additional growth rate in the daylight period, ~tso~ (~t~o~= actual growth rate imposed dilution rate) was different for each day, the maximum, 0.0107 h-1, being obtained on day 1, and the minimum, 0.0049 h - ~, on day 2 (Fig. 7). In addition, the biomass night losses also varied for each day. A maximum loss was observed on day 2 (20%) while a minimum was obtained on day 1 (9%) (Fig. 7). Elemental analysis of the culture broth, cell free culture medium and liophylised biomass were carried out in order to determine the variation of metabolic routes of the cells during the day. The carbon content of the broth was in the range 0.14 + 0.02% , being higher in the morning and lower in the afternoon in spite of the higher biomass concentration (Fig. 8). This can only be due to either a carbon accumulation in the cell free culture medium (supernatant) during the morning that is eliminated in the afternoon, or to a decrease during the day of the carbon content in the biomass. The elemental analysis of the supernatant showed carbon accumulation during the morning in the liquid phase and elimination during the afternoon, as well as a reduction of nitrogen and sulphur content during the day (Fig. 8). Mean values ranges were 0.017 + 0.003, 0.025 + 0.005 and 0.024 + 0.004% DW of carbon, nitrogen and sulphur, respectively. With regard to elemental analysis of the biomass, the carbon and oxygen content increased during the daylight period (37.3 +_ 0.6 and 39.0 +_ 0.7% DW), whereas the nitrogen and sulphur content decreased (4.6 _+ 0.2 and 1.10 + 0.06%DW) and hydrogen content remained constant (5.5 _+ 0.3% DW) (Fig. 8). Variations of the culture conditions also determined changes in the biochemical composition of the biomass. The pigment content, as chlorophylls and carotenoids, slightly decreased during the daylight period, being 0.36 + 0.02% at the beginning and decreasing to 0.32 _+ 0.02% at the end of daylight time (Table 1). This trend caused a de-

0.20 ~

9

_ 0.15 j

9149 9

e

e-

[_,

0.05

~J

0.00 ~ 0.0

~

~176

mo(~

~-0.5

, 1.0

I 9Carbon

1.5 2.0 -time, days A Nitrogen

,

1

., ,

2.5

3.0

3.5

mSulphur I

0.040 T A

0.03o

O

O

A 9

,.a

0.010 ~-

~

0.000

9

0 u

~

0.5

0.0

~, ,,J ,,,,l

9

1.0

1.5

2.0

2.5

3.0

3.5

Time, days 9% Carbon ~ % Nitrogen o % Sulphur / J

7.0 6.o

"~ 42 +

gas, ~

oo

~" 32 ~ 0 r

3.0~" 2.0

ee*

-

0.5

0.0 r

ooo 1.0

oo

o

. %

1.5 2.0 2.5 3.0 -time, days o % Oxy n O/o Hyd

A % Nitrogen

o % Sulphur

10~"

3.5

,,,,I = 1

Fig. 8. Variation of elemental composition of broth, cell free culture medium and liophylised biomass during the quasi steady state reached.

crease of the absorption coefficient of the biomass, K, the mean values varying from 0.041 + 0.001 m 2 g-1 at the beginning of the day to 0.038 + 0.001 m 2 g-1 at the end of daylight time (Table 1), because Ka is a function of pigment content (Molina Grima et al., 1994a,b,c). The protein content also decreased in the daylight period, from a mean value at the beginning of the daylight time of 30.3 + 0.6% to 27.0 ___0.6% at the end of the afternoon (Table 1). With regard to fatty acids, only the main ones were considered,

i

t

P2

Table 1 Variation of the biochemical composition and extinction coefficient of the biomass during the quasi steady state Date

2413 2413 2413 2513 2513 2513 2613 2613 2613

Hour

9.5 12.5 18.0 9.5 12.5 18.0 9.5 12.5 18.0

Chlorop (‘XDW)

0.339 0.284 0.261 0.300 0.342 0.333 0.358 0.303 0.314

Carot..(‘L,DW)

0.031 0.014 0.023 0.022 0.013 0.029 0.020 0.047 0.020

Pigments (‘%DW)

0.370 0.298 0.284 0.322 0.355 0.362 0.378 0.350 0.334

K , (mZ g ’)

0.042 0.037 0.036 0.038 0.041 0.041 0.042 0.040 0.039

Proteins (‘AtDW)

30.144 27.274 25.769 30.206 29.885 29.141 30.638 28.873 26.094

2 Fatty acids (‘LIDW)

_

_

16:n

1~:2

20:4

20:5

31.666 31.554 32.395 31.341 3 1.739 30.932 30.884 30.323 31.372

12.928 12.959 13. I48 12.100 I I ,836 I 1.654

38.592 38.290 37.901 37.589

16.812 17.197 16.554 18.970 23.522 20.355 21.754 21.997 20.448

________~ i6:n

18:2

214

1.669 I531

0.682 0.629 0.631 0.665 0.639 0.657 0.660 0.649 0.565

2.034

1.555

1.720 1.691 1.742 1.844

1.784 1.587

20:s

0.886 0.834 0.795 1.043 2.065 1.084 1.986 1.147 2.088 1.299 2.168 1.294 2. I57 1 . ~ 7 3 I ,035 I.858 1.819

1 1.058 I I .02 I

11.156

3X.818

37.064 36.304 36.659 37.024

_

_

~

N

W 4

280

M.M. Rebolloso Fuentes et al. /Journal of Biotechnology 70 (1999) 271-288

these being 16:0, 18:2, 20:4 and 20:5. The araquidonic acid (20:4) is the main fatty acid with 2%DW, the next being the palmitic acid (16:0) with 1.7%DW and eicosapentaenoic acid (20:5) with 1.1%DW (Table 1). All of the fatty acids content decrease in the daylight period, from 1.74 _+ 0.05, 0.67 _+ 0.0, 2.09 _+ 0.06 and 1.08 _+ 0.06% of biomass DW at the first hours in the morning to 1.63 _+ 0.05, 0.62 _+ 0.02, 1.93 _+ 0.06 and 0.99 +0.06% of biomass DW at the last hours in the afternoon, for 16:0, 18:2, 20:4 and 20:5, respectively. However, no variation of the fatty acids profile (as total fatty acids) during the day was observed (Table 1). The polyunsaturated fatty acids, araquidonic and eicosapentaenoic, represent more than 50% of total fatty acids (37.5% of araquidonic and 19.2% of eicosapentaenoic), the rest being mainly palmitic acid with 31.3% of total fatty acids (Table 1).

4. Analysis of the experimental results

r 2 = 0.9766

I =/0 exp( - K a ' P " C)

(6)

Ia__~= exp( -- Ka 9Paverage " C ) = m = cte

(7)

Iw

Secondly, the cells tend to adapt to the average irradiance inside the culture. Thus, the higher the external irradiance and biomass concentration, the lower the extinction coefficient of the biomass must be to keep the average irradiance constant. Eq. (5) makes it possible to determine the average irradiance inside the culture, Iav at any time during the quasi steady state directly from the external irradiance, Iw. With regard to the oxygen generation rate, Ro, it appears to be a function of the average lrrad~ance inside the culture (Fig. 10). Vonshak et al. (1985) also observed this dependence as a hyperbolic variation of the photosynthesis rate with irradiance in the laboratory thin layer cultures of P. cruentum, while Jensen and Knutsen (1993) observed this same behaviour with S. platensis. However, in outdoor cultures, the existence of photoinhibition has been observed (Fig. 4). In the present study, the photosynthetic activity of the cells was very high at first hours in the morning, indicating a fast response of Porphyridium to light 9

Although in outdoor chemostat cultures of microalgae the pH, temperature and dilution rate are controlled, a daily cyclic variation of the culture parameters takes place mainly due to variations in the solar irradiance. In addition to solar irradiance, the average irradiance inside the culture, Iav, defined as the mean light intensity a cell randomly moving inside the culture intercepts, also varies during the day. The external irradiance, biomass concentration and pigment content of the biomass and thus the average irradiance inside the culture are modified during a day and between different days. A variance analysis indicates that the oxygen generation rate is influenced by the average irradiance inside the culture rather than by the irradiance on the culture surface. In this sense, a linear relationship between I~v and I,,. is observed (Fig. 9). Iav = 0.089Iw + 4.05

between Iw and lav implies the quotient Iav/Iw is a constant and therefore the product K a ' P " C is also a constant during the quasi steady state. Thus, considering a mean light path, Pa,,erage, from Eq. (7) means that the product Ka" C must also be a constant.

(5)

The linear dependence of light intensity inside fermentor and outdoor light intensity has been previously reported (Hirata et al., 1996). This linear variation of Iav with Iw brings about two important points. Firstly, a linear relationship

400 7

~

w 200

i

.

S

100 l

O' 0

1000

2000

Iw, pE/m2s

3000

4000

Fig. 9. Correlation between the average irradiance inside the culture, Iav, and the external irradiance on the culture surface, Iw during the quasi steady state reached.

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288 0.0020 o.oo15 -~ oe

o.oolo

.~

-o

0.0005

0.0000

and regeneration of key components of the photosynthetic apparatus coexist and the available amount of functional photosynthetic pigments is the result of an equilibrium between that two processes.

# t.

*

9 -

9

-0.0005 ~ e 0

281

1O0

200

300

400

lav, pE/m2s Fig. 10. Variation of oxygen generation rate with the average irradiance inside the culture estimated by using Eq. (5). Line represents a smooth tendency curve.

availability. In the central hours of the day, when the solar irradiance is at the peak levels, the photosynthetic activity declined and then a slight increase again in the afternoon, when the solar radiation was reduced. This behaviour evidences the existence of photoinhibition that appears as a decrease in the efficiency of the culture to metabolize the available light when solar irradiance is increased, though the oxygen generation rate within the photobioreactor is not decreased because the loss in photosynthetic efficiency is compensated by a higher irradiance level. Different authors have referenced the existence of photoinhibition in outdoor cultures. Molina Grima et al. (1996a) observed the existence of photoinhibition in outdoor cultures in two ways. During the day, photoinhibition could be caused by an increase of the irradiance on the culture surface. With the position inside the culture, photoinhibition could arise caused by the strong irradiance gradient that exist in dense cultures. In this sense, Vonshak and Guy (1992) observed the existence of photoinhibition during the day in S. platensis cultures. Jensen and Knutsen (1993) demonstrated that photoinhibition is a reversible process in which degradation

5. D i s c u s s i o n

The influence of photoinhibition in the behaviour of outdoor cultures must always be considered. In outdoor cultures of P. tricornutum (Aci6n Ferndndez et al., 1998) and L galbana indoor cultures (Molina Grima et al., 1996b), the growth rate has been observed to be a function of average irradiance rather than external irradiance. Molina Grima et al. (1996a,b) report that the growth rate varies hyperbolically with the average irradiance inside the culture although the external irradiance might influence the parameters of the growth model. Additionally, Aci6n Fern~indez et al. (1998) proposed a mathematical growth model that considers the existence of photolimitation and photoinhibition, taking into account a decrease in the efficiency of the cells when external irradiance increased. Bearing these references in mind, the variation of oxygen generation rate with the average irradiance inside the culture has been fitted to a hyperbolic expression (Eqn. 8) for each single day.

Ro2

R~ = I~, + I~,, - R ~

(8)

Since the solar irradiance measured each day is different, the characteristic parameters of the model results are different (Table 2). The results show how the parameters of the model vary: n decreased while Ik increased linearly with the daily mean irradiance on the reactor surface, Iwm (Table

Table 2 Values of characteristics parameters of the model obtained by non-linear regression for each day during the quasi steady state

Day Iwm (laE 1 2 3

2000 1800 1600

m -2 s -1)

Ro2max (mol 0.002968 0.003238 0.001663

02 m -3 s -1)

n

Ik (laE m -2 s - l )

Ro2min (mol 02 m -3 s -1)

r2

0.837415 0.963006 1.325950

148.575 115.235 76.56

0.000482 0.000886 0.000082

0.99235 0.923979 0.959093

282

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

0.0020 1 Y = 0.91101x + 0.00005 "o 0.0015 t R2 = 0.93502

=

0.0005

.

99

*

1

o.

0.0000 1

-0.00__0.00050~40.00000.00050.00100.00150,0020 ROz experimental Fig. 11. Correlation between oxygen generation rate estimated by the model Eq. (9) and experimental oxygen generation rate.

2). Equation 8 can therefore be enhanced to take these linear relationships of n and lk with Iw. Doing so, the following model of instantaneous oxygen generation rate with external irradiance and average irradiance inside the culture is obtained. 9I( a + b / w )

Ro2max -av

R~ = (c + d" Iw)(~+ b. Iw) + I(~v+b. Zw)-

R~

(9)

Ro2max -- 0.003979 mol 02 m - 3 s - ~, a = 0.635, b = -0.000063 m2s ~tE-~, c = 706.65, laE m - 2 s-~, d = - 0.025471,

Ro2mi n

= 0.000317 mol 02 m - 3 S-1, r2._. 0.9317. The parameters of the model were obtained by non-linear regression, fitting to this equation all of the experimental data available (74 values). A representation of estimated oxygen generation rate Eq. (9) versus experimental oxygen generation rate show the adequacy of the model (Fig. 11). The maximum photosynthesis rate, 0.0040 mol O2 m - 3 s - 1 (600 ~tmol 0 2 h -~ mg Chlorophyl-~), is lower than the maximum value referenced for this strain, 1000 ~tmol 02 h-~ mg Chlorophyl-~ (Vonshak et al., 1985), this maximum being obtained under laboratory conditions. Ohta et al. (1992) observed a maximum CO2 consumption rate of 0.0044 mol m - 3 s - 1 in laboratory cultures Porphyridium under lightdark cycles. If an O2/CO2 ratio of one is accepted, the maximum photosynthesis rate obtained is the

same as predicted by the model. The minimum photosynthesis rate, 0.000317 mol O2 m - 3 s-1, is the respiration rate during the night, and although it has been considered to be constant, a certain influence of the daily culture conditions can be expected. In this sense, Torzillo et al. (1991a,b) and Molina Grima et al. (1994b,d) observed that the respiration rate is a function of the temperature and the irradiance during the daylight time. Moreover, the experimental results show that the higher the growth rates in the daylight time the lower the biomass losses during the night (Fig. 7). This same behaviour was observed in laboratory light-dark cycle cultures of Porphyridium (Ohta et al., 1992). Thus, the lower the growth rate the higher the carbohydrates synthesis and the lower the protein synthesis, the carbohydrates being consumed during the night by respiration. The obtained model Eq. (9) allows to determine the oxygen generation rate as a function of external and average irradiance inside the culture, thus being a useful tool in the design and scaleup of tubular photobioreactors. In this sense, one major problems in the scale up of tubular photobioreactors is to determine the acceptable maximum loop length, which will be limited by the maximum dissolved oxygen level that the cells can support without being affected by its toxicity. Therefore, for any given conditions of Iw and Iav, the model will estimate the oxygen generation rate. This, together with the liquid flow rate in the solar receiver, determines the maximum loop length admissible for a given maximum oxygen level. Alternatively, for a given length of the loop it is possible to calculate the liquid flow necessary to keep the dissolved oxygen under a given level and thus avoid its toxic effects. Moreover, by applying an oxygen mass balance to the overall reactor and by rejecting the accumulation term (Camacho Rubio et al., 1998), the model could be used to determine the mass transfer capacity necessary in the system in order to limit the dissolved oxygen level in the culture. O2 in - 02 out = 02 generated + 02 accumulated

(lo)

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288 y = 41572x + 67.71

140 _

120

160 140 .'~ =

R2 = 0.8895

.

;~ 100

_ tO*****

9 ,=

20

.q

0.~

9

60 !

. 9

40

" ,W*~.,.,

"

0

-0.0005

100 i

0.0005

0.0010

N(~, m~m~

I 9Carbonlosses,._o Dissolv~

020

0.111115 0.0020

oxygen]

Fig. 12. Variation of dissolved oxygen and carbon losses in the culture with the oxygen generation rate.

d[O2] d----~

Kla([02] - [O~=]) = Ro2 +

(1 l) (12)

Kla[02] - K/a[O~ ] = R% 1

[02] = ~a ~

(13)

+ [0~']

A high photosynthesis rate causes a high dissolved oxygen level in the system. On the other hand, the higher the volumetric mass transfer, Kla, the lower the dissolved oxygen. In this sense, the dissolved oxygen linearly increases with the oxygen generation rate (Fig. 12), the slope of this line being the inverse of the volumetric mass transfer coefficient of the system. Thus, the Kla of the system can be determined from the value of this experimental slope, resulting as 0.0023 s - 1, which is very similar to that experimentally determined (Kla=O.O029 s -~) by Camacho Rubio et al. 0.0020 0.0015

E

0.0010

C~ 0.0005 0.0000 -0.0005

-0.0005

|

0.0000

,

0.0005

,

0.0010

i

0.0015

0.0020

RO=, mol/mSs

Fig. 13. Correlation between carbon consumption rate and oxygen generation rate during the quasi steady state.

283

(1998) for this same culture system. A linear relationship between oxygen generation and carbon consumption is also observed, the ratio being 1.004 mol CO2 per tool O2 (Fig. 13). This value is higher than others referenced for different strains like Chlorella pyrenoidosa (Myers, 1980) being 0.70 mol CO2 per mol O2. The difference can be attributed to the different metabolic routes of each strain. In particular, P. cruentum trends to accumulate carbohydrates and excrete polysaccharides, thus increasing the value of CO2 consumption to the 02 generation ratio. From the linearity between carbon consumption and oxygen generation, two conclusions can be drawn. First, the oxygen generation and carbon consumption are coupled, the ratio being constant and equal to one all the time and independent of cell metabolism or culture conditions. Second, the obtained model also allows to determine the carbon requirements of the system, thus becoming a tool useful to study how the carbon losses can be reduced by optimising the amount injected. In this sense, it is observed (Fig. 12) that the carbon losses are a function of oxygen generation rate. A high oxygen generation rate causes a high carbon consumption and low carbon losses. This fact points out how the injection of pure CO2 allows for reliable pH control but causes a high loss of CO2. In order to reduce these values, Camacho Rubio et al. (1998) proposed to modify the composition of the gas phase, reducing the COz molar fraction and thus reducing the carbon dioxide oversaturation in the culture. However, this mode of operation causes an increase of the pH in the culture, which lowers the carbon availability and could reduce the yield of the system (Molina Grima et al., 1996a). With regard to the biomass productivity, the observed day to day variations of culture conditions determine different growth rates and values of maximum oxygen generation rate. However, these values are interrelated, the higher the growth rate in the daylight period, the higher the maximum oxygen generation rate (Fig. 14). This same behaviour was observed in Spirulina cultures (Guterman et al., 1990). The most significant variable determining both the growth rate in the daylight period and the maximum oxygen genera-

284

M.M. Rebolloso Fuentes et al. ~Journal of Biotechnology 70 (1999) 271-288 0.0022 y = 0.1216x + 0.0005

0

R 2 = 0.9627

o.ools

E

~ 0.0014

C 0.0010

--

0.004

,

,

,

,

0.006

0.008

0.010

0.012

I~ol, h "1

Fig. 14. Variation of maximum oxygen generation rate with the additional growth rate in the daylight time, ~tso=for 3 days during the quasi steady state.

tion rate is the mean average irradiance inside the culture in the daylight period. Moreover, both the growth rate and the maximum oxygen generation rate linearly increase with the mean average irradiance inside the culture in the daylight period (Fig. 15). The higher the solar irradiance or the lower the biomass concentration, the higher the mean average irradiance and light availability, thus the growth rate and photosynthesis rate being highest. Aci~n Fernfindez et al. (1998) observed that the growth rate of P. tricornutum varies hyperbolically with the mean average irradiance inside the culture, this relationship being approximately linear until Iav values of 200 gE m -2 s-1, with the slope strongly decreasing for irradiances above this value. The linear relationship between growth rate and mean average irradiance observed agreed with the linear relationship observed for P. tricornutum at low 0.012

0.0030

y = 0.00028x - 0.03985

0.010

R= = 0 . 9 ~

,,,,,,` ' ~

O.0025

0.008 0

~.oo6

0.0020 E

0.004

0.0015 n,,

0.002 R 2 = 0.981510

0.000 160

I

1

,

165

170 lawn, pE/rn=s

175

0.0010 180

Fig. 15. Variation of additional growth rate in the daylight period, ~tso~, and maximum oxygen generation rate with the mean average irradiance inside the culture, Iavm.

values of Iavm, thus indicating that the same behaviour could be accepted in this case although further research is necessary at different Iav values. With regard to elemental analysis of culture, supernatant and biomass, the carbon content of the supernatant showed a variation parallel to the solar irradiance during the day, an increase during the morning and a decrease in the afternoon (Fig. 8). However, Camacho Rubio et al. (1998) previously demonstrated that cultures operated with pH control by on-demand CO2 injection had a lower carbon content in the central hours of the day, due to the consumption by the cells, than in the first and last hours of the daylight period, by a reduction in photosynthesis and respiration The carbon content of the supernatant changes in a opposing way. Thus, at noon the carbon content of the supernatant is 0.200 g 1-~ whereas Camacho Rubio et al. (1998) estimate that there must be 0.072 g 1-1 of total inorganic carbon in the culture. The difference corresponds to the organic carbon excreted to the medium by the cells, 0.128 g 1 - 1 consisting mainly of exopolysaccharides. Vonshak et al. (1985) observed exopolysaccharide concentrations of around 0.24 g 1-1, double than the values measured in the present study, however this could be due to the low dilution rate, 0.02 h-1, and low pH, 7.5, at which the cultures were operated. In the first and last hours of the day, the carbon content in the supernatant decreased, 0.13 g 1-1, while the total inorganic carbon estimated by Camacho Rubio et al. (1998) increased, 0.077 g 1-1. This difference indicates a lower exopolysaccharides concentration in these hours, 0.053 g 1-1, and reflects that exopolysaccharides synthesis modifies during the day increasing at noon due to the existence of adverse culture conditions by photoinhibition. Thus, Vonshak et al. (1985) found that Porphyridium excretes big amounts of exopolysaccharides under adverse conditions as nitrogen limitation, a low growth rate, etc. The cellular polysaccharides are originated by the continuous excretion of polysaccharides, which form an envelope around the outer cell wall. The outer layer of this envelope is gradually sloughed off, enriching the growth medium and constituting the exocellular polysaccharide fraction (Vonshak et al., 1985).

M.M. Rebolloso Fuentes et al./"Journal of Biotechnology 70 (1999) 271-288

With respect to nitrogen, the second macronutrient, the amount of nitrogen supplied daily to the system with the medium is 27.4 g d - 1. However, the elemental analysis of the produced biomass indicated that only 16.03 g d-~ of nitrogen is taken up, thus the yield of this nutrient being 58%. In addition, the nitrogen content of supernatant decrease during the day, being a function of the daily variation of the cellular metabolism and not of the oxygen generation rate. Therefore, the results of elemental analysis of the biomass showed how during the illuminated period the metabolism is mainly directed to the synthesis of carbohydrates and lipids--carbon, oxygen and hydrogen content of the biomass increasing 1.2, 1.0 and 0.3%, respectively--while during the dark period, the accumulated carbohydrates were consumed and the metabolism was directed to the synthesis of proteins--nitrogen and sulphur content of the biomass increasing 3.7 and 0.2%, respectively--and structural lipids. The nitrogen is mainly taken during the afternoon and night when the protein synthesis is active. In this sense, the results confirm the idea of Professor Gudin (personal communication) that the nitrogen should be better supplied to the system during the night to avoid nitrogen limitation. However. in this research nitrogen limitation did not exist due to the excess of nitrogen supplied. A daily variation of cellular metabolism was observed in outdoor cultures of P. tricornutum (Aci6n Fernfindez et al., 1998), S. platensis (Bocci et al., 1988; Torzillo et al., 1991a,b) and Oscillatoria agardhii (Van Liere et al., 1979) and it is attributed to a response of the cells to the daily solar cycle, because the cells store energy during the illuminated period which is metabolised in the night period. The mean elemental composition of the P. cruentum biomass was estimated as C1Hl.v6N0.1180.olO0.79, a value similar to that proposed by different authors for microalgal biomass (Borowitzka and Borowitzka, 1988). The daily variation of the cellular metabolism also caused the variations on the fatty acids content of the biomass. The content of all the fatty acids decreases during the daylight time, the decrease being sharper for polyunsaturated fatty acids, 18:2, 20:4 and 20:5 (8-9%), than for satu-

285

rated and monounsaturated fatty acids, 16:0 (6%) (Fig. 14). In P. tricornutum (Aci6n Fernfindez, 1996) and I. galbana outdoor cultures (Molina Grima et al., 1995), an increase in the fatty acids content during the daylight period was observed, the increase being higher for the short-chain than for the long-chain fatty acids. This behaviour can be attributed to the different roles of the fatty acids in the cell metabolism. Short-chain saturated and monounsaturated fatty acids were found to be the main components of neutral lipids (storage lipids) (Molina Grima et al., 1994a,b, 1995), and the initial step in the path of fatty acid synthesis (Hodgoson et al., 1991). Thus, the amount of these fatty acids is subject to the daily cyclic variation of environmental conditions. On the other hand, polyunsaturated fatty acids are structural lipids, mainly found in glycolipids and phospholipids (Molina Grima et al., 1994a,b), and therefore, their contents are more related to state of growth than to short-term environmental variations. However, in this research a decrease in all the fatty acids content was observed. This decrease can be attributed to the accumulation of carbohydrates during the daylight period and highlights that the synthesis of lipids in P. cruentum takes place mainly in the darkness, when the carbohydrates accumulated are metabolised. Moreover, the lower decrease in the fatty acid content of 16:0 than 18:2, 20:4 and 20:5 indicates that the behaviour observed in P. tricornutum and I. galbana is also reproduced in P. cruentum, although variation of fatty acid profile during the day was not significant. With regard to the fatty acids profile (as total fatty acid), although the biomass was obtained in quasi steady state, the differences in the additional growth rate in each day determined a variation on the fatty acids profile. The higher the growth rate the higher the polyunsaturated fatty acid fraction and the lower the 16:0 and 18:2 fatty acid fraction (Fig. 16). This behaviour can be attributed to the reduction in the duplication time when growth rate increases, that increases the requirements of structural biomolecules while the storage of energy as lipids is reduced. This phenomenon causes a reduction in the fraction of saturated and monounsaturated fatty acids, and an increase of

286

M.M. Rebolloso Fuentes et al. / Journal of Biotechnology 70 (1999) 271-288 -~ 0.40

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6. C o n c l u s i o n s

In outdoor systems, daily variations of culture conditions take place due to the variation of solar irradiance. In order to respond to these variations, the cells adapt their behaviour. Some of these modifications are fast and are a direct function of culture conditions in a short time interval (seconds, minutes or a few hours). However, others are slower and are a function of culture conditions averaged over a long time interval (all the daylight time, all the night or all the day). Short term responses of oxygen generation rate, carbon consumption rate and exopolysaccharides production are observed whereas the growth rate and

metabolic routes show long-term responses. In this sense, in outdoor P. c r u e n t u m cultures, the oxygen generation rate is mainly determined by the average irradiance inside the culture, although the existence of photoinhibition, causing a reduction in the efficiency of the system to utilise the solar radiation, is observed. Considering this, a model for the photosynthesis rate as both oxygen generation rate and carbon dioxide consumption rate is obtained. This model allows to estimate the photosynthesis rate as a function of the solar irradiance--a function of system geometry and location of the factory--and average irradiance inside the culture--a function of the imposed dilution rate, biomass concentration and pigment content (Aci6n Fern~.ndez et al., 1 9 9 8 ) g a t which the cells are exposed to. Moreover, the linear relationship between the oxygen generation rate and carbon consumption observed allows the model to also estimate the carbon requirements of the system. The obtained model is therefore a useful tool in determining design parameters such as maximum solar receiver length of tubular external loops or the requirements of mass transfer in the system in order to avoid the deleterious effects of oxygen oversaturation. The elemental analysis of the biomass revealed how the daily variation of the cellular metabolism differs in the variation of the photosynthesis rate, indicating that the storage of energy by photosynthesis and its use by the cellular metabolism are not simultaneous. During daylight time, carbohydrates were accumulated by the biomass as energy storage, to be metabolised later during the night to synthesise proteins and structural lipids. This behaviour indicates that a supply of nitrogen during the night could be necessary, while the carbon injection can be switched-off in order to reduce the carbon losses.

Acknowledgements

This research was supported by the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT) (BIO-95-0692) (Spain), and Plan Andaluz de Investigaci6n II, Junta de Andalucia.

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

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S~nchez P6rez, J.A., Gim6nez Gim6nez, A., 1993. Cuantificaci6n de ~icidos grasos a partir de biomasa microalgal. Grasas y Aceites 44 (6), 348-353. Gudin, C., Chaumont, D., 1983. Solar biotechnology study and development of tubular solar receptors for controlled production of photosynthetic cellular biomass. In: Palz, W, Pirrwitz, D. (Eds.), Proceedings of the Workshop and E.C. Contractor's meeting in Capri. Reidel Publishing Company, Dordrecht, pp. 184-193. Gudin, C., Therpenier, C., 1986. Bioconversion of solar energy into organic chemicals by microalgae. Adv. Biotechnol. Process. 6, 73-110. Gudin C., 1989. An overview of microalgae biotechnology for fine chemicals. The first International Marine Biotechnology Conference, 3-6 September, Tokyo, Japan. Guterman, H., Vonshak, A., Ben-Yaakov, S., 1990. A macromodel for outdoor algal mass production. Biotechnol. Bioeng. 35, 809-819. Hansmann, E., 1973. Pigment analysis. In: Stein, J.R. (Ed.), Handbook of Psychological Methods, Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 359-368. Hemerick, G., 1973. Culture methods and growth measurements. In: Stein, J.R. (Ed.), Handbook of Physiological Methods. Cambridge University Press, Cambridge, UK, pp. 259-260. Hirata, S., Hayashitani, M., Taya, M., Tone, S., 1996. Carbon dioxide fixation in batch culture of Chlorella sp using a photobioreactor with a sunlight collection device. J. Ferment. Bioeng. 81 (5), 470-472. Hodgoson, P.A.. Henderson, R.J., Sargent, J.R., Leftley, J.W., 1991. Patterns of variation in the lipid class and fatty acid composition of Nannochloropsis oculata (Eustigmaphiceae) during batch culture. I. The growth cycle. J. Appl. Phycol. 3, 169-181. Jensen, S., Knutsen, G., 1993. Influence of light and temperature on photoinhibition of photosynthesis in Spirulina platensis. J. Appl. Phycol. 5, 495-504. Kates, M., Volcani, B.E., 1966. Lipid content of diatoms. Biochem. Biophys. Acta 116, 264-278. Lee. Y.K., 1986. Enclosed bioreactors for the mass cultivation of photosynthetic microorganisms: the future trend. TIBTECH 4. 186-189. Lee, Y.K., Low. C.S., 1992. Productivity of outdoor algal cultures in enclosed tubular photobioreactor. Biotechnol. Bioeng. 40, 1119-1122. Lee, Y.K., Low, C.S., 1993. Productivity of outdoor algal cultures in unstable weather conditions. Biotechnol. Bioeng. 41, 1003-1006. Lepage, G., Roy, C., 1984. Productivity of outdoor algal cultures in unstable weather conditions. J. Lipid Res. 25, 1391-1396. Little, A.D., 1953. Pilot plant studies in the production of Chlorella. In: Burlew, J.S. (Eds.), Algal culture from laboratory to pilot plant. Carnegie Institute of Washington, pp. 235-273.

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Molina Grima, E., Garcia Camacho, F., Sfinchez P6rez, J.A., Aci6n Fernandez, F.G., Fernandez Sevilla, J.M., Vald6s Sanz, F., 1994a. Effect of dilution rate on eicosapentaenoic acid productivity of Phaeodactylum tricornutum UTEX 640 in outdoor chemostat culture. Biotechnol. Lett. 16 (10), 1035-1040. Molina Grima, E., Garcia Camacho, F., Sanchez Perez, J.A., Urda Cardona, J., Aci6n Fernandez, F.G., Fern~indez Sevilla, J.M., 1994b. Outdoor chemostat culture of Phaeodactylum tricornutum UTEX 640 in a tubular photobioreactor for the production of eicosapentaenoic acid. Biotechnol. Appl. Biochem. 20, 279-290. Molina Grima, E., Garcia Camacho, F., S~inchez P6rez, J.A., Fernandez Sevilla, J.M., Aci6n Fernfindez, F.G., Contreras G6mez, A., 1994c. A mathematical model of microalgal growth in light limited chemostat culture. J. Chem. Tech. Biotechnol. 61, 167-173. Molina Grima, E., S~nchez P6rez, J.A., Garcia Camacho, F.. Garcia S~nchez, J.L., Aci6n Fernandez. F.G., Lopez Alonso, D., 1994d. Outdoor culture of Isochrvsis galbana ALII-4 in a closed tubular photobioreactor. J. Biotechnol. 37, 159-166. Molina Grima, E., S~,nchez P6rez, J.A., Garcia Camacho. F.. Garcia S~nchez, J.L., Fernandez Sevilla, J.M., 1995. Variation of fatty acid profile with solar cycle in outdoor chemostat culture of Isochrysis galbana ALII-4. J. Appl. Phycol. 7, 129-134. Molina Grima, E., S~nchez P~rez, J.A., Garcia Camacho. F.. Fernfindez Sevilla, J.M., Aci6n Fernfindez, F.G, 1996a. Productivity analysis of outdoor chemostat culture in tubular air-lift photobioreactors. J. Appl. Phycol. 8, 369-380. Molina Grima, E., Fernfindez Sevilla, J.M., Sfinchez P6rez, J.A., Garcia Camacho, F., 1996b. A study on simultaneous photolimitation and photoinhibition in dense microalgal cultures taking into account incident and averaged irradiances. J. Biotechnol. 45, 59-69. Myers, J., 1980. On the algae: thoughts about physiology and measurements of efficiency. In: Falkowsky, P.G. (Ed.), Primary Productivity in the Sea. Plenum Press, New York, pp. 1-15. Nichols, B.W., Appleby, R.S., 1969. The distribution and biosynthesis of arachidonic acid in algae. Phytochemistry 8, 1907-1915. Ohta, S., Chang, T., Aozasa, O., Kondo, M., Miyata, H., 1992. Sustained production of arachidonic and eicosapentaenoic acids by the red alga Porphyridium purpureurn cultured in a light/dark cycle. J. Ferment. Bioeng. 74, 398-402. Percival, E., Foyle, R.A.J., 1979. The extracellular polysaccha-

rides of Porphyridium cruentum and aerugineum. Carbohydr. Res. 72, 165-176.

Porphyridium

Qiang, H., Richmond, A., 1996. Productivity and photosynthetic efficiency of Spirulina platensis as affected by light intensity, algal density and rate of mixing in a flat plate photobioreactor. J. Appl. Phycol. 8, 139-145. Ramus, F., 1972. The production of extraceUular polysaccharides by the unicellular red alga Porphyridium aerugineum. J. Phycol. 8, 97-111. Rebolloso Fuentes, M.M., Aci6n Fernandez, F.G., S~nchez P6rez, J.A.. Gil Garcia, M.D., Guil Guerrero, J.L., 1998. Variacion de la composici6n nutritiva (composici6n centesimal, elementos minerales y ~cidos grasos) de la biomasa microalgal de Porphyridium cruentum. Food Sci. Technol. Int. (in press). Richmond, A., Boussiba, S., Vonshak, A., Kopel, R., 1993. A new tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol. 5, 327-332. Sukenik, A., Falkowski, P.G., Bennett, J., 1986. Potential enhancement of photosynthetic energy conversion in algal mass culture. Biotech. Bioeng. 30, 970-977. Torzillo, G., Sacchi, A., Materassi, R., 1991a. Temperature as an important factor affecting productivity and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors. Biores. Technol. 38, 95-100. Torzillo, G., Sacchi, A., Materassi, R., Richmond, A., 1991b. Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors. J. Appl. Phycol. 3, 103-109. Torzillo, G., Carlozzi, P., Pushparaj, B., Montaini, E., Materassi, R., 1993. A two plane tubular photobioreactor for outdoor culture of Spirulina. Biotechnol. Bioeng. 42, 891898. Tredici, M.R., Materassi, R., 1992. From open ponds to alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms. J. Appl. Phycol. 4, 221-231. Van Liere, L., Mur, L.R., Gibson, C.E., Herdman, M., 1979. Growth and physiology of Oscillatoria agardhii cultivated in continuous culture with a light-dark cycle. Arch. Microbiol. 123, 315- 318. Vonshak, A., Cohen, Z., Richmond, A., 1985. The feasibility of mass cultivation of Porphyridium. Biomass 8, 13-25. Vonshak, A., Guy, R., 1992. Photoresponse, photoinhibition and productivity in the blue-green alga, Spirulina platensis, grown outdoors. Plant Cell Environ. 15, 613-616. Whyte, J.C., 1987. Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aquaculture 60, 231-241.

iii

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I O U I t N A L

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Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 289-297

......

An integrated solar and artificial light system for internal illumination of photobioreactors James C. Ogbonna a

a,b,, Toshihiko Soejima a, Hideo Tanaka a,b

Institute of Applied Biochemistry, University of Tsukuba, I-I-1 Tennodai, Tsukuba 305-8572, Japan b CREST, Japan Science and Technology Corporation (JST), Tsukuba, Japan

Received 6 October 1998; received in revised form 23 November 1998; accepted 22 December 1998

Abstract Exploitation of photosynthetic cells for the production of useful metabolites requires efficient photobioreactors. Many laboratory scale photobioreactors have been reported but most of them are extremely difficult to scale up. Furthermore, the use of open ponds and outdoor tubular photobioreactors is limited by the requirement for large spaces and the difficulty in maintaining sterile conditions. In view of this, we have designed and constructed an internally illuminated stirred tank photobioreactor. The photobioreactor is simple, heat sterilizable and mechanically agitated like the conventional stirred tank bioreactors. Furthermore, it can easily be scaled up while maintaining the light supply coefficient and thus the productivity constant. A device was installed for collecting solar light and distributing it inside the reactor through optical fibers. It was equipped with a light tracking sensor so that the lenses rotate with the position of the sun. This makes it possible to use solar light for photosynthetic cell cultivation in indoor photobioreactors. As a solution to the problems of night biomass loss and low productivity on cloudy days, an artificial light source was coupled with the solar light collecting device. A light intensity sensor monitors the solar light intensity and the artificial light is automatically switched on or off, depending on the solar light intensity. In this way, continuous light supply to the reactor is achieved by using solar light during sunny period, and artificial light at night and on cloudy days. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Photobioreactor; Internal illumination; Indoor culture; Solar light; Artificial light

1. Introduction Solar light energy is one of the most a b u n d a n t natural resources on earth and since artificial light sources are expensive, it is desirable to utilize

* Corresponding author. Fax: + 81-298-534605. E-mail address: [email protected] (J.C. Ogbonna)

solar energy for photosynthetic cell cultivation. Thus, most commercial cultivation of photosynthetic cells is carried out in open ponds, utilizing solar light energy. However, the productivities of these o u t d o o r open ponds are very low (Terry and R a y m o n d , 1985; Laws et al., 1986) due to m a n y problems including the difficulties of controlling the culture conditions, c o n t a m i n a t i o n problems, and requirement for large area of land. Therefore,

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)0008 1-4

290

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 289-297

there is a need to develop a compact and sterilizable photobioreactor which can be efficiently illuminated by solar light. In order to achieve this, we have investigated growth kinetics of photosynthetic cells (Ogbonna et al., 1995b) and developed an index for quantitative evaluation of light conditions inside photobioreactors (Ogbonna et al., 1995c). Based on these, a method for designing and scaling up of internally illuminated photobioreactor was proposed and a photobioreactor which can be internally illuminated by either artificial or solar light energy was constructed (Ogbonna et al., 1996; Ogbonna and Tanaka, 1997). Day-night cycles and diurnal variation in light intensity is a major problem with the use of solar light energy. Depending on the location and season, the number of hours per day when the light intensity is high enough to support photosynthetic cell growth can be very short. In the absence of light energy or some other metabolizable organic carbon source in the medium, cells metabolize the cell components to obtain maintenance energy, thus leading to a decrease in cell weight. Both the productivity and biochemical composition of the cells were affected by light/dark cycles during the cultivation of Chlorella pyrenoidosa (Ogbonna and Tanaka, 1996). Depending on the growth phase and cultivation conditions, the decrease in the total biomass concentration during the night may be as high as 17% (Ogbonna and Tanaka, 1996). Results of other investigations (Grobbelaar and Soeder, 1985; Torzillo et al., 1991a,b) have also shown that as high as 35% of the biomass produced during the day may be lost through respiration at night. Furthermore, prolonged bad weather (cloudy and/or rainy days) often lead to total process failure when only solar light energy is used for cultivation. As a solution to this problem, we have reported that cyclic autotrophic/heterotrophic cultivation (whereby controlled amount of organic carbon source is added during the night period), can be used not only to prevent night biomass loss but to achieve continuous cell growth even under light/dark cycles (Ogbonna and Tanaka, 1996, 1998). However, this method can only be used for cells which can

grow heterotrophically. Furthermore, during the dark heterotrophic growth phase, the cellular concentrations of the photosynthetic metabolites decrease and recover only during the subsequent autotrophic growth phase. Thus there is an optimum ratio of autotrophic to heterotrophic growth phase within a cycle and this method would not work well if the dark period is not followed by an appropriate period of good sunshine. Thus, it would not work well in cases of prolonged period of bad weather. In this work, we have developed a system for internal illumination of indoor stirred tank photobioreactor using solar light. In order to overcome the problems of diurnal variation in solar light intensity and prolonged periods of bad weather, an illumination system with integrated solar and artificial light sources was developed. Solar light is used for illumination during the day but when the solar light intensity decreases below a set value (during cloudy days and at night) the system switches automatically to artificial light source, thus ensuring continuous light supply to the reactor.

2. Materials and methods

2.1. Photobioreactor A schematic diagram showing the concept and construction of the photobioreactor used in this study is shown in Fig. 1. The illumination capacity of a single light source is determined for the desired cell and process, using the reactor shown in Fig. I(A). The cells are cultivated in vessels of various diameters and the optimum light path (OL) which can be efficiently illuminated by the single light source is determined. Such a reactor with a single light source at the center is defined as a one-unit photobioreactor. A large photobioreactor with the same light condition as the single unit can be constructed by increasing the number of units in three dimensions (Ogbonna et al., 1996, 1997b). The optimum light path depends on the cell and the nature of product. Using a 4-W fluorescent lamp (light intensity = 163 ~tmol

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 289-297

291

•

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Fig. 1. Schematic diagrams of the set-up used to determine the optimum unit size (A) and a 4-unit photobioreactor (B) used in this study. Detailed description of this photobioreactor is given in the text.

m - 2 S-1), the optimum light path for production of Chlorella biomass was determined to be 2.3 cm and a 4-unit photobioreactor was constructed (Fig. 1B). By keeping the distance between two light radiators equal to 2 x OL (4.6 cm), the light condition (both the light energy supplied per unit volume and the light distribution inside the reactor) in the 4-unit photobioreactor was the same as that in the single unit. The working volume of the 4-unit photobioreactor is 3.5 1. Since the light radiators are not mechanically fixed to the reactor, the reactor can be heat-sterilized and illuminated after cooling by inserting the light radiators into the glass tubes. A modified impeller was installed to achieve good mixing and high rate of mass transfer inside the reactor. The glass tubes which house the light radiators also serve as baffle plates in breaking the gas bubbles and thus improving mass transfer and light refraction inside the reactor. Since the light radiators have no direct contact with the culture broth, there is no problem of cell adhesion on the light radiators.

2.2. Solar light collection and transmission into the photobioreactor Fig. 2 shows the schematic diagram of the device used for collecting solar light and distributing it inside the reactor. It comprised of solar light collectors each with 12 fresnel lenses of 105 mm in diameter. The total illumination surface for each ~Sun~

Lightcollectiondevice

,,-~-~ nb, r~

Photobioreactor

Fig. 2. Schematic diagram of the solar light collection device. The collected light is transmitted to the photobioreactor through the optical fibers.

292

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 289-297

solar light collector was 864 cm 2. It was covered by a transparent acryl dome which protects the lenses from dust and rain. The solar light collected by each lens was transmitted through an optical fiber (1 mm in diameter). The light collection device was equipped with a light tracking sensor so that the lenses rotate with the position of the sun. Approximately 38% of the collected photosynthetic active radiation was transmitted into the reactor. The solar light collection device is commercially available (Laforet Engineering and Information Service, Tokyo, Japan) under a trade name of 'Himawari'. In order to use this solar light collector for internal illumination of tank-type photobioreactor, six optical fibers (from six fresnel lenses) were made into a bundle and connected to a light radiator. Thus, there were two light radiators per solar light collector and thus two solar light collectors for the 4-unit photobioreactor. The light radiators were cylindrically shaped and made of vertically etched transparent glass or quartz. The light intensity is almost uniform throughout the illumination surface of the light radiators.

2.3. Integrated solar and artificial light illumination system Schematic diagram of the integrated solar and artificial light illumination system is shown in Fig. 3. A metal halide lamp was used as the artificial light source. Parabolic mirrors were used to produce parallel rays from the lamps which were then filtered and transmitted through the optical fibers to the light radiators inside the photobioreactor as described for the solar light. The same light radiators were used for both the solar light and metal halide lamp. A light intensity sensor monitors the solar light intensity and the artificial light is automatically switched on or off, depending on the solar light intensity. In this way, light energy is supplied continuously to the culture.

2.4. Micro-organism and medium composition Chlorella sorokiniana IAM C-212 formerly known as C. pyrenoidosa (Kessler and Huss, 1992) was obtained from the algal collection of the

Light tracking sensor

Fresnel lenses

Light collection device Control box

Optical fibers

Metal lamp

Fig. 3. A schematic diagram of the integrated solar and artificial light for internal illumination of the stirred tank photobioreactor.

Institute of Applied Microbiology, University of Tokyo, Japan. The medium was composed of (g 1-i) KNO3, 1.25; KHzPO 4, 1.25; MgSO4" 7H20, 1.25; CaC12, 0.04; FeSO4" 7H20, 0.002; and A5 solution, 1.0 ml 1-1. The composition of the A5 solution was given previously (Ogbonna et al., 1995a). The pH of the medium was adjusted to 6.8 before autoclaving at 121~ for 15 min.

2.5. Cultivation method Cells were activated by pre-cultivating them in 100-ml Roux bottles under continuous illumination at 36~ for 24 h. White fluorescent lamps were used as the light source and the light intensity at the surface of the bottle was 200 lamol rn- 2 s - 1. Aeration and mixing were achieved by sparging air enriched with 5% CO2 through a glass-ball filter, which was inserted to the bottom of the Roux bottle, at 0.3 vvm. The seed culture broth was used to inoculate the main culture in the photobioreactor. The agitation speed, aeration rate (with 5% CO2 in air) and cultivation temperature were 120 rpm, 0.3 vvm and 36~ respectively. Cultivation was done either with only solar light illumination or with the integrated solar and artificial light system. The artificial light was automatically switched on

J.C. Ogbonna et al. /'Journal of Biotechnology 70 (1999) 289-297

whenever the solar light intensity on the surface of the light radiators decreases below 50 lamol m - 2 s - l and off when the solar light intensity increases above this value. Cyclic autotrophic-heterotrophic cultivation was carried out as described in our previous papers (Ogbonna and Tanaka, 1996, 1998), using ethanol as the organic carbon source at night.

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2.6. Analytical methods Cell dry weight (DW) determinations were made according to the method described in our previous papers (Ogbonna et al., 1997a). When the cell concentration was very low, it was estimated by measuring the optical density at 680 nm (Spectronic 20A, Shimadzu Scientific Instruments, Japan). In the latter case, the OD readings were converted to dry cell concentrations using predetermined calibration curves. The light intensities were measured by an analogue photometer (LI185B, Licor, Nebraska, USA).

3. Results 3.1. Wavelength distribution in the radiated light A comparison of the wavelength spectrum of the direct sunlight and that of the radiated light inside the photobioreactor is shown in Fig. 4. The distribution patterns are similar in the direct sunlight and the radiated light. However, most of the infrared ( ~ 90%) and ultraviolet ( ~ 99.1%) wavelengths were removed in the radiated light due to chromatic aberration in the fresnel lenses. Also, by using lenses to channel the metal halide light to the optical fiber, all the ultraviolet and a significant percentage of the infrared wavelengths were filtered out before transmission into the photobioreactor (Fig. 5).

20~ /:f i

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An example of a time course of C. sorokiniana growth and the average light intensity during the day, when only the solar light was used for culti-

i ~

1000 1500 Wavelength (nm)

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2000

~

2500

Fig. 4. Wavelength distribution of the direct solar light and the transmitted light on the surface of the light radiators.

vation, is shown in Fig. 6. In this example, the first two days of good weather was followed by a sunny morning but cloudy afternoon, two rainy days, a cloudy morning but sunny afternoon, another two rainy days and two days of good weather. During the sunny days, the average light intensity at the surface of the glass tubes housing the light radiators was ~ 450 gmol m - 2 S - 1. This decreased to ~ 200 gmol m - 2 s-~, when only a part of the day was sunny. On rainy/cloudy days, the solar light intensity could be as high as 400 gmol m -2 s - l , but the light intensity at the 100 80

._ 40

i

20

3.2. Cultivation of C. sorokiniana using only the solar light collection device

t

0

x,

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400

600 500 Wavelength (rim)

700

800

Fig. 5. Wave length spectrum of the metal halide lamp transmitted to the photobioreactor.

