Advances in Applied Microbiology, Volume 42

ADVANCES IN

Applied Microbiology VOLUME 42

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ADVANCES IN

Applied Microbiology Edited by SAUL L. NEIDLEMAN Oakland, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 42

Academic Press San Diego London Boston New York Sydney Tokyo Toronto

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Copyright 0 1996 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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Academic Press Limited 24-28 Oval Road, London NWI 7DX, UK http://www.hbuk.co.uWapl International Standard Serial Number: 0065-2164 International Standard Book Number: 0- 12-002642-2 PRINTED IN THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 0 1 B B 9 8 7 6 5

4

3 2 1

CONTENTS

The insecticidal Proteins of Baci//usthuringiensis

P. ANANDA KUMAR. R . P. SHARMA.AND I. I1. I11. IV. V. VI .

VII . VIII .

v. s. MALIK

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Bt Toxins ................................ Structure of Bt Toxin Proteins and Genes . . . . . . . . . . . . . . . . . . . Screening for New Insecticidal Proteins and Genes . . . . . . . . . . . Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bt as a Biological Insecticide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance Development and Management Strategies . . . . . . . . . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 6 10 12 14 16 25 30 34

Microbiological Production of Lactic Acid JOHN

I. I1. 111. IV. V. VI .

H . LITCHFIELD

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganisms for Lactic Acid Production . . . . . . . . . . . . . . . . . Lactic Acid Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product Recovery and Purification ........................ Process Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 50 54 82 85 87 88

Biodegradable Polyesters

CH. SASIKALA Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Biodegradable Polyesters . . . . . . . . . . . . . . . . . . Biodegradable Natural Polyesters ......................... Biodegradable Synthetic Polyesters ....................... V. Poly(L-malate): A Potential Biodegradable Polyester . . . . . . . . . .

I. I1. I11. IV.

V

97 98 100 100 103

vi

CONTENTS

VI . PHAs: A Group of Polyesters Produced Naturally and Synthetically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Application of Biodegradable Polyesters . . . . . . . . . . . . . . . . . . . VIII . Biodegraduation of Polyesters . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

104 169 173 198

The Utility of Strains of Morphological Group II Bacillus

SAMUEL SINGER I. I1. 111. IV. V VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Utility According to Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Utility According to Strain and Species .................... Fermentation Processes. Toxins. and Products . . . . . . . . . . . . . . Past Needs and Future Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

219 221 222 236 250 256 259

Phytase

RUDYJ . WODZINSKI AND A . H . J . ULLAH I. I1. I11. IV. V. VI . VII . VIII . IX .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Phytic Acid and Hydrolysis Products . . . . . . . . . . Sources of Phytase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Phytase Synthesis .......................... Biochemistry of Phytase and Acid Phosphatases . . . . . . . . . . . . . Feed Studies with Phytase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economics and Potential Effect of Phytase on Pollution Abatement Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263 269 271 274 279 292 295 297 297 298

303 309

The I nsecticidal Proteins of Bacillus thuringiensis P. ANANDA KUMAR,*R. P. SHARMA,*AND v. s. MALIK~ *National Research Centre for Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012, India + US.Department of Agriculture Riverdale, Maryland 20737

I. Introduction 11. Classification of Bt Toxins 111. Structure of Bt Toxin Proteins and Genes IV. Screening for New Bt Toxin Proteins and Genes V. Mechanism of Action VI. Bt as a Biological Insecticide A. Construction of Novel Bt Strains by Conjugation B. Construction of Recombinant Bt Strains C. Construction of Transgenic Microbes D. Baculoviruses as Bt Vectors E. Insect-Tolerant Transgenic Crop Plants VII. Resistance Development and Management Strategies A. Rotations B. Mixtures of Toxins C. Refuges D. Toxin Doses E. Gene Expression VIII. Epilogue References

I. Introduction

Bacillus thuringiensis (Bt)is a gram-positive,aerobic, endospore-forming bacterium belonging to morphological group I along with Bacillus cereus, Bacillus anthracis, and Bacillus laterosporus (Parry et al., 1983). All these bacteria have endospores. Bt, however, is recognized by its parasporal body (known as the crystal) that is proteinaceous in nature and possesses insecticidal properties. These insecticidal proteins, synthesized during sporulation, are tightly packed by hydrophobic bonds and disulfide bridges. Various forms of true crystals have been observed using phase contrast microscope (Srinivas et al., 1995;Jung et al., 1995). The most common shape is a bipyramidal structure (Fig. 1).A Bt mutant defective in sporulation accumulates insecticidal proteins to form large crystal inclusion (Fig. 2) that remained encapsulated within the ghost 3 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 42 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

N

FIG.1. Scanning electron micrograph of Bacillus thuringiensis crystals: (A) bipyramidal crystals produced by a lepidopteranactive strain; (B) spherical crystals produced by a mosquito-active strain; (C and D) irregularly shaped crystals produced by nontoxic strains (mows indicate crystals).Reproduced with permission from Chilcote, C.N. and Wigley P.J. (1994). A@. Eco systems Environ. 49,51-52

W

FIG.2. Electron micrographs of a SpoOA mutant strain overproducing the CryIIIA crystal protein. Reproduced with permission from D. Lereclus, Institut Pasteur, Paris.

4

P. ANANDA KUMAR ET AL.

cell (Lereclus et al., 1915).The first record on Bt goes back to 1901, when Ishiwata discovered a bacterium from diseased silkworm larvae that he named Bacillus sotto (Ishiwata, 1901). Between 1909 and 1912, Berliner (1915), working at a research station for grain processing in Berlin, investigated an infectious disease of the Mediterranean flour moth (Ephestia kuehniella). The infected insects were originally obtained from a mill in the district of Thueringen. In a detailed report, Berliner (1915) described a spore-forming bacterium as the causative agent and designated it as B. thuringiensis. The first practical application of Bt was reported by Husz (1928) who isolated a Bt strain from Ephestia and tested it on European corn borer. This work eventually led to the first commercial product, Sporeine, which was produced in France in 1938 (Luthy et al., 1982). The development of potent organic insecticides, however, prevented the interest for biological alternatives for pest control to some extent. The pioneering research of Steinhaus (1951)on Bt and a growing realization that organic insecticides are deleterious to the environment and human health spurred a renewed interest in Bt in the 1960s. This led to the introduction of viable Bt biopesticides like Thuricide and Dipel. For many years, the inclusion body protein and spores were generally recognized as the two essential ingredients for most of the insecticidal activity of B. thuringiensis. Scientists at the Sandoz company and Asano and Hori (1995) discovered in the supernatant of the B. thuringiensis a growth medium potency-enhancing factor, Kurstakolin (Fig. 3), which enhances the insecticidal activity of B. thuringiensis cellular preparations by 30%. There are many subspecies and serotypes of Bt with a range of wellcharacterized insecticidal proteins or Bt toxins. Known Bt toxins kill subsets of insects among the Lepidoptera, Coleoptera, Diptera (Hofte and Whiteley, 1989), and nematodes (Feitelson et al., 1992). The host range of Bt has expanded considerably in recent years due to extensive

Cl,H*N,Otl FIG.3. Structure of Kurstakolin.

5

INSECTICIDAL PROTEINS OF B. th uringiensis

screening programs (Table I). By virtue of the lack of toxicity toward other species of animals, human beings, and plants, there is tremendous potential for exploiting Bt as a biological control agent ( Jones and Khachatourians, 1995; Salama et al., 1995; Bradley et al., 1995). Various aspects of Bt fermentation (Capalbo, 1995; Gangurde and Shethna, 1995),biology and genetics (Bulla et al., 1978; Aronson, 1986), molecular biology (Hofte and Whiteley, 1989; Yoshisue et al., 1995a; Dervyn et al., 1995), mechanism of action (Gill et al., 1992; Knowles, 1994), application as biopesticide (Gawron-Burke and Baum, 1991; TABLE I

HOSTRANGEOF Bacillus thuringiensis Susceptible families Order Insecta Lepidoptera

Diptera

Toxin

Example

&Endotoxin

Most lepidopteran families susceptible examples

&Endotoxin

Spingidae Pieridae Lymantriidae Tortricidae Noctuidae Culicidae Simuliidae Anisopodidae Chironomidae Psychodiae Sciaridae Tipulidae

Hawkmoths Cabbage worms Tussock moths Leafroller moths Cutworms/armyworms Mosquitoes Blackflies Gnats Midges Moth flies Black fungus gnats Craneflies

Muscidae Calliphoridae

Houseflies Blowflies

Thiringiensin Coleoptera

Family

&Endotoxin

Phthiraptera

Chrysomelidae

Leaf beetles

Philopteridae Trichodectidae

Bird lice Mammalian lice

Arachnida Acari

Thuringiensin

Dermanyssidae Tetranychidae

Animal mites Phytophagous mites

Nematoda Strongylida

?

Trichostrongylidae

Animal endoparasitic nematodes

?

Tylenchidae

Phytophagous nematodes

Tylenchida

6

P. ANANDA KUMAR ET AL.

Aronson, 1994, Pedersen et al., 1995; Farrar and Ridgway, 1995; Yang et al., 1995; Gibson et al., 1995; Li et al., 1995), and Bt transgenic plants (Peferoen, 1992; Kumar and Sharma, 1994) have been reviewed. Here, the classification and mode of action of Bt toxins are discussed. Strategies to screen new Bt straidgenes, expression of the toxin protein in transgenic microorganisms (Shin et al., 1995), and plants and various resistance management strategies in agricultural systems are examined. The review puts emphasis on agricultural application of Bt. II. Classification of Bt Toxins

A large number of Bt isolates are now available in laboratories around the world (Schnepf, 1995; Jung et al., 1995; Burtseva et al., 1995; Shin et al., 1995). New strains are being added every year. Bt strains can be characterized by a number of techniques including serotyping, crystal serology, crystal morphology, protein profiles, peptide mapping, DNA probes, and insecticidal activity. De Barjac first attempted to classify Bt toxins based on flagellar (H) agglutination (De Barjac and Bonnefoi, 1962). Recently, the classification of Bt based on H antigen was revised (De Barjac and Franchon, 1990) (Table 11). More than 40 H-serotypes are TABLE I1 CLASSIFICATION OF Bacillus th uringiensis

H-antigen 1 2

3a,3c 3a,3b,3c 3a,3d 3a,3d,3e 4a,4b 4a,4c 5a,5b 5a,5c 6

6a,6c 7

8a,8b 8a,8c 8b,8d

Variety

Toxicity"

th uringiensis finitim us alesti kurstaki sumiyoshiensis fukuokaensis sotto kenyae galleriae canadensis entomocidus oyamensis aizawai m orrisoni ostriniae nigeriensis

L ,D L L,D D L L,D L,C L L L,D L,D,C L (contin ues)

INSECTICIDAL PROTEINS OF B. thuringiensis TABLE 11-Contin ued H-antigen 9 L,D10a,10b 10a,10c

Variety

Toxicity"

to1worthi darmstadiensis

L.D

londrinalla,llb

toumanoffi lla,llc 12 14 15 16 17 18a,18b 18a,18c 19 20a,20b 20a,20c 21 22 23 24a,24b 24a,24c 25 26 27 28a,28b 28a,28c 29 30 31 32 33 34 35

36 37 38 39 40 41 42 43 44 45

kyush uensis thompsoni pakistani israelensis dakota indian (I tohokuensis kumamotoensis yosoo tochigiensis yunnanensis pondicheriensis colmeri shandongiensis japonensis neoleonensis novosibirsk corean ensis

L,D L,D13 D

C

L L

L C

silo

mexicanensis mon terrey jega thesan amagiensis

medellin toguchini cameroun leesis konkukian seoulensis malaysiensis andalousiensis oswaldocruzi brasiliensis h uazhongensis sooncheon jinghongiensis guiyangiensis higo roskildiensis

D

L, lepidopteran active; D, dipteran active; C, caleopteran active.

7

8

P. ANANDA KUMAR ET AL.

now available and in many of these the array of Bt toxin genes present in isolates from a particular serovar are the same (Rabinovitch et al., 1995). A notable exception is the presence of very different Bt toxin genes in subspecies morrisoni and tenebrionis within serotype 8a,b. Some of the serotypes are divided into subserotypes that can be differentiated by PCR (Bourque et al., 1993; Brousseau et al., 1993). However, a high level of sequence similarity among B.anthransis, B. cereus, and B. Thuringiensis does not permit construction of sequence-specific probes to be used in identification (Bourque et al., 1994). The most useful scheme for classification of Bt toxins is based primarily on homology of toxin gene sequences and the spectrum of insecticidal activity (Hofte and Whiteley, 1989; Ogiwara et al., 1995). A large number of distinct Bt toxin genes have been cloned and sequenced since the first report published in 1981 (Schnepf and Whiteley, 1981). Hofte and Whiteley (1989) have classified 42 Bt genes into 14 distinct types and grouped them into four major classes. The classes are cry1 (Lepidoptera specific), cry11 (Lepidoptera and Diptera specific), cry1IZ (Coleoptera specific), and cryIV (Diptera specific). Many more Bt genes have since been sequenced and analyzed. Following the analysis of toxin domains of 29 distinct Bt toxin proteins, Feitelson et al. (1992) added two new major classes, cryVand cryV1. Several novel genes were also added within the previously defined classes (Table 111).The nomenclature of Hofte and Whiteley (1989),based mainly on insecticidal activity, failed to accommodate genes that were highly homologous to known genes but with a different insecticidal spectrum. cryIZA and IIB were included in the Diptera-specific class because it is known that cryI1B is inactive against Diptera. cryIC is toxic to both Diptera and Lepidoptera (Smith and Ellar, 1994). Several genes with differing homology and bioactivity were named cryV, the next available Roman number in the original system (Gleave et al., 1992, Tailor et al., 1992). Based on amino acid identity of full-length gene products, Crickmore et al. (1996) have introduced a systematic nomenclature for classifying the cry genes and their protein products. Most cry genes retain the name assigned by Hofte and Whiteley with a substitution of Arabic for Roman numerals (e.g., cry1 Aa) to accommodate the newly discovered genes. Fifty genes comprising 16 homology groups are systematically arranged. Their dendrogram depicts the possible evolutionary relationships between the entire set of Bt toxins. Primary through quaternary ranks are based on 45, 75, and 95% level of sequence identity. Eighteen sets at the primary rank, CytA, CytB, and Cryl through -16, are defined into 4 homology groups. Cryl, -3, -4, -7, -8,-9 and -10 form the largest group. Cry2 and Cry11 are the second group. The third group is Cry5, -12, -13 and -14. The fourth group is the two Cyt proteins. The Cry6, -15, and -16 consist of unique proteins.

9

INSECTICIDAL PROTEINS OF B. th uringiensis

TABLE I11

Bacillus th uringiensis CRYSTAL PROTEIN GENES Gene designation

IC ID IE IF IG cryIIA IIB IIC cryIIIA IIIB IIIC(al,(bl cryIVA IVB IVC IVD cryv Genes not yet cloned

Predicted M,

Toxicity0

131-133 137 134 133 137 134 130

L L L L L L L

71 71 71

L,D L L

73 73 73

C C C

134 128 77 72 80

D D D L,C

130 100 40

? ? ?

D

L, Lepidoptera; D, Diptera; C, Coleoptera; Based on Hofte and Whiteley (1989)

Crickmore et al. (1996) define cry as a gene from B. thuringiensis encoding a parasporal inclusion protein that exhibits pesticide activity or is homologous to a known cry gene. 1. The mnemonic cry shall remain for the crystal-forming pesticidal genes from B. thuringiensis. The cry gene nomenclature shall be distinguished at all ranks on the basis of comparative amino acid sequence identity of the full-length gene products. 2. The primary rank of the nomenclature shall be Arabic numbers. The cry genes whose products share less than 45% amino acid homology shall be characterized by different Arabic numbers. 3. The secondary rank shall be an uppercase letter. The cry genes of the same rank whose products show less than 75% homology shall be separated into different secondary ranks. 4. The tertiary rank shall be a lowercase letter without parentheses. The cry genes whose products share less than 95% homology shall be given different tertiary ranks.

10

P. ANANDA KUMAR ET AL.

5 . The quaternary rank shall be allele numbers. The cry genes whose products differ in amino acid sequence, but are more than 95% identical to each other, shall be given separate quaternary ranks.

Crickmore et al. (1996) are the B. thuringiensis cry Gene Nomenclature Committee, a standing committee of the Bacillus Genetic Stock Center. They will assist workers in the field of B. thuringiensis genetics in assigning names of new cry genes and periodically review the literature of the cry genes. Ill. Structure of Bt Toxin Proteins and Genes

Bt toxin genes are usually plasmid borne (Gonzalez et al., 1995) but also chromosomally located (Carlson and Kolsto, 1993; Klier et al., 1982; Kronstad et al., 1983). The Bt toxin genes are encoded on plasmids of molecular weight 40-150 mDa (Carlton and Gonzalez, 1985; Jensen et al., 1995).Most of the plasmids are of low copy number. In addition to the toxin-encoding plasmids, there are often several other cryptic plasmids of 4-150 mDa whose function is not clearly known. Many of the plasmid-encoded toxin genes are bordered by transposons and/or insertion sequences (Delecluse eta]., 1990). Dervyn et al. (1995) examined the transcriptional regulation of the cryIVD gene operon from B. th uringiensis subspecies israelensis. Hofte and Whiteley (1989) compared sequences among a number of toxins with varying specificities and found five well-conserved regions designated blocks 1-5 (Fig. 4). Exceptions to this include CryIVC toxin of Bt subspecies israelensis and a novel toxin from subspecies thompsoni (Brown and Whiteley, 1992). Blocks 1 and 2 are very hydrophobic and are present as amphipathic a-helices with membranespanning potential. The protoxins designated CryIA-CryIG, CryIVA, and CryIVB contain 1100-1200 amino acids and the toxin is processed from within the amino half as shown in Fig. 4. The CryII, CryIII, and CryIVD protoxins are smaller, with processing to toxins as indicated. The carboxyl halves of the CryI, CryIVA, and CryIVB protoxins are also highly conserved except that there is a deletion of 26 amino acids in CryIA(b) protoxins. On the basis of the conservation of the defined blocks, it was postulated that all of the Bt toxins probably have a three-dimensional conformation similar to that of a CryIIIA toxin reported by Li et al. (1991) (Fig. 5 ) . According to this, the first 285 residues are present as a bundle of seven amphipathic a-helices, wherein six are arranged in a circle, and helix 5 is in the center (domain I). Residues 286-500 are organized as three p-sheets (domain 11) and contribute to the toxin specificity. The remaining amino acids are also present as p-sheets and arranged like a

INSECTICIDAL PROTEINS OF B. thuringiensis

Gene/ Protoxin Designation

Target Insects Lepidoptera cry (Diptera) Cry N A , B Diptera

11

Rotoxin

NH2

I

+ COOH

24-"i-a

I

H

Cry IVD

Diptera

I

/ ' / \

FIG.4. General structural features of protoxins as deduced from gene sequences and other related data. Protoxins designated CryIA-CryIG, CryIVA, and CryIVB contain 1000-2000 amino acids, and the toxin is processed from within the amino half as shown. The CryII, CryIII, and CryIVD protoxins are smaller, with processing to toxins as indicated (not known for CryIVD). Regions marked 1-5 are highly conserved among the CryI, CryIII, CryIVA, and CryIVB toxins and less so (pramarily regions 1 and 2) for the Cry11 and CryIVD toxins. The carboxl halves of the CryI, CryIVA, and CryIVB protoxins are also extensively conserved. A major difference is the deletion of 26 amino acids (h26) in most of the CryIA(b) protoxins. Other portions of the toxins are more or less conserved within a particular class (i.e., those designated CryI or CryII, etc.) but not between these classes. Reproduced with permission from Dr. Aronson.

sandwich (domain 111). All the three domains have specific functional roles. The first domain is required for toxicity, and domain I1 is important for specificity. Although the function of Domain I11 near the carboxyl end was not defined, it is speculated that it may have a role in the processing of protoxin (Aronson, 1994, Martens et a]., 1995) and channel-forming function (Chen et a]., 1993). Wu and Aronson (1992)induced localized mutagenesis in central helix of domain I and found loss of toxicity but not the capacity to bind midgut membranes. A synthetic peptide of helix 5 could insert itself into membrane and form ion channels that confirmed the importance of this helix (Gazit and Shai, 1995). Single site mutations in the conserved alternating arginine region affect ionic channels formed by CryIA(a), a Bt toxin (Schwartz et a]., 1995). The assembly and organization of the 01-5 and 01-7helices from the pore-forming domain of B. thuringiensis &endotoxin is relevant to a functional model for pore formation (Gazit and Shai, 1995). Similarly, a truncated peptide corresponding to the domain I of

12

P. ANANDA KUMAR ET AL.

Cry111 p2 was shown to be sufficient for membrane channel activity and ion efflux from artificial membrane vesicles (Van Tersch et al., 1994). Mutations in domain I reduced the irreversible binding of toxin to BBMV ( Chen et al., 1995). The evidence that domain I1 is involved in specificity comes from the structural comparisons of CryIA toxins and construction of hybrid genes to analyze specificity domains (Schnepf et a1.,1990; Ge et al., 1991). Chen et al. (1993) concentrated on the highly conserved block 4 of domain I11 and used site-directed mutagenesis to substitute other amino acids for arginine. Studies with these mutant proteins revealed that domain I11 is not only involved in structural stability and integrity of the toxin protein but also in function as an ion channel. Wabiko and Yasuda (1995) investigated the location of toxic border and the requirement of the nontoxic domain for high-level in vitro production of active toxin from B. thuringiensis protoxin. IV. Screening for New Insecticidal Proteins and Genes

As mentioned previously, the toxicity spectrum is being widened each year with the discovery of novel strains that are active against various organisms (Payne et al., 1995; Hickle and Payne, 1995; Kawalek et al., 1995). Following the early isolations of Bt from dead insect larvae, these bacteria have been found ubiquitously by using a novel enrichment technique that exploits unique germination properties of the spores (Martin and Travers, 1989) or by simply screening debris, such as soils, leaves, and dead larvae, for spore formers containing parasporal inclusions. An interesting example was the prevalence of isolates on the surfaces of leaves from various trees (Smith and Couche, 1991). One of the most important aspects about establishing a Bt collection is to have a methodology with which one can rapidly and accurately characterize the strain, the toxin protein, and the gene. This is especially important if the differences among endotoxin genes, carried by a certain strain, are critical for its specificity and toxicity. The bioassay analysis is an exhaustive and time-consuming process because it is necessary to screen all the isolates in all of the target insects. Various methodologies have been described to simplify this process. The important approaches are 1. Southern blot analysis in search of homologous genes (Kronstad and Whiteley, 1986); 2. Reactivity to different monoclonal antibodies (Hofte and Whiteley, 1989);and 3. Electrophoretic analysis of PCR products using specific primers (Carozzi et al., 1991).

FIG.5. A schematic ribbon diagram of the CryIIIA structure (Li et a]., 1991). Domain I, the putative membrane insertion domain, is a 7-helix bundle (left);domain 11, the putative receptor binding region, is an assembly of three (3-sheets (lower right); domain 111 is a (3-sandwich in which the C terminus is buried (upper right).

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INSECTICIDAL PROTEINS OF B. thuringiensis

13

Among the three approaches, PCR analysis is considered to be the best choice because it permits a rapid determination of the presence or absence of a sequence, it is highly sensitive, relatively fast, and can be used routinely. Carozzi et al. (1991) described the sequences of 1 2 PCR primers that can distinguish three major classes of Bt toxin genes (cry1, cry111, and cry1V). However, it is important to delineate the genes in each subgroup because of the differential insect toxicity. PCR analysis of three cry1A subgroups was reported (Bourque et al., 1993). This analysis did not allow for the identification of the remaining cry1 gene subgroups. Thus, it is important to develop a complete PCR set of primers that allows the identification of all reported cry genes. Bravo and co-workers (Ceron et al., 1994) at the National University of Mexico designed four oligonucleotides that can be used to identify the strains that carry any of the crylor cry111genes. These primers were selected from a highly conserved region between cryr’s or cry11I’s genes by computer analysis using a Gene work 2 program that allows simultaneous alignment af several gene sequences. The primers were able to amplify a region ranging between 272 and 290 bp from all the crylgenes and between 688 and703 bp from all cry111 genes. Strains with unique PCR product profiles were then characterized by using additional genespecific primers. A set of primers were designed that give a different molecular weight with each of the cry1 and cry111 genes. These primers were selected from the highly variable region among all genes. They were designed to be used in mixtures of six to eight primers per reaction. By using these primers, all the cry1 genes from cry1A to crylG,including subdivisions of cry1A genes as well as all the cry111genes from cry111A to cry111E,could be identified (Ceron et a1.,1994).The important feature of this screening method is that with each gene a precise molecular weight product is expected. Genes that produce different-sized products may represent novel genes. Using a similar approach, Kalman et al. (1993) found a novel cry1C gene. One limitation of the methodology, however, is that new genes from a Bt collection cannot be identified if the gene in question does not have any of the primer sequences. In addition, PCR screening does not provide information of the specific target insect of the novel gene. Bravo et al. (1992a) developed an immunocytochemical technique to identify proteins that may have potential toxicity toward selected insects. This was based on a clear correlation between binding of toxic protein to its specific receptor localized in the microvilli of the midgut cells and toxicity. By using this methodology, toxins that are highly toxic to Diatraea grandiocella, Spodoptera frugiperda, and Rhopalosiphum maidis (aphid) were found. The proteins to be tested should be

14

P. ANANDA KUMAR ET AL.

recognized by specific antibodies. They can also be labeled with biotin to be detected subsequently with streptavidin coupled to the peroxidase enzyme (Denolf et al.,1993). Another approach to identify a toxin protein is to analyze its effects on the permeability of brush border membrane vesicles. Changes in membrane permeability can be measured fluorometrically with a fluorescent dye sensitive to changes in membrane potential. Uemura et al. (1992) used membrane vesicles from Bombyx mori and found that toxic proteins were able to produce clear effect on ion transport, whereas nontoxic proteins did not do so. The novel toxins that are able to modify the permeability of the membranes from a selected larva are expected to have a higher potential of being toxic. Entomopathogenic bacteria belonging to Bacillaceae and the immunological relationship between their insecticidal toxins are being studied by cloning the toxin gene next to a Bacillus promoter in Escherichia coli. Recombinants are first screened with degenerate nucleotides probes based on the DNA sequence of the &endotoxin gene. Recombinant plasmids from positive clones are transferred into toxin minus Bacillus strains and the protein produced is screened with antibodies directed against toxin from the wild-type Bacilli strain. This method can be adapted for screening large number of isolates with a wide variety of degenerate oligonucleotides. V. Mechanism of Action

The target organ for Bt toxins is the insect midgut (Zimanyi et a]., 1995). The midgut of the lepidopteran larvae is a simple, tubular epithelium that dominates the internal architecture of the insect. The tissue is composed of two major cell types: a columnar cell with a microvillate apical border and a unique goblet cell, containing a large vacuolar cavity, linked to the apical surface by an elaborate and tortuous “valve” (Cioffi, 1979).The “Kf pump” is located in the apical membrane of the goblet cell, pumping K+ from the cytoplasm into the cavity and thence to the gut lumen via the valve. This electrogenic K+ transport is the predominant feature of the larval lepidopteran gut. Disruption of the activity of K+ pump as a result of toxin-induced pore formation in the plasma membrane of the columnar cells leads to osmotic imbalance. Another important feature of the midgut is that the pH of the lumenal fluid is about 12, which is essential for dissolving the crystalline Bt protoxins, usually soluble only above pH 9.5. The crystalline protoxins are inactive. They are solubilized and then activated by gut trypsin like proteases (Milne and Kaplan, 1993, Tojo and Aizawa, 1983), which typically cleave some 500 amino acids from

INSECTICIDAL PROTEINS OF 8.thuringiensis

15

the C terminus of 130-kDa protoxins and 28 amino acids from the N terminus, leaving a 65 to 55-kDa protease-resistant toxic active core comprising the N-terminal half of the protoxin (Hofte and Whiteley, 1989). The mature Cry1 A toxin is cleaved at the amino-terminal R2 arginine residue (Nagamotsu et al., 1984) and the carboxyl-terminal K lysine residue (Bietlot et al., 1989).A tightly bound 20-kilobaseheterogeneous DNA fragment is involved in the proper proteolytic processing of protoxin (Bietlot et al., 1993).The 70-kDa CryII, CryIII, and CryIVD proteins are naturally occurring truncated forms. The active toxins bind to specific receptors located on the apical brush border membrane of the columnar cells. Binding involves two steps, reversible (Hofmann and Luthy, 1986; Hofmann et al., 1988a) and irreversible (Ihara et al., 1993; Rajamohan et al., 1995). The irreversible step is followed by the insertion of the toxin into the apical membrane. Various studies revealed that there are many different toxin-binding protein receptors (Gill et al., 1992). Some of them were identified as 120 to 180-kDa glycoproteins (Garczynski et al., 1991; Knowles et al., 1991; Oddou et al., 1991). In Manduca sexta, a 210-kDa membrane protein is the CryIAb receptor (Vadlomudi et al., 1993, 1995). A 120-kDa aminopeptidase N has been reported as receptor for the Cry1 Ac toxin (Knight et al., 1994; Sangadala et al., 1994). Bacillus thuringiensis CryIAc 6-endotoxin-binding aminopeptidase in the M. sexta midgut has a glycosyl phosphatidylinositol anchor (Garczynski and Adang, 1995). After binding to the specific receptor, the toxin inserts irreversibly into the plasma membrane of the cell leading to lesion formation. Three models were proposed to explain the role of toxin receptor in pore formation (Knowles and Dow,1993). The first model envisages that the receptor is itself a transmembrane channel that is activated by the toxin in a manner analogous to the ligand gating mechanism employed by neurotransmitters. The second possibility is that the toxin and receptor together form a pore. The third model explains that the receptor catalyzes toxin association or insertion into the membrane and plays no further role in pore formation. The receptor may either simply act as a handle to which the toxin binds or induce a conformational change in the toxin, thus enabling it to insert into the membrane. The formation of toxin-induced pores in the columnar cell apical membrane allows rapid fluxes of ions. Different studies revealed that the pores are K+ selective (Sacchi et a1.,1986),permeable to cations (Wolfersberger, 1989),permeable to anions (Hendrick et al., 1989), or permeable to small solutes like sucrose, irrespective of the charge (Schwartz et al., 1991a). Using a simple light scattering assay, Carroll and Ellar (1993) found that the midgut membrane permeability in the presence of CryIAc was altered for cations, anions, and neutral solutes, as well as for water. It appears

16

P. ANANDA KUMAR ET AL.

that the toxin forms or activates a relatively large aqueous channel in the membrane. The model proposed by Knowles and Dow (1993) placed emphasis on the cessation of the K+ pump that leads to the swelling of columnar cells and osmotic lysis. The disruption of gut integrity results in the death of the insect from starvation or septicemia. There seems to be a different mechanism of action with respect to CryIIA toxins. Differences in the extent of solubilization may sometimes explain differences in the degree of toxicity among Cry proteins. Decreased solubility could be one potential mechanism for insect resistance (McGaugheyand Whalon, 1992).English et al. (1994)compared the differences in solubility, binding to the brush border membrane, and ion channels formed by CryIIA and CryIAc toxins in Helicoverpa zea. The results showed unique attributes in the mode of action of CryIIA, which was less soluble than CryIAc and failed to bind to a saturable binding component on the midgut brush border membrane. In addition, voltage-dependent, nonselective channels were formed by this toxin in planar lipid bilayers. This behavior was reminiscent of several other channel-forming protein toxins of bacterial origin such as the E.coli active colicins, which have a strong voltage dependence. It was suggested that the unique mode of action of CryIIA may provide a useful tool in managing field resistance to Bt toxins. Although the binding of the Cry toxins to receptors determines the insect specificity (Kronstad et al., 1983; Van Rie et al., 1990a),exceptions to correlation of binding and toxicity exist. CryIAc binds to ligand blots of Spodoptera exigua BBMV proteins without any toxicity to the insect larvae (Garczynski et al., 1991). CryIAb is more toxic to gypsy moth than CryIAc but does not bind as well to receptors on BBMV (Wolfersberger, 1990).Irreversible binding and ion-channel function directly correlate to toxicity in gypsy moth, thus unraveling the “Wolfersberger paradox” (Liang et al., 1995; Rajamohan et al., 1995; Liebig et al., 1995). VI. Bt as a Biological Insecticide

Bacillus thuringiensis is the most popular biological control agent with a worldwide projected sales of about $90 million during 1995 (Lambert and Peferoen, 1992). Sixty-seven registered B. thuringiensis products have more than 450 uses and formulations (Dean and Adang, 1992;Rowe and Margoritis, 1987). Bacillus thuringiensis is the major pesticide against gypsy moth in forests (Twardus, 1989). Bacillus thuringiensis subsp. israelensis (BTI) is extensively used to control mosquitoes and blackflies (Becker and Margalit, 1993; de Barjac and Sotherland, 1990)

INSECTICIDAL PROTEINS OF B. thuringiensis

17

Bacillus thuringiensis subsp. morrisoni and BTI carry four genes that encode mosquito and blackfly active toxins: crylVA, cryZVB, crylVC, and crylVD. BT also produces Cyt toxins that synergize the Cry toxins. Bacillus thuringiensis subsp. jegathesan encodes another potent mosquitocidal toxin immunologically related to CryIIA (Delecluse et al., 1995). Thus, Bt plays an important role not only in agriculture and forestry but also in the area of human health (Haider et al., 1986, 1987; Smith and Ellar, 1994; Orduz-Peralta et al., 1992). The Bt toxin normally accumulates during the stationary phase (Bechtel and Bulla, 1976) with exceptions (Krieg et al., 1980). The earliest commercial production of Bt began in France in 1938 under the trade name Sporeine (Luthy et al., 1982).During the 1960s, several industrial formulations of Bt were manufactured in the United States, France, Germany, and Soviet Union. The isolation of the highly potent kurstaki variety by Kurstak in 1962 and by Dulmage in 1967 (Dulmage,l970) provided a much-needed boost to the commercialization of Bt. The HD1 isolate of Dulmage is still the active ingredient in most Bt products used against caterpillar pests in agriculture, horticulture, and forestry. The discovery of new strains of Bt widened the toxicity spectrum of bioinsecticides. The use of conventional Bt insecticides, however, was found to have limitations like narrow specificity, short shelf life, low potency, lack of systemic activity, and the presence of viable spores (Lambert and Peferoen, 1992). These problems are now overcome by various approaches that utilize the tools of molecular biology and genetic engineering as well as conventional microbiological methods (Ben-Dov et al., 1995). A. CONSTRUCTION OF NOVELBT STRAINS BY CONJUGATION

The plasmid location of Bt toxin genes enabled the construction of novel Bt strains with microbial genetic approaches such as plasmid curing and conjugal transfer (Wiwat et al., 1995; Battisti et al., 1985). Conjugational transfer of native Bt plasmid between species of Bacillus is known to occur (Gonzalez et al., 1982; Reddy et al., 1987; Andrup et al., 1995). Expression of transformed plasmid-coded genes was analyzed by genotyping of crystal proteins and flagellar antigenicity. This particular set of studies employed conjugational transfer of an entire toxic polypeptide coding gene located on native plasmids. On the other hand, more versatility of the conjugational transfer-mediated approach was demonstrated with a mobilizable plasmid bearing a cloned gene coding for a variant Bt gene (Klier et al., 1983). Following the conjugational approach, scientists at Ecogen Corporation (USA)produced several bioinsecticides with broadened spectrum of toxicity (Gawron-Burke

18

P. ANANDA KUMAR ET AL.

and Baum, 1991). For instance, the product “Foil” is made from a strain that carries toxin genes active against European corn borer (Lepidoptera) and Colorado potato beetle (Coleoptera). Using the conjugational approach, Bora et al. (1994) transferred the cryIAa gene of Bt into Bacillus megaterium, which resides in the cotton phyllosphere. Leaf bioassays of cotton plants, inoculated with a single spray of the transcipient, showed that there was protection to the cotton plants from Helicoverpa armigera. Enhanced production of insecticidal proteins occurs in B. thuringiensis strains carrying an additional crystal protein gene in their chromosomes (Kalman et al., 1995). The conjugational approach to create novel Bt strains has certain limitations. Not all the Bt toxin genes are located on transferable plasmids. Second, the toxin protein with useful insecticidal activity may be synthesized at low amounts. Plasmid incompatibility could also be a problem. A significant advantage to the conjugal transfer approach is the simplified registration process for the Bt product. The U.S. Environmental Protection Agency treats transconjugants in a similar manner as it treats wild-type Bt isolates. Another interesting approach to expand the insecticidal host range of Bt is to make use of the in vivo genetic recombination property (Baum et al., 1990). Lereclus et al. (1992)used insertion sequence IS232 to deliver cryIZZA gene into an isolate producing CryIAc toxin. Expression of the introduced gene did not alter the composition of the polypeptides normally produced by the strain. Novel Bt mutants, defective in sporulation but overproducers of toxin, have been isolated (Lereclus et al., 1995). They can be used safely as a biopesticide in silkworm-rearing areas . These mutants were shown to achieve effective control of H. armigera in field-grown chick-pea (Satyanarayana, et al., 1995).

B. CONSTRUCTION OF RECOMBINANT BT STRAINS Development of novel cloning vectors for Bt has made possible the construction of improved Bt strains for use as microbial insecticides. The use of Bt as the host organism offers many advantages. Native Bt strains can stably maintain and efficiently express several homologous Bt toxin genes. The ability to maintain multiple Bt toxin genes in a single recipient broadens the insecticidal activity in an additive or synergistic manner. Multiple toxin genes with differing modes of action or receptor-binding properties may reduce the chances of insects developing resistance (Tabashnik, 1994). An essential element in the successful engineering of Bt strains is the availability of suitable cloning vectors. A number of convenient shuttle vectors, functional in E.coli and Bacillus species, have been constructed

INSECTICIDAL PROTEINS OF B. th uringiensis

19

using replication origins from resident Bt plasmid (Baum et al., 1990; Gawron-Burke and Baum, 1991). Considering the stability of resident Bt plasmids, shuttle vectors derived from resident plasmids might exhibit good segregational stability. Lereclus and Arantes (1992) selected a replication origin from a small cryptic plasmid of Bacillus subtilis (pHT1030) that exhibits excellent segregational stability. They constructed shuttle vectors (Lereclus et al.,1989; Arantes and Lereclus, 1991; Figure 6) to introduce new Bt toxin genes into Bt strains. For instance, cryIIIA gene from Bacillus tenebrionis, when introduced into Bt. kurstaki HD119, was highly expressed without affecting the level of expression of native cry genes (Game1and Piot, 1992). Shin et al., (1995) studied the distribution of cryV-type insecticidal protein genes in B. thuringiensis and cloned cryV-type genes from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. entomocidus. Wu and Federici (1995) improved production of the insecticidal CryIVD protein in B. thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein. Ely (1995) constructed insecticidal proteins from B. th uringiensis &endotoxin and An drocton us a ustralis neurotoxin AaHIT.

c. CONSTRUCTION OF TRANSGENIC MICROBES Cloned Bt toxin genes were introduced into a number of microbial hosts to create more stable and/or compatible agents for the toxin delivery. Monsanto scientists were the first to report the expression of the cryIAb gene in a root colonizing Pseudomonas at levels sufficient to kill lepidopteran larvae (Watrud et al., 1985). The gene was later cloned into Tn5 and transposed into the chromosome of six corn root-colonizing

[SspllBall] Scal

Accl

4

ori 1030 (2.6kb) I

1kb

[HpallBarnHI] ~c~~

*

[KpnllSspl] Hincll Scal

-

ErR (1.2kb)

--- -

~p~ ori EC. ( pUC19 : 2.7kb)

I

pHT304: 4 f l copies/chromosome pHT315: 15*5 copieslchromosome pHT370: 7 M O copies/chromosome

FIG.6. Vectors for cloning genes in B. thuringiensis and B. subtilis; Gene 1991 108: 115-119 (Reproduced with permission from D. Lereclus, Institut Pasteur, Paris).

20

P. ANANDA KUMAR ET AL.

strains of Pseudomonas jluorescens and Agrobacterium radiobacter (Obukowiczet al., 1986).Following this, many groups developed Psuedomonas strains carrying Bt toxin genes. The recombinant Psuedomonas is killed by a proprietary chemical treatment that crosslinks the bacterial cell wall to yield a nonviable encapsulated bacterium surrounding the crystal protein (Cell-Capproduct of Mycogen; Gaertner et al., 1993). Such a product is stable and safe for use in the environment. An interesting example of a toxin gene in a foreign bacterium is the introduction of the cryIAc gene into the plant endophyte, Clavibacter xyli subsp. cyanodonfis (Turner et al., 1991). Clavibacter resides in the xylem of Bermuda grass (Cynodon dactylon). It also colonizes the vascular system of corn when artificially inoculated. The recombinant bacterium can be inoculated into the stems to establish an endogenous supply of the toxin for protection against European corn borer. Recently, cryIAc was introduced into the chromosome of C. xyli by using an integrative plasmid vector that facilitates homologous recombination between the vector and the bacterial chromosome (Lampel et d.,1994). It is expected that this recombinant strain will show stability and in planta biological activity. Introduction of Bt genes into root-nodulating bacteria, thereby providing protection to nodules from soil-dwelling pests, was accomplished by Nambiar et al. (1990).Recombinant Bradyrhizobium carrying cryIVD was produced and used to infect the roots of pigeon pea. The root nodule infestation by the larvae of the dipteran species, Rivella angulata, was reduced by 40%. Bezdicek et al. (1994) introduced the cryIIIgene into Rhizobium leguminosarum and Rmeliloti by using a broad host range vector, pRK311, containing lacZ promoter or n i p promoter. The recombinant rhizobia expressed the toxin in sufficient quantities within root nodules to significantly reduce feeding damage by the nodule-feeding insects, Sitona lineatus on Pisum sativum and Sitona hispidulus on Medicago sativu. The pRK311 plasmid remained stable in the rhizobia that were either free living or within nodules of the legumes. The engineered strains of R.leguminosarum were equally competitive with the wild-type strain. Udayasuriyan et al., (1995)transferred an insecticidal protein gene of B. thuringiensis into plant-colonizing Azospirillum that may be used to control root-feeding insects. Mosquitocidal Bt toxin genes were also shuffled between Bacillus sphaericus and Bt subsp. israelensis to extend the host range of the bacteria for mosquito larvae (Bourgouin et a]., 1990; Bar et al., 1991). The cryIVA gene of Bt subsp. israelensis was also introduced into various unicellular cyanobacteria with the intent of providing a more accessible source of the toxin for filter-feeding dipteran larvae (Angsuthanasombat and Panyim, 1989; Chungiatupornchai, 1990; Soltes-Rak et al., 1993).

INSECTICIDAL PROTEINS OF B. thuringiensis

21

D. BACULOVIRUSES AS BT VECTORS It was demonstrated that insects are susceptible to a wide variety of virus infections (King et al., 1994).Many viruses are currently identified in insect hosts out of which baculoviruses carrying large, covalently closed, circular DNA genomes are useful as insect biocontrol agents. Two studies reported the insertion of Bt genes into the Autograph californica nuclear polyhedrosis virus genome (Martens et al., 1990; Merryweather et al., 1990). A full-length copy of the endotoxin coding sequence was inserted into the baculovirus genome in place of the polyhedrin gene-coding region. Martens et al. (1990) demonstrated that the protein produced in insect cells formed large crystals as inclusion bodies in the cytoplasm. When insect larvae were fed recombinant virus-infected cell extracts, the larvae perished. Merryweather et al. (1990)also cloned Bt gene upstream of the polyhedrin gene under the control of the AcNPV p l 0 promoter. When insects were given purified polyhedra in a bioassay, there was no effect on the larvae. It was possible that the protoxin produced by the virus remained as an intracellular protein and did not get processed and solubilized in the insect midgut for eventual toxicity. Addition of a suitable signal peptide sequence to the Bt toxin gene would facilitate secretion of the recombinant product from the cells into midgut and thereby improve its efficacy. E. INSECT-TOLERANT TRANSGENIC CROPPLANTS

An elegant, and perhaps the most effective delivery system for Bt toxins, is the transgenic plant (Stewart et al., 1995). The major benefits of this system are economic, environmental, and qualitative. In addition to the reduced input costs to the farmer, the transgenic plants provide season-long protection independent of weather conditions, effective control of burrowing insects difficult to reach with sprays, and control at all of the stages of insect development. The important feature of such a system is that only insects eating the crop are exposed to the toxin. Genetic transformation of almost all the major crop species is now feasible with the development of an array of techniques ranging from the Agrobacterium approach to electric discharge-mediated particle acceleration procedure (Finch, 1994). The first Bt-transgenic plants were made in 1987 (Barton et a1.,1987; Fischhoff et a]., 1987; Vaeck et al., 1987). The plants expressed fulllength or truncated Bt toxin genes (cryIA)under the control of constitutive promoters. The expression of the toxin protein was very poor in the tobacco plants and the mortality of M. sexta larvae was only 20%. Truncated cryIA genes coding for the toxic N-terminal fragment provided better protection to the tobacco and tomato plants. When compared to

22

P. ANANDA KUMAR ET AL.

the plants transformed with full-length genes, the plants expressing truncated genes were more resistant to the larvae, and the highest reported level of toxin protein expression was about 0.02% of total leaf-soluble protein. Despite these low levels of expression, many of the plants were shown to be insecticidal to the larvae of M. sexta. However, many of the noctuid lepidopterans, which constitute a very serious group of insect pests, need higher amounts of Bt toxins for effective control. Gene truncation as well as the use of different promoters, enhancer sequences, and fusion proteins resulted in only limited improvement in Bt gene expression (Barton et al., 1987; Carozzi et al., 1992; Vaeck et al., 1987). In 1990, researchers at Monsanto made a significant advancement in the expression of Bt genes in plants (Perlak et al., 1990). They noticed that Bt genes were excessively AT rich in comparison with normal plant genes. This bias in nucleotide composition of the DNA could have a number of deleterious consequences to gene expression because AT-rich regions in plants are often found in introns or have a regulatory role in determining polyadenylation. There are also instances in other eukaryotic systems in which AT-rich regions can signal rapid degradation of specific mRNAs. In addition, plants have a tendency to use G or C in the third base of redundant codons-A or T being rarer. Bt genes have the opposite tendency and because codon preference is thought to be linked to the abundance of the corresponding tRNAs, the overuse of rare codons would decrease the rate of synthesis of a Bt protein in plant cells. Perlak et al. (1991) followed two approaches to modify the crylAb and cryIAc genes. One approach included selective removal of DNA sequences predicted to inhibit efficient expression of Bt gene expression at both translational and mRNA levels by site-directed mutagenesis. These genes were termed partially modified (PM) genes. The other approach was to generate a synthetic gene with a fully modified (FM) nucleotide sequence, taking into account factors such as codon usage in higher plants, potential secondary structure of mRNA, and potential regulatory sequences. The PM-cryIAb gene is approximately 96 % homologous to the native gene with a GC content of 41%, with the number of potential plant polyadenylation signal sequences (PPSS)reduced from 18 to 7 and the number of ATTTA sequences reduced from 13 to 7. The FM-cryIAb is approximately 79% homologous to the native gene, with a GC content of 49% and the number of PPSS reduced to 1 and all ATTTA sequences removed. The toxin protein levels in transgenic tobacco and tomato harboring these modified genes increased up to 100-fold over levels seen with the wild-type Bt gene in plants. Perlak et al. (1990) made a gene construct in which the first 1359 nucleotides were derived from FM-crylAb gene and the remaining se-

INSECTICIDAL PROTEINS OF B. thuringiensis

23

quence from PM-cryIAc gene. The variant gene was placed under the control of CaMV 35s promoter containing a duplicated enhancer region. Cotton-variety Coker 3 1 2 was transformed and the transgenic plants were shown to have total protection from Trichoplusia ni (Cabbage looper), S. exigua, and H , zea (cotton boll worm). The maximum level of toxin protein was 0.1% of total soluble protein. The Monsanto group placed the FM-cryIAc gene under the control of Arabidopsis thaliana Rubisco small subunit promoter with its associated chloroplast transit peptide sequence (Wong et al., 1992).Transgenic tobacco plants expressing this gene provided a 10-to 20-fold increase in cryIAc mRNA and protein compared to gene constructs in which CaMV 35s promoter with duplicated enhancer region was used to express the same gene. The toxin protein was localized in the chloroplast and in the tobacco plants that produce the Bt protein nearly 1%of the total leaf protein had the highest levels of Bt toxin proteins yet reported. The enhancement of Bt toxin protein levels in tissues in which Rubisco expression is highest may lead to very effective control of certain insect pests that feed on leaves and other green tissues. Ciba Seeds, a subsidiary of Ciba Geigy Company, used microprojectile bombardment with two plasmids (pCIB4431 and pCIB3064) of a proprietary corn line to produce a commercial cultivar (Federal Register 60 FR 9656-9657 1995). Plasmid pCIB4431 contains two different tissue-specific promoters each fused individually to a copy of a synthetic cryIAb gene. The cryIA(b)gene encodes the first 648 amino acids, with an insecticidal-active (Koziel et a]., 1993)truncated product identical to that of the cryIAb gene of B. thuringiensis subsp. kurstaki strain HD-1 (Dulmage, 1970; Geiser et al., 1986; Hofte and Whiteley, 1989). The truncated synthetic gene accommodates the preferred codon usage for maize (Murray et al., 1989)that allows efficient expression of the cryIAb gene in plants (Perlak et al., 1991; Koziel et a]., 1993). The modified gene has about 65% homology at the nucleotide level with the native gene and GtC content has been altered from 38 to 65%. The transgenic plant produces a protein that is identical to the first 648 amino acids of the full-length 1155-amino acid CrylA(b)protoxin that occurs in nature. This truncated protein contains the portion of the native protein that is responsible for its insecticidal activity. The first promoter is derived from the corn phosphoenolpyruvate carboxylase (PEPC) gene (Hudspeth and Grula, 1989). It promotes expression of cryIAb in green tissue. The second pollen-specific promoter used is derived from a maize calcium-dependent protein kinase (CDPK) gene (Estruch et al., 1994). The combination of PEPC and pollen tissue-specific promoters provides high cryIAb gene expression in leaves and pollen, where it is

24

P. ANANDA KUMAR ET AL.

most effective in controlling European corn borer. PEPC intron 9 of the corn phosphoenolpyruvate carboxylase gene (Hudspeth and Grula, 1989) is located between the crylA(b) structural gene and the 35s terminator. Its presence also increases the expression level of the cry1A(b) gene (Luehrsen and Walbot, 1991). The 3' untranslated termination sequences (CaMV) 35s from the cauliflower mosaic virus (CaMV) is present adjacent to the PEPC intron 9. Its function is to provide a polyadenylation site and it has been described previously (Rothstein et al., 1987; Sanfacon et al., 1991). The activity of the pollen-specific promoter, associated with its native CDPK structural gene in maize, is not modulated by calcium levels in the plant. Rather, the catalytic activity of the mature CDPK protein in maize is affected by calcium levels. Therefore, fusion of this promoter sequence to the cry1Ab will not manifest in any changes in the calcium requirements of corn. High levels of CryIAb protein were obtained using both promoter configurations in the transgenic maize plants. Hybrid maize plants resulting from crosses of transgenic elite inbred plants with commercial inbred lines were evaluated for resistance to European corn borer (Ostrinia nubilalis) under field conditions. Plants expressing high levels of the insecticidal protein exhibited complete resistance to heavy infestations of the pest. A similar approach was followed by Fujimoto et al. (1993) to enhance cry1Ab gene expression in rice plants. Based on the codon usage of known rice genes, 66.6% of the codons in the coding region of the cry1Ab gene were altered. The overall G+C content of the modified gene was 59.2%, whereas that of the original gene was 37.6%. The monocotyledons, including cereals, have higher G+C contents than those from dicots. The level of expression of the modified gene in transgenic rice was 0.05% of total soluble leaf protein. The plants were significantly resistant to two lepidopteran rice pests, leaf folder (Cnaphalocrosis medinalis) and stem borer (Chilo suppressalis). Following the successful attempts to control lepidopteran insects by using FM-cry1A genes, synthetic cry111 genes were also made and expressed in tobacco and potato plants, primarily for the control of Colorado potato beetle (Coleoptera) (Sutton et al., 1992; Perlak et al., 1993). The Russet Burbank potatoes were protected from damage by all insect stages in the laboratory, and dramatic protection was discernible at multiple field locations (Perlak et al., 1993). Van der Salm et al. (1994) developed transgenic tobacco and tomato plants expressing two Bt genes, cry1Ab and cry1C, specific toward lepidopteran insects. Both of the genes were partially modified to remove sequence motifs that affect mRNA stability in plant cells. The expression of a cryIAb-cryIC fusion gene resulted in protection against S. exigua, Heliothis virescens, and M . sexta. This study demonstrated the potential

INSECTICIDAL PROTEINS OF B. thuringiensis

25

of expressing translational fusions not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies (see Section VIII). Recently, researchers at Calgene, in collaboration with Maliga and Svab (1993) (Waksman Institute), expressed a cryIA gene in tobacco chloroplasts using chloroplast transformation vectors and particle bombardment technique. The transplastomic tobacco expressed the Bt toxin at very high levels and achieved complete control of lepidopteran larvae (McBride et al., 1995). The advantages of such a system are manyfold: 1. The Bt gene does not need any modification because the chloroplast transcriptional and translational apparatus are typically prokaryotic; 2. It is possible to have many copies of the Bt gene in each cell; 3. The expression of the gene will be high if driven by promoters like rbcL and cab; and 4. Because chloroplasts are maternally inherited, there is no risk of pollen transfer of the Bt gene to related plant species or weeds. The disadvantage of this approach lies in its tissue specificity. For instance, stem and fruit borers cannot be controlled following this method.

Most of the transgenic plants developed so far contained the Bt toxin genes under the control of the powerful, constitutively active 35s promoter. However, expression of the Bt toxin gene throughout the plant growth and development and in tissues in which it is not needed may encourage resistance development by the target insect (Harris, 1991). Kumar and Sharma (1994) reviewed alternative approaches like using wound and light-induciblepromoters, tissue-specific promoters, and promoters responsive to chemical sprays being used in different laboratories. VII. Resistance Development and Management Strategies

Resistance will eventually develop as a result of widespread use of any biopesticide. Resistance to B. thuringiensis endotoxins has already been developed in the laboratory (Tabashnik, 1994) that can be initiated by alteration of the target of insect-toxin interaction (Gould et al. 1992; MacIntosh et al., 1991; Van Rie et al., 1990b). In insect-tolerant transgenic plants, solubility and proteolytic processing are bypassed because only the toxin-soluble core of the Cry protein is produced. Transgenic plants with multiple genes coupled with other management strategies might slow resistance development. Although resistant mosquitoes have been selected with individual toxins such as CryIIA, due to the combination of four Cry toxins and the spore, mosquito resistance will be hard to evolve against BTI (Georghiou, 1994; Goldman et al., 1986). Bt had been used as a biopesticide for more than two decades. Evolution

26

P. ANANDA KUMAR ET AL.

of resistance was presumed unlikely because of the lack of reports of substantial resistance development in open field populations (de Barjac, 1987). However, resistance to Bt was documented in field populations of diamondback moth (Tabashnik et al., 1990, 1991; Rabindra et al., 1995). These and many other reports confirmed doubts raised by the results of laboratory selection for resistance to Bt in several major pests (McGaughey, 1985; McGaughey and Beeman, 1988). Various aspects of insect's resistance to Bt viz. laboratory selection, resistance risk assessment, variation among conspecific populations, mechanisms, cross-resistance, genetics, stability, fitness costs, and management were recently reviewed (McGaughey, 1994; Tabashnik, 1994; Kennedy and Whalon, 1995). In this section, the mechanisms involved in resistance and strategies to manage its development are explored. Intensive selection pressure on insect populations inevitably leads to the development of resistance. The resistance could be achieved by different mechanisms ranging from the point of protoxin ingestion to the insertion of toxin in the membrane. The factors affecting the binding of toxin to the receptor would result in selective resistance. On the other hand, those steps utilized by all the toxins viz. proteolysis of protoxins, conformational alterations, and membrane insertion may lead to crossresistance. Studies revealed that midgut pH and the nature of proteases probably were not involved in achieving resistance (Kinsinger and McGaughey, 1979; Johnson et al., 1990). Reduced binding of Bt toxin to the brush border membrane of the midgut epithelium was identified as a primary mechanism of resistance in Plodia interpunctella (Van Rie et al., 199Oc) and Plodia xylostella (Bravo et al., 1992 a,b; Ferre et al., 1991). Studies with radioactive-labeled CryIAb showed that a 50-fold reduction in binding was correlated with a 100-fold reduction in toxicity of CryIAb in a resistant versus a susceptible strain of l? interpunctella (Van Rie et al., 1 9 9 0 ~ )A. strain of l? xylostella from the Phillippines showed a 200-fold resistance to CryIAb and little or no binding of the toxin to the midgut epithelial membrane compared to a susceptible strain. In contrast to the results for l? interpunctella and l? xylostella, two independent studies on H. virescens found no clear association between toxin binding and resistance to CryIAb or CryIAc (MacIntosh et al., 1991; Gould et al., 1992). The only evidence against involvement of the binding step in the mechanism of both resistance and specificity was presented by Wolfersberger (1990). He found that in Lymantria dispar there was a negative relationship between binding affinity and toxicity of two different Bt toxins toward a single strain of insect. That is, the more toxic protein is bound with less affinity than the less toxic one.

INSECTICIDAL PROTEINS OF B. th uringiensis

27

Wolfersberger'sresults are consistent with the idea that there could be differences in toxicity as well as differences in binding affinity. An observation of considerable significance was that of resistance development in P. interpunctella to multiple toxins (McGaughey and Whalon, 1992). Selection of rl interpunctella colonies resistant to Bt isolates, known to contain multiple protoxins, resulted in the isolation of colonies resistant to several toxins (McGaughey and Johnson, 1993). The apparent frequency of such resistant colonies appears to be too high for two or more independent mutations, each altering a specific receptor. It is possible that resistance is due to the mutation of one locus affecting the ability of a variety of toxins as in the H. virescens colony with broad resistance (Gould et al., 1992). It is also possible that these receptors may somehow interact or cluster, such that a single mutation affects the binding properties of several toxins (Aronson, 1994). With the realization that insects can develop resistance to Bt, attention is now being focused on developing deployment strategies that might delay or prevent its evolution. Theoretically, resistance to conventionally sprayed Bt could develop slower and be narrower in scope and easier to manage than resistance to synthetic organic insecticides because Bt has a shorter residual period and much narrower spectrum of biological activity. Expression of Bt toxins in other bacteria or addition of ultraviolet blockers to formulations can extend the persistance of Bt, making it comparable to organic insecticides (Tabashnik, 1994). Expression of Bt in transgenic plants may continuously select pests intensively for resistance because insects are exposed to Bt even when they are not causing economic damage (Mallet and Porter, 1992).Various strategies were suggested to tackle the problem of resistance development and have been summarized by Whalon and McGaughey (1993).These tactics were patterned after those used or proposed for use in managing chemical insecticide resistance and typically involve variations of the following: (i) rotation or alteration of toxins, (ii) mixtures or sequences of toxins, (iii) provision of refuges, (iv) ultrahigh doses of toxin, and (v) temporal and spatial expression of Bt toxin genes in transgenic plants. A. ROTATIONS

Rotation or alteration of Bt toxins, insecticides, and cultural or biological control strategies is probably the simplest approach to resistance management. Success with this tactic depends on restoring susceptibility when selection pressure is discontinued or changed to another gene, toxin, or insecticide. However, rotations among toxins that confer cross-resistance to each other have limited value (Gould, 1988;

28

P. ANANDA KUMAR ET AL.

Gould et al., 1992). Studies indicating considerable instability of resistance to Bt in l? xylostella (Hama et al, 1992) and H. virescens (Sims and Stone, 1991),and one case of negative cross-resistance in I! interpunctella (Van Rie et al., 199Oc),suggest that rotations might slow resistance development in certain situations. However, McGaughey and Beeman (1988) found that high levels of resistance in l? interpunctella were stable for long periods, and in such cases rotations may not be effective.

B. MIXTURES OF TOXINS Mixtures of toxins is also a relatively simple tactic that is possible in both conventional applications and transgenic plants. It is based on the idea that if resistance to each component in a mixture is rare, then individuals with resistance to all components will be exceedingly rare or absent. However, extensive cross-resistance among different Bt toxins may reduce the likelihood that mixtures will effectively control resistance (Gould et al., 1992). Many field populations of l? xylostella evolved resistance to Bt formulations that contain mixtures of up to five toxins (Tabashnik et al., 1990).In laboratory tests, l? interpunctella readily became resistant to a mixture of two Bt strains that contained at least six CryIA, CryIC, and Cry11 toxins (McGaughey and Johnson, 1992). Further research is needed to elucidate the patterns of response of different insect species to Bt mixtures before a suitable recommendation is made that assures prevention of resistance. C. REFUGES Facilitating the survival of susceptible insects is one of the best approaches to slow resistance development. Results from modeling studies demonstrated that refuges and immigration of susceptible insects into pest populations can slow the evolution of resistance (Tabashnik, 1990). This was supported by the results from laboratory experiments on H. virescens and l? xylostella (Gould and Anderson, 1991; Schwartz et al., 1991b). Spatial and temporal employment of refuges and factors affecting their efficacy need to be worked out at the field level. Spatial refuges facilitate random mating between susceptible and resistant adults and may limit movement of larvae between Bt-treated and untreated plants (Mallet and Porter, 1992). Spatial refuges can be provided among tissues within plants by ensuring tissue-specific expression of the Bt gene, among plants within fields by growing transgenic and nontransgenic plants in a defined ratio, or between fields in which neigh-

INSECTICIDAL PROTEINS OF B. t h uringiensis

29

boring fields are sown with plant varieties differing in their susceptibility to a given insect. D. TOXIN DOSES

There are two approaches dealing with high as well as low doses of Bt toxin application to circumvent resistance problems. The low-dose approach includes reduced rates and frequency of application, reduced thoroughness of application, and transgenic plants with low expression of toxin. This tactic aims to reduce populations only slightly or slow larval development to the point that the number of generations per year is reduced or natural enemies are more effective. However, this approach is not practical because farmers and pest managers prefer products that prevent any damage. Denholm and Rowland (1992) advocated a high-dose strategy in conjunction with untreated refuges as a potential means of managing resistance development in transgenic plants. This approach maintains that constitutive and continuous expression of Bt toxins in transgenic plants may be sufficient to kill all of the heterozygotes in a population (McGaughey and Whalon, 1992). This approach is not possible with conventional Bt applications because foliar applications never cover the entire plant and do not persist long enough to achieve "continuous" expression of Bt (Whalon and McGaughey, 1993). A high dose can be defined as that which consistently kills heterozygotes (Whalon and McGaughey, 1993).Determination of this dose is dependent on the genetics of resistance. It would be lowest in cases in which resistance is inherited recessively and highest in cases in which it is completely dominant. Because homozygous-resistant individuals are at a very low frequency early in the evolution of resistance and suitable refuges provide a continuous source of susceptible individuals, this tactic should be quite durable (Whalon and McGaughey, 1993). An extremely high dose or ultrahigh dose is possible where target insects are very sensitive and Bt expression in transgenic plants is very high (1%of total protein). This dose is sufficiently high to kill even homozygous-resistant individuals. However, doubts persist because doses as high as 268 g/liter of a B. thuringiensis subsp. kurstaki formulation could not kill resistant individuals of I? xylostella (Tabashnik et al., 1993). As discussed previously, binding affinity for toxins is a primary mechanism of resistance in I? xylostella. If binding affinity approaches zero, attempts to kill resistant insects with high doses may be futile (Tabashnik, 1994).

30

P. ANANDA KUMAR ET AL.

E. GENEEXPRESSION

Spatial, temporal, and inducible expression of Bt genes in transgenic plants is one of the features of management strategies. Continuous and constitutive expression of Bt genes results in significant selection pressure on pest populations. Tissue-specific (leaf, stem, root, boll, pod, or seed), stage-specific (vegetative or reproductive), and wound-specific promoters are now available that can be employed to rationalize Bt gene expression. Chemical sprays like that of salicylic acid can be used to induce Bt gene expression at will by using suitable promoters (Williams et al., 1992). All these approaches need to be experimentally verified in a thorough manner. Unfortunately, no transgenic plants have been experimentally evaluated with Bt-resistant insects. More work is needed to assess the role of behavior and other biological, ecological, and genetic factors in resistance development to Bt and to Bt transgenic plants. VIII. Epilogue

Both chemical and microbial insecticides are currently used for insect control. Among chemical insecticides organophosphates (Counter, Dyfonate, Lorsban, Thimet, Parathion, and Penncap),pyrethroids (Ambush, Pounce, and Capture),carbamate (Furadan)and others (Asana XL) are used. Although organophosphates and pyrethroids can be effective, careful insect surveillance is required. Applications must be carefully timed to reach certain insect populations before the insects bore into the stalk and other plant organelles, and repeated applications are often necessary. A class of insecticidal proteins, known as 6-endotoxins, are produced as parasporal crystals by B. thuringiensis in nature. These proteins are quite selective in their toxicity to specific organisms. The crystal proteins are typically produced as large protoxins. Following ingestion by a susceptible insect, the protoxin is solubilized in the alkaline insect gut, and then activated by digestive enzymes to yield a smaller protein. The activated protein binds to specific receptors in the insect midgut and brings about cell lysis by formation of pores. Cessation of feeding and death of the insects follow. These naturally occurring insecticidal proteins have been commercially produced and used as insecticides for decades. An extensive body of safety testing and experience supports their lack of toxicity to humans and animals and the absence of adverse effects on nontarget organisms and the environment. Bacillus thuringiensis var. kurstaki (Btk) preparations are registered for use on corn, vegetables, cotton, deciduous nuts, and fruits. As crystalline powder formulations, Btk has been used commercially as an in-

INSECTICIDAL PROTEINS OF B. thuringiensis

31

secticide under the trade name Dipel. Availability of recombinant DNA technology has provided the opportunity of expressing these biocidal proteins in various organisms (Table IV). The production of insect control protein by various crop plants represents a potentially important new option in pest control and an attractive alternative to external application of insecticides. Transgenic plants producing the insecticidal proteins are quite effective in controlling various crop pests, even though only minute quantities are produced (Table V). Plants are being engineered to preferentially express the insect control protein in desired tissues, while minimizing its production in other plant tissues in which it is not needed for control of the target pest. Transgenic plants hold great promise as an important new tool in integrated pest management programs. This technology allows the crop plant to deliver its own means of protection against insect attack. The expected result is a very specific and directed biological control method that is environmentally sound and that can be expected to reduce the need for manual and chemical inputs by the grower. Commercial bioinsecticide formulations are generally ineffective in controlling ECB on corn in which topical applications of the powder do not reach the inside of the plant tissue where the insects bore and feed (Bartels and Hutchison, 1995). Such transgenic crops provide farmers a means of controlling a serious insect pest that is not easily controlled by current chemical pesticides. Other advantages include: (i) reducing the risks associated with environmental spills or misapplication of chemical insecticides; (ii) eliminating unwanted effects on beneficial insect populations (which can be susceptible to conventional chemical applications)-these beneficial insects can, in turn, further reduce the reliance on chemical means of pest control; and (iii) reducing the consumption of fossil fuels required to deliver chemical inputs by machinery. Because of the environmental pollution and associated toxicity with chemical insecticides, biological insect control has a bright future. Various mutant forms of insecticidal proteins with improved biological activity will be created in the future by fusing diverse domains (Hon'ee et al., 1990) and in vitro mutagenesis of genes that codes for these biological agents (Aronson et al., 1995; Rajamohan et al., 1995). Mosquitoes and blackflies are vectors of a multitude of diseases of man and animals through transmission of pathogenic viruses, bacteria, protozoa, and nematodes. At the molecular level, the processed toxin binds to a specific receptor molecule located on the plasma membrane of the susceptible insect midgut. This initial binding could account for the specificity of the toxin. After binding to the receptor, the toxin creates small pores in the gut membrane leading to colloidal-osmotic lysis

32

P. ANANDA KUMAR ET AL. TABLE IV BT GENESEXPRESSED IN VARlOLJS ORGANISMS

Gene

Donor

Recipient plant

Institution

C T

Bt

Clavibacter

Crop genetics

clrIAlal

Bt

Cranberry

University of Wisconsin

Btk

Cotton

Agracetus

Btt

Potato

ARS

Bt

Corn Tobacco Corn Cotton Rapeseed Tobacco

Ciba-Geigy; Monsanto Rohm and Haas; Sandoz Ciba-Geigy; Northrup King Monsanto; Northrup King Agrigenetics Ciba-Geigy; North Carolina State University Campbell; Monsanto; Northrup King; Rogers NK Northrup King

crylAlbl

Btk

Tomato

c~yIA(c)

Btt

Corn

Bt

Corn Cotton

Btk

Potato Rapeseed Amelanchier laevis Apple Brassica aleracea Clavibacter Corn Cotton Poplar Rapeseed Spruce Tobacco Tomato Walnut

CTIB CI~IIA cryIlIA

Monsanto American Cyanamid; Miles; Monsanto Michigan State University University of Georgia Dow University of California/Davis Cornell University Crop Genetics Crop Genetics; Monsanto CalGene; Monsanto; Northrup King University of Wisconsin University of Chicago University of Wisconsin Ca 1Gene Agrigenetics; Campbell; Monsanto ARS; University of California/ Davis

Btt Btk

Potato Potato

ARS Monsanto

Bt

Eggplant Potato Potato Eggplant Potato

Rutgers University Monsanto ARS; Monsanto Rutgers University Frito-Lay: Monsanto

Btk Btt

INSECTICIDAL PROTEINS OF B. thuringiensis

33

TABLE V

BT ENGINEERED CROPSa Crop Corn

Rice Cotton Apple Potato Tomato Eggplant Canola (oilseed rape) Alfalfa Walnut Tobacco Poplar Spruce Cranberry

Companylinstitution Ciba-Geigy;DeKalb: Dow Elanco: Hunt-Wesson; Monsanto: Mycogen; North Carolina State University; Northrup King; Pioneer Hi-Bred; Rogers NK Seed Louisiana State University Agracetus: American Cyanamid; Calgene; Delta and Pine Land: Miles: Monsanto University of California Frito-Lay: Michigan State University: Monsanto; U.S. Department of Agriculture Campbell: Monsanto; Rogers NK Seed; Sandoz Rutgers University AgriGenetics; University of Chicago; University of Georgia Mycogen U.S. Department of Agriculture Agrigenetics; Calgene; Ciba-Geigy; North Carolina State University; Rohm and Haas University of Wisconsin University of Wisconsin University of Wisconsin

11 Since 1987, 14 crops and trees engineered to express the Bt toxin gene have been field tested in the United States by the companies and institutions shown in the table. Source: Applications and notifications submitted since 1987 to the U S . Department of Agriculture to field test genetically engineered plants. Other Bt plants are under development, but have not reached the field test stage.

and kills the larvae rapidly. The receptor for an insecticidal protein of B. thuringiensis has been cloned (Vadlomude et al., 1995). Rajamohan et al., (1995) and Chen et al., (1995) studied the binding of the toxin to the receptor. They showed that the binding is a two-step process in which the irreversible binding is directly correlated to insect toxicity and not the initial binding. The amino acids of CryIAb toxin involved in the irreversible binding to the receptor are F37, and G374 of CryIAb toxin. Rajamohan et al., (1994) also identified the amino acids (365-370) essential for the toxicity of another toxin, CryIAa, to B. mori. They also constructed several mutant toxins that increased toxicity, especially to gypsy moth (a forest pest insect) about 7-10 times more potent than the parental toxin. Hybrid wide-spectrum toxins, by switching the toxicity determining regions of different Cry toxins, may improve toxicity and yield a toxin with multiple insect specificity through protein engineering.

