Advances in Biochemical Engineering, Volume 003

Advances in Biochemical Engineering 3 Edited by T. K. Ghose, A. Fiechter, and N. Blakebrough With 119 Figures Springer...

Author: T.K. Ghose | A. Fiechter | N.Blakebrough

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Advances in Biochemical Engineering 3 Edited by

T. K. Ghose, A. Fiechter, and N. Blakebrough With 119 Figures

Springer -Verlag Berlin. Heidelberg. New York 1974

T. K. GHOSE Dept. of Chemical Engineering, Indian Institute of Technology, New Delhi/India A . FIECHTER Mikrobiologisches Institut der Eidgen. Techn. Hochschule, Zfirich/Switzerland N . BLAKEBROUGH The University of Birmingham Dept. of Chemical Engineering, Birmingham 15/Great Britain

ISBN 3-540-06546-6 Springer-Verlag Berlin • Heidelberg • N e w York ISBN 0-387-06546-6 Springer-Verlag N e w York. Heidelberg • Berlin

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. © by Springer-Verlag Berlin . Heidelberg 1974. Library of Congress Catalog Card Number 72-152360. Printed in Germany. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting and offset printing: Zechnersche Buchdruckerei, Speyer. Bookbinding: Bri~hlsche Universit~itsdruckerei GieBen.

Contents

Ciba-Geigy/Lepetit Seminar on Topics of Fermentation Microbiology CHAPTER 1 Genetic Problems of the Biosynthesis of Tetracycline Antibiotics Z. HOS'I"ALEK,M. BLUMAUEROVA,and Z. VAN~K, Praha (CSSR) With 22 Figures CHAPTER 2 Some Aspects of Basic Genetic Research on Fungi and Their Practical Implications K. ESSER, Bochum (Federal Republic of Germany) With 5 Figures CHAPTER 3 Microbial Oxidation of Methane and Methanol N. KOSARICand J. E. ZAJIC, London, Ontario (Canada) With 7 Figures

13

69

89

CHAPTER 4 Modelling and Simulation in Biochemical Engineering 127 H. W. BLANCHand I. J. DUNN, Zi,irich (Switzerland) With 26 Figures CHAPTER 5 Transient and Oscillatory States of Continuous Culture D. E. F. HARRISONand H. H. TOPIWALA, Sittingbourne, Kent (Great Britain) With 24 Figures

167

CHAPTER 6 The Significance of Microbial Film in Fermenters B. ATKINSONand H. W. FOWLER, Swansea (Great Britain) With 33 Figures CHAPTER 7 Present State and Perspectives of Biochemical Engineering I. MALEK, Praha (CSSR) With 2 Figures

221

279

Ciba-Geigy/Lepetit

Seminar on Topics of Fermentation Microbiology June 19-23, 1972, Zermatt (Switzerland)

The Microbiological Sections of the two pharmaceutical companies Ciba-Geigy Basel and Lepetit Milan held a joint Seminar on various microbiological topics in Zermatt, Switzerland, from June 19th to 23rd 1972. With the exception of the invited speakers, the participation was restricted to members of both companies and a few scientists from University Institutes associated with them. Dr. Ch. Stoll, Ciba-Geigy Basel, was in charge of the administrative part of the meeting whereas Dr. J. N~iesch from the same company was responsible for the scientific programme. The aim of this Meeting was to transmit to the academically trained personnel of both companies some of the progress made in genetics and molecular biology. After the biosynthesis of industrially interesting antibiotics, tetracyclines, rifamycins and/~-lactam antibiotics was outlined. Moreover the technique of continuous culture was delt with. This technique has various interesting applications for research and development, although it is relatively unknown in the field of antibiotics. It was the intention of the organizers of the meeting to concentrate on fundamentals rather than applied aspects of the subject. Application in the industrial laboratories and plants was worked out during the discussions. Considerable time was therefore reserved to analyze the various new aspects appearing in the course of the Seminar. This particular organisation proved to be very useful.

2

Ciba-Geigy/Lepetit

The scientific programme was organized as follows:

1. Genetics 1t. Introduction (G. Magni, Lepetit S.p.A., Milan, Italy) 12. Genetics of fungi (K. Esser, Ruhr University Bochum, West Germany) 13. Genetics of actinomycetes (D.A. Hopwood, John Innes Institute, Norwich, England) 2. Regulatory aspects of enzymes 21. Active and passive control of enzyme synthesis (R. Hiitter, ETH, Ziirich, Switzerland) 22. Structure of allosteric proteins and their regulation properties (G. N. Cohen, Institute Pasteur, Paris, France) 3. Continuous cultivation in application .for the study of biosynthesis and process development of antibiotics 31. Continuous fermentation of filamentous organisms (S.J. Pirt, Queen Elizabeth College, London, England) 4. Mechanisms of biosynthesis of antibiotics 41. Biosynthesis of tetracyclines (Z. Vanek, Institute of Microbiology Czech., Acad. Sci., Prag, CSSR) 42. Biosynthesis of rifamycins (G. Lancini, Lepetit, Milan, Italy) 43. Biosynthesis of/Mactam antibiotics (J. Ntiesch, Ciba-Geigy, Basel, Switzerland) Some of the contributions were prepared for publication. Summary and reference of these articles are given here whilst two of them are reported in extenso in this volume (Chapters 1 and 2). HOPWOOD, D.A.: Genetics of Actinomycetes. G. Sykes (Ed.). Actinomycetes. Symp. Soc. Appl. Bact., p. 9--31, 1973. Summary Genetic recombination has so far been discovered in members of four genera of actinomycetes: Streptomyces, Nocardia, Micromonospora and Thermoactinomyces. In the first three, there is reasonable evidence that some kind of "conjugation" process is responsible for gene transfer, whereas in Thermoactinomyces, transformation occurs. Transformation has not been unequivocally demonstrated in Streptomyces; results with Thermoactinomyces reveal one possible cause for this. By far the best known genetic system in the Actinomycetales is that of Streptomyces coelicolor A3(2), although studies of two other species, S.

Seminar on Topics of Fermentation Microbiology

3

rimosus and S. bikiniensis, have reached the stage of linkage analysis. Comparison of the linkage maps of these three species suggests that a common gene arrangement is (largely) conserved in the genus, even when strains have been subjected to enormous abuse by chemical and physical mutagens. Study of the fertility system of S. coelicolor A3(2) has reached the stage at which a donor (NF) and two classes of recipient strains (IF and UF) have been recognised. IF is the fertility of the wild-type, and NF arose from this by a chromosomal event (which may have been the integration of a plasmid); thus IF and NF segregate as "alleles" in a cross between these two fertility types. UF strains arise with a rather high frequency from IF strains by loss of plasmid SCPI, and can be reinfected with the plasmid by contact with an IF strain. The frequency of recombinant production in mixed cultures ranges from about 10-5 in the least fertile combination (UF x UF) to 1 in the most fertile (NF x UF). Streptomyces genetics has reached the stage where it can aid industrial strain-improvement programmes in several ways. One has been the provision of more efficient procedures for chemical mutagenesis. A second concerns the predictive power of a common linkage map, which should allow the use of markers of known map location without the necessity of mapping the markers in a new strain. An area which is still untried, but is on the threshold of feasibility, is the introduction of specific characters from a donor strain into a recipient strain on substituted plasmids, if not by transduction or transformation. Actinomycetes have much to offer to the study of differentiation in being the most complex prokaryotes in which genetic analysis is yet feasible. Spore delimitation in S. coelicolor and the three-dimensional geometry of the Thermoactinomyces vulgaris endospore are two topics which are currently under investigation.

HOTTER,R.: Passive and Active Control of Enzyme Synthesis. Regulation der Enzymsynthese bei Bacterien und Pilzen. Fortschr. Botan. 34, 309--323 (1972). Summary The levels of gene products, be it RNA or protein, are controlled in diverse ways. An intensive effort to elucidate the mechanisms of reyuIation has been made with the bacterium Escherichia coli. We wilt concentrate on this organism and focus our attention on some selected examples. The result will necessarily be a very schematic and 9eneralized picture of the real situation.

4

Ciba-Geigy/Lepetit

PIRT, S.J.: Continuous Fermentation of Filamentous Organisms. A monograph on continuous cultivation of microorganisms is in preparation. Summary

1. Uses of continuous flow culture in fermentation studies Limitations of batch culture - - special functions of the chemostat - difficulties in chemostat culture. Studies on effects of growth rate and environment on penicillin ferment a t i o n - the maintained state - - behaviour of microorganisms of growth rates.

2. Production of penicillin by tysine auxotrophs of Peniciltium chrysogemm7 Possible regulation of penicillin synthesis. Production and characterisation of lysine auxotrophs. Effects of lysine and :~-amino-adipic acid on penicillin production by parent and auxotrophic mutants.

LANCINI,G.: Biosynthesis of rifamycins. Lancini et al. Progr. Antimicrol. and Anticancer Res. 2, 1166 (1970); Lancini, G., White, R.J.: Proc. Biochem., p. 14, July 1973. N# ESCH, J.; Biosynthesis of/~-Lactam Antibiotics. Niiesch, J., Treichler, H.J., Liersch, M. (1970): Genet. Ind. Microorg. Van~k, Z., Hog~filek, Z., Cudlin, J. (Ed.), pp. 309--334. Prague: Academia 1973. Lemke, P.A., Brannon, D.R. (1972): Cephalosporins and Penicillins. Flynn, E.F. (FA.), pp. 370--430. New York and London: Academic Press 1972. Summary The /J-lactam antibiotics form a group of natural substances characterized by a bicyclic ring system: a four membered lactam ring fused with a five- or six-membered heterocycle. Although these compounds have many common features with regard to their structure, their biosynthesis as well as their biological activities, they can be divided into two groups on the basis of certain aspects of their biosynthesis. The penicillium-type comprises/?-lactam antibiotics with 6-amino-peniciltanic acid (6-APA) as nucleus and various N-acyl side chains. Generally of a nonpolar aliphatic or aromatic carboxylic acid type and L-a-amino acid. The synthesis of a specific penicillin is directly dependent on the N-acyl side chain precursor in the culture medium. In the absence of side chain precursor 6-APA may be accumulated. All producers in this groups are eucaryotic fungi.

Seminar on Topics of Fermentation Microbiology a) Penicillium-type R

Nucleus ( unvariable}

(N-Acyl side chain)

Producers

{variable)

(

6-AminopeniciIlanic acid }

Penicillium notatum

HOOC-CH{NH2} ICH213--CO-(L-~--aminoadipicacid ]

C~H3 CH3 s'~COOH

PeniciHium chrysogenum Other species of penicillia

CH3-CH2--CH=CH-CH2-co (flsy-hexenoic acid ) CH3(CH2 }3--CO-loctanoic Ocld )

Dermatophytes

C6H5-CH2-CO( phenylaceticacid)

Aspergil(us sp.

C6Hs-O-CH2-CO-{phenoxyacetic acid )

Malbranchea pulchella

b) Cephalosporium-type Nucleus (variable)

R1 (N-Acyl side chain} {unvariab{e)

R2

R3

Producers

CH3 CH3 HS , , ~ C O 0 1 ~ (6-aminopeniciilanic acid)

Cephalosporium sp.

\ N/ F [

Emericellopsis sp. Paeci|omyces persic i nus Streptomycetes

R,HN~

HOOC-CH (NH2)(CH2)3- CO, (D-ez'--aminoadipicacid}

%

H- -OCOCH3 CH2-R 3

H- -OH

coo.

S

\~"

R1HN

"O

HOOC-CH(NH~llCH.)~-CO- -OC~ I-OCOCH3 Cephafosporium acremoniul (D-(~-aminoadipic acid ) H- -OCONH2 Streptomycetes -OCH3 -OCONH2

6

Ciba-Geigy/Lepetit

The cephalosporium-type on the other hand is characterized by D-fiaminoadipic acid as the unique N-awl side chain. In contrast to the penicillium-type two nuclei are found, namely 6-APA and 7-aminocephalosporanic acid (7-ACA). Whereas in 6-APA the four membered lactam ring is fused with a five membered thiazolidine ring, 7-ACA is a bicyclic structure composed of the same lactam ring but fused with a six membered dihydrothiazine ring. Characteristic for the formation of this type offl-lactam antibiotics is its insensitivity to the addition of side chain precursors to the culture medium. Eucaryotic as well as procaryotic microorganisms are known as producers.

Cysteinyl moiety and sulfur metabolism (after Lemke and Brannon, 1972)

t

L-CYSTEINE

HS-~~L-~Serine

•

Sz05 -

L-Cystathionine

IT

L-Homocysteine

[ [

J S05 -

PAPS

, Choline-SOj

Choline

APS

SO2- (endo),

(endo) Dk-Methionine

l

Methioninepermeases

(exo) DL-Methionine

I Sulfatepermease SO2 (exo) Penicillium

Cephalosporium

~

L-2-Aminoadipic acid

or

C=O

COOH

,

CH2

Adenyl-aminoadipic acid semialdehyde

Adenosine

I

COOH Aminoadipic acid semialdehyde + L-glutamic acid

I CH2 I CH2 F

f I

CH2

CH2

[

Saccharopine

COOH

CH2

P

CH2

H--C--N-H

HOOC

;-"--~

CH2 ,

H2N--CH--COOH

Oxaloglutaric acid

COOH

I CH2 d

CH2

CH--COOH

I

O==C--COOH

.... '

H2N--CH--COOH

+

Adenosine

NN Adenyl-aminoadipic acid

I I

CH2

O

,

CH2

H--C~O

....

,

H2N--CH--COOH

H--C--OH

CH2

CH2

O

'

CH2

COOH

I CH2 I

CH2

Homoisocitric acid

'

CH--COOH

HO--CH--COOH[

H2N-CH--COOH

Homoaconitic acid

COOH

I CH: I

' CH2

C--COOH

CH--COOHj[

] 0

O==P--O-

I 0 r

CH2

CH2

I I

CH2

J J

'

CH2

,

CHz

[

[

Homocitrate

COOH

t CH2 I

CH2

CH2

"J{ ,

HO--C--COOH

H2N--CH--COOH

\

CH--COOH

H2N--CH--COOH

2-Ketoglutarate

COOH

r CH2 I

CH2

r

O~---C--COOH \

Acetat+

CH3--COOH

,

I

I [ CH2 [ NH/

CH2

CH2

2-Ketoglutarate

+

L-Lysine

'

CH2

[

,--

HzN--CH--COOH

2-Ketoadipic acid

COOH

I CH2 I

CH2

CH2

~=C--COOH

Biosynthesis of the presumable c o m m o n precursor amino acids of the fl-lactam antibiotics L-2-aminoadipyl moiety and L-lysine metabolism

8

Ciba-Geigy/Lepetit

All/~-lactam antibiotics seem to derive from the same three amino acids, L-~-aminoadipic acid, L-cysteine and L-valine. Obviously these precursors form a tripeptide which undergoes internal cyclization to the final compounds. In both types the e-amino-adipic moiety derives from the lysine biosynthetic pathway. Whereas in the penicillium-type of antibiotics this amino acid is in general replaced in the final product by a variety of N-acyl side chains the same amino

Valinyl moiety and L-valine metabolism TPP (CH3--CHO)

NADH

--H20

R--NH~ I

CH~

£o

CH3

I

H3C--C--OH

H3C--C--OH [ , H / COOH

COOH

I

CH3 /

r

H3C--C--H

OH 1_~

/=O

I

COOH

COOH

CH3 H3C--C--H [ tt --C--NH2 I

I

COOH

Pyruvate 2-Acetolactate 2,3-dihydroxy- 2-Ketoisovalerate L-Valine isovalerate t | i I I

L-Leucine acid is irreversibly bound as the D-isomer in the biosynthesis of the Cephalosporium-type. A further difference in biosynthesis between the two types is found in the formation of cysteine. In contrast to the Penicillium-type where inorganic sulfur is the optimal sulfur source for the cysteine which is subsequently incorporated into 6-APA, methionine is the optimal sulfur donor in the Cephalosporium-type. No fundamental differences could yet be detected with regard to valine. The condensation of the three amino acids and their cyclization is still a matter of speculation.

Formation of a common tripeptide and its cyclization to fl-lactam antibiotics (Hypothesis)

+H3N 9 9+H3N ÷H3N "CH3 L/xCHICH2)3C-O-P-O- Adenine ~.CHCH2SH ¢CHC~

+H~N

/

S..~

÷H3N~c/S~--N,~CO o-~×

N~J-~o Penicillium

+ N

m

~>..~A.coH_T__KS,h<:

-ooc

Cepholosporium

OOC~coH_~_KS., ~

o~...L_,~__Lcoo_ " , ~ , ~

o~J_,_~l.:coo +

_j~:~coo-~,~ "

0

"mN>...,,v.~coHT__FSh

f" CH2COO--ooc

o~.j_~/...~CH3,~ ' COOl

+H N~ O.11~'-N --'-"~COO3

v

A

s

.CON..T.~ h O~_L. ~ I(I,,~CH2OAc COO-

After A. L. Demain: In J. Snell (Ed.): Biosynthesis of Antibiotics, Vol. 1, pp. 29 (1966) and R.D.G. Cooper, S.L. Jos6: J. Amer. chem. Soc. 94, 1021 (1972).

94

Although no in vitro systems for the formation of fl-lactam antibiotics are yet available a few enzymatic reactions have been found in connection with the biosynthesis.

10

Ciba-Geigy/Lepetit

Known enzymatic reactions with real or hypothetical functions in the synthesis of fi-lactam antibiotics 1.

Peptide-symhetases

1.1. 6-(L-~-aminoadipyl)-L-cysteine + DL@eC)-valine crude extract ofC. acremonium

| ~

ATP PEP + PEP-kinase

6-(L-~-aminoadipyl)-L-cysteinyl-?-valine

1.2. DL-c~-aminoadipic acid + L-cysteine + L-valine crude extract of P. chrysooenum

/ ~

ATP PEP + PEP-kinase

6-(L-e-aminoadipyl)-L-cysteinyl-L-valine 2.

N-Acylases 0

H

0

II/

R--C--N--6-PA ~ 3,

II

R - - C - - O f t + 6-APA

AO,I- Transferases

3.1. OH

OH

*R--C--N

6-PA + R - - C - - N - - 6 - P A * ~ _ _ - , *R--C--N

H 1

O

+ R--C

H

6-PA* N--6-PA

II I

O

3.2. R--C--N--6-PA* + 6-APA ~

II

O

i

H

H

R - - C - - N - - 6 - P A + 6-APA*

lI

O

E

H

References

1. Reviews Abraham, E.P., Newton, G.G.: In: Antibiotics If. Gottlieb, D., Shaw, P.D. (Ed.), p. 1--16. Berlin - Heidelberg- New York: Springer 1967. Abraham, E. P.: In: Topics in Pharmaceutical Sciences. Perl-Man, D. (Ed.), p. 1 31. New York: Interscience Publisher 1968. Demain, A.L.: In: Biosynthesis of Antibiotics, Vol. 1. Snell, J. (Ed.), p. 29--94. New York: Academic Press 1966. Flynn, E.H.: Cephalosporins and Penicillins, 752 p. New York and London: Academic Press 1972.

Seminar on Topics of Fermentation Microbiology

2. Genetics Ball, C.: J. Gen. MicrobioL 66, 63 (1971). Elander, R. P.: Abhandl. Deut. Akad. Wiss. Berlin, K1. ffir Med. 2, 403 (1967).

3. Biochemistry Arnstein, H.R.V., Morris, D.: Biochem. J. 76, 357 (1960). Benz, F , Liersch, M., Nfiesch, J., Treichler, H.J.: Eur. J. Biochem. 20, 81 (1971). Loder, P.B., Abraham, E. P.: Biochem. J. 123, 477 (1971).

4. Regulation of Biosynthesis Goulden, S.A., Chattaway, F.W.: Biochem. J. 110, 55 (1968). Goulden, S.A., Chattaway, F.W.: J. Gen. Microbiol. 59, 111 (1969). Lemke, P.A., Nash, C.H.: Can. J. Microbiol. 18, 255 (1972).

1

CHAPTER

I

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics Z. HOg~ALEK, MARGITA BLUMAUEROVA, a n d Z. VAN~K With 22 Figures

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Selection of ltigh-Production Strains of S. aureq[aciens and S. rimosus a) Mutagen Treatment . . . . . . . . . . . . . . . . . . . . . b) Selection in Mutant Populations . . . . . . . . . . . . . . . . c) Hybridization . . . . . . . . . . . . . . . . . . . . . . . . d) Transduetion . . . . . . . . . . . . . . . . . . . . . . . . e) Increase of Resistance to its Own Metabolite . . . . . . . . . . 2. Isolation and Characterization of Mutants Blocked in the Biosynthesis of Tetracycline Antibiotics . . . . . . . . . . . . . . . . . . . a) Mutant Metabolites of S. aureofaciens . . . . . . . . . . . . . b) Mutant Metabolites of S. rimosus . . . . . . . . . . . . . . . c) Interspecific Cosynthesis . . . . . . . . . . . . . . . . . . . . 3. Genetic Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a) The Linkage Map of S. rimosus b) Analysis of Loci Controlling Tetracycline Biosynthesis ...... c) Interspecific Recombination . . . . . . . . . . . . . . . . . . 4. Genetic Control of the Biosynthetic Process . . . . . . . . . . . . a) Genes Responsible for the Biosynthesis of Tetracyclines . . . . . . b) Quantitative Aspects of the Biosynthesis of Tetracyclines ..... 5. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

15 15 17 18 21 2I 22 24 31 35 35 36 38 44 44 44 56 60 63

Introduction T h e g r o u p o f t e t r a c y c l i n e a n t i b i o t i c s is c o m p r i s e d o f c h l o r t e t r a c y c l i n e (CTC) p r o d u c e d by S t r e p t o m y c e s a u r e o f a c i e n s (Duggar, 1948) a n d oxytetracycline ( O T C ) p r o d u c e d by S t r e p t o m y c e s r i m o s u s ( F i n l a y et al., 1950).

14

Z. HO~'IALEK et aI.

The two antibiotics certainly represent the standard metabolites of these two species- in spite of their structural similarity and related biogenetic origin of the compounds one may discern the species specificity of several biosynthetic steps, namely the chlorination in position 7 of the tetracycline ring system of S. aureofaciens and the hydroxylation in position 5 ofS. rimosus. Another important antibiotic of the tetracycline series is tetracycline (TC) itself, produced by standard strains of S. aureofaciens or S. rimosus (Perlmann et al., 1960) as a minor metabolite, and by mutants of S. aureofaciens blocked in the chlorination step, as a major product (Doerschuk et al., 1959). The formation of TC has been described in a number of other producers but their taxonomy is an unresolved problem which will not be discussed here. The problems of genetic and metabolic regulation of the chlorination of the tetracycline nucleus were reviewed by Petty (1961). In addition to the above-named, both S. aureofaciens and S. rimosus produce a number of other compounds which occur under standard conditions as minor metabolites or are produced by biochemical mutants of the two species. A list of these compounds, some of known and some of unknown chemical structure, will be found in the second section of this chapter. Most of them are not active antibiotically but are worth investigating for a better understanding of the genetic control of the biosynthetic process. Immediately after their discovery, the tetracycline antibiotics became important fermentation products. This fact stimulated intense research into the process of biosynthesis of these compounds in a number of laboratories. More than twenty years of study have provided vast experimental material, including data on strain improvement, metabolism of the blocked mutants and on the genetic processes of the production strains. In the present paper we attempt to present an overall view of the genetic problems associated with the production of tetracycline antibiotics and, at the same time, to draw general conclusions on the genetic regulation of their biosynthesis.

R 7 Me OH R 5

OH

0

OH

Fig. 1. Structures of tetracycline antibiotics

N Me 2

0

CTC OTC TC

R5

R7

H OH H

CI H H

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

15

1. Selection of High-Production Strains of S. aureofaciens and S. rimosus a) M u t a g e n T r e a t m e n t Extensive selection work focussing on increasing the production of tetracycline antibiotics has been done mostly in industrial laboratories and the results have not been published. However basic information on the possibilities of strain improvement can be obtained from several papers describing the effects of physical or chemical mutagens on S. aureofaciens (Van Dyck and De Somer, 1952; Katagiri, 1954; Niedzwiecka-Trzaskowska and Stzencel, 1956; Wang, 1957; Alikhanian and Romanova, 1965; Alikhanian et al., 1957; 1959a; Goldat, 1958, 1961, 1965; Goldat and Sokolova, 1964; Blumauerovfi et al., 1973a) and on S. rimosus (Borenztajn and Wolf, 1956; Mindlin and Alikhanian, 1958; Alikhanian et al., 1959a; Htiber and Giinter, 1960; Goldat and Vladimirov, 1968). In spite of the fact that the work was carried out with different strains and under various experimental conditions, the results indicate that the most effective factor for increasing the productivity in both species is UV light, applied either alone or in combination with other mutagens. Thus, for example Blumauerovfi et al. (1973a) working with the production strain ofS. aureofaciens 84/25 in populations surviving after UV-irraTable 1. Comparison of spontaneous and induced variability of CTC production in S. aureofaciens (Blumauerovfi et al., 1973a) Spread in productivity (% of controlp Mutagen a

Frequency of superior producers (%)~

Total range

The most frequent class

40~110

80-100

5.3

UV light X-rays y-radiation

0---130 0-- 110 0--I 10

90--110 7 0 - 90 0 - 10

17.4 7.1 1.8

MNG N-mustard

0~120 0~110

40-- 80 80--100

9.4 5.8

None

a The following doses of mutagens resulting in less than t % of surviving conidia were used: UV light, 50~sec; )'-radiation and X-rays, 100 kR; N-methyl-N'-nitro-Nnitrosoguanidine (MNG), 1 mg/ml, for 60 min (in 0.02 M phosphate buffer, pH 6.7): N-mustard, 0.01 M, for 30 min {in 0.15 M phosphate buffer, pH 8.0). b The productivity of the parent strain is taken as 100 per cent. c Expressed as the percentage of total number of isolates tested.

16

Z. HO~1"ALEKet

al.

diation, obtained more than 17% variants with production exceeding the activity of the parent strain by 10--30%. Of the other tested mutagens the most effective one was N-methyl-N'-nitro-N-nitrosoguanidine while X-rays and nitrogen mustard were relatively ineffective. On the other hand, 7-radiation induced the highest number of inactive variants (Table 1). Goldat (1961) applied an eight-step selection procedure with UV light in combination with photoreactivation or with preceding treatment with sublethal doses of X-rays or ethylenimine, to achieve a five-fold increase of CTC production, in comparison with the activity of the parent low-production strain of S. aureofaciens 77 (Fig. 2). Working 77 (60O) 1 u.v i

536(t000) I

] UV

546(1000)

UV-~PR4.UV

112 (1200) [

El -~V

I

" 15(14001

1

XTUV

X

134 (1700)

2O5 (1700)

I

XTUV 16(2200) I

XTUV

542 (2260) I

El

E4f2-2 (2460) I

X-~ El -... UV

t 2185 (3000)

f

2201 (,:3500)

Fig. 2. Scheme of selection of new active variants in S. aureojaciens (after Goldat, 1961). Key: UV, UV light; PR, photoreactivation; EI, ethyleueimine; X, X-rays. Numbers in parenthesis represent the production level in ~tg CTC/ml with S. rimosus, Mindlin and Alikhanian (1958) applied repeatedly UV light to the parent production strain 8229 to obtain the variant LS-T 118 with production activity by 67% higher. Further UV-irradiation of S. rimosus LS-T 118 produced variant LS-T 293 which displayed

Genetic Problems of the Biosynthesisof TetracyclineAntibiotics

t7

only a little greater production activity than the parent strain but was more resistant to higher levels of inorganic phosphate in the fermentation media (Alikhanian et al., 1959b). Both species showed considerable morphological variability after treatment with mutagenic factors (alterations in size, shape, structure and pigmentation of colonies and in sporulation ability), most of the morphological mutants having a decreased antibiotic activity. On the other hand, most variants with greater biosynthetic activity did not differ morphologically from the standard type (Mindlin and Alikhanian, 1958). It thus appears that mutagens and doses bringing about maximum frequency of morphological mutants produce also the highest number of (--) variants and are not suited for selection work. S. aureofaciens is frequently reported as a typical example of a highly variable species of Streptomyces (Backus et al., 1954; Duggar et al., 1954; Kutzner, 1967). Its high spontaneous variability may frequently cause the instability of the production strains obtained through selection (Horvfith, 1954). Likewise, some strains of S. rimosus were observed to segregate spontaneously into morphological and nonproduction variants (Alikhanian et al., 1959a). Using multiple-step selection of producers of tetracycline antibiotics we found it useful to subject the isolates to natural selection before applying further mutagenic treatment. In this way we obtained not only a general stabilization of the given isolate but also frequently a further increase of its biosynthetic activity, as well as an increase of mutagen efficiency in the subsequent selection step. b) Selection in M u t a n t Populations To increase the efficiency of the selection procedure of S. viridifaciens and S. rimosus a method was developed, based on a selection of high-production variants from different types of mutant populations. The highest number of these variants was obtained among revertants of nonproduction mutants (i.e. mutants in which a previous mutagenic treatment has already caused mutation in loci controlling tetracycline biosynthesis). Some superior producers were also obtained by an indirect selection among auxotrophic mutants growing in the presence of high concentrations of the required growth-factor and among prototrophic revertants of induced auxotrophs (Dulaney and Dutaney, 1967; Dulaney, 1969). However, yield improvement through auxotrophy does not seem to be the way of choice with S. aureofaciens where most nutritional mutants show a very much decreased or completely lost antibiotic activity even in media with increased amounts of the given factor (Blumauerovfi et aI., 1973b).

18

Z. HO~ALEKet al.

c) Hybridization After the discovery of genetic recombination in S. rimosus (Alikhanian and Mindlin, 1957) and in S. aureofaciens (Alikhanian and Borisova, ! 961) the possibility of practical application of the recombination process was investigated as a new method for breeding the production strains in both species (J~irai, 1961; Borisova et al., 1962a;'Alikhanian et aI., 1959a; Mindlin et al., 1961a, 1961c; Vladimirov and Mindlin, 1967). The starting material for these experiments consisted of biochemical mutants (single auxotrophs or double mutants carrying at the same time markers of nutritional deficiency and resistance to streptomycin) derived after mutagenic treatment from various strains differing among themselves in their production capacity and some other characteristics. The nutritional mutations exerted usually a negative effect on antibiotic activity which, in most auxotrophs of S. aureofaciens and S. rimosus, attained only ~ - I 0 % or even less of the production level of the parent prototrophs. When estimating the biosynthetic activity of prototrophic recombinants selected from mixed cultures of pairs of biochemical mutants of S. aureofaciens different authors obtained different results. Recombinants obtained by Alikhanian and Borisova (1961) from crossing mutants derived from strains LS-536 and Bd (producing 850--1000 pg CTC/ml) generally showed a higher activity than the parent monoauxotrophs but mostly did not exceed the level produced by the prototrophic parents. Only the cross of arg-3 × ilv (as the only one of 11 tested arg × ilv crosses) gave rise to recombinants with greater activity. Similarly, Borisova et al. (1962a) obtained most recombinants with activity lower than or identical with that of the parent strains. An exception here were some recombinants from the arg x ala crosses, exceeding the activity of the production prototrophic parent LS-B 2201 (3,300 pg CTC/ml) by about 6% (Table 2). On the other hand, auxotrophic mutants isolated by Jfirai (1961) from six strains of S. aureofaciens (derived from LS-536 and LE-8234 and producing 1100--1690 pg CTC/ml) gave rise to three different types of recombinants: group I (11.5% of the total number of recombinants tested) produced 40~60% more of the antibiotic than the prototrophic parents, group II (31.7% recombinants) showed the same activity as the prototrophic strains and group III (56.8% recombinants) had an activity as low as the auxotrophic parent strains. The most active recombinants were obtained from the arg x met crosses. Hybridization experiments with S. rimosus were carried out with mutants derived from four production strains (101, 8229, LS-T 293 and BS-21). The recombinants obtained were usually more active than the parent auxotrophic mutants and frequently attained the productivity of the

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

19

Table 2. Productive activity of the original strains of S. aureofaciens, their biochemical mutants and prototrophic recombinants (after Borisova et al., 1962) Strain

Activity of original strain (gg/ml)

LS-B 2201 3300

Biochemical mutants

Prototr0phic recombinants

Code

Activity 0ag/ml)

Strains

50-- 70 250~ 370 60(0--1000

1 x 13 lx 8 220t x 2201 5 x 14 10 x 14 26 x 14

6(X)---3300 10(O2700 560--3500 25(~-2500 40(02000

5,8.10 13, 26 arg 1 thr 14 ala

C r o s s e d Activity mutants (gg/ml)

536

900

6 ilv

traces

2201x 536 14× 6 1x 6

500~-2300 1000--2700

Bd

900

4 his

traces

2201xBd

180~ 880 40(02500

14× 4 1x 4

original prototrophs; the most active recombinants (isolated only from the his x ilv cross) then produced 10.5--13.5% more OTC than the prototrophic parents (Alikhanian et al., 195%; Mindlin et al., 1961c). One of these most active strains, the LS-T Hybrid (derived from LS-T 293 and BS-21 which are parent strains of remote origin) was characterized by lower foam formation during submerged cultivation in rich media (Mindlin et al., 1961a). It appears that the results of hybridization of S. aureofaciens and S. rimosus are affected by a number of factors, the most important of these being the selective markers of auxotrophic partners used for crossing, and the production capacity and mutual relationships (related or remote) of the prototrophic parents of the auxotrophs (Jfirai, 1961; Vladimirov and Mindlin, 1967; Mindlin, 1969). If the original prototrophic strains are closely related and have similar characteristics it cannot be expected that crossing of their mutants will give rise to recombinants with markedly different properties. On the other hand, a crossing of strains originating from different parents and selected independently in different laboratories gives some hope of obtaining recombinants with higher production (Table 3). This assumption was supported by reciprocal crosses of active hybrids with one of the parent strains (Vladimirov, 1968; Mindlin, 1969). It should be noted here that certain differences in the properties of recombinants ofS. aureofaciens and S. rirnosus were observed, as concerns morphological properties and stability. With S. rimosus, recombinants selected from different combinations of crossed mutants as well as those

2650

19(10

LS-T 293

B S -21

Activity of original strain (pg/ml)

1234

101(2a)

Strain

1429

Activity of original strain (~tg/ml)

8229

Strain

7

115 0

49 his 24 arg

Activity (gg/ml)

1 ilv

Code

j

,

i

1361 x 870

310x870

} Crossed mutants

1 x 24

l x 49

I II III

I II III

Prototrophic recombinants i ...... i Crossed Recombinant | mutants type

1500 630 1800

2025 2925 1700

Activity (~tg/ml)

663 281 1269 I

lI Ill

705 1622 ! 555

Activity (gg/ml)

I II III

Recombinant type

Prototrophic recombinants

B. Crosses of high-activity strains

28

262 97

Activity (t.tg/ml)

Biochemical mutants

870 his

1361 thr 310 ilv

Code

Biochemical mutants

Table 3. Productive activity of the original strains of S. rimosus, their biochemical mutants and prototrophic recombinants (after Alikhanian et al., 1959a) A. Crosses of low-activity strains

7~ e~

.at

.N

b~

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

21

originating from the same cross differed in their morphology; moreover, each of the mutant combinations usually gave rise to at least 2--3 morphological types of recombinants (Alikhanian et al., 1959a; Mindlin et al., 1961c). On the other hand, prototrophic colonies selected from S. aureofaciens usually belonged to a single morphological type, similar to the parent strains. The offspring of these recombinants exhibited a segregation into 3~S different types of colonies resembling in phenotype one or the other auxotrophic parent, or the prototrophic parent strains, or showing some features of both. This segregation process continued even in other subcultures of the recombinants for a number of generations (Alikhanian and Borisova, 1961; Mindlin et al., 1961c). In our recombination studies with S. aureofaciens we obtained similar results. With S. rimosus such segregation patterns were observed only rarely, most of the recombinants being relatively stable (Alikhanian et al., 1959a; Mindlin et al., 1961c). The stability of the production strains is one of the prerequisites for their successful application in the industry. Jfirai (1961) or the other authors who obtained high-production hybrid forms of S. aureofaciens do not mention the stability of this improved production capacity. In view of the observed segregation of morphological variants from the recombination cultures one can assume, however, that the biosynthetic activity also underwent subsequent spontaneous changes and most likely gradually decreased. d) Transduction Alikhanian and Ilyina (1957) observed in experiments with Streptomyces olivaceus that the actinophage brings about a great increase of morphological variability. Mutagenic effects of the actinophage were also demonstrated in two production strains of S. aureofaciens; treatment with different phage types led to an increased variability in the production of the antibiotic-as compared with the untreated control by 6 0 - 200%, depending on the phage type and on the activity of the strain from which the phage had been isolated. The best results were obtained with mutant phages obtained after exposure to mutagens (Fedorova and Alikhanian, 1965). e) Increase of Resistance to its Own Metabolite Using S. aureofaciens, a relation between the CTC production and the interaction of the antibiotic with the protein-synthesizing system was demonstrated (Mikulik et aL, 1971a, 1971b). The results obtained with some other antibiotic producers indicate that the final concentration

22

Z. HOgt~,LEKet al.

of produced metabolite may be limited by a mechanism resembling feedback control (Dole~ilowi et al., 1966; Legator and Gottlieb, 1953, Malik and Vining, 1970; Gordee and Day, 1972). Differences in the biosynthetic activity of various strains of S. aureofaciens and S. rimosus might thus be affected by the genetically determined degree of their resistance to their own antibiotic. The possibility of increasing this resistance either by physiological adaption to gradually increasing concentrations of the antibiotic in the medium or by a mutation process was also checked as one of the ways toward improving the biosynthetic capacity of the production strains. Katagiri (1954) attained a 4-fold increase of productivity of S. aureofaciens by repeated transfers of the standard strain in a medium containing 200 400 lag CTC/ml. The resulting high-production variant R-9-70 was not stable and its biosynthetic activity gradually dropped to the original level. Veselova (1967) investigated the variability of different strains of S. aureofaciens and S. rimosus growing in media with an addition of CTC or OTC (200--1000 lag/ml) and compared it with the variability induced by mutagenic factors. The sensitivity of each strain to increasing concentrations of the antibiotic was in direct proportion to its production capacity. It was found that at a survival rate which in mutagen-treated populations led to increased frequency of low-production variants, tetracyclines resulted in an increased number of active forms. These results are explained by a lethal effect of the antibiotic which selectively deprives the natural population of low-active, sensitive forms. The method might thus be most effective during the initial stages of strain improvement or in cases when a population of a high-production strain was to be purified (e.g. during preparation of inocula for industrial-scale fermentations). 2. I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f M u t a n t s B l o c k e d in the Biosynthesis of Tetracycline Antibiotics A prerequisite for the study of tetracycline biosynthesis and its genetic control is the preparation of mutants of S. aureofaciens and S. rimosus with point mutations at different steps of the biosynthetic pathway. Alterations in the morphology and pigmentation of colonies are obviously used as the first criterion in the selection of these mutants in populations surviving after mutagen treatment (Alikhanian et at., 1961; McCormick et al., 1961; McCormick, 1969; Mindlin et al., 1968; Blumauerovfi et al., 1969b; Delid et al., 1969). For a qualitative analysis of the spectrum of metabolites produced by submerged cultures of mutants it was found most expedient to use paper chromatography

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

23

and subsequent detection of spots under UV light (Erokhina, 1965; Mindlin et aI., 1968; Blumauerovfi et al., 1969b). Basic information on the character and probable sites of genetic blocks in the biosynthetic pathway can be obtained by studying metabolic complementation (cosynthesis) in mixed cultures of pairs of blocked mutants, either under submerged conditions (Mindlin et al., 1966, 1968; McCormick et al., 1961; Blumauerovfi et al., 1969a) or by the agar method (Deli6, 1969; Deli6 et al., 1969; Pigac, 1969). The phenomenon of cosynthesis is explained by a mechanism of intercellular transmission of the intermediate or enzyme (coenzyme) essential for the biosynthetic pathway leading to tetracyclines (McCormick, 1966). It is an advantage of the agar method (Fig. 3) that one can determine which of the two participating mutants is the donor (secretor) and which is the recipient (converter) of cosynthetic activity. However, submerged cultivation and subsequent chromatographic analysis permit detection also of cosynthesis of antibiotically inactive substances (Blumauerovh et al., 1969a). Inactive mutant A

A

B. subtilis

I

Inactive mutant B

Mutant A

X ,

Mutant B

X

B

" :: -

Y ~ Y

Z ~' Z

antibiotic

Fig. 3. Detection of cosynthesis of tetracycline antibiotics by the agar method (Deli6, t969). Two inactive mutants were streaked on opposite halves of a plate containing agarized production medium (each mutant covering one half of the plate) about 1--2 mm apart. The plate was incubated 5--7 days at 28°C. A strip of agar was cut from the dish and placed on the surface of an agar plate containing the test organism Bacillus subtilis. After overnight incubation, any antibiotic activity was revealed as an inhibition halo near the middle of the strip. The mutant surrounded by the halo was evidently the one which converted to tetracycline a compound secreted by the other mutant

24

Z. HO~;~,~LEKet al.

A definitive characterization of mutants is possible only on the basis of understanding the structure of the corresponding mutant metabolites. Isolation and study of the blocked mutants of producers of tetracycline antibiotics were recently taken up in several laboratories; the results obtained are at different stages of development.

a) M u t a n t Metabolites of S. aureofaciens Standard strains of S. aureofaciens are characterized by the production of CTC (in mixture with about 5% TC) and of the antibiotically inactive glucoside aureovocin (Vokoun, 1970; Podojil et al., 1970). The position of its aglycon aureovocidin in the biosynthetic sequence is shown in the Fig. 16. Aureovocidin originates probably as a product of the overflow metabolism of 4-hydroxy-6-methylpretetramid,oxidation of ring A being apparently the rate limiting reaction of CTC biosynthesis under standard conditions (Fig. ll). Its glucosidation to aureovocin seems to be a detoxicating mechanism. The substrate specificity of enzyme system responsible for glucosidation and its general properties have been described by Hovorkov'~ et al. (1974) and Mat~jfi et al. (1973). Mutations leading to qualitative changes in the spectrum of standard products can be induced both in low- and in high-production strains rather easily (with a frequency of 5--30% of the surviving population, depending on the parent and mutagen used). Most frequently, however, only the ability to form aureovocin is lost, which is accompanied by a decreased production of tetracycline antibiotics. On the other hand, mutants with completely blocked synthesis of CTC and TC, or mutants producing novel substances different from the metabolites of standard strains, occur only at a low frequency (Blumauerovfi et al., 1971a, 1972). Of mutational changes resulting in the formation of qualitatively different metabolites, the most frequent is the block in the methylation at carbon-6 of the tetracycline skeleton (Fig. 10) (Blumauerovfi et al., 1971a, 1972). In many cases, however, the transmethylation activity is not completely lost: such leaky mutants then produce demethyltetracyclines as well as trace amounts of CTC, TC, or even aureovocin (Blumauerovfi et al., 1969b). The series of blocked mutants used by McCormick and co-workers for studying the biosynthesis of tetracyclines was obtained from a wild strain of S. aureofaciens A 377 (NRRL 2209 or ATCC 10.762) or from its derivatives. For induction of mutants, various types of radiation, chemical mutagens and physical manipulation (grinding) were used. UV light, X-rays and nitrogen mustard have been of particular usefulness as mutagenic agents (McCormick et at., 1961).

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

25

When cultivated on agar media, the wild parent strain A 377 forms lightyellow substrate mycelium (the reverse side of the colonies is yellow to yellow-orange or yellow-brown), white or light-grey aerial mycelium, light-grey spores and light-yellow to orange pigment (Duggar, 1948; McCormick et al., 1961); under submerged cultivation conditions the production may reach 20--300 lag CTC/ml. The nonproduction blocked mutants were either completely colourless or formed pigments different from those of the parent strain (dark-brown, dark-green, copper-red, etc.), or they differed in other characteristics, e.g., in the size or surface character of the colonies, formation of fluorescent diffuse zones (McCormick et al., 1961). Most mutant pigments belonged to tetracycline derivatives. Mutants blocked in the first step of biosynthesis, i.e. in the synthesis ofmatonamylCoA, form, in the place of tetracyclines, 2-acetyl-2-decarboxamidotetracyclines, possessing in position 2 an acetyl group (Miller and Hochstein, 1962; McCormick, 1965). The presence of the acetyl group deriving from acetoacetylCoA apparently has no influence on the further course of biosynthesis which is analogous to that during formation of the tetracyclines (McCormick, 1966). 2-acetyl analogues of tetracyclines were found also in cultures of production strains of S. psammoticus (Lancini and Sensi, 1964) and S. rimosus (Hochstein et al., 1960; Orlova and Smolenskaya, 1965, Frolova et al., 1971) as side products during the biosynthesis of TC or OTC; a strain of S. rimosus yields mutants producing increased amounts of 2-acetyl-2-decarboxamidooxytetracyclineand only very low concentrations of OTC (Hochstein et al., 1960). The formation of the 2-acetyl analogue of OTC can be suppressed by an addition of amides (Orlova, 1968). 2-acetyl-2-decarboxamidotetracyclines exhibit a similar antibacterial spectrum as the corresponding tetracyclines but they are 5 to 50 times less effective (Hochstein et al., 1960; Miller and Hochstein, 1962; Orlova and Smolenskaya, 1965). Genetic block in methylation at carbon-6 results in the formation of demethyltetracyclines (McCormick et at., 1957), and in combination with another block(s) -- in the accumulation of non methylated compounds, homologues of the methylated series (Fig. 18). Using mutants (e.g. ED 1639) blocked in the cyclization step of biosynthesis (Fig. 10) two biologically inactive compounds of the anthraquinoid type were isolated; protetrone (McCormick and Jensen, 1968) and its 6-methyl analogue (McCormick et al., 1968b) formed probably as by-products of oxidation of a hypothetical anthrone precursor of tetracyclines (Fig. 19). Protetrone is identical with the dark red-brown pigment of mutants which, in submerged cultures, is bound mostly in the mycelium (McCormick and Jensen, 1968).

26

Z. HOg'I'ALEKet al.

Mutant V 655 blocked before the 12a-hydroxylation and 7-chlorination of the tetracycline ring system produces 4-hydroxy-6-methytpretetramid (Fig. 11) at a concentration of up to 500 lag/ml (McCormick et al., 1965; McCormick and Jensen, 1965). The green pigment of this mutant was identified as the oxidation product of 4-hydroxy-6-methylpretetramid, the tetramid green (Fig. 15) (McCormick, 1965). The T 219 mutant and other strains blocked in the synthesis of 5a,6anhydrotetracycline accumulate metatetrene originating, probably spontaneously, from anhydrotetrene (McCormick 1965, 1966). The position of anhydrotetrene in the biosynthetic pathway is not very clear. So far no mutant has been prepared that would have a blocked capacity for hydroxylation in position 6 and that would thus accumulate large amounts of 5a,6-anhydrochlorotetracycline (McCormick, 1966). This is attributed to the high toxicity of anhydrochlorotetracycline (Goodman et al., 1955) and to its lethal effects on the producer. However, mutant 1 E 1407 was described that produce 4-amino-6-demethylanhydrochlortetracycline (McCormick et al., 1968a), and mutant 1 E 6113, producing 6-demethylanhydrochlortetracycline(McCormick and Jensen, 1969). The formation of N-demethylanhydrotetracyclines by S. aureofaciens BC-41 in the presence of L-ethionine was described by Miller et al. (1964). A block in the last step of biosynthesis connected with the loss of a hydrogenating enzyme or cofactor results in accumulation of dehydrotetracyclines (Fig. 12) (McCormick et al., 1958). Another group of mutants derived from strains producing demethyltetracyclines and blocked also in the last stage of biosynthesis is characterized by the production of pigments of naphthacenequinone type (McCormick and Gardner, 1963; McCormick, 1965). The colour of naphthacenequinones changes in dependence on pH from blue in the alkaline medium to red in the acid medium. A typical representative of these compounds is tetramid blue isolated from the E 504 mutant. From cultures of the pigment-free mutant W 5 the so-called cosynthetic factor I has been isolated. In a mixed culture of this mutant with strains producing dehydrochlortetracycline, it stimulated the synthesis of CTC; a similar effect was produced by adding the filtrate of the fermentation liquor of W 5 or of a pure preparation of this cofactor to cultures of complementary mutants (Miller et al., 1960; McCormick et al., 1960, 1961). The fluorescence, colour, solubility, composition and some other properties, e.g. the ability of reversible oxidation and reduction, indicate that the cosynthetic factor is probably related to flavines or pteridines. The results of the extensive work of McCormick's group, dealing with the characterization of mutants and identification of biosynthetic sequences using either cosynthetic experiments or transformation of

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

27

natural and synthetized intermediates were published in several excellent reviews (McCormick, 1965, 1966, 1969). Beside this there exist isolated publications on the preparation and properties of S. a u r e o f a c i e n s mutants blocked in the biosynthesis of tetracyclines. One of these mutants (obtained from the production strain LSB-2201 FU-57 after treatment with nitrosomethylurea) produced demethyltetracyclines and differed from the parental type in its morphological properties and in the production of a dark brown-red pigment (Makarevich et al., 1966). On applying nitrosomethylurea, the same parent strain yielded six mutants producing three new, hitherto unidentified, compounds with yellow-green fluorescence in UV light which can be distinguished chromatographically from the tetracycline antibiotics; one of the compounds was apparently antibiotically active (Gutnikova, 1966). Deli6 e t al. (1969) isolated blocked mutants from S. a u r e o f a c i e n s A-4 (wild type isolated from soil) after treatment with UV light, nitrous acid, ethyleneimine and 1,3-diepoxybutane. The most effective for their induction was UV light (0.1--1% surviving cells). Many of the mutants lost their ability to sporulate, and differed from the parent type in changes in pigmentation but some retained traces of antibiotic activity. The metabolites of mutants were not characterized further, nevertheless their mutual cosynthetic ability was studied. The results of these tests made it possible to divide the mutants into four complementation groups suggesting the probable sequence of geTable 4. Grouping of inactive mutants of S. aureofaciens A 4 (Deli6 et al., 1969) Attribution of mutants to complementation groups

Cosynthesis between groupsa

Mutants

Group

Groups

A

A

C

D

ctc-l,2,4 ctc-3 ctc-5 ctc-6, 7 ctc-8, 9,10

A B C D

A B C D

--

B --

C C

A ---

Doubtful

Complementation patternb:

C

B

A

D The sign (--) indicates no cosynthesis. Each letter indicates cosynthesis with a halo on the side of the strain of the corresponding group. b Non-overlapping segments correspond to complementing groups.

a

28

Z. HO~/~LEK et al.

netic blocks in the biosynthetic pathway (Table 4). Groups ctc B and ctc C (and possibly also ctc A) included mutants which were mutually complementary during the biosynthesis of the antibiotic and functioned in different combinations either as secretors or as converters. These mutants are probably blocked in their structural genes, controlling the main metabolic pathway during biosynthesis of CTC. On the other hand, the ctc D (and possibly ctc A) groups included mutants with no mutual complementarity with those of the preceding groups, acting always as secretors. These mutants are probably not formed by multiplepoint mutations covering several structural genes because they occur at relatively high frequency in comparison with the usually observed frequencies of single-point mutations of structural genes. The authors suggest that these mutants are blocked rather in the regulatory genes (or quite generally in genes controlling the onset of secondary metabolism, i.e. the shift of normal metabolic channels toward the antibiotic pathway) rather than in the structural genes of the secondary metabolic pathway itself. Another series of S. aureofaciens mutants was isolated from two standard strains of S. aureofaciens (84/25 and Bg) differing in their production capacity, after treatment with UV light, X-rays and 7-radiation, Nmethyt-N'-nitro-N-nitrosoguanidine, nitrosomethylurea, nitrous acid, hydroxylamine and nitrogen mustard (Blumauerovfi et at., 1969b). According to the differences in biosynthetic activity, the mutants were divided into twelve metabolic groups (Table 5). In eight of these, chromatography showed the production of novel compounds differing from the metabolites of the standard strains; in nine groups atypical pigments were also produced. The identification of these metabolites is still in progress, our preliminary results suggest that most of them belong to the tetracycline series. Comparison of the frequency of occurrence of the individual metabolic types in treated and untreated populations of both parent strains indicates a relative specificity of the mutagens used. Qualitative changes in metabolism resulting in the production of new compounds were induced with UV light, N-methyt-N'-nitro-N-nitrosoguanidine and nitrous acid. On the other hand, ?- and X-radiation induced only quantitative changes in the production of standard metabolites or a loss of their synthesis. Very specific was the inhibitory effect of the two types of radiation on the production ofaureovocin, accompanied by a decreased prod uction of tetracycline antibiotics induced at a 2 5 times higher frequency than after application of other mutagens (Blumauerovfi et al., 1971 a). Cosynthetic experiments (Blumauerovfi et al., 1969a) revealed that mutants can be complementary not only during biosynthesis of tetracycline antibiotics but also during production of other metabolites

~

I II III IV V VI VII VIII IX X XI XII

Bg 84/25

-÷

0 50--- 100 0 100 150 150 250 20 0 0

-+ + + --.

+

--

0

+ +

---

"

DTC

+ +

AVC

+ + + --+ -+ + + + -+ --. . . . . . . . .

CTTC TC

-~ -. .

+ + + ------

+ +

B

+ + + + + + + + + + +

+ +

C

--D, E --F, G F, G G, H K, J F, L, M L, M N, O, P, R S,T,U

Other

Unknown substances

S p e c t r u m of p r o d u c e d m e t a b o l i t e s

50--1500

400 2000

Tetracyclines (lag/ml)

light b r o w n - o r a n g e to b r o w n d a r k green yellow c r e a m or b r o w n i s h - c r e a m none red-violet red-brown red-orange b r o w n - g r e e n to b r o w n - b l a c k brown-violet grey-violet yellow-brown

b r o w n - o r a n g e to b r o w n b r o w n - o r a n g e to b r o w n

Pigment

343 5 20 147 54 5 2 5 6 2 2 1

----

N u m b e r of mutants tested

a T h e p r o d u c t i o n of s e c o n d a r y m e t a b o l i t e s was d e t e r m i n e d by p a p e r c h r o m a t o g r a p h y in the system c h l o r o f o r m - b u t a n o l - M c l l v a i n buffer (pH 4 . 5 ) - - 4 : 1 : 5, a n d by s p e c t r o p h o t o m e t r i c assay at 440 nm. A b b r e v i a t i o n s used: D T C , d e m e t h y l t e t r a c y c l i n e s : AVC, a u r e o v o c i n ; B-U, substances, the structure o f w h i c h has n o t been yet definitely established.

"U

~=

Metabolic type

Table 5. Characteristics of s t a n d a r d strains o f S. aureofaciens a n d their b l o c k e d m u t a n t s (Blumauerovfi et al., 1969b, 1971a) ~

:~ 9. o-~" g-

p~

~"

~,<

Pz w

30

Z. HO~TALEKet al.

Type t~ Tetracyctlnes ~r~ Substance A E~ SubstancesV,X,Y ~4utants UV45 8-96 Mixture B-t03 ~801/26Mixture Bq03 0202Mixture

RFI'°

¢~E

0.9-

[~Z. Substances H, Z Bq03 HA-V/382M~xture

<1')

0.81_ 0.7_

@B@

CT-

)r ()CT()

()x

tll~z -

0.6-

0.5 -

0.3 0.2 -

TC"

)T(

_9"Q° i '°i ~)

A

L

t~

EP

4)

0.1-

tnt~

Fluoreseention in UV-light: O yellow Q yellow green

(]]) yeltow orange

t~ brown

Q red

(~ orange

O blue, blueviolet

O violet

Fig. 4. Chromatographic analysis of substances produced by pure and mixed cultures of blocked mutants of S. aureofaciens {Blumauerovfiet al., 1969a). Key: CT, chlortetracycline; TC, tetracycline: EP, epimers: DT, demethyltetracycline; A, aureovocin; B-Z, substances, the structure of which has not yet been definitely established. Paper chromatography in the system chloroform-butanol-Mcllvain buffer pH 4.5 (4: 1:5) was used for the analysis

aureovocin and some novel compounds, probably intermediates or byproducts of the tetracycline pathway (Fig. 4). These results indicate that multiple blocks in this pathway are possible. In contrast with the results described by McCormick et at. (1960) all the types of cosynthesis, observed by Blumauerovfi et at. (1969a) took place only during mixed cultivation of both complementary mutants. The presence of any of the partners could not be replaced either by filtrates of its culture or by metabolites isolated from the mycelium. A similar finding was made by Shen and Shan (1957) in S. aureofaciens where an increased production of CTC in mixed cultures was accompanied by a substantial improvement of growth.

Genetic Problems of the Biosynthesisof TetracyclineAntibiotics

31

From the point of view of the present knowledge on the biogenesis of tetracyclines and its control (Van~k et al., 1971 ; Ho~filek and Van~k, 1973) it appears that the phenotypic expression of many mutants seemingly blocked in the secondary metabolic pathway can be actually due to the genetic changes in primary metabolism responsible for the supply of a specific precursor (acetylCoA) to secondary biosynthesis. This assumption is in agreement with the conclusions expressed by Deli6 et al. (1969) on the basis of cosynthetic experiments. Likewise, the concomitant characteristics of mutants, e.g. changes in morphology, growth rate, respiratory activity, ability to utilize carbohydrates, sensitivity to their own metabolites (Blumauerovfi, 1969) and in cell ultrastructure (Ludvik et al., 1971) suggest the possibility of multiple-point mutations, affecting simultaneously a greater number of biochemical functions of the organism. Subsequent changes in the biosynthetic activity of unstable mutants never led to complete quantitative reversal to the standard parental type and they thus appear to reflect spontaneous suppressions affecting the overall metabolic balance rather than back mutations of the originally altered genes (Blumauerov~ et al., 1972). b) Mutant Metabolites of S. rimosus The preparation, properties and the results of genetic studies of blocked mutants of S. rimosus were described by Alikhanian and co-workers in 1956~1970. The mutants were obtained from the production strain LS-T 118 with the aid of various mutagens, the most efficient being UV light. Good results were also achieved by using fast neutrons, diethyl sulphate and combined action of ethyleneimine and UV light. The mutants differed from the parent strain by a substantially reduced production of OTC, by morphological properties, changes in pigmentation of submerged cultures and by the formation of new compounds with characteristic fluorescence in UV light (Table 6). The structure of these compounds and their physico-chemical properties have not been described. In mixed cultures most mutants showed the capacity of mutual complementation during OTC biosynthesis; it is hence assumed that these mutants are genetically blocked in different loci controlling the biosynthesis of the antibiotic (Alikhanian et aI., 1961; Erokhina, 1965; Erokhina and Alikhanian, 1966; Mindlin et aI., 1966, 1968; Zaytseva et al., 1961; Boronin and Mindlin, 1970). The nonpigmented "white'" mutants of group 6 were cosynthetically active only in combination with the "black" mutants of group 3. The "white" mutants were found to produce the so-called X-factor. This compound which was found in a number of other actinomycete species probably has the function of a coenzyme; a stimulatory effect on the

10

7

3

5

3

11

1

2

3~

4

5

6d

None

Greenbrown

Dark brown

Dark brown to black

Dark crimson

Orange

Colour of culture fluid

0.1-~. 1.0

0.3---6.0

10----80

15--30

0.5---1.0

1--20

Antibiotic activity (gg/ml)

+

+

+

+

+

+

blue

yellow

violet

light blue

+

+

--

--

---

+

+

+

--

---

.......

+

. . . . . . . . . . . .

violet

+

.

.

.

.

.

.

.

. . . . . . . . . . . . . . . .

--

--

+

+

bluegreen

M u t a n t metabolites fluorescent in U V light b

+

---

yellow green

--

-

+

6

5 6

0.5--

6.0

1 5 0 ~ 200 10-- 80

2 0 0 - - 1000 4 0 ( 0 800 7~1200

100 600 2 0 ( 0 400 1 0 0 ~ 350 1-4

3 4 5 6 4 5 6

1.5 1 0 ( O 400 140--- 300 I ~ 450 0 . 5 - - 30

2 3 4 5 6

Compleorange mentary OTC group lag/ml

Cosynthesis

a Mutants were derived from S. rimosus LS-T 118 producing about 3000 lag OTC/ml. This strain formed a brown pigment under submerged conditions. b Production ( + ) or absence (--) of metabolites was detected by paper c h r o m a t o g r a p h y in system chloroform-nitromethane-pyridin (10:20:3) or butanol-acetic acid-H20 (16:4: 5). "Black" mutants. d "White" mutants.

Number of murants

Mutant group

Table 6. Characteristics of blocked mutants of S, rimosus described by Mindlin et al. (1968) and Mindlin (1969) a

7~

t-

..at

N

taO

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

33

biosynthesis of OTC was found also in partly purified preparations of the X-factor added to cultures of "black" mutants at the beginning of cultivation (Orlova et al., 1961, 1964). Cultures of "black" mutants were found to contain the antibiotically inactive compound Y.Its biosynthesis, just as that of OTC, was unfavourably affected by excess inorganic phosphate and by powerful aeration. Underoptimumconditions, compound Ywas produced at concentrations of up to 550 ~tg/ml (together with 15--30 ~tg OTC/ml), the maximum production having been reached after about 72 hours of cultivation. After adding a catalytic amount of the X-factor the production of OTC rose considerably with simultaneous disappearance of compound Y. Complementation took place even after transfer of the washed mycelium of the "black" mutant to the filtrate of a culture of a "white" mutant containing the X-factor. It is assumed that compound Y is either a precursor or a by-product during biosynthesis of OTC (Zaitseva and Orlova, 1962). Mutants of groups 3 and 6 thus could mutually complement each other during the formation of the antibiotic in a way similar to that described by McCormick (1966) for S. aureofaciens. It appears, however, that in other systems where different combinations of mutants of group 1 to 5 were tested, a different mechanism played a role during complementation. In all cases, the cosynthesis of OTC required direct contact between the two complementary types of mycelium. After plating the mixed cultures, as much as 10% of mosaic colonies were obtained, with mycelial sectors of both starting forms. The mosaic colonies were antibiotically active, in contrast to the colonies of pure mutant cultures. These results indicate that heterokaryons were formed in the mixed cultures. However, since the mixed cultures contained always besides heterokaryotic cells also the original homokaryotic mycelium of both blocked mutants, no full restoration of production comparable with that of the parent strain has been achieved (Mindlin et al., 1968). Data on the complementation activity of mutants obtained in the abovementioned cosynthetic experiments indicate the existence of at least two separate groups of loci controlling the biosynthesis of OTC (Mindlin et al., 1968). Using newly prepared series of inactive mutants (Boronin, 1970) the preliminary results were further supplemented by Boronin and Mindlin (1970): according to complementation patterns found with the agar method, the mutants were divided into several complementation groups, three of which have not been previously described. A new scheme of blocks in the sequence of OTC biosynthesis was advanced. Mutants of S. rimosus studied by Deli6 et al. (1969) and Pigac (1969) were isolated from the standard strain R-6 under the same conditions

34

z. HO~;J'ALEKet al.

as the above mutants of S. aureofaciens (p. 27). The cosynthetic ability of mutants was tested by the agar method. On the basis of the results the mutants were divided (according to their complementation pattern) into eight groups (Table 7). In analogy with the mutants of S. aureofaciens, Table 7. Grouping of inactive mutants of S. rimosus R-6 (Deli6 et al., 1969) Attribution of mutants to complementation groups Mutants

Cosynthesis between groups" GroupsE C B A F G H D

Group

otc-4, 5, 13,65, 123

A

E

otc-17 otc-15

B C

C B

otc-90, 98, t04, 118

D

A

otc-2

E

F

otc-10,91,94, II 1,112, 113, 114, 117 otc-8, 64, 105 otc-95, 96, 119, 120

F G H

G H D

--EEEEE CCC--C-B

Complementation patternb: E

C

B

A F

G H

D a,b See notes to Table 4. two different mutant classes were described. One of them (groups otc B, otc C, otc E, and perhaps otc A) included mutually complementary mutants (and hence apparently blocked in the secondary metabolic pathway) whereas the other class (groups otc D, otc F, otc G, otc H and possibly also otc A) included mutants of unknown character. This second class contained also up to 90% of the isolated mutants; it thus appears that only a small number of mutants are really blocked in the proper biosynthetic pathway.