J.C. Ogbonna et al. :/Journal of Biotechnology 70 (1999) 289-297

294

500 ~

5

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o

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Fig. 6. Time course of C. sorokiniana growth using only solar light transmitted through the optical fibers.

surface of the light radiators was almost zero. This could be due to light diffusion/dispersion which reduces the efficiency of light collection by the solar light collectors during rainy/cloudy weather. The pattern of cell growth followed that of the solar light intensity. The average linear growth rate during the first two days of good weather was 0.3 g 1-~ d-~ which corresponds to CO2 fixation rate of 0.54 g - C O 2 1 - 1 d - 1 . There was no significant increase in the cell concentration during the following bad weather but when the good weather returned, the cell concentration increased rapidly with an average linear growth rate of 0.48 g 1-1 d-1 and C02 fixation rate of 0.864 g C02 1-1 d-1.

3.3. Cyclic autotrophic-heterotrophic cultivation We have already shown that cyclic autotrophic-heterotrophic cultivation can be used to achieve continuous cell growth under light/ dark cycles (Ogbonna and Tanaka, 1998). As shown in Fig. 7, when artificial light was used at a definite cycles of 10 h light and 14 h dark, very high cell concentration with high protein content (56%) was achieved by adding controlled amounts of ethanol at night. This method has also been shown to work well for ~-tocopherol production by Euglena gracilis (Ogbonna and Tanaka, 1998). However, it is applicable only to cells with heterotrophic metabolism. Furthermore, when this cyclic autotrophic-heterotrophic cultivation was done using only the solar light, the protein con-

150

50 100 Cultivation time (h)

Fig. 7. Time courses of C. sorokiniana cell growth during autotrophic cultivation under light/dark cycles (O) and during cyclic autotrophic-heterotrophic cultivation (<)). Ethanol was used as the organic carbon source for the heterotrophic phase.

tent of the cells decreased sharply when night heterotrophic culture was not followed by autotrophic culture under bright sunlight due to cloudy weather (data not shown). Thus, the integrated solar and artificial light illumination system was investigated.

3.4. Cultivation of C. sorokiniana with the integrated solar and artificial light illumination system The results of cultivation of C. sorokiniana, using the developed solar and artificial light illu1.5 "7

400

~'= J O

= 1.0 t.. ,t=t r

"~ 0.5 Q

o

r 0 0

. . . . . 50 100 150 200 Cultivation time (h)

0 250

Fig. 8. Time course of C. sorokinkma growth using the integrated solar and artificial light illumination system.

J.C. Ogbonna et al. /Journal of Biotechnology 70 (1999) 289-297

mination system are shown in Fig. 8. The diameter of the light radiators used for the integrated system is bigger than that used when only solar light was used. Thus even on sunny days, the light intensity per illumination surface area was lower than those shown in Fig. 6. Although there was only two and half days of sunny weather, continuous cell growth was achieved since the artificial light source was automatically switched on when the solar light intensity was below the set value of 50 ~tmol m -2 s -1. The average light intensity from the artificial light source was only 60 ~tmol m -2 s -1, thus the average linear growth rate during the cloudy days and at night (0.11 g ld - l ) was lower than those obtained on sunny days (0.3 g 1-1 d-1).

4. Discussion

In order to increase illumination surface to volume ratio, photobioreactors with optical fibers have been reported. Mori (1985) reported a system where solar light was collected by fresnel lenses, transmitted through optical fibers and dispersed by 91 light radiators inside a photobioreactor. A similar type of reactor was constructed where light from a metal halide lamp was transmitted and distributed inside a photobioreactor by 661 light diffusing optical fibers (Matsunaga et al. 1991). These represented novel developments in photobioreactor design but there were some shortcomings such as complexity of design, difficulty of heat sterilization, lack of mechanical agitation and cell adhesion on the fiber surfaces. Furthermore, there was no criteria for the number and spacing of the fibers inside the photobioreactor. In order to overcome these problems, we have designed a photobioreactor which is simple, heat sterilizable and mechanically agitated like the conventional stirred tank bioreactors. Thus, there are no problems of contamination, mass transfer limitation, or cell adhesion on the light radiators (Ogbonna et al., 1996; Ogbonna and Tanaka, 1997). Since the light radiators are not mechanically fixed to the reactor, the same reactor can be used for efficient heterotrophic, mixotrophic or

295

photoautotrophic cultivations. Furthermore, the reactor can easily be scaled up while maintaining the same light supply coefficient and thus the productivity (Ogbonna et al., 1995c, 1997b). The results shown in this paper have demonstrated again that by using the type of system describe here (Fig. 2), it is possible to use solar light for cultivation of photosynthetic cells in indoor photobioreactors (Mori, 1985; Hirata et al., 1996). However, only a maximum of ~ 38% of the collected photosynthetic active radiation is transmitted into the reactor. Although this represents a significant improvement and thus resulted in much higher growth rate than that reported by Hirata et al. (1996), there is still a need to improve the efficiency of the solar light collection and transmission. More than 50% of the collected light are lost at the connection between the lenses and the optical fibers, ~ 5% along the fiber and the rest at the connection between the optical fibers and the light radiators. Laforet Engineering and Information Service is now developing more efficient solar collectors by using better lenses and improving the connection between the lenses and the optical fibers. A major advantage of this system is that most of the infrared and ultraviolet wavelengths were filtered out of the collected light before transmission into the reactor. It is significant to note that by using parabolic mirrors to produce parallel rays from the metal halide lamps, and transmitting them through the optical fibers to the light radiators, the infrared and ultraviolet rays were also significantly reduced (Fig. 5). Apart from avoiding possible physiological effects of infrared and ultraviolet wavelengths on the cells, filtering of these wavelengths also significantly reduces the amount of heat energy transmitted into the photobioreactor. Even at a maximum sunlight intensity, the temperature at the surface of the light radiators was below 38~ Thus, there was no problem of temperature control even with the internally illuminated photobioreactor. As shown in Fig. 6, when only the solar light is used for cultivation, there is almost no cell growth on cloudy or rainy days. Thus, prolonged period of bad weather can even lead to total failure of a culture. With the integrated solar and artificial

296

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light illumination system described in this paper, we can make maximum use of solar light and use artificial light only when there is no solar light (at night and on cloudy days). The extent to which artificial light is used depends on the nature of the process. Controlled intensity of artificial light can even be used to supplement solar light during the day so that a constant light intensity is supplied to the reactor all the time. On the other hand, the use of artificial light can be reduced to minimum by keeping the critical light intensity for switching to the artificial light low or by using a time control switch whereby the artificial light is supplied only for a desired period of time. The intensity of the artificial light source used in this study was low, thus the productivity on cloudy days was lower than the value observed on sunny days. Thus for cells which require high light intensity, there is a need to use artificial light of higher light intensity. This is currently under investigation. Cost consideration is a major issue in this type of device. The solar light collector is almost maintenance free and the electricity consumption is only 1.7 W h - 1 but the capital cost is still high. Thus, the cost of algal cultivation in bioreactors with the solar light collection device is still high compared with open cultivation ponds or tubular photobioreactors utilizing solar light. However, the use of open systems has been successful mainly with those cells which are not very sensitive to contamination because of their unique growth characteristics such as high pH and high salinity. A lot of useful photosynthetic cells grow only under mild conditions and their growth rates are usually very low so that they are very sensitive to contamination by predators and fast growing heterotrophs. For such photosynthetic cells, cultivation under sterile condition is very necessary and the type of system described in this paper would be very useful. Also, in some areas, insolation is very high during the winter season but the productivity in open systems would be limited by the very low temperature. By using closed systems equipped with the solar light collection device, efficient cultivation of photosynthetic cells during the winter seasons would be possible in such areas. Another major advantage of this system is the small ground area required. In comparison

with the ponds and horizontal tubular reactors which require large ground area per unit volume, the tank type reactors can be constructed high and thus, the reactor volume per unit ground area can be more than one order of magnitude higher than the cultivation ponds. Moreover, since the solar light collectors are installed on top of roofs and the photobioreactors can be installed even under ground, only a very small ground area would be required. A tank type photobioreactor equipped with solar light collection device is therefore potentially viable in areas where land cost is very high. There is little we can do about the d a y - n i g h t cycles, diurnal changes in solar light intensity and weather fluctuations. Thus, in order to reduce damage due to prolonged bad weather and for processes that require continuous illumination, there is a need to combine solar and artificial lights. This paper has demonstrated that this is possible and it is hoped that with intensification of screening for speciality products from photosynthetic cells, such a system can be used for practical production of high value metabolites. Moreover, demand for the solar light collectors is increasing steadily, especially from housing sectors. It is expected that if this trend continues, the prices of these solar collectors will drop significantly.

References Grobbelaar, J.U., Soeder, C.J., 1985. Respiration losses in

green alga cultivated in raceway ponds. J. Plankton Res. 7, 497-506. Hirata, S., Hayashitani, M., Taya, M., Tone, S., 1996. Carbon dioxide fixation in batch culture of Chlorella sp. using photobioreactor with a sunlight collection device. J. Ferment. Bioeng. 81,470-472. Kessler, E., Huss, V.A.R., 1992. Comparative physiology and biochemistry, and taxonomic assignment of Chlorella (Chlorophyceae) strains of culture collection of the University of Texas at Austin. J. Psychol. 28, 550-553. Laws, E.A., Taguchi, S., Hirata, J., Pang, L., 1986. High algal production rates achieved in a shallow outdoor fume. Biotechnol. Bioeng. 28, 191- 197. Matsunaga, T., Takeyama, H., Suso, H., Oyama, N., Ariura, S., Takano, H., Hirano, M., Burgess, J.G., Sode, K., Nakamura, N., 1991. Glutamate production from CO2 by marine cyanobacterium Synechococcus sp. using a novel

J.C. Ogbonna et al./Journal of Biotechnology 70 (1999) 289-297 biosolar reactor employing light diffusing optical fibers. Appl. Biochem. Biotechnol. 28/29, 157-167. Mori, K., 1985. Photoautotrophic bioreactor using visible solar rays condensed by fresnel lenses and transmitted through optical fibers. Biotechnol. Bioeng. Symp. 15, 331345. Ogbonna, J.C., Yada, H., Tanaka, H., 1995a. Effect of cell movement by random mixing between the surface and bottom of photobioreactors on algal productivity. J. Ferment. Bioeng. 79, 152-157. Ogbonna, J.C., Yada, H., Tanaka, H., 1995b. Kinetic study on light-limited batch cultivation of photosynthetic cells. J. Ferment. Bioeng. 80, 259-264. Ogbonna, J.C., Yada, H., Tanaka, H., 1995c. Light supply coefficient--a new engineering parameter for photobioreactor design. J. Ferment. Bioeng. 80, 369-376. Ogbonna, J.C., Tanaka, H., 1996. Night biomass loss and changes in biochemical composition of cells during light/ dark cyclic culture of Chlorella pyrenoidosa. J. Ferment. Bioeng. 82, 558-564. Ogbonna, J.C., Yada, H., Masui, H., Tanaka, H., 1996. A novel internally illuminated stirred tank photobioreactor for large-scale cultivation of photosynthetic cells. J. Ferment. Bioeng. 82, 61-67.

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Ogbonna, J.C., Tanaka, H., 1997. Industrial-size photobioreactors. CHEMTECH 27, 43-49. Ogbonna, J.C., Masui, H., Tanaka, H., 1997a. Sequential heterotrophic/autotrophic cultivation--an efficient method of producing Chlorella biomass for health food and animal feed. J. Appl. Psychol. 9, 359-366. Ogbonna J.C., Soejima T., Tanaka, H., 1997b. Development of efficient large scale photobioreactors; a key factor for practical production of biohydrogen. Proceedings of the International Conference on Biohydrogen, Kona, Hawaii, June, 1997. Ogbonna, J.C., Tanaka, H., 1998. Cyclic autotrophic/heterotrophic cultivation of photosynthetic cells: a method of achieving continuous cell growth under light/dark cycles. Biores. Tech. 65, 65-72. Terry, K.L., Raymond, L.P., 1985. System design for the autotrophic production of microalgae. Enzyme Microb. Technol. 7, 474-487. Torzillo, G., Sacchi, A., Materassi, R., 1991a. Temperature as an important factor affecting productivity and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors. Biores. Technol. 38, 95-100. Torzillo, G., Sacchi, A., Materassi, R., Richmond, A., 1991b. Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors. J. Appl. Psychol. 3, 103-109.

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Journal of Biotechnology 70 (1999) 299-312

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Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors G. Chini Zittelli, F. Lavista, A. Bastianini, L. Rodolfi, M. Vincenzini, M.R. Tredici * Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche dell'Universith di Firenze and Centro di Studio dei Microrganismi Autotrofi del CNR, P.le delle Cascine, 27, 50144 Firenze, Italy

Received 3 November 1998; received in revised form 8 December 1998; accepted 22 December 1998

Abstract Autotrophic microalgae cultures have been proposed as an alternative source of EPA, a nutritionally important polyunsaturated fatty acid that plays a key role in the prevention and treatment of several human diseases and disorders. The technology currently available is however, considered commercially not viable because of the low degree of control of algae cultures in outdoor open ponds. The use of closed reactors could overcome these limitations and bring EPA production by microalgae closer to becoming a reality. In this study, we have demonstrated the feasibility of outdoor cultivation of Nannochloropsis sp. in tubular reactors and the potential of this eustigmatophyte as an alternative source of EPA. Nannochloropsis sp. was cultivated in NHTRs of different sizes (from 10.2 to 610 l) from spring to autumn under the climatic conditions of central Italy. EPA productivity essentially reflected the productivity of the culture and reached its maximum in M a y - J u n e (mean monthly value: 32 mg 1-1 day-~). Although the fatty acid composition of the biomass varied significantly during the cultivation period, EPA content remained rather stable around the value of 4% of dry biomass. The transfer of the cultures from laboratory to outdoor conditions, the exposure to natural light-dark cycles, along with lowering the salt concentration from 33 g l-~ (seawater salinity value) to 20 g l - l , factors that caused lasting modifications in the fatty acid content and composition of Nannochloropsis sp., did not significantly affect the EPA content of the biomass. 9 1999 Elsevier Science B.V. All fights reserved. Keywords: Eicosapentaenoic acid; Nannochloropsis sp.; Microalgae mass cultivation; Tubular photobioreactors

Abbreviations: AA, arachidonic acid or 20:4
I. Introduction P U F A s o f the o)-3 series play a key role in the prevention a n d t r e a t m e n t o f a wide range o f h u m a n diseases and disorders and have been recognized as i m p o r t a n t dietary c o m p o u n d s . E P A (20:5o)3), in particular, has been shown to have

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00082-6

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several highly beneficial effects such as preventing atherosclerosis and cardiovascular diseases, lowering plasma cholesterol and triacylglycerol levels, and alleviating inflammatory conditions such as arthritis (Nordoy, 1991; Gill and Valivety, 1997). Recently, the potential of EPA as an antitumor agent has been also documented (Senzaki et al., 1998). At present, the only commercial source of EPA is marine fish oil, a rather unsatisfactory source because of problems of contamination, taste, odor and stability. In addition, the presence of considerable amounts of other PUFAs in the fish oil complicates the EPA purification process, resulting in high retail prices of the pure product (Yongmanitchai and Ward, 1989; Barclay et al., 1994). These factors have led to investigation of alternative EPA sources (Gill and Valivety, 1997). Several microorganisms have long been recognized as potential EPA producers (Radwan, 1991). Fungi, especially of the order Mucorales, and bacteria of the genera Shewanella, Alteromonas, Flexibacter and Vibrio can accumulate relatively large amounts of EPA (Yongmanitchai and Ward, 1989; Gill and Valivety, 1997; Leman, 1997), and have been indicated as promising sources of this long chain fatty acid. However, the ability of bacterial and fungal fermentations to compete economically with traditional sources of co-3 fatty acids is limited by low productivities and excessively long fermentation times (Barclay et al., 1994; Leman, 1997). Marine microalgae, such as Porphyridium cruentum (Cohen et al., 1988), Phaeodactylum tricornutum (Chrismadha and Borowitzka, 1994; Molina Grima et al., 1994a), Isochrysis galbana (Molina Grima et al., 1994b) and the eustigmatophytes Nannochloropsis oculata, Nannochloropsis sp. and Monodus subterraneus (Seto et al., 1992; Hu et al., 1997; Sukenik 1998) have also been proposed for commercial production of EPA under autotrophic conditions. Algae-based technologies in outdoor ponds, however, are considered commercially not viable for EPA production because of low cell concentrations, low production rates and a reduced degree of control of growth parameters (Barclay et al., 1994). For

this reason, some companies, such as Martek Biosciences, MD, and Omega Tech, CO, have developed processes based on cultivation of heterotrophic microalgae for the commercial production of EPA and DHA (Barclay et al., 1994). Heterotrophic algae production in fermentors has several potential advantages over autotrophic production in open ponds, including a higher degree of process control, easier maintenance of monoalgal cultures, lower costs for harvesting due to the higher cell concentration obtained, and significantly greater volumetric productivities (Barclay et al., 1994). Nannochloropsis is a marine eustigmatophyte currently cultivated in many aquaculture hatcheries as the basis of an artificial food chain. In Japan, N. oculata is the most important cultured feed for the rotifer Brachionus plicatilis and concentrated suspensions and frozen biomass of this rnicroalga are commercially available (Okauchi, 1991). Because of its high EPA content this eustigmatophyte is considered a good potential EPA source (Sukenik, 1998). Although the alga is grown extensively, its mass production in outdoor open ponds suffers from sudden culture collapse due to predation by protozoa (e.g. Paraphysomonas sp.), contamination by bacteria (e.g. Cytophaga sp.) and competition of cyanobacteria and other microalgae (Okauchi, 1991; Sukenik et aI., 1993a; Sukenik, 1998). Chlorination and the use of antibiotics do not prevent these problems (Okauchi, 1991; Sukenik 1998). Recently, GonenZurgil et al. (1996) have suggested the use of the herbicide DCMU to maintain the cultures free of contaminating algae, since Nannochloropsis shows a high resistance to this compound. Although the addition of DCMU at concentrations higher than 10-7 M to the cultures significantly decreases the growth of undesired microalgae, it does not solve the problem of bacteria and protozoa contamination and presents obvious negative implications. These limitations of open ponds could be overcome by use of closed reactors (photobioreactors). In closed systems, protection against contaminating microorganisms is much more easily obtained, and much higher cell densities and more efficient control of principal culture

G. Chini Zittelli et al. /Journal of Biotechnology 70 (1999) 299-312

parameters can be achieved so as to ensure a sustainable cultivation process (Tredici and Chini Zittelli, 1998). The aims of the present study were to evaluate the feasibility of outdoor cultivation of Nannochloropsis sp. in tubular photobioreactors and to assess the potential of this microalga as an alternative source of EPA.

2. Materials and methods

2.1. Description of the photobioreactors Single-tube, three-tube and eight-tube NHTRs (Tredici and Chini Zittelli, 1998) installed at the experimental station of Scandicci (Florence; latitude 43 ~ 46' N, longitude 11~ 15' E) were used for the outdoor cultivation of Nannochloropsis sp. The reactors consisted of flexible plastic tubes (length: 6.4 m; diameter: 43 mm; wall thickness: 0.15 mm)connected by two PVC manifolds. The upper, larger manifold acted as a degasser. The reactors were laid on a platform facing south and tilted to form an angle of 5~ with the horizontal. The platform was covered with a white corrugated plastic sheeting, which kept the tubes of the reactor well-aligned and at the proper inclination. Mixing and degassing of the culture suspension were achieved by continuously injecting air into each tube by means of a perforated pipe inserted into the lower manifold. In the eight-tube reactors two tubes were not aerated in order to increase the circulation speed of the culture suspension and to obtain a better mass transfer. The total volume of each system (including degasser and connecting tubing) was 10.2, 36.6 and 97.9 1 for the single-tube, three-tube and eight-tube reactors, respectively. Electrodes and sensors were inserted into the degassers. In September and October 1998, two larger eight-tube NHTR units (lengths: 11 and 44 m; volumes: 155 and 610 1) were installed at Azienda Agricola Montepaldi (San Casciano Val di Pesa, Florence, Italy; Fig. 1). For both reactors, the inclination of the platform was 7~ For the laboratory experiments a 1.5-m 2 alveolar panel was used (Tredici et al. 1991).

301

2.2. Organism and culture conditions The marine eustigmatophyte Nannochloropsis sp. was obtained from Dr Zmora of the National Center for Mariculture, Israel Oceanographic and Limnological Research (Eilat, Israel). Cultures were grown in artificial seawater (Tropic Marine Salt, Euraquarium, Bologna, Italy), enriched with f/2 medium nutrients (Ouillard and Ryther, 1962) at either 33 or 20 g 1-~ salinity. N a N O 3 and N a H 2 P O 4 w e r e added regularly to the cultures in order to prevent nutrient limitation. For laboratory experiments artificial seawater was autoclaved at 120~ for 20 min, whereas for outdoor experiments it was filtered through 10- and 1.5gm polypropylene filters (Domnick Hunter, UK). Sterile nutrient solutions were then added. The air injected into the photobioreactors was filtered through 1-~tm Polycap HD encapsulated filters (Arbor Tech, USA). Air-flow rate was maintained at 0.17 1 1-1 m i n - l , and gas hold-up was about

Fig. 1. Pilot eight-tube NHTR units installed at Azienda Agricola Montepaldi (San Casciano Val di Pesa, Florence, Italy).

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G. Chini Zittelli et al. Journal of Biotechnology 70 (1999) 299-312

5.5% in all the NHTRs but the pilot eight-tube units. In these reactors air-flow rate was maintained at 0.02 1 1-~ m i n - l , and gas hold-up was about 4%. In the alveolar panel air-flow rate was 0.4 1 1-1 m i n - l , and gas hold-up was about 2%. A control unit including a thermostat provided temperature regulation of the outdoor units by automatically activating water spraying onto the reactors, when the culture temperature exceeded the preset value of 25~ (optimum growth temperature of Nannochloropsis sp.). This cooling system was inadequate to maintain the culture temperature at the optimum value during the hours of strongest irradiation, particularly during the summer. During the night, the culture temperature was allowed to equilibrate to ambient. Under laboratory conditions, cultures were kept at a constant temperature of 25 + 0.2~ by circulating thermostating fluid from a water bath through a stainless-steel tube placed inside the reactor. Continuous illumination (100 ~tmol photons m - 2 s - ~) was provided on the reactor surface by banks of 58-W daylight fluorescent tubes. For both outdoor and laboratory experiments, culture pH was maintained at 7.8 + 0.2 by addition of pure CO_, to the air stream using a pH-stat system. A semicontinuous daily harvesting regimen was adopted in order to maintain a mean cell concentration of about 3.5 and 5.0 g 1-1 in the laboratory and outdoor cultures, respectively. The experiments started when quasi-steady state conditions were achieved. The biomass produced was harvested by centrifugation at 8000 x g using a Westfalia separator (model TA05-00-105, Westfalia Separator AG, Olde, Germany) and lyophilized for biochemical analysis.

measured with a pyranometric sensor (Kipp and Zonen, model CM 6, Delft, Holland) coupled with a Micros solarimeter (Model IS, Treviso, Italy). The photosynthetic photon flux density (PPFD) was measured using a Li-Cor quantum sensor (Model LI-190 SB, Li-Cor, Lincoln, NE) connected to a quantum/radiometer/photometer (Model LI-185 B). Fatty acid analyses of outdoor Nannochloropsis sp. cultures were made on samples collected at the end of the dark period, except that when otherwise stated. For fatty acid determination cells were harvested by centrifugation. Aliquots of the paste obtained (75% moisture) were lyophilized and the dried biomass (3% moisture) immediately analyzed. Fatty acids were extracted and methylated according to Bousfield et al. (1983). The methyl esters were analyzed with a gas chromatograph (GC 8380 Fisons Instruments) equipped with a hydrogen flame ionization detector and a SP 2380 column (30 m x 0.20 gm film thickness). Injections were made in split mode (split ratio 1:70, sample size 1.0 gl). Injector and detector were maintained at 250~ the oven temperature was programmed from 90 to 220~ at a rate of 3~ min-~. Carrier gas was helium at 1.5 ml min-~. Compounds were identified by comparing retention times of known standards. Quantification was based on the internal standard method.

3. Results

3.1. Transfer of Nannochloropsis sp. cultures from laboratory to outdoor conditions and its influence on fatty acid profile and content

2.3. Analytical procedures The productivity of the cultures was estimated by daily measurement of the dry biomass concentration. For dry weight determination, triplicate culture samples (2- 5 ml) were diluted (1:10) with distilled water and filtered through pre-weighed 1.2-gm membrane filters (Sartorius, Goettingen, Germany). Filtered cells were then quickly washed with 25 ml of distilled water and dried to a constant weight at 105~ Solar irradiance was

Laboratory Nannochloropsis sp. cultures were grown in a 1.5-m 2 alveolar panel (Tredici et al., 1991) under continuous illumination (100 rtmol photons m - 2 S - l ) and at the optimal growth temperature of 25 _+ 0.2~ A mean dry biomass productivity of 0.84 _+ 0.16 g 1-1 24 h - l and a mean EPA productivity of 36 + 4 mg 1-1 24 h-1 were attained. The total fatty acid content of the biomass was 13 + 0.4%, whereas the EPA content was 4.3 + 0.2%. At the end of August 1996, out-

G. Chini Zittelli et al. //Journal of Biotechnology 70 (1999) 299-312

door cultures were set up in single-tube NHTR systems (1NHTRs) using the laboratory culture as inoculum. The outdoor cultures, which were operated throughout September, achieved a mean productivity of 0.73 + 0.37 g 1-~ day-1. The transfer from laboratory to natural growth conditions caused significant and lasting modifications in the fatty acid composition of the biomass (Table 1). During the first 2 weeks of outdoor cultivation, the total fatty acid content of the biomass increased from 13%, typical of laboratory cultures, to 21%. This increase was essentially due to the increase of three fatty acids" 16:0 (from 3.2 to 6.7%), 16:lo)7 (from 3.1 to 5.4%) and 18" 16o9 (from 0.3 to 2%). In the second half of September the opposite trend was observed: the content of these three fatty acids, as well as that of total fatty acids, decreased steadily to the values typical of laboratory conditions. Among the PUFAs, 20:40)6 followed the general trend, first increasing from 0.7 to 1.2%, then decreasing to 0.8%, while EPA showed the opposite behavior although its content did not vary significantly from an average value of 4%. The considerable increase of saturated and monounsaturated fatty acids observed during the first 2 weeks of outdoor cultivation can be ascribed to the sudden exposure of laboratory cultures (cultures kept at low irradiance and optimal temperature) to high solar irradiances during the day and to low temperatures during the night and early morning (Fig. 2B,C). The opposite trend observed during the second half of the month can be related both to acclimation of the culture to outdoor conditions, and to lower solar irradiances and higher minimum temperatures during this period (irradiance decreased from about 20 to about 5 MJ m - 2 day-1, whereas the minimum culture temperature increased from 2 to 5 to 10~ It is worth noting that the highest total fatty acid content (about 21%) was reached between 9 and 11 September, i.e. after a period of 4-5 consecutive days characterized by relatively high diurnal irradiances (17-19 MJ m -2 day -~) and low night temperatures (2-5~ It is difficult to establish the separate influence of each of these two environmental factors, since in autumn both high diurnal irradiances and low night temperatures occur under clear sky conditions. The EPA

303

AI60

~ '~

2

40

~ ~"

20

~

9 0

_,--~ ,~ ~, .~_,v,

30 2oI

lo 0

1

,

1

,

i

,

i

,

l

,

i

,

1

i1

30 ~.o

zo 10 0

1

5

9

13

17

21

25

Day ofthe month Fig. 2. Biomass (It) and EPA ( 9 productivity of Nannochloropsis sp. cultures grown outdoors in 1NHTRs in September 1996 (A). The total daily solar irradiance (B) and the minimum ( Z ) and maximum (11) temperatures (C) of the cultures are also shown.

content of the biomass was not significantly affected by environmental conditions, although an initial decrease and a subsequent increase are evident. Consequently, the productivity of EPA depended essentially on the productivity of the culture, reaching a peak of 54 mg 1-1 day-1 after 1 week of cultivation and declining to about 20 mg 1-~ day-~ towards the end of the month (Fig. 2A).

3.2. Influence of the natural light-dark cycle on the fatty acid composition of outdoor Nannochloropsis sp. cultures Table 2 shows the variation of the fatty acid composition of a Nannochloropsis sp. culture grown outdoors in a 1NHTR unit during a period of 7 consecutive days in the second half of September 1996. Besides the decrease of saturated and monounsaturated fatty acid content that characterized this period (described in the previous paragraph) we could observe the influence of the natural light-dark cycle on the fatty acid composition of the biomass. The content of saturated and monounsaturated fatty acids decreased

W

0 P

Table 1 Variation of fatty acid content and profile ('YO of dry weight) of Nunnochloropsis sp. cultures after transfer from laboratory to outdoor conditions during August-September 1996" Fatty acid

2

e

Day of the month 28 August

31 August

3 Sept.

6 Sept.

9 Sept.

14:O

169 16:lw7 I 8: 1(1)9 18:206 20:4w6 20:503

TFA

0.72 3.16 3.09 0.33 0.61 0.65 4.29 13.0

0.79 3.52 3.27 0.53 0.38 0.99 3.96 13.7

0.77 3.79 3.52 0.63 0.43

1.16 4.26 14.8

I I Sept.