34

P. ANANDA KUMAR ET AL. ACKNOWLEDGMENTS

The authors are grateful to Professor Don Dean for providing the colored photograph and to Dr. Rajamohan for critical reading of the manuscript.

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Microbiological Production of Lactic Acid JOHN

H. LITCHFIELD

Battelle Columbus, Ohio 43201

I. Introduction A. Historical Background B. Chemical and Physical Properties C. Commercial Uses 11. Microorganisms for Lactic Acid Production A. Bacteria B. Molds 111. Lactic Acid Fermentation A. Raw Materials B. Process Systems C. Batch Processes D. Continuous Processes E. Process Kinetics and Modeling Studies IV. Product Recovery and Purification A. Precipitation and Acidification B. Centrifugation and MicrofiltrationKJltrafiltration C. Distillation D. Ion Exchange and Adsorption E. Reverse Osmosis F. Solvent Extraction and Extractive Fermentation V. Process Control A. pHContro1 B. Analytical Methods C. On-Line Control Systems VI. Economics References

I. Introduction

A. HISTORICAL BACKGROUND Why has there been a revival of interest in the microbiological production of lactic acid in the past decade? In the United States, until recently, microbiological production had been abandoned and supplanted by chemical synthesis. In this manner, lactic acid can be manufactured that has sufficient purity at a low enough cost suitable for synthesis of specialty food additives and polymers. With the advent of the Clean Water Act in the United States and promulgation of effluent limitations guidelines by the Environmental Protection Agency and related regulatory actions in other countries, 45 ADVANCES IN APPLlED MICROBIOLOGY,VOLUME 42 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TOHN H. LITCHFIELD

there is interest in the economic utilization of the large quantities of cheese whey produced by the dairy industry each year. In addition, the development of lactic acid-based polymers for specialty medical and environmentally biodegradable product applications also contributes to this revived interest in manufacturing lactic acid by microbiologicalprocesses. Lactic acid, 2-hydroxypropanoic acid or a-hydroxypropanoic acid, served as a food preservative in fermented milks, fruits, vegetables, meats, and breads since ancient times without any understanding of its chemical nature. Its discovery as the “acid of milk” by Carl Wilhelm Scheele in 1780 and subsequent work by Louis Pasteur, Joseph Lister, and Max Delbriick led to the identification of the lactic acid bacteria as the microorganisms involved in lactic acid fermentation (Benninga, 1990). Although early studies on lactic acid production by fermentation were conducted in Europe, the first commercially manufactured form of calcium lactate took place in the United States in 1883 at the Avery Lactate Company, Littleton, Massachusetts. This manufacturing process was based on U.S. patents granted to the founder, Charles E. Avery, and his associates who understood that the process was a microbial fermentation involving the conversion of sugars to lactic acid by a purified culture called “purified lactic ferment” (Benninga, 1990). Using a typical patent assigned to the Avery Lactate Company, Waite (1887) described the fermentation as shown in Table I. This patent mentions that a medium having a low nitrogen content is not as suitable for the production of the “lactic ferment” as a medium containing milk or other organic nitrogen sources. It is now known that the lactic acid bacteria have complex nutritional requirements for amino acids and vitamins that can be supplied by milk or other organic nitrogen sources such

TABLE I AVERY LACTATECOMPANY LACTICACIDFERMENTATION PROCESS^^

Medium composition Glucose (white), cane sugar or starch Calcium carbonate Ammonium sulfate Phosphoric acid “Lactic ferment” Process conditions Temperature Oxygen tension Time “From Waite (1887).

Weight (pounds) 100

50 0.5 0.02

Small amount 40-50°C Air excluded in sealed vessel 12-15 Days

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

47

as casein hydrolysates, yeast extracts, corn steep liquor, and malt sprouts. In addition to fermentation, lactic acid can be manufactured by a chemical synthetic process involving the hydrolysis of lactonitrile formed by the reaction of acetaldehyde with hydrogen cyanide (Benninga, 1990; Holten et al., 1971). Here, discussion is limited to microbiological processes. Numerous reviews of lactic acid fermentation processes have been published over the years and should be consulted for further background information (Atkinsonand Marituna, 1991;Benninga, 1990; Buchta, 1983;Lockwood, 1979; Prescott and Dunn, 1959; Schopmeyer, 1954; Vick Roy, 1985).

B. CHEMICAL AND PHYSICAL PROPERTIES Holton et al. (1971)provide an extensive coverage of the chemical and physical properties of lactic acid. The two optically active isomeric (enantiomeric) forms are designated L(+) or S(+) (dextrorotary) and D(-) or a(-) (levorotary) as shown below. COOH

COOH I HOCH

I

HCOH I

I

CH3 L(+) lactic acid

D(-)

CH3 lactic acid

Racemic DL lactic acid results from chemical synthesis from lactonitrile (Holten et al., 1971) or from D- and L-lactic dehydrogenase activity in some of the lactic acid bacteria (Dennis et a]., 1965). The 19th and early 20th century literature on lactic acid is confusing in that lowercase d and 1 are used to indicate dextrorotation (clockwise) and levorotation (counterclockwise),respectively, of the plane of polarized light by lactic acid solutions without indicating the molecular structure (Benninga, 1990).In this review, the L(+) and D(-) notation for the enantiomeric forms of lactic acid are used. Table I1 summarizes some of the properties of the different lactic acid enantomers. The specific optical rotations of the zinc salts of lactic acid enantiomers are opposite those of the free acids (Holten et al., 1971). Lactic acid readily forms the linear dimer lactoyl lactate and higher linear polymers in aqueous solutions in which the hydroxyl group of one molecule is esterified with the carboxyl group of another. In addition, a cyclic dimer, lactide, can also be formed by prolonged heating at 140°C at low pressures (10 mm) and is reversible by distillation. L, D, and DL lactides can be prepared born L(+) and D(-) lactic acid (Holten et al., 1971). Lactides are suitable monomers for the synthesis of biodegradable poly-

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JOHN H. LITCHFIELD

TABLE I1 SELECTED PROPERTIES OF LACTIC ACIDENANTIOMERS~

Enantiomer and Chemical Abstracts Service Registry No. Property

L(+) 79-35-4

Molecular weight Melting point (“C) Boiling point (“C) Optical rotation [ a ] 0 2 0 , degrees Acid Zinc salt Dissociation constant pK (25°C)

90.08 52.8-53.6

-

10326-41-7

D(-)

DL 598-82-3

90.08 52.8-53.6 103

90.08 16.8-33 82-85

-2.5 +8.18

-

+2.5 -8.2 1.90 x 10-4 3.79

-

3.83

1.38 X 10-4 3.73

acornpiled from Budavari et al. (1989), Holton et al. (19711, and Lockwood et al. (1965)

mers for medical, industrial, and consumer products (Lipinsky and Sinclair, 1986). C. COMMERCIAL USES Food-grade lactic acid meeting Food Chemicals Code I11 (1981) specifications is sold as 50, 80, and 88% USP grades. The 88% purity product is used in food, pharmaceutical, and industrial (including plastics) applications. Currently, the major applications of lactic acid and its compounds are as food additives. Table I11 presents a summary of typical food applications for lactic acid and its salts, which are generally recognized as safe by the Food and Drug Administration (FDA) in the United States (Code of Federal Regulations, 1994).Also shown are food additives formed by reaction of lactic acid with fatty acids such as calcium stearoyl-l-lactylate and sodium stearoyl lactylate and various lactylated fatty acid esters that have been cleared by FDA as dough improvers, emulsifiers, and plasticizers in foods. Lactate esters are also used as flavorings. Industrial (nonfood) applications of lactic acid are quite diverse and only representative examples are cited here. The original envisioned use by the Avery Lactate Company of lactic acid product in the 19th century was a substitute acidulant in baking powder. This application was replaced by applications in mordanting of textiles and in deliming baths in leather tanning (Benninga, 1990).

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

49

TABLE 111 FOODAPPLICATIONS OF LACTICACIDS AND LACTICACIDCOMPOUNDSa Compound (Chemical Abstracts Service Registry No.)

Food application

Code of Federal Regulations reference

L(+) lactic acid (79-35-4) D(-) lactic acid (10326-41-7) DL lactic acid (598-82-3)

Antimicrobial agent, curing and pickling, flavoring, enhancer, adjuvant, pH control, solvent and vehicle

2 1 CFR 184.1061

Calcium lactate (814-80-2)

Flavoring enhancer, firming agent, leavening agent, nutrient supplement, stabilizer, thickener Nutrient supplements and in infant formula

21 CFR 184.1207

Potassium lactate (996-31-6)

Flavor enhancer, flavoring agent, humectant, pH control

2 1 CFR 184.1639

Sodium lactate (72-17-3)

Flavor enhancer, flavoring agent, humectant, pH control, emulsifier

2 1 CFR 184.1768

Calcium stearoyl-2-lactylate

Dough conditioner in bakery products, whipping agent in egg products, conditioning agent in dehydrated potatoes

21 CFR 172.844

Sodium stearoyl lactylate

Dough conditioner, emulsifier, processing aid in baked products; emulsifier, stabilizer processing aid in milk or cream substitutes, snack dips, imitation cheeses, dehydrated potatoes

21 CFR 172.846

Lactylated esters of fatty acids

Emulsifiers, plasticizers, surface active agents in foods

2 1 CFR 172.848

Lactylated fatty acid esters of glycerol and propylene glycol

Emulsifiers, plasticizers, surface active agents in foods

2 1 CFR 172.850

Glycero-lacto esters of fatty acids

Emulsifiers, plasticizers, surface active agents in foods

2 1 CFR 172.852

Ferrous lactate (5905-52-2)

(25-383-997)

21 CFR 184.1311

aCode of Federal Regulations (1994).

Pharmaceutical applications of lactic acid and its compounds include uses as pharmaceutical intermediates particularly optically pure methyl, ethyl, and isopropyl lactate esters for synthesis of chiral molecules. Sodium lactate is used in parenteral and kidney dialysis solutions and calcium and magnesium lactates are used for treating mineral deficiencies (Purac, 1993).

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JOHN H. LITCHFIELD

In industrial applications, alkyl lactate esters, particularly ethyl and butyl lactates, are attractive alternatives to glycol ethers and other solvents owing to their lower toxicities (Bahner, 1994).Ethyl lactate [ethyl ester of L(+) lactic acid] has potential utility as a replacement for chlorinated hydrocarbon solvents in precision metal cleaning in the electronics, aerospace, and semiconductor industries (Hill and Carter, 1993). In cosmetics and toiletries, lactic acid and sodium and calcium lactates provide humectant properties. Acyl lactates prepared from the reaction of lactic acid with fatty acids ranging from capric to stearic acids are effective as emulsifiers, builders, and stabilizers in cosmetics and personal care products (R.I.T.A. Corporation, 1985). Lactic acid has been promoted as an “a-hydroxy acid” in skin care products for improving skin texture and appearance resulting from aging (Smith, 1993). Polylactide can be prepared from the lactide dimer. Also, lactideglycolide copolymers can be synthesized from lactide and the glycolide dimer of glycolic acid. These polymers are biocompatible, biodegradable, and resorbable materials useful in surgical sutures, staples, wound clips, implants, bone plates, and other medical materials (Lipinsky and Sinclair, 1986; Seeley, 1992). There has been increasing concern over the extent of degradability of commercial synthetic polymers in the environment, particularly in solid waste landfills. This need has led to the development of biodegradable lactic acid polymers for applications in food service plasticware and containers, disposable diapers, medical garments, personal hygiene products, and yard waste bags (Ecochem, 1992; O’Marro,1993; Seeley, 1992). Also, lactic and polymers have potential applications as controlled release systems for pesticides and drugs (Lipinsky and Sinclair, 1986). II. Microorganisms for Lactic Acid Production

A. BACTERIA A number of genera and species of bacteria convert carbohydrates to lactic acid. “Bergey’s Manual of Determinative Bacteriology,” 9th edition, classifies the genera of lactic acid bacteria into the following groups, with examples of representative species (Holt et a]., 1994). 1. Group 17-gram-positive cocci: Lactococcus spp., Enterococcus spp., Pedicoccus spp., Saccharococcus sp., Streptococcus spp. 2. Group 18-endospore-forming gram-positive rods and cocci: Bacillus spp., Sporolactobacillus sp. 3. Group 19-regular, nonsporing gram-positive rods: Lactobacillus spp.

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

51

Much of the scientific research on the lactic acid bacteria has been conducted in connection with their use as bacterial starter cultures in manufacturing cultured dairy products (cheeses and fermented milks) and other fermented foods (Gilliland, 1985; Friend and Shahani, 1985; Gasson and deVos, 1994; Harlander, 1992; Sanders et al., 1991). Also, several books cover the lactic acid bacteria and their applications (Foo, 1993; Salminen and Wright, 1993; Wood and Holzapel, 1992). The lactic acid bacteria are facultative anaerobes or microaerophilic from the standpoint of oxygen requirements. Tolerance to low pH (below 5.0) varies widely as is the case with temperature tolerance above 3740°C. Complex nitrogen sources providing amino acids and vitamins are required for growth and acid production (Kandler and Weiss, 1986). Those organisms are classified as (i) homofermentative-producing lactic acid without other metabolic products such as organic acids, alcohol, aldehydes, ketones, and carbon dioxide; or (ii) heterofermentativeproducing lactic acid along with other organic compounds and carbon dioxide. Fermentation of sugars by homofermentative strains takes place through the Embden-Meyerhof pathway with the theoretical conversion of 1mol of glucose to 2 mol of lactic acid. There is a net generation of two molecules of adenosine triphosphate (ATP)from substrate phosphorylation. Also, there is a reduction of two molecules of nicotine adenine dinucleotide to the reduced form that in turn is reoxidized in the reduction of pyruvate to lactic acid by lactic dehydrogenase (Garvie, 1980). Yields of lactic acid from glucose by homofermentative strains are generally 90% or greater of the theoretical yields. From an economic standpoint, homofermentative characteristics are an important criterion in strain selection. Some species of Lactobacilli considered as homofermentative with glucose as the substrate are actually facultatively heterofermative under aerobic conditions. For example, Lactobacillus plantarum produces only lactate from glucose under anaerobic or microaerophilic conditions but produces lactate, acetate, acetoin, and hydrogen peroxide aerobically. This shift in metabolic pattern is associated with increased activities of pyruvate oxidase and acetate kinase and slightly increased activities of a-acetolactate synthase and acetoin dehydrogenase in the presence of oxygen (Tseng and Montville, 1992). Most Lactobacilli that ferment lactose to lactic acid, such as L. delbrueckii subsp. bulgaricus, have lactose permeases and p-galactosidases. These enzymes mediate lactose transport into the cell and hydrolysis to glucose and galactose, respectively. These sugars are then metabolized by conventional pathways. However, L. casei and Lactococcus lactis contain a lactose-phosphoenolpyruvate-dependentphosphotransferase system effecting lactose transport into the cell and phosphorylation to

52

JOHN H. LITCHFIELD

lactose-6-phosphate. The hydrolysis of lactose-6-phosphate to glucose and galactose-6-phosphateis catalyzed by P-D-phosphogalactosidegalactohydrolase (Mercenier et a],, 1994). Some L. casei strains also have pgalactosidase activity. Various bacterial strains products L(+), D(-), or DL lactic acid. The development of specialty polymers from lactic acid mentioned previously has led to increased interest in the production of specific enantiomers. The dimers, LL, LD, and DL, can be used as building blocks of polymers having different physical properties. Table IV presents examples of lacTABLE IV TYPEOF FERMENTATION PATTERN AND LACTIC ACIDENANTIOMER FORMED BY SELECTED LACTIC ACID-PRODUCING BACTERIAAND MOLDS

Organism

Bacteria Bacillus coagulans

Bacillus laevolacticus Lactobacillus amylophilus Lactobacillus amylovorus Lactobacillus casei subsp. casei Lactobacillus delbrueckii subsp. bulgaricus (formerly L. bulgaricus) Lactobacillus helveticus Lactobacillus rhamnosus (formerly L. delbrueckii) Lactococcus lactis subsp. lactis and subsp. cremoris (formerly Streptococcus lactis and S . cremoris)

Type of fermentation pattern Facultative heterofermentative Facultative heterofermentative Homofermentative

Homofermentative

Lactic acid enantiomer

Reference Gibson and Gordon (1974); Nakayama (1983) Gibson and Gordon (1974); deBoer et 01. (1990) Kandler and Weiss (1986); Nakamura and Crowell (1979) Kandler and Weiss (1986)

Facultative heterofermentative Homofermentative

Kandler and Weiss (1986)

Homofermentative

Kandler and Weiss (1986)

Facultative heterofermentative

Collins eta]. (1989); Kandler and Weiss (1986)

Homofermentative

Holt et al. (1994)

Kandler and Weiss (1986)

(continues)

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

53

TABLE IV-Continued

Organism

Bacteria-Contin ued streptococcus faecalis Streptococcus thermophilus Sporolactobacillus in ulinis

Type of fermentation pattern

Lactic acid enantiomer

Reference

Homofermentative

Ohara and Yoshida (1993)

Homofermentative

Holt et al. (1994)

Homofermentative

Holt et al. (1994); Kosaki and Kawai (1985); Shi et al. (1990); Yanagawa (1990)

Molds Rhizopus arrhizus

Heterofermentative

Rhizopus delmar Rhizopus oryzae

Heterofermentative Heterofermentative

Rhizopus stolonifer Rhizopus sp. G-36

Heterofermentative Heterofermentative

Kristofikova et al. (1991); Soccol eta]. (1994) Soccol eta]. (1994) Hang (1989, 1990); Lockwood et al. (1936); Soccol et 01. (1994); Snell and Lowery (1964); Ward et al. (1938a) Soccol eta]. (1994) Homo and Uyeda (1972a,b)

tic acid enantiomers elaborated by selected bacteria of interest for lactic acid production. Strain selection can be based on the desired lactic acid enantiomer, substrate, temperature, pH and lactic acid tolerances, and yields and lactic acid productivities (g/liter/hr). For commercialscale production of L(+) lactic acid from glucose, L. rhamnosus (formerly L. delbrueckii) gives high yields at 45OC; however, this strain does not utilize lactose and cannot be used with cheese whey. Lactobacillus delbrueckii subsp. bulgaricus and L. helveticus produce D(-) and DL lactic acid, respectively, from lactose at 45°C and are suitable for fermenting cheese whey (Kandler and Weiss, 1986). Also, L. helveticus is tolerant to pH values below 5.0. In addition to the usual desirable characteristics for strain selection, resistance to bacteriophage attack is an important consideration. Lactococcus spp. Lactobacillus spp., and Streptococcus spp. are subject to bacteriophage infections that can disrupt fermentations. Klaenhammer and Fitzgerald (1994) discuss the various types of bacteriophage infections and development of phage-resistant strains.

54

JOHN H. LITCHFIELD

Roy et al. (1986) point out that the phages specific to L. delbrueckii subsp. bulgaricus are different from those active against L. helveticus. They conclude that the latter organism could be used as an alternative to the former in lactic acid production from cheese whey permeate in a yogurt or cheese factory environment. In recent years, there have been significant advances in our understanding of the molecular genetics of the lactic acid bacteria that have potential for strain improvement through genetic engineering (Gassonand de Vos, 1994). For example, studies have led to the cloning and sequencing of over 50 Lactobacillus genes (Mercenieret al., (1994). Furthermore,plasmids are present in Lactococcus spp. and many Lactobacillus spp., particularly L. casei, which offers an approach to strain modification through genetic engineeringhecombinant DNA techniques. In contrast to many other Lactobacillus spp., endogenous plasmids have not been found in L. delbrueckii subsp. bulgaricus. Consequently,transformation of this subspecies by extrachromosomal elements has not been successful to date (Mercenier et al., 1994).

B. MOLDS Prescott and Dunn (1959) state that molds in the genera Rhizopus, Mucor, and Monilia produce lactic acid but not those in the genera Aspergillus, Fusarium, or Pencillium. Table IV shows the various Rhizopus spp. that utilize glucose or sucrose aerobically to produce L(+) lactic acid (Goldberg and Stieglitz, 1986; Kristofikova et al., 1991; Lockwood et al., 1936; Musashino Kagaku Kenkyusho, 1994; Soccol et al., 1994; Ward et al., 1938a,b). Also, Rhizopus spp., such as R. arrhizus and R. oryzae, have amylolytic enzyme activity and convert starch or raw materials containing starch to L(+) lactic acid (Hang, 1989, 1990; Kristofikova et al., 1991; Yu and Hang, 1989). These organisms grow and produce lactic acid in media containing inorganic nitrogen sources, such as ammonium salts or nitrates and mineral salts, without supplements of amino acids and vitamins required by the lactic acid bacteria. The aerobic metabolism of glucose by Rhizopus spp. gives a theoretical yield of 1.5 mol of lactic acid per mole of glucose (Margulies and Visiniac, 1961). Ill. Lactic Acid Fermentation

A. RAW MATERIALS

Table V presents the various carbohydrate raw materials that have been investigated for lactic acid production by bacteria and molds. Examples are refined carbohydrates; glucose, sucrose, and starch; complex carbo-

TABLE V FOR THE MICROBIOLOGICAL PRODUCTION OF LACTICACID SELECTED R A W MATERIALS

Raw material

Refined carbohydrates Glucose

Lactose

Sucrose Starch

Microorganism

L. delbrueckii NRRL B-445 (L. rhamnosus) R. arrhizus, R.oryzae, R. stolonifer L. lactis subsp. cremoris L. delbrueckii subsp. bulgaricus (L. bulgaricus) B. coagulans L. delbrueckii (L.rhamnosus) L. amylophilus L. amylovorus L. delbrueckii subsp. bulgaricus L. lactis (S. lactis) and Aspergillus awamori R. arrhizus

Complex carbohydrates and wastes Barley, cassava, corn, oats, rice Cellulose Casein whey Cheese whey

Inskeep et al. (1952) Kristofikova et al. (1991); Snell and Lowery (1964); Soccal eta]. (1994) Nielsen et al. (1991) Venkatesh et al. (1993); Veringa (1994);Voelskow and Sukatsch (1984) Heriban et al. (1993) Benninga (1990) Mercier et al. (1992) Cheng et a]. (1991);Zhang and Cheryan (1994) Aries and Needle (1949) Kurusawa et ul. (1988) Kristofikova et al. (1991)

R. oryzae

Hang (1989);Yu and Hang (1989);Hang

L. delbrueckii and Trichoderma reesei L. delbrueckii subsp. bulgaricus L. delbrueckii subsp. bulgaricus

Abe and Takagi (1991)

(1990)

Lactobacillus casei Cheese whey permeate

Reference

L. delbrueckii subsp. bulgaricus

Burton (1937,1940); Prescott and h n n (1959) Campbell (1953);Keller and Gerhardt (1975); Stieber et a]. (1977) Rincdn et al. (1993); Whittier and Rogers (1931) Cox and MacBean (1977); Mehaia and Cheryan (1986,1987a,b)

L. helveticus

Jerusalem artichokes Molasses, blackstrap

L. rhamnosus; L. lactis Bacillus dextmlacticus

Aeschlimann and von Stockar (1989,1990); Denis et al. (1986);Gatje and Gottschalk (1991); Norton et 01. (1994a,b); Roy et al. (1986,1987a,b) Mulligan et al. (1991) Andersen and Greaves

Lactobacillus sp.

Prescott and Dunn (1959)

(1942)

(continues)

JOHN H. LITCHFIELD

56

TABLE V-Continued Raw material

Microorganism

Reference

Complex carbohydrates and wastes-Continued Molasses, wood, (from pulp, paper, and fiberboard) Municipal solid wastes (acid hydrolyzed) Potatoes

Sulfite waste liquor

Mixed Lactobacilli and yeasts

Griffith and Compere (1977)

Lactobacillus pentosus

McCaskey et al. (1994)

L. delbrueckii NRRL B-445 (L. rhamnosus) Lactobacillus sp. L. pentosus L. pentosus

Cordon et al. (1950)

Pseudomonas putida dehalogenase Methylobacillusflagellaturn

Hasan eta]. (1991)

Leonard et al. (1948)

Organic chemicals m-2-Chloropropionic acid 1,Z-Propanediol

Pseudomonas sp.

Arthrobacter oxydans

Dinarieva and Netrusov (1991) Shigeno and Nakahara (1991);Nakahara et 01. (1992) Yagi and Minoda (1979)

hydrates-cellulose, cereal grains, corn, Jerusalem artichokes, potatoes, and black strap molasses; and waste materials-cheese whey and permeate (ultrafiltrate), municipal solid wastes, sulfite waste liquor, and wood molasses. Also shown in Table V are organic chemical substrates (DL-2-chloropropionic acid and 1,2-propanediol) that can be converted to lactic acid by microorganisms or microbial enzymes. Complex materials may require either costly pretreatment or product recovery and purification processes or both. For example, if the organism does not have amylase activity, starch-containing substrates must be converted by amylolytic enzymes to glucose. For crude cellulosic substrates, physical, chemical, and/or enzyme pretreatment will be required before fermentation. Also, toxic residues, such as furfural and hydroxymethyl furfural, may be produced from pentoses and hexoses by pretreatment processes. In the United States in 1994, approximately 57 x lOg/lb (25.9 xi09 kg) of liquid whey containing 4 4 . 5 % lactose is produced from dairy prod-

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

57

ucts manufacture each year with only about half being converted to 1.88 x 109 lb (8.5 x 108 kg) of derived products such as dry and concentrated whey, whey protein concentrate, and lactose (American Dairy Products Institute, 1995;Yang et al., 1994).Consequently,there has been considerable interest in using cheese whey or cheese whey permeate from ultafiltration processes for lactic acid production by Lactobacillus spp. and Lactococcus spp. that ferment lactose (Denis et al., 1986). In the 1930s, several processes were developed for fermentation of cheese whey to lactic acid using L. delbrueckii subsp. bulgaricus (Burton, 1937, 1940;Whittier and Rogers, 1931).No supplemental nutrients were added to the whey in those processes. Subsequent work demonstrated the need for adding sources of amino acids, vitamins, purine, and pyrimidines depending on the specific requirements of the bacterial strain to obtain improved rates and yields. Typical supplements include corn steep liquor (Amrane and Prigent, 1994; Campbell, 1953), peptone (Acuna et al., 1994; Monteagudo et al., 1993),tryptone (Robison,1988),whey protein hydrolysate (Heriban et al., 1993; Leh and Charles, 1989b,c; Lund et al., 1992), and yeast extract (Aeschlimann and von Stockar, 1989, 1990; de Boer et al., 1990; Montelongo et al., 1993).Concentrations of these nutrient supplements vary widely and typically range from 0.5 to 3%. Enzyme-hydrolyzed whey protein supplementation of cheese whey permeate at levels up to 75% markedly improved lactic acid concentrations and productivities in batch fermentations with L. delbrueckii subsp. bulgaricus (Leh and Charles, 1989a,b,c).Similar results were obtained using whey protein hydrolysate alone with L. helveticus (Amrane and Prigent, 1994). Bailey et al. (1987, 1988) pointed out that fouling of heat-transfer surfaces can result from precipitation of residual proteins in cheese whey permeate during heat sterilization. They treated the cheese whey permeate medium with an acid protease at pH 4.0 to degrade residual proteins to peptides and amino acids. These hydrolysis products were readily utilized by L. casei without further supplementation with corn steep liquor or yeast extract. Liquid whey and permeate are not stable from a microbiological standpoint. Refrigerated transport and storage are necessary, which leads to problems resulting from the low water solubility of lactose that crystallizes out of the whey at low temperatures. Owing to this low solubility of lactose and dilute nature of liquid whey or permeate (7% solids), it is not economically feasible to collect and transport them from numerous widely dispersed cheese plants to a single fermentation plant. Ideally, a lactic acid fermentation plant should be located adjacent to a cheese

58

JOHN H. LITCHFIELD

manufacturing plant that discharges sufficient quantities of cheese whey throughout the year to permit economically viable operation. Glucose or sucrose are generally the preferred raw materials for cornmercial-scale lactic acid fermentation processes on the basis of cost, availability, purity, and ease of product recovery (Benninga, 1990). In the United States, glucose obtained from acid- or enzyme-hydrolyzed cornstarch produced in corn wet milling has been the usual raw material for commercial-scale processes. Producers in other countries, particularly in Europe and Brazil, have used sucrose (cane or beet sugar) on the basis of cost and availability in these regions. However, international sugar prices may fluctuate widely and have a severe impact on the economics of lactic acid production processes based on sucrose. Currently in the United States, Archer Daniels Midland's (ADM) fermentation plant at Decatur, Illinois produces lactic acid using glucose (dextrose) obtained from enzyme conversion of starch. Capacity is reported as 20-40 million lbs (9.1-18.2 million kg) per year but no details have been released on this process (ADM Corn Processing, 1993; Anonymous, 1992a, 1993a,b). Starch produced by corn wet milling is a potentially interesting raw material on the basis of cost and availability at large corn wet milling plants in the United States. Laboratory-scale fermentations have been reported for lactic acid production from starch by L. amylophilus (Mercier et al., 1992),L. amylovorus (Cheng et al., 1991; Zhang and Cheryan, 1991, 1994);Lactococcus lactis combined with Aspergillus awamorii (Kurasawa et al., 1988),and R. arrhizus (Kristofikova et al., 1991). However, none of these processes has been scaled up at the present time. B. PROCESS SYSTEMS

Factors affecting lactic acid concentrations, productivities, and yields include types of process (batch, fed-batch, or continuous), microorganism, strain, inoculum size and nutritional state, temperature, pH, substrate concentration and pretreatment, the presence of competing contaminants in nonsterile systems, the presence of bacteriophages, and inhibition by lactic acid with increasing concentrations and by toxic products from substrate pretreatment such as furfural and hydroxymethylfurfural. In addition, aeration is an important factor for aerobic Rhizopus spp.-based processes. Process control including pH control will be covered in a subsequent section. Table VI presents process conditions and lactic acid concentrations, productivities, and yields for typical laboratory, pilot plant, and commercial-scale processes involving a variety of organisms and substrates.

TABLE VI LACTIC ACID PRODUCTION BY BATCH AM) CONTINUOUS FREE-CELL FERMENTATIONS

Organism

and type of fermentation

Bacteria B. coagulans, batch B. laevolacticus, continuous

L. amylophilus,

Raw materials and concentrations (g/liter)

Sucrose, 120 Glucose, 50; yeast extract, 5 Glucose, 20

batch corn, 45

L. amylovorus, batch

Lactobacillus casei, batch

L. casei, Lactobacillus delbrueckii subsp.

Starch, enzyme thinned, 100; yeast extract, 3 Same, 20 Cheese whey peptone, 10; yeast extract, 30 Cheese whey

Fermentation conditions

5-liter fermenter, 55"C, pH 6.5, 34-48 hr 170-ml fermenter (gas lift), 30°C, pH 6.0 D,a 0.5 hr-l 5-liter fermenter, 30°C. pH 6.0, 24 hr 1-liter fermenter, 3OoC, pH 6.0, 72 hr 1.2-liter fermenter, 40%, pH 6.0,48 hr

Lactic acid productivity (P: g/liter/hr) and, concentration (C: g/liter)

Yield (g/g substrate, utilized)

P: 2.50;

0.92

c : 3.5 P: 13

0.94

P: 1.56;

0.93

c: 2 1

Reference

Heriban et al. (1993) de Boer et al. (1990) Mercier et al. (1992)

P: 0.55;

0.68

C: 31.2 P: 7.36; C: 120.7

0.94

Cheng et al. (1991) Zhang and Cheryan (1991) Rincon et al. (1990)

Z-liter fermenter, 40"C, pH 5.5, 25 hr 5-liter fermenter, 38"C, pH 5.4,30 hr

P: 5-6 C: 96.2 P: 2.1

0.90

2000-liter fermenter, 43"C, pH 5-5.8, 72 hr

P: 2-2.5

0.90

-

Whittier and Rogers (1931)

bulgaricus, continuous

(continues]

TABLE VI-Continued

Organism and type of fermentation Bacteria-Confin ued L. delbrueckii subsp. bulgaricus, batch

Q,

0

L. delbrueckii subsp. bulgaricus Semicontinuous Continuous L. delbrueckii NRRL B-445 (L.rhamnosus) Batch

Raw materials and concentrations (glliter) Cheese whey, 60; peptone, 5; yeast extract, 1 Cheese whey corn steep, liquor Cheese whey permeate, 150 Cheese whey (lactose, 50) corn steep liquor

Fermentation conditions

Lactic acid productivity (P: g/liter/hr) and, concentration ( C g/liter)

Yield (g/g substrate,

utilized)

Reference cuna et 01.

1.5-liter fermenter, 44"C, pH 5.8

C: 28

5000-gallon (189272), fermenter, 43.3"C, pH 5-6,24 hr 1-or 2-liter fermenter, 45"C, pH 5 . 6 , l l hr 9.5-liter fermenter, 43"C, pH 5.5

-

0.85-0.90

Campbell (1953)

P: 4.4; C: 115 P: 5; c: 60

0.99

Mehaia and Cheryan (1987a) Reddy et 01. (1976)

P 3.69-5.26

0.93

Sanchez-Podlech et al. (1990); Stein et al. (1991)

P: 1.77; c: 55

0.98

Keller and Gerhardt (1975)

C: 120-135

0.80-0.90

Inskeep et al. (1952)

Cheese whey (lactose, 43-50) Cheese whey (lactose, 50)

15-liter fermenter, 45%. pH 5.6 14-liter fermenter, 2 in series, 44OC, pH 5.5

Glucose, 150; malt sprouts, 0.375

30,000-gallon (113,562-liter) fermenter (9085 liters of medium), 48.9"C, pH 5.8-6.0,96-144 hr

0.87

(1994)

0.91

Continuous

Glucose, 50; yeast extract, 30

L. delbrueckii AICC 53197 (L. rhamnosus), batch L. helveticus

Glucose, 200; tryptone , yeast extract Cheese whey permeate (lactose, 39.2) yeast extract Cheese whey permeate, 54; yeast extract, 4 Cheese whey permeate, 120240 (threefold concentrate); yeast extract, 5 Cheese whey permeate, 60; yeast extract, 10

Continuous

OY CI

Continuous, two stages

Luctococcus Jactis subsp. cremoris 2487 1. Batch 2. Continuous

Cheese whey permeate, 65; yeast extract, 5

1.4-liter fermenter, 92"C, pH 6.0 D, 0.350.40 hr' 1-liter fermenter, 45%, pH 6.3,168-216 hr

P: 8.93; C: 22-25

0.74

Major and Bull (1985)

C: 14.05

0.94

Robison (1988)

400-ml Fermenter, (working volume). 42"C, pH 5.9,12 hr

P: 3.7; c : 35

0.93

Roy et al. (1986)

1.5-liter fermenter (working volume), 42%, pH 5.5, 7.5 hr Same as above D, 0.15-', 73 hr

P: 3.8; C: 40

Fermenters 1.Stage 1,1.5 liters 2. Stage 2, 2.5 liters 42%, pH 5.5,28 days

1.P: 8.27;

0.98

2. P: 7.64; C 41 1&2 combined P: 6.36 1.P: 3.41

Aeschlimann and von Stockar (1990)

0.86

Mulligan et al., (1991)

2. P: 4.38

0.91

Mulligan et aJ. (1991)

1.2.5-liter fermenter (2.0-liter working volume), 35%, pH 6.5 2. Three stage, 400-ml working volume/stage, 35"C, pH 6.1

P: 5;

0.83

C: 84.6

c:43.7

Aeschlimann and von Stockar (1989) Aeschlimann and von Stockar (1989)

~

~~

(continues)

TABLE VI-Continued ~~~~~~~~~~~

Organism and type of fermentation

Bacteria-Continued Lactococcus lactis subsp. loctis 9085, batch Molds Rhizopus R-41, batch R. arrhizus 8109, batch R. oryzae, batch

R. oryzae, batch

Fermentation conditions

Cheese whey permeate, 65; yeast extract, 5

3.5-liter fermenter (2.0-liter working volume), 30°C, pH 5.8

P 1.09

0.85

Mulligan et 01. (1991)

Glucose, 100

C: 78

>0.50b

KristoEkova et d. (1991)

Glucose, 100

7-liter fermenter (4.2liter,working volume), 28"C, pH 6, aeration 0.6 (v/v) min, 32 hr Same, 32 hr

c : 79

>0.50b

Glucose, 130

500-gallon (1893-liter)

c: 93

0.72

Kristofikova et al. (1991) Snell and Lowery,

1. Corn, 150 2. Barley, cassava,

~

*

Yield (g/g substrate, utilized)

Raw materials and concentrations (g/liter)

corn, oats, or rice, 100 a

Lactic acid productivity (P: g/liter/hr) and, concentration (C: glliter)