Genetic Problems of the Biosynthesis of TetracyclineAntibiotics

35

c) Interspecific Cosynthesis In cosynthetic experiments designed by McCormick et al. ( 1961) a certain possibility ofinterspecific complementation was found; thus, for example, formation of OTC was observed in mixed cultures of S. aureojaciens and S. rimosus, and formation of CTC during combined cultivation of S. aureofaciens with mutants of S. albus and S. platensis. The cosynthetic activity was also apparent in combination of inactive mutants of S. rimosus and S. aureofaciens, tested by Deli6 et al. (1969). Two mutants of S. rimosus, belonging to the groups otc E and otc B, yielded in combination with the mutant of S. aureofaciens of group ctc A a zone of antibiotic activity at the side of S. rimosus mutants which thus acted as converters. This result was predictable since the cte A gene is a late gene in CTC biosynthesis while the otc B and otc E are earlier genes in OTC biosynthesis. A cosynthesis between several wild strains of streptomycetes and inactive mutants of S. aureofaciens or S. rimosus was observed. In these experiments usually the highest cosynthetic activity ever observed was found, the wild-type strain acting as the secretor (similarly to the above-discussed class 2 of mutants of S. aureofaciens and S. rimosus).

3. G e n e t i c R e c o m b i n a t i o n The existing data on genetic recombination of producers of tetracycline antibiotics may be divided into several principal groups. Preliminary results obtained by the application of the recombination technique for strain improvement have already been discussed in the first section. Another group includes papers on the genome topology, i.e. on the construction of the linkage map of the chromosome on the basis of analysis of loci controlling the biosynthesis of essential metabolites (Ala~evi6, 1969a, 1973; Ala~evi6 et al., 1972; Friend and Hopwood, 1971). The third group of papers is focussed on the genetic analysis of loci controlling the secondary biosynthesis (Mindlin et aI., 1961b, 1966, 1968; Vladimirov and Mindlin, 1967; Boronin and Mindlin, 1971; Blumauerovfi et al., 1971b, 1972). Most of these investigations were conducted on the model of S. rimosus while data on the genome of S. aureofaciens are still rather scarce (Ala~evi6, 1969a; Blumauerov~i et al., 1971b, 1972). Interspecies recombination between S. rimosus and S. aureofaciens has also been described Ala~evi6, 1963, 1965a, 1965b, 1969b; Ala~evi6 et al., 1966; Polsinelli and Beretta, 1966; Blumauerov~i et al., 1971b).

36

Z. HO~I",~LEKet al.

a) The Linkage M a p of S. rimosus By adapting methods developed for S. coelicolor A 3/2/ (Hopwood and Sermonti, 1962; Hopwood, 1967; Sermonti, t 969), basic information on the genetic map of S. rimosus was obtained independently in two laboratories. Ala~evi6 (1969a, 1973) worked with auxotrophic mutants of a tow-production standard strain, S. rimosus R 7 (ATCC 10.970). Most of them had a decreased or practically nonexistent antibiotic activity but their products were not characterized in detail. A selective analysis of haploid recombinants obtained by four-point crosses showed a variety of recombinant phenotypes. In most cases an excess of prototrophs was obtained which distorted the results for the estimation of linkage relationships between genes. The main criterion used for the mapping was therefore based on data from heteroclone analysis. In the first stage of the work (Ala~evi6, 1969a) 10 markers were localized on the chromosome map by analyzing trios of markers from many heteroclones. For further more refined analysis, four-, five- or six-point crosses of multiple mutants were used (Ala~evi6, 1973). From the segregation data obtained, the arrangement of markers was deduced by checking all the possible permutations of the circular arrangement of the markers involved and by choosing the arrangement from which the segregants were formed by the minimum number of crosses. Although in some cases contradictory results were obtained, a preliminary map of the chromosome containing 24 markers

his.7~ ~.'~//his-12 2124',arg-13044 \\ ,// ,.,4,6 .,v,_._ ":// / • rib-4

pro-l/

\

(met-108)~

/ (rib-5)

/

pao-1 /J,'--" /~-

Fig. 5. Linkage map of Streptomyces rimo,sus R-7 (Alabevi6, 1973)

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

37

could be constructed. A clustering of some genes controlling the biosynthesis of histidine was established, six mutations appeared to be closely linked, similarly to the situation described previously by Piperno et al. (1966) in S. coelicolor. Likewise, three of the genes controlling the biosynthesis of arginine appeared to be in close linkage on the map of S. rirnosus (Fig. 5). This map is consistent with that constructed in another strain, S. rimosus R 6 (Ala~evi6 et aL, 1972). Friend and Hopwood (1971) used for their mapping studies auxotrophic mutants derived from the industrial production strain of S. rimosus. During crossing of these mutants they observed frequently variable numbers of heterokaryons which could be eliminated by a suitable choice of selected marker combinations. A map showing the linkage relationship of 24 marker loci (Fig. 6) was constructed on the basis

gtuB gLuA i ~ - - ~ .

//

/

metA hisA serA

// p a n A ~ ' ~ a r g A ,/ leuA / /proA /

\

\

~- ribB'~

X~ur.B\~

[ athA -~ J-cysD J II ~ /serC thiB~ , A~ \aae, X /leu~ / metB . x'b.....~ j . X / " itvB nicC hisD~hrA lysB guaA Fig. 6. Linkage map of Streptom),ces rimosus (Friend and Hopwood, 197I). The order of the bracketed loci is unknown. Certain loci have not been ordered relative to loci covered by broken lines. Loci are arbitrarily spaced at equal intervals

of results of selective analysis of haploid recombinants obtained both in four-point crosses of mutants and in multi-factor crosses between mutant and recombinant strains; heteroclones were not selected in these experiments. Mutants used in the two laboratories for genetic analysis were derived from different strains and in many cases displayed different growth requirements. From an incomplete characterization of the auxotrophic

38

Z. HO~A.LEKet al.

markers (defined usually only on the basis of the required final metabolites) it cannot be concluded with certainty whether in mutants with the same growth requirement an identical locus has been mapped, viz. the one controlling the same enzyme step of biosynthesis of the given primary metabolite. In spite of these facts, the identical sequence of at least nine markers suggests the similarity of the two genetic maps presented. Ala~evi6 (1973) as well as Friend and Hopwood (1971) observed a similarity between their map of S. rimosus and the linkage map of S. coelicolor A 3/2/(Hopwood, 1967). A similar arrangement of the markers was described by Coats and Roeser (1971) for Streptomyces bikiniensis var. zorbonensis UC 2989 (NRRL 3684), the only other industrial streptomycete where a tentative chromosome map has been constructed. These findings suggest a remarkable evolutionary stability of gene arrangements on prokaryote genomes (Friend and Hopwood, 1971). Ala~evid (I969a) reported also the results of some heteroclone analysis of S. aureofaciens. The pattern of segregation from the heteroclones seems to be comparable with that observed in S. rimosus. For any definitive conclusions, however, more data should be gathered, following the same markers introduced in crosses in different coupling arrangements. b) Analysis of Loci Controlling Tetracycline Biosynthesis To analyze the production of secondary metabolites, Blumauerovfi et at. (1972) suggested the following working procedure (Fig. 7). Mutants point-blocked at different steps of the biosynthetic pathway must be labelled in the next mutation step by supplementary nutritional or drug-resistance markers, essential for the detection of genetic recombination. For linkage mapping it is desirable to obtain from mutants representing various metabolic types, (from the point of view of production of secondary metabolites) the maximum number of doubly or multiply labelled strains which would retain the original biosynthetic activity of prototrophic parents and the markers of which would include loci from the widest possible region of the chromosome. One can except that an analysis of recombinants from crosses of such mutants will provide data on the localization of the reference markers on the chromosome map but will also suggest the possible linkage between these markers and loci controlling secondary biosynthesis. Some basic data on this linkage can be obtained also by the simplified method of crossing auxotrophic drug-resistant mutants and prototrophic drug-sensitive mutants belonging to different metabolic groups. The biosynthetic activity is then followed in prototrophic drug-resistant

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

39

parents J IStandard __~Mutagen /'/ ~

agen trMut eat m ent (Step2)

Mutants blocked in the biosynthesis of secondary metabolites

Mutagen l treatrnen~ (Step2 Monoauxotrophs or drug-resistant mutants

Mutsgen

~

Multipte-marked mutants

-arid]I

Three four- polnt

.ns:;..J

" oo.on,

Two-point crosses

I Prototrophic recom binants

,ns,,.,so,

......

IRec°mbinantsl I Segregants from 1 heteroclones l

Fig. 7. Working scheme for the study of the genetic control of the biosynthesis of secondary metabolites (Blumauerov~i et al., 1973a). In each step the biosynthetic activity should be proved in all the strains obtained recombinants (Mindlin et aL, 1961b, 1966, 1968; Boronin and Mindlin, 1971). In the course of the mapping experiments with S. rimosus (Ala~evi6, 1969a) the segregation of OTC production was also examined. However, the analysis of loci responsible for the biosynthesis of the antibiotic was impossible owing to the considerable interference of nutrition markers with productivity. The antibiotic activity was never found to be related to other phenotypic characteristics of the segregants; even strains with the same phenotype from the point of view of nutrition markers displayed in many cases different levels of productivity. Results of genetic analysis of blocked mutants of S. rimosus LS-T 118 carried out by Mindlin et al. (1966, 1968) and by Boronin and Mindlin (1971) indicate, on the contrary, the existence of at least two separate groups of loci controlling the biosynthesis of OTC, found in the region between the two loci for streptomycin resistance (Fig. 8). Mutation in one of the second-group loci, otc-6, appears to be responsible for

Z, HOg~ALEK et al.

40 o/c3

str

%

!

ot¢ll

l

i

98.4 98.2

ofc8

,I,

71.2

74.7

t

ofcl,5,7,9,10

i

i

98.0

ot¢lt otc8 otc4

otc3

%

oft4

i

99.5

92.6

otc! otc9 otclO of¢5 o~'7

t

t

76.7 77.5

t

!

91.0 92.0 92.6

i

I

str I

93.8 94.8

Fig. 8. Scheme of the linkage of the otc mutations with the str locus in S. rimosus (after Boronin and Mindlin, 197 l). The linkage values (obtained as results of crosses between different types of inactive mutants and two mutants serving as peculiar test strains) are ex pressed as the percentage of cotransfer of the respective loci

the loss of p i g m e n t a t i o n in t h e ~white'" n o n p r o d u c i n g m u t a n t s which is a c c o m p a n i e d b y increased sensitivity to O T C . R e c o m b i n a t i o n b e t w e e n loci f o u n d in different g r o u p s gave high yields o f r e c o m b i n a n t s with r e s t o r e d a n t i b i o t i c activity. O n the o t h e r hand, crossing of m u t a n t s b l o c k e d in t h e loci o f the s a m e g r o u p resulted in the f o r m a t i o n o f inactive r e c o m b i n a n t s (Table 8). This finding was c o n f i r m e d also d u r i n g r e c i p r o c a l crossing of different types of b l o c k e d m u t a n t s with the active strain (Table 9).

Table 8. Frequency of occurrence of active and inactive "dark" recombinants in a series of crosses between ala +str ~otc 6 ~ o t d 1--11) inactive mutants (belonging to different complementation groups)and ala str otc 6 o t c ( 1 - - I 1) + inactive "white" mutant 154 in S. rimosus (after Boronin and M indlin, 1971) ala ~sir recombinants ~

Partner in cross with 154 otc 6

739 otc 638 otc 382 otc 35 otc 93 otc 721 otc 29 otc 750 otc 30I otc

1 3 4 5 7 8 9 10 11

Active

Inactive

o t c 6 ~ o t c ( 1 - - t 1)+

otc6+ otc(1--11)

Number

%

Number

%

Relative frequency of linkage between otc and str markers

0 0 315 3 372 293 0 0 301

0 0 78.8 0.7 93.0 73.2 0 0 75.2

400 400 85 397 28 107 400 400 99

100.0 100.0 21.2 99.3 7.0 26.8 100.0 100.0 24.8

92.6 92.6 98.4 92.7 99.5 98.0 92.6 92.6 98.2

"~ There were 400 recombinants studied in each cross.

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

41

Table 9. Frequency of occurrence of active and inactive recombinants in a series of crosses between his+str+otc(1--11) inactive mutants (belonging to different complementation groups) and his str otc(1--11) ~ -- active mutant BS-49 in S. rimosus (after Boronin and Mindlin, 1971) his+str recombinants studied

Partner in cross with BS-49 otc(l--I l) + 739 otc 1 638 otc 3 382 otc 4 35 otc 5 93 otc 7 721 otc 8 29 otc 9 750 otc 10 36 ore 11

Active

Inactive

Number %

Number %

353 262 264 375 310 283 368 324 277

35 106 78 25 17 89 32 26 94

Total 388 368 342 400 327 372 400 350 371

91.0 71.2 77.5 93.8 94.8 76.1 92.0 92.6 74.7

9.0 28.8 22.5 6.2 5.2 23.9 8.0 7.4 25.3

The above results suggest that genes controlling certain (probably the final) steps of secondary biosynthesis, may be clustered like genes responsible for the biosynthesis of primary metabolites. The present experience obtained with S. aureofaciens indicates that the realization of each of the partial stages of studying the genetic control of secondary metabolism meets in this organism - - unlike S. rimosus - - with a number of difficulties following from specific peculiarities of its genome. The selection of suitable strains for recombination experiments is substantially limited first of all by the low yield of mutants, truly point-blocked in the secondary metabolic pathway, and by the frequent occurrence of leaky mutants, by irregular mutation patterns during isolation of auxotrophs, decreased viability and high instability of the mutants (Blumauerovfi et al., 1972). Most auxotrophs, independently of the experimental conditions under which they were prepared, required arginine only (Alikhanian and Borisova, 1961; Polsinelli and Beretta, 1966; Blumauerovfi et at., 1971a, 1972, 1973b) and were apparently blocked in the same locus as that controlling the final step of the biosynthesis of this amino-acid. To eliminate the at9 auxotrophs from populations surviving after mutagenic treatment, it was useful to employ a modified enrichment method using a complete synthetic medium without arginine (Blumauerovfi et al., 1973b). The spectrum of auxotrophic mutations different from ar9 was generally characteristic for each strain (Table 10). From the blocked mutants representing different metabolic types from the point of view

arg-77 his-I met-6 met-7 tyr-2 tyr-5

L- l L-t7 L-14 L-25 I-5 P-24

Arg; Cys; Gly/Ser; Ade

Arg; Cys; His; Ile+Val; Met; Gly/Ser; Tyr: Ade/Gua; Ura

Arg; Gly/Ser; Ade/Gua

Arg; Met

B-69

NMG-2

NMG-10

UV-0202

.

--

--

Arg: Met; Gly/Glu/Lys/Met/Thr/ Ser; Trp + Tyr; Ade: Ade/Gua

NMG-28

.

.

.

Ura Ala; Arg; Cys; Leu; Met/Hcs; Ade; Gua; Gua + Met/Cys

Mutations were induced by N-methyl-N'-nitro-N-nitrosoguanidine and by UV light. Both the delayed enrichment method and the total isolation were used for the detection of auxotrophs. b NRRL 2209, wild low-producing strain; 84/25, producing standard strain; the other parents represent different types of blocked mutants.

.

arg-19 ser-3

met-4

H-56

E-14 S-10

Arg; Leu; Gly/Ser/Thr; Met/Thi; Met/Thi/Glu; Met/Cys/Thr/Glu; N ic Arg; Gly/Ser/Thr; Ade/Gua

pur-I

H-12

none Arg; Gly/Ser; Ura Arg Arg Arg Arg; Ile+Val; Met; Phe

none Arg

-arg-8 ilv-2

C-22 NL-1

-

Arg; lle +Val

84/25

B-96

Arg; Ile; Ile+Val; Arg+Ile+Val; Leu; Lys/Pdx: Met; Thr: Ser/Gly: Guo

his-4

R-6

Arg: His; Ile+Val; Leu; Lys; Met; Gly/Ser; Ade; Pdx Arg; Lys

Additional requirement

Genotype marker

Requirement for

Parent strain

2nd step auxotrophs

1st step auxotrophs

NRRL 2209

Parent prototroph

Table 10. Growth requirements of anxotrophic mutants derived from different metabolic types of S. aureofaciens (Blumauero% et al,, 1972, 1973b)

7~

t-

N

Genetic Problems of the Biosynthesis of TetracyclineAntibiotics

43

of secondary metabolite production, it was usually impossible to isolate the identical types of auxotrophs essential for crossing with an exchanged arrangement of markers (Blumauerovfi et al., 1971a, 1972, 1973b). The unsuitability of S. aureofaciens for genetic analysis, caused mainly by the high instability of its mutants, is also mentioned by Ala&vid (1973). Most combinations of mutants used for the recombination experiments gave rise to heterokaryons only and frequently heterokaryons and recombinants were found simultaneously in the same population (Blumauerovfi et al., 1971b, 1972). The recombinants were usually unstable and exhibited different segregation patterns (see also p. 21). Crossing of mutants point-blocked at different steps of the biosynthetic pathway (e.g. during C6-methylation or during oxidation of the A ring of the tetracycline skeleton) and subsequently marked with nutrition markers, never yielded recombinants with the expected phenotype of the standard strain. Most recombinants showed a biosynthetic activity identical with one or other of the auxotrophic parents or resembled the original prototrophic mutants from which the auxotrophic mutants had been derived. It appears that the genes controlling the biosynthesis of tetracyclines are rather closely linked and are not localized near any of the hitherto examined nutritional markers. So far they were not found to be linked even to the loci for streptomycin resistance (Blumauerovfi et al., 1972). Several positive results of recombination (e.g., acquisition of the ability to produce one of the tetracycline derivatives or a complete restoration of the phenotype qualitatively identical with the standard strains) were obtained only in crossing mutants where the cause of the block in tetracycline biosynthesis is not yet understood and is apparently due to genetic changes of primary metabolism; one of the loci responsible for these changes appears to be closely linked to the locus for the biosynthesis of arginine. Even with the most active recombinants no complete quantitative recovery of the original production capacity of the parent standard strain has been achieved (Blumauerovfi et al., 1971b, 1972). It appears that repeated mutagenic treatment during several-step preparation of multiple mutants for the analysis of secondary metabolism can result in the accumulation of unknown blocks in primary metabolism affecting indirectly also secondary biosynthetic pathway. This multiple damage then cannot be repaired by a single recombination in one locus. This assumption is supported by the fact that many low-production mutants of S. aureofaciens exhibited negative complementation during recombination which led in the recombinant progeny to a complete loss of biosynthetic activity (Blumauerovfi et al., 1972).

44

Z. HO~ALEK et al.

c) Interspecific Recombination Most papers dealing with interspecific recombination between S. aureofaciens and S. rimosus emphasize the morphological markers or other taxonomic characteristics of the recombinants and do not provide any data on their biosynthetic activity (Ala~evid, 1965a, 1965b, 1969b). Only in a few preliminary experiments has it been found that, on crossing of mutants of S. rimosus producing trace amount of aureovocin with mutants of S. aureofaciens, recombinants could be obtained which phenotypically resembled S. rimosus but with aureovocin production increased to the level typical of S. aureofaciens (Blumauerovfi et al., 1971b).

4. G e n e t i c C o n t r o l o f t h e B i o s y n t h e t i c P r o c e s s The results of recombination experiments with S. rimosus reported in the previous section indicate the presence of several, little identified, loci on the chromosome which are responsible for OTC biosynthesis. So far it has not been possible to identify the nature of the biochemical reactions controlled by these loci or the proper order of biosynthetic reactions in the metabolic pathway. Our lack of information on the genetic control is not typical only of the tetracyclines. Of the entire breadth of natural compounds produced by actinomycetes there are scant data only on the position of genetic loci controlling the biosynthesis of the zorbomycin complex (Coats and Roeser, 1971) and of one of the pigments of S. coelicolor (Hopwood, 1965). There is much more information on the biosynthetic steps, enzyme reactions and the genetic control of formation of such metabolites as amino acids or nucleotides which permits a rational approach to obtaining high-production strains and to developing high-yield technological procedures. With natural substances, such as alkaloids, antibiotics or pigments, the situation is much more complicated. We have now at our disposal only fragmentary data on the biosynthetic pathway and on their intermediates; of the enzyme reactions participating in the biosynthetic pathways we are nearly ignorant. This state of affairs obviously forces all the work on optimization and intensification of the biosynthetic process to remain empirical. a) Genes Responsible for the Biosynthesis of Tetracyclines Van~k et al. ( 1971) created a theoretical concept of the genetic regulation of CTC biosynthesis. The work proceeds from the present knowledge of the chemistry of tetracycline antibiotics (McCormick, 1965, 1969;

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

45

Podojil et al., 1970). The authors divided the biosynthetic pathway of CTC into three sections. The first of these represents the conversion of glucose to acetylCoA, the second includes the carboxylation of acetylCoA to malonylCoA, condensation of the malonate units to a hypothetical nonaketide and its partial conversion to a tricyclic skeleton. The third section includes the subsequent transformation of the skeleton resulting in the formation of CTC. The glycolytic pathway leading from glucose to acetylCoA includes a total of ten reactions catalyzed by the appropriate enzymes. If it is taken into account that all the intermediates of the biosynthetic pathway may serve as substrates for other metabolic sequences it is perhaps a modest estimate to assume that more than 200 genes take part directly or indirectly in the transformation of glucose to acetylCoA. In the second section of the biosynthetic pathway from acetylCoA to the anthracene derivative one can envisage six enzyme reactions. AcetylCoA itself can be metabolized by at least 28 further enzyme systems. CTC belongs biogenetically among oligoketides. It is formed by condensation of 8 molecules of malonylCoA and of one molecule of malonamylCoA. The mechanism of formation of malonylCoA as a biosynthetic intermediate of tetracyclines is still a matter of discussion (B~hal and Van~k, 1970). The postulated pathway of malonylCoA is shown in Fig. 9. The condensation of malonamylCoA with other molecules of

pyruvate

,~ AcSCoA

/COS-E --------------i- CHE

+co2 ~ MaSCoA

""C00H

glutamate - ~ l u ~ 2- oxoglutarate---~ osporagine

-

CO-COOH [

/COSCoA --- CH 2 ~

CH2_ CONH2

CH2-'COS-E I C0-CHz-CONHz

/COS-E CH2

"~ CONH2

7~,~ 0 ~ S - E

\CONH 2

-3~o 0 " ~ ~ ' ~ L O S - E

Y

~

~I" ~ CONH2

Y

Y ~

0

0

0

0

0

0

0

~

C0NHz

0

Fig. 9. Postulated biosynthetic origin of the tricyclic nonaketide (Van~k et ak, 1971)

0

O

o

N

Me

OH

7. y OH

y OH

y OH

y

2H -H20

__

co~,..,~

'oH ;H ;H sH

"CONH2~_

/

A-ring cyCtlzation

OH

OH

y

OH

OH

OH

0

t

OH t OHYOH"rd 0 -co~.2

coNn~

"-< "-~ "CONH2

~retetram~d

OH

OH

~cm21h'q3rotetramid

~ C O O H

y

~

y y ~ y-co=H2

Me

to protetrone

Fig. 10. Biosynthesis of tetracycline-type corn pounds. Transformation of hypothetical tricyctic nonaketide (Van~k et aL, 1971)

0

Y Y "r CONm~

/

/

o y ~ ? ~ Y "T Y Y

Me--C6

B4

I-

.N u:

(~H ~H

~

-2H

6- cleoxytetramidg....

~ C O N H 2 ~

Me

0

--

0:~ ox00aHnhyd0ro~etra~yciine "~,~"

H20- C4a!2a

~_

C~ 0 ~ H

0

OH

Me CONH2

6- deoxychloraureovoddln

Me

OH d OH OH methyLchlorpretetramid

C

CI

4 ..... hydrOchlortetr'acycline

C{ hle

Fig. 1 l. Biosynthesis of tetracycline-type compounds. The outset of the methylated series (Van~k et al., 1971)

l

I (~H ~J

~ C O N ~ 2

0H--C~.

®

C6

~' C6

C5

C5

C4

C3

C2

~,C1

"-,4

£

;>

.e"

m

o

7 UU

o

O

O

/ 4 B1

OH

0

0

C0N H~"

a minoanhydroch[or tetra cyc[ine

OH

NH 2

2 ~ Me

OH

Me

O

()

NMe 2

anhyd rochlor tet racycLine

OH

C~

0NH 2

0H--C6

OH

NMe2

O

OH

0

0

O

CONH2

OH

4 - oxodebydrochior tet racydine

OH

Crl ~e

de__hhy.droch[ort et racycline

CI Me

2H

NMe2

0

OH

OH

0

~_~,

4 - oxochtor tet racydine

OH

CI Me

OH

cMor tet racycline

CI Me

CONH 2

OH

0NH2

Fig. 12. Biosynthesis of tetracycline-type compounds; chlortetracycline series (Van~k et al., 1971)

\

Me

/•[

NH 2

©

.--,_

>,

o

N :=

4~ O,O

0

NH2

4 " aminoonhydrotet rocyctine

OH OH 0

Me

2xMe

O

NMe2

onhydfotetfocycline

OH OH 0

Me

OH-C 6

OH 0

0

0

O

OH

4 - oxodehydrotetra cycline

Me OH

dehydrofetracycline

2H

0

4 - oxotetrocyclir."

Me OH

tetrocycline

Fig, 13. Biosynthesis of tetracycline-type compounds; tetracycline series (Vanfik et al., 1971)

B1

NH2

@

OH

>

B

50

Z, HO~fALEK et al.

malonate probably proceeds on a protein template, the hypothetical nonaketide then being cyclized and aromatized. The enzyme or rather enzyme complex catalyzing the reaction ("anthracene synthase") probably represents the first step of the biosynthetic pathway which is under the control of a specific locus. In contrast with the preceding reactions this is a rather specific process, typical of tetracycline producers. Fig. 10 shows two possibilities for the transformation of the hypothetical tricyclic derivative formed from the nonaketide by triple dehydration. In the initial stage, methylation proceeds in position 6 (branch B 1 and B 2) and is followed by cyclization of ring A (branch B I and B 3) and by the removal of the oxo group at Cs. It follows from Fig. 11 that in the following step, the biosynthetic pathway of tetracyclines is divided into two branches. The decisive step is a hydroxylation of ring A in position 4, followed by oxidation, hydration at Ca, and C12, and finally by chlorination in position 7. In the next step the tetracycline derivatives branch into two further routes (Fig. 12). On replacing the oxygen of ring A with an amino group, double N-methylation takes place and anhydrochlortetracycline is formed. The last biosynthetic steps are hydroxylation in position 6 and the final reduction which gives rise to CTC. Fig. 13 shows the metabolites formed under the conditions of blocked chlorination. The final product is TC. Fig. 14 shows the formation

@ NH2

2 x Me

OH-C6

C[

CONH2

Me

NMe2 H

"y"U'~'~'CONH2 OH 0

OH OH

C,I Me OH

NMe2

"U "r" " ~ " ~ "CONH2 OH 0

OH OH

methylchlor t e t r o m i d -

blue

el

el Me OH

0

~ c O ~ N H 2 OH 0

OH 0

chlorletrarnid - green

Fig. 14. Biosynthesisof chlortetramid compounds (Van~k et al., 197l)

51

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

® NH2

2 x Me

0H- C6

M=e

NIM%H

Me OH

N,MeaoH

CONH2 OH O

OH 0

OH OH

CONH2

OH OH methyltetramid - blue

BI

Me OH

_ _0

CONH 2

tetromid - ~reen

Fig. 15. Biosynthesis of tetramid compounds (Van~k et aL, 1971)

@ NH2

2xMe

OH-C6

CI

BI

Me OH

OH

~,, ~ C O N H z O H OH O OH OH chloraureovocidin

~,, ~

Me OH O H

OH

BI

CONH2 OH O OH OH aureovocidin

Fig. 16. Biosynthesis of aureovocin compounds (Van~k et al., 1971)

52

Z. HOg1"ALEKet al.

of two hypothetical intermediates, methylchlortetramid-blue and chlortetramid-green. If the metabolic blocks of the preceding diagram are joined by a block of chlorination, methyltetramid-blue and tetramidgreen are formed (Fig. 15). The whole diagram is supplemented by Fig. 16 where no oxidation of ring A takes place, and by Fig. 17 where no hydroxylation at C4 occurs.

® NH2

2xMe

OH-C-,6

CI Me OH BI CONH 2 OH 0 OH OH met hylhydroxychlor pretetra m id

Me

B!

CONH2 OH 0 OH OH me t hylhydroxypretetramid

Fig. 17. Biosynthesis of pretetramid compounds (Van~k et al., 1971)

Fig. 18 shows the beginning of the biosynthetic series in which methylation in position 6 was inhibited, the series being analogous with the methylated one. If the fourth ring of the hypothetical precursor is not closed, the number of intermediates and final metabolites is substantially reduced (Fig. 19). The enzymes modifying ring A cannot play a role here. It appears, however, that the enzyme hydroxylating tetracenes in position 6 is relatively nonspecific, this being evidenced by the two isolated metabolites of this series, "anthrone" and protetrone. Fig. 20 summarizes the enzyme reactions considered here, including all the intermediates and final products. The metabolites shown by full circles have already been isolated, the empty circles represent substances which are so far hypothetical. There is a total of 72 metabolites

O

H ik_Jl 'i ~ " ~ " ~ " ~ " ~ "CONH2 ~ OH 0 OH 0 ~

~

0

.0H IT

~

-

CONH2

~

chlorpretet

CL

OH

o.o

~OH bT

OH

OH

o

ramid

O

H CONH2

hyl- 6-deoxy-7- ¢hloraureovocidin

OH 0

o.o

~ ~. ~ ~ rf-%'Y Tf-"~T

ct

"

~6*demet

0

0

4- oxoanhydrodemethylchlortetracycline

OH OH 0

CI

cl

~

. . . . . hydrodemethyltetracycline --

T

H

~

CI

~

~

kk,.Ji,

~ ~ r K ' ~ T f ~T

H20--C4a,C12a

Fig. 18. Biosynthesis of tetracycline-type compounds. The outset of the non-methylated series (Van~k et al., 1971)

/

\

/o

o

/ ~

OH

-2H

..-/~/~..~"~/OH I f hi I f ~Tf'~T

OH--C4

@

-

C12

C12

- Cll

- C9

- C8

C7

5

o

>

----I.

F,

m

t~

O

F,

C)

54

Z. HO~,%~LEK et al.

of which 27 are already known and 45 are hypothetical. All are derived from a postulated tricyclic derivative by a combination of 11 enzyme reactions; methylation at C6, cyclization of ring A, removal of the oxo group from C8, hydroxylation at C4, oxidation of ring A, hydration at C~a and C~2,, chlorination at Cv, transamination, double N-methylation, hydroxylation at C6 and final reduction. The maximum number of metabolites is formed after hydroxylation at C6, which points to the relatively low specificity of the corresponding enzyme. The metabolic blocks at the level of these enzymes result in the production of the corresponding metabolites which accumulate in the culture medium.

®® OH-C4

-2H

Cl-C 7

NHz

2xMe

OH-C 6

H20"C,~,=zo

"r" ~T~ "~'C°NH2 OH

0

OH OH

"anthrone"

OH-C6 -2H

0 A"

~

C

OH ONH2

protetrone

Fig. 19. Biosynthesis of tricyclic compounds (Vanfik et al., 1971)

On the simplified assumption that one gene is responsible for the production of any one protein it could be speculated that in the final phase of biosynthesis (after condensation of the malonate units to the tricyclic nonaketide) there are at least 11 structural genes in action. On the basis of this consideration one can assume that some 300 genes participate directly or indirectly in the biosynthesis of the tetracycline molecule. A number of these can be detected and identified during

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics 04

EI t=

55

t

o

~I 7. O a

~ \

j° ~t ..............

o Nt-_e_ . . . . . . . . . . .

I

-

B2

I

i

! I ,o

I

\, A

. . . . . . . . . . . . .

LB__3_ . . . . . . . . . . . .

N J - ! 2_

.

.

.

.

.

.

.

.

.

.

I i I I I ! !

'°I-A

I.~'_~

'"

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

•

Fig. 20. Total scheme of the biosynthesis of tetracycline compounds (Van~k et al., 1971)

a genetic analysis as loci responsible for the biosynthesis of the antibiotic. The important genes responsible for the formation of tetracyclines may thus belong to different, frequently independent, metabolic pathways. A common feature of these loci is the final effect of their action-provision of intermediates for the biosynthetic process. Van~k et al. (1971) assume that the loci controlling the final biosynthetic reactions, i.e. the transformation of the hypothetical tricyclic nonaketide,

56

z. HOg~ALEKet al.

are grouped into the so-called ctc-operon, This idea is supported by the results of genetic analysis performed in S. rimosus (Boronin and Mindlin, 1971) and S. aureofaciens (Blumauerovfi et al., 1972).

b) Quantitative Aspects of the Biosynthesis of Tetracyclines As was shown in the first section, the most effective procedure for increasing the production of tetracycline antibiotics is still the mutagenic treatment of production strains. So far it has not been explained satisfactorily which genetic determinants are affected by the mutagenic treatment, whether we are dealing here with structural and regulatory genes of the biosynthetic pathway proper or with other specific genetic loci. To obtain a satisfactory answer to this problem one must first have a concrete idea of the genetic control of the biosynthetic process. The view is still accepted that the formation of natural substances is in principle a sequence of reactions which proceed independently of one

CH3

F CH2OH "l OH

a =

6-methytsalicylic acid

~ "OH m - cresol e~t CH3 HO~ OH toluquinol

CH3

L , . ~ / - OH

j

_--= ~L--~ COOH C

OH

b=

6-hydroxymethylselicylic 6 - formylsalicylic acid

a

b

OH m- hydroxybenzyl el olcohot CP~OH HO.~

0 ~'

OH gentisyl olcohol

CH2OH oCL °

0 toluquinone

COOH

CHO

gentisyI quinone

OH

C

rn- hydroxybenzaldehyde

el

~OH gentisaldehyde

m -hydroxybenzoic acid

el

COOH ~ HO~H

Ha ~

~ "OH 3 - hydroxyphthalic oci,

¢

gentisic acid

0 0 ~ 0

H

patuIin

Fig. 21. Postulated origin of patulin from 6-methylsalicylic acid in Penicillium urticae. Six types of diversifying reactions ( a - f ) mediated by sequentially induced enzymes, not having high substrate specificity, suffice to affords a large series of metabolites. Interplay of these reactions lead to a production of different metabolites by alternate sequence (after Bu'Lock et al., 1965)

Genetic Problems of the Biosynthesisof TetracyclineAntibiotics

57

another, in a random order and under catalysis of various enzymes induced by the accumulation of one of the biosynthetic intermediates. Such a view was presented by Bu'Lock et al. (1965) on the model of patulin biosynthesis by Penicillium urticae (Fig. 21). He is of the opinion that the formation of this compound is induced by the accumulation of 6-methylsalicylic acid in the medium; other intermediates are likewise attributed the role of inducers of the synthesis of further enzymes. The biosynthesis proceeds through the so-called diversifying reactions which are mediated by sequentially induced enzymes (Bu'Lock and Powell, t965). However, the most recent results of a study of the conversion of 6-methylsalicylic acid to patulin indicate that the process does not take place at random but that it obeys a firm inner order. Using a kinetic pulselabelling technique, Forrester and Gaucher (1972) demonstrated that

acetyl CoA+3 malony/CoA 3 CO- m''-NADPH*H~ + z'~NADP~ 4 CoASHCH3 COOH

H~.

~

6-rnethylsolicylic acid

1C83

COOH ~COOH "~'~'OR 3-hydroxyphthalicacid

COOH ~'~OH 6 ~ formylsalicylic acid OH NADP® NAOPH÷He CliO

ON

m - cresol

1

CN3 HO~]~O H toluquinol CH3

o

m- hydroxybenzyl ~ alcohol CH2OH HO,~

CH20H

0 toluquinone

- - -

m- hydroxybenzaldehyde 1 H~ HO

OH gentisyl alcohol

0 g~ntisyl quinone

COOH

m- hydroxybenzoicacid HO

CO~H

OH

OH

gentisaldehyde

gentisic acid

1

0

CHO HO,~ OOH ~

pre- patulin

~

O ~

OH

~.jO pa/ulin

Fig. 22. The preferred pathway for patulin biosynthesisin P. urticae is indicated by heavy arrows, with branch reactions indicated by light arrows and probable additional reactions by dashed arrows. The metabolites generally present in trace amounts if at all, are not connected by arrows (after Forrester and Gaucher, 1972)

58

Z. HO~I'~LEK et al.

the biosynthesis of patulin follows a rigorously determined metabolic sequence. Other minor metabolites considered before as intermediates represent merely the by-products of the principal biosynthetic pathway (Fig. 22). It is likely that CTC biosynthesis proceeds along similar lines, i.e. that the individual steps follow a defined sequence. It is thus logical to assume that the t t genes responsible for the final section of the biosynthetic pathway are clustered. In such a case it would be hardly predictable that a mutagenic treatment causing a block or retardation of one of these reactions would result in increasing the production of a standard metabolite, such as CTC for S. aureofi~ciens. A genetic block in the chlorination step will increase the yield of TC but at the expense of CTC production. Sometimes a metabolic block results in the formation of an intermediary metabolite at higher molar concentrations as compared with standard metabolites. This is apparently due to abolition of end-product control or to uncoupling of another, rate-limiting, reaction. As an example we may cite mutants of S. aureofaciens producing excessive amounts of dehydrochlortetracyclines (Growich and Miller, 1961). The metabolic block in the last step of the biosynthetic pathway of tetracyclines, i.e. final reduction (Fig. 12), makes impossible the end-product control of the whole process. It indicates a high structural (conformational) specificity of the feedback mechanism. In this connection it is interesting to mention that the antimicrobial activity of dehydrotetracyclines is fairly low as well. The only change in the biosynthetic pathway proper that would result in increased formation of a standard metabolite, is an acceleration of the reaction which is rate-limiting in the sequence. One may consider in this context an increased synthesis of one of the enzymes involved by mutation or an increase of the gene dose of the cell. Genetic changes in the control system of the metabolic pathway proper thus cannot be by themselves responsible for the broad variability in tetracycline production in different strains of a given species. For this reason, most microbial breeders still accept the views of classical genetics explaining the continual variability of quantitative features, such as the level of produced antibiotic, by the existence of a great number of genes controlling the realization of the genotype. The so-called polygenic inheritance is based on the view that the phenotypic expression of a character is controlled by a number of genes, each of which contributes to its formation but taken alone has a relatively little effect. Results of the study of tetracycline biosynthesis indicate that the level of production is considerably affected by the cultivation conditions and is closely associated with the overall metabolic activity of the

Genetic Problems of the Biosynthesisof TetracyclineAntibiotics

59

culture (Ho~[~ilek and Van~k, 1973). It appears that the productive activity can be affected even by genetic changes in the primary metabolism. The primary precursors of a number of natural substances represent common intermediates both of essential metabolites and of specific secondary metabolites of production strains. It has been shown experimentally that the rates of reactions of the alternative pathways of acetylCoA (i.e. above all the rate of energy metabolism) affects the quantitative character of CTC production (Ho~[filek et al., 1969). The antibiotic yield is thus controlled by genes regulating the tricarboxylic acid cycle and the respiratory chain. Retardation of this pathway through an interference affecting the rate of one of its numerous reactions, increases the possibilities of acetylCoA metabolism by the energetically relatively undemanding formation of the oligoketide chain. We feel that we may speak here of the polygenic character of determination of yield of natural substances but in a sense qualitatively different from the earlier views. One need not assume the involvement of the so-called genes of specific effect. The polygenic system determining the production as a quantitative feature comprises the structural and control genes both of the biosynthetic pathway proper and of the alternative pathways competing for a common precursor. A slowing-down of the metabolic flow of competing pathways results in a reinforcement of the biosynthetic pathway and in an increased synthesis of the antibiotic. The rate of formation of secondary metabolites in microorganisms is affected both by cultivation conditions and by induced hereditary changes of the producer genome. In particular with standard strains isolated from nature, the extent of cultivation conditions under which the product is synthesized is rather narrow; in some complex media, production may not take place at all. High production is usually due to changes in the regulatory mechanisms on the metabolic and genetic level. From the evolutionary point of view one may thus consider the so-called secondary metabolism, especially the biosynthesis of oligoketides, as a nonessential, probably detoxicating, shunt formation of organic polymers. These processes requiring not much energy have acquired prevalence only in selected strains of cultivated microorganisms. The above facts also indicate that one cannot draw a precise boundary between primary and secondary metabolism of natural compound producers. It is likely that a single general common metabolic pattern exists and that various regulatory mechanisms determine the flow of intermediates and the intensity of the individual competing metabolic pathways. The production activity is then the result of interaction of the various control levels. From this point of view then the terms secondary metabolism and secondary metabolite seem to be redundant. Van~k et al. (1973) hence suggest the use of the term excessive metabolites

60

Z. HO~'I'ALEK et al.

for compounds that are formed in a culture in amounts greater than under standard conditions. The expression reflects the quantitative peculiarities of their formation.

5. C o n c l u d i n g R e m a r k s The results so far obtained with different methods for increasing the productivity of S. aureofaciens and S. rimosus strains do not permit one to draw unequivocal conclusions on the most effective method described. For routine breeding, the application of mutagens remains the most suitable method. Hybridization might be used for obtaining strains carrying combinations of other desirable properties rather than for yield improvement. Greatest promise appears to be held by the approach employing mutagenic treatment in combination with one of the above methods. Strains obtained by hybridization might be used (because of their increased variability) as suitable starting material for mutagenic experiments (Borisova et al., 1962b; Goldat and Vladimirov, 1968). Inoculation of mutagen-treated populations into media with an addition of tetracyclines will increase the effectiveness of selection for high-production variants (Veselova and Komarova, 1968; Veselova, 1970). Likewise, mutagenic treatment of strains pretreated with tetracycline-containing agar assists in obtaining high-production strains (Katagiri, 1954). In spite of the success achieved recently with the improvement of biosynthetic activity of S. aureofaciens and S. rimosus, just as of other antibiotic producers, one cannot dismiss the fact that strain improvement is mostly based on a completely empirical approach, without deeper knowledge of the genome of the given microorganisms and of the control processes, a change of which is to be accomplished. In this context the fact should be stressed that studies of blocked mutants and cosynthetic experiments have provided new data for understanding the mechanism of biosynthesis of tetracycline antibiotics. Genetic work supplemented the results of precise chemical analysis yielded the basic information on the biosynthetic process, on the sequence of intermediates in the biosynthetic chain. Here, however, the possibilities of this methodical approach are exhausted and in this area no piercing insight into the genetic control of tetracycline formation has been achieved. Construction of the chromosome map and study of the localization of nutrition markers provide basic data on the genome topology but cannot by themselves elucidate the genetic basis of antibiotic biosynthesis. Even the existing attempts at defining the map position and linkage of determinants responsible for the biosynthesis of tetracyclines fail

Genetic Problems of the Biosynthesis of TetracyclineAntibiotics

61

to supply data for forming a concept on the genetic mechanisms controlling the production of these antibiotics as a qualitative feature. It appears that the genes responsible for the final stages of tetracycline biosynthesis are clustered and that the number of selective markers suited for linkage mapping is thus rather restricted. The selection of partners for crossing is the key problem of studying genetic control of the biosynthetic process. The fragmentary information about the chromosome and the fertility system of production strains and the lack of data on the basis of processes controlled by the individual loci, make it completely impossible to proceed systematically in recombination experiments; the selection of partners is carried out practically at random. Ala~evi6 (1969a, 1973) used auxotrophic mutants obtained after mutagenic treatment directly from standard strains. In these mutants the nutrition deficiency is usually coupled with the loss of productive activity. This fact does not mean, however, that the changes of antibiotic production are caused by metabolic blocks in the biosynthetic pathway proper but that we are rather dealing here with a disturbance of balance in primary metabolism. Detection of mutants with a defined block in a given segment of the biosynthetic chain is thus rather unpromising under these circumstances. On the other hand, during induction of double or multiple auxotrophs from prototrophic mutants blocked in a given step of the biosynthetic pathway (Blumauerov/t et al., 1972, 1973b) it is doubtless of advantage to understand the biochemical basis of the block. Nevertheless, the possibilities of further genetic analysis are rather limited even here since the multiple mutation process aids the formation of unstable, genetically-defective variants. The most recent, still unverified, experimental results indicate, however, that the biosynthesis of antibiotics may be under the control of extrachromosomal factors (e.g. the biosynthesis of kasugamycin, Okanishi et al., 1970). It is thus possible that during crossing of S. rimosus even episomal factors could be transferred, such as control the biosynthesis of OTC. This is suggested by the results of Boronin and Mindlin (1971) who reported that mutations in the otc 6 locus give rise, besides the loss in productive activity, even to the loss of resistance to the own antibiotic itself. In view of the fact that Boronin (1972) found that resistance to OTC can be cured in S. rimosus by acridine dyes, the question of localization of genetic determinants responsible for the final steps of OTC biosynthesis remains still open. Likewise, further experimental results, indicating the tight linkage of the locus responsible for the resistance to streptomycin with determinants for OTC biosynthesis, do not exclude the possibility of their extrachromo-

62

Z. HO~I"ALEKet

al.

somal localizationl Boronin and Mindtin (1971) did not specify more closely the character of streptomycin resistance in S. r i m o s u s mutants. Thus it is not known whether it is given by chromosomal mutation altering the properties of ribosomal proteins or by extrachromosomallycontrolled synthesis of an enzyme system inactivating streptomycin. Further perspectives for studying genetic control of biosynthesis of tetracycline antibiotics and practical application of the data obtained are difficult to assess at present. The decisive factor for further direction of research will lie in solving the question of genetic determinants responsible for the final steps of biosynthesis whether chromosomal loci or plasmid genes are involved. A systematic exploitation of the biosynthetic activity for attaining maximal yields of tetracyclines is clearly dependent on the degree of our understanding of the laws governing their formation. This is a question of knowledge of biosynthetic sequences as well as of regulatory mechanisms which, in their sum, are responsible for a certain level of production of the metabolite studied. The total amount of the biosynthetic capacity is not a limitless entity, the production activity being first of all determined by the balance of catabolic and anabolic processes, by the equilibrium attained between the energy metabolism of the cell providing the corresponding equivalents for the biosynthetic process and the secondary metabolic pathway itself. It is thus typical of the excessive formation of a given microbial product that, together with an intensification of the biosynthetic process, the other metabolic sequences and physiological functions of the organism are suppressed. The regulatory mechanisms functioning under certain standard conditions are modified either by a gene mutation or by extreme cultivation conditions or by combination of the two. So far only scant information is available on the enzyme systems participating in the production process. The knowledge of enzyme systems participating in biosynthesis will represent an important contribution to our understanding of the genetic regulation of the formation of tetracyclines. Genetic analysis should be aimed not only at the position of various symptomatically-labelled genetic loci but also at the linkage between the regulator and structural genes of the biosynthetic pathway. The papers describing the control mechanisms of the biosynthesis of industrially important metabolites still have a pioneering character and the practical utilization of the pertinent knowledge is still rather limited. With some optimism one can foresee in the near future a better understanding not only of the enzymes playing, directly or indirectly, an important role in the biosynthesis of tetracycline antibiotics but also of the mutual interactions of metabolic pathways and their participation in the biosynthetic process. A deeper understanding of genetic and

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

63

metabolic regulation is then likely to open the way toward m o r e rational techniques for obtaining high-production strains and toward an optimization of fermentation processes.

Acknowledgement. The preparation of this chapter was supported by the International Atomic Energy Agency Research Contract No 845/RB.

References Ala~evi6, M.: Nature 197, 1323 (1963). Ala~evi6, M.: Mikrobiologija Beograd 2, 143 (1965a). Ala~evi6, M.: Mikrobiologija Beograd 2, 159 (1965b). Ala~evi6, M.: In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala~evi6, M. (Eds.), p. 137. Zagreb: Yugoslav Acad. Sci. & Arts 1969a. Ala~evi6, M.: Mikrobiotogija Beograd 6, 9 (1969b). Ala~evi6, M.: In: Genetics of Industrial Microorganisms. Actinomycetes and Fungi. Van~k, Z., Hog[~ilek, Z., Cudlin, J. (Eds.), p. 59. Prague: Academia; Amsterdam: Elsevier 1973. Ala~evi6, M., Vegligaj, M., Pigac, J.: Genetika Beograd 4, 151 (1972). Ala~evi6, M., Vla~i6, D., Spada-Sermonti, I.: In: Antibiotics-Advances in Research, Production and Clinical Use, Herold, M., Gabriel, Z. (Eds.), p. 720. Prague: Czechoslovak Medical Press; London: Butterworths 1966. Alikhanian, S. I., Borisova, L. N.: J. Gen. Microbiol. 26, 19 (1961). Alikhanian, S. I., Goldat, S. Yu,, Teteryatnik, A. F.: Dokl. Akad. Nauk SSSR 115, 1015 (1957). Alikhanian, S. I., Ilyina, T. S.: Nature 179, 784 (1957). Atikhanian, S. I., Mindlin, S. Z.: Nature 180, 1208 (1957). Alikhanian, S. I., Mindlin, S. Z., Goldat, S. Yu., Vladimirov, A. V.: Ann. N. Y. Acad. Sci. 81, 914 (1959a). Alikhanian, S. I., Mindlin, S. Z., Orlova, N. V., Verkhovtseva, T. P.: Appl. Microbiol. 7, 141 (1959b). Alikhanian, S. I., Mindlin, S. Z, Zaitseva, Z. M., Orlova, N. V.: Dokl. Akad. Nauk SSSR 136, 468 (1961). Alikhanian, S. I., Romanova, N. B.: Antibiotiki 10, 1113 (1965). Backus, E. J., Duggar, B. M., Campbell, T. H.: Ann. N. Y. Acad. Sci. 60, 86 (1954). B~hal, V., Van~k, Z.: Folia Microbiol. (Prague) 15, 354 (1970). Blumauerovfi, M.: Ph. D. Thesis. Prague: Czechoslovak Acad. Sci. 1969. Blumauerov~i, M., Ho~[filek, Z., Mra~ek, M., Podojil, M., Vanfik, Z.: Folia Microbiol. (Prague) 14, 226 (1969a). Blumauerov~i, M., Mra~ek, M., Vondr~i~kovfi, J., Podojil, M., Ho~[filek, Z., Van~k, Z.: Folia Microbiol. (Prague) 14, 215 (1969b). Blumauerowi, M., Ismail, A. A., Ho~{~lek, Z., Van~k, Z.: In: Radiation and Radioisotopes for Industrial Microorganisms, p. 157. Vienna: International Atomic Energy Agency 1971a. Blumauerowl, M., IsmaiI, A. A., Ala~evi6, M., Ho~[~ilek, Z., Van~k, Z.: Folia Microbiol. (Prague) 16, 504 (1971b). Blumauerovfi, M., Ho~[~ilek, Z., Vanfik, Z.: In: Fermentation Technology Today. Terni, G. (Ed.), p. 223. Osaka: Soc. Fermentation Technology, Japan 1972.

64

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Blumauerov& M., Hog{~lek, Z., Vanfik, Z.: Stud. Biophys. 36/37, 311 (1973a). Blumauerovfi, M., Ismail, A. A., Ho~filek, Z., Callieri, D. A. S., Cudlin, J., Van~k, Z.: Folia Microbiol. (Prague) 18, 474 (1973b). Borenztajn, D., Wolf, Y.: Ann. Inst. Pasteur 91, 62 (1956). Borisova, L. N., Konyukhova, M. V,, Ivkina, N. S.: Antibiotiki 7, 685 (1962a). Borisova, L. N., Konyukhova, M. V., Ivkina, N. S., Oleneva, Z. G.: Mikrobiologiya 31, 850 (1962b). Boronin, A. M.: Genetika 6, 172 (1970). Boronin, A. M.: Abstracts. All-Union Conference on the Regulation of Biochemical Processes in Microorganisms (Pushchino 1972), p. 22, 1972. Boronin, A. M., Mindlin, S. Z.: Genetika 6, 72 (1970). Boronin, A. M., Mindlin, S. Z.: Genetika 7, 125 (1971). Bu'Lock, J. D., Hamilton, D., Hulme, M. A., Powell, A. J., Smalley, H. M., Shepherd, D., Smith, G. N.: Can. J. Microbiol. 11,765 (1965). Bu'Lock, J. D., Powell, A. J.: Experientia 21, 55 ([965). Coats, J. H., Roeser, J.: J. Bacteriol. 105, 880 (1971). Deli6, V.: In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala~evid, M. (Eds.), p. 177. Zagreb: Yugoslav Acad. Sci. & Arts 1969. Deli6, V., Pigac, J., Sermonti, G, : J. Gen. Microbiol. 55, 103 (1969). Doerschuk, A. P, McCormick, J. R. D., Goodman, J. J., Szumski, S. A., Growich, J. A., Miller, P. A., Bitler, B. A., Jensen, E. R., Matrishin, M., Petty, M. A., Phelps, A. S.: J. Am. Chem. Soc. 81, 3069 (1959). Dole~ilovfi, L., Spi~ek, J., Vondrfi&k, M., Pale~kovfi, F., Van~k, Z.: J. Gen. Microbiol. 39, 1 (1965). Duggar, B. M.: Ann. N. Y. Acad. Sci. 51, 177 (1948). Duggar, B. M., Backus, E. J., Campbell, T. H.: Ann. N. Y. Acad. Sci. 60, 71 (1954). Dulaney, E. L.: In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala&vid, M. (Eds.), p. 93. Zagreb: Yugoslav Acad. Sci. & Arts 1969. Dulaney, E. L., Dulaney, D. D.: Trans. N. Y. Acad. Sci. 29, 782 (1967). Erokhina, L. I.: Genetika I, 61 (1965). Erokhina, L. I., Alikhanian, S. I.: In: Antibiotics-Advances in Research, Production and Clinical Use. Herold, M., Gabriel, Z. (Eds.), p. 698. Prague: Czechoslovak Medical Press; London: Butterworths 1969. Fedorova, t. V., Alikhanian, S. I.: Antibiotiki 10, 579 (1965). Finlay, A. C., Hobby, G. L., P'an, S. Y., Regna, P. P., Routien, J. B., Seeley, D. B., Shull, G. M., Sobin, B. A., Solomons, I. A., Vinson, J. W,, Kane, J. H.: Science I l l , 85 (1950). Forrester, P. I., Gaucher, G. M.: Biochemistry II, 1102 (1972). Friend, E. J., Hopwood, D. A.: J. Gen. Microbiol. 68, 187 (1971). Frolova, V. I., Rosenfeld, G. S., Listvinova, S. N.: Antibiotiki 16, 687 (1971). Goldat, S. Yu.: Antibiotiki 3, 14 (1958). Goldat, S. Yu.: Tr. Inst. Mikrobiol. Akad. Nauk SSSR 10, 159 (1961). Goldat, S. Yu.: Genetika 1, 106 (1965). Goldat, S. Yu., Sokolova, R. V.: Antibiotiki 9, 126 (1964). Goldat, S. Yu., Vladimirov, A. V.: Genetika 4, 5 (1968). Goodman, J. J., Matrishin, M., Backus, E. J.: J. Bacteriol. 69, 70 (1955). Gordee, E. Z., Day, L. E.: Antimicrob. Agents Chemother. 1,315 (1972). Growich, J. A., Miller, P. A.: US Pat. 3007965 (1961). Gutnikova, M. N.: In: Supermutageny. Rapoport, I. A. (Ed.), p. 62. Moscow: Publ. House Acad. Sci. USSR 1966.

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

65

Hochstein, F. A., Schach von Wittenau, M., Tanner, F. W., Mural, K.: J. Am. Chem. Soc. 82, 5934 (t960). Hopwood, D. A.: Genet. Res. 6, 248 (1965). Hopwood, D. A.: Bacteriol. Rev. 31, 373 (1967). Hopwood, D. A., Sermonti, G.: Advan. Genet. 11, 273 (1962). Horvfith, J.: Acta Microbiol. Acad. Sci. Hung. 1, 131 (1954). Ho~[~lek, Z., Tint~rovfi, M., Jechov~, V., Blumauerovfi, M., Such~, J., Van~k, Z.: Biotechnol. Bioeng. 9, 539 (1969). Ho~[~dek, Z, Van~k, Z.: In: Genetics of Industrial Microorganisms. Actinomycetes and Fungi. Van~k, Z., Ho~[filek, Z., Cudlin, J. (Eds.), p. 353. Prague: Academia; Amsterdam: Elsevier 1973. Hovorkovfi, N., Cudlln, J., Mat~jt], J., Blumauerovfi, M., Van~k, Z.: Collection Czech. Chem. Commun. (1974), in press. Hiiber, J., Giinter, H.: Zentr. Bakteriol. Parasitenk., Abt. II 113, 672 (1960). Jfirai, M.: Acta Microbiol. Acad. Sci. Hung. 8, 73 (1961). Katagiri, I.: J. Antibiotics (Tokyo) 7, 45 (t954). Kutzner, H. J.: Zentr. Bakteriol. Parasitenk., Abt. II 121, 395 (1967). Lancini, G. C., Sensi, P.: Experientia 20, 83 (1964). Legator, M., Gottlieb, D.: Antibiot. & Chemotherapy 3, 809 (1953). Ludvik, J., Mikulik, K., Van~k, Z.: Folia Microbiol. (Prague) 16, 479 (1971). Makarevich, V. G., Laznikova, T. N., Gutnikova, M. N., Rapoport, I. A.: Antibiotiki ll, 980 (1966). Malik, V. S., Vining, L. C.: Can. J. Microbiol. 16, 173 (1970). Mat~jfi, J., Cudlin, J., Hovorkov~i, N., Blumauerov~, M., Van~k, Z.: Folia Microbiol. (Prague) (1974), in press. McCormick, J. R. D.: In: Biogenesis of Antibiotic Substances. Van~k, Z., H o ~ t e k , Z. (Eds.), p. 73. Prague: Publ. House Czechoslovak Acad. Sci.; London: Academic Press 1965. McCormick, J. R. D.: In: Antibiotics-Advances in Research, Production and Clinical Use. Herold, M., Gabriel, Z. (Eds.), p. 556. Prague: Czechoslovak Medical Press; London: Butterworths 1966. McCormick, J. R. D.: In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala~evi6, M. (Eds.), p. 163. Zagreb: Yugoslav Acad. Sci. & Arts 1969. McCormick, J. R. D., Gardner, W. E.: US Pat. 3074975 (1963). McCormick, J. R. D., Jensen, E. R.: J. Am. Chem. Soc. 87, 1794 (1965)~ McCormick, J. R. D., Jensen, E. R.: J. Am. Chem. Soc. 90, 7126 (1968). McCormick, J. R. D., Jensen, E. R.: J. Am. Chem. Soc. 91, 206 (1969). McCormick, J. R. D., Sjolander, N. O., Hirsch, U, Jensen, E. R., Doerschuk, A. P.: J. Am. Chem. Soc. 79, 4561 (1957). McCormick, J. R. D, Miller, P. A., Growich, J. A., Sjolander, N. O., Doerschuk, A. P.: J. Am. Chem. Soc. 80, 5572 (1958). McCormick, J. R. D., Hirsch, U., Sjolander, N. O., Doerschuk, A. P.: J. Am. Chem. Soc. 82, 5006 (1960). McCormick, J. R. D., Sjolander, N. O., Hirsch, U.: US Pat. 2998352 (1961). McCormick, J. R. D., Hirsch, U., Jensen, E. R., Johnson, S., Sjolander, N. O.: J. Am. Chem. Soc. 87, 1793 (I965). McCormick, J. R. D., Jensen, E. R., Johnson, S., Sjolander, N. O.: J. Am. Chem. Soc. 90, 2201 (1968a). McCormick, J. R. D., Jensen, E. R., Arnold, N. H., Corey, H. S., Joachim, U. H., Johnson, S., Miller, P. A., Sjolander, N. O.: J. Am. Chem. Soc. 90, 7127 (1968b).

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et al.

MikuHk, K., Karnetov/t, J., K~emen, A., Tax, J., Van6k, Z.: In: Radiation and Radioisotopes for Industrial Microorganisms, p. 201. Vienna: International Atomic Energy Agency t971a. Mikulik, K., Karnetov~i, J., Quyen, N., Blumauerovfi, M., Komersovfi, I., Van~k, Z.: J. Antibiotics (Tokyo) 24, 801 (1971b). Miller, M. W., Hochstein, F. A.: J. Org. Chem. 23', 2525 (1962). Miller, P. A., Saturnelli, A., Martin, J. H., Mitscher, L. A., Bohonos, N.: Biochem. Biophys. Res. Commun. 16, 285 (1964). Miller, P. A., Sjolander, N. O., Doerschuk, A. P., McCormick, J. R. D.: J. Am. Chem. Soc. 82, 5002 (1960). Mindlin, S. Z. : In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala~evi6, M. (Eds.), p. 147. Zagreb: Yugoslav Acad. Sci. & Arts 1969. Mindlin, S. Z., Alikhanian, S. I.: Antibiotiki 3, 18 (1958). Mindlin, S. Z., Alikhanian, S. I., Vladimirov, A. V., Mikhailova, G. R.: Appl. Microbiol. 9, 349 (1961a). Mindlin, S. Z., Kubyshkina, T. A., Alikhanian, S. I.: Antibiotiki 6, 623 (1961b). Mindtin, S. Z., Vladimirov, A. V., Borisova, L. N., Mikhailova, G. R.: Tr. Inst. Mikrobiol. Akad. Nauk SSSR 10, 187 (1961c). Mindlin, S. Z., Zaytseva, Z. M., Germanov, A. B., Shishkina, T. A.: In: AntibioticsAdvances in Research, Production and Clinical Use. Herold, M., Gabriel, Z. (Eds.), p. 695. Prague: Czechoslovak Medical Press; London: Butterworths 1966. Mindlin, S. Z., Zaitseva, Z. M., Shishkina, T. A.: Genetika 4, 126 (1968). Niedzwiecka-Trzaskowska, J., Stzencel, M.: Ann. Inst. Pasteur 91, Suppl. 12, 72 (1956). Okanishi, M., Ohta, T., Umezawa, H.: J. Antibiotics (Tokyo) 23, 43 (1970). Orlova, N. V.: Antibiotiki 13, 291 (1968). Orlova, N. V., Smolenskaya, N. M.: Antibiotiki 10, 210 (1965). Orlova, N. V., Smolenskaya, N. M., Zaitseva, Z. M.: Mikrobiologiya 33, 1032 (1964). Ortova, N. V., Zaitseva, Z. M., Khokhlov, A. S., Cherches, B. Z.: Antibiotiki 6, 629 (1961). Perlman, D., Heuser, L. J., Dutcher, J. D., Barrett, J. M., Boska, J. A.: J. Bacteriol. 80, 419 (1960). Petty, M. A.: Bacteriol. Rev. 25, 111 (1961). Pigac, J.: In: Genetics and Breeding of Streptomyces. Sermonti, G., Ala~evi6, M. (Eds.), p. 160. Zagreb: Yugoslav Acad. Sci. & Arts 1969. Piperno, R., Carere, A., Sermonti, G.: Ann. Ist. Super. Sanit/~ 2, 393 (1%6). Podojil, M., Van~k, Z., Vokoun, J., Cudlin, J., Blumauerovfi, M., Vondrfi~k, M., Hassal, C, H.: Abstracts. 1st internat. Symp. Genetics of Industrial Microorganisms, p. 106. Prague 1970. Polsinelli, M., Beretta, M.: J. Bacteriol. 91, 63 (1966). Sermonti, G.: Genetics of Antibiotic-Producing Microorganisms, p. 263. London: John Wiley & Sons 1969. Shen, S. C., Shan, W. C.: Mikrobiologiya 24, 458 (1957). Van Dyck, P., De Somer, P.: Antibiot. & Chemotherapy 2, 184 (1952). Van~k, Z., Cudlin, J., Blumauerov~,, M., Ho~ilek, Z.: Folia Microbiol. (Prague) 16, 225 (1971). Van~k, Z., Ho~[tilek, Z., Blumauerov~i, M., Mikulik, K., Podojil, M., B~hal, V., Jechovfi, V.: Pure Appl. Chem. 34, 463 (1973). Veselova, S. I.: Genetika 3, 73 (1967). Vesetova, S. I.: Antibiotiki 15, 219 (1970).