14 Sept.

17 Sept.

~~

~~

0.90 4.97 3.96 0.82 0.36 I .03 3.96 16.3

1.13 6.74 5.10 I .Y3 0.49 1.13 3.59 20.5

20 Sept.

23 Sept.

I .os 6.70 5.40 I .Y9 0.50 1.16 3.84 2 I .o

0.83 5.56 4.70 1.46 0.38 I .03 3.73 18.0

0.76 4.49 4.43 I .OY 0.36 0.91 3.82 16.1

25 Sept -~

~~~

0.63 3.45 3.82 0.76 0.32 0.89 3.85 13.9

0.49 2.72 3.29 0.66 0.32 0.83 4.20 12.7

0.51 2.79 3.30 0.54 0.32 0.83 4.3 I 12.8

Only the major fatty acids are shown ( >0.3'%1).The values of August 28 are those of the laboratory culture used as inoculum. The analyses were done at the end of the light period.

t,l

s

Table 2 Variation of fatty acid content and profile ( X , of dry weight) of Nunnochloropsis sp. cultures grown under natural light-dark cycle conditions in September 1996" Fatty acid

Day of the month

19 September ~

14:O 16:O 16:lw7 18:lw9 I8:2w6 20:406 20:503 TFA

20 September _____

21 September -~

22 September

23 September

~-

24 September

25 September _ _ ~ _ _

-~

L

D

L

D

L

D

L

D

L

D

L

D

L

D

0.71 3.71 4.20 0.97 0.36 0.87 3.48 14.5

0.63 2.94 3.82 0.94 0.44 0.88 3.82 13.6

0.63 3.45 3.82 0.76 0.32 0.89 3.85 13.9

0.57 3.02 3.67 0.77 0.42 0.81 4.09 13.5

0.59 2.98 3.73 0.80 0.34 0.85 4.05 13.5

0.57 2.50 3.53 0.70 0.41 0.79 4.09 12.7

0.58 2.78 3.52 0.64 0.33 0.78 4.02 12.6

0.47 2.24 3.13 0.61 0.39 0.80 4.32 12.1

0.49 2.72 3.29 0.66 0.32 0.83 4.20 12.7

0.47 2.35 3.15 0.61 0.41 0.81 4.54 12.5

0.56 3.43 3.43 0.60 0.31 0.86 4.20 13.6

0.50 2.80 3.29 0.47 0.38 0.74 4.35 12.8

0.51 2.79 3.30 0.54 0.32 0.83 4.31 I12.8

0.52 2.43 3.32 0.49 0.41 0.83 4.73 12.9

"Only the major fatty acids are shown (>0.3'%)).The analyses were done at the end of the light period (L) and at the end of the dark period ( D ) v) h,

W 0 vl

G. Chini Zittelli et al./Journal of Biotechnology 70 (1999) 299-312

306

significantly during the night (on average, 14:0 by 8.3%, 16:0 by more than 16%, 16:1037 by 5.4% and 18:1039 by 8%). A m o n g the P U F A s , 20:4036 did not change significantly, while 18:2036 and EPA increased by more than 24% and by 7%, respectively. This increase of both EPA and 18:2036 was relative, since it was due to the night consumption of storage material (carbohydrates, and saturated and m o n o u n s a t u r a t e d fatty acids) and not due to new synthesis (data not shown).

3.3. Prolonged outdoor cultivation of Nannochlorops& sp. & N H T R reactors under the climatic conditions of central Italy: biomass and EPA productivity Nannochloropsis sp. was grown outdoors in different types of N H T R systems uninterruptedly from March to September 1997 under the climatic conditions of central Italy (Florence). F r o m March to June and in September, 1 N H T R units were used; during the summer three-tube (3 N H T R s ) and eight-tube reactors (8 N H T R s ) were used. Table 3 shows the mean monthly productivities attained, together with the mean minimum and m a x i m u m culture temperatures, and the mean solar irradiance values recorded. The

highest volumetric productivities (0.7-0.8 g 1-1 d a y - 1 ) were obtained in late spring. The slightly lower productivities (0.5-0.6 g 1-1 d a y - l ) obtained in M a r c h - A p r i l might be due either to the lower solar irradiance available to the cultures (13.5 MJ m - 2 d a y - 1 in March and 15.3 MJ m - z d a y - ~ in April) compared to the M a y - J u n e period (17-19 MJ m - 2 d a y - 1) or to the excessively low temperatures during the night and the early morning (mean minimum temperatures of 2 - 3 ~ were recorded in the cultures during the spring). Since a mean monthly productivity of 0.73 g 1-1 d a y - ~ had been obtained in the previous September, with a mean minimum temperature of 10~ and at irradiance levels lower than 13 MJ m -2 d a y - 1 , it is likely that the lower performance of early spring cultures was due to the low night temperatures. Similar productivities obtained in May 1997 with a mean irradiance of 19 MJ m - 2 d a y - 1 , and in September 1996 with only 13 MJ m - 2 d a y - 1 , also indicate that solar irradiances higher than 13 MJ m -2 d a y - 1 do not significantly improve the productivity of Nannochloropsis sp. cultures under the conditions adopted. In the summer the productivity of cultures in the 3 N H T R and 8 N H T R units declined regularly to a minimum of 0.45 g 1-~ d a y - 1 . The

Table 3 Productivity of Nannochloropsis sp. cultures grown outdoors in NHTRs from March to September 1997 under the climatic conditions of central Italy (Florence)a Month

March April May June July

Reactor type

Mean volumetric productivity (g 1-1 day-l)

1 NHTR 0.51 + 0.36 1 NHTR 0.60 + 0.31 1 NHTR 0.76 + 0.39 1 NHTR 0.69 + 0.30 3 NHTR 0.56 + 0.30 3 NHTR 0.60 + 0.23 8 NHTR 0.56 + 0.26 3 NHTR 0.45 + 0.17 3 NHTR shaded 0.41 + 0.24 1 NHTR 0.57 + 0.20 w

August September

September 1996 1 NHTR

0.73 + 0.37

Culturetemperature (~ Min.

Max.

2.0 + 3.3 3.0 + 4.0 10.6 + 3.8 15.3 + 2.2 14.5 + 2.5 13.9 + 3.0 13.6 + 3.0 14.2 + 2.0 15.3 + 2.0 10.5 + 3.6

26.2 + 0.6 25.2 + 3.8 28.1 + 1.3 28.9 + 1.6 29.7 + 1.5 28.3 + 1.1 28.9 + 1.1 28.3 + 0.5 27.6 + 1.0 27.3 + 1.3

10.2 + 4.2

28.8 + 1.4

_

Solar irradiance (MJ m -2 day- 1)

13.5+ 4.1 15.3+ 6.1 18.9+ 4.4 17.4+ 5.7 21.0+ 2.5

m

17.6+ 3.3 15.9+ 3.2 12.7+ 5.1

The minimum and the maximum culture temperatures and the solar irradiance are also reported. Data of September 1996 is shown for comparison. Mean monthly values + S.D. are shown. a

307

G. Chini Zittelli et al./Journal of Biotechnology 70 (1999) 299-312

Fig. 3. A cluster of Nannochloropsis sp. cells. The exuded polysaccharide matrix was stained with alcian-blue (Crayton, 1982). Bar: 10 lam. Photomicrograph by C. Sili.

reason for this is not yet clear. Signs of stress, such as abundant exudation of polysaccharide and presence of numerous and very large cell clusters (Fig. 3), possibly due to high light intensity or to prolonged exposure to higher than optimal diurnal temperatures, were observed in summer cultures. An experiment carried out to compare shaded versus unshaded cultures did not give a clear response. The shaded cultures showed a better appearance (lower degree of aggregation and higher pigment content), but attained a lower productivity. In September, the productivity of Nannochloropsis sp. rose again to about 0.6 g 1-l

d a y - 1 . The fact that the productivity values of the previous September were not reached despite similar climatic conditions, suggests that the cultures had not yet fully recovered from the stress of the summer. The mean fatty acid content of the biomass and the mean monthly productivity of EPA for the entire cultivation season ( M a r c h - S e p t e m b e r 1997) are shown in Table 4. The culture used as inoculum, which had been grown in the laboratory as previously described, had a total fatty acid content of 12% and an EPA content of 3.6%. As observed in the previous autumn, the transfer from laboratory to outdoor conditions caused a significant increase of the total fatty acid content with a maximum of 26% in mid-April. The increase mainly involved 16:0, 16" 1037 and 18:1039. EPA content, except for a peak of 4.6% in June, was rather stable around 4% throughout the cultivation period. Exposure to low temperatures during the night and in the early morning (Table 3), after growth at optimal temperature in the laboratory, can again be considered the main cause of the phenomenon. In May, when minimum temperatures rose above 10~ the fatty acid profile and content showed values similar to those observed in the laboratory. EPA productivity essentially reflected culture productivity. A maximum of 32 mg 1-~ d a y - 1 and a minimum of 18 mg 1-1 d a y - 1 were achieved in M a y - J u n e and August, respectively.

Table 4 Fatty acid composition (% of dry weight) and EPA productivity (mg 1-~ day-~) of Nannochloropsis sp. cultures grown outdoors in NHTRs from March to September 1997 under the climatic conditions of central Italy (Florence)a

Fatty acid 14:0 16:0 16:1co7 18:lco9 18:2co6 20:4co6 20:5co3 TFA EPA productivity

March

April

May

June

July

August

September

0.75 3.53 3.97 1.31 0.58 0.86 3.90 15.3 19.9

1.06 7.46 8.05 3.38 0.52 0.85 3.95 25.8 23.8

0.91 5.10 5.31 1.29 0.38 0.60 4.22 18.2 31.5

0.82 4.71 5.01 1.22 0.32 0.68 4.64 17.9 32.0

1.03 4.12 4.32 0.61 0.70 1.11 4.18 16.6 23.4

0.90 3.83 3.44 0.54 0.56 0.90 3.96 14.5 17.8

1.20 4.63 4.63 0.70 0.74 0.89 3.98 17.1 22.0

a Only the major fatty acids are shown (>0.3%).

308

G. Chini Zittelli et al./Journal of Biotechnology 70 (1999) 299-312

In September and October 1998, Nannochloropsis sp. was grown outdoors in pilot 8 NHTR units (Fig. 1). The mean productivity was 0.4 g 1d a y - ~ and the EPA content of the biomass was 4%, with a mean EPA productivity of 16 mg 1d a y - 1. This experiment is still in progress. Contamination by protozoa or other microalgae was not a major problem in our NHTRs once efficient filtration systems for both the air and for the culture medium were adopted. Several bacterial species were observed in Nannochloropsis sp. cultures, but in very low concentrations. Moreover, the composition of the bacterial population associated with Nannochloropsis sp. cultures was rather stable and did not change significantly even after transfer from laboratory to outdoor conditions (Pastorelli et al., 1998). A noticeable exception occurred in the summer when, along with the abundant exudation of polysaccharidic material and massive formation of cell clusters, the bacterial load of the cultures increased by two orders of magnitude (data not shown).

3.4. Effect of salinity on productivity and EPA content of Nannochloropsis sp. cultures To evaluate the effect of a salinity value lower than that of seawater on culture productivity and fatty acid composition, Nannochloropsis sp. was grown outdoors in two 1 NHTR units at salt concentrations of 20 and 33 g 1-1. Neither the productivity (the mean monthly productivities were 0.69 __+0.4 at 20 g 1-1 and 0.71 + 0.4 g 1- 1 d a y - t at 33 g 1- ~ salinity), nor the total fatty acid content of the biomass (about 16.8% at both salt concentrations) was influenced by salinity in the range tested, although the fatty acid profile was modified (Table 5). At a salinity of 20 g 1-1 saturated fatty acids decreased by about 4% and monounsaturated fatty acids by about 23%, while PUFAs increased by more than 13%. EPA content rose from 4.3 to 4.8% of the dry biomass. Since the productivity of the culture was the same at both salinity values, EPA productivity was slightly higher at 20 g 1-1 (33.1 mg 1-1 day-1) than at 33 g 1-~ (30.5 mg 1- 1 day-1).

Table 5 Influence of salinity on fatty acid content and profile (% of dry weight) of Nannochloropsis sp. cultures grown outdoors in I NHTR units in May 1997a Fatty acid

14:0 16:0 16:1037 18:1o39 18:2036 20:4036 20:5033 TFA a

Salinity (g 1-l) 20

33

0.81 4.26 4.42 0.80 0.47 0.84 4.77 16.70

0.81 4.45 4.82 1.15 0.37 0.65 4.32 16.90

Only the major fatty acids are shown (>0.3%).

4. Discussion

Due to its high EPA content, Nannochloropsis has been proposed as a potential source of EPA for human consumption (Seto et al., 1984; Sukenik, 1991, 1998). Although in Japan Nannochloropsis is currently cultivated year-round in large outdoor tanks to provide a food chain for the production of fish larvae (Okauchi, 1991), data from outdoor mass cultivation of these microalgae is scanty. Boussiba et al. (1987) cultivated Nannochloropsis salina outdoors in 1- and 2.5-m 2 raceway-type ponds; prevention of contamination by diatoms and of predation by ciliates appeared to be the crucial factors for the successful operation of the cultures. Outdoor cultivation of Nannochloropsis sp. in raceway ponds with surface area ranging from a few square meters to 3000 m 2 was carried out at the National Center for Mariculture, Israel Oceanographic and Limnological Research, and at the Nature Beta Technology algae production plant, both located in Eilat, Israel (Sukenik et al. 1993a; Sukenik, 1998). The work done by Sukenik and co-workers at Eilat facilities has elucidated some fundamental aspects of the mass cultivation of this microalga and has confirmed the difficulty of maintaining monoalgal cultures outdoors for prolonged periods (Sukenik, 1998). The present work shows that photobioreactors have the potential to overcome the main limita-

G. Chini Zittelli et al./Journal of Biotechnology 70 (1999) 299-312

tions encountered in open ponds and may allow long lasting outdoor cultivation of this eustigmatophyte achieving a relatively high EPA productivity compared with other autotrophic systems. In fact, contamination by bacteria, protozoa or other microalgae has not been a major problem in the N H T R units used in our experiments. Cultivation of microalgae in photobioreactors is hampered by some problems, including overheating, fouling and accumulation of oxygen to toxic levels (Tredici and Materassi, 1992). Temperatures of up to 5~ over the optimal value (25~ were measured in our Nannochloropsis cultures during the hours of strongest irradiation, particularly in the summer. Prolonged exposure to supraoptimal diurnal temperatures, together with high irradiances at midday, were considered the main cause of the low performance of the cultures in July and August. Fouling and accumulation of oxygen to toxic levels have not been a particularly serious problem in the N H T R design. It must be pointed out, however, that during the summer, when maximum productivities were expected, the cultures performed poorly. Hence, it was not possible to demonstrate the ability of NHTR systems in avoiding build-up of toxic oxygen tensions during periods of high production rates for Nannochloropsis cultures. EPA productivity by Nannochloropsis sp. grown in 16 mm thick alveolar panels under laboratory conditions averaged 36 mg 1-1 24 h-1. Outdoors, in the NHTRs, mean monthly EPA productivity varied between 18 and 32 mg l-1 day-1 essentially following the trend of biomass productivity which in turn was dependent upon the season. This data is comparable to that obtained with the diatom P. tricornutum (Molina Grima et al., 1994a) and the freshwater eustigrnatophyte M. subterraneus (Hu et al., 1997). These two microalgae are considered among the most promising EPA producers under autotrophic conditions and are presently under investigation as part of the EC Brite-Euram project 'An integrated production system of highly purified eicosapentaenoic acid from microalgae' coordinated by Professor Molina Grima. The major fatty acids in Nannochloropsis are 14:0, 16:0, 16:1037 and EPA, whereas C18 fatty

309

acids and 20:4o36 are present in lower quantities (Teshima et al., 1983; Sukenik et al., 1989; Volkman et al., 1993). Fractionation of lipid extracts from Nannochloropsis sp. indicated that EPA is mainly associated with galactolipids (the major lipid constituents of the thylakoid membranes) and phosphatidyl glycerol, whereas the storage lipid triacylglycerol contains primarily 14:0, 16:0 and 16:1o37 (Sukenik et al., 1989, 1993b). The effect of environmental factors on lipid content and fatty acid composition has been investigated in various species of Nannochloropsis with the majority of the work conducted under laboratory conditions (Teshima et al., 1983; Seto et al., 1984; Sukenik et al., 1989, 1993a,b; Sukenik and Carmeli, 1990; Renaud et al., 1991; Sukenik, 1991). These studies have shown that light intensity and temperature, as well as nitrogen starvation, play a key role in regulation of the relative abundance of fatty acids and of the absolute quantity of EPA. The effect of temperature on EPA biosynthesis appears to be complex and has not yet been clarified. Seto et al. (1992) found that cells grown at 20~ contained 60% more EPA than cells grown at the optimal growth temperature of 25~ Similarly, Sukenik et al. (1993b) showed that cultures grown at a low temperature (20~ were characterized by a high rate of galactolipid synthesis and hence by a relatively high level of EPA, as compared with cells grown at a high temperature (30~ In contrast with these authors, Teshima et al. (1983) found maximal EPA synthesis at the optimal growth temperature of 25~ Although the effect of temperature is not easy to establish in outdoor cultures, where both temperature and irradiance vary during the day, the present work would suggest that minimum night temperatures play a key role in influencing the fatty acid level and composition. The main outcome of our work, in partial disagreement with conclusions drawn from most other studies, is that even when the fatty acid profile is deeply modified in response to an environmental stimulus (in our case temperature), the total content of EPA in the biomass is not significantly affected. Sukenik et al. (1989, 1993b) studied the influence of light intensity on lipid content and com-

310

G. Chini Zittelli et al. , Journal of Biotechnology 70 (1999) 299-312

position in Nannochloropsis sp. grown in the laboratory. Under light-limiting growth conditions, the cells were characterized by a high density of thylakoids with most of the lipid carbon allocated in the EPA-rich galactolipids. In contrast, cells grown under saturating light conditions accumulated carbohydrate and triacylglycerols as storage materials, resulting in a high content of 16:0 and 16:1037 and a reduction in the cellular EPA content of more than 50%. Similarly, Renaud et al. (1991) showed increasing saturation of the fatty acids of N. oculata with increasing irradiance using large-scale outdoor cultures. In the same organism, Seto et al. (1992) found no effect of high irradiances on EPA distribution and content. In our experiments, light intensity seems to play a minor role in fatty acid composition with respect to temperature. However, high irradiances might have been involved in causing excessive exudation and cell aggregation in Nannochloropsis sp. cultures. Sukenik and Carmeli (1990) investigated the synthesis of fatty acids in Nannochloropsis sp. grown in the laboratory under a 12:12-h lightdark cycle. These authors showed that storage triacylglycerol lipids are synthesized and accumulated during the light period and are rapidly consumed for cellular maintenance along with carbohydrates during the dark period. Galactolipids are also synthesized during the light period, but not turned over in the dark. Thus, the dark period is characterized by an increase of the relative proportion of EPA which is associated with the galactolipids. Our experiments confirm that Nannochloropsis sp. also uses fatty acids associated with storage lipid (mainly 14:0 and 16:0) during the dark period when cultivated under natural light-dark cycle conditions. Thus, as suggested by Sukenik and Carmeli (1990), the early morning is the preferred time for harvesting Nannochloropsis sp. biomass to obtain a high EPA content. Teshima et al. (1983) found no significant variation of the EPA content of N. oculata biomass with salinities between 4 and 30 g 1-1. Similarly, Renaud and Parry (1994) found that EPA content did not vary significantly with salinity in the range of 10-35 g 1-~ although

salinities from 20 to 25 g 1-~ were optimal for growth and production of EPA. Our results show that lowering salinity from 33 to 20 g 1slightly stimulates EPA accumulation and productivity. The effect of seasonal variations in temperature and light availability on fatty acid distribution was studied in outdoor mass cultures of Nannochloropsis sp. by Sukenik et al. (1993a). The highest cellular EPA content (3.8%) was observed during winter with water temperatures between 8 and 16~ and under low irradiances, i.e. in conditions of very low biomass productivity. In summer, when biomass productivity was highest, EPA content decreased to less than half the winter value. Maximum EPA productivity of our cultures was attained in late spring and early autumn, i.e. under relatively low irradiances and when prolonged exposure at supraoptimal diurnal temperatures could be prevented. The primary goals of algal biotechnology for production of EPA are high EPA productivity and high EPA content of the biomass. Most previous studies conducted with Nannochloropsis have shown that maximum EPA content is obtained under low light conditions and at temperature values slightly lower than the optimal temperature for growth, and have concluded that the conditions required to maximize EPA productivity are different from those needed to maximize EPA content (Sukenik, 1991). Our work has led us to a different conclusion: since the main environmental factors, such as temperature and irradiance, do not significantly influence the EPA content of the biomass in our photobioreactors, conditions for maximum EPA productivity are those required to maximize biomass production. Moreover, for establishing a reliable system for EPA production based on cultivation of Nannochlorops& in photobioreactors, high irradiances are not ideal or can even be detrimental, as is prolonged exposure to supraoptimal temperatures that cause a serious imbalance of algal growth. The potential of Nannochloropsis cultivation in closed systems would therefore be best realized in temperate countries.

G. Chini Zittelli et al../Journal of Biotechnology 70 (1999) 299-312

Acknowledgements We are deeply indebted to Drs Susan Blackburn and Laura Barsanti for critically reviewing the manuscript. This work was partly supported by EXENIA S.r.1. (Milano, Italy).

References Barclay, W.R., Meager, K.M., Abril, J.R., 1994. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. Appl. Phycol. 6, 123-129. Bousfield, I.J., Smith, G.L., Dando, T.R., Hobbs, G., 1983. Numerical analysis of total fatty acid profiles in the identification of coryneform, nocardioform and some other bacteria. J. Gen. Microbiol. 129, 375-394. Boussiba, S., Vonshak, A., Cohen, Z., Avissar, Y., Richmond, A., 1987. Lipid and biomass production by the halotolerant microalga Nannochloropsis salina. Biomass 12, 37-47. Chrismadha, T., Borowitzka, M.A., 1994. Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular photobioreactor. J. Appl. Phycol. 6, 67-74. Cohen, Z., Vonshak, A., Richmond, A., 1988. Effect of environmental conditions on fatty acid composition of the red alga Porphyridium cruentum: correlation to growth rate. J. Phycol. 24, 328-332. Crayton, M.A., 1982. A comparative cytochemical study of Volvocacean matrix polysaccharides. J. Phycol. 18, 336344. Gill, I., Valivety, R., 1997. Polyunsaturated fatty acids, part 1: occurrence, biological activities and applications. TIBTECH 15, 401-409. Gonen-Zurgil, Y., Carmeli-Schwartz, Y., Sukenik, A., 1996. Selective effect of the herbicide DCMU on unicellular algae--a potential tool to maintain monoalgal mass culture of Nannochloropsis. J. Appl. Phycol. 8, 415-419. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana (Hustedt) and Detonula confervacea (Cleve). Can. J. Microbiol. 8, 229-239. Hu, Q., Hu, Z., Cohen, Z., Richmond, A., 1997. Enhancement of eicosapentaenoic acid (EPA) and ~,-linolenic acid (GLA) production by manipulating algal density of outdoor cultures of Monodus subterraneus (Eustigmatophyta) and Spirulina platensis (Cyanobacteria). Eur. J. Phycol. 32, 81-86. Leman, J., 1997. Oleaginous microorganisms: an assessment of the potential. Adv. Appl. Microbiol. 43, 195-243. Molina Grima, E., Sanchez Perez, J.A., Garcia Camacho, F., Acien Fernandez, F.G., Fernadez Sevilla, J.M., Valdez Sanz, F., 1994a. Effect of dilution rate on eicosapentaenoic acid productivity of Phaeodactylum tricornutum UTEX 640

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in outdoor chemostat culture. Biotechnol. Lett. 16, 10351040. Molina Grima, E., Sanchez Perez, J.A., Garcia Camacho, F., Fernadez Sevilla, J.M., Acien Fernandez, F.G., 1994b. Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture. Appl. Microbiol. Biotechnol. 41, 23-27. Nordoy, A., 1991. Is there a rational use for n-3 fatty acids (fish oils) in clinical medicine.'? Drugs 42, 331-342. Okauchi, M., 1991. The status of phytoplankton production in Japan. In: Fulks, W., Main, K.L. (Eds.), Rotifers and Microalgae Culture Systems. Proceedings of a US-Asia Workshop. The Oceanic Institute, Honolulu, HI, pp. 247256. Pastorelli, R., Chini Zittelli, G., Tredici, M.R., 1998. Bacterial population associated with Nannochloropsis sp. mass cultures. Abstracts of the International Symposium 'Marine Bioprocess Engineering'. Noordwijkerhout, The Netherlands. November 8-11, 1998. Radwan, S.S., 1991. Sources of Cs0-polyunsaturated fatty acids for biotechnological use. Appl. Microbiol. Biotechnol. 35, 421-430. Renaud. S.M., Parry, D.L., 1994. Microalgae for use in tropical aquaculture II: Effect of salinity on growth, gross chemical composition and fatty acid composition of three species of marine microalgae. J. Appl. Phycol. 6, 347-356. Renaud. S.M., Parry, D.L., Luong-Van, T., Kuo, C., Padovan. A., Sammy. N., 1991. Effect of light intensity on the proximate biochemical and fatty acid composition of lsochrvsis sp. and Nannochloropsis oculata for use in tropical aquaculture. J. Appl. Phycol. 3, 43-53. Senzaki, H., Iwamoto, S., Ogura, E., Kiyozuka, Y., Arita, S., Kurebayashi, J., Takada, H., Hioki, K., Tsubura, A., 1998. Dietary effects of fatty acids on growth and metastasis of KPL-1 human breast cancer cells in vivo and in vitro. Anticancer Res. 18, 1621- 1627. Seto, A., Wang, H.L., Hesseltine, C.W., 1984. Culture conditions affect eicosapentaenoic acid content of Chlorella minutisshna. J. Am. Oil. Chem. Soc. 61, 892-894. Seto, A., Kumasaka, K.. Hosaka, M., Kojima, E., Kashiwakura, M., Kato, T.. 1992. Production of eicosapentaenoic acid by marine microalgae and its commercial utilization for aquaculture. In: Kyle, D.J., Ratledge, C. (Eds.), Industrial Application of Single Cell Oils. American Oil Chemists' Society, Champaign, IL, pp. 219-234. Sukenik, A., 1991. Ecophysiological considerations in the optimization of eicosapentaenoic acid production by Nannochloropsis sp. (Eustigmatophyceae). Bioresour. Technol. 35, 263-269. Sukenik, A., Carmeli, Y., 1990. Lipid synthesis and fatty acid composition in Nannochloropsis sp. (Eustigrnatophyceae) grown in a light-dark cycle. J. Phycol. 26, 463-469. Sukenik, A., Carmeli, Y., Berner, T., 1989. Regulation of fatty acid composition by irradiance level in the eustigrnatophyte Nannochloropsis sp. J. Phycol. 25, 686-692. Sukenik, A., Zmora, O., Carmeli, Y., 1993a. Biochemical quality of marine unicellular algae with special emphasis

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on lipid composition. II. Nannochloropsis sp. Aquaculture 117, 313-326. Sukenik, A., Yamaguchi, Y., Livne, A., 1993b. Alteratiofis in lipid molecular species of the marine eustigrnatophyte Nannochloropsis sp. J. Phycol. 29, 620-626. Sukenik, A., 1998. Production of eicosapentaenoic acid by the marine eustigrnatophyte Nannochloropsis sp. In: Cohen, Z. (Ed.), Chemicals from Microalgae. Taylor and Francis, London, in press. Teshima, S., Yamasaki, S., Kanazawa, A., Hirata, H., 1983. Effects of water temperature and salinity on eicosapenatenoic acid level of marine Chlorella. Bull. Jpn. Soc. Sci. Fisheries 49, 805. Tredici, M.R., Chini Zittelli, G., 1998. Efficiency of sunlight utilization: tubular versus fiat photobioreactors. Biotechnol. Bioeng. 57, 187-197.

Tredici, M.R., Materassi, R., 1992. From open ponds to vertical alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms. Appl. Phycol. 4, 221-231. Tredici, M.R., Carlozzi, P., Chini Zittelli, G., Materassi, R., 1991. A vertical alveolar panel (VAP) for outdoor mass cultivation of microalgae and cyanobacteria. Bioresour. Technol. 38, 153-159. Volkman, J.K., Malcom, R.B., Dunstan, G.A., Jeffrey, S.W., 1993. The biochemical composition of marine microalgae from the class eustigmatophiceae. J. Phycol. 29, 69-78. Yongrnanitchai, W., Ward, O.P., 1989. Omega-3 fatty acids: alternative sources of production. Process Biochem. 24, 117-125.

Biotechno,logy ELSEVIER

Journal of Biotechnology 70 (1999) 313-321

Commercial production of microalgae" ponds, tanks, tubes and fermenters M i c h a e l A. B o r o w i t z k a * Algae Research Group, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia

Received 16 October 1998; received in revised form 29 November 1998; accepted 22 December 1998

Abstract The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for 13-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20-40 1 carboys or in large plastic bags of up to approximately 1000 1 in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL TM, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as fiat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Photobioreactors; Chlorella; Spirulina; Dunaliella

1. Introduction Microalgal culture is one of the modern biotechnologies. The first unialgal cultures were achieved by Beijerinck (1890), with Chlorella vul* Fax: + 61-8-93606303. E-mail address: [email protected] (M.A. Borowitzka)

garis, and the use of such cultures for studying plant physiology was developed by W a r b u r g in the early 1900s (Warburg, 1919). Mass culture of microalgae really began to be a focus of research after 1948 at Stanford (USA), Essen (Germany) and Tokyo and the classic book edited by Burlew (1953) summarises many of these early studies. Interest in applied algal culture continued, especially with studies on the use of algae as photo-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00083-8

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321

314

synthetic gas exchangers for space travel and as microbial protein sources. The results of these studies can be found in several papers and books (Shelef and Soeder, 1980; Venkataraman and Becker, 1985; Wharton et al., 1988). Commercial large-scale culture of microalgae commenced in the early 1960s in Japan with the culture of Chlorella (Tsukada et al., 1977) followed in the early 1970s with the establishment of a Spirulina harvesting and culturing facility in Lake Texcoco, Mexico by Sosa Texcoco S.A. (Durand-Chastel, 1980). In 1977 Dai Nippon Ink and Chemicals Inc. established a commercial Spirulina plant in Thailand, and by 1980 there were 46 large-scale factories in Asia producing more than 1000 kg of microalgae (mainly Chlorella) per month (Kawaguchi, 1980) and in 1996 about 2000 t of Chlorella were traded in Japan alone (Lee, 1997). Commercial production of Dunaliella salina as a source of 13-carotene became the third major microalgae industry when production facilities were established by Western Biotechnology Ltd and Betatene Ltd in Australia in 1986. These were soon followed by other commercial plants in Israel and the USA. As well as these algae, the large-scale production of cyanobacteria (blue-green algae) commenced in India at about the same time (Venkatamaran, 1986). Thus in a short period of about 30 years the industry of microalgal biotechnology has grown and diversified significantly. The growth of commercial microalgae production is probably best illustrated by data on Spirulina production for which reasonably reliable figures are available (Fig. 1). The success of commercial large-scale production of microalgae depends on many factors, and one of these is the development of cost effective large-scale culture systems for the algae and the development of these has been, and continues to be, a gradual process. This paper will review some of the steps along the way and the future advances we can expect.

range in volume from about 102 1 (used for the production of organic compounds labelled with stable isotopes) to > 101~1 (used for the culture of D. salina). However, aside from the specialised small-scale ( < 1000 1) culture systems four types of culture systems predominate: large open ponds, circular ponds with a rotating arm to mix the cultures, raceway ponds and large bags. Other commercial large-scale systems include tanks used in aquaculture, the cascade system developed in Trebon, Czech Republic (Setlik et al., 1970) and heterotrophic fermenter systems used for the culture of Chlorella in Japan and Taiwan (Kawaguchi, 1980; Soong, 1980) and for the culture of Crypthecodinium cohnii in the USA (Kyle and Gladue, 1991; Kyle et al., 1998). Table 1 summarises the culture systems currently in use for commercial algal culture.

3000 Cuba Chile Myanmar

2500 -

[~ [~

raiwan Japan

2000 i

c c 0

c

0

India

Viemam China USA Thailand

Mexico

1500 -

0

lO0O

-

i

500

-

o

/

1970

1975

,l!iIill 1980

1985

1990

1995

2000

Year

2. Culture systems

Existing commercial microalgae culture systems

Fig. 1. Global production figures of Spirulina by country based on literature, company and trade information. Figures for 1997 and 1998 are estimates based on projected production figures provided by the producers.

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321

315

Table 1 Commercial microalgae culture systems currently in use and the algal species cultured Culture system

Algae

Tanks Extensive open ponds Circular ponds with rotating arm Raceway ponds

Many species (for aquaculture)

Cascade system with baffles Large bags

Chlorella spp. Many species (used for aquaculture) Chlorella spp., Crypthecodinium

Fermenters (heterotrophic) Two-stage system (indoors in closed reactor and then outdoors in paddlewheel ponds) a

Dunaliella salina Chlorella spp. Chlorella spp., Spirulina spp. Dunaliella salina

Approximate maximum volume (1)a 1• 1• 1.5 • 3•

104 109 104 104

3 x 104 1 x 103 > 103

cohnii Haematococcus pluvialis

Location

World Wide Australia Taiwan, Japan Japan, Taiwan, USA, Thailand, China, India, Vietnam, Chile, USA, Israel, China Czech Republic, Bulgaria World wide Japan, Taiwan, Indonesia, USA USA

These are order of magnitude estimates only.

There are several considerations as to which culture system to use. Factors to be considered include: the biology of the alga, the cost of land, labour, energy, water, nutrients, climate (if the culture is outdoors) and the type of final product (Borowitzka, 1992). The various large-scale culture systems also need to be compared on their basic properties such as their light utilisation efficiency, ability to control temperature, the hydrodynamic stress placed on the algae, the ability to maintain the culture unialgal and/or axenic and how easy they are to scale up from laboratory scale to large-scale. These properties are compared in Table 2. The final choice of system is almost always a compromise between all of these considerations to achieve an economically acceptable outcome. A common feature of most of the algal species currently produced commercially (i.e. Chlorella, Spirulina and Dunaliella) is that they grow in highly selective environments which means that they can be grown in open air cultures and still remain relatively free of contamination by other algae and protozoa. Thus, Chlorella grows well in nutrient-rich media, Spirulina requires a high pH and bicarbonate concentration and D. salina grows at very high salinity (Soong, 1980; Borowitzka and Borowitzka, 1988; Belay, 1997).

Those species of algae which do not have this selective advantage must be grown in closed systems. This includes most of the marine algae grown as aquaculture feeds (e.g. Skeletonema, Chaetoceros, Thalassiosira, Tetraselmis and Isochrysis) and the dinoflagellate C. cohnii grown as a source of long-chain polyunsaturated fatty acids.

3. Open-air systems Although much of the early work on microalgal mass culture focused on closed culture systems (see Burlew, 1953) all very large commercial systems used today are open-air systems (Table 1). The reason for this is simple economics; closed culture systems are very expensive and many of them are difficult to scale up. Furthermore, most closed systems are operated indoors with artificial lighting and this results in high energy costs whereas open air systems can utilise sunlight. However, only a small number of algal species can be grown successfully in open air systems. The four major types of open-air systems currently in use (shallow big ponds, tanks, circular ponds and raceway ponds) all have their advantages and disadvantages. The selection of a partic-

Table 2 Comparison of the properties of different large-scale algal culture systems Reactor type

Mixing

Light utilisa- Temperature tion efficiency control

Gas transfer

Hydrodynamic Species constress on algae trol

Sterility

Scale-up

Reference

Unstirred shallow ponds

Very poor

Poor

None

Poor

Very low

Difficult

None

Very difficult

Tanks Circular stirred ponds Paddle-wheel Raceway Ponds Stirred Tank reactor (internal or external lighting) Air-Lift reactor Bag Culture

Poor Fair

Very poor Fair-good

None None

Poor Poor

Very low Low

Difficult Difficult

None None

Very difficult Very difficult

Fair-good

Fair-good

None

Poor

Low

Difficult

None

Very difficult

Largely uniform

Fair-good

Excellent

Low-high

High

Easy

Easily achievable

Difficult

Borowitzka and Borowitzka, 1989 Fox, 1983 Tamiya, 1957; Stengel, 1970; Soeder, 1981 Weissman and Goebel, 1987; Oswald, 1988 Pohl et al., 1988

Generally uni- Good form Variable Fair-good

Excellent

High

Low

Easy

Difficult

Juttner. 1977

Low-high

Low

Easy

Difficult

High

Low high

Easy

Easily achievable Easily achievable Achievable

Baynes et al., 1979 Hu et al., 1996; Tredici and Zitelli, 1997 Richmond et al., 1993; Torzillo, 1997 Borowitzka, 1996

Uniform

Excellent

Good (indoors) Excellent

Tubular reac- Uniform tor (Serpentine type) Tubular Reac- Uniform tor (Biocoil tYF4

Excellent

Excellent

Low-high

Low-high

Easy

Achievable

Reasonable

Excellent

Excellent

Low-high

Low-high

Easy

Achievable

Easy

Flat-Plate reactor

Difficult

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321

ular system is also influenced by intrinsic properties of the alga as well as local climatic conditions and the costs of land and water. For example, the Australian producer of D. salina, Betatene Ltd, uses very large ponds of up to 250 ha in area at their plants at Whyalla and Hutt Lagoon, which are unmixed other than by wind and convection. This is possible since land costs are low, water (seawater) is free other than for pumping costs, and the climate is close to optimum so that production can be achieved all year round. Furthermore, the company has a very efficient harvesting system (Borowitzka, 1991; Schlipalius, 1991). On the other hand, the other Dunaliella producers in the USA (no longer in operation) and in Israel use paddle-wheel driven raceway ponds to achieve higher cell densities (Ben-Amotz, 1995). This is because the cost of land is relatively high at both of these sites and site preparation costs are also significant. As well as this they require the addition of NaC1 to the medium which is a significant additional cost (the cultures operate at between 15 and 25% NaC1). These factors mean that pond area and volume must be minimised and the cell density in the culture must be maximised in order to have an economical process. The culture of Spirulina and Chlorella also requires a well mixed system such as a raceway system to achieve high growth rates and to minimise the risk of overgrowth by other algae. The only successful extensive culture system for these algae was the 'Caracol' used by Sosa Texcoco in Mexico for the culture of Spirulina maxima. The Caracol was a giant spiral-shaped solar evaporator of 3200 m diameter with a surface area of 900 ha within Lake Texcoco where Spirulina grows naturally. This system had to be abandoned eventually due to the increased contamination resulting from urban and industrial development in Mexico City. The greater susceptibility to contamination by other algae and/or protozoa also means that Spirulina and Chlorella must be grown in batch or semi-batch mode with periodical reseeding of ponds with new inoculum (Kawaguchi, 1980; Belay, 1997). Furthermore, many of these algal plants are located in regions with climatic conditions which do not allow year round produc-

317

tion. This means that productivities have to be maximised to offset the high capital costs of the ponds and associated production systems such as harvesters and dryers. The problems with open air pond systems are that the productivity achieved is less than that theoretically possible and that it is difficult to control the culture environment which is exposed to the elements. The pond depth is a compromise between the need to provide adequate light to the algal cells (i.e. the shallower the more light is available to the cells) and the need to maintain an adequate water depth for mixing and to avoid large changes in ionic composition due to evaporation. Thus most paddle-wheel raceway ponds are between 20 and 30 cm deep and the very large (up to approx. 250 ha) unmixed ponds used in the culture of Dunaliella in Australia the depth may be up to 50 cm deep. This means that, in almost all cases, the algae are light limited and the maximum biomass achieved on a regular basis is between approx. 0.1 and 0.5 g dry weight I-~. The relationship between pond depth, culture density and productivity has been most extensively studied for Spirulina (cf. Vonshak, 1997) and is important for maximising the biomass output. The algae are also CO2 limited, however the addition of CO2 to these large ponds is usually inefficient and uneconomical except in the case of Spirulina culture where it is essential to maintain high alkalinity (Belay, 1997). The only open-air system which achieves significantly higher sustainable cell densities is the cascade system developed by Setlik et al. (1970) and which is still in use in Trebon, Czech Republic for the culture of Chlorella. In this system the culture depth is less than 1 cm and cell densities up to about 10 g 1-~ can be maintained while maintaining high growth rates (Doucha and Livansky, 1995). The Trebon system is very expensive with the base of the sloping culture surface made of glass. However, improved materials mean that a similar system could now be constructed at significantly lower cost (Doucha, personal communication). Furthermore the central European location of Trebon means that there is a relatively short annual culture period. Preliminary calculations indicate that this system would be very competi-

318

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321

tive with paddle-wheel raceway systems if located in a sunnier, warmer climate and if constructed of lighter and cheaper materials. A similar system comprising a 0.5 ha sloping, plastic lined pond was used to produce Chlorella near Dongara, Western Australia, for several years. This system had an average productivity of about 25 g m - 2 day-1 and could be operated in semi-continuous mode for the whole year due to the near optimal Climate. Unfortunately technical problems with further scale-up eventually led to the closure of this plant.

4. Closed systems Despite the success of open systems, future advances in microalgal mass culture will require closed systems as the algal species on interest do not grow in highly selective environments. Furthermore, many of the new algae and algal products must be grown free of potential contaminants such as heavy metals and micro-organisms. The concept of closed systems has been around for a long time (Little, 1953; Jfittner, 1977; Pirt et al., 1983) however their high cost has largely precluded their commercial application until recently. Algae can be grown in closed systems either photoautotrophically, mixotrophically or heterotrophically. Heterotrophic cultivation on acetate or glucose as carbon sources has been used for some time for Chlorella (Kawaguchi, 1980; Soong, 1980) with approximately 550 t produced in Japan in 1996 (Lee, 1997). For a short period Tetraselmis was also grown heterotrophically in the UK (Gladue, 1991; Laing and Verdugo, 1991), however the production costs for the latter proved uneconomical and the spray dried product was inferior to photoautotrophically grown algae. More recently, heterotrophic culture is being used for the dinoflagellate, C. cohnii by Martek Inc. in the USA to produce long-chain polyunsaturated fatty acids (Kyle et al., 1992, 1998). Heterotrophic culture has several advantages: (i) fermentation systems are well understood and there is wide experience in their design and operation; and (ii) high cell densities of between 20 and 100 g 1-1 can be

achieved reducing harvesting costs and the capital costs of the cultivation vessels (Radmer and Parker, 1994; Running et al., 1994). However, there are disadvantages as well. The main disadvantages are that heterotrophic cultivation is not possible for all microalgae and that the chemical composition of the algae often changes under heterotrophic conditions. Closed photoautotrophic culture systems are widely used in the aquaculture industry for the production of a range of algal species. The most widely used large-scale (up to about 1000 1) system is the 'big bag' system (Baynes et al., 1979; Watson, 1979). These systems use large sterile plastic bags of about 0.5 m diameter fitted with a system for aeration. Although most of these systems are operated in batch mode, semi-continuous systems have also been developed. A variant on this system has been developed by Cohen and Arad (1989) using multiples of narrower bags; however there appears to be no commercial production as yet using this system. The main problem with the big bag system is that the system has to be operated indoors for temperature control and that the relatively large diameter of the bags and the need for artificial lighting results in light-limited cultures. The operation of these systems is also labour intensive and the cultures are generally inadequately mixed, leading to recurrent culture 'crashes'. This makes the algae expensive to produce. Costs are about $US 50 kg-~ of dry algal biomass (Fulks and Main. 1991) for a large, specialised oyster hatchery in the USA with many smaller hatcheries having costs of up to $US 300-600 kg-1 (Coutteau and Sorgeloos, 1992; Borowitzka, 1997). This is much greater that the estimated production costs for Chlorella, Spirulina and Dunaliella of between $US 9 and 25 kg -1 (Borowitzka, 1992; Tanticharoen et al., 1993). In recent years there have been several major advances in the design and operation of closed photobioreactors for algal culture and several systems are likely to be commercial realities in the near future. The two basic designs are the flat plate reactors (Pulz, 1994; Hu et al., 1996; Tredici and Zitelli, 1997) and the tubular photobioreactors (Miyamoto et al., 1988; Richmond et al.,

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321

1993; Borowitzka, 1996; Torzillo, 1997). The fundamental principle in all of these designs is to reduce the light path and thus to increase the amount of light available to each cell. These reactors are also well mixed to ensure optimum light availability to the cells and to enhance gas exchange. The optimum thickness of the algal culture in these reactors is between 2 and 4 cm. Pilot scale units of up to about 1000 1 have been operated successfully for Spirulina, Chlorella and several marine microalgae, and at least one pilotscale system is in use in Potsdam, Germany for the production of Spirulina and another 6000 1 unit in Elbingerode, Germany is growing Chlorella using waste CO2 from a lime factory (Menz, 1998). These closed reactors have several advantages such as 'clean' algal culture, high light utilisation efficiency leading to high productivities as high sustainable biomass, temperature control and the ability to be used outdoors in natural daylight. This means that a much wider range of species can be cultivated as contamination is avoided and the reactors can be operated over a much wider climatic range that the open-air systems. There is also a greater ability to control the culture conditions so that the final product is of more consistent composition and quality (e.g. Chrismadha and Borowitzka, 1994). Finally, these systems are amenable to operation in continuous culture mode. Continuous culture and good control over the growth environment results in a consistent product quality and the higher operating cell densities also mean that harvesting costs are reduced and there is a smaller requirement for land. The challenge now is to reduce the construction costs of these systems further to make them more economically competitive. The BIOCOIL TM appears the most promising design at present (Robinson et al., 1988; Robinson and Morrison, 1992). The BIOCOIL is a helical tubular photobioreactor consisting of a photostage of small diameter clear plastic tubing (between 2.4 and 5 cm diameter) would helically around a tower (Borowitzka and Borowitzka, 1989). Several parallel bands of tubes are connected via manifolds to a pumping system which may be an airlift or pumps such as centrifugal,

319

diaphragm or lobe pumps, the type of pump used depends on the species of alga grown. If required a gas exchange tower may also be incorporated in the circuit. Temperature control is either by a heat exchanger or by evaporative cooling by flowing water over the photostage surface. We have grown a wide range of marine microalgae including Tetraselmis spp., Isochrysis galbana, Phaeodactylum tricornutum and Chaetoceros spp. as well as Spirulina at high productivities in pilot scale BIOCOILs of up to 700 1 for periods greater than 4 months in semi-continuous culture. The design of the BIOCOIL ensures uniform mixing and minimises adhesion of the algal cells to the inside of the tubes. The BIOCOIL can also be scaled up easily and the whole culture process can be automated thus minimising labour costs and improving reliability. Depending on need, the system can be designed to be operated as an axenic culture. Not all algal species are suitable for culture in this system. Some especially delicate species are damaged by the circulation system and other species may be too 'sticky' for normal operation. However, systems such as the BIOCOIL greatly expand the suite of algae which can be grown on a large scale and provide the opportunity to develop new algae and algal products such as bioactive molecules (Borowitzka, 1995). They will also serve to reduce the costs associated with the production of algae such as those species used in aquaculture.

5. Conclusions

Commercial microalgal culture is a well established industry. Most of the culture systems in use today are open air and relatively unsophisticated. However, over the last 50 years great advances have been made in our understanding of the biology of the algae and in the engineering requirements of large-scale algae culture systems. This has led to the development of several types of closed photobioreactors which will enable the commercialisation of new algae and algal products in the next decade.

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References Arthur D. Little Inc., 1953. Pilot-plant studies in the production of Chlorella. In: Burlew, J.S. (Ed.), Algal Culture. From Laboratory to Pilot Plant. Carnegie Institution, Washington, DC, pp. 235-272. Baynes, S.M., Emerson, L., Scott, A.P., 1979. Production of algae for use in the rearing of larval fish. Fisheries Research Technical Report 53, 13-18. Beijerinck, M.W., 1890. Kulturversuche mit Zoochloren, Lichenengonidien und anderen niederen Algen. Bot. Ztg. 48, 725-785. Belay, A., 1997. Mass culture of Spirulina outdoors--The Earthrise Farms experience. In: Vonshak, A. (Ed.), Spirulina platensis (Arthrospira): Physiology, cell-biology and biotechnology. Taylor and Francis, London, pp. 131-158. Ben-Amotz, A., 1995. New mode of Dunaliella biotechnology: two-phase growth for 13-carotene production. J. Appl. Phycol. 7, 65-68. Borowitzka, L.J., 1991. Development of Western biotechnology algal beta-carotene plant. Bioresource Technol. 38, 251-252. Borowitzka, L.J., Borowitzka, M.A., 1989. Industrial production: methods and economics. In: Cresswell, R.C., Rees, T.A.V., Shah, N. (Eds.), Algal and Cyanobacterial Biotechnology. Longrnan Scientific, London, pp. 294-316. Borowitzka, M.A., 1992. Algal biotechnology products and processes: matching science and economics. J. Appl. Phycol. 4, 267-279. Borowitzka, M.A., 1995. Microalgae as sources of pharmaceuticals and other biologically active compounds. J. Appl. Phycol. 7, 3-15. Borowitzka, M.A., 1996. Closed algal photobioreactors: design considerations for large-scale systems. J. Mar. Biotechnol. 4, 185-191. Borowitzka, M.A., 1997. Algae for aquaculture: Opportunities and constraints. J. Appl. Phycol. 9, 393-401. Borowitzka, M.A., Borowitzka, L.J., 1988. Dunaliella. In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Microalgal Biotechnology. Cambridge University Press, Cambridge, pp. 27- 58. Burlew, J.S. (Ed.), 1953. Algae Culture. From Laboratory to Pilot Plant. Carnegie Institution of Washington, Washington, DC. Chrismadha, T., Borowitzka, M.A., 1994. Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular photobioreactor. J. Appl. Phycol. 6, 67-74. Cohen, E., Arad, S., 1989. A closed system for outdoor cultivation of Porphyridium. Biomass 18, 59-67. Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirement for live algae in the hatchery and nursery of bivalve molluscs: an international survey. J. Shellfish. Res. 11,467-476. Doucha, J., Livansky, K., 1995. Novel outdoor thin-layer high density microalgal culture system: Productivity and operational parameters. Algol. Stud. 76, 129-147.

Durand-Chastel, H., 1980. Production and use of Spirulina in Mexico. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 51-64. Fox, J.M., 1983. Intensive algal culture techniques. In: McVey, J.P. (Ed.), CRC Handbook of Mariculture Crustacean Aquaculture. CRC Press, Boca Raton, FL, pp. 15-41. Fulks, W., Main, K.L., 1991. The design and operation of commercial-scale live feeds production systems. In: Fulks, W., Main, K.L. (Eds.), Rotifer and Microalgae Culture Systems. The Oceanic Institute, Honolulu, HI, pp. 3-52. Gladue, R., 1991. Heterotrophic microalgae production: potential for application to aquaculture feeds. In: Fulks, W., Main, K.L. (Eds.), Rotifer and Microalgae Culture Systems. The Oceanic Institute, Honolulu, HI, pp. 275-286. Hu, Q., Guterman, H., Richmond, A., 1996. A flat inclined modular photobioreactor for outdoor mass cultivation of photoautotrophs. Biotechnol. Bioeng. 51, 51-60. Jiittner, F., 1977. Thirty liter tower-type pilot plant for the mass cultivation of light- and motion-sensitive planktonic algae. Biotechnol. Bioeng. 19, 1679-1687. Kawaguchi, K., 1980. Microalgae production systems in Asia. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass Production and Use. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 25- 33. Kyle, D.J., Gladue, R.M., 1991. Eicosapentaenoic acids and methods for their production. World Patent 9,114,427. Kyle, D.J., Boswell, K.D.B., Gladue, R.M., Reeb, S.E., 1992. Designer oils from microalgae as nutritional supplements. In: Bills, D.D., Kung, S.D. (Eds.), Biotechnology and Nutrition. Butterworth-Heinemann, Boston, pp. 451-468. Kyle, D.J., Reeb, S.E., Sicotte, V.J., 1998. Dinoflagellate biomass, methods for its production, and compositions containing the same. USA Patent 5,711,983. Laing, I., Verdugo, C.G., 1991. Nutritional value of spraydried Tetraselmis suecica for juvenile bivalves. Aquaculture 92, 207-218. Lee, Y.K., 1997. Commercial production of microalgae in the Asia-Pacific rim. J. Appl. Phycol. 9, 403-411. Menz, K., 1998. Biotechnologische Nutzung von Kohlendioxid als Rohstoff mit Hilfe yon Mikroalgen. Forschung Technik und Innovation (Preussag) 23, 85-89. Miyamoto, K., Wable, O., Benemann, J.R., 1988. Vertical tubular reactor for microalgae cultivation. Biotech. Lett. 10, 703- 708. Oswald, W.J., 1988. Large-scale algal culture systems (engineering aspects). In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Microalgal Biotechnology. Cambridge University Press, Cambridge, pp. 357-394. Pirt, S.J., Lee, Y.K., Walach, M.R., Pirt, M.W., Balyuzi, H.H.M., Bazin, M.J., 1983. A tubular bioreactor for photosynthetic production of biomass from carbon dioxide: design and performance. J. Chem. Tech. Biotechnol. 33B, 35-58. Pohl, P., Kohlhase, M., Martin, M., 1988. Photobioreactors for the axenic mass cultivation of microalgae. In: Stadler, T., Mollion, J., Verdus, M.C., Karamanos, Y., Morvan,