fermenter (1514-liter working volume), 37°C. 48 hr to 44%, 56 hr, pH ~ 6 . 0 , aeration 0.17 (v/v] min 500-ml Erlenmeyer flasks, 100 ml, medium, 30°C, 96 hr

~~~~~~~~~

D, distribution rate. Values based on theoretical aerobic conversion of 1 mol glucose to 1 . 5 mol lactic acid.

Reference

(1964)

1.C: 53.2 2. -

1.0.44 2.0.230.74

Hang (1989) YU and Hang (1989)

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

63

On a commercial scale, lactic acid is manufactured by batch fermentation processes on the basis of ease of process control. Although considerable research has been conducted on continuous processes to achieve improved productivities, such processes have not been reported to be practiced on a commercial scale. 1. Equipment

During the 19th and early 20th centuries, wood fermenters were used for commercial production. However, wood deteriorates severely over time as a result of calcium lactate penetration into the pore structure. Also, cracks in wood fermenters are difficult to sanitize and provide harborage for contaminating organisms (Benninga, 1990; Schopmeyer, 1954). Owing to the corrosive nature of lactic acid, 316 low-carbon stainless steel is preferred for fermenter construction. 2. Sterilization

In most commercial-scale fermentations, clean, aseptic, but nonsterile conditions are employed with lactic acid bacteria such as L. rhamROSUS (formerly L. delbrueckii) that grow above 45°C. The fermenters are cleaned, steamed, and may be treated with disinfectants for boiling water to minimize contamination. With R. oryzae, temperatures and pH values are approximately 35OC and 5 or 6, respectively. In this case, the medium and fermenter must be sterilized to prevent the growth of bacterial, yeast, or mold contaminants. In any case, contamination with extraneous microorganisms can be a problem in commercial-scale fermentations. Some of the results reported in the older literature may have involved mixed Lactobacillus spp. and not pure cultures. Also, contamination by the butyric acid bacterium Clostadium butyricum has been a problem in nonsterile Lactobacillus fermentations under anaerobic conditions (Benninga, 1990). 3 Temperature Control, Agitation, and Aeration Temperature can be controlled by circulating water at the desired temperature for the organism used through stainless steel fermenter coils. Conventional lactic acid bacterial fermentations are “free-cell”in that the cells can be maintained in suspension in the production medium by mixing. Examples in pilot plant and commercial-scale processes include pump circulation from the bottom to the top of the fermenter (Inskeep et a]., 1952) or by mechanical agitators (Burton, 1937; Campbell, 1953). Mechanically agitated small-scale fermenters (20 liters or less) are widely used in laboratory-scale investigations (Table VI).

64

JOHN H. LITCHFIELD

Most strains of the lactobacilli are microaerophilic and some oxygen is required for growth. However, they may vary in their sensitivity to oxygenation by aeration. Kemp and West (1959) reported that L. delbrueckii NRRL B-445 (now L. rhamnosus) was relatively insensitive to oxygen concentration in the medium. However, as mentioned previously, facultatively heterotrophic species may shift from homofermentation to heterofermentation with increasing dissolved oxygen concentrations (Thomas et al., 1979; Tseng and Montville, 1992). The R. oryzae fermentation is aerobic and combined agitation and aeration is employed in fermenters (Snell and Lowery, 1964). If the culture becomes oxygen limited, ethanol is produced (Soccol et al., 1994). 4. Inoculum Development

Relatively little published information exists on inoculum sizes for pilot plant or commercial-scale lactic acid fermentations. In the former American Maize Products Co. process, the L. rhamnosus (formerly L. delbrueckii) inoculum was developed in two stages. A 375-gallon (1420-liter) culture volume was used to inoculate 600 gallons (17413 liters) of medium in a second stage, three of which were added to the production fermenter medium to give a final volume of 24000 gallons (90850 liters) (Inskeep et al., 1952). In all cases, the inoculum medium had the same composition as the production medium. However, no information was provided on cell concentrations in the inoculum. In the Miles Laboratories patent using R. oryzae, Snell and Lowery (1964) gave an example of 28 liters of germination medium containing 1.62 x 108 sporedliter as an inoculum for 1514 liters of production medium. With cereals or cassava as raw materials in a laboratory-scale fermentation, Yu and Hang (1989) found that increasing the inoculum size of R. oryzae spores from 1x 107 to 3 x 107 per 100 ml of medium did not give increased lactic acid production. In laboratory-scale studies, with lactic acid bacteria, typical initial cell counts are in the 105-107 colony-forming unit (cfu)/ml range. For example, with a cheese whey permeate medium, Roy et al. (1986) reported initial L. helveticus counts of 106-107 cfu/ml. Mehaia and Cheryan (1987a), with this same type of medium, used initial L. delbrueckii subsp. bulgaricus concentrations of 1or 2 g/liter (dry weight basis) corresponding to lo5-lo7 cfu/ml. Inoculum size had a significant effect on lactic acid concentrations and yields obtained with L. casei (Hujanen and Linko, 1994; Siimes et al., 1992a),L. delbrueckii subsp. bulgaricus (Borzani et al., 1993),L. helveticus (Chiarini et al., 1992),and L. plantarum (Lievense et al., 1990). In addition, the nutrient content of the inoculum medium has an important

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

65

influence on lactic acid production. Amrane and Prigent (1994) observed the most rapid production of lactic acid by L. helveticus on batch fermentation of cheese whey permeate supplemented with suitable peptides when the inoculum was grown in a medium containing yeast extract as a source of vitamins, purine, and pyrimidines. Nakajima et al. (1994) concluded that L. casei produced a “growth factor” in the inoculum medium that was utilized during growth in the production medium. However, they did not determine the identity of this growth factor. C. BATCHPROCESSES

Table VI shows typical conditions, lactic acid productivities and concentrations, and yields based on substrate utilization obtained in batch free-cell fermentations ranging from laboratory to commercial scales. In the former American Maize commercial-scale process, lactic acid concentrations reached 120-135 g/liter from 150 g/liter glucose with yields in the range of 0.80 to 0.90 of lactic acid/g substrate utilized (Inskeep et al., 1952). In this case, the lactate concentrations on the 96- to 144-hr fermentation were limited by the solubility of the calcium lactate formed. With R. oryzae, in the Miles Laboratories process a concentration of 93 g/liter was obtained from 130 g/liter glucose with a yield of 0.72 compared with a theoretical yield of 0.74 (Snell and Lowery, 1964). Only a few laboratory-scale batch bacterial fermentations reported in the scientific literature achieved lactic acid concentrations above 100 g/liter owing to the relatively low substrate concentrations used, generally in the range of 2 to 10% (Table VI). GonCalves et al. (1991) obtained 14 % lactic acid from an initial 20% glucose concentration in a batch fermentation with L. delbrueckii NRRL B445 (L. rhamnosus). With 15% cheese whey permeate, Mehaia and Cheryan (1987a) reported 11.5% lactic acid with L. delbrueckii subsp. bulgaricus. A 1 2 % lactic acid concentration was obtained using L. amylovorus and a medium containing 10% enzyme-thinned starch and 3% yeast extract (Cheng et al., 1991). In this case, the yield of 1.20 based on apparent starch utilization was adjusted to 0.94 to reflect the addition of water to starch during hydrolysis and the utilization of yeast extract as an additional carbon source. This observation of a yield above 100% points out the importance of examining yields reported in the literature to determine if they include utilization of supplemental nutrients in addition to the primary substrate. Endproduct inhibition occurs during lactic acid fermentations with increasing lactate concentrations. This effect has been observed in fermentations with various Lactobacillus spp. (Friedman and Gaden, 1970; Gatje and Gottschalk, 1991), Streptococcus faecalis (Ohara, et al., 1992a,b,c),

66

JOHN H. LITCHFIELD

and L. lactis subsp. cremoris ( S . crernoris) (Bibal et al., 1989). This inhibition is a function of increasing concentrations of undissociated lactic acid and no pH decrease during the fermentation (Gatje and Gottschalk, 1991). Furthermore, Gonqalves et al. (1991) observed that increasing glucose concentrations inhibited growth of and lactic acid production by L. delbrueckii NRRL B 445 (L. rhamnosus). D. CONTINUOUS PROCESSES

Productivities of batch fermentations with lactic acid bacteria are generally lower than desirable from an economic standpoint. Consequently, there has been considerable research on continuous fermentation processes to improve system productivities (Aeschlimann et al., 1990; Aeschlimann and von Stockar, 1989, 1991; de Boer et al., 1990; Keller and Gerhardt, 1975; Major and Bull, 1985; Mulligan et al., 1991; Shindo et al., 1989; Whittier and Rogers, 1931). 1. Continuous Stirred Tank Reactor Systems (CSTR)

Table VI also shows some examples of continuous lactic acid free-cell fermentation processes using various organisms and raw materials. Whittier and Rogers (1931) described a process for continuous fermentation of cheese whey with L. casei or L. delbrueckii subsp. bulgaricus. However, the productivity was low, although they obtained a yield of 0.90. Hanson and Tsao (1972) investigated the continuous fermentation of glucose to lactic acid by L. delbrueckii NRRL B-445 (L. rharnnosus) in CSTR compared with batch fermentation. A maximum lactic acid yield of 0.90 was obtained at pH 5.85 in batch fermentations, but there was no dependence of yield on pH in continuous fermentations. In another investigation of continuous fermentation of glucose by L. delbrueckii NRRL B-445, Major and Bull (1985) achieved maximum lactic acid and biomass productivities of 8.93 and 1.40 g/liter/hr, respectively, at dilution rates (D) between 0.35 and 0.40 h r l . Lactic acid and biomass yields were constant over a wide range of D values (0.05-0.50 hr') . Cheese whey has been converted to a feedstuff enriched in nitrogen content for feeding ruminant animals by fermentation of the lactose in whey by lactic acid bacteria. The lactic acid is neutralized with ammonia to form ammonium lactate (Keller and Gerhardt, 1975; Gerhardt and Reddy, 1979). Also, ammonium lactate can be used as a feedstock for producing lactic acid and its various salts. Keller and Gerhardt (1975) used a two-stage continuous fermentation of cottage cheese (acid)whey by L. delbrueckii subsp. bulgaricus with pH

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

67

control by ammonium hydroxide to produce an ammonium lactateenriched product as a ruminant feed supplement. The residual lactose was reduced to less than 0.1% in this system at pH 5.5 and a 31-hr retention time compared with 0.7% in a single-stage system with a 15-hr retention time. Ammonium lactate was produced by batch and continuous fermentation of cheese whey permeate with S . cremoris (L. lactis subsp. cremoris) (Mulligan et al., 1991). With three CSTRs in tandem, 1.6- and 1.3-fold increases in productivities were achieved with a 7.5-hr retention time over single-stage and two-stage CSTRs, respectively. However, a process cost analysis indicated that the cost of the yeast extract (Amberex 1003) supplement was 31.7% of the final product cost, which made this product uncompetitive with soybean meal on an equivalent crude protein basis. Continuous fermentation of cheese whey permeate supplemented with skim milk and yeast extract by L. helveticus at a D value of 0.15 h r l gave a lactic acid productivity of 5 g/liter/hr compared with a batch value of 3.5 g/liter/hr (Aeschlimann and von Stockar, 1989).A two-stage system of two chemostats in series resulted in a combined system productivity of 6.36 g/liter/hr at a system D value of 0.20 h r l with a 50% reduction in residence time over that of the single-stage chemostat (Aeschlimann et al., 1990). Semicontinuous fermentations were conducted on cheese whey supplemented by hydrolyzed milk and vitamins by L. delbrueckii subsp. bulgaricus. In this process, 10-80% of the volume of the medium was added as the inoculum for the next fermentation cycle and the balance made up with fresh medium (Sanchez-Podlechet al., 1990; Stein et al., 1991). Productivities increased with increasing volume fractions of inoculum for a given number of fermentation cycles, with a typical value of about 5 g/liter/hr for 40-50% inoculum addition and 5-9 fermentation cycles. An important factor affecting lactic acid productivity is the maximum cell concentration in cfu/ml or equivalent g (dry weight)/liter reached during the growth phase of a batch fermentation. For the Lactobacilli, lactic acid accumulation limits the cell concentration to 1010 cfu/ml even when the pH is maintained near the optimum value by continuous neutralization of lactic acid (Hayakawa et al., 1990). Changes in cell morphology including elongation may occur during batch and continuous lactic acid fermentations, which make estimation of cell populations by cell counts problematical. Examples include L. delbrueckii subsp. bulgaricus (Rhee and Pack, 1980; Stein et al., 1989) and L. helveticus (Aeschlimann and von Stockar, 1990; Norton et al., 1993; Roy et al., 1987a). Optical density measurement of biomass

68

JOHN H. LITCHFIELD

may be an alternative to cell counts if extraneous solids in the medium can be separated from the cells during sampling. The productivities of lactic acid fermentations can be improved by using continuous bioreactor systems that allow higher cell concentrations than obtainable free-cell conventional batch and continuous bioreactors. These systems include the following types: 1. Membrane recycle bioreactors in which a CSTR is coupled with a flat, hollow fiber or crossflow membrane unit in a semiclosed loop providing for cell and lactic acid separation and recycle of the cells to the bioreactor (Aeschlimann and von Stockar, 1991; Bailey et al., 1987, 1988; Boyaval et al., 1987; Hayakawa et al., 1990; Hjorleifsdottir et al., 1990,1991;Major and Bull, 1989;Ohleyer et al., 1985a,b; Taniguchi et al., 1987; Vick Roy et al., 1982, 1983). 2. Tubular bioreactors providing turbulent flow and gradients along the direction of flow with decreasing substrate concentration as product formation increases (Kulozik et al., 1992); 3. Immobilized cell bioreactors in which cells are entrapped in ceramic, glass, polymeric, or membrane solid supports in packed columns or in fluidized beds (Boyaval and Goulet, 1988; Demirci et al., 1993; Dinarieva and Netrosov, 1991; Groboillot et al., 1993; Gonqalves et al., 1992; Guoqiang et al., 1991; Hang et al., 1989; Krischke et al., 1991; Kurosawa et al., 1988; Norton et al., 1994a,b; Ohara et al., 1993b; Roukas and Kotzekidou, 1991; Roy et al., 1987a; Stenroos et al., 1982; Tamada et al., 1992; Tipayang and Kozaki, 1982; Tuli et al., 1985). 4. Aggregated cell systems in which culture conditions are adjusted to form aggregated bacteria in a gas lift-type bioreactor (deBoer et al., 1990). 2. Continuous Membrane Cell Recycle Systems

Table VII shows the results obtained with typical continuous membrane cell recycle bioreactor systems. The beneficial effects of high cell concentrations on lactic acid productivity obtained with this type of bioreactor are apparent from the results of Vick Roy et al. (1983). A CSTR with a 100,000 molecular weight cutoff (MWCO) ultrafiltration unit gave a lactic acid concentration and productivity of 35 g/liter and 76-g/liter/hr, respectively, from glucose at a L. delbruecki NRRL cell concentration of 54 g (dry wt)/liter). Lactic acid and cell mass yields (g/g glucose) were 0.96 and 0.09 compared with batch fermentation values of 0.90 and 0.16, respectively. With this same strain, Major and Bull (1989) observed a shift toward decreasing molar ratios of 1actate:ethanol plus acetate as a result of glucose limitation in a CSTR-hollow fiber membrane system. There were

TABLE VII MEMBRANE RECYCLE BIOREACTOR SYSTEMS FOR LACTICACID PRODUCTION

Organism

Type of membrane

L. amylovorus

Hollow fiber

L. casei

Cross-flow

L. delbrueckii subsp. bulgaricus

Hollow fiber Hollow fiber

L. delbrueckii (L. rhamnosus)

Substrate Starch (liquefied) Cheese whey permeate Cheese whey permeate Cheese whey permeate Glucose

NR, not reported.

c: 43

Hollow fiber

Glucose

Ultrafiltration with electrodialysis Ultrafiltration system Filter bed bioreactor

Cheese whey permeate Cheese whey permeate Glucose

P: 15.8; C: 15 P: 21.2;

Flat sheet

Glucose

S. faecalis

P: 8.4; c:42 P: 12; C: 48.3 P: 85; P: 84; c : 117 P: 65; c : 59 P: 38; C: 40 P: 160; c : 57 P: 10.112.1; C: 34-40 P: 22; C: 85

Lactose

L. helveticus

Lactic acid productivity (P:g/liter/hr) and concentration (C:g/liter)

c:10

Yield (g/g) substrate utilized) 0.88-0.92 >0.99 0.99 0.99 0.95 0.59 0.98

Reference Zhang and Cberyan (1994) Bailey et al. (1987, 1988) Mehaia and Cheryan (1986) Mehaia and Cheryan (1987a) Ohleyer et al., (1985a) Ohleyer et al. (1985a) Ohleyer et al. (1985a)

0.99

Major and Bull (1989)

0.81

Boyaval et al. (1987)

0.70

Aeschlimann and von Stockar (1991) Ohara et al. (1993b)

NRO

70

JOHN H. LITCHFIELD

higher lactate productivities with this system (10.1-12.0 g/liter) depending on recycle ratio than with the chemostat (CSTR) (8.3 g/liter/hr). Mehaia and Cheryan (1987a,b),obtained a lactic acid productivity of 84.2 g/liter/hr with a hollow fiber membrane system from cheese whey permeate at a L. delbrueckii subsp. bulgaricus concentration of 63 g/liter compared with 5.5 g/liter/hr at a cell concentration of 7 g/liter for the batch process. Subsequently, Zhang and Cheryan (1994) used this same type of continuous-membrane bioreactor for converting starch to lactic acid by L. arnylovorus. There were no differences in productivities with membranes of 30,000 and 500,000 MWCO. Consequently, they concluded that large pore microfiltration membranes could be used giving a higher flux in liters per square meter per hour (liters/mz/hr) and reducing capital and operating costs. Boyaval et d. (1987) coupled a continuous bioreactor with an ultrafiltration module (20,000 MWCO) and an electrodialysis unit for improving productivity and product concentrating cheese whey permeate fermentation by L. helveticus. They reported a lactic acid productivity of 22 g/liter and an outlet concentration of 85 k 5 g/liter with a D value of 0.88 hr-1 and a cell concentration of 64 g (dry wt)/liter. Also, with cheese whey permeate supplemented with yeast extract, Aeschlimann and von Stocker (1991) used a continuous-membrane bioreactor with a 100,000 MWCO to increase lactic acid productivities with L. helveticus over batch values. They found that 60% of the yeast extract in the medium could be replaced by skim milk with only a 25% decrease in productivity. In patents assigned to Engenics, Inc., Bailey et d. (1987, 1988) described a membrane cell recycle bioreactor process in which the cell mass concentration of L. casei in the reactor was maintained at 60 g/liter giving a lactic acid productivity of 12 g/liter/hr from the proteasetreated cheese whey permeate feed. They stated that any commercially available crossflow microfiltration or high MWCO ultrafiltration systems could be used without affecting the conversion of cheese whey permeate to lactic acid. Polymeric membranes are not resistant to the temperatures required for heat sterilization. Ultimately, they become fouled with small particles and require cleaning under conditions leading to weakening or failure of the membrane structure. Tanaguchi et al. (1987) used a heat-sterilized ceramic crossflow microfilter (0.2m pore size) in a continuous membrane bioreactor and obtained concentrations of L. casei and S. cremoris (L. lactis subsp. crernoris)with lactose as the substrate of 49.0 and 81.5 g (drywt)/liter, respectively. With

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

71

a sterilizable sintered carbon-zirconium oxide membrane (150,000 MWCO) module in the bioreactor system, Hayakawa et al. (1990) maintained a L. casei concentration of 40 g (dry wt)/liter (101' cfu/ml). The advantage claimed for this filter system is that particles that plug the membranes can be burned out in a combustion furnace without affecting the flux. 3. Tubular Bioreactors

According to Kulozik et al. (1992), a continuous tubular bioreactor for lactic acid production having plug flow and operated at high cell densities with cell recycle avoids the problem of lactic acid inhibition. By simulating the tubular bioreactor with a CSTR cascade (seven effects), a lactic acid concentration of 68 g/liter was obtained with a biomass concentration of 2 1 g (dry wt)/liter in 90 min of operation.

Immobilized Cell Systems Immobilization of bacteria and molds by entrapment in polymeric materials or attachment as biofilms on solid supports has been investigated for improving lactic acid productivities over those of free-cell processes. Cell immobilization avoids the need to separate cells from the fermentation medium and avoids membrane fouling encountered in recycle systems. Also, immobilized Lactobaccilus systems have been operated on a recycle basis for extended periods up to 157 days (Linko, 1985; Stenroos et a]., 1982). Table VIII summarizes typical studies of lactic acid production by immobilized cell systems. A number of investigators described the use of entrapment of living cells in polymeric beads including agar (Tuli et al., 1985),calcium or sodium alginates (Boyaval and Goulet, 1988;Gouqaing et al., 1991; Hang et al., 1989; Kurosawa et al. 1988; Roy et a]., 1987a; Roukas and Kutzekidou, 1991; Stenroos et al., 1982), carrageenan combined with locust bean gum (Audet et al,, 1988; Lacroix et al., 1990; Norton et al., 1994b), and polyacrylamide or polyvinyl alcohol gels (Mitsubishi Chemical Industries, 1982). However, with entrapment, lactic acid may soften materials, such as calcium alginate, leading to leakage of cells into the medium as a result of growth within beads. This softening and leakage can also result in plugging packed column reactors. Also, entrapped cells in the interior of polymer beads may not receive adequate amounts of nutrients for optimum metabolic activity. Any crosslinking agents used for immobilization must be nontoxic to the cells. Entrapment immobilization systems using natural polymers, such as agar, alginates, and carrageenan, are difficult to sterilize and maintain 4.

TABLE VIII IMMOBILIZED CELLSYSTEMS FOR LACTICAcn, PRODUCTION ~~

Organism Pore cultures L. casei

Type of bioreactor and immobilization support Continuous stirred tank (none) Fluidized bed, sintered glass beads

u

Substrate and concentration (glliter) Cheese whey permeate (lactose, 40) Cheese whey permeate (lactose, 40)

N

Stirred tank, alginate beads Stirred tank, polyacrylamide beads Stirred tank, agar beads

L. delbrueckii (L. rhamnosus)

Packed bed column, sodium alginate beads Biparticle fluidized bed, column, recycle carregeenan beads, activated carbon, or polyvinylpyridine beads

Glucose, 30 Cheese whey permeate (lactose, 45) Cheese whey permeate (lactose, 45) Glucose, 48 Glucose. 50

Lactic acid productivity (P: g/liter/br) and concentration ( C g/liter)

Yield

(Pk substrate utilized)

Reference

P: 5.5

1.00

Kritschke et 01. (1991)

1. D=0.4; P: 10.0 2. D=1.0;

0.93

Kritschke et al. (1991)

0.50

P: 13.5 P: 1.60

0.9w.99

P: 0.54; C: 26

0.90

Guoqiang et al. (1991) Tuli ef al. (1985)

P 0.70; c: 33

0.90

Tuli et 01. (1985)

0.87a

Stenroos et aJ. (1982) Davison and Scott (1992); Davison et d.(1992); Kaufman et al. (1994)

P 3.3; C: 46 P: 0.37: C 5.0

(0.97)b 1.0

L. helveticus

u

w

R. oryzae

Mixed cultures L. casei and Strep tomyces viridosporus Mixed Lactobacilli

Continuous recycle, tubular packed bed, sintered glass beads Continuous packed bedcolumn, calcium alginate beads Continuous three-stage packed bed column, sodium alginate beads Continuous stirred tank, 28 years 1st Stage: K-carrageenan-locust bean gum beads 2nd stage: free cells from 1st stage Stirred batch, calcium alginate Stirred batch, polyethylene glycol 400dimethylacrylate polymer cubes Continuous biofilm, polypropylene composite chips Fixed film column, gelatin crosslinked with glutaraldehyde-coated stoneware berl saddles

Glucose (variable)

P: 20.1; C: 51.4

0.76

GonGalves e l 01. (1992)

Cheese whey permeate (total solids, 65) Cheese whey permeate (lactose, 38.5) Cheese whey permeate

P: 8

0.50

Boyaval and Goulet (1988)

P: 2.6

0.82

Roy e l al. (1987a)

P: 13.5

0.95

Norton et al. (1994b)

0.72

Hang et al. (1989)

Glucose, 70

P: 2.6; C: 62.4 C: 50

0.71

Tamada et al. (1992)

Glucose, 20

C: 13

l:oo

Demirci et al. (1993)

Acid whey

P: 0.45-0.90; C 17 C: 31,32

0.18

Compere and Griffith (1975) Griffith and Compere (1977)

Glucose, 30

Wood molasses

>0.50

[continues)

TABLE VIII-Con tinued

Organism

Type of bioreactor and immobilization support

Mixed culturesContinued 5’.lacfis (L. lacfis) Continuous stirred coimmobilized tank, calcium with A. awamori alginate beads L. casei coimmobiStationary flask, calcium alginate lized with L. l a d s beads a

L(+) lactic acid.

b

Total lactic acid.

Substrate and concentration (g/liter)

Lactic acid productivity (P: g/liter/hr) and concentration [C: glliter)

Yield (g/g

substrate utilized)

Reference

Starch, 20-75

P: 0.34-0.43; C: 25

0.66

Kurosawa e f al. (1988)

Deproteinized cheese whey

P: 0.86;

0.97

Roukas and Kotzekidou

C: 41.3

(1991)

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

75

free of contamination. Yeasts can grow in calcium alginate in mobilized lactic acid bacteria (Champagne et al., 1989). Also, bacteriophage development can occur in these systems (Champagne et al., 1988). Boyaval and Goulet (1988) found that a packed bed of L. helveticus entrapped in calcium alginate beads plugged after a week of fermentation of cheese whey permeate. This plugging was attributed to decalcification of calcium alginate and leakage of the cells. Also, pH control was a problem in packed bed cell reactors, with alginate-immobilized L. casei (Guoqiang et al., 1991). Groboillot et al. (1993) microencapsulated L. lactis by using emulsificatiodinterfacial polymerization to form crosslinked chitosan membranes. This technique enabled the preservation of cell viability, whereas reagents and procedures used in previous attempts to microencapsulate lactic acid bacteria were toxic to the cells. Norton et al. (1994a) employed a two-stage bioreactor system for a stable lactic acid fermentation of cheese whey permeate by L. helveticus over a 91-day period. The first stage consisted of cells immobilized in K-carrageenan-locust bean gum beads and the second stage free cells continuously released from the first stage. They determined that yeast extract supplementation could be reduced from 10 to 1 g/liter to uncouple biomass from lactic acid production and improve yield. To overcome the fragility of alginate gels, Tamada et al. (1992) prepared a polymer support for immobilizing R. oryzae by y-ray-induced polymerization of polyethylene glycol 400 dimethacrylate. The specific lactic acid production rate of 0.24 g/g (dry wt)/hr was 1.8 times that of the free-cell culture. As an alternative to entrapment, lactic acid bacterial biofilms can be developed by adsorption onto inert materials such as sintered glass (GonGalveset al., 1992; Kritschke et al., 1991), ceramic materials (Griffith and Compere, 1977), or polypropylene composite chips (Demirci et a]., 1993). Such biofilm systems have the advantages of sterilizability,low cost, and do not require a large initial cell population for immobilization. Stoneware berl saddles were employed in fixed-film column reactors for producing lactic acid from acid whey or wood molasses using mixed Lactobacilli (Compere and Griffith, 1975; Griffith and Compere, 1977). With L. rhamnosus (L. delbruecki NRRL B-445), sintered glass beads yielded the highest lactic acid productivities in continuous packed column reactors compared with Raschig rings (sintered glass),porous glass beads, or irregular ceramic particles (Gonplves et al., (1992). Porous sintered glass beads were used in a continuous immobilized L. casei subsp. casei reactor with cheese whey permeate as the substrate (Kritschke et al., 1991).

76

JOHN H. LITCHFIELD

Composite chips prepared by extrusion of polypropylene with soy hulls-soy flour or soy hulls-zein were used effectively in lactic acid production by a biofilm mixed culture of L. casei and S . viridosporus (Demirci et aJ., 1993). The apparent lactic acid productivities shown in Table VIII for the various immobilized cell processes are lower than those shown in Table VII, although lactic acid concentrations obtainable are similar in many cases. High cell concentrations can be obtained in immobilized cell systems. However, it is difficult to determine actual cell concentrations in numbers or in cell dry weight on or in the immobilization medium. Gonqalves et aJ. (1992) obtained a L. rhamnosus cell concentration of 34 g/liter on sintered glass beads as determined by the difference between the dry weight of cells plus support medium and the support medium only. Kritschke et aJ. (1991) estimated L. casei biomass on porous sintered glass beads to be in the range of 86 to 94 g (dry wt)/kg of beads by ATP measurement of cell activity. Gonqalves et aJ. (1991) point out that most of the high lactic acid productivity data obtained with membrane recycle systems have been obtained over short time runs of 24 hr or less compared with longer runs with immobilized cell systems. Subsequently, Xavier et aJ. (1995) operated a continuous membrane recycle bioreactor with a tubular ceramic membrane ultrafiltration over a 90-hr period with L. rharnnosus (L. delbrueckii NRRL B-445). A dilution rate of 0.40 hr-1 gave lactic acid productivity and concentrations of 36 g/liter/hr and 9.0 g/liter respectively, with a cell concentration of 136 g(dry wt)/liter. The yield was 0.82 based on glucose utilized. Comparative productivities in batch, continuous (CSTR), and immobilization systems were 3, 6, and 20 g/liter/hr, respectively. A biparticle fluidized bed bioreactor is under development at Oak Ridge National Laboratory, Oak Ridge, Tennessee, for continuous simultaneous fermentation production and separation of lactic acid (Davison and Scott, 1992; Davison and Thompson, 1992; Kaufman et aJ., 1994; Scott, 1993). The fluidized bed column reactor consists of L. delbrueckii NRRL B-445 (L.rhamnosus) cells immobilized in calcium alginate beads and beads of a polyvinyl pyridine resin (Reillex 425). In the fermentation, the resin was added to the top of the column and recovered with the sorbed lactic acid at the bottom with an overall yield of 1.0 g lactate/g glucose consumed. The time for the fermentation in this system was reduced to 24 hr compared with 46 hr for the immobilized cell reactor without the resin. Cell aggregates of B. Jaevolacticus gave productivities of 13 g/liter/hr at a cell concentration of 25 g/liter in an anaerobic gas-lift reactor (de Boer et a]., 1990). The advantages claimed for this approach were

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

77

use of a simpler reactor configuration than either membrane cycle or immobilized cell reactors and the ease of separating the aggregated biomass from the production medium. 5 . Dialysis Systems

Dialysis systems have been investigated for recovering lactic acid from fermentations and overcoming its inhibitory effects on lactic acid bacteria by maintaining low lactate concentrations in the medium. In conventional dialysis fermentations, a dialysis membrane unit permeable to lactose and small molecules but impermeable to cells and proteins is coupled with batch (Friedman and Gaden, 19701, continuous (Coulman et al., 1977; Stieber et al., 1977; Stieber and Gerhardt, 1979), continuous with cell recycle (Stieber and Gerhardt, 1981a), or immobilized cell bioreactors (Stieber and Gerhardt, 1981b). Table IX shows the results of typical dialysis fermentations for lactic acid production using various organisms and substrates. In general, higher concentrations of substrate can be used and higher concentrations of lactic acid can be obtained with dialysis processes compared with batch processes. Also, the lactic acid produced initially is purer than that in conventional batch processes that require extensive product purification steps. Using a sweet whey feed containing 240 g lactose/liter, Stieher et al. (1977) ran a nonaseptic continuous dialysis process with L. delbrueckii subsp. bulgaricus for 94 days to give a 97% conversion into products. The pH was maintained at 5 . 3 with ammonium hydroxide that yielded ammonium lactate. However, membrane fouling required cleaning the dialyzer every 4 weeks. Conventional dialysis is limited by diffusion rates through the membrane. Also, large-volume dialysis units even greater than the volume of the fermenter vessel would be required in a commercial-scale unit. Consequently, electrodialysis has received attention as a method for improving dialysis-fermenter processes. In electrodialysis, lactate ion passes through the anion-exchange membrane under the influence of an applied DC electric current and is removed in the anode compartment. Results obtained in typical electrodialysis fermentations are also shown in Table IX. Electrodialysis units with an ion-exchange membrane are coupled with a fermenter (Hongo et al., 1986; Ishizaki et al., 1990; Nomura et al., 1991; Yao and Toda, 1990; Yen and Cheryan, 1991), combined with a microfilter (Ishizaki et al., 1990; Nomura et al., 1991;Vonktaveesuk et al., 1994), or combined with an ultrafiltration unit (Boyaval et al., 1987). Electrodialysis has been combined with immobilized growing L. delbrueckii cell systems for lactic acid production (Nomura et al., 1987).

TABLE IX DIALYSIS SYSTEMS FOR LACTICACIDPRODUCTION

Organism L. delbrueckii subsp. bulgaricus L. delbrueckii (L. rhamnosus)

L. helveticus

L. lactis (S. lactis)

Dialysis system Dialysis, continuous

Substrate

Lactic acid productivity (P: g l l i t e r h ) and concentration C: glliter)

Yield (gk substrate utilized

Reference

Cheese whey

P: 11.7;

0.97

Stieber et al. (1977)

NRa

Friedman and Gaden

0.91

NRa

Hongo et al. (1986) Nomura et al. (1987)

0.81

Boyaval et al. (1987)

C:80

Batch with dialysis

Glucose

Electrodialysis batch Electrodialysis coupled with immobilized cell bioreactor Electrodialysis with ultra6ltration coupled with continuous stirred tank bioreactor Built-in electrodialysis system

Glucose Glucose

Periodic electrodialysis

Glucose

P: 8 ; c: 35 C: 82.2 P: 5.3; C: 70.2

Cheese whey permeate

P: 22; C:85

Glucose

P: 2.4; C: 60b 1oc P: 5.1; C: 60b, 20c

NR, not reported. Total lactate. c Lactate in fermenter medium. a

b

(1970)

>0.99

>0.99

Ishizaki et al. (1990) Vonktaveesuk et al. (1994)

79

MICROBIOLOGICAL PRODUCTION OF LACTIC ACID

Also, using electrodialysis seed cultures free of inhibitory lactic acid for inoculating the production fermenter reduced the lag phase of L. lactis (Yamamoto et al., 1993). Electrodialysis has been applied to fermentation of cheese whey permeate (Boyaval et al., 1977; Yen and Cheryan, 1991). Yen and Cheryan (1991, 1993) reported the effective separation of lactic acid from cheese whey permeate fermentation broth and from model lactic acid solutions also containing glucose and lactose. In the latter case, increasing the current in model systems gave a higher extent of lactic acid separation but with increased power and energy consumption (Yen and Cheryan, 1993). Siebold et al. (1995) described a three-stage bioreactor ultrafiltration electrodialysis system for lactic acid production and recovery. A 10-kDa ultrafiltration membrane removed cells and 83% of the proteins and the salt circuit retained amino acids. The overall yield for the system was 69.5%.

E.

PROCESS

KINETICS AND MODELING STUDIES

Table X summarizes some of the numerous kinetics and modeling studies of bacterial lactic acid fermentations. The widely used model of TABLE X KINETICAND MODELING STUDIES OF THE LACTIC ACIDFERMENTATION

Organism

L. amylophilus L. casei L. delbruecki subsp. bulgaricus

L. delbrueckii subsp. bulgan'cus

Substrate

Type of process

Glucose, starch Cheese whey Cheese whey permeate Cheese whey

Semicontinuous

Cheese whey or permeate

Dialysis, continuous

Lactose

Batch and continuous Dialysis and continuous

Cheese whey permeate

Lactose

Batch Batch Batch

Batch and continuous

Reference Mercier et al. (1992) Rincdn et aJ. (1993) Leh and Charles (1989a) Borzani et 01. (1990, 1993) Coulman e t a ] . (1977); Keller and Gerhardt (197 7); Stieber and Gerhardt (1979, 198la,b); Stieber et al. (1977) Venkatesh et al. (1993) Coulman eta]. (1977); Stieber and Gerhardt (1979, 198la,b); Stieher et al. (1977) Venkatesh et 01. (1993) [continues]

80

JOHN H. LITCHFIELD TABLE X-Continued

Organism

L. delbrueckii subsp. bulgaricusContinued

L. delbrueckii (L. rhamnosus) L. delbrueckii (L. rhamnosus)

Substrate

Type of process

Cheese whey permeate

Dialysis and continuous

Lactose

Batch and continuous Dialysis, batch, and continuous Batch and continuous

Glucose G1u cose

Extractive L. helveticus

Cheese whey permeate

Batch

Continuous, immobilized cell Continuous, cell recycle, electrodialysis Batch Batch and continuous

L. plantarum S. cremoris (L. lactis subsp. cremoris)

Cucumber juice Glucose, galactose, lactose

S . faecalis

Glucose

Continuous

Streptococcus 10-1 (L. lactis 10-1)

Glucose

Batch

Reference Couhnan eta]. (1977); Stieber and Gerhardt (1979, 1981a,b); Stieber et al. (1977) Venkatesh et al. (1993) Friedman and Gaden (1970) GonCalves et 01. (1991); Hanson and Tsao (1972); Luedeking and Piret (1959a,b); Tsao and Hanson (1975); Yeh et al. (1991) Yabannavar and Wang (1991a) Amrane and Prigent (1994); Roy et al. (1987b) Norton et al. (1994b) deRaucourt et al. (1989a,b) Passos eta]. (1994) Jorgensen and Nikolajsen (1987);Nielsen et 01. (1991a,b);Nikolajsen et al. (1991);Rogers eta]. (1978) Ohara and Hijama (1996); Ohara et 01. (1992a,b,c) Ishizaki and Ohta (1989); Ishizaki et al. (1989,1990, 1992)

Luedeking and Piret (1959a,b) for fermentation of glucose by L. delbrueckii NRRL B-445 (L. rhamnosus) has growth- and non-growthassociated components. It was approximated in batch fermentations of cheese whey permeate by L. helveticus (Roy et al., 1987b), lactose by L. delbrueckii subsp. bulgaricus (Venkatesh et al., 1993), and cucumber juice by L. plantarum (Passos et al., 1994). The Luedeking and Piret model has been modified to take into account the inhibition of lactic

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acid production by increasing concentrations of lactic acid (Friedman and Gaden, 1970),particularly undissociated lactic acid (Gonplves et al., 1991; Yeh et al., 1991), and inhibition of growth from increased substrate (glucose) concentration in addition to lactic acid inhibition (Gonqalves et al., 1991). Alternative models for batch lactic acid fermentations include an uncompetitive inhibition model for glucose fermentation by Streptococcus 10-1 (L. lactis) (Ishizaki and Ohta, 1989; Ishizaki et al., 1989, 1992) and a logistic model for glucose or hydrolyzed starch fermentation by L. amylophilus (Mercier et al., 1992). Several investigators have found that the Luedeking-Piret model does not fit continuous lactic acid fermentation data as well as those from batch processes (Rogers et al., 1978;Jorgensen and Nikolajsen, 1987). In the case of the continuous fermentation of glucose by S . cremoris (L. lactis subsp. cremoris),modifications include introducing a negative term for the retarding of lactic acid concentration with increasing glucose concentrations (Jorgensenand Nikolajsen, 1987)and a two-compartment model dividing the biomass into active and inactive structural parts (Nielsen et al., 1991a,b; Nikolajsen et al., 1991). For the Streptococcus faecalis continuous fermentation, equations were developed relating the specific consumption of glucose and the specific production of lactic acid to the specific growth rate that fitted the experimental data better than the Luedeking-Piret model (Ohara et al., 1992a,b). In modeling an extractive fermentation of glucose with immobilized L. delbrueckii NRRL B-445 (L. rhamnosus), Yabannavar and Wang (1991a)found that the decreased pH and development of inhibitory lactic acid concentrations in the gel beads were limiting factors. However, they concluded that mass transfer of the substrate in the immobilization medium was unlikely to be limiting. A kinetic analysis of continuous lactic acid production from cheese whey permeate by immobilized L. helveticus revealed low specific lactic acid production rates for entrapped cells compared with those for free cells in the bioreactor (Norton et al., 1994b). This low rate was attributed to inhibition by substrate, lactic acid, and pH gradients within the immobilization beads. These kinetic and modeling evaluations of lactic acid fermentations are based on laboratory-scale studies with concentrations of various substrates and supplemental nutrients, such as peptones and yeast extract, that may not be representative of commercial practices. Owing to lack of published data on modern commercial-scale lactic acid fermentations, it is not possible to determine which of these models are predictive of the performance of scaled-up processes.

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IV. Product Recovery and Purification

Lactic acid, to be suitable for manufacturing stearoyl lactylates as food additives or plastics for medical applications, must be free of impurities such as residual sugars, Maillard reaction products between carbohydrates and amino acids, other organic acids, organic nitrogen compounds, and heavy metals. Consequently, the recovery and purification processes on completion of the fermentation can be quite complex. Furthermore, product recovery costs for fermentation lactic acid are significant and may constitute 50% of the final cost of the product (Evangelista et al., 1994). A. PRECIPITATION AND ACIDIFICATION

Inskeep et al., (1952) described the recovery and purification of lactic acid in the former American Maize Products Co. L. delbrueckii (L. rhamnosus) process based on glucose. This process involved heating the medium containing 0.1% glucose or less, adjusted to pH 10, to 2.2"C (180°F) to kill bacteria separating the calcium lactate by filtration, acidification with sulfuric acid removing calcium sulfate by filtration, multiple vacuum concentration, and activated carbon decolorization steps. Heavy metals were removed by precipitation with sodium sulfide. In the Miles Laboratories process based on R. oryzae, L(+) lactic acid was recovered by filtering the medium with no detectable residual glucose to remove the mycelium followed by acidifying with sulfuric acid and concentration by evaporation (Snell and Lowery, 1964). Further purification was achieved by ion exchange and activated carbon treatment. Because the production medium contained ammonium sulfate and mineral salts with no organic nitrogen sources, contaminants leading to discoloration were minimized. However, the fermentation must be controlled to minimize fumaric acid formation. B.

CENTRIFUGATION AND

MICROFILTRATION/ULTFUFILTRATION

There is considerable interest in processes for recovering and purifying lactic acid from fermentation media that avoid excessive heating, which leads to costly steps to remove the resulting impurities. Cell recycle systems based on membrane separation and recycle of bacterial cells and dialysis and electrodialysis systems for product separation were discussed previously in this chapter. Both microfiltration (Bailey et al., 1987,1988) and ultrafiltration (Tejayadi and Cheryan, 1988) have been used in downstream purification of lactic acid fermentation broths. Bailey et al. (1887, 1988) describe the use of a continuous centrifuge or a 0.2-pm ceramic crossflow microfilter to separate bacterial cells from

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a hydrolyzed cheese whey permeate medium after lactic acid fermentation. Crossflow ultrafiltration membranes could also be used. However, ceramic filters are preferable because they can be heat sterilized and are resistant to chemicals employed in cleaning. As far as can be determined, there have been no reports of membrane separation of cells from lactic acid fermentations on a commercial scale. C. DISTILLATION

Crude lactic acid can be purified by distillation of lactate esters. Schopmeyer (1954) states that reacting the lactic acid with methanol is the most practical method. A continuous esterification, distillation, and hydrolysis process involving methyl lactate has been practiced commercially (Schopmeyer and Arnold, 1944; Schopmeyer, 1954). A recent patent assigned to E. I. duPont de Nemours & Co. describes the recovery of lactate ester from a concentrated fermentation broth by continuous acidification with sulfuric acid and simultaneous esterification with isobutanol or 2-methyl-2-butanol and distillation (Cockrem and Johnson, 1993). Hydrolysis of the ester gave greater than 97% optically pure lactic acid.

D. ION EXCHANGE AND ADSORPTION Ion exchange has replaced compounds such as sodium sulfide for removal of heavy metals from lactic acid fermentations. When concentrated lactic acid is added to ion-exchange resin systems, lactate is replaced progressively by chloride and sulfate ions over time. This contamination with anions can be avoided by using dilute crude lactic acid in the feed to the resin (Benninga, 1990). Evangelista et al. (1994) evaluated the sorption of lactic acid from solutions having different pH values by weak, moderate, and strong base anion-exchange resins. Because lactic acid fermentations are ordinarily conducted at pH values of 5 or 6, above the lactic acid pK, of 3.86 the fermentation broth should be acidified for effective use of weak and moderate base resins. However, the strong base resin evaluated had a constant sorption capacity over the pH range of 2 to 6 but required a stronger eluant for desorption of lactic acid. Srivastava et d . (1992) coupled an anion-exchange packed column with a fermenter for lactic acid production from sucrose by L. delbrueckii NRRL B-445 (L. rhamnosus). The recirculation of the fermentation broth through the resin to the fermenter minimized the inhibition of the fermentation by lactic acid and gave a 5.32-fold improvement in productivity over the batch fermentation.

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After cell separation from batch fermentation of glucose by L. casei subsp. casei (ATCC 393), L. rhamnosus ATCC 7469 and DSM 2024, or L. salivarius ATCC 11741, lactic acid was initially extracted from the broth with a strong anion exchanger (Amberlite IRA-420) in the carbonate form (Vaccari et al., 1993). Ammonium lactate was formed by percolating ammonium carbonate solution through the resin. It was converted to lactic acid by treatment with a strong cation-exchange resin (Amberlite IR-120) in hydrogen form. A 99% pure lactic acid was obtained that contained small amounts of phosphate.

E.

REVERSE

OSMOSIS

Reverse osmosis (RO) has also been evaluated for recovering lactic acid from fermentation broths (Smith et al., 1977; Schlicher and Cheryan, 1990). In experiments with model solutions and L. delbreuckii subsp. bulgaricus, cheese whey permeate fermentation broths cell concentrations had little effect on performance. However, pH of the feed had a marked effect with greater than 90% rejection of lactic acid from a cellulose acetate membrane at pH 5.6 and 6.2. It was concluded that RO could effectively concentrate lactic acid from 10 to 1 2 0 g/dm3 at a 6.9 Mpa (6000 psig) transmembrane pressure at lower energy use than multiple-effect evaporation.

F. SOLVENT EXTRACTION AND EXTRACTIVE FERMENTATION Various solvent extraction processes have been used for purifying lactic acid after cell removal (Schopmeyer, 1954; Vick Roy, 1985). A continuous countercurrent solvent extraction procedure based on isopropyl ether was described by Jenemann (1933)in a patent assigned to du Pont. This process has been practiced on a commercial scale with modifications by Croda Bowmans Chemicals Ltd. in the United Kingdom (Benninga, 1990). Bailey et al., (1987, 1988) used the tertiary amine Adogen 364 in 60-75% isobutyl heptyl ketone as the preferred system for extraction of lactic acid from cheese whey permeate fermentation after removal of L. casei cells and suspended solids. Wang et al. (1991) proposed using a hollow fiber hydrophobic membrane between the solvent and aqueous phases of a nondispersive extraction process for lactic acid recovery. The solvent system trioctyl phosphine oxide (TOPO)in kerosine, while effective for extraction, clogged the membrane with TOPO crystals when it was exposed to air. Using model lactic acid solutions, a quarternary ammonium salt (ALIQUAT 336) gave the best extraction at pH 5 or 6 and .35"C, the usual conditions for lactose fermentation by L. casei (Lazarova and Peeva, 1994a). The optimum concentration of this ex-

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tractant for liquid membrane extraction of a 3 g/liter lactic acid feed solution was 5% vol in n-octane (Lazarova and Peeva, 1994b). Extractive fermentation involves using a water-immiscible phase for removing fermentation products such as lactic acid in situ. Lactic acid inhibition of the fermentation can be reduced by this method. Yabannavar and Wang (1987, 1991a,b,c)developed an extractive fermentation system for removing lactic acid continuously from glucose fermentation by L. delbrueckii (L. rhamnosus). The extractant system showing the least toxicity to the cells was 15% Alamine 336 (a tertiary amine) in oleyl alcohol. The cells were protected from the solvent by immobilization. The lactic acid productivity was 1 2 g/liter (gel)/hr compared with 7 g/liter (gel)/hr for a control fermentation without solvent. A final product concentration of 90 g/liter was obtained by back extraction with sodium hydroxide. In the extractive fermentation of glucose by L. delbrueckii NRRL B-445 (L. rhamnosus), amines, such as Adogen 464, Aliquat 336, Tri-n-octylamine (TOA), and TOPO, were toxic to the cells (Seevaratnam et al., 1991). The hydrophobic resin Bonopore in paraffin oil showed no toxicity in batch cultures. However, the yield of lactic acid was lower than that of a conventional batch fermentation that may have resulted from absorption of essential nutrients by the Bonopore resin. An emulsion liquid membrane system consisting of the amine Alamine 336 and the surfactant Span 80 in n-heptane paraffin was evaluated for extracting lactic acid from L. delbrueckii NRRL B-445 fermentation broth after cell removal (Scholler et al., 1993). Alamine 336 had a lower selectivity for lactic acid than desirable owing to its possible binding to other competing solutes. In a patent assigned to Purdue Research Foundation and Reilly Industries,Iyer et al. (1993)described the use of a solid-phasepolymer having tertiary amine groups in an exbactive fermentationto absorb lactic acid. Either Lactobacillus spp. or R. oryzae can be used in this fermentation. Dissing and Mattiesson (1994) investigated an aqueous polyI(ethy1eneimine) (PE1)-(hydroxyethy1)-cellulose(HEC) two-phase system for the extractive fermentation production of lactic acid from glucose by L. l a d s . Lactic acid partitioned into the PET-rich bottom phase, whereas the cells accumulated in the HEC top phase or at the interface. V. Process Control

For many years, in commercial-scale lactic acid fermentations, the course of the process was monitored by taking periodic samples and measuring pH and titratable acidity and reducing sugar concentrations by manual laboratory methods; limited control of pH was achieved by

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adding calcium carbonate to the production medium to yield calcium lactate. Temperature control was mentioned previously. A. PHCONTROL Continuous pH control has beneficial effects on lactic acid yield and substrate conversions and has been widely practiced in laboratory-scale batch and continuous fermentation [with a variety of Lactobacillus spp. and substrates (Aeschlimann and von Stocker, 1989; Aeschlimann et al., 1990; Amrane and Prigent, 1994; Finn etal., 1950; Gatje and Gottschalk, 1991; Kempe et al., 1950;Mehaia and Cheryan, 1987a; Roy etal., 1986). Without pH control, the lactic acid concentrations were less than half that obtained with pH control in the case of L. helveticus batch cultures in a cheese whey permeate medium, although satisfactory biomass concentrations were obtained (Amrane and Prigent, 1994). As mentioned previously, pH control is also important in continuous membrane cell recycle, immobilized cell systems dialysis and electrodialysis processes. Extractive fermentations with various extractants or anion-exchange resins also control pH by removing lactic acid as it is formed. A feed-forward neural network used stored information on satisfactory reference fermentations to predict pH and final fermentation time in batch-uncontrolled pH lactic acid fermentations (Latrille et a].,1993). For a 3-hr prediction horizon, future pH values were predicted with a mean error of 0.05 pH by sliding the reference fermentation pH-fermentation time curve along that of the actual fermentation. However, there is a lack of published information on continuous pH control in commercial-scale Lactobacillus fermentations. In the former American Maize process, continuous pH control was abandoned owing to variations in the line voltage and fouling of the electrodes (Inskeep et a]., 1952).

B. ANALYTICAL METHODS In recent years, high-performance liquid chromatography (HPLC)has been applied to the determination of lactic acid (McFeeters, 1993; Olieman and deVries, 1988). Also, enzymatic methods for determining L(+) or D(-) lactic acids using the L(+) or D(-) lactic dehydrogenases are now available in commercial test kits (Boehringer-Mannheim Corporation, 1995; Sigma Chemical Company, 1994). An automatic HPLC monitoring system has been developed for lactic acid fermentations consisting of a crossflow filter, dilution system, and an automatic sampling system coupled with the HPLC unit (Ohara et al., 1993a). Lactic acid and glucose concentrations were measured every

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30 min during the fermentation of 120 g/liter glucose by S . faecalis with a final lactic acid concentration of 100 g/liter.

C. ON-LINE CONTROL SYSTEMS Several on-line analysis systems have been developed for monitoring lactic acid fermentation including flow injection analysis (FIA) (Nielsen et al., 1989)and Near Infiared Spectroscopy (NIR) (Vaccari et al., 1994) for determining lactic acid, glucose, and biomass concentrations. Also, a glucose oxidase probe system (Shi et al., 1992) and Fourier transform infrared spectroscopy (FTIR) (Fairbrother et al., 1991) have been used in lactic acid fermentation monitoring. On-line optimizing control systems offer the opportunity to maintain lactic acid fermentations at optimum conditions. Shi et al., (1990) were able to employ an adaptive on-line optimization strategy to maintain constant cell and lactate concentrations in the fermentation of glucose by S. inulinis ATCC 15538 using a continuous-membrane bioreactor. They found that a lactic acid productivity of 20 g/liter/hr could be maintained for a 40- to 50-hr operation with lactate control at 20 or 30 g/liter/hr. On-line advisory knowledge-based advisory control systems have been applied to diagnosing and controlling lactic acid fermentations (Nakajima et al., 1992, 1994; Pokkinen et a]., 1992; Siimes et al., 1992a,b). A fuzzy-expert knowledge-based supervisory control system detected abnormal inoculum conditions for subsequent lactic acid production by L. casei (Nakajima,et d.,1994). Although these process control systems appear promising, there have been no published reports on their use in commercial-scale lactic acid fermentations. VI. Economics

As mentioned previously, the major use for lactic acid in the United States is as a food additive and preservative. Lactate esters as alternative solvents to glycol ethers and lactic acid-derived polymers are emerging uses. U.S. consumption of lactic acid in 1994 was estimated to be between 42 and 45 million lbs/year (19.1-20.4 kg/year), half of which was imported, with a growth of 3 4 % per year (Bahner, 1994). The world demand is estimated as 120-130 million lbs/year (54.5-59 kg/year) (Anonymous, 1992a). The worldwide growth is believed by some observers to be 12-15% per year (Bahner, 1994). The major U.S. producer of fermentation lactic acid is Archer Daniels Midland Co., Decatur, Illinois with an estimated capacity of 2 0 4 0 million lbs/year (9.1-18.2 kg/year) (Anonymous, 1993a).In 1993, Ecological Chemical Co. (Ecochem),a joint venture of E.I. duPont de Nemours and

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Con Agra, operated a fermentation plant on a pilot plant scale at Adell, Wisconsin using cheese whey. However, full-scale production has not taken place (Anonymous, 1993b). In 1994, Sterling Chemicals, Houston, Texas was the U.S. producer of synthetic lactic acid with an annual capacity of 19-20 million lbd(8.6-9.1 kg) (Bahner, 1994). The lactic acid imported into the United States manufactured by Purac at plants in Brazil, The Netherlands, and Spain (Bahner, 1994). There is an indication that China could become a major exporter of lactic acid in the future, although only small quantities are currently exported to Japan, Western Europe, and Southeast Asia (Anonymous, 1992b). In December 1994, market prices in the United States for both fermentation and synthetic food grade 50 and 88% lactic acid were $0.71 and $1.15 per pound ($1.56-$2.53/kg), respectively, in tank car lots, FOB works. Technical grade 88% lactic acid was quoted at $1.12 per pound ($2.4 7/kg) (Anonymous, 1994). The future demand for lactic acid in the United States will depend on the growth of new uses including lactic acid esters and lactic acid polymers. Fermentation lactic acid will have to compete with synthetic lactic acid in quality and price to participate in this growth. REFERENCES Abe, S., and Takagi, M. (1991). Biotechnol. Bioeng. 37, 93-96. Acuna, G.,Latrille, E., Beal, C., and Corrieu, G. (1994). Biotechnol. Bioeng. 44,1168-1176. ADM Corn Processing (1993). “Lactic Acid,” Decatur, IL. Aeschlimann, A., and von Stockar, U. (1989). Biotechnol. Lett. 11,195-200. Aeschlimann, A,, and von Stockar, U. (1990). Appl. Microbiol. Biotechnol. 32, 398-402. Aeschlimann, A., and von Stockar, U. (1991). Enzyme Microbiol. Technol. 13, 811-816. Aeschlimann, A., Di Stasi, L,. and von Stockar, U. (1990). Enzyme Microbiol. Technol. 12,926-932. American Dairy Products Institute (1995). “Production of Whey and Modified Whey Products.” Chicago, IL. Amrane, A,, and Prigent, Y. (1994). Appl. Microbiol. Biotechnol. 40,644-649. Andersen, A. A., and Greaves, J. E. (1942). Ind. Eng. Chem. 34, 1522-1526. Anonymous (1992a). Chemical Marketing Reporter (Aug. 3), p. 14. Anonymous (1992b). China Chemical Reporter, No. 17, p. 4. Anonymous (1993a). Chemical Marketing Reporter (March I),p. 7. Anonymous (1993b). Chemical Marketing Reporter (June 71, p. 18. Anonymous (1994). Chemical Marketing Reporter (Dec. 1 2 ) , p. 30. Aries, R. S.,and Needle, H. C. (1949). U.S.Patent No. 2,588,460. Atkinson, B., and Marituna, F. (1991). “Biochemical Engineering and Biotechnology Handbook,” 2nd Ed., pp. 87-88, 246, 336, 346-348, 798. Stockton Press, New York. Audet, P., Paquin, C., and Lacroix, C. (1988). Appl. Microbiol. Biotechnol. 29,11-18. Bahner, B. (1994). Chemical Marketing Reporter (Mar. 21), p. 14.

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Holt, J. G., Krieg, N. R., Sneath, P. H. A,, Staley, J. T., and Williams, S. T. (1994). “Bergey’s Manual of Determinative Bacteriology,” 9th Ed., pp. 5 2 8 4 2 9 , 5 4 0 , 566, 568. Williams & Wilkins, Baltimore. Holten, C. H., A. Miiller and D. Rehbinder (1971).“Lactic Acid-Properties and Chemistry of Lactic Acid and Derivatives.” VCH, Weinheim, Germany.

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Biodegradable Polyesters CH. SASIKALA AND CH. V. RAMANA Microbial Biotechnology Laboratory Department of Botany Osmania University Hyderabad 500 007 (A.l?),India

I. Introduction 11. Classification of Biodegradable Polyesters 111. Biodegradable Natural Polyesters IV. Biodegradable Synthetic Polyesters V. Poly-L-malate: A Potential Biodegradable Polyester VI. PHAs: A Group of Polyesters Produced Naturally and Synthetically A. Terminology B. Chemical Synthesis of PHAs C. Naturally Occurring PHAs D. Physical Properties of PHAs VII. Application of Biodegradable Polyesters VIII. Biodegradation of Polyesters A. Biodegradation of PHAs B. Biodegradation of Synthetic Polyesters References

I. Introduction

Waste disposal is becoming an increasingly difficult problem as available landfill areas diminish. Plastic currently accounts for about 7% by weight (18% by volume) of municipal solid waste, with half of this plastic waste used in packing (Thayer,1990).The disposal of plastics has become a worldwide environmental problem (Hanggi, 1995) and there is a need for the development of biodegradable polymers, a waste management option for polymers in the environment (Swift, 1992a),and in many cases the demand is driven by legislation (Leaversuch, 1987). Biopolymers is a term often used to refer to those polymers formed in nature during the growth cycles of all organisms; hence, they are also referred to as natural polymers (Lenz, 1993). Biopolymers include polysaccharides, starch, and cellulose from plants and microbial carbohydrates like xanthan (cellulose-type main chain and trisaccharide graft chains containing glucuronic acid), dextran (a-1,6-glucopyranoside polymer), gellan (a tetrasaccharide polymer with glycerol substituents), curdlan (P-1,3-glucopyranoside polymer), and pullulan (which is composed of 1,6-linkedmaltotriose units and produced by fungi),and proteins 97 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 42 Copyright 0 199fi by Academic Press, Inc. All rights of reproduction in any form reserved.

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and polyesters. Essentially all biopolymers are susceptible to enzymatic degradation because the enzymatic polymerization reactions responsible for their synthesis in nature have closely related counterparts in nature for their enzymatic depolymerization: "What nature creates, nature can destroy" (Lenz, 1993). Polyesters, a group of polymers formed due to the polycondensation of carboxylic acids with hydroxy alcohols, are of high industrial significance and interest has increased in recent years to exploit the biodegradable polyesters (Hanggi, 1995; Page, 1995), which cover a wide range of aliphatic polymeric ester compounds (natural and synthetic) degraded through biological means. There is an increasing demand to use biologically produced polymers, particularly polyesters (Brandl et al., 1990), making it a multibillion dollar per year industry in the future (Lindsay, 1992). In this review, we discuss various aspects of biodegradable polyesters.

I I . Classification of Biodegradable Polyesters Degradable plastics can be classified based on (a) the origin of the polymer and (b) the nature of degradation. The former can be categorized mainly into two categories (Fig. 1)based on whether they were polymerized synthetically or biologically (Huang et al., 1990; Swift, 1992a,b). Based on the nature of degradation (Brandl et d., 1995a),plastics can be categorized into three categories: whether the plastic is degraded chemically, physically, or biologically (Fig. 2). Biological degradation is generally referred to as "biodegradation"because the name indicates the involvement of biological material in the process of degradation. This occurs under either aseptic (sterile condition without any microbial action) or septic (systems with microbial activities such as activated BIODEGRADABLE POLYESTERS

4

4

Synthetic Polyesters

Biopolyesters I

A

Cutin

Suberin

Bacteria

c--i

PHAs

Poly (L-Malic acid)

FIG. 1. Classification of biodegradable polyesters based on the origin of polyester.

DEGRADABLE POLYMERS

3-

J-

Physical

r - 5Thermal

Mechanical

1

+ Biological

1

All Polymers All Polymers

3-

Micrrbial

Completely degradable polymers

~

Degradable additives

Asrtic

Resorbable Polymers

4

4

Oxidation

1

Oxidizable Polymers

Photochemical

*

1

Solubilization

Photo- PhotoSoluble sensitive sensitive Polymers additives copolymers

FIG. 2 . Schematic classification of degradable plastics based on the type of degradation.

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sludge, soil, etc.) conditions. Biodegradable plastics used under aseptic conditions in which the polymers are degraded by hydrolytic reactions and/or bioresorbed under those conditions are required for various medical applications. In contrast, under septic conditions, microbial enzymatic degradation of the polymer results in either complete or partial degradation of the polymer. Ill. Biodegradable Natural Polyesters

These are polyesters that occur naturally due to the synthesis by plants, insects, and microorganisms. They play a vital role in these living organisms, helping as a protective mechanism or serving as reserve food material. Polyesters are distributed widely in nature; examples include cutin and suberin in plant cuticle (Kolattukudy,1980) and brood cells of Colletes bee (Hefetz et al., 1979), respectively, which control water permeation and prevent pathogen penetration, poly(P-hydroxybutyrate) (PHB)and other poly(hydroxya1kanoates)(PHAs) in bacteria and algae that serve as storage materials (Dawes and Senior, 1973), and polyL-malate, which is found as a metabolite in Penicillium cyclopium (Shimada et al., 1969). Cutin (Fig. 3), a biopolyester composed of hydroxy and epoxy fatty acids, is the barrier between the aerial parts of higher plants and their environment. Suberin (Fig. 4), a polymer containing aromatics and polyesters, functions as a barrier for underground parts, wound surfaces, and in a variety of internal organs (Kolattukudy, 1980). The major function of these polymers is that they constitute the structural component of extracellular biological barriers, which because of their metabolic inertness do not require constant rebuilding. Several plant pathogens and saprophytes can grow on cutin and suberin as the sole source of carbon. Cutinase produced by certain plant-pathogenic fungi is required for penetration of the plant cuticle (for review, see Kolattukudy, 1988). The other natural polyesters are discussed in greater detail in this review. IV. Biodegradable Synthetic Polyesters

Apart from the biopolymers, interest in the possible biodegradation of synthetic polymers has developed only in recent years and primarily in response to the growing problem of the waste disposal of plastics. There is a limited number of synthetic polyesters that are truly biodegradable. The only synthetic high polymers found to be biodegradable are those with aliphatic ester linkage in the main chain

BIODEGRADABLE POLYESTERS

101

FIG. 3. Cutin (a natural complex polyester occurring in higher plants). Major monomers: C,, family, CH,(CH,),,COOH, HO-CH,(CH,),,COOH, HO-CH,(CH,),CHOH(CH,),COOH [y = 8, 7, 6, or 5; x + y = 13);C,, family:-CH,(CH,),CH=CH[CH,),COOH, HO-CH,(CH,), HO-CCH,)7CIH-CfI(CH,),COOH. CH=CH(CH,),COOH, HO-CH,(CH2),CH~H(CH2),COOH, 0 OH OH

(Diamond et al., 1975; Tokiwa et al., 1990a). The prominent ones include poly (ecaprolactone), (PCL),poly(&valerolactone), poly(trimethylene carbonate), poly(lactide), poly(glycolide), poly(ethy1ene oxide), poly (vinyl alcohol), poly(tetramethy1ene succinate), and a synthetic copolyester, poly(P-methyl-6-valerolactone-co-L-lactide). Poly (ecaprolactone) is the choice synthetic polyester because of its good mechanical properties, its compatibility with many types of polymers, and because it is one of the more hydrophobic of the commercially available biodegradable polymers (Koenig and Huang, 1995). A novel method for its synthesis has been described (Duda and Penczek, 1995).Poly(glyco1ide) (Craig et al., 1975),a copolymer of glycolide and lactide, and poly(dioxanone) (Doddi et al., 1977) polyesters are being used as absorbable surgical sutures. L-Lactide (LA) is a well-known monomer used extensively for the synthesis of bioabsorbable polymers for biomedical applications (Gilding and Reed, 1979; Jamshidi et al., 1988;Reeve et a]., 1994).It was used in the synthesis of copolyesters by

102

CH. SASIKALA AND CH. V. RAMANA

I / " "

0-CH-CHCH~OC-CH-CH -@OH OCH3 CH3O

on

FIG. 4. Suberin (a natural complex polyester occurring in higher plants). Major monomers: CH,(CH,),COOH, CH, (CH2),CH20H, CHOH(CH,),COOH, HOOC(CN2), COOH. Phenolics: m = 18-30; n = 14-20.

ring-opening copolymerization with -DL-P-methyl-S-valerolactone(MV) (Fig. 5). The reaction was catalyzed by tetraphenyltin resulting in the formation of a copolyester poly(P-methyl-6-valerolactone-co-L-lactide) [P(MV-co-LA)](Nakayama et a!., 1995). Although the homopolymer poly(MV) was hardly hydrolyzed by the fungal lipase and poly(LA) exhibits a low rate of hydrolysis because of its high crystallinity, amor-

Poly(p-methyl-8-valerolactone-co-L-lactide)

FIG. 5. Synthetic preparation of copolyester poly(p-methyl-6-valerolactone-L-lactide) by copolymerizing p-methyl-6-valerolactone with L-lactide (feed ratio 50/50 mol/mol) catalyzed by tetraphenyl tin (SnPh,) catalyst.

BIODEGRADABLE POLYESTERS

103

phous P(MV-co-LA)showed 85% hydrolysis within 1 8 days and had better hydrolysability than the homopolymers (Nakayama et al., 1995). Polytetramethylene succinate [(-OCH,CH, * CH, * CH, OCO * CH,CH, * CO-),I (PTMS),a biodegradable polyester (Pranamuda et al., 1995) synthesized from 1,4-butanedioland succinate, has atbacted much attention in recent years due to its high melting point (T,= 113°C). This polyester is the best choice when compared with poly(e-caprolactone), which has a low melting point (T, = 62OC) thus limiting its use in a range of application, as compared with PHB, which has a higher melting point (T, = 175OC) but is highly crystalline. V. Poly-L-malate: A Potential Biodegradable Polyester

Poly (L-malic acid) [P(L-MA)]denotes a family of polyesters derived from L-malic acid as the building unit. By chemical synthesis, three kinds of P(L-MA)have been obtained (Fig. 6), depending on the molecular position of the ester bond. They are the ci type (I) (Ouchi and Fujino, 1989), the p type (11) (Vert, 1987), and a mixture of ci and p types (111) (Ohtani et al., 1987) (Fig. 6).Natural P(L-MA)was detected in Penicillium cyclopium as an inhibitor of acid protease (Shimada et al., 1969) and in

H-CO-FH-CH2-CO@O-$ COOH

H-C Hz-C%OH

I

CO-&O-$H-C?-CO~;OH COOH

Branched

(IV) (Nagata eta/., 1993)

FIG. 6. Natural and synthetic poly(L-malic acid).

104

CH. SASIKALA AND CH. V. RAMANA

Physarum polycephalum, a slime mold, as an inhibitor of homologous DNA polymerase (Fischer et al., 1989). The type of polyester has not been identified for l? cyclopium, but has been identified as the P type for l? polycephalum (Cammas et al., 1993) and the a-branched poly(P-LMA) type (type IV) from Aureobasidium sp. (Nagata et al., 1993). At neutral pH, ~(P-L-MA), is completely ionized (Seki et al., 1984).The function of P(P-L-MA),which is concentrated in the plasmodia of cell (Holler et al., 1992b), is to act as a transport and binding agent for the cell cycledependent storage of nuclear protein and as an agent, or precursor thereof, for the induction of the formation of sclerotia or spores at times of unfavorable growth conditions (Fischer et al., 1989; Holler et al., 1992a,b: Windisch et al., 1992). This compound has been receiving attention in the fields of molecular biology, pharmacy, and surgery and in the polymer industry (Vert, 1987). A 68-kDa extracellular glycoprotein from l? polycephalum that hydrolyses specifically P(P-L-MA)has been purified and characterized (for review, see Korherr et al., 1995). VI. PHAs: A Group of Polyesters Produced Naturally and Synthetically

PHAs are a group of polyesters having a general structure

where R is the n-alkyl pendent group of variable chain lengths and has many biological functions as summarized in Fig. 7. The short-chain (130-1 70 monomer units) complexed poly(P-hydroxybutyrate), is a ubiquitous constituent of cells associated with the plasma membranes of bacteria, plant tissue, mitochondria, and microsomes of animal cells and has many physiological functions (Reusch, 1992,1995). On the other hand, the high-molecular-weight PHAs, synthesized by

Energy Reserve FIG. 7. Functions of PHAs.

105

BIODEGRADABLE POLYESTERS

many microorganisms and found as inclusion bodies in bacteria, serve as an important storage material (Dawes and Senior, 1973) and it is these polyesters that are of high industrial significance. Apart from naturally occurring PHAs, many chemically synthesized PHAs have gained importance in recent years as biodegradable plastics. Bacterially produced polymers are (R)-3-hydroxyalkanoates that are optically active, easily biodegradable thermoplastic with a melting temperature around 18OOC and show properties similar to those of some of the synthetic nonbiodegradable polyesters (Table 1). Many reviews have been published on the occurrence, metabolism, metabolic role, and industrial use of bacterially produced polyesters [Dawes and Senior, 1973; Shively, 1974; Howells, 1982; Winton, 1985; Holmes, 1985b, 1988; Dawes, 1988;

TABLE I PROPERTIESOF SOME OF THE PHAS COMPARED WITH THE SYNTHETIC POLYESTERS, POLYPROPYLENE (PP), POLY(ETHYLENE TEREPHTHALATE) (PETP)AND NYLON 6,6 (N6,6)a P(HB-HV) Property

PHB

PHV

(4-29%)

PHO

PP

PETP

N6,6

Crystalline melting point (“C]

175

107

157-102

61

176

267

265

30-50

40-60

Crystallinity (%) Molecular weight M, (x105)

80

80

69-39

30

70

1-8

2

6

5

2-7

Glass transition temperature Tg(“C)

5-15

-16

2 to -8

-35

-10

69

50

Density (g cm-3)

1.250

1.2

1.2

1.0

0.905

1.385

1.14

Water uptake (wt %)

0.2

0.0

0.4

4.5

Flexural modules (GPal Tensile strength, (MPa) Extension to break (YO)

4.0

1.7

2.9

2.8

38

70

83

100

60

LJV resistance Solvent resistance Biodegradability Oxygen permeability (crn3m-2atn-ldap*)

40 6

Good

Good

Poor

Poor

+ 45

+

36.22

6-1 0

8-10

300-450

400

Good

Good

Poor

+

+

Good

1700

Source: King (19821, Winton (19851, Howells (1982), Brand1 et a1. (1990), Pearce and Marchessault (1994a,b), Jesudason and Marchessault (1994), and Poirier et a]. (1993).

106

CH. SASIKALA AND CH. V. RAMANA

Byrom, 1987, 1992; Marchessault et al., 1988; and from 1990 to 1995 (see Table II)].

TABLE I1 SOME OF THE IMPORTANT REVIEW ~~~~

ARTICLES PUBLISHED DURING1990-1995

ON

PHAS

~

Brief description/topics covered in the review

Reference

Occurrence, isolationlanalysis, metabolism, metabolic role and industrial uses of bacterial PHAs

Anderson and Dawes (1990)

Microbial formation, characterization, properties and biodegradation of PHAs

Brandl et 01. (1990)

An overview on the novel microbial polymers

Dawes (1990) Lenz et al.(1991)

Polyesters production by microorganisms Structure and organization of PHA-biosynthetic genes from a wide range of different bacteria is provided

Steinbuchel et a]. (1992)

Occurrence and possible role of PHAs in oxygenic phototrophic bacteria (cyanobacteria]

Stal (1992)

Storage polymers in prokaryotes

Dawes (1992)

Industrial production of PHB

Hrabak (1992a)

The mechanism of biodegradation of natural and synthetic polymers (includes polyesters)

Lenz (1993)

Thermal, crystallization behavior, mechanical properties, morphology and biodegradability of polymer blends containing PHAs Chemical synthesis of biodegradable polymers

Verhoogt et al. (1994)

Requirements of bacterial polyesters to be used as substrates for conventional plastics in future

Takashi (1994) Hanggi (1995)

Production of PHAs particularly by using trangenic plants

Poirier et a]. (1995)

Diversity of bacterial PHAs

Steinbuchel and Valentin (1995)

Analysis of PHAs produced by free-living nitrogen fixing microorganisms (Note)

Itzigsohn et al. (1995)

Strategies for the sustainable production of new biodegradable polyesters in higher plants. Degradation and applications of PHAs

van der Leij and Witholt (1995)

A dynamic and versatile molecule-PHA Bacterial production of PHAs and several processes recently developed and employed for their production

Reusch (1995)

Physical properties of bacterial PHAs Occurrence, synthesis, and production of PHAs by anoxygenic phototrophic bacteria

de Koning (1995)

Brandl et al. (1995a) Lee and Chang (1995b)

Sasikala (1996)

BIODEGRADABLE POLYESTERS

107

A. TERMINOLOGY

Many new natural and synthetic hydroxyalkanoic acids have been recently discovered and there is an overlapping of the terminology used by various researchers. To overcome the ambiguities, Steinbuchel et al. (1992) have recommended the following terminology (abbreviations to be used for individual hydroxyalkanoates are given in Table 15) 1. Poly(3-hydroxyalkanoates)[P(3HA)] is used for general homopolyesters. 2. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]is used for copolyesters. 3. Poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-3-hydroxyvalerate) [P(~HB-co-~HP-co-~HV)] is used for terpolyesters. 4. Short chain length (SCL), medium chain length (MCL), and long chain length (LCL) refer to those hydroxyalkanoates that consist of 3-5, 6-14, and >14 carbons, respectively, and these are used as subscripts, for example, PHA,,,, PHA,, and PHA,,,.

B. CHEMICAL SYNTHESIS OF PHAs

The synthesis of structural and stereochemical isomers of PHAs can be carried out by the ring-opening polymerization reactions of appropriate four-membered p-lactones in the presence of a catalyst (Table 111). These synthetic analogs are useful for understanding the physical properties and biological activity (including biodegradation) of PHAs from bacterial origin. Both homopolymers (PHB and PHV) (Teranishi et al., 1974; Iida et al., 1977; Kricheldorf et al., 1994) and copolymers [P(HBco-HV]] (Bloembergen et a]., 1987) could be chemically synthesized from p-lactones and their molecular weight is comparable with those of natural PHAs (Table IV). Extensive studies have been performed on anionic ring-opening polymerization of p-propiolactone (Inove et al., 1961; Ohse et al., 1962; Shiota et al., 1967; Yamashita et al., 1968) and c-caprolactone (Perret and Skoulios, 1972). In the synthetic polymerization reaction, ring opening of a lactone may proceed by bond breaking either between the carbonyl carbon and oxygen atom of the p-lactone ring (acyl cleavage, path b in Fig. 8) attacked by strong nucleophiles, resulting in retention of configuration as shown in path (a) (Fig. 8),or by bond breaking between the P-carbon and oxygen atom (alkyl cleavage, path a in Fig. 8) attacked by weak nucleophiles, which could lead to either inversion of configuration (path b) or recemization. Physicochemical and biological properties of polymers are often dependent on the composition and distribution of enantiomeric units in the polymer chain.

TABLE 111 POLYMERIZATION OF p-ALKYL-p-PROPIOLACTONES WITH VARIOUS CATALYSTS

Alkyl CH,

Catalyst (mol ratio)

Polymerization time (days)

Total yield

(%I

7

36

AlEt,-HZOepichlorohydrin

M"

Melting temperature PC)

Reference

nd

nd

Teranishi et al. (1974)

(1:l:l)

AlEt,-H,Ooxetane (1:l:l)

7

nd

48

nd

Teranishi et al. Teranishi et al.

(1974)

7

53

nd

nd

5

60

36,700

277K

7

78

400,000

160-170

7

63

410,000

163

Gross et al.

5

72

20,000

nd

Zhang et al.

7

62-100

vary

nd

(1:O. 7)

5

a4

34,000

135.6

Kemnitzer eta]. (1992) Tanahashi and Doi (1991)

AlEt,-H,O

7

30

240,000

nd

Zhang et al.

[-AlEtO-],

14

50

190,000

nd

Zhang et al.

Ethylaluminoxane (EAO) Al(0-i-Pr), (alluminum triisopropoxide) tert-Butylaluminoxanes 1-Ethoxy-3chlorotetrabutyl distannoxane Methylaluminoxane

14

51

100,000

nd

Gross et al.

A1Et3-H,0

(1974)

(1:l) (1:0.6)

Iriondo et al. (1995)

Iida et al. (1977)

R,Al-H,O

(1988)

(1:l)

ZnEt,-H,O

(1990)

(1:0.6)

(1990)

(1:l)

(1990) (1988)

15

100

nd

nd

Kricheldorf et UI.

2

25-100

vary

nd

(1988)

Lenz et al. (1995)

4

(hr)

434,000

99

nd

Hori et 01. (1995)

7

55

vary

vary

Hocking and Marchessault

4

60

nd

nd

>90

>100,000

vary

Kemnitzer eta]. (1993a) Hori et ul.

(1993)

Distannoxane

16 (fd

(1993)

(continues)

108

TABLE 111-Continued

Alkyl

C,H,

i-C,H,

Polymer izationtime (days)

Total yield

(%I

M"

Melting temperature VC)

KOH

24

61

nd

nd

H,O-AlEt, (1:l) (EtAlO),

13

9

nd

nd

7

78

400,000

102-110

AlEt,-H,O

30

49

nd

nd

AlEt3-H,0epichloro hydrin (1:1:1) (EtAlO),

30

31.5

nd

87-89

22

49

100,000

72-79

(cY,P,y,G-Tetraphenylporphinato) aluminum chloride (TPPAlC1) (EtAlO),

13

100

2,800

nd

32

21

10,000

69-73

TPPAlCl

14

86

6,200

nd

4 (hr)

99

279,000

119

(hr)

91

166,000

47

4 (hr)

96

313,000

113

Catalyst (mol ratio)

(1:1)

t-C,H,

Copolymers P((R)-~-HB-co-~HV) distannoxane catalysts

4

P((R)-~HB-co-~HV) distannoxane catalysts

Reference Deffieux and Boileau (1976) Teranishi et al. (1974) Iida et al. (1977) Teranishi et al. (1974) Teranishi et al. (1974) Iida et al. (1977) Yasuda et al. (1983)

Iida et al. (1977) Yasuda et al. (1983) Hori et al. 1995 Abe et al. (1995a) Hori et al. (1995)

Note. nd, Not determined.

Cleavage of ecaprolactone occurs only at the acyl-oxygen bond (Fig. 8, path b) and propagation thus proceeds through alkoxide group (Deffieux and Boileau, 1976). f3-Propiolactone polymerizes to form a liquid or solid polyesters of low molecular weight by heating up to 130-150°C in the presence of ferric chloride or 60-80°C in the presence of sulfuric acid (Gresham et al., 1948) or by the action of strong alkali at room temperature (Deffieux and Boileau, 1976),high-molecular-weight polyesters 109

110

CH. SASIKALA AND CH.

V. RAMANA

TABLE IV

MOLECULAR WEIGHT ANALYSIS OF SYNTHETIC AND NATURAL PHAsa ~

M,x 10-3

M,x 10-3

MJM,

n (dl/g)

glmol

n (dllg)

glmol

Natural Synthetic

2.17 1.46

293 177

3.3 1.4

161 21.4

2.1 5.6

Natural Synthetic

1.78

190

1.99

52.1

3.4

96-4 94-6 92-8 83-17 80-20 68-32 5-95 0-100

Natural Synthetic Natural Synthetic Natural Natural Natural Synthetic Synthetic Synthetic

2.17 1.46 1.03 1.69 1.16 3.42 1.59 1.49 1.64 1.78

293 177 113 213 131 526 197

P(3HB-co-HP) 57:43 9O:lO

Natural Natural

PHA PHB

PHV

P (HB-co-HV) 100-0 Yo

(mol YO)

181

205 228

21.4

2.1 5.6

51.7 35.5 33.8 52.1

3.6 3.5 4.1 3.5

28.0 36.5

2.4 2.1

161

source: Bloembergen et ol. (1989a,b);Shimamura et al. (1994),and Hiramitsu and Doi (1993).

by y-ray irradiation at room temperature, or by using various metal alkoxides at room temperature (Table 111). Inove and co-workers (1961) were the first to propose the use of organometallic compounds as catalysts for polymerization. In regard to the catalytic activity for the lactone polymerization, they classified organometallics into the following three groups: Group I. Catalyst available without any cocatalyst (e.g., compounds of Li, Na, K, and Mg). Group 11. Catalyst available only in the presence of cocatalyst (e.g., compounds of Zn, Cd, and Al). Group 111. Ineffective compounds (e.g., compounds of Hg and Sn). Only a very limited type of organoaluminum catalyst, not organozinc catalyst, gave an isotactic polymer to a varying degree of stereospecificity. Typical anionic catalysts gave no polymer and typical cationic and

BIODEGRADABLE POLYESTERS

111

R p-Propiolactone

FIG. 8 . Ring-opening polymerization reactions of p-propiolactones resulting in various stereochemical isomers of PHAs.

some coordinated catalysts gave only amorphous polymers (Teranishi et al., 1974). Crystalline isotactic poly(P-alkyl-P-propiolactones) were obtained by the fractionation of the raw polymer obtained from D , L - ~ alkyl-p-propiolactones(alkyl:methyl, ethyl, and isopropyl) with AlEt,-H,O and AlEt,-H,O-epichlorohydrin catalyst systems (Teranishi et al., 1974). (a-P-y-E-Tetraphenyl porphinato)aluminum chloride [(TPP)AlCl] was a very good catalyst for the polymerization of P-propiolactone and p-butyrolactone, which gave corresponding polyesters with narrow molecular weight (Yasuda et al., 1983).The polymerization of p-lactone with (TPP)AlCl proceeds as follows (Fig. 9): (i)the first step in the polymerization is the attack of chlorine on the carbon atom adjacent to the ester oxygen, (ii)leading to the insertion of p-lactone with alkyl-oxygen scission into the aluminum-chloride bond to give a (tetraphenyl porphinato)aluminum carboxylate, and (iii) three attacks subsequently as a nucleophilic on p-lactone with alkyl-oxygen scission to regenerate a (porphinato) aluminum carboxylate. Repetition of this reaction leads to the living polymerization of p-lactone with the aluminum carboxylate group as the growing and (iv) to give the polymer with narrow molecular weight distribution (Yasuda et al., 1983). (TPP)AlCl was also used as a catalyst for the production of polyester-polyether or polyester-polyester block copolymers with narrow molecular weight distribution by adding epoxide or p-lactone as the second monomer to this living polyester (Fig. 10) (Yasuda et al., 1984).

s

R TPPAlCl

TPPAL-0-C-CH2CHCI

*Qo0

isl" 0

e - Lactone

0 3 CD

n

(4) FIG. 9. Polymerizationof p-lactone using (a,P,y,G-tetraphenylporphinato) aluminum chloride [(TPP)AlCl]catalyst.

Epoxide ? q B TPPAISTO-COCH~CH0x0-CH~CH CI L

L,

,

I

3.

Polyester - Polyester Block Copolymers

V

113

BIODEGRADABLE POLYESTERS

Aluminum-based catalyst produced a highly stereoregular, crystalline polymer fraction, whereas the zinc catalyst produced only atactic, amorphous P([R,S]-HB)(Gross et al., 1988).P([R,Sl-HB)synthesized from the recemic P-lactone has, of course, a diastereomeric relationship to the optically pure polymer produced by bacteria from P-hydroxybutyric acid (Gross et al., 1988). High-molecular-weight PHB and PHV were synthesized from racemic P-butyrolactone and p-valerolactone, respectively, with an oligomeric alumoxane catalyst obtained from a reaction of AlEt, with H,O (Bloembergen et al., 1989a). In this chemical synthesis of PHV (Bloembergen et al., 1989a), propionaldehyde and malonic acid were reacted first to form p-pentenoic acid via the Knoevenager reaction, followed by P-brominationand lactonization yielding p-valerolactone,which when polymerized by a suitable catalyst yielded PHV as shown in Fig. 11. Copolymer P(HB-co-HV) with varied monomeric compositions of HB/HV can be prepared (Bloembergenet al., 1987,1989b)fiom (k)p-butyrolactone-(*) p-valerolactone mixtures by using alumoxane catalyst from a 1:l molar ratio of AlEt, and H,O (Fig. 12). This synthetic approach gives P(HB-co-HV)samples with the composition range 0-100% HV.

CY.

C ~ C H ~ C H OC H ~ ( C O O H ) ~ +

Propionaldehyde Malonic acid

co2

A

> C~CH~CH=CHCOOH

1

HBdg)

C H3CH2CHBrCH2COOH

I cH3cH2r7 3-bromopentenoicacid

C HCkj/ H2

O-Valerolactone -0

Poly (p-hydroxyvalerate)

FIG. 11. Stepwise reaction pathway of synthetic preparation of p-valerolactone and its polymerization to poly(p-hydroxyvalerate).

CH. SASIKALA AND CH. V. RAMANA

114 CH3

Butyrolactone

AIEt3/ H20 (1: 1)

Valerolactone

--/i~ 44EL c H2-c00

Wooj Y

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)

FIG. 12. Synthetic preparation of copolyester poly(3-hydroxybutyrate-co-hydroxyvalerate) by copolymerizing (+)P-butyrolactone with (*)P-valerolactone in the presence of alumoxane catalyst.

The mode of ring opening for an AlEt,-H,O (1:l) catalyst involved primarily cleavage of the bond between the @-carbonand oxygen of the lactone (alkyl cleavage), with 93% R and 7% S configuration. In contrast, ethylaluminoxane catalyst proceeded primarily by bond breakage between the carbonyl carbon and oxygen of the lactone (acyl cleavage) with 15% R and 85% S configuration and the mode of ring opening with the ZnEt,-H,O (1:0.6) catalyst occurred by acyl cleavage with 100% S configuration (Zhang et al., 1990). Similarly, aluminum triisopropoxide [Al(O-i-Pr),]catalyst occurred by acyl cleavage (Kricheldorf et al., 1988). The polymerization of racemic @-butyrolactonewith AlEt,-H,O catalyst leads to a mixture of crystalline isotactic PHB (with a melt temperature at 16O-17O0C) and amorphous atactic PHB (Yamashita et al., 1963; Agostini et al., 1971; Shelton et al., 1971; Teranishi et al., 1974; Iida et al., 1977). On the other hand, the ZnEt,-H,O catalyst produces only amorphous atactic PHB (Iida et d, 1977; Tanahashi and Doi, 1991). The first synthesis of PHB having properties similar to those of the natural polymer (crystallinity, melting point, and morphology) has been reported by Agostini et ~ l (1971) . and Shelton et al. (1971). Random stereopolymers of PHB can be prepared from @-butyrolactone using a diethyl zinc-water catalyst system (Kemnitzer et al., 1992). The PHB stereoisomers produced had a R repeat unit composition of 50-90%. Sn(IV) organometallic compounds were also used for ringopening polymerization of lactones for the production of PHB (Kemnitzer et al., 1993b). The synthesis of P[(R)-HB] by ring-opening polymerization of R-or S-butyrolactone using aluminum, or zinc-based catalysts yields a low-molecular-weight polymer, whereas with distannoxane catalysts, a high-molecular-weight (Mn>lOOOOO)polymer was prepared (Hori et al., 1993).

115

BIODEGRADABLE POLYESTERS

Although synthetic PHAs have received little attention, it is interesting to prepare such polymers for at least two reasons. If they are prepared synthetically, it is possible, in principle, to obtain a great variety of sterocopolymers. The following structures can thus be considered: -RRSRSSSR (atactic) (Structure 1) -RRRRRR-(isotactic, polyenantiomer) (Structure 2) -RRRRRR-(isotactic, polyracemate) (Structure 3)

-ssssssIntermediate structures between Structures (1)and ( 2 ) or ( 2 ) and (3) can also be produced. In addition, PHB synthetic analogs have been viewed as interesting model systems to investigate effects of stereochemical and morphological parameters on the biodegradation kinetics. Some of the patents on the synthetic preparation of PHAs are shown in Table V. C. NATURALLY OCCURRING PHAs 1. Occurrence of PHAs among Various Microorganisms

Since the first discovery of PHAs in Bacillus megaterium (Lemoigne, 1927), a number of other bacteria have been shown to produce polyesters within the cells under certain stress conditions. As many as 60

different bacteria have been shown to accumulate PHAs (Brandl et al., 1990) phylogenetically representing both Eubacteria and Archaebacteria (Fernandez-Castillo et al., 1986). Apart from the PHA accumulation by

TABLE V PATENTS ON CHEMICAL SYNTHESIS OF PHAs Assignee

Patent number

Year

Kumagai and Doi

JP 06,65,345

1994

Hori et al.

EP 612 780

1994

Brief Description Chemical synthesis of polyester films using zinc-based catalyst Production of highmolecular-weight PHB by ring opening polymerization of p-butyrolactone i n the presence of tin compounds

116

CH. SASIKALA AND CH. V. RAMANA

bacteria, transgenic plants can also accumulate these polymers in considerable amounts.

a . Phototrophs. Oxygenic phototrophic bacteria (previously referred as blue-green algae or cyanobacteria), which derive energy from sunlight, fix carbon dioxide via the reductive pentose phosphate pathway and deposit a part of the fixed carbon as glycogen as a carbodenergy reserve. Because these bacteria do not possess the tricarboxylic acid cycle (TCA cycle),which helps in the accumulation of PHAs in other bacteria, PHA is not a common energy storage compound in this group of organisms. Nevertheless, certain oxygenic phototrophic bacteria accumulate PHB in small quantities, which was first observed by Carr (1966) in Chlorofloeafritschii. Later, a number of other oxygenic phototrophic bacteria were also found to accumulate PHB (Table VI). The PHA content (Table VII) in this group of microorganisms is usually less than 1% (Suzuki et al., 1994; Capon et a]., 1983) and not more than 2.5-9% (of cellular dry wt) (Vincenzini et al., 1990; Stal, 1992), and only one type of polyester is known to occur [i.e.,PHB] (for review, see Stal, 1992). The other group of photosynthetic microorganisms that can accumulate PHAs are the anoxygenic phototrophic bacteria (APB). Studies with a number of isolates of APB (Liebergesell eta]., 1991) suggest that PHA formation is a common phenomenon among this group of microorganisms. However, considerable variation in the PHA accumulation was observed in different species of APB and the purple nonsulfur bacteria are certainly better producers of PHAs compared to purple sulfur and TABLE VI OCCURRENCE OF PHAs IN OXYGENIC PHOTOTROPHIC BACTERIA^

(Capable of PHA accumulation) Chlorogloea fritschii, Aphanocapsa sp., Aphanothece sp., Microcoleus chthonoplastes, Gloeothece sp., Nostoc spp., Microcystis ueruginosa, Oscillatoria sp., 0.Iimosa, Spirulina platensis, S. subsalsa, S. maxima, S. laxissima, S. jenneri, Anacystis cyanea, Microcoleus sp., Lyngbyu aestuarii, Schizothrix sp.. Calcicola, Trichodesmium thiebautii [Incapable of PHA accumulation) Anabaena variubilis, Oscillatoria limnetica, Microcoleus chthonoplastes, Phormidium spp., Plectonema boryonum, Crinalium epipsammum, Synechococcus sp., Nostoc sp., Trichodesmium erythraeum 0 Source: Carr (19661, Campbell et al. (1982), Jensen (1980), Jensen and Baxter (19811, Jensen and Sicko (19711, Rippka et al. (1971), Stal et al. (1990), Capon et al. (1983), Siddiqui et 01. (19921, Oren and Shilo (1979), Vincenzini stal. (19901, Allen (1984), Sicko-Goad (1982), and Stal (1992).

117

BIODEGRADABLE POLYESTERS

green sulfur bacteria (Table VII). Among the purple nonsulfur bacteria, Rhodobacter sphaeroides showed a limited flexibility in its ability to form PHAs with varying compositions (Brandl et a]., 1991) compared to TABLE VII POLY (P-HYDROXYALKANOATE) CONTENT OF PHOTOTROPHIC ~

~

BACTERIA^

~~~

PHA content of cellular dry wt)

Composition 3HB

3HV

6.0 0.3 0.4

100 100 100

0 0

(Oh

Organism

(Oxygenicphototrophic bacteria) Sprillunia platensis S. laxissima S. jenneri (Anoxygenicphototrophic bacteria) Purple nonsulfur bacteria Rb. capsulatus Rb. sphaeroides Rps. palustris Rps. acidophila Rps. blastica Rps. viridis R. fulvum R. molischianum R. rubrum R. centenum Rm. vannielii Rc. gelatinosus Rc. tenuis Purple sulfur bacteria Chromatium vinosum C. min u tissim urn C. purpuratum C. okenii C. warmingii Lamprocys tis roseopersicina Thiocapsa pfennigii Amoebobacter roseus A . pendens Thiocystis violacea Ectothiorhodospira mobilis E. vacuolata E. shaposhnikovii

Carbon source

co, co, CO,

0

Acetate Acetate Acetate Valerate Acetate Acetate Acetate Propionate Acetate Butyrate Valerate Acetate Valerate

33.7 69.9 15.2 51.8 31.5 2.0 37.2 16.5 41.2 30.0 12.2 34.1 22.1

97.0 92.7 100 8.9 96.0 100 100 100 95.8

2.1 7.3 0 91.1 4.0 0

32.4 97.4 29.4

67.6 2.6 70.6

Acetate Acetate Acetate Acetate Acetate Acetate

58.0 36.0 8.9 12.8 22.9 27.1

100 100 100 100 100 100

Acetate Acetate Acetate Acetate Acetate

36.2 32.6 30.8 83.0 57.5

100 100 100 100 100

Acetate Acetate

35.6

100 100

29.3

0

0 4.2

0 0

0 0 0 0

0 0 0

0 0

0 0

~

nSource: Liebergesell et a1. (1991), Brandl et a1.(1989, 1991), Ulmer et al. (1994), and Philippis et al. (1992).

118

CH. SASIKALA AND CH. V. RAMANA

Rhodospirillum rubrum (Brandl et al., 1989; Gross et al., 1989a,b). Anoxygenic phototrophic bacteria can accumulate three different types of PHAs: (i) homopolyesters in which the monomers are 3-hydroxyalkanoates (3HA) of various chain lengths (Stanier et al., 1959; Ulmer et al., 1994; Liebergesell et al., 1991); [ii) copolyester P(3HB-co-3HV) in which the monomers are 3HB and 3HV (Brandl et al., 1989);and (iii) terpolyester P(3HB-co-3HP-co-3HV)in which the monomers are 3HB, 3HP, and 3HV (Ballistreri et al., 1995).

b. Chemotrophs. A number of chemotrophs accumulate considerable amounts of PHAs (Table VIII). A large number of Pseudomonas spp. accumulate PHAs (Haywood et al., 1989b; Timm and Steinbuchel 1930; Table 1x1, which has taxonomic significance (Huisman et al., 1989).The type of PHAs produced [Table X) and some of the hydroxy monomers produced by different Pseudomonas spp. are shown in Table IX. The uniqueness of this group of microorganisms is their ability to synthesize a variety of PHAs containing functional groups, such as phenyl (Fritzsche olefin (Preusting et al., 1990), chloride (Doi and Abe, 1990), et al., 1990~1, and fluoride (Abe et al., 1990), obtained from the growth of I? oleovorans on functionalized substrates. One of the most extensively studied species is I? oleovorans. This organism, apart from accumulation of substantial amounts of PHAs (Table IX) as intracellular granules, has the ability to synthesize a wide range of monomer units incorporated into PHAs. Hydrogenophage pseudoflava [formerly Pseudomonas pseudoflava) was able to accumulate large amounts of copolyesters when grown on mixed substrates of glucose and lactose in a batch fermentation [Choi et al., 1995). Staphylococci are gram-positive chemoorganotrophic facultative anaerobic bacteria; mostly opportunistic pathogens of human and animal skin can accumulate PHB in their cells. High levels (about 1.33 pg/mg dry wt) of PHB accumulated in the cells of S. xylosus, S. aureus, and S. epidermidis (Szewczyk and Mikuki, 1989).Accumulation of PHB in the cells of Staphylococci was first demonstrated by Ivler (1965) and was confirmed by many others (Mikucki et al., 1989; Szewczyk and Mikucki, 1989; for review, see Szewczyk, 1992). Alcaligenes eutrophus is the most extensively studied organism of both basic and applied research on PHAs. This organism can accumulate up to 96% (w/w) of PHA (Pedros-Alio ef a]., 1985). Alcaligenes latus is another species that has received attention in recent years for its ability to accumulate PHAs (Table XI). These organisms can accumulate (a) homopolyesters in which the monomers are of 3HA, 4HA, and 5HA; (b) copolyesters of P(3HB-co-3HV),P(3HB-co-3HV1, or (3HP-co-HV); and (c) terpolyesters of P(3HP-co-3HB-co-3HV)when supplied with various single or mixed carbon sources (Table XI).

119

BIODEGRADABLE POLYESTERS TABLE VIII CONTENT OF CHEMOTROPHS~ POLY(p-HYDROXYALKANOATE)

Organism

Alcaligenes eutrophus A . hydrogenophilus A. latus Aquaspirillum autotrophicum Azotobacter sp. Bacillus megaterium Bacillus cereus Bacillus sp. Beggiatoa sp. Chromobacterium violaceum Corynebacterium hydrocarboxydans Haloferax mediterranei Hydrogenophage pseudoflava Janthin obacterium lividum Legionella pnemophila Leptothrix sp. Methylobacterium sp. Methylobacterium rhodesian urn Methylocystis Methylosinus Micrococcus halodenitrificans Mycoplane rubra Nocardia lucida Paracoccus denitrificans Pseudomonas oleovorans I? pseudoflava I? capacia Rhodococcus sp. Seliberia carboxydohydrogen a Streptomyces sp. Thiobacillus sp.

Carbon source

PHA content (% of cellular dry wt)

Composition 3HB

Gluconate Valerate Gluconate Valerate

91.5 92.6 75.7 44.6

Valerate Valerate Glucose Glucose Acetate Valerate

67.3 40 13 25 57.0 43.8

Acetate Starch Glucose + butyrolactone Valerate Complex Pyruvate Methanol Methanol

43.0 67.0 47.0 70.0

Methane Methane Glucose

70.0 25.0 21.0

Methanol Acetate Gluconate

25.0 20 78.9

100 47 100

Valerate Glucose Glucose Acetate

49 43 64 20.0 30.0

Vary

Glucose Valerate

35.0 87.7

3HV 0 77.1

100 22.9 100 66.1

33.9

4.7

95.3

Vary 50 100 100

50

0

100

21

50

50

67.0 71.0

87.0

0

55.8

28.3

71.7

100

0 0

0

9.0 0

53 0

45

30 31

8.4

69

91.6

Source: Kannan and Rehacek (1970),Gude et al. (1981),Williamson and Wilkinson (1958),Powell al. (1983), Asenjo and Suk (19861, Haywood et al. (19911,Steinbuchel et al. (1993); Volova e t a ] . (1994a,bl, B. A. Ramsay et al. (1990), Breuer et al. (19951, Choi et 01. [1995),and Foellner et al. (1995). et

TABLE IX COMPOSITION OF PHAS ACCUMULATED BY PSEWJOMONAS SPP. FROM VARIOUS CARBON SOURCESa ~~~

~~

~

3-Hydroxyacid monomers in PHA (mol %] PHA Species

Carbon source

(%Wt/Wt)

C4

l? cepocia

Propionate

44.0

70.0

l? fluorescens

1,3-Butanediol 3-Hydroxybutyrate Octanoate Glucose Pyruvate

15.1 17.9 30.5 37.5 46.3

17.7 15.1

f? morginalis

1.3-Butanediol Octanoate

11.9 31.4

I? mendocina

1,3-Butanediol Octanoate

19.3 13.5

Hexane

2 11.4 25.3 24.3 21.9 14.3 5.8 5.0

cr

N 0

r! oleovorans

Heptane Octane Nonane Decane Undecane Dodecane Hexanoate

1.1

7.8

1.2 3.9

C5

C6

C7

c8

C9

C10

-

-

1.5 3.5 4.2 1.6 3.9

9.6 15.7 95.8 9.4 19.1

71.2 65.7 68.5 72.7

5.9 11.7

33.7 76.8

52.6 6.4

1.5 3.6

19.9 69.6

77.4 23.0

-

-

-

-

-

100

-

11

-

89

-

-

-

10

66

24

2 72 81.5

31 22 17.5

36 3 0.3

3

-

-

-

-

-

-

-

C11

C12

Heptanoate

22

-

<1 0.8

<1 1.2

-

Octanoate

41

<1

49

-

75 86.1 5

17 4.3

Nonanoate

6 9.6 <1

Decanoate

37

<1

6.1 44 62.9

2.8 47 11.1

Octanol Nonanol Decanol 5-Phenylvalerateb

15 33 6 25

-

1.1 7 19.6 6

91

3

-

-

-

63

37

-

-

Glucose

16.9 8.5

-

<0.1 1

6.9 11.0

74.3 66.0

Fructose Glycerol Octanoate Oleate

24.5 22.0 22.3

-

0.5 1.7 6 4.4

12.6 21.4 92.0 33.5

70.8 63.6 2.0 32.2

75.0 23.0

9.0 7.0

-

I! putida

I? resinovomns

Octanoate Hexanoate

37.2

1.0 8.0

15 62

0.2

-

OLageveen et al. (19881, Brand1 eta]. (19881,Fritzsche and Lenz (19901, Huijberts et 01. (1992,1994),Gross et al. (1989a),Haywood et et al. (19951, Ramsay et al. (1989, 1992), and Eggink et al. (1992). 100% 3-Hydroxy-5-phenylvalerate (Fritzsche and Lenz, 1990; Curley et al., 1996).