Genetic Problems of the Biosynthesis of Tetracycline Antibiotics

67

Veselova, S. I., Komarova, L. V.: Genetika 4, 100 (1968). Vladimirov, A. V.: Genetika 4, 53 (1968). Vladimirov, A. V., Mindlin, S. Z.: Genetika 3, 152 (1967). Vokoun, J.: Ph.D. Thesis. Prague: Czechoslovak Acad. Sci. 1970. Wang, E. L.: J. Antibiotics (Tokyo) Ser. A 10, 254 (1957). Zaitseva, Z. M., Orlova, N. V., Mindlin, S. Z., Alikhanian, S. I., Khokhlov, A. S., Cherches, V. Z.: Dokl. Akad. Nauk SSSR 136, 714 (196t). Zaitseva, Z. M., Orlova, N. V.: Mikrobiologiya 31,449 (1962). Dr. ZDEN~K HO~TALEK Dr. MARGITABLUMAUEROV,~ Dr. Z. VANI~K Czechoslovak Academy of Sciences Institute of Microbiology Budejovick~ 270 Praha 4 - KR• ((~SSR)

CHAPTER

2

Some Aspects of Basic Genetic Research Fungi and Their Practical Implications 1

on

KARL ESSER 2' 3 With 5 Figures

Contents 1. Fungi Used in Industrial Practice to Manufacture Certain Products, M a k i n g Use of Their Particular Metabolic Characteristics .... 2. M u s h r o o m s Serving as Food . . . . . . . . . . . . . . . . . . I. Control of Recombination by Sexual Systems . . . . . . . . . . . 1. Monoecism a n d Dioecism . . . . . . . . . . . . . . . . . . 2. Incompatibility . . . . . . . . . . . . . . . . . . . . . . . 3. Heterokaryosis . . . . . . . . . . . . . . . . . . . . . . . II. The Production of M u t a n t s

70 71 72 74 74 75

. . . . . . . . . . . . . . . . . . .

76

. . . . . . . . . . . . . . . . . .

81

III. The S y n d r o m e of Senescence

IV. Conclusions for Breeding Research with Useful Fungi . . . . . . . i. Conservation of Yield by Regular Regeneration . . . . . . . . . 2. Increase of Yield by a C o m b i n e d Programme of Mutation, Selection and R e c o m b i n a t i o n . . . . . . . . . . . . . . . . . . . . . 3. Adoption of New Strains for the Production of Useful Fungi . . .

84 85

Conclusion

86

References

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83 84

87

Translated with the kind permission of the editor and publisher from the original G e r m a n version, published in: J a h r b u c h 1971/72; Der Minister f'tir Wissenschaft und Forschung des Landes Nordrhein-Westfalen - L a n d e s a m t ftir Forschung . Westdeutscher Verlag, Opladen, pp. 79-98. We are indebted to N. A. Bush for the translation. 2 To my friend J. R. Raper (Harvard University, USA) on completion of his 60 th anniversary. 3 The original experimental work mentioned in this article was carried out with the support of the ,,Landesamt f'tir Forschung" (NRW, Germany).

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O v e r the last few decades fungi have steadily gained i m p o r t a n c e as objects in basic genetics research. Like the microbes (viruses and bacteria) they have contributed to our knowledge on structure and function of the genetic material on a molecular level. A p a r t from these p r o b l e m s of general biological importance, of which e v e r y b o d y has increasingly b e c o m e a w a r e under the catchword of "molecular genetics", there are still other aspects of "fungal genetics", which although less popular, are still of great i m p o r t a n c e for a concerted cultivation of useful fungi. U n d e r the term of"useful fungi" we should c o m b i n e two groups (independent of their t a x o n o m i c classification):

1. Fungi Used in Industrial Practice to Manufacture Certain Products, Making Use of Their Particular Metabolic Characteristics Like yeasts and bacteria in alcoholic and lactic fermentation respectively fungi are of importance in many fermentative processes. Some of the Penicillium species are responsible for the aromatic substances in cheese (Camembert, Roquefort etc.). Some processes are based on the fact that fungi, after an oversupply of carbohydrates, develop primary metabolites (e.g. organic acids) to such an extent that they are no longer transformed by the intermediary metabolism and are excreted into the nutrient medium. In using certain mutants, amino-acids can be accumulated by blocking a particular reaction step in a biosynthetic sequence, thus avoiding further processing of the amino-acid concerned. The so-called biological transformation reactions represent another application making use of the ability of many fungi to transform organic compounds specifically in one single-step process into other compounds. In some steroid-transforming reactions (inhibitors of ovulation, anti-arthritics, etc.) an essential synthetic step, the l lc~-hydroxylation, is carried out by the fungus Curvularia, in a single-step process, which otherwise would encounter technical difficulties. Last not least it may be mentioned that a large number of antibiotics are synthesized by certain fungi. The production of 6-amino penicillanic acid by certain mutants of Penicillium chrysogenum has in recent years gained increased importance; this product serves as starting substance for subsequent in vitro production of the so-called semi-synthetic penicillins. However, it cannot be overlooked in this connection that besides the fungi bacteria play a role in these industrially important metabolic conversions which is at least equal to that of the fungi (see Rehm [t] and Z/ihner [2] for additional references).

Some Aspects of Basic Genetic Research on Fungi

71

2. Mushrooms Serving as Food Apart from the production of "champignons" (Agaricus bisporus), mushrooms have so far not been cultivated in Europe 4 for human nutrition on an industrial scale although they have a higher protein content than other vegetables [3]. This is mainly due to an almost total lack of commercial cultivation of other mushroom species, as truffles (Tuber aestivum, Tuber brumale etc.). A beginning has been made in the United States and recently in Hungary with semi-industrial production of the oyster mushroom, Pleurotus ostreatus, a wood-destroying fungus [4, 5]. The aims of breeding research with regard to useful fungi are, on the one hand, to obtain stable strains which maintain their productivity in the course of prolonged vegetative propagation, and, on the other hand, to increase the yield. This objective differs in no way from the general principles of any animal- and plant-breeding. However, the modern methods used in these fields have little counterpart in the cultivation of fungi. In animal- and higher-plant-breeding "positive mutations", obtained either by artificial induction or by isolation from natural strains, are combined with higher breeds by means of genetic recombination, whereas the classical principle of selection is to a large extent relied upon in genetic research and development of mushrooms. In the search for more efficient strains one restricts oneself to the isolation of new strains from nature. If and when productivity decreases, the strain concerned is eliminated and a start with fresh isolates is made. This rather primitive approach, that differs markedly from the procedures used in other areas of genetical research, appears to be justified by reduced cost. No doubt it is more economical, especially in small production plants, to work on the "seek-and-throw-away" principle than to entertain a laboratory for basic research. However, such a procedure does not necessarily pay off, because it is not predictable whether and when a change in yield will occur either through spontaneous mutation or through "ageing" (see page 81 and following) and this can lead to costly losses, especially in the fermentation industry. In addition a planned cultivation with a view to increasing yield is impossible on this basis. It may be seen from the foregoing that it cannot be the purpose of this treatise to show in comprehensive form the genetic facts gained with fungi--this has already been done, see Esser and Kuenen [6] , but to indicate in condensed form some findings in the genetics of fungi whose application should permit a concerted breeding of useful fungi. 4 In Japan the Shii-take mushroom (Lentinus edodes) has been cultivated for centuries. The annual exports (mainly in dried condition) are 5000 to 6000 tons.

72

KARL ESSER

I. Control of Recombination by Sexual Systems Recombination of genetic material is a basic phenomenon causing a constant rearrangement and reorganization of genetic information. It is mainly based on a steady alternation of karyogamy and meiosis, occurring in the course of sexual propagation. It is a peculiarity of the fungi that also those species which have lost their ability of sexual propagation during their evolution (.fungi imperfecti) can recombine their hereditary material to a limited extend via so-called parasexual processes. A great number of species that are of industrial importance belong to the imperfecti, e.g. the penicillin producers.

fimgi

The mechanism of recombination on the molecular level is not yet known in detail, despite many efforts [6]. However, this does not concern breeding research in practice because recombination is exclusively used as a method for the incorporation of hereditary factors of economic importance. In this area of biology it is essential to know those parameters that are required for the realization of recombination, e.g. for initiation of sexual or parasexual processes. Apart from environmental factors (composition of the culture medium, light, temperature etc.) there are intrinsic genetic factors. The latter are, on the one hand, the genes controlling the development of the sexual organs (morphogenetic genes) and therewith the normal life cycle and, on the other hand, hereditary factors controlling the physiological conditions that lead to karyogamy and consequently to recombination. As there are numerous fungi (e.g. edible mushrooms) showing no sexual differentiation, the last mentioned gene category is of special importance, because those hereditary factors which exert their influence in the frame of the so-called breeding systems eventually decide whether and to what extent recombination is possible and can take place. In this connection it must be mentioned that the parasexual cycle of the imperfect fungi is also controlled by the breeding systems {for details see Esser and Kuenen [6]). There is great confusion in the scientific literature on the nomenclature of sexual systems. For a better understanding it would therefore seem advisable first to deal with the various systems (see Fig. 1). A more detailed description is given elsewhere [7].

fungi, 5ordorioetc,)

MONOECISM

( oIl setf-compotible

OIOECISM physiologicd

IPhycomvces)

HETEROGENICINCOMPATIBILITY (sexual ond vegetotivephose, e.g. Podospororoces)

HOMOGEBICINCOMPATIBILITY I Neurosporo,Schizophyllurn)

HETEROGENICINCOMPATIBILITY (vegetutivephose,e.g. Podospora, Neurosporo,Aspergitlus ond fungi imperlecti)

perTect fungi

iutoIqenes by non olteliccomplementotiooin

tteterokorvosis moy concur the defeds of struc-

lncompotibIespecies, fungi imperfedi )

Fig. 1. Actions and interactions of breeding systems in fungi. The enclosed rectangle in the centre shows the main systems. Heterokaryosis is illustrated at the right-hand side. For each system representative organisms are indicated in brackets. The rectangles in the region of the different systems represent single individuals. The rectangles concerning heterogenic incompatibility are an exception to this insofar as they symbolise single Podospora races. The sexual symbols indicate nuclei with male and female sexual potency respectively. Differences in the genetic information of the nuclei are marked black and white. As no sexual differentiation can be attributed to the nuclei in physiological dioecism these are marked with black and white circles. The bold arrows indicate the direction of karyogamy respectively heterokaryotisation. The blocked arrows indicate the impossibility of karyogamy and heterokaryotisation respectively. Interactions between various systems are indicated with thin arrows. For more details see text (from Esser [7])

hibit 14oryogomy

rnuto~ecl to sterilHv moy interfere wilhoil systems ond in-

st~ctu[o~ genes

rnorphotogicol ( AchLyo)

HETEROKANYOSIS

(Setf-cornpotibleondsetf-

O

~r

o

@

>

Go o

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KARL ESSER

1. Monoecism and Dioecism Monoecism and dioecism are the basic sexual systems. They are based on the ability of an organism to contribute one or two nuclei to karyogamy. F r o m this simple criterion of sexuality it follows that a monoecious individual can function both as a nuclear donor and a nuclear acceptor. An organism able to function only as d o n o r or as acceptor is called dioecious. Among dioecious fungi there are species which, because of the formation of sexual organs, show a clear polarity of male and female individuals (= morphological dioecism) and others without morphologically developed sexual organs, and only recognizable by their sexual reaction (= physiological dioecism). Apart from the hereditary factors causing the normal morphological differentiation, there are no special genes for monoecism. As opposed to the higher plants and animals, dioecism in fungi is not determined by sex chromosomes, but by single genes.

2. Incompatibility Incompatibility is observed if the nuclei cannot fuse during normal sexual reaction. This prevention of k a r y o g a m y is not due to defects of the nuclei concerned leading to sterility. In accordance with genetic principles underlying this sexual barrier, one distinguishes between two systems: a) Homogenic incompatibility: K a r y o g a m y does not occur between nuclei bearing the same incompatibility factors. In the slmplest case a gene pair + and - is sufficient for the control of sexual reaction. If both nuclei have the factor + or the factor - , karyogamy does not take place; it only occurs when one nucleus contains the + gene and the other the - gene. For information on more sophisticated systems, which, however, all work on the same principle, see Raper [8], Esser and Kuenen [6]. b) Heterogenic incompatibility: K a r y o g a m y does not occur between nuclei bearing different incompatibility factors. In contrast to homogenic incompatibility, a differing constitution of at least two gene loci is required for this kind of incompatibility. Heterogenic incompatibility is not only limited to the sexual cycle, but also takes place as a so-called vegetative incompatibility after the fusion of hyphae. This is very c o m m o n in fungi and is lethal for the participating cells. This defensive reaction prevents mixed growth of the species concerned. It is initiated even by a single gene difference.

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The difficulties occurring occasionally in fermentations using so-called mixed cultures (i.e. producing consecutive metabolic steps in transformation reactions) may partly be traced back to heterogenic incompatibility. Heterogenic incompatibility seems to be a basic biological phenomenon. The incompatibility known as histo-incompatibility of tissues after organ transplantations both in man and animals is based on the same mechanism of incompatibility of genetically different nuclei. The immunological response occurring in higher organisms is far more complicated than that of fungi possessing no lymphatic system. However, it has been shown with fungi (see Blaich and Esser [9]) that enzymatically active proteins are involved in the incompatibility reaction : a model for a basic study of histo-incompatibility is offered therefore by fungal systems.

3. Heterokaryosis Heterokaryosis, the occurrence of genetically differing nuclei in the same cytoplasm is a phenomenon specific to fungi and very common there. It is initiated through fusion of hyphae, as mentioned in the preceding paragraph, Heterokaryosis strictly speaking is not a breeding system as are those mentioned above. However, it plays a role in those species whose life cycle is not introduced by the fusion of sexual organs and gametes respectively, but through hyphat fusion. It is significant in imperfect fungi, which have lost their ability to multiply sexually during evolution and can only exchange their genetic material with the aid of the parasexual cycle. Consequently there are no special genes responsible for heterokaryosis. Its control is taken over by the hereditary factors responsible for the incompatibility. The compilation of these sexual systems may appear rather complicated to the non-geneticist. Nevertheless there arises the question in what way they achieve the control of recombination. First, a general point:recombination of genetic material has no practical effects on organisms subjected to a continuing inbreeding. It follows that every system inhibiting or reducing inbreeding increases the effectiveness of recombination through outbreeding. In nature this is controlled by the individual and mutual effects of the various propagation systems illustrated in Fig. 1. 1. First of all inbreeding is reduced by dioecism and homogenic incompatibility. Both systems inhibit self-fertilization and allow karyogamy to take place only between genetically different individuals. 2. Inbreeding is promoted by monoecism. Species that are self-fertile tend to form local races which, for lack of exchange of genetic material, develop in different directions in the course of evolution. This tendency is still enhanced by heterogenic incompatibility inhibiting heterogamy in monoecious organisms.

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3. The effect of heterokaryosis is less evident than that of the sexual systems. However, it has to be considered that hyphal fusions and the subsequent exchange of nuclei should not be underestimated in fungi which live under natural conditions, because "foreign" nuclei have a relatively easy access to the germ line. Its importance for the imperfect fungi has already been mentioned---it is the only possibility for the occurrence of a recombination via the parasexual cycle. 4. The effects of any breeding system can be partly or wholly abolished by sterility genes, which cause the Jack or the non-function of sexual organs or cells respectively. Such deficiencies can be overcome if, after heterokaryosis, genetically different defects are complemented by the effect of corresponding unmutated genes. 5. It must be pointed out that in most fungi sexual or parasexual cycles and consequently recombination are rarely controlled by a single breeding system but rather by a concerted action of various systems. The most widely spread mutual effect is the prevention of monoecism by homogenic incompatibility. By this the same outbreeding effect is bestowed into a self-fertile species as by dioecism. Heterogenic incompatibility can be superimposed on monoecism, just as dioecism can reinforce homogenic incompatibility. Therefore potential recombination may be restricted to single biotypes, splitting the species into several "independent" strains. As heterogenic incompatibility is not restricted to the sexual cycle but also occurs in the vegetative phase, its effect extends also to heterokaryosis, thereby limiting the exchange of genetic material in the fungi imperfecti.

II. The Production of Mutants Mutations leading to discontinuous hereditary changes of the genetic material can take place both spontaneously and by induction by mutagenic agents. As mentioned above effective breeding cannot rely solely upon the occurrence of spontaneous mutations and their natural selection, but requires a systematic application of mutagenic agents. In the literature much information is available on general methods and special techniques for induction and selection of mutants 5 Isee literature index by Calm [10] and Hopwood [11]). In this context however we only intend to show some fundamental principles that could be of practical interest. 5 Information on the current state of the literature on mutagenicity is given by ,,Zentrallaboratorium ftir Mutagenit~itspriifung der Deutschen Forschungsgemeinschaft", 78 Freiburg i. Br., Breisacher Str. 33, Germany.

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t. Fungal structures to be treated with mutagenic agents should contain no more than one nucleus. This condition is fulfilled with most of the asexually developed spores of the conidial type (e.g. in AsperyilIus, Penicillium, Neurospora-~microconidia). When using spores resulting from a sexual process (as ascospores and basidiospores), it must be noted that some of these, although originally uninucleate, will become multinucleate in the course of subsequent nuclear divisions. If a fungus does not fulfill this condition and if multinucleate spores or even hyphal fragments must be used, mainly heterokaryotic mycelia are obtained after treatment with mutagens, in which advantageous characteristics can be dominated by others. This difficulty can be overcome by analysis of the progeny of these heterokaryons, e.g. by plating sexual spores or by multiplication of uninucleated hyphal tips. In this way if necessary a broad spectrum of mutants can be isolated from a single heterokaryon generated by mutagenic treatment, albeit with considerable technical effort. 2. Choice of adequate mutagenic agents must be adapted to the fungal structure to be treated. Ultra-violet irradiation of fungal spores which, because of melanine deposits, have black cell walls is very inefficient. Such walls consequently absorb the rays to a high degree. 3. The application of selective methods not only saves time, but also represents the most efficient way to obtain specific mutants. In contrast to the geneticist, who is mainly concerned with obtaining mutants bearing "biochemical defects", the breeder searches for "positive" mutants with better yields. For this breeder the selection techniques for mutants with nutrient deficiencies, familiar in basic research, cannot be used. To obtain a positive selection (e.g. increase of a particular metabolite), it is recommended to let the mutagen-treated material germinate on an agar medium containing specific dyes as indicators for the desired products. According to the pigment formation, a quantitative assay and therefore selection is possible. Since only the fruit bodies are of the edible fungi generally used, a selection according to their characteristics is easily made. 4. In recent years it has been shown that chemical mutagens are far more potent than any kind of irradiation for induction of biochemical as well as morphological mutations. For example, deletions, translocations or inversions of chromosomes occur more sparingly. The chance of obtaining point mutations is greater and, not least, one has a good idea on the nature of the changes that are caused by the chemical mutagens to the DNA-molecules (lit. in Drake [12]). 5. The main problem in the production of biochemical and morphological mutants is not the initiation of mutation and selection, but rather to obtain stable and viable types where mutations are fixed in the

78

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genetic material in such a way as to be constantly inherited after many nuclear divisions. For example, mutated genes tocalised in surplus chromosomes (aneupIoidia) can be lost after a number of nuclear divisions.

Fig. 2. Spore tetrads of the Ascomycete Sordaria macrospora. In the course of meiosis the colour genes of the spores (lu + = yellow spores) are split into typical patterns from which the localisation of the genes can be deduced (details from Esser and Kucnen [6])

It is almost trivial to mention that all strains showing altered characteristics after mutagen treatment m a y or m a y not be real mutants. They m a y actually- represent only variations, as their altered phenotype can be the result of various processes (e.g. aneuploidia or heterokaryosis). Genuine mutants can only be obtained if the genetically altered nucleus has experienced a meiotic division. Owing to c h r o m o s o m a l recombination occurring during meiosis, all "'unbalanced" genomes are eliminated. Moreover, after completion of this division, one can start from one unique nucleus ensuring that the produced strain is not heterokaryotic. In order to fulfill these criteria, the simplest method is a back-cross of the variant with the original strain. Based on the segregation pattern

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79

of the progeny (most fungi are haploid) first information on the nature of the m u t a t i o n is obtained. It soon becomes evident whether mutations in one or m o r e genes c h r o m o s o m i c alterations have occurred. Where analysis of ordered tetrads--four products of meiosis (see Fig. 21 is applicable--the most complete information is obtained. In such a case even some indication of the approximate localisation of the mutational site within the chromosomes is obtained. The m e t h o d of back-crossing also makes it possible, by meiotic recombination to trace back in multi-mutations each mutated gene a m o n g the progeny. For this very purpose the use of tetrad analysis is advantageous. It saves time as in most cases the analysis of a few tetrads is sufficient to obtain the desired information.

Fig. 3. Cross of sterile mutants of the Ascomycete Sordaria macrospora. Formation of fruiting bodies (black dots) occurs only in the contact zone isee Esser and Straub [18] for more detailed information) Back-cross analysis with some m o n o e c i o u s fungi is difficult as one c a n n o t generally distinguish between fruiting bodies originated t h r o u g h self-fertilization from one o f the two partners and those originating from a cross. This difficulty can be o v e r c o m e in two ways: a) cross a m o r e or less sterile m u t a n t with the wild type yielding fruiting bodies only in the cross area (see Fig. 3); b) by using colored spores as genetic

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KARL ESSER

markers. Thus fruiting bodies originating from a cross can easily be distinguished from those arising after self-fertilization (Fig. 2). It should be mentioned that many industrially applicable fungi are imperfect. Therefore genetic analysis ~,ia meiotic recombination is not possible. Fixation of mutations can only be obtained through the parasexual cycle ~ia mitotic recombination: however, the use of gene markers (color of the conidiospores) is invaluable.

Fig. 4. Pleiotropic mutant zomlm of the Ascomycete Podosporu anserimt (left half)~ the right half shows for comparison the wild strain. Apart from physiological changes te.g. production increase for the enzyme tyrosinase), morphological changes have also occurred in zonata as a resutt of a point mutation within a single gene: rhythmic growth, lower growth rate, failure to form fruiting bodies ~compare the dot-shaped fruiting bodies present in the wild strain) caused by the lack of female sex organs 6. When dealing with initiation and incorporation of mutations, the phenomenon of pleiotropy must not be disregarded. Pleiotropy is the occurrence of various apparently uncorrelated characteristics after mutation of a single hereditary factor. Strains with such "genetic syndromes" appear fairly frequently during a systematic search for mutants. They mostly show a synchronous alteration of morphological and physiological characteristics (e.g. change of growth characteristics, loss of sexual organs or sexual spores, defects in nutrient requirements etc.: see also Fig. 4~.

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81

Sometimes these alterations are associated with increased yield or production (see Fig. 4). When the development ofa favourable characteristic is accompanied by other changes, it is necessary to investigate further before adopting the new strain. Determination of the genetic constitution, by genetic analysis, is important to produce a stable genome containing the favourable genes. Back-crossing tests can be adopted as well. The most reliable criterion for the proof of pleiotropy is, however, the somewhat time consuming back-mutation test. Only single-factor mutants offer a significant chance of reproduction of the original phenotype.

III. The Syndrome of Senescence Every mycologist knows that most fungi (especially Phycomycetes and Ascomycetes) are subject to ageing after prolonged exclusively vegetative propagation. This senescence sometimes occurs much more quickly than in the laboratory after continuous cultivation in a fermenter or in the production of edible mushrooms. Generally, senescent strains first lose their ability to multiply sexually. The subsequent growth anomalies lead frequently to a cessation of hyphal growth and therefore to the death of the strain ~Fig. 5). But even before this final state of ageing sets in, physiological anomalies occur in parallel with the morphological changes. Losses of ability to produce a particular metabolite are notorious. Such senescence syndromes are generally explained as genetic changes of nuclei which accumulate as a consequence of numerous spontaneous mutations and which can be repaired with corresponding back-crossing. This is, however, not the only cause of senescence. There are examples lEsser and Kuenen [6]) where degeneration symptoms occurring in the course of ageing can only be eliminated through exchange of cytoplasm. This can take place in the course of sexual multiplication after crosses using a young strain as a female parent and, as male parent, gametes of a senescent strain devoid of cytoplasm. After genetic analysis of the progeny, young strains with the original characteristics are again obtained. With imperfect fungi nuclei from ageing mycelia can be incorporated in the same way by heterokaryosis in healthy strains. Tile original nucleus, together with the functioning plasma, is thereafter regained by the use of the methods dealt with in the preceding section. From this example of regeneration of senescent strains by cytoplasmic exchange it can be concluded that senescence in the cases concerned is not determined by a change of genetic information located in the nucleus but by cytoplasmic factors. The nature of these factors however is still unknown.

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Fig. 5. Young (left) and senescent tright) strains of the Ascomycete Podospora anserina, The age of the two cultures is 6 days

A more detailed analysis of the genetic background of senescence syndromes has shown that simple cytoplasmic heredity is not always the only factor involved. Some types of senescent hyphae are able to "infect" young hyphae if brought in contact over a cytoplasmic bridge via heterokaryosis, From this it may be concluded that senescence is initiated by distinct particles of the cytoplasm, It is assumed that these particles, whose nature is yet unknown, are able to multiply themselves, in a manner analogous to the behaviour of viruses. Merely by a single contact they can affect the whole of the young material after a relatively short time. The cause of their spontaneous appearance is supposed to be the existence of two modifications, similar to the temperate bacteriophages: a kind of resting phase while integrated in the nucleus and after spontaneous release as infectious rapidly multiplying particles in the cytoplasm respectively. The assumption that infectious senescence is caused by specific virus-like particles is not mere speculation. In recent years experimental data have been accumulated proving that fungi can ~'fatl ill" from viruses just like other living beings (Hollings etal. [13], Hollings [14], Banks [15]).

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IV. Conclusions for Breeding Research with Useful Fungi Any successful breeding of piants or animals presupposes not only the control of external growth parameters essential for optimal yield, but also knowledge of the internal conditions of growth and development and its manipulation. In the special case of breeding useful fungi, in addition to knowing the conditions for cultivation (e.g. substrate, light, air humidity, pH, temperature) the genetic factors coding for the normal life cycle and for the recombination of the genome must also be known, in order to have entire control over the system. As already intimated, in the breeding of fungi emphasis has mainly been given to the control of favourable culture conditions. Economic reasons, as well the lack of data from basic research, have led to neglect of the genetic parameters. There are also technical difficulties in investigating the genetics of fungi. In contrast to higher organisms sophisticated microscopical methods (isolation of spores) are required for the analysis of fungal progeny. However, it should not be overlooked that fungi have a number of advantages: 1.they can be cultivatedunder controlled laboratory conditions;2. short generation times enables the production of many generations per annum: 3. special methods such as tetrad analysis allow conclusive results to be obtained in respect of number and localisation of genes with even a small number of descendents; 4. there is a substantial reduction in analytical work on the progeny, because of the haploid character of cultured fungi. Any segregation is recognised in the first generation, therefore eliminating the need for time-consumingtest crossings. From the information given in the preceding sections concerning genetic control of recombination, induction and selection of mutants and the genetic basis of senescence the following conclusions for fungal breeding may be drawn:

t. C o n s e r v a t i o n o f Yield by R e g u l a r R e g e n e r a t i o n Isolating new clones from vegetative spores to regain the original strains in case of decreasing yields represent a suboptimal method. Many fungi cannot produce such spores. Furthermore the chromosomal and extrachromosomal deficiencies accumulated in the course of numerous vegetative cycles cannot be eliminated without drawbacks by this technique. Successful regeneration of inefficient strains is achieved by initiating meiotic or mitotic recombinations and by testing the strains obtained from single spores. Restitution of the original genotype on the chromoso-

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KARL ESSER

mal and e x t r a c h r o m o s o m a l lcvcls is offered during these processes, in which the "'original" nucleus is sorted out from the mass of mutated nuclei or of mutated cytoplasm. This m e t h o d requires, a p a r t from the knowledge of the physiological conditions of the life cycle, familiarity with the genetic systems controlling the breeding systems. Our experience with "'laboratory" strains leads us to recommend that stock cultures be maintained at 4~C in a refrigerator. Regeneration by meiotic (or parasexual) passages should be undertaken at the first sign of production deficiencies. In the final stages of senescence regeneration is often very inefficient.

2. Increase of Yield by a Combined Programme of Mutation, Selection and Recombination T h e search for m o r e efficient strains by using either selection techniques after application of mutagens or isolation by the classical m e t h o d from nature is uneconomical. O n l y a c o m b i n a t i o n of both methods will pay off in the long run. C o n t i n u o u s regeneration of the genetic material through r e c o m b i n a t i o n is require& and this in turn presupposes knowledge and the use of parameters for n o r m a l development and for breeding systems. This may be illustrated by an example: By the use of selective techniques it has been possible with the "~champignon" (Aqaricus bisporus) to obtain strains producing fruiting bodies of the size of a child's head offering entirely new applications also in respect of flavour C~mushroom steaks") isee Sengbusch [3]). As there has so far been no definite model of cytological events taking place during spore formation, and as the breeding system of the champignons is, genetically, not under control, it has hitherto not been clearly determined how or whether this most valuable characteristic is fixed in the genome. As the big fruiting bodies are sterile and as, moreover, a decrease in size occurs rather regularly after vegetative growth, no commercial application of such strains has up to now been possible. In the meantime, however parallel with these investigations carried out by Fritsche of the'Sengbusch group the life cycle and genetics of the champignon have been elaborated in a laboratory dealing with basic research on mushrooms (see Raper [16]). Application of this knowledge may soon emerge as an example of a successful cooperation of theory and practice.

3. Adoption of New Strains for the Production of Useful Fungi T h e ill-considered application of antibiotics leads to increased resistance of sensitive microbes and consequently to an inefficient therapy. This not only calls for a cultural improvement of the k n o w n antibiotic pro-

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ducers but also for a permanent search for new strains. This is considerably facilitated if the basic of an ontogenetical and genetical knowledge of the production strains is at hand. With these facts in mind some predictions may be possible concerning the genetic control of the life cycles of "newcomers". In this way, for example, the Basidiomycete Oudemansiella mucida, which produces an antibiotic effective against mycosis, was carefully examined biologically and genetically, before being used in mass production. This was in order to start right at the level of clinical testing with a continuous breeding to maintain and raise the yields (see Musilek [17]). There appear to be vast unknown possibilities for bringing into play new strains in the production of edible mushrooms, since, apart from the champignon (Agaricus bisporus), commercial production of mushroom species is insignificant. This cannot however be put down to a lack of demand for other mushroom species. Laboriously mushroom gatherers during the season and imports for rest of the year try to fill the gap, but lack of scientific background prevents the design of any efficient production strain. Which enterprise would venture to make big investments when there is the risk that its organisms suddenly cease producing under the influence of unknown factors? The following examples show how the production programme may be expanded: The oyster mushroom ( Pleurotus ostreatus) belongs to the group of wood-destroying mushrooms which can cover their entire energy requirements from wood. In respect of taste and flavour the fruiting bodies of this mushroom can quite compete with the champignon. Also from the economical point of view its cultivation would pay, as scrap from the timber industry (cuttings, chips, sawdust) could be used. The first tests in Hungary mentioned before were restricted to merely infecting wood logs Ipoplar, beech, hornbeam) in the open with mycelia and harvesting in autumn the fruiting bodies grown each year. Transfer of the production into plants (as with the champignon) should bring about several harvests per annum. As this mushroom is rather easy to handle in the laboratory, the cultivation necessary for production should not encounter significant difficulties. On the other hand some technical difficulties would have to be overcomc in the utilisation of the truffle (e.g. 7bber aestirum) and morel (Morchella esculenta), because of the development cycle of these mushrooms. As opposed to the champignon and the oyster mushroom which belong to the group of the Basidiomycetes. these are Ascomycetes. As with the Basidiomycetes a so-called dikaryotic mycelium can be used as a spawn, from which fruiting bodies continually develop under appropriate culture conditions without further sexual processes. The Ascomycetes require a sexual process for the formation of each single fruiting body for which two genetically different strains (showing homogenic incompatibility) must be brought together. By adoption of appropriate genetic methods it should be possible to obtain well-balanced heterokaryons which could be applied for the inoculation of the substrate, as with champignon spawn. It must, however, be mentioned that the nutrient requirements and culture conditions of truffle and morel are by no means clarified.

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In this connection one may mention the cultivation of orchids in commercial enterprise which could also only be realised after knowing the culture conditions (above all breeding for seeds having no food reserve, such as endosperm or cotyledon of seeds) and production of hybrid forms based on genetic methods. This application seems to have borne fruit economically, judging by the price reductions effected in this field in the last years.

Conclusion Basic genetic research on mushrooms has revealed some aspects which could be of interest to the mycologist involved in the production of useful fungi and which could contribute to increased yields of material. Above all it is a question of the genetic factors controlling the recombination of hereditary factors, and of problems in the production of mutants and the syndrome of the ageing of fungal cultures. F r o m consideration of these aspects, it is feasible to conceive concerted cultivation on the basis of a systematic production and selection of mutants and their planned incorporation in the genetic material through recombination processes, as is usual with animals and higher plants. Increase of yield should eventually compensate the cost of such an '~applied basic genetic research".

References 1. Rehm, H. J.: Industrielle Mikrobiologie. Berlin--Heidelberg--New York: Springer 1967. Rehm, H. J.: EinfiJhrung in die industrielte Mikrobiologie. Berlin--Heidelberg-New York: Springer 1971. 2. Z~hner, H.: Biologie der Antibiotica. Berlin Heidelberg--New York: Springer 1965. 3. Sengbusch, R. v.: Champignon 10, 1----37(1970). 4. Block, S. S., Tsao, G., Han, L,: Mushroom Science IV, Proc. IV. Intern. Conf. Copenhagen 1959, pp. 309--325. 5. V~ssey, E.: Z. f. Pilzkunde 34, 125--d36 (1968). 6. Esser, K., Kuenen, R.: Genetik der Pilze. Berlin--Heidelberg--New York: Springer 1965. 7. Esser, K.: Mol. Genet, ll0, 86--100 (1971). 8. Raper, J. R.: Genetics of sexuality in higher fungi. New York: Ronald Press 1966. 9. Blaich, R,, Esser, K,: Mol. Genet. 109, 186 192 (1970). 10. Calam, C. T.: In: Methods in microbiology. Norris, J. R., Ribbons, D. W. (Eds.), Vol. 3 A, pp. 435--459. New York: Acad. Press 1970. I I. Hopwood, D. A.: In: Methods in microbiology. Norris, J. R., Ribbons, D. W. (Eds.), Vol. 3 A, pp. 363--433. New York: Acad. Press 1970. 12. Drake, J. W.: The molecular basis of mutation. San Francisco: Holden-Day 1970.

Some Aspects of Basic Genetic Research on Fungi 13. 14. 15. 16. 17.

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Hollings, M., Gandy, D. G., Last, F. T.: Endeavour 22, 112--117 t1963). Hollings, M.: Mushroom Sci. 6, 255--262 (1967). Banks, G. T.: Nature 222, 89--90 (t969). Raper, C. A.: Abstr. I. Intern. Mycol. Congr. Exeter 1971. Musilek, V., Cern& J., ~a~ek, V., Semerd~ieva, M., Vondr~ek, M.: Folia Microbiol. (Prague) 14, 377-387 (1969). 18. Esser, K., Straub, J.: Z. Vererbungslehre 89, 729-746 (1958).

Professor Dr. KARL ESSER Lehrstuhl fiir Allgemeine Botanik Ruhr-Universit~it Bochum (Germany) D-463 Bochum, Postfach 2148

CHAPTER

3

Microbi al Oxidation of Methane and Methanol N. KOSAR1C a n d J. E. ZAJIC With 7 Figures

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . a) Historical Developments . . . . . . . . . . . . . . . . . . . b) Classification of Methane Oxidizing Bacteria . . . . . . . . . . 2. Liquid Media . . . . . . . . . . . . . . . . . . . . . . . . . pH and Temperature . . . . . . . . . . . . . . . . . . . . . . 3. Composition of the Gas Phase . . . . . . . . . . . . . . . . . . 4. Oxygen and Methane Requirements . . . . . . . . . . . . . . . . 5. Biochemistry of Methane Oxidation . . . . . . . . . . . . . . . . a) Autotrophy-- Heterotrophy . . . . . . . . . . . . . . . . . . . b) Methane Oxidation Pathways . . . . . . . . . . . . . . . . . c) Nitrogen Metabolism and Denitrification in Methane- and Methanol Oxidizers . . . . . . . . . . . . . . . . . . . . . . . . . . d) Sulfate Reduction in Relation to Methane Oxidation ....... 6. Biosynthesis of Cell Components . . . . . . . . . . . . . . . . . 7. Present and Possible Future Applications . . . . . . . . . . . . . a) Single-Cell Protein Production . . . . . . . . . . . , ...... Quality of the SCP Derivcd from ttydrocarbons . . . . . . . . . b) Removal of Methane from ('otll Nliiacs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Petroleum Prospecting d) Microbial Fuel-Cell . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89 90

90 96 101 102 103 103 108 108 109 110 112 1 t3 115 115 120 12t 121

122 122

Introduction Methane and methanol fermentations have been a subject of study e v e r since t h e d i s c o v e r y o f m e t h a n e - o x i d i z i n g b a c t e r i a b y S 6 h n g e n (1906). I n t e r e s t h a s i n c r e a s e d c o n s i d e r a b l y t h e last d e c a d e , p a r t i c u l a r l y b e c a u s e

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N. KOSARIC and J. E. ZAJIC

of the widespread availability and low costs of these compounds and their potential for use as energy or in either synthetic or biosynthetic reactions. Applied microbiological developments are gaining in importance as they represent a new possible way to produce enough high-quality protein for the human population which is increasing explosively. Although protein production from methane and methanol has not yet been fully developed, there is no doubt that of the available raw materials which possess purity, availability and can be used without pre-processing, both methane and methanol offer immediate value in food production. In addition, they do not need degrading prior to use as fermentable substrates. Gaseous hydrocarbons are relatively pure when recovered from nature and require very little processing to high purity. This review covers the accumulated information regarding methaneand methanol-utilizing microorganisms, basic characteristics of these microbes and conditions for growth, as well as the mechanisms and biochemical pathways involved in the conversion of methane to cellular components. An outlook into possible applications and future prospects is also included.

1. Microorganisms a) Historical Developments The first report of methane-oxidizing bacteria was made about 70 years ago by S6hngen (1906). He reported the isolation of a pure culture capable of oxidizing methane which he named Bacillus methanicus. The organism was described as a Gram-negative rod forming pink pigmented colonies on salt-water-washed agar. The organism was later renamed Methanomonas methanica by Orla-Jensen (1909). Since then and particularly in the past decade, studies on methane fermentations have been developed considerably. Over 450 soil isolates of bacteria, yeasts and fungi were made by Zajic (1964). Of these, most isolates had at least some ability to oxidize methane and all had the ability to utilize methane as a sole carbon and energy source. Some bacterial genera, known to subsist upon methane are Pseudomonas, My~vbacterium, :Methanomonas, Desulphovibrio, Bacillus, Clostridium, Methanobacterium and Methylococcus. Some of the more significant developments are reviewed in the following. Hutton and Zobell (1949)described methane oxidizers isolated from marine sediments, brackish water and surface soil. A new species, Methanomonas carbonatophila, requiring carbon dioxide to initiate growth, was tentatively identified by Hutton (1948), although the claims of

Microbial Oxidation of Methane and Methanol

9I

pure cultures in the early studies must be regarded with some reserve. The bacteria were described as Gram-negative non-spore-forming rods. A more complete description of S6hngen's bacterium, Methanomonas methanica, was undertaken by Dworkin and Foster in 1956. The isolate was obtained from triturated leaves and stems of Elodea and represented Gram-negative rods, usually occurring singly and staining unevenly intracellularly with basic dyes, giving the cells a mottled appearance. Sevenday-old cells, measuring from 0.6 to 1.0 lam, were highly motile and possessed a single polar flagellum. The cells were pinkish, the colour being concentrated more in the central portion of the colony. The pigment was produced intracellularly and the properties resembled the general characteristics of the carotenoid pigments. An "active factor" which increased growth was extracted from agar, suggesting a polysaccharide type of material but it was shown that calcium pantothenate was capable of partially replacing the agar extract. Approximately maximum growth rates were observed at methane concentrations from 1 0 - 9 0 % while a 3% concentration was found to be decidedly below optimum. Oxygen was apparently toxic at air concentrations; while an atmosphere containing 15% 02 seemed to be optimal, there seemed also to be a definite requirement for exogenous carbon dioxide, the optimal initial concentration in the gas phase being 0.3%. Concerning other organic substances as carbon sources, only methane and methanol supported growth. Glucose appeared to stimulate growth in the presence of methane. On the basis of the results, Dworkin and Foster concluded that their isolate coincided with SiShngen's, except for growth requirement. They abandoned the genus Methanomonas reclassifying the organism as Pseudomonas methanica. Further studies of Methanomonas methanica by Leadbetter and Foster (1958) ted to the description of four groups of pigmented forms, other morphological and physiological characteristics of the bacteria being the same. No growth factor was required, and liquid medium containing Ca ÷ ÷ supported rapid and abundant growth. In 1962, Johnson and Temple reported the isolation of a strain of Pseudomonas methanica that possessed several characteristics different from Dworkin and Foster's strain. No requirement for growth factors and no inhibition by high oxygen concentration was observed. The isolate utilized nitrate, glutamate, tryptone or ammonium sulphate as a nitrogen source. The organism grew in liquid media as a membranous petlicle "producing a flocculent deposit rather than giving uniform turbidity". A gas mixture comprising 02, CH4, CO2 and N2 was used. Opposite to the observation by Dworkin and Foster, 45% oxygen concentration was better for

92

N. KOSAR1Cand J. E. ZAJIC

growth than 15% concentration. With a concentration of methane at 25% a slightly better growth was observed compared to the 45% methane concentration. In 1964, Davis et at. isolated from several sources methane-oxidizing bacteria capable of fixing atmospheric nitrogen. The organisms were Gram-negative, non-spore-forming, motile rods, 2 - 4 gm in length. Most commonly, light yellow colonies were formed. The name, Pseudomonas methanitrificans was given to the organism. It did not utilize ethane, propane, n-butane and n-tetradecane. Polyhydroxybutyricacid accumulated in the lipid fraction, while a maximum amount of fixed nitrogen was 0.13 mg/ml. Brown, Strawinsky and McCleskey (1964) reported an isolation and characterization of Methanomonas methanooxidans. The organism depended on methane and methanol as carbon and energy sources utilizing both organic and inorganic nitrogen sources. The organism was described as a Gram-negative, non spore-forming rod, 1.5-3.0 gm by 1.0 ~m in size, being motile by means of a single polar flagellum. In growing cultures, the oxygen and methane were consumed at a molar ratio of approximately 1.1:1 respectively, while this ratio increased in resting cells up to 1.7: 1. Resting cells were unable to oxidize organic compounds other than methane, methanol, formaldehyde and formate. Foster and Davis (t966) reported the isolation from sewage of a new" coccus-shaped bacterium capable of aerobic growth at the expense of methane or methanol in a mineral salts medium. The organism did not grow at the expense of any of the conventional substrates or homologous hydrocarbons. The organism (1.0 Jam in diameter) was Gram-negative~ non motile and thermotolerant growing well at 50~C, optimally at 37 C but the growth ceased at 55 C . The cell colonies were approximately I mm in diameter, colourless to ivory, smooth, rounded and even-bordered. Rod-like forms were not observed. Most of the cells had a distinctively diplococcoid arrangement and were encapsulated. The capsular polysaccharide was found to be practically insoluble in water under growth conditions but dissolved at 100 C . The molar ratio of methane to oxygen uptake was similar to that of M. methanooxidans (l:l). The organism was also able to oxidize ethane and propane but only methane or methanol supported growth. Foster and Davis proposed the name Methylococcus capsulatus for this new isolate. Wolnak et al. (1967) reported the isolation from soil and the plant Elodea of a comparatively large bacillus (5--I5 ~am long and 2--3 fa wide). It appeared to be different from other previously described methane-metabolizing organisms. It thrives in a medium of mineral salts saturated with a gaseous mixture of 40% methane, 40% oxygen, 15%

Microbial Oxidation of Methane and Methanol

93

nitrogen and 5% carbon dioxide. It is moderately to strongly Gram-negative with a dark blue nucleus-type stained area. Poly-/J-hydroxybutyrate formation was investigated but it did not appear that the organism contained it. In 1970, Hazeu and Steennis reported the isolation and characterization of two vibrio-shaped methane-oxidizing bacteria. The strain A, that was isolated from soil, was non-motile and contained only trace amounts of poly-/J-hydroxybutyricacid in the lipid extract (approximately 60%). The strain grew equally well on nitrate or ammonia as a nitrogen source. Strain B was motile by means of 1--5 flagella in young cultures when grown at 22--25:C in liquid media or on plates. It also had a preference for ammonia and contained 41--55% lipid of which 25 to 90% was poly-//-hydroxybutyric acid. Other characteristics of both strains were similar. They were slightly curved rods (1.5~2.2 tam by 0.8--1.1 pm), Gram-negative, catalase-positive, producing colonies of 0.5--2 mm in diameter, that were round, flat, smooth and creamcoloured. Optimum temperatures for growth were 30--37':C and pH near neutrality. Neither was able to grow on complex media, both were able to oxidize normal primary alcohols (C1 -Clo) but only methane and methanol supported growth. The name Methylovibrio soehngenii was proposed for this species. Mixed methane-oxidizing bacterial cultures were isolated by Sheehan and Johnson (1971) from activated sludge sampled from the inlet end of an aeration tank of a municipal sewage-treatment plant. The culture consisted of two types of Gram-negative non spore-forming rods resembling Pseudomonas. The organisms were isolated at 45 ~'C, one of them representing a short, almost coccoid rod measuring 0.6 by 1.0 to 1.3 gm; the other being a longer but thinner rod, 0.3 by 1.5 to 3.0 gm. The organisms were grown continuously under aseptic conditions in a medium containing a simple inorganic salt mixture and 35----37% methane, 3--5% COz and 60% air. The only report on growth of fungi on natural gas, methane or ethane is by Zajic, Volesky and Wellman (1969). They isolated a fungus which grows well on a mineral salts solution with natural gas as a carbon source. It was identified as a Graphium species. The fungi were isolated after selection by continuous enrichment techniques performed in a stirred tank-type fermentor at 28~C. The pH varied from 2.7 3.5 and the dry weight of microbial tissue was 65--275 mg/1. Also present in the continuous culture was an acid-tolerant bacterium, which, when isolated, grew well on natural gas, methanol and ethanol. Ethane is the best substrate for this isolate of Graphium. However, it co-oxidizes methane in the presence of ethane. Propane and butane are also used as energy sources.

94

N, KOSARICand J. E, ZAJIC

Two different fungi were isolated by streaking the effluent on agar plates of C-medium (Coty's medium) and incubating the plates in enclosed desiccators supplied with 40% natural gas and 60% air. The Graphium isolated was not solely dependent on natural gas for growth and several carbohydrates were found to be able to substitute the gaseous carbon source. In submerged culture, the best growth was obtained with dextrose and sucrose. These substrates give a lower yield than when grown upon natural gas. Large colonies developed on the solid C-medium being, after 10 days, from 2 to 4 cm in diameter. When grown on solid media, hyphae were hyaline, regularly septate, and 1.5 to 4.5 gm wide. With natural gas as the carbon source, the vacuolation of the hyphae was very marked. Also conidia were produced abundantly both in liquid and on solid media. To summarize the findings regarding methane- and methanol-utilizing bacteria, the following can be stated: a) They are all Gram-negative, strictly aerobic. b) True methane oxidizers have an obligate requirement for methane or methanol as the carbon and energy source for growth but methanoloxidizing microbes do not necessarily require methane. c) They have no absolute requirement for any organic growth factors or organic nitrogen sources. d) Most of the microorganisms investigated were able to oxidize other substrates (e.g. hydrocarbons or alcohols) but these did not support the growth per se. e) The majority of the bacteria form a resting stage that might be metabolically different. f) They have a complex internal membranous structure (Davies and Whittenbury, 1970; Whittenbury, Phillips and Wilkinson, 1970). g) Many of the strains are high in lipid content, a considerable part of the lipid extract representing poly-/~-hydroxybutyric acid. h) There is a tendency for many methane-oxidizing microbes to be isolated as mixed culture with the contaminating organism being a methanol oxidizer. i) Most true methane oxidizers are relatively poor growers, i.e, require 6 or more days for maximal growth. j) A variety of pigments is produced, most of which are poorly characterized. k) The relationship of the methane-oxidizing microbes to the methanoloxidizing microbes is poorly understood. A tabulated summary of organisms is given in Table I.

Microbial Oxidation of Methane and Methanol

95

Table 1. Methane- and methanol-oxidizing bacteria and fungi Organism

Investigator

Bacillus methanieus Bacillus hexacarbovorum Bacterium methanicum Bacterium fluorescens liquefaciens (alias Bacillus Pseudomonas fluorescens ) Bacillus methanicus Methane bacterium Methane-oxidizing bacteria Methanomonas methanica Methanomonas methanica M ethanomonas carbonatophila M ycobacterium methanicum and M ycobacterium flavum var. methanicum Methane-oxidizin9 bacteria Methanomonas methanica Methanomonas methanica Methanomonas methanooxidans Pseudomonas methanica (SBhngen) var. fufva var. fusca var. incolorata Pseudomonas methanica and related strains Pseudomonas PRL-W-4

S6hngen, 1906 St6rmer, 1908 Munt~ 1915 Aiyer, 1920

Pseudomonas methanica Pseudomonas AM-t Pseudomonas methanica Chromobacterium M ycobacterium rubrum and M ycobacterium lacticolum Pseudomonas methanitrificans Methanomonas methanooxidans Pseudomonas radiobacter and Pseudobacterium Methylococeus capsulatus Bacillus spp. Methylovibrio soehngenii Mixed bacterial culture Graphium spp. Chlorella

Haseman, 1927 Tausz and Donath, !930 Mogilevskii, 1940 Bokova et al., 1947 Slavnina, 1948 Hutton, 1948 Nechaeva, 1949 Hutton and Zobell, 1953 Dworkin, 1955 Davis, 1956 Brown, 1956 Dworkin and Foster, 1956 Leadbetter and Foster, 1958 Kaneda and Roxburgh, 1959 Harrington and Kallio, 1960 Quayle and Peel, 1960 Johnson and Temple, 1962 Elizarova, 1963 Kersten, 1964 Davis, Coty and Stanley, 1964 Stocks and McCleskey, 1964 Bogdanova, 1965 Foster and Davis, 1966 Wolnak et al., 1967 Hazeu and Steennis, 1970 Sheehan and Johnson, 1971 Zajic, Volesky, Wellman, t969 Enebo, 1967

96

N. KOSARI(' and J. E. ZAJIC

b) Classification of M e t h a n e Oxidizing Bacteria Few attempts have been made to organize the bacteria found to oxidize methane and methanol. Some of the earlier studies provided a very complete amount of information but the question of whether the reported cultures were pure remains open. A subdivision of microorganisms capable of growth on C1 compounds was attempted by Quayle (I963). Three groups were envisaged. Group A comprised photosynthetic and chemosynthetic authotrophs. Group B represented microorganisms capable of aerobic growth on reduced C1 compounds while Group C comprised organisms deriving their energy anaerobically by organic dismutation of methanol, formic acid and carbon monoxide. Ribbons (1968) suggested that the reports in the literature conceal numerous examples of the use of mixed cultures and found that it was extremely difficult to isolate methane oxidizers in pure culture. He suggested therefore that the true methane utilizers live in very close association with symbiotic microorganisms which usually form pink colonies on plates, cannot use methane but can grow on methanol. The properties of the most common contaminant were identical with those of Pseudomonas methanica. There was no report of these statements in the more recent literature. Two more serious and recent classification attempts, one by Ribbons etal. (1970) and by Whittenbury etal. (1970) are worth mentioning.

Table 2. A simplified basis for the classification of methane-oxidizing bacteria (after Ribbons et al., 1970) Motility

Capsule

Organism Morphology

and flagellation

or slime

Colony Colour

soluble or Pigments spore

Methylo- vibrioid sinus Methylo- pleomorphic cystis Methylo- rod monas rod-coccoid Methylo-rod bacter rod-coccoid Methylo- diplococci coccus or chains

+PT

+

W-Y

variable (brown)

~--

+

W

+p

variable

_+P

variable

W,Y P, R W-B

+

W

PT --- polar tufts of flagella P - - polar flagellum W-Y white to yellow

W --- white Y yellow P pink

Water

variable (green) variable (yellow)

Cyst

spore lipid cyst immature cyst cyst immature cyst

R - - red W-B .... white to brown

Microbial Oxidation of Methane and Methanol

97

Ribbons et al. presented a simplified basis for the classification of methane-oxidizing bacteria, as presented in Table 2. Whittenbury etal. isolated more than 100 Gram-negative, strictly aerobic methane-utilizing bacteria. All used only methane and methanol and were classified into five groups on the basis of morphology, fine structure and type of resting stage formed (exospores and different types of cysts). All the organisms were also catalase- and oxidase-positive and methanol was extremely toxic to many strains when added to the medium even at 0.01% (w/v). A pH of 6.6-6.8 was found to be optimal for growth rate and yield. More carbon dioxide was formed from methanol than from methane and the organisms grown on methanol gave about 20% lower yield compared to methane. Ethanol and ethane did not support growth but were oxidized. In terms of morphology, rods, cocci, vibroid and pear-shaped organisms of various sizes and dimensions were found. Some possessed capsules and flagella, and resting stages of three types were formed. A complex fine structure was characteristic of all isolates. The groups were:

Vibroid :

M eth ylosinus M eth yloc yst is

Rod/Coccoid:

Methylomonas Methylobacter Methylococcus

All these groups were subdivided on the basis of various characteristics. Group "M ethylosinus" (subgroups "trichosporium'" and "sporium") The organisms were generally rod-shaped in the non-sporing stage but were occasionally of bizarre form with a polar tuft or flagella. They stained by the polysaccharide stain of Hotchkiss (! 948) but their capsules were not stained. Exospores were produced by budding off the non-flagellated poles assuming a pear shape (Methylosinus trichosporium) or vibrio-shape (Methylosinus sporium). All strains possessed the same complex paired membranous system and divided at 5 to 6 hours under optimal growth conditions. The main differences between the two subgroups were cell shape and size, spore morphology and pigment production. Group "Methylocystis" (subgroup " parvus') One strain was isolated which was non-motile and non-spore-forming. A non-heat-resistant cyst was also formed.

98

N. KOSARICand J. E. ZAJIC

Group "Methylomonas" (subgroup "methanica", "albus", "streptobacterium', "agile", "rubrum'" and " rosaceus") All the organisms were rod-shaped and possessed a complex membrane (Type II) represented in a series of bundles composed of disc-shaped membrane vesicles distributes through-out the cell (see Davies and Whittenbury, 1970). Many of the strains were capsulated. In all subgroups some strains formed a resting stage which was not resistant to desiccation but survived in absence of methane for 4 - - 5 weeks, while vegetative cells survived only 3--4 days. Subgroup "rnethanica" strains were identified as Pseudomonas methanica as described by Leadbetter and Foster (1958).

Group "Methylobacter" (subgroup "chroococcum", "'boris', " capsulatus" and "vinelandii") Many resembled "Methylomonas" strains morphologically and were rodshaped in all stages of their growth cycle. Some were similar to the large-cell-forming species of Azotobacter, changing from rod form to coccal and intermediate forms and back to rod form. Also slime and capsule formation were similar to the Azotobacter. All strains possessed a type II membranous system, some being polarly flagellated. The minimum generation time was about 4 hours for all strains.

Group "~Methylococcus" (subgroup " capsulatus" and "minimus") These were non-motile cocci possessing capsules and a type II membrane. Resting stages were formed by both and the minimum generation time was 3.5--4 hours. The division to subgroups was on the basis of morphology and ability to grow at 37 and 45 °C. Subgroup capsulatus includes Methylococcus capsulatus isolated by Foster and Davis (1966). Comparing the isolates with previously described species, two were considered to be identical, as mentioned above. None were identified as Methanomonas methanooxidans (Brown etal., 1964). The "Methylosinus" strain may have been similar to Pseudomonas methanitrificans (Coty, t967). Other strains were also suspected to be identical with the Leadbetter and Foster (1958), particularly the "'Methylobacter vineIandii" strains. Table 3 presents a summary classification.

5

'minimus'

.

+

. + . +

.

.

.

.

.

.

.

+

+

. --

.

--

--

.

---

.

--

.

.

.

.

.

.

.

.

.

.

--

. --

.

+

.

.

+

+ +

---

.

.

.

.

.

. .

.

.

.

.

.

.

.

G r o w t h on methanol (0.1% w/v)

.

.

.

.

.

.

.

--

--

--

+

.

+ +

--.

.

.

.

.

.

.

+

---

+

---

--

+ ----

-+

3 •5

3 •5

5 4 4 3

4

4

3.5

4

3 -5 3

5

5 5

--

--

+ P + P

--

+ P

+ P

+ P

--

+ P + P

--

+ PT + PT

G r o w t h o n C H ~ enh a n c e d (0.1% w/v) S h o r t e s t , ", division Motility a n d Yeast time (hr) flagellation extract Malate

+

+

+ +

+

+

+

+

+ --

+ *~

+ *" + *a

Capsule formed

--

--

--

-----

G - S +~ ---~

--

-Br-BI +~

Water soluble pigment

W

W

---

---

PP --W-Br t b Y W - B r t b ----W - B r t ~ ----

PP

R

W

W

Oc-Pi W

W

W-Y W-Bu

Colony colour -

PT = polar tufts of flagella W = white W - B r = white to b r o w n P = p o l a r flagellum Oc-Pi = y e l l o w - o c h r e t o pink Br-B1 = b r o w n to black W - Y = white to yellow R = red G - S = green to s a p p h i r e W - B u = w h i t e to buff P P = pale pink "* C a p s u l e s u n d e r electron m i c r o s c o p e consisted of s h o r t fibres r a d i a t i n g f r o m cell wall. N o s t r u c t u r e w a s seen in capsules of o t h e r o r g a n i s m s . bt B r o w n c o l o u r restricted to colonies c o n t a i n i n g cysts. ~ Pigment p r o d u c e d o n iron-deficient m e d i u m .

3

'capsulatus'

'Methylococcus'

'vinelandii"

'capsulatus"

'boris'

'chroococcum'

9 5 4 5

.

2

"rosaceus"

'Methylobacter'

---

7

+

'rubrum"

.

5

4

--+

+

+ +

'streptobacterium'

30 3

t

9 12

No. of strains

'ayile'

~albus'

'methanica'

"Methylomonas'

~parvus"

'Methylocystis'

'sporium"

'trichosporium'

'Methylosinus'

G r o u p and s u b g r o u p

G r o w t h at "37 45 ~

Table 3. Properties of s u b g r o u p s of m e t h a n e - u t i l i z i n g bacteria (after W h i t t e n b u r y , Philips a n d Wilkinson, 1970)

d

[]

(a), (b), etc. hx hh

t

~x

0.1

0.5

1.0

S6hngen (1906)

0.1

0.1

0.5

1.0"

1.0"

Hutton (1948)

(a)

0.001 t

0.09 0.21 0.2 hh

2.0

Dworkin Foster (1956)

0.05

0.2 hh

0.5

I,O

Brown (1956)

0.001

0. t

0d

0.5 hh

1,0 1,0

[ 16.7] hx (b) [0.16]

[0.66] d

0.4 0.6 0.2 hh

0.1

Johnson Hamer Templc et al. (1962) (1967)

either NH~CI, KNO3 or (NH4)2SO4 used as nitrogen source trihydrate salt used refer to the text hexahydrate salt used heptahydrate salt used refers to mg values dihydrate

MgNHuP04.6If20 NH4 CI NaNO~ KN03 (NHa)2S04 K2HP04 K He P04 Na2 H P04 Mg S04 Ca SO4 Ca ('0.~ Ca C12 Na Ct Fe S04 Fe Cl~ Other CuSO4 "5 H20

.............................

Component

Table 4. Media for growth of methane-oxidizing bacteria (composition in grams;/liter)

0.5

1.0 1.0

Coty (1969)

(c) 0.0004

(d)

0.1 0.1 0.002 hh 0.001

0.09 0.0004 0.2 hh 0.02 d

2.0

Wolnak et al. (1967)

(e) 0.004

0.014 hh

1.6 1.16 0,08 hh

1,18

Sheehan Johnson (1970)

N >

?,-

r'~

:>

Microbial Oxidation of Methane and Methanol

101

2. Liquid Media Simple inorganic salts media are used by most investigators, the only carbon source being methane or methanol. Nitrates and/or ammonium salts are commonly taken as the source of nitrogen with sodium and potassium phosphates used as sources of phosphorus. Some of the usual media are presented in Table 4. In his pioneering work, S6hngen (1906) used a very simple medium while subsequent investigators added various other combinations of mineral salts to supply nitrogen, phosphorus and minerals. Magnesium was added in most of the applied media which indicates its requirement for growth, whilst convincing proof for the necessity of addition of trace elements is lacking. Manganese, cobalt, copper, zinc, boron and molybdenum have been supplied in trace quantities in a number of media (Dworkin and Foster, Hamer et al., Wolnak etal., Sheehan and Johnson, etc.). Wolnak et aL (1967) examined the effect of trace elements and found that Cu + was not required by a methane-oxidizing bacillus in amounts greater than those present as contaminants in the salts and water used to make up the medium. Higher concentrations of copper (greater than 0.0125 ~JgCu + +/ml) definitely inhibited cell growth. Similar results were obtained with zinc. The results with cobalt were more positive and the fermentation with 5--75 ~tg Co + + was completed in about 85 to 90hours compared to 110 to 115 hours for the control. Sheehan and Johnson (1970) found no conclusive evidence for Co ++ requirement while a specific requirement for Ca + +, Cu + +, (MOO4)--, Zn + + and Mn + + was determined, by the following method: When the methane- or oxygen-limited continuous fermentation had been at steady state for at least two residence times at cell concentration greater than 6.0 g dry wt/liter, the feed line to the fermentor was switched to a medium from which the metal ion, being tested, was omitted. As the fermentation broth became limiting by a decrease in the concentration of the metal in question, the dissolved oxygen concentration in the fermentor rapidly increased and the cell concentration slowly decreased. When the required metal was added directly to the fermentor an increase in growth rate and decrease in dissolved oxygen level was observed. On this basis, a medium for continuous bacterial growth to support a concentration of 12 grams of dry weight cells/liter was as follows: g/l KII2PO~ 0.67 Na2HPO4 0.22 NaNo3 9.55 MgSO4-7H20 0.32

102

N. KOSARICand J. E. ZAJIC FeSO4.7H20 Ca(NO 3)2-4H20 CuSO4.5H20 ZnSO4.7H20 MnSO4.HiO Na2MoO4-2H20 COC12.6H20 Conc. H2SO4 (36 N)

0.029 0.18 9.1 × 10 - 3 1.1 x 10 -3 1.5 × 10 - 3 6.4 x 10 4 4.5 x 10 -s 2.7 ml

Concerning nitrogen sources, nitrate, rather than ammonium ion was reported to be beneficial in widening the pH range for optimal growth from 6.0 to 6.6 to 6.6 to 8.0, but it was disadvantageous in extending generation times (Dworkin and Foster, 1956 and Vary and Johnson, 1967). Whittenbury, Phillips and Wilkinson (1970) in describing more than 100 methane-utilizing bacteria, reported the use of ammonium salts by all organisms. Nitrates were used by the majority of strains while urea, casamino acids and yeast extract were only used by some. All formed non-inhibitory concentrations of nitrite from ammonia which was also previously recorded by Hutton and Zobell (1949). Organisms using nitrate reduced some to nitrite, but were unable to grow anaerobically on methane with nitrate as an alternative electron acceptor to oxygen. While the pH value in nitrate cultures remained around neutrality, the growth was partially inhibited in ammonium salts cultures as the pH fell to 5.0 or lower. At a controlled pH (with KOH), growth rates were similar with nitrate and ammonium salts and growth yields were higher with ammonium salts. Eroshin, Harwood and Pirt (1968) investigated the influence of aminoacids, carboxylic acids and sugars on growth of Methylococcus capsulatus on methane. Using the increase in colony diameter on Petri dishes as a growth criterion, they found that L-amino acids and carboxylic acids prevented growth in low concentrations. Threonine, leucine, histidine and glycine were especially inhibitory (0.1% was sufficient to suppress growth completely). Glutamic acid at 0.1% concentration completely suppressed growth but at 0.005% markedly stimulated growth, similar effects being shown by lysine, methionine, tryptophane, tyrosine, proline and citric acid. Among the sugars, glucose was the most potent growth inhibitor while maltose and sucrose had practically no effect. p H and Temperature In general, pH values near neutrality seem to be optimal. Dworkin and Foster (1956) reported that pH optima for Pseudomonas methanica varied with different sources of nitrogen. With ammonium sulphate,

Microbial Oxidation of Methane and Methanol

103

growth was best over a narrow, slightly acidic p H range of 6.0 to 6.6. With sodium nitrate the range was considerably wider, spreading from pH 6.6 to at least p H 8.0. This could not be confirmed by Johnson and Temple (1962) as their strain of P. methanica exhibited a broad p H o p t i m u m (6.6 to 9.4) with a m m o n i u m sulphate as nitrogen source. In the experiments with Graphium grown on natural gas (Volesky and Zajic, 1971), a m m o n i u m sulphate was found to be far superior to sodium nitrate over a p H range of 3.5 to 6.0. F o r steady state cultivation, a p H between 4.0 and 5.1 was most effective. Both methane- and methanol-oxidizing bacteria are reported to be mesophiles. O p t i m u m temperatures are between 25.2 and 37°C and little or no growth is observed above 37 °C.

