Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum

109

Biochimie, 69 (1987) 109- 115 © Soci6t6 de Chimie biologique/Elsevier, Paris

Research article

Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum Ghassan MATTA-EL-AMMOURI, Rachid JANATI-IDRISSI, Anne-Marie JUNELLES, Henri PETITDEMANGE and Robert GAY Laboratoire de Chimie Biologique L Universit~ de Nancy L B.P. 239, 54506 Vandoeuvre Les Nancy Cedex, France (Received 5-9-1986, accepted after revision 18-11-1986)

Summary -

The actions of butyric and acetic acids on acetone-butanol fermentation are investigated. Production of butyric and acetic acids are controlled by the extracellular concentrations of both acids: acetic acid added to the medium inhibits its own formation but has no effect on butyric acid formation, and added butyric acid inhibits its own formation but not that of acetic acid. The ratio of end metabolites depends upon acetic and butyric acid quantities excreted during the fermentation. In contrast to acetic acid, which specifically increases acetone formation, butyric acid increases both acetone and butanol formations. Acetate and butyrate kinase activities were also examined. Both increase at the start of fermentation and decrease when solvents appear in the medium. Coenzyme A transferase activity is weak in the acidogenic phase and markedly increases in the solvent phase. Acetic and butyric acids appear to be co-substrates. On the basis of these results, a mechanism of acetic and butyric acid pathways, coupled to solvent formation by C. acetobutylicum glucose fermentation is proposed. Clostridium acetobutylicum / acetone / butanol / kinase

R ~ s u m ~ - E f f e t s d e s a c i d e s a c ~ t i q u e et b u t y r i q u e sur la f o r m a t i o n de I ' a c ~ t o n e et du b u t a n o l par Clostridium acetobutylicum. Les actions des acides ac~tique et butyrique sur la fermentation

ac6tono-butylique sont recherch6es. La production des acides ac6tique et butyrique par Clostridium acetobutylicum est contr616e par les concentrations extracellulaires des deux acides : l'acide ac6tique ajout~ au milieu de culture inhibe sa propre formation mais n 'a pas d'effet sur celle de l'acide butyrique; de m~me l'acide butyrique ajout6 dans ie milieu de culture inhibe sa propre formation mais non celle de l'acide ac6tique. Les acides ac6tique et butyrique sont des cosubstrats; l'utilisation de i'acide ac6tique par la cellule augmente sp6cifiquement la quantit6 d'ac6tone form6e aiors que celle de l'acide butyrique augmente les productions d'ac~tone et de butanol. Les activit6s de l'ac~tate kinase et de la butyrate kinase ont 6t6 6tudi6es, les activit6s sp6cifiques de ces deux enzymes sont maximales au d6but de la fermentation et d6croissent lors de la formation des solvants. A l'inverse, l'activit6 de la coenzyme A transf~rase impliqu6e darts la rem6tabolisation des acides est faible lors de la phase acide de la fermentation et elle augmente significativement lors de la phase solvantog~ne. Sur la base de ces r6sultats, il est propos6 un m6canisme d'utilisation des acides par la cellule coupl~ gt la formation des soivants lors de la fermentation du glucose par C. acetobutylicum. Clostrisdium acetobutylicum / acdtone / butanol / kinase

110

G. Matta-EI-Ammouri et al.

Introduction Butyric and acetic acid effects, as well as the p H action on solvent productions, have been studied b y several authors [ 1 - 8 ] but the final results are sometimes contradictory. Andersch et al. [1], observed that butyric acid furthers butanol production, in agreement with other results [4]. In 1983, M o n o t et al., [2] showed that, at low p H and high glucose concentration, solvent production occurs when butyric and acetic acid concentrations in the m e d i u m become critical. Martin et al. [5] studied acetic and butyric acid effects on solvent production in a synthetic medium by batch fermentation in the presence o f a non limiting glucose concentration. The increase in acetone concentration by acetic acid addition was observed, whereas the butanol concentration remained stable. Under the same conditions, butyric acid very slightly modified b u t a n o l and acetone concentrations. Yu and Saddler [6] studied acetic and butyric acid effects on the a c e t o n e - b u t a n o l fermentation when D-xylose was the c a r b o n substrate. They noticed that b o t h acids increased solvent production, particularly when the acids were added at the inoculation time. F o n d et al. [7] worked under conditions o f n o r m a l l y weak solventogenesis (i.e., by a semicontinuous fermentation). Their final results showed that both acids induced the solventogenetic phase and markedly increased solvent production in a non specific way, because b o t h acetone and butanol were favored. Recently [8], we studied the acetic acid effects on solvent production. Using a b a t c h fermentation, with a non limiting glucose concentration and continuous acetic acid addition, we succeeded in increasing acetone production, without modifying butanol production. This result clearly demonstrates that, under o u r experimental conditions, only acetic acid specifically induced acetone production. The aim o f the present investigation was to study the effects o f butyric acid on metabolite f o r m a t i o n by C. acetobutylicum, and also the effects o f b o t h butyric and acetic acids on some enzyme activities involved in cellular metabolism, such as: acetate and butyrate kinases and coenzyme A transferase.

Materials and methods Microorganism The organism used was Clostridium acetobutylicum strain 77, a mutant obtained from C. acetobutylicum ATCC

824 [9] by the procedure of Hermann et al. [10]. Characteristic properties of this mutant have been described [9]. Spores of the strain 77 were stored at 4°C in RCM medium (reinforced clostridial medium, Oxoid).