*

al. (i989a), Lee

122

CH. SASIKALA AND CH. V. RAMANA TABLE X

TYPESOF PHAS PRODUCED BY PSEUDOMONAS OLEOVORANS DEPENDENT ON SUBSTRATE 1. 2. 3. 4.

5.

A homopolymer from a single good substrate-I? oleovorans with 5phenylpentanoate (Fritzsche et al. 1 9 9 0 ~ ) . A random copolyester from a single good substrate-4 oleovorans with n-octanoate (Brand1 et al., 1988). A random copolyester from two good substrates-I? oleovorans with n-octanoate + n-nonanoate (Ballistreri et al., 1990). A mixture of two different polymers from two good substrates-I? oleovorans with 5-phenylpentanoate + n-nonanoate (Kim et al., 1991). A random copolymer from a good + a poor (nonproducing) substrate-I? oleovorans with n-octanoate + 6-methyloctanoate (Fritzsche et al., 199Ob).

Free-living and symbiotic nitrogen-fixing bacteria can also accumulate PHAs in considerable amounts (Table XII) and Azotobacter beijerinckii (Dawes and Senior, 1973; Jackson and Dawes, 1976; Senior and Dawes, 1971, 1973; Ritchie et al., 1971; Senior et al., 1972; Stockdale et al., 1968; Ward et al., 1977) and Az. vinelandii (Manchak and Page, 1994; Chen and Page, 1994; Page and Cornish, 1993; Page, 1989,1990, 1992a,b,c, 1993; Page et al., 1992; Page and Knosp, 1989;Jurtshuk et al., 1968) have been extensively used in the past few years for studies on PHB synthesis, enzymology, and control. A stable capsule-negative mutant of Az. vinelandii strain UWD (ATCC 53799) was derived as a result of transformation of Az. vinelandii UW (capsule-negative strain; ATCC 13705) with Az. vinelandii 113 DNA (strain 113 was derived from a capsule-forming ATCC strain 12837 by treating with N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis), This strain produced large quantities of PHB during exponential growth (Page and Knosp, 1989) and the kinetic properties of the enzymes (3-ketothiolase, acetoacetyl-CoA reductase, and P-hydroxybutyrate dehydrogenase) involved in the PHA synthesis were studied (Manchak and Page, 1994). This mutant has a defective NADH oxidase and PHB biosynthesis acts as an alternative electron sink with growth and simultaneous accumulation of PHB (65-70% PHB dry wt after 24 hr of aerobic growth) without nutrient limitation. Furthermore, this strain seems to be useful for the production of PHAs particularly from unrefined sugars and molasses (Page 1992a,b; Page et al., 1992). Nitrogen-fixing Azospirillum brasilense accumulated only homopolymers up to 70% of cell dry wt (Okon and Itzigsohn, 1992; Itzigsohn et al., 1995). Other nitrogen fixers, viz. Rhizobium meliloti (58% w/w; Tombolini and Nuti, 1989), Rhizobium japonicum (Stovall

TABLE XI COMPVSITION OF PHAS ACCUMULATED BY ALCALIGENES SPP. FROM VARIOUS CARBVN COMPOrnvDS

PHA Species

A . eutropbus

(YOwtlwt)

C3

C4

C5

C6

Glucose Fructose Sucrose (S) S + 3-hydroxy propionate (1:l) Pentanoate 4-chloro butyrate

16.9 24.5 60.0 29.0

-

-

<0.1

-

100 26

-

36.0 27.0

-

10 89

90

-

4-HydrOxy

30.0

-

43.0 8.0 29.0

Carbon source

butyrate (4HB) 4HB+HB 1,4-Butanediol 4-chloro benzoate + benzoate 5-ChlOrO benzoate 5-chloro benzoate + pentanoate 4-hydrOXy butyrate + pentanoate

A . latus

Sucrose ( S ) S + 3-hydroxy propionate (1:1)

4-Hydroxyacid monomers in PHA (mol YO)

3-Hydroxyacid monomers in PHA (mol %) C7

C8

C10

c4

c5

11

-

67

33

-

-

82 75 63

18 25 37

-

1.0

-

24

25

-

19.0

-

26

65

-

18.0

-

32

23

-

45

-

60.0 26.0

100 29

-

5-Hydroxyacid monomers in PHA (mol %) c5

74

-

71

Source: Shimamura et al. (1994,19951, Hiramitsu and Doi (19931,Kunioka et al. (1988,3989),Doi et al.(1987a,1988a,1989b,1990a),and Steinbuchel (1995).

124

CH. SASIKALA AND CH. V. RAMANA TABLE XI1 PRODUCTION OF PHAS BY SOME OF THE NITROGEN-FIXING BACTERJA

Organism

Azotobacter vinelandii (mutant)

Carbon

PHA

Hv

(YO dry wt)

(molY01

Reference

Beet molasses, (BM) BM + propionate BM + butyrate BM + valerate BM + hexanoate BM + heptanoate BM + octanoate Glucose Glucose (G)

63

0

61 66 65 65 64 63 77 50

0 0 16 0 4.7 0 0 0

G + valerate

94

18

G1u cose G + propionate G + butyrate G + valerate G + hexanoate G + heptanoate G + octanoate G + nonanoate G + 2-pentenoate Maltose Corn syrup Cane molasses Raw sugar Molasses

76 75 78 78 77 75 79 73 57 30 41 59 37 66

0

Corn syrup Black strap molasses Cooking molasses Refiners molasses Beet molasses Malt extract Alpechin

57 54 54 56 60 66 70

Glucose

42

0

Martinez-Toledo et 01. (1995) Stockdale et al.

70

0

Nuti et al.

70 22

0

Ward et al. (1977) Stockdale et al.

Page (1992a)

Page (1992b) Stockdale et 01. (1968)

Page and Manchak (1995) Page eta). (1992)

0 0 16 0 5.3 0

Trace 12

0

Page ( 1 9 9 2 ~ )

0 0

0 0

Page and Cornish (1993)

Az. chroococcum

0 0

Page (1989)

(1968) (1972)

Az. beijerinckii Glucose Az. macrocytogenes Glucose

0

(1968)

(continues)

125

BIODEGRADABLE POLYESTERS TABLE XII-Continued Organism

Carbon

PHA (YOdry wt)

Hv (mol YO)

Reference

Az. agilis

Glucose

49

Stockdale et al.

Az. insigne

Glucose

23

Stockdale et al.

Az. paspali

Fructose

Beijerinckia la ticogenes B. indicus

Glucose

16

Stockdale et al.

Glucose

21

Stockdale et al.

B. mobile

Glucose

37

Stockdale et al.

B. puminensis

Glucose

6

Stockdale et al.

Derxia gummosa

Glucose

26

Stockdale et al.

(1968) (1968) 9.08

Itzigsohn et al. (1995)

(1968) (1968) (1968) (1968) (1968)

and Cole, 1978; Karr et al., 1983),Rhizobium phaseoli (Bonartseva et al., 1995), a number of Rhizobium sp. (Stam et al., 1986; Tombolini and Nuti, 1989), Bradyrhizobium japonicum (McDermott et al., 1989),and a variety of nodulating bacteria (Bonartseva et al., 1994), can also accumulate PHAs in considerable amounts. PHB accumulation by carbohydrate-utilizing halophilic archaebacteria has also been reported (Fernandez-Castillo et al., 1986). Among the halophilic bacteria, Halobacterium mediterranei accumulated about 38% (w/w) PHB even at 15% salts concentration (for review, see Rodriguez-Valera and Lillo, 1992). Methylotrophs of both types I and I1 (which assimilate carbon via the ribulose monophosphate and the serine pathways, respectively), viz. Pseudomonas methanica (Kallio and Harrington, 1960), Methylosinus trichosporium (Weaver et al., 1980), Methylobacterium rhodesianum (Hilger et al., 1991; Breuer et al., 1995), Methylocystis ~ Q W U S(Hou et a]., 1979), are able to synthesize PHB.

c. Higher Plants. It has been possible in recent years to produce PHAs using higher plants. Although plants are well known for products, such as starch, sucrose, fatty acids, or cellulosic material, production of PHB in higher plants has been demonstrated (for reviews, see Poirier et al., 1995b, 1992a,b, 1993). PHB formation had been made possible in

126

CH. SASIKALA AND CH. V. RAMANA

plants by expressing the PHB biosynthetic genes of the bacterium Alcaligenes eutrophus in the plant Arabidopsis thaliana. Of the three enzymes required for PHB synthesis from acetyl-CoA, only the 3-ketothiolase (phb A gene) is endogenously present in plants. The other genes encoding the acetoacetyl-CoA reductase (phb B gene) and PHB synthase (phb C gene) were introduced into plants to complete the PHB biosynthetic pathway (Fig. 13) (Poirier et al., 1992a). In such plants, PHB was synthesized as granules of 0.2-1 p,m in diameter surrounded by an electron-dense layer thought to be mainly composed of phospholipids, and the granules could be detected in the vacuole, nucleus, and cytoplasm, accounting for up to 100 p,g/g fresh wt (Poirier et al., 1992b) and up to 14% of dry weight as 0.2- to 0.7-pm granules within plastids (Nawrath et al., 1994a,b). Polymer from transgenic plants appears to have a chemical structure identical to that of PHB produced by bacteria; however, it varies with respect to the molecular weight distribution (studied in vitro using suspension cultures derived from PHB producing A. thaliana) (Poirier et al., 1995a).Plant-produced PHB was much broader than that of typical bacterial PHB. Reduction in growth and accumulation of PHB by transgenic plants resulted in a decrease in the seed yield that was due to a high level of acetoacetyl-CoA reductase (Poirier et al., 1992a,b). Although production of PHB was made possible with transgenic plants, several problems need to be overcome before the agricultural production of PHB can begin (for reviews, see Poirier et al., 1993, 1995b; van der Leij and Witholt, 1995).

Acet yl-CoA

Malonyl- CoA

i\1

Flavonoids

Isoprenoids

FIG. 13. PHB biosynthetic pathway expressed in Arabidopsis thaliana plants. The Alcaligenes eutrophus genes phaA, phaB, and phaC encoding the 3-ketothiolase,acetoacetylCoA reductase, and PHA synthase, respectively,are shown in boxes, along with the products.

127

BIODEGRADABLE POLYESTERS TABLE XI11

ISOLATION, AND ANALYSIS OF PHAs DETECTION, ~~

Analysis

In situ staining of PHA granules Sudan black B Malachite green Nile blue A In vivo I3C[NMR]studies Isolation of native polymer granules Sonically disruption Repeated centrifugation Differential and density gradient centrifugation Extractionhcovery of PHAs Chlorinated hydrocarbons Chloroform

Methylene chloride 1,2-Dichloroethane 1,1,2-Trichloroethane + water Chloroform with methanol, ethanol, acetone, or hexane Sodium hypochlorite

Dispersion of sodium hypochlorite and chloroform Acetone Ethylene carbonate and propylene carbonate Enzymes and surfactants Analysis of PHAs Spectrophotometric method Gas chromatography (GC) GC-mass spectrometry

Pyrolysis-GC

Reference Smibert and Krieg (1981) Sun et a]. (1995), Kushnaryov et 01. (1980) Ostle and Holt (1982), Kitamura and Doi (1994) Poirier eta]. (1992a), Barnard and Sanders (1988) Fukui et al. (1976) Ritchie and Dawes (1969), Merrick and Doudoroff (1964) Griebel et al. (1968), Nickerson (1982) Walker et al. (1982), Vanlautem and Gilain (1982), J. A. Ramsay et al.(1994) J. A. Ramsay et al.(1994) Barham and Selwood (1982), Holmes and Jones (1980), J. A. Ramsay et al. (1994) Vanlautem and Gilain (1987) Stageman (1985) J. A. Ramsay etal. (1990), Berger et a]. (1989), Hahn etal. (1995) Hahn et al.(1993, 1994, 1995)

Baptist (1962), Brand1 et al. (1988) Lafferty and Heinzle (1977, 1978) Holmes and Lim (1985) Law and Slepecky (1961) Braunegg et a]. (19781, Huijberts et a]. (1994), Riis and Mai (1988) Findlay and White (1983), Morikawa and Marchessault (1981), Karlsson e t a ] . (1994) Helleur (1988) (continues)

128

CH. SASIKALA AND CH. V. RAMANA TABLE XIII-Continued Analysis

Fast atom bombardment-mass spectrometry (FAB-MS) NMR techniques

Fluorescence properties exhibited by PHB HPLC

Reference Ballistreri et al. (1989, 1990, 1992, 1995), Ulmer et a]. (1994) Hocking and Marchessault (1995), Spyros and Marchessault (1995), Jacob et al. (1986),Doi et al. (1986a,b,c),Nedea et al. (1991, 1992), Spyros et 01. (1995), Huijberts et al. (1994) Turchetto and Cesaro (1995) Ballistreri et al. (1995)

2. Extraction and Characterization of Poly(hydroxya1kanoates)

Poly(P-hydroxyalkanoate) formed by bacteria and plants can be detected, isolated, and analyzed as summarized in Table XIII. In general, the PHAs can be extracted from lyophilized dry cells by Soxhlet solvent extraction and determined gravimetrically (Williamson and Wilkinson, 1958; Law and Slepecky, 1961) and calculated as the percentage of cellular dry weight. Characterization of the purified polymer can be quantitatively determined after methanolysis in the presence of 3 or 5% (v/v) sulfuric acid, by gas chromatography, high-pressure liquid chromatography, or by nuclear magnetic resonance spectroscopy (see Table XI11 for more details). Large-scale separation and purification of PHAs from microorganisms by various methods has been patented by many workers (Table XIV). 3. Biosynthesis of Bacterial Poly(hydroxya1kanoates)

a. Diversity of Bacterial Poly(hydroxyalkan0ates). Until the 1970s, 3HB was considered to be the only constituent of bacteria PHAs. In the 1980s, PHAs having other monomers besides 3HB were shown to be accumulated by many bacteria with the addition of precursor substrates in the medium. Today, we know of 90 or more different monomers of PHA that are biosynthesized by different bacteria (for review, see Steinbuchel and Valentin, 1995) representing both natural and laboratory samples (Table XV). In brief, these include 1. 3HA monomers 2. Unsaturated 3HA monomers with one or two double bonds 3. 3HA with methyl group at various positions

TABLE XIV PATENTED METHODS FOR EXTRACTION OF PHAS Assignee

Patent number

Year

Brief description

Baptist

US 30 36 959

1962

Extraction of PHB from bacterial cells using pyridine and other solvents.

Lafferty and Heinzle

us 4 101 533

1978

Isolation of PHAs using cyclic carbonates.

Holmes and Jones

EP 46 335

1980

Extraction of PHB using chlorinated compounds from Azotobacter beijerinckii

Barham and Selwood

EP 58 480

1982

Extraction of PHB using 1,2dichloroethane.

Vanlautem and Gilain

US 4 310 684

1982

Walker et al.

EP 46 017

1982

Extraction of PHAs using chloroform. Extraction of PHAs using chloroform.

Holmes

EP 91 224

1983

Holmes et al. Holmes and Lim

US 4 393 167

1983

EP 145 233

1985

Isolation of PHI3 from micro organisms by subjecting to proteolytic enzyme (alcalase) digestion and/or a surfactant digestion to solubilize the cell material. Nuclic acids present were also denatured by heating the suspension to 80°C prior to or during digestion.

Vanlautem and Gilain

US 4 705 604

1987

Extraction of PHAs using azeotrophic mixtures.

Holmes and Lim

US 4 910 145

1990

Aqueous enzymatic digestion method of extracting PHAs has been developed by ZENECA as an alternative to the extraction. This process involves the thermal treatment of PHA-containing biomass, followed by enzymatic treatment, and finally washing with an anionic surfactant to dissolve non-PHB cell material.

Method for extracting PHB from Alcaligenes eutrophus by autolyzing the suspension.

(contin ues)

TABLE XIV-Con tinued Assignee

Patent number

Year

Brief description

Tokiwa et 01.

US 5 124 371

1992

Antonio and Francisco

EP 622,462

1994

Production and procedure for extraction of PHAs from halophilic bacteria using lysis or rupture of halobacteria developed in high salt concentrations.

MacDonald and John

WO 94,24,302

1994

Production and process for recovery of plastic produced by microorganisms. Plastic was recovered from microorganisms by solubilizing nonplastic material with an oxidizing agent in the presence of a chelating agent.

Greer

WO 94,10,289

1994

Use of hydrogen peroxide during the purification process of PHA from A . eutrophus was described.

Yamamoto et al.

Tp 7 79 787

1995

Matsushita et 01.

Tp 7 79 788

1995

Matsushita et al.

Tp 7 135 985

1995

Separation and purification of PHAs from microorganisms using surfactants that help in retaining the amorphous states of PHAs. Extraction of PHAs from microorganisms. Production and extraction of PHA from Protomonas extorquens

4. non-3HA monomers such as 4HB, 4HV, 4HHx, 4HH, 4H0, 4HD, 5HV, 5HHx, and 5HDD 5 . 3HA monomers with various functional groups that include carboxyl, benzyl, acetoxyl, phenoxy, cyano, and nitrophenoxy, phenyl, cyclohexyl, epoxy, or halogen groups

Three types of polyesters, SCL, MCL, and LCL PHAs, are generally synthesized by many bacteria when grown on various carbon sources. The SCL monomer units include 3-hydroxypropionate, 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate,4-hydroxyvalerate, and 5-hydroxyvalerate, whereas the MCL monomer units have three to nine more carbon 130

TABLE XV VARIOUS HYDROXY ALKANOATE MONOMERS DETECTED IN PHA ISOLATED FROM MICROORGANISMS Hydroxy alkanoate detected in PHA isolated from microorganisms Name

Molecular Formula

(Saturated hvdroxv alkanoates) COOHCH,CH,OH 3-Hydroxypropionic acid

Abbreviation

Reference

Shimamura et al. (1994), Hiramitsu and Doi (1993), Doi et al. (1990~) 3-Hydroxybutyric acid 3HB Finlay and White (1983) COOHCH,CH(OH)CH, 3-Hydroxyvaleric acid 3HV Ballistreri et al. (1995) COOHCH,CH(OH)CH,CH, 3-Hydroxyhexanoic acid 3HHx Lageveen et aJ, (1988), COOHCH,CH(OH)(CH,), Sato eta]. (1992) Wallen CH3 and Rohwedder (1974) or 3-hydroxycaproic acid 3HC Gagnon et aJ. (1992a) 3-Hydroxyheptanoic acid 3HH Finlay and White (1983), COOHCH,CH(OH)(CH,), Kunioka et a]. (1988) CH, COOHCH,CH(OH)(CH,), 3-Hydroxyoctanoic acid 3H0 De Smet et al. (1983), Findlay and White CH, (1983),Sat0 et al. (1992), Gagnon et aJ. (1992a) 3HN Lageveen eta]. (1988) COOHCH,CH(OH)(CH,),CH, 3-Hydroxynonanoic acid 3HD Lageveen et a]. (1988), COOHCH,CH(OH)(CH,),CH, 3-Hydroxydecanoic acid Gagnon et al. (199Za) COOHCH,CH(OH)(CH,),CH, 3-Hydroxyundecanoic acid 3HUD Lageveen et al. (1988) COOHCH,CH(OH)(CH,),CH, 3-Hydroxydodecanoic acid 3HDD Lageveen et al. (1988) 3-Hydroxytetradeconoic COOHCH,CN(OH)(CH,),, acid 3HTD Lee et aJ. (1995) CH, 3-Hydroxyhexadeconic COOHCH,CH(OH)(CH,) acid 3HHD Lee eta]. (1995) CH, 4-Hydroxybutyric acid 4HB Kunioka et al. (1988), COOH(CH,),OH Choi et al. (1995) 4-Hydroxyvaleric acid 4HV Valentin et aJ. (1992) COOH(CH,),(OH)CH, 4-Hydroxyhexanoic acid 4HHx Valentin et aJ. (1994) COOH(CH,),(OH)CH,CH, 4-Hydroxyheptanoic acid 4HH Steinbuchel and COOH(CH,),(OH)(CH,),CH, Valentin (1995) 4H0 Steinbuchel and COOH(CH,),(OH)(CH2),CH, 4-Hydroxyoctanoic acid Valentin (1995) 4-Hydroxydecanoic acid 4HD Eggink eta]. (1995) 5-Hydroxyvaleric acid 5HV Doi et 01. (1987a) 5% Steinbucher and 5-Hydroxyhexanoic acid Valentin (1995) 6-Hydroxydodecanoic 6HD Steinbucher and acid Valentin (1995) (Unaturated hydroxy alkanoates) COOHCH(OH)CH, 3-Hydroxy-2-butenoicacid Davis (1964) COOHCH,CH(OH)CHCCH, 3-Hydroxy-4-pentenoic Ballistreri e t a ] . (1995) acid 3Hp

,

(continues)

131

TABLE XV-Continued ~~

~

Hydroxy alkanoate detected in PHA isolated from microorganisms Molecular Formula (Unsaturated hydroxy alkanoates) COOHCH,CH(OH)CHCHCH,

Name

Reference

COOH(CH,),CHCHCH,CH(OH) (CHzl,CH,

3-Hydroxy-4-transhexenoic Fritzsche et al. (199Oa) acid 3-Hydroxy-4-cis-hexenoic Fritzsche et al.(199Oa) acid 3-Hydroxy-5-hexenoicacid Fritzsche et al. (199Oa) 3-Hydroxy-5-trans-octenoic Fritzsche et al. (199Oa) acid 3-Hydroxy-5-cis-octenoic Fritzsche et al. (199Oa) acid 3-Hydroxy-7-octenoicacid Fritzsche et al.(199Oa) 3-Hydroxy-8-nonenoicacid Lageveen et al.(1968) 3-Hydroxy-9-decenoicacid Lageveen et 01. (1988) Eggink et al. (1990) 3-Hydroxy-5-cisdodecenoic acid 3-Hy droxy-B-cisEggink et al. (1990) dodecenoic acid Eggink et al. (1990) 3-Hydroxy-5-cistetradecenoic acid Choi et al. (1994) J-Hydroxy-7-cistetradecenoic acid Eggink et al. (1995) 3-Hydroxy-5,8-cis,cistetradecenoic acid Eggink et 01. (1995) 6-Hydroxy-3-cisdodecenoic acid Eggink et al. (1995) 12-Hydroxy-94soctadecenoic acid

(Methyl hydroxy alkanoates) COOHCH(OH)CH,

Lactic acid

COOHCH,CH(OH)CH,CHCH, COOHCH,CH(OH)(CH,), CHCHCH,

COOHCH,CH(OH)(CH,),CHCH, COOHCH,CH(OH)(CH,),CHCH, COOHCH,CH(OH)(CH,),CHCH, COOHCH,CH(OH)CH,CHCH (CHz)&H, COOHCH,CH( OH)(CH,),CHCH (CHz)JH, COOHCH,CH( OH)CH,CHCH (CHz),CH, COOHCH,CH(OH)(CH,),CHCH (CH2)5CH3

COOHCH,CH(OH)CH2CHCHCH, CHCH(CH,),CH, COOHCH,CHCHCH,CH(OH) (CH2)5CH3

COOHCH,CH(OH)CH(CH,)CH, COOHCH(CH,)CH(OH)CH,CH, COOHCH,CH(OH)CH(CH,)CH, COOHCH,CH(OH)CH(CH,) CH,CH, COOHCH,CH(OH)CH,CH (CHJCH, COOHCHZCH(OH)(CH2),CH (CHJCH, COOHCH(CH,)CH(OH)CH, CHC(CH,)CH, COOHCH,CH(OH)CH(CH,) (CH2)3CH3

COOHCH,CH(OH)CH,CH(CH,) (CH,)zCH,

3-Hydroxy-2methylbutyric acid 3-Hydroxy-2methylvaleric acid 3-Hydroxy-4methylvaleric acid 3-Hydroxy-4methylhexanoic acid 3-HY~TOXY-5methylhexanoic acid 3-HY&OXY-6methylheptanoic acid 3-Hydroxy-2,6-dimethyl5-heptenoic acid 3-Hydroxy-4methyloctanoic acid 3-Hydroxy-5methyloctanoic acid

132

Valentin and Steinbuchel (1993) Sat0 et al.(1992) Sato et al. (1992) Findlay and White (1983)

Fritzsche et al. (199Ob) Fritzsche et al.(199Ob) Findlay and White (1983) Steinbuchel and Valentin (1995) Fritzsche et al. (199Ob) Fritzsche et al.(199Ob)

TABLE XV-Continued Hydroxy alkanoate detected in PHA isolated from microorganisms Molecular Formula

Name

(Methyl hydroxy alkanoates) continued COOHCH,CH(OH)(CH,),CH(CH,) 3-HydrOxy-6methyloctanoic acid ~,CH, COOHCH,CH(OH)(CH2),CH 3-Hydroxy-7methyloctanoic acid (CHJCH, J-Hydroxy-6COOHCH,CH(OH)(CH,),CH(CH,) methylnonanoic acid 3-Hydroxy-7COOHCH,CH(OH)(CH,),CH(CH,) methylnonanoic acid CH,CH, COOHCH,CH(OH)(CH,),CH(CH,) 3-Hydroxy-8methylnonanoic acid CH, 3-Hydroxy-7COOHCH,CH(OH)(CH,),CH(CH,) methyldecanoic acid (CHJzCH, 3-Hydroxy-9COOHCH,CH(OH)(CH,),CH(CH,) methyldecanoic acid CH3 3-Hydroxy-7-methylCOOHCH,CH(OH)(CH,),CHC 6-octenoic acid (CHJCH, (Dicarboxvlic and ether hvdroxv - - alkanoates) COOHCH,CH(OH)COOH Malic acid 3-Hydroxysuccinic COOHCH,CH(OH)COOCH, acid methyl ester 3-Hydroxyadipinic COOHCH,CH(OH)(CH,),COOCH3 acid methyl ester COOHCH,CH(OH)(CH,),COOCH, 3-Hydroxysuberic acid methyl ester 3-Hy droxyazelaic COOHCH,CH(OH)(CH,),COOCH, acid methyl ester 3-Hy droxysebacic COOHCH,CH(OH)(CH,),COOCH, acid methyl ester COOHCH,CH~OH)(CHZ),COOC,H, 3-Hy droxysuberic acid ethyl ester 3-Hydroxysebacic COOHCH,CH(OH)(CH,),COOC,H, acid ethyl ester 3-Hy droxypimelic COOHCH,CH(OH)(CH,),COOC,H, acid propyl ester 3-Hydroxysebacic COOHCH,CH(OH)(CH,),COOC,H, acid benzyl ester 3-Hydroxy-8-acetoxy COOHCH,CH(OH)(CH,),OCOCH, octanoic acid 3-Hydroxy-9-acetoxy COOHCH,CH(OH)(CH,),OCOCH, nonanoic acid (Aromatic hydroxy alkanoates) Phenoxy-3-hydroxyCOOHCH2CH(OH)CHOC,H, butyric acid Phenoxy-3-hydroxyCOOHCH2CH(OH)(CH,),0C,H, valeric acid Phenoxy-3-hydroxy COOHCH2CH(OH)(CH,),0C,H5 heptanoic acid Phenoxy-3-hydroxyCOOHCH,CH(OH)(CH,),OC,H, octanoic acid

133

Reference Fritzsche et al. (199Ob) Fritzsche et al. (lggob), Findlay and White (1983) Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995) Steinbuchel and Vdentin (1995)

Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995) Fischer et al. (1989) Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995) Lenz et a]. (1994) Lenz et a]. (1994) Lenz et al. (1994) Lenz eta]. (1994) Lenz eta]. (1994) Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995) Lenz eta]. (1994) Lenz et al. (1994) Steinbuchel and Valentin (1995) Ritter and von Grafin Spee (1994) Steinbuchel and Valentin (1995) Steinbuchel and Valentin (1995)

(continues

TABLE XV-Continued Hydroxy alkanoate detected in PHA isolated from microorganisms Molecular Formula

(Aromatichydroxv alkanoates) COOHCH,CH(OHj(CH2)6C,H, COOHCH,CH(OH)CH,OC,H, CN COOHCH,CH(OH)(CH,),OC,H, CN COOHCH,CH(OH)(CH,),OC,H, CN COOHCH,CH(OH)(CH,),OC,H, NO, COOHCH,CH(OH)(CH2)2C,H, COOHCH,CH(OH)CH,C,H,,

(Dihydroxyalkanoates) COOHCH,CH(OH)(CH,),OH COOHCH,CH(OH)(CH,),CH(OH) (CHJSW COOHCH,CH(OHICH,CHCHCH, CH(OH)(CHJ,CH,

(Epoxy hydroxy alkanoates) COOHCH,CH(OH)CH(O)CH (CHJ,CH,

COOHCH,CH(OH)(CH,),CH(O) CH(CH,),CH, COOHCH,CH(OH)CH,CHCHCH, CH(O)CH(CH,),CH, COOHCH,CH(OH)(CH,), CHCHCH,CH(O)CH(CH,)CH, (Cyanohydroxy alkanoates) COOHCH,CH(OH)(CH,),CN COOHCH,CH(OH)(CH,),CN

(Halogenatedhydroxyalkanoates) COOHCH,CH(OH)(CH,),F COOHCH,CH(OH)(CH,),F COOHCH,CH(OH)(CH,),CI COOHCH,CH(OH)(CH,),Cl COOHCH,CH(OH)(CH,l,Br COOHCH,CH(OH)(CH,),Br COOHCH,CH(OH)(CH,),Br

Name

Reference

Phenoxy-3-hydroxynonanoic para-Cyanophenoxy3-hydroxybutyric acid para-cyanophenoxy3-hydroxyvaleric acid para-Cyanop henoxy3-hydroxyhexanoic acid para-Nitrophenoxy 3-hydroxyhexanoic acid 3-Hydroxy-5-phenylvaleric acid 3-Hydroxy-5-cyclohexylbutyric acid

Ritter and von G r d n Spee (1994) 0. Kim etal., (1995)

3,12-Dihydroxy dodecanoic acid 3,6-Dihydroxydodecanoate 3,8-Dihydroxy-5-cistetradecenoic acid

Lenz et al. (1992)

3-Hydroxy-4,5epoxydecanoic acid 3-Hydroxy-6,7, epoxydodecanoic acid 3-Hydroxy-8,9-epoxy5,B-cis-tetradecanoic 3-Hydroxy-12,13-epoxi9-cis-octadecanoic acid 7-Cyano-3-hydroxyheptanoic acid 9-Cyano-3-hydroxynonanoic acid 3-Hydroxy- 7-fluoro-

heptanoic acid 3-Hydroxy-9fluorononanoic acid 3-Hydroxy-6chlorohexanoic acid 3-Hydroxy-8chlorooctanoic acid 3-Hydroxy-6bromohexanoic acid 3-Hydroxy-8bromooctanoic acid 3-Hydroxy-11-bromoundecanoic acid

0. Kim et al., (1995)

0. Kim etal., (1995) 0. Kim et al., (1995)

Fritzsche et al. (199Oc), Kim et al. (1991) Lenz et al., (1992)

Eggink et al. (1995)

Eggink et al. (1995) Eggink et al. (1995) Eggink et al. (1995) Eggink et al. (1995) Lenz et al.(1992) Lenx et al. (1992) Abe et al. (1990) Abe et al. (1990) Lenz et al. (1990) Doi and Abe (1990) Lenz et al. (1990) Lenz et al. (1990) Lenz et al. (1990)

BIODEGRADABLE POLYESTERS

135

atoms than the SCL monomer units. Accumulation of polyesters with SCL monomer units is commonly observed with A. eutrophus (Song et al., 1993a,b), I! pseudoflava (Bertrand et al., 1990), and R. rubrum (Brandl et al., 1989; Ulmer et al., 1994). Polymers with MCL and LCL monomer units are commonly observed with I? oleovorans (Brandl et al., 1988; Gross et al., 1989a; Huisman et al., 1989, 1992; Lageveen et al., 1988; Lenz et al., 1992; Hazer and Lenz, 1994).I! putida (de Waard et al., 1993; Huijberts et al., 1992). I! citronellolis (Choi and Yoon, 1994), and other fluorescent Pseudomonas strains (Husisman et al., 1989). Careful analysis of the monomer unit composition of PHAs enables us to determine the pathway through which the substrates fed were metabolized to synthesize these PHAs (de Waard et al., 1993; Huijberts et d., 1992).

b. Biosynthesis of PHB and Other PHAs. Poly(P-hydroxybutyrate) is the best known member of the PHA series of polyesters and is known to be produced as intracellular energy and carbon reserves that are later utilized by the bacteria for cellular growth (Dawes and Senior, 1973). Most of the bacteria accumulate PHB as storage reserve, whereas the oxygenic phototrophic bacteria accumulate mostly glycogen as their reserve. The nature of the carbon source and the conditions of growth influence the flow of carbon into these different reserves. When substrates are photometabolized via acetyl-CoA, without the intermediate formation of pyruvate, PHB accumulation predominates, whereas substrates, which are metabolized via pyruvate, led to the formation of glycogen (Stanier et a]., 1959). In most of the organisms investigated to date, PHB is synthesized from acetyl-CoA by a sequence of three reactions catalyzed by 3-ketothiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9), acetoacetyl CoA reductase (hydroxybutyrl CoA dehydrogenase; EC 1.1.1.36), and PHB synthase. However, purple nonsulfur anoxygenic phototrophic bacteria (and also the purple sulfur bacterium Chromatium vinosum; Liebergesell and Steinbuchel, 1992) and some of the syntrophic bacteria like Syntrophomonas wolfei (McInerney et al., 1992) differ from other bacteria in which two stereospecific (Stern et al., 1955) enol-CoA hydratases are also involved (Stern et al., 1956; Moskowitz and Merrick, 1969), thus making the synthesis occur in five steps. The enol-CoA hydratases catalyze the conversion of ~-(+)-3-hydroxybutyryl-CoA via crotonyl CoA to ~-(-)-3-hydroxybutyryl-CoA, which is polymerized to yield PHB. The overall pathway that leads to PHB synthesis from acetate by R. rubrum can be represented as follows (Merrick and Doudoroff, 1961; Moskowitz and Merrick, 1969):

136

CH. SASIKALA AND CH. V. RAMANA Acetate -+ acetyl CoA -+ acetoacetyl CoA + L-(+)-p-hydroxybutyryl CoA -+ crotonyl CoA -+ D-(-)-P-hydroxybutyryl CoA 4 poly(P-hydroxybutyrate

In the absence of CO,, acetate that is assimilated is converted to PHB (Stanier et al., 1959; Schmidt and Kamen, 1970); however, for the utilization of PHB by the bacterial cells, CO, was found to be essential (Stanier et al., 1959). A part of the acetate may undergo oxidation through the Kreb's cycle, and the remaining acetate is reduced to PHB according to the reaction light

9n A c e t a t e d 4 ( C 4 H , O , ) ,

+ 6nH,O

however, in the presence of molecular hydrogen, utilization of acetate by R. rubrum cells is much more rapid without any partial oxidation of the acetate (Fig. 14) and 86% of acetate passes into PHB synthesis (Stanier et al., 1959). 2n Acetate + nH,

%(C,H,O,),

+ ZnCO, + ZnH,O

Synthesis of PHB from acetate is a reductive process, whereas that from butyrate is oxidative. Because the reducibility of butyric acid is

Light-

HSCoA

- HSCoA poly- p-Hydroxybutyric acid FIG. 14. Acetate-hydrogen reaction in purple bacteria as postulated by Stanier et al. (1959). PN, pyridine nucleotide.

BIODEGRADABLE POLYESTERS

137

higher than that of PHB, when butyric acid polymerizes the excess hydrogen with the CO, forms a polysaccharide in R. rubrum cells in addition to PHB (Stanier et al., 1959), Zn C,H,O, -+2(C,H,O,),

+ 4n (HI

4n (H) + CO, -1(CH,O),

+ nH,O

2n C4H802+ nCO, +2(C,H,O,),

+ (CH,O)n

Other substrates, such as pyruvate (Schon and Voelskow, 1976; Voelskow and Schon, 1978), acetone (Karayiannis and Madigan, 1990; Madigan, 1990), producer gas (CO t H,O) (Maness and Weaver, 1994), and methane (Asenjo and Suk, 1986), can also be biosynthesized to PHB. Anoxygenic phototrophic bacteria such as R. sphaeroides, when grown chemoautotrophically (dark H, + CO,), can also accumulate PHB; however, the yields are less than 1% (of cell dry wt) (Madigan and Gest, 1979). Under dark anoxic conditions, in this group of microorganisms, the storage carbohydrates or externally supplied sugars can be converted into PHB. The process of formation of PHB through fermentation of stored carbon (glycogen) to PHB by C. vinosum (Stal, 1992) or from exogenously supplied sugars to R. capsulatus (Madigan et al., 1980) has been well studied. Elemental sulfur acts as an electron acceptor in the conversion of storage carbohydrates to PHB and CO, by Chromatiurn sp. according to the following equation (van Gemerden, 1968): (C,H,,O,),

+ nH,O

+(C,H,O,)n

+ 2n CO, + 6n[H1

3nS + 6n[H] -13n H,S

(C8H,,0,)n

+ nH,O +3nS -+(C,H,O,),

+ 2n Co, + 3n H,S

Such conversion of storage carbohydrates to PHB is a process from which the phototrophic bacteria like Chromatiurn can derive ATP in the dark (van Gemerden, 1968). In general, the PHA biosynthetic route was linked to (i) the P-oxidation cycle (Fig. 15) in which the substrates are oxidized, resulting in a monomer containing two or more carbon atoms less than those used during growth (Brand1 et al., 1988; de Waard et al., 1993; Lageveen et al.,

138

CH. SASIKALA AND CH. V. RAMANA

NADH* NAD

OH 0

L(+) R

e S C o A

FIG. 15. Proposed relationship between @-oxidationand PHA synthesis in Pseudomonos putida and I! oleovorans. 1, Acyl-CoA synthetase; 2, Acyl-CoA dehydrogenase; 3 , Enol-CoA hydratase; 4,3-hydroxyacyl CoA dehydrogenase; 5,3-ketoacyl-CoA thiolase; 6, 3-hydroxyacyl-CoA epimerase; 7, PHA polymerase. a, Alkane hydroxylase; b, alkanol dehydrogenase; c, alkanal dehydrogenase. Modified from de Waard et ol. (1993) and Lageveen et ol. (1988).

1988; Choi and Yoon, 1994); (ii) the de novo fatty acid biosynthesis (Fig. 16) in which the substrates get reduced, resulting in monomer units with two or three additional carbon atoms (Eggink et a]., 1992; Huijberts et a]., 1992); or (iii)a process of direct oxidation (Fig. 17) in which PHA contained 3-hydroxy acids of the equivalent carbon chain length to that of the growth substrate (Haywood et a]., 1989a). In conclusion, a comprehensive view of central carbon metabolism of most of the bacteria that can lead to the biosynthesis of PHB from various carbon sources can be seen from Fig. 18. Theoretical yields of PHB from several carbon sources have been estimated from biochemical pathways leading to PHAs (Yamane, 1992).

139

BIODEGRADABLE POLYESTERS Acetyl-COA

1

A

5

OH &CoA

J

B

OH

R&Q*

-FHA polymerase

PHA

FIG. 16. Postulated relationship between fatty acid biosynthesis and PHA formation on nonrelated substrates. (A) 3-Hydroxyacyl intermediates that occur during elongation and diversification of the fatty acid biosynthesis. (B) The synthesis of PHA (modified from Huijberts et ul., 1992).

Biosynthesis of copolyesters from Alcaligenes, viz. P(3HB-co-3HP) (Fig. 19), P(3HB-co-3HV), P(3HB-co-3HHx) (Fig. 20), and R. ruber (Anderson et al., 1995), and the production of PHAs from unusual carbon compounds by I? oleovorans, viz. n-alkenes (Fig. 21), have also been studied extensively. For a better understanding of the control of PHA biosynthesis, it would be useful to measure the intracellular metabolite concentrations (Bowien et al., 1974; Cook and Schlegel, 1978; Ruhr and Schlegel, 1975) and their respective enzymes (Mansfield et al., 1995; Floccari et al., 1995; Breuer et a]., 1995; Motthes and Babel, 1995) involved in the PHAs biosynthesis. The key enzymes involved in the PHA biosynthesis are 3-ketothiolase, acetoacyl-CoA reductase, and PHA synthase. 3. KETOTHIOLASE.This enzyme has been purified and characterized from various PHA-synthesizing bacteria (Table XVI), with CoA as the key effector metabolite (Oeding and Schlegel, 1973). The diversion of acetyl-CoA into tricarboxylic acid cycle (Fig. 18) is affected by modulation of citrate synthase activity of NADH; the acetyl-CoA concentration increases, whereas that of CoA decreases, and polymer synthesis via 3-ketothiolase proceeds (Dawes, 1992).

140

CH. SASIKALA AND CH. V. RAMANA

Octanoic acid

C%(CH2)&OOH c

OCtanyl CoA

O

A

~

H

OctanoylCoA ~ synthase

CY(CH~)&O-S-COA

FA D * q O c t a n o y l C o A dehydrogenase FADH2 c H$H&CH=CHCO-S -COA Octanvl enovlCoA EnoylCoAhydrat ase

7 -

Keb-octanoym

Ct5(CH2& C y CO-S-CoA ~$pH$-Octanoyl-CoA

reductase(NADP1

NAP

D-pW-IydroxvoctanQyl

C~(CH2)4CHOHCH2CO-S-~A

PHO FIG. 17. Biosynthesis of poly(3-hydroxyoctanoate) starting with octanoic acid as sole substrate.

p-Ketothiolases are homotetrameric enzymes with molecular masses ranging from 160 to 190 kDa, having similar kinetic regulatory properties (Table XVI), and inhibited by coenzyme A in A. eutrophus (Oeding and Schlegel, 1973),Azotobacter beijerinckii (Senior and Dawes, 1973), and Zoogloea rarnigera (Nishimura et al., 1978). This enzyme has been extensively studied with respect to the C-C bond-forming sequence (Davis et a]., 1987b), N-terminal amino acid sequence (Davis et a]., 1987a; Masamune et a].,1989),active site residues of the enzyme (Davis et al., 1987a, and gene coding the enzyme (Peoples et a]., 1987). ACETOACYL-COA REDUCTASE (HYDROXYACYL-COA DEHYDAOGENASE). This enzyme catalyses the oxidation reduction reaction between p-hydroxybutyrate and acetoacetate and plays a key role in the PHA synthesis and degradation. This enzyme has been purified and characterized from different bacteria (Table XVII). Two acetoacetyl-CoA reductases with different substrate and coenzyme specificities have been isolated from

141

BIODEGRADABLE POLYESTERS StorageCarbohydrates

1 1

Fructose

5.

1

Fructose-I-P

Glucose

.1

J

Glucose-6-P 5Fructose.6-P,--,Fruct0~.1-6-diP

t t

F’ropionate

---~

c,

LPHBg

FIG. 18. Composite reactions of central metabolism in bacteria and the formation of PHB. ED, Entner Doudoroff pathway; Em,Embden-Meyerhof pathway; Calvin, Calvin reductive pathway; TCA, tricarboxylic acid cycle; L, light; D, Dark. (1) Acyl-CoA synthetase (it is whether specific synthetase exists for all substrates (Steinbuchel and Schlegel, 19911, (2) p-ketothiolase, (3) NADH-dependent acetoacetyl-CoA reductase, (4) Enoyl-CoA hydratase (forming butyryl CoA), (5) Enoyl-CoA hydratase (forming D-(-)-P-hydroxybutyryl CoA), (6) PHB synthase, (7) butyryl-CoA dehydrogenase, (8) enzymes involved in the p-oxidation pathway. Based on information from Schubert et al. (1988), Steinbuchel and Schlegal(1991), Liebergesell and Steinbuchel(1992),and Steinbuchel et al. (1992).

different bacteria (Saito et al., 1977; Haywood et al., 1988b; Shuto et al., 1981;Moskowitz and Merrick, 1969). The NADH reductase is active with CoAs, whereas the NADPH reductase was a range of ~-(+)-3-hydroxyacyl active with only C, and C, D-(-)-3-hydroxyacyl CoAs (Fukui et al., 1987; Saito et al., 1977). This enzyme is a tetramer, with identical subunits with molecular masses ranging from 85 to 140 kDa.

3- HP-COA

(R)-~)-~-HB-COA

3-HP FIG. 19. Metabolic pathway of P(3HB-co-3HP) biosynthesis by Alcaligenes latus (Shimamure et a/., 1994) and A . eutrophus (Hiramitsu and Doi, 1993). X = A . eutrophus grew on 3-hydroxypropionic acid (3HP) (Nakamura et a/., 1991), whereas A . lotus did not grow (Shimamura eta/., 1994). AcCoA, acetyl-CoA; AcAcCoA, acetoacetyl-CoA; 3HB, 3-hydroxybutyrate; TCA, tricarboxylic acid cycle; 3HP, 3-hydroxypropionate.

IP(3HH-co-3HB)J

t

t

FIG. 20. Biosynthesis of P(3HB-co-3HHx)and P(3HB-co-3HV)from alkanoic acids of even carbon number (C,,,) and odd carbon number (C,,,), respectively, by Aeromonas caviae (Doi et a/., 1995). Czn(n - 5) and AC,, acyl-coenzyme A; TCA, tricarboxylic acid cycle.

143

BIODEGRADABLE POLYESTERS

0 CH3-(Ch)n-CH-CH2

f

/ \

,

CH$H$n-

OH ?H 6H-C H2

1

C %s-(CH~)~-CH= CH2 1- A lkene

Product (1)

1 poxidation 1 qH

\

C H35-(CH21ni2C H2- C H-C H2- CO-SCOA

OH-CH2-(CH$n-CH= C H2

(21

1

5 p-oxidation 1 ?H C H2=CH-(CH2)ni CH- C H2-CO-SCOA

0 FIG. 2 1 . Postulated degradative routes of 1-alkenes in Pseudomonas oleovorans (Lageveen et a] . , 1988). (1)Epoxide produced and released into the medium, (2) saturated 3-hydroxyalkanoate-CoA, (3) 3-hydroxyalkenoate compounds. Both (2) and (3) can be incorporated into PHA.

PHA SYNTHASE (POLYMERASE). This enzyme is associated with the PHA granules isolated from different bacteria and helps in the polymerization reaction from hydroxyalkanoic acids as substrates (Fig. 22). It has been suggested that the cellular PHA polymerase may determine the type of monomer unit of PHAs (Doi, 1990; Huijberts et al., 1992; Huisman et al., 1991; Gerngross et ~ l .1994). , PHA polymerase has not yet been purified in large quantities to homogeneity, and no information is available on the tertiary or quaternary structure of this enzyme. Currently, it is not very clear whether there is only one polymerase functioning in the formation of different PHAs or whether different polymerases exist. The PHB synthase may be strictly specific for 3-hydroxyacyl-CoA as observed in anoxygenic phototrophs (Liebergesell et al., 1991) and A . eutrophus in which the substrate specificity of PHB synthase permits only C, and C, 3-hydroxyacid units to be incorporated into PHA (Haywood et al., 1989b). However, with certain other bacteria, such as R. rubrum and Pseudomonas spp., the PHA polymerase may not be very substrate specific, therefore allowing the incorporation of a variety of different monomers into the polymer (Brand1 et al., 1989; Gross et al., 198913). Three different types of PHA synthases have been distinguished (Steinbuchel et ~ l .1992): , Type I PHA synthases are of high molecular

TABLE XVI

PROPERTIES OF p-KETOTHIOLASES INVOLVED IN PHA SYNTHESIS IN DIFFERENT BACTERIA

K, (WM Thyolysis Bacterium

Molecular Mass Subunit ( m a ) composition

AcAcCoAo

CoA

Condensation of Acetyl CoA

Optimum PH

Reference

Alcaligenes eutrophus

170

x4

44

16

1100

ndb

Haywood et al.(1988b)

Bradyrhizobium japonicum

180

x4

19

30

104

7.8

Suzuki et al. (1987)

Syntmphomonas wolfei

160

x4

23

3.6

290

nd

McInerney et al. (1992)

Zoogloea ramigera

162

x4

10-24

8.5

330

nd

Davis et al. (1987a,b), Nishimura et al. (1978)

I&

a AcAcCoA,

b nd.

acetoacetyl-CoA, Not determined.

TABLE X W PROPERTIES OF

Bacterium

w

$

Molecular Subunit mass compo(kDa) sition

~HYDROXYBUTYRATE DEHYDROGENASE (EC 1.1.1.30) FROM SEVERAL BACTERIA

Oxidation

HI3

NAJJ

Reduction AcAcCoA NADH

Optimum Temperature0C

PH 0

R

Reference

Azospirillum brasilense Azotobacter vinelandii Paracoccus denitrifcans

100

x4

1000

nd

nd

nd

36

8

nd

Tal et al. (1990a)

nd

nd

nd

80

nd

54

37

7.5

nd

Jurtshuk et al. (1968)

132

x4

nd

nd

nd

nd

nd

8.5

7.0

Matyskova et al. (1985), Kovar et al. (1886)

Pseudomonas lemoignei Rhodobacter sphaeroides Rhodospirillum rubrurn Staphylococcus xylosus Syntrophomonas wolfei Zoogloea rarnigera

nd

nd

600

nd

200

nd

nd

8.0

nd

Dela6eld et 01. (1965)

85

x4

410

80

280

54

nd

8-9.2

6-6.8

Bergmeyer et al. (1967)

nd

nd

840

70

71

nd

nd

6.8-8.5

6.2-6.8

Shuster and Doudoroff

140

x4

39

0.445

5.47

1.02

37

8.6

6.7

Szewczyk and Rozalska

nd

nd

nd

nd

nd

nd

nd

nd

nd

Amos and McInerney

(1962) (1994) (1993)

112

x4 ~

320

57

150

nd

15 ~~

~

8.0 ~

5.5-7.5 ~

~

~

Nakada et 01. (1981) ~

Note. HB, D(-)P-hydroxybutyrate; AcAcCoA, acetoacetyl-CoA; 0, oxidation reaction; R, reduction reaction; nd, not determined.

_

_

_

_

_

146

CH. SASIKALA AND CH. V. RAMANA

FIG. 22. Reaction catalyzed by PHA synthase. x = 1-3; R,various straight chain, branched, unsaturated or substituted alkyl side chain (Steinbuchel, 1991; Steinbuchel et ul., (1992).

weight (about M,, 61,000 and above) and use hydroxy fatty acids of SCL ((2,-C,) as substrates, type I1 synthases use MCL (C6-C14) as substrates, and the low-molecular-weight (M, less than 40,000) enzymes of the type I11 synthases use SCL as substrates. A mutant strain of Bacillus megaterium isolated by the treatment of N-methyl-N'-nitro-N-nitrosoguanidine did not synthesize any PHB because of no or extremely low synthase activity and had no effect on other PHA-related enzymes (Floccari et a]., 1995). The PHA-biosynthetic genes have been cloned and analyzed at the molecular level (for review, see Steinbuchel et al., 1992) from C. vinosum (Liebergesell and Steinbuchel, 1992), I?. rubrum and Rhodobacter sphaeroides (Hustede et al., 1992; Liebergesel et al., 1993; Eilert and Alexander, 1993),A. eutrophus (Peoples and Sinskey, 1989a,b;Park et al., 1995a),Z. ramigera (Peoples and Sinskey, 1989c),I? oleovorans (Huisman et al., 1991, 1992), I? aeruginosa (Timm and Steinbuchel, 1992). Thiocystis violacea (Liebergeselland Steinbuchel, 1993), Escherichia coli (Rhie and Dennis, 1995; Lee and Chang, 1995a; Kalousek and Lubitz, 1995), and the genes and the enzymes they are coding for are summarized in Fig. 18.

Biopolyester Granules and Their Biochemistry Poly(P-hydroxyalkanoates)are formed within the cell cytoplasm as granular inclusions and some of the studies performed in relation to the PHA granules are summarized in Table XVIII. These granules can be observed under the light microscope as refractile bodies (Shively, 1974; Dawes and Senior, 1973), which may or may not be crystalline in the living cell and can be stained by Nile blue (Kitamura and Doi, 1994), Malachite green (Sun et al., 1993), or by Sudan black (Smibert and Krieg, 1981). 4.

147

BIODEGRADABLE POLYESTERS TABLE XVIII STUDIES ON THE STRUCTURE OF PHA GRANULES ISOLATED FROM BACTERIA Organism

Study/major findings

Reference

Bacillus cereus

Membranes of granules contain approximately 11% of the dry weight of a mixture of triglycerides, free fatty acids, and traces of phospholipids

Williamson and Wilkinson (1958)

Rhodospirillum rubrum Bacillus cereus and B. megaterium Ferribacillus ferrooxidans

Membrane-4.5 nm thickness

Boatman and Douglas (1963)

Membrane of 150-200 (1A = 0.1nm)

A thickness

Membrane-2.5-3.5 nm thickness

Lundgren et al. (1964), Pfister and Lundgren (1964) Wang and Lundgren (1969)

Caulobacter sp. Bacillus

Membrane around the PHB granule Poindexter (1964) Merric and Doudoroff Polymerization and depolymerization of PHB are attached to (1961, 1964) the native granule

Lampropaedia hyalina Bacillus megaterium

Membrane around the PHB granule Doudoroff (1966) Native granules contain protein and phosphorus

Doudoroff (1966)

B. megaterium

Native PHB granules contain 2% (w/w) of protein and 0.5% (w/w) of lipid

Griebel et al. (1968)

Bacillus megaterium B. megaterium

Azotobacter beijerinckii Escherichia coli Bacteria in general Pseudomonas oleovorans

Only PHB synthase

Ellar et 01. (1968)

Polymerization and depolymerization proteins are associated with the granules

Griebel and Merrick (1971), Griebel et al. (1968)

PHB synthesis associated with purified native granules

Dawes and Senior (1973)

PHB membrane structure

Reusch et al. (1986)

Only PHA synthase

Masamune et al. (1989) Fuller et 01. (1992)

PHO-containing granule consists of three major components: polyester, protein, and water. Two PHA synthases plus PHA depolymerase plus 18 kDa protein plus 43 kDa protein plus phospholipids

Fuller et al. (1994)

Structural study of isolated PHB granules

L,auzier et 01. (1992a,b)

(continues)

148

CH. SASIKALA AND CH. V. RAMANA TABLE XVIII-Continued

Organism

Study/major findings

Reference

Only PHA synthase

Gerngross et al. (1993)

PHA synthase and phospholipids

de Koning and Maxwell (1993)

PHA synthase plus other proteins and phospholipids PHA synthase, GA 14 protein and phospholipids

Hocking and Marchessault (1994a)

Chromatium vinosum

Enzyme PHA synthase was found to be associated on the surface of the PHB granules under stress conditions, whereas under nonstress conditions it was located within the cytoplasm

Liebergesell et al. (1994)

Pseudomonas oleovorans, A. eutrophus, B. megaterium, and a few other bacteria

Four classes of structurally and functionally different granuleassociated proteins are distinguished: Class I: Composed of PHA synthases Class 11: Composed of PHA depolymerases Class A new protein referred to as "Phasins" Class IV: Other proteins A model for the distribution of protein and lipid components associated with the ordered macromolecular complex on the surface of PHO inclusion bodies

Steinbuchel et al. (1995)

Alcaligenes eu troph us Bacteria in general

Rhodococcus ruber

Pieper-Furst et 01. (1994)

m:

Pseudomonas oleovorans

Methylotrophic bacteria using serine pathway Acinetobacter sp.

A . eutrophus

Rhodococcus ruber

.!hart

et 01. (1995)

%o dominant low-molecularweight proteins of 11and 20 kDa are associated with PHB granules

Foellner et al. (1995)

Four predominant proteins of 64, 41,38, and 13 kDa were associated with PHB granules 24-kDa protein (M, = 23963) referred to as GA24 protein was isolated from granules

Schembri et 01. (1995)

Function of the GA14 protein during biosynthesis of PHA and the molecular basis of its bonding to PHA granules

Wieczorek et al. (1995)

Pieper-Furst et al. (1995)

BIODEGRADABLE POLYESTERS

149

The granules of PHAs are about 0.2-0.7 pm in diameter, with about 8-12 granules per cell (Dunlop and Robards, 1973);however, sometimes the PHA granules occupy almost the entire cytoplasm of the cell, with a size of 2-3.5 pm in diameter (Hustede et al., 1992; Page et al., 1995). Cells of Azotobacter vinelandii UWD incubated for 18-24 hr were most frequently 2 or 3 pm diameter spheres containing up to 20 PHB inclusions per cross section or a calculated 100 inclusions per cell volume. These inclusions tended to be of small size (0.5 pm diameter) and become fewer and larger in older cells. The most striking feature of these pleomorphic cells was the apparent extrusion of polymer from the cells that may be due to the polymer expansion (Page et a]., 1995). The granules are surrounded by a membrane that does not show the typical bilayered structure of a biomembrane as observed under electron microscope, and this layer was assumed to be hydrophilic on the outer side, and hydrophobic on the inside, because it functions as a layer separating two phases (Mayer, 1992)and the membrane is composed of lipid and protein representing about 0.5 and 2%, respectively, of the granule weight (Griebel et al., 1968; Dawes and Senior, 1973). Furthermore, it was suggested that this membrane contains the PHA-synthase or polymerase system (Brand1 et al., 1990). Poly(P-hydroxybutyrate)granules of C. vinosum were associated with at least four granular proteins (Liebergesell et al., 1992). Two proteins represented the expression of PHA synthase activity, whereas the specific function of the third and fourth proteins was not clear. Three major classes of PHA granule-associated proteins have been identified in bacteria (Schembri et al., 1995): PHA synthase, PHA depolymerase, and phasins. Other proteins that constitute a major proportion of the total protein surrounding the surface of PHA granules have been loosely grouped into a fourth class (Steinbuchel et al., 1995). Recombinant microorganisms contained at least two different active polymerases, one polymerase of the original bacterium and the other is due to the introduction of the foreign gene. These recombinant bacteria could produce a blend of PHB-PHO homopolymers, and the PHAs were stored in separate granules within the producing cells (Preusting et al., 1993~). Two granule populations observed in vivo in the recombinant A. eutrophus are likely to be the direct result of the nanoscale polymer incompatibility of PHA and PHO polymers as demonstrated by the thermal analysis of artificial polymer granules of PHB and PHO (Horowitz and Sanders, 1994a,b).Thus, in the cytoplasm of recombinant A. eutrophus, PHA polymerases covalently attached to PHB will tend to self-associate with each other but not with the host PHA polymerases which are specific for medium chain length hydroxyacids and will covalently attach to PHO.

150

CH. SASIKALA AND CH. V. RAMANA

Poly(p-hydroxybutyric acid) granules of R. rubrum undergo a rapid enzymatic digestion when incubated either in the soluble fraction of the extracts or in buffer, yielding f3-hydroxybutyric acid, suggesting that not only the synthase enzyme but also the depolymerizing enzymes are associated with PHB granules (Merrick and Doudoroff, 1964).Therefore, the regulation of the intracellular polymerase and depolymerase system should be clarified not only from the point of view of the enzymatic mechanism but also to obtain maximum polymer synthesis and production (Foster et al., 1994). Phasins are a newly defined class of proteins thought to have a similar function to oleosins of triacylglycerol inclusions in seeds and pollen of plants (Murphy, 1993). This type of protein has been identified from A. eutrophus (Wieczorek et al., 1995). R. ruber (Pieper-Furst et al., 1994, 1995), and Acinetobacter spp. (Schembri et al., 1995) and has been shown to influence the size of intracellular PHA granules. The polyester storage granules in bacterial cells resemble a chemical emulsion polymerization (de Koning and Maxwell, 1993). In the first stage of granule formation, soluble PHA polymerase molecules acquire surfactant-like properties as the result of covalent attachment to newly synthesized hydrophobic PHA. These "surfactant" molecules self-associated to form micellar structures, which are then enlarged through further polymer biosynthesis to form full-sized granules (Horowitz and Sanders, 1994a). In vivo PHB has a density around 1.16 g ~ m - whereas ~, the isolated PHB was about 1.24 g cm-3; the difference in the two densities was attributed to a water content of 40% in the granules (Mas et al., 1985). Initially, the native PHB granules were believed to be crystalline like the isolate polymer (Ellar et al., 1968);however, findings (Barnard and Sanders, 1988, 1989; Lauzier et al., 1992b; Kawaguchi and Doi, 1990; Amor et al., 1991) demonstrate that PHB in vivo is in the amorphous fluid (mobile) state at ambient temperatures. Due to their natural origin, PHAs have an exceptional stereochemical regularity, which enables the polymer to crystallize (Preusting et al., 1990) by specific treatments during isolation procedures (Kawaguchi and Doi, 1990; Barnard and Sanders, 1989; Harrison et al., 1992; Amor et al., 1991; Calos and Kennard, 1994). However, in an exceptional case with an recombinant E. coli strain, the nascent PHB granules were found to be crystalline in differential scanning calorimetric analysis (Hahn et a]., 1995). The in vivo amorphous state of the granules was explained to be due to the result of the slow nucleation kinetics that often govern the behavior of small, independent particles (de Koning and Lemstra, 1992: Bonthrone et al., 1992). This model was supported by the preparation of artificially prepared PHA granules that mimic the amorphous phy-

151

BIODEGRADABLE POLYESTERS

sical state of native PHA storage granules (Horowitz and Sanders, 1994a, 1995; Marchessault et al., 1995; Horowitz et al., 1993) coated with a detergent or phospholipid layer in place of native protein-lipid coat, which remained amorphous for long periods in aqueous suspensions but crystallized rapidly on disturbance of the protective surface layer (Lauzier eta]., 1992c, 1994). 5. Regulation of Polyester Accumulation in Microorganisms

The PHA inclusion bodies are generally accumulated by the bacteria during metabolic stress caused by nutrient-limiting conditions in the presence of excess carbodenergy source and may account for up to 90% of cell dry weight. Metabolic stress is mainly due to the limiting of certain compounds (Table XIX), and polymer production can be achieved (Fig. 23) either simultaenously with the production of microbial biomass (Gross et al., 1989a; Brandl et al., 1989, 1991) or in a serial process in which microorganisms are first grown with a carbon source to obtain large

TABLE XIX FACTORS REGULATING PHA ACCUMULATION IN MICROORGANISMS Regulation of PHA accumulation

Organism

Reference

~~

I. Compounds whose limitation leads to PHA accumulation Doi et 01. (1995) Aeromonas caviae Carbon Dunlop and Robards (1973) Spirillum sp. Nitikin et al. (1986) Hyhomicrobium sp. Azospirillum Tal and Okon (1985) brasiliense Doi et al. (1987b) Ammonia (nitrogen Alcaligenes starvation) eutrophus Lafferty et 01. (1984) A. latus Tal and Okon (1985) A z . brasiliense Suzuki et al. (1986) Pseudomonas sp. Brandl et al. (1988), F! oleovorans Lafeveen et a]. (1988) Ramsay eta]. (1989) I? cepacia Brandl et al. (1989) Rhodospirillum rubrum Brandl et al. (1989, 1991) Rhodobacter sph aeroides Rhodopseudomonas Philippis et al. (1992) palustris Spirulina maxima Philippis et al. (1992) (continues)

152

CH. SASIKALA AND CH. V. RAMANA TABLE XIX-Continued

Regulation of PHA accumulation Ammonia (nitrogen starvation) contin ued

Iron Magnesium Manganese Oxygen

Phosphate

Organism

Reference

Methylocystis parvus Methylobacterium rhodesianum Thiosphaera pan totropha Rhizobium sp. Pseudomonas sp. Pseudomonas sp. Rhizobium sp. Pseudomonas sp. Az. brasiliense Az. vinelandii Az. beijerinckii Rhizobium sp. Methylobacterium rhodesian um

Asenjo and Suk (1986) Breuer et al. (1995)

R. rubrum

Brandl et al. (1989)

Rb. sphaeroides A. eutrophus Caulobacter crescen tus Spirulina maxima Rhodopseudomonl 3s Pal ustris Methylobacterium rhodesian urn Potassium Bacillus thuringiensis Sodium Rhizobium sp. Sulfate Pseudomonas sp. R. rubrum Rb. sphaeroides 11. Environmental factors regulating PHA accumulation

Bonnet-Smits et al. (1988) DeVries et al. (1986) Suzuki et al. (1986) Suzuki et al. (1986) DeVries et al. (1986) Suzuki et al. (1986) Tal and Okon (1985) Ward et al. (1977) DeVries et al. (1986) Breuer e t al. (1995)

Asenjo et al. (1995) Poindexter and Eley (1983) Philippis et al. (1992) Philippis et al. (1992) Breuer et al. (1995) Wakisaga et al. (1982) Natarajan et al. (1995) Merrick (1988) Brandl et al. (1989)

Light intensity

Spirillunia sp. Rb. sphaeroides

Philippis et al. (1992) Brandl et al. (1991)

Temperature PH

S. maxima

Philippis et al. (1992) Macrae and Wilkinson (1958), Hashimoto et al. (1993)

111. Physiological factors regulating PHA accumulation

Grobth rate CIN ratio Complex nitrogen

Escherichja coli

Preusting et al. (1991) Park et al. (1995b) Lee and Chang (1995a)

BIODEGRADABLE POLYESTERS

153

I Microorganism 1 d

4 Cell growth first (Biomass)

PHA formation (A) PARALLEL PROCESS

PHA producing substrate and under nutrient limitation

\1

(6)SERIAL PROCESS FIG. 23. Microbial PHA formation. (A) A substrate is utilized simultaneously for both cell growth and PHA formation. (B) Polymer producing substrate is added to the culture after growth.

amounts of biomass, then the medium is depleted of an essential nutrient and a polymer-forming substrate is added (Liebergesell et al., 1991; Doi et al., 1986c, 1987a,b; 0. Kim et al., 1995). The yields are usually very high in the latter process because the substrate is converted directly to polymers and essentially only little additional cell growth occurs. In the majority of bacteria, polymer was formed at the stationary phase when a high concentration of carbon was used in the medium. PHA content of 40-70°/o of cellular dry weight was detected under nitrogen-limiting conditions in R. rubrum (Brandl et al., 1991),R. sphaeroides (Brandl et al., 1991; Gross et al., 1992), R. capsulatus, R. vannielii (Karayiannis and Madigan, 1990), P: oleovorans (Ramsay et al., 1991; Gagnon et al., 1992b), P: resinovorans (Ramsay et al., 1992), I? cepacia (Frederick et al., 1994; Ramsay et al., 1991,1989),A . eutrophus (Valentin et al., 1995; I. Y. Lee et a]., 1994b;Cho et al., 1994),Methylobacterium extorguens (Taidi et al., 1994),R. ruber (Williams et al., 1994), and S. wolfei (Amos and McInerney, 1990). The type of nitrogen source used during growth (ammonium chloride or dinitrogen) has not caused any significant variation in the intracellular concentration of PHB in various purple nonsulfur anoxygenic phototrophic bacteria and oxygenic phototrophs (Philippis et a]., 1992). However, with other bacteria it was found that nitrogen-fixing cells are better producers of PHB than ammonia-grown (nitrogenase-repressed) cells (Ward et al., 1977; Stevenson and Socolofsky, 1966). This was explained (Page 1992b)in Az. vinelandii to be due to the greater use of the tricarboxylic acid cycle by nitrogen fixers than ammonia-grown cells, resulting in an increased activity of isocitrate dehydrogenase providing

154

CH. SASIKALA AND CH. V. RAMANA

NADPH for nitrogen fixation (Benemann et al., 1971) and for PHB synthesis (Haywood et aJ., 1988a). In cells of anoxygenic phototrophic bacteria exposed to nitrogen starvation, the type of nitrogen source used for growth caused significant differences in PHB synthesis. When diazotrophically growing cultures were transferred from an atmosphere of nitrogen to argon (i.e., N limitation), the cells with active nitrogenase began to produce large amounts of hydrogen with no effect on PHB synthesis. In contrast, when nitrogen starvation arose in a culture grown on ammonium (in an atmosphere of argon), protein synthesis suddenly ceased, and a significant PHB accumulation took place. Addition of new ammonium chloride resulted in a marked decrease in PHB content (Philippis et aJ., 1992). Rhodobacter capsulatus (Klein et al., 1991) and syntrophic cocultures of a green sulfur bacterium (ChJorobium vibrioforme) and a sulfur-reducing bacterium (Desulfuromonas acetoxidans) could not accumulate PHA from acetate (10-15 mA.4) under nitrogen limitation but produced excess amounts of hydrogen gas with simultaneous formation of polysaccharide (20-40% of cell dry wt) (Warthmann et aJ., 1992). Thus, a low concentration of organic carbon under nitrogen limitation favors hydrogen formation by nitrogen-fixing bacteria and a concentration as high as 100-130 mMof substrate was determined to be optimal for PHA stimulation (Brand1 et al., 1991; Hashimoto et al., 1993). The effect of complex nitrogen sources on PHB synthesis was examined (Lee and Chang, 1994, 1995a). PHB synthesis was generally promoted by supplementing the medium with a small amount of complex nitrogen sources. Supplementation with 0.2% ( w h ) tryptone, casamino acids, or casein hydrolysates promoted PHB synthesis to a greater extent. The reasons for this enhancement was explained by considering the primary metabolism of E. coli as follows (Lee and Chang, 1995a): 1. The synthesis and accumulation of PHB is dependent on the amount of acetyl-CoA available, which is routed to other directly competing pathways such as the formation of acetate, citrate, or fatty acids; thus, in normal medium the amount of acetyl-CoA available for PHB synthesis would be less than that in semidefined or complex medium because acetyl-CoA must be used to synthesize the biosynthetic intermediates and to generate energy. 2. In a complex medium, many of the intermediary metabolites, such as amino acids, vitamins, and fatty acids, are provided such that the cells accumulate more PHAs.

