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 -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

.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

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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

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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.

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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

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76

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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

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References

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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.

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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

74

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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75

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.

Some Aspects of Basic Genetic Research on Fungi

77

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

KARL ESSER

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

Some Aspects of Basic Genetic Research on Fungi

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

80

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~.

Some Aspects of Basic Genetic Research on Fungi

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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KARL ESSER

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]).

Some Aspects of Basic Genetic Research on Fungi

83

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-

84

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-

Some Aspects of Basic Genetic Research on Fungi

85

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.

87

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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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

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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.

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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.

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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

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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 . ~ 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

--- O 0 8 . E (.3

o 0.19

o

~ 0.04 ' o o 0.13 O.

~. 9

~_

10

~ 11

&0.22 E 0.08 . o

~ 12

~

13

n

, 14

~

15

•

131

o

switch to glucose

LD

-20.16 0.04 . d o -

0.10 14

15

16

17 Time (dcIys)

18

19

20

Fig. 9. Effcct of switching carbon sources between glucose and ×ylose on a chemostat culture of Escherichia coil E], Optical density; O , xylosc; A , glucose. (From Standing et al., 1972)

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

0.48[

0.44

25i35°C

r

=

0

x$R l/

0.4(~

~.r =3.33

0.36 0 I

0.4Z

I

....

35°~25° 0~ i

2 Time(h) l

I,

4

I

I

b

0"83

0.L xsR 0.36

0

5

10

Time(h}

15

20

Fig. 10a and b. Transient response of a chemostat culture of Klebsiella aerogenes to (a) a stepwise increase and (b) a stepwise decrease in temperature. Open circles arc experimental points: solid lines are theoretical curves calculated from an Arrhenius-type relationship, r = first-order time constant (h). (After Topiwala and Sinclair, 1971)

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

D . E . F . HARRISON and H. H. TOPIWALA (a)

40

-

~

~

2.0 1.6

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_~ 60. ~ 50.

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~ 40, -2o ~

~

~\ "\~'--

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. . . . .

~

~--&

~

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......??.-.

I~

m o E

C

0

,

1.2 I.C

"~

o.e

8 o.o 0,4

0.2

Time

of aerobiosis

(h)

Fig. 1 t a - - c . Response, on reaeration, ofa chemostat culture of Klebsiella aeroqenes grown anaerobically for 74 h at D=0.15 h i. (a) E:], Cell dry-weight concentration: solid line, dissolved oxygen; broken line, CO2 production, (b) A , Y~ . . . . . ; /x, Yo2: O, Q o~ (potential); 0 , Q o~ ( in situ), (c) O, acetate: 0 , ethanol; E:], butanediol: II, pyruvate. (From Harrison and Loveless, 1971 b)

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

2O

"a "6 15 E E

5

I

I

i

i

i

0

1

2

3

4

!

~

i

5 6 7 Time Anerobic (h)

!

8

1'0

1'1

Fig. 12. Response of CO2 production on making an aerobic/anaerobic transition in a glucose-limited chemostat culture of Klebsielta aerogenes. Growth rate=0,17 h-5. (From Harrison and Loveless, 1971 b)

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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0

30

60

90

120 z

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/290

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320

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350

380

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Fig. 13a--c. Changes in intracellular concentrations of metabolic intermediates and adenine nucleolides following interruption of oxygen supply to a chemostat culture of Klebsiella aerogenes. (a) 0 , phosphoenol pyruvate; D, citrate; (b) A, glucose-6-phosphate; 0 , fructose-diphosphate plus triosephosphate (expressed as Cs units), (c) O, ATP; 0 , ADP: L-I, AMP. Arrows denote times of ceasing and restarting oxygen supply. (From Harrison and Maitra, 1969)

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

201

(a) 4OO

=

300

2ooi-

/

~

/

0 6.8(~

:=~ 6.7C 6.6( -~

E

10C

6o 60

-~ ~ ~

4o

2o o

;o

1\

1~o

18o

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