3. Composition of the Gas Phase Various methane: air ratios have been utilized and some reported compositions are presented in Table 5. Table 5. Gas phases for growth of methane oxidizers Organism

CH4

Volume per cent Air 02

C02

Methanomonas methanica

33.3

66.7

--

--

98

--

2

--

50

--

40

10

65

--

30

5

40

60

--

--

25

20~

45

10

33.3

66.7

--

--

(S6hngen, 1906) Bacillus methanicus

(Miinz, 1915) Methanomonas carbonatophila

(Hutton and Zobell, 1949) Methanomonas methanooxidans

(Brown, 1958) Pseudomonas methanica

(Dworkin and Foster, 1956) Pseudomonas methanica

(Johnson and Temple, 1962) Mycobacterium methanicum

(Nechaeva, 1949) a N2

used instead of air.

4. Oxygen and Methane Requirements As mentioned in the preceding paragraph, various concentrations of methane and oxygen have been used for fermentation studies. There is a definite relationship between methane and oxygen consumption

104

N. KOSARICand J. E. ZAJ1C

and the biomass production. However, as mentioned by Dworkin and Foster {1956), compositions and volumes of the gas phase reported in the pioneering work are unreliable as the experiments were performed under unhomogeneous physiological conditions and were performed in the apparatus devised by S6hngen, "one of the worst for studies of gas metabolism". Another comment regarding stationary cultures is that they are characterized by surface: volume ratios extremely unfavourable for diffusion of gases relative to consumption of gases by the bacterium (Finn, 1954). The true gas concentration effects and maximal growth rates can be obtained only under homogeneous conditions assuring uniform exposure of all cells at all times to a given gas phase. Dworkin and Foster {1956), using 40% methane and 60% air for primary enrichment cultures, found that 15% oxygen was optimal with pure cultures. However, oxygen was apparently toxic at the concentration at which it exists in air, a finding that Johnson and Temple (1962) were unable to confirm. Hutton (1948), working with Methanomonas carbonatophiIa investigated the rate of methane utilization in various CH4/Oz/CO2 mixtures. An atmosphere consisting of 10% CO2, 40% oxygen and 50% methane was recommended for cultivating methane-oxidizing bacteria (Hutton and Zobell, 1949). Bewersdorff and Dostalek 1971) studied the influence of gas phase on biomass production during logarithmic growth in batch cultures and at steady-state conditions at D=0.08 in continuous culture. Using relative proportions of CH4:O2 of 0.5:1, 1:1, 1:15, they found no significant differences in absolute values of oxygen and methane consumption {1.7 moles O2/mole CH~ in all cases). On this basis they concluded that the gas mixture for cultivation on CH~ should contain 1 volume of CH4 and 1.7 volumes of 02. In order to adjust the 02 level at a value not higher than 12% (danger of explosion) the optimum mixture according to this demand contained CH~ ......... 7.0 liters air ...... 59.5 liters N2 --- 33.5 liters 100.0 liters which corresponds to 7.0% v/v CH4 and 11.9% v/v O2. Leadbetter and Foster (1958) observed that the average molar ratios of metabolized gases are quite different from the equation: CH~+ 202--+CO2 + 2 H 2 0 which is usually employed to depict the overall metabolism of methane by bacteria. The average observed moles used by P. methanica in gaseous systems containing 50% CH4 and 50% air were 1.0CH4+0.4002-*0.21 CO2. The discrepancies between

Microbial Oxidation of Methane and Methanol

105

the actual molar consumption of 02 and production of CO2 per mole of methane consumed and between the theoretical values calculated on the basis of complete oxidation of methane, indicate an exceptionally high conversion ofmethane-C to organic-C. These data are in accordance with similar findings by Dworkin and Foster in 1956. According to Brown et al. (1964) with growing cultures of Methanomonas methanooxidans, methane and oxygen consumption and C02 production was 1.0:1.1:0.2 respectively, e.g. 1 CH4 + 1.1 02--,0.2 CO2 + cells + organic material. As no significant accumulation of partially oxidized organic material was indicated, approximately 80% of methane carbon is converted to cell carbon, which suggests that methane oxidizers are extremely efficient organisms in conserving substrate carbon. In resting cells, the O2/CH4 ratio was about 1.7:1 indicating that methane carbon is not being fixed as cell carbon but is going to CO2. A comparison of the stoichiometry of methane and methanol utilization and the efficiency with which they were converted into biomass, was also carried out on a number of strains isolated by Whittenbury, Phillips and Wilkinson (1970). Organisms (non-capsulate, non-slime-forming) and carbon dioxide were the only end-products of methane and methanol utilization detected. The results were similar in both batch and continuous cultures and are expressed by the following two equations: Methane . - 1CH~+(1.0- 1.1)O2-+(0.2-0.3)CO2+ 1.1 g cells, Methanol - - 1CH3OH + (1.0 - 1.1)O2 --~ ( 0 . 5

-- 0.6)CO2

+ 0,4g cells.

On a molar basis, methanol yielded about 20% less dry weight of organisms than methane, implying that energy useful to the organisms is released in the initial oxidation of methane. These results confirm Johnson's (1967)assumption that 47% of bacterial dry weight is carbon and that carbon-containing products other than organisms and CO2 were not formed under these culture conditions. With slime-forming strains, Whittenbury et al. (1970) found lower bacterial dry weights (less than 1.0g/g methane) and the CO2 production was higher than that given above. Variations in the stoichiometry of methane utilization also occurred under nitrogen limitation and with resting cells. In the former case, 02 consumption and CO2 production were lower per mole of methane utilized and the organisms became packed with lipid inclusions (mainly poly-/~-hydroxybutyrate), tn the latter case, 02 consumption and CO2 production increased per mole of methane utilized. The results reported by Whittenbury etat. confirmed the ones recorded by Brown etal. (1964) but were not in agreement with the findings

106

N. KOSARICand J. E. ZAJ|C

of Leadbetter and Foster (1958). Their results (0.4 moles 02 utilized/mole of CH4) seem to be low and their assumption that 0.5 moles of 02 was required in the initial conversion of one mole of methane to methanol, seems not to be justified. Klass, Iandolo and Knabel (1969) discussed the key process factors in the microbial conversion of methane to protein, working with an obligate heterotrophic methane bacterium designated as 1 GT-10. The organism metabolizes methane in a mineral salts medium and produces about a pound of dry biomass/lb of methane consumed. They recognized the difficulty in determining the stoichiometry of a hydrocarbon fermentation process because of the vast number of reactions that occur simultaneously, and they developed an overall, empirical stoichiometry. A modified Darlington's equation (1964) for biomass production from hydrocarbons was used and represented as: 6.25CH4 + 7.9202

~

C3.92H6.5001.92 + 2.33CO2 + 9.25H20.

The stoichiometry of this equation corresponds to 63.4% of the theory for complete oxidation to CO2 and was supported by experimental data. The stoichiometry (presented graphically in Fig. l) clearly shows that more oxygen is required for microbial oxidation of methane than of

02 REQUIRED/HYDROCARBON CONSUMED weight units 20

2,1

1.0

I.I

22

23

2.4

2,5

2.6

e~ 0

CO z

1.2

1.3

1.4

1.5

1.6

PRODUCED/HC CONSUMED

Fig. 1. Stoichiometry of microbial conversion of paraffins to (after Klass, Iandolo and Knabel, 1969)

C3.92H,5.5o01.92

Microbial Oxidation of Methane and Methanol

107

the higher paraffins, which is well-known and documented. Also less COz is produced with methane, which leads to the conclusion that better carbon utilization should result with methane compared to higher hydrocarbons. The product yields, observed by Klass et al. (1969) were up to 1.441b of dry product/lb of methane consumed with relatively low concentrations of CO2 in the effluent gas. According to the empirical equation above, the stoichiometric amounts of oxygen and methane (which have different solubilities in water and salt solutions) have to be supplied in the liquid media. The curve, presenting the solubility relationships is presented in Fig. 2. ~ENR~"S C()NSTA'NTS #ROM INTL. CRIT TABLES -

36

III, 2 5 4 - 2 6 1 ( 1 9 2 8 )

--

~20°C \ ~'-~25* C ~30°C

E

c; c

X m

0-c. _ 25"C ~-;

I=J

>, 0 r.n

o~ CC 0 L)

~-~t r9%. / ~

d

35.c

'oc

20 4 0 60 80 I 0 0 CH4 IN AIR OR CH4- 02 MIX,VOL, %

Fig. 2. Methane and oxygen dissolved in water in equilibrium with methane air or methane oxygen mixtures (after Klass et al., 1969) The figure can be used to determine the feed gas composition that provides stoichiometric amounts of dissolved methane and oxygen. The data of Johnson and Temple (1962) best fitted with the theoretical predictions. At concentrations less than 42.6% methane (relative to the total methane and oxygen in the feed gas) it is expected that the oxygen will be present in stoichiometric excess of the dissolved methane.

t08

N. KOSARlCand J. E. ZAJI¢

According to Klass et al., the doubling time of the organism was reduced from 22 to 16 hours simply by changing the feed from 90% methane and 10% Oz to a mixture of 1 : 1 CH4 to O2. Concerning the pressure, solubility of the gas mixture is approximately doubled with a doubling in the partial pressure. However, biological differences between methane utilizers, the consumption of oxygen along with endogenous substrates by resting cells and sometimes observed toxicity of excess oxygen, make it difficult to predict the exact response of methane utilizers to changes in the partial pressures of methane and oxygen. A material balance using methane oxidation by microorganisms was also discussed by Smirnova (1971). Gas mixtures with O2/CH4 ratios of 0.6, 0.7, 1.0, 1.2, 1.5, 2.0, 2.3 and 3.7: were investigated. With increased ratio in the gas phase, the hydrocarbon conversion to biomass decreased. Best biomass yields relative to methane utilization (100% efficiency) and to oxygen utilization (30% efficiency) were obtained at an O2/CH4 ratio of 1.5. In this case, to produce ling biomass, 1.0rag methane and 3.3 mg of oxygen were utilized.

5. B i o c h e m i s t r y of M e t h a n e O x i d a t i o n Methane and methanol oxidation seems to be quite unique compared to microbial oxidation of higher hydrocarbons and there was quite an interest in the past to elucidate the operating pathways and mechanisms. One particular characteristic of the organisms involved is that all methane-oxidizing bacteria studied thus far can grow only in the presence of methane and methanol. Other organic compounds, above C~, cannot be utilized as the sole carbon and energy source but they may be co-oxidized (Leadbetter and Foster, 1958, 1960). a) A u t o t r o p h y - - H e t e r o t r o p h y Much controversy also exists as to whether methane-oxidizing bacteria should be classified as autotrophs or heterotrophs. Most of the discrepancy was derived from different interpretations of autotrophy/heterotrophy in the older literature. Autotrophy used to be defined as growth on one carbon compound, and this definition automatically led to the classification of the methane oxidizers as autotrophic. However, today's definition of an autotroph is the ability to grow at the expense of COz as the exclusive carbon source for cell synthesis. The metabolism of C~ compounds in autotrophic and heterotrophic microorganisms has been reviewed by Quayle (1961) and Silverman (1964). From the work presented in the literature, it is obvious that

Microbial Oxidation of Methane and Methanol

109

there still exists a controversy since the question of heterotrophic and autotrophic assimilation of carbon in methane-oxidizing bacteria has not been completely resolved. There is, however, much evidence that most of the organisms are obligate heterotrophs (Leadbetter and Foster, 1958; Harrington and Kallio, 1960; Wolnak etal., 1967; Johnson and Quayle, 1964; Coty, 1969; Zajic, 1964; Wilkinson, 1971; and others). If both mechanisms are considered (as proposed by Leadbetter and Foster, t958), two separate pathways could be involved. Autotrophy a) C H 4 + 2 H 2 0 - * C 0 2 + 8(H) b) 4(H) + CO2 ~ ( C H 2 0 ) + H20 c) 4(H)+ 0 2 - - , 2 H 2 0 Net Reaction: CH4 + O2-*CH2O + H2O

Heterotrophy a) b) c) d)

CH4(+ 02 or H 20) -, C (Int. oxid. level)+ "Active" H2 C(Int. oxid. level)+ "Active" H2--* (CH2O) C(Int. oxid. level) + H20-'*C02 q-"Active" H2 "Active" H2 + 02 --* H 2 0

Net Reaction: 2CH4 + 302--* (CH20) + CO2 + 3 H 2 0

b) Methane Oxidation Pathways It is generally accepted that the steps involved in methane oxidation are the following: CH4 --* CH3OH --* HCHO ~ HCOOH --* CO2 methane methanol formaldehyde formic acid The evidence for this sequence is supported by various authors using P. methanica and P. methanooxidans (Brown and Strawinski, 1957, 1958; Kallio and Harrington, 1960; Leadbetter and Foster, 1959, 1960). Brown and Strawinski reported the identification of several intermediates in the oxidation of methane by resting cell suspensions of Methanomonas methanooxidans. In the presence of iodoacetate, which acts as an oxidation inhibitor, methanol was produced. When sodium sulfite was used as a trapping agent there was an accumulation of formaldehyde while formate accumulated in the absence of any blocking agents. The results were supported by Leadbetter and Foster, who studied Pseudomonas methanica. Also, Harrington and Kallio provided evidence that methanol is oxidized to formaldehyde via a catalase-linked peroxidase in Pseudo-

110

N. KOSAR1C a n d J. E. ZAJIC

monas methanica. Formaldehyde was subsequently oxidized by a substrate specific aldehyde dehydrogenase requiring NAD + and glutathione. The Harrington and Kallio strain was apparently dependent upon methanol and could not oxidize methane. Kaneda and Roxburgh (1959) however, demonstrated an alcohol-dehydrogenase dependent upon NAD + and glutathione in a methanol-oxidizing pseudomonad, but were unable to demonstrate an aldehyde-dehydrogenase. Serine was the first stable product they claimed. Anthony and Zatman (1965) demonstrated an unusual alcohol-dehydrogenase in a methanol-oxidizing pseudomonad which would use only phenazinemethyl sulphate as a hydrogen acceptor. Zajic (1969, i966) demonstrated the absence of an absolute requirement for exogenous CO: for methane-oxidizing microbes and a formation of free gaseous hydrogen during the oxidation of methane by Pseudomonas methanica. The hydrogen was postulated to arise from formate through the action of formic-hydrogen-lyase. Work by Johnson and Quayle (1964) tends to confirm the sequence: C H 3 O H ~ H C H O - - * H C O O H - - * C O z + H 2 0 . In cell-free extracts of P. methanica, Protaminobacter ruber and two other pseudomonads, complete oxidation of methanol to carbon dioxide was achieved and three enzyme systems were demonstrated. 1. An alcohol-dehydrogenase dependant upon NAD + and specific for methanol (found in all species except in one pseudomonad). 2. An aldehyde-dehydrogenase found in one pseudomonad and inhibited by thiol-binding agents and EDTA. 3. An NAD +-linked formate dehydrogenase specific for formate, inhibited by iodoacetate, C N , Fe + + and Cu + +. In summary, the pathways shown in Fig. 3 tbr methane oxidation seem to have been established. c) Nitrogen Metabolism and Denitrification in Methane- and Methanol-Oxidizers The best-known denitrifying systems are found in anaerobic bacteria such as the autotrophs Thiobacillus denitrificans and Thiobacillus novellus and the heterotroph Clostridium nitrificans. In these microbes NO~ acts as the oxidant in place of oxygen. Hutton and Zobell (! 953) reported a nitrifying system in methane-oxidizing bacteria and Davis etal. (1964) identified certain natural isolates as Pseudomonas methanitrificans which fix atmospheric nitrogen. Zajic and Smith (1966) showed that P. methanica (Temple) possesses an active denitrifying system. Denitrification was determined in a mineral salts medium with 0.2% KNO3. The specially adapted test flasks were

Microbial Oxidation of Methane and Methanol

111

FORMIC ALCOHOL DEHYDROGENASE co ~' DEHYDROGENASE ~ , ~-2 02 CH4

I....o+o+ .AD" I ~

"J

CH30H

SO.

"

%7' +A

HCHO ~

t

HCOOH

i

l

I

1,,I

.~HYDROGEN

i ALDEHYDE m DEHYDROGENASE ALCOHOL C021+H2i PEROXIDASE

LYAS

Fig. 3. Pathways for methane oxidation

gassed with 30% CH4, 30% 02 and 0--40% CO or C 0 2 and were then inoculated with 5.0% by volume of an active broth culture of P. methanica. Either hydrogen or helium gas was used to adjust the gaseous volume to 100%. Nitrate reduction was found not to be suppressed by oxygen, and free nitrogen was rapidly produced from nitrate by the bacterium. It was also found that 20% or greater concentrations of either CO or CO2 (vol/vol) inhibited the denitrification. Similar experiments were conducted on two methanol-oxidizing cultures in the Kerr-McGee culture collection and both possessed an active denitrifying system. Maximal rate of nitrogen production was in all cases observed between 24 and 48 hours. The overall reaction can be presented as: 3CH4 + 2NO~- ~ 3 C 0 2 + N2 + 6H2 - 129 Kcal which supports the theory that anaerobic microbes probably exist that are capable of coupling denitrification and methane oxidation using NO~- as a terminal electron acceptor. Hansen and KaUio (1957) found that NO£ would not act in an anaerobic atmosphere as an oxidant with cultures of Pseudomonas stutzeri for oxidizing dodecane and l-dodecene. Free nitrogen was formed, however, when intermediates such as dodecanol, dodecanal and dodecanoic acid were tested anaerobically in the presence of NO~-. Other reports in the literature indicate that anaerobic oxidations of hydrocarbons might be of greater importance than previously anticipated (Dutova, 1963; Zajic, 1966). Nitrous oxide has also been postulated as a logical intermediate in the denitrification processes primarily because it is evolved from soils

112

N. Koshmc and J. E. ZAJIC

where denitrification is occuring and also because some microbes convert it to nitrogen. It was demonstrated by Zajic that denitrifying products synthesized by P. methanica were nitrite and nitrogen, and nitrous oxide was also identified. A general scheme for the nitrogen metabolism of methane oxidizers is presented in Fig. 4.

N2

| P. METHANffRIFICANS FIXATION Y PEPTONE TRYPTONE N,+ ' CELLULAR NITROGEN ' P. METHANICA DENITRIFICATION :'

AMINOACID YEAST EXTRACT

i

NO;.q

,i

I

NO2 ( NITRIFICATIONNH3 METHANOMONAS CARBONATOPHILA

Fig. 4. Nitrogen metabolism of methane oxidizers

d) Sulfate Reduction in Relation to Methane Oxidation Sulphate reduction is usually anaerobic while all of the reported methaneoxidizing bacteria isolated to date are aerobic. They derive their energy from the following exergonic reaction: CH4+202-~COz+2H20-

195.5 Kcal.

Depending upon the ionic species formed from sulfate reduction, it is at least theoretically possible that the sulfate reducers, like the denitritiers, can oxidize methane. Calculations made by Baas-Becking (1957) show favourable thermodynamics for the two potential reactions: 1. Fe + +SO2 - +

CH4

2. S O 2 - + C H 4 ~ H S

--~

FeS + C 0 2 -t- 2 H 2 0

-

19.2 Kcal,

+HCO3 +H20-4.5Kcal.

Both these reactions are weakly exergonic but they could support microbial growth (Zajic, 1966). Sorokin (1957) has attempted to grow sulfatereducing bacteria such as Desutfovibrio at the expense of methane. Both pure and mixed cultures were tested without success. However, sulfatereducing bacteria which oxidize light and heavy crude oils have been reported (Zheludev, 1959; Dutova, 1963; Rosenfeld, 1947; Novelli and

Microbial Oxidation of Methane and Methanol

113

Zobell, 1944). Anaerobic bacteria in general have been implicated in oil decomposition (Ekzertsev, 1958; Shmonova, 1964) especially in desulfurization.

6. B i o s y n t h e s i s o f Cell C o m p o n e n t s Quayle and co-workers (Johnson and Quayle, 1964; Kemp and Quayle, 1965, 1967; Lawrence, Kemp and Quayle, 1970) produced convincing evidence for a unique C1 pathway in P. methanica resembling the photosynthetic dark reaction of higher plants. C14-1abelled methane, methanol and bicarbonate were fed to methane-grown cells and C14-methanol to methanol-grown cells. They found that over 90% of the radioactivity appeared in the phosphorylated compounds at the earliest time of sampling. Glucose and fructose phosphates constituted 70---90% while phosphoglycerate accounted for 2--t7% of the phosphorylated compounds. Other compounds, labelled to a lesser extent were glycine, serine, glutamate, aspartate, malate, citrate and alanine. When C14-1abetled bicarbonate was added to cells growing on methane, malate and aspartate were the earliest to become labelled. No carboxydismutase activity was found in cell-free extracts. All of these results support Leadbetter and Foster's postulation in 1958 that the organism is heterotrophic. In autotrophic metabolism, the labelled phosphate is mainly phosphoglycerate, carboxydismutase is the key enzyme formed and the products of bicarbonate incorporation are usually rapidly labelled phosphates. It was concluded that the C1units such as CO2 are reduced within the cell and form intermediates which enter metabolic pathways at oxidation levels between methanol and formate or alternately the CO2 is fixed directly. They postulated that carbon dioxide incorporation might be concerned mainly with the synthesis of C4 compounds from C3 compounds. Patel and Hoare (1971) studied Methylococcus capsulatus in order to determine the biochemical basis for its obligate methane- or methanoldependence. They found M. capsulatus growing on methane able to assimilate acetate but it was incorporated into the lipids and only four amino-acids: glutamine, proline, arginine and leucine. A particulate NADH-oxidase and a soluble NAD-specific formate dehydrogenase and low levels of most enzymes of the TCA cycle were identified. However, no ~-ketoglutarate dehydrogenase activity was detectable. Ribbons, Harrison and Wadzenski (1970) published an excellent review on the metabolism of single-carbon compounds. They have calculated the standard free energy changes for the individual oxidative steps in methane oxidation:

114

N. KOSARICand J. E. ZAJIC

AG+ pH 7 Kcal mole- 1

CH4 + 1/202 -* CH3OH CH30H + 1/202 ~ H C H O + H20 HCHO + 1/202 -* H C O O - + H + H C O O - + H + + 1/202--*CO2 + H20 CH4 + 202 -"+CO2 + 2H20

-

26.12 44.81 - 57.15 - 58.25 - ! 86.33 -

They propose that the methane or methanol oxidizers are more efficient than the autotrophs since they incorporate carbon into cellular constituents at the oxidation level of formaldehyde. The methane oxidizers need only expend 1 mole of ATP per 3 carbon atoms of formaldehyde incorporated into the triose phosphate pathway (see below) while autotrophs must expend 3 moles of ATP plus 2 moles NADPH per mole of CO2 incorporated into the triose phosphate pathway. These energetics must be established. There seem to exist at least three distinct pathways that would account for the synthesis of cell constituents from reduced C1 units. The first, which represents a special case, is an autotrophic pathway which has so far been observed only in Pseudomonas oxalaticus and which was elucidated by a well-established reaction sequence (Kornberg and Elsden, 1962; Quayle and Keech, 1959a, 1959b, 1960, 1961).

CH4,-~CH30H-'='-HCHO'~HCOOH~ C02+H20 37SE 5-P

/

I TRANsALDOLAsE I

3~%PIMERIZATION

I

I

I TRANSKETOLASE I I 'REARRANGEMENTS"]/

i,-,,,

p~AOp

FRUCTOSEI.I.6-DIP TRIODE3-P

~TRIOS~P I

CELL I CONST,,TUENTSI

Fig. 5 Allulose pathway for cell synthesis

A second pathway which was elucidated by the results obtained by Quayle and his associates is presented in Fig. 5. The pathway was established on the basis of the findings that labelled formaldehyde condensed with ribose-5-phosphate to give allulose-6-

Microbial Oxidation of Methane and Methanol

115

phosphate which then underwent epimerization at C3 to produce fructose-6-phosphate. From the evidence available, the authors suggested a modified phosphogluconate pathway resulting in the net formation of a molecule of triose-3-phosphate from three molecules of formaldehyde and one of ATP, as presented above. The third serine pathway has been observed in M. methanooxidans (Lawrence, Kempand Quayle, 1970). They noted that the earliest labelled intermediate was a tetrahydrofolate derivative. This has lent support to the two possible reaction sequences postulated by Large and Quayle (1963) as shown in Fig. 6.

CH,OHI" X . HCHO!

HCOOH ~ CH, NH~ i Ns,,o~ ~ L

y

CINE

f METHYLENE~ TETRA HYDROFOLATE CELL <-OXA <--PEP ~-- ~--SERINE CONSTITUENT DE NOVOSYNTHESISOF GLYCINE

CYCLICREGENERATIONOF GLYCINE CH ~'

HCHO/ HCOOH/~

"ac02 ~

CHN. j "...,o J

OXALOACETATE

SERINE A

Jl

, ~/

"LYC,NE" C?.A..ENT

METHYLENETHFA

~" CELL CONSTITUENT

Fig. 6. Serine pathways for cell synthesis

7. Present a n d Possible F u t u r e A p p l i c a t i o n s a) Single-Cell Protein Production Hydrocarbon fermentations are becoming increasingly important particularly for protein and food production. However, although microbes and their metabolites have been consumed for centuries in the form of alcoholic beverages, beer, cheese, bread, etc., a massive acceptance

116

N. KOSARICand J. E. ZAJIC

of single cell proteins derived from hydrocarbons has not yet been achieved. The reason is primarily due to the lack of data concerning biological, nutritional and toxicological effects that might develop from the use of this type of food. These data are however, accumulating particularly for animal feed (Shacklady, 1969). There are several outstanding advantages of microbial proteins over their agriculturally-derived counterparts, such as: a) Production of microbial proteins derived from hydrocarbons is independent of agriculture, climatic conditions or high-quality soil. Consequently, a plant producing SCP could be created anywhere in the world which is of particular interest to some protein-deficient and agriculturallyunderdeveloped areas. b) The growth rate and consequently the mass doubling time of microorganisms is significantly in their favour over any other biological system. The mass of proteins and the area required for their production is best illustrated by the example given by Humphrey (1967) who estimates a required area for culture tanks of 1/2 square mile to be sufficient to produce 50% of today's world protein needs. c) The other advantage is the abundance of the raw materials at a relatively low cost. Table 6 adapted from Wang (1968) demonstrates thischaracteristic. Table 6. Approximate SCP process costs (after Wang, 1968) SCP type

Yeast (50% protein)

Bacteria (70% protein)

Substrate

Gas Oil

n-Alkane

Methane

Gas Oil

n-AIkane

Cell yield (lb cells/lb substrate) Substrate costs Agitation costs Aeration costs Refrigeration

0.80

1.00

0,60

0.90

1.00

1.25 1.00 0.62 1.28

2.00 t.00 0.50 1.03

0.42 0.70 0.83 2.00

I. 11 1.00 055 1.15

2.00 1.00 0.50 1.03

Recovery costs Equipment costs

0.25 2.00

0.25 2.00

0.90 3.00

0.90 1.00

0.90 t.00

Subtotal

6.40

7.70

7.90

5,70

6.40

COSTS

These data are based on 15-g cell/litre concentrations in fermenter. Doubling times of 2, 4 and 6 hr were assumed for bacteria on n-alkane and gas oil, for yeast on n-alkane and gas-oil, and for bacteria on methane, respectively. Equipment costs are based on steriliseable fermenters and seven-year amortisation. Costs are given in c/lb. cell.

Microbial Oxidation of Methane and Methanol

117

The characteristics of the present processes for hydrocarbon fermentations for food or feed production can be summarized as follows: I. Generally, high yields of biomass are obtained and usually amount to 90--100% compared to 50--60% in the case of carbohydrate fermentations. A table (Table 7) representing a summary of the yields, according to Wilkinson (1971) is presented. Table 7. The yield of methylobacteria from methane (taken from Wilkinson, 1971) Organism

Yield Reference ( g. cells/g, methane)

Pseudomonas methanica Methanomonas methanooxidans Methylococcus capsulatus Mixed culture 1 GT-IO(?"methylosinus") Various methylobaeteria

56% 110% 110%

65% 114% 110%

Dworkin & Foster (1956) Brown et al. (1964) Foster & Davis (1966) Vary & Johnson (1967) Klass et al. (1969) Whittenbury et al. (1970b)

2. The processes are designed by various petroleum companies to utilize waste liquid or gaseous petroleum fractions. Owing to ease of removal of unused methane, many regard methane as the "'ultimate hydrocarbon source to make single-cell protein", (Coty, 1969). 3. Compared to carbohydrates as substrates, more oxygen is needed for the biosynthesis of the cell mass, the demand for methane being approximately 5 times greater than that in cell formation from carbohydrates and 3 times greater than that from liquid hydrocarbons (Vary and Johnson, 1967). Should this be generally the case, this will be obviously a limiting factor. Since aeration and energy for agitation to assure sufficient oxygen transfer to the growing culture is considerably larger. Another consequence of this operation is the additional heat generated by the impeller, which adds to the heat produced in the exothermic hydrocarbon fermentation. The total heat evolved may be three times the heat generated by an equivalent carbohydrate fermentation (Volesky and Zajic, 1971). The cost of the process is also strongly influenced by this factor as an adequate cooling system must be supplied to maintain the optimum temperature for microbial growth. Thermophilic strains could be developed but operation at higher temperatures could introduce some other disadvantages like diminished solubility of the hydrocarbons in the fermentation liquid and consequently a poorer mass transfer to the growing cell.

118

N. KOSARIC and J. E. ZAJIC

A general flow diagram for SCP production from gaseous (and alternatively liquid) hydrocarbons is presented in Fig. 7. The whole process is essentially composed of five main operations.

Inorganic salts storage tanks i

L~iquid H C storage tank ant/roam ~

J J [ J

]

L,~ Mokeup~

/

.oter'aL

H

Exhaust gas

J J~

/

SUBSTRATEPREPARAT~/ON Gaseous

Hydrocarbon

~

|

,--4-4 R

I---

:

.¢~

-----~ II ld°°lhng , ter

~t

+ was,water ,

ol

" ~". . ~--C I ~,microblol r'--"Imass

L__

~'

f \wosneal f-----Scell I

SEPAR~T/ON-'s--~or°t°r~

separator

seporo

Refriget'ation unit

~

•

Purge

I.--. t recycle__

F L---C:~--~----I;E..~.,E. Medium ¢ III ; sterilizer

Air

By-products recovery

IFIj Fermentationtiqu~

ttU,

.o,o

prep°to "o to.k o,,o.

J

-

J [__~

FERMENTATION ~ A

Microbial mass

,,,,,,

Solvent

[HYdr°-I ~1=°'"°4 I I JsOlvent I l,=~bool ~ i--~l IVraction-I l°~.t?°l ~ I I I at°r I l~Tl-~ I I l I ~' I I=°~'vo=n~ V I ]" ~ C >J Microbial Lipids and Hydrocarbons

SOLVENT EXTRACT/ON

lDrlerl [ J \ /

\ /

[ 1"

Packaging unit

Dr"led

~ 00000 m,J~Packed ", '' Product

Dried

Product

DRYING AND PACKAGING

Fig. 7. Hydrocarbon fermentation flow diagram

1. Preparation and sterilization of the liquid medium and preparation of the microbial culture. 2. Fermentation with adequate agitation, cooling and aeration facilities. 3. Separation of the product from the residual fermentation liquid. Product is washed with water and the medium can be recycled to the fermenter or used for byproducts recovery. 4. In the case of liquid hydrocarbon fermentation, a solvent extraction step is included. The solvent could be hexane and it is recirculated back to the system. 5. Drying and packaging of the final product. Besides SCP, other products or byproducts such as vitamins could be recovered which presents another potential.

FAO

ILEU 4,2 LEU 4,8 LYS 4.2 MET 2.2 PHE 2.8 THR 2.8 TRY 1.4 VAL 4.2 TOTAL PROTEIN

Essential amino-acids

3.9 7.1 5.4 1.0 4.2 4.4 1.3 6.8 24--80%

5.5 7.9 8.2 2.5 4.5 4.8 1.2 5.5 45---50%

4.5 7.0 7.0 1.8 4.4 4.9 1.4 5.4 68--70%

Yeast BP

Algae

Yeast

n-Paraffins

Carbohydrates

Table 8. Essential amino-acids in SCP

3.6 5.6 6.5 2.0 2.9 4.0 0.9 4.5 50---70%

Bact. Nestle 5.0 7.9 4.3 3.0 5.2 4.9 2.7 6.2 74%

Sheehan

6.1 9.1 5.3 3.4 6.2 4.5 -8.5 59%

Vary-Johnson

methane (bacteria)

Microbial proteins grown on:

5.1 8.1 8.5 1,8 4.6 1,7 -6.6 52%

Zajic-Volesky

natural gas (fungi)

0

0

0

120

N. KOSARICand J. E. ZAJIC

Quality of the SCP Derived from ttydrocarbons Nutritional quality of single-cell proteins derived from hydrocarbons has been extensively evaluated by BP Proteins Limited (Shacklady, 1969a, t969b; Groot et aI., t970) who made a tong term study of all possible toxicological, biological and nutritional effects of SCP derived from petroleum. No acute or chronic ill effects were observed by feeding this material in l 0--15 % concentrations of the total diet to rats, poultry and pigs. No results for humans are reported. Concerning SCP derived from methane, similar results could be expected as with SCP derived from higher hydrocarbons. The quality of the product can be partially assessed by the results obtained from chemical analyses, e.g. fats, carbohydrates, proteins and vitamins. Of particular interest in this respect is the protein and essential amino-acid content that is quite favourable, and comparable to the FAO standard or conventional protein sources. Some comparisons are given in Table 8. Wolnak et al., (1967) reported 32 and 36% of protein for two methane-oxidizing cultures, fat being 5.2% and 2.8% and carbohydrates 60% and 59% respectively. They also determined the vitamins present in the biomass (see Table 9). Table 9. Vitamin analysis in SCP (after Wolnak eta[., 1967) Concentration in mg/Ib Vitamin

dried ),east

skim milk

bacteria grown on methane

Thiamine Riboflavin Niacin Panthothenic acid Choline Vitamin B ~2 Pyridoxine

43.0 t 4.0 180 49. t 2000 0

1.5 ! 0.0 5.7 l 6.0 3000 0.3 --

9.0 23.2 79.8 12.0 4840 4.8 71.8

25.2

Although the chemical analysis is in favour of SCP use as food, it does not give a full assessment of the product regarding its nutritional quality. Elaborate long-term studies on possible toxic or other effects should be completely investigated before this new type of food can be commercially used. Some caution regarding chemical results might be appropriate also. Although the protein values are quite impressive, the amount of crude protein is in most cases derived from total nitrogen analysis, usually done by the Kjehldahl method, which does not necessarily have to be a component of proteins alone. Nucleic acids, purines,

Microbial Oxidation of Methane and Methanol

121

pyrimidines, amino sugars and other minor cell constituents contain nitrogen as well and it is known that these compounds are, to a significant extent, present in single-cell organisms. Concerning available information on the biological value of SCP, a possible problem might be developed which could be manifested in apparently low digestibility (74% compared to 96% for casein). Also very high levels in the diet should be avoided as digestion problems have been observed on subjects consuming algal SCP. However, it is not known whether the same problem would develop with other forms of SCP as well. Also, allergenicity to the single-cell proteins should be investigated, and is particularly possible in infants suffering from lesions of the gastrointestinal tract which is a common situation in the less-developed parts of the world. In addition to these, one has to be prepared to face also some unknown problems associated with any unusual and poorly-characterized food source. b) Removal of Methane from Coal Mines A potential use of microbes to remove methane from coal mines has been tested by Yurovskii and co-workers as early as 1939. They used enrichment cultures obtaines from sewage, which consumed methane from a gas atmosphere composed of 15.0% CH4, 0.5% CO, 30.0% N2 and 54.5% O2. Gas uptake took place in a simulated coal mine. As suggested by Silverman (1964), application of methane-oxidizing bacteria to the walls or roofs of coal mines might be one method to apply. Another approach would be to inject methane-oxidizing bacteria and an oxygen supply into bore-holes in advance of the working face, which would have the advantage of removing methane before it enters the working areas. An obvious disadvantage of this method is the addition of oxygen into methane that is already under pressure. If cell-free methane oxidation were developed it might be possible to use enzyme preparations for methane removal, which might lead to its transformation to some nonexplosive product without addition of oxygen. An unexplored potential is in the utilization of methane gas produced by anaerobic fermentations of all types of waste products (e.g. for SCP production). This potential has unlimited scope as the organic wastes available are not being handled in terms of their energy potential. c) Petroleum Prospecting There have been various reports in the literature that deal with geomicrobiological prospecting methods based on methane concentrations in oil-bearing soil (Davis, 1957; Mogilevski, 1940; Strawinski, 1954). How-

122

N. KOSARrCand J. E. ZAJIC

ever, methane measured in soils that are not associated with petroleum is in the order of 5 ~ 1 5 ppm (Subbota, 1947a; Kartsev et al., 1959) and methane-oxidizing bacteria are wide-spread in nature which leads to the conclusion that the presence of methane-oxidizing bacteria is not always indicative of the presence of petroleum or natural gas (Davis and Updegraff, 1954). For this purpose other hydrocarbon oxidizers (e.g. specific to ethane and propane) are looked upon for petroleum prospecting. d) Microbial Fuel Cell Silverman (1964) brought forward a few interesting ideas regarding use of methane oxidizers for microbial fuel ceils visualized as an anode consisting ofsubstrate plus microbe, an electron conductor and a cathode consisting of a molecular oxygen electrode. The principle involved is the use of the cellular biochemical electron-transport system during the aerobic biological oxidation of the organic matter. While there are no reports in the literature of the use of methane oxidizers in this respect, ethane-oxidizing bacteria such as Nocardia have been used in an ethane fuel cell (Davis and Yarbrough, 1962). Several practical applications for a methane fuel cell are foreseen by Silverman (1964). It might function as a sensitive methane detector in coal mines or as an automatic safety cutoff for mining machinery where methane is present. Other prospects like efficient large-scale conversion of cheap fuel to electrical energy have to be investigated further. If methane became limiting in supply, adding methane-forming bacteria that are capable of forming methane from CO + H2 or C02 q-H2 would be used to synthesize the substrate needed by the methane-oxidizing bacteria.

References Aiyer, P.: Mere. Dept. Agr. India. Chem. Set. 5, 173 (1920). Baas-Becking, L. G. M.: Acta Biotheoret. 12 (2), 71 (1957). Bewersdorff, M., Dostalek, M.: Biotech. Bioeng. 13, 49 (1971). Bogdanova, U. M.: Mikrobiologiya 35, 197 (1965). Bokova, E. N., Kuznetsova, V. A., Kuznetsov, S. 1.: Doklady Akad. Nauk SSSR 56, 755 (1947). Brown, L. R.: Thesis. Louisiana State University 1956. Brown, L. R., Strawinski, R. J.: Bacteriol. Proc. 18 (1957). Brown, L. R., Strawinski, R. J.: Bacteriol. Proc. t22 (t958). Brown, L. R., Strawinski, R. J., McCleskey, C. S.: Can. J. Microbiol. 10, 791 (1964).

Microbial Oxidation of Methane and Methanol

123

Coty, V. F.: Biotech. Bioeng. Symp. 1, 105 (t969). Darlington, W. A.: Biotech. Bioeng. 6, 241 (1964) (Listed by Klass et al., 1969). Davies, S. L., Whittenbury, R.: J. Gen. Microbiol. 61,227 119701. Davis, J. B., Coty, V. F, Stanley, J. P.: J. Bacteriol. 88, 468 II964). Davis, J. B., Updegraff, D. M.: Bacteriol. Rev. 18 [4), 215 ~1954t. Davis, J. B.: Md. Eng. Chem. 48, 1444 (1956). Davis, J. B.: U. S. Pat. 2777799, Jan. 15, 1957. Davis, J. B., Yarbrough, H. F., Jr.: Science 137, 615 (1962). Davis, J. B., Coty, V. F, Stanley, J. P.: J. Bacteriol. 88, 468 (1964). Davis, J. B.: Petroleum microbiology. London: Elsevier Publishing Co. 1967. Dutova, E. N.: Tr. Vses. Nauchn. Issled. Geol. Inst. 109, 367 (1963). Dworkin, M.: Thesis. University of Texas 1955. Dworkin, M., Foster, J. W.: Nov. Comb. J. Bacteriol. 72, 646 (I 956). Ekzertsev, V. A.: Mikrobiologiya 27, 626 (1958). Elizarova, T. N.: Mikrobiologiya 32, 1091 (1963). Enebo, L.: Acta Chem. Scand. 21,625 (1967). Eroshin, U. K., Harwood, J. H., Pirt, S. J.: J. Appl. Bacteriol. 31, 560 (1968). Finn, R. K.: Bacteriol. Rev. 18, 254 (1954). Foster, J. W.: In: Oxygenases. Osamu Hayashi (ed.), p. 241. Academic Press 1962. Foster, J. W., Davis, R. H.: J. Bacteriol. 91, 1924 (1966). Groot, A. P., Til, H. P., Feron, V. J.: Food Cosmet. Toxicol. 8, 267 (1970). Hamer, G.. Hed6n, C. G.. Carenberg, C. O.: Biotech. Bioeng. 9. 499 11967). Hansen, R. W., Kallio, R. E.: Science 125, 1198 (1957). Harrington, A. A., Kallio, R. E.: Can. J. Microbiol. 6, 1 (1960). Haseman, W.: Biochem. Z. 184, 147 (1927) (Listed by Davis, 1967). Hazeu, W., Steennis, P. J.: Antonie van Leenwenhock. J. Microbiol. Serot 36, 67 (1970). Heptinstall, J., Quayle, .I.R.: Biochem. J. i 17, 563 ~1970). Higgins, I. J., Quayle, J. R.: Biochem. J. 118 (2), 28 P (1970). Hotchkiss, R. D.: Arch. Biochem. Biophys. 16, 131 (1948). Humphrey, A. E.: Biotech. Bioeng. 9, 3 (1967). Hutton, W. E.: Thesis. University of California at Los Angeles 1948. Hutton, W. E., Zobell, C. E.: J. Bacteriol. 58 (4), 463 (1949). Hutton, W. E., Zobell, C. E.: J. Bacteriol. 65, 216 (1953). Johnson, J. L., Temple, K. L.: J. Bacteriol. 84, 456 (1962). Johnson, M. J.: Science 155, 1515 (1967). Johnson, P. A., Quayle, J. R.: Biochem. J. 95, 859 (1964). Kallio, R. E., Harrington, A. A.: J. Bacteriol. 80 (3), 321 t1960). Kaneda, T., Roxbrugh, J. M.: Can. J. Microbiol. 5, 87 (1959). Kartsev, A. A., Tabasaranskii, A. Z., Subbota, M. I., Mogilevski, G. A.: Geochemical methods of prospecting and exploration of petroleum and natural gases. Berkeley, Calif.: Univ. Calif. Press 1959. Kemp, M. B., Quayle, J. R.: 1965. Biochem. Biophys. Acta 107, 174 (1965). Kemp, M. B., Quayle, J. R.: Biochem. J. 102, 94 (1967). Kersten, D. K.: Mikrobiologiya 33, 31 (1964)(Listed by Coty, 1967). Klass, D. L., Iandolo, J. J., Knabel, S. J.: Chem. Eng. Progr. Symp. Ser. 93, 72 (1969). Kornberg, H. L., Elsden, S. R.: Advan. Enzymol. 23, 401 (1962). Large, P. J., Quayle, J. R.: Biochem. J. 87, 387 i1963). Lawrence, A. J., Kemp, M. B., Quayle, J. R.: Biochem. J. 116, 631 (1970). Lawrence, A. J., Quayle, J. R.: J. Gen. Microbiol. 63, 371 (1970).

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Leadbetter, E. R., Foster, J. W.: Arch. Mikrobiol. 30, 91 0958). Leadbetter, E, R., Foster, J. W.: Nature 4696, 1428 (1959). Leadbetter, E. R., Foster, J. W.: Arch. Mikrobiol. 35, 92 (1960). McKenna, E. J., Kallio, R. E.: Ann. Rev. Microbio]. 5 (19), 183 (1965). Mogilevski, G. A.: Razvedka i Okhrana Nedr. 12, 32 (1940) (Listed by Davis, 1967). Miintz, E.: Thesis. Halle: Friedrichs University 1915. Nechaeva, N. B.: Mikrobiologiya 18, 310 (1949) (Listed by Coty, 1967). Novelli, G. D., Zobell, C. E.: J. Bacteriol. 47, 447 (1944). Orla-Jensen, S,: Zentr. Bakteriol. Parasitenk., Abt. II. 22, 305 (1909). Patel, R. N., Hoare, D. S.: J. Bacteriol. 107, 187 (1971). Quayle, J. R., Keech, D. B.: Biochem. J. 72, 623 (1959a). Quayle, J. R., Keech, D. B.: Biochem. J. 72, 631 (1959b). Quayle, J. R., Keech, D. B.: Biochem. J. 75, 515 {1960). Quayle, J. R., Peel, D.: Biochem. J. 76, 3 P (1960). Quayle, J. R., Keech, D. B., Taylor, G. A.: Biochem. J. 78, 225 (1961). Quayle, J. R.: Ann. Rev. Microbiol. 15, t19 (1961). Quayle, J. R.: J. Gen. Microbiol. 32, 163 (1963). Quayle, J. R.: Symposium on Microbiology, London, Sept. 19--20. Society of Chem. Industry, London 1 (1967). Ribbons, D. W.: J. Inst. Petrol. 47 (t968), Ribbons, D, W., Harrison, J. E., Wadzinski, A. M.: Ann. Rev, Microbiol. 24, 135 (1970). Rosenfeld, W. D.: J. Bacteriol. 54, 664 (1947). Shacklady, C. A.: Food Manuf. 44 (4), 36 (1969a). Shacklady, C. A.: Voeding 30 (10), 574 (1969b). Sheehan, B. T.: Ph. D. Thesis. University of Wisconsin 1970. Sheehan, B. T., Johnson, M. J.: Appl. Microbiol. 21 (3), 511 (1971). Shmonova, N. I.: Tr. Vses. Neft. Nauchn.-Issled. Geologorazved. Inst. 227, 64 (1964). Silvcrman, M. P.: U. S. Bur. Mines, Inform. Circ. 8246, 1 (1964). Slavnina, G. P.: Mikrobiologiya 17, 76 (1948) (Listed by Davis, 1967). Smirnova, E. S.: Izv., A. S. USSR 3, 423 (1971). SiShngen, N. L.: Zentr. Bakteriol. Parasitenk.i Abt. II. 15, 513 (1906). Sorobin, Yu.: I. Dokl. Akad. Nauk SSSR 115, 816 (1957). Stocks, P. K., McCleskey, C. S.: J. Bacteriol. 88, 107I (t964). St6rmer, K.: Zentr. Bakteriol. Parasitenk., Abt. II. 20, 282 (1908). Strawinski, R. J.: U. S. Pat. 2665237. Jan., 5 (1954). Subbota, M. I.: Neft. Khoz. 25, 13 (1947). Tausz, J., Donath, P.: Z. Physiol. Chem. 190, 14l (1930) (Listed by Davis, 1967). Van der Linden, A. C., Thijsse, G. J. E.: Advan. Enzymol. 5 (27), 469 (1965). Vary, P. S., Johnson, M. J.: AppL Microbiol. 15, 1473 (1967). Volesky, B., Zajic, J. E.: Appl. Microbiol. 21 (4), 614 (1971). Wang, D. I. C.: Chem. Eng. Ang. 99 (1968), Whittenbury, R.: Process Biochemistry, January, 51 (1969). Whittenbury, R., Phillips, K. C., Wilkinson, J. F.: J. Gen. Microbiol. 61, 205 (1970). Wilkinson, J. F.: Symp. Soc. Gen. Microbiol. 21, 15 (1971). Wolnak, B., Andrcen, B. H., Chisholm, J. A., Saadeh, M.: Biotech. Bioeng. 9, 57 (1967). Yurovskii, A. Z., Kapilash, G. P., Mangubi, B. V.: Ugol. 7, 48 (1939). Zajic, J. E.: Develop. Ind. Microbiol. 6, 16 (1964).

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Zajic, J. E., Smith, N. D.: Unpublished report (1966). Zajic, J. E., Volesky, B., Wellman, A.: Can. J. Microbiol. 15 (10), 1231 (1969). Zheludev, A. N.: Izv. Voronezhsk, Gos, Ped. Inst. 27, 169 (1959). Dr. N. KOSARIC Associate Professor Chemical and Biochemical Engineering Faculty of Engineering Science The University of Western Ontario London, Ontario (Canada) N6A 3K7

Dr. J. E. ZAJIC Professor and Assistant Dean Chemical and Biochemical Engineering Faculty of Engineering Science The University of Western Ontario London, Ontario (Canada) N6A 3K7

CHAPTER 4

Modelling and Simulation in Biochemical Engineering H. W. BLANCH a n d I. J. DUNN With 26 Figures

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Models for Microbial Growth . . . . . . . . . . . . . . . . . . 2. Growth Models in Stirred Reactors Using the Monod Relationship . . 3. Transient Response of a Plug-Flow Reactor . . . . . . . . . . . . 4. The Influence of Mixing on the Transient Performance of Continuous Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Transient Response of the Turbidostat . . . . . . . . . . . . . . . 6. Enzymatic Reaction within a Porous Support . . . . . . . . . . . . 7. Unsteady-State Formulation of Section 6 . . . . . . . . . . . . . . 8. Tubular Immobile-Enzyme Reactor . . . . . . . . . . . . . . . . 9. Control of Activated-Sludge Reactors . . . . . . . . . . . . . . . 10. Comments on the Future . . . . . . . . . . . . . . . . . . . . Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127 129 130 134 135 140 143 148 t53 157 162 163 165

1. Introduction Increasingly the biochemist, microbiologist a n d biochemical engineer are interested in describing their experiments with m a t h e m a t i c a l models. U s u a l l y the p r o b l e m s are of sufficient complexity to require a c o m p u t e r solution. T h e present paper is intented to serve as a n i n t r o d u c t i o n to m a t h e m a t i c a l m o d e l l i n g a n d c o m p u t e r s i m u l a t i o n in biochemical systems. Shown, by way of example, are some of the areas to which

128

H.W. BLANCHand I. J. DUNN

mathematical description can be applied and how solutions may be obtained easily using digital simulation programming techniques. Simulation can be defined in the present context as the description of a space- or time dependent physical system by the use of differential equations, computer methods are generally required for their solution. Several important steps can be recognized in any simulation development. First, the physical problem must be clearly defined and carefully analyzed. It may consist of many separate processes, and each must be understood in relationship to the other. This detailed knowledge of the system is necessary because equations must be formulated for each process and assembled in their natural cause-and-effect order. Successful construction of the mathematical model depends on an insight into the physical nature of the problem. Frequently the effort required to simulate a system can be justified by the amount of physical understanding that is derived. The second step is to write the equations. The transformation of physical concepts into mathematical terms requires practice and will always remain demanding. The third step is the solution of the differential equations. For all but the trivially simple problems, a computer solution is required. In the past, this step has required the talents of those who are trained in numerical analysis and computer programming. Fortunately these difficulties have essentially been eliminated by the development of digital simulation languages. The books by Franks (1967) and Chu (1969) provide an introduction to these techniques. Simulation languages are available for almost all scientific computers and exist in various degrees of sophistication. The most widely used are MIMIC (CDC and UNIVAC) and CSMP (IBM). Other such as LEANS and PACTOLUS are suitable for smaller machines and require more programming effort. One program, LEANS III, is now available to solve even three-dimensional transient problems. The early digital simulation programs were developed in order to make the digital computer as easy to program as the electronic analogue computer. A block diagram was needed for these programs which was very similar to the wiring diagram of an analogue computer solution. Larger simulation programs require only that the equations be written in FORTRAN-like notation. The actual arrangement of the step-by-step calculations is taken care of automatically by the simulation program. A number of sophisticated integration methods may be chosen by the user. The input of data and output of printed and plotted results are particularly simple to program. It can be said when using a digital simulation language that the program follows simply and directly from the mathematical model.

Modelling and Simulation in BiochemicalEngineering

129

Models for Microbial Growth Recently there has appeared a great variety of kinetic models to describe microbial growth and related processes. Earlier models were used mainly to improve knowledge of fermentation kinetics in both batch and continuous cultures (Luedeking and Piret, 1959; Hockenhull and MacKenzie, 1968), and simple mechanistic models (Maxon and Chen, 1966) allowed information on the biochemical pathways to be collected. Further models were used to represent industrial fermentation processes, and gave better control possibilities. More recently optimisation stages have been introduced which demonstrated the practical advantages of modelling for fermentation processes (Constantinides et al., 1970; Blanch and Rogers, 1972). The types and purposes of mathematical models have been briefly summarised by Calam et al. (1971). These authors pointed out that the construction of a model (in their case, for the griseofulvin fermentation) and its comparison with experimental results demonstrated that such a modelling procedure can illuminate the ways in which fermentations behave and indicate new areas for investigational work. Models of microbial growth have been divided into several different classifications. Koga et al. (I967) suggested that three categories could apply: engineering, population and biochemical models. The first deals with the microbial system at an averaged macroscopic level, the second considers the population as heterogenous, and the last category is concerned with models of the overall reaction patterns in the cell. However, whatever type of model is considered, all result in multivariable, nonlinear systems of differential equations. In simpler models these equations may be algebraically soluble, although the solutions are quite often so cumbersome that they cannot be readily used. Rapid digital solution of even these simple equations is thus advantageous. As the models increase in complexity only numerical solutions may be possible, this being especially the case in studies of transient behaviour of culture systems. The use of digital simulation languages allow rapid solutions to be obtained without the need to fully comprehend the complexities of the numerical techniques involved. Such approaches have not been often applied in fermentation, but examples have demonstrated the usefulness of the technique (Calam et al., 1971; Koga et al., 1967). The purpose of this paper is thus to solve a variety of biochemical problems in order to demonstrate the ease with which the computer solution follows from the mathematical model. In some of the more complex examples considerable care is taken to give a clear understanding of the physical problem and its mathematical description. All the pro-

130

H.W. BLANCHand I. J. DUNN

grams are written using the MIMIC digital simulation program. The computation times given in the examples are for a CDC 6400/6500 machine.

2. Growth Models in Stirred Reactors Using the Monod Relationship The continuous flow system used for continuous cultivation is generally an agitated vessel, so well-mixed that one assumes complete mixing (i.e. "perfect" backmixing). The general parameters of growth considered in this system are, in the simplest case, dry matter, substrate and product. The simple chemostat can thus be described by the following mass balance equations: dX - ~X-DX, dt

dS_D s dt -

R--DS--Y

(1)

1

X

'1~ '

--=dP (kl + k 2 p ) X _ D P " dI

(2) (3)

Here the mass balance for the product considers P as a result of growth and non-growth-associated rates of production, kl is the non-growth associated coefficient and kz the growth-associated coefficient. Such a model has been found applicable in a fairly wide variety of processes (Humphrey, 1966), and one may set either of these coefficients equal to zero in extreme cases. The growth rate (kt) is a function of the substrate concentration, and this relationship has been usually taken as the Monod relationship; /~

~'~max S _

-

-

(4)

Ks+S

This relationship is most frequently used for steady-state continuous cultures, and also in transient situations (e.g. in continuous-flow systems and batch growth), although there is evidence that in these situations some modifications are necessary to predict the observed response (Young et al., 1970). Other expressions have been proposed for cases where this simple relationship has been found to be invalid, such as inhibition by either substrate or product (Ramkrishna et at., 1967).

Modelling and Simulation in Biochemical Engineering

131

These usually involve the addition of terms to the denominator in Equation (4). Analytical solutions for these later more complex models become very difficult for steady-state cases, and the possibilities of multiple steady-states arise. In transient cases only numerical solutions are usually possible. As a simple example showing the ease of programming in MIMIC language, batch growth is considered and the growth rate expressed as a function of substrate concentration according to the Monod model. The equations describing growth in batch cultures using this relationship are: dX #,,~. X . S d~ = Ks+ S ' dS -1 d~t = Y

#max'S'X Ks+ S '

dP

~max" X - S

-d~ = k , . X + k ~ .

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(5)

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Fig. I. Program and constants for section 2

The program for the solution of these equations is shown in Fig. 1, and the graphical output from the MIMIC program in Fig. 2 shows a typical batch growth curve, without however the lag or decline phases,

132

H.W. BLANCH and I. J. DUNN

which are not allowed for in the equations proposed. This type of problem may also be readily solved by analogue means; the digital output and ease of programming make even such a simple case worth-

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,

,

,

•

•

8

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8

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+

+•.,

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°

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•

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,

, . * , . , * . * , . . . . , . , • •

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.

+.,,,.,.,

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+

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,

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*

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....

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*

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.

.

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.........

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* .

. . . . . . . . . .

.

. . . . . . . . . . . . . . . . . . . . . . .

•

.

C+C

.

+.,...,

.

,

,,.,**,*,*,.*

.

*A+

. ,

.

~*~

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' " ' * * *

•

,

•

. . . . . . . . . .

•

....

,*

.

•

. +A+

++

~O~OOOO+,,, +

.

, ++A

,

,

+B

•

,

.

,

iO+O000*..,

•

• ....

A+A

,

.

,

,..C~+o,.+÷o, •

*

. . . . . . . . . . . . . . . . . . . . . . .

G*

.

.

. . . . . . . . . . . ,

,* .~

5+00C0.++.*+*....

*

.

. ..............

.

,*,°,,°°

.....................

Fig. 2. Output of Example l, showing dry weight (X 1), substrate (X 2) and product (X 3) as functions of time

while however. A simple extension of this problem can be seen as the "start-up" of continuous-culture operation by the addition of the flow terms, the equations then becoming similar to Equations (1), (2) and (3).

Modelling and Simulation in Biochemical Engineering ***

133

HIMIC SOORC~-LAN&JAGE PROGRAM * * *

CON(UH,KS,Oe¥) CON{ALPHA~OETA) CON(XL0,X20~X~0) PLUG FLOW MODEL SIMULATION BY N*CSTR IN SERIES ORIV Xll XZl X3L ORIV XiZ XZ2 X3Z ORIv X13 X23 X33 ORIV XtW X2~ X3~ ORIV Xl~ X25 X35 ORIV X16 XZ6 X36 ORIV

CSP(0,,;0,pQ,jXILjX2t~XS~) RSP{OXIt,O~ZI~OXSt) I~T(DXlt~X%O) INT(~X~t,XZ0) INTIDKStsXSO) ESP(XIIyXZZ,X31~Xt2jXZZ*X3Z) RSP(OXIZ,OX2Z,OXSZ) INI(DXIZyQ.} INT(DXZZ)Q,) INT(~X3210,) PL0(T~XtI~X21,X31) CSP(X121X2Z~X32~XI3}X~SjX33) pSP(D~IO,OXZO~OX33) ItlliDXlSlO.) INTi0123~Q,} INT(OX33jO.I CSP(X~3,X23~X33~XI~yXZ~X3~) RSP(~XI~OX~W~DXS~I INT(DXt~,0.) ItJI(UX~4~0,) INI(QX34~0.) CSP(X~XZW,XS~XlS~X25~X35} RSP(~XIS~OXZ~OX35) INT(0X15~0,) INT(~X~5~0.) INT(SX35~O,} CSP(XtS=X25,X~5~XIS~X~6~X36} RSP(OXI6~DXZ6eOXS6) l~T(OXt6~0.} INT(0X~6~0.) IHT(~X~6~O,) CSP(XI6~X26~X36~Xt/~X2/~X37)

RSP(OXlI~OX~I~OXST) Xt/ X27 X3l ORIV xi8 X28 X38 ORIV X19 X29 XS9 ORIV X110 X210 x310

DRIV OXl DX2 Oxs ORIV

ItIT(DX%l,Q,) IhT(Dx27~0,) INT(OX3I~O,} ESP{X~7~X2?~X37~X18~XZ~X38} RSP(~X%8~DXZd~X~8) INT(OXi6~0.) INT(OX~@,0.) Irdr(OX35,0,) CSP(XtB~XZ~X38=XI~X29)X39} RSP(DXIg~OX~g~DX39) I~I(OXLg~0,} INT(DX29~O.) INI(0X39~0,) ~ CSP(X%9~X29~X39~XII0~X~I0~X3¢0) RSP{DXII0,OXZIG~X3~0) INY{DXII0~B.) INTtOX~I0,O,) INT(OX3%0~O,) FIN(T~80.) PLO(I,XilO~X~I0~X3%~) 8SP(XIN%,XINZ~XINS~X[~X2~X3) D~(XINI-XL)+UH~XI~X2/(KS+X2) O*(XIN2-X2)-UH~Xt*X21(Y~(KS~X2)) ALPHA~OXI÷SEIA~XL÷O'(XIN3-XS) ESP(DXt~OXZ~DXS) ENO

UM 3,Q00O~E-01

KS 5.~0QOSE-0Z

ALPHA 2,00000E-01

~ETA 5,0QOOOE-03

xIO t . OOOOOE-01

xZQ 9,OO000E+OO

• **~COMPUIING lIME S

O I.SGQ00E-Ol

X30 I,SCOOOE-03

33,6W5 SECONDS**'*

Fig. 3. Program and constants for example of section 3

Y 8. G~0OOE-O~

134

H.W. BLANCH and I. J. DUNN

3. T r a n s i e n t R e s p o n s e of a P l u g - F l o w R e a c t o r Although experimentally no great use has been made of the tubular or plug-flow fermenter in biological systems, it has been theoretically analysed for steady-state behaviour (Powell and Lowe, 1964) using the Monod equation to express/~ as a function of substrate concentration. The transient behaviour of the system is described by partial differential equations (see Fredrickson et al., 197 I), which may become more complex when axial diffusion is considered:

+~+r+ C~+) = D+\+Z+] + ~ X +++ B:X210 C=X31~

0+00~ 0.0~00

.00~3 l*OOO0

,0~0 ~.O~6D

T o.ooOO+

. . . . . .