A variety of commercially available complex nitrogen sources [fish peptone (FP);proteose peptone No. 3 (PP3),yeast extract (YE), casitone,

BIODEGRADABLE POLYESTERS

155

proteose peptone, phytone, and tryptone; casamino acids: bactopeptone; neopeptone: gelatin and beef extract] were tested for their effect on PHB formation by Az. vinelandii UWD using glucose as the carbon source (Page, 1992b). A high initial concentration of complex nitrogen sources (0.5%) of FP, PP3, or YE significantly increased PHB yield. With increasing C:N ratios and lowering of the growth rate, the production of PHB increased (Dierstein and Drews, 1974). Polyester formation, composition (Tables IX and XI), and molecular weight (Table XX) were affected by different carbon sources used for growth. Propionate and pyruvate were used as potential PHA-producing carbon sources. Acetate concentration of between 100 and 130 mMwas found optimal with respect to both cell and polymer formation in R. rubrum (Brandl et al., 1991). Use of fatty acids alone to promote PHA copolymer formation in Az. vinelandii inevitably resulted in decreased polymer yield. However, valerate and other short-chain, uneven-length fatty acids promoted the formation of the PHA copolymer P(HB-co-HV)in Az. vinelandii UWD growing in glucose medium (Page and Manchak, 1995). The uptake of valerate was inducible, being repressed by acetate but not by glucose. A likely route that would direct valerate into PHA synthesis involved the P-oxidation pathway (Page and Manchak 1995). Lactones such as y-butyrolactone, y-valerolactone, and higher analogs did not support the cell growth of Hydrogenophage pseudoflava when used as the sole carbon source (Choi et al., 1995). However, cofeeding of lactones with glucose enhanced the utilization of lactones for both copolyester accumulation and cell growth (Doi, 1990: R. A. Ramsay et al., 1990). Chemostat studies revealed that oxygen limitation initiated the accumulation of PHA in Az. beijerinckii (Ward et d.,1977; Senior et a]., 1972) and that relaxation of an oxygen limitation had led to an immediate decrease in PHB content (Senior and Dawes, 1973; Jackson and Dawes, 1976). Furthermore, nitrogen-grown organisms like Az. beijerinckii displayed a steady decrease in polymer content with increasing oxygen concentration (Ward et d.,1977). For phototrophs, decreasing light intensity stimulated the formation of PHAs in R. rubrum (Gobel, 1978); however, in Rhodobacter sphaeroides, such a stimulation in PHA accumulation was not observed, and instead polymer content was decreased at very low light intensities (Brandl et al., 1991). The effect of pH was tested on the production of PHB by many bacteria (Macrae and Wilkinson, 1958) and was observed to be optimum at pH 7.0 and 8.0 (Hashimoto et al., 1993). PHA content was also determined as a function of growth rate (Preusting et al., 1991) and at various temperatures (Philippis et al., 1992).

TABLE XX MOLECULAR WEIGHT AND THERMAL PROPERTIES OF A FEWNATURAL PHAs

PHAS Organism

Carbon Source

A. eutrophus

Sucrose (S) 3-hydroxypropionate (3HP)

+

(% mol ratio)

Thermal Molecular weight properties M, MjM, T,,,("C)

P(3HB-3HP)

(1OO:O)

768,000

1.9

177

365,000 203,000 293,000 180,000

2.1 2.9 2.2 2.9

155 150 85

Glucose

(9O:lO) [81:19) (74:26) PHB

Glycerol

PHB

78,000

3.4

s+3HP

P(3HB-3HP) (80:ZO)

280,000

2.1

143

280,000 110,000 143,000 621,000

2.4 2.9 1.4 8.3

155 61 77

Methanol

(57:43) (22:88) (0O:lOO) PHB

Succinate

PHB

87,000

3.9

l? oleovorans

Hexanoate

a

150.000

2.2

4

150,000 94,000 85,000 46,000 148,000

2.5 2.2 2.2 2.0 1.45

l? cepacia

Heptanoate Octanoate Nonanoate Decanoate Octanoate (OA) + CPH (1:l) OA + CPV (1:l) OA NPH (1:l) Propionate

79,400 94,200 137.000

2.01 1.88 3.9

I! putida

OA + CPH (1:l)

0

75,200

1.89

OA + CPV (1:l)

0

OA

0

91,700 74,000 800,000

1.98 1.83 2.83

A. latus

Methylobacterium

Reference

Hiramitsu and Doi (1993)

Taidi et al. (1994) Taidi et al. (1994) Shimamura eta]. (1994)

Taidi et al. (1994)

extorquens

Rhodococcus SP

+

+ NPH (1:1)

Glucose

a a a a

0

a

P(3HB-3HV)

Taidi et al. (1994) Brand1 et al. (1988)

H. Y.Kim eta]. (1995) Ramsay et 01. (1989)

P(3HB-3HV) (17:83)

H. Y. Kim et al. (1995) 91.6

Haywood et al. (1991)

Note. CPH, para-cyanophenoxyhexanoate; CPV, para-cyanophenoxyvalerate;NPH, para-nitrophenoxyhexanoate. Composition of PHA varies.

157

BIODEGRADABLE POLYESTERS

The molecular weight of microbially produced PHAs is greatly affected by either the culture conditions or the procedures employed during their isolation (Table XXI). Molecular weight and copolymer composition of PHAs greatly depend on various cultural conditions (Anderson and Dawes, 1990) as observed in Pseudomonasputida (Hori et al., 1994b),Az. vinelandii (Manchak and Page, 1994; Chen and Page, 1994),and M. extorquens and A. eutrophus (Anderson et al., 1992; Taidi et al., 1994). Molecular weight of PHB produced during batch culture declined with glucose (Ballard et al., 1987) and fructose (Bradel and Reichert, 1993) for A. eutrophus and octanoate for l? putida (Hori et al., 1994) or remained unchanged when supplied with butyrate as a sole carbon for A. eutrophus (Shimizu et al., 1993). The polydispersity (MJM,) of the polymer remained constant in the studies of Kawaguchi and Doi (1992) and Mansfield et al., (1995), whereas an increase in M,/M, was noted by Brand1 and Reichert (1993). More details are given in Table XX on the molecular weight and thermal properties of PHAs by

TABLE XXI SOME OF THE

FACTORS AFFECTING THE MOLECULAR WEIGHT OF MICROBIAL PHAS

Factors affecting molecular weight ~

Reference

~

Culture conditions

Varies with different organisms Environmental factors pH, temperature Carbon concentration Type of carbon

Combined nitrogen C/N ratio Mg and P limitation Growth phase

Monomer composition in the copolyester Isolation procedures

Ballard et al. (1987) Suzuki et al. (1988) Taidi et 01. (1994), Anderson et al. (1992) Haywood et al. (1991), Taidi et al. (1994), Anderson et al. (1992) Anderson et al. (1992) Hori et al. (1994a) Asenjo et al. (1995) Mansfield et al. (1995), Bradel and Reichert (1993) Doi et al. (1995) Berger et al. (1989), Hahn et al. (1995), B. A. Ramsay et al. (1990), Nuti et al. (1972), Lundgren et al. (1965), Lehrle et al. (1995)

158

CH.

SASIKALA AND CH. V. RAMANA

different group of bacteria as influenced by carbon source and copolymer composition. 6. Microbial Production of Polyesters

Production of PHAs begins with the cultivation of proper microorganisms. Alcaligenes eutrophus (Holmes et a]., 1982a,b, 1983a,b, 1984; Holmes, 1985b) is the organism of choice for large-scale production of PHAs by a subsidiary of ICI, Ltd., in Great Britain, and by many other workers (Takashi and Masako, 1994; Hiroshi et al., 1993; Hughes and Richardson, 1982; Eggink et al., 1994), and it was found to accumulate over 90% (of cell dry weight) PHA. Alcaligenes latus DSM 1124 can also accumulate about 80% (of cell dry weight) PHB and is being experimented with on a large scale (Hrabak, 1992a). Apart from production of PHB, this organism can also produce copolymers P(3HB-co-3HV) and P(3HB-co-4HB)and terpolymer P(3HB-co-3HV-co-4HB)on a large scale (Hrabak, 1992a,b). Pseudomonas oleovorans is a potential organism for the production of unusual PHAs on a large scale because this organism has the ability to grow and accumulate unusual PHAs from long-chain carbon sources (Doi et al., 1995; Gagnon et al., 1992b). Production of PHB by recombinant E. coli harboring the A. eutrophus PHA biosynthetic genes has been investigated by several workers (S. T. Lee et al., 1994; Fidler and Dennis, 1992; Hahn et al., 1995; Middelberg et al., 1995; Haigermoser et al., 1993; Kim et al., 1992; S. Y. Lee et al., 1994a,b,c).Such recombinant strains accumulate as high as 88 g/liter PHB in 42 hr by fed-batch cultures (Kim et al., 1992). Rhodospirillum rubrum is the phototrophic microorganism of choice for many laboratories (Brand1 et al., 1989; Ulmer et al., 1994; Maness and Weaver, 1994). This organism is very useful for the production of desired polyester because it can accumulate a number of PHAs, and quantitative studies on polyester production have been performed (Weaver and Maness, 1993). On an industrial scale, the use of anoxygenic phototrophic bacteria could harness sunlight as an energy source for the production of PHAs (Sasikala,1996) along with many other valuable chemicals (Fig. 24). The only other photosynthetic bacteria are the oxygenic phototrophs, but their PHB content is very low (Capon et al., 1983; Vincenzini et al., 1990; Stal, 1992) and not attractive for applied work. Although some of the halobacteria can also accumulate PHAs in considerable amounts (Fernandez-Castillo et al., 1986), the major disadvantage with this group of organisms is that they are extremely halophilic, which creates problems in industrial processes (RodriguezValera and Lillo, 1992), including exocellular polysaccharide produc-

BIODEGRADABLE POLYESTERS

1

Solar Energy

159

I

Hormones

Waste Treatment FIG. 24. Multiple utilization of the group anoxygenic phototrophic bacteria. This group of microorganisms derives energy from sunlight and uses organidinorganic wastes as electron donors: this property can be exploited for the production of many valuable compounds including PHAs (Sasikala and Ramana, 1995a,b).

tion by Haloferax mediterranei (Lillo and Rodriguez-Valera, 1990). Methylotrophs are also being used for the mass production of PHB (Suzuki et al., 1986). An efficient production process for PHAs can be achieved by considering the kinetics of PHA accumulation in fermenters (Yo0 and Kim, 1994; Heinzle and Lafferty, 1980; Lee and Yoo, 1991; Byrom, 1992; Mulchandani et al., 1989; Braunegg et al., 1995; Belfares et al., 1995). Normally, a two-stage culture is employed in which a high biomass content was achieved in the first stage for subsequent PHA synthesis in the second stage (Braunegg et al., 1995). The following are very important factors for PHA production on a large scale: 1. High cell density is essential for higher productivity of the PHAs (Yamane and Shimizu, 1984).To obtain high cell densities of I! oleovoruns, a reactor was designed that allowed very efficient oxygen transfer (Preusting et a]., 1993b). 2. Concentration of carbon source should be maintained at the optimum value for efficient production of PHA (Lee and Yoo, 1991; Suzuki et al., 1986). 3. Polymer accumulation under nitrogen limitation.

Two different terpolyesters were obtained by fermentation of R. rubrum fed with either 4-pentenoic acid alone or with an equimolar mixture of 4-pentenoic and valeric acid (Ballistreri et al., 1995). When grown on 4-pentenoic acid, R. rubrum PHA oligomers of the general formulas

160

CH. SASIKALA AND CH. V. RAMANA

(HV),, (HV)x-l,(HP), (HV),-, (HP),, (HV),-, (HB), and (HV),-, (HP)(HB) were obtained in a high percentage. The terpolyester of HV/HP/HB having a monomeric ratio varying from 82:15:3 to 85:13:2 was obtained. In contrast, when grown on an equimolar mixture of 4-pentenoic and valeric acid R. rubrum produced terpolymer ( H V ) , (HP), (HB), with a ratio of 90:7:3 (Ballistreri et aJ., 1995). When provided with propionate and pyruvate, a copolymer with a very high content of 3HV was produced by I?. rubrum (Brand1 et aJ., 1991). The addition of certain chemicals to the fermentation medium has not only improved the polymerization but also controlled the polymer structure. Thus, the addition of phenyllactic acid in the medium during growth of Azotobacter chroococcum has influenced the polymerization of PHB and cell elongation (Nuti et al., 1972). The presence of poly(ethy1ene glycol) in the fermentation medium of A. eutrophus was shown to serve as a highly interactive as well as reactive additive that is also powerful for PHA structural control (Shi et al., 1995). Although batch fermentative production of PHAs (I. Y . Lee et al., 1994a) was the choice of many workers, and recently many new batch fermenters with on-line monitoring of fermentations are being used (Kim et al., 1994; Meier-Schneiders et aJ.,1995), continuous cultivation of microorganisms may be a better choice for the production of polyester on a large scale (Preusting et aJ.,1993a; Asenjo et a]., 1995). Biopolymer production by using solid-state fermentations has not been attempted so far, but in our laboratory work has been initiated on the production of PHAs using solid-state fermentation technologies. Use of cheap carbon substrates would lower the cost of microbial production of PHAs (Ramsay et aJ., 1993a). Some of the cheap carbon sources used so far for the production of PHAs are listed in Table XXII. Use of plant oils such as castor oil and euphorbia oil permits formation of PHAs containing novel functionalized constituents (Eggink et al., 1993,1995). Pseudomonas aeruginosa was able to metabolize euphorbia oil and the presence of the main component, vernolic acid, results in the incorporation into PHAs of 3-hydroxy fatty acids with epoxy groups at different positions that are formed as a result of the p-oxidation pathway of vernolic acid (Eggink et aJ., 1995). In PHAs formed from castor oil, 7% of the monomers appear to be derived from ricinoleic acid (12,hydroxy-9-cis-octa decanoic acid). Incorporation of epoxy and hydroxy monomers into PHA is of great importance because it allows further chemical modification and crosslinking of PHAs thereby extending the potential application of these polymers. However, the yields of such polymers are very low (7-15% of dry weight). Aeromonas caviae, a facultative anaerobic bacterium grown on glucose and sucrose, did not accumulate polyesters; however, when grown on

161

BIODEGRADABLE POLYESTERS TABLE XXII

PRODUCTION OF PHAs FROM ORGANIC WASTES AND OTHER CHEAP COMPLEX ORGANIC CARBON SOURCES

Organism

Carbon source used for polymer production

PHA (% dry wt) ~~

Alcaligenes AK201 Alcaligenes latus A . eutrophus Azotobacter vinelandii

Lactococcus lactis followed by Alcaligenes eutrophus Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas pseudoflava Paracoccas denitrificans

Palm oil Molasses Beet sugar Vegetable oil Beet molasses Cane molasses + fish peptone (Fp) Corn syrup + FP Malt extract + FP Glucose + FP Beet molasses + sucrose Sugars + FP Xylose Castor oil Euphorbia oil Hemicellulose Hemicellulose Ethanol

Reference ~

~~

63.8

Majid et al. (1994)

80

Hrabak (1992b) Eggink et al. (1993) Page (1989, 1992a,c) Page (199Zb)

65

70 69 77 74 85

55

Page and Cornish (1993), Page (1993) Page (1990) Page et 01. (1995) Tanaka et al. (1995)

20-30 20-30 60 25

Eggink et al. (1995) Eggink et al. (1995) J. A. Ramsay et al. (1995) Ramsay et al. (1993b)

66 80

50

Yamane et 01. (1996)

oleic acid and olive oil, polyester accumulation was observed (Doi et al., 1995). When grown on unusual carbon sources, particularly the higher fatty acids, n-alkanes, and n-alkenes, many bacteria are known to incorporate LCL polyester (Lageveen et al., 1988; Findlay and White, 1983). The biosynthesis of PHAs from alkanes (Fig. 15) and alkenes (Fig. 21) by Pseudomonas spp. was discussed previously. A two-stage culture method employing Lactococcus lactis 10-1 in the first stage and Alcaligenes eutrophus in the second stage was developed for the production of PHAs horn xylose via L-lactate (Tanaka et a]., 1995a). First, xylose was converted into L-lactic acid and acetic acid by L. Iactis and the organic acids were subsequently converted to PHAs by A. eutrophus (Tanaka et al., 1995b).Pseudomonas cepacia was used for the production of PHAs from xylose, a major hemicellulosic sugar of hardwoods (Ramsay et a]., 1995). Use of nylon manufacturing wastes was recommended as a suitable carbon source for the production of PHAs (Ramsay et al., 1986).Much of the work on the production (Table XXIII), extraction

162

CH. SASIKALA AND CH. V. RAMANA TABLE XXIII ON THE PRODUCTION OF PHAs BY BACTERIA SOMEOF THE PATENTS

Assignee

Patent number

Year

Brief description

Baptist

US 3 044 942

1960

Baptist

US 3 072 538

1963

Baptist

US 3 121 669

1964

Production of PHB.

Baptist and Werber

US 3 182 036

1965

Production of PHB by Rhizobium spp. and plasticized with 10-50% by weight of esters, polyesters, chlorinated polyphenols, polyethers, or cyanocompounds.

Lafferty

GO 2 733 202

1978

Production of 3HB by fermentation with a mutant strain of Clostridium butyricum.

Lafferty

GO 2 733 203

1978

Procedure for isolating mutant bacterial strains for producing PHB.

Powell

EP 15 669

1980

ICI list of PHA-producing microorganisms and a microbiological process for PHB production.

Hughes and Richardson

EP 46 344

1982

Holmes et al.

EP 52 459

1982

Production of PHB with Alcaligenes eutrophus under N and P limitation with glucose as C source. At steadystate conditions, 11.92g cell dry wt was recovered/liter containing 55 mol% of PHB. Production of high-molecular-weight copolymers of PHAs using a mutant strain of Alcaligenes eutrophus.

Holmes et al.

EP 69 497

1983

Holmes et al.

Us 4 477 654

1984

Hughes and Richardson

U s 4 433 053

1984

Production of PHB using Rhodospirillum rubrum and Bacillus megaterium and the process of extraction from bacterial cells using organic solvents. Isolation of bacterial strains capable of producing polyesters.

Production of high-molecular-weight copolymers using Alcaligenes eutrophus with propionic acid as carbon source. Production of copolymers using Alcaligenes eutrophus amounting to 70% of cell dry weight with molecular weight ~ 0 0 , 0 0 0and containing about 30 mol% of 3Hv. Fermentative production of PHB. (continues)

163

BIODEGRADABLE POLYESTERS TABLE XXIII-Continued Assignee

Patent number

Year

Brief description

Metha et al.

IN 170 614

1992

Bacillus thuringiensis obtained from diseased Heliothis arrnigera larva was used for the production of PHB from glucose and peptone. PHB was isolated from the biomass by ultrafiltration and drying as powder.

Weaver and Maness

US 5,250,427

1993

Production of PHA from carbon monoxide using photosynthetic bacteria.

Hiroshi et al.

JF' 05,310,897

1993

PHA produced from Alcaligenes eutrophus using glucose as substrate was disolved in CHCl,, applied to a glass plate, and dried to form a 10-pm film with a contact angle 87" against water. A printing paper coated with the copolymer solution was water repellent and showed good printability.

Takashi and Masako

JP 06,62,831

1994

A bioreactor was described for the manufacture of biopolyesters. Both polyester-producing biomass and polyester production can occur simultaneously in the reactor. A PHB-producing organism (A.eutrophus) was located at the bottom of the bioreactor where the culture medium, having controlled carbon and nitrogen, was supplied, and the PHB was recovered from the top of the bioreactor.

Tooya

JP 06,126,297

1994

Wastewater and wastegases containing sulfur compounds such as hydrogen sulfide, metal sulfates, and/ or organic compounds are treated with hydrogen-generating photoautotrophic microorganisms and the generated hydrogen was mixed with carbon dioxide and treated with hydrogen-consuming chemotrophic bacteria for simultaneously treating the wastes and generating useful materials.

Hubhs et al.

WO 94,00,506

1994 (continues)

164

CH. SASIKALA AND CH. V. RAMANA TABLE XXIII-Continued

Assignee

Patent number

Year

Brief description

John

WO 94,12,014

1994

Eggink et al.

WO 94,09,146

1994

Dennis and Slater

US 5,371,002

1994

Gonzalez et al.

ES 2 061 405

1994

Minagawa et al.

JP 7,155,192

1995

Production of PHB from methanol using Protomonas (Methylobacterium) extorquens at pH 5.5 yielded 35.9 g bacteria/liter having 38.5% PHB.

Jens et al.

DE 4 320 223

1995

A method for improving the economics of PHA formation by lowering the cost of the starting materials was described.

Production of PHAs by transgenic cotton plants. Manufacturing of PHB using glycerol as carbon source by a mutant strain of A. eutrophus. Production of PHA copolymers by a recombinant strain of Escherichia coli. Production of PHAs by a mutant strain of A.chroococcum from waste olive exudate as substrate.

(Fig. 25), and purification processes (Table XIV) of microbial PHAs on a large scale has been patented by many workers. 7. Polyesters from Activated Sludge and from Natural Environments

Poly(hydroxya1kanoates)can be isolated from samples collected from natural carbon-rich environments with a high microbial number (Wallen and Rohwedder, 1974; Findlay and White, 1983; Sat0 et al., 1992; Odham et al., 1986). Various monomers of PHAs, such as 3HB, 3HV, and 3HHx (Wallen and Rohwedder, 1974), 3HB, 3HHx, and 3H0 (Odham et al., 1986), and 3HB, 3HV, 3-hydroxy-2-methylbutyricacid, and 3-hydroxy-2-methylvaleric acid (Sat0 et al., 1992), were isolated from activated sludge collected from sewage plants. PHAs isolated from estuarine sediment contained about 30% (w/w) of PHB and five other 3HAs (Findlay and White, 1983). Determination of PHA content of planktonic bacteria in freshwater can help in assessing the nutritional status of natural microbial planktonic communities (Lopez et al., 1995; Mas-Castella and Guerrero, 1995a,b; Herron et al., 1978; Nickels et al., 1979; Findlay and White, 1983; Balkroill et al., 1988; Blenkinsopp et al., 1991). In planktonic systems, however, only Del Don et al. (1994) reported an appreciable amount of PHA in cells

165

BIODEGRADABLE POLYESTERS

[

Cell Biomass with PHA J.

u u 1 J.

Reflux in hot methanol to remove lipids and phospholipids Cell extraction with solvents like chloroform or methylene chloride Cell debris is removed by filteration

.L

rupture

Enzyme treatment

Washing

a Precipitationof PHB

Drying Extrusion Comminution

Vacuum drying

1

[

Polymer Chips

I1

FIG. 25. Recovery and separation of PHAs from cell biomass and subsequent conversion to polymer chips.

harvested from a freshwater lake. The specific PHA content of the bacterioplankton from Lake Cis0 (Spain] was measured at different depths (Mas-Castella and Guerrero, 1995a). Phototrophic bacteria reach large populations in this lake and the PHA concentration changed seasonally according to their biomass. During summer stratification of the lake, phototrophic bacteria formed a metalimnetic peak. Bacterial counts were maximal at depths of 1.5-2.0 m, whereas the PHA specific content reached a maximal value at 3.0-3.5 m.

D. PHYSICAL PROPERTIES OF PHAs Poly(p-hydroxybutyrate) has a number of interesting characteristics and can be used in various ways similar to many conventional synthetic plastics now in use (TableI). It can be molded, reinforced with inorganic fillers, spun into a fiber, or formed into a film with excellent gas-barrier

166

CH. SASIKALA AND CH. V. RAMANA

properties (Lageveen et a]., 1988). Although these properties of PHB as a biodegradable thermoplastic material have captured attention for more than two decades, this polymer is reported to be relatively hard and brittle apart from its thermal instability at temperatures above melting point (about 180°C) and its thermal degradation during processing is significant (Kunioka and Doi, 1990; Tanahashi and Doi, 1991; Lehrle and Williams, 1994). Furthermore, PHB is subjected to a detrimental aging process that hampers its application possibilities (de Koning et al., 1992). Although moulded PHB shows ductile behavior upon storage at ambient temperature, an aging process embrittles the material. To date, most attempts to overcome the brittleness of PHB have focused on incorporating comonomers (Gagnon et al., 1992b). de Koning et al. (1994) and de Koning and Lemstra (1993) have demonstrated that by using a simple annealing treatment PHB homopolymer can be toughened while subsequent aging is prevented to a larger extent. In contrast to PHB, copolymers containing primarily P-hydroxybutyrate and p-hydroxypentanoate[P(p-hydroxybutyrate-co-p-hydroxypentanoate)] have many desirable physical properties (Barham et al., 1992), and a number of reports on bacterial production of copolymers have been made (Nakamura et al., 1991,1992;Doi et al., 1990b, 1987b; Amos and McInerney, 1991). The production of copolymers has been commercialized and they are now available in the market as a biodegradable plastic under the trade name Biopol, produced by M/s. Imperial Chemical Industries (Europe) with Alcaligenes sp. The physical properties of copolymers vary considerably with comonomer content (Luzier, 1992). Results based on both experimental and theoretical approaches indicate that P(3HB-co-3HV)sform isomorphic crystals (Bloembergen et al., 1986; Bluhm et al., 1986) in which some of the minor comonomer components cocrystallize with the major comonomer components. It is believed that the degree of crystallinity of the copolyester of 3HB and 4-hydroxybutyrate, i.e., P(3HB-co-4HB),decreased with an increase in the amount of the 4HB component, and that P(3HB-co-4HB)with a 4HB content of more than 50 mol% showed no crystalline properties (Nakamura et al., 1994). P(3HB-co-82% 4HB) shows a sharp melting endotherm at 44°C that is close to the melting point of P(4HB) homopolymer (54OC). Synthetic chemists play a very important role in improving the quality of the biopolyester in addition to learning about their structures, chemical properties, and biological activities (Seebach, 1992). One such method of improving PHAs is achieved by blending, which provides a potential method for optimizing mechanical properties without com-

167

BIODEGRADABLE POLYESTERS

promising on ease of processing (Organ, 1994). Several studies have been performed on blending PHB or P(3HB-co-3HV)(Table XXIV) with other dissimilar compounds, e.g., P(3HB-co-3HV)with ethylene-vinyl acetate (Gassner and Owen, 1992), polysaccharides (Holland et al., 19901,PHB blended with poly(ethy1eneoxide) (Avella and Martuscelli, TABLE XXIV

BIODEGRADABLE POLYESTER BLENDS Polyester

Blended compound

Reference ~

P(CL)

Starch derivatives

Phenoxya Polypropylene, polystyrene, nylon 6, poly(ethy1ene terphthalate), PHB PHB

Poly(viny1 phenol) (PVP) Poly(propy1ene) Poly(ethy1ene oxide) (PEO)

Poly(viny1 acetate) (PVAc) Poly(viny1 chloride) (PVC) Poly(viny1 alcohol) (PVA) Poly(epich1orohydrin) (PECH)

Phenoxy Ethylene propylene rubber

(EI" Poly(viny1idene fluoride) (PVDF) Poly(ethy1ene glycol) (PEG) Poly(methy1 methacrylate) (PMMA) Poly(l,4-butylene adipate) (PBA) Ethylene vinyl acetate (EVA) Modified EPR rubbers, grafted with succinic anhydride (EPR-@A); dihutyl maleate

~~

Koenig and Huang (1995), Tokiwa et al. (1990), Koenig and Huang (1992) Coleman et al. (1992) Tokiwa and Iwamoto (1994)

Moskala et al. (1985), Iriondo et al. (1995) Graebling and Bataille (1994) Avella and Martuscelli (1988), Avella et al. (1991,1993), Kumagai and Doi (199Pa) Greco and Martuscelli (1989) Dave et al. (199Oa) Azuma et al. (1992) Dubini et al. (1993), Sadocco et al. (1993), Scandola (1995) Coleman et 01. (1992), Harris et al. (1982) Greco and Martuscelli (1989), Abbate et al. (1991) Marand and Collins (1990), Edie and Marand (1991) Kumagai and Doi (1993) Lotti et al. (1994), Scandola (1995) Kumagai and Doi (199Zb) Abbate et al. (1991) Abbate et al. (1991)

(continues)

168

CH. SASIKALA AND CH. V. RAMANA TABLE XXIV-Continued

Polyester PHB continued

Blended compound (EPR-g-DBM);a modified EVA polymer containing OH groups (EVAL) Poly(cyclohexy1 methacrylate) (PCHMA) Poly((R,S)-lacticacid) P((R,S)-LA) Ethyl cellulose (EtC) Cellulose acetate butyrate (CAB) Cellulose acetate propionate (CAP) P(HB-HV)

Poly(P-propiolactone) (PPL) Poly(ethy1ene adipate) (PEA) Poly(ecapro1actone) (PCL)

PHB (natural) P(3HB-co-3HV)

Acylglycerols (glycerol tri acetate, glycerol monostearate glucerol tristearate) PHB (synthetic) PVC Acrylonitrile-butadiene styrene (ABS) copolymer Styrene-arylonitrile (SAN) copolymer Polystyrene (PS) Polyethylene (PE) EVA Polypropylene Polysaccharides

Cellulose ester Carboxymethyl cellulose PCL, poly(L-lactide) (PLL), poly(D-lactide) (PDL) Starch granules PCL PEG “Polyhydroxyether of bisphenol A.

Reference

Lotti e t a ] . (1994), Scandola (1995) Koyama and Doi (1995) Scandola Scandola Scandola Scandola

(1995)

et al. (1992), (1995) et al. (1992)

Kumagai and Doi (1992c), Organ and Barham (1993), Barham et al. (1992) Kumagai and Doi (1992~) Kumagai and Doi ( 1 9 9 2 ~ ) Kumagai and Doi (1992b), Abe et al. (1994a) Abe et al. (1994b,c)

Pearce e t a ] . (1992) Dave et al.(199Oa,b)

Bhalakia et al. (1990) Bhalakia et al. (1990) Gassner and Owen (1992) Chatterjee and Salanitro (1992) Tanna et al. (1992), Ramsay et ol. (1993b), Mayer et al. (1990), Yasin et al. (1989) Lotti and Scandala (1992) Lauzier e t a ] . (1993) Dave et ol. (1990a,b,c) Ramsay et al. (1993b) Gada et al. (1994) Gada et al. (1994)

BIODEGRADABLE POLYESTERS

169

1988),poly(viny1acetate) (Greco and Martuscelli, 1989),poly(methacrylates) (Lotti et al., 1994; Canetti et al., 1994), cellulose derivatives (Scandola et al., 1992) or blends of microbial copolymers [e.g., blends of PHB with P(3HB-co-3HV)(Organ, 1994)], or P(HV) with P(3HB-co3HV) (Pearce and Marchessault, 1994a). Such blending has not only improved the quality of the polyester but also accelerated the hydrolytic mode of degradation in nature (Sato et al., 1994; for review, see Verhoogt et a]., 1994; for patents, see Table XXV). Apart from the production of PHA blends, synthetic chemists play an important role in the synthesis of PHA conjugates to obtain materials with improved properties (Yalpani et al., 1991). LCL polyesters are good thermoplastic elastomers (Marchessault et al., 1990a,b)from the shape of stress, strain, curves, and values of the initial modulus for representative samples. Thus, unlike PHB and P(HB-coHV), which are crystalline thermoplastic and somewhat brittle, these LCL polyesters have the potential for commercial applications requiring biodegradable plastic with tough elastomeric properties (Jesudason and Marchessault, 1994).

VII. Application of Biodegradable Polyesters

There is a great demand for the biodegradable polymers in the years to come with a view of having a clean environment (Hrabak, 1992a; Hanggi, 1995). Biodegradable polyesters are being used for various applications (Table XXVI) and many of the products that are of commercial importance are being patented (Table XXVII). Apart from using PHAs as biodegradable plastics in industries, the 3-hydroxybutyric acid monomer that is a chiral molecule (the natural polymer consists purely of D-(-) monomers) can be used as basic molecules for the TABLE XXV SOMEPATENTS OF BLENDED PHAs Assignee

Patent number

Year

Brief description

Holmes et al.

EP 52 460

1982

Polymer blends of PHB with chlorinated polyethylene

Chatterjee and Salanitro

US 5 135 966

1992

Preparation of blends of P(HB-co-19% HV) and polypropylene with and without a metal stearate

170

CH. SASIKALA AND CH. V. RAMANA TABLE XXVI PRACTICAL APPLICATIONS OF PHA" ~

Medical applications 1 Surgical pins, sutures, staples, and swabs 2 Wound dressing 3 Blood vessel replacements 4 Bone (orthopedic) replacements and plates 5 Stimulation of bone growth by piezoelectric properties 6 Biodegradable carrier for long-term dosage of drug and medicines Industrial applications 1 Biodegradable carrier for long-term dosage of herbicides, fungicides, insecticides, or fertilizers 2 Packing containers, bottles, wrappings, bags and films, fiber-reinforced biodegradable bicycle helmet, autoseparative filter 3 Disposable items suchas diapers or feminine hygiene products 0 Source: Brandl et al. (1 990, 1995a), Boaree et al. (1993), Foster and nghe (1 994). Kammish (1993) and Mochizuki et al. (19931.

TABLE XXVII PATENTED PRODUCTS MADEOF PHAs Assignee

Patent number

Year

Brief description

Baptist and Frank

U s 3 107 172

1963

Molded product from bacterial cells containing PHB.

Holmes

EP 104 731

1984

Process for spinning PHB into oriented fibers.

Holrnes

GB 2 160 208

1985

Medical dusting powders are formulated with a sterile particulate (average < 30 )L particle size) PHB containing >80 mol% of PHB units.

Rosetta and William

US 05,266,422

1993

Compounds and method for providing a solid conductive electrolyte component containing a PHA and a salt of conductive material for batteries was described.

Nozawa et al.

JP 05, 209,314

1993

Polyester fibers were manufactured by melt-spinning polyester composed of 3HB and 4HB unit at 162-180°C. A. eutrophus produced copolymer that had 5 8 . 3 ~ 1 0 4 molecular weight with 87.3 mol% 3HB and 12.7 mol%

(continues)

171

BIODEGRADABLE POLYESTERS TABLE XXVII-Continued ~

Assignee

Patent number

Year

Nozawa et al. continued

~~

Brief description 4HB. The polyester was spun to give fibers showing tensile strength 0.16 g/denier and elongation at rupture 119%.

Ito et al.

JP 05,230,351

1993

Polyester resins containing of hydroxyalkanoate units and copolymers blended with poly(ecapro1actone) gave sheets with good yield, strength, and rigidity.

Mochizuki et 01.

JP 05,93,317

1993

Conjugate polyester was prepared by melt spinning together poly(E-caprolactone) and/or poly (P-propiolactone) and PHAs. The fibers thus produced are useful for diapers, sanitary napkins, and disposable wiping cloths. Poly(ecapro1actone) and pivalic acid and copolymer were together melt spun at 1:1 weight ratio, drawn, crimped, and cut to give staple fibers, which completely degraded on embedding in soil for 2 months.

Brunger and Kemmish

WO 93,10,308

1993

Disposable biodegradable articles (containers) useful as a substitute for polystyrene foam containers, comprise a core of a material that has good dry strength and poor wet strength and a coating of a biodegradable polymer (3HB-co-3HV)was prepared.

Buckmann and Ballard

WO 93,17,064

1993

Block copolymers containing alky(ene) side chains (C5J in one block and hydroxyalkanoic acid residues as side chains in the other block are useful as compatibilizers for polyolefin-poly(hydroxy alkanoic acid) blends. The blends thus obtained have good biodegradability and are useful as films. (contin ues)