M.A. Borowitzka /Journal of Biotechnology 70 (1999) 313-321 H., Christiaen, D. (Eds.), Algal Biotechnology. Elsevier, London, pp. 209-217. Pulz, O., 1994. Open-air and semi-closed cultivation systems for the mass cultivation of microalgae. In: Phang, S.M., Lee, K., Borowitzka, M.A., Whitton, B. (Eds.), Algal Biotechnology in the Asia-Pacific Region. Institute of Advanced Studies, University of Malaya, Kuala Lumpur, pp. 113-117. Radmer, R.J., Parker, B.C., 1994. Commercial applications of algae--opportunities and constraints. J. Appl. Phycol. 6, 93-98. Richmond, A., Boussiba, S., Vonshak, A., Kopel, R., 1993. A new tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol. 5, 327-332. Robinson, L.F., Morrison, A.W., 1992. Biomass production apparatus. USA Patent 5,137,828. Robinson, L.F., Morrison, A.W., Bamforth, M.R., 1988. Improvements relating to biosynthesis. European Patent 261,872. Running, J.A., Huss, R.J., Olson, P.T., 1994. Heterotrophic production of ascorbic acid by microalgae. J. Appl. Phycol. 6, 99-104. Schlipalius, L., 1991. The extensive commercial cultivation of Dunaliella salina. Bioresour. Technol. 38, 241-243. Setlik, I., Veladimir, S., Malek, I., 1970. Dual purpose open circulation units for large scale culture of algae in temperate zones. I. Basic design considerations and scheme of pilot plant. Algol. Stud. (Trebon) 1, 11. Shelef, G., Soeder, C.J. (Ed.), 1980. Algae biomass. Production and use. Elsevier/North Holland Biomedical Press, Amsterdam. Soeder, C.J., 1981. Productivity of microalgal systems. U.O.F.S. Publ., Series C 3, 9-15. Soong, P., 1980. Production and development of Chlorella and Spirulina in Taiwan. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 97-113. Stengel, E., 1970. Anlagentypen und Verfahren der technischen Algenmassenproduktion. Ber. Deutsch. Bot. Ges. 83, 589-606.

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Tamiya, H., 1957. Mass culture of algae. Ann. Rev. Plant Physiol. 8, 309-344. Tanticharoen, M., Bunnag, B., Vonshak, A., 1993. Cultivation of Spirulma using secondary treated starch wastewater. Aust. Biotechnol. 3. 223-226. Torzillo, G., 1997. Tubular bioreactors. In: Vonshak, A. (Ed.), Spirulina platensis (Arthrospira): Physiology, cell-biology and biotechnology. Taylor and Francis, London, pp. 101115. Tredici, M.R., Zitelli, G.C., 1997. Cultivation of Spirulina (Arthrospira) platensis in flat plate reactors. In: Vonshak, A. (Ed.), Spirulina platensis (Arthrospira): Physiology, cellbiology and biotechnology. Taylor and Francis, London, pp. 117-130. Tsukada, O., Kawahara, T., Miyachi, S., 1977. Mass culture of Chlorella in Asian countries. In: Mitsui, A., Miyachi, S., San Pietro, A., Tamura, S. (Eds.), Biological Solar Energy Conversion. Academic Press, New York, pp. 363-365. Venkatamaran, L.V., 1986. Blue-green algae as biofertilizer. In: Richmond, A. (Ed.), CRC Handbook of Microalgal Mass Culture. CRC Press, Boca Raton, FL, pp. 455-471. Venkataraman, L.V., Becker, E.W., 1985. Biotechnology and utilization of algae--The Indian experience. Department of Science and Technology, New Delhi. Vonshak, A., 1997. Outdoor mass production of Spirulina: The basic concept. In: Vonshak, A. (Ed.), Spirulina platensis (Arthrospira): Physiology, cell-biology and biotechnology. Taylor and Francis, London, pp. 79-99. Warburg, O., 1919. (0ber die Geschwindigkeit der Kohlens~iurezusammensetzung in lebenden Zellen. Biochemische Zeitschrift 100, 230-270. Watson, A.S., 1979. Aquaculture and Algae Culture. Process and Production. Noyes Data Corporation, NJ. Weissman, J.C., Goebel, R.P., 1987. Design and analysis of microalgal open pond systems for the purpose of producing fuels. Solar Energy Research Institute, Report SERI/ STR-231-2840, 1- 214. Wharton, R.A., Smernoff, D.T., Averner, M.M., 1988. Algae in space. In: Lembi, C.A., Waaland, J.R. (Eds.), Algae and Human Affairs. Cambridge University Press, Cambridge, pp. 485-509.

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Journal of Biotechnology 70 (1999) 323-333

Specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana under medium duration light/dark cycles" 13-87 s Marcel Janssen a,,, Tjibbe Chris Kuijpers a Bram Veldhoen a, Michel Brik Ternbach a, Johannes Tramper a, Luuc R. Mur b, Ren6 H. Wijffels a a Food and Bioprocess Engineering Group, Department of Food Science, Wageningen Agricultural University, PO Box 8129, 6700 EV Wageningen, Netherlands b Amsterdam Research Institute for Substances in Ecosystems, University of Amsterdam, Nieuwe Achtergracht 127, I018 WS Amsterdam, Netherlands

Received 19 October 1998; received in revised form 26 November 1998; accepted 22 December 1998

Abstract

The specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana decreased under square-wave light/dark cycles of medium duration, 13-87 s, in comparison to continuous illumination. Three experiments were done in three different turbidostats at saturating and sub-saturating light intensities during the light period, 240-630 ~tmol m - 2 s-1. Within each experiment the light intensity during the light periods of the intermittent light regimes was equal and this intensity was also applied under continuous illumination. The specific growth rate decreased proportional or more than proportional to the fraction of time the algae were exposed to light; this light fraction ranged from 0.32 to 0.88. We conclude that under these light regimes the chlorophyta C. reinhardtii and C. sorokiniana are not able to store light energy in the light period to sustain growth in the dark period at the same rate as under continuous illumination. C. reinhardtii increased its specific light absorbing surface by increasing its chloropyll-a content under light/dark cycles of 13 s duration and a light fraction of 0.67 at 240 ~tmol m - 2 s-1; the chloropyll-a content was twice as high under intermittent illumination in comparison to continuous illumination. The combination of a higher specific light absorption together with a lower specific growth rate led to a decrease of the yield of biomass on light energy under intermittent illumination. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Chlamydomonas reinhardtii; Chlorella sorokiniana" Light/dark cycles; Intermittent illumination; Specific growth rate

* Corresponding author. Tel.: + 31-317-483396; fax: + 31317-482237. E-mail address: [email protected] (M. Janssen)

1. Introduction

The possibility of using photo-autotrophic micro-organisms has been recognized in biotechnol-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00084-X

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ogy. Microalgae are produced on a commercial scale for the production of single-cell protein, polysaccharides, health-food compounds such as polyunsaturated fatty acids and vitamins. In addition, algae are capable of accumulating heavy metals (Becker, 1994). Other promising applications of photo-autotrophic micro-organisms are the production of hydrogen gas from water and (sun)light (Rao and Hall, 1996; Schulz, 1996) and the fixation of carbon dioxide (Yoshihara et al., 1996; Akimoto et al., 1997; Hu et al., 1998). Large-scale cultivation of photo-autotrophic micro-organisms, however, is still limited by scaleup problems and economical considerations. Development of cheaper and more efficient photobioreactors is a key issue. Light energy often limits reactor productivity and therefore light should be used at the highest possible efficiency. But in high-density cultures only algae close to the illuminated surface, the so-called photic zone, are exposed to light. Algae outside the photic zone receive no light. Moreover, a considerable part of the light energy absorbed in the photic zone will be dissipated as heat because the capacity at which algae fix light energy is limited. This heatdissipated light energy cannot be used anymore by the algae outside the photic zone. It has been shown that faster mixing of highdensity cultures increases the yield of biomass on light energy at high light intensities (Laws et al., 1983; Hu et al., 1996; Hu and Richmond, 1996). Mixing results in movement of algae through the culture and, as such, they are exposed to an intermittent light regime. In other words, algae are alternately exposed to light and no light at all. As a result of faster mixing, algae are exposed to high light intensities more often, but the time of exposure is shorter. These shorter light/dark cycles probably have caused the reported higher biomass yields on light energy because less energy is wasted in the photic zone (Richmond, 1996). The duration of the light/dark cycles in the above studies was in the range of 0.2-1 s. For providing these fast fluctuations reactors were developed with a short light path and a fast mixing rate provided by intense aeration. It would be interesting to investigate the effect of longer light/dark cycles on algal productivity. Light/dark

cycles of medium duration, 10-100 s, can be applied more easily in closed photobioreactors. Reactor types in which the suspension is circulated in a manner resembling plug flow, such as air-lift loop reactors, are suitable for this. Only part of the reactor volume needs to be exposed to (sun)light. The algae are intermittently illuminated as they circulate through the reactor. Such systems can be better temperature controlled, as the dark part of the reactor is not exposed to sunlight and can be cooled more easily. Furthermore, the dark/light cycle can be controlled well by controlling the superficial liquid circulation velocities. Finally, there is a lot of knowledge available on hydrodynamics, mass transfer and scale up of air-lift systems (Chisti, 1989). The influence of medium duration light/dark cycles is not clear. In short term oxygen evolution experiments with non-acclimated Chlorella and Scenedesmus no influence of fluctuating light, of equal time-averaged irradiance, (1-0.0038 Hz, 1263 s cycle duration) was found on specific oxygen production and carbon fixation (Grobbelaar, 1989, 1991; Grobbelaar et al., 1992). However, an increase in specific oxygen production was observed after 3 h of exposure to fluctuating light and apparently physiological changes took place under fluctuating light (Grobbelaar, 1991). Others observed a decrease of the specific growth rate of the chlorophyta Chlamydomonas, Oocystis, Dunaliella, Chlorella and Scenedesmus at frequencies of 0.2-0.1 Hz (5-10 s cycle duration) in comparison to continuous illumination of the same time-averaged irradiance (Walsh and Legendre, 1982; Qu~guiner and Legendre, 1986; Nedbal et al., 1996). On the contrary, Pirt (1986) suggested that Chlorella sorokiniana was able to store enough ATP and reducing power to sustain maximal growth for about 9 s in darkness. In addition, Merchuk et al. (1998) showed that the red microalga Porphyridium sp. could sustain maximal growth in darkness for 6 s during light/dark cycles of 27 s. It is also suggested that photosynthesis in green plants is limited by carbon fixation and that short dark periods (up to 30 s) following a saturating light fleck can result in enhanced carbon fixation (Sharkey et al., 1986a,b; Stitt, 1986).

M. Janssen et al./'Journal of Biotechnology 70 (1999) 323-333

With respect to photobioreactor design it would be very interesting to elucidate the effect of medium duration light/dark cycles on algal productivity. Therefore we measured the specific growth rates of two green algae (chlorophyta) under square-wave light/dark cycles. For comparison the specific growth rate was also determined under continuous illumination. We used Chlamydomonas reinhardtii and Chlorella sorokiniana and they were cultivated in three different systems. The cycle duration ranged from 13 to 87 s and the fraction of time the algae were exposed to light during a cycle (e) ranged from 0.32 to 0.88.

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2.1. Experiment 1" C. reinhardtii 2.1.1. Organism and medium A C. reinhardtii wild type strain, coded 21 gr, was kindly provided by Dr C. Vilchez from the University of Huelva (Spain). The organism was cultivated in a Sueoka high salt (HS) medium as described by Harris (1989). The medium has the following composition (amounts in g 1-1): NH4C1, 0.5; MgSO4.7H20, 0.02; CaClz.2H20, 0.01; K2HPO4, 1.44; KH2PO4, 0.72 and 5 ml of Htitner's trace elements solution (Harris, 1989). Pure cultures were maintained on agar slants containing Sueoka medium + 0.84 g 1-1 NaHCO3 + 1.75% w/v agar. The cultures were exposed to very mild daylight at room temperature.

2. Materials and methods

Three different cultivation systems were used in three different experiments: (1) C. reinhardtii was cultivated in two air-lift loop reactors under continuous illumination and under a 8.5/4.4 s light/ dark cycle at 240 ~tmol m -2 s - l ; (2) C. sorokiniana was cultivated in a bubble column under continuous illumination at four different light intensities. At the highest and lowest intensity a 30/10 s square-wave light/dark cycle was applied; (3) C. sorokiniana was cultivated in a flat panel reactor under five different square-wave light/dark cycles at 250-290 lamol m - 2 S-1 which are presented in Table 1. Within each experiment the light intensity during the light periods of the intermittent light regimes was equal.

Table 1 Experimental design specific growth rate determination of C. sorokiniana in fiat panel turbidostat under different light/dark cycles Cycle duration, tc (s)

Light fraction, e (-)

25 25 56 87 87

0.32 0.88 0.60 0.32 0.88

2.1.2. Light intensity We used 2-re PAR (Photosynthetic Active Radiation, 400-700 nm) sensors made by I M A G - D L O (Wageningen, The Netherlands) to measure the photon flux density in lamol m - 2 s-1. The photon flux density is used to calculate the average light intensity as is explained later. 2.1.3. Reactor Chlamydomonas was cultivated under non-aseptic conditions in two glass 0.6 1 air-lift loop reactors (Fig. 1). The reactors were equipped with a water jacket connected to a temperature-controlled water bath (25~ The pH was maintained at 7.0 by automatic addition of 1 N sodium-hydroxide solution. The applied gas-flow rate was 30.9 1 h-~: the gas was a mixture of air and carbon dioxide (3% v/v CO2). The downcomer of one reactor was sealed with aluminum foil to create a dark part and the algal suspension circulated between this dark part and the illuminated part. The circulation time (tc) through the loop and the residence time in the downcomer (td) of the reactor liquid were determined by following the movement of a small colored K-carrageenan gel bead (2.6% w/v). tc was 12.9 s and td was 4.4 s leading to a light fraction (E) of 0.66 ( = { t c - td}/tc). The reactors were placed in a closed cabinet with seven fluorescent light tubes placed horizontally at one side (Philips TLD HF, 32W, color

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M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

~6

ily and it is a compromise between a low level of mutual shading and a cell density high enough for measurements of biomass characteristics and accurate turbidostat control. All these operations were monitored by a datalogger (CR10, Campbell Scientific Ltd., UK). I e

O"

-3

||

o

o

o~

7"---8

Fig. 1. Air-lift loop reactor used for cultivation of C. reinhardtii (front view), (1) gas inlet; (2) water jacket; (3) quantum sensor; (4) pH electrode; (5) suspension outlet; (6) medium inlet; (7) direction of suspension flow; (8) riser; (9) downcomer. 94). Both reactors were placed close to the tubes. One fluorescent tube (Pope FTD, 18W, color 94) was placed vertically at the opposite side of each ALR. The photon flux impinging at a certain height on the surface of the riser of one of the reactors was measured in four directions. The sum of these fluxes was divided by 2 to obtain an estimate of the average flux crossing a flat surface from both sides, called the average light intensity (I); I was 240 ~tmol m - 2 S-1. The reactors were illuminated 24 h a day; no day/night cycle was applied. The reactors were operated as turbidostats. Each reactor was therefore equipped with a quantum sensor positioned behind the riser (Fig. 1) and this sensor was facing the wall with the horizontal fluorescent tubes. When the biomass density increased the photon flux impinging on the sensors decreased. On the moment the photon flux was lower than the set point, 70% of the maximal flux measured when the reactor contained medium without algae, the reactors were automatically diluted until the set point was reached again. This set point was chosen arbitrar-

2.1.4. Specific growth rate The reactors were inoculated with pure C. reinhardtii cultures, maintained on agar slants and they were operated as turbidostats for 66 days. The dilution rates of the reactors were determined by daily measurement of the diluted culture volumes. Since the reactors were operated as turbidostats the biomass density in each reactor was constant and the specific growth rate was equal to the measured dilution rate. 2.1.5. Photosynthetic activity Photosynthetic activity (Po2) was measured in a small reaction vessel with a water jacket connected to a temperature-controlled water bath (25~ The vessel was placed in a closed cabinet. The reaction vessel was equipped with a Clarktype oxygen electrode (YSI 5331, Yellow Springs Instruments Co., USA), connected to a read-out unit (YSI 5300) which in turn was connected to a pen-recorder. The reaction vessel was surrounded by four halogen lamps (Osram 44875 M F L Natura, 12V/ 35-50W/30 ~ which were connected to a power supply equipped with two timers to turn on and off the lamps alternately to apply a light/dark cycle. The light intensity in the vessel could be adjusted by means of neutral density filters (Schott NG-filters, Schott Glaswerke, Germany). The photon flux in the vessel was measured by placing a quantum sensor in the vessel. The photon flux was measured in eight directions. The sum of these fluxes was divided by 4 to obtain an estimate of the average flux crossing a flat surface from both sides, called the average light intensity (I). For each measurement at one light intensity a fresh 6 ml sample was used consisting of 4 ml 75 mM phosphate buffer of pH 6.8 and 2 ml (diluted) culture suspension from the turbidostats; the oxygen activity in the sample was lowered by

M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

purging nitrogen gas; extra carbon dioxide was added as sodium bicarbonate (25 ~tl, 0.56 M). First, the oxygen evolution rate was measured under illumination and after this dark respiration was recorded when it had reached a constant rate. The photosynthetic activity was calculated by adding the measured dark respiration rate to the oxygen evolution rate.

2.1.6. Chlorophyll-a A known volume of the algal suspension (containing 24-98 g of chlorophyll-a) was filtered through a glass-fiber filter (Whatman GF/F) coated with a thin layer of CaCO 3. The filter was put in a glass tube with screw-cap. The extraction was done with 10 ml of 90% v/v ethanol and was facilitated by heating the solution for 3 min (78~ in closed tubes. The tubes were stored in the dark at 4~ to complete extraction overnight. After centrifugation the chlorophyll-a concentration was determined according to Nusch (1980). 2.1.7. Dry weight A known volume (20-50 ml) of the algal suspension was filtered through a pre-dried and preweighed glass-fiber filter (Whatman GF/F). The biomass on top of the filter was washed by filtering demineralised water. The filter with biomass was dried at 105~ and allowed to cool in a desiccator. After this the filter was weighed again. 2.2. Experiments 2 and 3: C. sorok&&na 2.2.1. Organism and medium The chlorophyt C. sorok&iana (CCAP 211/8k) was obtained from the Culture Collection of Algae and Protozoa (Ambleside, UK). This organism was also used by Lee and Pirt (1981) and was called Chlorella vulgar& (Sorokin strain). We used the same A9 medium as described by Lee and Pirt (1981). Since this medium was developed for high density cultures we decided to half the concentration of most components. Only the buffer concentration was maintained at 19.5 mM during the experiments in the bubble column. During the experiments in the flat panel reactor the phos-

327

phate buffer was decreased to 4.85 mM and the urea concentration was also decreased from 33 mM in the bubble column to 10 mM in the flat panel. The medium was not autoclaved but was stored in the dark as four separate solutions. Prior to an experiment the solutions were combined to obtain medium for one week. Growth of contaminant organisms in the medium did not occur in this period.

2.2.2. Reactor C. sorokiniana was cultivated in two types of reactors with different light sources. The first type was a glass bubble column with 380 ml liquid volume with a gas inlet of sintered glass in the bottom. This bubble column was illuminated from two opposite sites by a series of six halogen lamps (Osram Decostar Titan, 12V/50W/60 ~ placed on top of each other to illuminate the whole of the column length. This reactor was illuminated 24 h a day; again no day/night cycle was applied. A 2-rt PAR sensor was continuously placed in the center of the bubble column containing the growing culture suspension The signal of this diode was used to apply a turbidostat control. The photon flux in the center of the column was allowed to drop to 70% of the value measured when the reactor contained medium without algae. The average light intensity in the bubble column was measured in an empty column without liquid. The photon flux density was measured on five different heights and at each height in four directions. The sum of these 20 measurements was divided by 10 to obtain an estimate of the average flux crossing a flat surface from both sides and this was called the average light intensity (I). The second type of reactor was a plexiglass flat panel of 225 ml liquid volume and a depth ( = light path) of 1 cm. The height and width of the rectangular panel was 28 cm and 10 cm, respectively. At the bottom gas was injected via an inlet of sintered glass. The complete reactor surface was illuminated by a screen (9 x 28 cm) of 578 light emitting diodes (LEDs). The emission wavelength of the LEDs (Kingbright, type L 53 SRC E) was 660 nm. Matthijs et al. (1996) concluded

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M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

there was no need for an additional supply of blue light when using these red LEDs for cultivation of Chlorella pyrenoidosa. The photon flux density leaving the reactor at the opposite side was continuously measured by a 2-~ PAR sensor (LICOR, type LI-190 SA) and this sensor was used to apply turbidostat control. The photon flux leaving the reactor was allowed to drop to 50% of the value measured when the reactor contained medium without algae. The average light intensity in the reactor was measured as the photon flux leaving the reactor, which contained medium without algae. In this flat panel reactor C. sorokiniana was illuminated according to a 14/10 h day/night cycle. Both reactors were equipped with a water jacket connected to a temperature-controlled water bath (35.5-37.5~ Carbon dioxide was added by bubbling the reactors with a carbon dioxide/air mixture, 3 - 5 % v/v CO2. The pH was controlled between 6.5 and 7.2 by automatic addition of 1 N sodium hydroxide or hydrochloric acid. The light sources of both reactors were connected to power supplies each equipped with two timers to turn on and off the lamps alternately to apply a light/dark cycle. 2.2.3. Specific growth rate 50 ml batch cultures were inoculated from pure cultures maintained on agar slants. The batches were cultivated under low light ( < 100 ~tmol m - 2 s-1) and used to inoculate the turbidostat reactors. The turbidostats were operated non-aseptically for one or two weeks under a certain light regime. During this period the specific growth rate was determined over 3 - 5 days by daily measurement of the diluted culture volume. Since the reactors were operated as turbidostats the biomass density in each reactor was constant and the specific growth rate was equal to the measured dilution rate. The growth rate measurements on the different days were used to calculate the 95%confidence interval. After each experimental run at a certain light regime the reactors were emptied, cleaned and inoculated for a new run at another light regime.

3. Results and discussion 3.1. Experiment 1: C. reinhardtii

The air-lift loop reactors were inoculated and 3 days later, denoted as day 0, the biomass density had reached the set point at which the reactors were automatically diluted. From day 0 until day 66 the reactors were operated as turbidostats and the dilution rate was followed. After about a week a considerable amount of algae attached to the reactor surface. The PAR-sensors controlling the dilution rates were placed behind the riser. In the risers attachment of algae and concomitant biofilm growth were negligible due to the rising gas bubbles. As a consequence, the biomass density in the suspension was maintained constant. Attached algae were detached once or twice a day resulting in a temporal increase of biomass density and, consequently, optical density. After detachment the dilution rate increased until the turbidostat set point was reached again. The extra volume diluted in the hour following detachment was estimated and subtracted from the volume diluted daily. Detaxzhment was done by scraping the inner surface with a magnet or with a rubber ring connected to a stick. The magnet was moved around by another magnet outside of the reactor. The specific growth rate of both cultures was measured almost continuously by following the dilution rate. The average specific growth rate of the intermittently illuminated culture, 0.11 h - ~, was 69% of the average growth rate of the continuously illuminated culture, 0.16 h - 1 (Table 2). The specific growth rate of 0.16 h - 1 is higher than the maximal growth rate we measured in another experiment (not shown) with C. reinTable 2 Average specific growth rate (/~) and dry weight concentration (DW) in the continuously and intermittently illuminated cultures during days 0-66. STD represents the estimated standard deviation

Continuous Intermittent

/1 (h- ~)

DW (g 1- ~) average (n)

STD

0.16 0.11

0.22 (6) 0.16 (6)

0.02 0.02

M. Janssen et al. ,'Journal of Biotechnology 70 (1999) 323-333

hardtii, 0.14 h-1. The light intensity of 240 lamol m -2 s -1 obviously is sufficient for maximal growth and this is said to be saturating. The intermittently illuminated culture was exposed to light 66% of the time. Hence the specific growth rate was proportional to the fraction of time the algae were exposed to light. Although the specific growth rate of both cultures is measured, we do not know whether the biomass yield on light energy has changed without information about the amount of light energy that is absorbed by the biomass. The overall biomass yield on light energy (Y~,E) in dry weight per amount of absorbed photons (g mol-1) is calculated according to Eq. (1). x./~ Y~,F-=E. e

[ggmol-1]

329

1.0

0.8

--

-

ratio chl-a content continuous 9intermittent

oo 0 . 6 !

o 0.40.20.0 0

I

I

I

I

I

I

10

20

30

40

50

60

70

Time (days) Fig. 2. Ratio of the chlorophyll-a (chl-a) content of algal biomass from the continuously and intermittently illuminated cultures during the period of operation (e).

(1)

Yx, E represents the ratio of biomass production over energy consumption, including maintenance requirements. The volumetric light absorption rate of the algal suspension (E in gmol 1-~ h-~) is not known. But E will be the same under continuous and intermittent illumination because the optical density of the cultures in both turbidostats was equal. The biomass densities (x) of both cultures were determined several times as dry weight per volume during the 66 days experimental period and are presented in Table 2. The densities of the continuously and intermittently illuminated cultures were 0.22 and 0.16 g 1-~, respectively. The ratio of the yield under continuous and intermittent illumination now can be calculated and was 1"0.76. Yx, E decreased under intermittent illumination in comparison to continuous illumination. The specific light absorption of the intermittently illuminated culture obviously is higher because the biomass density is lower in comparison to continuous illumination. This is caused by a difference in the chlorophyll-a (chl-a) content of both cultures. The chl-a content of the continuously illuminated culture was twice as low as the chl-a content of the intermittently illuminated culture, 0.51 _+ 0.057 (95% confidence interval). This ratio was determined several times during the experimental period and is presented in Fig. 2.

The intermittently illuminated culture acclimated to this light regime by increasing its chl-a content, enabling the algal cells to intercept a larger part of the photon flux. This was also reported in other investigations (Qu6guiner and Legendre, 1986; Shin et al., 1987). The higher chl-a content, however, could not prevent the decrease of the specific growth rate of C. rein~ hardtii under intermittent illumination in comparison to continuous illumination and consequently also y,. E decreased. Increased pigmentation is also found during the process of acclimation to low light intensities, which is called photoacclimation (Falkowski and LaRoche, 1991). Grobbelaar et al. (1996) already concluded that one of the most important factors influencing photosynthetic rates under continuous or intermittent illumination was whether algae were low or high light acclimated. Low light/dark frequencies (20 s period) were perceived as low light conditions and high light/dark frequencies (20 ms period) as high light conditions. Our results are in agreement with these findings since we applied medium frequency light/dark cycles and C. reinhardtii showed low light acclimation. Next to the specific growth rate also the photosynthetic activity (Po2), oxygen production per dry weight, was measured for both C. reinhardtii cultures. This was done at several light intensities to obtain a so-called photosynthesis-irradiance

330

M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

(PI) curve. Po2 of the intermittently illuminated culture was determined under intermittent illumination and Po2 of the continuously illuminated culture was determined under continuous illumination. The results are graphically represented in Fig. 3. The data were fitted with the hyperbolic tangent model (Chalker, 1980), see Eq. (2). PO2

~-- P 0 2

....

"

tan h ( P o~

.I)

[ m g g - l h -1]

9m a x

(2)

In this model, I is the light intensity during the light period in the case of intermittent illumination. At increasing light intensities the slope decreases and the activity increases until the maximal activity (Po2, max) is reached. The fitted constants, the initial slope of the PI curve, and Po 2,m a x are presented in Table 3. These constants were used to calculate the specific oxygen production rates in the turbidostats on a dry weight basis. The light intensity in the turbidostats is 240 ~tmol m - 2 S - - 1 or lower. At this light intensity the Po2 is 248 and 212 mg O2 g-1 h-1 for the continuously and intermittently illuminated cultures under continuous and intermittent illumina-

300 -

..

_-, _-

"7 180 "~

O

j

--'-" 240 -

120 60 0

0

I

1

I

I

1

300

600

900

1200

1500

I (~tmol.m'2.s- 1) Fig. 3. Photosynthetic activity (Po2), oxygen production per dry weight, versus light intensity (I) for the continuously (O) and intermittently ( 9 illuminated cultures under continuous and intermittent illumination (8.5/4.4 s light/dark cycle), respectively. Samples from the turbidostats were analyzed on days 64 and 66, respectively. The light intensity (I) on the x-axis represents the intensity in the light period; the solid lines represent a curve fit with the hyperbolic tangent model.

Table 3 Po2 . . . . and 95% confidence intervals as determined from the data presented in Fig. 3 by a curve fit of the hyperbolic tangent model. The chl-a content on dry weight basis of the continuously and intermittently illuminated cultures was 10.3 and 20.2 mg g - l , respectively

Continuous Intermittent

~(mggh - l (mol m -2 s - l ) - ! )

(mg g-1 h - l )

1.49 + 0.43 1.19 + 0.22

297 + 37 270 _+ 21

P o 2, m a x

tion, respectively. The lower photosynthetic activity per dry weight of the intermittently illuminated culture under intermittent illumination corresponds to the lower specific growth rate of this culture. The decrease of the photosynthetic activity under intermittent illumination, however, is not proportional to the decrease in specific growth rate under intermittent illumination compared to continuous illumination. Under these circumstances specific growth rate and oxygen production do not seem to be linearly correlated. 3.2. Experiments 2 and 3: C. sorokiniana

The specific growth rate of C. sorokiniana was measured under continuous illumination at four different light intensities in the bubble column. The results of these measurements are shown in Fig. 4. As expected the specific growth rate increased with an increasing light intensity. At 630 ~tmol m - 2 s - 1 a specific growth rate of 0.27 h-1 was measured. Lee and Pirt (1981) measured a maximal specific growth rate of 0.235 h-1 for C. sorokiniana and 630 pmol m - 2 s-1 obviously is saturating. At 630 lamol m - 2 S-1 C. sorokiniana was cultivated under a 30/10 s light/dark cycle and the average specific growth rate was 0.18 h-1. This corresponds to a decrease of 33% in comparison to continuous illumination. Lee and Pirt (1981) and Pirt (1986) suggested that under this light/ dark cycle C. sorokiniana would be able to maintain the maximal growth rate. We were not able to confirm their conclusion and we found a reduction of the specific growth rate proportional to the fraction of time the culture was exposed to

M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

light. Unfortunately the variation of the measured growth rates was considerable. This was caused by attachment of algae to the reactor surface and concomitant biofilm growth. C. sorokiniana was also cultivated under intermittent illumination at a low light intensity of 58 ~mol m - 2 s-1. Again a 30/10 s light/dark cycle was applied and the average specific growth rate was 0.060 h-1 in comparison to 0.077 h-1 under continuous illumination. Although the confidence intervals of the averages are big, the specific growth rate decreased by 22% under intermittent illumination in comparison to continuous illumination. Similar experiments were done in the other turbidostat, the flat panel illuminated with the LED screen, at 250-290 ~tmol m -2 s-~. The results of these experiments are shown in Fig. 5. The specific growth rate under a large light fraction of 0.88 was 0.15 h-~ and this is low in comparison to the growth rates measured in the bubble column. The introduction of a 10 h night period caused a reduction in the specific growth rate. Furthermore, the light intensity could be just too low to support the maximal growth rate and maybe is sub-saturating. In Fig. 5 the specific growth rate is plotted as a function of the light fraction (e). The dotted line 0.35

0.28 0.21 "7 =t. 0 . 1 4 continuous illumination intermittent illumination

0.07 -

0.00 0

i

i

I

l

~

100

200

300

400

500

600

700

I (~tmol.m'2.s"1) Fig. 4. Specific growth rate (p) of C. sorokiniana under continuous illumination (O) and intermittent illumination (A), 30/10 s light/dark cycle. Error bars represent the 95% confidence intervals. The light intensity (I) on the x-axis represents the intensity in the light period; the intensity during intermittent illumination was increased by 10 lamol m - 2 s-~ in this graph to allow better comparison of the intervals.

331

25 /

0.20 -

J

0.16 -

56 s / / 1

0.12 -

/// 87s

::s

I

0.08 0.04 -

0.00 0.0

I

I

I

0.2

0.4

0.6

i 0.8

1.0

e(-) Fig. 5. Specific growth rate (it) of C. sorokiniana under intermittent illumination of different cycle duration (tr and light fraction (e) at 250-290 lamol m - 2 s-~, presented as a function of e (O). tr is given in seconds in the graph. The error bars represent the 95% confidence intervals.

represents the specific growth rate when it would be proportional to e. We assumed that the maximal growth rate could be obtained by extrapolating the line from the origin to the point corresponding to the highest growth rate at a light fraction of 0.88 and cycle duration of 25 s. All measured growth rates are on top or below the dotted line. At a low light fraction of 0.32 the growth rate decreased even more than proportional to e. Although large variations occurred in the growth rate measurements all three experiments using C. reinhardtii and C. sorokiniana showed that the specific growth rate decreased proportional or more than proportional to the fraction of time the algae were exposed to light under intermittent illumination in comparison to continuous illumination. We conclude that these algae are not able to store light energy during the light period to sustain growth in the dark period at the same level as under continuous illumination as was suggested by Lee and Pirt (1981) and Pirt (1986). At higher light intensities leading to photoinhibition ( > 600 lamol m - 2 s - 1 ) , algae could react quite differently to intermittent illumination. Under these intensities the dark period could provide time to recover from photoinhibitory damage as was suggested by Merchuk et al. (1998).

332

M. Janssen et al./Journal of Biotechnology 70 (1999) 323-333

In this study accurate determination of growth rates in dilute culture suspensions was negatively affected by a t t a c h m e n t of algae to glass or other surfaces in the reactor. This is an i m p o r t a n t aspect, which should be kept in mind when designing experiments. Wall growth itself and also the influence of wall growth on measurements should be minimized. Finally, we found that the overall biomass yield (specific growth rate over specific light absorption rate) can vary significantly under different light regimes. The overall biomass yield of C. reinhardtii under an 8.5/4.4 s light/dark cycle was considerably lower than the yield under continuous illumination. As a result, the dark period will have to be shorter than 4.4 s for optimal light utilization efficiency. Moreover, it is not possible to increase the efficiency of C. reinhardtii by applying light/ dark cycles of m e d i u m duration, 10-100 s, at a saturating light intensity of 240 ~tmol m - 2 S-1.

Acknowledgements We are grateful to S O N for providing a scholarship for this research within the f r a m e w o r k of the p r o g r a m for ' Y o u n g Chemists', no. 4030-08.

Appendix A. Nomenclature PAR

L,E

E P%

Photosynthetic active radiation, all photons between 400 and 700 nm light intensity or [gmol rn -2 s - l ] p h o t o n flux density in P A R range fraction of time [-] algae were illuminated during light/ dark cycle yield of biomass [g lamol-1] on light energy biomass density [g 1-1] specific growth [h-1] rate volumetric light [lamol 1-~ h-~] absorption rate specific photosynthetic activity [mg g-1 h-1]

Po 2, max m a x i m a l specific

photosynthetic activity in PI curve initial slope of PI curve

[mg

g-1

h-l]

[mg g-1 h-1 (~tmol m -2 s-1)-1]

References Akimoto. M., Yamada, H., Ohtaguchi, K., Koide, K., 1997. Photoautotrophic cultivation of the green alga Chlamydomonas reinhardtii as a method for carbon dioxide fixation and alpha-linolenic acid production. J. Am. Oil Chem. Soc. 74, 181-183. Becker. E.W., 1994. Microalgae: Biotechnology and Microbiology. Cambridge University Press, Cambridge. Chalker. B.E., 1980. Modeling light saturation curves for photosynthesis: an exponential function. J. Theor. Biol. 84, 205-215. Chisti, M.Y.. 1989. Airlift Bioreactors. Elsevier, London. Falkowski, P.G., LaRoche, J., 1991. Minireview: acclimation to spectral irradiance in algae. J. Phycol. 27, 8-14. Grobbelaar, J.U., 1989. Do light/dark cycles of medium frequency enhance phytoplankton productivity? J. Appl. Phycol. 1, 333- 340. Grobbelaar, J.U., 1991. The influence of light/dark cycles in mixed algal cultures on their productivity. Bioresour. Technol. 38, 189-194. Grobbelaar, J.U., Kroon, B., Burger-Wiersma, T., Mur, L.R., 1992. Influence of medium frequency light/dark cycles of equal duration on the photosynthesis and respiration of Chlorella pyrenoidosa. Hydrobiologia 238, 53-62. Grobbelaar. J.U., Nedbal, L., Tich~, V., 1996. Influence of high frequency light dark fluctuations on photosynthetic characteristics of microalgae photoacclimated to different light intensities and implications for mass algal cultivation. J. Appl. Phycol. 8, 335-343. Harris, E.H., 1989. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego. Hu. Q., Richmond. A., 1996. Productivity and photosynthetic efficiency of Spirulina platensis as affected by light intensity algal density and rate of mixing in a flat plate photobioreactor. J. Appl. Phycol. 8, 139-145. Hu, Q., Guterman, H., Richmond, A., 1996. A flat inclined modular photobioreactor for outdoor mass cultivation of photoautotrophs. Biotechnol. Bioeng. 51, 51-60. Hu, Q.. Kurano, N., Kawachi, M., Iwasaki, I., Miyachi, S., 1998. Ultrahigh-cell-density culture of a marine green alga Chlorococcum litterola in a flat-plate photobioreactor. Appl. Microbiol. Biotechnol. 49, 655-662. Laws, E.A., Terry, K.L., Wickman, J., Chalup, M.S., 1983. A simple algal production system designed to utilize the flashing light effect. Biotechnol. Bioeng. 25, 2319-2335. Lee, Y.K., Pirt, S.J., 1981. Energetics of photosynthetic algalgrowth: influence of intermittent illumination in short (40 s) cycles. J. Gen. Microbiol. 124, 43-52.

M. Janssen et al. ,/Journal of Biotechnology 70 (1999) 323-333 Matthijs, H.C.P., Balke, H., Hes, U.M.V., Kroon, B.M.A., Mur, L.R., Binot, R.A., I996. Application of light-emitting diodes in bioreactors: flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa). Biotechnol. Bioeng. 50, 98-107. Merchuk, J.C., Ronen, M., Giris, S., Arad, S.M., 1998. Light/ dark cycles in the growth of the red microalga Porphyridium sp. Biotechnol. Bioeng. 59, 705-713. Nedbal, L., Tich~, V., Xiong, F., Grobbelaar, J.U., 1996. Microscopic green algae and cyanobacteria in highfrequency intermittent light. J. Appl. Phycol. 8, 325333. Nusch, E.A., 1980. Comparison of different methods for chlorophyll and phaeopigrnent determination. Arch. Hydrobiol. Beiheft 14, 14- 36. Pirt, S.J., 1986. The thermodynamic efficiency (quantum demand) and dynamics of photosynthetic growth. New Phytol. 102, 3-37. Qu6guiner, B., Legendre, L., 1986. Phytoplankton photosynthetic adaptation to high frequency light fluctuations simulating those induced by sea surface waves. Mar. Biol. 90, 483-491. Rao, K.K., Hall, D.O., 1996. Hydrogen production by cyanobacteria: Potential problems and prospects. J. Mar. Biotechnol. 4, 10-15. Richmond, A., 1996. Efficient utilization of high irradiance for production of photoautotrophic cell mass: a survey. J. Appl. Phycol. 8, 381-387.

333

Schulz, R., 1996. Hydrogenases and hydrogen production in eukaryotic organisms and cyanobacteria. J. Mar. Biotechnol. 4, 16-22. Sharkey, D.S., Seemann, J.R., Pearcy, R.W., 1986a. Contribution of metabolites of photosynthesis to postillumination CO2 assimilation in response to lightflecks. Plant Physiol. 82, 1063-1068. Sharkey, D.S., Stitt, M., Heineke, D., Gerhardt, R., Raschke, K., Heldt, H.W., 1986b. Limitation of photosynthesis by carbon metabolism. II. O2-insensitive CO2 uptake results from limitation of triose phosphate utilization. Plant Physiol. 81, 1123-1129. Shin, C.N., Rhee, G.-Y., Chen, J., 1987. Phosphate requirement, photosynthesis and diel cell cycle of Scenedesmus obliquus under fluctuating light. Can. J. Fish. Aquat. Sci. 44, 1753-1758. Stitt, M., 1986. Limitation of photosynthesis by carbon metabolism. I. Evidence for excess electron transport capacity in leaves carrying out photosynthesis in saturating light and CO 2. Plant Physiol. 81, 1115-1122. Walsh, P., Legendre, L., 1982. Effets des fluctuations rapides de la lumi~re sur la photosynth6se du phytoplancton. J. Plankton Res. 4, 313-327. Yoshihara, K.I., Nagase, H., Eguchi, K., Hirata, K., Miyamoto, K., 1996. Biological elimination of nitric oxide and carbon dioxide from flue gas by marine microalga NOA-113 cultivated in a long tubular photobioreactor. J. Ferment. Bioeng. 82, 351-354.

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JOU

reNAl.

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 335-342

Modelling of a continuous pilot photobioreactor for microalgae production Daniel Baquerisse b,,, St6phanie Nouals a,b Ars6ne Isambert b, Patrick Ferreira dos Santos a, G6rard Durand b a Thallia Pharmaceuticals S.A, l'Orke d'Ecully, 5 chemin de la ForestiOre, 69130 Ecully, France b Ecole Centrale Paris, Laboratoire de Chimie et Gknie des Prockdks, Grande voie des vignes, 92295 Ch6tenay-Malabry, France

Received 8 October 1998; received in revised form 26 November 1998; accepted 22 December 1998

Abstract

In this study, a model of a continuous pilot photobioreactor for microalgae production is proposed. Three aspects have been studied: the modelling of kinetic growth, the gas-liquid transfer and the hydrodynamics in the photobioreactor. The modelling of each aspect has been developed with the dynamic simulation software SpeedUp, after experimental studies, then validated step-by-step. The connection of these three aspects aims to predict and optimise biomass production of the pilot plant. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Porphyridium purpureum; Tubular photobioreactor; Growth model; Gas-transfer modelling; Hydrodynamics modelling

I. Introduction

Modelling of biotechnological processes becomes an important aid. It allows the best understanding of biological phenomena, hydrodynamics, thermodynamics and the interactions of these domains. Given a set of input data, a model is used to predict the output response of the process. A model can be used to solve differ-

* Corresponding author. Fax: + 33-1-41131554. E-mail addresses: [email protected] (D. Baquerisse),

[email protected] (S. Nouals)

ent types of problems like design, economical optimisation, process synthesis, and control. The aim of our work is to describe with a set of equations the behaviour of a microalga in a tubular photobioreactor. It implies the comprehension of the effects of different variables on microalgal growth. Mixing effects in the reactor and gas-liquid mass transfer must also be taken into account. This study includes an experimental part which allows the determination of interactions between different variables and the growth rate. A hydrodynamic study has been done to analyse the flow inside the reactor. At last, a gas-liquid transfer study allows to control variables of interest in growth modelling.

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00085-1

336

D. Baquerisse et al./'Journal of Biotechnology 70 (1999) 335-342

2. Material and methods

2.1. Alga and growth conditions Porphyridium purpureum 1380-1A strain, cultured in the photobioreactor and used for each experimental study, has been obtained from the Sammlung von Algenkulturen Pflanzenphysiologister Institut der Universit/it G6ttingen. The cells have been maintained in liquid culture, on Hemerick medium (Hemerick, 1973). The algal suspensions have been subcultured, by transferring cells to fresh medium every two weeks to maintain cells in exponential growth phase. 2.2. The photobioreactor The reactor we used is a continuous pilot-scale tubular photobioreactor of the Thallia Pharmaceuticals company. It is made of a horizontal snaked tube (length: 84 m; internal diameter: 2.4 cm). This one is connected to an airlift which is composed of an ascending and a descending tube (height: 2.5 m) separated by a 7 1 broth recipient. The total volume of this culture system is 50 1. The horizontal tube is artificially illuminated under white light (halogen lamp) with an intensity of 500 ~tE m - 2 s-1 and a 14/10 photoperiod. In addition, the horizontal tube is immersed in water of which the temperature is controlled around 25~ pH is also controlled by injection of carbon dioxide at the horizontal tube entry.

2.3. Growth kinetics experiments For growth kinetics experiments, we have chosen to work in batch mode with small volume photobioreactor (2.5 1). These thermoregulated double-jacket cylindrical reactors have been illuminated from three sides by fluorescent cool tubes (OSRAM L30W/77 for pink light and OSRAM L30W/72 for white light). An air feed enriched in carbon dioxide has been permitted the supply of carbon and the agitation of the culture. The aim of this experimental work has been to study the effects of different operational conditions on the growth of P. purpureum. In a first time, we studied the effects of: the airflow rate,

the percentage of carbon dioxide in the air and the incident light intensity. Each study on influencing variables has been realised separately, i.e. each factor has been varied on a range centred on its optimal value for the growth of P. purpureum. In the same way, the other operational conditions have been maintained at their optimal value. The optimal values for the growth of P. purpureum and for our little volume reactors are: T = 25~ air-flow = 2.5 --2 V.V.H., %CO2 in the air-flow---2, /in = 120 ~tE m s -1, pH 7 (experimental optimisation done by Thallia Pharmaceuticals Company). Each factor has been varied on an efficient range to obtain a limitation and inhibition of the growth around their optimal value.

2.4. Daily measures A cellular count on Malassez cells and a measure of the Optical Density at 760 nm (which is correlated at the turbidity of our alga) (Dermoun et al., 1992), have been done every day. In the same way, we have measured the incident and outgoing light intensity with a Licor Quantum Photometer and the dissolved carbon dioxide concentration with a probe INGOLD.

2.5. H)'drodynamics experiments The hydrodynamics experiments are implying the experimental determination of the residence time distribution (RTD). For its modelling, the photobioreactor has been separated in two parts, hydrodynamically different (horizontal tubes and airlift). To realise our hydrodynamics study, we have done an impulse of NaC1 to allow normal operation of the photobioreactor during our experiments. Two different gas flow rates have been studied (180 and 540 1 h-1) to obtain different liquid speeds inside the photobioreactor. NaC1 has been measured by use of silvermetry.

2.6. Mass transfer of carbon dioxide in the airlift experiments In our case, gas-liquid mass transfer of C O 2 is

D. Baquerisse et al./ Journal of Biotechnology 70 (1999) 335-342

of prime importance, because the CO2 is the main carbon source. In the aim to study the CO2 mass transfer efficiency, it has been necessary to determine the volumetric mass transfer coefficient KLa (CO2) which permits to characterise the CO2 transfer rate between the gas and liquid phases. According to the literature, volumetric mass transfer coefficients depend on the physical properties of the liquid, the liquid flow and on system and gas injector geometries (Nielsen and Villadsen, 1994a). Our photobioreactor is composed of two parts geometrically and hydrodynamically different, the air-lift and the snaked tube. Thus, the determination of the KLa (CO2) is necessary. In this paper, we present only the study in the air-lift. The calculation of KLa of CO2 has been done from the determination of the K,a of 02: /

KLa(C02) =

.

/ D~

. KLa(02)

(1)

Dco 2

To use this method, we have verified that the absorption of CO2 could be considered as a purely physical process (Talbot et al., 1991; Molina Grima et al., 1993). KLa of 02 has been obtained by the dynamic method with a probe INGOLD (Leveau and Bouix, 1988). A study of the effect of the airflow rate on KLa of CO2 has been done, coupled to a study of the viscosity effect. Three airflows have been studied and repeated twice for different viscosity, i.e. for different microalgal concentrations in the reactor (Table 1).

2. 7. The modelling software The software used for modelling and simulation is the dynamic simulation software SpeedUp, produced by Aspentech.

3. R e s u l t s and discussion

3.1. Growth kinetics Experiments done in batch process lead to growth curves similar to the one presented on Fig. 1. The analysis of exponential phase growth curve has allowed us to obtain the maximal specific growth rate, /z, which is the key variable of the modelling. In the aim to integrate at our modelling reactor internal variables, we have studied the evolution of incident and outgoing light intensity for each experiment. As Fig. 2 shows, the whole incident light intensity is captured by the culture when cell density is greater than 8 • 106 cells ml-~. Thus, we have defined a variable, which allows us to determine the amount of light intensity accessible per cell. This variable, E, has been defined by Krystallidis (1994): E = (/~N -- IOVT) " A

Experiments

Air-flow(l h - l )

Viscosity (cpoises)

1 2 3 4 5 6

540 540 380 380 170 170

1.209 1.264 1.220 1.272 1.206 1.296

(2)

V'X The treatment of all experimental data has allowed us to see that two internal variables, E and the concentration in dissolved CO2, had effects of limitation and inhibition on the growth rate and so an optimal value. So, the equation proposed by Steele (1977) has appeared as an adequate modelling in our case: I

Table 1 Operational conditions for the mass transfer experiments

337

H -- ]'/max /'opt

i 9e ~ l S ~ ]

(3)