+ ÷

.

÷o...,

................

.,..

.0$&0 %QOQQ . .....

.0200

5.gO00 •

*,,,.o,.+,,,..,

.......

*..+,.*,,

.

............................

,,,

....................................

.,++....,,.,

.....

...,.,.,.,,

+ ++

N

E

.DL2~ 3.~00~

. ,**.,,,.,

............................

,

(8)

~o ooo~ ..........

: .........

: .........

+ *+

+ ....

: .........

,,,,

. . . . . . . . .

+BBB~B*B

. . . . . . . . . . . . . . . .

•

,

.

2.C

B+8+BBBS+BBa

......

+ .............

++

.

++

. , . , , , , + ,

....

,

*

,

...

,

.

:

. . . . . . .

,

,...,

. . . . . .

,

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+

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,

•

•

•

•

. . . . . . . .

,.~-,,*+

....

•

*+ ,

, **

. . . . . . A+A

+

,

+

.

++ . . . . . . . . . . . . .

+,,

,.*

. +k

C++

, ++ ..... e++

.

.+*

.

.

• .....

,

+

•

•

•

.

. . . . . . . . . . . . . . . . . . . . . . . .

.

.

, , . . + ÷ . . , . + .++ .

, . . . , , + o ,

....

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+ +

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,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

•

.

, . . . . . . . . . .

.

.

•

,

++C

, .....

, . . . . . .

A++

• ÷+~,

•

A++

....

' * ' ' ' ' ' ' ' + ° ' ' ' ' •

. ++ +++

• . . . . . . . . . . . . . . . .

+++ • ++

+*

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

' * ' ' ' + * * * ' ' * * '

,

++ +.++

•

.

. . . . . . . . . . . . . . . . . . . . . . . . . . +*+C

+*A

.

•

•

++,,++~

+*+

+

,

, + , , . , , , ° . o , . , . . . , , , + . , . , , ~ , , +

++C .

++ . . . . . . . ++÷. +*

•

,

,.

++C

++

,

+ . + . . . . . . . . . . . . . . . . . . . .

.

. . . . . .

**C

a+*

.

.

. C++ . C+*

4 + +.

. . . . . . . . . . . .

.

.

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.

. . . . . . . . . . . . . . . . . . . . . . . . . + .

++

•

.

,

60,0+0+*

+.*,,,*

,

+++

+

+ +

. . . . . .

: .................................................

C++

....

+,+..° +

•

...........

•

,

++

+ +

Be+

"

.

.

++. ++

•

. , . , , * , . * , +

°

++.

++

++,O00Q+,.****

•

~B~a~Baa

: ...................

. . . . . . . . . . . . . . . . . . . . . . . . . .

*+

+

~a °~

•

: .........

+++++~

•

50+OOO~+

.

.,,....,..daUB÷e*

BaB~Baa

•

+

+

a

• . . . . . . . . . .

*

aeaBa

,+÷~ +++~

+ +

BB~B~B~B+SB'*+BB

. .............. .

;

*

, B

,

...................... ~

+

: ................... •

• . . . . . . . . . . . . . . . . . . .

BBBBBS~BB~

+

: .........

•

•

30.ODO~+,++.+C

; .........

•

•

+

; .........

.

+

Zo.O~O0+

: .........

. ++*

•

+

•

, , , * , , , , , , . . . + , * .

• ....

, , . , . , , + , + , , + *

*

•

++ *

+* . . . . . . . . . . . .

"

+ *

*

C*+ . . . . . . . . . . . . . . . . . . . .

,

.,+o,,

"

....

, * * - , * . - , * , .

Fig. 4 Plot of dry weight (X 110), substrate (X 210) and product (X 310) versus time, from the 10TM stage of the multistage culture

Modelling and Simulation in Biochemical Engineering

135

(here vf is the velocity of flow through the tube, Da the effective axial diffusion coefficient and Z the axial distance from the inlet of the tube). The solution of these equations (similar ones for S and P can be postulated) can be effected by finite differencing one variable. The solution of the unsteady-state formulation in Section 7 of enzymic reaction within a porous support demonstrates this approach. The corresponding steady-state equations can be handled using an iterative approach as seen in Section 6. In this example, however, one may conveniently obtain solutions by considering the plug-flow system as being approximated by a series of continuous-stirred tank reactors. Such an approximation is discussed by Levenspiel (1962). Using the same basic growth model as in section 2, the transient solution for the plug-flow system was obtained by considering a 10-CSTR'S-in-series model. This approach provides no extra difficulties in programming, only the computational time increases. The growth kinetics are the same for each stage, and the equations describing growth are written in a MIMIC subroutine which is called for each stage, as shown in Fig. 3. The output from the last stage (Fig. 4) consists of first a lag, an early substrate peak, and then slowly increasing concentrations of cells and product. The attainment of steady state is fairly slow. Transient damped oscillations were observed in product concentration in the second and third stages, and in general an overshoot occurred in product concentration before the attainment of steady state. The ease of programming is clearly demonstrated; as an analogue solution would prove laborious and require a considerable number of integrators and related circuitry.

4. T h e I n f l u e n c e o f M i x i n g o n t h e T r a n s i e n t P e r f o r m a n c e of Continuous Cultures The use of radial-flow impellers (either paddle or turbine type) in fermentation practice gives rise to the formation of two regions of mixing, one above and one below the impeller. As the fermentation vessel is usually considered to be perfectly mixed, it may be necessary to re-evaluate this assumption in the light of the observed mixing patterns. Several authors (Sinclair and Brown, 1970; Fan et al., 1970), have considered the effects of imperfect mixing on chemostat behaviour. The ideal tank reactor is replaced by a more complex and hopefully more realistic model. One such simple signal-flow model is that of Sinclair and Brown. These authors considered the tank as divided into two regions at the level of the impeller. The fluid within each regions is

136

H.W. BLANCHand I. J. DUNN

assumed to be perfectly mixed, but the transfer of material between regions is limited. It is assumed that nutrient solution and cells both enter and leave from the upper portions of the culture (Fig. 5). This model may then be easily applied to the calculation of both steady-state and transient behaviour of the system. In order to demonstrate the ease of application in even a complex growth situation, a simple refinement to the Monod relationship can be considered by the addition of terms to the mass balances for cell material and substrate to account for maintenance requirements of the organism. This allows a wider simulation of the experimental results. The maintenance requirements of an organism may result in the consumption of exogenous substrate;

SR, F

REGION ABOVE IMPELLER ~V X1, S . P~

i REGIOBELOW N

NPELLER

(1-~)V Xv S2, P2

Fig. 5. Mixing model of Sinclair and Brown

however earlier models allowing for this consumption were not very suitable as they predicted substrate consumption even when none was present; a more realistic model was proposed by Ramkrishna et al. (1966), in which the equations d X _ ~ .... X S / ( K , + S ) -

&

K'~,X/(K'+S)-

(9)

OX,

1 --=dS D(S _S)_~I~m,xXS/(K,+S)_aK,]~LcXS/(K,+S dt

)

(lo)

were used for continuous-culture operation. The increasing complexity of this model makes calculation of even steady-state results difficult.

MIMIC SOURCE-LANGUAGE PROGRAIt + * *

SO~ 9,SQOOOE+QO

OEIA Z,5@OOOE-QI

UC 1.03000E-~t

A 8,00000E-0%

X~O S,O0000E-02

ALOHA Z,5OOOOE-0%

F ~.O0000E~O0

R E,OOODOE~O~

AS "5*QOQOOE-0%

P?O 5,OOOOOE-Q3

P~O 5*OGOOOE-O~

KS i*DDOOQE-01

V l.~OOOOEtO%

Fig. 6. Program and constants for example of section 4

StO 9,5000OE+O0

XtO ~.OPOOOE-02

PLO{T;XI~S%~RI) PLO(T;X2;SZIR2) E~ID Y %.OOOOOE~03 SR t,OODOOE*Ot

~I(A*V) U;4*XO*SZ/(KS*SZ) F/((t.-A)*V) ,~I((Z.-A)*V) AI-A2*Xt-A3~X%+A3~XO-KI~UC~X~Sl/(K~*S%) ~I-KI~UC~XO~S2/(K%÷S2)-B~X2~B3mXI -AZ/Y-ASmK%~UC~St~XII(Kt~SI)~A2~(SR-S~)-A3~(SI-S2)

-qIIY-AS'KI~UC~S~X~I(KI~SE)~83~(S~-S~) ALPHA*AA*BETA~X%-(AZ+A3)~PA~A3~PE ALPHA~B%+3ETA~X2+B3~PA-B3~P~ IqT(OX1~XtO) I'IT(DX~XO0) I:qT(nSt~SLO) IqT(~SO~S~0) I'I[(O#$,PIO) [NT(OPO~PZO} FIn{t~25.0)

UH'X~SL/{KS~SE)

A3 Ot B2 ~3 OXt OX2 0S% 03~ OPt O~2 X% X2 S~ S2 R& P2

CQtI({IHtY~KS~SRt Cq~}( XtO~SIO,PZO) CQN(X2Q~S~O,P2Q) CQN{ALFHAIBETA) COI4(Kt~UC~AS) mAC(F~R~V)

AI

,JH 1.03000E~60

C C

~*"

MOOEL OF SI!ICLAIR AN3 ~ROWNFOR TRANSIENTANALYSIS OF OUTPUT DUE T9 I~4FERFECT HIXING

*~COHPUTING TIME ;

2,75~ SECON~S~**~

-4

=--£

e~

qQ

0

138

H.

W.

BLANCH

and

I. J. D U N N

ROll PLOT

e=X~

o,oooo OoOOJJ o,0oo0

m:Sl ¢=pl.

2.oo00 2.0000 t,oo00

T o.++0o;~ . . . . . . . .

: .........

+ ++

. .

;:

.

~.o000 q.ooO0

: ......... . .

: .........

. .

.

6.0000 6.0000 3,0000

2,04~0

: .........

. .

.

.

i .........

. .

.

: .........

. .

.

.

8.0000 a+O0oo 4,0000

. .

: ......... . .

.

.

iO.OoO0 10,0oo0 ~,0000

: .........

: . . . . . ~...2

. .

.

.

B+

• .

+

.

. . . . . . . . . ~o • + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +. • + . . . . . . . . . +, ,+

.

.

. . . . . . . . . . . . . . . . . . . . . .

.

.

5+o~oa,++

.

.

.

.

.

.

.

:

.

.

to,~060

..

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.

~+

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+

. .

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.

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.

.

: o . . . . .

.

.

.

.

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.

.

++0 +++

.

:

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d: *

...... : .........

.

.

.

.

.

: . . . . . . . . . . . . . .:.

+i: + "'"

++

.

.

.

.

.

++

+

, ...........

. ...... .

•

,

•

++++A

+ •

.

+

.

.

i .........

i ........

•

,

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. ................

•

,

,

, + .........

.

..........." ++

++ ++

.

, . . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . .

.

.

C+C . ++

÷+

.

+ .....

++ ++ ......

.

.

,

• . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+c

+~A

i .........

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++G

+ ,+

,

9+BB,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ++ . . . . . . . . . . . . . . . •

,,,

+B+

. . . . . . . . . . . . . . . . . e...++.+C++ ~+ ++ •

+

....

B++

~ + + + + C

.

.

,++

++

+B~B8 . . . . . . . . . . . . . . . . 8+8B

. BBBBB, aB+OB .......

.

+÷

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + .......

A~++CCS

i .........

:

,

:

.

.

,++++CC

.

+.

,

,++++ . . . ++++

. . . . +++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a++++C

.

.

. . +. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +9°

, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

aO.CO0o

.

.

:

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , .........

,

.

. . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.o.++

•

I++0+0~

.

.+

.4 .........

i .........

+,

,

+

:-

+C

•

: ...... i,-i ......... +

i .........

! .........

i

-

-

•

°

•

N.

~.+~+:

Fig.

7a.

.........

Output

: .........

from

~. . . . . . . . .

i ...................

Example

3,

i ..................................................

showing dry weight

(X

1),

substrate

{S

1)

and product (P 1) concentrations against time from the upper region in the vessel Applying this growth model to the proposed mixing model yields the following equations describing growth in the upper region: K'ttcS1 K'+S 1

dX* = PmaxS~. X1 dt Ks+S ~ ×

dS 1 dt

(11)

X1 -(F/~ V) XI +(:'/~ V)[X2 - Xl], 1

flmaxS1 • X

1-

aK'&S 1 K'+S1

Y Ks+S1 x X+(F/~z V)(SR - S O - r f ( S 1--52)/~ V,

d P l - k l t ~ m a x S 1 X t F-k2XI--(F+r,)/o~V.P+ +(rt/c~V)P 2 . dt Ks+S 1

(12)

(13)

Modelling

and

Simulation

in B i o c h e m i c a l

Engineering

139

Similar expressions can be derived for the lower mixing region. These equations can be simply programmed (see Fig. 6), and the transient solutions obtained• The steady-state results have been discussed by Sinclair and Brown• At high dilution rates differences in the transient behaviour of the two regions become most apparent, this also being a function of the rate of interchange between the regions. Typical results are shown in Fig. 7. As can be seen, with an overflow device and

RUN

a=x~ B=8~

Q . ~ 0.05a3

C=P8

Z,~OO0 ~.OQ00

T 0•~000; . . . . . . . . .

; .........

::: .......

: .........

.

N

o

~.0000 ~.0~08

: .........

.

.

,~.

**

A*

: .........

: .........

: .........

: .........

: .........

: .........

: .........

i .........

i .........

i .........

i ...... :":

.

.

: .........

: ..... :

: .........

.

+*l

.

.

•

•

.

•

•

: .........

: .........

: .........

: .........

: .........

: .......

.

.

•

.

•

.

.

°

•

•

.

**

.

**g

C+~

•

,

•

•

• •

• •

........

8.*

, *GC

.

.

. .............

*IAA

AAAAA

.

.

. * ......

,

.

.

"

..........

, •

•

.

°

,

AA

, •

*~O 8B

, AAAA

A• AAA

°•,

•

•

•

•

•

•

8

e

9

CCCCC ~

8*

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5

B

•B

B*B

*C~CC

,

C

B

B

BB

B

. . . . . . . . . . . . . . . . . . . . . ~ . ÷. C. C . . . . . . . . . . . . . . . . .B .A * • + B A * •

•+*

.

;;~ ..........

a** *B*

.

:

.

.

B** • . . . . . . . . . . . . . . . . . B.B + B . . . . . . . . . . . . . . . . . . . . .

°

•

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C÷+, CC*C

•

•

.

: .........

: .........

**

*C•

• ..........

•

BBB*

.

BBOBO 8BB

.

.

. .

•

.

. .

•

. .

. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

*

A

A

A•A

•

•

•

CCCC

.

*

CCC CC B B . , • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. ... ... . . . C÷CC . . . . . . . . . C**

A

* A

A

.

A•A

A

•

,

•

~B B

~B.O000•.*~ • *

.

: .........

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*g.

C~

: o

•

*

. to,oooo

ZO•~O lo.o~oo

: .........

: ...................

. . . . ~....2"'":: . . . . . . . •

8.0oo0 8. o~0o

: .........

.

i .........

~.ooo~ . . . . . , . . . . . . . . . . . . . . . . . . • •

6, O00~ 6oO0~o

:.........

A

A A

•

, ....... .

A A

.

.

.

.

.

•

A

.

A

a

A A

. * . . . . . . . . . . . . . . . . . . . . . . . . . *.* ..: . . . . ÷ • o

.~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,*

.

"*

.

.

. .

.

zo.oooo

. .

°.

.....

, , , , , .

.....

~*. °

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

÷*

.

÷C

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8,

*,.

.

.

.

•

,

,

~ .

:

* ,.~

• • , . . . . . . . . . . . . . . . . . . . . . . .

*

. . . . . . .

•

.*

.

.

.

•

.

.

÷÷

.

.

.

÷

:;

:

:

:

:

:

:

i

:

•

•

1"

.

*.. * .

.÷ ........................................ .* . .

zs.oeoo•,

. . . . . . . .

. .

.

.°

.

.

.

.

.

.

.

°....••..•......o.......°•, .

.

.

.

.

.

.

.

Fig. 7b. Plot of growth parameters was almost entirely consumed, and

.

.

.

÷

,

.

.

.

from the lower mixing a higher dry weight was

..

............................. • •

.

.

.

.

.

.

.

region. The observed

.

.

.

•

.

.

.

. ÷.

substrate

substrate feed both in the upper region, sampling during a transient from the lower region would give a different kinetic pattern, especially for a non-growth-associated product.

.

•

140

H.W. BLANCHand I. J. DUNN

5. Transient Response of the Turbidostat Although not so widely used as the chemostatic type of operation of continuous culture, the turbidostat may offer advantages for the investigation of particular problems (Watson, 1972). The operation of the turbidostat is described by the same set of differential equations, but the flow rate of the incoming substrate is controlled by the concentration (more correctly, the turbidity) of the population in the vessel (see Fig. 8). This control is usually on-off or proportional in practice, but E SR

I TURBID0- l_

I METER

J-

X,S.P I

CONTROL CIRCUIT

Fig. 8. Diagram representing the operation of the turbidostat. The control circuit shows a P + I controller action

more sophisticated control would be simple to implement. In general the controller equation for turbidostat operation can be written in MIMIC language simply as: P+I

control

E PI F

X-XS K C , E + (1/T1) , (LIM (E, O.O,E), 0.0) F S W(E,O.O,O.O, P I)

Modelling and Simulation in Biochemical Engineering

141

It is necessary to limit the output F (i.e. the flow rate) when the error term E (the difference between the measured variable X and the set point X S) is negative. No flow should result, allowing the culture to grow batchwise.

***

C C

HIMIC ]0URGE-LANGUAGE PROGRA~ * ~ *

TRANSIENT R~SPONSE OF TUR~IOOSTAT T0 ST~P C~ANGES IN SUOSTRATE CONCENTRATION

C C

CON(VARINT} CON(V~A~O,Y} CON{XO,SO~PO) CON(X$~KS~I PAR{SRI~SR~KO~TSW~TI) C GROWTH EQUATIONS C gx OS OP HONO0

F~X/~ U~X -u,x/Y • F~(SR-S)IV A~U~X + B~X - FeP/V -

RELATIO~

C U

UM*S/(~S

+ S)

CONTROL ~dUATIONS

C PI

X - XS FSNIE,O,0~J.O~PI) KC~E ~ ( I N T ( L l f l ( E e O . O ~ E ) ~ O . O ) ) / I I

C

C C

INTROOUCESTEP CHANGE SR

FSW(T-ISW~SRI~SR2~SR2)

INTEGRATION AHO OUTPUT C X $I S P

It4T(OX~xo)

]NT(DS~SO) LIM(SIt3.O~SI)

~NI(OP,PO) FIN(T.50.O) PLO(TvXjSjP~F) EHO

VARINT 1.000¢0E+00 V 1,00000E+O%

A loSOOQOE-uX

9 I°SQO~OE-~I

XO 2*O0000E+OO

SO 8.00000E,00

PO %.OOOOOE-O1

KS 1,OOGOGE-Ol

UM 5.00G;OE-~Á

XS 9,OOOOOE*O0

Y

* ~ C O M P U T I N G TIME !

1 2 . 3 9 Z SEGONO$*~+

Fig. 9a. Program and constants for example of section 5

In order to illustrate the use of the turbidostat under transient conditions, the simple growth model described by Equations (1), (2) and (3) was coupled with various types of controller action. The system was allowed to reach steady-state operating conditions, corresponding to the desired value of the turbidity (XS). A load change was then introduced by a function switch (FSW) after an elapsed time; the change being a concentration difference in the inflowing medium. The response of the turbidostat was then followed till the new steady-state had been obtained.

142

H.W.

SR2 1o50~0E+01

S~i

I,O00OOE*01

KC

BLANCH

TSW ;•50000E*OI

Z,~OOOOfl~O

and

I . J+ D U N N

TT 0.00000~+00 RUP+

PLOT

A=X B=S C=P

O=r

O•D~OO

XoOO0O

2,00o~

+ 0.00~0;. . . . . . . . . ~. . . . . . . . . : ......... +*v

•

+

++

+

,

• A+~A

++

+

+ o

3

•

•

•

.

A~

o

[8

*C+

+ +,,, +

,

+ , + . . . . . . . . . . . . . . . . . . .

÷ o * . . . . . . . . . . . + +

• , . . . . . . . . . .

..... ++O

+

•

*++ • .......

+9 +

: .........

•

o

++

+ ......

•

+O

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

•

OOO0000°000

*

,

.OD o 0 O

., + C C + **•.°+C

+

,

. ....

,

. . . . . . . . . . . . . . . . . . . . . . . . . .

.+ .++ •

•

++ +

•

+ . . . . . . . .

•

+

, .

+ +

. ......

*°°*.+C

...... +

• +

++•+,

+c +C

•

• + • *

. . . . . . . . . . . .

Co+

*+

A+A

•

C+ +

° •

O+

o++ . o

.

,

, , ......

*,o

•

,

OOOO

, • . , o o , , , , , , , o o o , , , , , ° , ,

•

.

,

: .........

; .........

: ..........

.

OOO~O

++

+,*°,

......

,,°

....... ,

,

o~o . . . . . . . . .

A+

° .....

.

OO00O

A+

*

• * .....

,

.

o+**o

O++

. . . . . . . . .

H•--

+

~*Cc

°

+ .....

,

.

, .......

+

. +

C++ •++

°

........

.

.

0.ooo.o . . . . . c+c . . . . . . .

•

,,°°

. +.

°

.

' +~.

; .................

, ....

++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C+°

•+A

•

++

.,+B

+~,O0oo

c

+ .......

oo o°o:

•

B+ ~+•

•

,

H~

0

+ •

....

+

..... +. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : ........ ~. . . . . . . . . . . . . . . . . . . . : . . . . . . .

•

, ...

,

,

+

. . . . . . . . . . . . . . . . . . . . . . . . . .

,

,

•

C++ ,

,

, .......

•

,

. 0+

•

.

+

..oooo

.,.°,

+

* •

1. . . . . . . . .

• . , . . . . . . . . . . . . . . . . . .

...... •

C+C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *++

+ +

..........

o ,+,

+++

, . . . . . . . . .

,

•

o

,

++

,

+

, ,,.

+

,+

,

,

,

"

,

.

+ +

•

,

,

°

;Z

,

.......

. , . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+ +

•

, o,

,

+

+

. . . . . . . . . . . . . . . . . . .

,

,

+ + . . . . . . . . . +

++C

+••.

~+

:

,

++C ....... • ++C

+ . . . . . . . . . . . . . . . . . . .

iS,0000+

,

+

,

++ ,

÷ . . . . . . . . . . . . . . . . . . +

+

;

•

+

+ +

; .........

+

+

+

am

°

+ + ....... *

+

+ • . . . . . . . . . . . . . . . . . . .

•

A*

C+*

+

B

5,0QO~

.~; ......... 88

8 B ~ B

+ +

+C+ • .... ++C•.o C++

BOB w

B.8

•

+

•

g

4•A A A A ,

+

.

a

: .......

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

,

++~ +

•

A

B,•B..ABA~B~.+

~•o0o0

: .........

.

a

AA

. . . . . . . . . . .

+

10.++0++

~,oo00

: ......... . •

9CCC+C ,B

. + . . . . . . . . . . . . . .

....

: .........

, *

CC+O

+•

+3~

+

; ......... ,

AAAAA C++i

+ . . . . . . . . .

fi.o+oo+

**A

•

+A +

, .

.

+

.

.

+A . . . . . . . + +

, •

÷ +

°

+ ....... 1 ,

+ +

:

.

.

O++ ÷+ +* O+,

°

. ° , o , , , , . , , , , 0 + , . , , , ° . , . , • , • ....

+* ,+

•

,0+ , + • . . . . . . . . . .

,

.

: .........

+,,,°,,.,

°+

, .

,

O+ +

F i g . 9 b . Dry weight (X), substrate (S), product (P), and flow rate (F) in a turbidostat+ After ! 5 h o u r s a step change in SR w a s introduced, The overshoot in dry weight is clearly seen with proportional control

With only proportional control an offset was observed, this being reduced by selecting larger values of the controller gain (K C). Overshoot in product concentration occured (Fig. 9)• Such offset may be undesirable in some processes, and thus integral action was added to the control equation• With the use of computer-controlled fermentations, such control actions can be seen as very simple to implement• The response of this system to the imposed load change is shown in Fig. 10. The use of MIMIC to test the different types of control action for a particular process can thus been seen as advantageous, allowing decisions regarding suitability of controllers to be made on the basis of the results obtained.

Modelling and Simulation in Biochemical Engineering SRI

SR~

~C

143

T~

II

PLOT

A=X e=S

C=P O=F T

o . o11oo O.~QB~ ~.OQbC O.~u(~(~ .

,.,,0+.ccc .

.

.

.

3 • ~

Z•OOGO .

3•0~00

8.0ouO 8.0000 ~.odo0 k*QOOO *

..~

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

Ao

* +.,,,

,

GO

C

AAA .

•

e

O

B

O.

•:

a

C C C C

.

~

.

.

AA .

C . . . . . . . . . . . . . . . . . . B . , .. B .

*

~ • 9~e.o ,~• OttO0

~,uOoo

. . . . . . . . . . . . . . .

.CO •

•

.

~,t~ooo ~,,ot160

ccccc.

* +

~.O~Oo..+

z,o~Ou Z,O00O :1,, 0 Q;,'O 3..QQgO .

AA

C C C * *I~**B,

,at)

•

.

• A

A

*

.

.

A

A

.

A

A

C ,B B B .B*C,.C* .C,,

. B

d

B

OU

•

C

ElO

a

*,'~ A a A. C,,C*, .......

.

B~B"

• . . . . . . .

,

* ,

eso

C

C

.

. . . . . . . . .

rib

.

, *

• •

, w

• ........

•

.

,C

:

•

•

,

,

: : .........

•

: : .........

: : .........

°

,

0

•

:

:

.

.

.

:

•

,

.

.

: : .........

••

÷

i

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.

.

•"

•

". . . . . . . . .

+.° . . . . .• . . . . . . . . . . . . .

:

B;

.

+

o ....

.

A

B

B

~B

•

.

.

.

.

.

. .

a

. •

,

.

.

.

: .........

.

: .........

: .........

•

.

: .........

: ...... ;: :.

•

•,

•.

.

. , .......

.

** + t *÷ *

.,

A÷+

•

.

• . . . . . . . . . . . . . . . o. . . . . . . . . .

*,-.-**,,**

......

.

**+,,

.......

,

.

o+t~B,

• .......

.

B*

°

o

: .........

: .......

. . . . . . . . . . . . . . . . . . ° .. . . . . . .

°

~+: .........

+o *°,+

+

o÷

+C

,e

+* ~ .....

....

**

, •

• .,,

,

+,

,,,o,*

, ,

...... * .

i)+

, .

~++

*d,, +B +

*

. •

.....

4-, t + C~. . . . . . . .

,,,,~

*

.

. . BOB* . . . . . . . . . . . . . . . . . o . ,.,

.

• ....

+ ,~.

.

. . . . . . . . . . . . . ,., . . . . .

*÷*

* *'

*

, .........

•

::. :: :.::..:

÷ * +.

+

.

°

.

: .........

*f

,

.

.

,,°

•

.......

: .........

,

°°°;o.. . . . . .:

.

.

, ,

.

•

"5:

, ,C~-+,**,

:

.

.......

.

.

BBB+ ,

.......

,

:

.

.

.

. BOBB

°

,,

....... A

°°:oLoo

? °° OEIDD~

*, .A

•+÷

.

Bd~

.

. . . . . . . . . . . . . . . , .. . . . . . . . . . . . .

.

.

.

. . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . .

~O,O@OO,.,,.o . .

.

. •

BB

.

.

•

Z~,OOOO.,,,,°

.

B

,

,

• .

•

.

, . . . . . . . . . . . . . . .,o . ,

.

.

•

tt

,

+ . 6 . B, . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . .• .. ... . . . . . .

.

•

• ,,,,

.

- .

÷• *

1 5 , ~OIIO . . . . .

.

.

* .......

A.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. ~., ).o 0 . . . . . . . . . . 8~-

: ':.: ...... : .............

-

- . . . . . . . . .

.

Od •

, A.,A,*A.,.A..A

C.

1 o ,OOraO $0.0005 5,11o0~ 5.~5~0

¢-

*,,,,..,-*. ~ *

+, *+.

+

÷

* ,÷, * +

•

•

~-C

: .........

: ...................

,

•

, . . . . . . . . . . . . . ,. *

. .....

*

+

~

+,, +

•

:

+ * ....

•

~ ,

+

* *o*,

,

.....

,

o+, +•

, •

.....

, +0

,* ,*

° ,..,*

•

i-* '~

• .

....

++

,~' *,,,,,°+,,

• * .....

o •

+., +

Fig. 10. Analogous plot to that of Fig, 9 (b), with P + I control. The step change was introduced after 15 hours. No offset was observed

6. Enzymatic Reaction within a Porous Support This example concerns the "porous catalyst diffusion problem" which has been extensively investigated and summarized in the books by Satterfield and Sherwood (1963), and Peterson (1965). Goldman and coworkers (1971) have considered this problem for the specific case of enzyme kinetics. A catalyst, in this case an enzyme, is supported on a porous solid• In order for reaction to occur the reactant must diffuse through the porous lattice to the reaction site. The process of diffusion accompanied by reaction produces concentration gradients within the solid• As a result of the lower reactant concentrations at

o

144

H . W . BLANCH a n d I. J. D U N N

the interior reaction sites the overall rate is lower than one would calculate based on the exterior reactant concentration. Thus the "effectiveness" of the catalyst is measured by the ratio of the observed overall reaction rate to the reaction rate which would exist if the catalyst interior were at the exterior concentration conditions. Not only is the observed reaction rate changed, but also the sensitivity of the rates on concentration and temperature change is masked by the diffusion process. This fact is of obvious importance to the kineticist.

/

Support~

So~ob, P,o,,i .,,...~ t~ot~ Exterior Flu id SO, PO

So s_

in t2tsDii~fusion

X=O

°

~ "I PO

........

/

t Diffusion out of Solid

ire

~'

/

/

/

f

L~x

I

x~L

0

Fig. 11. Enzymereaction on porous support with substrate and product diffusion In order to predict these effects quantitatively it is necessary to integrate the mass balance within the catalyst using the appropriate boundary conditions. The mathematical model for the process is a steady-state mass balance which equates the supply of reactant which is provided by diffusion to the amount consumed by reaction. In expressing the reaction rate term, a quasi-homogenous form is used which neglects the fact that a surface reaction is involved. Thus for the geometry of a flat slab, as shown in Fig. 11. D

d2 S

sT

- r=O.

(14)

Modelling and Simulation in Biochemical Engineering

145

The boundary conditions are: S=So,

atx=L, atx=O,

dS/dx=O.

These correspond to a known external concentration and a symmetrical profile at the centre. The reaction rate r may be any appropriate rate expression. In this example, the Michaelis-Menten form with product inhibition is used as considered by Goldman et al., 1971, r=

(

kES p)

Km 1 + ~ -

(15)

+S

where k,E,K,, and KI are constants and P is the product concentration. Since the reaction rate is dependent on the product concentration, P, a relationship for the product concentration is needed. This is available from the fact that the supply of reactant by diffusion is equal to the loss of product by diffusion. For the stoichiometry S ~ P , this is expressed dS dP for any point by, Ds dx Dp dx which gives on integration, P = - S/D~ + constant or

If the concentration of product is very low at the exterior, that is P = O , at x = L P = ( S o - S ) / Df - . (16) Thus, from a knowledge of the exterior concentrations, the ratio of diffusivities for product and substrate, and the value of S at any point, the corresponding value of P can be obtained. Writing the model in terms of dimensionless variables that range between O and 1 allows extraction of the dimensionless parameters. Defining the new variables as follows: -~ = S/S o , P = P/So, :~ = x/L

146

H.W. BLANCHand I. J. DUNN

gives,

d2 "~

L2T

d-22

DsS o

=0

(17)

where, T=

_(

k E'S

Km 1.0+

Sop)+ S

So

KI ]

with boundary conditions, for S: at 2 = 1 ,

(18)

S=I,

~g at 2=0,

dx

0

for P: P = (1.0- S) / Ds

(19)

The effectiveness factor is defined as follows: EF=(rate of reaction within pellet)/(rate of reaction at outside conditions). In order to obtain the solution, which gives S and P as a function of x, two boundary conditions are needed, one for S and one for dS/dx. However S is known only at x = L and d S / d x only at x=0. The solution of such a "split boundary value" problem requires guessing a boundary value at either end, integrating to the other boundary and comparing the values obtained there with the calculated and the required boundary values. Thus in this example, we start from if=0, use d~/dxt ......o, guess S at ~7=0, and integrate to ~ = 1.0. The calculated values of S at .~= 1 is compared with the required condition of ~ - 1 . The new guess is provided by the "half-interval method" which starts with a given range of Sx=o values and narrows the range by dividing it in half after each iteration. The direction, whether higher or lower, which the next guess takes, depends on the value of Sx = 1.o. This simple convergence technique is useful for the many physical problems whose models are well-behaved enough to exhibit a definite trend when the initial condition is increased or decreased. The revised MIMIC program provides an automatic iteration according to the parameter RESET. When convergence is obtained RESET takes a value of zero, which effects one more iteration with a graphical plot.

Modelling and Simulation in Biochemical Engineering

147

The program as shown in Fig. t2 follows simply and directly from the mathematical model. Only one subtle change was incorporated to obtain satisfactory convergence; this was necessary to avoid negative values of P which occurred if S became greater than 1, and was handled easily by using absolute values of P in the rate expression. Understanding the logical functions which are used to program the half-interval method requires consultation of a programming manual, but one sees by the number of instructions that the program can be quite simply written.

**~

MIHZC SOOECE-LANGUAGE PROGRAM * * *

C

SUPPORIEO ENZTME KINETICS With POROUS ~IFFUSIOR CON(K,E,KM,KIePQt$O) CON{OT.ERRINI.DSTART) CON(ER~ICMAX.ICMIN) CON(DO,L) CON(OSOXO) PAR(ICSIRESET) C OINENSIONLESS NOIATION O$OX INT(R*L~LIOS~OSOXO) $ INT(OSOX.ICS) R ((K'EIS@)'S)I(IKMISO)'(1,0÷ISOIKI)'ABS(P))*S! P (L,O*O)/2.0 X T ENO FIN(X,1. 0 ] C EFFECTIVENESS FACTOR EF O$*SG.DSOX/(L~L*RO) RO [K=E~SO}I{KM*{I,0e(PO/KI))+SO| HALF ~NTERYAL ITeRATiON HEIHOO C CQRV FSWIABS(S-I.0)-ER.TRUE~TRUE.FALSE) STOP AND|END*CO,V} 5TOP RE$[T 0.O ICS (ICNAX~ICHINI/2.0 HIGH FSW(S-I.~FALSE.FALSE.IRUE~ MOOIFI AtaO(END.HZGH) MODIFt ICMAX ICS NOT(HIGH) LO~ HODIF2 AND(ENOtLOW} MOOIF2 ICHIN IC$ OUT[X~$~PtDSOXtOPOX) OUT(.EF.QS) PLO(X.S.P} scalo,o2,O,Ol.O,ol) Z~R(O,O,O.O,O.O) TTP{CONC PROFILES POROUS ENZYHEI TTY(SUOSIRATE AND PRODUCI CONC) TTW(OISTANCE) ~uO(X,OSOX~ ENO

R 8.7O~O0E~0~

£ 1.~000~E-03

KM 3*Z000OE-0Z

OI 1,0OOOOE-O2

ERRINT I*GOODEE-~6

OSTART I*90ODOE-0~

ER 1,0O0~0E-0Z

ZCMAX

bOJOooE*o0

0s 3.0QO~0E-06

L 1,0~00GE-0Z

gI

Ca

9o~000E-03

0,

S0 1.90000£-01

ICMIN G, *e**COMPUTZNG tiME !

8.8?3 SECONDS****

OSDX0 0.

Fig. 12. Program and constants for example of section 6

Concentration profiles for the substrate and product for this example are shown in Fig. 13. The constants used here were chosen from an example presented by Goldman et al., 1971.

H. W. BLANCHand I J DUNN

148

C~NC PROFILES POROUS ENZYME

~9N PCOT

SUBBTRA~ aMO PROOUCT GOnG

A=S ~=P

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,

Concentration profiles for example of section 6

Unsteady-State Formulation of Section

6

OcassionaUy a complex steady-state iteration problem, such as the one solved in section 6, will not converge to satisfy the split-boundary conditions. This can happen with a complex rate expression or when simultaneous balances are solved. In such cases the matter of convergence can be avoided completely by reformulating the problem in the unsteady-state form. The boundary conditions can then be satisfied during the approach to the steady-state by another means. The problem in this form becomes an initial-condition problem instead of a two-point boundary-value problem.

Modelling and Simulation in BiochemicalEngineering

149

Recast in dynamic form the model of section 6 becomes: aS (~2S~ 8---[= Ds [ 8 ~ - ] - r , 8 P Dv {¢~2p'~ 8t = \ 8 x 2 ] +r.

(20)

(21)

What was formerly an ordinary differential equation in one independent variable becomes now a partial differential equation with the additional time dimension. These equations need two conditions in terms of x and one in terms of t. These conditions are provided by the boundary conditions: 8S 8x

8P 8x

=

0

at

x=L,

P=Px and S=SI at x = 0 and the initial condition: S and P = 0 for all x when t = 0. Note that now, because of the transient nature of the problem, we cannot use the simple steady-state relation for P as a function of S. Instead the simultaneous unsteady-state mass balance for P must be solved. Solution of these partial differential equations by simulation programming requires that all but one independent variable be removed by finite-differencing. Replacing the continuous variable x by N increments, the model becomes a set of ordinary differential-difference equations of the form: dS, Ds dt - (DX) ~ ( S , + 1 - 2 S , + S , - 1 ) - r , ,

(22)

dP, Dp dt - (DX) T

(23)

(P.+l-2Pn+Pn-1)+rn

where D X is the increment length and n refers to the nth increment. The boundary conditions are satisfied by setting the exterior concentrations equal to the known constant values, St and PI. At the other boundary the zero concentration gradients are satisfied by setting SN+ 1= SN and PN + 1= PN.

150

H.W. BLANCH and I. J. DUNN

The equations are rewritten in dimensionless form by defining, S, P~ P.=~, DX

DX-

L

In this form they become,

dS. Ds (S.+1-2S.+S._1) 1 - £ i = Lr (D X) 2 + ~L,

(24)

dP, D. ( P . + , - 2 P . + P . _ 0 1 dt = ~(DX) 2 + ~T.

(25)

where

kES, $I\,

(26)

+

+S"

Finite Elements

13 ~ I s I~ I~ I~ L~ ISff

j S~ [ss

I%lS 7 Concentration Profi{e

U•s9

lS1o $11,,

I

I

0

L

=X

Fig. 14. Diagram of the finite differencing scheme in example of section 7

Sn, I -

s°.,

Sn,

Sn I

-is.

~

BaLance

=

[

So.,

iSn

Botance

'I

Fig. t5. Coupling among the finite elements in example of section 7

r

Modelling and Simulation in Biochemical Engineering ~+*

NI~IC SOURCE-LANGUAGEPROGRAN" + +

SUPPORTED ENEYME KINETICS RIr~ POROUS DIFFUSION TRANeIENI F~NITE DIFFERENCE FUR~ CON(K~E~KMeK~) CO~{SO,POIL.S¢CtPIC| GONISS~OP~DW| CON(~tP~| COM(OT+OSTART.ERRINT} CON|SIIeSIZtSIS+SI;~S[~SIE) CON(SIT,SIS,EIg,SIIO) GONIPI£tPIZ,PI3,PIW,PIS,PI@) CON(PIT,PEE,PIg~PII~) C INTEGRATIONS THROUGHTEN FINIT~ OIFFERENCES PBAL CSP(PG*PI,P~,RI) RSP(P~) PI INT(PSIePE~| SBAL CSP(SO,S£~SZIRI~ RSP|S01~ S~ INT(SD~SE~| RATE CSP(PI,S~) RSP(Rt) PeAL CSP(P~Pe~PS~RZ) RSP(POZ} P2 INT(ROZ,PIZ) SeAL CSP(SttSZ,S3*RZ) RSPtSO2) $2 INT(SOZtSIZ) RATE CSPIP2~S~} RSP(RZ) C PeAL CSP(PZtPStPk~R31 NSP(PO3) P3 ENT(PD3tPI3) SEAL CSP(SZ~S]tS~IR3| RSP(SDS| $3 INT(SOS+SIS) C RATE CSPIP3,S3) RSP(R3) PSAL CSP(P3IP~+PelR~I C RSP(PO~) P~ INT(PDk~PI~) SBAL CSP(S3~S~SetR~I RSP(SO~) Sq INT(SO~SI~) RATE CSP(PW~S~) RSP(R~I PeAL CSP(P~tPStPEtRS) RSP(POS) PS INT(POSIPIS) SeAL CSP(SNtSS~SEtRS| RSP(SOS) S5 INT(EOS,SI~) GSP(PS,SS} RATE RSP(Re) PBAL CSP|PE,PSePTtR6) RSP(POG) P5 I~TIPDStPIS) SBAL CSP(SS,SE.ST~R6] RSP($S6) SO INT(SO6+SIE) RATE CSP(PE~SE| RSP(RE} PBAL CSP(PB,P?~P8eRTI RSP(PS?) PT INT(POTIPIT) SeAL CSP(SStSZ,SO,RT~ RSP(SOZ) NH K E ~.20000E-02 8,?0O00E+O0 1,~0OfiGE+03 SO l,O00OOE+O0

O,

L 1,03000E-02

OS Z,30000E-06

OP ~,60000E'06

OX 1,00000E'01

SI 1,000~OE-01

O*

OT 1.OOO~Oe+O0

OSTART 1,OOOOOE-05

P0

SiC O,

SIT 0o

KI ~.~OOOOE-O3 SIC I,O0000E-Ot

SI3 ~* SI9

t6,3S3 SECONU$~

O*

516 O.

.

PIW 0,

PI9 0,

0 SIC

SItO Oo

PI3

PIG 0.

SIk

O.

O,

0.

PIC O*

ERRINT

PIe

PIT 0.

INT(SSItSII) CSP(P~,S7) RSPCRT) CSPtP~tPSePg,RB) RSP(PSS) P6 INT(POStPIe} SeAL CSP(SZ~Se,S~RS) REP(S08) BE INTiS08+SIS) RATE CSP(Pe~se) RSP(Re) PeAL CSP(PS~Pg~PIO~Rg) RSP(P09) P9 INTiPSg~PIO) SeAL CSPISB~Sg,StO~Rg) RSP(S09} S9 INT(SOg+SIg) RATE CSPIPStSg) RSP(Rg) PSAL CSPIPg+P~O+PIItRLO) RSP|PSIO] PtO INT(POIGtPIlO) SeAL CSP(S~,SIO,SlI,RIO) RSP(SOiS| StO INT(S010~SII0) RATE CSP(P1A+SIO) RSP(RIO| BOUNOARYCONOITION~ $11 510 P11 P1O EFFECTIVENEgS FACTOR O A S E O ON GRADIENT AT SURFACE EF (DS/(K~L+L|)~(SO-S1}/(OX~RO) ~ASEOON REACTION IN EACH ELEMENT EFE OX=SI=(RI*R2÷R3÷R~*Re~RE+RT~Re+Rg*R1QI/RG RO (K~E~SI)/{KH~(I,O÷(PI/KI))~SI) SUBPROGRAHS RATE 8SP|P~S) R (K~E~S/SI)/((KH/$I)~(~*O~SI~P/KI|~S) RATE ESP(R) PeAL ~SP(PA~PBtPCtRB) POER OP~(PC-2.0~PB+PA)/((OX~OX)~(L~L)) ~RB ReAL ESP(POER) SEAL 8SP(SAISS,SC~B) SSER DS~(SC-2,0~ES+SA)/(iDX " S X ) ~ { L ~ L ) ) ' R B SeAL ESPKSSER} FIR(T~Ze.O) OUT(T~SttSS,S%O) OUT(~PItPB~PI0) OUT[~EF) OUT(*RljRS~R%0) OUT{ tEFF) PLO(TtSltS~S~O) SOA(2*Q~O,OI~O.OItA,Ot) ZER(0.0,&,O~0,Sv0.0) TTP(TRANSIENT SUSSTRATE PROFILES) TTX(TIHE) TTY(DINEHSIORLESS COHC) PLO(T~PI~PS~PIOI ENO PBAL

• ~COMPUTING l i N E :

S~8 0.

PI1 0.

S? RATE

PI

511 S.

151

PIG 0o

PI10 0°

Fig. 16. Program and constants for example of section 7

PIS O.

152

BLANCH a n d

H.W.

I. J.

DUNN

The finite-differencing scheme with the boundary conditions is shown in Fig. 14. In Fig. 15 an information-flow diagram illustrates the simultaneous solution of the set of N difference equations. It is apparent that the accuracy of solution depends on the number of finite increments used. Also apparent is that considerably more computational effort is required for the transient problem because of the large number of coupled equations. This approach in reformulating the problem would only be taken if convergence could not be obtained for the steady-state balances or if dynamic information were required. Fortunately the programming of this problem with a simulation language does not become too difficult. In Fig. 16 the program is shown. Notice that for each of the ten increments the mass balances for ;~ and P T~A~SZENI SJBSTRATE PROFILES OZHENSIONEES$ COHC

RJN

1

R~0T

I

A=SI 0,00oo 0,0000 .

e=s5

C=S10 T

.2o00 ,ZOOO

o.oo,o:.;. . . . .

.

.

8

.~000 .

: ......... 6

,6o£0

-~000

.

~.: .........

• CC •

.

: .........

.

.

.

: ........

.~

~ B

~

•

.

,

CC

.

•

•

CO.

................

•

,

•

,

CC

...................... •

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.~

....

,

.

.

.

.

*

,

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CC

+.

. . . . . . . .

, ,

*

.

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............

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. ....

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. .........

:

. . . . . . . . . . . . . . . . . . . . . .

.

~.~oo

.

:

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+

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.

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i .........

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: ...................

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:c

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.

°"~

. ..... ..............

;; .......................

÷

:

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................. +

6

,

,*

:

+

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.

*

; .............

...............................

•

o,

+

•

. . . . . . . . . . . . . . . . . . . . . . . . .

*

•

...........

+ AA

, •

.,..

°

~o.~o.

• +

*o,~,.*.°

•

+ ........

*o ............

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

::......... ::........ ~:!! ........ ::......... ::

. .

•

86

• *

*

* ...........................

BS ,

1.0000 1.0000

~A

BB.

...............

*,*.-,,

,

. .

Be ............

CC

•

.................

•

B8

°,CC

CC ~0.00Q0

6B

,

Cc, . . . . . . . . . . . . . . . • CC . CC

~

,

,

.?'"'::°i °°......... oo ::......."°~°: ............................. °. • : ZO*O000o,,

.

.

,

cc

*~0~0 .SDO0 .

,~000

.

: .........

, . ......

..,

•

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.........

o,,.,

•

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? .........

) .........

•

: .............................. •

? .........

? .........

i .........

:

Fig. 17. Plot showing development of concentration with time at three fixed points in porous support

Modelling and Simulation in Biochemical Engineering

153

are called from subprOgrams using CSP and RSP statements. Also included as a separate subprogram is the reaction-rate term. The solution gives the transient changes in concentrations for each length increment. Thus, the development of the concentration profiles from given initial conditions can be determined as well as the final steady state. Comparison of the profiles of Fig. 17 with those of Fig. 13, in section 6, reveals that close but not exact agreement is obtained. For an accurate estimate of the effectiveness factor finer-mesh increments are needed. Instead of using the same increment size throughout the porous slab, a more effective approach would be to use smaller increments where the gradients are steep and larger ones where the profiles flatten out.

8. T u b u l a r I m m o b i l e - E n z y m e R e a c t o r The possibility of immobilizing enzymes on solid supports leads to the consideration of their use as catalysts in continuous-flow reactors. Much of the well-developed technology for packed-bed catalytic reactors can be used in the design of enzyme-catalyzed reactors. The phenomena of porous-catalyst diffusion, transport to the catalyst surface, and fluid mixing within the reactor are as common to conventional catalytic reactors as they are to new developments in enzyme reactors. To illustrate the use of digital simulation programming in this area, the problem which was solved by Kobayashi and Moo-Young (1971) is considered. The problem consists of predicting the conversion in a catalytic packed-bed tubular-flow reactor in which the mass-transport rates to the catalyst surface are important. It is well-known that the axial mixing effects which are found in packed-bed reactors can be described with a dispersion model. Discussion of this approach can be found in the texts by Levenspiet (1962) and Aris (t 969). The one-dimensional dispersion model has the form of an axial-diffusion term which is superimposed on a convective plug flow. Written as a steady mass balance for the substrate reactant the equation is:

Do \ dZ2/i -v dZ-

-r=0.

(27)

For a reactor which does not allow diffusion past the entrance and exit the following boundary conditions are used:

vSR=vS--DJ~Sz at Z=O, dS dZ - 0 at Z=LR

154

H . W . BLANCH and I. J. DUNN Tubular Reactor So

1 I L

m I.........

0

---"-Z

Convect ion Convection and Diffusion

SO

Zero Gradient no Diffusion

0

"X Concentration Profile

I

L

Fig. 18. Concentration profiles in a tubular reactor with dispersion

where SR is the entering substrate reactant concentration. These boundary conditions are illustrated in Fig. 18. Again as in section 6 we have a split-boundary-value problem. The reaction rate term, r, in the above equation is actually a mass-transfer rate to the surface of the solid support. The reaction proceeds on the surface at a rate which is governed by the concentration drop f'f~'X'\Diffusion

Film

/ Substrate Concentration

I,

---11

Exterior

I

t

1

t

Liquid

SO

I I J

HS 1

Concentration Gradients Distance -.._ Fig, 19. Concentration gradients for the case of external-transfer control

Modelling and Simulation in BiochemicalEngineering

155

across the diffusion film. For the surface rate process, Michaelis-Menten kinetics are assumed, and adsorption equilibrium is assumed to exist between the surface film and the immobilized enzyme. Porous-diffusion effects are neglected; this is valid for a small particle size catalyst with large pores. Fig. 19 illustrates this process. In formulating a model for the influence of mass transfer, steady-state is assumed to exist between the mass-transfer rate and the reaction rate. Equating these provides a relation for the film concentration, S*. Thus, k3EHS*(1-e) kLa(S-S*) = K,,+HS* (28) where the quantity H S* is the concentration of the adsorbed substrate. Conveniently this relation is a quadratic equation in S*, which can be solved analytically. This will not be the case for more complex kinetics, and in general S* will have to be obtained by iteration. Defining the following dimensionless variables: ~= S/SR, 2* = Z/LR,

'd* = S* /SR

we write the dispersion model in dimensionless form: d2S

dS

Pe d22

1

dZ

W(S-S*)=O,

Pe = Peclet Number -

(29)

LRV Da' kLa LR

W = mass group transfer -

gU

In these terms the boundary conditions become, --1.0 P~ d Z

d~

--0.0

dZ

at

at

Z=O,

2=1.0.

Solving for S* in dimensionless form from equation 28 we have

S-S* =

fl+ S*

(30)

156

H, W. BLANCH and I. J. DUNN

where:

c('- kaE(1-~:) kcaSR

'

Km

[~ -

HSR

which gives the quadratic,

~*~+ (/Y+~'- ~)~*-/~=0. Solving, ~*--

02)

2

The problem has now been reduced to only four dimensionless parameters. The solution by MIMIC programming for particular values of the parameters is shown in Figs. 20 and 21. As in section 6 the half-interval method is used to converge on the split-boundary values. The integration ~ C C

MIMIC SOURCE-LANGUAGEPROGRAM ~ ' *

TUBULARSUPPORTED ENZYME REACTOR DISPERSION MODEL CON(P.A,O.WI CON(ICMAX, ICMIN) CCN(ER,OT,STOP) PAR(IS,IOS,RESET} US INT(P-(OS*W*{S-SE)}.IOS) S INTID$,IS) SE (S-B-A*SQRTllB+A-S)'t~+A'S)+~.~ "0~S))12'~ Z T END FINIZ, L.L) END ICON CSP(O3,1CMAX,ICHIN~IS) END RSPIlS,IOS,STOP,IC~AX,ICHIN} STOP R~SET J.~ ICON 3SP(AOS,AICMAX~AIGMIN,AIS) ASTOP FSW(A~3IAOSI-ER,TKUE.IRU£,FALSE} HIGH FSH(AOS,TRUE,FALS~,FALSE) tOW NOT{HIGH) HIGH AICHIN AIS LOW AICHAX AIS AIS (AICMAX+AECMIN)/~.~ AIOS

P~IAIS-~,E)

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~SP(AIS,AIOS,ASTOP,AICMAX~AICMIN} OUT(Z.S.US,IS.IOS~STOP) PLO(Z,3)

SOA(~.0Z,0.011 TIP(REACTOR GONG PROFILEI TTX(DISIANOE) ITY(SUaSTRATE GONG) END

p

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Fig. 20. Program and constants for example of section 8

I Z . Z / B SEOON O s ' ' ~

Modelling

and

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in Biochemical

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is started from the exit of the reactor with an estimate of S[~=o0. The derivative at this point is calculated from the entrance boundary condition. The convergence is checked against the zero exit derivative condition. The solution exhibits extreme sensitivity to the initial conditions, and as a consequence, about 30 iterations were required to obtain convergence with a 1% error.

9. Control of Activated-Sludge Reactors Wastewater treatment facilities operate under unusually difficult conditions with regard to the large fluctuations of concentration and flow which occur in the feed streams. Any analysis of their operation must

,

158

I7t. W . B L A N C H a n d t. ,t. D U N N

recognize the dynamic nature of these processes if a specified effluent quality is to be maintained. In this example we are concerned with a well-mixed activated-sludge tank reactor which is controlled with a sludge recycle stream. Continuous measurement of the exit organic substrate concentration provides an error signal to a proportional controller which regulates the recycle flow rate. The system is shown in Fig. 22. Feed

Effluent

$1, X1

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l t I [ I {

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t

I

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Fig. 22. Activated-sludgesystem with controlled recycle The physical arrangement of an aerated reactor, followed by a clarifier with recycle of some of the organisms is common to all activated-sludge treatment processes. The usual aim of the biological process is the removal of organic carbon from the incoming wastewater. This is accomplished through the assimilation of organic material by a suitable aerobic microbial population. Sufficient quantities of air and other nutrients provide a growth environment which leads to rapid carbon consumption and large increases in microbial mass. Some of the active biological material (activated sludge) must be recycled to provide the adequate populations that are necessary whenever incoming flow rates and carbon concentrations increase. Most sewage treatment facilities rely on experienced men, who operate the process, to make manual changes in the recycle flow rate based on a desired carbon concentration in the effluent stream. Relatively little progress has been made in developing rate equations for activated sludge systems. Steady-state systems are treated with some degree of confidence with the Monod equation which has also been

Modelling and Simulation in Biochemical Engineering

159

used by Andrews (1969) to describe rapid transient conditions. To describe the dynamics of pure chemostatic cultures, Young, Bruley and Bungay (1970) have considered refinements which involved a delay in the Monod growth constant. Gaudy and Gaudy (1972) discussed in some detail the special considerations which have to be made when an activated-sludge system is operating under transient conditions. The dynamic-lag approach of Young et al. is taken here for the activatedsludge kinetics. The system as shown in Fig. 22 can be described by the following simultaneous differential equations: A transient organism balance on the reactor vessel, dX1 d - (RXR-(Fo+R)XD/V+Px.

(33)

A transient substrate balance on the reactor vessel, d S, Fo d~- -- V (SO

Px

--S1)--Y'

(34)

P 1Grow'hi. Rate ~i __ FO

Fluctuation#-n Feed ~ S

k_____I G,ow,h I

Balance °e. Ix'l R

XR

IC°n'tantl

IXI

G,ow,h J ~0 -~ Constant Log

S1

.,._JClarifie r I

XRI DynamicsI _ k ] Controt [ ~ - ' ] l 7 Equation

L ~ Substrote

---~, Ba once SI ~P(from growth rate brock) Fig. 23. Information flow diagram for activated-sludgesystem model

160

H. W, BLANCHand I. J. DUNN

Assumed here is that the substrate concentration in the recycle streams is the same as that which leaves the reactor vessel. The M o n o d rate equation, including a death term, and a first order lag in growth constant, P x = [dl X1 KaXa, (35) -

-

#,,S~

(36)

#o = K s + S~ ' dlh _

(#o-/~)

dt

(37)

To

a clarifier dynamic relation including a concentrating factor, dXR dt •~

CX~-XR Tc

-

(38)

HIMZG SOURCE-LANG~ADZ PROGRAM * * *

CGN(XA~,S~,FO,$Q,V) CCN{UM,~S,Y,KD,[D)

C C

C O G

C

C

CON(RQ~S~eC,TC) CDN(CIjERRINTj3STARI) PAR(KC) A~TIVATEDSLUDGE REACTOR WIT4 CONTROLLED RECYCLE CELL BALANCE DXZ (R~XR-(F~R)eXil/VfP XI INT(OX~XiQ) SUOSIRATE~ALANCE (FOIV)~{SO-S~}-P/¥ OSl SI IHTIDSL~S%~) GLARIFiER OY~AHIC~ XR [HYI(C'XL-XR)/IC~D*XI~) G~OXTHRATE RITH T I ~ DE~AY P U%~X~*KD*X~ U@ U~(S~LI(KS*SiL I ! SIL LIMISl~.O~3Ofi.g) CONTROL EQUATIONS g S~L-SO rG RO*KC'~ R L~M(RC~b.Q~OB*O} LOAD FLUCTU~IIONS FSIN 50QQ.+~QO~.~SIN(2.0~3'AT FO LIMIFS~N~SQ~O.Q~£OOOO.Q) 50

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V 2..O00QOE*Ok TG

Modelling and Simulation in Biochemical Engineering

161

a proportional control equation for the recycle flow rate,

R= Ro+ Kc(S~-Sa).

(39)

An information-flow diagram for this mathematical model is shown in Fig. 23.

~=S1L

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............

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. A

. ....................... ~ ~ A :,

.

..................................

: ......................................

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•

An+~

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................

.: .........

: .........

Fig. 25. Plot of effluent carbon concentration (mg/1) versus time (hrs) for the case of recycle without control

The computer program for this system of differential equations is shown in Fig. 24. Part of the program generates 24-hour fluctuation cycles for the incoming substrate concentration and flow rate. These take the form of limited sine waves. Figs. 25 and 26 show the effect of the recycle control on the organism substrate concentration. Proper choice of the controller constant significantly reduces the substrate fluctuations in the effluent stream.

H. W. BLANCHand 1. J. DUNN

162

PLOt

l~5~L

O. JO~O ,

T

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E

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....

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*

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a~g

,...,...,.,.,

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;O,oO0~

.

: .........

30,~OOQ~+~

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~

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a

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: .........

: .........

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: .........

: .........

: ........

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. . . . . . . . . . . . . . . . . . . . . .

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.........

50*~O0**A,AA*A,*A* •

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............

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.......

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.

.

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.........

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.........

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.........

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.

.

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Fig. 26. Plot of effluent carbon concentration (rag/l) versus time (hrs) for the case of recycle with proportional control

lO. C o m m e n t s on the Future Mathematical modelling techniques are taught as part of the normal course of study to most engineering students throughout the world. From this teaching effort have come a number of excellent books which make it possible for practicing engineers and scientists to acquire a background in the subject without formal training. Computer programming is a compulsory subject in all modern engineering programs and is increasingly common to many other scientific educational programs. We can expect that the young biochemical engineers of the future will not hesitate to call on the computer for solutions to their problems.

Modelling and Simulation in Biochemical Engineering

163

What about those who completed their education before 1960? H o w can they best begin to acquaint themselves with the computer so that they are able to express their physical problems in computer language? This paper attempts to answer this question and is intended as an introductory point from which the non-mathematical and non-computeroriented biochemical engineer can begin to consider the use of mathematical models. It is demonstrated here that simulation languages have eliminated the tedium of computer programming. We can therefore expect to see a general increase in the level of mathematical-modelling competence a m o n g practicing biochemical engineers. This development will hopefully lead to a softening of the rather sharp lines which sometimes exist between the experimentalists and the theoreticians and will likely result in greater cooperation a m o n g the people of unusually diverse backgrounds who m a k e up the interdisciplinary field of biochemical engineering.

Nomenclature a C D D. Dp Ds DX

DX E F Fo H k kl k2 k3 kLa

K' Kc

K,. Ks

L LR

P P Pe P~ P.

P.

constant in maintenance model, dimensionless, clarifier concentration factor, dimensionless, dilution rate, "F i effective axial diffusion coefficient. L z T- i, diffusion coefficient for product, L27 ~ ~, diffusion coefficient for substrate, L2"F 1, distance finite increment, L, dimensionless distance finite increment, enzyme concentration, M L 3, flow rate of medium, L s T~ 1 inlet feed flow rate, L 3 7~ i partition coefficient, dimensionless, reaction rate contant, 7~ 1, kinetic constant, T- l, kinetic constant, M L 3 T- 1, kinetic constant, T- 1 mass transfer coefficient based on total volume of reactor, 7"- 1, saturation constant in maintenance model, M L-3, proportional controller constant, L6 M - 1 7~ 1 Michaelis-Menten constant, M L- 3, saturation constant, M L - s , distance from slab center to surface, L, reactor length, L, product concentration, M L- 3, dimensionless product concentration, P/So, dimensionless Peclet Number, Lv/D., external product concentration in porous support, M L-3, product concentration in nth element, M L -3, dimensionless product concentration, P./SI,

164 Po r r~ R F r. F,

H.W. BLANCH and I. J. DUNN product concentration in exterior liquid. M L-3, reaction rate, M L 3 T- i, rate of interchange between volumes in mixing model, T 1 recycle stream flow rate, L 3 T ~, reaction rate in terms of dimensionless concentrations, M L - 3 7"- ~, reaction rate in nth element, M L - 3 T - 1 reaction rate in nth element in terms of dimensionless concentrations,

ML 3T1 Ro S S* S*

Sd Sx $1 S,, ~. So

SR t

7~, 7}~ V v vr W x X 2 Xt

XR Y Z :~ ~' fl e /~ #~ ~1 #,, ~Lo

steady-state recycle flow, L ~ T~ 1, reactant substrate concentration, M L-3, reactant substrate concentration at catalyst surface, M L - 3 dimensionless substrate concentration, S/S~, dimensionless substrate film concentration. S*/SR. desired value of effluent concentration. M L ~, external substrate concentration in porous support, M L - 3 substrate concentration in reactor. M L 3 substrate concentration in nth element, M L-3, dimensionless substrate concentration. S~/S~, substrate concentration in exterior liquid, ML 3, substrate concentration in inflowing medium. M L 3, time, T, time constant in clarifier dynamic equation, T, time constant in growth constant lag equation, T,, reactor volume, L 3, linear flow velocity within the voids, L/Z velocity flow through plug flow reactor, L/T, dimensionless parameter, kLaL/ve, distance variable, L, celt concentration, M L " 3 dimensionless variable, x/L, cell mass concentration in reactor, ML 3 cell mass concentration in recycle stream, M L -3, cell-yield factor, dimensionless, axial distance from inlet, L, fraction of effective volume above the impeller, dimensionless, dimensionless parameter, K3Etl-e)/kLaSR, dimensionless parameter, K,./HSR, void fraction, dimensionless, specific growth rate, 7"- 1 constant in maintenance model, 7~ 1 delayed Monod growth constant, 7"-t maximal specific growth rate, 7"- ~, instantaneous Monod growth constant, T-1.