172

CH. SASIKALA AND CH. V. RAMANA TABLE XXVII-Continued

Assignee

Patent number

Year

Brief description Use of Biopol as a waste-ink absorbent. Preparation of PHA porous films with good heat resistance and mechanical properties

~~~

~

Isao

EP 613 927

1994

Hirashi et al.

EP 629 662

1994

Nakagawa et al.

JP 6 293 113

1994

Fukconishi and Sato

JP 6 322 198

1994

Wada et al.

JP 6 313 63

1994

Takashi et al.

JP 6 329 774

1994

Liggat and O'Brien

WO 94 28 049

1994

Preparation of biodegradable paper-based laminates by coating polycaprolactonebased dispersions or emulsions on paper substrates. Preparation of biodegradable, transparent, and waterresistant polymer compositions with excellent mechanical and gas barrier properties. Use of polycaprolactone-starch blended sheets as food packing material. PHA useful for films, moldings, fibers, etc., are prepared by condensing lower alcoholic esters of C3-5 hydrocycarboxylic acids [containing no asymentric C atomuin monomer nucleus) in the presence of transesterification catalysts at >7OoC. Heat treatment restores 150% of the ductility of the original nonaged polyester. Heating Biopol for 5 sec to 30 hr at 8O-15O0C,preferably by IR radiation, displayed improved resistance to applied stress compared to the untreated samples.

chemical production of complex chiral pharmaceutical or agrochemical agents (Hrabak, 1992b). An important property of PHB is that it is biocompatible, which means that it can be implanted in the body and will not cause inflammations. PHB is degraded very slowly inside the body over a period of months to years, unlike, for example, polylac-

BIODEGRADABLE POLYESTERS

173

tic acid (Hrabak, 1992a). PHB was also found to be an excellent material for the slow release of pharmaceuticals (Collins et ul., 1989; Hrabak, 1992a). Biopolyesters like PHAs have to pass the following requirements to be accepted on a large scale (Hanggi, 1995): 1. They have to fulfill an urgent market need (need/demand). 2. They must compete with the present plastics in terms of quality

and processing performance (performance). 3. They must meet the requirement for the registration as food packages (registration). 4. They should be available at a competitive price (price). 5. New and efficient composting systems have to be installed in urban areas (composting infrastructure). The price of PHB at present is far beyond the accepted levels: however, mass production can be lowered in such a way that PHB can meet accepted price limits (Hanggi, 1995).

VIII. Biodegradation of Polyesters Biodegradation: Chemical degradation caused by biochemical reactions, especially those catalyzed by enzymes produced by microorganisms under either aerobic or anaerobic conditions. (Lenz, 1993).

A committee of the American Society of Testing and Materials has set specific conditions and developed specific test procedures for evaluating both the aerobic and the anaerobic biodegradation of plastics in the environment. This has become necessary because of the increasing industrial interest and application of biodegradable polymers as well as the growing importance of plastic waste management and the confusion over the basic concepts of biodegradation (Lenz, 1993). The Agency of Industrial Science and Technology in Japan (Masuda, 1991) expresses the degree of biodegradability in percentage (DB)by the following equation: DB =BOD-!? x 100 TOD

where BOD is the biological oxygen demand of the sample (in mg), B is the oxygen consumption of the culture medium containing a standard activated sludge (in mg), and TOD is the theoretical oxygen demand as calculated for the complete oxidation of all organic carbon in the sample (in mg). Some of the test methods used for the determination of biodegradability of plastic materials are shown in Table XXVIII.

TABLE XXVIII TESTMETHODS FOR THE DETERMINATION OF BIODEGRADABILITY OF PLASTIC MATERIALS

Method

Surface growth

Indicator Surface growth of microorganisms

Microbial growth Biomass

Observation or measurement Visual

SEM Turbidometric Protein content Phospholipid content Gravimetric

Polymer utilization

Weight loss

Polymer utilization Change in polymer characteristics

Turbidity Turbidity Tensile strength

Visual Tubidometric Different material tests

Molecular weight

Chromatographic

Organisms or system

Aspergillus niger Penicillium funiculosum Chaetomium globosum Gliocladium virens Aureobasidium pullulans Pseudomonas aeruginosa Aerobic mixed culture Pure or mixed cultures

Pure or mixed cultures or natural ecosystem Pure or mixed cultures Cell-bee enzyme exbact Pure or mixed culture or natural ecosystem

Streptornyces spp.

Phanerochaete chryosporium

Test Laboratory Field

Reference

-

ASTM (1986a)

ASTM (1986b) Ramsay et al. (1993a)

X

-

X

X

X

X

X

X

-

X

X

X

-

X

Aminabhavi et al. (1990)

Brand1 and Puchner (1992), Mergaert et al. (1992a) Gilmore et al. (1990) Gilmore et al. (1990) Sawada (1994), Kaplan et 01. (1994)

ASTM (1994~)

Microbial activity

Clear zone test

Gas production

Titrimetric

Sewage sludge

X

-

ASTM (1994a)

Gas production Oxygen utilization -doGas production Oxygen utilization Gas production

GC Electrochemical

Sewage sludge Pure or mixed cultures

X

-

X

-

ASTM (1994b) MITI (1983, Nr117)

-doTitrimetric Manometric

Sewage sludge Compost Various fungal cultures

X

-

Radiometric

ASTM (1994d) ASTM (1994e) Tilstra and Johnsonbaugh (1993) Albertsson (1989)

Turbidometric

Visual

X

-

X

-

Pure or mixed cultures or samples from natural ecosystem Acidovomx delafieldii

X

-

X

-

Pseudomonas maculicola

X

-

Masao and Yukie (1994), Brand1 et al. (1995b) Foster et al. (1995)

Note. x, Tested;-, not tested; GC, gas chromatographic; ASTM, American Society for Testing and Materials; MITI, Agency of Industrial Science and Technology, Japan; SEM,scanning electron microscope.

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CH. SASIKALA AND CH. V. RAMANA

A. BIODEGRADATION OF PHAs Considerable work has been carried out on the biodegradation of PHAs (Schink et al., 1992; Timmins et al., 1994; Timmins and Lenz, 1994) under both field and lab conditions and under various environmental conditions (Table XXIX). Both extracellular (Kasuya et al., 1995) and intracellular (Saito et al., 1995) degradation of PHAs was studied. However, the majority of the studies were concentrated on extracellular degradation of PHAs because of applied interest with regard to environmental pollution. Among various PHAs, the majority of such work is concentrated on the degradation of PHB and the copolymer, P(3HBco-3HV). Very little work was carried out on the degradation of other PHAScL,PHAMC-, and PHALCL. Most of the work performed on PHA degradation was on bacterial PHAs and degradation of synthetic PHAs was only sparingly studied (Jesudason et al., 1993; Brandl et a]., 1995b). Chemically modified linear substituted 3HB or cyclooligo 3HB were also tested for biodegradation (Brandl et a]., 1995b). Degradation experiments were carried out under natural environments (Gilmore et al., 1993, 1994; Loetter, 1987; Brandl and Puchner, 1992; Mergaert et al., 1992) and also under laboratory conditions (Mergaert et al., 1993; Ramsay et al., 1993b). Although degradation of P(HB-co-HV)was studied in a number of environments (Mergaert et al., 1992a, 1993), the majority of the studies were in the compost (Gilmore eta]., 1992; Mergaert et a]., 1992b, 1994a; Budwill et al., 1992). Mergaert et al. (1992b) found that PHB and P(HB-co-HV) were degraded in all natural environments studied, i.e., soils (hardwood, pinewood, sandy, clay, and loamy), waters (freshwaters and seawater), and composts, although the rates varied depending on the environment and increased with temperature. Under anaerobic conditions, methanogenic degradation of PHAs was also known (Budwill et a]., 1992). Biodegradation of plastic bottles, commercially available polymers made from Biopol, and dogbone-shaped or injection-molded polymer from Zeneia bioproducts were also studied in an aquatic ecosystem under in situ conditions (Brandl and Puchner, 1992; Mergaert et al., 1995). Principally, the following approaches were used to show bacterial PHA degradation: 1. As weight loss of PHB strips incubated in natural environments (Hartley 1977; Marchessault et al., 1994; Brandl and Puchner, 1990; Malik and Claus, 1978; Matavulj and Molitovis, 1991a; Mergaert et al., 1992a,b) or under lab conditions (Hocking eta]., 1994; Jesudason et al., 1993; Mergaert et al., 1993). 2. As loss of turbidity on PHB-containing simple mineral agar plates (Chowdhury, 1963; Griebel and Merrick, 1971; Saito et al., 1989).

TABLE XXIX

BIODEGRADATION OF SOME OF THE PHAs BY WHOLE CELLOR EXTRACELLULAR ENZYMES PHA

Nature of PHA

Biodegradation tedorganism

A number of substituted PHAs

Powdered polymeric material

Acidovoran delafieldii (WC, CFE)

P(3HB), P(3HB -co-~HV)

Polymeric films Injections, dogboneshaped polymer

In situ natural waters

Various polymer blends, of PHAs Binary blends of P((R)-3HB)and P((R,S)-lacticacid) Wheat starch granules and P(3HB-c0-19.1% 3HV) blends PHB

Isothermally crystallized Films

Activated sludge

Activated sludge

Alcidovoran delafieldii (CFE)

Test assay

Reference Brandl et al. (1995b)

Modified turbidometric assay (clear zone test) Mass loss Decrease in mass, molecular weight Tensile strength Weight loss and sample thickness Weight loss

Koyama and Doi (1995)

Hori et al. (1995) Mergaert et al. (1995)

Scandola (1995)

Films

Alcaligenes eu trophus (CFE)

Weight loss

Ramsay et al. (1993)

Bottles and films

In situ lake conditions

Weight loss

PHB, ~(HB-CO-HV)

Dry powder

Gas production

PHA LCL

Tensile test pieces Polymer latex

Anaerobic sewage sludge with methanogenic bacteria In situ compost Pseudomonas maculicolo (WC)

Brandl and Puchner (1990, 1992) Budwill et al. (1992)

Weight loss Clear zone technique

Mergaert et al. (1994b). Foster et al. (1995)

Note. WC, whole cell; CFE, cell-free enzyme.

178

CH. SASIKALA AND CH. V. RAMANA

3. As loss in turbidity on PHB containing “polymer overlayer plates” (Delafield et al., 1965a; B. A. Ramsay et al., 1993b, 1994). 4. Quantitatively as extracellular depolymerase activity causing a decrease in turbidity of insoluble PHB (Tanio et al., 1982; Stinson and Merrick, 1974; Shirakura et al., 1983; Nakayama et al., 1985). 5. Spectrophotometrically by quantitative determination of crotonoic acid as the product of PHB degradation (Law and Slepeky, 1961; Wong and Evans 1971; Shiraki eta]., 1995; Saito et al., 1993; Tanio et al., 1982; Shirakura et al., 1986). 6. Scanning electron microscopically by recording changes of material exposed to microbial enzymatic attack (Brand1 and Puchner, 1992; Matavulj and Molitoris, 1991d, 1992; Marchessault et al., 1994). 7. By gas chromatography and high-pressure liquid chromatography (Braunegg et al., 1978; Mergaert et al., 1994a; Findlay and White, 1983). 8. By nuclear magnetic resonance studies (Doi et al., 1989a,c). 9. Detection of depolymerase-producing colonies was also carried out immunologically (Saito et al., 1989) in a recombinant E. coli transformed with PHB depolymerase from Alcaligenes faecalis. 10. Titration of free acid released (Shirakura et al., 1986). 11. By enzymatic (~-(-)-3-hydroxybutyrate dehydrogenase) (EC 1.1.1.30) assay of ~-(-)-3-hydroxybutyrate formed (Shirakura et al., 1986; Saito et al., 1992; Nakada et al., 1981). 12. As a decrease in molecular weight (Mergaert et al., 1993). 13. As a loss of mechanical properties (Mergaert et al., 1993).

Use of agar overlay plates (Delafield et al., 1965a)is the method of choice for isolation of PHA-degrading microbes from various environments (Mergaert et al., 199213, 1993, 1994b). Using this method, solidified mineral agar base-supplemented yeast extract and casein hydrolysate are overlaid with approximately 1 mm of the same medium supplemented with the PHA powder (0.25-1%). On this medium, degrading colonies were surrounded by clear zones due to the hydrolysis of the water-insoluble polymer by extracellular depolymerase (Foster et al., 1995; Mergaert et al., 1994b).Even though the isolation of PHA-producing microorganisms was achieved by other ways, this method was nevertheless used for determining the depolymerization capability of the isolates (Gilmore et al., 1992). Matavulj and Molitoris (1991a, 1992) have developed similar simple methods for the screening of marine as well as terrestrial fungi for the degradation of PHAs and PHA-based plastics that are based on the formation of a halo around the fungal colony in a solid medium containing PHB as the principal carbon source. This method using agar overlay plates was extensivly useful for PHA,,, and a number of organisms were isolated that could degrade such PHAs; however, microbes capa-

179

BIODEGRADABLE POLYESTERS

ble of degrading PHALCL were isolated only recently, which is at least partially due to the inability to produce a suspension of PHALcLfor the agar overlay method. B. A. Ramsay et al. (1994) have developed a simple method for the preparation of an autoclavable, PHALCL colloidal suspension, which was used as a substrate for enzymatic degradation and to prepare agar overlay plates for isolating microbes producing extracellular PHALCL depolymerase. PHAs are susceptible to enzymatic and hydrolytic degradation in biological environments by a number of microbes (Table XXX). Gilmore et al. (1992) have concluded that the PHB and P(3HB-co-3HV)are degraded by a combination of biological and chemical mechanisms. TABLE XXX MICROORGANISMS CAPABLE OF DEGRADING PHAs Organism

Acidovorax facilis Acidovorax delafieldii Acremonium sp. Alcalienes faecalis Alteromonas haloplanktis Arthrobacter aurescens Arthrobacter viscosus Aspergillus sp. Aspergillus fumigatus A spergill us penicilloides Bacillus megaterium Bacillus polymyxa Cladosporium sp. Clavibacter michiganense subsp. insidiosum Comamonas testosteronii Comamonas acidovorans Cytophage johnsonae Eupenicillium sp. llyobacter delafieldii Mucor sp. Paecilomyces marquandii Penicillium adametzii Penicillium chermisinum Penicillium daleae Penicillium funiculosum Penicillium janthinellum

Reference Mergaert et al. (1992a), Jendrossek et al. (1993a) Chowdhury (1963) Mergaert et al. (1992a) Tanio et al. (1982) Mergaert et al. (1995) Mergaert et al. (1995) Mergaert et al. (1992b) Mergaert et al. (1992a) Mergaert et al. (1992a) Mergaert et 01. (199Za) Mergaert et 01. (1993) Mergaert et al. (1992a) Jendrossek et al. (1995a), Matavulj and Molitoris (1992) Mergaert et al. (1995) Mukai et al. (1993), Kasuya eta]. (1994) Mukai et al. (1994) Mergaert et al. (1993) McLellan and Halling (1988) Janssen and Harfoot (1990) Matavulj and Molitoris (1992) Mergaert et 01. (1992a) Mergaert et al. (1992a) Mergaert et al. (1992a) Mergaert et al. (1992a) Brucato and Wong (1991) Mergaert et al. (1995)

(continues)

180

CH. SASIKALA AND CH. V. RAMANA

TABLE XXX-con tin ued Organism

Penicillium ochrochloron Penicillium restrictum Penicillium simplicissimum Polyporus circinatus Pseudomonas sp. Pseudomonas cepacia Pseu domonas chlororaphis Pseudomonas fluorescens Pseudomonas lemoignei Pseu domonas picketii Pseudomonas stutzeri Pseudomonas syringae Pseu dom on as syringae pv. maculicola Pseudomonas vesicularis Staphylococcus epidermidis Streptomyces sp. Variovorax paradoxus Verticillium leptobactrum Vibrio ordalii Xanthomonas maltophilia Zoogloea vamigera

Reference Mergaert et al. (1992a) Mergaert et al. (1992a) McLellan and Halling (1988) Matavulj and Molitoris (1992) Mergaert et al. (199Za) Mukai et al. (1994) Mergaert et al. (1995) Schirmer et al. (1993,1995) Delafield et al. (1965) Mukai et al. (1994) Mukai et al. (1994) Mergaert et al. (1993) Mergaert et al. (1995) Mukai et al. (1994) Mergaert et 01. (1995) Mergaert et al. (1992a) Mergaert et al. (1992a) Mergaert et al. (1992b) Mergaert et al. (1995) Mergaert et al. (1992b) Jendrossek et al. (1993a)

Although hydrolytic degradation physically weakened the material and reduced the molecular weight of the polymer making more chain ends available for enzymatic attack, microbes enzymatically degraded the polymer and metabolized the products. Bacteria, fungi, and actinomycetes are known to biodegrade PHA, although their composition and number varied depending on environmental conditions. Nishida and Tokiwa (1993a,1994),who established a method for evaluating the distribution of polymer-degrading microorganisms in different environments, found that PHB-degrading microorganisms are distributed widely and that they represent 0.8-11.0% of the total number of microbes in the environment. A maximal number of PHB-degrading microorganisms were isolated from neutral soils (Mergaert et al., 1992a,b). Among 325 microbes isolated from soil that degraded PHB in vitro were 154 bacteria, 77 streptomycetes, and 94 molds (Mergaert et al., 1992a). Most gram-negative bacteria were isolated from neutral soils and were identified as Acidovoraxfacilis or Varivoraxparadoxus (Mergaert et al., 1992b, 1993). Acidvorax facilis was the dominant PHB-degrading gram-negative

BIODEGRADABLE POLYESTERS

181

bacterium isolated from soil, water as well as compost (Mergaert et al., 1992a, 1993, 1994a). In contrast to soil, V paradoxus was isolated only once from compost (Mergaert et al., 1994a). Unlike neutral soils, acid forest soils contained relatively few PHB-degrading gram-negative bacteria (Mergaert et al., 1993). Among gram-positive bacteria, although Bacillus megaterium was predominant in neutral soils and compost, Bacillus polymyxa was isolated only from acidic soils (Mergaert et al., 1992b, 1993, 1994a). About one-third of the prokaryotic isolates from soils as well as compost had the typical morphology of Streptomyces (Mergaert et al., 1992a, 1994a). Mergaert et al. (1992b, 1993), who studied PHB and P(HB-co-HV) degradation under natural environments as well as laboratory conditions in various soils, have opined that the species from which extracellular PHA depolymerase was studied may not be representative of the P(3HB)- and P(3HB-co-3HV)-degrading microflora in soils because these species (A.faecalis and Penicillium lemoignei) were not found in their study or were encountered only once (Comamonas sp.) Mergaert et al. (1994b) could isolate only three strains of P(3HB)-degrading molds from the composts compared to 61 gram-negative and 10 gram-positive bacteria and 35 actinomycetes. However, Matavulj and Molitoris, (1993) recorded high numbers of P(3HB)degrading fungi in compost and correlated this with high degradation of Biopol samples in this environment. Gilmore et al. (1992) found that of the microbial isolates from film surfaces, only fungi possessed P(3HB-co-HV) depolymerase activity, but mixed cultures of bacteria and fungi cleared P(3HB-co-3HV)overlayer plates more rapidly. The species of fungi encountered among molds were Aspergillus f u migatus, Aspergillus penicilloides, Paecilomyces marquandii, Acremonium sp., Penicillium simplicissimum, Penicillium restrictum, Penicillium ochrochloron, Penicillium daleae, Penicillium chermisin um, Penicillium adametzii, and Verticillium leptobactrum. Aspergillus fumigatus predominated in soils at the end of the polymer degradation experiments at 40°C (Mergaert et al., 1992a). Verticillium leptobactrum was isolated from compost alone, being absent in both acidic and neutral soils (Mergaert et al., 199Za). The species of fungi showing the highest Biopol degradation activity among 111tested are: deuteromycetes: Penicillium simplicissimum, €? atrovenetum, and Trichoderma polysporum; basidiomycetes: Collybia peronata. Lentinus edodes, Pleurotus ostreatus, and Serpula lacrymans; ascomycetes: Nectria episphaeria; and zygomycetes: Mucor hiemalis and Mucor sp. (Matavulj and Molitoris, 1991b, 1992). All chytridiomycetes tested (Phlyctochytrium africanum and Phlyctochytrium palustre) and

182

CH. SASIKALA AND CH. V. RAMANA

all myxomycetes tested (Dictyostellumdiscoideum and Physarum polycephalum) proved to be active (Matavulj and Molitoris, 1991b). Quantitative analysis of microbial population adhering to the degraded Biopol samples revealed that actinobacteria and Pseudomonas and Bacillus strains dominated among the bacteria, whereas the active fungal population consisted of deuteromycetous fungi mainly of the genera Aspergillus, Penicillium, Cladosporium, Cephalosporium, and Verticillium (Matavulj and Molitoris, 1993). Although some microorganisms were ubiquitous in composts (Acidovorax facilous and Streptomyces spp.), others were characteristic of a given compost heap, e .g .,Alterom onas h a1oplanktis for compost A, Pse u dom onas a1caligen es for compost B, and B. megaterium for compost C (Mergaert et al., 1994b). Few microbes were isolated from the copolymer alone: these include Cytophaga, Comamonas, and Variovorax (Mergaert et al., 1994b). Microflora-degrading PHAs also varied with temperature (Mergaert et al., 1992b). P(3HB)- and P(3HB-co-3HVJ-degrading microflora consist of a wide range of different organisms at mesophilic temperatures, whereas only a few species are involved in degradation at higher (4OoC), temperatures (Mergaert et al., 1992b, 1993). At 15"C, the microflora consisted mainly of gram-negative bacteria and streptomycetes, whereas at 28°C gram-positive bacteria and molds were frequently isolated. At 40"C, molds and streptomycetes predominated (Mergaert et al., 1992b). B. A. Ramsay et al. (1994) have isolated six cultures producing extracellular PHALCL depolymerase from a composted hydrocarbon-contaminated soil. All were pseudomonads (of RNA group 1)or related bacteria, which is in contrast to the PHAScL-degradingcapability, which is widespread among a number of gram-positive as well as gram-negative bacteria in addition to Streptomycetes and molds (Mergaert et al., 1992a, 1993, 1994a). The restricted ability to degrade PHA,,, polymers can be explained as due to the restricted ability to produce such polymers by different bacteria, and it was found that these polymers are only degraded by those bacteria that can accumulate such polymers (Schirmer et al., 1993; B. A. Ramsay et al., 1994; Kanesawa et al., 1994; Foster et al., 1995). However, all the bacteria capable of accumulating PHALcL polyester did not possess LCL depolymerase activity (B. A. Ramsay et al., 1994). A close relationship between certain lipases and extracellular PHAL,, depolymerases was suggested (B. A. Ramsay et al., 1994) because all the Pseudomonas isolates producing extracellular PHAL,, depolymerases grew exceptionally well on canola oil. The biodegradation of PHAs varied depending on several factors (Table XXXI), viz. temperature (Mergaert et al., 1992b, 1993), monomer composition and relative quantities (Mergaert et al., 1992b, 1993), ex-

BIODEGRADABLE POLYESTERS

183

TABLE XXXI SOMEOF THE IMPORTANT FACTORS THAT AFFECT THE RATEOF BIODEGRADATION OF NATURAL AND SYNTHETIC POLYMERS--THE TESTSTO BE PERFORMEDO Material processing-Type of processing, surface characteristics, material thickness, additives used, fillers, and coatings. Polymer structure-Particularly the hydrophilicity and the presence of functional groups in, or immediately on, the main chain along with the bond type between monomers, molecular weight and molecular weight distribution, and steric configuration. Physical and morphological state of the polymer-Particularly whether it is crystalline (the degree and form of crystallinity) or amorphous (glass transition temperature), porosity. Physicochemical parameters of the ecosystem-Temperature, pH, humidity, oxygen availability, light, etc., and nutritional requirement of the microorganisms. Microbial population-Number, in particular.

types and interactions of fungi and bacteria,

Surface-to-volume ratio, test sample size, and purity of the polymer sample. Duration of the test. Method of contact between the sample and the microorganisms (or enzymes)Whether in solution, as a gel or slurry, by soil burial of a solid, etc. Products formed by biodegradation and their effect on environment, if any? Toxicity tests on microorganisms, animal life, or humans.

Is the synthetic polymer, in the end, converted completely to CO, and water? OSource: Cook (1990),Lenz (19931,Shimamura et 01. (1994),Doi et a1. (19951,Brand1 et al. (1990,1995a), and Palmisano and Pettigrecu (1992).

posure protocol (Gilmore et al., 1992), oxygen (aerobidanaerobic conditions (Ramsay et al., 1993b; Chowdhury, 1963; Galvin, 1990; Gilmore et al., 1992), blending with polysaccharides, polymer thickness, and exposure surface (Ramsay et al., 1993b), time (Mergaert et al., 1993; Gilmore et al., 1992), environment (Mergaert et al., 1993), film-making technique (Marchessault et a]., 1994), origin of the polyester (Araki and Hayase, 1979; Marchessault et al., 1994), and crystallinity, tacticity, and diad content (Araki and Hayase, 1979; Marchessault et al., 1994; Hocking et al., 1995). Temperature had an effect not only on the microbes that degrade PHAs but also on the rate of degradation. Mergaert et al. (1992) have found that the rate of PHB and P(3HB-co-HV)copolymer degradation increased with temperature. Hydrolysis of polymers only occurs at temperatures higher than 28°C within 98 days (Mergaert et al., 1992b, 1993,1994b).

184

CH. SASIKALA AND CH. V. RAMANA

The monomer composition of PHA is an important factor that affects the rate of degradation of PHA. Generally, copolymers were found to be more biodegradable than the homopolymers, although an observation to the contrary was also reported (Doi et al., 1990b).Mergaert et al. (1992a) found that the copolymer P(3HB-co-3HV)degraded much faster than the homopolymer. After 358 days in a freshwater canal, 34% mass loss was recorded for the homopolymer and 77% for the P(3HB-co-lO% 3HV) samples, whereas the P(3HB-co-20% 3HV) samples had completely disappeared. In contrast, Doi et al. (1990b) found that PHB homopolymer chloroform cast films degrade faster than P(3HB-co-71% 3HV) copolymer. Jesudason et al. (1993) proposed that this observed differences in susceptibility toward enzymatic attack may be the consequence of limited accessibility of the depolymerase to the ester linkages by the presence of the bulkier ethyl side chains along the polymer backbone. Doi et al. (1990a) reported a four- or fivefold lower rate constant for enzymatic degradation of P(HB-co-HV)compared with PHB with samples that were found to have comparable molecular weights and similar crystallinities. Among the copolymers, P(3HB-co-4HB) and P(3HB-co-3HV),the former was found to be more rapidly biodegraded (Doi eta].,1990a). It was proposed that the rapid degradation of P(3HBco-4HB) compared to P(3HB-co-3HV)was due to the lower steric bulkiness of 4HB units compared to those of 3HB and 3HV. The studies of Jesudason et al. (1993) and Hocking et al. (1995) have revealed that chain accessibility and stereochemical susceptibility are the two factors determining the rate of degradation of PHA by PHA depolymerases. The relative compositions of monomers in a copolymer also influence the rate of biodegradation. In in vitro degradation experiments using the enzyme of A. faecalis (Doi et al., 1990a; Saito, 1990), the rate of degradation of copolymers with different HV contents decreased in the following sequence: P(HB-co-45'/0 HV) = P(HB-co-41% HV) >> P(HB-co-lZ.B% HV) = PHB > P(HB-co-S% HV) > P(HB-co-71Yo HV)

Mergaert et al. (1993) found that copolymer, PI~HB-co-~HV), with 20% HV content degraded faster than that with 10% HV content. Marchessault et al. (1994), in a degradation study in activated sludge comparing homopolymer with 5 and 2 1 % 3HV-containing copolymer, could not find a clear relationship between the degradation rate of PHA and the HV content in the copolymer P(HB-co-HV).However, the work of Mergaert et al. (1992b) shows that in relative ratios of degradation,

BIODEGRADABLE POLYESTERS

185

PHB and P(HB-co-HV)are highly dependent on the environments used and can be quite different from data obtained in in vitro tests. Although in in vitro tests single enzymes are used, in natural environments the composition of the degrading microflora and their enzymes varies. Exposure protocol also had an affect on biodegradability of the copolymer. Gilmore et al. (1992) exposed individual samples of P(HBco-HV) to compost for periods of time then removed them for the study rather than reexposing a few samples several times during the study. This was done to avoid the possibility of delays in biodegradation that might have occurred during reestablishment of biofilms on the surfaces of washed samples. It was indeed found that the weight loss per month of reexposed P(HB-co-HV)samples over the first 4 months was always less than the calculated weight loss per month of samples that had not been removed and reexposed. The work of Chowdhury (1963) indicated that PHA degraded more rapidly under anaerobic conditions. In contrast, Ramsay et al. (199313) have observed that under aerobic conditions the solvent-cast PHB film had totally degraded, whereas under anaerobic conditions only 3YO (w/w) of the original weight was lost in 15 days. Depolymerase activity was also much higher under aerobic conditions compared to anaerobic conditions. Budwill et al. (1992) observed methanogenic degradation of PHA and are of the opinion that this anaerobic digestion of waste plastic made from PHA may be a feasible solution to the waste problem with the added benefit of energy conservation due to methane recovery. Although microbial biomass can degrade PHAs completely to CO, and water, it takes months or weeks for biodegradation to occur and in instances such as municipal composting a quicker rate of biodegradation is required. To achieve such quicker biodegradation, Ramsay et al. (1993b) tried blending P(HB-co-HV)with wheat starch. Blends of starch and P(HB-co-HV) (19.1 mol% HV) showed an increased rate of degradation as the starch content increased from 0 to 50%. When starch was added to P(HB-co-HV)polymer it was lost (maybe due to its solubility in water and/or its enzymatic hydrolysis) more rapidly than P(HB-coHV) and its removal resulted in a larger exposed surface for the degradation of the P(HB-co-HV).Films (150 km thick) containing 50% (w/w) starch had a 100% weight loss in fewer than 8 days, whereas a month was required for total weight loss when only P(HB-co-HV)was present. Their studies showed that the addition of starch to P(HB-co-HV)leads to a completely biodegradable material whose degradation rate can be tailored by adusting the starch/PHA ratio in addition to reducing the cost. Other polysaccharide blends are also known to result in similar accelerated effects on PHA degradation (Yasin et al., 1989).

186

CH. SASIKALA AND CH. V. RAMANA

Thicker (800 pm) samples of PHA-starch blends took more time ( 2 1 days) to show complete weight loss than thinner (150 pm) films that had 100% weight loss in fewer than 8 days. Apart from thickness, surface area of the polyester is an important factor determining the rate of microbial degradation of polyesters (Ramsay et al., 1993b). Biodegradation resulting in mass loss occurs at the surface of the sample, with intermediates being rapidly metabolized by the degrading microorganisms. Gilmore et al. (1992) and Mergaert et al. (1993) found that weight loss of plastics (degradation) occurred linearly with time. The environment also had an effect on the biodegradation of PHAs. Among various environmental conditions tested, the highest Biopol degradation was found in greenhouse peat in which fungi dominated, and the lowest degradation was observed in active sludge in which, under semianaerobic conditions, bacteria dominated (Matavulj and Molitoris, 1 9 9 1 ~ )When . PHB and P(HB-co-HV)degradation was studied under different environments (hardwood forest soil, sandy soil, pinewood forest soil, clay soil, and loamy soil), it was found that degradation (weight loss) was dependent on the soil used (Mergaert et al., 1993). However, it was not clear whether the differences in the polymer degradation rates in the different soils are attributable to the differences in the compositions of the degrading microflora. Crystal structure and molecular weight influence the microbial degradability of polyesters (Jarrett et al., 1985; Nishida and Tokiwa, 1993b; Pranamuda et al., 1995). The effect of the degradation on the molecular weight and mechanical properties of the polymer has been studied (Mergaert et al., 1995). There seems to be no correlation between loss of mats and decrease in molecular weight of PHA films either under natural (Mergaert et al., 1992a, 1993, 1994a, 1995) or laboratory (Doi et al., 199Oc) conditions. The results of the study on the effect of polymer composition and film morphology on PHA biodegradation showed that film-making technique has a significant effect on the rate of microbial degradation of the polyester. Hot-pressed latex films that retain much of the nascent structure are readily susceptible to microbial attack and more readily degraded than solvent-cast films that are crystalline (Marchessault et al., 1994). The results of their study suggest that latex films that retain the granular texture should be ideal for degradable packaging applications. The biodegradability of a coated paper using activated sludge gives promise that this approach could lead to a readily manufactured packaging material with built-in degradability of the coating but allownig for recycling of the cellulose fibers. Bacterial PHB is more readily degraded than synthetic PHB (Marchessault et al., 1994). Araki and Hayase (1979) studied biodegradability of syn-

BIODEGRADABLE POLYESTERS

187

thetic PHB (Marchessault et al., 1994). Araki and Hayase (1979) studied biodegradability of synthetic P-substituted poly(P-hydroxypropionate) with fungi and found degradation to occur only after the molecular weights of these polymers were reduced to less than 50,000 g/mol using chemical treatments on polymers with initial molecular weights greater than 1,00,000 g/mol. The relative degradability of the PHB stereoisomers was studied with a PHB depolymerase enzyme isolated from Pseudomonas funiculosum (Kemnitzer et al., 1992). This enzyme has been shown to catalyze the hydrolysis of (R)-PHBbut does not show activity for the enantiomeric substrates (S)-PHB. Synthetic PHB of intermediate tacticity exposed to depolymerase enzyme biodegraded more extensively than highly isotactic synthetic PHB (Marchessault et al., 1994).Similarly, isotactic fractions were found to be less prone to enzymatic attack than atactic fractions (Araki and Hayase, 1979) and the higher degradability of atactic fractions was due to higher accessibility and chain mobility (Araki and Hayase, 1979). Hocking et al. (1995) explained that atactic recemic PHB shows the greatest degradability, highly isotactic recemic PHB degrades less well due to high crystallinity, and syndiotactic recemic PHB shows minimal degradation due to both high crystallinity and low isotacticity (Hocking et al., 1995). PHA depolymerases from several microorganisms have been purified, well characterized (Table XXXII),and patented (Table XXXIII). Studies on PHB depolymerase of a few microorganisms have revealed that their activities and properties differ (Delafield et ~ l .1965a,b; , Chowdhury, 1963; McLellan and Halling, 1988;Nakayama et al., 1985; Shirakura et al., 1986; Tanio et al., 1982; Shiraki et al., 1994). Most PHA-degrading bacteria apparently have only one PHA depolymerase, which is specific for either PHA,,, or PHAMcL(Schirmer et al., 1993; Jendrossek et al., 1995a). Pseudomonas lemoignei was found to have at least five PHA depolymerases (Briese et al., 1994). Schirmer et al. (1995) have classified various microorganisms from natural habits capable of degrading PHAs: group I comprises only P(3HB)-degradingbacteria represented by A. faecalis and Comamonas sp.; group I1 comprises only P(6HH) by Xanthomonas campestris; group In comprises only P(3HO)by four unidentified strains; group IV comprises P(3HB)and P(3HV)by l? lemoignei; group V comprises P(3HB) and P(6HH) by three unidentified strains; group VI comprises P(6HH) and P(3HD-co-3HO)by three unidentified strains; group VII comprises P(6HH),P(3HO),and P(HD-co-HV)by seven strains of Pflpuorescens; group VIII comprises P(3HB),P(HO), and P(3HD-co-3HO)by two unidentified strains; and the other groups (groups IX-XI, consisting of both PHAMcLand PHA,,,, are hydrolyzed by several bacterial strains. It is not clear whether these microorganisms secrete several PHA depolymerases or only one enzyme with a broad specificity toward different polymers.

TABLE XXXII PROPERTIES OF PHA DEPOLYMERASE ISOLATED FROM SEVERAL BACTENA Molecular mass (kDa)

Structure

90

Monomer

48-50

Monomer

nd 44-45

Monomer Monomer

Comamonas testosteronii

50

Penicillium funiculosum

36

Bacterium

Alcaligenes faecalis (marine) A. faecalis

Kmo

Optimum Temperature (C)

pH

Inhibitor

Product of hydrolysis

Reference

55

9.0

PMSF

Dimeric and trimeric esters

Kita et a/. (1995)

13.3

nd

7.5

PMSF, deoxycholate , DFP, Triton

Dimeric and trimeric esters

Saito eta/. 1989), Tanio eta/. (1982)

20

nd

nd

DTT

nd

29-35

9.4

DTT

3-HB-dimer 3-HB

Shivakura et al. (1986) Jendrossek eta/. (1993b)

nd

nd

nd

9.5

nd

Mukai et a/. (1993)

Monomer

170

nd

6.0

nd

Brucato and Wong (1991)

nd

nd

45

9.8

DFP, DTT, PMSF, Tween 20 DAN, DTT DFP, EPNP, HgC12, PMSF, Triton X-100. Tween 80 DFP, DTT, PMSF

3-HB (dimer)

Chowdhury (1963)

Dimer

nd

30-32

8.5

DTT, EDTA, HgC12, KCN, MIA, NaN3

3-HO-dimer

Schirmer et al. (1993)

x-100 CI

m

m

A. faecalis

Comamonas sp.

Pseudomonas. sp. nd Pseudomonas fluorescens

25

I? lemoignei

Y

02

W

nd

nd

nd

nd

8.0

CETAB, EDTA, SDS, Triton X-100, Tween 80 PMSF, DTT, DFT

3-Hl3, dimer

3-HB, dimeric and trimeric esters

Nakayama et aJ. (1985)

Delafield et aI. (1965)

(trimer)

I? lemoignei

Isoenzymes A1 = 45 A2 = 54 A3 = 49 A4 = 58

Monomer

73-131

I! lemoignei

54

Monomer

65

65

8.0

PMSF,DTT

3-HV

Muller and Jendrossek (1993)

P. pickettii

40

nd

nd

40

4.4

DEP, DTT, PMSF

3-HB (dimer)

Yamada e t a ] . (1993)

I! stutzeri

48

nd

nd

55

9.5

DEP,DTT, PMSF, Tween 20

3-HB dimer, trimer

Mukai et al. (1994)

8.0

Note. nd, Not determined; DAN, diazonorleucine methyl ester: DFP, diisopropyl fluorophosphate; DTT, dithiothreitol; CETAB, cetyltrimethyl m m o nium bromide: EDTA, ethyldiaminetetraacetate: EPNP, l,l-epoxy-3(p-nitrophenoxy) propane: MIA, monoiodo acetate; PMSF, phenylmethylsulfonyl5uoride. "F d .

190

CH. SASIKALA AND CH. V. RAMANA TABLE XXXIII PATENTS ON THE BIODEGRADATION OF PHAs

Assignee

Patent number

Year

Brief description

Saito and Saegusa

JP 06,86,681

1994

PHA depolymerase gene of Zoogloea ramigera was cloned, sequenced, and its expression in E. coli was observed

Toyoda et al.

JP 7,153,180

1995

Purification of PHB degrading enzymes from Pseudomonas sp.

The PHA depolymerase protein (Schirmer et aJ., 1995) and structural gene (Saito et al., 1989; Jendrossek et al., 1993b, 1995b; Briese et aJ., 1994b; Kuruso et al., 1994) have been well studied. It was found (Shiraki et al., 1995) that the molecular mass (40-50 ma),effect of nonionic detergent diisopropyl fluorophosphate, dithiothreitol (inhibition), and trypsin (abolition of PHB depolymerase activity but not the D-3hydroxybutyric acid dimer hydrolase activity of all the enzymes) of all five PHB depolymerases of bacteria isolated from various sources resemble those of the A. faecalis enzyme. Analysis of N-terminal amino acid sequence of the purified enzymes revealed that these enzymes, including A.faecalis T1 enzyme, fell into three groups (Shiraki et al., 1994). The properties of PHB depolymerases of both Aspergillus fumigatus MZA and P Jemoignei were similar except that the A . fumigatus system is more affected than the I? Jemoignei system (Hocking et aJ., 1995) by the presence of the S repeat units and the maximum degradation rate for the P lemoignei system was double that of the A . fumigatus system. It was suggested (Saito et al., 1993) that in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers but is necessary for the hydrolysis of hydrophobic and solid substrate (Fukui et al., 1988). Participation of the active serine in the PHB depolymerase mechanism has been implicated (Tanio et aJ., 1982) because the enzyme was inactivated by phenylmethanesulforyl fluoride and diisopropyl fluorophosphate (DFP). The participation of serine had been confirmed by studies on the trypsin-digested radioactive DFP-labeled PHB depolymerase enzyme (Saito et al., 1993). The PHB depolymerase seems to have an overall similarity to bacterial extracellular hydrolases like chitinases and cellulases (Saito et al., 1993).

BIODEGRADABLE POLYESTERS

191

The depolymerase activity was largely associated with the PHB granules. PHB depolymerase attacked from the chain ends (exo attack) or by endo (random) cleavage of polyester (Abe et al., 1995b). Biological degradation of exogenous PHA is caused by the hydrolysis of the polymer by the extracellular PHA depolymerase to monomeric or oligomeric products (Schirmer et al., 1995). Studies of the extracellular depolymerase and oligomer hydrolases from A . faecalis have shown that the mechanism of PHB depolymerization under aerobic conditions is a twostep process (Shirakura et al., 1986). First, PHB depolymerase hydrolyzes the water-insoluble PHB substrate into water-soluble oligomers and traces of monomers (Tanio et al., 1982). Second, the slightly waterinsoluble PHB oligomers are reduced to monomers (Fig. 26) by an oligomers hydrolase (Shirakura et al., 1986). D-(-)-3-Hydroxybutyrate oligomer hydrolase [(R-R)-3-(R)-3-hydroxybutanoyloxy)butanoate hydroxybutanoyl hydrolase, EC. 3.1.1.221 was purified and some of the properties of the enzyme were studied (Shirakura et al., 1983). The intermediate products of PHB degradation by aerobes have been reported to be monomeric 3-hydroxybutyrate (Chowdhury, 1963), dimeric 3HB (Merrick et a]., 1962; Tanio et al., 1982), and mixtures of monomer, dimer, and trimer (Delafield et al., 1965b). Janssen and Schink (1993) have elucidated the pathway of anaerobic PHB degradation by Ilyobacter delafieldii, an obligately anaerobic bacterium. They detected monomeric 3HB as an intermediate (dimers or higher polymers were not assayed) that was then fermented to butyrate, acetate, and hydrogen (Fig. 27). The reducing equivalents (NADH) formed could be disposed of by the evolution of hydrogen or by a reductive pathway in which 3-HB-CoA was dehydrated to crotonyl-CoA and reduced to butyryl-CoA. Budwill et al. (1992) concluded that overall, the biodegradation of PHB and P(HB-co-HV) under methanogenic conditions would most likely involve the initial hydrolysis of PHA to its monomeric units. The

Depolymerase Trimers+

Monomers

.1

.+Dimers

Cell Growth (Biomass) FIG. 26. Degradation products of the enzyme PHA depolymerase.

192

CH. SASIKALA AND CH.

V. RAMANA

3 HE

Ext racellular Intrac ar acetate

1

AcAc CoA

acetate butyrate

2H'

Acetyl CoA

GASH

Acetate FIG. 27. Pathway of PHB and 3-hydroxybutyrate fermentation to acetate, butyrate, and H, by Ilyobacter delafieldii (anaerobic bacterium) (Janssen and Schink, 1993). PHB,

poly(3-hydroxybutyrate); 3HB, 3-hydroxyhutyrate; 3HB-CoA, 3-hydroxy-butyrate coenzyme A; AcAc-CoA, aceto acetyl-coenzyme A; Ac-CoA, acetyl-coenzyme A. 1 , AcetylCoA-3-hydroxybutyrate CoA transferase (EC 2.8.3.-); 2, butyryl-CoA-3-hydroxybutyrate CoA transferase (EC 2.8.3-); 3,3-hydroxybutyryl-CoA-dehydrogenase (NAD) (EC 1.1.1.35); 4 , Acetyl-CoA-acetyltransferase (thiolase) (EC 2.3.1.9); 5, 3-enoyl-CoA hydratase (crotonase) (EC 4.2.1.17/55); 6, butyryl-CoA dehydrogenase (DCPIP) (EC 1.3.99.2/3); 7, ferredoxin:NAD oxidoreductase (EC 1.18.1.3);8, hydrogenase ( M V )(EC 1.18.99.1); 9, phosphate acetyltransferase (EC 2.3.1.8); 10,acetate kinase (EC 2.7.2.1); 11,butyryl-CoA acetate CoA transferase (EC 2.8.2.8).

conversion of the monomers (e.g., 3HB) to acetate and hydrogen by hydrogen-producing acetogenic bacteria (such as S. wolfei) in syntrophic association with hydrogen-using methanogens would follow. Ultimately, methanogens would complete the degradation of acetate derived from the PHAs, yielding CH, and CO,. The enzymatic degradation of P[(R)-3HB] by depolymerase takes 1988). In the first place in two steps (Saito et al., 1989, 1993; Fukui et d., step, the enzyme gets adsorbed onto the surface of the polymer film via

BIODEGRADABLE POLYESTERS

193

the binding domain and in the second step it hydrolyse the polymer chain into water-soluble products by the catalytic domain. Abe et al. (1995b) have proposed a model of enzymatic hydrolysis of PHB films by PHB depolymerase. In their model, they suggested that the binding domain of depolymerase adheres selectively to the PHB crystalline phase on the surface of PHB film, and that the catalytic domain hydrolyzes predominantly PHB chains in the amorphous phase on the surface and subsequently erodes PHB chains in the crystalline phase. Field degradation tests of biodegradation of dog bone-shaped PHA test pieces (homopolymers of 3HB as well as copolyesters of 3HB and 3HV) have been performed under natural conditions by measuring mass loss after incubation of the polymeric material in ecosystem such as soil, freshwater, seawater, and compost (Mergaert et al., 1992a). Biodegradation of PHB, P(3HB-co-lO% 3HV), and P(3HB-co-40% HV) was investigated in situ in natural waters (Mergaert et al., 1995). Polymers from Zeneca Bioproducts, whether as injection molded or dog bone-shaped, were less biodegraded, after 6 months of submersion, and the mass loss was less than 7%. Bottles made from Biopol were utilized to study the degradation of PHA under in situ conditions in freshwater lake and sediment (Brandl and Puchner, 1992). The studies of Brandl and Puchner (1990, 1992) on the degradation of commodity bottles (shampoo bottles) and films made from Biopol have clearly demonstrated that in an aquatic ecosystem (water column as well as sediment) under in situ conditions (i.e., under extreme conditions of low temperature, high hydrostatic pressure, no sunlight, and seasonal variations of oxygen concentration) plastic goods made from Biopol are degraded and a life span of 5-10 years for commercially available shampoo bottles made from PHA (Biopol) was estimated. Although extracellular biodegradation of PHA, which is important with regard to environmental degradation of these polymers, has been extensively studied, only a few reports are available on intracellular degradation of PHA that may be important in relation to mass production of PHA (Foster et al., 1994; Hocking et al., 1995). Such studies on intracellular degradation of PHA were conducted on B. magaterium, R. rubrum Merrick and Doudoroff, 1964),A. eutrophus (Hippe and Schlegel, 1967; Saito et al., 1995), and Z. ramigera I-16-M (Saito et al., 1992). The soluble enzyme fraction from polymer-depleted cells of R. rubrum contained a thermostable activator and a thermolabile depolymerase. The granulebound PHB depolymerization system of Z. ramigera required high ionic strength and high pH for digestion of PHB. PHB depolymerase from A. faecalis was cloned and expressed in E. coli (Saito et al., 1989).

194

CH. SASIKALA AND CH. V. RAMANA

B. BIODEGRADATION OF SYNTHETIC POLYESTERS

In addition to the synthetic PHAs, a number of other synthetic polyesters (Table XXXIV) are also known to be biodegraded (Darby and Kaplan, 1968),which mainly include poly(ecapro1actone) (Benedict et al., 1983),poly(ethy1ene adipate) (Tokiwa and Suzuki, 1977a),poly(1actide) (Evans and Sikdar, 1990),poly(glyco1ide)(Frazza and Schmi, 1971), PTMS (Pranamuda et d., 1995), and a variety of polyester blends (Verhoogt et d.,1994). However, most synthetic polyesters (and in general polymers) are not inherently biodegraded, both because they have not existed in nature long enough for depolymerase to have evolved to exploit them as a carbon source and because their structure units bear lit-

TABLE XXXIV

BIODEGRADATION (HYDROLYSIS) OF VARIOUS SYNTHETIC POLYESTERS BY FUNGAL LIPASES~ Degradation potentialc Polyester Poly(ethy1ene adipate) Poly(ethy1ene suberate) Poly(ethy1ene azelate) Poly(ethy1ene sebacate) Poly(ethy1ene decamethylate) Poly(tetramethy1ene succinate) Poly(tetramethy1ene adipate) Poly(tetramethy1ene sebacate) Poly(hexamethy1ene sebacate) Poly(hexamethy1ene adipate) With hydroxy terminals With HHPA terminals With HHPA-glycidil ester terminals Poly(2,2-dimethyltrimethylene succinate) Poly(2 ,2-dimethyltrimethylene adipate) Polyglycolide Copolyester of glycolide and lactide (molar ratio (92:8)) Polypropiolactone Poly(DL-P-methylpropiolactone) Poly(D-p-methylpropiolactone) (PHB) Polycaprolactone (PCL)

Mnb 2,720 4,050 4,510 1,570 1,610 4,240 1,790 2,440

Penicillium sp. Hi-

+

Hi-

+

+ + + +

5,820 3,980 4,170 6.830

tHHitH-

+ +

+

Rh. delemar +tt

tk t k t

+

+ +

tt

+t+

+++ ++

+ +

+

-+

+ +t

+

2,370

2,020

Rhizopus arrhizus

+

+ -

4,270 8,190

+ -

>25,000 6,740

+t

+

tk

+

-

+

Hi-

+ (continues)

195

BIODEGRADABLE POLYESTERS TABLE XXIV-Continued Degradation potentialc

Polyester Polycaprolactone diol With hydroxy terminals With HHPA terminals With HHPA-glycidil ester terminals Copolyester of ecaprolactoneadipic acid-hexamethylene diol With hydroxy terminals With HHPA terminals With HHPA-glycidil ester terminals Poly(cis-2-butene adipate) Poly(cis-2-butene sebacate) Poly(trans-2-butene sebacate) Poly(2-butyne sebacate) Poly(hexamethy1ene fumarate) Poly(cis-butene fumarate) Poly(tetramethy1 cyclobutane succinate Poly(cyc1ohexane dimethyl succinate Poly(cyc1ohexane dimethyl adipate) Poly(tetramethy1ene temphthalate) Poly(ethy1ene tetrachlorophthalate) Poly(2,2-dimethyltrimethylene isophthalate) Bisphenol A polycarbonate Poly(p-hydroxybenzoate) Poly(3,5-dimethyl-phydroxybenzoate) Copolyesters of PCL and Poly(ethy1ene terephthalate) (molar ratio 90:lO) Copolyamide ester of PCL and nylon-12 (9O:lO) Poly(p-methyl-evalerolactoneco-L-lactide)

Mnb

Penicillium sp.

2,860 8,980

U

12,480

tt

2,200

U

6,230 6,380

++

Rhizopus arrhizus

Rh. delemar

+

+

*

t k

2,700 6,190 3,560 4,930

+tt

U

+ +

3,440

+ + + + +

-

-

3,910

+

+

+

3,250

+

+

+

1,670

+ + +

+

tt

+tt

OTokiwa and Suzuki (1977a,b, 1978), Tokiwa et 01. (1986), and Nakayama et 01. (1995). b M n , number average molecular weight. CDetermined by measuring the water-soluble total organic carbon (TOC). +, TOC 10-1000 ppm; ++, TOG 1001-2000 ppm; +++, TOC 2001 and above; -, unable to hydrolyze. Figures not mentioned were either not analyzed or not reported.

196

CH. SASIKALA AND CH. V. RAMANA

tle resemblance to compounds that normally occur in the metabolic cycles of living systems (Timmins and Lenz, 1994). Among the synthetic polyesters, the degradation of PCL was extensively studied. PCL-degrading microorganisms are distributed widely in the environment and they represent 0.2-11.4% of the total number of microorganisms in the environment (Nishida and Tokiwa, 1993a, 1994). PCL has good mechanical properties and is compatible with many types of polymers because it is one of the more hydrophobic of the commercially available biodegradable polymers (Koleske, 1978). PCL was found to be biodegraded even after extensive crosslinking with organic peroxides (Jarrett et al., 1985). However, increasing crosslink density does slow the rate of degradation. Poly(ecaprolactone), when buried in soil, is completely degraded after about 1year (Potts et al., 1972). A long methylene chain promotes biodegradability by imparting flexibility to the polymeric chain. Therefore, poly(6-valerolactone) and poly(e-caprolactone),with four and five methylene units, respectively, in their backbones are more biodegradable than poIy(P-propiolactone), which has two methylene units (Nakayama et al., 1995). However, a much longer methylene chain might cause a decrease in biodegradability because of a decrease in the density of ester bonds in the main chain. Furthermore, substituted groups were also found to decrease the susceptibility of polymers to biodegradation (Nakayama et al., 1995). Poly(MV) is an ideal polyester for degradable packaging applications. The biodegradability of a coated paper using activated sludge gives promise that this approach could lead to a readily manufactured packaging material with built-in degradability of the coating but allowing for recycling of the cellulose fibers. Bacterial PHB are more readily degraded than synthetic PHB (Marchessault et al., 1994). Araki and Hayase (1979) studied biodegradability of synthetic P-substituted poly(P-hydroxypropionate)with fungi and found degradation to occur only after the molecular weights of these polymers were reduced to less than 50,000 g/mol using chemical treatments on polymers with initial molecular weights greater than 100,000 g/mol. The relative degradability of the PHB stereoisomers was studied with a PHB depolymerase enzyme isolated &om P funiculosum (Kemnitzer eta].,1992). This enzyme has been shown to catalyze the hydrolysis of (R)PHB, but does not show activity for the enantiomeric substrates (S)-PHB. Synthetic PHB of intermediate tacticity exposed to depolymerase enzyme biodegraded more extensively than highly isotactic synthetic PHB (Marchessault et al., 1994). Similarly, isotactic fractions were found to be less prone to enzymatic attack than atactic fractions (Araki and Hayase, 1979) and the higher degradability of atactic fraction was due to higher accessibility and chain mobility (Araki and Hayase, 1979).Hocking et al.,

BIODEGRADABLE POLYESTERS

197

(1995) explained that atactic recemic PHB shows the greatest degradability, highly isotactic recemic PHB tensile strength on environmental exposure. High-molecular-weight (40000 and 80000 g mol-1) PCL has been blended with high-amylose corn starch (70% amylose) (Tokiwa et al., 1990b; Koenig and Huang, 1992) and the various properties were compared with similar blend films made with P(HB-co-16% HV) (Koenig and Huang, 1995). Similarly, PCL/PHB (Kumagai and Doi, 1992a,b,c)and PHA/polyolefin (Gilmore et al., 1993) blends are easily biodegradable. Lipases are the enzymes that are known to be involved in the hydrolysis of aliphatic polyesters (Tokiwa et al., 1986; Tokiwa and Suzuki, 1978). PHA depolymerase, the PHA-hydrolyzing enzyme, cannot hydrolyze synthetic polyesters; conversely, lipase cannot hydrolyze PHAs (Tanio et al., 1982). However, strain HT-6, an actinomycete isolated from soil sample, could degrade both PHB and synthetic (PTMS and PCL) polyesters, suggesting that the enzyme produced by this strain has a wide range of substrate specificity (Pranamuda et al., 1995). The substrate specificities of extracellular lipases purified from different bacteria and of extracellular PHA depolymerase and esterase of various polyesters were analyzed (Jaegar et al., 1995). All lipases and the esterase of I? fluorexens, but not of the PHA depolymerase tested, hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerase. Most lipases were able to hydrolyze polyesters consisting of an w-hydroxyalkanoates, viz. P(6-HHx) or P(4-HB), resulting in dimeric esters. Polyesters containing side chains in the polymer backbone, such as P(3HB) and other P(3-HAs), were not or were only slightly hydrolyzed by the lipases tested (Jaeger et a]., 1995). Biodegradability of synthetic polyesters by Rhizopus arrhizus lipase decrease with increasing melting point (Tokiwa et al., 1990b, Tokiwa et al., 1986). Partially purified lipase of R. arrhizus (Tokiwa et al., 1986), R. delemer (Tokiwa and Suzuki, 1977b), and Penicillium sp. (Tokiwa and Suzuki, 1974,1977a) showed a broad substrate specificity for polyesters (Table XXXIII) and the enantiopreference and biological function of lipase of Candida rufosa has been reviewed (Cygler et al., 1995). Melting point (Tokiwa et al., 1986) and particle size (Tokiwa and Suzuki, 1978) greatly influenced synthetic polyester hydrolysis. As with PHAs, with synthetic polyesters copolymerization enhanced the biodegradability. This is evidenced (Nakayama et al., 1995) by the higher degradability of the copolymer, poly(P-methyl-6-valesolactoneco-L-lactide), when compared to the homopolymers, poly(P-methyl-8valerolactone), which is hardly biodegradable with fungal lipase, and poly(L-lactide), which exhibits a low rate of hydrolysis because of its high crystallinity.

198

CH. SASIKALA AND CH. V. RAMANA Synthetic Chemical Reactions

PRODUCT UTILIZATION

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Sludge

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\1

.1

Higher Plants

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Medical Pharmaceutical Argonomic Bottling App l i c a t i o n v u J

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DISPOSAL

Microbial Action

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C02 + H2O FIG. 28. Production, utilization, and mineralization of biodegradable polyesters.

An overview on the production, utilization, and mineralization of polyesters is shown in Fig. 28. Biodegradable polyesters have potential applications in the future. However, much work has to be done on the commercialization of these polyesters, particularly in reducing the production cost. ACKNOWLEDGMENTS Sasikala thanks the U.G.C., Government of India, New Delhi, for the award of Research Scientistship and Ramana thanks the C.S.I.R., New Delhi, for the award of Pool Scientistship.

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Walker, J., Whitton, J. R., and Alderson, B. (1982). Eur. Patent Appl. 46,017. Wakisaga, Y., Masaki, E., and Nishimoto, Y. (1982). Appl. Environ. Microbiol. 43,1473. Wallen, L. L., and Rohwedder, W. K. (1974). Environ. Sci. Technol. 8, 576-579. Wang, W. S., and Lundgren, D. G. (1969). J. Bacteriol. 97,947-950. Ward, A. C., Rowley, B. I., and Dawes, E. A. (1977). J. Gen. Microbiol. 102,61-68. Warthmann, R., Cypionka, H., and Pfennig, N. (1992). L.Arch. Microbiol. 157,343-348. Weaver, P.F., and Maness, P. C. (1993). U.S. Patent 5,250,427. Weaver, T. L., Patrick, M. A., and Dugan, P. R. (1980).J. Bacteriol. 124,602-605. Wieczorek, R.,Pries, A., Steinbuchel, A., and Mayer, F. (1995).J. Bacteriol. 177,2425-2435. Williamson, D. H., and Wilkinson, J. F. (1958). J. Gen. Microbiol. 19, 198-209. Williams, D. R., Anderson, A. J., Dawes, E. A,, and Ewing, D. F. (1994). Appl. Microbiol. Biotechnol. 40,717-723. Windisch, C., Miller, S., Reisner, H., Angercr, B., Achhammer, G., and Holler, E. (1992). Cell Biol. Int. Rep. 16, 1211-1215. Winton, J. M. (1985). Chem. Week. Aug. 28,55-57. Witholt, B., and Lageveen, R. G. (1992). U.S. Patent 51,35,859. Wong, P. P., and Evans, H. J. (1971). Plant Physiol. 47,750-755. Yalpani, M., Marchessault, R. H., Morin, F. G., and Monasterios, C. J. (1991). Macromolecules 24,6046-6049. Yamada, K., Mukai, K., and Doi, Y. (1993). Int. J. B i d . Macromol. 15,215-220. Yamamoto, O.,Myata, Y., and Yanagi, S. (1995). Jpn. Kokai Tokkyo Koho JP 7 79 787. Yamane, T. (1992). FEMS Microbiol. Rev. 103,257-264. Yamane, T., and Shimizu, S. (1984). Adv. Biochem. Eng. Biotechnol. 30, 147-194. Yamane, T., Chen, X. F., and Veda, S. (1996). FEMS Microbiol.Lett. 135,207-211. Yamashita, Y., Tsuda, T., Ishida, H., Clchikawa, A., and Kuriyama, Y . (1968). Makromol. Chem. 113,139-146. Yamashita, Y., Ishikawa, Y., Tsuda, T., and Miura, S. (1963). Kogyo Kagaku Zassi 66,11&114. Yasin, M., Holland, S. J., Jolly, A. M., and Tighe, B. J. (1989). Biomaterials 10,400-411. Yasuda, T., Aida, T., and Inoue, S. (1983). Macromolecules 16, 1792-1796. Yasuda, T., Aida, T., and Inoue, S. (1984). Macromolecules 17,2217-2222. Yoo, S.,and Kim, W. S. (1994). Biotechnol. Bioeng. 43,1043-1051. Zhang, Y., Gross, R. A., and Lenz, R. W. (1990). Macromolecules 23, 3206-3212.

The Utility of Strains of Morphological Group II Bacillus SAMUEL SINGER Department of Biological Sciences Western Illinois University Macomb. Illinois 61 455

I. Introduction 11. General Considerations A. Bacterial Candidates B. Rearing and Fermentation Process Details 111. Utility According to Target A. Mosquito Larvae (Aedes,Anopheles, and Culex) B. Biting Blackflies (Simuliumvitattum) C. Cigarette Beetle (Lasioderma serricorne) D. Indian Meal Moth (Plodia interpunctella) E. Zooparasitic Nematodes (Trichostrongylus colubriformis) F. Phytoparasitic Nematodes (Heterodera glycines) G. Snails (Biomphalaria glabrata) H. Zebra Mussels (Dreissena polymorpha) IV. Utility According to Strain and Species A. Bacillus alvei B. Bacillus brevis C. Bacillus circulans D. Bacillus laterosporus V. Fermentation Processes, Toxins, and Products A. Fermentation Processes B. Toxins C. Products VI. Past Needs, and Future Needs A. What Is Biological Control? B. Is Bt Enough? C. Where to Now? References

I. introduction Over the past 25 years or so, the author has had the opportunity not only to investigate Bacillus sphaericus as a potential bacterial larvicide (Singer, 1973, 1987, 1988, 1990), but on several occasions to also examine other potential bacterial biological control agents (Singer 1974, 1981). Among the many strains of Bacillus that the author has examined as freshly obtained field samples, several showed sparks of biological activity that were interesting enough to have a series of students 219 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 42 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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investigate them further. It should be noted that they were not of the activity of a B. sphaericus or a B. thuringiensis, (Bt),which show LC,,’s [dilution of the final whole culture (FWC) killing 50% of the target animals] of 106-109, but rather they showed low to modest activity that one might coax up to an LC,,of perhaps 103-104.This activity was of further interest because the targets were not the usual diptera or lepidoptera. Also of interest to the author was the fact that many of them were isolated initially in and among the field samples from which more active B. sphaericus strains were to be found (Singer, 1973). Often, they kept insinuating themselves into ongoing projects. On hindsight, the mind was lured away to the potential riches of B. thuringiensis and B. sphaericus, ignoring the possible value and utility of these “other” isolates. In the present mid-1990s, we hear the hint that “Bt is enough” and that with the advent of the miracles of recombinant DNA technology we can fashion the candidate Bt genomes into products to fit whatever target we wish to address (Feitelson et al. 1992; Lambert and Perferen, 1992). In the process, we forget that there are many bacterial populations out there in the wild that, with perhaps a little effort, would yield a series of new beginnings toward products, particularly those aimed at the biological control of a variety of pests. This assumes of course that the biological control effort is meaningful, still exists, and has not been wiped out by the “miracles of chemistry” (in the case of pests of agriculture), wiped out by the “miracles of immunology” (in the case of vectors of human disease), or wiped out by the “combined miracles of chemistry and engineering” (in the case of the intrusion of nonindigenous species into our aquatic systems, such as the zebra mussel). Drawing on experiences from the past 25 years in his laboratory, the author will illustrate the utility of many of the morphological group I1 strains of Bacillus. In the past, there was insufficient data or insight to warrant individual publication of much of what will be presented. In addition, the strains were thought to be lost when they would not perform as initially promised. One generally does not report what one cannot share with one’s colleagues. With strides made in the author’s laboratory concerning work with molluscicidal strains of Bacillus and with the recent preliminary information gained from the study of at least one of the toxins responsible for molluscicidal activity (Singer et al., 1994a), perhaps some insight has been gained into explaining what had been seen on the several occasions in the past when we interfaced with these strains. In this chapter, the discussion is divided into “utility according to target” and “utility according to strain and species.” The former is for those interested in the bottom line, “is he talking about targets I may be interested in,” and the latter for those interested in the microbiology, systematics,

UTILITY OF STRAINS OF GROUP XI Bacillus

221

and fermentation practices (as they exist) of each of these potential agents. First, the general considerations that laid the background for this chapter need to be examined. II. General Considerations

A. BACTERIALCANDIDATES 1. The Morphological Groups of Bacillus

Although it is not in vogue at present, it has always been convenient for me to examine newly isolated and previously unidentified strains of Bacillus in terms of Smith’s three morphological groups (Smith et al., 1952). The grouping is based on the position of the endospore in the cell (sporangium) and whether the cell, as a consequence, appears to be swollen. In morphological group I, the endospore does not seem to swell the cell and is centrally or subterminally located. In morphological group 11, the endospore is centrally or subterminally located with the endospore seemingly swelling the cell, causing a spindle-like shape. In morphological group 111, the endospore is terminally located, round, and swollen. Once the morphological group was determined, it was only a matter of time (at our convenience) when the species identification would be elaborated. Bacillus thuringiensis is an example of morphological group I, and B. sphaericus is an example of morphological group 111. The morphological group I1 is the subject of this chapter. 2. Sources of the Strains of Bacillus Being Examined

As mentioned previously, many strains were isolated by the author from field material (usually from dead, dried mosquito larvae). Many strains came from national and international culture collections accessioned into the author’s in-house collection. The isolated strains were identified to species using a biotype approach (Knight and Proom, 1950; Proom and Knight, 1955). Some of the early isolates carried an “intermediate” designation, such as “alvei/circulans,” which was the custom at that time (Knight and Proom, 1950; Proom and Knight, 1955). Subsequent work with PAGE clarified some of these designations. The strains of each of the species under discussion are described in the following paragraphs. For B. alvei strains, the 42, 46,II, and 111 series and their derivatives were isolated by the author; strains 2771,2772, and 2198 were courtesy of Dr. S. R. M. Bushby and the Wellcome Research Laboratories (WRL), Beckenham, UK; the remaining strains were courtesy of Dr. L. K. Nakamura, Northern Regional Research Laboratories (NRRL) Peoria, Illinois.

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SAMUEL SINGER

Bacillus brevis strains 2904, 2918, 2922, 2932, 2934, and 3010 were courtesy of WRL (see above); strain 42-14-B2 was isolated by author; and the remaining strains were courtesy of (NRRL) (see above). Bacillus circulans strains 3987, 3986, 3370, and 2628 were courtesy of WRL (see above); and the 42,46,11, and I11 series and their derivatives were isolated by the author. For Bacillus laterosporus, the series of strain BI/a was isolated by the author; the remainder were courtesy of Dr. A. A. Yousten, Virginia Polytechnic Institute, Blacksburg, Virginia (Favret and Yousten, 1985). The B. thuringiensis and B. sphaericus strains are part of the author’s in-house culture collection. B. REARINGAND FERMENTATION PROCESS DETAILS Rearing of each of the targets is detailed under Section I11 at the appropriate target. The fermentation process details are presented under Section IV at the appropriate bacterial species and summarized under Section V. Ill. Utility According to Target

The targets under discussion are presented in the order in which they were examined. There were two periods of research activity in terms of the targets: from 1970 to about 1979 (targets A-D) and from about 1988 to the present (1995)(targetsE-H). The renewed interest was due for the most part to the association of the author with Bone and with Bone’s work on zooparasitic nematodes during the middle and late 1980s and early 1990s (Bone, 1989; Bone and Singer, 1989,1990,1991;Bone and Tinelli, 1987). Renewed interest was also due to the receipt of strains from Ghana that were truly molluscicidal (Singer et al., 1988, 1994a).

A. MOSQUITO LARVAE(Aedes,Anopheles, and Culex) The morphological group I1 strains were first noted when many of them were isolated initially in and among the field samples from which more active B. sphaericus strains were to be found (Singer, 1973). Liu (1972), Chao (1973), and Ralston (1979) examined these isolates in an attempt to circumscribe the activity and to initiate a crude fermentation process that allowed for the preparation of some of these materials, some of which was briefly reported (Singer 1974, 1979, 1981). 1. Rearing

Our standard rearing and bioassay methods for Culex quinquefasciatus, as well as Aedes aegypti and Anopheles albimanus have been described previously (Singer, 1973, 1974, 1981).

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223

2. Biological Events and Effects

As part of an ongoing World Health Organization (WHO)program, 2 accessions (WHOARC reference Nos. 1242 and 1246), originally collected in Rangoon, Burma, had been sent to the author by Dr. John D. Briggs (at that time, Director of WHOARC at Ohio State University, Columbus, Ohio) as well as 15 accessions (WHOARC reference Nos. 1321/I-XV) sent from Delhi, India. The active strains of morphological groups I1 and I11 were isolated from these dead larvae. The events and activity of the morphological group I11 strains have been reviewed previously (Singer, 1990). Over 100 bacterial strains were isolated from the dead larvae of the Rangoon accessions, 1242 and 1246. These 100 isolates included 35 strains from the Delhi accessions, 1321 (the I1 and I11 series). In general, when tested against Culex larvae, the activities of these morphological group I1 isolates showed LC,, values from 102to 1 0 4 with values of 3 x 102-103 being most common. Against Aedes larvae, the LC,, values ranged from 1:50 to 103, with values of 102-103 being most common. Examination of these strains against Anopheles larvae showed similar values to the Culex values. Examples of biological activity of strains from accessions 1242,1246, and 1321 are shown in Table I. The B. alvei strains appeared to be the most active. Their activity, illustrated by B. alvei/III3D, appeared to be influenced by the media (raw material media) used in the fermentation process (Table 11). The toxic effects initially appeared to be due to a heat-labile toxin (Singer, 1973). Subsequent unreported studies indicated the presence of additional heat-stable toxins (Ralston, 1979). TABLE I INITIAL ACTIVITY OF 24-HR FINAL WHOLE CULTURES OF NINEMORPHOLOGICAL GROW11 STRAINS OF Bacillus GROWN IN BRAIN-HEART INFUSION BROTHvs Culex Quinquefasciatus

Strain From Accession 1242 (Rangoon) B. alvei (aberrant) 42-F-3 B. brevis 42-14-B-2 B. circulans/pulvifaciens Intermediate 42-G-1

From Accession 1246 (Rangoon) B. circulans 46-B-2 B. alvei (aberrant) 46-F-3 B. alvei (aberrant) 46-C-3 B. circulans/alvei Intermediate 46-J-3 From Accession 1321 (New Delhi) B. alvei 111-2-E B. a h e i 111-3-D

" LC,,, dilution of the final whole culture killing 50%

LC,,-ln 165 600 210 200

500 140 70

aoo 600 of the test animals Culex quinquefasciatus.

224

SAMUEL SINGER TABLE I1 GROWTH AND INSECTICIDAL ACTIVITY 01: B. alvei 111-3-D GROWNIN RAWMATERIALS MEDIA 24 hr

16 hr

Media

TVC

RM-1

3.0 x lo9 2.0 x 107 2.0 x 109

RM-2 RM-3

LC,,-1 1600 200

iao

TVC 2.5 x lo8 6.0 x 109 4.5 x 1 0 7

LC,,-' 1000 580 30

~~

Note. TVC, total viable count; LC,,, dilution of the final whole culture killing 50% of the test animals Culex quinquefasciatus. RM-1: 2% fish meal, 1.0% soybean oil meal, 0.1% CaCO,, and 0.1% vitamin mix; RM-2: 1.0% glycerol and 1.0% nonfat dried milk; RM-3: 1.0% nonfat dried milk.

B. BITINGBLACKFLIES (Simulium vitattum) Some of the morphological group I1 strains shown previously to be active against Culex larvae also appeared to be active against the biting blackfly Simulium [some of whose species act as the vector for onchocersiasis (river blindness)] (Jaska, 1977; Singer, 1981). Bacillus alvei strains were found to be the most active. 1. Rearing

The Simulium larvae used were collected locally, between October and April, from below a spillway in one of our local state parks. The native blackfly species used was an overwintering larvae type, S. vittatum (Fredeen, 1973). The larvae were collected 2 days prior to the bioassay date. The rocks to which the larvae were attached were gathered and placed in collection tanks that were 5-gallon plastic-lined buckets filled with river water. Tarshis and Adkins (1971) outlined a 6-hr critical time interval for larval survival in nonaerated water. The larvae were returned to the laboratory within this time period. The field-collected rocks were placed in the center of the holding tanks, which were 5-gallon aquaria lined with inert plastic inserts. Air supply lines surrounded the inner periphery of the aquaria. The holding tanks were filled with river water. In the 2-day period, the larvae released from the rocks and attached to the inert plastic liners because this was the location of aeraters in the aquaria. The bioassay design was a serial (decimal) dilution system in 25 x 200mm test tubes. This tube size was chosen because it allowed a 6-in. water depth for the 50-ml bioassay water sample. FWC samples were decimally diluted 102-106. Ten blackfly larvae of varying sizes were re-

225

UTILITY OF STRAINS OF GROUP I1 Bacillus

moved from the inert plastic and placed into the bioassay tube. Aeration was provided by means of a Pasteur pipet connected via a syringe to an air supply line. The larval diet was 25 parts Purina dog chow (60 mesh), 10 parts brain-heart infusion broth, and one part brewer’s yeast powder (Tarshis, 1968) and it was added at the start of each test. A 12-hr photoperiod was used and the test was performed at 25OC. The numbers of surviving larvae were recorded at 2 and 5 days. Water level adjustments and diet addition were performed at 2 and 4 days. Control mortality was adjusted by Abbott’s formula. LC,, was expressed in terms of the dilution of the FWC material causing death of 50% of the test animals. In the cadaver bioassay procedure, two infected surface-sterilized larvae were placed in a test tube containing 10 uninfected larvae. The control tube had 2 uninfected larvae. The test was performed in triplicate. 2. Biological Events and Effects

From in-cycle experiments (Jaska,1977),it was determined that 1 2 hr of a standardized fermentation process was the optimum time for maximum larvicidal activity. Of the group I1 isolates active against Simulium, strains of B. alvei were the most active. Subisolates from the B. alvei cultures were obtained and tested against Simulium (Table 111).There appeared to be an

TABLE I11 OF GROWTH AND LARVICIDAL ACTIVITY OF A COMPARISON 1 2 HR FINAL WHOLECULTURES OF SUBISOLATES OF Bacillus alvei III3DT-1 AND B. alvei III3DT-1A GROWN IN BHI+

Subisolate

TVC

TVSC

LC50

2.9 x 1090 3.1 x 109 2.6 x 109 2.7 x 109 2.6 x 109

0

0 2.8 x 105 7.6 x 105 2.8 x 105

10-3.70 104.22 10-2.65 104.50 10-2.84

2.8 x 2.9 x 2.6 x 2.9 x

5.8 x lo5 2.6 x lo6 2.9 x 105 2.6 x 105

B. alvei III3DT-1 0

il i2 05 1

B. alvei III3DT-1A 1 il 2 i2

109 109 108 109

10483

10-3.39 10-2.95 10-2.83

Note. LC,,, the dilution of the FWC material killing 50% of the test larvae Simulium vittotum. BHI+, brain-heart infusion plus the vitamins biotin, thiamin, Ca pantothenate, and nicotinic acid. TVC, total viable count; TVSC, total viable spore count. OAll figures are the means of two observations.

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SAMUEL SINGER

effect of media on larvicidal activity (Table IV). In other unreported studies, the cell (pellet) fraction showed activity, whereas the supernatant fraction of the FWC did not. Larvicidal activity against S. vittatum occurred in a range of 5 to 23OC. Transmission of the cell-bound toxin of B. alvei was aided by two factors: (i)the multipliction of B. alvei in the larval cadavers, and (ii) the cannibalistic tendency of the larvae (Table V). Preliminary histopathology revealed no gross tissue damage prior to death of the larva, which was similar to the effect of B. sphaericus on mosquito larvae. Strains of B. sphaericus, used as a control, showed no toxic effect against S. vittatum larvae. Favret and Yousten (1985), investigating the insecticidal activity of strains of B. laterosporus, report that strain NRS 590 showed pathogenic activity against larvae of S. vittatum. TABLE IV A COMPARISON OF GROWTH AND LARVICIDAL ACTIVITY OF 2-HR FINAL WHOLE CULTURES OF SUBISOLATES OF Bacillus a h e i III3DT-1 GROWN IN VARIOUS MEDIA Isolate il il il il 0 0

Medium

_ _ _ _ _ _ ~

PCPC+ PC+ with 1% glucose PC- with 1%glucose BHIt with 0.5% CaCO, BHI+ with 150 W M shake flask

PH

TVC

5.20 8.2 5.3 4.8 8.4 8.2

3.1 x 109 3.0 X lo7 3.3 x 109 2.4 x 107 3.3 x 109 3.0 x 109

TVSC

LC50

2.3 x 105 3.0 X lo5 0 0 0 0

10-381 10-493 104 05 10-0 4 9 1 0 4 18 10-3 01

~

Note. PC, plate count medium minus vitamins: PC+, plat count medium plus vitamins (for vitamins see Table 111 note). BHI+, brain-heart infusion medium plus vitamins, TVC, total viable count; TVSC, total viable spore count; LC50, the dilution of the FWC material killing 50% of the test larvae Simulium vittatum. 0 All figures are the means of two observations.

TABLE V LARVICIDAL ACTIVITY OF Bacillus alvei III3D-INFECTED Sirnuliurn vittatum CADAVERS Day 2 Strain number T-lAl T-lo5 CI

Tube 1 Tube2 5.6' 5.0

5.6 3.8

Day 5

Tube 3

Control

3.3

10 10

0

Values corrected by Abbott's formula.

Tube 1 Tube2 3.3 0

5.0 1.3

Tube 3

Control

0

10 10

0

UTILITY OF STRAINS OF GROUP I1 Bacillus

227

C. CIGARETTE BEETLE(Lasioderma serricorne)

The cigarette beetle (Lasioderma serricorne) and the Indian meal moth (Plodia interpunctel1a)were the subject of a study of the potential affect of the morphological group I1 strains on stored grain insects. This study was undertaken by the author at about the same time that the Simulium studies were taking place. In this section, the effects on the cigarette beetle are discussed. Two earlier studies were performed by Brooks (1975) and by Bradshaw (1979), and a more recent study was performed by Matheny (1992). Brooks examined a large number of strains of Bacillus from among the three morphological groups, including several strains of B. cereus isolated from dead cigarette beetles provided by Dr. w. R. Kellen. Brooks found only minimal activity, with LC,, values no greater than 102. From among the B. cereus samples however, 11strains designated Bl/a Ol-Bl/a 11were isolated that Bradshaw (1979) later found to have some activity against L. serricorne (Table VI). 1. Rearing

The cigarette beetle was reared in glass jars containing a commercial insect diet (Adkisson-Vanderzant Wheat Germ Insect Diet), which consisted of 24% wheat germ, 8% wesson salts, 28% vitamin-free casein, 28% sucrose, and 12% alphacel. The sterile covered jars were filled TABLE VI COMPANSON OF COLEOPERAN ACTIVITY AGAINST NEONATE vs OLDER LARVAE OF Lasioderma serricorne

Strain number

Neonate larvae

Older Larvae

Blla 01 Blla 02 Blla 03 Blla 04 Blla 05 Bl/a 06 Blla 07 Blla 08 Blla 09 Blla 10 Blla 11

190 185 250 234 130 148 325 173 135 270 210

58 130 58 198 52 230

44 60 60 135 30

Note. LC,, is the reciprocal of the dilution of the final whole culture killing 50% of the test insects.

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SAMUEL SINGER

one-third full of the diet and 50-100 larvae were put into each jar. In about 4-12 days, the larvae pupated and emerged as adults. Six to 18 days later, small larvae were observed in the diet. Eggs and neonate larvae were used in the subsequent experiments. 2. Biological Events and Effects

The Bl/a Ol-Bl/a 11 strains were each identified as an intermediate strain designated as B. laterosporus/alvei (Bl/a).The cultures were active only against neonate larvae (Table VI). In-cycle studies (sampled during the fermentation cycle) showed that larvicidal activity of the bacilli began when the culture was 1 2 hr old, was maximum at 18 hr, and remained active for at least 48 hr. The larvicidal activity was associated with the cell itself and not the supernatant (no exotoxin produced). A related morphological group I1 Bacillus, B. pulvificiens , also killed larvae of the cigarette beetle by a nondialyzable and heat-labile factor, extractable from the whole cells (Jackson and Long, 1965).The activity of the cultures was increased when the isolates were taken from dead infected (previously treated) larvae and passed serially through two neonate larvae (Singer, 1981). Matheny (1992) examined primary powders from 29 strains of B. laterosporus for coleopteran activity against L. serricorne. Of the B. laterosporus strains tested, strains NRS 590, NRS 1111, and NRS 1338 were the most active showing coleopteran activity with LC,, values in some cases as high as 3 x 1 0 4 . D. INDIANMEALMOTH(Plodia interpunctella) During the study of the effect of the morphological group I1 strains of Bacillus on the Indian meal moth (r! interpunctella) as part of a study of stored grain insects, these cultures generally were found to be not very active against the moth. The LC,, values of B. alvei 46-11-5, B. alvei II12E,B. brevis 42-14-B2, and B. circulans 46-B1 were less than %o. E. ZOOPARASITIC NEMATODES (Trichostrongylus colubriformis)

In the late 1980s, the author collaborated with Dr. Leon W. Bone, then of the USDA/ARS Regional Parasite Research Laboratory in Auburn, Alabama. Initially, primary powders of Bt and then B. sphaericus strains were sent to be used by him in his on-going work with the zooparasitic nematodes (Bone, 1989; Bone and Singer, 1989, 1990, 1991) and later strains of B. laterosporus were used. The latter work culminated in a patent on the control of zooparasitic nematode, ova/larvae by strains of B. laterosporus (Bone and Singer, 1991).

UTILITY OF STRAINS OF GROUP I1 Bacillus

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1. Rearing

The ruminant zooparasitic nematode ( T colubriformis) was maintained by Bone in male, cross-bred goats that weighed about 20 kg at infection. Animals were killed 2 1 days after infection and the eggs were removed from rectal feces, surface-sterilized, and placed in media according to the procedures described by Bottjer et al. (1985). Ovicidal activity was determined as described by Bone and Singer (1991). 2. Biological Events and Effects

Four of 27 strains of B. laterosporus showed nematicidal activity against I: colubriformis (Table VII) (Bone and Singer, 1991). The nematicidal activity appears to be related to the cellular fraction of the FWC. F. PHYTOPARASITIC NEMATODES (Heterodera glycin es)