Our choice was done on an extension of such an equation in the case of E and dissolved carbon dioxide concentration. Many authors (Frohlich et al., 1983; Engasser, 1988; Cornet et al., 1992; Krystallidis, 1994; Nielsen and Villadsen, 1994b) have established models in the case of a specific growth rate influenced by more than one substrate. For all these models, the specific growth

D. Baquerisse et al./Journal of Biotechnology 70 (1999) 335-342

338 40

1,6

35

1,4

30

1,2

25

1,0

20

0,8

15

0,6

10

0,4

!

~ ,,o ~ * s

~m ~ ~

0,2 -

0

7,

-

,

,,

,,

200

300

.

100

.....

,

0,0

400

Time (h) 1"-*- Cells ~

OD (760 nm) I

Fig. 1. Typical growth of P. purpureum, strain 1380-1A on Hemerick medium. 140 120

!

m,,. - - w

m - . .

.

m - .

.

a .

l

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.

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~

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cc

9

300

Time (h)

Fig. 2. Evolution of luminous intensity coming in and out the 2.5 1 photobioreactor.

i

400

D. Baquerisse et al./Journal of Biotechnology 70 (1999) 335-342

3.2. Hydrodynamics

rate is obtained by multiplying functions of limitation of the different substrates. Therefore, we have decided to write the complete form of our modelling as follows: fl = flmax

"F,(E)'F2(C02)

The flow has been modelled by a cascade of continuous stirred tank reactors. Indeed such a modelling is able to represent the hydrodynamics of the photobioreactor. The analysis of the experimental curve by calculation of variance and residence time gives the number of reactors in cascade J that allows to represent the flow (Villermaux, 1993). This determination is valid for a symmetrical response of the type of this we obtain for the horizontal part of the photobioreactor. We present in Fig. 4 a response curve of the horizontal part of the photobioreactor to an impulse. It is shown that the deviation between experimental and simulated curves is low and included inside the uncertainty of the chloride concentration measures by silvermetry. When we considered the vertical part of the photobioreactor (airlift), we have had a response that is not symmetrical. The proposed modelling

(4)

We describe only the function of E, the function of C O 2 is similar: E

E . e(1._k_~op)

(5)

F,(E) = ~opt

339

This modelling needs to fit three parameters ]-/max, Eopt, C02opt. We have done this fitting with the batch experiments realised with 2.5 1 photobioreactors. We present here a validation example on a batch experiment, i.e. on an experiment not used to fit our model parameters (Fig. 3). The correlation coefficient of the model with the data used for fitting parameters and validation experiments is RZ = 0.97.

16 14

12 L.., Q

10

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r,j

9

10

9

20

9

w

30

40

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50

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60

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Fig. 3. Validation curve of the proposed growth model.

70

80

90

D. Baquerisse et al. /Journal of Biotechnology 70 (1999) 335-342

340 0,54 !

0

E r,j o

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9

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240

260

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280

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320

Time (s) Experimental - - - - Simulation

Fig. 4. Validation curve of the hydrodynamics model based on a cascade of CSTR.

in this case is inspired of the airlift geometry. A large volume continuous stirred tank reactor (which corresponds to the broth recipient) is included in the modelling to obtain the good response of the airlift. A good accuracy between the experimental and the simulated curves has been obtained by this way. 3.3. Carbon dioxide mass transfer in the airlift

Four experiments were used to model KLa of CO2. We have used a model found in the literature and adapted on the transfer of CO2 in airlift systems (Lenglace, 1986) which is the following: KLa = ~ " F~" q "/

(7)

The parameters ~, fl and ~ were regressed with a good correlation R 2 - 0.99 (Fig. 5). The modelling obtained has been validated on the other experiments. Fig. 6 shows a good accuracy between the results simulated by the modelling and experimental results R 2 = 0.98.

4. Conclusions

The described model seems to reproduce correctly the experimental results. The modular aspect of the photobioreactor modelling is interesting because it allows us to complete the modelling if necessary. For example, the growth modelling is going on with studying the effect of temperature on the growth rate. We are thinking of studying as well the effect of the photoperiod. Indeed, the industrial reactor of the company for which we are working, will be under natural conditions. It could complicate the growth modelling since the literature shows that according to the photoperiod, cells can split up during the dark period (Dermoun, 1987). A model such the Droop one, which is a structured model and relates the specific growth rate (/1) to the intracellular concentration of the limiting nutrients ('cell quota', q) will have perhaps be considered (Droop, 1973, 1983). The mass transfer study might be also completed by the KLa determination in the horizontal tube of the photobioreactor. So, we will have a

D. Baquerisse et al./Journal of Biotechnology 70 (1999) 335-342

341

1,2E-02

y-x 1,0E-02

.,-,, !

R 2 -

r.~

0,9997

'

-

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,

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,

,

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Kla

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(S-1)

Fig. 5. Parameters mass transfer modelling fitting: comparison between experimental and simulated KLa.

1,0E-02

8,0E-03

Y = 1,0974x

,

"-

!

6,0E-03

4,0E-03 ~ v..,.I

2,0E-03

0,0E+00 , 0,0E+00

,

,

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, ,6,0E-03

'I

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Experimental Kla (S-1) Fig. 6. Mass transfer modelling validation: comparison between experimental and simulated KLa.

1,0E-02

342

D. Baquerisse et al. ,/Journal of Biotechnology 70 (1999) 335-342

c o m p l e t e m a s s transfer m o d e l allowing to simulate the CO2 profile along the p h o t o b i o r e a c t o r .

Appendix A. Nomenclature V.V.H

KLa CO 2 Dco 2

002 02 # I

/opt Iouf A V X E

EOPT

C02 OPT ~, ~, ~,

Fo r/

gas flow rate per liquid culture volu m e a n d per h o u r mass transfer coefficient (s -~) c a r b o n dioxide c a r b o n dioxide diffusion coefficient (m 2 s - ' )

oxygen diffusion coefficient (m2 s-') oxygen specific growth rate (h-') maximal specific growth rate (h-') light intensity (E m -2 s-') optimal light intensity (E m -2 s-') incident light intensity (E m -e s-') outgoing light intensity (E m -2 s-') reactor area (m2) culture v o l u m e (m 3) cell c o n c e n t r a t i o n (106 cells m l - ' ) a m o u n t of light intensity accessible per cell (E s - ' 10 -6 cells) o p t i m a l a m o u n t o f light intensity accessible per (E s-1 10-6 cells) o p t i m a l c a r b o n dioxide c o n c e n t r a t i o n (mol 1-l) c o n s t a n t s o f the KLa m o d e l l i n g air flow rate (1 h - ~ ) viscosity (poiseuille)

References Comet, J.F., Dussap, C.G., Dubertret, G., 1992. A structured model for simulation of cultures of the cyanobacterium Spirulina platensis in photobioreactors: II. Identification of kinetic parameters under light and mineral limitations. Biotechnol. Bioeng. 42, 826-834. Dermoun, D., 1987. Ecophysiologie de Porphyridium cruentum. Validation exprrimentale d'un modrle de croissanceEtude de la production de polysaccharides. PHD thesis of

the Universit6 de Technologie de Compirgne, Compirgne, France. Dermoun, D., Chaumont, D., Thebault, J.M., Dauta, A., 1992. Modelling of growth of Porphyridium cruentum in connection with two interdependent factors: light and temperature. Bioresour. Technol. 42, 113-117. Droop, M.R., 1973. Some thoughts on nutrient limitation in algae. J. Phycol. 9, 264-272. Droop, M.R., 1983. 25 years of algal growth kinetics. Botanica Marina 26, 99-112. Engasser, J.M., 1988. Modelisation des Processus de Fermentation. In: Scriban, R. (Ed.), Biotechnologie. Lavoisier Tec et Doc, Paris, pp. 301-326. Frohlich, B.T., Webster, I.A., Ataai, M.M., Shuler, M.L., 1983. Photobioreactors: models for interaction of light intensity, reactor design and algal physiology. Biotechnol. Bioeng. Symp. 13, 331-350. Hemerick, J., 1973. Culture methods and growth measurements. In: Stein, J.R. (Ed.), Handbook of Physiological Methods. Cambridge University Press, Cambridge, pp. 250-260. Krystallidis, A., 1994. Application du grnie des procrdrs aux biotechnologies marines--Etude de faisabilitr, modrlisation et simulation dynamique d'un procrd6 de culture de micro-algues. PHD thesis of the Ecole Centrale Paris, Chgttenay-Malabry, France. Lenglace, C., 1986. Transferts de matirre dans un biorracteur. PHD thesis of the Institut Polytechnique de Lorraine, Nancy, France. Molina Grima, E., Sanchez Prrez, J.A., Garcia Camacho, F., Robles Medina, A., 1993. Gas-liquid transfer of atmospheric CO2 in microalgal cultures. J. Chem. Tech. Biotechnol. 56, 329-337. Nielsen, J., Villadsen, J., 1994a. Mass transfer. In: Bioreaction and Engineering Principles. Plenum, New York, pp. 296313. Nielsen, J., Villadsen, J., 1994b. Modeling of Reactions Kinetics. In: Bioreaction and Engineering Principles. Plenum, New York, pp. 163-224. Leveau, J.Y., Bouix, M., 1988. Le transfert d'oxygrne. In: Scriban, R. (Ed.), Biotechnologie. Lavoisier Tec et Doc, Paris, pp. 243- 245. Steele, J.H., 1977. Microbial kinetics and dynamics. In: Lapidus, L., Amundson, N.R. (Eds.), Chemical Reactor Theory. Prentice Hall, Englewood Cliffs, NJ, pp. 405-483. Talbot, P., Gortares, M.P., Lencki, R.W., De la Noue, J., 1991. Absorption of CO2 in algal mass culture systems: a different characterization approach. Biotechnol. Bioeng. 37, 834-842. Villermaux, J., 1993. Bilans de population et ditribution de temps de sdjour. Moddlisation de l'~coulement et du mdlange dans les rracteurs rrels. In: Grnie de la rraction chimique--Conception et fonctionnement des rracteurs. Lavoisier Tec et Doc, Paris, pp. 159-233.

Biotecbnology ELSEVIER

Journal of Biotechnology 70 (1999) 343-349

Opportunities for marine bioprocess intensification using novel bioreactor design" frequency of barotolerance in microorganisms obtained from surface waters Phillip C. Wright a,* Colin Stevenson b Eileen McEvoy a j. Grant Burgess ~ a Department of Mechanical and Chemical Engineering, Heriot-Watt University, Riccarton, Edinburgh EHI4 4AS, Scotland, UK b CoBoco (UK) Limited, Aberdeen ABI5 6FZ, Scotland, UK Department of Biological Sciences, Heriot-Watt UniversiO', Riccarton, Edinburgh, EH14 4AS, Scotland, UK

Received 29 October 1998; received in revised form 27 November 1998; accepted 22 December 1998

Abstract In the context of marine biochemical systems, opportunities exist for the development of novel reactors, with optimization and conversion of current technologies having the potential to yield more efficient units. A limiting factor in the widespread commercial acceptance of a large range of marine metabolites is the efficient production of, for example, sufficient quantities of antibiotics and nutraceuticals to allow for structural analysis and clinical testing. Conventional methods utilised for physical and chemical process intensification require careful analysis of their potential application to shear-sensitive bioprocess systems. Stress induction, for example, provides one route to marine bioprocess intensification due to the expression of metabolites not otherwise possible. Use of high pressure as a stressing agent and/or intensification tool is discussed, and its potential, demonstrated by showing the existence of barotolerant (at 120 MPa) marine microorganisms obtained from shallow surface waters ( < 1.5 m deep), is shown. Microorganisms associated with the surface of, for example, seaweed show a greater likelihood of being barotolerant. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: High hydrostatic pressure; Stress induction; Bioprocess intensification: Bioreactor: Barotolerance

I. Introduction The oceans, due to their vastness and range of biodiversity, offer the potential for innovative bioprocess engineering, particularly in the areas of novel antibiotic/drug development (Fenical, 1997) * Corresponding author. Tel.: + 44-131-4495111; fax: + 44131-4513129. E-mail address: [email protected] (P.C. Wright)

and nutraceuticals (Brower, 1998). Due to the typically low energy density found in bioreactors there is every likelihood that these bioprocesses can probably be intensified. In nonbiological systems, chemical engineers use increased turbulence, temperature and pressure to achieve the desired intensification. M a n y different reactor configurations are used to do this, including bubble columns (airlift), stirred tanks and

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00086-3

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P.C. Wright et al./Journal of Biotechnology 70 (1999) 343-349

three-phase fluidized beds (Wright and Raper, 1996). However, in biological systems turbulence is not a good option for living materials and much more care must therefore be taken (Chisti and Moo-Young, 1996). With the discovery and increased research into extremophiles, the conventional restrictions of low to moderate bioreactor temperatures and pressures faced by engineers may be circumvented. One particular route to bioprocess intensification (the enhancement of bioprocess productivity) is the harnessing of extremophiles. However, despite the increasing interest and number of these organisms being discovered, the step-change in bioprocess intensity/efficiency that beckons has still not been realised to any great degree. This was highlighted at the recent Extremophiles '98 conference in Japan, where the rate of discovery and characterisation of the mechanisms of extremophiles, rather than process applications, were the main focus of the registrants (Cowan, 1998). It is also obvious from the wider literature that the main thrust of extremophile research rests with (hyper)thermophiles. Screening for thermophiles and hyperthermophiles from the ocean depths is already a widespread practice, with thermostable enzymes such as amylases, proteases and glucosidases being the target (Prieur, 1997). Comparatively little work has been done on barotolerant/barophilic microorganisms.

1.1. High pressure Use of high pressure has some very exciting bioprocessing applications, as new deep-sea microorganisms, particularly members of the Archaea, are being discovered, with the potential for a range of applications in both aerobic and anaerobic environments (DeLong, 1997). One reason for the lack of knowledge on behaviour and novelty of microorganisms obtained from the deep sea is that little enrichment culturing work has been undertaken at very high hydrostatic pressure, despite this being the very environment common to these systems (Prieur, 1997). Both Nelson et al. (1992) and Kato et al. (1998) conducted experiments on marine bacterial strains

taken from large depths. Both found that the growth of these organisms were greater at elevated pressures than at lower pressures. Nelson et al. (1992) reported no growth was achieved at atmospheric pressure for the samples taken from the Mariana Trench. In a significant proportion of conventional bioreactor systems, high pressure or high temperature can have detrimental effects on growth of a wide range of microorganisms. For example, Hauben et al. (1997) reported that pressures in the range of 20 to 130 MPa could inhibit cellular growth, while higher pressures can result in cell death. A significant proportion of the high pressure biotechnology research has been carried out into the food sterilisation and the properties of the resultant treated product (see, for example, Pothakamury et al., 1995; Hill, 1997). Ath6s et al. (1997) carried out experiments on [3-galactosidase derived from Escherichia coli, Aspergillus oryzae and Kluyveromyces lactis, and noted that an important decrease in catalytic activity was observed above 300 MPa, and led to a quite complete inactivation at 500 MPa. However, showing important implications for improved bioprocess intensification, it was found by the same authors (Ath6s et al., 1997) that in the moderately high pressure range of 50 to 250 MPa, the biocatalysed (via [3-galactosidase) lactose hydrolysis reaction could be carried out at a higher temperature, thereby offering enhanced reaction rates. This often synergistic relationship between the important intensification bioprocess variables has also been noted elsewhere (Nelson et al., 1992; Michels and Clark, 1997).

1.2. Aims and scope of present research The scope of this work was to examine a range of options for bioprocess intensification, particularly those arising out of stress induction. A number of high pressure biological systems were examined utilising microorganisms obtained from a range of specific ecological niches. Several different methods have been used to study the effects of pressure on the growth of deep sea microorganisms (Yayanos, 1995; Michels

P.C. Wright et al./Journal of Biotechnology 70 (1999) 343-349

and Clark, 1997), with the pressure being applied either hydrostatically, or hyperbarically. However, no studies have systematically compared the pressure tolerance of marine bacteria recovered from surface waters as distinct from those recovered from deep sea habitats. In this work, a high pressure batch bioreactor (HPBB) was used throughout to create a maximum hydrostatic pressure of 120 MPa and to survey the pressure tolerance of a number of marine bacterial strains from surface waters (1-5m depth: < 50 Pa). In addition, the comparative barotolerance of strains isolated from open water and algal biofilms was investigated. Deep sea strains were not studied, as the aim was to survey barotolerance of strains from surface waters.

2. Materials and methods

2.1. Escherichia coli EMG-23 A standard strain of E. coli EMG-23, to act as a control, was inoculated into 5 ml of nutrient broth and incubated at 25~ with shaking, for 24 hours. Prior to pressure treatment this inoculated solution was further diluted by adding 2 ml of media to give a final cell concentration of approximately 5 x 108 cell m l - 1. A 300 pl control sample was taken from the cell suspension. A 3 ml aliquot from the cell suspension was then added to the HPBB, which was constructed from stainless steel and consisted of a maximum working volume of 3.9 ml. Hydrostatic pressure was supplied to the bacterially inoculated broth by use of an electrically driven piston. The maximum allowable pressure within the cell was 120 MPa, and this pressure was used throughout all experiments. High pressure cycling was used, as it has been shown that this methodology may lower the ability of the biological species to withstand high pressure (Mozhaev et al., 1994; Hill, 1997). After the E. coli inoculated broth was fed into the HPBB, it was subjected to the maximum pressure of 120 MPa for 5 min at a constant temperature of 25~ The pressure was then reduced to 0 MPa and the broth allowed to rest for 5 min. The time for pressurising and de-pressuris-

345

ing was 1-2 s, and therefore, could be classed as essentially instantaneous. This multiple pulse treatment (high pressure cycling) was carried out until the E. coli was subjected to 80 min of pressure. During the test, 300 pl samples of the broth were taken at time intervals of 5, 10, 20, 40 and 80 min.

2.2. Isolation and culture of mar&e stra&s Marine agar (MA) (Difco, Detroit, USA) and marine broth (MB) (Difco) were prepared in accordance with the manufacturers instructions. The 50% MA and 20% MA contained 18.7 and 7.6 g of Difco Marine Broth 2216 per litre of distilled water, respectively (Boyd et al., 1998; MearnsSpragg et al., 1997, 1998). All media were autoclaved prior to use (121 ~ for 15 rain). Specimens of the seaweed Rhodymenia palmata were collected from sites on the South East Coast of Scotland. Portions of the plant were rinsed with sterile sea-water (2 x 10 ml) and a small area was swabbed with a sterile cotton-tipped swab. The swab was then used to directly inoculate plates. For each specimen a range of media were used for isolation (Boyd et al., 1998). The plates were then incubated at room temperature. Colonies were removed and sub-cultured when the plates showed good growth. Colonies were picked on the basis of colony morphology so as to maximise the diversity of strains isolated. Subculturing provided pure isolates that were stored as stabs in MA. These strains were Rhodymenia epiphytes and designated surface strains since they were associated with surface biofilms. Other strains were isolated from water (1-5 m depth) which were not associated with surfaces. G strains were all recovered from sea-water (G548, G538, G51, G55, and G518), whereas KBRP strains were removed from the seaweed biofilms (KBRP1, KBRP4, KPRP16 and KBRP13). All strains used are currently unidentified, however, each strain number does represent a species obtained from a specific ecological niche. Their natural distribution in the environment has not been investigated. Marine strains were inoculated and grown for 5 days, at 25~ in 5 ml of marine broth, with

346

P.C. Wright et al./Journal of Biotechnology 70 (1999) 343-349

shaking. The same experimental technique was used for the marine strains as for the E. coli, except that the samples were spread on a marine agar plate. The samples were then incubated for 1 day at 25~ High pressure suspended cultures proceeded in exactly the same manner as the E. coli.

2.3. Population determination The 300 ~tl pressure-treated samples of either the marine strains or E. coli were diluted to give a range of dilutions down to 10-9 cells/ml. Subsequently, 100 lal samples of these dilutions were then spread onto marine agar plates and incubated at 37~ for 24 h. After the incubation period the colonies on the plates were counted. Non-pressure treated samples of identically inoculated solutions were tested in exactly the same manner to provide a standard. A minimum of two tests were performed for each process condition, thus ensuring reproducibility.

3. Results and discussion

3.1. Barotolerance of marine strains The behaviour of liquid supported marine strains subjected to high pressure is presented in Fig. l(a) and (b). (a) Depicts strains obtained from suspended sea-water (strains G55, G518, G538, G548 and G51). (b) Depicts strains obtained from seaweed surfaces (strains KBRP1, KBRP13, KBRP4 and KBRP16). It is interesting to observe that barotolerance is greater in those bacteria obtained from the biofilms on the surface of seaweed, rather than those obtained from open water. This is particularly interesting because these normally surface associated strains were exposed to the pressure cycling whilst suspended in culture, and not attached to a surface. The major exception to this observation is strain G538, which still showed strong survival even after 80 min of exposure (see Fig. la). Strain G55 has been removed from (a) as it showed no survival at the 5 min sample time.

3.2. Barotolerance of Escherichia coli (EMG-23) The results for the liquid-supported E. coli (Fig. 2) show that E. coli appears to be more pressure tolerant than the marine strains. After 20 rain of exposure to 120 MPa, it can be seen that there has been no significant decrease in the number of colonies initially present. This trend continued up to the maximum pressurised time tested, 80 rain. This was not entirely unexpected, as other strains of E. coli (LMM 1010, LMM 1020 and LMM 1030) have shown barotolerant behaviour at pressures over 220 MPa (Hauben et al., 1997). An obvious implication from this demonstration of barotolerance is the use of this variant of E. coli for expression of foreign proteins at high pressure.

3.3. Implications for process intensified bioreactor design The findings in this paper are important from the standpoint of effective bioprocess engineering because so many of the tested strains are barotolerant. In many reaction systems the application of pressure may lead to enhanced yields of target metabolites within the framework of Le Chfttelier's principle, i.e. high pressure will favour an equilibrium state that results in a negative volume change. In addition, the findings presented here form the first stage of an approach to utilising pressure as a stressing agent. This knowledge can then be coupled to reactor design to lead to an improved, and more intensive, bioprocess. As a lead on to a future bioprocess, strain KBRP1, as shown in Fig. l(b), is barotolerant and it has also produced antimicrobial compounds during bench scale operation. For a significant proportion of bioprocessing operations, the use of barotolerant rather than barophilic microorganisms may be preferred, because isolation and enrichment of barophilic cultures is much more difficult in an engineering sense, as they must remain at these significantly high-pressure levels ( > 50 MPa; Kato et al., 1998).

P.C. Wright et al./Journal of Biotechnology 70 (1999) 343-349

4. Conclusions A number of techniques are being explored as possible routes to bioprocess intensification. To date, most of the techniques fall into the category

347

of alterations to pure transport properties, such as increased turbulence or mass transfer enhancement via bubble size reduction. In this paper, it is discussed that more emphasis could be put onto examination of stress induction as a route to a

100 90 80 70 ~

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- - o - - KBRP13

Fig. l. Behaviour of marine strains under cyclic high-pressure (120 MPa)" (a) sea-water strains; (b) solid surface associated strains.

348

P.C. Wright et al. /Journal of Biotechnology 70 (1999) 343-349

100

..

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more 'intensive' bioprocess. In addition, in line with predictions from Le Chfitelier's principle, bioprocess improvements may be achievable in reaction systems having a negative volume change. The ultimate aim is enhanced bioproduct generation. Manipulation of barotolerant/barophilic properties was suggested as a strong example of this methodology. As demonstrated by the example microorganisms presented here, a number of barotolerant species may be found at non-extremophile conditions. From our sample group it can be seen that surface strains are more barotolerant, even when grown in suspensions, than strains obtained from open water. As a lead on to a future bioprocess, strain KBRP1 has also produced antimicrobial compounds and is being investigated as a potential model species.

Acknowledgements Phillip Wright would like to thank Pfizer Ltd for provision of an Academic Travel Award, The

British Council (Amsterdam) and the N W O (Netherlands Organisation for Scientific Research) for provision of a fellowship to The University of Groningen, as well as The Royal Society for financial support. Colin Stevenson would like to acknowledge The Department of Mechanical and Chemical Engineering (HWU) for the provision of a Vacation Scholarship. Grant Burgess thanks the Natural Environment Research Council (NERC) and The British Council (Tokyo) for financial support.

References Athes, V., Degraeve, P., Cavaill6-Lefebvre, D., Espeillac, S., Lemay. P., Combes, D., 1997. Increased thermostability of three mesophilic b-galactosidases under high pressure. Biotechnol. Lett. 19 (3), 273-276. Boyd, K.G., Mearns-Spragg, A., Brindley, G., Hatzidimitrou, Rennie, A., Bregu, M., Hubble, M.O., Burgess, J.G., 1998. Antifouling potential of epiphytic marine bacteria from the surfaces of marine algae. In: Le Gal, Y., Mueller-Fuega, A. (Eds.), Marine Microorganisms for Industry. Editions IFREMER (Institut Frangais de Recherche pour l'Ex-

P.C. Wright et al./Journal of Biotechnology 70 (1999) 343-349 ploitation de la Mer). Plouzane, France, ISBN 2-90543494-5, pp. 128-136 Brower, V., (August) 1998. Nutraceuticals: Poised for a healthy slice of the healthcare market? Nature Biotech 16, 728-731. Chisti, Y., Moo-Young, M., 1996. Bioprocess intensification through bioreactor engineering. Trans. IChemE. 74 (A), 575-583. Cowan, D.A., 1998. Hot bugs, cold bugs and sushi. TIBTECH 16 (6), 241-242. DeLong, E.F., 1997. Marine microbial diversity: the tip of the iceberg. TIBTECH 15 (6), 203-207. Fenical, W., 1997. New pharmaceuticals from marine organisms. TIBTECH 15 (9), 339-341. Hauben, K.J.A., Bartlett, D.H., Soontjens, C.C.F., Cornelis, K., Wuytack, E.Y., Michiels, C.W., 1997. Escherichia coli mutants resistant to inactivation by high hydrostatic pressure. Appl. Environ. Microbiol. 63 (3), 945-950. Kato, C., Li, L., Nogi, Y., Nakamura, Y., Tamaoka, J., Horikoshi, K., 1998. Extremely barophilic bacteria isolated from the Mariana Trench, challenger deep, at a depth of 11 000 meters. Appl. Environ. Microbiol. 64 (4), 15101513. Mearns-Sprang, A., Boyd, K.G., Hubble, M.O., Burgess, J.G., 1997. Antibiotics from surface associated marine bacteria. Fourth Underw. Sci. Symp. Society for Underwater Technology, London, ISBN 0 906940 31 1, 147-157.

349

Mearns-Spragg, A., Bregu, M., Boyd, K.G., Burgess, J.G., 1998. Cross-species induction and enhancement of antibiotic production by epiphytic bacteria from marine algae and invertebrates after exposure to terrestrial bacteria. Lett. Appl. Microbiol. 27, 142-146. Michels, P.C., Clark, D.S., 1997. Pressure-enhanced activity and stability of a hyperthermophilic protease from a deepsea methanogen. Appl. Environ. Microbiol. 63 (10), 39853991. Mozhaev, V.V., Heremans, K., Frank, J., Masson, P., Balny, C., 1994. Exploiting the effects of high hydrostatic pressure in biotechnological applications. TIBTECH 12 (12), 493501. Nelson, C.M., Schuppenhauer, R., Clark, D.S., 1992. Highpressure high temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth. Appl. Environ. Microbiol. 58 (5), 1789-1793. Hill, S., 12 April 1997. Squeezing the death out of food. New Sci. 1997, 29-32. Pothakamury, U.R., Barbosa-C~inovas, G.V., Swanson, B.G., Meyer, R.S., 1995. The pressure builds for better food processing. Chem. Eng. Prog. March, 45-53. Prieur, D., 1997. Microbiology of deep-sea hydrothermal vents. TIBTECH 15 (7), 242-244. Wright, P.C., Raper, J.A., 1996. A review of some parameters involved in fluidized bed bioreactors. Chem. Eng. Technol. 19 (1), 50-64. Yayanos, A.A., 1995. Microbiology to 10500 meters in the deep sea. Annu. Rev. Microbiol. 49, 777-805.

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Journal of Biotechnology 70 (1999) 351-356

Effect of light-path length in outdoor fiat plate reactors on output rate of cell mass and of EPA in Nannochloropsis sp. Ning Zou, Amos Richmond * Microalgal Biotechnology Laboratory, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede-Boker Campus 84993, Israel Received 12 October 1998; received in revised form 4 December 1998; accepted 22 December 1998

Abstract The effect of light-path length (i.e. reactor width or thickness) of flat plate glass reactors on outdoor production of eicosapentaenoic acid (EPA) and cell mass of Nannochloropsis sp. was tested, using a range of light-paths from 1.3 to 17.0 cm. Volumetric productivity of cell mass and optimal, as well as maximal cell density which represents the highest sustainable cell density under the experimental conditions, decreased with increase in light-path. Daily areal output rate (g dry weight m - 2 d a y - ~) increased with increased light-path, in contrast with results obtained in similar reactors with Spirulina cultures, in which areal output rates increased when the light-path was reduced. Maximal areal productivity of Nannochloropsis sp. (12.8 and 22.4 g ash-free dry weight per day per m 2 of irradiated reactor surfaces, in winter and summer, respectively), reflecting maximal efficiency in light utilization, was obtained with the long light-paths, i.e. 10.4 and 17.0 cm. Increasing the light-path from 1.3 to 17.0 cm resulted in an increase in areal EPA productivity, from 66.7 to 278.2 mg m -2 day -~ in winter and from 232.1 to 515.7 mg m -2 day -~ in summer. This enhancement in areal productivity of EPA stems from increased productivity of cell mass which was associated with the increase in light-path. We concluded that the optimal light-path, which must be defined for each algal species, represents an important parameter which determines optimal culture density (i.e. resulting in the highest output rate of cell mass per irradiated reactor surface), as well as productivity of cell mass and cell products. Under our conditions the optimal light-path for culturing Nannochloropsis in vertical reactors was ca 10 cm. 9 1999 Elsevier Science B.V. All rights reserved.

Keywords: EPA; Light-path; Nannochloropsis sp.; Outdoor cultures; Photobioreactor; Productivity

1. Introduction Light limitation to growth c a n n o t be described solely in terms o f the light flux impinging on the reactor surface, n o r in terms o f the average light * Corresponding author.

flUX available for each cell. Particularly in ultrahigh density cultures, the light regime is the m o s t significant rate-limiting factor of photoautotrophic productivity of cell mass ( H u et al., 1996b, 1998). This s o m e w h a t elusive p a r a m e t e r concerns to overall characteristics involved in the cells' exposure to light, as reflected in light inter-

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N. Zou, A. Richmond/Journal of Biotechnology 70 (1999) 351-356

mittence due to mutual shading as well as in the intensity and duration of the light flashes, shaping together a dominant factor of the light regime, i.e. the characteristics of the light-dark ( L - D ) cycle to which the cells are exposed, in moving back and forth from the dark to the lit volumes in the reactor (Hu et al., 1998). Together with the population density and the rate of mixing, the length of the light-path (i.e. the width of a flat plate reactor) represents an essential parameter in mass cultures, affecting the light regime through its effect on the L - D cycle. The role of the light-path in optimizing reactor performance was recognized by Hu et al. (1996a), who studied the detailed interactions between optimal cell concentration and light-path length in Spirulina platensis cultures grown in flat plate reactors. In the framework of our efforts to optimize biotechnological aspects involved in mass production of Nannochloropsis, we attempted to delineate the optimal light-path in flat plate reactors for maximal areal output rates of cell mass and of EPA.

2. Materials and methods

.

Culture control: pH was monitored by a microprocessor pH meter (WTW). Dissolved oxygen (DO) was monitored by YSI Model 58. Light intensity was measured with a quantum sensor Li-Cor model Li-185A. Temperature was maintained at 29 + 2~ using tap water spray for evaporative cooling on the reactor front and back panels, pH was maintained between 7.2 and 8.0 by adding 1% CO2 into the compressed air stream used for mixing the culture. Flow rate of the CO2-enriched air was 0.84 1 min-1 1-1 culture.

2.2. EPA analysis The pellet obtained by centrifuging the culture at 2000 rpm for 15 rain was freeze dried (Cohen, 1994). To 25 mg freeze-dried pellet were added: (a) 0.25 mg C17 standard solution (Sigma) and (b) 2 ml H2SO4:methanol solution (2% H2SO4). After scrubbing with Ar gas, the sample was stirred and heated to 80~ for 1 h. Subsequently, 1 ml H20 (to stop the reaction) and 1 ml hexane were added, mixing well with vortex. Centrifuging at 3500 rpm for 5 min resulted in two layers, the upper layer filtered through glass fiber and dried with N2. The sample was redissolved in 100 ~tl hexane and finally chromatographed, using 8590 gas chromatography (GC).

2. I. Growth conditions 1. Organism: Nannochloropsis sp. obtained through the courtesy of Mr Odi Zemora, Oceanographic Inst., Eilat. 2. Reactors: flat plate glass reactors (Hu et al., 1996a), with the following light-paths: 1.3, 2.6, 5.2, 10.4 to 17.0 cm. The total irradiated surfaces (i.e. the front and back panels) of all reactors were identical, 0.52 m 2. 3. Growth medium: unbuffered artificial sea water medium, as follows: 2.7% NaC1, 6.6 g 1MgSO4.7H20, 5.6 g 1-1 MgClz.6H20 , 1.5 g 1-1 CaClz'2H20, 1.45 g 1-1 KNO3, 0.12 g I-1 KHzPO4, 0.04 g 1-1 NaHCO3, 0.01 g 1-1 FeC13.6H20, 0.078 g 1-~ Na2-EDTA, 0.01 mg l-1 CuSO4.5HzO ' 0.022 mg 1-~ ZnSO4"7H20, 0.01 mg 1-~ COC12"6H20, 0.18 mg 1-~ MnC12-4H20, 0.006 mg 1-1 Na2MoOa'2H20.

2.3. Determination of cell mass and cell concentration Cell mass was estimated by measuring: (a) total organic carbon (TOC) in the pellet, obtained by 3500 rpm centrifugation of a culture sample for 6 min, and/or (b) by determining ash free dry weight (AFDW). The latter method was used in winter, the former having been used in summer. The ratio of TOC/AFDW was measured several times in duplicates for different reactors throughout the summer, and it ranged between 0.49 and 0.59 (0.04 standard deviation). TOC was thus multiplied by 1.89 to convert it to AFDW. TOC measurement: samples were diluted according the cell density (to about 1 x l0 s cells m l - ~). One ml diluted sample was centrifuged at ca 3500 rpm for 6 min, adding thereafter HgSO4

N. Zou, A. Richmond/Journal of Biotechnology 70 (1999) 351-356

353

centrations were maintained. Cultures with cell concentrations which yielded, at steady state, maximal output rates of cell mass were thereby identified as optimal.

(0.2 g), 1 ml DDW, 1 ml H2SO4, 0.5 ml AgzSO4, and 1 ml 0.25 N K2Cr20 7 to the pellet suspension. Another 1 ml H z S O 4 w a s then added to the test tube, which was incubated at 150~ in an oven for 1 h. When cooled, the mixture was poured into flasks for titration. Three drops of Ferroin solution (1/40 mol/1 redox indicator) were added and the sample was finally titrated with 0.1 N Fe(NH4)2(SO4)2"6H20. Dry weight was measured using preweighted Whatman GF/C (0.45 ~tm) 47-ram ~ glass fiber filters. Cells were washed twice with acidified water (pH 4.0) followed with distilled water. The filtered cells were dried at 105~ overnight. Ash content in these samples was ca 6% of the total dry weight (Hu and Richmond, 1994). Cell count was determined using a 0.0025 mm 2 cytometer, with a light microscope ( x 128 magnification). Optimal cell density for each reactor with a given light-path was determined in continuous cultures along which several steady-state cell con-

3. Results and discussion

The length of the light-path exerted a strong effect on the optimal cell density, defined as that cell concentration which results in the highest areal output rate of cell mass (g A F D W m - 2 day-~) (Fig. 1). As expected in a light-limited system, the shorter the light-path, the higher the optimal cell density became. Through the year, however, no striking differences in optimal cell density were observed in a given light-path, except for the 1.3-cm reactor. In the latter the optimal cell density in summer was distinctly the highest of the year (Fig. 1). We suggest this very significant increase in optimal cell density observed for the 1.3-cm reactor only was due to light intensity:

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N. Zou, A. Richmond/Journal of Biotechnology 70 (1999) 351-356

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in the very short light-path reactor, it represented, in summer, photoinhibitory radiation for Nannochloropsis sp. This light stress could be eliminated only by increasing areal cell density (reducing thereby the intensity of the light climate for the single cell) associated with longer lightpaths. This effect was elucidated for Spirulina platensis cultures which yielded increased productivity upon exposure to very high radiation levels, provided cell density was increased appropriately, in adjustment to increased radiation (Hu et al., 1997). A similar surge in optimal cell density was not observed in the other reactors with longer light-paths, because the D - L cycle frequency (Hu et al., 1998) in the longer light-paths was obviously lower, whereas areal cell number was higher, compared with the 1.3-cm reactor. Cells in the longer light-path reactors were thus exposed to lesser radiation dose, which was not photoinhibitory. The light-path greatly affected the productivity of cell mass (Fig. 2). Volumetric productivity increased ca 7-fold with a 13-fold decrease in lightpath (from 17.0 to 1.3 cm). In contrast, areal productivity (which relates to the irradiated panels of the reactors measuring 0.52 rn2), sharply increased with the increase in light-path, reaching its peak with the 10.4-cm reactor and becoming slightly lower as the light-path was further increased to 17.0 cm (Fig. 2). Clearly, the high

radiation existing in summer (e.g. ca 1800-2100 ~tE m - 2 S-1 for some 5 h at midday) was best utilized in reactors with the longer light-path. These reactors exhibited on the one hand a larger number of cells per irradiated surface (Fig. 3) and on the other hand affected a decrease in the frequency of the L - D cycle (Zou, 1996; Hu et al., 1998; Tredici and Zittelli, 1998). Thus for the slow growing Nannochloropsis cells, the light regime prevailing in association with the more narrow light-paths (to which Spirulina cultures responded well by enhanced productivity (Hu et al., 1996a; Richmond, 1996)), could not be effectively used by Nannochloropsis. This species reached peak areal productivity, reflecting highest net photosynthetic efficiency, only when light per cell was in effect reduced, as a result of being both distributed to a larger number of cells per given irradiated area (cells m - 2 ) and by being exposed to a lower L - D cycle frequency existing in long light-path reactors, compared with the narrower light-paths. The great increase in volumetric productivity caused by reduction of the light-path (Fig. 2) was due to a surge in the growth rate which resulted from reduction in the extent of light-limitation, an advantage of short light-paths. Nevertheless, the overall lower productivity of the short light-path reactors indicated a less efficient use of radiation, 250 (45.0)

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N. Zou, A. Richmond/Journal of Biotechnology 70 (1999) 351-356

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as was readily evident in the reduced areal productivity obtained in these reactors. Cell weight of Nannochloropsis may represent an important parameter in production of food chain components for aquaculture. The lightpath, conceivably through its effect on the light regime, exerted a significant effect on cell weight. This effect, however, was evident only in the 10.4and 17.0-cm reactors and mainly through the spring and summer (Fig. 4). The season of the year, in contrast, affected a large increase in average cell weight (Fig. 4), which may be attributed to the average diurnal culture temperature as well as to the light regime, both factors changing markedly from winter to summer. In addition to environmental factors, cell concentration exerted a clear and consistent effect on cell weight in mass cultures outdoors (Fig. 5). Roughly, doubling cell concentration in a 17.0-cm reactor in winter (from 3.5 to 6.6 x 108 cells per ml) resulted in a reduction of ca 50% in cell weight (Fig. 5). Cultures in other reactors exhibited similar relationships yielding a correlation coefficient (R) of 0.8. Special attention was given in this work to the effect of the light-path on cell content and culture productivity of eicosapentaenoic acid (EPA), an essential nutritional component of Nannochloropsis as an aquacultural feed (Sukenik, 1991; Renaud et al., 1991). The length of the light-path

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was not found to exert a significant effect on cell EPA content, which was however, greatly influenced by the season of the year. The lowest cell EPA was found in winter and the highest in summer, irrespective of the light-path (Fig. 6). The light-path exerted, in contrast, a significant effect on the volumetric productivity of EPA, particularly in summer (Fig. 7). This effect was most prominent in the smaller light-paths, up to 5 cm, above which the light-path did not have a significant effect on EPA productivity. The high EPA production figures reported in this work (i.e. 35.5 mg 1- ~ day-1 and 515.7 mg m -2 d a y - l ) are worth noting, representing the highest reported to 0.3

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N. Zou, A. Richmondi"Journal of Biotechnology 70 (1999) 351-356

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EPA, and to Ms Larissa Shemtov for assistance in measurement of TOC. Ben Frihoff and Donghong Sun provided excellent technical assistance.

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date for Nannochloropsis (Sukenik, 1991). This high EPA productivity is accomplished, according to our interpretation, by use of flat plate glass reactors which facilitate, particularly in the short light-path reactors, maintaining cultures of ultrahigh cell densities (i.e. over 10 g AFDW cell mass l-l), yielding record volumetric productivity. These reactors have two important advantages responsible for the record areal productivity of EPA: being made of fully transparent glass and placed vertically, they are fully exposed from all surfaces to both direct beam and diffuse as well as reflected light. In addition, a simple, yet efficient mixing system readily provides the high turbulence mandatory for a high photosynthetic efficiency and productivity in ultrahigh density cultures.

Acknowledgements The authors wish to acknowledge the advice given by Professor Zvi Cohen. Our thanks to Ms Shosh Didi for assistance in measurement of

References Cohen, Z., 1994. Production potential of eicosapentaenoic acid by Monodus subterraneus. J. Am. Oil Chem. Soc. 71, 941 - 945. Hu, Q., Richmond, A., 1994. Optimizing the population density in Isochrysis galbana growth outdoors in a glass column photobioreactor. J. Appl. Phycol. 6, 391-396. Hu, Q., Guterman, H., Richmond, A., 1996a. A flat inclined modular photobioreactor for outdoor mass cultivation of photoautotrophs. Biotechnol. Bioeng. 51, 51-60. Hu. Q., Guterman, H., Richmond, A., 1996b. Physiological characteristics of Spirulina platensis (cyanobacteria) cultured at ultrahigh cell densities. J. Phycol. 32, 1066-1073. Hu, Q., Hu, Z.Y., Cohen, Z., Richmond, A., 1997. Enhancement of eicosapentaenoic acid (EPA) and r-linolenic acid (GLA) production by manipulating algal density of outdoor cultures of Monodus subterraneus (Eustigmatophyta) and Spirulina platensis (Cyanobacteria). Eur. J. Phycol. 32, 81-86. Hu, Q., Zarmi, Y., Richmond, A., 1998. Combined effects of light intensity, light-path and culture density on output rate of Spirulina platensis (Cyanobacteria). Eur. J. Phycol. 33. 165-171. Renaud, S.M.. Parry, D.L., Thinh, L.V., Kuo, C., Padovan, A., Sammy, N., 1991. Effect of light intensity on the proximate biochemical and fatty acid composition of Isochrvsis sp. and Nannochloropsis oculata for use in tropical aquaculture. J. Appl. Phycol. 3, 43-53. Richmond, A., 1996. Efficient utilization of high irradiance for production of photoautotropic cell mass: a survey. J. Appl. Phycol. 8. 381 - 387. Sukenik, A.. 1991. Ecophysiological considerations in the optimization of eicosapentaenoic acid production by Nannochloropsis sp. (Eustigmatophyceae). Bioresource Technol. 35 (3), 263-269. Tredici, M.R., Zittelli, G.C., 1998. Efficiency of sunlight utilization: tubular versus flat photobioreactors. Biotechnol. Bioeng. 57 (2), 187-197. Zou, N., 1996. Development and application of algal photobioreactors. Ph.D. dissertation. Oceanology Institute of Chinese Academy of Sciences, China.

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Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 357-362

Methanol as alternative carbon source for quicker efficient production of the microalgae Chlorella minutissima" Role of the concentration and frequence of administration K. Kotzabasis a,, A. Hatziathanasiou a,b, M.V. Bengoa-Ruigomez b,c, M. Kentouri a,b p. Divanach b a Department of Biology, University of Crete, PO Box 1470, 71110 Heraklion, Crete, Greece b Institute of Marine Biology, Department of Aquaculture, PO Box 2214, 71003 Heraklion, Crete, Greece c Department of Animal Biology and Ecology, Unit'ersio'of Basque Country, Leioa, Vizcaya, Spain

Received 13 October 1998; received in revised form 8 November 1998; accepted 22 December 1998

Abstract

Autotrophic cultures of the marine microalgae Chlorella minutissima were performed at 13 000 lux continuous illumination in 1 1 chambers fertilised with 0.25 g 1-1 F2 medium and different doses of methanol. This was administered in two ways during two parallel experimental series of 10 days: 0.05, 0.1, 0.5, 1.0 and 5.0% methanol (v/v) in one unique dose at the beginning of the culture and 110 of these (i.e. 0.005.0.01, 0.05, 0.1 and 0.5% methanol (v/v)) in daily doses for the 10-day culture period. Low concentrations of methanol induced a faster increase of cell density and dry weight than control, while high concentrations induced symptoms of toxicity. The higher cell densities and quicker growth were observed in the experiments with daily administration of 0.005 and 0.1% (v/v) methanol, while those with one dose presented an initial boosted growth but a final cell density lower than control. The role of methanol as alternative carbon source for microalgae, as well as its possible impact on the quality of biomass production and on the environment, are discussed. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Methanol; MeOH; Chlorella minutissima; Carbon source

1. Introduction Methanol (MeOH) is one of the simplest organic components recently proved to enhance biomass production in various photosynthetic organisms. In C3 plants, recurring periodic sprays of 10-50% methanol (v/v) were documented to * Corresponding author.

increase biomass production up to 100% ( N o n o m u r a and Benson, 1992; Fall and Benson, 1996). In cultures of the microalga S c e n e d e s m u s obliquus, low methanol concentrations under high light intensities proved to induce strong biomass production (Kotzabasis et al., 1999). The mode of action of methanol, which depends on concentration, light intensity and cell concentration, is not yet fully understood. A posi-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00088-7

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K. Kotzabasis et al./Journal of Biotechnology 70 (1999) 357-362

tive effect is observed exclusively under autotrophic (photosynthetic) conditions, while under heterotrophic growth it is toxic (Nonomura and Benson, 1992; Kotzabasis et al., 1999), as with other heterotrophic organisms (Corpe, 1985; Gluth and Hanke, 1985; Rahaman et al., 1990). Methanol is a natural metabolic product of plants (Nemecek-Marshall et al., 1995). Obendorf et al. (1990) suggested that its presence in leaves was the result (unique source) of the pectin methyltransferase activity, as coproduct of the demethylation of pectin constituents (galacturonan methyl ester) in the wall of meristomatic cells during their differentiation (Levy and Staehelin, 1992). Experiments with 14C-methanol suggested that its possible metabolism in higher plants was a four step oxidative pathway with successive formation of formaldehyde, formic acid and carbon dioxide (CH3OH -~ HCHO ~ HCOOH ~ CO2) (Colas des Francs-Small et al., 1993). The fast metabolic transformation of methanol into CO2 and the subsequent assimilation of CO2 by the photosynthetic Calvin-Benson cycle, was an explanation of the possible positive methanol role on biomass production (Fall and Benson, 1996). As toxicity of methanol for many organisms (Corpe, 1985) depends on both dose and exposition time, the present study aimed to provide biotechnological elements for the role of concentration and frequency of administration of methanol on the growth of Chlorella minutissima, a marine microalga widely used in aquaculture.

2. Materials and methods

2.1. Organisms and growth conditions The experiments were conducted at the hatchery unit of the Institute of Marine Biology of Crete (IMBC). The marine microalga was the C. minutissima (Foti and Novak) strain isolated from Heraklion bay, which naturally dominated all the cultures in non-sterile conditions and is now routinely cultivated at IMBC. The cultures (1 1 volume) were carried out in transparent cylindrical plastic columns (8.5 cm diameter, 30 cm high), at a constant temperature

of 26 + I~ under continuous illumination (13 000 lux) provided by a double line of fluorescent lamps (Sylvania cool white 60 W). The rearing medium was brackish water 25 ppt salinity from a littoral well, filtered successively through 20, 5 and 1 ~tm porosity cylindrical filters (Filtri Atlas, Mod 3/4 senior 3P ATO), then sterilised by chlorination (1 ppt sodium hypochloride, 50 g active chlorine per 1 over 16 h without aeration) and finally neutralised (1.4 ppt sodium thiosulphate over 6 h with strong aeration) for inactivation of remaining hypochloride. The medium was fertilised with 0.25 g 1-~ of F2 inorganic combination (F media algal salt type II, M. & M. Suppliers, USA) and homogenised by bubbling air (400 ml min-~) from the bottom. All cultures (1 1 volume) were inoculated the same day with 3 x 106 Chlorella cells per ml (about 250 ml added) from a exponential growth phase culture filtered through a 5 ~tm sock filter. The mother culture was held in 200 1 polyethylene bags under 10000 lux continuous light source with filtered, enriched (0.25 g 1-~ of Trovital (Bayer) medium) and treated (10 ppm antibiotic, Oxytetracycline) well brackish (25 ppt salinity) water. Two series of experiments were conducted.

2.2. Main experiments (MeOH doses) The main experiments were organised into two parallel series aiming to duplicate five different doses of methanol and two ways of administration. In the first, the doses (i.e. 0.05, 0.01, 0.5, 1 and 5%) were added once at the beginning of the experiment, while in the second, 1/10 of the previous doses (i.e. 0.005, 0.001, 0.05, 0.1 and 0.5%) were added daily over 10 days in such a way as to allow a comparison of the similar final quantities added to the culture.

2.3. Additional experiment (limiting factor) In order to postulate which of the three factors (light or methanol or fertiliser) was the possible limiting factor, the experiment which revealed the best microalgal production (0.005% MeOH day-~) (A-culture) was repeated and its respective rates of photosynthesis and respiration were com-

K. Kotzabasis et al./Journal of Biotechnology 70 (1999) 357-362

pared to those of an homologue with a parallel addition of 0.25 g 1-~ inorganic medium F2 every 48 h (B-culture).