Acknowledgement: This work has been partly (H.W.B.) supported by the Kommission zur F6rderung der wissenschaftlichen Forschung, Project No. 91.

Modelling and Simulation in Biochemical Engineering

165

References Andrews, J. F.: J. Sanitary Engineering Division, ASCE 95, No. SA I, p. 95 (1969). Aris, R.: Elementary Chemical Reaction Analysis. Prentice-Hall 1969. Blanch, H. W., Rogers, P. L.: Biotechnol. Bioeng. 14, 151 (1972). Calam, C. T., Ellis, S. H., McCann, M. J.: J. Appl. Chem. Biotechnol. 21, 181 (1971). Chu, Y.: Digital Simulation of Continuous Systems. McGraw-Hill 1969. Constantinides, A., Spencer, J. L., Gaden, E. L.: Biotechnol. Bioeng. 12, 803 (1970). Fan, L. T., Erickson, L. E., Shah, P. S., Tsai, B. I.: Biotechnol. Bioeng. 12, 1019 (1970). Franks, R. E.: Mathematic Modeling in Chemical Engineering. Wiley 1967. Fredrickson, A. G., Megee, R. D., Tsuchiya, H. M.: Advan. Appl. Microbiol. 13, 419 (1971). Gaudy, A. F., Gaudy, E. T.: In: Adv. Biochem. Eng. 2. Ghose, T. K, Fiechter, A., Blakebrough, N. (Eds.), Springer (1972). Goldman, R., Gotdstein, L., Katchalski, E. Ch. I.: In: Biochemical Aspects of Reactions on Solid Supports. G. P. Stark (Ed.), New York: Academic Press 1971. Hockenhull, D. J. D., MacKenzie, R. M.: Chem. Ind. 607 (1968). Humphrey, A. E.: 9th Int. Congr. Microbiol., p. 183 (1966). Kobayashi, T., Moo-Young, M.: Biotechnol. Bioeng. 13, 893 (1971). Koga, S., Berg, C. R., Humphrey, A. E.: Appl. Microbiol. 15, 683 (1967). Levenspiel, O.: Chemical Reaction Engineering. New York: Wiley and Sons 1962. Luedeking, R., Piret, E. G.: Biotechnol. Bioeng. 1,343 (1959). Maxon, W. D., Chem, J. W.: J. Ferment. Technol. 44, 255 (1966). Peterson, E. E.: Chemical Reaction Analysis, Prentice-Hall 1965. Powell, E. O., Lowe, J. R.: In: Continuous Cultivation of Microorganisms. Malek, I., Beran, K., Hospodka, J. (Eds.), New York: Academic Press 1964. Ramkrishna, D., Fredrickson, A. G., Tsuchiya, H. M.: J. Gen. Appl. Microbiol. 12, 311 (1966). Ramkrishna, D., Fredrickson, A. G., Tsuchiya, H. M.: Biotechnot. Bioeng. 9, 129 (1967). Satterfietd, C. N., Sherwood, T. K.: The Role of Diffusion in Catalysis. Addison Wesley 1963. Sinclair, C. G., Brown, D. E.: Biotechnol. Bioeng. 12, 1001 (1970). Watson, T.: J. Appl. Chem. Biotechnol. 22, (2), 229 (1972). Young, T. B., Bruley, D. F., Bungay, H. R.: Biotechnol. Bioeng. 12, 747 (1970). Dr. HARVEY BLANCH Mikrobiologisches Institut Eidg. Technische Hochschule CH-8006 Ziirich, Weinbergstr. 38

Dr. I. J. DUNN Technisches Chemisches Labor Eidg. Technische Hochschule CH-8006 Ztirich, Universit~itsstr. 6

CHAPTER

5

Transient and Oscillatory States of Continuous Culture D . E. F. HARRISON a n d H . H. TOPIWALA With 24 Figures

Contents 1.

Introduction

. . . . . . . . . . . . . . . . . . . . . . . .

2. 2.1. 2.2. 2.3. 2.4.

Predictions from ldealised Models . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . Models . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple Steady States and Their Stability . . . . . . . . . . . Transfer-Function Analysis of Stability and Transient-Response Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Theoretical Transient Response and Approach to Steady State . . . 2.5.1. M o n o d Model . . . . . . . . . . . . . . . . . . . . . . . 2.5.2. Substrate-Inhibition Model . . . . . . . . . . . . . . . . . . 2.5.3. Phase-Plane Analysis . . . . . . . . . . . . . . . . . . . . . 3. 3.1. 3.2. 3.3. 3.4. 3.5.

4. 4.1. 4.1.1. 4.1.2. 4.1.3. 4.1.4. 4.1.5. 4.2. 4.3.

Transient Responses of Microbial Steady State . . . . . . . . . Dilution Rate . . . . . . . . . Feed Substrate Concentration . . Temperature . . . . . . . . . Dissolved-Oxygen Tension . . . General Discussion . . . . . .

Cultures to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Perturbations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

of . . . . . .

the . . . . . .

Oscillatory Phenomena in Continuous Cultures of Microorganisms Oscillations Derived from Equipment Artifacts . . . . . . . . . Temperature . . . . . . . . . . . . . . . . . . . . . . . . pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stirring-Rate and Oxygen Transfer Coefficient ( K L a ) . . . . . . . Foaming and Antifoam . . . . . . . . . . . . . . . . . . . . Discontinuous Feed . . . . . . . . . . . . . . . . . . . . . Oscillations Derived from Feedback Between Cells and Environmental Parameters . . . . . . . . . . . . . . . . . . . . . . . . . Oscillations Derived from Intracellular Feedback Regulation . . .

168 169 169 169 171 177 178 178 179 182

182 182 185 187 189 t92 195 196 196 197 t98 198 200 203 206

168 4.4.

D.E.F.

HARRISON a n d H. H. TOPIWALA

4.5.

O s c i l l a t i o n s D e r i v e d f r o m I n t e r a c t i o n s Between D i f f e r e n t Species in Continuous Culture . . . . . . . . . . . . . . . . . . . . . 211 S y n c h r o n o u s D i v i s i o n o f Cells . . . . . . . . . . . . . . . . . 215

5.

Conclusion

Nomenclature References

. . . . . . . . . . . . . . . . . . . . . . . . .

216

. . . . . . . . . . . . . . . . . . . . . . . . . .

217

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

217

1. I n t r o d u c t i o n In the early years following Monod's pioneering work (Monod, 1950) continuous culture was employed primarily as an experimental tool to obtain steady states of microbial growth. It is now fully established not only as an experimental technique but also as an industrial unit process. Recently, interest in the unsteady-state or dynamic behaviour of continuous culture has grown. A knowledge of system dynamics is required in order to design effective control systems. Control strategy is necessary for the attainment and maintenance of steady-state conditions as industrial processes are never free from fluctuations in input variables. To rationalise the study of dynamic responses, mathematical models must be constructed and analysed. This approach develops a theoretical framework necessary for an understanding of the underlying mechanisms. The first part of this article deals with the construction of dynamic models of continuous culture systems and their analysis, and illustrates relative importance of the hydrodynamic and biological lags. The second part examines the dynamic responses of some actual biological systems. This account is not intended to be an exhaustive review of all reported transient phenomena; since this would serve little purpose. Rather, it describes examples which typify the structured complexities of the dynamic biological system. Oscillatory phenomena in continuous culture can be particularly revealing of the nature of the regulatory mechanisms intrinsic in a biological system. They are an important consideration both from an experimental standpoint and for the control engineer. Some of the oscillatory phenomena reported in continuous culture systems are discussed together with the possible underlying mechanisms.

Transient and Oscillatory States of Continuous Culture

169

2. P r e d i c t i o n s f r o m Ideatised M o d e l s 2.1. Introduction Attention is now directed to the dynamic behaviour of continuous cultures from the theoretical point of view. The type of analytical approach employed comprises a study of the attainable steady states under specific operating conditions, determination of whether a steady state is truly steady or is characterised by stable oscillations, and of how systems respond to disturbances. The techniques generally used to predict the transient behaviour and stability of continuous cultures have been extensively used in chemical-reactor studies (Bilous and Amundson, 1955; Aris, 1969; Perlmutter, 1972; Friedly, 1972). Some of the techniques will be discussed briefly here to indicate their application to a simple continuous culture represented as a lumped-parameter system, though no attempt will be made to be mathematically rigorous. Techniques are also available to handle more complex models such as those represented by partial differential equations (Hlav~i~ek et al., 1972). The continuous-culture system considered in this article will consist exclusively of a single-stage, well-stirred, continuous-flow system (CSTR) without recycle or feedback. It should be realised that very often continuous cultures are operated with some type of superimposed control e.g. control of turbidity or substrate level by manipulation of dilutionrate. This will introduce additional lags (Sinclair et al., 1971) which may impose instabilities not present in the open-loop response, though in general the aim of control strategy is to have more stable operation. Edwards et al. (1972) have shown that feedback control of substrate concentration improves the stability of continuous cultures growing on inhibitory substrates. 2.2. Models The starting-point of any theoretical study of continuous-culture stability is a realistic dynamic model of the system. Microbial systems are by nature complex and varied. There is no reason to suppose that a single model will be adequate for describing all continuous-culture systems. Since different models will not have the same stability criteria, one should be cautious in drawing conclusions about their dynamic behaviour. Most theoretical dynamic studies of continuous cultures have been based on models which have been formulated from steady-state or equilibrium kinetics and have not been very successful in describing dynamic behaviour. Thus the relevance of the study of dynamic behaviour

170

D.E.F. HARRISONand H. H. TOP1WALA

and optimal control of continuous culture without experimental verification of the models must be seriously questioned. The mathematical models used to study the dynamics of the system in this section will be 'unsegregated' (Tsuchiya and Fredrickson, 1966). These models assume that growth dynamics can be described in terms of generalised variables representing average properties. Growth is treated as a deterministic process rather than a stochastic process and variation in properties of individual cells is neglected. This is a valid approach provided the number of individuals in the population under consideration is sufficiently large (Kozheshnik, 1971; Megee, 1971) so that random deviations from the mean average out. Thus interactions in microbial systems can be described in a similar manner to those found in homogenous chemical reactions. A microbiological system can be defined in terms of a vector state-space whose elements (x3 represent various component concentrations, physical parameters, temperature, pressure, stoichiometric constants, etc. The biological rate expression for any material component (yj) can be expressed as a continuous function: R ~ = f ( y l . . . . yk . . . . T, pressure, mass-transfer terms, pH, ...),

(1)

j = 1,2,...,k. Rj is the rate of overall biochemical reaction producing or consuming component yj per unit volume per unit time. in most models of microbiological systems, the time (t) does not occur explicitly in rate expressions, though formulation of ~structured" models allows for the dependence of growth on the history of the system. A mass balance on any single component yj in a continuous culture (Fig. 1) yields the equation: d Vi _ -~i{- - Rj + D (y }- yj)

(2)

provided Rj is assumed uniform throughout the culture and the growth process has negligible effect on fluid density. Equation (2) can be modified in the case of components present in more than one physical phase to include transport terms. The detail of the kinetic model, i. e. the nature of the Rj terms, determines the theoretical stability of the system. Because of the complexity of microbiological growth, the rate expressions are mostly semi-empirical and their formulation entails abstraction from and simplification of real behaviour. R~ represents a macroscopic or phenomenological process and should not be taken as a microscopic description of control circuits within an individual cell. The problem is one of deciding the level

Transient and Oscillatory States of Continuous Culture

171

1 yj (concentration)

Dilution rate: D = F / V

V (volume)

F (flow rate) (y)j (concentration)

Fig. 1. Schematic diagram of a single-stagecontinuous culture of complexity of rate expression necessary for the correct interpretation of dynamic behaviour of the continuous culture. In general the model of the continuous culture system as defined by equations (1) and (2) is represented by a set of first-order, non-linear differential equations and can be conveniently written in matrix notation as;

=f(x)

(3)

where x and f are n-vectors. 2.3. Multiple Steady States and Their Stability The equilibrium condition of the state-space dynamic model represented by the differential equation (3) is given by solution of: f~)=0.

(4)

Any point _~ which satisfies equation (4) is termed a singular point or steady state. A solution may not always exist, or more than one solution may satisfy the equilibrium conditions as is found, for instance, in substrate-inhibition models (Andrews, 1968). In practice, steady states which give non-feasible or physically unacceptable values e.g. negative values for material components, are neglected. The stability of a particular steady state can be found by the Liapunov indirect method (MacFarlane,

172

D . E . F . HARRISON and H. H. TOPIWALA with negat ve real parts only; stable and damped system

> time

(b)

positive real parts only; unstable system

I= time

~I

(c)

x' ~ ' ~

complex with negativereal parts; stable system

(d)

complex with positive real parts; unstable system

x,

Fig. 2. Stability of the non-linear system to a small perturbation of the steady state. Theoretical response as determined by the eigen-values of the linearised model equations

Transient and Oscillatory States of Continuous Culture

173

1970; Friedly, 1972) which establishes the stability (or instability) of the non-linear differential equations by examining the stability of the singular point for the locally approximating set of linear equations. To linearise, the model equation (3) can be expanded in a Taylor series about the singular point to obtain: dx - -- J x ' + N ~ _ ' ) dr'

(5)

where x' is the perturbation variable,_J the Liapunov first-approximation matrix and _N(x') a matrix which represents higher-order terms. The stability of the steady state to small perturbations is determined by the eigen-values of matrix J. A necessary and suffÉcient condition for the sufficiently small perturbation to die away is that all the eigen-values of J have negative real parts. If the eigen-values consist only of negative real parts the system response will be overdamped. However if any eigen-value has a positive real part the steady state will be unstable i.e. a small perturbation will lead the system away from that steady state. Fig. 2 shows the nature of stability as determined by the eigenvalues. If_Jhas any pure imaginary eigen-values the stability or instability of the steady state will be determined by the higher-order terms in _N(y.'). It should be noted that this approach to the study of stability does not require exact solution of the non-linear differential equation (3). To illustrate the above general approach to a more specific problem let us take the most common model of continuous culture given by the equations: dx dt

ds

d--t =

(6)

= #(s)x-Dx,

D (SR - s)

/~ (s) x

y

(7)

Here x and s refer to the biomass and substrate concentrations in the culture. SR is the substrate concentration in the feed stream which contains no biomass, /~, an arbitrary function of s, is defined as the specific growth-rate and Yas the constant yield-factor. If x and s represent steady state values, the J matrix for the linearised form of above differential equations is given by: /~(~)-D

j

(8)

u(~-) Y

-Ui2,)

÷

174

D.E.F. HARRISONand H. H. TOPIWALA

The two eigen-values of the J matrix are given by: 21=-D

and

; t z - y ds~'

(9)

Examination of 2~ and 22 yields the condition that the non-trivial steady state (~ > 0, ~< S•) will be stable to small perturbations if:

ds--~ > 0,

(10)

i.e. the steady state will be stable to small perturbations provided the rate of change of specific growth rate with respect to the substrate is positive, the rate of change being evaluated at the steady state. In the case of the Monod model, where the specific growth rate, /~, is taken to be a simple hyperbolic function, the non-washout steady state will always be stable. The eigen-values of the linearised Monod model can be obtained from equation (9) as:

21=-D

and

where

22= _(I~,,,-D)[SR(It,,-D)-KsD] tlmKs ~,, s

(11) (l la)

P - K~+s"

Since 2~ and 22 consist only of negative real parts for the non-trivial steady state, the model will not give rise to oscillations when subjected to small disturbances. Furthermore, the speed of response of the system as it returns to the steady state will be characterised by two exponentially decaying modes which will be associated with ,~t and 22, respectively. To illustrate the relative importance of these two response modes Fig. 3 shows computed numerical values of the eigen-values plotted as function of the dilution rate. The values were obtained using the parameter combination:

SR=3g/1-1,

K~=0.012g/1 -l,

kt,,=l.0h

l

which represents approximately the situation studied by Topiwala and Sinclair (1971) for a glucose-limited chemostat culture. It is evident from Fig. 3 that two widely differing modes of response could exist depending upon the operative dilution rate. Interestingly, ,i~ which represents the mixing lag is much smaller than )~2 at low dilution rates but the trend reverses as D approaches/~,~. This aspect has been elaborated by Perram (1973) who found that longer periods were necessary for attainment of steady state when D was in the neighbourhood of/~,,. Jefferson and Smith (1973) included a disturbance vector in their perturbation analysis of the Monod model to find which mode is activated

175

Transient and Oscillatory States of Continuous Culture

7"50~7.

t ~=~'L -3`2 (SR=0.3 g/I)

.ci

T

I i

~v---

-;~2 (SR = 3.o)

t e= g

u3

3£

1°l 0.2

0,4

0.6 Dilution

0,8 rate,

D

1.0 ~ (h-l)

Fig, 3. Relative importance of the two eigen-values which determine the speed of response for the Monod chemostat model, Computed numerical values of the eigen-values plotted as a function of the operating dilution-rate. The values were obtained using the parameter combinations: SR= 3 g I ~: Ks=0,012 g 1 ~; ,u,, = 1.0 h - 1

by a particular disturbance. They showed that responses of substrate and biomass concentrations to flow and temperature disturbances are likely to be fast, whereas response to disturbance of inlet substrate concentration will be slow. Forms of/~ have been proposed as in the case of substrate inhibition (Andrews, 1968; Edwards, 1970) and oxygen limitation (Degn and Harrison, 1969) which do not meet the stability criteria of equation (10). The substrate-inhibition model as represented by Fig. 4b has been

176

D. E, F, HARRISONand H. H. TOPIWALA

studied in some detail because of its reievance in biological waste-treatment processes. It is evident from the shape of the /~ versus s curve that the condition represented by equation (10) will be violated if the culture is operating at a steady-state substrate concentration in excess of that represented by sl in the figure. Provided that the dilution rate is such that washout does not occur and the feed substrate concentration

:3_

~ o

~

~'~

unique steady state

(a)

V

Substrate c o n c e n t r a t i o n , s

stable steady state = -

unstable steady state

sI

v

Substrate c o n c e n t r a t i o n , $

Fig. 4a and b. Effect on available steady states of substrate/specific growth rate relationship based on: (a) A Monod-type hyperbolic model, (b) A model including a substrate-inhibition function

Transient and Oscillatory States of Continuous Culture

177

(SR) is in excess of s l, this model gives two possible steady states for every value of dilution rate (D =/~), but only that steady state corresponding to the lower substrate concentration and to the left of sl in Fig. 4b is stable. The Liapunov direct method described above does not reveal how a system will respond to finite disturbances from a steady state. It is often required to study the response of the system which is initially well away from a steady state e.g. start-up of continuous culture after a period of batch operation. The response is of particular interest when multiple steady states exist since the system has a possibility of attaining any of the multiple steady states. In such cases a knowledge of regions of stability or attraction around the steady state will prove useful, since the system will only go to a particular steady state if it was initially within a region of stability around it. Liapunov and other tracking functions have been used in some cases (Perlmutter, 1972) to define regions of stability, but a generally applicable analytical method is not yet available. 2.4. Transfer-Function Analysis of Stability and Transient-Response Technique In control theory, models represented by linear differential equations are often handled by the Laplace-transform method. The system dynamics are examined by analysing the input/output transfer function. Stability criteria such as those due of Nyquist, Bode and Routh-Hurwitz are then applied. In cases where transfer functions cannot be obtained from mechanistic models they are determined experimentally by frequency-forcing or pulse-testing. In continuous culture systems pH, temperature, inlet substrate concentration and dilution rate have been used as forcing functions (Young et al., I970; Gilley and Bungay, 1968; Zines and Rogers, 1970). For instance, Fuld et al. (1961) studied the bacterial density response to sinusoidal forcing of pH. The response was shown to be a first-order lag. However, Powell (1961) has criticised the experimental evidence presented and questioned the wisdom of employing a complex medium with unknown limiting substrates. There are some important practical considerations to be borne in mind when determining transfer-function models in continuous culture. The transfer-function relationship between feed and effluent composition variables will be influenced by the mixing lag and the kinetic lag. Since residence times of magnitude greater than one hour are not uncommon in continuous cultures, the frequency-response technique using sinusoidal variations in input variables may present practical difficulties.

178

D.E.F. HARRISONand H. H. TOPIWALa

Pulse-testing is more convenient for continuous culture systems but here again the long mixing lag could swamp any effects due to shorter kinetic time constants.

2.5. Theoretical Transient Response and Approach to Steady State In the previous section we discussed the stability of continuous cultures in small regions around the steady states. To observe the exact transient behaviour of such a non-linear system when large perturbations or changes in operating conditions occur, e.g. major shift from one dilutionrate to another, the non-linear differential equations have to be solved. Analytical solutions are rare and recourse has to be made to computer solutions. Both analog and digital computer solutions of differential models have been obtained (Ramkrishna et al., 1967; Megee, 1971; Young et al., 1970). Two articles in this series (Nyiri, Vol. 2; Blanch and Dunn, Vol. 3) deal with various aspects of modelling and digital simulation in biochemical engineering. Usually the models considered have been based on Monod-type kinetics as represented by equations (6) and (7) and the response of the time-dependent variables to changes in system variables such as dilution rate, inlet substrate concentration and temperature have been examined. 2.5.1. Monod Model Fig. 5 shows generalised transient responses of the Monod model to stepwise increases in dilution rate and SR. For this model the transient response of cell concentration to a stepwise change in SR or D is always over-damped provided the system is in a steady state prior to the disturbance (Sinclair, 1964). The response of substrate concentration (s) to a change in S~ shows a single overshoot or undershoot depending on whether it is an upward or downward change. After any disturbance the system returns to a unique steady state corresponding to the new values of the operating conditions. The initial condition of the system does not affect the final steady state according to this unstructured model. Theoretical transient responses of some continuous culture models other than the simple Monod model have also been obtained. Slight modifications of the Monod model such as inclusion of terms to account for endogenous respiration can lead to such transient-model behaviour as damped oscillations (Koga and Humphrey, t967). Models which are more structured to account for the 'physiological state' of the culture may also give similar dynamic behaviour.

Transient and Oscillatory States of Continuous Culture

179

(a)

x

m

E t~

0

/

E 8

~a

E

0

time

Fig. 5a and b. Generalised transient responses, predicted by a Monod-type substrare/specific growth-rate relationship to:(a)A stepwise increase in inlet substrate concentration (S~): (b)A stepwise increase in dilution-rate (D)

2.5.2. Substrate-Inhibition Model In the section on stability we discussed the unstable nature of one of the two possible steady states which can be obtained with the substrateinhibition model (Andrews, 1968; Edwards, 1970). To study the response of the cell and substrate concentrations after a major operational change the solution of the following differential equation can be considered: dx at ds

d-t =

IImXS

(Ks+s)(1 +s/Ki) D ( S R -- s)

-- Dx,

(13)

~tm x s

Y(K~+s)(1 +s/K~) "

04)

180

D.E.F. HARRISONand H. H. TOPIWALA

The response of the system, starting from the two different steady states, to a change in dilution rate as obtained by a digital computer solution is shown in Fig. 6. It is clearly seen that washout occurs in the case where the system is at the unstable steady state prior to the stepwise change, but not when the system is originally at the stable steady state.

step change in dilution rate

D=0.08

|

t

D =0-18 10.0

20

A

7,5 --~

---= t5 = .o

8 8

\ /

5

2,5

A

0 Time, t (h)

Fig. 6. Response of organism (x) and substrate (s) concentrations to a stepwise change in dilution rate, as predicted by the inhibition model (equations 13 and 14), starting from: i) a stable steady state (Curves A), ii) an unstable steady state (Curves B). Parameters employed for the digital simulation were: SR=20: K~=0.O02; Ki=7.37: pro=0.2:Y-0.5

Transient and Oscillatory States of Continuous Culture

18t

(a)

(b) lit

T

state

Y x~

Steady

state

T / Fig. 7a--c. Examples of generalised phase-planes of state-variables x~ and x2 around the steady state. (a) A system displaying damped oscillations as a umque steady state is approached, (From Himmelblau and Bischoff, 1968). (b) A system displaying limit-cycle behaviour. (c) An overdamped system; no oscillations are obtained as the unique steady state is approached (From Aris, 1969)

182

D.E.F. HARRISONand H. H. TOPIWALA

2.5.3. Phase-Plane Analysis Many models of continuous culture systems are formulated in terms of only two dependent variables (e.g. average cell and substrate concentrations) and are ideally suited for phase-plane analysis (Koga and Humphrey, 1967). The state variables are plotted against one another through a series of states from initial to final conditions. Fig. 7 shows generalised trajectory paths for three models with typically different behaviour. In the figure only trajectories associated with a single steady state or single limit cycle are shown, In reality a complete phase plane for any continuous-culture model will have at least two steady states since the possibility oPwashout' or zero population always exists. Phase planes of models with multiple steady states and limit cycles have been computed by other workers (Yano and Koga, 1969; Edwards et al., 1972; Friedly, t972). The single-limit-cycle behaviour (Fig. 7) is of particular interest since it predicts continuous oscillations and all trajectories, regardless of initial condition will approach it. The trajectories inside the limit-cycle spiral outwards while those outside spiral inwards to the limit cycle. The significance of this type of behaviour is that a fermenter operating under these conditions would exhibit continuous oscillations in the state variables even though parameters such as temperature, pH, medium flow rate, etc. remained constant. Predator prey kinetics often show this type of behaviour (Fredrickson et al., 1973).

3. T r a n s i e n t R e s p o n s e s of M i c r o b i a l C u l t u r e s to P e r t u r b a t i o n s of t h e S t e a d y State 3.1. Dilution Rate The most common perturbation that is produced in a chemostat culture is, perhaps, the stepwise change in dilution rate brought about by raising or lowering the medium feed rate. This, in effect, imposes on the organism a change in growth rate. The simplest response would be that predicted by the unstructured Monod-type growth model described above (Eq. 6, 7, 11 a). It is implicit in this model that growth is regulated only by the concentration of the growth-limiting substrate and that, even while growing slowly, the microbes possess all the requisite cell constituents for growth at the maximum growth rate and can accelerate to a higher growth rate instantaneously when the substrate concentration in the culture fluid is raised. However, cell division and metabolism are regulated by complex control systems (Goodwin, 1963) and so this is probably not a reasonable assumption. The RNA content of bacteria

Transient and Oscillatory States of Continuous Culture

183

increases steeply with growth rate (Tempest, Hunter and Sykes, 1965) and, from work with magnesium-, potassium- and phosphate-limited cultures (Tempest, Dicks and Hunter, 1966) it is difficult to avoid the conclusion that the correlation between growth rate and RNA content of bacteria is not fortuitous; the RNA (ribosome) content may regulate protein synthesis and thus growth rate of the organisms. If this is so, then, in order to increase its growth rate, an organism would require a finite time to produce more RNA before the rate of protein synthesis and thus growth rate could be accelerated. This was the finding of Kjelgaard, Maaloe and Schaechter (1958) from experiments carried out on batch cultures, in which increases in growth rate were obtained by transferring organisms to richer medium. Mateles et al. (1965) studied the transient response to an increase in dilution rate of nitrogen-limited chemostat cultures of E. coli. Their findings (summarised in Table l) were at variance with those of Kjelgaard et al. (1958) in that they found that for a small increase in dilution rate the culture growth rate could accommodate immediately. They also carried out experiments with radioactively-labelled 1-1eucine and uracil to further demonstrate that protein synthesis increases immediately on increase in dilution rate. However, with more substantial increments in dilution rate a considerable time lag was found before the growth rate increased to match the new value of dilution rate. A similar pattern of results was found by Harrison and Wren (unpublished data) for the methanol-utilising bacterium Is. extorquens; small changes in dilution rate were accommodated immediately, but larger increments caused a pronounced lag Flow rate increased

1.4 ~x

'~ 1.2 ....

S ~-'~'~

1.0 0.8 ~>. ~ 0.6 "o

-~ 04 0.2

2

4

6

8

10

12 1L 16 18 20 22 24 26 28 30 Time {h ) Fig. 8. Effect of a stepwise increase in dilution-rate, from 0.045 to 0.148 h i on a continuous culture of Pseudomonas extorquens growing on methanol

184

D.E.F. HARRISONand H. H. TOPIWALA

in the concentration of organisms (Fig. 8) indicating a finite time delay before the organisms reached the new growth-rate. In this culture, where the substrate was potentially toxic, this effect was most important since, with a larger increase in dilution rate, the lag in population concentration was larger and was accompanied by an accumulation of methanol. If methanol reached a critical inhibitory level the organism could not attain the new growth rate demanded of it and washout of culture ensued. The apparent contradictions in the results discussed above may be reconciled if it is assumed that the cells at any particular growth rate contained RNA in a slight surplus over that required to meet the immediate protein-synthesis requirements. A small increase in dilution rate could then be accommodated whereas larger shifts would require synthesis of more RNA so that a finite delay would occur before attainment of the new growth rate. It should be possible to predict the rate of RNA-synthesis from the time course of the transient but this does not seem yet to have been studied. Besides the changes in cell-RNA-content, there may be other cell constituents which must adapt before growth rate may increase; enzymes involved in the metabolism of the growth-limiting substrate may be subject to regulation by the concentration of that substrate which will itself vary with growth rate, so that at low growth rates the enzyme may be present at concentrations insufficient to sustain maximum metabolism of the substrate, but at higher growth rates, higher enzyme concentrations may be induced by exposure to high concentrations of the substrate. Johnson (1967) reported that in oxygen-limited yeast cultures the apparent affinity for oxygen of the whole cells and the maximum respiration rate varied with growth rate and he attributed this to changes in the cytochrome-oxidase concentration which he considered to be rate-limiting. Table 1.Response to stepwise increase in dilution rate of a nitrogen-limited chemostat culture of Escherichia coli (After Mateles et al., 1965) Change in D(h- 1) from

to

0.286 0.316 0.525 0.492 0.410 0.377 0.388

0.466 0.503 0.710 0.777 0.830 0.845 0.870

Time to reach new steady state (h)

Maximum growthrate attained during change (h 1)

0 0 0 1.25 3.00 4.60 12.50

0,466 0.563 0.710 1.0 0.91 0.93 1.00

Transient and Oscillatory States of Continuous Culture

185

The response to a decrease in dilution rate would be expected to follow the predictions of the unstructured model with regard to cell concentrations notwithstanding changes in RNA or enzyme constitution, because the lower growth rate could be attained instantaneously. However, it should be borne in mind that, even though a transient state is not apparent in relation to the organism or substrate concentration, the cells themselves will require a finite time to reach the physiological state appropriate to the lower dilution rate and, for this reason, a transient condition will persist for a period which will depend on the rate of turnover of the responding cell components, e.g. RNA content. 3.2. Feed Substrate Concentration A sudden change of concentration of the growth-limiting substrate in the feed medium to a chemostat culture should have an immediate effect on the growth rate according to a simple unstructured Monod-type growth model. Thus the effect of a sudden increase in substrate level or a pulse of substrate added directly to the growth vessel will be similar to that of a sudden increase in dilution rate. The use of a substrate pulse for producting an increase in growth rate has been used by some workers in preference to a stepwise change in dilution rate for studying transient behaviour during acceleration of growth rate. Nagai et al. (I 968) produced accelerated growth of Azotobacter vinelandi by the pulsing of glucose to glucose-limited chemostat cultures, and measured nucleic acid contents during these transitions. These workers used the term 'unbalance growth' for the condition when the growing cells were undergoing a change of composition so that some components were increasing at a rate different from that of the total cell mass, balanced growth being the condition when all components were increasing at the same rate and cell composition remained constant. In their studies Nagai et al. found that the RNA content increased faster than cell mass during the phase of unbalanced growth following a pulse of substrate to a substrate-limited culture. Use of the term "unbalanced growth" for this period of adaptation would seem somewhat misleading as it implies a loss of equilibrium and control within the cell when, rather, what is happening in the transition, may be a controlled change from one equilibrium state within the cell to another. The reported responses of cells to a sudden increase in substrate concentration in general, are similar to those for a sudden increase in dilutionrate. The cells can accommodate immediately to small changes in substrate concentration but a lag period is characteristic of a large increase. For example in studies of continuous cultures of E. coli, Harvey (1970) found that for cells grown at dilution rates above 0.3 h-1 there was

186

D . E . F . HARRISON and H. H, TOPIWALA

no immediate increase in the growth rate when the cells were abruptly exposed to excess glucose, but a lag of 1--2 h before the growth rate increased. However, in cells grown at dilution rates less than 0.3 h 1, the growth rate accelerated immediately on exposure to excess glucose, but did not attain the maximum growth rate. The transient behaviour of cultures grown on t w o different carbon substrates simultaneously is of particular interest. Such a system might be said to model some of the complex situations that occur, for instance, in activated sludge waste water treatment. Standing, Fredrickson and Tsuchiya (1972) grew cultures of E. coli on mixtures of glucose and xylose. In batch culture a clear diauxic effect was obtained with these two substrates (glucose was used first and then xylose was metabolised after a lag period), but in chemostat cultures fed glucose and xylose together, both substrates were utilised completely. When the culture was grown for some time on glucose alone and then the feed was switched to xylose as sole carbon source, there was a transient fall in cell population lasting for about a day before it recovered to the previous level (Fig. 9). Xylose accumulated during the fall in cell population and disappeared from the culture fluid as the population recovered. 0.25,

switch to xy[ose

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Transient and Oscillatory States of Continuous Culture

187

On carrying out a similar switch from xylose back to glucose, however, there was no lag in organism density but a smooth transition. This clearly demonstrates the difference between the transients caused by switching to a substrate (xylose) for which the metabolising enzymes are inducible and those obtained on switching to a substrate (glucose) for which the metabolising enzymes are constitutive. When a sudden increase in dilution rate was applied to a culture growing on glucose and xylose together there was a temporary fall in population during which xylose, but not glucose, concentration in the culture fluid increased. Tile recovery times in each of these transients was very long, from half to two days. This suggests a rather slow response for simple enzyme induction by the substrate, and probably the situation was complicated by repression of xylose utilisation by glucose. Clearly, the different interactions which may exist between the various substrates and cells must be considered when modelling transient responses in multisubstrate systems and relaxation times in such systems may be expected to be somewhat longer than for single-substrate systems. 3.3. Temperature Temperature might be expected to influence a continuous culture through altered growth rate, yield coefficient or affinity for substrate. Other physiological and metabolic functions of the cell, such as content of carbohydrate, lipid and RNA, may also be sensitive to temperature (Rose, 1969). The response of steady-state growth rate to temperature has been found to follow a simple Arrhenius relationship (Ingraham, 1958; Ng et al., 1962; Topiwala and Sinclair, 1971). From this, a sudden change in temperature to a growing culture might be expected to produce an immediate change in the maximum growth rate. However, in studies on E. coli by Ryu and Mateles (1968) and on Aerobacter 1 aerogenes by Topiwala and Sinclair (1971), it was found that there was a lag before the growth rate accelerated to the expected new value on subjecting the organisms to a sudden increase in temperature. Fig. 10a shows the response to such a change in temperature applied to a glucose-limited culture of Aerobacter aerogenes, compared with the expected response from the Arrhenius relationship. That there should be a delay in response to a stepwise change in temperature is possibly not very surprising in the light of the discussion above. Controls similar to those involved in regulating the growth rate during an increase in dilution rate would presumably apply here: an increase in temperature allows an acceleration i This may by synonymus with Klebsielta aerogenes, but we have retained the nomenclature used in the original publications cited.

188

D . E . F . HARRISON and H. H. TOPIWALA

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Transient and Oscillatory States of Continuous Culture

189

in growth-rate which probably requires the synthesis of greater amounts of RNA and/or some other regulatory molecules whose concentrations are limiting growth at the lower temperature. The response of Aerobacter aerogenes to a sharp decrease in temperature is shown in Fig. 10b. In this case there was a much smaller lag and a fairly smooth transition to a lower concentration of organisms. Thus, although steady-state responses to temperature may show a relatively simple Arrhenius type of relationship, the transient response indicates a more complicated underlying regulatory mechanism. 3.4. Dissolved-Oxygen Tension Most microorganisms are insensitive to changes in dissolved-oxygen tension over a wide range of values i.e. I5--150 mm Hg (Harrison, 1972, 1973). At very high oxygen tensions the metabolism of most microorganisms is inhibited to some degree (Harrison, 1972). At low oxygen tension the response to changes in dissolved-oxygen tension may be very complex, especially in facultative organisms (Harrison and Pirt, 1967; Harrison, MacLennan and Pirt, 1969; Harrison and Loveless, 1971). Clearly it is not feasible to consider here all the possible transients which may result from changes in dissolved oxygen in continuous cultures of microorganisms, as an almost infinite variety of adaptations to oxygen tension is demonstrated by different organisms (Harrison 1972, 1973). However, the facultative anaerobe, Klebsiella aerogenes, provides a convenient example for generalised discussion of transient responses to dissolved oxygen as this organism demonstrates responses at various metabolic levels and over various time periods (Harrison and Pirt, 1967; Harrison and Maitra, 1969; Harrison, MacLennan and Pirt, !969; Harrison and Loveless, ! 971 b). Fig. 11 shows some of the metabolic changes observed during the transition from anaerobiosis to aerobiosis in a continuous culture of K. aerogenes. The response is complex, which is not surprising as the adaptation from anaerobic to aerobic growth must demand considerable reorganisation of metabolism. On re-aerating the anaerobic culture there were immediate changes in such parameters as Qo2, Qco:, and pyruvate and acetate production. These occurred within the first 15 minutes and were, therefore, probably a result of rapid feedback regulation of metabolism not requiring protein synthesis. This was followed by more gradual changes in Qo2 and the disappearance of fermentation products over the next 8 hours during which period the yield coefficient from oxygen was lower than in the aerobic steady state indicating less efficient growth. Irrespective of whether the culture was grown under anaerobiosis for 4.5 or 74 hours the time required to attain

190

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Transient and Oscillatory States of Continuous Culture

191

a steady state after re-aerating the culture was 8 to 9 hours, although the pattern of metabolism in the transition was affected by the length of the anaerobic period. Thus two levels and speeds of response could be distinguished: (1) Those of short duration i.e. 10 to 15 minutes, probably resulting from regulating mechanisms not involving induction or repression of enzyme synthesis, but rather mass action and allosteric control of enzyme reaction rates; (2) those of longer duration i.e. 1 to 8 hours probably involving induction and repression of protein synthesis. A clear example of the slower type of response was also demonstrated by the CO2 production during an aerobic/anaerobic transition in a continuous culture of K. aerogenes (Fig. 12). In this case there was a distinct lag of 8 hours before carbon dioxide production increased sharply, presumably indicating an induction period for elaboration of fermentative enzymes. Apart from control by feedback regulation of enzyme activity and enzyme synthesis there is yet another longer-term response possible to changes Oxygen feed off 25

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192

D.E.F. HARRISONand H. H. TOPIWALA

in growth conditions; selection of mutant strains. A chemostat culture must be regarded as a continuous selection process (Novick and Szilard, 1950). Any change in growth conditions will change the selection pressures on the culture and favour different strains more fitted to the new conditions. This has been demonstrated in chemostat cultures of K. aerogenes grown under anaerobic conditions when it was found that the relative amounts of the various metabolic products formed changed slowly but significantly during 700 hours of growth (Harrison, 1966). Also, when K. aerogenes was maintained under high (greater than 150 mm Hg) dissolved-oxygen tension for over 10 generations a yellow mutant strain was selected which did not subsequently revert on lowering the dissolved-oxygen tension again (Harrison, MacLennan and Pirt, 1969). 3.5. General Discussion The list of possible transient responses in continuous culture is, of course, inexhaustible. Any change in a parameter which affects cell metabolism in any way will lead to transient phenomena. The degree and duration of the perturbation will depend on the sensitivity of the microorganism to the change. We have discussed a few examples of transient behaviour above to illustrate some typical aspects of response by microorganisms. Examples could equally be cited of response to changes in pit, osmolarity, light intensity, etc. The conclusions to be drawn from the above examples are: 1. Even in the simplest types of transients, such as response to a change in dilution rate or substrate concentration, a simple, unstructured growth model cannot adequately describe the behaviour of the culture. 2. Even where the steady-state response shows a good fit to a simple model, the transient response may, and usually does, reveal a greater degree of structuring. 3. The duration of a transient response may vary from a matter of seconds to days. in the case of a response dependent on simple physicochemical effects, the transient may last less than a second; in the case of response by regulation of enzyme activity, the response will be complete in a few minutes; for enzyme induction and repression, the transient condition may endure for several hours and many generations may be required to complete a response by selection of mutant strains. These considerations beg the questions "What is meant by ~steady state' in a continuous culture?" and "How long must a culture be maintained after a perturbation before it can be assumed to have reached a 'steady state?'" We would suggest that there is no absolute answer to either question. To take an extreme view, a true steady state probably never

Transient and Oscillatory States of Continuous Culture

193

exists in a biological system (otherwise there would be no evolution of species). In truth, it is necessary to define what is meant by ~steady state' in the context of the particular study. In most cases concerning continuous culture studies 'steady state' seems to be defined as a constant cell population maintained over a period of several generations. When defined in this way, only the most rapid selection processes need to be considered and steady states should be obtained after sufficient time has been allowed for appropriate induction and repression of enzymes. Exactly what this period should be cannot be stipulated for all systems. Rather it is necessary to decide with respect to which parameters and within what limits the steady state is to be defined and to follow the transition after perturbation until these parameters are constant within the stated limits. In practice, steady states are rarely defined in such terms in reports of continuous culture experiments although such a definition must be tacitly assumed. As explained in section 2.3 above, where there is more than one steady state available under one set of conditions, a small perturbation may be sufficient to cause a transition from one steady state to another. It is difficult to find authentic accounts of such responses in the literature. In the case of complex mixed cultures of microorganisms, many steady states may be possible under any given set of conditions and the actual steady state reached may depend very much on the past history of the culture and the perturbations experienced. This is highly relevant to the case of activated-sludge waste-water treatment systems where it has proved difficult in the past to reconstruct the same steady states under apparently identical conditions. We have discussed above some of the types of regulatory mechanisms that are revealed through studies of transient conditions in continuous cultures. The nature and mechanisms of such regulatory controls are still, for the most part, not at all well understood. However, studies of transitory responses to perturbation of the steady state provide a means, probably the best, of defining regulatory mechanisms involved in cell metabolism. In a well-regulated system the parameters under control will deviate very little from their regulated position so that steady-state measurements reveal little about the regulatory mechanisms. By perturbing the system, however, the regulated parameters may be induced to change in a way that reveals aspects of the control mechanisms. An example of the application of this principle to continuous culture is the study of control of respiration of K. aerogenes by Harrison and Maitra (I 969). Harrison and Maitra followed the changes in various co-enzymes and metabolic intermediates during transient states in respiration rates caused either by lowering the oxygen tension of a continuous culture of K. aerogenes or by giving a pulse of substrate to cultures

194

D . E . F . HARRISON and H. H. TOPIWALA

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Transient and Oscillatory States of Continuous Culture

t95

in a steady state. A typical result is shown in Fig. 13. The rapid return of the levels of the coenzymes to their previous steady states was interpreted as indicating tight control over the coenzyme content in these cells. The response of glucose-6-phosphate (G 6 P) concentration to the decrease in dissolved-oxygen tension was to fall rapidly and then recover. On resumption of the supply of oxygen, G 6 P concentration returned approximately to its initial value. This pattern was reversed for the fructose-diphosphate plus triosephosphate concentrations, which rose when that of G 6 P fell, and fell, after a short delay, when that of G 6 P rose. This is the well-known cross-over pattern reported for the Pasteur effect in yeast (Ghosh and Chance, 1964) and was interpreted as indicating a site of glycolytic control at the phosphofructokinase reaction. Other similar results obtained by Harrison and Maitra (1969) implicated the adenylate coenzymes in the regulation of respiration in this organism. Such transient studies using continuous culture offer wide scope for the investigation of regulatory mechanisms in growing systems. The technique has, as yet, been little exploited.

4. O s c i l l a t o r y P h e n o m e n a in C o n t i n u o u s C u l t u r e s of Microorganisms The chemostat is basically a system for producing a steady state in growing cultures of microorganisms. According to the model of Monod, under conditions of steady medium feed to a culture of a single organisml grown with a single limiting substrate, with constant volume, the organism and substrate concentrations should arrive at stable levels unique for the particular growth conditions. We have argued above that in the simple, unstructured model, oscillations in organism population and substrate concentration would not be expected. However, from the many reports in the literature, it is clear that both damped and continuing oscillations are not unusual in continuous cultures (Harrison, 1973 b). Microorganisms possess many feedback-control systems and oscillations are, of course, a frequent characteristic of such systems. Oscillations which are not caused by imperfect control of culture conditions quite probably arise from feedback interactions. Feedback interactions in a continuous culture may occur: (1) between a cell and an environmental parameter; (2) between linked intracellular reactions; (3) between different interacting populations. Examples of all these types of oscillations have been reported in the literature and some are cited below. Synchrony of division is, in effect, an example of feedback interaction between linked intracellular reactions but is dealt with separately as representing a special type of interaction.

196

D, E. F. HARRISONand H. H. TOPIWALA

4.1. Oscillations Derived from Equipment Artifacts When oscillations are detected in continuous culture systems it is necessary to show whether the oscillations are genuine metabolic fluctuations or merely a reflection of periodic changes in some equipment function. Oscillations may arise as a result of poor feedback regulation of parameters such as temperature, pH, stirring speed, foaming and volume control. Small oscillations in any such environmental function may be amplified through the response of cells to such changes, to give large fluctuations in other culture parameters. For instance, it has been found (Harrison and Wayne-Smith, unpublished data) that in cultures of Pseudomonas extorquens growing on formate, oscillations in pH (generated by feedback titration with alkali linked to the output of a pH electrode) of less than 0.1 unit amplitude, about pH 7.0, caused fluctuations in respiration rate of _+6% of the total rate which could, in turn, be reflected in oscillations in oxygen tension of as much as _+25% of saturation. In the discussion of transient response above it emerged strongly that microbial cultures are characterised by lags in their response to environmental changes. Thus, any oscillations in continuous-culture control equipment are likely to produce oscillations in cell parameters which are out of phase with the imposed oscillations. The frequency of the response should be similar to that of the imposed oscillations, but the cells are never, in fact, in equilibrium with their environment. It can be envisaged that such continual inducing of different metabolic or physiological states in the cells may be manifested in overall cell properties such as yield coefficient. The impact of fluctuations in an environmental parameter will depend on the sensitivity of the culture over that particular range. Below we shall consider some of the possible sources of periodic fluctuations imposed by the equipment on continuous cultures and their possible impact on the culture. 4.1.1. Temperature Most laboratory continuous culture systems are equipped with simple on-off temperature control and, commonly, the culture temperature fluctuates over a temperature range of one or two degrees. Temperature has an influence on most cell properties but the sensitivity of microorganisms to temperature generally follows a double Arrhenius-type curve shown in Fig. 14. Clearly any small changes in temperature over the sensitive region (see Fig. 14) will have a drastic effect of growth rate, and fluctuations caused by temperature control over this region may

Transient and Oscillatory States of Continuous Culture

197

create oscillations of large amplitude in cell density or metabolic rate. Over most of the growth temperature range, however, the culture would be relatively insensitive to imposed oscillations in temperatures with an amplitude of less than 1.5.

0.!

E

0.2

0.0032

0,0033

0.0034

~/K Fig. 14. Reciprocal plot of temperature against maximum growth rate of a chemostat culture of KIebsieIla aerogenes. 0 , experimental points: solid line is best-fit curve based on Arrhenius-type relationship. (From Topiwala and Sinclair, 1971)

4.1.2. pH The responses of cell growth and metabolism to pH usually follow an inverted U-shaped curve and over most of the range of pH the cell metabolism may be relatively insensitive to small changes in pH. However, there is often quite a sharp change in cell metabolism at about pH 7.0. For instance, in anaerobically-grown cultures of K. aero9enes at pH 6.5 the metabolic products are mostly ethanol and butanediol,

198

D.E.F. HARRISONand H. H. TOPlWAI~A

together with small amounts of acetic acid. When grown at pH 7.4, however, the major products from glucose are acetate and formate (Harrison and Pirt, 1967). Even small fluctuations in pH at values around neutrality could thus cause oscillations of large amplitude in cell metabolism. Also at the extremes of the pH range for growth, cell metabolism is likely to reflect oscillations in pH about the control set-point. 4.1.3. Stirring-Rate and Oxygen Transfer Coefficient (KLa) Fluctuations in stirring rate are likely to affect the mixing and gas transfer rate of a fermentation. Mixing fluctuations are unlikely to be a serious problem except in very poorly-stirred cultures. Gas-transfer fluctuations, however, will certainly manifest themselves as changes in dissolved-oxygen tension. Provided the dissolved-oxygen tension is above the "critical value" (Harrison, 1972) this is not likely to cause any great effect on cell metabolism. However, if oxygen is limiting, or nearly so, for growth, then even small changes in Kx, a caused by fluctuations in stirring will be reflected in oscillations in culture metabolism and growth. The KLa of a culture may also change as a result of changes in other physical parameters such as pH, temperature and viscosity. Foaming will of course affect KLa; KLa can also be quite sensitive to salt concentrations so that the periodic addition of acid and alkali during pH control may cause fluctuations in oxygen uptake rate. 4.1.4. Foaming and Antifoam Significant foam formation in a continuous culture system is certain to lead to fluctuations in culture parameters: a large foam head over a culture represents a two-phase system which is unlikely to reach a stable equilibrium. Foaming is often combated by addition of chemical antifoam agents. Where these are added continuously to the medium they are not likely to lead to fluctuations in the steady state although they may have an influence on cell growth. However, it has been found more satisfactory either to add antifoam in response to formation of a foam-head, detected by means of a conductivity probe, or to make regular timed additions throughout the life of the culture. Antifoam agents, particularly those based on polypropylene glycol or silicones, are generally considered non-toxic. This is not however to say that they are without effect on microorganisms. Antifoams are, after all, surface-active agents and so may be expected to influence cells through their membrane functions. Periodic addition of polypropylene glycol 8000 to continuous cultures of K. aeroyenes was found to give rise

Transient and Oscillatory States of Continuous Culture

199

to oscillations in dissolved oxygen tension as shown in Fig. 15 (Harrison, 1966). At a pH of 6.0 the addition of antifoam coincided with a rise in oxygen tension but at a pH of 7.4 a sharp fall in oxygen tension immediately accompanied antifoam addition. In this case it was not determined whether the antifoam affected cell respiration rate or gas transfer-rate through its effect on bubble coalescence and mass transfer co-efficient (Benedek, 1970). However, it has been shown (Harrison and Maitra, 1969) that silicone antifoams can cause a temporary stimulation of respiration rate. ~.rn.\

2 P-m.~

(mm Hg) "133 ~-114

76 57 38 19

o

Fig. 15. Effect of periodic additions of antifoam agent (Polyglycol P 2000) on the dissolved oxygen tension in a nitrogen-limited chemostat culture of Klebsiella aerogenes. 'A' indicates addition of 0,05 ml antifoam to 1.5 1 culture, Growth rate=0.10 h - 1; pH = 6.0. (From Harrison, I966)

200

D.E.F. HARRISONand H. H. TOPIWALA

4.1.5. Discontinuous Substrate Feed A good example of the way in which an oscillation originating from an equipment artifact can fundamentally affect culture properties is that demonstrated in cultures of a methanol-utilising Pseudomonas by Meers (1973b). He found that pulse-feeding substrate to a culture gave rise to oscillations in respiration rate, oxygen tension and pH (Fig. 16a). Further it was found (Fig. 16b) that the yield coefficient of the culture varied with the pulse frequency even though the average flow rate or the dilution rate of the culture was kept constant. The explanation of this would appear to lie in the observation by Harrison et at. (1972) that the methanol-utilising organisms are poorly regulated in terms of energy conservation, so that whenever they are exposed to methanol in excess of that immediately required to satisfy the carbon and energy demand of synthetic pathways the excess methanol is rapidly oxidised to the detriment of the yield coefficient. Thus, a pulse-feed in which methanol is alternatively available in excess and limiting amounts, would give rise to a lower yield than a steady-state feed at a limiting rate (Meets, t973 b). Intermittent addition of a substrate can be caused by either a discontinuous flow rate or by an unmixed feed stream. Discontinuous flow rates are frequently encountered in laboratory fermenters where the flow rates are small and the feed enters in discrete drops. The situation is worsened by feed pumps which do not have a stead},' continuous delivery. On an industrial scale, components of a single feed stream are often not premixed, causing pulsed entry of the various nutrients even though the flow rate is steady. Megee (1971) studied the effect of drop feed using the Monod kinetic model and noted the oscillating behaviour of limiting-substrate concentration. Topiwala et al. (unpublished data) have extended this type of theoretical approach to examine the effect of dropwise addition on a model for a system in which the yield is substrate-sensitive. The model assumes that cell metabolism responds instantaneously to exocellular substrate concentration and that substrate concentration in excess of a constant value (So) gives a significantly reduction in yield. It is assumed that when one drop of substrate enters the fermenter, one drop of culture leaves the fermenter. Between the drops, the fermenter operates as a batch culture. The differential equations representing the model are: dx p,,xs dt = (K~+s) D(t)x, (15) ds dt

=

tlmXS D(t)(Sg-s) -(K~+s)Y(t)

(16)

Transient and Oscillatory States of Continuous Culture

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Time (see)

(b) 45

=o ca

E

g

¢n

8

35

>-

o

s'o

~o

1~o

200

Interval between methanol additions (sec)

Fig. 16a and b. Effect of drop-feeding methanol to a methanol-limited chemostat culture of a Pseudomonas sp. (a) Fluctuations in dissolved-oxygen tension (DOT); pH and supernatant methanol concentration, (b) Effect of frequency of drops on yield coefficient. (From Meers, 1973b)

202

D.E.F. HARRISONand H. H. TOPIWALA

with the following parameter values: Y-0.5 when S<So,

Y=0.1 when S>So,

Average dilution rate (D) Fermenter liquid volume Spherical drop volume Maximum specific growth rate ll,, Monod saturation constant K~

s0=0.33

× 10 - 3

g/l,

= 0.05 h - 1 = 2.0 1, =6.5 x 10 5 1, =0.8 h ~, =0.005 g/l- ~.

The results of the simulation are shown in Fig. 17. Once the dropwise feed is commenced the lilniting-substrate concentration shows oscillations of significant amplitude depending upon the inlet substrate concentration. The biomass concentration shows a gradually decreasing trend due to the substrate-sensitive yield factor. The oscillations in biomass concentration due to this type of drop addition are of extremely small amplitude and will not be detected in experiments. Drop feed

Continuous flow ~12 ~

=

~

8

g <~-,65 x 10- 3 h r ~ .28 x 10 -~ hr 10.001

Variable yield model SR = 20

10.00

8

9.~99 0.8

g A

0.6

8 ~ 0.4

x<..

~0.2

0

02

0.4

0.6

F%.._ 0.8

1.0

1.2 1,4 1.6 Time (x 10 -'3) h

I\-.j 1.8

2.0

8R='°l'v..-. 2.2

2.4

2.6

Fig. 17. Computed effect of dropwise medium feed to a chemostat culture in which the yield coefficient is dependent on substrate concentration (for details see text)

Transient and Oscillatory States of Continuous Culture

203

For further refinements of the model, diffusional and metabolic tags may be incorporated. It is interesting to note that pressure and mixing effects in a large industrial fermenter can cause cells to be exposed to a continuously varying substrate environment similar to that discussed above.

4.2. Oscillations Derived from Feedback Between Cells and Environmental Parameters In this situation a feedback loop exists between cell metabolism and the environment such that cell metabolism has an impact on its environment which, in turn, affects cell metabolism. A simple and common example of such a system is one in which the pH of a weakly buffered culture is not controlled. The culture grows and produces acid so that the pH falls until it reaches a level which inhibits metabolism. Metabolism slows, or stops, and the pH again increases, owing to the influx of fresh medium, until the metabolic rate increases again. Such a system can give rise to damped or continuous oscillations depending on the lags in the system. A more complex example is that which was shown to cause the oscillations in oxygen tension and respiration rate in cultures of Klebsietla aeroyenes at low oxygen tension shown in Fig. 18 (Harrison and Pitt, 1967). These oscillations have been modelled by Degn and Harrison (1969). This model is a specific example of the generalised system given by equation (3). In this model three criteria must be met in order to produce sustained, undamped oscillations: 1. A region of negative slope should exist in the relationship between respiration rates and oxygen tension in the liquid (c.f. eq. 10). This type of relationship exists in K. aerogenes growing in chemostat culture (Harrison and Pirt, 1967); the Qo2 of the culture is independent of oxygen tension at oxygen tensions above 10 mm of mercury but increases when the oxygen tension falls below 2 mm Hg (Fig. 19). 2. A time dependence of the respiration rate of the culture should exist. The simplest mechanism for time dependence would be that the respiration rate of the culture depends on the concentration of a substrate which becomes depleted at high respiration rates and replenished at low respiration rates. Glucose, which was the sole carbon source supplied to these cells, did not fulfil this role as it was always present in excess but possibly a metabolic intermediate which varied in concentration with respiration rate acted in this way. Alternatively, a time-dependent function might derive from a buildup of some inhibitor of respiration which is removed during the phase of low respiration rate, or by changes

204

D. E. F. HARRISONand H. H. TOPIWALA 16

{a)

12 E E

14 12 10

(b)

E E

24 8

¢a 4 O 2 ,

2

i

,

4

6

8

lo

12

!

1~

'

1~

'

18

Time (h}

Fig. 18a and b. Tracing of dissolved-oxygen tension obtained in nitrogen-limited chemostat cultures of Klebsiella aerogenes.(a)D =0.20 h - 1: (b) D=0.40 h- t Lines A, traces obtained when oxygen was in excess; Lines B, traces obtained under limited-oxygen tension. (From Harrison and Pirt, 1967)

in cell concentration. Cell concentration fell during the high-respiration phase and increased during the low-respiration phase. 3. Limitation of the transport of oxygen from the gas to the liquid phase. This condition is not related to any physiological or biochemical mechanism in the cells. The resistance to diffusion at the gas/liquid interface is represented by the constant KLa. Using this mathematical model, it was shown that, when appropriate values were given to the three parameters: the negative slope of the

Transient and Oscillatory States of Continuous Culture

205

respiration/oxygen tension curve; the time dependence of respiration rate: and the transfer rate of oxygen from the gas to the liquid phase, continuing oscillations could result. Of course, if any of the parameters fell outside a certain range the oscillations would not occur. It was found that on increasing the rate of stirring in a culture, which, in effect, lowered the diffusional barrier to oxygen, stable oscillations could not be obtained (Harrison, unpublished data). The frequency and slope of the oscillations was affected by the growth rate of a culture presumably by altering one or both of the first two conditions. 110

11.0

1 O0

10.0

90

9.0

A 8O

8.o

,3

70

7.0 E m

g ~ 60

6.0

O m "6 5.0 E vE

y

g ~. 50 × o

40 o

a 30

20

4.0

3.0 g 2.0

/

10

1.0

/ / / 10

20

30

40 50 60 70 80 90 100 110 Partial pressure of o x y g e n in the gas phase ( m m . Hg)

120

130

140

Fig. 19. Effect of varying oxygen supply and thus dissolved oxygen tension, on the respiration rate (Qo:) of a chemostat culture of Klebsiella aerogenes. ©, dissolved oxygen tension: 0 , Qo~. (From Harrison and Pirt, 1967) Another type of interaction between feedback and environment would be the case where the concentration of an extracellularly-produced metabolite regulated cell metabolism. An oscillation which possibly fits into this category is that observed by Sikyta and coworkers (Sikyta and Slezak, 1965; Sikyta et al., 1966) in the concentration of pyruvic acid produced by E. coli grown in chemostat culture. Pyruvic acid was the only parameter found to oscillate and this described large, spiked

206

D . E . F . HARRISON and H. H. TOPIWALA

0.3-~

30

c

E

~20

0.25C3 -x

u

-

x

-

.x-

C o~ X3

.o

0.t--

2 10

\ J

1

© i

7

2

..

i

8

10

Time ( h ) Fig. 20. Oscillations in pyruvate concentration obtained in continuous culture of Escherichia coli. Q, pyruvate: x, bacterial cells. (From Sikyta, 1965)

fluctuations at hourly intervals (Fig. 20). A most plausible explanation of the oscillations is that the pyruvate is produced initially at a high rate but that eventually production is repressed by the high concentration of pyruvate itself. Pyruvate is then oxidised rapidly until the concentration falls sufficiently to derepress enzymes for pyruvate production. For these oscillations to be so persistent and show no damping, there must be significant time lags in the system so that the fcedback regulation continuously overshoots. 4.3. Oscillations Derived from Intracellular Feedback Regulation Rhythmic phenomena are common in biological systems and many of these have been found to have their origin at a physiological level. Rhythmic fluctuations in metabolism generated by intracellular feedback systems are believed to play an important role in the physiology of higher animals, for instance in nervous control and in biological-clock mechanisms (Hasting, t960). Such oscillations are found not only in higher animals (Frenkel, 1965) but also in yeast (Pye, 1969). In bacteria, cell division is, in reality, a culmination of a whole series of rhythmic reactions (Goodwin, 1963). With the many feedback reactions present in a microbial cell, the presence of oscillations in some metabolic parameters is predictable. However, oscillating intracellular parameters would only be detectable in continuous culture if a high degree of synchrony existed between the oscillations within the individual cells. Unless the oscillating systems in the

Transient and Oscillatory States of Continuous Culture

207

individual cells could interact through a common environmental parameter. the oscillations as detected in the whole culture would be highly damped as the cells lost synchrony. Most metabolic pathways in microorganisms are subject to feedback control through induction and repression of enzymes. Oscillations in an induced system might be expected, especially if a product of the pathway acts as a feedback repressor. In such systems it might be predicted that the specific rate of enzyme production would give damped oscillations after the particular pathway had been induced. In fact, there are few studies where specific enzyme production rates have been carefully followed in growing cells. One such study carried out on exponentially growing cells was that by Knorre (1968). Knorre found oscillations in the rate of production of/3-galactosidase after induction of this enzyme with lactose. In both constitutive and inducible strains of E. coli, four oscillations with a periodicity of one hour were obtained before damping was complete. Knorre (1968) proposed a model for these oscillations whereby the content of messenger-RNA for the enzyme was controlled by induction and catabolite repression. Another type of feedback regulation found in cells is that based on allosteric control of enzyme activity. This type of control renders a response on a much shorter time-scale than induction and repression of enzymes - - thinutes rather than hours. Allosteric control of phosphofructokinase by adenylnucleotides was implicated in the mechanism of oscillations in the rate of glycolysis of resting suspensions of the yeast, Saccharomyces carlsbergensis, when the organism made the transition from aerobiosis to anaerobiosis (Pye, 1969). An example of such oscillations caused by intracellular feedback regulation in growing cells is perhaps given by the short-period oscillations in the NADH fluorescence obtained in a continuous culture of K. aerogenes when the culture was subjected to an anaerobic shock (Harrison, 1970). That these oscillations were completely unconnected with the oscillations obtained in oxygen tension discussed above (Fig. 18) was shown by their high frequency and the fact that they occurred at oxygen tensions well above the critical dissolved-oxygen tension. The damping of these oscillations became less on repeating the anaerobic shock, and eventually completely undamped oscillations were obtained (Fig. 21). These oscillations continued for several days when the culture was left undisturbed, but could be stopped, at will, by interrupting the medium supply to the culture for a prolonged period (more than half an hour). These oscillations in NADH fluorescence were found to be accompanied by oscillations of the same frequency in oxygen tension (Fig. 21), the amplitude of which indicated an oscillation in respiration rate represent-

208

D.E.F. HARRISONand H. H. TOPIWALA

0,2m~rmles NADH/ml

-&

L

I

E E NADH

=:

I-

o"

"--~

5 rain ~1~

~ IncreasedNADH

Fig. 21. Oscillations in NADH fluorescence and dissolved-oxygen tension obtained in a glucose-limited chemostat culture of Klebsiella aerogenes. (From Harrison et al., 1969)

ing less than one per cent of the total respiration rate of the culture. Respiration was not a prerequisite for the oscillations in pyridine-nudeotide as oscillations also occurred under anaerobic conditions, albeit with a lower frequency. In fact, the frequency of the oscillations varied with growth conditions as shown in Table 2. Various metabolic intermediates and coenzymes were measured during such oscillations but the only one to show any sign of oscillating at a similar frequency to N A D H was ATP (Fig. 22).