Heterodera glycines, the soybean cyst nematode, is considered to negatively impact soybean production around the world. Soybeans are widely grown as a source of oil and high-protein meal, grossing an estimated $12 billion annually. It is believed the soybean cyst nematode has cost some states in the United States losses of up to $100 million annually. Above-ground symptoms are apparent in soybean plants in which high populations of nematodes are present. The plants appear stunted and chlorotic, which gives them a “yellow dwarf” appearance. The estimated loss in plant yield can range from slight to greater than 90% depending on the degree of infestation, secondary infection, environmental conditions, and the race of nematodes infesting the plant. TABLE VII ACTIVITY OF A COMPARISONOF NEMATICIDAL PHMARY POWDERS OF Bacillus laterosporus AGAINST Trichostrongylus colubriformis Strain ATCC 64 Bon 705 CCEB 629 NRS 1647 23 other powders0

Ovicidal toxicity, LC50 (pg total protein/ml) 0.24 6.90 0.67 0.22

Note. LC,,, pg total proteidml killing 50% of the nematodes (protein is that contained in primary powder as measured by Lowry procedure.)-, No activity detected. 0 See particular species under Section IV.

230

SAMUEL SINGER

Heterodera glycines’ life cycle begins as a protective cyst in which 200-500 eggs develop. The cysts drop off into the soil where emerging

soybean roots stimulate hatching. The second larval stage emerges from the protective cyst and invades roots near the tips and feeds on the cortical and stellar tissues. On maturity, the male emerges from the root while the female remains sessile. Her posterior end extends through the epidermis of the root and is fertilized by the free-living males. The cysts and eggs are spread to uninfected soil by means of soil distribution primarily through soil erosion, farm equipment, and wind. Many agents for soybean cyst nematode control have been studied but have been shown to be ineffective or impractical for agriculture. Currently the most popular control technique involves 2-year crop rotation to reduce soybean cyst nematode infestation and the use of nematode-resistant biovars. Because strains of B. laterosporus had been shown to be active against zoopathogenic nematodes (Bone and Singer, 1991), and because soybeans are such an important local cash crop, the effect of some of the morphological group I1 strains of Bacillus was examined for nematicidal effects against the soybean cyst nematode, H. glycines. Initial studies (Cadwallader and Singer, 1992) involved a laboratory model Caenorhabditis elegans in two tests, an agar-diffusion test and a larvicidal-dilution test. In addition, a greenhouse screen was used to examine the nematicidal activity of primary powders of the Bacillus strains in the soil environment. Responses in the greenhouse test did not necessarily follow the responses in the two C. elegans tests (Cadwallader and Singer 1992; Singer et al., 1994b). What follows considers mainly the greenhouse test results. Further details will be included in a paper that is currently in preparation relative to these phytoparasitic nematode experiences. 1. Rearing and Bioassay

Greenhouse studies were performed to examine the inhibitory effect of infection of soybean root systems using the various strains of the four Bacillus species (B. alvei, B. brevis, B. circulans, and B. laterosporus) using their primary powders against H. glycines (Cadwallader, 1992). It was necessary for three components to be brought together: the candidate strain of Bacillus, the soybean cyst nematode, and the soybean plant. Each component had a series of technical difficulties that had to be mastered before an effective greenhouse screen could be performed. The greenhouse screen enables the assessment of toxin activity of the various strains of Bacillus in a soil environment. Treated and untreated plants were handled in a similar manner, and at the end of 30-35 days

UTILITY OF STRAINS OF GROUP I1 Bacillus

231

a comparison was made of the number of H. glycines cysts on the roots. For each powder tested, a dilution was set up at 10-3powder to soil (wt/wt). For each powder tested in triplicate, a negative control (minus the bacterial powder) was planted using the same soil mixture. Each well contained 60 g of soil mixed with 60 mg of bacterial powder. A soybean seed was planted in each well. Three days after planting, an inoculum of crushed H. glycines cysts, females, and root exudate was prepared. The cysts were collected from the soil around the root. These were pooled with fragments of the roots containing cysts and females. These were all crushed gently with a small tissue grinder. The emulsions were used to infect both the treated and untreated plants. One milliliter of the suspension was released 2 or 3 cm into the soil by use of a 5-ml pipete. All specimens were inoculated evenly throughout the wells. The remaining crushed roots containing egg masses were also deposited equally among the wells. The soybean plants were maintained under 12-15 hr of light per 24 hr at 17-24OC and 60-70% relative humidity in the greenhouse for 30-35 days. At 30-35 days, the entire root system was dislodged from the growth well, leaving the root and soil intact. A gentle stream of water was poured over a mesh sieve (400 Fm). After the removal of the adhering soil, the root system was placed in a 500-ml beaker with diluted methylene blue. The latter step aided in counting. The root system was then examined and the cysts were counted for each experimental series. A parasitic index (PI) was then determined for each point of the experiment. The PI was calculated as the mean number of treated cysts over the mean number of untreated cysts x 100. 2. Biological Events and Effects

Sixteen strains from four Bacillus species were examined for nernaticidal effects against H. glycines (Table VIII). Twelve showed excellent results (PI < 60%)with 4 strains showing a perfect PI of loo%, 4 showed good results (PI SO-SO%), and 1 was not active at all. Two successive 30-day plantings in soil containing a rifampin-resistant strain of one of the active cultures showed no significant loss of activity in suppression of infestation of H. glycines or in the total viable count of the culture retrieved from the soil from the 30- to the 60-day assay. G. SNAILS (Biomphalaria glabrata)

Schistosomiasis is a tropical disease that is transmitted by snails. This disease affects over 300 million people, most in developing tropical countries. Its total public health impact is surpassed only by that of

232

SAMUEL SINGER TABLE VIII SUMMARY OF GREENHOUSE STUDY OF PRIMARY POWDERS,

Bacillus STRAIN vs Heterodera glycines Strains

PI

Relative activity

Bacillus alvei 2771 III3DTiA III3F 46-C-3 46-11-4

0.0 0.0

Excellent Excellent Excellent Excellent Excellent

76.2 46.7 17.0

Good Excellent Excellent

62.9 55.8 40.0 66.7

Good Excellent Excellent Good

0.0 220.0 47.4

Excellent NA Excellent

27.0 18.6 0.0

B. brevis ss-86-4 2904 2904

B. circulans 3986 42-G1 46-J3 46-B1

B. laterosporus 064 629 1111

B. laterosporus 1647 1645RA 1645RA

8.3 10.7 15.0°

Excellent Excellent Excellent

Note. PI, parasitic index (treatedhntreated x 100):excellent, <60%;good, 60-80%; NA, >80%. RA, rifampin resistant. OResults after 60 days.

malaria and tuberculosis (WHO, 1984). Biomphalaria glabrata is the intermediate host for the disease and is therefore a primary control target. Copper- and tin-based chemical molluscicides initially used to control the transmission of the disease no longer can be used due to their level of toxicity and their persistence in the environment (McCullogh and Mott, 1983). 1. Rearing and Bioassay

The snail rearing and bioassay are described in detail elsewhere (Singer et al., 1994a). The test snail B. glabrata as well as the diluent were maintained in deionized water containing 1 g/liter of synthetic sea salts (Carolina Biological). The snails were maintained in 35-liter

UTILITY OF STRAINS OF GROUP I1 Bacillus

233

aquaria and fed lettuce on demand. For each bioassay (in 3.5-oz disposable plastic cups), four dilutions (1/30,1/100, 1/300,1/1000)of test material and 3-10 snails, 3-5 mm in diameter (as available), were used. All test materials were incubated at room temperature (25°C) and the surviving snails were counted at Days 3 and 6 of the treatment. For the LC50 calculation (the dilution of the FWC, primary powder, toxin extract, or cell component that killed 50% of the test snail population) as well as the 9 , values were obtained by plotting the linear regression of mortality after 6 days incubation of the snails vs the dilution of the material being tested. The 9 indicates the reliability of the LC,,, Only P values >0.70 (a conservative value) were used. 2. Biological Events and Effects

Over the past 20 years, the author's laboratory has examined thousands of isolates for molluscicidal activity against the target snail B. glabrata without successfully noting any molluscicidal strains. In 1986, Dr. A. A. Yousten (Virginia Polytechnic Institute) provided cultures isolated by Ms. Janet Ofori (Institute of Aquatic Biology, Achimote, Ghana). After reisolating (cloning) the material, the molluscicidal activity of the Bacillus spp. cultures provided by Dr. Youston was confirmed. The initial activity of the cultures (LC,, is the dilution of the final whole culture killing 50% of the test animals) was low, with LC,, values of 1/30-1/300. Subsequent fermentation development raised this value to LC,,-1/3000 (Singer et al., 1994a).Based on classical biotyping, the three active strains, SS86-3, SS86-4, SS86-5 (the author's accession numbers) were identified as Bacillus brevis (Singer et al., 1988). Once the molluscicidal strains were identified, it was incumbent to test other B. brevis cultures in the author's collection. Of the 41 cultures tested, all 41 have given some indication of molluscicidal activity (Singer et al., 1994a). In addition to B. brevis, strains of several other species were shown to be molluscicidal to our snail target species. Table IX illustrates these effects with one strain for each of the four species. The fermentation process and toxin(s) produced by these strains are discussed under sections IV, V, and VI as appropriate.

H. ZEBRAMUSSELS (Dreissena polymorpha) The zebra mussel (D.polymorpha), a native mussel of Europe's Black and Caspian Seas, was accidentally introduced into North American waters in the mid-1980s. It is believed that zebra mussels (probably the larvalheliger stage) arrived in the ballast water of an ocean-going ship when environmental conditions, such as temperature and food supply,

234

SAMUEL SINGER TABLE IX A COMPARISON OF MOLLUSICIDAL ACTIVITY OF FINAL OF STRAINS FROM EACHOF FOURSPECIES WHOLECULTURES OF Bacillus AGAINST Biornphalaria glabrata (SNAIL] Strain

Mollusicidal activity

Bacillus alvei III3DTlA First experiment Second experiment B. laterosporus 1111 First experiment Second experiment

+++ ++ ++

++

B. circulans 46-J3 First experiment Second experiment B. brevis SS86-4 First experiment Second experiment

++ +++ ++ ++ ~~

Note. +++, Excellent [LC,, > 10-3); ++, good [LC,,-10-2-10-3),

were favorable for the establishment of a healthy population of the mussels (Griffith et al., 1991).Since the discovery of the zebra mussel in Lake St. Clair in June 1988, their number and distribution have increased throughout every waterway east of (and including) the Mississippi River and as far south as the deep waters around New Orleans. itself. The female zebra mussel is prolific, producing up to 1 million eggs in several spawns per year. The larval stage is planktonic and readily disperses through water currents. The adult is capable of attaching to any hard surface, accumulating in the hundreds to hundreds of thousands per square meter of hard surface. When this starts to block water intake pipes it costs each effected plant thousands of dollars to get rid of them. Existing control methods make use of chemicals and a manual removal approach. Biocontrol methods would be preferred for rational ecological reasons, particularly to spare or replace some of the more noxious materials that might be used. In 1991, representatives of the USEPA-Gulf Breeze, Florida Research Laboratories, asked the author to undertake a search among molluscicidal bacterial strains for cultures that could be used potentially in a biological control program against the zebra mussel. It had been previously

UTILITY OF STRAINS OF GROUP 11 Bacillus

235

shown by Molloy et al. (1994) that various B. thuringiensis and B. sphaericus primary powders had little or no affect against the zebra mussels. 1. Rearing and Bioassay

The rearing and bioassay procedures mainly follow those of Stoeckel and Garton (1993).

a. Adult Zebra Mussels. Healthy zebra mussel adults were obtained locally (mainly from the Illinois River) and maintained in our laboratory in 35-liter aquaria equipped with power filters. Ammonia levels were checked regularly. Preserved algae in the form of a concentrated diatom sludge, Diet B (Coast Seafood Company, Bellevue, WA), was provided daily as a food source for the zebra mussel. The bioassay was similar to that using the snail (see Section II1,G) except that the assay used glass specimen dishes each containing six 3- to 5-mm zebra mussels. Incubation temperature was 18°C. Surviving mussels were counted at Days 3 and 6 following treatment. b. Larval Stage (Veliger)Zebra Mussels. Veligers were obtained by artificially inducing spawning in healthy adults by using serotonin. For veliger bioassays, veligers were collected from the bottom of the spawning tank and diluted to approximately 10 veligerdml. Two milliliters of the veliger suspension was added to each of five wells of a microtiter plate for each experiment and veligers were counted and the numbers recorded; this was considered “zero time.” Addition of the treatment powder followed immediately. One milliliter of diluted powder (diluted in synthetic spring water) was added to each test well. Controls consisted of sterile water substituted for the bacterial dose. Surviving veligers were counted at 3 and 5 hr. The veliger counts were normalized and then LC,, and 3 values were calculated as in the snail bioassay (see Section 111,G). 2. Biological Events and Effects

The application of strains of morphological group I1 Bacillus from four separate species, B. alvei, B. brevis, B. circulans, and B. laterosporus (as illustrated in Table X), was successful against small adult zebra mussels. The effect of these strains is not apparent against the large zebra mussels (10-mm animals and above), but the strains are quite active against the smaller (3-5 mm) animal (Singer et al., 1994~). Although the veliger bioassay is still under development, initial efforts indicate that strains of the four Bacillus species are active at LC,, values of approximately lo8 (as

236

SAMUEL SINGER TABLE X STRAINS OF MORPHOLOGICAL GROUPI1 Bacillus SHOWING MOLLUSCICIDAL ACTIVITY"

Bacillus alvei 2771 III3DTlA B. brevis SS86-4 2904 B. circulans 42-G1 46-J3 46-B1 B. laterosporus 1111 11 Against small adult Dreissena polymorpho (zebra mussel).

illustrated in Table XI) after only 3-5 hr,rather than the 3-6 days for activity to appear when used against small adult zebra mussels. IV. Utility According to Strain and Species

After briefly examining the utililty of the morphological group I1 strains of Bacillus according to target (Section 111),the bacterial biology of the strains and their utility according to strain and species will be discussed (Table XII). In the following subsections, in addition to a brief introduction, the systematics of the specific strains, specifics of their TABLE XI STRAINS OF MORPHOLOGICAL GROWI1 Bacillus SHOWING AGAINST VELIGER-STAGE MOLLUSCICIDAL ACTIVITY Dreissena polyrnorpha (ZEBRA MUSSEL) Strain

Bacillus alvei III,DTIA B. brevis 2904 B. circulans 42-G1 B. laterosporus 1647

LC,,-'=

>1 x

1 0 8 after

3-5 hr

A A A A

Note. A, observed activity; LC,,, concentration of final whole culture that kills 50% of the test organisms Veliger-stage Dreisseno polyrnorpha (zebra mussel).

TABLE XI1 TARGETKANDIDATE OVERVIEW ______

~______

~~ ~

Diptera larvae Candidate

~~

Cxq

Aea

~~

Stored grain

sv

Ana

~~

Ls

~

Molluscs

Nematodes

Pi

TC

Hg

Bg

Dp(A1

Dp (V)

_____

Note. Numbers in parenthesis are number of active strains to date; A, biologically active:-,data not available. Cxq, Culex quinquefasciatus; Aea, Aedes aegypti; Ana, Anopheles olbimanus; Sv, Simulium vittatum; Ls, Lasioderma serricorne; Pi, Plodia interpunctella: Tc, Trichostrongylus colubriformis; Hg, Heterodera glycines; Bg, Biomphalaria glabrata; Dp(A),Dreissen polymorpha (small adult); Dp (V),Dreissen polymorpha (veliger). If known and unless otherwise stated, all of the above targets are egg or larval stages.

238

SAMUEL SINGER

fermentation process (an overview of what is known), and their biological events and effects are discussed. It is intended that the latter will bring together what is known of the biological effects for each of the strains according to the particular species. Section V consists of a more general discussion comparing the strains and species across the four species in terms of their fermentation processes, their “toxins,”and their potential for products. First, an examination of some background in systematics is needed [mainly derived from a previous discussion (Singer, 1988)l. At the most elementary teaching level, the beginning bacteriology student determines the reactions of a series of “biochemical”tests (e.g.,does the unknown utilize lactose with the production of “acid and gas” in 24 hours?). Systematics is concerned with the scientific study of the diversity of organisms and the relationships among them. To this end, chemical profiling or fingerprinting methods [auxanotyping (biotyping), serotyping, phage typing, fimbriation typing, etc.] have been classically used to detect phenotypic variation. To rationally utilize the information generated by modern techniques, computer-based numerical taxonomy approaches have been developed to handle the ever-growing database. To illustrate this at a simple phenetic level [the utilization of carbon and nitrogen sources (Bell,1987; Singer and Bell, 1987)],30 strains of Bacillus were examined using 28 carbonhitrogen (C/N) sources. Carbonhitrogen utilization tests were performed according to Gordon et al., (1973). The results were examined by numerical taxonomic analysis, and the phenetic similarities found were used to construct a matrix of similarity from which a phenogram (dendrogram) was developed (Fig. 1).The genetic relatedness between the strains was estimated by making a phenogram from an unweighted-pair-group-cluster-analysis-with-~ithmetic-averages (UPGMA) (Sneath and Sokal, 1973). This was done by comparing percentage similarities of the various classes of characteristics used (Lessel and Holt, 1970). The percentage similarity between each strain was calculated by dividing the number of similar characteristics between two strains by the total number of characteristicsstudied. The organisms were then sorted into phenetic groups by use of the “highest-link’’criterion (Gower and Ross, 1969; Qieg and Lockhart, 1966). Genetic diversity at a locus (h)among electrophoretic types was calculated by the following equation: h:1-C x2 [nl(n-1)1, where x is the frequency of the allele at that locus, n is the number of isolates in the sample, and nl(n-1) is a correction for bias in small samples (Selander et al., 1986). Mean diversity per locus is comparable to heterozygosity estimates in diploids.

-

I

&

2918 B. brevis 46-11-4 g. 46-C-3 g.

7

-

-

082 B lateros r u s 590 B: 661 g. lateroswrus

a.

I

HD-1 thurinqiellsis 2904 B. brevis 2934 B. brevis 1761 B. circulans 2718 B. circulans 2201 g. circulans

240

SAMUEL SINGER

Phenetic differences found between strains of B. laterosporus compared to the other group I1 bacilli tested supported the concept of B. laterosporus as a tightly knit, easily distinguishable group. The remaining groups were more loosely clustered. Morphological group I11 B. sphaericus strains (used as controls) were clustered away from the morphological group I1 strains as well as from the morphological group I B. thuringiensis strains (except for Bt HD1). Although DNA is the source of all taxonomic information, proteins, the secondary macromolecular product of DNA, are usually easier to work with (Goodfellow and Minnikin, 1985). At this secondary or epigenetic level, comparative analysis of specific homologous proteins can provide an accurate means of establishing relationships among bacteria. More complex epigentic analyses involve the comparison of groups of proteins by gel electrophoresis and regulatory mechanisms governing the synthesis of enzymes operating in given pathways. Changes in amino acid sequences of specific proteins can reflect evolutionary divergence between organisms (Goodfellow and Minnikin, 1985). One can measure the amino acid sequence of a specific homologous protein from representative strains or one can use serological techniques (Schleifer and Stackebrandt, 1983),but the most powerful, relatively simple, yet cost-effective method is that of protein electrophoresis. The two general approaches for using protein electrophoresis are (i)determination of specific protein patterns using the SDS-PAGE method (Dent and Williams, 1985), and (ii) examination of specific enzyme patterns (zymogram) (Williams and Shah, 1980);both methods are of great taxonomic value. Baptist et al., (1978) used electrophoresis of limited numbers of enzymes to differentiate Bacillus species. According to these workers, if a sufficient number of enzymes are compared (more that five), then the results will show that two individuals from different species will differ in electrophoretic mobility by about 50% or more, whereas two members of the same species will usually differ by 20% or less. To compare similarities and differences among the strains, the TAXAN numerical taxonomy program (Information Resources Group, Maryland Biotechnology Institute, University of Maryland) was used. This program provided a similarity matrix and the dendrogram that reflected the percentage similarity among strains. The phenograms (also called dendrograms) that follow for each of the particular species will involve the previously mentioned electrophoresis examination of specific enzyme patterns (ca. 15 enzyme systems) usually by polyacrylamide gel electophoresis (PAGE). For further specifics, see Singer (1988).

UTILITY OF STRAINS OF GROUP I1 Bacillus

241

The order of the species discussed is alphabetical. A. Bacillus alvei Morphological group I1 of Bacillus contains many true insect pathogens such as the agents of milky disease of the Japanese beetle (Popillia japonica), B. lentimorbis and B. popilliae, as well as the causative agent of American foulbrood of honeybee hives, B. larvae. Similarly, B. alvei was once considered to be the causative agent of European foulbrood of honeybee hives but this has since been disproved (Priest, 1993). It is therefore not surprising to find among these strains agents for insect (more correctly, invertebrate) biological control-toxinogenic agents. 1. Systematics

Ms. Gi Kyung Han in our laboratory recently had the opportunity to do a multilocus enzyme analysis of B. alvei strains (Fig. 2). The study of the genetic relationship among 30 strains of B. alvei was performed by surveying 15 enzyme-staining systems utilizing PAGE. A total of 39 loci were generated by the 15 enzyme systems. A dendrogram summarized the genetic similarity among the 30 strains. A genetic diversity was calculated for each locus. The population structure of B. alvei was found to be clonal (Fig. 2). The average genetic diversity over all loci [mean genetic diversity (E)] was calculated for the 30 strains of B. alvei as 0.41 (Table XIII). It appears that this species is not tightly clustered. This type of comparatively high value of diversity has been explained (Selander et al., 1986) as reflecting a large effective population size or an early evolutionary origin of extant strains.

TABLE XI11 OF MEANGENETICDIVERSITY OF A COMPARISON SEVERAL Bacillus SPECIES

Taxon

Mean genetic diversity h

Bacillus alvei Bacillus brevis Bacillus laterosporus Larvicidal B. qJhaericus

0.410 0.463 0.154 0.125

- A2 A

A3

B B2

386 4009 1306 4186 1649

1180 2771 2772 111-3-F IIIZE

2. Fermentation Process

A modification of Dulmage's B 4 medium (Dulmage et al., 1990) was used in an inoculum buildup and fermentation process as previously described for B. brevis (Singer, 1988). (See Section IV,B and Section V for a more detailed discussion of the fermentation process for the morphological group I1 strains.) 3. Biological Events and Effects

Of the B. alvei strains tested, some appear to be biologically active against the diptera Culex, Aedes, Anopheles, and Simulium, the phytoparasitic nematode Heterodera, and the molluscs Biomphalaria and Dreissena (Table XIV). There appears to be little or no activity against the stored grain insects Lasioderma and Plodia nor against the zooparasitic nematode Trichostrongylus. There is variation in biological activity against the targets tested and among the strains tested.

243

UTILITY OF STRAINS OF GROUP I1 Bacillus TABLE XIV

COMPARISON OF BIOLOGICAL ACTIVITY OF Bacillus alvei AGAINST EIGHTPESTTARGETS Stored grain

Diptera larvae Strain

Cxq Aea

42-F-3 46-C-3 46-F3 46-11-4 46-11-5d 111-2-E 111-3-D 111-3-DT1 III-3-DT1A 111-3-F 2771 2772 2198

++ ++ ++ ++ ++ ++ + 0

Ana

Sv

Ls

Pi

Tc

++ +++

++

++ ++

Hg

Molluscs Bg Dp(A) Dp (Vl

+++

++

0

++ ++ ++ ++

Nematodes

+

+ +

+++ +++

+

+++ 0

+++ +++ +++ +++

+++ ++

+++

+++ ++

Note. +++, Excellent (LC,,>10-3); ++, good (LC,,= 10-"10-3): +, poor (LC5,<1O-Z): 0, inactive: open spaces, data not available. Where LC,, values are not available (e.g., H . glycines), see appropriate footnote from a previous table (e.g., Table VIII for H. glycines). LC,,, concentration of final whole culture that kills 50% of the test organisms. Cxq, Culex quinquefasciatus; Aea, Aedes aegypti; Ana, Anopheles albimanus: Sv, Simulium vittatum; Ls, Lasioderma serricorne;Pi, Plodia interpunctella: Tc, Trichostmngylus colubrifonnis: Hg, Heterodem glycines: Bg, Biomphalario glabrata: Dp. (A), Dreissen polymorpho (small adult):Dp. (V), Dreissen polymorpha (veliger).If known and unless otherwise stated, all of the above targets are egg or larval stage. For source of strains and cultures, see text and titles of specific dendrograms.

B. Bacillus brevis Gramicidin and gramicidin S, and other small peptide antibiotics, are what B. brevis is probably best known for. Much of what is known about peptide antibiotic biosynthesis has come from the study of gramicidin S (Zuber et al., 1993).The B. brevis group (which includes B. laterosporus) is widely distributed in the environment with the soil being the primary habitat and marine and freshwater habitats secondary (Priest, 1993). Because the only use that has heretofore been considered for B. brevis is for the production of gramicidin and tyrocidin, little has been done in terms of examining its role in the environment (Priest, 1993). 1. Systematics

The genetic relatedness of 7 1 strains of B. brevis [the result of two studies (Boyd and Singer, 1990; Madjid and Singer, 1992)l was examined by using a PAGE multilocus enzyme study of the strains. From the

244

SAMUEL SINGER

dendrogram generated (Fig. 3), the strains were seen to be divided into two clusters, clonal groups I and 11, with 75% similarity. Clonal groups I and I1 could each be further divided into three subclonal groups. Subgroup IA could be separated from subgroups IB and IC at 81% similarity. Subgroup IB contained both the type culture 604 and the three Ghanian molluscicidal strains, SS86-3, -4, and -5. Subgroup IIC could be separated from IIA and IIB at 76% similarity and subgroup IIA from IIB at 86% similarity. From the above, one would think that B. brevis should be considered a tightly clustered species. However, the mean genetic diversity value (6) of 0.463 belies this. 2. Fermentation Process

A modification of Dulmage’s B4 medium (Dulmage et al., 1990) was used in an inoculum buildup and fermentation process as previously described for B. brevis (Singer, 1988; Singer et al., 1994a).To determine when the molluscicidal toxin was first produced, a series of in-cycle experiments was performed in which whole culture samples were taken at various times during the fermention (Singer et al., 1994a). When 0.5% fructose was used, molluscicidal activity (toxin production) appeared as early as 8 hr, peaked at 1 2 hr, and declined rapidly by 24 hr. All of these events took place before the initiation of sporulation, which occurred at about 24 hr. The total viable count also appeared to be lowest at 1 2 hr, as was the pH. These declines in activity, population density, and pH were not as pronounced when 0.5% phenylalanine was used as an additive. Molluscicidal activity did not appear to be reduced during sporulation, even though there appeared to be a dip in activity after 16 hr. Two factors might account for the presence of a lower amount of toxin after 1 2 hr of fermentation: the possible susceptibility of the toxin to protease or to oxidation. Both possibilities were confirmed (Singer et al., 1994a). In the absence of the addition of one of several sugars such as fructose, or one of several amino acids such as phenylalanine, no detectable molluscicidal activity was seen. In the presence of fructose or phenylalanine or one of their congeners, molluscididal activity is seen. What is the role of fructose and/or phenylalanine in the fermentation? They appear to be “secondary factors” as classically described by Margalith (1964). One of the characteristics of a secondary factor is that it itself is not essential for growth but influences and changes the outcome of the process in other ways (e.g., the role of 0, in the Pasteur effect). The addition of either fructose or phenylalanine (or their congenors) may have an antioxidant effect, preventing the oxidation of the protein (toxin) (by causing a low oxygen tension inside the cell) as long as the “fermentation” is

YOSimilarity r 100

1

1

1

1

95

90

85

80

I

1

75

70

in 0

376 846

856 1158 S1206 616 780 782 799 809 859 1157 S614 S622 5612 1219 1365 5606 561 1 5605

664 803 1029A 1212 640 1366 1334 S603 S761 1214 1359 1127 1222 S641 779 818 781

16%

'6% 11

815

U29 18 51373 S378 L135 1027

C

1 1

75%

L1202 -J U2904 u2a33 SS86-3 SS86-4 SS86-5

U2954

-8 78%

-

_J

s379

5380 A 81874 84389 84558 51204 L771 8691 S1208 S1207 8439

1

A

81%

FIG. 3. Dendrogram consisting of 71 strains of Bacillus brevis. Percentage similarity based on PAGE rnultilocus enzyme analysis of strains.

246

SAMUEL SINGER

proceeding and using up either of these two additives. All of this allows the fermentation process to proceed to the point where it becomes more convenient to harvest the toxic product (Singer et al., 1994a). 3 . Biological Events and Effects

There was only one B. brevis strain that showed up in the initial isolates from Burma and India (the 42,46, and I11 series), and it (42-14-BZ) was not particularly active in the early examinations against some of the 11 pest targets (Table XV). In contrast, once the Ghanian isolates were identified and a fermentation process was developed, it encouraged the author to investigate not only the use of B. brevis strains against molluscs (such as the Biomphalarian snail, and later against the zebra mussel), but also a phytoparasitic nematode. Against these targets, there appeared to be excellent activity (Table XV). A more detailed discussion of the molluscicidal toxin is presented under Section V. C . Bacillus circulans

Bacillus circulans strains were always the “next group to be worked on.” As a consequence, a PAGE multilocus enzyme study on these strains has yet to be performed. Bacillus circulans is one of those very heterogenous groups that should be split into several species (Priest, 1993; Logan, 1994). The group has been shown to produce small peptide antibiotics and several enzymes (Zuber et al., 1993; Ferrari et al., 1993).The species B. alvei and B circulans each have strains that are capable of producing motile colonies. If you have never seen motile colonies, you are in for a treat. Try to do a total viable count when each colony can move and deposit small satellite colonies along the way! 1. Systematics

Figure 1is a phenogram based on carbonhitrogen utililzation, which shows that the B. circulans strains clustered and were separated away from the other Bacillus groups. The members from the 42 series and from the 46 series isolates clustered fairly well with the known (confirmed) B. circulans cultures (strains 1761, 2718, and 2201). 2. Fermentation Process

A modification of Dulmage’s B4 medium (Dulmage et al., 1990) was used for the inoculum buildup and fermentation process as previously described for B. brevis (Singer, 1988). (See Section IV,B and Section V for a more detailed discussion of the fermentation process for the morphological group II strains.)

247

UTILITY OF STRAINS OF GROUP I1 Bacillus TABLE XV

COMPARISON OF BIOLOGICAL ACTIVITY OF Bacillus brevis AGAINST EIGHTPEST TARGETS Diptera larvae Strain

Cxq Aea

Ana

42-14-B2 B444 B541 B439 S1207 S1208 B691 L771 s1204 B4558 B4389 S380 w2954 SS86-5 SS86-4 SS86-3 W2904 L1202 L604(t) S1203 L135 s210 S1610 S1373 W2918 S1609 1211 1127 664

++ ++

++

S1206

Stored grain Sv

Ls

Pi

o

+

Nematodes Tc

Hg

Molluscs Bg D p ( A ) Dp (V)

++ +++ ++ ++ + ++ +++ ++

+++ +++

+ + ++ +++ +++ +++ ++ +++ +++ ++ +++ ++ + + + ++ ++ +++ + ++ +++ +++ ++

+++

+++

Note. +++, Excellent (LC,,>lO-3): ++, good (LC,,= 10-2-10-3); +, poor (LC,,<10-2); 0, inactive; open spaces, data not available. Where LC,, values are not available (e.g., H . glycines), see appropriate footnote from a previous table (e.g., Table VIII for H . glycines). (t),Type strain. LC,,, concentration of final whole culture that kills 50% of the test organisms. Cxq, Culex quinquefasciatus; Aea, Aedes oegypti; Ana, Anopheles albimonus; Sv, Simulium vittatum; Ls, Losioderma serricorne; Pi, Plodia interpunctella: Tc , Trichostrongylus colu briformis; Hg , Heterodera glycines; Bg , Biomph alaria gla bra to: Dp. (A),Dreissen polymorpha (small adult): Dp. (V),Dreissen polyrnorpha (veliger). If known and unless otherwise stated, all of the above targets are egg or larval stage. For source of strains and cultures, see text and titles of specific dendrograms.

248

SAMUEL SINGER

3. Biological Events and Efiects

The biological events using the B. circulans strains are similar to those of B. alvei. Of the B. circulans strains tested, some appear to be biologically active against the diptera Culex, Aedes, and Anopheles, the phytoparasitic nematode Heterodera, and the molluscs Biomphalaria and Dreissena (Table XVI). There appears to be little or no information available concerning the stored grain insects Lasioderma and Plodia, the zooparasitic nematode Trichostrongylus, or the dipteran Simulium. One can, however, see that there is a variation in biological activity against the targets tested. In addition, there is variation among the strains tested.

D. Bacillus laterosporus According to Favret and Yousten (1985, B. laterosporus (like B. alvei) was at one time thought to be a pathogen of honeybees but has since been shown to be a saprophyte living off dead bee larvae. This species

TABLE XVI COMPARISON OF BIOLOGICAL ACTIVITY OF Bacillus circulans AGAINST EIGHTPESTTARGETS Diptera larvae Strain 46-J-3a 42-G1

Cxq Aea

Ana

Sv

Ls

Pi

++ +

42-G-1Ab 46-B1 46-B2 2628 3370 3986 3987

Stored grain

++ ++ + + +

o o

+

o

++

+

+

Nematodes

Tc

Molluscs

Hg

Bg D p ( N Dp (V)

+++ ++ +++ ++

+++ ++ ++ +++ ++

+++

++

++

Note. +++, Excellent (LC,,>iO-3); ++, good (LC,,= 10-2-10-3); +, poor (LC,,
UTILITY OF STRAINS OF GROUP I1 Bacillus

249

is also distiguishable from the other morphological group I1 species by the production of a canoe-shaped parasporal body that cradles the spore and is firmly attached to it. This gives the appearance of being laterally displaced. It is a little disconcerting to suddenly come upon a crosseyed (or wall-eyed) pair when examining them microscopically! 1. Systematics

The relationships between insecticidal (to mosquito larvae) and noninsecticidal strains of B. laterosporus were examined using PAGE multilocus enzyme analysis of B. laterosporus strains (Fig. 4) (Saari 1989;

9141 Shi3 NI 1645 707 Shil

--

-

Shi2 Shi5 661 NI 1338 705 706 64 6457 1111 1267 1647 342 NI

-

-B 2 B

Shi4 NI

629 N 712 NI 708 NI 1642 NI 1648 NI 1644 NI 1649 NI

I

I

4I

L

1

A

FIG.4. Dendrogram consisting of 29 strains of Bacillus laterosporus. Percentage similarity based on PAGE multilocus enzyme analysis of strains.

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SAMUEL SINGER

Singer and Saari, 1990). The genetic relatedness was determined by zymogram analysis. A UPGMA analysis of similarity divided the strains into two major clusters, clonal groups A and B (with 75% similarity), whereas B divided into two subgroups B 1 and B2 (showing 87% similarity), Clonal subgroup B2 was occupied by all the insecticidal strains tested but also included three noninsecticidal strains. Clonal group A, which was occupied by four noninsecticidal strains, was quite different from other strains and showed few polymorphic characteristics. These experiments indicated that B. laterosporus was tightly clustered. This was confirmed by the mean genetic diversity of all the strains being at 0.145 for the 2 1 loci from the 1 3 enzyme systems (Table XIII). 2. Fermentation Process

A modification of Dulmage’s B4 medium (Dulmage et al., 1990) was used for the inoculum buildup and fermentation process as previously described for B. brevis (Singer, 1988). (See Section IV,B and Section V for a more detailed discussion of the fermentation process for the morphological group I1 strains.) 3. Biological Events and Effects

The biological events using the B. laterosporus strains are similar to those of the other three species, B. alvei, B. brevis, and B. circulans. However, more complete data are available concerning several of the 11pest targets examined. Of the B. laterosporus strains tested, some appear to be biologically active against the diptera Culex, Aedes, and Simulium, both the phytoparasitic nematode Heterodera and the zooparasitic nematode Trichostrongylus, the molluscs Biomphalaria and Dreissena, and the stored grain insect (beetle) Lasioderma (Table XVII). There appears to be little or no information available regarding the stored grain insect Plodia and the dipteran Anopheles. However, one can see that there is a variation in biological activity against the targets tested. In addition, there is variation (from activity to no activity) among strains tested. V. Fermentation Processes, Toxins, and Products

A. FERMENTATION PROCESSES

Two major components need to be considered when examining a fermentation process: the bacterial candidate cultures being used and the process itself.

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251

TABLE XVII ACTIVITY OF Bacillus COMPARISON OF BIOLOGICAL Diptera larvae Strain 590 9141 Shid 1645 707 Shil Shi2 Shi5 661 1338 705 706 064 6457 1111

1267 1647 342 Shi4 882 340 629 708 712 1642 1648 1644 1643 BlIaOl, to Bl/all

Cxq Aea

++ ++ ++ 0

++ ++ ++ ++ 0 0

++ ++ ++ ++ ++ ++ ++ ++ 0 0 0 0

0 0 0 0 0 0 0

Ana

latt?rOSpOrUS AGAINST

Stored grain Sv

Ls

++

++ + + + + + + + + ++ + + + + ++ + + + + + +

+ + + + +

+ +

Pi

EIGHT PEST TARGETS

Nematodes Tc

Hg

Molluscs Bg DP(A) D P W

0 0 0 0 0

0 0 0 0

+++

+++ +++ +++ +++ 0 0 0

+++

+++

+++ ++

+++ +++

+++ ++

+++

0 0

0 0

+++

0

0 0 0 0 0 0

++

Note. +++, Excellent (LC,,>10-3); ++, good (LC,,= 10-2-10-3); +, poor (LC5,<10-z); 0 , inactive; open spaces, data not available. Where LC,, values are not available (e.g., H. glycines),see appropriate footnote from a previous table ( e g , Table VIII for H . glycines). LC,,, concentration of final whole culture that kills 50% of the test organisms. Cxq, Culex quinquefasciatus; Aea, Aedes aegypti; Ana, Anopheles albimanus; Sv, Simulium vittatum; Ls, Lasiodermo serricorne; P i , Plodio interpunctella; Tc, Trichostrongylus colubriformis; Hg, Heterodera glycines; Bg, Biomphalaria glabrata; Dp. (A),Dreissen polymorpha (small adult); Dp. (V), Dreissen polymorpha (veliger). If known and unless otherwise stated, all of the above targets are egg or larval stage. For source of strains and cultures, see text and titles of specific dendrograms.

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1. Bacterial Candidate Cultures

In terms of the morphological group I1 strains of Bacillus examined in this chapter there appear to be several from each of the four species available for potential use against most of the targets (Table XII). At this stage of the investigations, none of the strains and none of the species appear to be universally toxic. It appears as if there is differential activity. Whether dealing with a new single class of toxin or several toxins remains to be determined. The numbers of strains shown indicate only those showing positive activity, As subsequent tables indicate (Tables XI1 and XIV-XVII), many strains were not tested. Most of the isolated strains, even though they originally indicated an intermediate taxonomic position, appeared to fit in well with their purported species when examined using PAGE zymograms (Figs. 14).Considering the close taxonomic (systematics)position of these particular four species, it is not surprising that active strains are found in all four of them. For example, B. laterosporus and B. brevis are reported to be quite close (Priest, 1993; Logan, 1994; Logan and Berkeley, 1984). Similarly, B. alvei and B. brevis (Priest, 1993; Logan, 1994; Logan and Berkeley, 1984) appear to be closely related. Strains of B. circulans form such a loose and large cluster group (Priest, 1993; Logan, 1994; Logan and Berkeley, 1984) that it would not be surprising to find them closely associated with the other three species examined. What better proof of a close relationship than finding original isolates of an “intermediate” nature? Today, this can better be explained by invoking the idea of plasmids carrying many characteristics and spreading them among these “species” leading to a potpouri of phenotypes that lead to ‘‘intermediate strains,” which is Gordon’s concept of a “spectrum of strains” (Gordon et al., 1973). The tables showing a comparison of biological activity of strains from each of the species against the 11targets used (Tables XIV-XVII) show many empty spaces, indicating that information is lacking. These empty spaces suggest that we have only scratched the surface of our investigation of these cultures. Similarly, we have explored only one or two examples of each of the potential target areas that we might have. For example, there was only one lepidopteran examined ( P interpunctella). Favret and Yousten (1985) also report the lack of activity of their B. laterosporus strains against the cabbage looper (another lepidopteran). 2 . Fermentation Process

The initial effort was to prepare material for testing against a target, at first a final whole culture preparation, then a primary powder. In the initial investigations (Sections 111,A-D), the usual laboratory media, such

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253

as nutrient, plate count, or brain-heart infusion media, were adequate. In some instances, raw material media (Table 11) were tried and differences in biological activity were noted. As the effort became more intensive (Sections 111,E-H), modifications of Dulmage’s B4 medium (Dulmage et al., 1990) were used. In the initial studies of the Ghanian B. brevis strains (Singer et al., 1994a) there were few fermentation problems at the shake flask level, but at the small fermentator level there were some difficulties in preparing final whole culture and as a consequence a potential problem in preparing primary powder. It was thought at the time that this difficulty may relate to the addition of fructose to the fermentation medium (Singer et al., 1994a). Fructose was one of many sugars tested and for which this effect was noted (Singer et al., 1994a). If the sugar (fructose) was omitted from mB4 medium, there was no molluscicidal activity. If one of these sugars was added there was activity, but the addition of the sugar to the fermentor resulted in an immediate drop in pH, which also resulted in a sizable decline in population. These effects were not seen in the shake flask. Several amino acids, particularly phenylalanine, could substitute for the sugar and yield molluscicidal activity with less of an effect on pH and on the bacterial population in the fermentor. It should be explained that the additives (the sugar or amino acid) were added in addition to a raw material carbodnitrogen source, PROFLO (an enzymatic digest of cottonseed meal). The aforementioned additives were not required for good growth of the fermentor population. An explanation postulated to explain the effect of these additives lies in their role as secondary factors as classically described by Margalith (1964). (see Section IV, B for details). Whether the same events take place with the Bacillus strains other than B. brevis remains to be explored. The question could be asked as to how many biologically active peptides and proteins have been missed in view of the above effect?

B. TOXINS Toxins from strains of three of the morphological group I1 species have been examined (Table XVIII). We examined B. alvei (reviewed here), B. brevis toxin (Singer et al., 1994a), and a B. laterosporus toxin (Bone and Singer, 1991). Examination of a B. circulans toxin as well as a reexamination of a B. laterosporus toxin are currently in progress. The most important bit of information bearing on the discussion in this chapter is that none of the previously mentioned toxins resemble the B. thuringiensis or the B. sphaericus crystalline toxins (6-endotoxins). The B. brevis toxin (Singer et al., 1994a) is molluscicidal, with an LC,, value of 1rJ.gtoxin proteidml. It may be described as a small, pro-

254

SAMUEL SINGER TABLE XVIII SUMMARY TO DATEOF BIOLOGICAL ACTIVITY FACTORS (TOXINS) FROM Bacillus MORPHOLOGICAL GROW I1 STRAINS

Bacillus alvei Target: Strain: Toxin characteristics:

B. brevis Target: Strain: Toxin characteristics:

Biomphalaria glabrata 2771a

MW, 5.6 and 9.0 kDa (per PAGE): HPLC signature, 167.76 sec; best LC,,, 0.6 pg toxin proteidml. Properties: heat stable, oxygen sensitive, trypsin reduces activity: lysozyme reduces activity (of FWC): site of toxin: associated with cell wall fraction.

Biornphalaria glabrata SS86-4"

MW, 5.3 and 8.7 kDa (per PAGE); HPLC signature, 148.37 and 163.96 sec; best LC,,, 1.00 pg toxin proteidml. Properties: heat stable, oxygen sensitive, trypsin reduces activity: lysozyme reduces activity (of FWC): site of toxin: associated with cell wall fraction.

B. circulans No information available. B. laterosporus (Bones and Singer, 1991) Target: Trichostrongylus colubriforrnis Strain: 629a Toxin characteristics: MW, 2.9 kDa (per HPLC); best LC,,, 0.22 pg total protein/ml. Properties: trypsin reduces activity; lysozyme enhances activity: heating increases lethality. astrain used for toxin characterization.

teinaceous, heat-stable, oxygen-sensitive entity associated with the particulate portion of the cell wall fraction of B. brevis that is formed prior to sporulation, whose HPLC signature shows major peaks at 148.37 and 163.96 sec and consists of two bands of approximately 5 . 3 and 8.7 kDa on PAGE gel. The B. alvei toxin is quite similar to the B. brevis toxin, with minor differences in molecular weight and HPLC signature. The only striking difference noted so far is the detection of only one HPLC peak for B. alvei (Table XVIII). In our own work and in the few references noted here, the biological activity against all of the targets, with one exception, always related to the particulate cell fraction. The one exception is the activity of soluble, filtered, supernatant material of some of these strains against mosquito

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larvae (Section IILA). This, in a sense, is supported by a more recent series of experiments (not described here) in which the molluscicidal final whole culture cell particulate (pellet) and primary powders, when tested against C. pipiens larvae, showed no larvicidal activity. Also of interest is that gramicidin S (typically a B. brevis product) was quite active against both the snail and the Culex larvae, whereas bacitracin was inactive against both targets. The B. brevis toxin is described as “proteinaceous” because the presence of affiliated material, such as carbohydrate, lipid, or associated metals, has not as yet been ruled out. Also interesting is the effect of lysozyme on both the B. alvei and the B. brevis toxic material (which may bear on the toxin(s) composition). Lysozyme markedly reduces the activity of the FWC activity but has no apparent effect on the methanolextracted toxin. The activity of the B. laterosporus toxin is enhanced by lysozyme. The B. alvei and the B. brevis toxins are heat stable for 60 min at 100°C, which is probably in keeping with their small size, whereas heating increases the lethality of the B. laterosporus toxin (Bone and Singer, 1991). C. PRODUCTS

The following is a consideration of the utility of the morphological group I1 strains of Bacillus in terms of useful products and in terms of bacterial biology. 1. Useful Products

The activity of B. alvei, B. laterosporus, and B. circulans strains against the soybean cyst nematode, Heterodera, appears to be of the most immediate short-range usefulness. Next follows the same group of strains plus the B. brevis strains against both the zebra mussel D.polymorpha and the schistosomiasis vector, B. glabrata. A study testing the efficacy, particularly of the B. alvei strains, against zooparasitic nematodes should prove most enlightening. Despite the miracles of the chemical ivermectin against onchocerciasis (river blindness), one should reinforce the (single agent) biological control arsenal of B. thuringiensis by the addition of one or more of the B. alvei cultures. The potential utility of these strains against the mosquito larvae, or against the stored grain complex Lepidoptera or Coleoptera, merits further investigation. Targets were chosen ad lib or as opportunities presented themselves. Therefore, it is suggested that there may be many other economically

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SAMUEL SINGER

choice targets out there, particularly in the area of the aquatics. We hear of the arsenal of medicines to be found in marine habitats. In a parallel vein, freshwater habitats should not be neglected. If this century was the century of soil microbiology, the next may well be that of aquatics, whether marine or freshwater. 2. Bacterial Biology

In light of all of the above, efficacy in terms of a smaller size target or at least a more sensitive part of the life cycle should be seen. Also, the most sensitive stages need to be used when testing against nontarget organisms. It took the author a while before understanding the obvious, that not all Bacillus toxins are obtained the same way. Not all Bacillus toxins are crystals or are made during or part of sporulation. As mentioned in a previous section, there is a question as to how many toxins have been missed because of an insufficient understanding of the biology of the fermentation process. As researchers become more rigid and more directed, they are apt to make more of these errors of “innocence.” Researchers also spend so much time with the engineering part of development that they sometimes ignore the biology of the bacterial (or microbial] candidate. In Figs. 1-4, “species” containing clonal groups that represent emerging species are shown. Identification of the bacterial culture is important for patent purposes. It remains to be determined what molecular biology and genetic engineering can do. Now that the author as the “pathfinder,” have shown the potential biological activity of this orphan group, the molecular biologists need to examine the possibility of inserting these new toxin genes into other “winners” such as Bt, B. sphaericus, or vice versa. VI. Past Needs and Future Needs

In the past, before Rachel Carson’s “Silent Spring,” the biological (bacterial) control of pests, the use of biorational agents, was only an emerging concept in the minds of some. Along came the crystaliferous Bt’s and B. sphaericus. Several decades later, another group of potential agents is on the stage (fretfully so) that is not crytaliferous but still offers the qualities of being biorational agents. The morphological group I1 strains of Bacillus and their toxin(s) are not “crystalliferous” [despite the fact that B. laterosporus has a crystal (Favret and Yousten, 198511 but represent a whole new class. In this last section, the questions, what is biological control, is Bt enough, and where to now are discussed.

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A. WHATIS BIOLOGICAL CONTROL?

Which biological control are we talking about? There are at least three varieties that come under consideration, and the three are not necessarily mutually exclusive. These are 1. The use of parasitoids and their relatives (for want of a better description), favored by the agriculturists. 2. The use of fermentation products (e.g., Bt, B. sphaericus) favored by many in industry. 3. The use of the molecular biological refashioned agent or host (plant/animal) or vector, favored by the rest of industry and by the avant-garde group of the WHO’STDR.

What is happening in these three areas? The first and last are doing well, the middle one is in retreat. Like most things philosophical or political, anecdotal events sometimes better illustrate the situation than do “facts.” The use of parasitoids (e.g., wasps) as biological agents to control pests is almost ancient. Its success has probably been in the smaller customized areas, and not broadly like the successful application of Bt. Its proponents have been successful politically, and in keeping its practioners funded (particulary in the USDA). Bacillus thuringiensis (and to a lesser degree B. sphaericus) have become mature commodities. As such, they deserve the best development, which today means the full genetic and molecular biological gamut. This is fine, but even here there are limits. These older cows can be milked just so far. Why depend just on Bt? Although B. sphaericus was on the scene before the isolation of Bti, “corporate strategy” devined that Bti be developed first. Eventually, B. sphaericus development (in the United States) reached the point of having EPA approval for trials; then stasis set in. Meanwhile, in Europe it was found that B. sphaericus was very useful along the Rhine to get rid of wetland mosquitoes so that one could raise the value of the accompanying real estate. What started as a program to assist the emerging third world nations found success in first world economics. Actually, this is not bad if it leads to the availability of a less expensive product that emerging nations could afford. Once available, it too (B. sphaericus material) should get mature commodity treatment. The question though is what comes next, because with B. sphaericus the potential product reserve has essentially been exhausted. In the author’s opinion, fungi, viruses, nematodes, protozoa, etc., cannot do it like endospore-forming bacteria can.

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As mentioned previously the use of fermentation products, such as Bt, is in retreat. As indicated in the previous paragraph, after B. sphaericus the pot is empty. There has been a calamitous reduction by both industry and the federal funding agencies in support of the search for these new agents. Among the many, two reasons are most plausible. The first is that there no longer are vocal partisans the likes of Angus, Briggs, Dulmage, Heimpel, (even deBarjac) making waves. Equally important, we are not training academic disciples of the above partisans to go forth and explore for these new agents. Industry will not do it. Apparently, neither will the federal funding agencies. In some cases, it is defeat by composition of panels. In other cases, it is a new generation that is not paying attention to the haranguing arguments of the old vocal partisans. As Tom Angus might say, “They have had their day, eh!?” B. Is BT ENOUGH?

A. Arata and J. Hamon (WHO) might say, “the bottom line is how many fewer cases of a target tropical disease do we have as the result of application of your agent or technique.” Are we starting to see successful genetically altered (by recombinant DNA technology) Bt products? There has been increased understanding, but have there been new products with great impact? It may be too soon to judge. Most things must eventually bend to the law of diminishing returns, even an industrialist’s dream of an endless stream of altered Bts. Is Bt enough? Possibly, for the short term. C. WHERE TO Now?

Product-wise, the strains and species mentioned in this chapter would appear to offer great utility against a series of different economic targets. One must consider not only pests of agriculture and vectors of human diseases, but also the pests circumscribed by the terms “nonindigenous” species, such as the zebra mussel. Where to look for these new agents? In the aquatic as well as in the soil habitats. Scientifically (bacterial biology), we need to answer the questions: What is the mode of action (histopathology) of the toxin against its invertebrate target? What is the toxin’s normal function within the bacterial cell? What is the composition of the toxin-protein or protein-complex?

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Because the latest taxonomic research (Logan 1993) places some of these endospore-forming bacilli with nonspore formers, do these other gram-positive or even gram-negative bacteria exhibit similar biological activity? The author will be tackling some of these questions for a while; perhaps he will see you along the way! ACKNOWLEDGMENTS The author thanks 40+ M.S. graduate students past and present, whose determination made progress possible with the morphological group I1 and group 111studies during these past 25 years. I also thank the Western Illinois University Research Council for local funding and USEPA for national funding, particularly during these past 4 years of our molluscicidal efforts, with particular thanks to Drs. Fred Genthner and David Yount (of EPA) for their support and encouragement.

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Ferrari, E., Jarnagin, A. S., and Schmidt, B. F. (1993). In "Bacillus subtilis and Other GramPositive Bacteria" (A. L. Sonenshein, J. A. Hoch, and P. Losick, eds.). American Society for Microbiology, Washington, DC. Fredeen, F. J. H. (1973). "Black Flies," p. 19. Canada Department of Agriculture, Ottowa, Canada.. Goodfellow, M., and Minnikin, D. E. (1985). In "Chemical Methods in Bacterial Systematics" (M. Goodfellow and D. E. Mininikin, eds.), pp 1-15. Academic Press, London. Gordon, R. E., Haynes, W. C., and Hov-Nay Pang, C. (1973). "The Genus Bacillus." Agricultural Handbook No. 427. USDA, Washington, DC. Gower, J. C., and Ross, G. J. S. (1969). Appl. Stat 18, 54-64. Griffith, R. W., Schloesser, D. W., Leach, J. H., and Kovolak, W. P. (1991). Can. J. Fish Aquat. Sci. 48, 1381-1388. Jackson, R. H. and Long, M. E. (1965). Biochim. Biophy. Acta 100,418-425. Jaska, J, M. (1977). Unpublished Master's Thesis, Western Illinois University, Macomb. Knight, B. C. J. G., and Proom, H. (1950). J. Gen. Microbiol. 4, 508-538. Krieg, R. E., and Lockhart, W. R. (1966). J. Bacteriol. 92, 1275-1280. Lambert, B., and Perferen, M. (1992). BioScience 42,112-122. Lessel, E.F., and Holt, J. G. (1970). In "Methods for Numerical Taxonomy" (W. R. Lockhart and J. Liston, eds.), pp. 50-58. American Society for Microbiology, Maryland. Liu, B. Y.-M. (1972). Unpublished Master's Thesis, Western Illinois University, Macomb. Logan, N.A. (1994). "Bacterial Systematics," pp. 164-197. Blackwell, London. Logan, N. A., and Berkeley, R. C. W. (1984).J. Gen. hlicrobiol. 130,1871-1882. Madjid, A., and Singer, S. (1992). Annu. Meeting Am. Soc. Microbiol. gznd, p. 290. Anaheim, CA. Margalith, P. (1964). Adv. Appl. Microbiol. 6, 69-90. Matheny, M. L. (1992). Unpublished Master's Thesis, Western Illinois University, Macomb. McCullogh, P. F. S., and Mott, K. E. (1983). Document WHO/SHCIST0/83.72. Molloy, D. P., De Lucca, A., and Griffin, B. (1994). VIth Int. Colloq. Invertebr. Pathol. Microbial Control, Montpellier, France.[Abstract CP4 No. 6, pp. 26-27] Priest, F. G. (1993). In "Bacillus subtilis and Other Gram-Positive Bacteria" (A.L. Sonenshsein, J. A. Hoch, and P Losick, eds.), pp. 3-16. American Society for Microbiology, Washington, DC. Proom, H. and Knight, B. C. J. G. (1955). J. Gen. Microbiol. 13, 474-480. Ralston, D. G. (1979). Unpublished Master's Thesis, Western Illinois University, Macomb. Saari, R. (1989). Unpublished Master's Thesis, Western Illinois University, Macomb. Schleifer, K. H., and Stackebrandt, E. (1983). Annu. Rev. Microbiol. 37, 143-187. Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour, M. N., and Whittam, T. S. (1986). Appl. Environ. Microbiol. 51, 873-884. Singer, S. (1973). Nature 244, 110-111. Singer, S. (1974). Dev. Ind. Microbiol., 187-194. Singer, S. (1979). Dev. Ind. Microbiol. 20, 117-122. Singer, S. (1981). In "Microbial Control of Pests and Plant Diseases 1970-1980" (H. D. Burges, ed.), pp. 283-298. Academic Press, London. Singer, S. (1987). In "Biotechnology Advances i n Insect Pathology and Cell Culture" (K. Maramorosh, ed.), pp. 133-163. Academic Press, New York. Singer, S. (1988). Adv. Appl. Microbiol. 33, 47-74.

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Singer, S . (1990). In "Bacterial Control of Mosquitoes and Black Flies: Biochemistry, Genetics, and Applications of Bacillus sphaericus" (H. de Barjac and D. Southerland, eds.), pp. 221-227. Rutgers Univ. Press, New Brunswick, NJ. Singer, S. and Bell, K. D. (1987). Annu. Meeting Am. Soc. Microbiol., 87th, p. 183, Anaheim, CA. Singer, S., and Saari, R. (1990).Annu. MeetingAm. Soc. Microbiol. SOth, p. 247, Anaheim, CA. Singer, S., Doherty, K. A., and Stambaugh, A. D. (1988). Annu. Meeting Am. SOC. Microbiol., 88th, p. 76. Anaheim, CA. Singer, S., Bair, T. B., Hammill, T. B., Berte, A. M., Correa-Ochoa, M. M., and Stambaugh, A. D. (1994a). J. Ind. Microbiol. 13, 112-119. Singer, S., Cadwallader, H., Owens, A., and Rives, J. (1994b). VIth Int. Colloq. Invertebr. Pathol. Microbial Control, p. 281, Montpellier, France. Singer, S , Genovese, E. E., and Flaharty, T. (1994~).VIth Int. Colloq. Invertebr. Pathol. Microbiol Control, Montpellier, France. [Abstract CP4 No. 7, p. 281 Smith, N. R., Gordon, R. E., and Clark, F. E. (1952). "Aerobic Spore-Forming Bacteria." Agriculture Monograph No. 16., U.S. Department of Agriculture. Sneath, P. H. A., and Sokal, R. D. (1973). "The Principles and Practice of Numerical Classification." Freeman, San Francisco. Stoeckel, J. A., and Garton, D. W. (1993). Proc. Third Int. Zebra Mussel Conf.,Section 2 , 53-57. Tarshis, I. B. (1968). Ann. Entomol. Soc. Am. 61,1072-1083. Tarshis, I. B. and Adkins, T. R., Jr. (1971). Ann. Entomol. Soc. Am. 64,1194-1195. Williams, R. A. D., and Shah, H. N. (1980). In "Microbiological Classificatin and Identification (M. Goodfelow and R. G. Board, eds.), pp 299-318. Academic Press, London . World Health Organization (WHO) (1984). Document TDR/BCV-SCH/SIH/84.3, WHO, Geneva. Zuber, P., Nakano, M. M., and Marahiel, M. A. (1993). In "Bacillus subtilis and Other Gram-Positive Bacteria" (A. L. Sonenshein, J. A. Hoch, and P. Losick, eds.). American Society for Microbiology, Washington, DC.

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Phytase RUDYJ. WODZINSKI~ AND A. H. J. ULLAH+ *Department of Molecular Biology and Microbiology University of Central Florida Orlando, Florida 32826 tSouthern Regional Research Center Agricultural Research Service United States Department of Agriculture New Orleans, Louisiana 702 79 I. Introduction A. Thirty+ Years of Basic and Applied Scientific Research Necessary to Generate a Product 11. Importance of Phytic Acid and Hydrolysis Products A. Role in the Plant B. Role in Nutrition 111. Sources of Phytase A. Plant Sources B. Bacterial Sources C. Fungal Sources IV. Regulation of Phytase Synthesis A. Effect of Source of Cornstarch and Phosphorous Concentration B. Effect of Medium Ingredients, Inoculum Size on Pellet Formation, and Phytase Yields C. Mutation Studies V. Biochemistry of Phytase and Acid Phosphatases A. Purification and Characterization B. Sequence Studies C. Active Site Determinations D. Enzyme Engineering Studies E. Cloning of Phytase and Acid Phosphatases F. Immobilization Studies with Phytase and Acid Phosphatase VI. Feed Studies with Phytase A. Early Studies with Poultry B. Recent Studies on Phytase as a Feed Additive VII. Economics and Potential Effect of Phytase on Pollution Abatement VIII. Future studies IX. Summary References

I. Introduction

A. THIRTY+YEARSOF BASICAND APPLIEDSCIENTIFIC RESEARCH NECESSARY TO GENERATE A PRODUCT 1. Overview of Product Development

The research on phytase (Fig. 1)spans 87 years from its discovery by , until its commercialization in Europe in 1993-1994 Suzuki et ~ l .(1907) by Gist-brocades. Commercialization required not only a practical use and delivery of the enzyme but also the ability to produce the enzyme 263 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 42 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

264

HZW,

RUDY J. WODZINSKI AND A. H. J. ULLAH

< ?;: H

H

Phytase

(-> on

H

+ 6H3PO4

H -2

myo-inositol 1,2,3,4,5,6-herakis dihydrogeo phosphate (Phytic acid) structure suggested by Anderson 1914. C6HlsO24P6

-

HS

4

H

Inositol

MW=659.86

FIG.1. Hydrolysis of phytic acid to inositol and phosphoric acid by phytase.

economically. The milestones are shown in Table I. Commercialization was not possible until methods were available to develop and produce high yields of the enzyme in microbial culture or in plants. Research to produce high yields of the enzyme was initiated whenever new techniques were discovered that had potential for progress. After the “new techniques” were applied to phytase production and it was determined that further progress was unlikely, research was suspended until newer techniques that appeared promising were developed. The story of commercialization of phytase is illustrative of the development of many products that are produced by microorganisms. They require fundamental discovery of new principles as well as applied long-term research. It is possible to use the phytase “story” to identify the principles that are germane for the commercial development of microbial products. 2 . Early Research

The International Union of Biochemistry (1979) lists two phytases: a 3-phytase, EC 3.1.3.8, which hydrolyzes the ester bond at the 3 position of myoinositolhexakis phosphate to D-myoinositol 1,2,4,5,6-pentakisphosphate + orthophosphate and a 6-phytase, EC 3.1.3.26, which first hydrolyzes the 6-position of myo-inositolhexakis phosphate to Dmyoinositol1,2,3,4,5-pentakisphosphate +- orthophosphate. Subsequent ester bonds in the substrate are hydrolyzed at different rates. The 6-phytase dephosphorylates phytic acid completely. However, the 3-phytase of at least one of the phytases described does not hydrolyze the phosphomono ester. Posternak (1903) described phytic acid for the first time. Suzuki et al. (1907) were the first to describe the enzymatic activity of rice bran phytase and to prepare an extract that retained its activity. Dox and Golden (1911) demonstrated that aspergilli produce phytase. Early studies to identify organic phosphorus compounds in plant material were

PHYTASE

265

TABLE I IN THE COMMERCIALIZATION OF PHYTASE MILESTONES

1903

Posternak-Describes phytic acid.

1907

Suzuki et a1.-Describe

and extract rice bran phytase.

1911

Dox and Golden-Demonstrate phytase in aspergilli.

1913

Plimmer and Anderson-Identify plant material.

1914

Anderson-Determines

organic phosphorous compounds in

the structure of phytic acid.

1959

Casida-Lists 20 soil fungi that have phytase activity.

19621971

International Minerals and Chemicals initiates first commercial attempt to develop phytase as a product.

1967

Ware and Shieh-Patent

1968

Shieh and Ware-Screen over 2000 isolates for phytase activity. Isolate Aspergillus niger NRRL 3135 syn A . fi’cuum produces phy A and phy B at the highest yield ever reported in a nongenetically modified strain. Nelson et a1.-Feed phytase-treated soybean meal and document that hydrolyzed phytin is assimilated efficiently by broilers.

1968

acid phytase.

1971

Nelson et a1.-Direct feeding of supplemental A . niger NRRL 3135 phytase to broilers in experimental and practical diets is titered.

1969

Shieh et a1.-Partial purification characterization and regulation of A. niger NRRL 3135 phytase.

1984

Southern Regional Research Center Agricultural Research Service, United States Department of Agriculture begin basic studies on phytase.

1988

Ullah-Purified, characterized, and determined the partial amino acid sequence of A. niger NRRL 3135 phyA.

1993

Ehrlich et al.-Cloned and sequenced the gene for A. niger NRRL 3135 phyB.

1994

Ullah et a].-Cloned and sequenced the gene for A . niger NRRL 3135 metallo pH 6.0 acid phosphatase.

1987

Alko Ltd. (Finland)(PanLabs) initiates project to commercialize phytase.

1993

Piddington et al.-Cloned and sequenced phytase from A. niger var. awarmori

1987

Gist-brocades (The Netherlands)initiates project to commercialize phytase. of efficacy of phytase in broilers and pigs.

1990

Simons et a].-Demonstration

1991

Van Gorcom et a1.-Application for patent on the overproduction of phyA by cloned strains of A. niger NRRL 3135 and A. niger CBS 513.88 that has a glucoamylase promoter and in which synthesis is not controlled by levels of P. Extracellular yield of phyA increased by 50-fold.

1992

Ecological benefits of the use of phytase to abate phosphorous excretion by monogastric animals.

1993

Pen et al.-Expression

1994

Beudeker and Pen-Expression (Brassica napus).

of fungal phytase phyA in tobacco.

of fungal phytase phyA in canola

266

RUDY J. WODZINSKI AND A. H. J. ULLAH

made by Plimmer (1913)and Anderson (1914a,b,c,d).They recognized the importance of phytin in corn soybean diets. Many early attempts to determine the structures of phytin were made. Eventually, the Anderson (1914d) structure, which is considered correct at this time, was developed (Fig. 1).Reddy et al. (1982) reviewed the structure of phytin and evaluated the data for each of the proposed structures. It was recognized early that phytin was an effective chelating agent that bound metals and proteins in animal feedstuffs. As such, it is considered an antinutrient for monogastric animals. It binds calcium, iron, zinc, and protein. Most of the phosphorus of the phytic acid is not available to monogastric animals. A controversy as to whether phytin was hydrolyzed at significant rates in the intestines of rats, poultry, pigs, and man continued for many years. Many attempts were made to determine whether animals produced phytase in the intestine. Attempts were also made to feed live yeast that produced phytase to animals to aid in the hydrolysis of phytase in viva Results were unequivocal in the early research. 3. Research at International Minerals and Chemicals (IMC) in the 1960s

The first concentrated effort to make phytase a commercial product started in 1962 at International Minerals and Chemicals in Skokie, Illinois in which approximately 1 2 man years were expended on the project. The group in Microbial Biochemistry initiated the project to screen for microorganisms that produce phytase. Tsuong Rung Shieh screened over 2000 organisms for phytase activity. He isolated an organism from the soil in a flowerpot that produced the highest yields of phytase. Although many attempts have been made, no one has discovered a naturally occurring organism that produces more phytase in liquid culture. This organism was originally identified on the basis of its poor conidiagenesis, no known sexual cycle, and ability to produce oxalic acid rapidly as Aspergillus ficuum by Kenneth Raper at the University of Wisconsin. It was deposited as NRRL 3135. Aspergillus ficuum was previously placed in the taxonomic group Aspergillus niger. Centraalbureau voor Schimmelcultures designated NRRL 3135 as A. niger van Tieghem. Nuclear DNA reassociation studies suggest that A. ficuum is not a valid species and the A. niger designation should be conserved (Frisvad et al., 1990; Peterson, 1992). There are many references on phytase that refer to this organism. In this review, if we can identify with surety that this particular strain was used by the author, we will always refer to it as A. niger NRRL 3135 regardless of what designation the author used. Shieh et al. (1969) continued to develop the strain by optimizing the media and the conditions for production of the enzyme. The strain was

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267

of special interest because one of the phytases (phyA) that it produced had two pH optima: one at pH 2.5 and one at pH 5.5. It also produced a second acid phosphatase with an optimum pH of 2.0 (phyB) that had activity on phytase. The intent of the project was to replace the feeding of inorganic phosphate by feeding phytase to monogastric animals. In 1962, the value of phytin-phosphorus (phytin-P) that was excreted by poultry was approximately $50 million. It was necessary to test the feasibility of feeding phytase to broiler chicks to determine whether the enzyme would retain its activity when passed through the crop and intestine of the chicken. The pH optima of the enzyme approximated the pH of the chick digestive track and promised that the enzyme would be active in the crop of the chicken and at least partially in the intestine. Nelson et al., (1968a) also tested the organism for its ability to grow in both moistened soybean meal and cotton seed meal. Feed studies were performed in chicks with meal that was pretreated with phytase. It was established (Nelson et al., 1968a) that the phytin-P present in soybean meal was available to the chick and deposited in their bones if phytase was used to hydrolyze it. The microbial biochemistry group at IMC scaled the process to the 114 L stage. Enough enzyme was produced and it was tested for use in monogastric animals by the animal nutrition group. The enzyme was fed to chicks and Nelson et al., (1971) established that when phytase was fed to chicks, the phytin-P present in soybean meal and corn was made available and it was deposited in bones of the chick. Unfortunately, the yields of phytase at this time were not high enough to produce a product that would be competitive with the feeding of inorganic phosphorus. The yield would have to be increased, by one estimate, about 250-fold just to break even. It was well known, at that time, that the phosphorus that was excreted by the chicks in the chicken manure would be mineralized by soil organisms and that it presented a pollution problem. Several attempts (media and applied genetics studies) were made to increase the yields of phytase to make it economic. However, they were unsuccessful and the project was terminated by IMC in 1968. 4. Research at the United States Department of Agriculture (USDA),

Agricultural Research Service (ARS), Southern Regional Research Center (SRRC) in the 1980s and 1990s In the 1980s, methodology became available that permitted the cloning of genes into microorganisms. Of special significance was the methodology that permitted the use of efficient promoters in gene constructs that increased yields significantly. This was recognized and in 1984 the technology that was developed at IMC was transferred to the

268

RUDY J. WODZINSKI AND A. H. J. ULLAH

SRRC, ARS, and USDA. A research team, that expended approximately 16 man years, was formed at SRRC that isolated, characterized, and sequenced the phytases (phyA and phyB) and acid phosphatase produced by A. niger NRRL 3135. Their fundamental research provided the technical basis for the cloning studies and gene sequencing that followed at Gist-brocades, Pan Labs (Alko), and USDA. 5. Research in Finland and The Netherlands

At approximately the same time, Gist-brocades in the Netherlands initiated a project to abate pollution while trying to improve the yields of A. niger NRRL 3135. They expended approximately 40 man years. They cloned phyA along with an amyloglucosidase promoter into A. niger NRRL 3135. They increased the yield approximately 52-fold. They also cloned the enzyme along with a amyloglucosidase promoter and the A. niger NRRL 3135 leader sequence into A. niger CBS 513.88. They increased the yield of phytase by 1400 in one of the wild-type nonproducers (VanGorcom et a]., 1991). In one of the trials listed, one of the cloned strains produced 270 pM/ml/min when the enzyme is assayed at 37°C. If one adjusts the reaction rate by multiplying by 3.7 to 58°C (Ullah, 1987),recalculates the unit to nKat, and divides by the specific activity of the pure enzyme (2100 nKat/mg protein), then approximately 7.9 g/liter is secreted into the medium by this bioengineered strain. The nutrition group at Gist-brocades tested the enzyme extensively in poultry and swine. They have received approval in Austria, Belgium, Brazil, Bulgaria, Canada, Denmark, Germany, Norway, Finland, France, Greece, Ireland, Italy, Korea, Luxembourg, The Netherlands, Portugal, Spain, Switzerland, Taiwan, and the United Kingdom for use of the enzyme (Natuphos) as a feed additive. The FDA has approved a GRAS petition for use of phytase in food. Phytase is being marketed as a food additive in the United States as of January 1996. Also, at approximately the same time, Alko in Finland began researching phytase in aspergilli. Their intended main use is in baking processes and animal feeds. They enlisted the group at Pan Labs (Bothell, WA) to increase the yield of the phyB enzyme by cloning procedures. It is the purpose of this review not only to detail the events that were necessary to make phytase an economic reality but also to indicate that it is a combination of basic and applied research over long period of times that is often required to bring projects to fruition. One can extrapolate the cost of performing this type of research by adding up the man years expended in research and multiplying a current cost factor per man year. The cost of discovering the fundamental required knowledge, developing a process for production of the en-

PHYTASE

269

zyme, and demonstrating the efficacy (applications) and safety required not only the 68 man years documented previously but also the man years expended in various government, academic, and industrial research laboratories inherent in the research papers listed in the references that have direct application to solving the problem. It is noteworthy that it was the cooperation of industrial, government, and academic researchers that was necessary for capitalization of the results. It is the synergistic interactions of these, along with the generation of new methodology, that assures the introduction of new products in molecular biology and microbiology. 11. Importance of Phytic Acid and Hydrolysis Products

A. ROLEIN THE PLANT Phytic acid (myoinositol1,2,3,4,5,6-hexakis dihydrogen phosphate) (Fig. 1)is a major component of plant-derived food. It is the primary source of inositol and storage form of phosphorus in plant seeds that are used as animal feed ingredients (oilseed meals, cereal grains, and legumes) (Maga, 1982). Approximately 75% of the total phosphorus in cereals (Common, 1940), legumes (Nelson et al., 1968b), and seeds (de Boland et al., 1975; Erdman, 1979) exists as phytic acid phosphorus. Cottonseed meal, wheat, corn, rice, oats, soybean meal, and other plant-derived feedstuffs contain various amounts of phytate (Anderson, 1914a, b, c, d; Common, 1940; Erdman, 1979; Nelson et al., 196813; Rackis, 1974). Reddy et al., (1982) reviewed the topic exhaustively. Early speculation on the role of phytin-P in the plant centered on its use as a storage product. It was believed that large amounts of phosphorus were stored in the seed and that phosphorus was liberated on germination and incorporated into ATP. Recent studies have established the role of inositol phosphate intermediates in the transport of materials into the cell. Their role, especially that of inositol triphosphates, in transport as secondary messengers and in signal transduction in plant and animal cells is a very active area of research (Berridge, 1989, 1993; Berridge and Irvine, 1989; Morre et al., (1990); Boitano et al., 1992; Allbritton et al., 1992; Iino and Endo, 1992). There are many applications of phytic acid. Graf (1986) reviewed and listed 59 different applications including rust removal, prevention of dental caries, and its use as a hypocholesteromic agent. All of the applications center on the properties of phytic acid to chelate metals, interact electrostatically with proteins, its high affinity for hydroxyapatite, and interact with miscellaneous targets (Graf, 1986).

270

RUDY J. WODZINSKI AND A. H. J. ULLAH

The applications of phytic acid and especially the inositol intermediates impinge on eventual applications of phytase. “Of broad nutritional and medical significance will be the preparation of myo-inositol, di, tri, tetra and pentaphosphate esters and their investigation of their interactions with different polyvalent cations” (Graf, 1986). Phytic acid or inositol intermediates have been implicated in starch digestibility and blood glucose response (Thompson, 1986),as an antioxidant (Graf et al., 1987), in the lowering of cholesterol and triglycerides (Jariwalla et al., 1990),in tumor formation (Shamsuddin et al., 1988,1989; Shamsuddin and Ullah, 1989; Ullah and Shamsuddin, 1990), in the treatment of Alzheimers disease (Sabin, 1988, 1989),in the treatment of Parkinson’s disease (Sabin, 1992),and in the treatment of multiple sclerosis (Sabin, 1993).The preparation of these intermediates by immobilized phytase is provided under Section V, F

B. ROLEIN NUTRITION I. Phosphorous