supply (IL 760) and a photomultiplier (IL 780). The spectrum of irradiation was exempt of infrared due to 2% CuSO4 filters (4 cm width) inserted into the light beam. The cell density was adjusted to 10 ~1 of PCV m l The daily growth rate, /1 = (log (Nf/Ni))/(tfti), was calculated using the formula of Vonshak (1993). Comparisons of ~ were performed between the 4th and 5th as well as between the 0 (inoculation day) and 5th day of culture. For statistical analysis, analysis of variance (one-way ANOVA) among the replicates were carried out.

2.3.1. Method At 09:00 daily, all cultures conditions were checked, pH was measured with an electronic device (WTW pH metre 320), 10 ml samples were taken for counting and analysis and the eventual required doses of methanol were added to the corresponding columns. The rnicroalgal cell density was determined by counting culture aliquots in a Malassez chamber under an Olympus CH-2 microscope. The samples were centrifuged at 1400 • g for 5 min and the packed cell volume (PCV) was measured in hematocrit tubes. Pigments were estimated after exhaustive extraction of an aliquot from the pellet (algae) with hot methanol in darkness until it was colourless. The total amount of Chlorophyll (Chl) was measured by photometry using the method of Holden (1965) and then used for the estimation of the rates of photosynthesis and respiration per unit of Chl. Maximum photosynthetic and respiration rates were determined by polarography at 30~ using a Clark type electrode system (Hansatech, GB). Light intensity (470 W m - 2 ) was provided by two lamps ENX360W/82V and estimated using a sensitive photoradiometer (International Light, Newburyport, MA, 01950) consisting of a control box (IL 1700), a power

3. Results

3.1. Main experiment (Fig. 1) All duplicates presented high intragroup homogeneity. In the control, the two duplicates presented a short (less than I day) lag phase and an logarithmic phase (/~- 0.79) leading to a plateau ranging between 116 and 119 millions cell m l on day 8. In the experiments with one initial dose of methanol, the culture with 5% MeOH presented from the beginning evident symptoms of toxicity, leading to the end of the culture in 6 days. All other cultures presented a classic development scheme without significant intergroup differences

140

140

140

120

120

120

100 o 80

100 80

100 80

60 40 20

60 40 20

8 60 40 20 0 1

,I 3 5

,, 7

~ m e (days)

A

, 9

0 11

359

0 1

3

5

7

Time (days)

B

9

11

1

3

5

7

9

11

~ m e (days)

C

Fig. 1. Cell concentration (million cells m l - l ) of autotrophic C. minutissima cultures incubated without methanol (A), with one initial dose of various methanol concentrations (B) and with l0 daily additions of 1/10 of the previous various methanol concentrations (C). Legend B: (11) 0.05; ( 0 ) 0.1; (A) 0.5; (O) l; and ( ~ ) 5. Legend C (ll) 0.005; ( 0 ) 0.01; (A) 0.05; (O) 0.1; and

([]) 0.5.

360

K. Kotzabasis et al./Journal of Biotechnology 70 (1999) 357-362

Table 1 Growth constant factor /z between the 1st and 5th and between the 4th and 5th day in the different cases of cultures Methanol additions

gl-5

g4-5

Control 1 x 0.05% 1 x O.1% 1 x 0.5% 1 x 1% 1 x 5% 10 x 0.005% 10x0.01% 10 x0.05% lOxO.l% 10x0.5%

0.79 0.95 0.94 0.95 0.94 -0.29 1.01 0.96 0.96 0.91 0.84

0.25 1.45 1.02 1.00 1.18 - 1.45 2.05 1.31 1.82 1.26 0.80

(Fig. 1), neither during the exponential phase until day 5 (/~ ranging from 0.94 to 0.95) nor during the plateau phase on day 6 and 7 which ranged between 75 and 95 million cells m l - 1 according to cultures. Later, all cultures, except that with 0.05% MeOH which remained stable, presented a decrease of cell density. In the experimental series with daily additions of MeOH, the cultures presented significant intergroup differences, with evidence of inverse correlation Maximum Cell P r o d u c t i o n - - D o s e on day 5 (Table 1). The best results were observed in the duplicates with daily doses of 0.005% (final dose 0.05%) which reached 136 million cells ml-~ on day 9. The worst were observed in the duplicates with daily doses of 0.5% MeOH (final dose 5%) in which the micro algae began to develop correctly (even better than other cultures) during the first 3 days, but then (i.e. after a dose of about 1.5%) presented characteristics of toxicity and stabilised to a low plateau of about 30-40 million cells ml-1. The culture with 0.1 and 0.05% MeOH per day (1 and 0.5% final dose) presented good growth (/z = 0.91 and 0.96) during 6 or 7 days, reaching a maximum of 100 and 115 million cells m l - 1 and, just after, began to decrease or to fluctuate, which are clear indices of toxicity. The culture with 0.01% M e O H / d a y presented an evolution close to that of the culture with 0.005%/ day, but reached a plateau of lower maximum density at about 117 million cells m l -

3.2. Control experiment

In this experiment, the culture with parallel addition of 0.25 g 1-1 inorganic medium F2 every 48 h (B-culture) only reached 110 million cells ml -~, i.e. an additional 15% increase of the biomass production compared with the culture without an extra adition of F2 (i.e. A-culture). The maximum photosynthetic rate measurements of this culture after 10 days growth was 0.091 lamol 02 lag C h l - 1 h - 1, i.e. about six times higher than the corresponding one of A-culture (i.e. 0.014), while the respiration rate was 0.013 i.e. 50% higher than the corresponding one of A-culture (i.e. 0.06).

4. Discussion

This study was aimed at understanding the conditions for use of methanol in Chlorella culture and to find the best conditions of growth for optimisation of biomass production at constant light. The results showed that the mode of action of MeOH on C. minutissima was complex, depending on dose, frequency of addition and photosynthetic capacities of micro algae, which themselves in turn depended on incidental illumination of culture (available light) and on the changes o f mutual shadowing effect (i.e. on density) of the culture when growing (available light per cell). With 13 000 lux incident light and 3 million cells ml-1 in the culture, one dose of 5% MeOH was toxic and leads to a quick death of the culture, while similar additions of 0.1, 0.5 and even 1% enhanced growth. But the daily repetition of a 0.5% dose, which was well supported (enhancing growth) during the first 3 days, developed symptoms of instability when the culture reached 38.7 million cells m l - l and of toxicity at 54.3 million cells m l - ~, leading to a regressed plateau phase of about 30 million cells ml-1. This plateau was the cell response/adequation to the MeOH equilibrium between the daily input and the air stripping output of bubbling. Similar observations were made with the daily repetition of a 0.1% dose, which was well supported (enhancing

K. Kotzabasis et al./Journal of Biotechnology 70 (1999) 357-362

growth) during 7 days, but began to be toxic (decrease of the cell density) when the culture reached 109 million cells ml-1. These results are in agreement with the observations of Corpe (1985), Rahaman et al. (1990) and Gluth and Hanke (1985) on the toxicity of methanol for many heterotrophs. More precise knowledge on the conditions of use of methanol for C. minutissima culture will be available when its mechanisms of metabolisation by the unicellular green microalga are known. Mainly if the pathway proposed by Colas des Francs-Small et al. (1993) [CH3OH ~ HCHO-~ HCOOH -~ CO2] is valid and the formaline intermediate has a toxicity before the positive effect of assimilation of the end product CO2 by the Rubisco (Calvin cycle) then this leads to the formation of organic molecules and therefore to the biomass production. In our conditions, the best growth results at low algal density (i.e. at high illumination /cell) were obtained with medium doses of 0.5 and 1% MeOH, while those of maximum cell densities were obtained with the lower doses of 0.05 and 0.1% MeOH per day. The decreasing capacities of methanol utilisation by algae when density increases is due to the interaction boosting effect/ toxicity in relation to light availability. The additional (B-culture) experiment, which leads to a low (15%) increase on biomass production (by reference to the A-culture) with a higher (six times) increased photosynthetic rate, showed clearly that the main limiting factor of our experiment was light intensity. The corresponding increase of respiration rates, which was proof of an increased carbon conversion efficiency (Hemming et al., 1995), suggested that under higher light intensities, the potentialities of using higher doses of methanol and inorganic medium would yield a better biomass production. The use of thin solar photobioreactor in very sunny regions could be a solution to this problem if the methanol input during the day light phase is managed correctly. The toxicity of methanol for a majority of heterotrophic organisms (Corpe, 1985; Gluth and Hanke, 1985; Rahaman et al., 1990), associated with its growth value for photosynthetic organisms, gives the (rare) possibility to promote its use

361

as both a booster/fertiliser of phytoplankton and a regulator of grazers, i.e. to find its applications in cultures where there is a risk of contamination, mainly the outdoor non sterile ones. Moreover, the extremely low amounts of methanol needed for the enhancement of algal production, compared to the high amounts for higher plants (about 50%), suggests that its use in algal cultures, as opposed to higher plants, will match international environmental principles. Further experiments with other species (i.e. Isochrysis galbana) should be carried on to observe the role of methanol on the culture of not dominant species.

References Corpe, W.A., 1985. A method for detecting methylotrophic bacteria on solid surfaces. J. Microbiol. Methods 3, 215221. Colas des Francs-Small, C., Ambard-Bretteville, F., Small, I.D., Remy, R., 1993. Identification of a major soluble protein in mitochondria from non photosynthetic tissues as NAD-dependent formate dehydrogenase. Plant Physiol. 102, 1171-1177. Fall, R., Benson, A.A., 1996. Leaf methanol--The simplest natural product from plants. Trends Plant Sci. 1,296-300. Gluth, G., Hanke, W., 1985. A comparision of physiological changes in carp, Cyprinus carpio, induced by several poilutans at sublethal concentrations. 1. The dependency on exposure time. Ecotoxicol. Environ. Saf. 9 (2), 179-188. Hemming, D.J.B., Criddle, R.S., Hansen, L.D., 1995. Effects of methanol on plant respiration. J. Plant. Physiol. 146, 193-198. Holden, M., 1965. Chlorophylls. In: Goodwin, T.W. (Ed.), Chemistry and Biochemistry of Plant Pigments. Academic Press, London, pp. 461-488. Kotzabasis K., Hatzantona A., Hatziathanasiou A., BengoaRuigomez M.V., Kentouri M., Divanach P., 1999. Effect of methanol on the microalgal Scenedesmus obliquus growth response under autotrophic, heterotrophic and mixotrophic conditions of the culture (in preparation.). Levy, S., Staehelin, L.A., 1992. Synthesis, assembly and function of plant cell wall macromolecules. Curr. Opin. Cell Biol. 4, 856-862. Nemecek-Marshall, M., MacDonald, R.C., Franzen, J.J., Wojciechowski. C.L., Fall, R., 1995. Methanol emission from leaves: Enzymatic detection of gas phase methanol and relation of methanol fluxes to stomatal conductance and leaf development. Plant Physiol. 108, 1359-1368. Nonomura, A.M., Benson, A.A., 1992. The path of carbon in photosynthesis: methanol and light. Res. Photosynth. 3, 911-914.

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Obendorf, R.L., Koch, J.L., Gorecki, R.J., 1990. Methanol accumulation in maturing seeds. J. Exp. Bot. 41,489-495. Rahaman, A., Saha, N.C., Kaviraj, A., 1990. Micronucleous test on the peritheral erythrocytes of Oreochromis mossombicus exposed to liqued wastes of quinidin. In: Manna G.K., Janna B.B. (Eds.), Impacts of enviroment on animal and aquaculture, Kalyani-India-University of Kalyani, pp. 305-307.

Vonshak, A., 1993. Microalgae: laboratory growth techniques and the biotechnology of biomass production. In: Hall, D.O., Scurlock, J.M.O., Bolhar-Nordenkampf, H.R., Leegood, R.C., Long, S.P. (Eds.), Photosynthesis and production in a changing environment. A field and laboratory manual, pp. 337-335.

IOUINAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 363-371

Fishing products from the sea rational downstream processing of marine bioproducts L.A.M. van der Wielen

a,,,

L.K. Cabatingan a,b

a Kluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands b Department of Chemical Engineering, University of San Carlos, Cebu, Philippines

Received 18 November 1998; received in revised form 25 November 1998; accepted 22 December 1998

Abstract

In this paper, the implications for process design for various classes of marine bioproducts are reviewed. The potential of a thermodynamic framework to structure and quantify downstream processing alternatives for a wide range of marine bioproducts is demonstrated and the application of the framework is illustrated for two process options. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Fishing products; Marine bioproducts; Rational downstream processing

1. Introduction

At least as far back as the beginning of recorded history, the sea has always been an important source of man's food as well as feed for other land animals that also eventually end up on man's table. The sea's highly diverse life forms of fish, shell-fish, mammals, algae, etc. has stimulated local culinary traditions and has generously provided the raw materials for clothing and housing. Owing perhaps to the great difficulty involved in the catching of some marine organisms, all parts of the organism so obtained were in most cases utilized to their fullest extent. This implied * Corresponding author. Tel.: + 31-15-2782361; fax: + 3115-2782355. E-mail address: [email protected] (L.A.M. van der Wielen)

an industry based on the fractionation processes. One of the truly large scale industries that exploited marine life started and nearly ended with the catching and fractionation of whales into their main products--liver oil, meat and balenes (Zorgdrager, 1720). But the days of Moby Dick have ended. Nowadays, with the renewed industrial interest for deriving products from the seas, there is a need to find different ways to develop more sustainable processes relative to those in the early days of M oby Dick. It is evident that marine bioprocess engineering will play an important role in this development. This role will comprise not only the design, optimisation and control of final production processes, but also contribute to the development of a systematic and quantative approach to this developing field. Essential steps in the engineering ap-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All fights reserved. PII: S0168-1656(99)00089-9

L.A.M. van der Wielen, L.K. Cabatingan /Journal of Biotechnology 70 (1999) 363-371

364

proach are to relate the required raw (F-feed) and auxiliary (A- matter and energy) material streams to a desired product stream (P). This is shown schematically in Fig. 1. Whatever does not end up in the product stream is considered waste (W). These basic input-output data of process alternatives must then be recalculated into some key numbers or criteria that quantify process performance. Some general criteria that determine the quality of the final process are also given in Fig. 1. In order to compare process alternatives, it is evident that these criteria should be quantified on the basis of mass or money per unit of product. A first step to analyse the requirements for the development of sustainable processes for marine bioproducts, is to classify the various products into categories with common problems and challenges. Using this classification, we will shortly discuss opportunities in the downstream processing of natural and recombinant marine bioproducts in each of the classes. We will do so on the basis of a general methodology to quantitatively characterise the thermodynamic properties of the marine bioproducts to enable a rapid development and screening of downstream processing alternatives.

Criteria

Process Quality

capa~ty rate

A auxiliary F raw materials

yield and s~ectivity

n~eriais

energy cor~mption

waste ~ a t i o n P product

robuslne~ safety

economic feasibility

Wwaste II

unit operation

comparison requires quantification (to give it a number)

Fig. 1. Input and output streams of a process and performance criteria.

2. Classification

A convenient manner to classify marine bioproducts is by the degree of (increasing) complexity of the final product with respect to molecular composition or structure. For instance, novel biopharmaceuticals may have a relatively complex molecular structure when compared to petrochemicals or to most synthetic fine chemicals, but they are in general substantially less complex than food products. In the latter case, the product quality is determined by its molecular composition, where the presence of the absence of trace flavour or aroma components make the difference between a desired fresh and natural taste or a completely undesired sensation, as well as by its microstructure, which may range from creamy to slimy. Complexity may in this case also include criteria such as functionalities such as catalytic activities. On this basis, we can distinguish the following four categories of marine bioproducts: 1. Bioactive compounds such as biopharmaceuticals and food additives. 2. Novel biomaterials that may range from (modified) marine substances to marine-inspired, artificial structures. 3. 'Extreme' biocatalysts such as enzymes with specific capabilities or with activities at very unusual conditions. 4. Food products such as whole fish, shell fish and algae.

2.1. Processing implications Each of these classes leads to a number of implications and constraints for the processing conditions and process design. A crucial implication is whether an actual marine environment is actually required for obtaining the desired product quality or for meeting economic or technological feasibility criteria. In the case of edible marine products, the natural taste and freshness of the product are key aspects of product quality. Also the abundant availability of inexpensive space, water and, in some cases, nutrients, makes the sea environment attractive. This is in particular true for bulk products such as food proteins in

L.A.M. van der Wielen, L.K. Cabatingan /Journal of Biotechnology 70 (1999) 363-371

Present focus: screening and cultivation

Fig. 2. General outline of a cultivation based downstream process (after Wesselingh et al., 1994). fish. In these cases, the harvesting technologies should be able to deal efficiently with huge amounts of sea water with dilute products without being voluminous themselves. But the natural marine environment may also lead to a limited harvesting possibilities when the producing organisms are rare or not living under ambient conditions (polar regions, hot wells, bottom of the ocean). Production conditions are also relatively poorly controlled in natural environments as well as in aquacultures, which may lead to an undesired variation in amount, quality and production rate. In these cases, modern biotechnological methods that use genetically modified micro-organisms in controlled cultivations and modern fermenters provide a serious option. In these cases, the specific aspects of processing marine biomass are unimportant and the downstream processing is expected to follow the typical rationale of modern fermentation products (Wesselingh et al., 1994), which is shown in Fig. 2. It is important to realise that both biomass concentration as well as expression level of the product in marine organisms are sometimes prohibitively low, when compared to typical fermentation situations for conventional antibiotics. This vast difference can be illustrated with the example by Munro and coworkers (Battershill et al., 1998) of the promising anticancer drug halichondrin B, a polyketide component, which is produced in a relatively rare sponge species. In that case, 1 tonne of natural sponges yielded 80 mg of bioac-

365

tive material. For example, when we assume an annual productivity as low as 100 kg halichondrin B, the required amount of sponge biomass is 1.25 • 106 tonnes per year. Production in bacteria or fungi, with final product concentrations ranging from 1 to 10 g 1-1 of fermentation broth, will require 100-10 m 3 fermentation broth which is easily produced in relatively small industrial fermenters. It is also evident that the initial downstream processing steps will be five orders of magnitude smaller than for a comparable 'wild type' production. With these implications and aspects in mind, each of the four classes of marine bioproducts is discussed in some more detail.

2.1.1. Bioactive products The interest of the pharmaceutical and fine chemical companies for the marine biotechnology is mostly driven by nature's diversity in novel molecular structures with unexpected yet beneficial functions and activities. These may lead to novel pharmaceuticals with anti-cancer, anti-inflammatory and antibiotic properties (Fenical, 1998). Sofar, bioactive compounds comprise a whole range of polar (saponins, amino acids, alkaloid salts, polyhydroxy steroids), medium polar (such as peptides, peptide-derivatives, polyketides) and apolar (terpenes, terpenoids, cyclodepsipeptides, long chain fatty acids) (Levring et al., 1969; Riguera, 1997). Another interesting field is that of food additives such as natural pigments and colourants, vitamins, anti-oxidants, stabilisers such as alginates and other biopolymers. Probably the most mature area of marine bioproducts is that of long chain, poly-unsaturated fatty acids (PUFA's) and there derivatives. These essential fatty acids can hardly be produced in higher organisms but are produced in various micro-algae and some fungi and bacteria. Higher organisms such as fishes efficiently accumulate valuable PUFA's, often in an esterified form. In this case, the nutritional value may be even subject to its (potential) pharmaceutical effect and the compounds may be characterized as nutriceuticals. Tentative production volumes are in the order of 100 kg year-~ for truly small scale biopharmaceuticals and at least in the order of 1000 ton year-1 of broad spectrum antibiotics and for PUFA's.

366

L.A.M. van der Wielen, L.K. Cabat&gan /Journal of Biotechnology 70 (1999) 363-371

It is evident that in this class of marine bioproducts, controlled production is crucial. Variable product quality will reduce the overall efficiency of production and one may anticipate that this could lead to rejection by the FDA and other regulatory bodies. In case of biopharmaceuticals, septic operation of the process is essential, which may prohibit--for example--aquaculturing techniques. Given the required product purity, a strong emphasis will be laid on efficient purification (fractionation) technologies such as chromatography, extraction and crystallization. 2.1.2. Biomaterials Marine products can be used as auxiliary materials, with interesting functionalities for processing other products. One of the well known examples is the use of inexpensive, dead marine biomass for removing toxic heavy metal ions from industrial waste effluents. From this point of view, the use of marine algae (that have a naturally high polyelectrolyte content with weak acid groups) as biosorbent materials is probably best documented (Kratochvil and Volesky, 1998). In this case, inexpensive marine production and harvesting of the materials is probably one of the most attractive alternatives. Sustainable process development will imply controlled harvesting methods, in addition to sound 'replanting' schemes. Functional biopolymeric materials have been reported in the framework of mussel adhesive proteins (Jacobs et al., 1998) that have a substantial potential as biodegradable adhesive materials in surgery and in case of functional fibers from shell fish' chitin (Hirano and Kim, 1998) with wound healing properties. These proteins and polysaccharide derivatives can be extracted from relatively cheap and readily available natural marine sources. Depending on the particular application, yield of this harvesting may become a crucial parameter in process development. The further (bio-) chemical modification as well as the efficient formulation into the desired product form seem to be bottle necks in the process design. Another area is that of nano-structured, composite biomaterials that are found in some marine shells. In some of these materials, one can find a

complex layered structure of bio-organic and inorganic polymers such as the exoskeletons of diatoms (Vrieling et al., 1998), leading to entirely new product properties. Although shells that contain these materials can sometimes be obtained in relatively huge quantities, it is virtually impossible to modify these in such a manner that they can be applied directly in different products. In this case, biotechnological production implies that artificial production that yields the proper form and shape of the end product, must at least be inspired on the biological systems and most likely on a combination of self-assembly and biological action. The production will most entirely rely on the selective synthesis and downstream processing will most likely play only a minor role. 2.1.3. 'Extreme' biocatalysts An important category of marine bioproducts concerns biocatalysts that have special selectivities or that are still active under extreme conditions such as encountered in cold polar seas, in hot wells with unconventional chemical compositions and from organisms that live extremely isolated from other populations (bottom of the ocean). However, it is certainly not attractive to produce these biocatalysts at the original, extreme conditions. The underlying genetic information is much more interesting than using the original producer at industrial scale. The genetic information c a n - in principle--be cloned into more familiar microorganisms. A illustrative example is provided by cold-adapted proteases from arctic krill, that exhibit interesting properties for application as low temperature wound debriding agents or for use in detergents for low-temperature washing (J.A. Asenjo and B. Andrews, personal communication). These proteins are cloned with success into yeasts and can be efficient recovered by using available methods for processing broth streams from conventional fermentations. Recently, Schweder et al. (1998) described other cold-adapted, extracellular enzymes such as pectinases, glucanases and amylases. 2.1.4. Food In this category, we aim at conventional products that are mainly intended for direct and indi-

L.A.M. van der Wielen, L.K. Cabatingan ,/Journal of Biotechnology 70 (1999) 363-371

rect use in food and feed and more rarely, in pharmaceutical products. The degree of processing should preferably be minimal, in order to maintain the label of being 'natural' and 'fresh'. Therefore, production is mostly in the natural marine environment, a way that also appears in most cases to be the cheapest. In some isolated cases, the control over this natural environment is improved by using protected zones such as in case of aquacultures. Fig. 3 summarises the anticipated emphasis with respect to steps in the down stream processing for each of these classes of products. Because formulation depends strongly on the actual application of a product, it is hardly possible to comment general tools for this area. However, given the expected importance of harvesting and purification technologies in the field of marine bioprocessing, we will comment in the following sections a general thermodynamic tool for the quantification of molecular properties of complex compounds such as most marine bioproducts. In addition, we will speculate on some possible processing options that may prove to be relevant in case of commercial breakthrough of marine bioproducts.

Processingimplications i purify 'formulate i

ii

b ~

,,,

x

X

!

eldl'l~e

|

X

i i

-t

,

z

~

X

'

X

!

!

X

),

1 ! /

X

(

Y

i

Fig. 3. Schematic summary of anticipated bottlenecks and focus points (capital X) in the development of downstream processes for marine bioproducts.

367

3. Thermodynamic properties

Selection of purification systems for these structurally related species is troublesome and is not obvious in case of multi-step separation processes. A quantitative comparison of alternatives is required, which asks--in t u r n - - f o r quantification of molecular properties and operating conditions. Intuitive qualitative concepts, based on substantial empirical experience have been and are being used to quantify and rank molecular properties and operating conditions of processes in order to facilitate the selection of process alternatives. Clearly, identifying generalizing quantitative principles in the determination of thermodynamic properties can assist in translating this valuable knowledge into quantitative tools, such as computerized models. We will illustrate this approach using relatively small biomolecules that may carry several charges and can be overall neutral (zwitterionic species) but also may have a net charge (ions). Typical examples of molecules are amino acids that are models for more complex molecules such as antibiotics and small peptides. With respect to peptides, this work is restricted to medium-sized peptides which are composed of less than approximately eight amino acid residues, such that conformational changes are not too important. Larger species may exhibit a phase behaviour that is strongly affected by conformational changes, that could be induced by the environment. This complicates the interpretation and is subject of ongoing research. The general thermodynamic framework can of course be extended to more relevant classes of marine biomolecules, but the lack of a sound data base does not allow such an elaboration. A starting point for developing a generalizing methodology, is to recognise common conditions. One of the obvious observations is the diluted and usually aqueous environment in which dissolved bioproducts and their contaminants occur. Although the data base with (asymmetric) activity coefficients of biomolecules is limited to amino acids, it is now clear that the departure from the infinite dilution standard state are practically negligible. Selecting a thermodynamic framework

368

L . A . M . van der Wielen, L.K. Cabatingan / Journal o f Biotechnology 70 (1999) 3 6 3 - 3 7 1

'1 deal' s o l u b i l i t y miscible cosolvent (1)

vaine (3) solubility input10~r

1\

'pure' water (2)

same

crystal s

mi xt ure of solvents 1,2

10" 3

-

I~4

-

input

X3

lO-S

straight line

o.o

H20

I

~

x~

'T"

i

"1'

1.o

EtOH

Gude, Van der Wieten and Luyben Fluid Phase Eq. 116,110-7,'96 I ECR 35, 4700-12/96

Fig. 4. E s t i m a t i n g ideal solubilities o f biomolecules (valine) in mixtures of solvents (water and ethanol).

that recognises this aspect is expected to generate realiable descriptions for not too different systems, but also be a sound basis for developing theories that describe phase behaviour in more foreign systems realistically. Secondly, the deviations of these conditions are expected to be captured by relatively simple models, with parameters that are relatively easily transferred to different systems. In earlier work, we have derived such a framework and demonstrated its versatility to correlate phase behaviour of biomolecules in a wide variety of fairly different systems (solubility, liquid-liquid partitioning, sorption, ion exchange). For example, the solubility of a sparingly soluble solute (i) in liquid mixtures Aim .sat that are considered ideal in this framework, can be estimated simply as the logarithmic average of the solubilities of the solute in each of the 'pure' solvents ( j ) in the mixture, weighted by their respective solute-free mole fraction x):

In A" isat m =

E Xj J

ysat ln..,j

The usefulness of the simplified model is demonstrated in Fig. 4 for the solubility of valine in water-ethanol mixtures. For real liquid phase mixtures of biomolecules, it should be noted that the mixed solvent solubilities are usually very low. Therefore, the asymmetric activity coefficients of the solute are expected to be close to unity, as is

demonstrated to be true for amino acids in water (Cohn and Edsall, 1943). In earlier work (Gude et al., 1995, 1996), we have derived an expression for the excess Henry's constant based on a FloryHuggins type combinatorial expression combined with a Margules residual term. Similarly, in thermodynamically ideal systems, the partition coefficient of solute s in a mixture of partially miscible solvents (e.g. hexane-methanol-water) can be estimated on the basis of the following simple equation: In K~ h = y" ( x ~ - x~)In x,jsat J In case of extraction systems with aqueousmixed organic solvent systems, the solvents are usually selected such that the solubility of the solvents in the aqueous phase is small. Therefore, it can be assumed that the composition of the aqueous phase remains constant and the partition coefficient of the solute as a function of the solvent composition can crudely be estimated as follows: . sat

In g'".~, a

.m In ~"J/" "Xsj, ideal

_.

. ?(j j

with

f("j/a

~tXs, ideal

=

A sj

ysat -- sw

where K,j is the partition coefficient of the solute in the water--single solvent j system and x TM is the solubility of solute s in water. Hence, a first estimate of partition coefficients in partially miscible solvents can be obtained already when only the solubilities in each of the solvent components is known. It can be demonstrated that this approach is easily extended to aqueous two-phase systems (Eiteman and Gainer, 1990), ion exchange equilibria (Dye et al., 1990; van der Wielen et al., 1996) and many other situations (van Berlo et al., 1997 van der Wielen and Rudolph, 1999). In real systems, the phase behaviour departs in a smooth manner from this 'logarithmic interpolation' rule. It can however, be demonstrated that the deviation from the logarithmic interpolation' model shows a regular behavior in homologous series of solutes and solvents. As a matter of fact, the extent of deviation can be correlated to the polarity of the compound relative to a reference species. This constructs a scale that ranks components in a homologous series by their increasing

L.A.M. van der Wielen, L.K. Cabatingan /Journal of Biotechnolog), 70 (1999) 363-371

hydrophobicity. This scale can be correlated to other existing hydrophobicity scales (log P, solubility parameter, Hansch parameter). The advantage of this framework is that the phase behaviour of a complex molecule can be estimated on an absolute minimum of experimental data. On this basis, fairly accurate predictions can be made that drastically reduce the amount of costly experimental work as well as minirnise the required amount of the scarce marine bioproduct and that maximise the amount of information.

Extraction COz c o n d e n s e s b e l o w critical point in most organic solvents

0.5 t........ ~ 0. 4 1

Yco

::

-~_ ~

0,f l|v# \\T/

~176

"

.....

~ 40

y" ", , . r

I

;

"

\ ] .\

:t: o "

20

2,0

w[%]

000 40o0 5oo0 60o0 7oo0 time [s]

Fig. 5. Comparison of measured (dashed curves) and calculated (solid curves) gradient elution of chloromethylated phenols in heptane/methanol/water systems in a CPC apparatus. Data: van Buel et al. (1997).

tl

40 ~2~

60

9

70

~ "9

1

lo s lO3

~' lOa

I

solvent

supercritJcal regime

The thermodynamic framework presented above provides a simple yet powerful tool, for example, for optimization of multi-component organic solvent blends such as used for the extraction or liquid-liquid chromatography of biomolecules. An example of the successful use of the model in the development of gradient separations for liquid-liquid chromatographic techniques was given by van Buel et al. (1997). They demonstrated a drastic reduction in laborious experimental work by using the model for the chromatographic separation of chloromethylated phenols in heptane/methanol/water systems in a centrifugal partition chromatographic (CPC) apparatus. Some complex gradient patterns were demonstrated to be optimal for the separation of mixtures of these components which included combinations of multiple steps and linear gradients in solvent composition. An example is given in Fig. 5.

solubility

107

operatingregimee~ceedsthe

4. Application in some purification processes

369

! "'

pamty I

X,co~

I

sCO2

# Van H~serna, Van de" Wielen and Luyben, IECR37, 748-58, 1998

Fig. 6. p-T diagram showing the supercritical region of CO 2 and a schematic diagram of the solubility of a biomolecule in a binary mixture of solvent and CO2.depending on the polarity of this solute.

This model may also prove to be very helpful in estimating the feasibility of attractive and clean techniques such as the application of supercritical carbon dioxide (sCO2) for the replacement of organic solvents in these processes. CO2 condenses at pressures below its critical pressure in most organic solvents such as alcohols. Therefore, the CO2-content will increase to unity when the pressure in the system increases towards the critical pressure of sCO2. This is shown in the righthandside panel of Fig. 6, as the correspondence of the solute-free mole fraction of CO2 (solvent is balance) at the lower horizontal axis and the system pressure at the upper horizontal axis. In addition, we use the common rule of thumb that polar components dissolve more readily in polar solvents such as low molecular weight alcohols, whereas, apolar components dissolve better in apolar solvents such as sCO2 or heptane. On the basis of the framework, we can estimate that -depending on the polarity of the solute- its relative solubility (S/So) will show a similar relation to solvent (alcohol-CO2) composition as is predicted by the 'logarithmic interpolation' model (or a more rigorous variety). This is shown schematically as the curves calculated with the 'ideal model' in the righthandside diagram of Fig. 6. Because the pure desired product is usually a solid, a typical process will in most cases involve

370

L.A.M. van der Wielen, L.K. Cabatingan /Journal of Biotechnology 70 (1999) 363-371

an extraction or leaching step, followed by a crystallisation step. Economic feasibility as well as environmental legislation will require splitting and recovery of both solvents, which is relatively easily done using pressure moderation. Both CO2 and alcohol are reused, after compression and distillation respectively. The corresponding conceptual flow sheets are shown in Fig. 7 for polar as well as apolar species.

general and downstream processing of its products in general.

Acknowledgements One of the authors (LKC) would like to gratefully thank the Commission on Higher Educat i o n - F u n d for Assistance to Private Education (CHED-FAPE).

5. Conclusions It is evident that many other examples and speculations can be given for the downstream processing of marine bioproducts and that the requirements for the design of clean, compact and efficient marine bioprocesses are far from exhaustive. Clearly, other downstream processing technologies may finally turn out to be much more promising then those that are used as an illustration in this work. Also, the preliminary classification as well as the relatively simple thermodynamic framework should only be regarded as initial attempts to support and structure marine biotechnology from the point of view of process design. But the necessity to exploit the natural marine resources for society in a sustainable manner will without doubt lead to the further development of marine bioprocess engineering in ~lar ~:ies c o 2 mcyae

~

waste t

| i

~uct ~

waste

nm-~ar ~ ~ EtOH

feed l leoclp I ` I

sol~ waste t

[ ~

recyO~~solvent recovery

~oduct crystals

waste

Fig. 7. Conceptual flow sheets for the leaching/extraction of polar as well as apolar species using an alcohol such as ethanol in combination with CO2 and recycling of both solvents.

References Battershill, C.N., Blunt, J.W., Dumdei, E.J., Lill, R.E., Munro, M.H.G., 1998. Bioactivity down-under: discovery and development. In: Wijffels, R.H., et al. (Eds.), Proceedings of the International Symposium on Marine Bioprocess Engineering. Working Party on Applied Biocatalysis of the European Federation of Biotechnology, Noordwijkerhout, p. 4. van Berlo, M., Gude, M.T., van der Wielen, L.A.M., Luyben, K.Ch.A.M., 1997. Solubilities and partitioning of glycine in water + ethanol + butanol solutions. Ind. Eng. Chem. Res. 36 (6), 2474-2482. van Buel, M.J., Gude, M., van der Wielen, L.A.M., Luyben, K.Ch.A.M., 1997. Gradient elution in CPC. J. Chrom. 773, 13-22. Cohn, E.J., Edsall, J.T., 1943. Proteins, Amino Acids, Peptides as Ions and Dipolar Ions. Reinhold. Dye, S.R., DeCarli, J.P., Carta, G. II, 1990. Equilibrium sorption of amino acids by a cation exchange resin. Ind. Eng. Chem. Res. 29, 849-857. Eiteman, M.A., Gainer, J.L., 1990. Peptide hydrophobicity and partitioning in poly(ethylene glycol)/magnesium sulphate aqueous two-phase systems. Biotechnol. Prog. 6, 479-484. Fenical, W., 1998. New pharmaceuticals from marine organisms. TIBTech 15, 339-341. Gude, M.T., Meuwissen, H., van der Wielen, L.A.M., Luyben, K.Ch.A.M., 1996. Partition coefficients and solubilities of ~-amino acids in aqueous 1-butanol solutions. Ind. Chem. Eng. Res. 35, 4700-4712. Gude, M.T., van der Wielen, L.A.M., Luyben, K.Ch.A.M., 1995. Phase behavior of s-amino acids in multicomponent aqueous alkanol solutions. Fluid Phase Equilibria 116, ll0-117. Hirano, S., Kim S.K., 1998. The preparation and applications of functional fibers from crab shell chitin. In: Wijffels, R.H., et al. (Eds.), Book of Abstracts, International Symposium on Marine Bioprocess Engineering. Noordwijkerbout, p. 56. Jacobs, D.I., Haemers S., van der Leede, M.C., Frens G., Duine J.A., 1998. Mussel adhesive proteins, possibilities

L.A.M. van der Wielen, L.K. Cabatingan /'Journal of Biotechnology 70 (1999) 363-371

for an adhesive insurgery. In: Wijffels, R.H., et al. (Eds.), Book of Abstracts, International Symposium on Marine Bioprocess Engineering. Noordwijkerhout, p. 76. Kratochvil, D., Volesky, B., 1998. Advances in the biosorption of heavy metals. TIBTech 16, 291 - 300. Levring, T., Hoppe, H.A., Schmid, O.J., 1969. Marine Algae. Cram, de Gruyter, Hamburg. Riguera, R., 1997. Isolating bioactive compounds from marine organisms. J. Mar. Biotechnol. 5, 187-193. Schweder, T., Le, V.T., Borriss, M., Helmke, E., 1998. Coldadapted enzymes from psychrophilic marine micro-organisms. In: Wijffels, R.H., et al. (Eds.), Book of Abstracts, International Symposium on Marine Bioprocess Engineering. Noordwijkerhout, p. 70. Vrieling, E.G., Beelen T.P.M., van Santen, R., Gieskes, W.W.C., 1998. Diatom skeletons as a silica source. In: Wijffels, R.H., et al. (Eds.), Book of Abstracts, Interna-

371

tional Symposium on Marine Bioprocess Engineering. Noordwijkerhout, p.80. Wesselingh, J.A., Krijgsman J., Vonk P., 1994. Downstream processing of a product in biotechnology (unfortunately in Dutch). Delft University Press. van der Wielen, L.A.M., Jansen, M.L., Luyben, K.Ch.A.M., 1996. Multicomponent ion exchange equilibria of weak electrolyte biomolecules. In: Greig, J.A. (Ed.), Ion Exchange: Developments and Applications. SCI Publishers, Cambridge, UK, pp. 290-292. van der Wielen, L.A.M., Rudolph, E.S.J., 1999. On the generalization of thermodynamic properties for selection of bioseparation processes. J. Chem. Tech. Biotechnol. 74 (3), 275-283. Zorgdrager, G.C., 1720. Bloeyende Opkomst der Aloude en Hedendaagsche Groenlandse Visscherij. Amsterdam.

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Journal of Biotechnology 70 (1999) 373-377

....

The preparation and applications of functional fibres from crab shell chitin Shigehiro Hirano a,* Tamayo Nakahira a Masuo Nakagawa a, Son Ki Kim b Chitin/Chitosan R&D, 445-Sakuradani, Tottori 680-0853, Japan b Youngdeok Chitosan Co., Ltd., Youngdeok, Kyoung-buk, South Korea a

Received 1 October 1998; received in revised form 24 November 1998; accepted 22 December 1998

Abstract

Novel chitin-silk fibroin fibres and chitin fibres were prepared by an environmental friendly wet-spinning method. Each aqueous solution of sodium chitin (N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The tenacity and elongation values of the chitin-silk fibroin filament decreased with an increase of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed that both the chitin filament and the chitin-silk fibroin (67:33, w/w) filament had vertical strips with faint scale structures on their surfaces. Some applications of these staple fibres were also reported. 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Chitin fibres; Chitin-silk fibroin fibres: Chitosan: Fibroin; Textile; Wound dressing

I. Introduction

One of the tasks in marine bioprocess engineering is to exploit the novel ecological and environmental friendly materials of marine wastes. Crab and shrimp shells are abandoned in their processing companies. These shells consist of chitin, proteins and calcium carbonate as the main structural components. Chitin and cellulose have a linear (1 -~ 4)-linked ]3-D-glycoside structure, and silk fibroin is a linear structure. Cellulose and silk are used in a large quantities, but chitin fibres are little used in the textile industries because of (1) * Corresponding author.

the difficulty in collecting a large amount of chitin resources and (2) no development of economical spinning methods for their physically strong filaments. Recently, several methods have been used to attempt to prepare chitin fibres: (1) a chitin solution in LiC1-N,N-dimethylforamide was spun into n-butanol (Nakajima et al., 1984); (2) each of sodium chitin xanthate (Noguchi et al., 1973; Hirano et al., 1997) and sodium chitin salt solutions (Hirano and Midorikawa, 1998) in aqueous N a O H was spun into an aqueous H2SO 4 solution containing Na2SO4 and ZnSO4; and (3) a chitin solution in formic acid-dichroloacetic acid was spun into acetone (Tokura et al., 1979). However, no report has dealt with any chitin-silk

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S. Hirano et al./Journal of Biotechnology 70 (1999) 373-377

fibroin blend fibres. The choice of environmentally friendly spinning and coagulating solutions is also essential in the fiber spinning. In the present method, sodium chitin salt is used in place of sodium chitin xanthate, which has been prepared with the use of harmful carbon disulfide (Hirano et al., 1997). The aqueous saturated ammonium sulfate solution is well known for the isolation, fractionation and purification of enzyme proteins in biochemistry (Green and Hughes, 1955). We now report that an aqueous acidic solution of saturated ammonium sulfate is usable as a novel coagulating solution for the spinning of chitin-protein blend fibres and chitin fibres. Some preliminary applications of these fibres are also described.

2. Materials and methods 2. I. Mater&& Chitosan was prepared from crab shell chitin by treatment with aqueous 40% sodium hydroxide (Horton and Lineback, 1965). The chitosan was dissolved in aqueous 2% acetic acid, filtered through a glass filter, and neutralized with aqueous sodium hydroxide, and a purified chitosan was obtained as a precipitate, which had a degree of substitution (d.s.) 0.10 for NAc and molecular weight (MW) 15 x 10 4 estimated from the viscosity in aqueous 2% acetic acid. Chitin

(N-acetylchitosan, d.s. 1.0 for NAc) was prepared by chemical N-acetylation of chitosan with acetic anhydride in MeOH (Hirano et al., 1976). Cocoons of silk worms (Bombyx mori) were obtained from the Department of Agronomy, Tottori University. 2.2. Methods F T I R spectra (KBr) were recorded on a Jasco FTIR 5300 spectrometer (Jasco, Tokyo). The filament titer [denier (g per 9000 m)], tenacity (g d e n i e r - l ) and elongation (%) values at dry conditions were analyzed on a Vibroscop Micro (Lenzing Technic Instrumental, Austria), and the filament tenacity and elongation values on a Virodyn 400 (Lenzing Technic Instrumental). Scanning electron microscope (SEM) analyses were performed on a scanning electron microscope (JSM-6301F; Jeol, Tokyo). Elemental analyses were performed at the Micro-analytical Center of Kyoto University, Kyoto, Japan. 2.3. Silk fibroin Silk worm's cocoons (72 g), which were cut into several pieces by a knife, were suspended in 0.2% soap solution (2.0 1) containing sodium carbonate (10 g). The mixture was allowed to boil by stirring for 20 min and a sericin protein was solubilized to afford a crude silk fibroin as a precipitate. The fibroin precipitate was washed with water several

Table 1 Some properties of chitin filaments and chitin-silk fibroin filaments at dry state Filamenta

Chitin (%)

Fibroin (%)

Titerb (denier)

Tenacityb (g denier-l)

Elongation b (%)

153-3-1 153-3-2 153-3-3 153-6-2 Chitin Ac Chitin Bc

67 80 94 100 100 100

33 17 6 0 0 0

18.5 9.70 3.24 3.10 3.18 3.47

0.10 0.67 1.05 1.09 0.79 0.83

0.71 4.20 8.44 10.7 27.1 29.8

Chitin-silk fibroin filaments (153-3-1, 153-3-2 and 153-3-3) and chitin filament (153-6-2). b An average value of 3-5 analyses. c Chitin A was prepared from sodium N-acetylchitosan salt (Hirano and Midorikawa, 1998), and chitin B was prepared from sodium N-acetylchitosan xanthate (Hirano et al., 1997) with use of an aqueous 10% H2SO 4 solution containing 32% N a z S O 4 and 1.3% Z n S O 4 as a coagulating solution. a

S. Hirano et al. Journal of Biotechnology 70 (1999) 373-377

375

and hung in ventilated open air at room temperature for several days. The concentrated fibroin solution in the tube was filtered through a glass filter, and a silk fibroin solution (about 10%) was obtained.

2.5. Sodium chitin salt solution A powdered sample (1.0 g) of chitin (N-acetylchitosan) was swollen by stirring in aqueous 46% N a O H (7.6 ml) at room temperature overnight. To the mixture were added several pieces of crushed ice by stirring to give rise to a solution (25 ml) of 4% sodium chitin salt in aqueous 14% N a O H (Hirano et al., 1991). A small amount of insoluble material was removed by filtration through a flannel filter, and a clear alkaline chitin solution was used as a dope for spinning.

2.6. Spinning

Fig. 1. A SEM photograph of a chitin-fibroin filament. The chitin-fibroin filament (153-3-1) of 18.5 denier shows a 46 ~m diameter (scale bar, 10 Jam).

times, and air-dried. The dry fibroin was suspended in 750 ml of a chloroform-methanol (2:1, v/v), and the suspension mixture was stirred at room temperature for 70 h to extract lipids and pigments. The product was air-dried to afford the purified silk fibroin in 69% yield (50 g).

2.4. Aqueous silk fibroin solution The purified silk fibroin (6.0 g), obtained as already described, was dissolved in aqueous 10 M LiBr'HzO (60 ml) by stirring at room temperature, and the fibroin solution was put into a dialysis membrane tube and dialyzed against running water for 1 day, and then in distilled water for 1 day to remove LiBr. The tube was taken out

An aqueous 4% sodium chitin salt solution in 14% N a O H (25 ml), obtained as already described, was mixed with 5.0, 2.0 and 0.6 ml of aqueous 10% silk fibroin solution. Each of the dopes was treated at a reduced pressure for a few minutes to remove air-bubbles, and spun through a viscose-type spinneret (12.5 • 18 • 0.3 mm 3 in size, 300 holes, 0.10 mm in the hole diameter; Japan Nozzle Company, Kobe, Japan), into a coagulating bath containing aqueous 10% sulfuric acid saturated with ammonium sulfate (ca. 43%) at room temperature (20-25~ The filaments were cut into about 25 cm lengths, dipped in the same coagulating solution at room temperature overnight, and washed several times with aqueous 30% methanol to remove ammonium sulfate. The fibres were dipped in aqueous 60% methanol and then in 100% methanol at room temperature overnight. To a suspension mixture of the staple fibres in methanol (100 ml g-~ fibres) was added acetic anhydride (1 ml g-~ fibres) for their complete N-acetylation. After remaining at room temperature overnight, the stable fibres were taken out and washed with methanol thoroughly, and air-dried (Hirano et al., 1997). Chitin-silk fibroin staple fibres (153-3-1): Vmax(KBr) cm-~: 1647 and 1554 (C--O and NH for N-acetyl and peptide), ~ 1034 (C-O).

376

S. Hirano et al. Journal o[ Biotechnology 70 (1999) 373-377

Chitin-silk fibroin staple fibres (153-3-2): Vmax(KBr) c m - 1. 1650 and 1557 (C--O and NH for N-acetyl and peptide), ~ 1053 (C-O). Chitin-silk fibroin staple fibres (153-6-2): Vmax(KBr) c m - 1:1660 and 1560 (C--O and NH for N-acetyl and peptide), ~ 1070 (C-O). Chitin staple fibres (153-6-2)" Vma• ) cm -l" 1660 and 1562 (C--O and NH for N-acetyl), ~, 1074 (C--O). Anal. Calc. for [C8H~3NOs"0.97H20]" C, 43.50; H, 6.78; N, 6.35. Found. C, 43.86: H 6.85, N, 6.30.

3. Results and discussion

3.1. Spinning and coagulating solutions An aqueous 4% sodium chitin salt (alkaline chitin) solution in aqueous 14% NaOH was used as a dope for spinning, and its regeneration reaction was performed in an aqueous 10% H2SO4 solution saturated with ammonium sulfate at room temperature. No heating treatment is required in the present method, although heating at 40-50~ was used in the previous methods (Hirano et al., 1997). The aqueous solution of ammonium sulfate is well known in biochemistry as a salting-out salt of active enzyme proteins without their denaturing (Green and Hughes, 1955).

tents up to 33% (Table 1). The tenacity value of the chitin filament prepared by the present method was 1.3 times and the elongation value was one-third of those prepared in an aqueous 10% H2SO4 solution containing 32% NazSO 4 and 1.3% ZnSO4 as a coagulating solution (Hirano et al., 1997; Hirano and Midorikawa, 1998). These data indicate that the present new coagulating solution improves the filament physical properties. In addition, silk fibroin is almost insoluble in the aqueous ammonia solution saturated with ammonium sulfate at room temperature, although the silk protein is soluble or denatured in the previously reported coagulation solutions (Noguchi et al., 1973; Tokura et al., 1979; Nakajima et al., 1984; Hirano et al., 1997; Hirano and Midorikawa, 1998).

3.3. SEM photographs As shown in SEM photographs (Fig. 1), the chitin filament (153-6-2) in 3.1 denier showed a 17 lam diameter, and the chitin-fibroin (67:33, w/w) filament in 18.5 denier showed a 46 gm diameter. Both the filaments had vertical strips on their surfaces with a faintly scaly structure. The scaly structure on the filament surface is considered to be formed during the neutralization and dehydration processes of the filaments.

3.2. Fiber properties A partial N-deacetylation (5-7'70) was found in the fibers under the present conditions (Hirano and Midorikawa, 1998). Therefore, the fibers were retreated with acetic anhydride in MeOH at room temperature (Hirano et al., 1997). White chitin and chitin-silk fibroin staple fibres were obtained. The chitin staple fibres showed C--O and NH absorptions of N-acetyl at 1660 and 1562 cm-1, and the chitin-fibroin staple fibres showed C--O and NH absorptions of N-acetyl and peptide bonds at 1647-1660 and 1554-1560 cm I in the FTIR spectra. These fibres were stable up to 250~ In the chitin-fibroin filaments, both the filament tenacity and elongation values decreased almost linearly with an increase of fibroin con-