F "~ 6 E C)3 E

../

0.2 m~ f. e NADH

~

~

~

~

~

I~TP

o

E E Time --~D, Fig. 22. Oscillations in NADH fluorescenceand ATP content of Klebsiella aerogenes grown in chemostat culture

Transient and Oscillatory States of Continuous Culture

209

Table 2. Effect of growth conditions on the frequency of oscillations in pyridine nucleotide in a continuous culture of K. aerogenes Growth rate (h- 1)

Dissolvedoxygen availability

Period of Oscillations

(rain) 0.04 0.10 0.20 0.36 a

excess excess excess limited anaerobic excess

--3.4 2.4 3.0-- 5.0~ 12.0 2.3

Varied with degree of oxygen deficiency excess oxygen = > 20 mm Hg limited oxygen = <0.5 mm Hg

Oscillations of similar frequency were also observed in the flavoprotein absorption in non-growing cell suspensions of K. aerogenes (Degn and Harrison, 197t). It is doubtful, however, whether these oscillations were homologous to those obtained in NADH-fluorescence in growing cultures. There is a strong similarity between the oscillations obtained in N A D H in continuous cultures of K. aerogenes and the oscillations reported in N A D H fluorescence in resting whole yeast cells. However, oscillations in the yeast were of glycolytic origin whereas the oscillations in pyridinenucleotide observed in continuous cultures of K. aerogenes could be demonstrated not to be of glycolytic origin because the cells were respiring on succinate as sole carbon source. The involvement of both N A D H and ATP in such oscillations possibly indicates allosteric feedback mechanisms of a type similar to those proposed to explain glycolytic oscillations in yeast (Chance, Schoener and Elsaesser, 1965). It would be difficult to explain these high-frequency oscillations of N A D H in cultures of K. aerogenes on the basis of feedback interaction between cell and environment, as they were obtained under such a wide variety of environmental and physiological conditions. The oscillations were quite insensitive to changes in external parameters such as pH, temperature, oxygen tension and medium supply rate. Therefore, it would seem quite likely that these oscillations do, in fact, represent examples of oscillations created by intracellular feedback loops. However, if this be so then one is led to the interesting conclusion that these oscillations are perpetually occurring within individual cells but are brought into synchrony by the anaerobic shock. A synchronising mechanism must exist for oscillations to be maintained for such long

210

D.E.F. HARRISONand H. H. TOPtWALA

periods of time and this implies a form of cell communication. Repeating the anaerobic shocks might serve to strengthen any synchronising mechanism. On four separate occasions oscillations were generated in a culture spontaneously, without the aid of anaerobic shocks, thus demonstrating negative damping. The self-generating ability of oscillations is an important observation, for, if the oscillations are indeed truly of intracellular origin and not caused by some feedback with an extracellular parameter, then it indicates a definite tendency for the culture to become synchronised spontaneously. In theoretical studies involving mathematical analysis, eletronic experiments and digital-computer simulation, Winfree (1967), found that, for populations of generalised relaxation oscillators, threshold conditions might exist for mutual synchronisation in any of a variety of modes. He proposed that, in communities of independently-oscillating individuals, even weak interactions may give rise to entrainment of the oscillations such that the whole population is brought into synchrony. Evidence for such synchronising interactions has been found in the case of glycolytic oscillations in the yeast S. carlsbergensis (Pye, 1969). It is therefore possible that the effect of anaerobic shock in the cultures of K. aerogenes was to bring into synchrony independently-oscillating cells and that continuing anaerobic shocks serve to strengthen the synchronising interaction, eventually bringing the whole population into synchrony. As yet this idea remains pure conjecture but, should it be true, its ramifications for continuous culture may be great. If there is a possibility of synchronising various activities of individual cells in a population then assumptions about average properties of average populations may become completely invalid. Most of the oscillatory systems discussed so far have resulted from a perturbation of a feedback loop caused by a change in a physical aspect of the cell's environment. Such a perturbation, however, may result also from a chemical agent which interferes with metabolic regulation. Damped oscillations in metabolic functions, as a response to the administration of drugs, have been reported in higher animals (Hollman and Neubar, 1967). Experiments in which inhibitors are added to growing cultures are rare, and there has been but one report of oscillations in continuous culture in response to exposure to a chemical inhibitor. This observation was made by Dean and Moss (1971) during studies of the effect of barbiturate drugs on K. aerogenes. Their observations are of particular interest as examples of oscillations in continuous culture, in that they were obtained in a turbidostat culture and the oscillations were in the specific growth rate of the culture. Regular, sinusoidal oscilla, tions were obtained on exposing steady-state cultures continuously to barbitone. The oscillations were highly damped and a new steady state

Transient and Oscillatory States of Continuous Culture

21I

was obtained, at a lower growth rate, after 3 to 4 complete cycles. The period of the oscillation was about two generations. The authors do not offer a detailed model of mechanism for the oscillations but there seems to be a strong interdependence between growth rate and uptake of the drug which might form a feedback loop with the requisite overshoot properties. 4.4. Oscillations Derived from Interactions Between Different Species in Continuous Culture The occurrence of oscillations in biological populations caused by predator/prey relationships is a well-known phenomenon and there have been many mathematical models proposed for such oscillations (Canale, 1970; Fredrickson et al., 1970). Although continuous culture would seem to present an ideal tool for studying simple predator/prey relationships between microbes under conditions which eliminate other environmental variables, there have been surprisingly few studies employing continuous culture to investigate predator/prey relationships in microbial systems. Two such studies of the interrelationships between bacteria and protozoa in continuous cultures have been reported by Curds and Cockburn (1971) and Tsuchiya et al. (1972). Tsuchiya et al. (1972) studied the relationship between the predatory amoeba, Dictyostelium discoideum and the bacterium E. coil when the organisms were grown together aerobically in a mixed continuous culture. The system may be represented: glucose ~ E. coli ~ Dictyostetium discoideum The culture was run in a chemostat with glucose as the limiting substrate. The authors studied the effects of environmental factors, including temperature and holding time in the culture vessel, on the population of the two organisms and on glucose concentration. Oscillations of large amplitude were found in all three monitored parameters (Fig. 23). The shape of the oscillations varied with growth conditions, but under all conditions the oscillations eventually damped out to give a steady state. Tsuchiya et al. (1972) considered their oscillations in the light of the Lodka-Voltaira model for predator/prey systems. They found that although their experimental data were somewhat more irregular than those predicted by the model, the agreement was reasonable. However, the decay of oscillations would not be predicted by this model although this was a constant feature of the experimental system. It is not clear from their work why the experimental behaviour should depart from the model in this manner. However, the authors reported that there

212

D. E. F. HARRISONand H. H. TOPIWALA

,% ×

8

u.i g

0

E 6 O U m

4

C5 2 c5 0

2

Z,

6

8

10

12

14 16 18 Time (days)

20

22

24

26

22, 30

32

Fig. 23. Changes in numbers of amoebae (Dictyostelium discoideum), bacteria (Escherichia coli) and glucose during continuous culture. Temperature; 25; holding time, 8 h. (From Tsuchiya et aL, 1972)

was some growth of organisms on the wall of their fermenter, and this may have had a stabilising effect upon the population density (Topiwala and Hamer, 1971). The authors found that the environmental factors had a strong effect on the behaviour of the oscillating population, and proposed a relationship between the holding times and substrate concentration that would give either continuing oscillations, damped oscillations or no oscillations depending on the cultural conditions (Fig. 24). The relationship between a phage and its host may be considered that of a parasite/prey system. Oscillations are not usually obtained and no steady-state relationship exists between a phage and a sensitive culture: a sensitive culture is lysed completely by the phage. Bacteriophages generally replicate at a very high rate compared with the doubling time of the host and the nmnber of infected particles produced is extremely large so that there would be no recovery of the type present in the more normal predator/prey relationship which leads to steady-state systems. After infection, the population of a sensitive culture decreases,

Transient and Oscillatory States o]"Continuous Culture

•

20 r-

E

~os

I

213

Continuing oscillations

Damped \ cdiations No . .

o "1-

10

~

Predatorwashesout Total washout

...........

0

I,,

I

0.05 0.10 Concentration of substrate in feed (mg/ml)

Fig. 24. Predicted behaviour of simultaneous continuous culture of a predator and a prey. (From Tsuchiya et al., 1972)

usually in two or more steps corresponding to infection and burst cycles. The culture may subsequently recover owing to selection of a lysogenic or resistant strain of the organism. The situation in a lysogenic strain infected with a phage is not so simple. Noack (1968) Woposed that two steady states were possible under the same conditions in a population of lysogenic bacteria: an upper state characterised by a maximal cell volume and a lower state with a heavily cell volume attributed to the autocatalytic increase of the induction of phage multiplication due to a process of superinfection. Low dilution rates would favour the lower state and high dilution rates the upper state. Noack's

214

D.E.F. HARRISONand H. H. TOPIWALA

model would predict considerable hysteresis so that under suitable conditions, both states are possible over the whole range of dilution rates. Noack showed the presence of two such stable states in the culture of E. coli K. 12 (CI 857). The existence of two possible stable states under one set of conditions satisfies one criterion for producing stable oscillations as explained above but, of course, other conditions, such as the presence of a time-dependent factor, must be satisfied. Noack did not report spontaneous oscillations between the two stable states, but damped oscillations were observed about the lower state after an induced transition from upper to lower states. The various types of interrelationships to be found in mixed populations of microorganisms have been reviewed by Meers (1973). Reports of oscillations in mixcxt continuous cultures other than predator/prey systems are few. In the case of straightforward competition between different species for common substrates, of course, no steady state would be predicted for a mixed population unless both had identical affinities for the substrate, or identical maximum specific growth rates (Meers, 1973). Where there is some degree of symbiosis, the interrelationship should be one of positive feedback rather than negative feedback and would be unlikely to lead to stable oscillations. Exceptions to this have been reported where symbiosis is coupled to a negative feedback. Contois and Yango (1964) reported oscillations in a mixture of strains of Lactobacillus which interacted through variations in the pH of the culture which was not controlled. One strain grew and lowered the pH to a level which inhibited its own growth but permitted growth of a second strain. The second strain, on growing, raised the pH of the culture allowing the population of the first to dominate again. Bergter and Noack (1966) observed oscillations in population in a mixed culture comprising Aerobacter aerogenes and an unnamed Gram-negative organism. The culture was limited by nitrogen source and glucose was the only carbon source supplied. A. aeroqenes grew on the glucose and produced organic acids which were necessary for the growth of the other organism. However, this organism then competed successfully for the nitrogen, causing a decrease in the growth of A. aerooenes and thus the supply of its own carbon source, this, inturn, leading to cycling of populations. No doubt the possibilities for oscillating populations in mixed cultures are legion. Such cyclic phenomena are of interest not only from an academic point of this but also because the mechanism of the oscillations can reveal the underlying interrelationships in mixed cultures. Such systems may, of course, be useful in modelling microbial interreactions in natural environments e.g. in the skin, soil, activated sludge, etc.

Transient and Oscillatory States of Continuous Culture

215

4.5. Synchronous Division of Cells The cell-division cycle is most obviously an example of rhythmic behaviour and temporal control in microorganisms. Much has been written on the subject of the cell-division cycle and it suffices here to refer the reader to one of the recent reviews of the topic (Padilla, Whitson and Cameron, 1969). In fact, there appears to be little agreement as to the exact mechanism regulating DNA replication and cell division. As a purely phenomenological model the three-step process described by Helmstetter (1969) provides a useful concept of temporal control in a bacterial-division cycle. This is simply expressed as:

I+C+D=ta where I is the time between initiation of sequential chromosome replications (time for the accumulation of an initiator complex); C is the time for a round of chromosome replication; D is the time between the end of a round of replication and the following cell-division; ta is the generation time. This model has been used to explain the absence of immediate acceleration of growth rate following a stepwise increase in nutrient supply, the rate of cell division remaining unchanged for the time period (C + D) after the increase as only ! is changed, C and D remaining constant. The division cycle in cells in a population can be brought into synchrony in a number of ways, usually by arresting growth and then suddenly accelerating cell division. Studies on synchronously growing cultures have shown that, besides DNA replication and cell-wall formation, other cell components and functions are also subject to temporal control e.g. certain enzymes, RNA synthesis and respiration rate. Therefore, synchronisation of division would be accompanied by oscillations in culture parameters such as oxygen tension, substrate level, rate of pH change, etc. However, under conditions of unrestricted growth, microbial cells usually lose synchrony of division after 3 to 4 generations. Thus, in a conventional chemostat system synchronisation of cell division would not be expected to occur unless some re-synchronising factor was applied. Continuing oscillations seem unlikely to arise as a result of spontaneous synchronisation of cell division in continuous cultures, although the model of Winfree (1967), discussed above, might suggest a mechanism whereby weak interactions could possibly lead to synchrony m cellular processes. In order to study cells growing in synchrony, modifications have been made to the conventional continuous culture systems to re-synchronise cell division at regular intervals. Dawson (1965) made use of periodic starvation and nutrient supply to phase the division of yeasts. Division time in this case was controlled by feeding sufficient nutrients appropriate

216

D, E. F. HARRISONand H. H. TOPIWALA

for one cell cycle at intervals corresponding to the required division time. However, it should be noted that this situation does not correspond to that in a normal chemostat as the cells are alternately exposed to excess nutrient and then starved to hold back division, whereas in the chemostat the cells are never exposed to excess nutrients nor starved, but the whole division cycle is controlled by the rate of availability of nutrients. One example of an effect on culture metabolism of periodic exposure to excess substrate has been given above, in discussing the effect of dropwise addition of a substrate to a culture. Clearly, other transient responses may be induced by alternating excess nutrient and limiting nutrient states which are not necessarily related to the division cycle per se.

5. Conclusion This paper set out to provide a systems approach to dynamic modelling of continuous cultures. From the examples of observed transient behaviour it is clear that a simple unstructured model of microbial growth rarely gives a good fit to experimental data. The deviations from the predictions of unstructured models indicate complex regulatory mechanisms at several metabolic levels. Examination of transient behaviour in continuous cultures can provide valuable insight into the mechanisms of regulation in biological systems. There are many reports of oscillations in continuous culture systems which are clearly not a function of equipment artifacts. Such oscillations may be indicative of complex feedback control loops within the organism. Because of the widespread presence in biological systems of feedback loops, there are bound to be oscillations in some parameters when an attempt is made to impose a steady state on a growing microbial culture. Thus "steady state" is a relative term and its limits must be defined. Oscillations between acceptable limits are unlikely to be very disadvantageous in a continuous culture system provided their occurrence is appreciated. In fact, biological steady states may owe their stability to oscillations in less important cell functions. Gross fluctuations of important parameters, such as occurs frequently in mixed-population systems with a predator/prey relationship, are, of course, highly undesirable, for most purposes, and the conditions likely to produce such oscillations need to be understood by all workers in the field. The study of transient and oscillatory behaviour in continuous-culture systems is not only a necessity for the design and efficient running of commercial systems, but also a powerful research tool for the study of regulatory mechanisms in microorganisms.

Transient and Oscillatory States of Continuous Culture

217

Nomenclature D

.f(s) J_

Ki KLa Ks N~

QCOz Qo~ R] S SO~ S1

SR t T X X, S, l~ X XI YJ

y~ Y # 2

Dilution rate, T-1, n-dimensional vector of vector functions, Liapunov first-approximation matrix, Kinetic constant in substrate-inhibition model, M L-3, Overall mass-transfer coefficient, 7-1, Saturation constant for Monod relationship, ML-3, Matrix of higher-order terms in linearised model, Specific carbon dioxide production-rate, 7~ 1, Specific oxygen uptake-rate, T 1, Reaction rate, M L - a T - 1 , Substrate concentration, in culture, M L-3, Fixed-value substrate concentrations, M L 3, Feed substrate concentration, M L 3, Time, T, Temperature, K, Biomass concentration in culture M L-3, Steady-state values, n-dimensional vector of state variables (xl, x2, ...), n-dimensional perturbation vector, Concentration of component j in continuous culture, M L 3, Concentration of component j in the feed stream to the continuous culture, M L 3, Cell yield-factor, dimensionless, Specific growth-rate, T~ 1, Maximum specific growth rate, T- 1, Eigen-value, T- i.

References Andrews, J. F.: Biotechnol. Bioeng. 10, 707 (1968). Aris, R.: Elementary Chemical Reactor Analysis. Englewood Cliffs, N. J.: PrenticeHall, Inc. 1969. Benedek, A. A.: Ph.D. Thesis. University of Washington 1970. Bergter, F., Noack, D.: Stud. Biophys. I, 257 (1966). Bilous, O., Amundson, N. R.: A. I. Ch. E. J. 1, 513 (1955). Canate, R. P.: Biotechnol. Bioeng. 12, 353 (1970). Chance, B., Schoener, B., Elsaesser, S.: J. Biol. Chem. 240, 3170 (1965). Contois, D. E., Yango, L. D.: Abstr. Papers. Am. Chem. Soc. 148th Meeting, 17 Q (1964). Curds, C. R., Cockburn, A.: J. Gen. Microbiol. 66, 95 (1971). Dawson, P. S. S.: Can. J. Microbiol. 11, 893 (1965). Dean, A. C. R., Moss, D. A.: Biochem. Pharmacol. 20, 1 (1971). Degn, H., Harrison, D. E. F.: J. Theoret. Biol. 22, 238 (1969). Degn, H., Harrison, D. E. F.: Biochem. Biophys. Res. Commun. 45, 1554 (1971). Edwards, V. H.: Biotechnol. Bioeng. 12, 679 (1970). Edwards, V. H , Ko, R. C., Balogh, S. A.: BiotechnoL Bioeng. 14, 939 (1972). Fredrickson, A. G., Jost, J. L., Tsuchiya, H. M., Hsu, P.: J. Theoret. Biol. 38, 487 (1973).

218

D . E . F . HARRISON and H. H. TOPIWALA

Fredrickson, A. G., Megee. R. D., Tsuchiya, H. M.: Adv. Appl. Microbiol. 13, 419 (1970). Frenkel, R.: Biochem. Biophys. Res. Commun. 21,497 (1965). Friedly, J. C.: Dynamic Behaviour of Processes. Englewood Cliffs, N. J.: PrenticeHall, Inc. 1972. Fuld, G. J., Mateles, R. I., K usmierick, B. W.: Soc. Chem. Ind. (London), M onogra ph 12, 54 (1961). Ghosh, A., Chance, B,: Biochem. Biophys. Res. Commun. 16, 174 (1964). Gilley, J. W., Bungay, H. R.: Biotechnol. Bioeng. 10, 99 (1968). Goodwim B. C.: In: Temporal Organisation in (?ell. London: Academic Press 1963. Harrison, D. E. F.: Ph.D. Thesis. University of London 1966. Harrison, D. E. F., Pirt, S. J.: J. Gen. Microbiol. 46, 193 t1967). Harrison, D. E. F , MacLennan, D. G., Pirt, S. J.: In: Fermentation Advances, D. Perlman (ed.), p. 117. New York: Academic Press 1969. Harrison, D. E. F.~ Maitra, P. K.: Biochem. J. 112, 647 {1969). Harrison, D. E. F.: J. Cell Biol. 45, 4574 (t970). Harrison, D. E. F., Loveless, J. E.: J. Gen. Microbiol. 68, 35 (1971) (a). Harrison, D. E. F., Loveless, J. E.: J. Gen. Microbiol. 68, 45 (1971) (b). Harrison, D. E. F.: In: Environmental Control fo Cell Synthesis and Function. Dean, A. C. R., Pirt, S. J., Tempest, D. W., p. 417. Academic Press 1972. Harrison, D. E. F., Topiwala, H. H., Hamer, G.: Proceedings of 4 th Fermentation Symposium. Kyoto in press (1972). Harrison, D. E. F.: Critical Rev. Microbiol. 2, 185 (1973). Harrison, D. E. F.: In Biological and Biochemical Oscillators. Chance, B., Pye, E. K., Gosh, A., Hess, B. New York: Academic Press 1973 (b). Harvey, R. J.: J. Bacteriol. 104, 698 (1970). Hasting, J. W.: Ann. Rev. Microbiol. 14, 297 (1960). Helmstetter, C. E.: In: The Cell Cycle. Padilla, G. M., Whitson, G. L., Cameron, I. L., p. 15. New York: Academic Press 1969. Himmelblau, D. M., Bischoff, K. B.: Process Analysis and Simulations. John Wiley & Sons, Inc., New York (1968). Hlav~i~ek, V., Sinkule, J., Kubicek: J, Theoret. Biol. 36, 283 (1972). Hollman, S., Neubar, J.: Hoppe-Seyter's Z. Physiol. Chem. 348, 877 i1967). Ingraham, J. L.: J. Bacteriol. 76, 75 (1958). Jefferson, C. P., Smith, J. M.: Chem. Eng. Sci. 28, 629 (1973). Johnson, M. J.: J, Bacteriol. 94, 101 (1967). Kjelgaard, N. O., Maaloe, O., Schaechter, M.: J. Gen. Microbiol. 19, 607 (1958). Knorre, N. A.: Biochem. Biophys. Res. Commun. 31,812 I1968). Koga, S., Humphrey, A.: Biotechnol. Bioeng. 9, 375 (1967). Kozheshnik, Y.: Biofizika 16, 270 (1971). Macfarlane, A. G. J.: Dynamical System Models. London: G. G. Harrap & Co. Ltd. 1970. Matetes, R. I., Ryu, D. Y., Yasuda, T.: Nature 208, 263 (1965). Meers, J. L.: Critical Rev. Microbiol. 2, 139 (1973). Meers, J. L.: J. Gen. Microbiol., in print {1973b). Mcgee, R. D.: Ph. D. Thesis. Minneapolis: University of Minnesota 1971. Monod, J.: Ann. Inst. Pasteur 79, 390 (1950). Nagai, S., Nishizawa, Y., Endo, I., Aiba, S.: J. Gen. Appl. Microbiol. 14, 12t t 1968). Ng. H., Ingraham, J. k., Mart, A. G.: J. Bacteriol. 84, 331 (1962). Noack, D.: Z. Allgem. Mikrobiol. 8, 161 t1968).

Transient and Oscillatory States of Continuous Culture

219

Novick, A., Szilard, I.: Proc, Nat. Acad. Sci. U. S. A. 36, 708 (1950). Padilla, G. M., Whitson, G. L., Cameron, I. K.: The Cell Cycle. New York: Academic Press 1969. Perlmutter, D. D.: Stability of Chemical Reactors. Englewood Cliffs, M. J.: PrenticeHall, Inc. 1972. Perram, J. W.: J. Theoret. Biol. 38, 571 (1973). Powell, E. O.: Soc. Chem. Ind. (London), Monograph 12, (1961). Pye, E. K.: Can. J. Botany 47, 271 (I969). Ramakrishna, D., Fredrickson, A. G., Tsuchiya, H, M.: Biotechnol. Bioeng. 9, 129 (1967). Rose, A. H.: In: Fermentation Advances. Perlman, D., p. 157. New York: Academic Press 1969. Ryu, D. Y., Mateles, R. I.: Biotechnol. Bioeng. 10, 385 (1968). Sikyta, B.: Suomen Kemistilehti, A. 38, 180 (1965). Sikyta, B., Slezak, J.: Biochem. Biophys. Acta. 100, 311 (1965). Sikyta, B., Slezak, J., Brookes, R., Heden, C. G.: Studia Biophys. 1, 151 (1966). Sinclair, C. G., King, W. R., Ryder, D. N., Topiwala, H. H.: Biotechnol. Bioeng. 13, 451 (1971). Sinclair, J.: B. Sc. Thesis. Cambridge, Massachusetts: M. I. T. 1964. Standing, C. N., Fredrickson, A. C., Tsuchiya, H. M.: Appl. Microbiol. 23, 354 (1972). Tempest, D. W., Dicks, J. W., Hunter, J. R.: J. Gen. Microbiol. 45, 135 (1966). Tempest, D. W., Hunter, J. R., Sykes, J.: J. Gen. Microbiol. 39, 355 (1965). Topiwala, H. H., Hamer, G.: Biotechnol. Bioeng. 13, 919 (1971). Topiwala, H. H., Sinclair, C. G.: Biotechnol. Bioeng. 13, 795 (1971). Tsuchiya, H. M., Drake, J. F., Jost, J. L., Fredrickson, A. G.: J. Bacteriol. 110, 1147 (1972). Tsuchiya, H. M., Fredrickson, A. G., Aris, R.: Advan. Chem. Eng. 6, 125 (1966). Winfree, A. T.: J. Theoret. Biol. 16, 15 (1967). Yano, T., Koga, S.: Biotechnol. Bioeng. 11, 139 (1969). Young, T. B., Bruley, D. F., Bungay, H. R.: Biotechnol. Bioeng. 12, 747 (1970). Zines, D. O., Rogers, P. L.: Biotechnol. Bioeng. 12, 561 (t970). Dr. D. E. F. HARRISONand Dr. H. H. TOPIWALA Shell Research Limited Sittingbourne, Kent (Great Britain)

CHAPTER 6

The Significance of Microbial Film in Fermenters B. ATKINSON and H . W . FOWLER With 33Figures

Contents 1. Formation of Microbial Film . . . . . . . . . . . . . . . . . . . 2. General Characters of Microbial Film . . . . . . . . . . . . . . . 3. Control of Microbial Film Thickness ............... 4. Kinetics of Microbial Film . . . . . . . . . . . . . . . . . . . . 5. Microbial Film in Fermenters . . . . . . . . . . . . . . . . . . . 6. Future Potential . . . . . . . . . . . . . . . . . . . . . . . . Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

224 232 240 246 260 267 270 274 275

Fermenters may be divided into two groups, depending upon whether the microbial mass appears to be in the form of flocs or films. However, a working hypothesis for the interaction between microorganisms and surfaces suggests that any surface in contact with a microbial suspension will, in time, become biologically active due to the adhesion of microorganisms and that these isolated points of growth eventually unite to form a continuous layer of microbial mass. Although little is known about the factors affecting the adhesion of microorganisms to surfaces, it seems unlikely that the hydrodynamic forces normally found in a fermenter would effect complete removal of the microbial layer, that is, sterilisation of the surface. The performance of some fermenters is dominated by the kinetics of microbial mass in the form of films and Table 1 summarises the characters of a number of fermenters of this type that have commer-

222

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cial application. In the laboratory, microbial film fermenters provide a useful tool for the study of microbial kinetics, since certain operating characteristics are easier to control in a film fermenter than in a conventional stirred-tank fermenter (see section 4). Examples of the use of film fermenters for laboratory purposes are summarised in Table 2. Practical observations of growth in Continuous Stirred-Tank Fermenters (CSTF) often fail to agree with theoretical predictions. Various explanations offered for this discrepancy suggest that the theory has failed to account for incomplete mixing (Iterbert et al., 1956; Sinclair and Brown, 1970), evaporation of water (Topiwala, 1970), submerged surface growth (Larsen and Dimmick, I964; Topiwala and Hamer, 1971) and liquid entrainment and re-seeding from wall growth above the surface (Hamer, 1972). Since the submerged surfaces in any fermenter are likely to be covered by microbial film, it follows that surface films can make a significant contribution to the overall performance of a fermenter, even when it would appear that biomass in the form of microbial floc predominates.

1. F o r m a t i o n of M i c r o b i a l F i l m Since film is the basis of some forms of fermenter and may make a significant contribution to the performance of others, for example, the CSTF, it follows that a knowledge of the factors affecting the adhesion of microbial mass to surfaces is of considerable importance. Despite the ubiquitous nature of biological film, very little attention has been given to the reasons for the formation of microbial film on a submerged surface and the mechanisms whereby microorganisms adhere to the surface. The majority of publications relate to aquatic microbiology and particularly to marine microbiology where the formation of microbial film is considered to be a preliminary to the attachment of higher organisms, a problem of great economic importance in the 'fouling' of ships and submerged surfaces (ZoBell, 1946). Studies of the microbial population in rivers has shown that very few organisms are in free suspension, but are associated with solid surfaces, such as silt particles. For example, Heukelekian and Dondero (1964) report bacterial counts in the Nile of the order of 10--20 x 10 6 per ml, of which only 0.02-0.04 per cent were in free suspension. This effect of solid surfaces has long been recognised; Russell (t89t) pointed out that the bacterial distribution in the Bay of Naples showed much higher counts in the bottom deposits than near the surface.

The Significance of Microbial Film in Fermenters

225

Whipple (1901) observed that bacteria multiplied faster in small containers than in larger ones (Table 3) and suggested that this was due to the availability of oxygen. Similar results (Table 4) were obtained by ZoBell and A n d e r s o n (1936), w h o avoided the problems of oxygen availability by using completely filled, stoppered bottles and terminating their experiments before all the oxygen was consumed. These data are expressed in Table 5 relative to Table 3. Bacterial counts obtained in water stored for 24 hours in partially filled containers of various volumes. Initial count =77 bacteria/ml (Whipple, 1901) Container Volume

Bacterial count (organisms/ml)

1 gallon 1 quart 1 pint 2 ounces

300 900 7,020 41,400

Table 4. Number of bacteria found in sea water, which initially contained 276 bacteria per ml, after two weeks storage in the dark at 16°C in completely filled glass-stoppered bottles of different capacities. The area of glass exposed to the water and the ratio of volume in ml of water to the area of glass surface are also given (ZoBell and Anderson, 1936) Volume of Sea Water in ml

Solid Surface in cm 2

Ratio of mt: cm 2

Average Number of Bacteria per ml

Oxygen Consumed rag/1

14 120 1,225 13,220

37 148 640 3174

1 : 2.64 1 : 1.23 1 : 0.52 l : 0.24

1,863,000 1,070,000 553,000 261,000

3.42 2.53 1.31 0.97

Table 5. Relative specific surfaces, relative bacterial counts and relative oxygen uptakes for containers of various volumes Data of ZoBell and Anderson (1936)

Data of Whipple t1901)

Relative Specific Surface

Relative Specific Surface

Relative Bacterial Count

Relative Oxygen Uptake

Relative Bacterial Count

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1.0

1.0

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2.1 4.1 7.2

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1.6 2.0 4.4

3.0 23.4

11.0

138

226

B. ATKINSONand H. W. FOWLER

the values for the largest container, that is, the one with the smallest surface/volume ratio. The fact that both the bacterial count and the oxygen consumption increased is an indication that the surface/volume ratio was an important factor in the experiments. The results of Whipple (1901) are also included in Table 5 using the same basis for calculation and, since the relative bacterial counts again depend upon the relative specific surface, it is improbable that oxygen was limiting. Thus, the two sets of experimental data indicate that the surface/volume ratio is likely to be an important experimental parameter in determining the performance of a fermenter. ZoBell (1943) confirmed these observations by increasing the surface area by including a number of pieces of thin-walled glass tubing or glass beads within containers. Bacterial activity, as shown by oxygen consumption, was enhanced by the presence of additional surface in the form of glass tubing, but the inclusion of glass beads showed less effect. Further experiments with other materials, such as sand and kieselguhr, showed that increased surface area alone is not necessarily advantageous

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The Significance of Microbial Film in Fermenters

227

but that the distribution of the surface in the nutrient medium is of great importance. Thus, solids in the form of fine particles introduce a large surface area, but settle to a static bed with fine pores which render much of the surface ~inaccessible' to nutrients or organisms. Heukelekian and Heller (t940) investigated the effect of added surface area in a fixed liquid volume, when the substrate concentration was varied, with the results shown in Fig. 1. Escherichia coli was inoculated into glucose and peptone medium of concentrations varying from 0.5 to 100 ppm. Under the conditions of the experiment, growth of the E.coli did not occur when the nutrient concentration was 0.5 ppm, but the introduction of glass beads into medium at this concentration resulted in considerable growth of organisms. The effect of the beads was significant at concentrations up to 25 ppm. According to ZoBell (1943), the beneficial effect of solid surfaces in dilute media results from concentration of nutrients at these surfaces. Kriss

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228

B. ATKINSONand H. W. FOWLER

and Markianovich (1959) suggest that, not only does concentration of organic matter occur at the solid-liquid interface, but that breakdown of large molecules may also take place in this region, resulting in forms more easily assimilable by heterotrophic microorganisms, for example, the denaturation of proteinaceous material at phase boundaries. ZoBell (1943) points out that there is a possible further advantage to cells adjacent to a surface in the retardation of diffusion of metabolites and exo-enzymes from the interstices between the cell and the surface. These interstices may also aid the production of optimum oxidationreduction or other physico-chemical conditions, as well as providing protection from Brownian movement or hydrodynamic shear forces (Fig. 2). Once the microbial film is established, adhesion may be increased in various ways (ZoBeli, 1943), for example, by the formation of a slime film or the secretion of cement-like materials, some of which show considerable chemical resistance. Certain varieties of organisms form 'stalks' or 'holdfasts' which are responsible for the attachment of the cell to a surface. While the above sequence of events may offer an explanation for the formation of film on new surfaces in dilute solutions, microbial film may develop in conditions where there is no substrate limitation. Nordin et al. (1967) investigated the adhesion of C h l o r e l l a to glass and measured the charges on the algal and glass surfaces. Electrolytes were used to change the charges and some results are given in Fig. 3 and Table 6.

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229

The Significance of Microbial Film in Fermenters Table 6. (Nordin et al., 1967) Reference to Figs. 3 and 5

FeC13 concentration

Charge on algal and glassware surfaces

Algal adhesion to glass

1

0 to 5 x 10- 6 M

Very little adhesion of algae to glass

2

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Chlorella less positive; glass strongly positive Chlorella slightly negative; glass strongly positive

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Ferric chloride was used as the electrolyte since ferric iron is necessary for the growth of this organism. When the charges were opposite, adhesion of the algae to the glass surface was very strong, but could still be considerable when charges were of the same sign, provided that there was a sufficient difference in zeta potential. The importance of ionic strength on the adhesion of microorganisms to solid surfaces is endorsed by experiments carried out by Meadows (1964). Freshly recovered sea sand was washed with solutions of electrolytes of the same ionic strength as sea water (0.7 M) and with non-electrolytes of the same osmotic pressure as sea water (1.0M). Removal of bacteria was measured by determination of the optical density of the washings (Table 7). Most bacteria were removed by distilled water and by the solutions of non-electrolytes, with sodium sulphate as the only electrolyte removing substantially more organisms than sea water.

230

B. ATKINSON and H. W. FOWLER

Table 7. Optical densities of solutions of electrolytes and non-electrolytes which have been shaken with sand (Meadows, 1964) Test Solution

Optical Density of Washings relative to Sea Water 3.49 2.89 2.24 2.18 1.61 1.60 1.53 1.53 1.50 1.00

Distilled water Glycerol Sucrose Na2SO4 MgCI, NaCI MgSO4 KC1 CaCI2 Sea Water

Daniels and Kempe (1966) used six bacterial species to study adsorption of bacteria on to anion and cation exchange resins. Adsorption data for Pseudomonas ovalis on an anionic exchange resin are given in Fig. 4. These data indicate total adsorption at a pH of 3.9; below this value little adsorption occurs, while at larger pH values the extent of adsorption is insensitive to pH. Since most organisms exhibit a positive charge below their isoelectric point, the lack of adsorption at low pH coincides with the region of lowest zeta potential. The reduced adsorption at values of pH above 3.9 is difficult to interpret from these data, due to the changes in ionic strength which results from the experimental procedure and lack of data on the effect of pH on the charge on the ion exchanger.

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4

5

6

pH

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8

9

10

11

12

Fig. 4. Equilibrium adsorption of Pseudomonas ovalis onto an anion exchange resin (based on the data of Daniels and Kempe, t966)

The Significance of Microbial Film in Fermenters

231

Thus bacteria are adsorbed on to anion or cation resins, depending upon the pH being above or below the critical pH described by the authors as an apparent isoelectric point. A total of 96 different bacteria were isolated by ZoBell (t943) from marine materials and used to inoculate sea water into which sterile, chemically clean glass slides were inserted. Large numbers of bacteria were found on slides from 29 of the cultures, virtually no bacteria were found on slides from 47 cultures and a variable number of bacteria on slides from 20 cultures. No relationship was found between the variation in the attachment propensities and characters such as Gram-reaction. Zvyagintsev (1959) tested some 30 different organisms for adsorption on 5 different glasses and found no clear pattern; some species were not adsorbed by glass at all, while others were adsorbed strongly. Others were adsorbed only by certain glasses and adsorption could be affected by using glass cleaned only by washing or which had been defatted by storage in 96 % ethanol. The results of these workers indicate that there can be considerable variation in the adhesive properties of different organisms on different surfaces. However, Nordling et al. (1965) observed that tissue cells adhered readily to glass that had been dried with ethanol-ether and sterilised by dry heat, whereas attachment was poor if the glass was steam-sterilised in an autoclave at 120"C. On the other hand, adhesion was good if the glass was autoclaved at 120 °C under water rather than in steam. This discussion began with a hypothesis which suggested that any submerged surface exposed to microorganisms will be covered, eventually, by microbial film. It has been shown that published observations account for the adsorption of organic materials which encourage surface growth of microorganisms in media of low nutrient concentration. At higher nutrient concentrations, organisms can still become attached to the surface as a result of a difference in charge between the organism and the surface. Such charges may be influenced by the conditions prevailing in the environment, for example, pH, electrolyte concentration and properties of the surface. These charges can set up considerable adhesive forces, as indicated by the results of Nordin et al. (1967) shown in Fig. 5 where, given appropriate conditions, the Chlorella was not washed off the glass surface when the average liquid velocity through the test cell exceeded 3 m s- 1. In other circumstances, the majority of the algae was removed by an average liquid velocity as low as 1.6 x 1 0 4m s- 1. Hence, while fluid forces alone will not control microbial film, the evidence suggests that an appropriate selection or treatment of surface, control of ionic strength of the environment and a satisfactory fluid

232

B. ATKINSON and H. W. FOWLER 100F

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velocity might result in a situation in which microorganisms will, or will not, adhere to the surface. However, complex biological and physico-chemical factors are involved and there is no simple explanation for the phenomenon of adhesion of microorganisms to surfaces, but it is clear that such knowledge is essential if microbial film is to be controlled or exploited in fermenters.

2. General Characters of Microbial Film Measurements have been made of the thicknesses of the microbial films which occur in various biological reactor configurations and some results of a number of studies are given in Table 8. To simplify comparison, the weights of wet film have been converted to kg m- 2 where necessary, since this is approximately equal to the film thickness expressed in millimetres. The numerical values that have been reported cover a considerable range from approximately 3 × 10- 5 to 4 kg m- 2 but may be divided into four groups as indicated in Table 9. These groups show a rough correlation between the weights and the general characters of the film. The thickest films arise where mixed populations of microorganisms are involved, particularly in units for waste water treatment. Such films are commonly zoogloeal in character and may contain a variety of organisms. For example, Unz and Dondero (1967) obtained samples of

~ (kgm-a) b

Film thickness controlled by scraping above fibreglass meshes of various thicknesses Maier, Behn and Gates (1967)

Dissolved oxygen and substrate usage constant when wet film thickness exceeds 0.07 mm Kornegay and Andrews (1968)

1.4

1.4 mm

25--1000 mg 1- ~

0.48

0.48

Wet film thickness

Simulation of the trickling filter Glucose and minerals Mixed population Plastic plate

27---440 mg 1- ~

0.25

Kinetics of fixed film biological reactors Glucose and minerals Mixed population Plastic reactor annular form with rotating drum Wet thickness and dry weight of unit volume of wet film Up to 0.25 mm 0.095g cm-S

Biological Film Reactor

Film Fermenters

Atkinson, Daoud and Williams (1968) Atkinson and Daoud (1970)

Tomlinson and Snaddon (1966)

Respiration ceases approximately 0.2 mm below film surface

3.8

Film thicknesses controlled by scraping

1.15

BOD 200 mg 1- i

2.0

2,0 mm

Weight of wet film in tube and mean wet film thickness 139--467 g 1.15 3.8 mm

Oxidation of sewage by films Sewage Mixed population Perspex tube coated with kieselguhr

Rotating Tube Reactor

Up to 10000 mg 1- ~

0.073

0.073 mm

Wet film thickness

Diffusion effects within microbial films Glucose and minerals Mixed population Roughened Aluminium glass plate plate

Biological Film Reactor

" The term "mixed population" is used to describe a mixed population of organisms in equilibrium with the environment, that is, the fermenter is operated under non-aseptic conditions. Values for film weights and thicknesses have been converted where necessary to wet film weight in kg m- 2 for comparison. If it is assumed that the density of microbial film is I000 kg m - a, wet film weight (kg m - 2)=film thickness (mm). Wet film weights have been calculated from dry film weights on the basis of 9.5 % w/w dry solids [Kornegay and Andrews 11968), Rincke and Wolters ( 1971)]. Bacterial counts have been converted to wet weights on the assumption that each cell weighs 10- ~e g.

Reference

Inlet Substrate Concentration Remarks

'Fypieal Values ij" of ltold-up i Converted | to Wet Film ] Weight

quoted

Methods of Quantifyin9 Microbial Hold- Up

Objective of Work (Title of Publication) Substrate Organism" Surfaee on which Film Grown

Annular Biological Reactor

Table 8. Summary of Experimentation on Microbial Films

O

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Reference

Inlet Substrate Concentrat ion Remarks

Typical Values of Hold-up

,{kg m--2) u

Converted to Wet Film Weight

"As quoted

1,7

BOD 250 300 mg 1- J 0,24 0.51 mm on plastic packing 1.3--1.7 mm on slag and rock Bruce and Merkens ( 1 9 7 0 )

0.24

Mixed population Slag, rock and various plastic packings Mean thickness of wet film and average weight of wet film per unit bed volume 0.24 1.7 mm 2,9- 9.5kgm 3

Oryanism ~ Surface on which Film Grown

BOD up to 3000 mg t Dry film approximately 9.5 % w/w of wet film Rincke and Wolters (1971)

Bruce, Merkens and Macmillan (1970)

Mixed population Plastic in form of tubes and corrugated sheets Wet Coat Thickness (WCT) which includes Film Flow Thickness (FFT) also dry weight of surface coating (TS) WCT 0.35--3,57kgm -~' FFT 0.232-----1.02 kg in - a TS 0.0007--0.138 kg m - 2 (WCT-FFT) 0.118----2.55 kg m e 0.118--2.55

Molasses and mineral

Technology of plastic media for trickling filters

Trickling Filters

BOD 250---300 mg 1 ~ Further report on work by Bruce and Merkens (1970)

Up to 2,0

1 mm on plastic packings 1---,2 mm on slag and rock

Mixed population Slag, rock and various plastic packings Mean thickness of wet film

Sewage

Substrate

Methods of Quant(/),in~ Microbial Hold- Up

Research and development on high-rate biological filtration Sewage

Trickling Filters

Studies of highrate biological filtration

Trickling Filters

Packed Bed Fermenters

Objective of Pcbrk ( Title of Publication)

Table 8. (continued)

330 m g l

Monadjemi and Behn (1971)

180

0.705

6.7 mg c m z

Glucose and minerals Mixed population 4 inch diameter tube, packed with table tennis balls Dried slime weight per unit area of surface

Oxygen uptake in a model trickling filter

Packed Bed

Borchardt (1971)

0.194

18gft 2

Weight of scrapings of wet film

Mixed population Rotating discs, 4 ft diameter

Sewage

Biological waste treatment with rotating discs

Bio-Disc

Rotating Disc Fermenter

©

=

z

> ~q 7:

i,o 4~

The Significance of Microbial Film in Fermenters

235

oo~

"~

"~ o -~

x

0

"O

Fn~

o ×

I

o

'.,0 ~ - ~

~ -

.~

2~

~ ....

~

06

0

-

~

~

~ ~ ~ ~

,......

~

.•

236

B. ATKINSON a n d H. W. FOWLER

. ~

~,

. ~ "--

.o-

,~ ~,

~-.~

?

0

~

o

~

~._

0

~,-~ ~_

~

0 -~

~

x

o 0

m

o ~

._ "a

0 b~

--

d ! ° ~.~ B

"~ ,,,

o~

~=

? o

0 x

0

0

>

0

O ~,.o

'aO

#:~- ~,-

= = =

N "= .~

.;%

=~

"z

~'~

~ , ~ -" ,.

"~ ~

.~'.~

.~

%

237

The Significance of Microbial Film in Fermenters Table 9. Groups of Microbial Films summarised from Table 8 Group

Description

Film Thickness (mm)

I II

Uncontrolled zoogloeal film Zoogloeal film, subject to mechanical or hydrodynamic control Pure cultures in CSTF Casual deposition

0.2 --4.0 0.07 --0.2

III IV

0.001--0.01 < 0.001

microbial mass from 51 waste water treatment plants and were able to isolate 203 viable bacterial strains. In all of the samples, more than 60% of the organisms were zoogloeal-forming strains and in some cases 80--100 % of the organisms were of this type. The second group contains films of similar origin, but the film thickness is controlled, either mechanically or by liquid shear forces. Films in the third group are those found in CSTF, usually with pure cultures, and often represent only a monolayer of organisms. Munson and Bridges (1964) point out that such films are usually invisible in the presence of suspensions, but that wall films can often be seen to cover glass surfaces once the suspension has been removed. On some occasions, these authors found that massive 'sticky' wall growths of

03

E5

~20

ppm

o

~2 t

0ppm J

150 100 50 50 100 150 200 Above film In film Distance ( ~ m )

Fig. 6. Dissolved oxygen profiles in a microbial slime at two different glucose concentrations (Bungay et al., 1969)

238

B. ATKINSONand H. W. FOWLER =

o

80

5 E

60

g cl 40

/

-~ 20 I

~ o

O.l

J

L

i

L

[

1

02 0.3 0./. 05. 0.6 0.7 0.8 Mean thickness of film (mrn)

i

0.9

Fig. 7. Effectof film thickness on substrate usage (Tomlinsonand Snaddon, 1966) Escherichia coti could occur. It is of interest to note that this work was concerned with a study of ~take-over' by prototrophic mutants in the culture and that the proportion of prototrophs in the wall-adhering organisms was 10 or more times greater than among those in suspension. This suggests a variation in adhesion between auxotrophs and prototrophs resulting in different film thicknesses. Films in the fourth group arise from casually deposited organisms which lead to isolated colonies and thence to approximately monolayer films in due course. Since microbial film thicknesses of several millimetres are common, Bungay et al. (1969) have used a microprobe technique to determine oxygen profiles through such films (Fig. 6). These profiles indicate that respiration may cease in the depths of thick films and that the depth where this occurs is between 50 and 150 lam depending upon the substrate concentration. This observation is confirmed by the fact that the rate of consumption of substrate attains a constant value, while the film thickness continues to increase. Results from Tomlinson and Snaddon (1966) and Kornegay and Andrews (1968) are given in Figs. 7 and 8 and these indicate an 'active' film thickness of the order of 70--100!am. The agreement between these three sets of results is worth noting, since it will be seen from Table 8 that the investigations used completely different reactor configurations, nutrient media and experimental techniques, while the organisms were mixed populations derived from natural sources in different countries. The dissolved oxygen profiles given by Bungay et al. (1969) provide an indication of microbial activity within a thick film at two different substrate concentrations. The results of Tomlinson and Snaddon (1966) and Kornegay and Andrews (1968) serve a similar purpose for film that is increasing in thickness. While the measures of substrate concentration used in each case are not

The Significance of Microbial Film in Fermenters

239

;000

~-

d

~, 250

0

50

100 150 200 250 Film thickness (~.m)

300

Fig. 8. Effect of film thickness on the rate of substrate removal (Kornegay and

Andrews, 1968) strictly comparable, it is interesting to note that there is an increase in the depth of penetration of the nutrient into the microbial film as the concentration outside the film is increased; this trend is illustrated in Fig. 9. This 'Penetration Depth" is a parameter of some significance, since it represents the distance through which mass transfer takes place to maintain the organisms in an active state. Hence there is likely to be an optimum film thickness, approximately equal to the penetration depth, which leads to the maximum rate of substrate uptake for a particular substrate concentration at the interface between the microbial film and the nutrient medium. 150

fx f f

:L ~100 ¢-

/ f~

A~

/

f

1 /

f f ~o x j~

"6 50

J x Bungay

etat,(1969)

Kornegay and A n d r e w s (1968) A Tom{inson and Snaddon (~966) •

100

200 300 400 500 Inlet substrate concentration (mg [-1)

Fig. 9. Penetration depths into microbial film

240

B. ATK1NSONand H. W. FOWLER

When the film thickness exceeds the penetration depth, there is no advantage in terms of substrate uptake but there may be a considerable disadvantage since, as the film/continues to grow, gaseous respiration products in the depth of the film come out of solution and form bubbles of gas (Fig. 10). These impair the adhesion between the

'Solid' microbial fi~m

Liquid medium

death,autolysis a n d \ ]'~

gaseousproducts~ ~ l

Gas bubbtes ~ 1

Fig. lO. Conditions within a 'thick" microbial film

microbial film and the support surface, so that ultimately the film falls away under the action of its own weight or as the result of hydrodynamic forces. The residual layer starts to grow, but it is usually of low viability and the resulting film is often thinner than that present originally. This reduction in film thickness probably results from the presence of dead organisms or the breakdown of film due to endogenous respiration, which weakens the adhesive forces.

3. Control of Microbial Film Thickness It is apparent from the discussion in the previous sections that the presence of microbial film is usually advantageous, except in some laboratory fermenters where it can lead to complications in the interpretation of experimental results. In any event, it is necessary either to control the film thickness to a value that does not exceed the penetration depth or to aim at complete elimination.

The Significance of Microbial Film in Fermenters

241

Microbial films are likely to be self-regulating to some extent, since excess film sloughs off. In some circumstances, this situation may be completely acceptable and, in general, this is dependent upon whether the fermentation is a batch or continuous operation. When the fermentation is under batch conditions the accumulation of film usually presents no problems, since the process is in an unsteady state. In fact, there are advantages in transferring microbial mass from the film into suspension (Chue, 1973). The results of Bungay et al. (1969), illustrated in Fig. 6, show that an oxygen profile exists through the film and that there must also be a similar variation in substrate concentration. Hence, if a portion of the film is detached, the organisms may be removed from an environment of low substrate concentration to one of higher concentration in the bulk of the liquid. The only factor which may be disadvantageous to a batch fermentation is that biochemical products from the depths of the film are released, which may have a deleterious effect on viable organisms or on the medium. In the case of continuous fermentation, the objective is to attain a steady-state situation with an equilibrium film thickness, whereby the accumulation by deposition or growth is balanced by removal of film. Clearly, control by self-regulation is not acceptable, since the portions of film that are detached may be large and cause blockages elsewhere in the fermenter; furthermore, this random breaking away leads to variable film thicknesses. In addition, there is the possibility of the same problem of released products referred to in connection with batch fermenters. These disadvantages would prevent the use of a self-regulating mechanism in fermenters operating under aseptic conditions and present design and operational problems in any fermenter, since they can lead to considerable variation about a mean performance. Only in waste water treatment plants is self-regulation of practical value since the large size of trickling filters moderates the effect of the variation in film thickness and the open structure of the packing reduces the risk of blockage. In addition, removal and break-up of film in trickling filters is assisted by the presence of higher organisms, such as worms, insects and larvae (Hawkes, 1963). In view of the desirability of controlling the microbial film thickness and the disadvantages of self-regulation, it is useful to consider alternative methods of control. The work of Nordin et at. (1967), which was discussed in section I, showed that microbial mass could only be swept off surfaces by liquid shear forces when the environment was adjusted to reduce the adhesive forces. Fig. 11 illustrates an impeller taken from a 20-1itre fermenter after 3 months of continuous operation with a mixed population of microorganisms with zoogloeal properties; the impeller was rotating at 500 r.p.m. Growth also occurs in

242

B. ATKINSONand H. W. FOWLER

Fig. 11. Growth on an impeller (rotation speed 500 r.p.m.) transfer lines containing flowing fluids, both in industry and in the laboratory. These observations indicate that the hydrodynamic shear forces normally encountered in a fermenter cannot control the thickness. Mechanical control by scraping of the surface has been used effectively (Maier et al., 1967), (Atkinson et al., 1968) and presents no operational problems when used under non-aseptic conditions. The use of such mechanical procedures under aseptic conditions is much more difficult, but rotating rubber wipers have been used by Anderson (1953) and a similar method with an oscillating movement has been described by Northrop (1954). There are advantages in terms of reliability, flexibility and simplicity if the mechanical action can be provided without recourse to direct me-

The Significance of Microbial Film in Fermenters

I°° O°°o o

o

o o

o

oooo°

o

o°

o oo o o

° °o o o o o o oo o o °o o o o o o o o

o Oo

o

243

o o

Fig. 12. CSTF containing inert particles

T

f

,.

0

0 0 0

o O°o°g ~00c~ ~)Ooo b ~

0000%

o

0

_

o

v~ 0 0~,~

~oooO~

"0 O0 U 0 ~ 0 0 O 0 V_ VO000~

o o 8 oo

00°0

0 0 0o

o 0

o

o 0

0 0

0

0

0

OgOo ~

0

(1}

(2)

(3)

Fixed bed

Fluidised bed

Elutriotlon

(2)

(3)

log.,4P

log flow rote

Fig. 13. Effect of flow rate on the particles in a tubular fermenter

244

B. ATKINSONand H. W. FOWLER

chanical linkages. This may be brought about by the abrasive forces arising from physical contact between solid surfaces and such contacts may be achieved by using relatively small, discrete, biochemically-inert particles as the support surfaces for microbial growth. If these particles are maintained in suspension, frequent collisions between the particles cause the biological film to attain a thickness that is essentially constant. Under these conditions, 'excess' microbial film is removed mechanically from the surfaces and is transferred as flocs to the liquid phase. Two arrangements are possible for fermenters of this type; a quantity of the inert particles can be added to a CSTF configuration (Fig. 12) or alternatively the particles can be arranged in the form of a bed and fluidised (Fig. 13). Discussion in section 1 suggested that control of film by physicochemical methods might be feasible. Clearly, there are problems associated with the proposal, since the conditions that are required may be inappropriate for the fermentation, but preliminary experimentation has shown some interesting results. A CSTF was operated with a mixed population of organisms with zoogloeal characteristics and the vessel included a baffle system that could be removed, so that individual baffles acted as small test surfaces for the determination of microbial film thickness. Table 10. Microbial film thicknesses obtained in a CSTF Material of surface

pH

Film thickness (glnj

Glass Glass Glass PTFE Stainless steel

7 9 5 7 7

891 194 3 89 88

The fermenter was operated at three different conditions of pH with glass baffles and at one pH level with PTFE and stainless steel surfaces. To ensure that there was a minimum level of adhesion, glass beads were included to provide continuous attrition. Fig. 14 illustrates the baffle system with growth at pH 7 before the beads were used. Fig. 15 was photographed after the beads had been brought into use and when the pH was 9. The results are summarised in Table 10 and confirm the influence of the physico-chemical conditions of the environment on the thickness of the film. Of particular interest are the low values obtained for FI'FE and stainless steel compared with glass at the same pH.

The Significanceof Microbial Film in Fermenters

245

Fig. 14. Extensivegrowth over glass slides and frame (pH=7, no beads) The results for different pH values illustrate the influence of an alteration in one of the factors contributing to the equilibrium film thickness. In the presence of the beads, the forces of attrition remain the same, but there is a reduction in the accumulation of film by growth, due to the change in pH. It is possible, therefore, that a fermenter utilising additional surface, with some degree of attrition taking place and with physico-chemical control of film thickness, could have great advantages in terms of the power requirements to achieve film control, as well as increased flexibility as regards configuration. It has to be borne in mind that few of the methods of control described above lead to a predetermined film thickness and that, at present, the objective is the attainment of an equilibrium film thickness of unknown proportions which leads to steady-state operation in continuous fermentation.

246

B. ATKINSONand H. W. FOWLER

Fig. 15. Growth over glass slides and frame (pH = 9, beads in suspension)

4. K i n e t i c s o f M i c r o b i a l F i l m Reference has been made in section l to the work of Whipple (1901), ZoBell and Anderson (1936) and ZoBell (1943) which showed that the numbers of microorganisms that grew in containers of various sizes increased as the surface/volume ratio was increased. The effect of surface on bacterial activity was shown by the relative oxygen uptake (Table 5) whence it can be inferred that increase of surface leads to increased uptake of nutrient, that is: R r oc A (1) where R r is the rate of nutrient removal in the fermentation vessel and A is the area of biologically active surface. This relationship has been confirmed by Atkinson (1973); surface area in a CSTF was increased by the introduction of glass ballotini and a nutrient medium was used which contained excess glucose. Results are given in Fig. 16 and indicate a relationship between substrate uptake and surface area that is essentially linear. Although it is satisfactory to consider the uptake of substrate by microbial flocs in terms of a specific rate of substrate removal (that is, to express results on a basis of unit microbial mass), it is more con-

The Significance of Microbial Film in Fermenters

247

1,0 0.8 0.6 fr O.Z, o

Fixed surfaces

0.2

Experimental

~

I . . . . . 1,.

results

L=O25mm I

I

2000 4000 6000 8000 Total surface in Fermenter (cm2)

Fig. 16. CSTF with particles added

effect of area

venient to use unit area of support surface in the case of microbial film. Thus: N -- Rf

A

(2)

w h e r e N is flux o f s u b s t r a t e at the ' s o l i d ' - l i q u i d interface.

- -

"T 4:

1250

~I000

.~. 500

2~

2so I

~

I

l,

t00 200 300 400 Substrate concentration (mg 1-1)

5~ 500

Fig. 17. Effect of concentration on the rate of substrate removal by a microbial film (Kornegay and Andrews, 1968)

Data obtained by Tomlinson and Snaddon (1966) and Kornegay and Andrews (1968), shown in Figs. 7 and 8, indicate a relationship between the uptake of substrate and the film thickness, so that: 1 N = g(L).

(3)

1 g(X1 ,X2,X3,..., Xn) represents a known or unknown function of xl,x2,x3, etc., which in this case defines N.

248

B. ATKINSONand H. W. FOWLER

Kornegay and Andrews (1968) also found that the rate of substrate removal depended on the concentration of the substrate (Fig. 17), that is: N = g(C*)

(4)

where C* is the substrate concentration at the 'solid'-liquid interface between the microorganisms and the aqueous solution. From the complex dependence of substrate uptake on film thickness and concentration, together with the concentration profile (Fig. 6) measured by Bungay et aI. (1969), it may be suggested that the uptake of substrate by microbial film involves both diffusion and biochemical reaction. A model for a single cell has been proposed by Powell (1967) which was based on the principles of diffusion with biochemical reaction and employed a series model, with a metabolic region surrounded by a diffusion zone. While such a model will provide an adequate theoretical description of the situation, difficulties arise since it becomes necessary to invoke geometric parameters with no physical analogue and, as a result, such expressions are inconvenient in quantifying the problem. Hence, a more fruitful approach is one based upon simultaneous diffusion with biochemical reaction and it is reasonable to anticipate that rate equations resulting from a model based on these principles, and which can be shown to describe the overall rate of substrate consumption by groups of cells in the form of flocs or films, will also describe the single-cell situation of the Powell model. The reactions involved in microbial and biochemical systems have been shown to follow empirical equations that have become well known, for example, the Michaelis-Menten equation for enzyme kinetics (Laidler, 1958), the Monod equation for microbial growth (Monod, 1949), and the equation describing 'active" transport by bacterial permeases (Cohen and Monod, 1957). All of these equations have the form: r -

~C

(5)

3+C

where r is rate of substrate uptake per unit area, C is substrate concentration and e and/~ are rate equation coefficients. Commonly, such equations are approximated, for example: if

C>0

r=~

C_=0

r=0.

(6)

Substantial inaccuracies may result from the use of such approximations. For example, if a concentration profile within a microbial film (Fig. 18), curve [a]) was obtained using the technique of Bungay et at. (1969), the local rates of reaction (Fig. 18, curves [b] and [c]) could

The Significance of Microbial Film in Fermenters

l

249

(¢) "-,\ \

\

\

XXb()

\

0

x -------,-

p

Fig. 18. Concentration and local rate of reaction profiles within microbial film. Curve (a) based on experimental concentration profile observations of Bungay et al. (1969) Curve [b) local rate of reaction based on equation (5) Curve (c) local rate of reaction based on equation (6)

§

Increasmg C

c o u C~

,~ c ~

pl P2 P3 P4 P5 Distance into microbial f i l m

Fig. 19. Concentration profiles in microbial film (p~, p2, etc. represent penetration depths into the film)

250

B. ATKINSONand H. W. FOWLER

be calculated using either equation (5) or (6). The flux of substrate entering a film is given by the integral under curves [b] and [c] of Figure 18. The difference between these two integrals can be very large and this emphasises the need for caution in the use of approximations such as equation (6) in problems involving diffusion with biochemical reaction. The dependence of N on L and C* (equations (3) and (4)), together with the observations of Bungay et al. (1969) shown in Fig. 6, suggests that Fig. 19 may represent the form of the concentration profiles obtained as the bulk concentration of substrate in the liquid (and hence C*) is increased. The intercepts between the concentration profiles and the abscissa (pl, p2, etc.) represent the penetration depths referred to in section 2 (Fig. 9).

C* / V i a b t e micro-organism / [nterceHular gel M e t a b o l i c zone _Transport zone

Support surface

I }

L

x L

Substrate so[ution

Fig. 20. Model for a microbial film

It follows that only a mathematical solution of the complete problem is of any real utility. Unfortunately, this involves the numerical solution, or an analytical solution based upon infinite series, of a second order, non-linear differential equation. While such solutions are formally correct, they are of limited value for design purposes or in the interpretation of experimental data. Consequently, it is desirable to seek a functional relationship that will describe adequately the numerical solution over the full range of the independent variables. In order to describe the problem in mathematical terms, it is necessary to set up a physical model and Atkinson and Daoud (1968) propose a model (Fig. 20) in which individual organisms consist of two zones,

The Significance of Microbial Film in Fermenters

251

an outer transport zone and an inner metabolic zone. The organisms are considered to be discrete and viable and to be dispersed homogeneously throughout a biochemically inert, intercellular gel structure. This is analogous to the situation in some chemical processes, where the reactions are carried out using heterogeneous catalysts (highly porous particles, with large internal surface areas coated with catalyst). Thus both systems consist of a network of inter-connected inactive channels, through which the reactants must diffuse before reaching the 'active' surface where the reaction occurs. The theory of heterogeneous catalysis is well-established, so that use may be made of the analogy to describe diffusion with biochemical reaction. Providing that a single limiting substrate is involved, a differential mass balance may be applied to the microbial film of Fig. 20, in which molecular diffusion of the substrate is equated with its removal by the microorganisms. This leads to: dZC De ~fx2 = a r

(7)

where De is an effective diffusion coefficient. Now r is given by equation (5) so that: dZ C De d x 2

ao~ C f l . + ~ - O.

(8)

Although the mechanism of transport in the outer zone of the organism may be unknown, the use of equation (8) can be justified. If entry is due to active transport by permeases, an equation of the form of the second term has been shown to apply (Cohen and Monod, 1959). In the event that transport in the outer region is by molecular diffusion, this zone can be included in the first (diffusion) term and the second term then describes the conditions in the metabolic zone, where ~he Michaelis-Menten equation can be assumed to apply. Boundary conditions for the situation shown in Fig. 20 are: x=L

x=0

C = C*

(9)

dC --=0. dx

The first condition specifies the substrate concentration at the 'solid'liquid interface and represents the concentration to which the film is exposed, while the second indicates that the support surface is impermeable to the substrate. The solution to equations (8) and (9) is more convenient if expressed in dimensionless terms, since this enables the effective number of parameters

252

B. ATKINSONand H. W. FOWLER

to be reduced from six to three. Hence ec uations (8) and (9) become: d2f dX 2

M2f 1+ Bf

X=I

f=l

- 0

(lO)

df

x=o j-x=O where f =

C;

X

= ZL. M=L

and

a~

B

fl

The parameter M is a dimensionless microbial film thickness and B is a dimensionless substrate concentration. A further convenience can be obtained by defining two biological rate equation coefficients, whereby: k2 = ~f~e'a:~ 1

(11)

k3=~.