Ruminant animals sustain the microflora that enzymatically release inorganic phosphorus from phytic acid. However, phytic acid-phosphorus is essentially unavailable to monogastric animals: humans, chickens, and pigs (Nelson et al., 1968a, b). They produce little or no phytase in the intestine and the phytin-P is excreted. An external source of phosphorus must be supplied in sufficient quantity to meet their daily mineral requirements (Nelson, 1967; Nelson et al., 1968b). Phytic acid that is present in the manure of these animals is enzymatically hydrolyzed by soil- and waterborne microorganisms. The released phosphorus is transported into rivers and lakes and can cause eutrophication in aquatic environments in which phosphorus is limiting. When it is introduced in high quantities, excessive algal growth and oxygen depletion ensue. Phytic acid chelates divalent and trivalent cations. It binds Ca2+,Zn2+8 Mg2+ and Fe3+and trace minerals Mn2+’Cu2+,and Mo+(Erdman, 1979; Maga, 1982; Nelson et al., 1967, 1968b, Rackis 1974). It also binds proteins. Complexes may be formed with all of these constituents of the monogastric diet that renders them unavailable to the animal. Because all of these are required nutrients for monogastrics it influences the concentration that must be fed to the animals. Many have described phytinP as an antinutrient factor. The antinutritive properties and its value as a possible phosphorus source, have stimulated researchers to develop

PHYTASE

271

a method to remove phytic acid in a manner that is economically competitive with mineral supplementation. One method for phytic acid removal is enzymatic treatment with phytase. Phytate phosphorus is decreased 81% in a mixture of ground maize, soybean meal, and wheat bran when treated with the native plant phytases (Zhu et al., 1990). The tibia bone ash weights of the chicks fed the treated feed was higher than those of the control chicks (Zhu et al., 1990). Although some feed ingredients contain native phytase, it is not a consistent source of the enzyme. The pH and temperature during feed processing may inactivate the enzyme (Maga, 1982; Nelson, 1967). The feeding of a phytase produced by a microorganism may ameliorate these problems. Ill. Sources of Phytase

A. PLANT SOURCES

Suzuki et al., (1907) were the first investigators to make a preparation of phytase activity. They detected activity in rice and wheat bran. They also isolated inositol as a product of the reaction. The occurrence of phytase in germinating plants has been exhaustively reviewed by Reddy et al. (1982) and Gibson and Ullah (1990). They note that phytase has been isolated and/or characterized in cereals, such as triticale, wheat, corn, barley, and rice, and beans such as navy beans, mung beans, dwarf beans, and California small white beans. Eeckhout and De Paepe (1994) measured phytase activity, phytin-P, and total P in 39 different samples of cereals, cereal by-products, oil meals, legume seeds, roots and tubers, and other plant byproducts. Only rye (85 nKat/g), triticale (28 nKat/g), wheat (20 nKat/g), and barley (10 nKat/g) were “rich” in phytase. Wheat byproducts, fine bran meal (76 nKat/g), or pellets (43 nKat/g), middlings (73 nKat/g), feed flour (56 nKat/g), and bran (49 nKat/g), also contained phytase. There was no correlation between the phytin-P content or total P with the phytase levels present in the plant-derived material. If one calculates the amount of phytase necessary to hydrolyze all of the phytin-P in a commercial diet and compares it with the amount of naturally occurring phytase in plants, it is evident that the commodity would have to be 40% of the diet to hydrolyze all of the phytin-P. This is not practical. Cloning of the A. niger phyA gene into plants that are used in practical diets along with efficient promoters circumvent the problem (see Section V,E,2).

272

RUDY J. WODZINSKI AND A. H. J. ULLAH

Houde et al., (1990) purified phytase from canola seed. However, the purified “phytase” from canola had 232 times more activity with pyrophosphate than it had with sodium phytate. The pH optimum of the plant enzymes range from 4.0 to 7.5. Most of the enzymes have a pH optimum between 5.0 and 5.6. They have an optimum temperature of 45 to 60°C and a Km of 2.22 x 10-4to 0.99 x lO-3mM. The seeds of higher plants generally contain 6-phytase. Hayakawa et al., (1990) presented data that rice bran F2 enzyme is able to dephosphorylate at the myoinositol2 position. The levels of phytase in plants increase by several orders of magnitude during germination. B. BACTERIAL SOURCES

Phytase has been detected in Aerobacter aerogenes (Enterobacter aerogenes) (Greaves et al., 1967), Bacillus subtilis (Powar and Jagannathan, 1982), Bacillus subtilis (natto) N-77 (Shimizu, 1992), Escherichia coli (Greiner et al., 1993), Klebsiella aerogenes (Tambe et al., 1994), and Pseudomonas sp. (Irving and Cosgrove 1971) (Tables 11,111,IV).The only bacterial organism that produces extracellular phytase is B. subtilis. When phytase is produced by bacteria, the yields are low and the pH optimum is neutral to alkaline that precludes their use as feed additives. C. FUNGAL SOURCES

A number of surveys of microorganisms have been made for phytase production. The organisms that have been reported to produce extracellular phytase are shown in Table 11. Shieh and Ware (1968) screened 2000 cultures from 68 soil samples and enrichments for phytase production. Howson and Davis (1983) surveyed 84 fungi from 25 species for phytase production. The incidence of phytase production is highest in aspergilli. The yields of phytase produced extracellularly are shown in Table 111. Of all the organisms (plants, bacteria, and fungi) surveyed, A. niger NRRL 3135 produces the most active extracellular phytase in cornstarch (Shieh and Ware, 1968) and semisynthetic (Howson and Davis, 1983) media. It produces 110 nKat P/ml. Aspergillus niger NRRL 3135 produces two different phytases, one with pH optima at 5.5 and 2.5 and one with a pH optimum of 2.0. Later, these enzymes were designated phyA and phyB, respectively. A variety of organisms produce phytase intracellularly (Table IV). A comparison of the properties of phytases that have been highly puri-

2 73

PHYTASE TABLE I1 PRODUCTION OF EXTRACELLULAR PHYTASE BY MICROORGANISMS ~~

Organism Fungi Aspergillus spp. *3 Aspergillus amstelodami A . chevalieri A . candidus A . niger syn A . ficuum A. Pavus A . flavus A . niger A . niger A. niger A. repens A. sydowi A . terreus A . terreus A. vesicolor A . vesicolor A. wentii Botrytis cinerea Geotrichum candidum Mucor spp. Mucor Priformis M. Racemosus Penicillium spp. Rhizopus oryzae R. oligosporus R. stolonifer Saccharomyces cerevisiae Schwanniomyces occidentalis syn. S. castelli

Bacteria Bacillus subtilis B. subtilis (natto) N-77

No. of cultures tested 3

No producing acid phytase

1 10 6 1 7 22 4 3 1 2 5 3 1 5 1 37 4 5 58 5 1 4 8 1

3 3 1 3 1 2 4 1 3 21 3 3 1 1 2 1 1 2 1 1 2 4 1 2 1 2 6 1

1

1

1

1

5

2 5

Reference Shieh and Ware (1968) Howson and Davis (1983) Howson and Davis (1983) Howson and Davis (1983) Howson and Davis (1983) Shieh and Ware (1968) Howson and Davis (1983) Skowronski (1978) Howson and Davis (1983) Shieh and Ware (1968) Howson and Davis (1983) Howson and Davis (1983) Yamada et al., (1968) Shieh and Ware (1968) Howson and Davis (1983) Shieh and Ware (1968) Howson and Davis (1983) Howson and Davis (1983) Howson and Davis (1983) Shieh and Ware (1968) Howson and Davis (1983) Howson and Davis (1983) Shieh and Ware (1968) Howson and Davis (1983) Howson and Davis (1983) Howson and Davis (1983) Howson and Davis (1983) Segueilha et al. (1992)

Powar and Jagannathan, (1982). Shimizu (1992).

fied is shown in Table V. The extracellular phytases have molecular weights ranging from 36 to 85 m a ; a Pi of 4.0-6.3, a Kcatof 5.5-6209 per second, a K, of 27-5000 micromol, an optimum pH of 2.5-7.5, and an optimum temperature of 35-63°C.

274

RUDY J. WODZINSKI AND A. H. J. ULLAH TABLE 111 YIELDS OF

EXTRACELLULAR PHYTASE PRODUCED IN CULTURE FILTRATES OR SUPERNATES ~~

Organism Fungi A. awamorii ATCC 11382 A. awamorii ATCC 11358 A. carbonarius NRRL 368 A. carbonarius PCC 1040 A. niger syn A.ficuum NRRL 3135 A. niger syn A. ficuum WB 320 A. niger syn A. ficuum WB 364 A. niger syn A. ficuum WB 4016 A. niger syn A. ficuum WB 4541 A. niger syn A. ficuum WB 4781 A . niger joponicus saito ATCC 1034 A. niger ATCC 9142 A. niger ATCC 10864 A. niger var. cinnamomeum NRRL 348 A. niger NRRL 326 A. niger NRRL 330 A. niger NRRL 337 A. niger NRRL 372 A. niger NRRL 4361 A. niger van Tieghem 1 A. niger K (soil isolate) A. niger x (soil isolate) A . satoi A. tubingensis NRRL 4875

nKat1 ml

pmP1 minlml

19.9 28.6 16 13

1.20 1.72 0.99 0.78 6.62 0.52 0.78 0.57 0.52 0.625 0.31 1.09 0.42 0.73 0.36 0.42 0.26 0.52 0.36 1.83 2.08 2.60 0.94 1.25

Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968) Shieh and Ware (1968)

110 8.6 13 9.46 8.6 10.4 5.1 18.1 7.0 12 6.0

7.0 4.3 8.6 6.0 13.8 34.5 43.2

Reference

Bacteria Bacillus subtilis

4.0

0.24

Powar and Jagannathan

Bacillus subtilis (natto)

1.8

0.11

Shimizu (1992)

(1982)

IV. Regulation of Phytase Synthesis

A. EFFECTOF SOURCE OF CORNSTARCH AND PHOSPHOROUS CONCENTRATION

Ware and Shieh (1967) related an invention in which the parameters for maximum acid phytase production by A. niger NRRL 3135 were listed. They discovered that the available inorganic phosphorus content of the medium controlled the synthesis of the enzyme. Maximum yields (113 nKat P/ml in shake flasks in 5 days) were obtained if the inorganic phosphorus content was controlled in the range of 0.0001% to about

2 75

PHYTASE TABLE IV PHYTASE DETECTED IN CELLS OR WHOLECULTURE

Aspergillus clavatus J239 A. pavipes Fla. A-14 A. flavus A. nidulans QM-329 A. niger NRRL 67 A. niger P-330 A . niger A . oryzae QM-228 A. phoenicus QM 329 A. repens QM-44c A. terreus Fla. C-93 A. tarnarii Jl008 A. Ustus QM-385 A. spp. 1* A. spp. 2* A. spp. 3* A. spp. 4* A. spp. 5 * Mucor spp. Penicilliurn spp. P-320 Rhizopus spp. Klebsiella aerogenes Pseudornonas spp.

Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Dox and Golden (1911) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959). Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Casida (1959) Tambe et al. (1994) Irving and Cosgrove (1971)

TABLE V PHYSICOCHEMICAL AM) KINETICPARAMETERS OF MICROBIAL PHYTASES AND ACIDPHOSPHATASES

Enzyme

Mr (ma)

A. ficuum phyAo A. ficuum phyBb A.ficuum,pH6.0APaseC E. coli phytase P2d E. coli, pH 2.5 APasee B. subtilisf

85 68 85 42 45 38

Km 4.5 4.0 4.9 6.0 6.3 6.3 ~~

~ U l l a hand Gibson (1987). bullah and Cummins (1987b). c u l l a h and Cummins (1988b). dGreiner et al. (1993). eDassa et al. (1980). fShimizu (1992).

Gat (PI

Pi

348 628 260 6209 2698 5.5 ~

27 103 200 130 5000 500

K,,,IK,

optimum Optimum temperaPH ture

1.29 x lo7 5.0; 2.5 6.1 X106 2.5 1.3 x 106 6.0 4 . 7 7 ~ 1 0 ~ 4.5 5.4 x1O5 2.5 1.1X lo4 6.0

58 63 63 35 37 60

2 76

RUDY J. WODZINSKI AND A . H. J. ULLAH

0.005% by weight determined as phosphorus (Fig. 2). Shieh and Ware (1968) demonstrated the interaction of total phosphorus content with the carbon source in the medium and with the type of cornstarch that was used as the carbon source to phytase yield. The optimal conditions for phytase production were 0.4 mg/100 ml phosphorus and 8% w/v cornstarch (Hubinger). They noted that repression of phytase synthesis at high levels of P was a general phenomenon because it was observed with all molds and yeasts that produced phytase. The relative amounts of the pH 2.0 (phyB) and pH 5.5 phytase (phyA) differed depending on the amounts of inorganic phosphorus in the medium (Shieh et al., 1969). The regulatory effect of high P on phytase synthesis was confirmed by Howson and Davis (1983). Han and Gallagher (1987) also confirmed that high P concentrations inhibited phytase synthesis by A. niger NRRL 3135. When they used high amylose cornstarch (Hylon V National Cornstarch and Chemical Corp. Bridge Water, NJ), 1-5 mg P/100 ml was needed for maximum phytase production (44 nKat/ml in 7 days), whereas 8 mg P/100 ml was required for maximum cell growth. They also noted that the source of phosphorus played a role in the concentration of P required for maximum phytase synthesis. If the source of P was sparingly soluble, higher initial concentrations of P would produce the same levels of phytase. At 5 mg P/100 ml in the medium, growth was suboptimal but phytase production was optimal. It should be noted that the relative yields in their study are consistent with those of other studies. However, the yields of enzyme are low under the culture conditions that they used-oxygen starvation (50 ml in 125-ml flask at 200 rpm) and prolonged incubation (7-1 7 days). Utt (1987) tested the effect of initial P concentration of eight different sources of commercial cornstarch on yields of phytase. Two treatment

7

P

*

v)

120

I

100

-

80

-

I

r

Cell Weight

60-

I.&1.2 9 1.1

g

40-

2

20-

0 0

5

10 15 20 25

PHOSPHORUS (ma%)

FIG.2. Effect of initial phosphorus concentration in cornstarch medium on growth and production of phytase by A . niger NRRL 3135. From Shieh and Ware (1968).

277

PHYTASE

methods were examined for their effectiveness in releasing P from cornstarch. Cornstarch preparations were autoclaved at 121°C for 15 min in basal fermentation medium and at 121OC for 24 hr in 2 NHC1. Significant differences between treatment methods occurred. Significantly higher levels of P were detected in the samples that were hydrolyzed for 24 hr as compared to those of the 15-min hydrolysis. Some commercial sources of cornstarch contain as much as 2.4 times as much P as other sources. One source had 8.25 p,g P /g of cornstarch. In a medium that contains 80 g/liter of cornstarch, the level is significant (660 p,g /liter) if one is attempting to control the level of P at 4 mg/liter. The levels of P in each of the samples were adjusted with P to 4 mg/liter and tested for phytase production. If total P content of the medium was the only factor in the cornstarch that affected yield, each of the conditions should have produced the same yield of phytase. Differences in the amount of phytase produced were as great as 5.2-fold depending on the specific cornstarch used. The source of cornstarch in which high yields of phytase yields (132 nKat/ml in shake flasks in 5 days) were produced consistently was Corn Products 3005 cornstarch (Fig. 3). Gibson (1987)also confirmed that synthesis of phytase was controlled by the levels of phosphorus in the medium. She compared the production of enzyme from different sources of starch and speculated that the phosphoester linkage in some starch sources may be more resistant to cleavage than others, resulting in a low but steady supply of phosphorus, conversely some phosphoester linkages might be hydrolyzed at a

150

,

ir 7

-

6

.: 120

E

Hc

5

90

4 pH

3

2

0

0

50

100 150 200 250 HOUR

FIG.3. Production of phytase by A . niger NRRL 3135 in 14-L fermenter with the following conditions: cornstarch medium (4mg/100 ml total P) (Shieh and Ware, 1968); 28C; aeration, 0.18 liters/min; 1 5 psi; agitation, 350 rpm. From Utt (1987).

278

RUDY J. WODZINSKI AND

A. H. J.

ULLAH

faster rate than others increasing the concentration of P in the medium and repressing synthesis of the enzyme. She also demonstrated that covalently bound P is present in Hylon V cornstarch and in potato starch as glucose-6-phosphate and glucose-3-phosphate, These phosphodextrins are effective substrates for the enzyme. If commercial cornstarch is used as a substrate in a medium to produce phytase using a strain of microorganism in which phytase synthesis is repressed by P, it is necessary to test that particular source of cornstarch for its effect on yield of the enzyme. The question of phytase repression by high levels of P becomes moot in strains that are bioengineered for phytase production. Van Gorcom et al. (1991) removed the repression of phytase synthesis in genetically engineered A . niger NRRL 3135 and A. van Tieghem. Their work is discussed under Section V,E,2. B. EFFECTOF MEDIUM INGREDIENTS, INOCULUM SIZE ON PELLET FORMATION, AND PHYTASE YIELDS

When a simple sugar, such as glucose or fructose is used as a sole source of carbon for phytase production by A. niger NRRL 3135, mycelial pellets are formed and the enzyme is made in low yields (Shieh and Ware, 1968). Han and Gallagher (1987) also observed these phenomena. However, if they used a medium containing surfactant (sodium oleate, 0.5% v h ) , growth was dispersed and phyA yields were 4.7-fold higher than in controls in a 17-day incubation. The organism has also been propagated and phytase produced in submerged liquid fermentation and semisolid fermentation with cottonseed meal and soybean meal (Han, 1989; Han and Wilfred, 1988). The organism produces glucoamylase (Vandersall et al., 1995) and a-galactosidase (Zapater et al., 1990) extracellularly. If either the inoculum size is too small, or if a relatively low viscosity medium (one devoid of cornstarch) is used, the organism forms pellets. Pellet formation may be minimized in shake flasks if at least 1x 1 0 7 conidia are added per 50 milliliters of medium or if mass mycelial transfers are used [at least 10% (v/v) inoculum]. Pellet formation may also be minimized by the shearing action of the agitation system that breaks u p aggregates of hyphae (Smith and Berry, 1974) and other factors such as pH, metal ion concentration, and complex or synthetic medium (Righelato, 1975).The property of lowered extracellular enzyme yields, when pellets are formed, appears to be a general characteristic of the organism. Reduced yields of a-galactosidase have also been observed when the organism formed pellets in a medium designed for production of that enzyme (R. J. Wodzinski, unpublished results).

PHYTASE

2 79

C. MUTATION STUDIES

Published results of mutation studies are sparse. Utt (1987) devised a medium that produced microcolonies and permitted large number of colonies per plate. However, the only mutants of A. niger NRRL 3135 isolated had limited increases in yield of phytase (17%). The increase in yield was probably due to an increase in phyB and not phyA. Chelius and Wodzinski (1994) isolated a mutant from the same wild-type strain that had a 3.3-fold increase in phyA activity, an 80% decrease in the pH 6.0 optimum acid phosphatase; and the same levels of phyB. The isolate may be a phytase catalytic mutant as well as an overproducer of phyA. The mutant approach is hampered by the lack of a powerful selection method that would differentiate between the phytases and phosphatases produced in a preliminary screen. Classical mutation approaches for increasing yield of secreted proteins have value. Dunn-Coleman et a!., (1991) increased the yield of chymosin fivefold after the gene for the enzyme was cloned into A . niger var. awamori along with a glucose promoter. They noted an increase in secreted protein. A combination of cloning techniques with classical mutagenesis probably alters genes or properties of the secretion system that are not fully understood and that resist a purely rational approach to increasing yield. V. Biochemistry of Phytase and Acid Phosphatases

A. PURIFICATION AND CHARACTERIZATION The activity of orthophosphoric monoester phosphohydrolase (acid optimum) has been described in a variety of species and tissues. To keep our focus on microbial enzymes, we will not describe in any detail the acid phosphatases produced in mammalian tissues. However, the mammalian acid phosphatase cannot be ignored altogether because there is sequence homology at the active site with the microbial phytase and acid phosphatases (Ullah and Dischinger, 1993b). Furthermore, an X-ray-deduced three-dimensional structure of recombinant rat prostatic acid phosphatase has been determined (Schneider et al., 1993). The physicochemical and catalytic properties of phytases and acid phosphatases of microbial origin are summarized in Table V. Aspergillus niger NRRL 3135 phytase (pH 5.0 optimum phyA) and acid phosphatases [pH 2.5 optimum (phyB)and pH 6.0 optimum] are secreted proteins. The relative amounts of these proteins in the media are considerably higher when grown under phosphate starvation in starch media. One estimate from the purification tables (Ullah and Gibson, 1987; Ullah and Cummins, 1987b, 1988b) and the enzymes' K,, indicated that approximately 50%

280

RUDY J. WODZINSKI AND A. H. J. ULLAH

of the total secreted proteins are phyA, phyB, and the pH 6.0 optimum acid phosphatases. About 40% of the protein, from the enzyme activity data, was identified as pH 6.0 optimum acid phosphatase; phyA and phyB were about 5% each. It was estimated that to purify these enzymes to near homogeneity, only 5- to 25-fold purification of each enzyme was needed. The secreted glycoproteins of A. niger NRRL 3135 are stable for many months at 5OC and they lack intrinsic protease activity (A. Ullah, unpublished observation). A combination of these properties made it possible for investigators to purify these proteins at room temperature. The three enzymes are acidic proteins with Pi values below 5.0. Therefore, all three enzymes can be concentrated by passing the culture filtrate onto a strong cationic exchanger at pH 2.8 or below. Subsequent purification steps involve additional ion-exchange chromatography and chromatofocusing. Biochemical and kinetic characterizations were performed on the enzymes recovered from the terminal step. The determination of molecular mass of the fungal phytase and acid phosphatases was determined by either gel filtration or SDS-PAGE. The enzymes are microheterogeneous because they are differentially glycosylated. The molecular masses of the monomeric form of phyA, phyB, and pH 6.0 optimum acid phosphatase were estimated by SDS-PAGE to be 85, 65, and 85 kDa, respectively. The Pi's of the native fungal phytase, pH 2.5 optimum acid phosphatase, and pH 6.0 optimum acid phosphatase were estimated to be 4.5, 4.0, and 4.9, respectively. The hydrolysis pathway for phytate was only elucidated for the pH 5.0 optimum phytase (phyA);the enzyme was labeled 3-phytase (EC 3.1.3.8) (Ullah and Phillippy, 1988). The Km and Kcatof fungal phytases for myoinositol hexa-, penta-, tetra-, and triphosphates have been reported (Ullah and Phillippy, 1994).The KcatlKm Values indicate that the preferred substrate for both phyA and phyB is myoinositol hexaphosphate. Therefore, it is justifiable to designate these enzymes phytase. An extracellular phytase and an extracellular acid phosphatase were purified from Aspergillus oryzae K 1 (Shimizu, 1993). Unlike A. niger NRRL 3135 phytase and pH 6.0 optimum acid phosphatase, which are monomers, the active enzymes of A. oryzae are dimers. The molecular masses of A. oryzae phytase and acid phosphatase are 60 and 70 kDa, respectively. The Pi, temperature optima, and pH optima of A. oryzae enzymes are very similar to those of the A. niger NRRL 3135 phosphomonoesterases. A significant sequence homology is expected for this class of extracellular proteins. An extracellular phytase from B. subtilis (natto) N-77 was purified 322fold by gel filtration and DEAE chromatography (Shimizu, 1992). The molecular mass of the active monomeric form was judged to be 36 kDa by SDS-PAGE. The protein's Pi was estimated to be 6.25. Like the fungal en-

PHYTASE

281

zymes, the optimum temperature for the bacterial phytase was about 60°C. Two periplasmic phytases, P1 and P2, were purified from E. coli to near homogeneity (Greiner et al., 1993). The active species were judged to be monomer with a molecular mass of 42 kDa. Both enzymes are very specific for phytate. The hydrolysis pathway for phytate was deduced for PZ; the enzyme was identified as 6-phytase (EC 3.1.3.26). The pH optima for P2 was 4.5 for phytate and 3.5 for the synthetic substrate, p-nitrophenyl phosphate. Like B. subtilis phytase, the E. coli enzyme is also a weakly acidic protein with a Pi of 6.0. Both chemical and kinetic properties of P2 point to identity with an acid phosphatase (Dassa et al., 1982). A phytase was purified from yeast (Nayini and Markakis, 1984);however, purity and molecular data were not reported. The pH and temperature optima were 4.6 and 45OC, respectively. Although the enzyme can degrade a variety of inositol phosphatases-di-, tri-, tetra-, penta-, and hexaphosphate-inositol monophosphate was hydrolyzed by this enzyme at an accelerated rate. From the kinetic data, it is reasonable to class the enzyme as a myoinositol monophosphatase. It is notable that A. niger NRRL 3135 (phyA) shows poor substrate acceptability to myoinositol monophosphate. Therefore, the yeast enzyme is very different from the A. niger NRRL 3135 phytase in terms of substrate specificity. Both E. coli and A. niger NRRL 3135 also produce acid phosphatases (Dassa et al., 1980; Ullah and Cummins, 1988b). The bacterial enzyme resides at the periplasm; the enzyme has high affinity (Km =2.7 ~ h for f Jpnitrophenyl phosphate (Dassa et al., 1980).The fungal enzyme, pH 6.0 optimum acid phosphatase, is a highly glycosylated extracellular metalloenzyme (Ullah et al., 1994).This copper-containing phosphomonoesterase is a poor acceptor of myoinositol phosphates; based on KcatIKm ratio, phytate is hydrolyzed at an efficiency of 0.06% compared to phyA (100%). The KcatIKm ratio for the lower forms of myoinositol phosphates is one order of magnitude lower than that of hexaphosphate. The substrate accommodation data for the fungal pH 6.0 optimum acid phosphomonoesterase clearly suggest the enzyme to be an acid phosphatase. B. SEQUENCE STUDIES

The primary structure of A. nigerNRRL 3135 phytase (phyA) was determined from both the cloned DNA (GenBank Accession No. M94550) and chemical sequencing (Ullah and Dischinger, 1993a). The sequence deduced from DNA is in full agreement with the chemically deduced protein sequence. Phytase sequence has also been deduced from the cloned DNA of A. niger strain van Tieghem (Van Hartingsveldt et al., 1993) and is identical. A third phytase sequence was obtained from A. niger var. awamori (GenBank Accession No. L02421) and revealed 97.2%

282

RUDY J. WODZINSKI AND A. H. J. ULLAH

homology to A. niger NRRL 3135 phytase (Fig. 4). The substituted amino acids in A . niger var. awamori revealed that of a total of 13 substitutions only 1 at the penultimate C-terminal end was nonhomologous. The other 1 2 substitutions in A. niger var. awamori phytase were all conservative replacements predicting a very similar tertiary structure for both of the proteins. The primary structure of A. niger NRRL 3135 phyB and A. niger var. awamori phyB was elucidated from the cloned DNA and a partial sequence was verified by chemical sequencing (Ehrlich et al., 1993; 10 20 30 40 50 60 AFphya MGVSAVLLPLYLLSGVTSGLAVPASRNQSSCDTVDQGYQCFSETSHLWGQYAPFFSLANE

.......................................................... ............................................................

ANphya MGVSAVLLPLYLLAGVTSGLAVPASRNQSTCDTVDQGYQCFSETSHLWGQYAPFFSLANE 10 20 30 40 50 60

70 80 90 100 110 120 AFphya SVISPEVPAGCRVTFAQVLSRHGARYPTDSKGKKYSALIEEIQQNATTFDGKYAFLKTYN

........................................................ ............................................................

ANphya SAISPDVPAGCRVTFAQVLSRHGARYPTESKGKKYSALIEEIQQNVTTFDGKYAFLKTYN 70 80 90 100 110 120

130 140 150 160 170 180 AFphya YSLGADDLTPFGEQELVNSGIWYQRYESLTRNIVPFIRSSGSSRVIASG~IEGFQST

.......................................................... ............................................................

ANphya Y S L G A D D L T P F G E Q E L V N S G I K F Y Q R ~ S L T R N I I P F I R I E G F Q S T 130 140 150 160 170 180

190 200 210 220 230 240 AFphya KLKDPRAQPGQSSPKIDWISEASSSNNTLDPGTCTVFEDSELADTVE?NFTATFVPSIR

........................................................... ............................................................

ANphya KLKDPRAQPGQSSPKIDWISEASSSNNTLDPGTCTWEDSELADTVEANFTATFAPSIR 190 200 210 220 230 240

250 260 270 280 290 300 AFphya QRLENDLSGVTLTDTEVTYLMDMCSFDTISTSTVDTKLSPFCDLFTHDEWINYDYLQSLK

........................................................... ............................................................

ANphya QRLENDLSGVTLTDTEVTYLSFDTISTSTVDTKLSPFCDLFTHDE~IH~Y~SLK 250 260 270 280 2 90 300

310 320 330 340 350 360 AFphya KYYGHGAGNPLGPTQGVGYANELIARLTHSPVHDDTSSNHTLDSSPATFPLNSTLYADFS

........................................................... ............................................................

ANphya KYYGHGAGNPLGPTQGVGYANELIARLTHSPVHDDTSSNHTLDSNPATFPLNSTLYADFS 310 320 330 340 350 360

370 380 390 400 410 420 AFphya HDNGIISILFALGLYNGTKPLSTTTVENITQTDGFSSAWTVPFASRLYVEMMQCQAEQEP

............................................................ ............................................................

ANphya HDNGIISILFALGLYNGTKPLSTTTVENITQTDGFSSAWTVPFASRLYVEMMQCQAEQEP 370 380 390 400 410 420

430 440 450 460 AFphya LVRVLVNDRWPLHGCPVDALGRCTRDSFVRGLSFARSGGDWAECFA

............................................. ..............................................

ANphya LVRVLVNDRWPLHGCPIDALGRCTRDSFVRGLSFARSGGDWAECSA 430 440 450 460

FIG.4 Comparison of deduced phytase sequence from cloned phyA DNA of A. niger NRRL 3135 (AFphyA) (GenBank Accession No. M94550) with cloned DNA of A. niger var awomori (ANphyA) (GenBank Accession No. L02421) (97.2% homology).

283

PHYTASE

Piddington et al., 1993). The phyB from A . niger NRRL 3135 showed 99% homology to the corresponding protein from A . nigervar. awarnori. Surprisingly, phyA revealed only 23.1% homology to the phyB (Fig. 5). The primary structure of the bacterial phytases has not been deduced 10 20 30 40 50 MGVSAVLLPLYLLAGVTSGLAVPASRNQSTCDT-VDQGY-QCFSETSHLWGQYAPFFSLA

. . . . . . . . . ... . . . . . . ... . . . . . . . . ... . . . ... . . .

MPRTSLLTLACALATGASASYG~~IPQSTQEKQFSQEFRDGYSILKH-YGGNGPY-SER 10 20 30 40 50 60 70 80 90 100 110 NESAISPDVPAGCRVTFAQVLSRHGARYPTESKGKKYSAL1EEI-QQI"TFDGKYAFLK

...... ......... ...... ..............................................

VSYGIARDPPTGCEVDQVIMGERYPSPSAGKSIEEALAKVYSINTTEYKGDLAFLN 60 70 80 90 100 110 120

130

140

150

160

TYNYSLGAD-------DLTPF-GEQELVNSGIWYQRYESL--TRNIIPFIRSSGSSRVI

. ... . . . ... . ... . . . . . . ...... . .. . .

.. .. .

.......

DWTYYVPNECYYNAETT SGPYAGLL~AYNHGNDYKARYGHLWNGETWPFF -sSGYGRVI

r20

130

140

150

160

170

170 180 190 200 210 220 ANphya ASGEKPIEGFQSTKLI
. . . ..... ........

.... ...........................

AFphyb ETARKFGEGFFGYNYS-------- TNAALNIISESEVMGADSLTP-TCDTDNDQTTCDNL 180 190 200 210 220 230 240 250 260 270 280 ANphya EANFTATFAPSIRQRLENDLSGVTLTDTEVTYLMDMCSFDTISTSTVDTKLSPFCDLFTH ~-

.. . . . . . . . . ........................

.....

. . . . .... . -----F SNWINAFTQ

AFphyb TYQLPQ-~KV~-ARLNSQNPGMNLTASDVYNLIVMASFEI.NW 230 240 250 260 270

280

290 300 310 320 330 340 DEHlIHYDYLQSLKKYYGHGAGNPLGFTQGVGYANELIARLTHSPVHDDTSSNHTLDSNPA

... . . . . . . .... . . .....................................

DEWVSFGYVEDLNYYYCAGPGDKNMAAVGAWANASLTLI.NQGPKEAGP----------2 90 300 310 320 330 350 360 370 380 390 400 TFPLNSTLYADFSHDNGIISILFALGLYNGTKPLSTTTVENITQTDGFSSAWTVPFASRL

. ... . ... ... . ... . ... . . . . . . . . . . . . . . . . . . . . . . . .. ... ---____ LFFNFAHDTNITPILAALGVLIPNEDLP---LDRVAFGNPYSIGNIVPMGGHL 340

350

360

370

380

410 420 430 440 450 YVEMMQCQA----EQEPLVRVLVNDRWPLHGCPIDALGRCTRDSFVRGL----------

.... .......

. . . . . . ..............................

TIERLSCQATALSDKGTYPFNDCTSGPGYSCPLANYTSILNKNLPDYTTT

390

400

410

420

430

440

460 -----SFARSGGDW--------------AECSA- --- ...... ...

CNVSASYPQYLSFWWNYNTTTELNYRSSPIACQEGDAMD 450 460 470

Comparison of chemical sequenced phytase from cloned phyB DNA of A . niger NRRL 3135 (AFphyB) (Ehrlich et al.;l993) with cloned DNA of A . niger var awamori (ANphyA) (GenBank Accession No. L02421),(23.1% homology).

284

RUDY J. WODZINSKI AND A. H. J. ULLAH

chemically;however, the primary structure of pH 2.5 optimum acid phosphatase from E. coli was deduced from the cloned DNA (Dassa et al., 1990). When sequence alignment was performed between a truncated A. niger NRRL 3135 phyB (-44 residues from the N terminus) and E. coli pH 2.5 acid phosphatase, the result showed only 15.1% homology (A. H. J. Ullah, unpublished data). Although phytase activity had been reported in yeast (Nayini and Markakis, 1984), the enzyme was not purified to homogeneity or any sequence reported to this date. However, acid phosphate genes phol and pho5 from yeast were cloned and sequenced (Elliott et al., 1986; Bajwa et al., 1984). Protein sequence alignment of A. niger NRRL 3135 phyA and phyB with the deduced primary structure of phol and pho5 of yeast showed remarkable homology for cysteines, certain hydrophobic residues, and the active site residues (Ullah and Dischinger, 1995). C. ACTIVESITEDETERMINATIONS

The active site of phytases shows remarkable homology to the active site residues of the members of a particular class of acid phosphatase termed "histidine phosphatase" (Van Etten et al., 1991; Ullah et al., 1991). Chemical probing at the active site of human prostatic acid phosphatase suggested that an arginine residue is involved in catalysis (Van Etten, 1982). A similar observation was also made in A. niger NRRL 3135 (Ullah et a]., 1991).Sequence similarity search among diverse phosphate metabolizing enzymes, i.e., fructose 2,6-biphosphatase, phosphate glycerate mutase, and acid phosphatase, led to the identification of a tripeptidic region with the sequence RHG (Bazan et al., 1989). When A. niger NRRL 3135 N-terminal amino acid sequence was compared with these enzymes, it was noted that the conserved tripeptidic region was also present not only in fungal phyA but also in the N-terminal region of phyB (Ullah and Dischinger, 1993b). Further chemical probing of the fungal phytase also suggested a sensitive histidine at the active site (Ullah and Dischinger, 1992). On close examination of the active site residues of phyA and phyB in A. niger NRRL 3135, pH 2.5 optimum acid phosphatase in E. coli, pho3 and pho5 gene products in yeast, human prostatic, and lysosomal acid phosphatase, it was observed that the most conserved sequence is RHGXRXP (Table VI). The acid phosphatases and phytases containing this active site motif in the N-terminal segment of the protein are grouped under "histidine phosphatase"; a survey of the protein and DNA databases revealed 14 members belonging to this group of acid phosphatases (Table VII). The positive charge of the guanido group of arginine is probably responsible for the recognition and anchoring of the negatively charged phosphate group to the proximity of a histidyl residue in the active site. The phosphate group is transiently

PHYTASE TABLE VI OF FUNGAL PHYAAND PHYBACTIVE-SITE SEQUENCE WITH PUBLISHED ALIGNMENT ACIDPHOSPHATASES, PHOSPHOGLYCERATEMUTASE,AND FRUCTOSE-2,&BIPHOSPHATASE ~~~

AfphyA AfphyB YscACP3 YscACP5 YscpACPl ECACP HuPACP HuLACP RatACP RtF2,6BP YePGM HuPGM-M HuBPGM

(52) (52)

(46) (46) (29) (6) (1) (1) (1) (250) (1) (1) (1)

C C C C C L K R R

R V T E V D EMK EMK K I K K L E E L K S L R S L R P

F T Q Q Q S F F F R * * P * M A T H * * S K Y

A V L L V V V V V S

K R K

~~

Q V L I M V Q M L Q M L H T L V I V T L V T L L T L L I Y L L V L L V M L I M

S R K R A R A R Q R S R F R Y R Y R C R V R V R L R

H H H H H H H H H H H H H

G G G G G G G G G G G G G

A R Y P T D S K G K E R Y P S P S A G K E R Y P T Y S K G A E R Y P T V S L A K S R N P T G G N A A V R A P T K A T Q L D R S P I D T F P T D R S P V K T Y P K D R S P V K AY P K E S E L N L R G R I Q S E W N E K N L F E T T W N Q E N R F E G A W N K E N R F

transferred to the histidine group to form an unstable phosphoenzyme complex before hydrolytic cleavage to form orthophosphate (Van Etten, 1982). This mechanism is reasonable because it is known that a phosphate group attaches to the solitary histidine residue in phosphocarrier protein, HPr of gram negative bacteria (Anderson et al., 1993). In A. niger NRRL 3135 phyA inactivation of tryptophan led to catalytic demise (Ullah and Dischinger, 1992). Of the four tryptophans, only Trp25 and Trp267 are in the hydrophilic region; the other two residues are in the hydrophobic region and may not play a role in active site formation.

D. ENZYME ENGINEERING STUDIES Any future improvement of the kinetic parameters and thermo- and pH stability of phytase will depend on understanding the three-dimensional structure of the biocatalyst. Unfortunately, the tertiary structure of the protein has not been determined. Crystallization of the heavily glysosylated fungal phytase and acid phosphatases is extremely difficult (A.H.J. Ullah, unpublished data). Efforts are now under way to express the cloned gene in bacteria to obtain the unglycosylated form of phytase. If the nonglycosylated isoform of fungal phytase can fold properly in bacteria and exhibit enzymatic activity, then subsequent crystallization and structure elucidation will be meaningful and yield information that could be used to fine tune the three-dimensional structure of the protein. Despite these difficulties, current research in structure refinements is proceeding in three areas: increasing thermostability,

TABLE VII A SURVEY OF MICROBIAL AND MAMMALIAN ACIDOPTIMUM HISTIDINE PHOSPHATASES

Enzyme

co

Source

EC No.

NCBI Seq. ID

Gene loci

AA

RHG motif

HDmotif

432

Present

Present

Dassa et al. (1990)

467

Present

Present

Ullah and

Reference

Periplasmic phosphoan hydride phosphohydrolase, 6-phytase, pH 2.5 optimum APase

E. coli

3.1.3.2, 3.1.3.26

130735

3-Phytase

A . niger NRRL 3135

3.1.3.8

464382

3-Phytase

A. niger

3.1.3.8

484414

phyA

467

Present

Present

Van Hartingsveldt et al. (1993)

3-Phytase

A. niger (awamori)

3.1.3.8

166518

phyA

467

Present

Present

Piddington et al. (1993)

3-Phytase, pH 2.5 optimum acid phosphatase

A. niger NRRL 3135

3.1.3.2, 3.1.3.8

464385

phyB

479

Present

Present

Ehrlich et al. (1993)

3-Phytase

A. niger (awamori)

3.1.3.8

464384

phyB

479

Present

Present

Piddington et al. (1993)

Acid phosphatase

Schizosaccharomyces pombe

3.1.3.2

130719

phol

453

Present

Present

Elliot et al. (1986)

Acid phosphatase

Sacchoromyces cerevisiae

3.1.3.2

130721

pho3

467

Present

Present

Bajwa et al. (1984)

phyA

Dischinger (1992)

m

Acid phosphatase, thiamine repressible

Schizosaaccharomyces pombe

3.1.3.2

400839

Human prostatic acid phosphatase

Homo sapien

3.1.3.2

189620

Rat prostatic acid phosphatase

Rat

3.1.3.2

206028

381

Present

Absent

Roiko et al. (1990)

Mouse lysosomal, acid phosphatase

Mouse

3.1.3.2

52871

421

Present

Present

Geier et al. (1991)

Human lysosomal acid

Homo sapien

3.1.3.2

130727

423

Present

Present

Pohlmann et al. (1988)

Rat

3.1.3.2

130729

423

Present

Present

Himeno et al.

pho4

463

Present

Present

Yang and Schweingruber (1990)

386

Present

Present

Van Etten et al. (1991)

phosphatase Rat lysosomal acid phosphatase N

m u

(1989)

288

RUDY J. WODZINSKI AND A. H. J. ULLAH

reducing the molecular mass, and constructing a chimeric enzyme with the acid phosphatase. Enzyme engineering studies with the acid phosphatase, lacking phytate hydrolysis, may include incorporation of the phytase active site sequence in the protein to impart phytate binding and catalysis. The other protein engineering effort may include alteration of the glycosylation signals to understand the role of glycosylation in secretion and stability of the protein. Because data are lacking on the X-ray-deduced three-dimensional structure of any microbial acid optimum phosphomonoesterase, computer-assisted modeling cannot be performed on fungal phytase or acid phosphatase. However, the crystal structure of recombinant rat acid phosphatase containing the active site sequence RHGXRXP had been determined to 3 A by protein crystallographic methods (Schneider et al., 1993). The protein is built up of two domains: an a / p domain consisting of a seven-stranded p-sheet with helices on both sides of the sheet and a smaller a domain. The topology of one of the domains of acid phosphatase is very similar to the structure of phosphoglycerate mutase (Campbell et al., 1974). The Garnier analysis of phytase also shows similar arrangements of alternating a / p structure (Fig. 6). It is reasonable to predict that the overall structure of fungal phytase will parallel the structure of rat acid phosphatase. Computerassisted homology modeling of phytase based on the structure of rat acid phosphatase could not be performed at this time (A. H. J. Ullah, unpublished observation).

E.

CLONING OF

PHYTASE AND ACIDPHOSPHATASES

1. USDA Studies

The group at the Southern Regional Research Center started purification and characterization of the fungal phytase in 1984. The N-terminal and cyanogen bromide fragments of the purified protein were obtained and reported at the poster session of the 9th Enzyme Engineering Conference at Santa Barbara, California in 1987. Researchers from the Gist-brocades were also present at the meeting. Therefore, the sequence information, so vital for the gene cloning, became available to the Gist brocades researchers. The full account of the N-terminal and intersequences was, however, published a year later (Ullah, 1988). The phyA gene from A. niger NRRL 3135 was partially cloned in a A g t l l expression library as identified by immunoprobe and sequence verification (Mullaney et al., 1991). The full-length gene was cloned subsequently and the sequence submitted to the GenBank (Accession No. M94550). A second phytase gene (phyB) from A. niger NRRL 3135 was cloned; this DNA fragment codes for a 479-amino acid enzyme and was found to contain four exons (Ehrlich et al., 1993). An acid phosphatase from A. niger

289

PHYTASE Garnier plot of A. ficuum phytase (phyA)

. 10 20 30 40 50 . 60 LAVPASRNQSSCDTVDQGYQCFSETSHLWGQYAPFFSLANESVI~PEVPAGCRVTFAQVL

helix EE E E E EEEEEEEEE sheet EEEE turns TTTTTTTTTTTTTTTT TTTTTTT T T T TTT coil c ccc cccccccc c

.

.

.

.

70 80 90 100 110 120 SRHGARYPTDSKGK~C~SALIEEIQQNATTFDGKYAFLKT~SLG~DLTPFGEQEL~S

helix HHHHHHHH HH HHHHHH sheet EE EEEE turns TTTT TTTTTTT TTTT TTTT TTT TT coil c ccc cccc c ccccc

.

130 140 150 160 170 180 GIKF;QRYESLTRN~VPFIRSGSSRVIASGKKF~EGFQSTKL~P~QP~SSPKIDW helix H HHHH sheet EEEEE EEEEEE EEE EE E EEEE turns TT TTTTTTT TTT TTT T TT T coil cc cccc c ccc cc ccc

. 190 200 . 210 . 220 . 230 . 240 ISEASSSNNTLDPGTCTVFEDSELADTVEANFTATFVPSIRQRLENDLSGVTLTDTEVTY HHHHHHHHHHHHHHH HHHHH

helix sheet EEE EEEEE turns T TTT TT cc c cc coil

EEEE

T

ccccc

EEEEE TTTT T C

. 250 260 . 270 . 280 . 290 . 300 LMDMCSFDTISTSTVDTKLSPFCDLFTHDEWINYDYLQSL~YYGHGAGNPLGPTQGVGY

H H H HHH helix HHHHH H E E E E EEEEE EEEE sheet T TTTT TT TTTTTTT T TTTT turns ccccc cc C cccccc cc coil

.

.

.

.

310 320 330 340 350 360 ANEL1ARLTHSPVHL;DTSSNHTLDSSPATFPLNSTLYAFSHDNGIISILFALGLYNGTK

. . . . _ . . . . . . . . . helix nnnnnnn

sheet turns coil C

T

T TTT

ccc c

370

T

T

EEE T TT

cc ccc ccc 380

EEEE

ccc

390

EEEEEEEE TTT TTTTT

cc

cc 400

410

420

PLSTTTVENITQTDGFSSAWTVPFASRLYVEMMQ~QAEQEPLVR~LVNDRWPL~GCPM helix HHHHHHHHHHHHHHHH EEE EEEEE EEEEE EEEE EEEE sheet TTTT TT T T TTT turns coil cc c c cc ccc ccc

.

430 440 ALGRCTRDSFVRGL~FARSGGDWAE helix HH EEEEE sheet EEE TTTTT T turns coil cccc ccccc

FIG.6. Secondary structure of phyA as predicted from Garnier plot.

NRRL 3135 that could not be readily separated from pH 2.5 optimum phytase/acid phosphatase (phyB) was cloned and was shown to be homologous to Penicillium chrysogenum PHOA (Ehrlich et d., 1994). Very recently, a gene coding for the metalloenzyme pH 6.0 optimum acid

290

RUDY J. WODZINSKI AND A. H. J. ULLAH

phosphatase was cloned (Mullaney et al., 1995) and shown to be homologous to the aphA gene. The encoded amino acid sequence verified by the chemically deduced sequencing (Ullah et al., 1994). 2. Dutch Studies

The Dutch group initiated the cloning work in late 1980s after the biochemical and sequence information was published by the USDA group (Ullah, 1988b).The researchers from the Gist-brocades and TNO Medical Biology cloned and sequenced the gene, and overexpressed the phytase gene (phyA)from A. niger NRRL 3135. The gene was isolated using degenerate oligodeoxyribonucleotidesdeduced from phytase amino acid sequences. Nucleotide sequence analysis of the cloned DNA indicated an open reading frame coding for a 467-amino acid protein and included in the DNA is an intron of 102 base pairs in the 5' part of the gene (GenBank Accession No. 216414). The gene was identical to the one cloned by the USDA group (GenBankAccession No. M94550).However, the major contribution of the Dutch group was the understanding of the expression of the phytase gene at the mRNA level by inorganic orthophosphate levels. The researchers have detected a transcript of 1.8 kb after cell growth in low-phosphate medium. Transcription of phyA initiates at least seven start points within a region located 45-25 nt upstream from the start codon. In transformants of A. niger, expression of multiple copies of phyA gave up to about 10-foldhigher phytase activities than the level produced by the native wild-type strain (Van Hartingsveldt et al., 1993). In other studies, the Dutch group has also expressed the fungal phytase (phyA) in tobacco seeds. The protein was expressed as 1% of the soluble proteins in mature tobacco seeds (Pen et al., 1993). Detailed descriptions of the gene construct used for transformation of tobacco have been published. The molecular weight of the phytase produced in seeds was 6 7 kDa compared to 80 kDa in A. niger. Differences were due to glycosylation. When the cloned plant phyA was deglycosylated and compared to deglycosylated A. niger phyA, the molecular weights were identical. The phyA gene has been cloned into canola (Brassica n a p ) (Beudeker and Pen, 1994). The transgenic plant seeds of both species have been tested as a source of phytase for monogastric animals and are efficacious. 3. Pan Labs Studies

The Pan Labs research with fungal phytase and acid phosphatase stems from the interest of Alko Ltd., Rajamaki, Finland. These groups cloned the genes for phytase and pH 2.5 optimum acid phosphatase from A . niger var. awamori (Piddington et a]., 1993). The genes were isolated from the genomic DNA using oligodeoxyribonucleotide probes based on the protein sequences reported by the USDA group. A potential 1 0 2 - b ~

PHYTASE

291

tial 102-bp intron was identified between the start codon and the N-terminal amino acid residue. Furthermore, a 19-residue signal peptide was identified in the N-terminal segment. The same genomic library was also probed with oligodeoxyribonucleotidesbased on the pH 2.5 optimum acid phosphatase sequence. One of the clones contained a 2.1-kb fragment that hybridized strongly to two oligonucleotideprobes based on different peptides from the same protein. This 2.1-kb fragment contained the sequence for 1 2 previously sequenced peptides including the N-terminal peptide sequence. The researchers identified intron boundaries through the isolation and sequencing of pH 2.5 optimum acid phosphatase-encoding cDNA. Unlike the phytase gene (phyA),three short introns were revealed by the sequencing. The resulting translated sequence codes for a 479amino acid protein including a 19-amino acid signal peptide. The gene coding for the acid phosphatase (aph)is very similar to a gene coding for the second phytase (phyB)in A. niger NRRL 3135 (Ehrlich et a]., 1993). STUDIES WITH PHYTASE AND ACIDPHOSPHATASE F. IMMOBILIZATION Fungal phytase and acid phosphatases are hydrolytic enzymes with a high catalytic turnover number-typically the range being from 220 to 1000 per second (Ullah, 1988a, Ullah and Cummins, 1987b, 1988b). Thus, these categories of enzymes are the ideal candidates for immobilization and construction of packed-bed bioreactors. Phytases act on myoinositol hexaphosphate to degrade sequentially to liberate orthophosphates, the byproduct being myoinositol penta-, tetra-, tri-, di-, and monophosphate. An efficient bioreactor of the immobilized phytase could be used to produce various species of myoinositol polyphosphates and treat soybean milk to lower the content of myoinositol hexa-, and pentaphosphate. The higher form of myoinositol phosphates, i.e., hexa-, and pentaphosphate, may act as metal chelators, and thus interfere with the mineral nutrition in monogastric animals. The enzymatic action of immobilized phytase on phytate may render the molecule to be a nonchelator by conversion to a lower form of myoinositol tetra-, and triphosphates. Aspergillus niger NRRL 3135 phyA was covalently immobilized on Fractogel TSK HW-75 F. A packed-bed bioreactor was constructed with the immobilized enzyme. The catalytic parameters and stability of the immobilized enzymes were determined. No shift in pH optima was observed for the covalently attached phyA compared to the soluble enzyme. The temperature optima shifted from 58" to 65OC. Despite the increase of Km for phytate and a downward shift in catalytic activity, the immobilized enzyme can hydrolyze over 50% of the orthophosphate from phytate after repeated passage through the bioreactor (Ullah and Cummins, 1987a). When the product of the bioreactor

292

RUDY J. WODZINSKI AND A. H. J. ULLAH

was characterized by an HPLC on an anion-exchange column, myoinositol penta-, tetra-, tri-, di-, and monophosphates were detected in the eluate. After exhaustive hydrolysis of phytate by the bioreactor, only myoinositol di-, and monophosphates were detected in the eluate (Ullah and Phillippy, 1988). To improve the kinetic parameters of the immobilized biocatalyst, fungal phytase was also covalently attached to glutaraldehyde-activated silicate (Ullah and Cummins, 1988a). About 20% of the total phytase was bound to the activated resin, and the catalytic activity of the bound protein was reduced fourfold. This may have resulted from extensive crosslinking of the phytase to the silicate. In an attempt to immobilize phytase through its carbohydrate moieties rather than protein backbone, the biocatalyst was immobilized on crosslinked agarose (Dischingerand Ullah, 1992).When immobilization was achieved through the protein backbone, myoinositol di- and monophosphates were the predominant species generated on substrate passage. By contrast, when immobilization was performed using carbohydrate side chain, a maximum of 31% of the available phosphate was cleaved. This may be a consequence of a difference in conformation of the catalytic active center, which resulted from differential crosslinking of the enzyme through its carbohydrate moieties. The bioreactor's output and the Kcatindicate a diminished catalytic performance. Similar diminished activity was also observed with the immobilized phytase when the attachment was made through the protein backbone. The results of the immobilization study indicate that the active catalytic center of the enzyme may be distorted by extensive crosslinking of proteins to the matrix. Because the native phytase is heavily glycosylated, prevention of extensive crosslinking is difficult. By using site-directed mutagenesis, it should be possible to remove most glycosylation to produce an enzyme that can be immobilized by a few carbohydrate chains while retaining a high level of activity. VI. Feed Studies with Phytase WITH POULTRY A. EARLYSTUDIES

Studies on enzymatic treatment of feed using microbial phytase sources have demonstrated that this method increases the bioavailability of proteins and essential minerals and provides levels of growth performance as good as or better than those with phosphate supplementation. Nelson et al. (1968a) were the first to pretreat a corn-soya diet with culture filtrate containing phytase A . niger NRRL 3135 to a corn-soya diet and fed it to 1day old chicks. The chicks showed increases in bone ash due to the phytin-P released from the dietary substances by the enzyme. When the

PHYTASE

293

phytin-P of either soybean meal or cottonseed meal was hydrolyzed by the phytase in the culture filtrate, it was necessary to increase the moisture level to distribute the enzyme within the meal (T. R. Shieh, personal communication). The cost of heat energy to dry the treated meal to prevent subsequent spoilage in a proposed process was higher than the value of phosphorus released. This approach was abandoned. Nelson et al. (1971) added graded levels of solvent-precipitated A. niger NRRL 3135 phytase to an experimental and to a commercial corn soybean meal diet and fed it to chicks. They measured bone ash and feed-to-gain ratios. They reported that the enzyme was active in the animals and that they were able to incorporate the released phytin-P into bone. The feed-to-gainratios were higher than would be expected. They concluded that chicks could utilize the P from phytin-P as well as supplemental P. They also titered the enzyme in the chick and presented data that indicated the units of phytase that would be required to attain maximum phosphorus release from the diets. The maximum amount of phytin-P (2.1-3.0 g / kg of diet) was released if 1500-2000 pm P/hr/ml of phytase was added per kilogram of diet. Nelson et al. (1968~)determined the effect of feeding phytase on calcium requirements. They concluded that if 90% of the phytin-P was hydrolyzed by phytase, the amount of calcium from natural ingredients required in the diet was at least one-third less. The superior activity of A. niger NRRL 3135 phytase and its practical application to animal feed for the removal of phytic acid has been demonstrated in many experiments (Han, 1989; Han and Wilfred, 1988; Howson and Davis, 1983; Nelson et al., 1971, 1968a; Rojas and Scott, 1968).

B. RECENTSTUDIES ON PHYTASE AS A FEED ADDITIVE Simons et al. (1990),Jongbloed and Kemme (1990), and Jongbloed et al. (1992), in The Netherlands, collaborated to reaffirm the pioneering studies of Nelson et al. (1968a,b, 1971) of phytase fed to broilers. They not only confirmed the earlier work with the feeding of solvent precipitated enzyme to chick diets, but they also extended the data and were the first to demonstrate the efficacy of feeding phytase to swine. They concluded that the addition of enzyme (1000 pm P/hr/ml of phytase/kg of diet) was sufficient to provide levels of performance equal to or better than that attained by adding supplemental inorganic phosphate to a broiler diet. If the amount of enzyme was increased to 1500 pm P/hr/ml of phytase/kg of diet, the results indicated improved performance compared with birds fed control diets. It is interesting to compare these feed studies with those of Nelson et al. (1971). The units of enzyme required for releases of phytin-P in commercial type diets is in

294

RUDY J. WODZINSKI AND A. H. J. ULLAH

fairly close agreement. They extended their studies and measured the amount of phosphorus in the feces of the broilers fed enzyme. When microbial phytase was fed to low-P diets for broilers, the availability of P increased to 60% and the amount of P in the droppings decreased by 50%. They established the potential benefits that the use of phytase in commercial diets would have on abating phosphate pollution in soil and water pollution. Royal Gist-brocades, Delft, The Netherlands has conducted and/or has furnished phytase (Natuphos produced by cloning phyA from A. niger NRRL 3135 into A. niger CBS 513.88) to various researchers for poultry feed studies. The results (Farrell et al., 1993; Von Schoner et al., 1992, Schoner, 1992, Broz et al., 1994) have reaffirmed the earlier studies that the phytin-P in feed ingredients is utilized by monogastric animals fed phytase. They have also extended the studies to layers (Van der Klis and Veersteegh, 1991), ducks and ducklings (Farrel et al., 1993), and swine (Beers and Jongbloed, 1992; von Pallauf et al., 1992; Ketaren et al., 1993; Hoppe et al., 1993; Mroz et al., 1994). Alko Biotechnology has supplied phytase, Finase F, produced by their cloned version of A. niger NRRL 3135 to various researchers who tested its effect in swine (Young et a]., 1993; Lei eta]., 1993a, 1994; Cromwell et al., 1993). The results utilizing their preparation are consistent with the results using phytase derived from other sources. Lei et al. (1993b) demonstrated that the addition of phytase to the diets of weanling pigs improved the bioavailability of zinc and phytate phosphorus. In some of the studies, data are presented that allow the relationship between units of phytase and P released to be calculated. It is noteworthy that the units of enzyme required for hydrolysis of a set amount of phytinP are fairly consistent. One can estimate within a fairly narrow range the amount of phytase required to hydrolyze the phytin-P present in almost any diet that is used commercially to rear animals. Depending on the specific diet used in the study, 380-1000 Fm P/hr (6308-16600 nKat) of phytase is required to replace 1 g of P supplied from an inorganic source. This value holds from the earliest research using phytase from the original strain to the cloned strains. It is true in broilers, layers, swine, etc. Because some types of diets produce beneficial effects, such as calcium and iron availability, it is necessary to empirically test the absolute levels required if least-cost formulations are to be achieved. Additionally, the new studies have measured the concentration of phosphorus in the intestinal tract and feces. As such, these studies are very useful in quantifying the effect of feeding the enzyme to poultry and swine and the effect it has on pollution abatement. If a sufficient quantity of enzyme is fed, the calculated P agrees with the decrease in P excreted.

295

PHYTASE

VII. Economics and Potential Effect of Phytase on Pollution Abatement

One of the driving forces in the development of phytase as a product is the awareness of the environmental effects of the release of concentrated pollutants, especially in intensive agricultural production of animals. In various European countries, especially in The Netherlands and Germany, government has mandated that the amount of manure that may be landspread is governed by the actual needs of the soil and the crop and not by some arbitrary stocking rate (Dunn, 1994). If a livestock producer wishes to increase the size of its animal production unit without increasing the acreage of land on which the manure is spread, it is necessary to decrease the total phosphate and other nutrients in the manure. This necessitates either a more efficient method to utilize the phosphorus supplied to the animal or the necessity to remove the phosphorus from the excrement before discharge to the environment. These mandates in the late 1980s caused a resurgence in the interest in phytase. The economics of the situation was changed. The value of phytase was no longer restricted to the value of phytin-P released or some nebulous benefit from the enzyme preparations not yet identified. A flurry of research activity occurred If phytase was used as a feed ingredient in the diets of all of the monogastric animals reared in the United States, it would release phosphorus that has a value of $1.68 x 108 (Table VIII). These data are based on

TABLE VIII

ESTIMATEOF PHYTASE MARKET VALUE IN ANIMAL FEEDS

Animal

No. in United States in1992n

lo9

Average livewt.

(kg)

kg of feed/ animal

Phytin-P conc. in feed

kg Phytin

(%)

utilized per animal

kg Phytin-P hydrolyzed/ year

Total value of phytin-P

($1

Broilers

6.14 x

0.21

0.008

4.91 x 107

Layers Ducks

3.64 x lo8 1 . 8 0 ~ 1 0 ~2.95

36.4/year 0.18 7.08 0.21

0.066 0.015

2.40 X lo7 2.70 X lo5

3.32 X 3.70 X

Turkeys

2 . 8 9 108 ~

26.4

0.21

0.055

lo7 lo5 1.59 X lo7 2.19 X lo7

Pigs

5 . 7 8 ~ 1 0 ’ 80.4

0.21

0.56

3.24 x 107

4.44 x 107

2.01

9.91

3.8

265

Total ONational Agricultural Statistics 1993.

* 134.082 Tons.

1.22 x

losb

6.75 x 107

1.68 x

lo8

296

RUDY J. WODZINSKI AND A. H. J. ULLAH

the most recent livestock production statistics available from the USDA, the current value of inorganic phosphate, the amount of feed required to produce the animals, etc. The animal feed studies discussed previously have provided the quantitative data on the amount of supplied dietary phosphate assimilated by animals and the diminished amount of phosphorus excreted in the manure. Hoppe et a]., (1993); Hoppe (1992); Von Schoner and Hoppe (1992); and Von Schoner et a]., (1992) have summarized and conservatively estimated the amount of phosphorus that is assimilated and not excreted when phytase is used in the diets of poultry and swine [e.g., the feeding of phytase to swine caused an average reduction of P excreted in all trials of 56 f 9% (Hoppe, 1992)l. When those data (Hoppe et a]., 1993; Hoppe, 1992; Von Schoner and Hoppe, 1992; Von Schoner et a]., 1992) are used as a basis of estimating the reduced phosphate levels excreted in manure and are multiplied by the number of monogastric animals reared in the United States, 8.23 x 1 0 7 kg of P would be precluded from entering the environment (Table IX).

TABLE IX

EFFECTOF USE OF PHYTASE ON ABATEMENT OF PHOSPHATE POLLUTION

No. in United States Animal

in1992"

Average livewt. (kg)

Broilers

6 . 1 4 ~ 1 0 ~ 2.01

Layers

3.64 x lo8

Ducks

1.8 x 1 0 7

2.95

Turkeys

2.89 x108

9.91

Pigs

5.78~10'

80.4

kg of feed/ animal

gp excreted/ animal if supplemented with P

gp excreted/ animal if supplemented with phytase

kg PI Yew not excreted

14.5

8.4

1391

80.5/

year

year

year

27 (Estimate)

15.6 (Estimate)

2.81 X

101 (Estimate)

58.5 (Estimate)

1.69 x 107

271 (Estimate)

177

7.08 26.4 265

Total National Agricultural Statistics 1993. 90,000 Tons. Note. 134,082 of P=total value of $1.68 x lo8.

3.75

x 107

3.8 36.41

2.20 x 107

5.62 x

lo5

lofi

8.23 x 107b

PHYTASE

297

VIII. Future Studies

Future studies will probably be concentrated on (i) enzyme engineering to improve the heat stability of the enzyme, reduce the molecular mass, and construct a chimeric enzyme with the acid phosphatase; (ii) elucidation of the 3-dimensional structure of the enzyme and precise glycosylation of the enzyme especially in different plant and microbial systems; (iii) increasing yields in microbial and plant systems by use of various promoters and leader sequences: (iv) application research to find additional uses of the enzyme; (v) basic research on inositol intermediates in plant and animal systems that may create demand for an immobilized enzyme to produce those intermediates; (vi) additional titration of the enzyme in animals for use of least-cost formulations; and (vii) research on delivery systems for the enzyme for use in animal feeds (cloning of the enzyme into various plants high in phytin-P that are used in commercial diets). IX. Summary

Of all the sources of phytase that have been studied (plant, animal, and microorganisms), the highest yields are produced by a wild-type strain A. niger NRRL 3135 (12.7 mg P/hr/ml= 6.8 p m P/ml/min = 113.9 nKat/ml) in a mineral salt medium in which total phosphate (4 mg %) is limiting for growth and cornstarch and glucose are the carbon sources. Synthesis of the enzyme is repressed by phosphate in the wild-type strain. Aspergillus niger NRRL 3135 produces two phytases one with pH optima at 2.5 and 5.5 (phyA) and one with an optimum at pH 2.0 (phyB). It also produces a pH 6.0 optimum phosphatase that has no phytase activity. These three glycoproteins have been purified to homogeneity, characterized, sequenced, and cloned. The sequences have been compared to each other, other phytases, and to known phosphatases. Their homology has been determined. The active sites of phytases show remarkable homology to the active site residues of the members of a particular class of acid phosphatase (histidine phosphatase). The most conserved sequence is RHGXRXP. Phytase has been covalently immobilized on Fractogel TSK HW-75 F and glutaraldehyde-activated silicate. It has been immobilized on agarose. Losses of activity have been noted on immobilization but these may be minimized by future research. It should be possible to commercially produce and recover penta-, tetra-, tri-, di-, and monoinositol phosphates using immobilized phytase if markets develop for those products.

298

RUDY J. WODZINSKI AND A. H. J. ULLAH

Phytase (phyA) from A. niger NRRL 3135 has been cloned into an A . niger glucoamylase producing strain CBS 513.88 using a construct that has a glucoamylae promoter and an A. niger NRRL 3 1 35 leader sequence, and that is devoid of phosphate repression. The yield of the secreted enzyme was increased 52-fold above that of wild-type A. niger NRRL 3135. The bioengineered organism produces 270 pm P/ml/min (4500 nKat/ml) which is approximately 7.9 g/liter in the medium. The yield of the secreted enzyme was increased 1440-fold above that of wild type CBS 513.88. Commercial preparations of the cloned enzyme are available. Phytase (phyA)has been cloned into tobacco and canola. The enzyme is localized in the seed and expressed at high levels. Feeding of the seed to animals has made the phytin-P in the commercial diets available to the animals. The efficacy of feeding phytase to monogastric animals (poultry and swine) has been established. The amount of enzyme that is necessary to be added to commercial diets has been titred for broilers, layers, turkeys, ducks, and swine. The units of enzyme required are related to the phytin-P content in the diet. The use of the enzyme as a feed additive has been cleared in 22 countries. If phytase were used in the diets of all of the monogastric animals reared in the U. S. , it would release phosphorus that has a value of $1.68 x 108 per year. The FDA has approved the enzyme preparation as GRAS. The effect of feeding phytase to animals enables assimilation of the P found in feed ingredients and diminishes the amount of phosphate in the manure and subsequently entering the environment. The effect of feeding phytase to animals on pollution has been quantitatively determined. If phytase were used in the diets of all of the monogastric animals reared in the United States, it would preclude 8.23 x 1 0 7 kg P from entering the environment. REFERENCES Allbritton, N. L., Meyer, T., and Stryer, L. (1992). Science 258, 1812-1815. Anderson, J. W., Pullen, K., Georges, F., Klevit, R. E., and Waygood, E. B. (1993). J. Biol. Chem. 268,12325-12333. Anderson, R. J. (1914a). J. Bid. Chem. 17, 141-150. Anderson, R. J. (1914b). J. B i d . Chem. 17, 151-163. Anderson, R.J. ( 1 9 1 4 ~ )I.. Bid. Chem. 17, 165-170. Anderson, R. J. (1914d). J. B i d . Chem. 17, 171-190. Bajawa, W., Meyhack, B., Rudolph, H., Schweingruber A. M., and Hinnen, A. (1984). Nucleic Acids Res. 20, 7721-7729. Bazan, J. F., Fletterick, R. J., and Pilkis, S. J. (1989). Proc. Nutl. Acad. Sci. USA, 86,9642-9646.

PHYTASE

299

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INDEX

targets, 222 toxins, 253-256 Bacillus thuringiensis, 257 application, 4-6 as biological insecticide, 16-1 7 description, 1 - 4 genes, see Cry genes novel strains, construction, 17-18 protein structure, 10-14 recombinant strains, construction, 18-19 resistance, 25-27 resistance management via refuges, 28-29 via rotations, 27-28 via toxin mixtures, 28 toxins classification, 6-10 delivery systems, 21-26 dosage, 29-30 mechanisms of action, 14-16 vectors, baculoviruses as, 21 Bacteria, see also Archaebacteria in lactic acid production, 50-54 nitrogen-fixing, 1 22-1 2 5 PHAs, biosynthesis, 128-135 phytase sources, 272 Baculoviruses, as Bt vectors, 2 1 Batch processes, 65-66 Beetles, see Cigarette beetles Biodegradable polyesters, 169-1 73 application, 169-173 classification, 98-100 granules, 146-151 microbial production, 158-164 natural, 100 synthetic, 100-104 terminology, 107 Biodegradation, 173-176 under aerobic conditions, 185 approaches, 176-179 bacterial, 176-179 Biopol, 181-182 enzvmatic, 179-180 extracellular, 176

A

Acetoacyl-CoA reductase, 140-142 Acidification, 82 Acid phosphatase active site determinations, 284-285 characterization, 279-281 cloning, 288-291 enzyme engineering studies, 285-288 immobilization studies, 291-292 sequence studies, 281-284 Activated sludge, 164-165 Aedes aegypti, 222 Aeration, 63 Agitation, 63 Alcaligenes eutrophus, 118 copolyesters, 139 Anopheles albimanus, rearing, 222 Archaebacteria, carbohydrate-utilizing halophilic, 125 Aspergillus niger active site determinations, 284-285 mutation studies, 279 phytase isolation, 278-279 phytase production with, 266-268 sequence studies, 281-284

B Bacillus brevis, 243-246 Bacillus circulans, 246-248 Bacillus laterosporus, 248-250 Bacillus megaterium, 115-1 16 Bacillus sphaericus, 257 Bacillus spp. B. alvei systematics, 241-243 biology, 256 candidate cultures, 252 fermentation, 252-253 general considerations, 2 2 1-2 22 Indian meal moths and, 228 products from, 255-256 sources, 221-222 strain comparison techniques, 236-241 303

304

INDEX

Biodegradation (cont.) factors effects, 182-187 hydrolytic, 179-180 methanogenic, 176 in natural environments, 181 PHA depolymerases in, 187-194 polymer composition and, 1 8 7 synthetic polyesters, 194-198 temperature and, 183-184 Biological controls, 257-258 Biological insecticides Bacillus thuringiensis as, 16-17 Biomphalaria glabrata, 231-233 Biopolymers, description, 97-98 Biting blackfly larvae, 224-226 Bt, see Bacillus thuringiensis P-Butyrolactone polymerization, 114-115 C

Centrifugation, 82-83 Cigarette beetles, 227-228 Clean Water Act, 45 Conjugation, in Bt strains construction, 17-18 Continuous membrane cell recycle systems, 68-71 Continuous stirred tank reactor systems, 66-68 Copolymer P(HB-co-HV),polymerization, 113-1 14 Cornstarch, phytase synthesis and, 274-278 cry genes, 8-10 in Bt strain construction, 18-19 expression in plants, 22-25 insect-tolerance and, 31-33 screening, 12-14 structure, 10-12 in transgenic microbe construction, 19-20 Culex quinquefasciatus, rearing, 222 Cutin, 100

D Dialysis systems, 77-79 Distillation, lactic acid, 83

Dreissena polymorpha, 2 33-23 6 Dutch cloning studies, 290

E &Endotoxins, 30

F Feed studies, 292-295 Fermentations B O C ~ ~spp., ~ U 252-253 S B. alvei, 242 B. brevis, 244-246 B. circulans, 246-247 B. laterosporus, 250 extractive, 84-85 lactic acid acidification, 82 adsorption, 83-84 aeration, 63-64 agitation, 63-64 bacteria, 50-54 batch processes, 65-66 centrifugation, 82-83 continuous membrane cell recycle systems, 68-71 continuous stirred tank reactor systems, 66-68 dialysis systems, 77-79 economics, 87-88 equipment, 63 history, 45-47 HPLC, 86-87 immobilized cell systems, 71-77 inoculum development, 64-65 ion exchange, 83-84 microfiltration, 82-83 modeling studies, 79-81 molds, 54 on-line control systems, 87 pH control, 86 process control, 85-86 process kinetics, 79-81 process systems, 58-64 purification, 82 raw materials, 54-58 reverse osmosis, 84

INDEX solvent extraction, 84-85 sterilization, 63 temperature control, 63-64 tubular bioreactors, 71 ultrafiltration, 82-83 PHAs, 159-161 Fungi, see also specific types phytase sources, 272-273 G

Granules, 146-1 51

H Heterodera glycines, 229-231 High-performance liquid chromatography, 86-87 HPLC, see High-performance liquid chromatography Hydrogenophage pseudoJava, 118 Hydroxyacyl-CoA dehydrogenase, 140-142

I Immobilization, 291-292 Immobilized cell systems, 71-77 Indian meal moths, 228 Inoculum development, 64-65 Insecticides, see Biological insecticides Insect tolerance Crygenes and, 31-33 in plants, 21-26 Ion exchange, 83-84

K

305

adsorption, 83-84 aeration, 63-64 agitation, 63-64 bacteria, 50-54 batch processes, 65-66 centrifugation, 82-83 continuous membrane cell recycle systems, 68-71 continuous stirred tank reactor systems, 66-68 dialysis systems, 77-79 economics, 87-88 equipment, 63 history, 45-47 HPLC, 86-87 immobilized cell systems, 71-77 inoculum development, 64-65 ion exchange, 83-84 microfiltration, 82-83 microorganisms, 50-54 modeling studies, 79-81 molds, 54 on-line control systems, 87 pH control, 86 precipitation, 82 process control, 85-86 process kinetics, 79-81 process systems, 58-64 purification, 82 raw materials, 54-58 reverse osmosis, 84 solvent extraction, 84-85 sterilization, 63 temperature control, 63-64 tubular bioreactors, 71 ultrafiltration, 82-83 properties, 47-48 P-Lactone, polymerization, 111-1 13 Lasioderma serricorne. 227-228

P-Ketothiolases, 140 M

L Lactic acid commercial uses, 48-50 distillation, 83 fermentation acidification, 82

Microbes, see Transgenic microbe construction Microfiltration, 82-83 Microorganisms, in phytase production, 266-268 Molds, in lactic acid production, 54 Mosquito larvae, 222

306

INDEX

Moths, see Indian meal moths Mussels, see Zebra mussels N

Nematodes phytoparasitic, 229-231 zooparasitic, 228-229 Nitrogen, PHB synthesis and, 154-155 Nutrition, phytic acid in, 270-271

0 Osmosis, see Reverse osmosis

P Pan Labs cloning studies, 290-291 Pellet formation, in phytase synthesis, 278-279 PHAs, see Poly(hydroxya1kanoates) PHA synthase (polymerase), 143-146 PHB, see Poly(P-hydroxybutyrate) pH control, 86 Phosphorous, phytase synthesis and, 274-278 Phototrophs, 116-118 Phytase active site determinations, 284-285 Aspergillus niger producing, 266-268, 272-273 bacterial sources, 272 characterization, 279-281 cloning, 288-291 enzyme engineering studies, 285-288 feed studies, 292-295 fungal sources, 272-273 immobilization studies, 291-292 microorganisms producing, 266-268 plant sources, 271 pollution abatement and, 295-296 product development, 263-269 sequence studies, 281-284 synthesis cornstarch source effects, 274-278 medium ingredient effects, 278-279 pellet formation effects, 278-279

phosphorous effects, 274-278 yields, 278-279 Phytic acid in nutrition, role, 270-271 in plants, role, 269-270 Plants, see also Transgenic plants phytase sources, 271-274 phytic acid in, role, 269-270 Plodia interpunctella, 228 Pollution abatement, 295-296 Poly (r-caprolactone), 101-103 Polyesters, see specific types Poly(P-hydroxyalkanoates), 146 granules, 149-151 Poly(P-hydroxybutyrate) in activated sludge, 164-165 in Alcaligenes eutrophus, 118 in Bacillus megaterium, 115-116 biodegradation under aerobic conditions, 185 approaches, 176-1 79 bacterial, 176-179 Biopol, 181-182 enzymatic, 179-180 extracehlar, 176 factors effects, 182-187 hydrolytic, 179-180 methanogenic, 176,176-179 under natural environments, 181 PHA depolymerases in, 187-194 polymer composition, 187 temperature and, 183-184 bios ynthesis, 128-1 3 5 , 135-1 38 with acetoacyl-CoAreductase, 140-142 with Alcaligenes copolyesters, 139 with hydroxyacyl-CoA dehydrogenase, 140-142 with P-ketothiolases, 140 PHA synthase (polymerase), 143-146 carbohydrate-utilizing halophilic archaebacteria, 125 characterization, 128 chemical synthesis, 107-115 chemotrophs, 118-125 descriptions, 104-107 extraction, 128 fermentation, 159-161 in Hydrogenophage pseudoflava, 118 microoganism accumulation, 151-15 8

INDEX in natural environments, 164-165 in nitrogen-fixing bacteria, 122-125 in phototrophs, 116-118 physical properties, 165-169 in Pseudomonas oleovorans, 158 in Rhodospirillum rubrum, 118,158-159 in transgenic plants, 125-126 Poly(P-hydroxybutyric acid) biosynthesis, 154-155 granules, 150 Poly-L-malate, 103-104 Polymerization P-butyrolactone, 114-115 copolymer P(HB-co-HV),113-114 p-lactone, 111-113 P-propiolactone, 109-1 11 Polytetramethylene succinate, 103 Precipitation, 82 P-Propiolactonepolymerization, 109-111 Proteins, see Toxin proteins Pseudomonas oleovorans, 158 Pseudomonas pseudoflava, see Hydrogen oph age pse u dofla va

R Rearing processes biting blackfly larvae, 224-226 cigarette beetle larvae, 227-228 mosquito larvae, 222 snails, 232-233 soybean cyst nematode, 230-231 zebra mussels, 233-236 zooparasitic nematodes, 229 Refuges, 28-29 Resistance Bacillus thuringiensis, 25-28 via refuges, 28-29 via rotations, 27-28 via toxin mixtures, 28 Reverse osmosis, 84 Rhodospirillum rubrum, 118,158-1 59 Rotations, in resistance management, 27-28

307

Snails, 231-233 Solvent extraction, 84-85 Soybean cyst nematode, 229-231 Sterilization, lactic acid, 63 Synthetic polyesters, biodegradation, 194-198 Systematics Bacillus alvei, 241-243 B. brevis, 243-244 B. circulans, 246 B. laterosporus, 249-250 definition, 238

T Toxin proteins, 30 structure, 10-14 Toxins Bacillus spp., 253-256 B. th uringiensis classification, 6-10 Cry gene structures, 10-14 delivery systems, 21-26 mechanisms of action, 14-16 dosages, 29-30 genes, see Cry genes mixtures, i n Bt resistance management, 28 Transgenic microbe construction, 19-20 Transgenic plants insect-tolerant , 2 1-26 in pest management programs, 31 PHA accumulation, 125-126 Trichostrongylus colubriformis, 228-229 Tubular bioreactors, 71 U

USDA cloning studies, 288-290 W

Waste disposal, 97 S

Sim u h m vita ttum, 2 24-226 Sludge, activated, 164-165

Z Zebra mussels, 233-236

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CONTENTS OF PREVIOUS VOLUMES Volume 32 Microbial Corrosion of Metals Warren F? Iverson Economics of the Bioconversion of Biomass to Methane and Other Vendable Products Rudy]. Wodzinski, Robert N. Gennaro, and Michael H. Scholla The Microbial Production of 2,3-Butanediol Robert J. Magee and Nain Kosaric Microbial Sucrose Phosphorylase: Fermentation Process, Properties, and Biotechnical Applications ErickJ. Vandamme, Jan Van Loo, Lieve Machtelinckx, and Andre De Laports Antitumor Anthracyclines Produced by Streptomyces peucetius A. Grein INDEX

Volume 33 The Cellulosome of Clostridium thermocellum Raphael Lamed and Edward A. Buyer Clonal Populations with Special Reference to Bacillus sphaericus Samuel Singer

Foam Control in Submerged Fermentation: State of Ine Art N. l? Ghildyal, B. K. Lonsane, and N. G. Karanth Applications and Mode of Action of Formaldehyde Condensate Biocides H. W Rossmoore and M. Sondossi Occurrence and Mechanisms of Microbial Oxidation of Manganese Kenneth H. Nealson, Bradley M. Tebo, and Reinhardt A. Rosson Recovery of Bioproducts in China: A General View Xiong Zhenping INDEX

Volume 34 What’s in a Name?-Microbial Secondary Metabolism J. W Bennett and Ronald Bentley Microbial Production of Gibberellins: State of the Art I? K. R. Kumar and B. K. Lonsane Microbial Dehydrogenations of Monosaccharides Milos Kulhcinek Antitumor and Antiviral Substances from Fungi Shung-Chang Jong and Richard Dona vink

Molecular Mechanisms of Viral Inactivation by Water Disinfectants R. B. Thurman and C. J? Gerba

Biotechnology-The Golden Age V S. Malik

Microbial Ecology of the Terrestrial Subsurface William C. Ghiorse and John Z Wilson

INDEX

309

310

CONTENTS OF PREVIOUS VOLUMES

Volume 35 Production of Bacterial Thermostable aAmylase by Solid-state Fermentation: A Potential Tool for Achieving Economy in Enzyme Production and Starch Hydrolysis B. K. Lonsane and M. V Ramesh

High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins Erik l? Lillehoj and Vedpal S . Malik INDEX

Volume 37 Methods for Studying Bacterial Gene Transfer in Soil by Conjugation and Transduction G. Stotzky, Monica A. Devanas, and Lawrence R. Z e p h

Microbial Degradation of the Nitroaromatic Compounds Frank K. Higson

Microbial Levan Youn W. Han

An Evaluation of Bacterial Standards and Disinfection Practices Used for the Assessment and Treatment of Stormwater Marie L. O’Shea and Richard Field

Review and Evaluation of the Effects of Xenobiotic Chemicals on Microorganisms in Soil R. J. Hicks, G. Stotzky, and l? Van Voris Disclosure Requirements for Biological Materials in Patent Law Shung-Chang Jong and Jeannette M. Birmingham

Haloperoxidases: Their Properties and Their Use in Organic Synthesis M. C. R. Franssen and H. C. van der Plas Medicinal Benefits of the Mushroom Ganoderma S. C. Jong and J. M. Birmingham

INDEX

Volume 36

Microbial Degradation of Biphenyl and Its Derivatives Frank K. Higson

Microbial Transformations of Herbicides and Pesticides Douglas J. Cork and James l? Krueger

The Sensitivities of Biocatalysts to Hydrodynamic Shear Stress Ales Prokop and Rakash K. Bajpai

An Environmental Assessment of Biotechnological Processes M. S. Thakur, M . J. Kennedy, and N . G. Karanth

Biopotentialities of the Basidiomacromycetes Somasundaram Rajarathnam, Mysore Nanjarajurs Shashirekha, and Zakia Bano

Fate of Recombinant Escherichia coli K-12 Strains in the Environment Gregg Bogosian and James F: Kane Microbial Cytochromes P-450 and Xenobiotic Metabolism F. Sima Sariaslani Foodborne Yeasts T Deak

INDEX

Volume 38 Selected Methods for the Detection and Assessment of Ecological Effects Resulting from the Release of Genetically Engineered

CONTENTS OF PREVIOUS VOLUMES Microorganisms to the Terrestrial Environment G. Stotzky, M. W Broder, J. D. Doyle, and R. A. Jones Biochemical Engineering Aspects of Solid-state Fermentation M. I/: Ramana Murthy, N. G. Karanth, and K. S. M. S. Raghava Rao The New Antibody Technologies Erik I! Lillehoj and Vedpal S. Malik Anoxygenic Phototrophic Bacteria: Physiology and Advances in Hydrogen Production Technology K. Sasikala, Ch. R Ramona, l? Raghuveer Rao, and K. L. Kovacs INDEX

Volume 39 Asepsis i n Bioreactors M. C. Sharma and A . K. Gurtu Lipids of n-Alkane-Utilizing Microorganisms and Their Application Potential Samir S. Radwuan and Naser A . Sorkhoh Microbial Pentose Utilization Prashant Mishra and Ajay Singh

311

Volume 40 Microbial Cellulases: Protein Architecture, Molecular Properties, and Biosynthesis Ajay Singh and Kiyoshi Hayashi Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective D. K. O’Toole Challenges in Comnmercial Biotechnology. Part I. Product, Process, and Market Discovery Ales Prokop Challenges i n Commercial Biotechnology. Part 11. Product, Process, and Market Development Ales Prokop Effects of Genetically Engineered Microorganisms on Microbial Populations and Processes in Natural Habitats Jack D. Doyle, Guenther Stotzlq Gwendolyn McClung, and Charles W Hendricks Detection, Isolation, and Stability of Megaplasmid-Encoded Chloroaromatic Herbicide-Degrading Genes within Pseudomonas Species Douglas J. Cork and Amjad Khalil INDEX

Medicinal and Therapeutic Value of the Shiitake Mushroom S. C. Jong and J. M. Birmingham Yeast Lipid Biotechnology Z. Jacob Pectin, Pectinase, and Protopectinase: Production, Properties, and Applications Takuo Sakai, Tatsuji Sakamoto, Johan Hallaert, and Erick J. Vandamme Physicochemical and Biological Treatments for Enzymatic/Microbial Conversion of Lignocellulosic Biomass Purnendu Ghosh and Ajay Singh INDEX

Volume 41 Microbial Oxidation of Unsaturated Fatty Acids Ching T. Hou Improving Productivity of Heterologous Proteins in Recombinant Saccharomyces cerevisiae Fermentations Amit Vasavada Manipulations of Catabolic Genes for the Degradation and Detoxification of Xenobiotics Rup Lal, Sukanya Lal, l? S. Dhanaraj, and D. M. Saxena

312

CONTENTS OF PREVIOUS VOLUMES

Aqueous Two-Phase Extraction for Downstream Processing of EnzymedProteins K. S . M. S. Raghavarao, N . K. Rastogi, M. K. Gowthaman, and N . G. Karanth Biotechnological Potentials of Anoxygenic Phototrophic Bacteria. Part I. Production of Single Cell Protein, Vitamins, Ubiquinones, Hormones,

and Enzymes and Use in Waste Treatment Ch. Sasikala and Ch. V Ramana Biotechnological Potentials of Anoxygenic Phototrophic Bacteria. Part 11. Biopolyesters, Biopesticide, Biofuel, and Biofertilizer Ch. Sasikala and Ch. V Ramana INDEX

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ISBN 0-L2-002642-2