Fig. 2. A photograph of a sock prepared experimentally by knitting with N-acetylchitosan-silk fibroin staple fibres (1533-3) containing 6"' silk fibroin.

S. Hirano et al./ Journal of Biotechnology 70 (1999) 373-377

3. 4. Some applications The chitin fibres may be usable as functional and ecological friendly natural fibres in biomedical, biotechnological and cosmetic fields. Cellulose fibers (rayon) do not have these biofunctions of chitin. In addition, no toxic carbon disulfide was used in the present method, and toxic sulfur compounds are free in the fibres. The chitin fibres were digestible in animal and plant tissues (Hirano et al., 1996, 1997; Hirano and Midorikawa, 1998). Chitin fibres are usable as a digestible surgical suture and a wound dressing material in medical (Hirano, 1996), veterinary (Shigemasa and Minami, 1995) and botanical (Hirano et al., 1996) fields. The novel chitin-silk fibroin fibres have been tried as a new textile material to knit socks in our laboratory (Fig. 2). Their blend fibers (Hirano et al., 1997) may be usable as novel functional biomaterials.

4. Conclusion

As demonstrated in the present study, an aqueous 10% sulfuric acid solution saturated with ammonium sulfate is usable as a novel coagulating solution at room temperature. Not only chitin-silk fibroin filament, but also chitin filament can be regenerated by this method. Acidic, alkaline and neutral solutions saturated with ammonium sulfate are also usable as the coagulating solution for preparation of a series of the other blend fibres including chitin-protein, cellulose-protein, chitin-glycosaminoglycan, cel-

377

lulose- glycosaminoglycan, and chitosantropocollagen blend fibres. These results will be published elsewhere by our laboratories. References Green, A.A., Hughes, W.L., 1955. Protein fractionation on the basis of solubility in aqueous solutions of slats and organic solvents. Methods Enzymol. 1, 67-90. Hirano, S., 1996. Chitin biotechnology applications. Biotechnol. Ann. Rev 2, 237-258. Hirano, S., Midorikawa, T., 1998. Novel method for the preparation of N-acylchitosan fiber and N-acylchitosancellulose fiber. Biomaterials 19, 293-297. Hirano, S., Ohe, Y., Ono, H., 1976. Selective N-acylation of chitosan. Carbobydr. Res. 47, 315-320. Hirano, S., Inui, H., Mikami, T., Ishigami, Y., Hosamori, H., 1991. Am alkaline chitin solution and some O-alkylchitin derivatives. Agric. Biol. Chem. 55, 2627-2628. Hirano, S., Kitaura, S., Sasaki, N., Sugiyama, M., Hashimoto, K., Tanitani, A., 1996. Chitin biodegradation and woundhealing in tree bark tissues. J. Environ. Polym. Degr. 4, 261-266. Hirano, S., Usutani, A., Midorikawa, T., 1997. Novel fibres of N-acylchitosan and its cellulose composite prepared by spinning their aqueous xanthate solution. Carbohydr. Polym. 33, 1-4. Horton, D., Lineback, D.R., 1965. N-Deacetylation. Methods Carbobydr. Chem. 5, 403-406. Nakajima, M., Atsumi, K., Kifune, K., 1984. Development of absorbable sutures from chitin. In: Zikkakis, J.P. (Ed.), Chitin, Chitosan and Related Enzymes. Academic Press, Orlando, FL, pp. 227-237. Noguchi, J.. Wada, O.. Seo, H., Tokura, S., Nishi, N., 1973. Chitin and chitosan-cellulose fibres. Kobunshi Kagaku 30, 320-326. Shigemasa, Y., Minami, S., 1995. Applications of chitin and chitosan for biomaterials. Biotechnol. Genetic Eng. Rev. 13, 383-420. Tokura, S., Nishi, N., Noguchi, J., 1979. Studies on chitin. Preparation of chitin fibres. Polym. J. 11, 781-796.

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Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 379-391

Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils A. Robles Medina *, L. Esteban Cerdfin, A. Gim6nez Gim6nez, B. Carnacho P/tez, M.J. Ibfifiez Gonz/dez, E. Molina Grima Grupo de Biotecnologia de Microalgas, Departamento de Ingenieria Quimica, Universidad de Almeria, E-04071 Almeria, Spain

Received 9 October 1998; received in revised form 30 November 1998; accepted 22 December 1998

Abstract This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. The lipase Novozym 435 (Novo Nordisk, A/S) from Candida antarctica was used to catalyze this reaction. The main factors influencing the degree of esterification and triglyceride yield were the amount of enzyme, water content, temperature and glycerol/fatty acid ratio. The optimum reaction conditions were established as: 100 mg of lipase; 9 ml hexane; 50~ glycerol/PUFA concentrate molar ratio 1.2:3; 0% initial water; 1 g molecular sieves added at the start of reaction; and an agitation rate of 200 rpm. Under these conditions, a triglyceride yield of 93.5% was obtained from cod liver oil PUFA concentrate; the product contained 25.7% eicosapentaenoic acid and 44.7% docosahexaenoic acid. These optimized conditions were used to study esterification from a PUFA concentrate of the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. With the first, a triglyceride yield of 96.5%, without monoglycerides and very few diglycerides, was obtained after 72 h of reaction; the resulting triglycerides had 42.5% eicosapentaenoic acid. A triglyceride yield of 89.3% was obtained from a P. cruentum PUFA concentrate at 96 h of reaction, which contained 43.4% arachidonic acid and 45.6% EPA. These high triglyceride yields were also achieved when the esterification reaction was scaled up 5-fold. 9 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Lipase-catalyzed esterification; Polyunsaturated fatty acids; Fish oil; Microalga oils; Triglycerides

1. Introduction It is by now well established that the polyunsaturated fatty acids (PUFA), especially eicosapentaenoic (EPA), docosahexaenoic ( D H A ) and * Corresponding author. Tel.: + 34-950-215065; fax: + 34950-215484. E-mail address: [email protected] (A. Robles Medina)

arachidonic (AA) acids, are beneficial to health. EPA was recently shown to have such highly beneficial effects as preventing coronary heart diseases (Weylandt et al., 1996), hypertriglyceridemia (Rambjor et al., 1996), blood platelet aggregation and lowering blood cholesterol, thus reducing the risk of arteriosclerosis (Simopoulos, 1991), inflammation (Ziboh, 1991) and various carcinomas (Braden and Carroll, 1986). D H A is important in

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A. Robles Medina et al. / Journal of Biotechnology 70 (1999) 379-391

the development of the central nervous system of infants (Innis, 1991). AA is an essential fatty acid in human nutrition (WHO/FAO, 1977) and a biogenetic precursor of the biologically active prostaglandins and leucotrienes with important functions in the circulatory (Sing and Chandra, 1988) and central nervous systems (Innis, 1991). AA is necessary for visual acuity and better cognitive development of the infant after birth (Koletzko et al., 1996) and as a component of mature human milk, it is potentially valuable as an ingredient in various formulations of artificial baby food (Koletzko et al., 1989). At present, pharmaceutical applications of PUFA are in the developmental stage. A few companies market PUFA, but only as PUFA-rich oils. Some commercial products are EPAX TG (Pronova Biocare A.S., Sandet]ord, Norway) of which EPA represents 5-20% of the total fatty acid content in TG and DHA is present in variable amounts, ARASCO (Martek Bioscience Corporation, Columbia, MD), a single-cell oil containing 40% AA and SUN-TGA (Suntory Limited, Tokyo, Japan), which has a minimum AA content of between 22 and 37%. These oils are encapsulated in gelatin and are marketed as health food supplements. The only commercial sources of n-3 PUFA (EPA and DHA) and AA are fish oils and animal viscera, respectively, e.g. AA obtained from pig liver (containing less than 0.5% AA on dry wt. basis) (Shimizu et al., 1987) and the fungus Morteriella alpina 1S-4 (Shinmen et al., 1989). However, many microalga species have also been found to be rich in oils containing various amounts of PUFA. Phaeodactylum tricornutum is a potential source of EPA because it is a fastgrowing microalga with low content in other PUFAs such as DHA and AA, which has important advantages in simplifying recovery (Yongmanitchai and Ward, 1991). Porphyridium cruentum UTEX 161 (Nichols and Appleby, 1969; Yongmanitchai and Ward, 1991) is a potential source of AA as well as EPA. PUFA may be administered as free fatty acids (FFA), as ethyl esters or as TG. FFA seem to be the best absorbable pharmaceutical form for preventing cardiovascular diseases (Leaf and Kang,

1996) and the ethyl ester is used for treating pancreatic cancer cachexia (associated chronic weight loss) (Wigmore et al., 1996). The authors had previously developed a three-step method to obtain highly pure PUFA from cod liver oil and the marine microalgae Isochrysis galbana (Molina Grima et al., 1994 and Robles Medina et al., 1995), P. tricornutum (Cartens et al., 1996; Molina Grima et al., 1996) and P. cruentum (Gim6nez Gim6nez et al., 1998). However, for other applications, the most appropriate pharmaceutical form might be the more natural TG, such as the highly pure trieicosapentanoyl glycerol (tri-EPA) used as an infusion to study thrombotic disorders in humans (Hamazaki et al., 1988). Boswell et al. (1996) have established that TG is the most adequate form to include AA and DHA into the baby formulations, because both PUFA are present in the human milk as TG. Many lipases catalyze esterification reactions in organic solvents. Better TG synthesis from DHA ethyl ester has been obtained using the lipase sp 435 from Candida antarctica immobilized on macroporous acrylic resin; more than 95% of DHA was converted to TG by esterification at 50~ for 23 h (Kosugi and Azuma, 1994). In a previous paper (Esteban Cerd~m et al., 1998), the esterification of glycerol and n-3 PUFA from cod liver oil was studied using three commercially available lipases: Lipozyme IM from Mucor miehei, Novozym 435 from C. antarctica and lipase PS from Pseudomonas. Maximum synthesis of TG was obtained with lipase Novozym 435, which proved to be highly active. In that work, the authors studied the experimental conditions required to obtain strong esterification and a large TG yield (Esteban Cerdfin et al., 1998). In this work, the goal has been the production of commercially viable EPA-rich TG from cod liver oil and the microalga P. tricornutum and TG rich in EPA and AA from the microalga P. cruentum by esterification of these PUFA with glycerol by lipase-catalyzed reactions. To the best of our knowledge, no similar attempts have been made to use PUFA or lipids from microalga as a source of PUFA-rich TG.

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2. Materials and methods

was dehydrated by heating at 120~ stant weight.

until con-

2.1. Chemicals and materials 2.2. Cod liver oil saponification, fatty acid extraction from microalga biomass and preparation of PUFA concentrates

Lipase Novozym 435 was donated by Novo Nordisk A/S (Bagsvaerd, Denmark). It was derived from C. antarctica and the gene coding for the lipase was transferred to the host organism Aspergillus oryzae. This lipase, containing 2-3% water, was immobilized on a macroporous acrylic resin. 1,3 positional specificity of Novozyrn 435 is observed in some reactions while none is observed in others. Analytical grade glycerol and hexane were obtained from Sigma Chemical (St. Louis, MO) and Panreac (Barcelona, Spain). Molecular sieves of 4-,~ were obtained from Sigma. Water was purified in a Milli-Q system (Millipore Co., Bedford, MA). For some experiments the lipase

Commercial cod liver oil (Acofarma, Barcelona, Spain) was saponified to obtain FFA as described elsewhere (Robles Medina et al., 1995). Lyophilized biomass of the microalgae P. tricornutum (9.4% of total fatty acids d. wt.) and P. cruentum (4.1% of total fatty acids d. wt.) were used as oil-rich substrates that contain large portions of EPA and AA (Table 1). Cells were grown in an outdoor tubular photobioreactor as described elsewhere (Aci6n Fernfindez et al., 1998; Camacho R ubio et al., 1999), harvested by cen-

Table 1 Fatty acid composition (% of total fatty acids) of extracts and PUFA concentrates from cod liver oil, P. tricornutum and P. cruentum biomass Fatty acids

Cod liver oil Extract a

14:0 16:0 16:1n7 16:2n4 16:3n4 16:4nl 18:0 18:1n9 18:1n7 18:2n6 18:3n3 18:4n3 20:3n6 20:1n9 20:4n6 (AA) 20:4n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) Others

3.5 10.8 7.4 1.0

2.7 18.2 5.2 1.8 0.8 2.7

P. tricornutum

PUFAb 0.8 0.2 0.7 2.7 0.6 0.6 0.4 0.7 0.6 10.6

Extract c 5.9 14.5 21.4 10.0 5.0 1.2 0.3 0.7 0.8 2.7 0.4 0.2 0.3

P. cruentum

PUFAd 0.2 7.0 15.0 10.5 2.5

2.3 0.5 0.5 0.3

Extracff

PUFAd

0.5 26.4 0.9

0.1 0.1 0.1

0.7 0.6 1.2 8.1

5.6

12.0 1.0 10.1 1.3 12.2 9.4

0.8 2.2 25.7 1.3 45.7 6.4

3.2 0.6 24.3 2.2 1.0 5.3

5.5 0.8 45.8

26.4 0.1 27.5

41.8

1.9 7.4

0.4 7.5

0.2 4.4

47.6

Fatty acids obtained by saponification of cod liver oil. b Urea/fatty acid ratio 4:1, crystallization temperature 4~ solvent methanol. c Fatty acids extraction by direct saponification of lyophilized biomass. Fourteen ml ethanol (96%) and 1.6 g KOH (85%) per gram biomass, 1 h, 60~ d Urea/fatty acid ratio 4:1, crystallization temperature 28~ solvent methanol. a

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A. Robles Medina et al. / Journal of Biotechnology 70 (1999) 379-391

trifugation and stored at - 1 8 ~ until used. The extraction of fatty acids from biomass has previously been described (Molina Grima et al., 1996; Gim6nez Gim6nez et al., 1998). The PUFA concentrates were obtained from fatty acid extracts by the urea method as described by Robles Medina et al. (1995).

2.3. Esterification reactions A typical reaction mixture for glyceride synthesis in the assays carried out to optimize the reaction conditions consisted of: glycerol, 41.7 mg; PUFA concentrate, 0.4 g; hexane, 9 ml; water, 48 gl (0.5% on total volume); lipase, 100 rag. This reaction mixture was placed in 50 ml Erlenmeyer flasks with silicone-capped stoppers under argon atmosphere to avoid degradation of PUFA. The suspension was incubated at 50~ and agitated in a water-bath shaker at 175 rpm. 1 g of molecular sieves was added after 24-h incubation to remove the water formed during the reaction. The reaction was stopped by addition of acetone/ethanol mixture (1:1 v/v) after which the lipase was separated by filtration and the volume of filtrate was adjusted to 25 ml by addition of acetone/ethanol. This mixture, product of the reaction, was stored under argon atmosphere at - 2 0 ~ until analysis. This typical assay was appropriately modified for studying the influence of temperature, water content, glycerol/fatty-acid ratio and fluid dynamics.

2.4. Identification of reaction products and estimation of the degree of esterification and percentage and yield of triglycerides Reaction products (FFA, monoglycerides, MG, diglycerides, DG and triglycerides, TG) were identified by thin-layer chromatography (TLC) followed by quantitative gas chromatography (GC). TLC plates were G-25 precoated silica-gel (Aldrich Chemical Co., Milwaukee, WI) activated by heating at 105~ for 30 min. The samples were spotted directly on the plate with authentic standards. After adding 0.2 ml of reaction product mixture the plate was developed in chloroform/ acetone/methanol (95:4.5:0.5 v/v/v). Spots of each lipid were visualized by spraying the plate with

iodine vapor in a nitrogen stream. Fractions corresponding to each lipid type were scraped from the plates and methylated according to the method of Lepage and Roy (1984). Methylation and methyl ester analysis by GC have been described elsewhere (Molina Grima et al., 1994). The degree of esterification (ED) represents the percent of initial fatty acids consumed in the reaction mixture, i.e. fatty acids in the glyceride mixture and initial fatty acid ratio, which was determined by TLC-GC and by titration of FFA with 0.025 N NaOH. The acidity of the reaction mixture was first determined without fatty acids to correct the volume of NaOH solution consumed in the titration of the reaction mixture. ED in this work is the average of the values obtained by both methods. The percentages of the individual glycerides over total glycerides (MG/GLY, DG/GLY and TG/GLY) and TG yield have also been calculated from the results of TLC-GC analysis. So, for example, the TG/GLY ratio is the fatty acid present as TG and the total fatty acids esterified by glycerol and the TG yield is the ratio of fatty acids present as TG to initial fatty acid in the reaction mixture obtained by multiplying ED by TG/GLY. The deviations and errors in the TG yields are firstly due to the complex and long analytical method necessary to obtain these TG yield. The TG yields have been represented in Figures with error bars which correspond to the ED obtained by titration and TLC-GC. Regarding reproducibility, the experiments in which results were clearly different from the general trend were repeated. Also some esterification reactions were repeated being the deviations lower than 8%; these experiments are indicated in the caption of Figures.

3. Results and discussion

In previous work (Esteban Cerdfin et al., 1998), the authors studied the experimental conditions for obtaining high degrees of esterification and high TG yield using a PUFA concentrate obtained from cod liver oil. For this reaction it is important to include organic solvents in the reaction systems in order to perform the conversion of

383

A. Robles Medina et al. , Journal o f Biotechnology 70 (1999) 379-391

100

80 7O ~, 60 50

T = 400C

N

T = 50~

40

T = 60~

30 20 10 0 0

8

16

24

32

40

48

56

64

72

80

88

TIME

96

(h)

Fig. 1. Effect of temperature and time on the TG yield. Esterification conditions: 9 ml hexane, 41.7 mg glycerol, 400 mg PUFAs (glycerol/fatty acid ratio 1:3 molar), 0.5% (v/v) water, 1 g molecular sieves added at 24 h, 100 mg of lipase N-435 and 175 rpm. Esterification reactions at 24 and 96 h were made twice with the deviations being lower than 8%.

lipophilic compounds effectively and avoid the degradation of PUFA. The authors ascertained that the most important variables for obtaining high TG yields were the amount of enzymes, water content, temperature and glycerol/FA ratio. The optimum conditions obtained for high TG yields were: glycerol/FA 1:3 molar, 9 ml of hexane, 0.5% (v/v) of initial water, 1 g of molecular sieves added after 24 h of reaction, 60~ and 100 mg of Novozyn 435 lipase from C. antarctica. These conditions permitted an equilibrium ED of 87% and a TG yield of 85% to be obtained after 48 h (a relatively short reaction time). In this work, a more detailed study of the influence of temperature, initial water content and glycerol/ FA ratio has been carried out in order to increase the equilibrium ED and TG yield. The influence of the agitation rate on the esterification rate was also studied using P U F A concentrates from cod liver oil. The optimized conditions were applied to obtain TG highly enriched in EPA and AA from P. tricornutum and P. cruentum microalgae. Table

1 shows the fatty acid composition of the extracts and P U F A concentrates obtained from the cod liver oil and microalgae used in glycerol esterification.

3.1. Effect of temperature on esterification Fig. 1 shows that before adding molecular sieves (1 g at 24 h), the higher reaction rate was obtained at 50 and 60~ After adding molecular sieves, a slightly higher rate was obtained at 60~ Before the molecular sieves are added, the water produced in the reaction is not eliminated and this water hydrolyzes the glycerides formed producing free fatty acids and diminishing the T G yield. After 24 h the water is eliminated by the sieves and a strong increase in the rate is observed; although no significant differences were obtained at the three temperatures tested, it seems that the higher the temperature, the faster the reaction rate is. It is likely that this result was partly due to the fact that immobilization and the organic solvent

384

A. Rohles Medina et al.. Journal of Biotechnology 70 (1999) 379-391

conferred this enzyme greater thermostability (Dordick, 1989). Because the equilibrium TG yield does not depend on temperature and because the differences between reaction rates at the temperatures tested are not significant. 40 or 50~ may be preferable in order to preserve the stability of the PUFA. 3.2. Effect o f the initial ~t'ater co!lte, t and the addition time o f molecular sieres oll TG synthesis

The initial water content affects the esterification reaction equilibrium because water increases hydrolysis of TG, however the enzyme molecule requires a hydrating layer as the primary component of the enzymatic microenvironment (Zaks and Klibanov, 1985). This layer acts as a buffer between the enzyme surface and the bulk of the reaction medium. It is possible to keep an enzyme fully hydrated and active with xvater only comprising a small portion of the reaction volume. Water requirements for enzymes in organic media

vary greatly, therefore each enzyme must be examined at various levels of hydration (Dordick, 1989). Esteban Cerdfin et al. (1998) demonstrated that low water content is preferable for T G synthesis, since high TG yields were obtained at the lowest initial water content tested (0.5% v/v). In this work, experiments were performed with an even lower initial water content. Fig. 2 shows that the highest TG synthesis rates were obtained with dehydrated lipase and without adding additional water. The rate of T G formation decreased after 48 h because the equilibrium TG yield had been attained. These results and the ones obtained in a previous work (Esteban Cerd~in et al., 1998) indicate that very little water content is necessary to maintain enzymatic activity. Any appreciable water content diminishes the TG yield because water is a product of the esterification reaction and equilibrium is displaced to the reactants, so to maintain the enzymatic activity only a few molecules adsorbed around the active site of the lipase are required.

100 T 9O + 80 +

7o + ~ 6o - , - Dehyd. 4 - Original

50 40

0.25%

30

0.50%

20 10

0

8

16

24

32

40

48

56

64

72

80

88

96

T I M E (h)

Fig. 2. Effect of water content and time on the TG yield. Esterification conditions: 9 ml hexane, 40~ 41.7 mg glycerol, 400 mg PUFAs (glycerol/fatty acid ratio 1:3 molar), 1 g molecular sieves added at 24 h, 100 mg of lipase N-435 and 175 rpm. All the analysis by TLC and titration of the products of esterification reaction were done twice.

A. Robles Medina et al. Journal of Biotechnology 70 (1999) 379-391 Table 2 Effect of the addition time of molecular sieves on the ED and the triglyceride synthesis ~ Molecular sieves (g)

Addition time (h)

ED (%)

T G yield ("/,,)

0 1 1 1 1 1 1

0 4 8 12 16 24

27.6 86.8 86.7 79.6 85.7 84.8 87.8

85.9 85.5 78.0 84.7 83.5 85.2

+ + + + + + +

1.5 2.5 1.2 1.1 0.7 1.8 0.4

+ + + + + +

5.0 2.4 2.5 2.0 3.2 1.0

385

must be added at the beginning of the reaction in order to obtain a high initial rate of esterification (Fig. 2) and not close to reaction equilibrium, as indicated by Ergan et al. (1988). Table 2 also shows that ED was low with no molecular sieves and drastically increased with addition of 1 g.

3.3. E//ect o/gl)'cerol P UFA ratio on glyceride s~'~tthesis

a Esterification conditions: 9 ml hexane. 50~ 41.7 mg glycerol, 400 mg P U F A s (glycerol fatty acid ratio 1:3 molar). without water, 100 mg of lipase N-435, esterification time 96 h and 175 rpm.

Figs. 1 and 2 show that the rate of TG formation increased after 24 h when the molecular sieves were added and the water produced during esterification was eliminated. Table 2 shows that the time the molecular sieves are added has little effect on the equilibrium TG yield obtained after 96 h of reaction. Therefore, the molecular sieves

In previous work (Esteban Cerd/m et al., 1998) the stoichiometric glycerol/FA ratio of 1"3 molar was established as optimum for obtaining a high TG yield. Excessive glycerol increased the ED because the esterification equilibrium is displaced toward product formation, however, the TG yield decreased because a large amount of M G and DG were synthesized due to the excess glycerol. A similar result was obtained by Ergan et al. (1990) with Lipozyme IM-20. In this work, a slight excess of glycerol that would carry the esterification equilibrium to the formation of glycerides without decreasing the TG yield was tested. Fig. 3 shows that the TG yield did increase slightly when the

100 -

95 90

§

__r e _

§

--I-

85

I

*~ 80 t t

,.1 75

!

r.r,1

!

70

i

:! i

"

!

[" 65

9 9

. .

.

9: . . . .

60

..:.

i .....

55 50

,

,,

.:. ,,,

......

9 .: .: .i .. i: ' .........: ....... :_ _,

1"3

i

I! i

:.:! ii i i I .:

t

......

+10%

t

| .....

+20%

t

t

+30%

GLYCEROL

:., ..,:

: ..

i, .,.

,,,i~

t

+40%

I

+50%

IN E X C E S S ( % )

Fig. 3. Effect of the excess of glycerol over the stoichometric ratio on the T G yield. Esterification conditions: 9 ml hexane, 50~ 400 mg P U F A s , 0% water, 1 g molecular sieves added at 0 h. 100 mg of lipase N-435. esterification time 96 h and 200 rpm. All the esterification reactions were carried out in duplicate.

386

A. Robles Medina et al. . Journal o f Biotechnology 70 (1999) 379-391

Table 3 Scaling up 1-5 of the esterification reaction Time (h)

24 48 72 96

Scale la

Scale

5b

ED (%)

TG yield (%)

ED (%)

TG yield (%)

94.0 92.1 96.2 95.7

85.2 85.4 90.0 93.5

98.1 98.8 98.5 95.7

85.7 86.7 94.2 92.6

+ 1.4 + 1.8 _+ 1.1 + 0.2

+ 2.8 + 3.4 _+ 2.0 + 1.0

_+ 1.4 + 1.4 _+ 1.2 _+ 1.8

+_ 2.9 + 2.5 _+ 2.4 _+ 3.6

a Esterification conditions: 9 ml hexane, 50~ 50.0 mg glycerol, 0.4 g PUFAs (glycerol/FA ratio 1.2:3 molar), 0% water, 1 g molecular sieves added at 0 h, 100 mg of lipase N-435 and 200 rpm. b Esterification conditions: 45 ml hexane, 50~ 250.0 mg glycerol, 2.0 g PUFAs (glycerol/FA ratio 1.2:3 molar), 0% water, 5 g molecular sieves added at 0 h, 500 mg of lipase N-435 and 200 rpm.

glycerol/FA ratio did from the stoichiometric ratio (1:3 molar) to a 20% excess of glycerol over the stoichiometric ratio, whereas further increase in the amount of glycerol caused the TG yield to decrease.

3.4. Effect of the agitation rate on the TG yield Fig. 4a shows that between zero and 24 h (i.e. before adding the molecular sieves), the rate of TG synthesis was higher at 200 rpm than at 175 and 100 rpm. After 24 h TG synthesis rates at 175 and 200 rpm are similar and higher than at 100 rpm. Equilibrium was first reached at an agitation rate of 200 rpm. Fig. 4b shows the effect of agitation rate on the TG yields when molecular sieves are added at the commencement of the reaction. Only a very slight difference in rate is observed at 100 and 200 rpm. This demonstrates that within the range of the study, the agitation rate has little influence on the reaction rate, i.e. mass transfer does not exert much control on the reaction rate. Some authors have also noted that when vigorous stirring of the reaction mixture is maintained, the reaction rate is slow enough for intraparticle (Reyes and Hill, 1994) and external mass transfer limitations (Luck et al., 1988; Miller et al., 1991) to be negligible for these macroporous particles where lipase is immobilized. With no molecular sieves (between 0 and 24 h, Fig. 4a) at low agitation rates (100 and 175 rpm), the water liberated remains close to the active site of lipase and the reaction rate is low. When the

sieves are added after 24 h, the water is removed more quickly, as the agitation rate is much higher. Therefore, the agitation rate affects more to the rate of withdrawal or elimination of water due to the molecular sieves than to the rate of transfer of the reactants between the lipase and the reaction mixture. The esterification reaction was repeated under the best experimental conditions but using five times more hexane, glycerol, PUFA, molecular sieves and lipase than under the optimized conditions. Table 3 compares the ED and TG yields obtained on both scales. The results were virtually identical, suggesting an absence of solid-liquid mass transfer limitation because of an exceptionally slow enzyme reaction. This happened even though the mixture consistence resembled a thick slurry.

3.5. Esterification of glycerol and PUFA concentrates from P. tricornutum and P. cruentum biomass The best experimental conditions for synthesis of previously obtained TG (9 ml hexane, 50~ 0% initial water. 1.2:3 mol/mol glycerol/FA ratio, 1 g of molecular sieves added at the beginning of the reaction, 100 mg of lipase and 200 rpm) were applied to study the variation in the ED and TG yield from P U F A concentrates obtained from the microalgae P. tricornutum and P. cruentum over reaction time. Table 4 shows that the ED was over 94% with PUFAs from both microalgae after

A. Robles Medina et al./Journal of Biotechnology 70 (1999) 379-391

387

a)

100 90 80 70 60 5O + 40

1O0 rpm 175 rpm 200 rpm

30 20 10 0m

0

8

16

32

24

40

48

56

64

72

80

88

96

~ M E (h) 100

b)

-

90 8070*~ 60 ,d

200 rpm

50

1O0 rpm

P, r~ 40 [,,, 30 20 I0 ,

0

8

t

t

16

24

32

40

48

9t

- I ..

~

56

64

72

~ .....

80

I

I

88

96

~ M E (h) Fig. 4. Effect of the rate of agitation and time on the TG yield. Esterification conditions: (a) 9 ml hexane, 50~ 41.7 mg glycerol, 400 mg PUFAs (glycerol/fatty acid ratio 1:3 molar), 0.5% (v/v) water, 1 g molecular sieves added at 24 h and 100 mg of lipase N-435; and (b) 9 ml hexane, 50~ 50.0 mg glycerol, 400 mg PUFAs (glycerol/fatty acid ratio 1.2:3 molar), 0% (v/v) water, 1 g molecular sieves added at the start of reaction and 100 mg of lipase N-435. All the analysis at 24 h and 100 and 175 rpm and all the analysis at 96 h were made in duplicate.

388

A. Robles Medina et al.

Journal qf Biotechnology 70 (1999) 379-391

Table 4 Esterification of glycerol and PUFAs from P. tricormmml and P. cruentum biomass ~ Time (h)

24 48 72 96

P. tricornutum

P. cruentum

ED (%)

TG yield ('I',,)

ED (%)

TG yield (%)

95.2 95.9 98.3 97.8

92.5 93.2 96.5 95.7

94.7 96.8 94.1 99.4

75.7 80.9 82.6 89.3

+ + + +

1.3 1.1 1.5 0.7

+ + + +

2.6 2.3 2.9 1.5

_+ 1.0 _+ 1.2 _+ 2.5 + 0.7

_+ 2.0 _+ 1.6 _+ 4.4 + 1.6

a Esterification conditions: 9 ml hexane. 50~ 50.0 mg glycerol. 400 mg PUFAs (glycerol/FA ratio 1.2:3 molar), 0% water, 1 g molecular sieves added at 0 h, 100 mg of lipase N-435 and 200 rpm.

24 h. With PUFAs from P. tricornutun~, the equilibrium TG yield of 96.5% was reached at 72 h of reaction. This was similar to that obtained with P U F A concentrate from cod liver oil (93.5'~,,. Table 3). As the fatty acid compositions of cod liver oil and P. tricornutum PUFA concentrates (Table 1) differ fundamentally in their EPA and DHA contents, this would seem to indicate that the composition does not affect the esterification reaction. The TG yield obtained with PUFAs from P. cruentum were lower (89.3% at 96 h). However, the fatty acid composition of synthesized TG (Table 5) and PUFA concentrates are similar (Table 1), indicating that lipase N-435 did not appear to be acyl-specific and all the fatty acids were incorporated into the TG in the same proportion. PUFA concentrates from P. tricortlutum and P. cruentum (Table 1) also contain other substances apart from fatty acids (such as unsaponifiable lipids) not present in the PUFA concentrate from cod liver oil which do not affect the esterification rate and/or enzyme activity. This differs slightly from the results obtained by Osada et al. (1990), who reported certain differences in the degree of PUFA incorporation into glycerides (based on the number of carbon and double bond numbers) with some lipases. Some differences were found in the incorporation of AA, EPA and D H A with TOYO lipase (from Chromobacteriunl viscosum), for instance. A lower TG yield was also obtained for EPA than AA and DHA. Furthermore, with lipase from Candida c)'limh'acea, the degree of incorporation of DHA into glycerides was lower than AA and EPA and the TG yield was also lower for DHA (Osada et al., 1990).

Fig. 5 shows the detailed changes in composition of lipids during the course of the esterification reaction. In the initial 24 h, rapid consumption of PUFA and production of TG were observed. With PUFA from P. tricornutum Table 5 Fatty acid composition (% of total fatty acids) of triglycerides synthesized by esterification of glycerol and PUFA concentrates (Table 1) from cod liver oil, P. tricornutum and P. ~'rttentttm biomass a Fatty acid

Cod liver oil

P. tricornu-

P. cruentum

gum

14:0 16:0 16:1n7 16:2n4 16:3n4 16:4nl 18:0 18:1n9 18:1n7 18:2n6 18:3n3 18:4n3 20:3n6 20:1n9 20:4n6 (AA) 20:4n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) Others

0.9 0.2 1.2 1.5 0.6 0.6

0.7 1.0 1.1 10.6

8.8 17.0 10.9 2.6

0.4 0.2 0.3

2.6

5.2

1.0 2.1 25.7 1.5 44.7

5.4

43.4

42.5

45.6

2.2

0.1

6.6

8.0

4.8

~' Esterification conditions: 9 ml hexane, 50~ 50.0 mg glycerol, 400 mg P U F A s (glycerol/FA ratio 1.2:3 molar), 0% water. 1 g molecular sieves added at 0 h, 100 mg of lipase N-435, 96 h and 200 rpm.

A. Robles Medina et al.

Journal

of Biotechnology

389

70 (1999) 379-391

a) 100

--

.~

-

_-

,

--

90

70 ~

60

+ DG - - s - TG

50

~ 30 ~ 20 10 0

0

12

24

36

48

60

72

84

96

T I M E (h) 100 ~

b)

90

8o 70

60

- - 4 - MG

50

+FFA DG

~Z 40 ~

30

~

20

--r162 9 TG

10 0 0

12

24

36

48

60

72

84

96

T I M E (h) Fig. 5. Composition of glycerides synthesized by esterification of glycerol and PUFAs from (a) P. tricornutum', and (b) P. cruentum. Esterification conditions: as in Table 4. All the analysis at 96 h were made in duplicate.

390

A. Robles Medina et al. ,' Journal of Biotechnology 70 (1999) 379-391

(Fig. 5a) the formation of M G was not detected and the m a x i m u m D G / G L Y ratio was 2.8% at 24 and 48 h of reaction. However, with P U F A from P. c r u e n t u m (Fig. 5b) more M G and D G were synthesized (the m a x i m u m D G / G L Y ratio was 17.7% at 24 h). As this lipase does not appear to be acyl-specific, the explanation for this is not yet clear. In previous work, (Esteban Cerdfin et al., 1998) the sensitivity of D G / G L Y to the glycerol/fatty acid ratio had been analyzed, finding that a larger proportion of D G / G L Y was obtained when the glycerol/FA ratio was increased from 1:4 to 24:1. Because of this, a 1.2:3 glycerol/FA ratio was maintained in both microalgae P U F A concentrates, although it is likely that other reasons could be responsible for the differences observed and perhaps more research is needed to justify them. Finally, compared with the results published by Esteban Cerdfin et al. (1998), the initial rates of P U F A consumption and T G formation observed were faster in this production profile because no water was added and the molecular sieves were added at the beginning of the reaction.

Appendix A. List of abbreviations AA DG DG/GLY DHA ED EPA FA FFA GC MG MG/GLY N-435 PUFA TG TG/GLY TLC

Arachidonic acid Diglyceride Percentage of diglycerides over total glycerides Docosahexaenoic acid Degree of esterification Eicosapentaenoic acid Fatty acid Free fatty acid Gas chromatography Monoglyceride Percentage of monoglycerides over total glycerides Lipase Novozym-435 de Novo Nordisk Polyunsaturated fatty acids Triglyceride Percentage of triglycerides over total glycerides Thin layer chromatography

References 4. Conclusions F r o m the above results it may be concluded that TG, without M G and DG, containing highly concentrated EPA, D H A and AA may be easily synthesized by lipase N-435 in hexane. Using P U F A concentrates from P. t r i c o r n u t u m and P. c r u e n t u m biomass, it has been possible to obtain a T G with 42.5% EPA and very low AA and D H A content and T G with 43.4% AA and 45.6% EPA, respectively. It should also be remarked that a T G yield of over 95% was obtained from P. t r i c o r n u t u m P U F A concentrate._

Acknowledgements This research was supported by grants from the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT), BIO 95-0652.

Acien Fernandez, F.G., Garcia Camacho, F., Sfinchez P6rez, J.A.. Fern/mdez Sevilla, J.M., Molina Grima, E., 1998. Modeling of biomass productivity in tubular photobioreactors for microalgal cultures: effects of dilution rate, tube diameter and solar irradiance. Biotech. Bioeng. 58, 605614. Boswell. K., Koskelo, E.-K., Carl, L., Glaza, S., Hensen, D.J., Williams, K.D., Kyle, D.J., 1996. Preclinical evaluation of single-cell oils that are highly enriched with arachidonic acid and docosahexaenoic acid. Food Chem. Toxicol. 34, 585-593. Braden, L.M.. Carroll, K.K., 1986. Dietary polyunsaturated fat in relation to mammary carcinogenesis in rats. Lipids 21. 285-288. Camacho Rubio, F., Aci6n Fernandez, F.G., Sfinchez P6rez, J.A., Garcia Camacho, F., Molina Grima, E., 1999. Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture. Biotech. Bioeng. 62, 71-86. Cartens, M., Molina Grima, E., Robles Medina, A., Gim6nez Gim6nez, A., Ib~ifiez Gonz/tlez, M.J., 1996. Eicosapentaenoic acid (20:5n-3) from the marine microalga Phaeodactvlum tricornutum. J. Am. Oil Chem. Soc. 73, 1025-1031.

A. Robles Medina et al. /Journal of Biotechnology 70 (1999) 379-391

Dordick, J.S., 1989. Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol. 11, 194-211. Ergan, F., Trani, M., Andr6, G., 1988. Solvent free triglyceride synthesis using LipozymeTM IM-20. Biotechnol. Lett. 10. 629-634. Ergan, F., Trani, M., Andr6, G., 1990. Production of glycerides from glycerol and fatty acid by immobilized lipases in non-aqueous media. Biotech. Bioeng. 35, 195-200. Esteban Cerd~.n, L., Robles Medina, A., Gim6nez Gimenez, A., Ib~ifiez Gonz~ilez, M.J., Molina Grima, E., 1998. Synthesis of PUFA enriched triglycerides by lipase catalyzed esterification. J. Am. Oil Chem. Soc. 75, 1329-1337. Gim6nez Gim6nez, A., Ib~,fiez Gonz~ilez, M.J., Robles Medina, A., Molina Grima, E., Garcia Salas, S., Esteban Cerd~in, L., 1998. Downstream processing and purification of eicosapentaenoic (20:5n-3) and arachidonic acids (20:4n6) from the microalga Porphyridium cruentum. Bioseparation 7, 89-99. Hamazaki, T., Fischer, S., Schweer, H., Meese, C.O., Urakaze, M., Yokoyama, A., Yano, S., 1988. The infusion of trieicosapentaenoyl-glycerol into humans and the in vivo formation of prostaglandin 13 and tromboxane A 3. Biochem. Biophys. Res. Comm. 151, 1386-1394. Innis, S.M., 1991. Essential fatty acids in growth and development. Prog. Lipid Res. 30, 39-103. Koletzko, B., Decsi, T., Demmelmair, H., 1996. Arachidonic acid supply and metabolism in human infants born at full term. Lipids 31, 79-83. Koletzko, B., Schmidt, E., Bremer, H.J., Haung, M., Harzer, G., 1989. Effects of dietary long-chain polyunsaturated fatty acids on the essential fatty acid status of premature infants. Eur. J. Pediatr. 148, 669-675. Kosugi, Y., Azuma, N., 1994. Synthesis of triacylglycerol from polyunsaturated fatty acid by immobilized lipase. J. Am. Oil Chem. Soc. 71, 1397-1403. Leaf, A., Kang, J.X., 1996. Cardiac arrhythmia. Abstract of International Conference on Highly Unsaturated Fatty Acids in Nutrition and Disease Prevention, Barcelona, p. 41. Lepage, G., Roy, C., 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res. 25, 1391-1396. Luck, T., Kiesser, T., Bauer, W., 1988. In: Applewhite, T.H. (Ed.), Proceeding of the world conference on Biotechnology for the fats and oils industry, American Oil Chemists Society, Champaign, IL, pp. 343-345. Miller, D.A., Prausnitz, J.M., Blanch, H.W., 1991. Kinetic of lipase-catalised interesterification of triglycerides in cyclohexane. Enzyme Microb. Technol. 13, 98-103. Molina Grima, E., Robles Medina, A., Gim6nez Gim6nez, A., S~inchez P6rez, J.A., Garcia Camacho, F., Garcia Sfinchez, J.L., 1994. Comparison between extraction of lipids and fatty acids from microalgal biomass. J. Am. Oil Chem. Soc. 71, 955-959. Molina Grima, E., Robles Medina, A., Gim~nez Gim6nez, A., Ib~ifiez Gonz~ilez, M.J., 1996. Gram-scale purification of eicosapentaenoic acid (EPA 20:5n-3) from wet Phaeodactv-

391

lum tricornutum UTEX 640 biomass. J. Appl. Phycol. 8, 359-367. Nichols, B.W., Appleby, R.S., 1969. The distribution and biosynthesis of arachidonic acid in algae. Phytochemistry 8, 1907-1915. Osada, K., Takahashi, K., Hatano, M., 1990. Polyunsaturated fatty glyceride synthesis by microbial lipases. J. Am. Oil Chem. Soc. 67, 921-922. Rambjor, G.S., Walen, A.I., Windsor, S.L., Harris, W.S., 1996. Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans. Lipids 31 (Suppl. S), 45-49. Reyes. H.R.. Hill, C.H. Jr., 1994. Kinetic modeling of interesterification reactions catalyzed by immobilized lipase. Biotechnol. Bioeng. 43, 171 - 182. Robles Medina. A., Gimenez Gim6nez, A., Garcia Camacho, F., S~inchez P6rez, J.A., Molina Grima, E., Contreras G6mez, A., 1995. Concentration and purification of stearidonic, eicosapentaenoic and docosahexaenoic acids from cod liver oil and the marine microalga Isochrysis galbana. J. Am. Oil Chem. Soc. 72, 575-583. Shimizu, S., Shinmen, Y., Kawashima, H., Akimoto, K., Yamada, H., 1987. Production of C-20 polyunsaturated fatty acids by fungi. In: Proceeding of World Conference on Biotechnology for the Fats and Oils Industry, AOCS, Champaign, IL pp. 1000-1006. Shinmen. Y., Shimizu, S., Akimoto, D., Kawashima, H., Yamada. H., 1989. Production of arachidonic acid by Mortierella fungi. Selection of a potent producer and optimization of culture conditions for large-scale production. Appl. Microbiol. Biotechnol. 31, 11-16. Simopoulos, A.P., 1991. Omega-3 fatty acids in health and disease and in growth and development. Am. J. Clin. Nutr. 54, 438-463. Sing, M.D., Chandra, M.D., 1988. Biochemical and cellular effects of fish and fish oils. Prog. Food Nutr. Sci. 12, 371-419. Weylandt, K.H., Kang, J.X., Leaf, A., 1996. Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids. Lipids 31. 977-982. WHO FAO, 1977. Dietary fats and oils in human nutrition. Report of an expert consultation. FAO, Rome, Italy. Wigmore, S.J., Ross, J.A., Falconer, J.S., Plester, C.E., Tisdade, M.J., Carter, D.C., Faron, K.C.H., 1996. Effect of PUFA on the progress of cachexia in patiens with pancreatic cancer. Nutrition 12, 27-30. Yongmanitchai, W., Ward, O.P., 1991. Screening of algae for potential alternative sources of eicosapentaenoic acid. Phytochemistry 30, 2963-2967. Zaks, A., Klibanov, A., 1985. Enzyme-catalyzed processes in organic solvents. Biochemistry 82, 3192-3196. Ziboh, V.A., 1991. o33 polyunsaturated fatty acid constituents of fish oil and the management of skin inflammatory and scaly disorders. In: Simopoulos, A.P., Kifer, R.R., Martin, R.E., Barlow, S.M. (Eds.), Health Effects of o33 Polyunsaturated Fatty Acids in Seafoods. Word Rev. Nutr. Diet., 66. 425-435.

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JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 393-402

Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen L. Vandanjon

a,,, p. Jaouen b N. Rossignol b F. Qudm6neur b J.-M. Robert c

a Laboratoire PolymOres et ProcOdOs (L2P), UniversitO de Bretagne Sud, 4 rue Jean Zay, F-56325 Lorient codex, France b Laboratoire de GOnie des ProcOdOs (LGP), Institut des Substances et Organismes de la Mer (ISOMer), Boulevard de l'UniversitO, BP 406, F-44602 Saint-Nazaire codex, France c Laboratoire de Biologie Marine (LBM), Institut des Substances et Organismes de la Mer (ISOMer), Rue de la Houssinikre-BP 92208, F-44322 Nantes codex 3, France

Received 9 October 1998; received in revised form 24 November 1998; accepted 22 December 1998

Abstract The marine microalga Haslea ostrearia, also called << blue navicula >>, presents the unique peculiar property among the diatoms, to produce at its extremities a blue hydrosoluble pigment called <<marennine >>. It is presented the concentration and the desalting of the exocellular pigment by membrane processes (ultrafiltration, nanofiltration, reverse osmosis). Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting. It is shown the effect of velocity and pressure on performances of nanofiltration membranes. Permeation flux superior to 100 1 h - l m - 2 (at 14.105 Pa) are obtained with the Kiryat Weizmann membrane MP 20 (polyester coated with a polyacrylonitrile layer, cut-off 450 Da). For the desalting of the blue pigment solution, nanofiltration membranes present a few advantages: a low salt rejection (less than 10% at 14.105 Pa) and a high pigment rejection (the nanofiltration membrane MP 20 retains more than 95% of the pigment). This membrane used in diafiltration mode allows an acceptable speed of desalting (700 g of salt eliminated per hour and per m 2 at 25.105 Pa for a concentration of 18 g of salt per litre of solution). 9 1999 Elsevier Science B.V. All rights reserved. Keywords: Concentration; Desalting; Haslea ostrearia; Nanofiltration; Pigment

I. Introduction

* Corresponding author. Tel.: + 33-2-97874531; fax: + 332-97874588. E-mail addresses: [email protected] (L. Vandanjon), [email protected] (P. Jaouen), [email protected] (J.-M. Robert)

The aim of the study is to develop the industrial application of the marine d i a t o m H a s l e a o s t r e a r i a Simonsen, also called << blue navicula >>, responsible for the greening of the oysters in France. This microalga presents the unique peculiar prop-

0168-1656/99/$ - see front matter 9 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00092-9

394

L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393-402

erty among the diatoms, to produce at its extremities a blue hydrosoluble pigment called <<marennine >> (Robert, 1983). Ultrastructurally, the diatom shows marked changes depending on the stage of cell blueing by accumulation of marennine (Nassiri et al., 1998). The interest of this pigment is notably linked to:

9 its colouring properties: the pigment might be used as a new blue natural dye in the food and agriculture fields, 9 its ability for the greening of oysters: the only current industrial application is at the SOPROMA Company in Bouin (Vend6e, Atlantic coast, France), where the production of the diatom is performed in batch cultures, using tanks of 6 m 3 in volume under carefully-controlled incubation conditions, 9 its antiproliferative properties since aqueous extracts of the pigment show inhibitory effects both in vitro and in vivo against solid carcinoma lines (Belt et al., 1997) In this study, it is presented the concentration and the desalting of the exocellular pigment by membrane processes: performances of ultrafiltration, nanofiltration, reverse osmosis membranes are compared. Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting.

250 ml erlenmeyer flasks filled with 150 ml of ES 1/3 medium (first preculture). The temperature of incubation was 16~ and the light intensity was 3 x 1016 quanta cm -2 s -1 with a 14/10 h light/ dark cycle. The erlenmeyer flask contents were then inoculated into 25 1 flasks containing 20 1 of ES 1//3 medium (second preculture). After incubation of these larger flasks for ca 7 to 10 days in identical temperature and light conditions, their contents were transfered into 500 1 tanks filled with 400 1 underground sea-water from the Bouin district (Rouillard, 1996). In these natural incubation conditions, cell suspensions with concentration ranging between 60 and 100 x 103 cells m l - ~ were obtained after incubation for 5-12 days. The algal mass collected by centrifugation, with a continuous centrifuger CEPA-PATBERG, LE (clarification cylinder type K, inox), was then collected and the blue coloured medium containing the exocellular marennine was recuperated for concentration and desalting by membrane processes.

2.2. Experimental set-up and membranes The experiments are carried out on two pilote plants: Millipore (Prolab Bench Top) and Gamma Filtration (Microlab 80S) built on the same principle (Fig. 1). The both pilote plants are constituted from a centrifugal pump allowing liquid recirculation and

2. Materials and methods

2.1. Biological material The study was performed using an axenic strain of H. ostrearia isolated in the Marine Biology Laboratory from the oyster-pond waters of the Bouin district (Vend6e, France). The clone cells were characterized by an average model length of 65 gm. The algal cultures were maintained by weekly transfer to fresh ES 1/3 medium (Robert, 1983; Lebeau et al., 1999). Algal precultures were precultivated by applying a two steps procedure, before to be used for mass production of the diatom. Cells from the clone pool were first precultured for ca 7 days in

Permeate

[Heatexchanger Throttling valve

Flowmeter

V

Outlet pressure

Filtration module

Feeding tank Recirculating pump

z_._..x

Feedingpump

z_._.a

Inlet pressure Flow control valve

Fig. 1. Schematic representation of the pilote plant.

L. Vandanjon et al. Journal of Biotechnology 70 (1999) 393-402

395

Table 1 Characteristics of membranes used Trade Mark

Manufacturer

Geometry

Technique

Cut-off

Area (m 2)

Material

Iris 3028

Tech-Sep, Or61is Millipore Kiryat Weizmann Kiryat Weizmann

Flat

UF

3 kDa

6.9 x 10- 3

Polyethersulfone

Spiral Tubular

RO NF

<200 Da 450 Da

0.3 47 x 10- 3

Tubular

NF

450 Da

47 • 10 -3

Polyamide Polyester coated with a polyacrylonitrile layer Polysulfone on polypropylene

R 45P M P T 20 M P T 31

a volumetric pump assuming the feeding of the recirculation loop. In this way, pressure and tangential velocity adjustments are independent. Transmembrane pressure is measured upstream and downstream of the permeation module by manometers. A pressure of 50 • l0 t Pa can be reached in the Millipore installation and 45 • 10 -~ Pa in the Gamma Filtration installation. A freezing group Mouvex RFA-30 allows to maintain a constant temperature of 25~ The filtration module is either flat or tubular. Four membranes are tested: one ultrafiltration (UF) membrane, one << loose >> reverse osmosis (RO) membrane and two nanofiltration (NF) membranes. These membranes and their characteristics are presented in the Table 1. The UF and N F experiments are carried out by total recycling of the retentate and the permeate. Steady flux and retention rates of the membranes are measured after 120 min. Concentration of the blue pigment solution is carried out with a <> RO membrane. Working pressure is 41 • 105 Pa and temperature is kept constant at 25~ The concentrated solution is desalted by diafiltration using the same << loose >> RO membrane in a continuous mode (Fig. 2).

Qv

"

QR

Qv : Volumetric flow of permeate

The concentration of pigment in solution is determinated by spectrophotometry (Secomam SG 1000). Optical density is measured at 663 nm, which corresponds to the maximum absorbance of the marennine (Robert and Hallet, 1981). Salinity is determinated by measuring resistivity (Tacussel CD 60) of the marennine solution.

3. Resuits~discussion

The pigment << marennine >> seems to be composed of a mix of macromolecules of different sizes. Size repartition of pigment molecules has been estimated by using six UF membranes (bearing negligible adsorption rate) of different molecular cut-off comprised between 1 and 300 kDa (Vandanjon, 1997). By relying on the model proposed by Ferry (1936), we have shown that a large part of the pigment has a molecular weight comprised between 3 and 7 kDa (Fig. 3). So the ultrafiltration (low cut-off) could be convenient a technique for the concentration and the desalting of marennine solution. High purity molecules can be obtained by using multistage diafiltration (Muller, 1996). However, the existence of a small fraction of pigment whose molecular weight is close to hundreds Dalton involves the use of nanofiltration or reverse osmosis membranes for the recovery of the totality of the marennine.

(= volumetric flow of solvent) QR : Recirculating flow

Fig. 2. Diafiltration in a continuous mode.

3.1. Performance of ultrafiltration membrane (low cut-off) The operating conditions are as following:

396

L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393-402 %

70 61%

60 50 40 30 17%

20 10

8%

5%

5%

4%

[ 0,85

3,3

7

20

J

35

Molecular weight (kDa) Fig. 3. Molecular weight repartition of marennine molecules. 9

Pressure: 4 x 105 Pa Tangential velocity: 1 m s - ] Results are presented on Fig. 4. The ultrafiltration membrane 3028 3 kDa presents a high rejection percentage for the pigment (90%) and a low rejection percentage for the salts. This membrane could be convenient for the desalting of pigment solution by diafiltration. 9

3.2. Performance of << loose >> reverse osmosis membrane

3.2.2. Comments 9 Steady flux of 34 1 m - 2 h-~ is obtained after 26 h. 9 Pigment is highly concentrated while salt concentration is constant. 9 There is a gap between the VRF (VRF = 70) and the CF of pigment (CF = 50). It can be explained by the loose of pigment in the permeate and the fixation of pigment on the membrane separators.

400

3.2.1. Concentration of the pigment solution In order to obtain a high volumic reduction factor (VRF), a volume (Vo) of 350 1 of culture medium containing the hydrosoluble pigment is concentrated. The <> (culture medium) presents the following characteristics: 9 Optical density (at 663 nm): 0.024 (length of the optical path = 1 cm) 9 Salt concentration: 30 g 1-1 9 pH8 COncentration is achieved with the << loose >> reverse osmosis membrane Millipore R45P. Fig. 5 presents the evolution of the permeate flux and the concentration factor (CF) of pigment and salts.

--

100

Pigment rejection eq , cO

3= ._~

200

50

--

.2 u.

.~, O

Salt rejection 0

--"

'

-

J

loo

i

!

~

20O

Time (min) Fig. 4. Evolution of permeate flow and rejection percentage for an ultrafiltration membrane (cut-off 3 kDa).

L. Vandanjon et al.

Journal

o/"

50~

Biotechnology 70 (1999) 3 9 3 - 4 0 2

- -9 50

o~

397

P=41.105Pa T = 25 ~

~0

L__ 40

E

t_

"7 ,.I:7, ~ , 30---

A

/

jzx

~,

20--

,/

1,-, O

/ r--30

Lx

VRF = Vo/Vf o .,.a r

ao~176

CF = Cf/Co

.1=

,~igment

,---- 2 0

V o : Initial volume of blue water

r O

Vf: Final volume of blue water

L)

Co : Concentration at the initial time '---- 1 0 o/

l'-

Salts 0

c

!

c

Cf: Concentration at the end time

I 0

9 q,

I

1--o

10

20

30

Time (h) Fig. 5. Concentration of marennine with a <> reverse osmosis membrane. 3.2.3. C o n t i n u o u s

diafiltration

Diafiltration with the membrane R45P is achieved at a constant volume (5 1) and a constant pressure (10 x 105 Pa). Fig. 6 presents the evolution of the salt concentration and the rejection rates for salts and pigment in function of time and added pure water volume. It can be noticed that the salts are not totally eliminated after 13 h of diafiltration. Experimental curves are compared to the theoritical model developed in diafiltration (Mulder, 1991). After integrating the overall mass balance sheet:

(m 3 s - 1); TR(%) = 100" [1 -(Cp/C0)], salt rejection rate: and V0, initial volume of blue water (m3). If the salt rejection rate is constant with time, diafiltration experimental curves are close to the theoritical model. In the present case, the gap between theoritical curve and experimental curve is due to a salt rejection rate which is not perfectly constant (evolution of the osmotic pressure during diafiltration, adsorption phenomenom). However, it is possible to estimate the number of diavolumes (DV) necessary for desalting the solution with the relation (Tutunjian, 1985): In

dC -

(1)

Vo " --d~ = Qv " C p

DV =

Co TR

the following equation is obtained: C = C0 9e x p [ -

Qv .(1 - T R / I O 0 ) .

100 t/Vo]

(2)

with: t, filtration time (s); C, salt concentration in the coloured water at the time t (kg m - 3); Co, salt concentration in the coloured water at the initial time (kg m - 3 ) ; Cp, salt concentration in the permeate (kg m - 3 ) ; Qv, volumetric flow of permeate

Cf

(3)

1

with G, salt concentration in the coloured water at the end time (kg m - 3 ) . With the << loose >> reverse osmosis membrane (salt rejection rate -~ 60%), it is possible to know the number of diavolumes for desalting from 15 to 1 g l - I :

L. Vandanjon et al. Journal of Biotechnology 70 (1999) 393-402

398

are presented on Fig. 7 (membranes MPT 20 and MPT 31). With the low pressures (AP < 105 Pa), filtrate flux increases linearly and does not depend from the recirculation velocity (Darcy's law). Then flux increases slowly (primary polarization), but it is noticeable that it is strongly influenced by recirculation velocity (Brun, 1989). For these nanofiltration membranes, it can be considered that beyond 20 x 105-25 x 105 Pa, increase of the pressure has no more significant influence on the filtrate flux. However, fluxes superior to 50 1 m -2 h-~ at 30 x 105 Pa can be considered as acceptable performances. The ultrafiltrate fluxes versus tangential velocity are often represented according to the relation (Qu6m6neur and Schlumpf, 1980):

DV = In(1/15)/(60 - 100). 100 = 6.8 On the experimental curve (Fig. 6), it is necessary to add 35 1 of pure water to 5 1 of <> for desalting the solution, i.e: DV = 35/ 5=7. These results confirm that the model is correct whenever the salt rejection rate is constant.

3.3. Performance of nanofiltration membranes 3.3.1. Permeation flux A few sets of experiments by total recycling of the retentate and the permeate are carried out at different pressures and velocities. For each couple pressure-velocity, the stabilization of the flux is reached after about 2 h. These points allow to draw steady flux versus pressure at different tangential velocities. Results 1 6 -

a

='~

--~_

J = :~ 9u"

. . . . . . .

_~ _:

,_.

(4) :.

--

-

z

~

-

100

Pigment rejection "7

12

o

o

v

o .E

8--

~

0

~

~

-50

O ~ v,,,~ ,4-,,)

o O eO

perimental

_

Theoritical "".........~ model 0

'"

'

I-

'

4

!

'

I 12

8

Time (h) 0 0

~

~

l

7

.

.

.

.

.

14

21 |

28 i

,

35 I

42 l

....

49 :

Added pure water (litres) Fig. 6. D i a f i l t r a t i o n o f m a r e n n i n e s o l u t i o n with a << loose >> reverse o s m o s i s m e m b r a n e .

L. Vandanjon et al. , Journal o f Biotechnology 70 (1999) 3 9 3 - 4 0 2 40-I

~'

~

Pure water flux

9 - eo-

- - -o --o

399 9 ....

.

.

.

.

~1~-100

_...Q

Pigment 1 O0 ~

. - - - - ,~.,.~.~

,

I

?'E

,

i

50 ~

_ u = 2 m/s

,/

'

~

,,'"

.'" . ~ J m . .

i

Re = 29,000

o. Membrane MPT20 _.,. Membrane MPT31

u = 1 rrds Re = 14,500

//"'-.~,...__---.z-u

.' ~

,

,--

2o

MembraneMPT 31

....

: _ ~

= 0 5 m/s

-

Re = 7,250

-,.- -- "-.'~=ln~s . . . .

o .,..,

u = 0 5 m/s

--50

20--

j ~ 01..o.

r~

o

I 0

6

lO

20

3o

ot D

~

0

(D

-

Salts

__6.__.o_. . . . . . . . . . .

E

Q....

0

Pressure (10 5 Pa)

0 Fig. 7. Steady flux versus pressure for nanofiltration membranes.

J, ultrafiltrate f l u x ( m 3 m - 2 s - ] ) z(, 1l, constants; and u, fluid velocity (m s-1). In order to verify the validity of the Eq. (4) in nanofiltration, it is drawn on Fig. 8 the logarithmic permeate flux versus the logarithmic tangential velocity under a pressure of 25 x 105 Pa. For each membrane, a straight line is obtained with:

4.B

M

r.-

4.4 ---=

MPT 20 + 4.2--

4.0

.

I -0.8

0"

.

'

.

.

I -0.4

3.8-

.

.

10

20

30

Pressure (105 Pa) Fig. 9. Evolution of salt and pigment rejections with the pressure.

whose equation follows: In J = In z~+ n 9In u

(5)

In turbulent flow, the following values of the slope n can be determined graphically: Membrane MPT 20: n = 0.13 Membrane MPT 31: n = 0.18 These values are inferior to the most common values of the literature: n = 0.69-0.91 (Blatt et al., 1970; Zaitoun, 1979; Aimar, 1992). Generally, the coefficient n depends on hydrodynamic characteristics of the module geometry (flat, tubular, spiral etc.) and of the couple membrane-foulant (Gekas and Hallstr6m, 1987; Nabetani et al., 1990). But the very low concentration of natural pigment and the high salinity of the marennine solution may be responsible for these results.

3.3.2. Selectivio' of nanofiltration membranes

--

~ / / M P M P T

31

.

'

,,,

' 0.0 In U

I 0.4

'

I 0.8

Fig. 8. Experimental validation of the relation J--z~. u" in nanofiltration. Graphic determination of the exponent n.

From a practical or economical point of view, it is interesting to carry out desalting by diafiltration after concentration without any changing of material (pilot-plant and membrane). The << ideal >> membrane should retain the maximum of pigment and be as permeable as possible for the salts. Fig. 9 describes performance, in term of salt and pigment rejection, of two nanofiltration membranes MPT 20 and MPT 31.

400

L. Vandanjon et al./Journal of Biotechnology 70 (1999) 393-402

The membrane MPT 20 retains the fast totality of the pigment and its permeability for the salts remains high at high pressure. Rejection rate (TR) seems to be independent of the pressure, it would correspond to a mass transfer mechanism intermediate between capillar type mechanism (TR decreases with pressure) and diffusional type mechanism (TR increases with pressure). On the contrary, the nanofiltration membrane MPT 31 presents a rejection rate that increases with the applied pressure. This behaviour is characteristic of a diffusional type mechanism: TR(%) = 100.

1 - A . ( A P - Arc)

(6)

with: A, solvent permeability of the membrane; and B, solute (salts) permeability of the membrane. With this type of nanofiltration membrane, close to reverse osmosis membranes, an increase of the pressure induces an increase of the permeate flux and the salt rejection rate; concurrently, it is observed a light decrease of the pigment rejection. That means that high permeate flows during concentration will induce loss of pigment and will penalize the next step of desalting. So it will be necessary to find a compromise in the aim to optimize the overall process of concentrationdesalting.

The most efficient membrane for desalting is the ultrafiltration membrane 3028 3 kDa. Salts can easily pass through the membrane but the low pigment retention makes the membrane unusable for a diafiltration. At the opposite, the membrane R45P retains perfectly the pigment but its high salt rejection induces a long and difficult desalting. The nanofiltration membrane MPT 20 constitutes the best compromise in term of desalting and pigment rejection and it may be used efficiently in diafiltration mode.

4. Conclusion

Nanofiltration seems to be the best performing technique for desalting the blue coloured solution. The model used in diafiltration with a << loose >> reverse osmosis membrane may be used with nanofiltration membranes, whether salt rejection rate is about constant. In these conditions, it is possible to calculate, from laboratory scale experiments, performances of pilot-plant or industrial process. In this study, we have shown the effect of velocity and pressure on performance of nanofiltration membranes. Flows superior to 50 1 m - 2 h-1 (at 30 x 105 Pa) are obtained with the Weizmann membrane MPT 20 (in polyacryloni-

3.4. Comparison of membranes for diafiltration Diafiltration is carried out at a constant volume: Vo = 5 1. Three membranes are tested functionning in diafiltration mode: 9 an ultrafiltration membrane Or61is-Iris 3028 3 kDaatAP-4x 10SPa, 9 a nanofiltration membrane Kyriat Weizmann MPT 20 at A P - 2 5 x 105 Pa, 9 a <> reverse osmosis membrane Millipore R45P at A P - 10 x 105 Pa. With the aim to evaluate the efficiency of the three membranes (ultrafiltration, nanofiltration and reverse osmosis) to carry out a diafiltration, it has been drawn on Fig. 10 the graphic of desalting speed versus salts concentration in the retentate.

: ~,

1000

-

-

E ,s::

3028 3 kDa (4.105 Pa) MPT20 450 Da (25.105 Pa) R45P (10.10 s Pa) /

"7

UF low c ~ / ~ j

500 - -.=

.

NF

10

15

{/) r

0 9

-

0

5

20

Salt concentration in the retentate (g.1 l ) Fig. 10. Compared efficiency of the membranes for the elimination of the salts.

L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393-402

trile coated on polyester, cut-off 450 Da). Higher fluxes could be obtained by optimizing hydrodynamic conditions or the couple tangential velocity/pressure. For the desalting of the blue-green pigment solution, nanofiltration membranes present a few advantages over loose reverse osmosis and ultrafiltration: a low salt rejection rate (less than 10% at 14 bars) and a high pigment rejection (the nanofiltration membrane MPT 20 retains the fast totality of the pigment). This membrane used in diafiltration mode allows to obtain an acceptable speed of desalting (for example, 700 g of salt eliminated per hour and per m 2 at 25 x 105 Pa for a concentration of 18 g of salt by litre of solution). So the nanofiltration membrane MPT 20 is interesting in the aim of a simultaneous concentration-desalting (without changing of processunit or membrane) of the marennine solution produced by H. ostrearia. The diafiltration does not allow to eliminate entirely the salts, but it does not constitute an obstacle in the aim of food enhancement of the marennine. Indeed, legislation makes difficult the commercialization of new natural dyestuffs (Labatut and In, 1990). In these conditions, non purified marennine should preferently be enhanced as a food ingredient. However, a high degree of purity is necessary for the use of marennine for therapeutical application in cancerology for example (Riou et al., 1993). The dialysis technique is then well suited for the final elimination of the salts when using small volumes of concentrated pigment solution. One further development of this study is the membrane photobioreactor which combines biological reaction and separation. Biological reaction: the research work consists in optimizing culture conditions of H. ostrearia for the highest pigment production: choice of the clones, study of the shear effects caused by pumps and valves in a recirculating loop (Vandanjon et al., 1999), addition of nutriments. Separation" for the extraction of pigment, two stages of membranes are necessary. Ultrafiltration (cut-off 40 kDa) can be used for the extraction of the exometabolite from the culture in a continuous mode (Rossignol et al., 1999). Then after-

401

wards, the marennine solution is concentrated and desalted by nanofiltration. References Aimar, P., 1992. Limiting flux in membrane separations: a model based on the viscosity dependency of the mass transfer coefficient. Chem. Eng. Sci. 47 (3), 579-586. Belt S.T., Robert J.-M., Roussakis C., Rowland S., 1997. Chemotherapeutic compounds from microalgae. Patent n~ ISOMer (University of Nantes, Nantes, France) and University of Plymouth, Plymouth, UK. Blatt, W.F., Dravid, A., Michaels, A.S., Nelsen, L., 1970. Solute polarization and cake formation in membrane ultrafiltration. In: Membrane Science and Technology, vol. 47. Plenum Press, New York. Brun J.-P., 1989. Proc6d6s de s6paration par membranes. Masson, Paris, 270 p, ISBN 2-225-81573-9. Ferry, J.D., 1936. Ultrafilter membranes and ultrafiltration. Chem. Rev. 18, 373-455. Gekas, V., Hallstr6m, B., 1987. Critical literature review and adaptation of existing Sherwood correlations to membrane operations. J. Membr. Sci. 30, 153-170. Labatut, M.-L., In, T.. 1990. La couleur au naturel. Biofutur November, 37-42. Lebeau, T., Junter, G.-A., Jouenne, T., Robert, J.-M., 1999. Marennine production by agar-entrapped Haslea ostrearia Simonsen. Biores. Technol. 67 (1), 13-17. Mulder, M., 1991. Basic Principles of Membrane Technology. Kluwer, Dordrecht. Muller A., 1996. Proc6d6 d'obtention d'~-lactalbumine de haute puret6: 6tapes 616mentaires du fractionnement des prot6ines de lactoserum et mise en cascade. Ph.D. thesis, ENSAR Rennes, France. Nabetani, H., Nakajima, M., Watanabe, A., Nakao, S.I., Kimura, S., 1990. Effect of osmotic pressure ans adsorption on ultrafiltration of ovalbumine. AIChE J. 36 (6), 907-915. Nassiri, Y., Robert, J.-M., Rinc6, Y., Ginsburger-Vogel, T., 1998. The cytoplasmic fine structure of the diatom Haslea ostrearm (Bacillariophyceae) in relation to marennine production. Phycologia 37 (2), 84-91. Qu~m~neur. F., Schlumpf, J.-P., 1980. Traitement des huiles solubles par ultrafiltration. Entropie 93, 22-29. Riou, D., Roussakis, C., Biard, J.-F., Verbist, J.-F., 1993. Comparative study of the antitumour activity of bistramides A, D and K against a non-small cell broncho-pulmonary carcinoma. Anticancer Res. 13, 2331-2334. Robert J.-M., 1983. Fertilit6 des eaux de claires ostr6icoles et verdissement: utilisation de l'azote par les diatom6es dominantes. Ph.D. thesis, Nantes University, Nantes, France, 281 p. Robert, J.-M., Hallet, J.-N., 1981. Absorption spectrum in vivo of the blue pigment <<marennine >> of the pennate diatom Nat'icula ostrearia Bory. J. Exp. Botany 32 (127), 341-345.

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Rossignol N., Vandanjon L., Jaouen P., Qu6meneur F., 1999. Membrane technology for microalgae harvesting: compared performances of cross-flow microfiltration and ultrafiltration. Aquacult. Eng. (submitted for publication). Rouillard I., 1996. Optimisation de la production en masse de Haslea ostrearia Simonsen sur une eau souterraine sal6e: importance de la souche et des conditions de culture: comparaison avec Skeletonema costatum (Grev) Cleve. Ph.D. thesis, Nantes University, Nantes, France, 270 p. Tutunjian, R.S., 1985. Scale-up considerations for membrane processes. Biotechnology 3, 615-626.

Vandanjon k., 1997. Etude d'un proc6d6 de valorisation d'une microalgue marine: concentration et purification par techniques '/i membranes d'un pigment naturel produit par la diatom6e Haslea ostrearia. Ph.D. thesis, Nantes University, Nantes, France, 257 p. Vandanjon, L., Rossignol, N., Jaouen, P., Robert, J.-M., Qu6m6neur, F., 1999. Effects of shear on two microalgae species. Contribution of pumps and valves in tangential flow filtration systems. Biotechnol. Bioeng. 63 (1), 1-9. Zaitoun A., 1979. Osmose inverse et ultrafiltration en milieu organique. Equations de transport. Application ~ l'ultrafiltration des huiles moteur. Ph.D. thesis, INP Lorraine, France.

JOURNAL

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Marine bioprocess engineering" the missing link to commercialization Oskar R. Zaborsky Department of Chemistry, College of Natural Sciences, University of Hawaii, 2525 Correa Road--HIG 131, Honolulu, HI 96822, USA

Received 21 November 1998; received in revised form 7 December 1998: accepted 22 December 1998

Abstract

Success of US biotechnology has been and continues to be dependent on new discoveries and their timely transformation into useful products through bioprocess engineering and a systems approach. Bioprocess engineering is an essential element of 'generic applied' or 'precompetitive' research. For marine biotechnology, like biopharmaceutical biotechnology, bioprocess engineering represents the key. The many hundreds of tantalizing bioactive compounds discovered and isolated from varied marine organisms over the past decades have led to only minimal commercialization due to the limited availability of the compounds in question. To address international competitiveness and the revitalization of key US industries, the National Science Foundation launched the Engineering Research Centers Program in the mid 1980s. The essential feature of this program is a partnership among academia, industry and the government to develop next-generation technology through cutting-edge research, relevant education and innovative technology transfer. MarBEC (Marine Bioproducts Engineering Center) is a recently established multi-disciplinary engineering-science cooperative effort of the University of Hawaii and the University of California at Berkeley. Additional partners include three federal laboratories--Argonne National Laboratory, the Edgewood Research, Development and Engineering Center and the Eastern Regional Research Center of the US Department of Agriculture--and the Bishop Museum. MarBEC's research program consists of four major thrusts: Production Systems; Marine Bioproducts and Bioresources; Separation and Conversion; and Bioproduct Formulation. 9 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Commercialization; Marine bioprocess engineering; Missing link

I. Introduction

Biotechnology is a critical high technology recognized by many countries worldwide including the United States, Japan, Germany, England, France and China. While there are still some skeptics about biotechnology's delivery of prod-

UCtS in relation to its promises, significant success stories are already on hand especially in the biopharmaceutical sector. More than 60 biopharmaceutical products have reached the US market in 16 years and worldwide product sales were $13 billion in 1997 (Thayer, 1998a). Three drugs are above $1 billion in annual sales and another seven

0168-1656/99/$ - see front matter 9 1999 Published by Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00093-0

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O.R. Zaborsky /Journal of Biotechnology 70 (1999) 403-408

exceed $250 million. Indeed, without modern biotechnology there would be no life-saving drugs such as erythropoietin, inteferon, colony stimulating factor, glucocerebrosidase, dornase alpha or tissue plasminogen activator. Further, recombinant vaccines and monoclonal antibodies are prevalent among products currently available or in development. In total, more than 350 biotechnology drugs are in development, with the leading targets being cancer, infectious diseases, AIDSHIV related diseases, heart disease and neurologic disorders. Indeed, biotechnology, a 'dream machine' for US entrepreneurs, is now poised to explode into reality not only in the biomedical arena but also agriculture and the chemical industry. In fact, some major chemical firms are becoming life sciences companies through biotechnology (Thayer, 1998b).

2. Marine biotechnology Marine biotechnology, i.e. the application of modern biotechnology to marine organisms and processes, is an area of significant industrial importance whose ramifications will reach almost every major industrial sector including health, environment, energy, food (aquaculture and agriculture), chemicals, advanced materials and national defense (Bioscience, 1996; Oceanography, 1996; Attaway, 1997; Attaway and Zaborsky, 1993; Zaborsky, 1993). In part, the varied nature of areas impacted by marine biotechnology makes it difficult to track and appreciate its full ramifications. Marine biotechnology, like general biotechnology, will no doubt have its most immediate impact in the industrial sectors of drugs, industrial specialty bioproducts and foods/feeds, especially aquaculture. The importance of marine biotechnology has been recognized by a number of organizations including the National Science and Technology Council (NSTC)--Office of Science and Technology Policy (NSTC, 1995), the National Research Council (NRC) of the National Academy of Sciences and National Academy of Engineering (NRC, 1985) and the World Bank (Zilinskas and Lundin, 1993). The NSTC concluded that marine

biotechnology is one of four 'second wave' biotechnology areas that deserves priority attention from federal agencies. In particular, the priorities included: 9 Develop fundamental understanding of genetic, nutritional and environmental factors that control production of primary and secondary metabolites in marine organisms, the basis for new and improved products; 9 Identify bioactive compounds and determine mechanisms of action and function and provide models for new lines of active materials for application in medicine and chemical industry; 9 Develop bioremediation strategies for application in coastal oceans, where multiple uses (including wastewater disposal, recreation, fishing and aquaculture) demand prevention and remediation of pollution; develop bioprocess strategies for sustainable industrial processes; 9 Use tools of modern biotechnology to improve health, reproduction, development, growth and overall well-being of cultivated aquatic organisms; promote interdisciplinary development of environmentally sensitive, sustainable systems that will enable significant commercialization of aquaculture; and 9 Improve understanding of microbial physiology, genetics, biochemistry and ecology to provide model systems for research and production systems for commerce and contribute to understanding and conservation of the seas. Recent activities in marine biotechnology related to drug discovery have been described (Cragg et al., 1997; Fenical, 1997; Wallace, 1997). Activities in Hawaii have focused on aquaculture, specialty bioproducts and bioactive agents effective against cancer and infectious diseases. Most noteworthy developments have included the isolation and synthesis of cyptophycins (a potentially effective class of anti-cancer agents) by Moore and Patterson (Smith et al., 1994), the production of nutritional supplements such as Spirulina pacifica and also astaxanthin by the Cyanotech Corporation and the culturing of varied marine organisms for food applications by firms located at Kona on the Big Island. In terms of research,

O.R. Zaborsky /Journal of Biotechnology 70 (1999) 403-408

many efforts are underway including those of our group in the production of biological hydrogen (Zaborsky, 1998), a collaborative effort with Professor Tredici at Florence employing his specially designed tubular photobioreactors (Szyper et al., 1998) and marine-derived enzymes. Our research group has just established a major culture collection, the Hawaii Culture Collection that houses the former Mitsui-Miami and NREL-Golden collections (http://www.hawaii.edu/hicc). Additionally, the University of Hawaii has exceptional marine and ocean research vessel operations for acquiring even more biological resources from extreme environments such Loihi, an active undersea volcano 20 miles off the Big Island that in time will be the newest island in the Hawaiian archipelago.

3. Bioprocess engineering m the critical link

One of the other three biotechnology areas identified, namely manufacturing/bioprocessing, represents bioprocess engineering as had been also advocated by a NRC study (NRC, 1992). Success of US biotechnology has been and continues to be dependent on new discoveries and their timely transformation into useful products through bioprocess engineering and a systems approach. Without doubt, bioprocess engineering has been the key to success in the commercialization of biotechnology in biopharmaceuticals. Bioprocess engineering, of course, is an essential element of 'generic applied' or 'precompetitive' research (as it is now more commonly referred to), i.e. research that is beyond basic science but that is before development and traditional engineering applications. Generic applied research represents research that addresses generic issues or bottlenecks that go beyond the means of any one company or even any one industry. The production technology for recombinant proteins, the separation of chiral bioproducts or the precise delivery of bioproducts are some examples. While generic applied research or precompetitive research has been supported through a number of programs and special initiatives by the US federal government throughout the past two

405

decades, these efforts have been very vulnerable to the whims of political changes at the presidential or congressional levels. One of the very noted early programs was the Research Applied to National Needs (RANN) program at the National Science Foundation (NSF) in the mid 1970s. This program was dedicated to the application of science and engineering research to national needs such as energy, materials and chemicals, the environment and human safety. Current generic applied research programs addressing key technology areas include the Department of Commerce's Advanced Technology Program (ATP), including its catalysis and biocatalysis thrusts and the now ubiquitous Small Business Research Innovation Research (SBIR) Program that advances technological innovation and commercialization by providing phased funding to small businesses in a number of areas including biotechnology and marine biotechnology. Generic applied research has been recognized for its importance in translating basic research to commercial products but also its vulnerability (Zaborsky, 1984, 1985). To address international competitiveness and the revitalization of key US industries, NSF launched the Engineering Research Centers (ERC) Program in the mid 1980s. The essential feature of this program is a partnership among academia, industry and the government to develop next-generation technology through cuttingedge research, relevant education and innovative technology transfer. A recent analysis of this program has shown it to be quite positive in achieving its objectives (Parker, 1997). For marine biotechnology, like biopharmaceutical biotechnology, bioprocess engineering represents the key from discovery to commercialization. As an example, the many hundreds of tantalizing bioactive compounds discovered and isolated from varied marine organisms by many over the past decades have led to only minimal commercialization due to the limited availability of the compounds in question for clinical trials or further modification by chemical or biological means--a limit to production caused by our limited marine bioprocess engineering skills (Rouhi, 1995).

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O.R. Zaborsky Journal of Biotechnology 70 (1999) 403-408

4. New opportunities in marine biotechnology: marine bioprocess engineering Several years ago at the Edinburgh, UK and Greifswald, Germany meetings, this author advocated three major areas of attention and opportunity for marine biotechnology. These were: 9 Marine Bioprocess Engineering Project--with the objective of developing reliable bioprocess technologies for marine organisms (microalgae) and marine-derived bioproducts. This also included efforts to establish an international 'marine biosolar' facilities network, with a demonstration facility in Hawaii. 9 Industrial Marine Biodiversity Project--with the objective being to establish an inventory of marine biodiversity (especially the neglected microbes) and crystallize current efforts through an international network of collaboration. 9 Marine Genome Project--with the objective of mapping and sequencing commercially and scientifically significant marine genomes. As with other genome projects, the objective included organizing an international effort. While some activities have been initiated in all three areas, the one of highest priority has been the pursuit of marine bioprocess engineering through a dedicated center that is now named the Marine Bioproducts Engineering Center (MarBEC).

5. MarBEC MarBEC is a multi-disciplinary engineering-science cooperative effort of the University of Hawaii at Manoa, the lead organization and the University of California at Berkeley. Additional partners include three federal laboratories--Argonne National Laboratory, the Edgewood Research, Development and Engineering Center and the Eastern Regional Research Center of the US Department of Agriculture--and the Bishop Museum. MarBEC was officially established on 2 November 1998 and will be a $4-7 million per annum operation once fully functional. NSF funding is $12.4 million for a 5-year period. How-

ever, the full life time of such a center with NSF funding is expected to be 10 years. MarBEC is also expected to become self-sufficient in support. MarBEC's mission is to develop the engineering technology and science base for producing highvalue marine bioproducts essential to the chemical, pharmaceutical, nutraceutical and life sciences industries. Equally important, MarBEC's mission is to produce a new cadre of engineers--i.e. marine biotechnology engineers who will be essential not only to industry but also to government and academia. In essence, MarBEC's mission entails advanced manufacturing of bioproducts that are dependent on marine organisms, solar energy and the technological base offered by modern biotechnology. MarBEC's bioproducts of interest all have multiple uses and multi-million or even billion dollar markets, with significant growth potential. These include carotenoids, polyunsaturated fatty acids, enzymes and bioactive agents. While the importance of marine biotechnology had been recognized, the engineering component had not been. Quite frankly, it is very fragmented throughout academic institutions in the US Equally, education in marine biotechnology engineering is basically non-existent, with no single curriculum dedicated to engineering aspects of marine biotechnology existing at any major US university. Key engineering challenges include: (a) production systems--especially the design, development, modeling and evaluation of sustainable production systems employing phototrophic organisms, development of metabolic engineering and immobilized cell and enzyme bioreactors, environmental aspects of large-scale photobioreactors and near-shore and open-ocean production systems; (b) separation and conversion--more cost-effective separation technologies for labile and chiral bioproducts; and (c) bioproduct formulation, especially in reference to stability and functionality. MarBEC's research program consists of four major thrusts: Production Systems; Marine Bioproducts and Bioresources; Separation and Conversion; and Bioproduct Formulation. These four thrusts are integrated, focusing on engineering yet capturing relevant marine and biological sciences.

O.R. Zaborskv ' Journal o f Biotechnology 70 (1999) 403-408

Each research thrust has near-, mid- and longterm areas of interest to industry and participating faculty. A crucial point, however, is that these four interrelated thrusts are components of a matrix focused on economically significant marine bioproducts. The research conducted will be through multi-disciplinary teams employing the latest advances in science and engineering. Value-added benefits include: a team-work environment, the ability to address cross-cutting engineering issues, a systems approach, the opportunity to tackle long-term engineering challenges and the integration of key disciplines in engineering and marine/biological sciences. Most importantly, MarBEC has a 'critical mass' of expertise that can be called upon to tackle the major barriers or pursue opportunities in a timely fashion. The goal of the education program is to produce the next generation engineer at various levels, especially at the graduate, undergraduate level and post-graduate (industrial) levels. At the undergraduate level, this will be accomplished by introducing marine biotechnology engineering concepts and research project results in appropriate courses at both universities. At the graduate level, the education goal will be accomplished by offering specific courses and specialized short courses dealing with advanced topics. Furthermore, graduate and undergraduate students will be actively engaged in research projects. At the post-graduate level, summer institutes geared to industrial participants will be held. At all levels, research and education will be linked. MarBEC has an industrial collaboration program which aims to establish an effective partnership with industrial firms located in Hawaii, the US mainland and leading technology countries in the Pacific Rim and Europe. The strategy is to have a constant dialogue with industry at every level and opportunity at the research project, research program and center level. MarBEC's outreach program is designed to influence research and education in marine bioproducts engineering in Hawaii, the US mainland and elsewhere where appropriate. As such, MarBEC will work with other organizations to achieve this goal in a bi-directional mode; actions

407

and benefits must flow in both directions. MarBEC's strategy is to work at different levels with organizations within the state, the nation and the world. Important outreach programs are the Marine Options Program and the Sea Grant College Programs of both Hawaii and California. MarBEC is also reaching out to international research organizations. Already in existence are collaborative efforts with organizations in Japan (Tokyo University of Agriculture and Technology) and Europe (University of Florence). As with other ERCs, MarBEC is guided by an Advisory Board consisting of distinguished members of academia, government and industry who can provide counsel on its future direction. MarBEC also has an Industrial Research Advisory Board (IRAB) consisting of experts in the relevant fields of research from member companies. IRAB, in concert with both universities, will establish the formal policies and operating guidelines for industrial participation. A special feature of MarBEC's technology transfer program is its strong linkage to key Hawaii business, financial and economic development organizations as well as to the technology transfer programs of the federal laboratories that will provide a multiplier effect to MarBEC's efforts. In summary, MarBEC builds upon past accomplishments and current research efforts at both universities, yet forges into uncharted areas of immense scientific, engineering and business opportunities of benefit to many.

6. Conclusions

Bioprocess engineering is the critical link that transforms basic research discoveries into commercial reality. Of course, many other aspects come into play when success is achieved such as a market need and timing. As with biopharmaceutical biotechnology, however, bioprocess engineering skills are the critical edge in marine biotechnology. MarBEC represents a new paradigm for research, education and technology transfer in marine biotechnology and a framework for economic development by working with industry in

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Hawaii, the US mainland, Europe and Asia. MarBEC represents a window to 21st century opportunities of the Pacific--but more importantly a launching pad for real action.

References Attaway, D.A., 1997. A Report on Marine Biotechnology in the National Sea Grant College Program, Washington, DC. Attaway, D.A., Zaborsky O.R. (Eds.), 1993. Marine Biotechnology, Pharmaceutical and Bioactive Natural Products, vol. 1. Plenum, New York. Bioscience, 1996. Marine biotechnology, special issue, 46 (4). Cragg, G.M., Newman, D.J., Weiss, R.B., 1997. Coral reefs, forests and thermal vents: the worldwide exploration of nature for novel antitumor agents. Semin. Oncol. 24, 156163. Fenical, W., 1997. New pharmaceuticals from marine organisms. Trends Biotechnol. 15, 339-341. NRC-1985, 1985. National Research Council--National Academy of Sciences, Marine Biotechnology: Basic Research Relevant to Biomaterials and Biosensors. NRC-1992, 1992. National Research Council--National Academy of Sciences--National Academy of Engineering, Bioprocess Engineering: Putting Biotechnology to Work. NSTC-1995, 1995. National Science and Technology Council-Office of Science and Technology Policy, Biotechnology for the 21st Century: New Horizons. Washington, DC. Oceanography, 1996. Marine Biological Diversity Special Issue, 9 (1).

Parker, L., 1997. The Engineering Research Centers (ERC) Program: An Assessment of Benefits and Outcomes. National Science Foundation, Washington, DC. Rouhi, A.M., 1995. Supply issues complicate trek of chemicals from sea to market. C&EN 11 (20), 42-44. Smith, C.D., Zhang, X., Mooberry, S.L., Patterson, G.M., Moore, R.E., 1994. Cryptophycin: a new antimicrotubule agent active against drug-resistant cells. Cancer Res 54, 3779-3784. Szyper, J.P., Yoza, B.A., Benemann, J.R., Tredici, M.R., Zaborsky, O.R., 1998. Internal gas exchange photobioreactor: development and testing in Hawaii. In: Zaborsky O.R., Benemann J.R., Miyake J., Matsunaga T., San Pietro, A. (Eds.), BioHydrogen. Plenum, New York (in press). Thayer, A.M., 1998a. Great expectations. C&EN 8 (10), 1931. Thayer, A.M., 1998b. Living and loving life sciences. C&EN 11 (23), 17-24. Wallace, R.W., 1997. Drugs from the sea: harvesting the results of aeons of chemical evolution. Mol Med Today 3, 291-295. Zaborsky, O.R., 1984. A critique of government funding of biotechnology: basic and applied research. Chem Econ. Eng. Rev. 16, 9-10. Zaborsky, O.R., 1985. Technology development: the missing link. Visions 2, 6-8. Zaborsky, O.R., 1993. Marine biotechnology. Genet. Eng. News 13, 10. Zaborsky, O.R., Benemann J.R., Miyake J., Matsunaga T., San Pietro A. (Eds.), 1998. BioHydrogen. Plenum, New York. Zilinskas, R.A., Lundin C.G., 1993. Marine Biotechnology and Developing Countries. World Bank Discussion Papers, Washington, DC.

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Journal of Biotechnology 70 (1999) 409-411

Author Index Aci6n Fern~ndez, F.G., see Rebolloso Fuentes, M.M. (70) 271 Armstrong, E.D., McKenzie, J. and Goldsworthy, G.T., Aquaculture of sponges on scallops for natural products research and antifouling (70) 163 Asada, Y., see Miyake, J. (70) 89 Asian, K., see Eroglu, i. (70) 103 Baquerisse, D., Nouals, S., Isambert, A., dos Santos, P.F. and Durand, G., Modelling of a continuous pilot photobioreactor for microalgae production (70) 335 Bastianini, A., see Chini Zittelli, G. (70) 299 Battershill, C.N., see Munro, M.H.G. (70) 15 Beelen, T.P.M., see Vrieling, E.G. (70) 39 Bengoa-Ruigomez, M.V., see Kotzabasis, K. (70) 357 Blunt, J.W., see Munro, M.H.G. (70) 15 Borghi, A., see Sponga, F. (70) 65 Borowitzka, M.A., Commercial production of microalgae: ponds, tanks, tubes and fermenters (70) 313 Bowles, R.D., Hunt, A.E., Bremer, G.B., Duchars, M.G. and Eaton, R.A., Long-chain n - 3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production (70) 193 Boyd, K.G., see Burgess, J.G. (70) 27 Bregu, M., see Burgess, J.G. (70) 27 Bremer, G.B., see Bowles, R.D. (70) 193 Burgess, J.G., Jordan, E.M., Bregu, M., Mearns-Spragg, A. and Boyd, K.G., Microbial antagonism: a neglected avenue of natural products research (70) 27 Burgess, J.G., see Wright, P.C. (70) 343 Cabatingan, L.K., see van der Wielen, L.A.M. (70) 363 Camacho P~ez, B., see Robles Medina, A. (70) 379 Cavaletti, L., see Sponga, F. (70) 65 Chini Zittelli, G., Lavista, F., Bastianini, A., Rodolfi, L., Vincenzini, M. and Tredici, M.R., Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors (70) 299 Chisti, Y., see Molina Grima, E. (70) 231 Chisti, Y., see S~.nchez Mir6n, A. (70) 249 Ciciliato, I., see Sponga, F. (70) 65 Contreras G6mez, A., see S~.nchez Mir6n, A. (70) 249 Cs6g6r, Z., Melgar, D., Schmidt, K. and Posten, C., Production and particle characterization of the frustules of Cyclotella cryptica in comparison with siliceous earth (70) 71 PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 4 5 - 5

de Beukelaer, P.B., see Osinga, R. (70) 155 de Rijk, T.C., see de Swaaf, M.E. (70) 185 de Swaaf, M.E., de Rijk, T.C., Eggink, G. and Sijtsma, L., Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii (70) 185 Divanach, P., see Kotzabasis, K. (70) 357 dos Santos, P.F., see Baquerisse, D. (70) 335 Duchars, M.G., see Bowles, R.D. (70) 193 Duckworth, A.R., see Munro, M.H.G. (70) 15 Dumdei, E.J., see Munro, M.H.G. (70) 15 Durand, G., see Baquerisse, D. (70) 335 Eaton, R.A., see Bowles, R.D. (70) 193 Eggink, G., see de Swaaf, M.E. (70) 185 Erard-Le Denn, E., see La Barre, S. (70) 207 Eroglu, I., Aslan, K., Gfindfiz, U., Yficel, M. and Tfirker, L., Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor (70) 103 Eroglu, |., see Sediroglu, V. (70) 115 Ero(glu, |., see Yi~it, D.O. (70) 125 Esteban Cerdfin, L., see Robles Medina, A. (70) 379 Etoundi, P., see Helmholz, H. (70) 203 Fern~ndez, F.G.A., see Molina Grima, E. (70) 231 Fern~ndez Sevilla, J.M., see Rebolloso Fuentes, M.M. (70) 271 Gad, G., see Liebezeit, G. (70) 77 Garcia Camacho, F., see Molina Grima, E. (70) 231 Garcia Camacho, F., see Shnchez Mir6n, A. (70) 249 Garcia Sanchez, J.L., see Rebolloso Fuentes, M.M. (70) 271 Garcia-Jimenez, P., Marian, F.D., Rodrigo, M. and Robaina, R.R., Sporulation and sterilization method for axenic culture of Gelidium canariensis (70) 227 German, J.B., see Wood, B.J.B. (70) 175 Gieskes, W.W.C., see Vrieling, E.G. (70) 39 Gim6nez Gim6nez, A., see Robles Medina, A. (70) 379 Goldsworthy, G.T., see Armstrong, E. (70) 163 Grimson, P.H.K., see Wood, B.J.B. (70) 175 Gromek, E., see Turkiewicz, M. (70) 53 Gfindiiz, U., see Eroglu, I. (70) 103 G(indiiz, U., see Sediroglu, V. (70) 115 Gfindfiz, U., see Yi~it, D.O. (70) 125

410

Author Index

Hatziathanasiou, A., see Kotzabasis, K. (70) 357 Helmholz, H., Etoundi, P. and Lindequist, U., Cultivation of the marine basidiomycete Nia vibrissa (Moore & Meyers) (70) 203 Hickford, S.J.H., see Munro, M.H.G. (70) 15 Hirano, S., Nakahira, T., Nakagawa, M. and Kim, S.K.. The preparation and applications of functional fibres from crab shell chitin (70) 373 Hunt, A.E., see Bowles, R.D. (70) 193 Ibfifiez Gonzfilez, M.J., see Robles Medina. A. (70) 379 Isambert, A., see Baquerisse, D. (70) 335 Janssen, M., Kuijpers, T.C., Veldhoen, B., Ternbach. M.B.. Tramper, J., Mur, L.R. and Wijffels, R.H., Specific growth rate of Chlamydomonas reinhardtii and Chlorelhi sorokiniami under medium duration light/dark cycles: 13-87 s (70) 323 Jaouen, P., see Vandanjon, L. (70) 393 Jordan, E.M., see Burgess, J.G. (70) 27 Jozefowicz, M., see La Barre, S. (70) 207 Kalinowska, H., see Turkiewicz, M. (70) 53 Kentouri, M., see Kotzabasis, K. (70) 357 Kim, S.K., see Hirano, S. (70) 373 Kotzabasis, K., Hatziathanasiou, A., Bengoa-Ruigomez. M.V., Kentouri, M. and Divanach, P., Methanol as alternative carbon source for quicker efficient production of the microalgae Chlorella minutissima: Role of the concentration and frequence of administration (70) 357 Koulman, A., Pruijn, L.M.C., Sandstra, T.S.A., Woerdenbag. H.J. and Pras, N., The pharmaceutical exploration of cold water ascidians from the Netherlands: a possible source of new cytotoxic natural products (70) 85 Kreitlow, S., Mundt, S. and Lindequist, U.. Cyanobacteria--a potential source of new biologically active substances (70) 61 Kuijpers, T.C., see Janssen, M. (70) 323 Kiinnemann, T.D., see Liebezeit, G. (70) 77 La Barre, S., Singer, S., Erard-Le Denn, E. and Jozel'oxvicz. M., Controlled cultivation of Alexan~h'ium J~lmututll and [33p] orthophosphate cell labeling towards surface adhesion tests (70) 207 Lavista, F., see Chini Zittelli, G. (70) 299 Lazzarini, A., see Sponga, F. (70) 65 Liebezeit, G., Kiinnemann, T.D. and Gad. G., Biotechnological potential of North Sea salt marsh plants--a review of traditional knowledge (70) 77 Lill, R.E., see Munro, M.H.G. (70) 15 Lindequist, U., see Helmholz, H. (70) 203 Lindequist, U., see Kreitlow, S. (70) 61 Li, S., see Munro, M.H.G. (70) 15 Losi, D., see Sponga, F. (70) 65 Marian, F.D., see Garcia-Jimenez, P. (70) 227 Marinelli, F., see Sponga, F. (70) 65 Matsunaga, T., Takeyama, H., Nakao, T. and Yamazawa, A..

Screening of marine microalgae for bioremediation of cadmium-polluted seawater (70) 33 McEvoy. E., see Wright, P.C. (70) 343 McKenzie, J.D.. see Armstrong, E. (70) 163 Mearns-Spragg, A.. see Burgess, J.G. (70) 27 Meijer, E.M., see Osinga, R. (70) 155 Melgar. D.. see Cs6g6r, Z. (70) 71 Miyake. J.. Miyake. M. and Asada, Y., Biotechnological hydrogen production: research for efficient light energy conversion (70) 89 Miyake. M.. see Miyake, J. (70) 89 Molina Grima. E., Fernfindez, F.G.A., Garcia Camacho, F. and Chisti. Y.. Photobioreactors: light regime, mass transfer. and scaleup (70) 231 Molina Grima. E.. see Rebolloso Fuentes, M.M. (70) 271 Molina Grima. E.. see Robles Medina, A. (70) 379 Molina Grima. E., see Sfinchez Mirdn, A. (70) 249 Mundt. S.. see Kreitlow. S. (70) 61 Munro. M.H.G.. Blunt, J.W., Dumdei, E.J., Hickford, S.J.H., kill. R.E.. ki. S.. Battershill, C.N. and Duckworth, A.R., The discovery and development of marine compounds with pharmaceutical potential (70) 15 Mur. L.R.. see Janssen. M. (70) 323 Nakaga~a. M., see Hirano, S. (70) 373 Nakahira. T.. see Hirano, S. (70) 373 Nakao, T., see Matsunaga, T. (70) 33 Nouals. S.. see Baquerisse, D. (70) 335 Ogbonna. J.C.. Soejima, T. and Tanaka, H., An integrated solar and artificial light system for internal illumination of photobioreactors (70) 289 Ogbonna. J.C.. Tomivama. S. and Tanaka, H., Production of ~.-tocopherol bx sequential heterotrophic-photoautotrophic cultivation of Euelemi gracilis (70) 213 Ogi. T.. see Tsukahara. K. (70) 223 Osinga. R.. de Beukelaer. P.B., Meijer, E.M., Tramper, J. and Wijffeis. R.H.. Grov~th of the sponge Pseudosuberites (aft.) amtrc~.~i in a closed system (70) 155 Pomponi. S.A.. The bioprocess-technological potential of the sea t70) 5 Posten. C.. see Cs6g6r. Z. (70) 71 Pras. N.. see Koulman. A. (70) 85 Pruijn. L.M.C.. see Koulman, A. (70) 85 Quemeneur. F.. see Vandanjon, L. (70) 393 Rebolloso Fuentes. M.M.. Garcia Sfinchez, J.L., Fern/mdez Sevilla. J.M.. Acien Fernfindez, F.G., Sfinchez P6rez, J.A. and Molina Grima. E., Outdoor continuous culture of Pop7~l:.vridiul~ ~ruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters (70) 271 Richmond. A.. see Zou. N. (70) 351 Rinkevich. B.. Cell cultures from marine invertebrates: obstacles. new approaches and recent improvements (70) 133 Robaina. R.R.. see Garcia-Jimenez, P. (70) 227

Author hldex Robert, J.-M., see Vandanjon, L. (70) 393 Robles Medina, A., Esteban Cerdfin, L., Gimenez Gim6nez, A., Camacho Pfiez, B., Ibfifiez Gonzfilez, M.J. and Molina Grima, E., Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils (70) 379 Rodolfi, L., see Chini Zittelli, G. (70) 299 Rodrigo, M., see Garcia-Jimenez, P. (70) 227 Rossignol, N., see Vandanjon, L. (70) 393 S/mchez Mir6n, A., Contreras G6mez, A., Garcia Camacho, F., Molina Grima, E. and Chisti, Y., Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae (70) 249 Sfinchez Perez, J.A., see Rebolloso Fuentes, M.M. (70) 271 Sandstra, T.S.A., see Koulman, A. (70) 85 Sawayama, S., see Tsukahara, K. (70) 223 Schmidt, K., see Cs6g6r, Z. (70) 71 Sediroglu, V., Eroglu, |., Yticel, M., Tfirker, L. and Giindfiz, U., The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production (70) 115 Sijtsrna, L., see de Swaaf, M.E. (70) 185 Singer, S., see La Barre, S. (70) 207 Soejima, T., see Ogbonna, J.C. (70) 289 Sponga, F., Cavaletti, L., Lazzarini, A., Borghi, A., Ciciliato, I., Losi, D. and Marinelli, F., Biodiversity and potentials of marine-derived microorganisms (70) 65 Stevenson, C., see Wright, P.C. (70) 343 Takeyama, H., see Matsunaga, T. (70) 33 Tanaka, H., see Ogbonna, J.C. (70) 289 Tanaka, H., see Ogbonna, J.C. (70) 213 Ternbach, M.B., see Janssen, M. (70) 323 Tomiyama, S., see Ogbonna, J.C. (70) 213 Tramper, J., see Janssen, M. (70) 323 Tramper, J., see Osinga, R. (70) 155 Tredici, M.R., see Chini Zittelli, G. (70) 299 Tsukahara, K., Sawayama, S., Yagishita, T. and Ogi, T., Effect of Ca 2+ channel blockers on glycerol levels in Dunaliella tertiolecta under hypoosmotic stress (70) 223 Ti.irker, L., see Eroglu, |. (70) 103 Tiirker, L., see Sediroglu, V. (70) 115 Tfirker, L., see Yi~it, D.O. (70) 125 Turkiewicz, M., Gromek, E., Kalinowska, H. and Zielifiska, M., Biosynthesis and properties of an extracellular rnetalloo

411

protease from the Antarctic marine bacterium Sphingomonas paucimobilis (70) 53 Turner, M., see Wood, B.J.B. (70) 175 Vandanjon. L.. Jaouen. P., Rossignol, N., Qu6meneur, F. and Robert, J.-M., Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen (70) 393 van der Wielen. L.A.M. and Cabatingan, L.K., Fishing products from the sea--rational downstream processing of marine bioproducts (70) 363 ,,an Santen. R.A.. see Vrieling, E.G. (70) 39 Veldhoen, B.. see Janssen, M. (70) 323 Vincenzini. M.. see Chini Zittelli, G. (70) 299 Vrieling, E.G.. Beelen. T.P.M., van Santen, R.A. and Gieskes, W.W.C.. Diatom silicon biomineralization as an inspirational source of new approaches to silica production (70) 39 Wijffels. R.H.. see Janssen, M. (70) 323 Wijffels, R.H.. see Osinga, R. (70) 155 Woerdenbag. H.J.. see Koulman, A. (70) 85 Wood. B.J.B.. Grimson, P.H.K., German, J.B. and Turner, M.. Photoheterotrophy in the production of phytoplankton organisms (70) 175 Wright. P.C.. Stevenson, C.. McEvoy, E. and Burgess, J.G., Opportunities for marine bioprocess intensification using novel bioreactor design: frequency of barotolerance in microorganisms obtained from surface waters (70) 343 Yagishita. T.. see Tsukahara, K. (70) 223 Yamazawa. A.. see Matsunaga, T. (70) 33 Yi~it. D.O.. G/indtiz. U., Tiirker, L., Yficel, M. and Erofglu, [., Identification of by-products in hydrogen producing bacteria: Rhodobacter sphaeroides O.U. 001 grown in the waste water of a sugar refinery (70) 125 Yficel. M., see Eroglu. i. (70) 103 Yficel, M.. see Sediroglu, V. (70) 115 Yticel. M.. see Yi~it, D.O. (70) 125 Zaborsky. O.R.. Marine bioprocess engineering: the missing link to commercialization (70) 403 Zielifiska, M.. see Turkiewicz, M. (70) 53 Zou, N. and Richmond. A., Effect of light-path length in outdoor flat plate reactors on output rate of cell mass and of EPA in Nannochloropsis sp. (70) 351

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JOURNAL

OF

Biotechnology ELSEVIER

Journal of Biotechnology 70 (1999) 413-414

Subject Index Acetic acid, (70) 175 Adhesion, (70) 163 Adsorption, (70) 33 Agar diffusion method, (70) 61 Alexandrium minutum, (70) 207 Algae, (70) 175 Antarctic marine bacteria, (70) 53 Antibiotic, (70) 27, 61 Antibiotic activities, (70) 65 Anticancer, (70) 15 Antifungal, (70) 61 Antimitotic, (70) 15 Aplidium glabrum, (70) 85 Aquaculture, (70) 5 Artificial light, (70) 289 Ascidian, (70) 85 Autotrophy, (70) 175 Axenic culture, (70) 227 Bacteriorhodopsin, (70) 115 Barotolerance, (70) 343 Biodiversity, (70) 15, 65 Biogenic silica, (70) 39 Biomineralization, (70) 39 Bioprocess intensification, (70) 343 Bioreactor, (70) 185, 343 Bioremediation, (70) 33 Blue-green algae, (70) 61 Ca 2 + channel blocker, (70) 223 Cadmium, (70) 33 Carbon dioxide consumption rate, (70) 271 Carbon source, (70) 357 Cell culture, (70) 163 Cell lines, (70) 133 Cell numeration, (70) 207 Cellulose acetate membrane, (70) 115 Chitin fibres, (70) 373 Chitin-silk fibroin fibres, (70) 373 Chitosan, (70) 373 Chlamydomonas reinhardtii, (70) 323 Chlamys opercularis, (70) 163 Chlorella, (70) 313 Chlorella minutissima, (70) 357 Chlorella sorokiniana, (70) 323 PII: S0168-1656(99)00146-7

Commercialization, (70) 403 Concentration, (70) 393 Continuous culture, (70) 271 Controlled proliferation, (70) 207 Corn steep liquor, (70) 213 Crvpthecodinium cohnii, (70) 185 Cultivation, (70) 71, 185 Cyanobacteria, (70) 61, 89 Cytotoxicity, (70) 85 Desalting, (70) 393 Diatoms, (70) 71 Didemnum lahillei, (70) 85 Docosahexaenoic acid. (70) 185, 193 Dual substrate, (70) 103 Dunaliella, (70) 313 Dunaliella tertiolecta, (70) 223 Eicosapentaenoic acid, (70) 193, 249, 299 Elemental analysis, (70) 271 Endotoxin, (70) 203 EPA, (70) 351 Ethanol, (70) 213 Euglena gracilis, (70) 213 Extreme environments, (70) 77 Fibroin, (70) 373 Fish farming, (70) 175 Fishing products, (70) 363 Fish oil, (70) 379 Fouling, (70) 163 Frustules, (70) 71 Gas-transfer modelling, (70) 335 Gelidium canariensis, (70) 227 Glucose, (70) 213 Glycerol, (70) 175, 223 Growth, (70) 155 Growth model, (70) 335

Halobacterium halobium, (70) 115 Haslea ostrearia, (70) 393 Heterotrophic culture, (70) 213 High hydrostatic pressure, (70) 343 Hydrodynamics modelling, (70) 335

414 Hydrogen, (70) 89, 125 Hydrogen production, (70) 103, 115 Hydrogen production factor, (70) 103 Hypoosmotic stress, (70) 223 Immobilisation, (70) 115 Indoor culture, (70) 289 Instantaneous fractional yield, (70) 103 Intermittent illumination, (70) 323 Internal illumination, (70) 289 Invertebrate cell culture, (70) 5 In vivo cultivation, (70) 155 Light/dark cycles, (70) 323 Light-path, (70) 351 Light regimen, (70) 231 Lipase-catalyzed esterification, (70) 379 Lipid composition, (70) 175 Marine, (70) 185, 193 Marine bioprocess engineering, (70) 403 Marine bioproducts, (70) 5, 363 Marine Chlorella, (70) 33 Marine epibiotic bacteria, (70) 27 Marine fungi, (70) 203 Marine invertebrates, (70) 133 Marine microalgae, (70) 33 Marine microorganisms, (70) 65 Marine pharmaceuticals, (70) 5 MarinLit, (70) 15 Mass transfer, (70) 231 Medium composition, (70) 185 MeOH, (70) 357 Metalloprotease, (70) 53 Methanol, (70) 357 Microalgae, (70) 231, 249 Microalgae mass cultivation, (70) 299 Microalga oils, (70) 379 Microbial antagonism, (70) 27 Microculture tetrazolium assay, (70) 85 Missing link, (70) 403

Nannochloropsis sp., (70) 299, 351 Nanofiltration, (70) 393 Nia vibrissa, (70) 203 North Sea, (70) 85 N - 3 polyunsaturates, (70) 193

Subject Index Photobioreactor, (70) 71, 103, 289, 351 Photobioreactors. (70) 231, 249, 313 Photoelectrochemical reactor, (70) 115 Photosynthetic bacteria, (70) 89 Photosynthetic culture, (70) 249 Physisorption. (70) 39 Pigment. (70) 393 Platyhelminths, (70) 133 Polyacrylamide gel, (70) 115 Poly-/~'-hydroxy butyric acid, (70) 125 Polymer therapeutics. (70) 15 Polyunsaturated fatty acids, (70) 379 Porphyridium cruentum. (70) 271 Porph.vridium purpureum, (70) 335 Productivity. (70) 351 Protist. (70) 193 Pseudosuberites (aft.) andrewsi, (70) 155 Psychrophilic enzymes. (70) 53 Radioactive labeling. (70) 207 Rational downstream processing, (70) 363 Renewable energy. (70) 89 Rhodobacter sphaeroides. (70) 103, 125 Rhodophyta. (70) 227 Salt marsh plants, (70) 77 Scale-up, (70) 249 Scaleup. (70) 231 Screening. (70) 33 Semiterrestrial, (70) 77 Siliceous earth. (70) 71 Silicon (bio)chemistry, (70) 39 Small angle X-ray scattering (SAXS), (70) 39 Solar energy. (70) 89 Solar light. (70) 289 Specific growth rate, (70) 323 Spirulina. (70) 313 Sponge aquaculture, (70) 15 Sponges, (70) 155 Sporelings. (70) 227 Stress induction, (70) 343 Suberites ficus, (70) 163 Sustainable use, (70) 5

Outdoor cultures, (70) 351 Oxygen generation rate, (70) 271

Textile, (70) 373 Thraustochytrid, (70) 193 z~-Tocopherol, (70) 213 Triglycerides. (70) 379 Tubular photobioreactor, (70) 271, 335 Tubular photobioreactors, (70) 299

Phaeodactylum tricornutum, (70) 249 Photoautotrophic culture, (70) 213

Waste water. (70) 125 Wound dressing. (70) 373