(12)

M = k2L,

(13)

B = k3 C*

(14)

Hence:

so that k2 has dimensions of L- 1 and k3 has dimensions of M - 1L 3. The dimensionless parameters k z L and k3C* defined in equations (13) and (14) are a direct consequence of the system of equations and their components k2 and k3 delineate the biological characteristics involved, while L and C* are physical variables. In a particular case, C* can be imposed arbitrarily and the control of L was discussed in section 3. In the absence of any 'solid'-phase diffusional resistance, the substrate concentration throughout the film is uniform and equal to C* so that the rate of uptake of substrate is given by: C*

N* =- (aL) f l + C * klLC* - 1+k3C*

where

kl -- a~

ft.

(15) (16)

(17)

The Significance of Microbial Film in Fermenters

253

Equation (16) can also be written as:

k3C*

(18)

N* ~- Nmax 1 +k3C*

klL

where

(19)

Nm,x = k3

kl is a further biological rate equation coefficient with dimensions of T 1, N* is a limiting value for the flux N for a given value of C* and N,,,x represents the maximum possible rate of reaction for a film of thickness L.

/ :;o! NmoxI- "]Oj

///

N

/

/

/ /

0.5

0.1 0.I

1

I0, k3C

I00

1000

Fig. 21. The biological rate equation for films

It is convenient to consider the flux, N, obtained by the solution of equation (10) as a deviation from the limiting value N*, so that: N = ), N*

(20)

where 0 < 2 ~<1 and is known as an 'effectiveness factor'. The solution to the problem may be expressed (Fig. 21) as: N

Nmax-g(kzL, k3C*).

(21)

254

B. ATKINSON and H. W. FOWLER

The above model leads to an effectiveness factor which is equal to or less than one since the substrate concentration in the film would be expected to be equal to or less than C* Beneficial conditions may arise within microbial film (Fig 2) and this would suggest that, in some circumstances, it is possible that the effectiveness factor could exceed unity Although this is a reasonable hypothesis, it is not supported by any direct experimental evidence and is outside the scope of the present model, but could be included if necessary By combining equations (18)--(21) the overall rate of substrate uptake by a microbial film may be expressed in mathematical terms as:

N = g(kl ,k2,k3,C*,L).

(22)

It is convenient to describe k~, k2 and k3 as Biological Rate Coefficients and to note that k3 is a kinetic coefficient only; k~ is a measure of the number density of the microorganisms within the microbial mass and the kinetic coefficients c~ a n d / / k2 contains the same factors as k~ but also includes the effective diffusion coefficient, so that: k2 = V D ,

(23)

Hence kz represents a diffusional limitation in the 'solid'-phase If this limitation is absent, the effectiveness factor will be equal to one and k2 will not appear in the rate equation. For individual organisms which are described by the Monod equation, the parameters kl, k2 and k3 are related to the Monod coefficients, as follows:

kl-

YoKm

k2 =

____Gmaxp°1 LV~ e

(24)

1

k3 - Km" In these particular circumstances equation (22) can be re-stated as:

N=g(G~po,D,,K,,,C*,L).

(25)

When an individual organism exhibits a diffusional limitation (Powell, 1967), no such simple relationships exist and the coefficients k~, k2, k3 have to be retained explicitly. Thus equation (22) represents the more general description of the problem

The Significance of Microbial Film in Fermenters

255

A number of rate equations that have been suggested for microbial film are summarised in Table 11. These range from the empirical expressions of Stack (1957) and Kornegay and Andrews (1968) to the pseudoanalytical equations ofAtkinson et al. (1974). The latter represents functional relationships, devised using the known asymptotes to the problem (Table 12), which provide a good fit (Fig. 21) of the numerical solution to equation (10). This Biological Rate Equation (BRE) is the algebraic analogue of equation (22). Table 11. Suggested forms of rate equations for microbial film Author

Basis

Equation

Stack (1957) N = E C *

Empirical

Atkinson

Diffusion with biochemical reaction

et al.

N

k, C*

=k2

(1967) Kornegay and Andrews (1968) Atkinson and Daoud (t968)

N - kl x

where: x = L when L < d ~ d i s t h e x = d when L > d J 'active' film thickness kl L C* N=)~ 1 +k3C*

for 4 ) < t ) k3C,>50

1

2=qS 4) -

for 4)> k 2L

(1 + k 3C*) °'576

Atkinson (1974)

Diffusion with biochemical reaction

tanh 4) for k3C*< 1 2 _....... ~.... 2= I

et al.

Empirical

k3C*

k3 iq--k73C*

N=2Nm.~

2=1

k3C*

1 +k3C*

tanhk2L( k2L

1 ) ' - - OR

4)R tanh ~bn

tanhkiL( keL k2L

1

) 1 , qSR~<1 )

t a n h ~ R -- 1 ,4)n>~l

Diffusion with biochemical reaction

Number of biological parameters

1 or2

256

B. ATKINSONand H. W. FOWLER

Inspection of Tables l l and 12 shows that the equation of Atkinson et al. (1974) is the more general case of that due to Atkinson and Daoud (1968); also that one of the asymptotic forms of the general equation corresponds to those proposed by Stack (1957) and Atkinson et al. (1967). In addition, it has been shown by How (1972) that the data of Kornegay and Andrews (1968) can be .described by means of the BRE. Table 12. Asymptotic forms of the Biological Rate Equations (Atkinson, 1974) Rate Equation

Conditions

Significance

klLC* N = i+kaC*

Small L

Applicable in the absence of "solid'-phase diffusional limitation

N = ~C*

Large L Small C*

A thick film gives rise to a'solid'-phase diffusional resistance and ultimately substrate concentration controls

N

Large L Small C*

The substrate concentration is not limiting and the rate depends on the film thickness

k3C* = Nmax l+k3C*

-

klL

k3 = Nmax

The Biological Rate Equation allows design equations to be developed on an analytical basis and assists in the quantitative interpretation of experimental data. Thus, for a given set of rate coefficients, the removal flux can be expressed in terms of the substrate concentration and the film thickness (Figs. 22 and 23). It will be seen that there is a 'critical' substrate concentration, beyond which the removal flux is essentially independent of concentration, that is, zero order with respect to substrate, and that this concentration is a function of film thickness. An additional feature of the Biological Rate Equation follows from the demonstration by Petersen (1965) that, for problems involving diffusion and chemical reaction, the mathematical solutions for 'slabs' and particles are similar, provided that a suitable characteristic dimension of the particle is selected. For a microbial floc, this may be defined as:

L-- --VP Ap

(26)

The Significance of Microbial Film in Fermenters

257

4.5 4.0 35 e4

3"0 / ~ x l O - 3 c m

i

E 2.5

03 2.0 o o x

/

kl= 2.13x10 -1 s-! k3=/~31 x106 mt g-1

/ /

1.5

Z

1.0 0.5 I 1

0

I 2

I 3

l 4

C*x t06,g m[-I

Fig. 22. NH3--N Removal flux

6.0 5.4

#

/,8

/

&.2

3.6

L=O.12 cm

/

kl =2.13 xlO_ t s_I 1<2=133.5 cm -I

/

k3=4.31xlO6ml g-1

,'., 'E 3.0 ,= 2/.. %x 1.8 Z

1.2 0.6 0

I

I

I

*

t

1

J

10 20 30 40 50 60 70 C*x106 g m[-1

Fig. 23. NH3--N Removal flux

258

B. ATKINSONand H. W. FOWLER

where Vp and Ap represent the volume, and the external surface area of a 'wet' microbial floc. For flocs, the use of a flux based on unit surface area is inappropriate and a relationship for the specific substrate removal (R) is preferable. Hence equations that have been derived for film may be applied to floc, with L replaced by Vv/A p and N by R, so that: R =,t~Rm,x k3C* (27) 1+ k 3 C * " The biological rate coefficients kl, k2 and k3 are of paramount hnportance in the biological rate equations. Since the actual values are specific for any microbe substrate system, it is useful to have an indication of the order of magnitude of these coefficients and Table 13 summarises some values that have been obtained for mixed microbial populations in a glucose medium. Full details of the practical determination of the biological rate coefficients have been given by Atkinson (1974). The methods are summarised in Table 14 from which it will be seen that values may be obtained by experimentation with either flocs or films. It will be apparent that only the Biological Film Reactor (Atkinson and Daoud, 1970) can be used to determine all three rate coefficients. This apparatus consists of an inclined plane, on which a film of microorganisms is grown, while the nutrients are provided by a liquid film in laminar flow over the surface. The thickness of the microbial film is controlled by mechanical scraping; a "thin" film can be obtained on a roughened glass plate and a "thick" film on a surface to which a perforated sheet is attached so Table 13. Experimental values of Biological Rate Coefficients for mixed microbi'A populations, obtained using a Biological Film Reactor Atkinson et al. (1968) Atkinson and Daoud (1970)

Atkinson et al. (1974)

RateLimiting Substrate

Glucose

Glucose

NH3-- N

kl (s -1)

1.08x10 1(18 C) 1.62x10 1(22~C)

2.91x10-1

2.13x10-1

k2 (cm ~)

1.455 X

--

--

k3 (cm3 g-l)

1.622 xlOs (18~'C) 1.706x105 (22~C)

10 2

1.18xl05

~4.31x106

259

The Significance of Microbial Film in Fermenters

Table 14. Summary of methods for determination of Biological Rate Coefficients (Atkinson, 1974) Biological Flocs

Biological Film

Apparatus

Batch Fermenter

CSTF

B i o l o g i c a l Fluidised Film Bed Reactor Fermenter

Conditions

Aseptic

Aseptic

Non-aseptic

Aseptic

Rate Coefficients

kl, k3

kl, k3

kl k2, k3

kl k3

Type of Rate Coefficients

Integral

Pseudoinitial

Pseudoinitial

Pseudoinitial

Mass Transfer Coefficients required

None

None

From theory

By experiment

"

k2 '

that organisms grow in compartments of known depth. Anaerobic conditions can be provided by enclosing the plane with a plastic cover and purging the equipment with nitrogen. The Biological Film Reactor represents a useful experimental method, with the use of a predetermined biological film thickness as a major convenience, as is the mathematical description of the liquid phase mass transfer phenomena (Atkinson et al., 1968). Hence no additional experimentation is required to estimate either the biological film thickness or the mass transfer coefficients. The reactor is applicable to anaerobic and aerobic studies but suffers, at present, from a restriction to non-aseptic conditions. The biological rate coefficients may also be obtained from experiments with microbial floc in either batch or continuous stirred tank fermenters. Difficulties are encountered, however, in the determination of k2, since this involves conditions imposing a 'solid'-phase diffusional limitation. This demands a detailed experimental knowledge of the floc size distribution, which is difficult to obtain. In addition, the requirements regarding hydrodynamic shear to achieve a sufficiently large floc size to determine k2, are in confict with those leading to complete mixing and satisfactory liquid-phase mass transfer coefficients. Batch experiments suffer from the additional problem of unsteady state operation, so that there is accumulation of products. Unfortunately, the lag phase of microbial growth prevents the use of initial rates of reaction to overcome this problem, as can be done in studies of enzyme kinetics. In the case of continuous experiments, the small size of laboratory fermenters

260

B. ATK[NSONand H. W. FOWLER

imposes low flow rates to avoid wash-out. This is especially true with anaerobic systems, where growth rates are relatively low. However, the determination of k~ and k3 is relatively straightforward and it is possible that the coefficients could be obtained by simple batch experiments with shaker flasks. The difficulty of obtaining k2 could be overcome by using an estimated diffusion coefficient with equation (23). Tomlinson and Snaddon (1966) found that the effective diffusion coefficient of oxygen in a bacterial film was about two-thirds of the value for the diffusion of oxygen in water. Bungay and Harold (t971) showed that the theoretical oxygen concentration agreed with experimental observations if the effective diffusion coefficient of oxygen in the film was about 80 per cent of that in water. Results from Atkinson et al. (1974) indicate that the effective diffusion coefficient of N H 3 - - N is about 70 per cent of the corresponding diffusion coefficient in water.

5. M i c r o b i a l F i l m in F e r m e n t e r s It has become traditional for many fermentation processes to be carried out in batch fermenters, since these offer economy and flexibility in dealing with small batches of different materials in a developing industry. Continuous operation is well established for certain processes, particularly those using microbial film with the substrate supplied by a trickle flow system. The trickling filter used for waste-water treatment is the principal example of this type and certain disadvantages associated with the general application of this method have been considered in section 3. Hence, the remainder of this discussion will be limited to procedures which, if microbial film is used, permit control of the film thickness and have the potential for aseptic operation. The application of the BRE to trickle flow systems has been discussed as a design exercise by Atkinson (1974). Currently, continuous processing is attracting interest for some products usually made by batch methods, for example in the brewing industry, and new materials such as microbial protein are appearing which require large-scale, continuous production. Continuous fermentation has a number of advantages (Righelato and Elsworth, 1970) and as the basic configuration of a batch fermenter is a simple stirred tank, in principle there is little difficulty in converting to continuous operation by fitting inlet and overflow devices. In practice, however, there is a critical flow rate at which the amount of microbial mass falls to zero and fermentation ceases. This is the situation known as wash-out, which is a major limitation to the use of the CSTF and is illustrated by curve Mm of Fig. 24. There are corresponding effects on productivity

The Significance of Microbial Film in Fermenters

261

Fractional viability

/~Mrn

/"-"N

S 1.0 Ci

'fi I" 0

FIV

( Fwlol V}

Fig. 24. The performance characteristics of a CSTF (curve Pr, Fig. 24) which also falls to zero and on the substrate concentration (curve C, Fig. 24) which rises to reach the inlet concentration. It should be pointed out that these curves are based on the assumption of a complete absence of surface, which cannot apply in practice. However, the general shape of these curves is characteristic of simple growthassociated systems, particularly the skewness of the productivity curve, indicating that the maximum productivity is obtained in a region where the microbial concentration is very sensitive to flow rate. Hence, to avoid the danger of wash-out, it is safer to operate at a flow rate which falls short of the maximum productivity. The problem can be alleviated by using a flow rate through the fermenter greater than washout, but introducing a second stage in which the microbial mass is concentrated, by sedimentation or centrifugation, and re-cycled to the fermenter. This is practised widely in the activated sludge process used for waste-water treatment (Abson and Todhunter, 1967) and has been developed for continuous brewing processes (Coutts, 1961). In the case of aseptic operation, however, the use of re-cycle is much more difficult; centrifugal methods are expensive and, since sedimentation is a slow process, large tanks with considerable hold-up are required. This difficulty has been overcome partially in the case of the Tower Fermenter (Greenshields and Smith, 1971) by the use of the internal sedimentation which is characteristic of fluidised systems (Fig. ! 3), although this is restricted to operation under plug-flow conditions. Even when adequate facilities are available for the concentration and re-cycle of microbial mass, wash-out is still a feature of the CSTF and the Tower Fermenter. In contrast fermenters based on trickle-flow systems in which microbial

262

B. ATKINSONand H. W. FOWLER

film predominates (Table 1) do not have a problem of wash-out which suggests that it might be advantageous to seek ideal fermenters which exploit this characteristic, while retaining the general properties of the CSTF. Section 3 has indicated the advantages of the use of a controlled thickness of microbial film on a support surface. At low flow rates microbial mass in this form shows little advantage over flocs but, as the flow rate is increased, there is a benefit if a substantial quantity occurs as film. Since both the CSTF and the Tower Fermenter are limited by washout and severe problems result with the continuous concentration of microbial mass, it follows that an ideal fermenter would be capable of operation over a wide range of flow rates without encountering washout and without the complication of concentration of the microorganisms during any re-cycle. In addition, the contents of the fermenter should be completely mixed, that is, the concentration is uniform throughout the system so that the outlet concentration is the same as that in the fermenter vessel. Very few processes are completely nonaseptic (waste-water treatment is the only significant example) so that ideal fermenter configurations must be suitable for attaining and maintaining strictly aseptic conditions. Microbial film can be controlled in thickness by particle-particle attrition (section 3) when suitable solid particles are introduced into a CSTF (Fig. 12) and this configuration shows the properties described above. Feed pH control t

[emperature control

-- Products

Fig. 25. The completelymixed microbial film fermenter(CMMFF)

The Significance of Microbial Film in Fermenters

263

Alternatively, a tubular fermenter may be used with the particles fluidised (Fig. 13) giving greater control of the attrition and hence of the film thickness. If a re-cycle system is incorporated (Fig. 25) the fermenter approaches the ideal system described above. Fermenters using microbial film in this way will be referred to as Completely Mixed Microbial Film Fermenters (CMMFF). Microbial film forms on the particles and biomass in excess of the equilibrium film thickness is recirculated as floc, so that the fermenter performance depends on the contributions of both film and floc. Combination of the substrate uptake by flocs and by films leads to:

R~.= RMm + NA~

(28)

where R, is the substrate uptake per unit liquid volume; Mm is the concentration of dry microbial mass and As is the area of support surface per unit liquid volume. Using equation (28) in conjunction with mass balances for substrate and microorganisms and the rate equations for fihn and floc discussed in section 4 leads to a mathematical description of the problem. The rate equations consist of (20) and (24) and, for the special simplifying case when the effectiveness factor is equal to unity, the mass balance equations become:

A'f + B'f,- 1= 0 where L =

(29)

C and 0 < f , < 1. The coefficients A' and B' are given by: (-1

A'=k3C,I1- ~,],

(30)

B'=I-k3G + l_[k3C1+fl,]

(31)

where:

17 ~'

fl,

-

-

-

+I,--

Gmax

,

k1L A~ Gm~x

(32)

(33)

In equation (3211, is an endogenous respiration coeMcient. The parameters ~' and fl' respectively reflect the influence of flow rate and biologically active area on the performance of the fermenter. The solution to equation (29) may be expressed as: f , = g(~',fl',k3 CI)

(34)

264

B. ATKINSONand H. W. FOWLER 1.0

-

D'=5

10

o/-/

L

I

20

25

Fig. 26. The effect of biologically active surface area on the performance of the CMMFF (k3 = 100)

CI

k3C~.

and this equation has been plotted in Fig. 26 for one value of These curves show that in the absence of surface area ( I f - 0 ) washout occurs, but as surface area is increased, substrate usage increases even at very high flow rates. The productivity of the fermenter, expressed as substrate consumed, can be related to substrate concentration by:

Pr = FC~ [ - ~]

(35)

Pr~d~=°~'(k3Cl}[1 -Gill

(36)

or

where Pl~a) is a dimensionless productivity, given by:

5

T •

Equation (36) has been plotted in Fig. 27 which emphasises further that wash-out can occur only in a fermenter which has either sterile surfaces or no surface at all. Since it is impossible for a fermenter to be completely devoid of surface, it follows that, in a mathematical sense, wash-out cannot occur in a real fermenter. In practice, in a conventional CSTF on the industrial scale, the surface/volume ratio is very small and a practical wash-out situation is obtained.

The Significance of Microbial Film in Fermenters

265

/

1600

,0'=1500 £?£~/./~"""--

o Locus of P r(d) max

y ,ooo

° o

Pr(d )

0 0

°-

o

0

0

0

o

,~"=100

~,..~30

I 20

I0

25

Fig. 27. The productivity of the C M M F F (k~ Ct = 100)

The curves of Fig. 27 still show a maximum as in the case of the CSTF (Fig. 24), but the maximum is less sharp and becomes of greater magnitude as the surface area is increased. Thus, it is not necessary to operate at a flow rate below a critical value as in the CSTF, in fact the CMMFF curves suggest that operation should be beyond the minimum flow-rate for any given value of surface area. It is worth noting that the conventional CSTF is a special case of the C M M F F when A = 0 and equation (29) reduces to: f~=l

or f ~ -

1 A'

(37)

This corresponds to the generally accepted theory for the CSTF (Herbert, 1956). For a real situation (A > 0) it is interesting to note the observations of Larsen and Dimmick (1964) mentioned in Table 8. A CSTF of small volume (18 ml) and hence with a relatively large surface area was operated at steady state. The dilution rate was increased for a short time and the flow was then stopped so that batch conditions prevailed. The results are shown in Fig. 28 and it will be seen that the experimental bacterial count was in excess of the theoretical growth rate, indicating many organisms resulted from surface growth. The authors calculated that 98 per cent of the cells in the liquid culture during the period of batch growth must have originated from the walls. As well as Serratia marcescens (Fig. 28), Larsen and Dimmick (1964) used eight other bacterial species and found that two of these showed

B. ATKINSON and H. W. FOWLER

266

~109 D,.

T:

Dilution rate

°108

~ t I

increased

bo

( ...'/M'a / "xl //. /

"S J~

E z 10'Z 42

I

Flow s t o p p e d _ ~ /

mum //growth rate in batch culture

I

,I

t

t

4.3

44 Time (h}

45

46

Fig. 28, Influence of change of dilution rate on the population density of a continuous culture of Serratia marcescens (Larsen and Dimmick, 1964)

only moderate adhesion to the walls, while two showed virtually no adhesion at all, approaching a wash-out situation. This emphasises the variation in the attachment properties shown by different organisms, as discussed in section 3. Two configurations are possible for the CMMFF, either a conventional form of CSTF with added particles (Fig. 12) or a tubular system (Fig. 25). The latter has been described by Atkinson and Davies (1972) 1.0 x

Experimental results

(1

Theory (k3C[= 170)

~,, o 30 000 b ~0 000 c 60 000

/ 500 Fig. 29, Experimental data for the CMMFF

1000

1250

The Significance of Microbial Film in Fermenters

267

and consists of a vertical cylindrical tube containing the fluidised, biologically active particles. A centrifugal pump provides recirculation of the liquid, and the recirculation ratio is large to fluidise the supporting particles as well as ensuring that the system is completely mixed. The theory for the C M M F F has been tested experimentally and the effect of added surface in a CSTF is shown in Fig. 16. The observations of C/C~ show a base value for the fixed surfaces and an increase in substrate uptake as area was increased by the addition of beads. The curve represents the theoretical results for a film thickness of 0.25 mm and, while this was not confirmed experimentally, it is of the correct order of magnitude. Experimental results for a tubular C M M F F are shown in Fig. 29 where theoretical curves again show satisfactory agreement. The values of fl' in the range 30,000 to 60,000 correspond to a biological film thickness of 0.35--0.69mm, again of the correct order of magnitude.

6. F u t u r e P o t e n t i a l The application of the principles of chemical reaction engineering to biochemical systems in the manner discussed in the preceding sections is relatively recent. Some experimental evidence has been obtained that provides encouraging support for the theory and it is appropriate to conclude by highlighting those aspects which have received some confirmation and to speculate upon the future. As in all chemical engineering activities, the theory should have a dual objective. Firstly to provide correlations for laboratory data, having application over wide ranges of all known variables, and secondly to provide methods for the utilisation of laboratory information in procedures for the design of industrial equipment. Because of the complexity of biological systems, the majority of the correlations that have been proposed have had an empirical basis. As shown in section 4, it is logical to describe microbial kinetics in terms of combined diffusion and biochemical reaction and there is every reason to believe that the resulting Biological Rate Equations are of considerable generality (Table 11). It has been shown that a number of empirical correlations reported in the literature are reduced forms of the BRE (Table 12). There are indications also that the biological rate coefficients should be interchangeable between film and floc. This is not unreasonable since it seems unlikely that the behaviour of a microorganism will be affected simply by the geometry of its environment. The geometry will, however, affect the conditions to which the organism is exposed and

268

B. ATKINSONand H. W. FOWLER

thereby lead to effects on the behaviour of the organism. These factors impose controls such as diffusional limitation that are accounted for in the BRE, which suggests that the basic theory should be applicable to all conditions of microbial growth. The generality of the BRE leads to advantages in the design of equipment and should offer the possibility of designing fermenters on engineering principles. It is common practice in chemical engineering to use data obtained in laboratory equipment for the design of plant having a different configuration. Thus, mass transfer coefficients may be obtained in the laboratory using a wetted-wall column and used for the design of plant based on packed beds. tn the same way, data from the Biological Film Reactor (BFR) may be applied to biological film grown in a packed bed (Atkinson and Williams, 1971). Similarly, there seems to be no reason why biological rate coefficients obtained with a BFR should not be applied to a CSTF, provided that allowance is made for the difference in physical arrangement. Practical demonstrations so far have been based on mixed populations of microorganisms grown under non-aseptic conditions, which raises the possibility of selective growth of different strains as the conditions are changed. Hence, investigations are in hand for the determination of rate coefficients for single strains grown under aseptic conditions. Apart from the Tower Fermenter (Greenshields and Smith, 1971) and the Pressure-Cyde Fermenter (ICI, Agricultural Division, 1973) there has been little change in industrial fermenter configurations for a number of years, but the use of microbial film on a support surface suggests new possibilities. To achieve a satisfactory continuous process it may be necessary to simulate the changes which take place in the equivalent batch fermentation. The reasoning behind this suggestion is associated with the apparent need to expose the organisms to a particular environmental history in order to produce a required biochemical product. This requirement is not met completely by the microbial film fermenter described in the previous section, since the film growth in different parts of the fermenter has different histories. However, the microbial mass, once it leaves the support surface, is exposed to exactly the same concentration variations as all the other flocs. For these reasons the microbial film fermenter may turn out to be applicable to all fermentations, but for the present it would appear sensible, at least for the more complex fermentations, to use the microbial film fermenter to generate microbial mass. This mass can then be fed to a tubular microbial floc fermenter. Such a configuration is illustrated in Fig. 30; this consists of two tubular sections of different diameters mounted above one another; the lower section consists of a controlled microbial film fermenter while the upper section is a simple tubular

The Significance of Microbial Film in Fermenters o

o

o

0

o

269

0 o

o 0

o 0

o o

0

o

o

0 o

o

/

f[ocs

o o~ o

o

o o

o o o

o o

o

o O o

O

o O O

o o

O

o

/ o

o

i0o0

1 %

o

o

B o0 cayactve o0O ° Jj'inert'particles

° 1°~9

po£_oo.

t Fig. 30. Fluidized-bed fermenter feeding a plug-flow fermenter

fermenter. The diameters of the two sections will usually be different, since they will have differing requirements as regards superficial flowrate. In the one case fluidisation of the support particles will dominate the design, while in the other the overall residence time of the nutrient medium will be all important. For a simple growth-associated system, Atkinson (1973) has given design equations which may be solved graphically as shown in Fig. 31. The use of microbial film on a support surface as in the CMMFF presents considerable opportunities in terms of improving'operating characteristics of fermenters. Control of film thickness has been discussed in section 3 and the use of physico-chemical control would appear to have considerable potential. All experimental procedures to date have used glass beads, but the use of other materials could lead to interesting possibilities. Thus, materials of different density would demand different fluidisation velocities and hence give higher or lower velocities over the particles as required. Alternative materials would have other surface properties, so affecting the adhesion. In fact, there would appear to be no reason why ion exchange resins should not be used as sup-

270

B. ATKINSON and H. W. FOWLER

1

g(C) I Area proportional Area proportional/ to volume of upper section CO

C'I

CI

C

Fig. 31. Design of an FBF feeding a PFF

port media, so that the film would be adsorbed under one set of conditions and released by a change of pH or ionic strength. The use of microbial film attached to a surface has a possible further advantage compared with floc systems, as the solid and liquid phases are more clearly defined. This is likely to simplify the rheological properties leading to a lower value of the apparent viscosity of the liquid phase and, in turn, to lower power requirements and improved aeration efficiencies. These suggestions appear to offer potential for the control of washout, improved flow characteristics, the simulation of batch conditions during continuous fermentation and, possibly, greater control of the metabolic pathways and of products. For example, a fermenter similar to that shown in Fig. 30 could be used for brewing, with the lower section aerated to encourage yeast growth, while anaerobic conditions were maintained in the upper portion to enhance ethanol production. Calculation of suitable design features for the C M M F F can be achieved by the use of the theory presented in section 5. The following example illustrates the ease with which the size of the support particles can be calculated for the tubular version of the CMMFF.

Example It is required to calculate the diameter of the spherical particles to be used in a tubular C M M F F with the following conditions:

The Significance of Microbial Film in Fermenters Liquid volume (V) Flow-rate (F) Inlet concentration (CI) Outlet concentration (C) Density of microbial mass (po)

271

= 10 1, = 5 1h- 1 = 0.6 g t- 1, 0.15 g 1-1, = 0.1 g cm 3

The organisms involved are a mixed population for which biological data have been given by Atkinson and Davies (1972). Yield coefficient (Y0) kl k3

= 0.73, = 0.1624 s- 1, = 1.7 x 105 cm 3 g - 1.

F r o m equations (24) and (32)

= VkYok~/

if

~c=O

5xO.l x 1.7x 105 3600x 10x0.73 x0.1624 = 20.1, fi -

C 0.15 = 0.25, Cx 0.6

k3C1 = 1.7× 105 x 0 . 6 x 10 -3 = t02. F r o m Fig. 26, when f i = 0 . 2 5 and ~ ' = 2 0 , f l ' = 1500. N o w from equations (24) and (33)

= 0.233 x 105 (AsL). Therefore _

As

fl'

15oo

0.233 x l0 s - 0.233 x l0 s = 0.065.

The quantity AsL represents the wet volume of microbial mass per unit liquid volume in the fermenter. The specific surface As is totally determined by the particle size, while the microbial film thickness (L) is

272

B. ATKINSON and H. W. FOWLER

likely to depend principally upon the particle size and density (as these determine the fluidising velocity) and to some degree on the growth rate. Fig. 32 relates A~L to the particle diameter, for various arbitrarily selected values of L, with As based upon a typical bed voidage of 50 %. 1.0

I00

I/1

.JD

0.1

10 ._~

'7 ol

1

0.01

J

AsL Z

E

k,=

-6

O1 k5

0.001

o

:z

0.0t

0.0001

0 011 0.2 0.3 0.4 015 0.~6 0.7 08 0.9 10 Particte diameter (cm) Fig. 32. Values of A~L for various particle sizes and microbial film thicknesses

Since A~L is the microbial hold-up in the fermenter it can be expressed in the usual units of dry weight per titre liquid volume. Thus, using po=0.1 g cm -3,

AsL = 0.065 = 6.5 g (dry wt.) 1and a dual scale has been provided in Fig. 32 for A~L. The lowest value of L reported for mixed populations is 73 lam for a scraped surface (Table 8) and this leads, from Fig. 32, to a particle diameter in the region of 0.6 cm. This diameter is typical of fluidised

The Significance of Microbial Film in Fermenters

273

bed operations and if the microbial film thickness were to exceed 73 ~tm, when using this diameter, it would simply mean that the required efficiency of 75 % would be exceeded. A less conservative estimate of the particle size requires experimental data on L as a function of particle size etc. Once the particle size has been determined, the number of particles, fermenter volume and diameter, and the circulating velocity for fluidisation can be obtained. The design exercise for the C M M F F discussed above can be generalized by the inclusion of equation (36).

Pqa) = d(k3 (71)[1 --f,]

(36)

on to data of the form shown in Fig. 27

Pq~) = g(o(,fl',k3Cl).

i.e.

'°°°rI

(38)

.~%/~/~-~/ /

o Locus of Pr(d)mox

I

.

o/'I"

////<_.>-I

,oool , 1 0

,,--o.. 10

a¢j

20

25

Fig. 33. The effect of area, flow rate and conversion on the productivity of the CMMFF (k3Ci = 100)

This is illustrated in Fig. 33 with lines of constant fr when k3 Ct is 100. The inter-relationship between conversion, productivity, area and flowrate are clearly evident from Fig. 33. In particular, it may be seen that for a given area the productivity can be enhanced at low flow rates with little reduction in conversion, while at high flow rates the conversion falls with flow rate while the productivity remains largely constant.

274

B. ATKINSONand H. W. FOWLER

Nomenclature

A A' Av A~ B B'

C Cx C* d

D. E

f

F

Gmax k~ k2 k3

Km L M

mm N

Nmax N* P Pr Pr~

R

external surface area of viable microorganisms per unit volume of microbial mass area of the biologically active surface in a fermenter dimensionless parameter (defined by equation (30)) external area of a single "wet' biological particle or floc area of support surface per unit liquid volume of fermenter dimensionless coefficient (defined by equation (10)) dimensionless parameter (defined by equation (31)) limiting-substrate concentration limiting-substrate concentration fed to a fermenter limiting-substrate concentration at the interface between "solid' microbial mass and the adjacent solution (Fig. 10) thickness of 'active' microbial layer effective diffusion coefficient of limiting substrate within microbial mass (defined by equation (7)) empirical coefficient (Table 11) dimensionlesssubstrate concentration (defined by equation (10)) dimensionless substrate concentration (defined by equation (29j) volumetric flow-rate to a fermenter maximum specific microbial growth rate biological rate equation coefficient (defined by equation (17)) biological rate equation coefficient (defined by equation (11)) biological rate equation coefficient (defined by equation (12)) Monod coefficient (defined by equation (24)) 'wet" mica-obial film thickness dimensionless coefficient (defined by equation (10)) concentration of'dry' microbial mass flux of substrate at the interface between microbial mass and the adjacent solution (defined by equation (2)) maximum flux of substrate (defined by equation (18)) rate of substrate uptake (given by equation (18)) penetration depth productivity dimensionless productivity local rate of substrate uptake per unit area of viable microorganisms specific rate of substrate removal by a microbial floc (rate of removal per unit microbial mass)

LLz ....... L2 L--ML

~

M L- ~ ML

s

L L2LT

........ ...... L 3T 1 TTL- 1 M - ~L 3 ML- 3

L ...... ML

3

M L 27"- 1 ML

2T- t

M L 2T-

L M L ST-

..... ML

T-

2T-

The Significance of Microbial Film in Fermenters Rf Rt,

V

v~ x

X

1Io 3( O~r

9' K

Po

overall rate of reaction overall rate of reaction per unit liquid volume in a fermenter liquid volume in a fermenter volume of a single 'wet' biological particle or floc distance coordinate dimensionless distance (defined by equation (10)) yield coefficient rate coefficient dimensionless flow-rate parameter (defined by equation (32)) rate equation coefficient dimensionless area parameter (defined by equation (33)) endogenous respiration coefficient effectiveness factor (defined by equation (20)) density of cells measured in terms of dry weight per wet volume

275 MT-1 ML-3T-'1 L3 L3 L

ML-eT-I

ML T

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References Abson, J. W., Todhunter, K. N.: In: Biochemical and Biological Engineering Science, Vol. I. Blakebrough, N. (Ed.). New York: Academic Press 1967. Anderson, P. A.: J. Gen. Physiol. 36, 733 (1953). Atkinson, B.: Biochemical Reactors. London: Pion Press 1974 (to be published). Atkinson, B.: Pure App. Chem. 36 (1973). Atkinson, B., Daoud, I.S.: Trans. Inst. Chem. Engrs. (London) 46, T19 (t968). Atkinson, B., Daoud, I. S.: Trans. Inst. Chem. Engrs. (London) 48, T245 (1970). Atkinson, B., Daoud, I. S., Williams, D. A.: Trans. Inst. Chem. Engrs. (London) 6, T245 (1968). Atkinson, B., Davies, I. J.: Trans. Inst. Chem. Engrs. (London) 511,208 (1972). Atkinson, B., Davies, I. J., How, S. Y.: The Overall Rate of Substrate Uptake (Reaction) by Microbial Film (to be published 1974). Atkinson, B., Swilley, E. L., Busch, A. W., Williams, D. A.: Trans. Inst. Chem. Engrs. (London) 45, T257 (1967). Atkinson, B., Williams, D. A.: Trans. Inst. Chem. Engrs. (London) 49, 215 (1971). Beaman, R. G.: In: Microbial Technology. Peppler, H. J. (Ed.). Rheinold 1967. Borchardt, J. A.: Biotechnol. Bioeng. Symp. 2, 131 (1971), Bruce, A. M., Merkens, J. C.: J. Inst. Water Pollution Control 2, 3 (1970). Bruce, A. M., Merkens, J. C., MacMillan, S. M.: Inst. PuN. Health. Engrs. J. 69, 178 (1970). Bungay, H. R., Harold, D. M.: Biotechnol. Bioeng. 8, 569 (1971). Bungay, H. R., Whalen, W. J., Sanders, W. M.: Biotechnol. Bioeng. I l, 765--772 (1969). Chue, V. T.: Ph. D. Thesis. University of Wales 1973. Cohen, C. N , Monod, J.: Bacteriol. Rev. 21, t69 (1957). Courts, M. W.: Australian Patent 216618 (1958); British Patent 872391-9, 872400 (1961).

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Daniels, S. L., Kempe, L. L.: Chem. Eng. Progr., Symp. Ser. 69, 62, 142 (1966). Dias, F. F, Dondero, N. C., Finstein, M. S.: Appl. Microbiol. 16, 1191 (1968). Etsworth, R.: Proc. Biochem. 5, 21 (1970). Freeman, R. R.: J. Biochem. Microbiol. Technol. Eng. 3, 339 (1961). Greenshields, R. M., Smith, E. L.: Chem. Engr. (London)249, 182 {1971). Hamer, G.: Biotechnol. Bioeng. 14, 1 (1972). Hawkes, H. A.: The Ecology of Waste Water Treatment. London: Pergamon Press 1963. Herbert, D.: Soc. Chem. Ind. (London), Monograph 12, 21 (1956). Herbert, D., Elsworth, R., Telling, R. C.: J. Gen. Microbiol. 14, 601 (1956). Heukelekian, It., Dondero, N. C.: Principles and Applications in Aquatic Microbiology. New York: Wiley 1964. Heukelekian, H., Heller, A.: J. Bacteriol. 40, 547 (1940). How, S. Y.: Ph.D. Thesis. University of Wales 1972. I.C.I. (Agricultural Division): I.C.I. Protein (Brochure). Billingham: Imperial Chemical Industries Ltd. (Agricultural Division) 1973. Kornegay, B. H., Andrews, J. F.: J. Water Pollution Control Federation 40, 460 (1968), Kriss, A., Markianovich, Y.: Microbiologiya 28, 399 (1959). Laidler, K. J.: The Chemical Kinetics of Enzyme Action. Oxford University Press 1958. Larsen, D. H., Dimmick, R. L.: J. Bacteriol. 88, 1380 (1964). Maier, W. J., Behn, V. C., Gates, C. D.: J. Sanit. Eng. Div., Am. Soc. Civil Eng. SA 91 (1967). Meadows, P. S.: J. Exp. Biol. 41,499 (1964). Monajemi, P., Behn, V. C.: Oxygen uptake and mechanism of substrate purification in a model trickling filter. 5th International Water Pollution Research Conference (1971). Monod, J.: Ann. Rev. Microbiol. 3, 371 (1949). Munson, R. J., Bridges, B. A.: J. Gen. Microbiol. 37, 411 (1964). Nordin, J. S., Tsuchiya, H. M., Fredrickson, A. G.: Biotechnol. Bioeng. 9, 545 (1967). Nordling, S., Pentinnen, K., Saxen, E.: Exptl. Cell Res. 37, 161 (1965). Northrop, J, tt.: J. Gen. Physiol. 38, 105 (1954). Petersen, E. E.: Chemical Reaction Analysis. Englewood Cliffs, N. J.: PrenticeHall 1965. Powell, E. O.: In: Microbial Physiology and Continuous Culture. Powell, E. O., Strange, R. E., Tempest, D. W. (Eds.). London: H.M.S.O. 1967. Righelato, R, C., Elsworth, R. : In: Advan. Appl. Microbiol. 13, 1970, Rincke, G., Wolters, N.: Technology of Plastic Medium Trickling Filters. 5th International Water Pollution Research Conference (t971). Russell, H. L.: Zeitschr. f. Hyg. Infektionskrankh. 11, 165 (1891). Sinclair, C. G, Brown, D. E.: Biotechnol. Bioeng. 12, 1001 (1970). Stack, V. T,: Sewage Ind. Wastes 29, 987 (1957). Telling, R. C., Radlett, P. J.: In: Advances in Appl. Microbiol., Vol. 13. Perlman, D. (Ed.). New York: Academic Press 1970. Tomlinson, T. G., Snaddon, D H. M.: Int. J. Air Water Pollution 10, 865 (1966). Topiwala, It. H.: Ph. D. Thesis. England: University of Manchester 1970. Topiwala, H. H., Hamer, G.: Biotechnol. Bioeng. 13, 919 (1971). Trudinger, P. A.: Minerals Sci. Eng. 3~ 13 (1971). Unz, R. F., Dondero, N. C.: Can. J. Microbiol. 13, 1671 (1967).

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Whalen, W. J., Bungay, H. R., Sanders, W. M.: Environ, Sci. Technot. 3, 1297 (1969). Whipple, G. C.: Tech, Quart. 14, 21 (1901). Wood, E. J. F.: Australian J. Marine Freshwater Res. 1, 85 (1950). ZoBell, C. E.: J. Bacteriol. 46, 39 (1943). ZoBetl, C. E.: In: Marine Microbiology. Waltham (Mass): Chronicia Botanica Company 1946. ZoBell, C. E., Anderson, D. Q.: Biol. Bull. 71,324 (1936). Zvyagintsev, D. G.: Microbiology 28, 104 (1959). R ATKINSON,Ph.D., Reader in Biochemical Engineering Department of Chemical Engineering University College of Swansea Singleton Park GB-Swansea, SA2 8PP H.W. FOWLER, B. Sc., Lecturer Department of Chemical Engineering University College of Swansea Singleton Park GB-Swansea, SA2 8PP

CHAPTER 7

Present State and Perspectives of Biochemical Engineering IVAN MALEK With 2 Figures

When evaluating the present state and future trends of biochemical engineering, it is indispensible to begin by answering the fundamental questions: 1. Is biochemical engineering a new, independent hybrid and integrated scientific discipline, i.e. a branch with its own theoretical basis, or is it only an agglomeration of other disciplines, especially of their practical applications? 2. If we accept that biochemical engineering can be regarded as an independent scientific discipline, there arises the question, whether it really represents a new discipline or a discipline of long standing whose importance has emerged only recently. If it is accepted as a new discipline what is the essence of its novel character? In particular does this lie in the theoretical background or in the practical applications? 3. If it is a new discipline, what are its roots and perspectives? 4. What is the relation of this branch to other related disciplines, as e.g. bioengineering (in the broad sense), biotechnology and microbial or chemical engineering? 5. And finally: What kinds of perspectives for further development appear in relation to its theoretical background in economic and social needs and finally in the education of experts and scientific specialists? With regard to first question, the appearance of"Advances in Biochemical Engineering" seems itself to be significant indication that it represents not only a set of freely cooperating branches, but that both scientific developments and the economic and social applications have produced a rather independent, compact branch, which of course naturally retains its ties with the basic parental disciplines which formed it. This may

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be supported also by the fact, that among the relevant periodicals, a profound differentiation is to be observed between those devoted mainly to the basic problems of this branch, as for example "Biotechnoiogy and Bioengineering", and those such as "Process Biochemistry", with an emphasis on process applications. In a similar way, this view is supported through the inception of symposia, such as the international fermentation symposia or through the interest in symposia devoted to some of the problems of the discipline, as in the case of the symposium on "Genetics of Industrial Microorganisms" (Prague, 1970), or the Symposium on "'Advances in Microbiological Engineering" held in Marienbad in 1972. It may finally be shown by the interest of scientific societies and international bodies such as IUPAC, IUBS (IAMS), which offer necessary facilities for the development of activities within the discipline. Nevertheless, in spite of the demand, there is lack of specialist education both for scientific work and for the application of results in this branch itself. Recognition of this fact may lead to efforts to find the most effective ways of education in this area. I believe strongly that we really are seeing a new discipline in statu nascendi, whose importance and necessity have been established through a rapid growth of processes based on biochemical knowledge. It may be said that such processes have been the more successful, the closer was the collaboration of biological and biochemical specialists with expert engineers. As an outstanding example there may be cited the production of antibiotics which it was possible to introduce as generally available medicines only when adequate new engineering processes for their production had been worked out. But, at the same time, it is to be concluded on the basis of experience to date, that even more could be achieved through still closer co-working. This view is supported, for example, by the logistics of continuous processes in the production of microbial products, not only in relation to fundamental knowledge --biological and biochemical on the one hand and engineering on the other but also through close collaboration in the search for adequate technical solutions. Perlman (1969), evaluating the stormy development of the fermentation industry, which can be considered as one of the practical projections of biochemical engineering, says that its technology "usually lagged behind the microbiological aspects". Perlman is only partially right: this lagging of technology behind the possibilities given by microbiological and biochemical discoveries is not of the essence of engineering and technology, but is very much due to lack of integration of microbiological and technological solutions. Mention can be made again of the successes achieved in the economical production of antibiotics, which was possible only as a result of that relatively close integration. These observations support the case for

Present State and Perspectives of Biochemical Engineering

28!

the development of biochemical (including microbiological) engineering as a separate discipline. It can be said, therefore, that this new branch arises first of all from the needs of industrial microbiology; the production processes involve a series of operations which are not integral parts of conventional chemical engineering (e.g. sterilization and sterile product management, aeration, work with microbial strains, disintegration of cells etc.) and are, at the same time, subject to a special scientific approach related to the kinetics of microbial processes, especially in connection with continuous processes, selection and genetics of microbes, etc. As I have already pointed out, their effective application can be achieved only through close coordination of all these individual processes and procedures and through a cooperative solution of problems. It is possible therefore to consider biochemical engineering as a rising independent discipline. But is it really a new branch or has it already existed de facto for a long time--as long as there have been fermentation industries--with its independence only beginning to emerge now owing to increasing need and the possibilities of new production processes based on biological processes? It is undeniable that some fermentations on which biochemical engineering is based were present from the very beginning of all rationally performed industrial fermentation processes. Processes originally based on accumulated empirical experience only met with difficulties when they had to be turned from small-scale productions into great industrial ones. These problems presented themselves at various levels and were nearly always associated with the biological and biochemical components of the process, which proved to be decisive: any technological improvements in equipment were of little effect when the problems of productive strains, including the isolation of pure cultures, cultivation, physiological needs and genetic selection, were not solved. Mention can be made of Pasteur's intervention or of the impressive results of genetic selection of microbial strains in connection with mass production of antibiotics. And, similarly, other clear-cut evidence is offered in connection with continuous production of microbial biomass and of other products, which could be safely introduced in practice only when the theory of cultivation of microbial populations was mastered. All this experience has shown that it is impossible to safeguard the development of industries based on biological and biochemical processes in terms of chemical engineering alone. This fact is very markedly pointed out in the Editorial in "Process Biochemistry" (July 1969): "to argue that the chemical engineer ... can readily deal with the enormous complexity involved in living cells is a gross over-simplification'. All the evolutionary processes leading to this recognition during the last 20 years are expressed

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in the best manner in the confession of Sir Harold Hartley, "one of the founding fathers of biochemical engineering", when he says, that he"was wrong in 1951 to base the development of industrial microbiology on chemical engineering alone. The first approach must come from the microbiologist who could discover the conditions permitting economical operation in the laboratory. It is t h e n for the chemical engineer to develop the complex design and control systems needed". Again I would like to stress that these affirmations do not emphasise the importance of the biological (and biochemical) aspect only, but above all the mutual coordination and integration of the biologist's and engineer's approach. And here the rise of the new branch follows the general laws applying to the rise of such hybrid scientific disciplines and is in full accordance with the basic features of scientific and technological progress. Outstanding progress in scientific knowledge is realized mainly along the borders of, and through active contact and communication among, two or more integrated and already established branches of science which (a) differ considerably in methodology and technology, and (b) need each other as they accumulate a wide range of mutually applicable knowledge and methods. To reach this level each of these disciplines has to develop a good theoretical basis. Without this, basic technical and methodological achievements may be interchanged, but a n e w discipline cannot come into existence. Only a good theoretical basis can guarantee that within the new hybrid branch all progressive elements of the initial disciplines are comprised, supplementing one another; or in other words, that one discipline is not a"servant" of the other. One example of this development is biochemistry which itself is one of the sources of our new branch. Biochemistry came into existence when the behaviour of biological systems could be linked to the existence of substances whose chemical nature could be elucidated. This gave rise to a new discipline, no longer biology or chemistry in their original forms, but embracing the methodological, technical and experimental attitudes of both initial scientific disciplines. At the beginning, this new" scientific discipline had to face many problems as there were no full-rank specialists; on the one hand there were biologists who had difficulties in mastering the processes of analytical chemistry, on the other hand there were chemists who tended to simplify the complex dynamics, history and multiple integration of biological systems. However, cooperation in solving problems resulted not only in understanding and in mutual influencing of thinking, but also gave rise to a new category of scientists-biochemists. I give this example as it may help to explain the rise and development of the new discipline--biochemical engineering. Biology and biochemis-

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try have achieved remarkable results: a deeper knowledge of the dynamics of living systems, particularly of populations of unicellular organisms, the mechanisms of energy transfer necessary for life processes, the elucidation of metabolic pathways, their kinetics and chemical character, and the systems of physiological and genetic regulation. A thorough study of these processes resulted in knowledge of laws on the macromolecular level, and in the ability to control, to a certain extent, the life processes of these organisms in desired ways. Many of these theoretical findings have proved to be extremely important for the solution of practical problems. An outstanding example is the discovery of antibiotics. Successful industrial production of these substances is based on both deep biological and biochemical knowledge and highly sophisticated technology and engineering. New perspectives have been given by the introduction of processes for the production of microbial protein and enzymes. On the other hand, chemical engineering also has made considerable progress in controlling the transfer of mass and energy in heterogenous systems, introducing a variety of control and automation elements for these systems and designing necessary equipment and instrumentation (Ghose, 1970). Chemical engineers have started to model and control biological processes by means of computers. But again here close cooperation with the biologists and biochemists and their knowledge about the physiological demands of the organisms involved has led to deeper study of some engineering problems as for example problems of efficient and economic aeration and agitation, studies of flow patterns inside process equipment etc. The cooperation of biologists, biochemists and engineers has resulted in considerable experience being gained. Again, we could return to the example of antibiotics, where industrial production was started only when biologists cooperated with chemical engineers who elaborated the technology; and vice versa: high productivity was reached when biologists--biochemists and geneticists--were able to develop highyielding strains of microorganisms. Similarly, the production of microbial biomass can be mentioned as an example. It developed dynamically after the theory of multiplication in microbial populations in continuous systems had been mastered, giving new possibilities for technical and physiological control. However, this knowledge would be useless without the successful solution of bioengineering problems. These examples clearly indicate the importance of mutual influence the conditions under which successful development of this new branch could be achieved. As emphasised, bioengineering must be based on profound theoretical knowledge, both biochemical and technological, and on complex analysis of the models employed. These models must

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take fully into account the dynamic properties of living systems which are connected with their life cycle (multiplication, metabolism, development of genetically fixed properties, differentiation). This is extremely important, when we design mathematical models (Tsuchiya, 1970). Therefore, all engineering approaches must not only be in accordance with the biological properties of the systems under consideration, but at the same time they must provide the optimum conditions for the formation of the desired product. Therefore not only knowledge of biological processes, but also the ability to transpose this knowledge on to the bioengineering level is required. It is often postulated that a system realized for example in continuous process "degenerates" after some time; the fault is assigned to the genetic instability of a biological object, though the actual responsibility for such a phenomenon can be an inadequate engineering approach also. On the other hand, inadequate knowledge of engineering theory, experience and equipment prevents full exploitation of the biological capabilities. I hope that in developing the answer to the second question I have answered also the third of our questions. To summarize only: On the biological and biochemical side, biochemical engineering can call upon a great supply of knowledge of life processes. In many processes, which serve as a basis for modern fermentations, the end-products (citrate, glutamate, glutarate, lysine) are either intermediates of metabolic cycles or are derived from them ("Process Biochemistry", January 1968, p. 3). That is why knowledge of the enzymatic systems associated with fermentative processes remains fundamental to all production processes. Lilly (1967) makes the following comment: "in many of (these) industrial processes the point where further progress will depend on our knowledge of the mechanisms controlling the metabolic activities of microorganisms may rapidly be reached". Although our knowledge of the processes controlling the synthesis of the enzymes involved, regulating their activity by activation and inhibition really is developing intensively, yet in relation to product formation we find repeatedly that we still have far from sufficient knowledge of the full system of regulation, differentiating the basic metabolic pathway into that of end-product formation. Despite intensive development of knowledge on the genetic regulation of basic metabolic processes at the level of molecular biology, these problems remain unclear as yet. An important theoretical and practical field for biochemical engineering is the research into continuous processes. Continuous production has been largely limited up to the present to biomass formation and to products immediately connected with it; even this gave an impulse for the study of some theoretical engineering questions and the quest for practical solutions, concerning the kinetics of non-homogenous sys-

Present State and Perspectives of Biochemical Engineering

285

]until t958

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[ ] i 0 ~ientiflc Publlcations~lO0) •

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1964 1965

1967

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Fig. 1. The number of publications and patents connected with the method of continuous cultivation of microbial (and tissue-culture) cells is presented as an illustration of the scientific explosion in recent years in one typical topic of biochemical engineering. Some irregularity in the apparent growth of the number of publications is caused by lags in the recording of some publications. [Based on material presented at the 1st Symposium on Continuous Cultivation of Microorganisms, Prague 1958, and on annual bibliographies published in Folia microbiologica, Vol. 5 - - 7, 9 - - 11, 13-- 15 ( 1960 - - 62, 1964-- 66, 1968-- 70)]

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Fig. 2. Publication of different aspects of the method of continuous cultivations in the years of"the publications explosion". [Based on Mfilek and Rieica, Annual bibliographical reviews in Folia microbiologica, VoL 10------11, 13 15 ( 1965-- 1966, 1968-- 1970)]

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terns, relation of liquid and gaseous phases, regulation of various parameters, etc. It can be said that the introduction of continuous large-scale production of biomass, some enzymes etc. would, in itself, have been an important impulse for forming biochemical engineering as a branch. Although research into continuous formation of some important secondary metabolites, comprising substances of such importance as antibiotics, is only in its very infancy, the solution of problems associated with this represents a very important stimulus for the further development of the new branch. Besides the development of multistage equipment with the necessary controls and possibilities for regulating the indMdual stages, further theoretical and design problems on tubular and multistage reactors arise. Certainly a relevant engineering problem here is the integration of the total continuous system, starting with the preparation of ingredients through to the continuous isolation and purification of the product. Important new engineering problems have been introduced also by research into the production and purification of enzymes. And what are the practical demands and perspectives for the application of biochemical engineering? Processes based on biochemical engineering research are the basis for extensive and extraordinarily important practical applications. First of all there is production of antibiotics, enzymes, amino-acids, citric and some other organic acids. A special sector here is the production of proteins especially through unicellular organisms --yeast, bacteria, algae etc. It is widely accepted today that, without the full development of this way, it would be impossible to secure sufficient quantities of valuable proteins for the rapidly growing demand of human populations. It can be said ~ith confidence, that the automated production of protein from various substrates will become a necessary supplement to traditional agricultural production in the future. It will of course be a task of biochemical engineering to bring these processes to the ultimate objective-protein acceptable directly in human nutrition. It is an open question to what extent biological and biochemical processes will be replaced by purely chemical syntheses. Though there is no doubt that many simple substances obtained by biological processes will be synthetized chemically, as for example ethanol, there are many others, even relatively simple chemical substances, where the biological and chemical methods are entirely competitive. In some processes (production of some amino-acids such as lysine, citric acid production, transformation of sterols etc.), the biological method is still preferable to the chemical one. For many of these substances the biochemical, biological and bioengineering processes have still not been fully developed. The industrial production of citric acid, in which the simple surface culture process is mostly employed because the continuous sub-

Present State and Perspectives of Biochemical Engineering

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merged method has not been completely mastered, may serve as an example. There is no doubt that biological processes find wide application in the production of more complex substances, and even recent dramatic developments in chemical synthesis do not warrant their synthetic production in the near future. This is the case in the production of proteinaceous substances, both of biomass as a source of nutrition, and various enzymes. Concerning the latter, not only a wide development, but also new, more effective forms for their application are expected. Similarly, a further development in the production of antibiotics and other biologically active microbial substances is to be expected since their biochemical production is more advantageous than the chemical one. Production of these substances can be made more economical by introducing more effective strains obtained by new isolation or by breeding, by a better knowledge of biosynthetic pathways, and by modifying engineering processes according to biological demands. An entirely open field in which empirical solutions of biological, biochemical and engineering problems have prevailed, and in which considerable progress can be achieved by a close coordination of both approaches, is neutralisation and utilization of waste (especially liquid), so that waste circulation loss may be reduced and the utilization of waste substances maximized. At present, an imperfect solution of the first requirement predominates; water is treated, but generally without taking into consideration the possibilities of utilization of the remaining organic substrate. The new discipline--biochemical engineering--could help to solve both theoretical and practical problems in this field. To conclude, we can say that biochemical engineering has a solid theoretical basis and wide possibilities of practical application. There is no doubt that entirely new possibilities will appear as this branch becomes fully developed. As mentioned above, besides the term "biochemical engineering" the term "bioengineering" is also used. The latter, as a rule, is more comprehensive as it includes medical engineering, sanitary (environmental) engineering, agricultural engineering, bionics, human factors engineering and biochemical (fermentation) engineering. This would suggest that biochemical engineering is a part of a wider branch-bioengineering. Logically this is so, but pragmatically biochemical engineering is an independent discipline having its own scientific tradition and concrete tasks. The term "biochemical engineering" is sometimes employed solely for microbial or fermentation engineering. Even if biochemical engineering consists mainly of these two branches, which decisively affect the educational demands in this field, it would be incorrect to adopt such a limited definition. It is assumed that biochemical engineering will

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include also research on isolated biochemical systems, such as enzymatic pathways, proteins of different quality and origin and their transformation according to the demands of man and his nutrition. In the total perspective, a wider application of ecological factors is to be taken into consideration. This part of my contribution deals with the perspective, social requirements and assurances for the development of biochemical engineering. The most important factor in the further development of biochemical engineering is the education of specialists biochemical engineers. I do not intend to give a detailed account of this problem, but to summarize the rules resulting from what I have said in the preceding paragraphs. Even if we consider that in the future also such specialists as biochemists, molecular and cell biologists, microbiologists, plant and animal biologists, chemists and chemical engineers, mathematicians and mechanical engineers will continue to participate in this branch, the key problem is the education of specialists in whose education all the above-mentioned disciplines would be integrated to a certain extent. This will not be easy and good results will be achieved only on the assumption that this integration will concentrate on the basic theoretical and methodological preparation, while further more practical specialisation will be differentiated. Owing to the intensive development of this branch which is in prospect, it will be necessary to provide sufficient support and suitable conditions, and to develop appropriate systems for full-time postgraduate studies and additional training. What are the actual perspectives of biochemical engineering? It seems that microbes, both pure and as mixed populations, will continue to be a source of valuable substances whose introduction into production will depend on biochemical engineering. Microbial biomass and protein, as well as other valuable substances isolated from biomass, will play an important role in human life. It may be emphasised that microorganisms comply with the demands of automated production much better than animals and plants used in traditional agriculture. We may assume that this trend will increase through the economic employment of scientific results, leading to better water and mineral economy and reduced transportation of large qualities of material. This production will probably become, no matter what the raw material used, a part of agro-industrial complexes built particularly in the vicinity of atomic power plants possibly connected with desalination of sea water. Further development stages of biochemical engineering will certainly help to master the production of fresh water algae and products from their biomass. Although it may appear at first sight that there are not too many scientific problems in this field,

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the completion of the production cycles will demand the solution of many scientific tasks, especially because this production will be connected with the utilization of different substances which at present are considered waste (plant cellulose, substances in waste water, etc.). Undoubtedly, attention will be paid to the production of antibiotics and other biologically active substances, not only to the substances acting against microbes, but also to substances for biological insect control etc. There is also a possibility of discovering cancerostatic and antiviral substances (despite the present experience being not as encouraging as originally supposed). These substances need not necessarily be of microbial origin; experience with the production of interferon proves that various possibilities are open. We realise more and more that from a wide selection of microbial metabolites only a few have been employed and that in some of them a new, until now unexpected, effect may be discovered. There is the question whether and to what extent microbes will be used in the production of essential amino-acids, but the fact that such processes have already been introduced commercially suggests the possibility of further developments, particularly if we are able to regulate the metabolic systems of microorganisms effectively. I have already stressed the importance and perspectives for the production of different enzymes, not only of those which are produced commercially (amylase, proteinase, cellulase, pectinase), but also of unusual enzymes which may prove to be useful. The discovery of 1-asparaginase effect may serve as an example. The development of biochemistry will no doubt bring about many surprises, particularly concerning the application of enzymes in water-insoluble forms. More effective utilization of microbial potentialities will be achieved by the application of continuous process to microbial systems which will retain their essential characteristics in such conditions. This will be made possible as mentioned above only by better control of the mechanisms affecting product formation. Biochemical engineering will have to pay even more attention than at present to the control of recycling and treatment of organic (and partly also inorganic) substances in order to maintain the natural equilibrium and to minimize the losses connected with losses connected with waste disposal. There are many other perspectives, some of which were discussed at the Fermentation Symposium, New Brunswick, N. J. in 1968: microbial transformation of natural polymers, alkanes, terpenes, and alkaloids, employment of sulphate-reducing bacteria etc. I do not wish to discuss in detail the trends analysed thoroughly by Hed6n (1969), and Langlykke (1969), both in Perlman (1969), pp. 861--881, 8 8 3 9 0 3 .

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It is not necessary to give a detailed account of the perspectives of the engineering part of biochemical engineering. Effective employment of computation can considerably increase the number of identified, monitored and controlled parameters and reveal relationships which require synchronous evaluation. More accurate knowledge will help in scaling-up laboratory data and bring about new solutions which may be completely different from those traditionally held to be unique. In conclusion, I would like to stress another important factor. When considering biochemistry and its role in different branches, we tend to see only those developmental aspects of biology and biochemistry which resulted in a detailed knowledge of molecular biology and of the control of the basic life cycle, both at the macromolecular level and at the level of cellular systems. Even if this is one of the basic aspects, vital for the development of biochemical engineering, we should not forget other aspects which are seemingly (or actually) less exact, but at least equally stimulating: the ecological attitude to the activity of living organisms in a natural environment and the improvement of biochemical and engineering knowledge applicable to this new branch. It is important to study the living associations in soil and water and in other natural substrates with respect to the specific requirements of biochemical engineering. Even if this opinion may not seem sufficiently proved, I believe that it represents one of the aspects which should not be neglected in the future consideration of biochemical engineering.

References Aiba, B., Humphrey, A. E., Millis, N. F.: Biochemical Engineering. Tokyo: Univ. Tokyo Press 1965. Commentary (Krebs and Fermentation Technology): Process. Biochem. 3 (1), 3 {1968). Editorial: Process. Biochem. 4 (3), 3 (1969). Ghose, T. K.: The Anatomy of Change, a Case for Sophistication. Inaugural Lectures-I, Delhi: Indian Institute of Technology, July 1970. Lilly, M. D.: Enzyme Synthesis in Microorganisms. Process. Bioehem. 2 (2), 17 (1967). Mfilek, I., Fencl, Z. {Eds.): Theoretical and Methodological Basis of Continuous Culture of Microorganisms. Prague: Publ. House Czechoslov. Acad. Sci. 1966. Perlman, D.: Fermentation Industry-- Evolution. Process. Biochem. 4 (6), 29 (1969). Perlman, D. (Ed.): Fermentation Advances. New York and London: Academic Press 1969. Tsuchiya, H. M.: Introductory Comments. Biotechnol. Bioeng. 12, 645 (1970). Academician IVANMALEK Na Dolin~fch 18 14700 Praha 48 -- Podolf (CSSR)

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