Medium The preculture medium contained the following components per liter of distilled water: (NH4)2SO4, 3 g; K2HPO4, 0.5 g; MgSO4.7H20, 0.2 g; MnCI2.4H20, 10 rag; (NH4)~MoTO24.4H20, 10 mg; FeSO4.7H20 , 10 rag; CaCO 3, 3 g; yeast extract, 4 g; glucose, 60 g. The growth temperature was 35°C. This medium, without CaCO3, was used as the culture medium. An exponential culture grown in the preculture medium was used as inoculum (10070, v/v) in a 2 ifermentor (Biolafitte). Continuous inflow of sugar or of sugar and acid was obtained by means of a peristaltic pump. This flow rate was maintained for 34 h. The culture was stirred at 200 rpm. The pH was maintained at 4.8 by the automatic addition of 6 M NaOH. Method of analysis Cell concentrations were estimated by cell dry weight measurement using a predetermined correlation between absorbance at 600 nm and cell dry weight. Analyses were made on supernatants of the samples previously centrifuged at 20000 x g for 10 min at 4°C. The concentration of residual sugars was determined according to the method of Miller et al. [11]. Concentrations of solvents (ethanol, acetone and butanol) and acids (acetic and butyric acids) were determined by injecting acidified supernatants into and Intersmat IGC 121 FL gas chromatograph equipped with a flame ionization detector. Separation took place in a 2 m long glass column packed with Porapak Q, 100/120 mesh. N 2 was used as the carrier gas. injector and detector temperatures were 220°C and column temperature was programmed from 175 to 225°C. The analysis of chromatographic data was carried out by an Intersmat ICR 1B Integrator. Enzyme assays Cells were harversted from the fermentor, centrifuged at 20000 × g for 15 rain at 4°C. The supernatant was used to determine the concentrations of the products of metabolism. The pellet was resuspended in a Tris-HCl (0.1 M, pH 7) degassed buffer and disrupted by sonication at 2°C for 4 min at 20 K cycles. The supernatant was collected from the cell lysate by centrifugation at 20000 x g for 20 min at 4°C. Proteins were determined by the method of Lowry et al. [12] using crystalline bovine serum albumin as the standard. Acetate and butyrate kinase activities were determined by measuring the rate of acetyl or butyryl phosphate produced as described previously [13]. The assay mixture contained, in a total volume of I ml: 100 mM, TrisHCI, pH 7.5; 6 mM MnSO4; 10 mM ATP; 700 mM NH2OH (pH 6.4); 400 mM potassium salts of acetic or butyric acids; crude cell extract and distilled water up to 1 ml. The reaction was initiated by addition of crude extract, incubated for 5 rain at 30°C and stopped by addition of 3 ml of FeCI3, 6 H20 (5070 w/v in 0.1 N HCI).

Effects o f butyric and acetic acids on acetone-butanol fermentation The amounts of acetyl hydroxamate or butyryl hydroxamate formed were determined at 540 nm using a standard curve obtained with purified acetyl phosphate, since acetyl hydroxamate and butyryl hydroxamate produce almost the same color, tool per mol [14]. Coenzyme A transferase activity was measured according to the method of Andersch et al. [15]. The assay mixture contained, in a total volume of 1 ml: 74 mM TrisHCI buffer, pH 7.6; 0.02 mM acetoacetyl-CoA, 0.08 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB); 50 mM potassium arsenate, pH 8.0; 0.2 M potassium acetate or potassium butyrate, pH 7.0; and cell extract (0.6-1.2 mg of proteins). The coenzyme A transfer was measured in a coupled assay [15]. The acetyl.CoA or butyryl-CoA formed was subjected to an arsenolysis with phosphotransacetylase or phosphotransbutyrylase and the coenzyme A liberated was determined with DTNB. For calculation of enzyme activity measured with DTNB, the molar extinction coefficient of 13.6 mM-~ cm-1 at 405 nm was used [16]. One unit of mzyme activity was defined as the amount of enzyme c~talyzing the conversion of 1/zmoi of reactant into product per min. Specific activity was expressed per mg of protein.

Results

Effects o f butyric acid on solvent f o r m a t i o n Many experiments were carried out in order to obtain the maximal utilization o f butyric acid as fermentation co-substrate. To avoid the deleterious effects o f butyric acid addition on the microbial activity, this acid was continuously fed to the culture medium: Table I, assays 2 and 4. This table summarizes the effects o f butyric acid on glucose fermentation by C. acetobutylicum under two different experimental conditions. The toxic concent r a t i o n o f solvents (21.4 g.l -~ o b t a i n e d in experiment 4) limits the glucose fermentation but

111

more solvents were produced with less glucose metabolized due to the butyrate utilization by the cells: assays 3 and 4. With 50 g.1-l o f glucose, the substrate utilization (47 g.l - I ) was not modified by added butyric acid but more solvents were produced in experiment 2. A batch fed 0.36 g . l - L h -1 o f butyric acid for 34 h had an increased acetone concentration, from 2.9 to 4.6 g.l -~ (i.e., 0.029 mol), whereas the butanol concentration rose from 10.2 to 12.6 g.1-1 (i.e., 0.032 tool) (Table I assays 1 and 2). Using fermentation carried out with 66 g.l- ~ o f glucose (Table I, assays 3 and 4), a batch fed 0.36 g.1-1.h-1 of butyric acid for 34 h had an increased acetone concentration: from 3.4 to 4.6 g.l- 1 (i.e., 0.020 mol); whereas the butanol concentration rose from 13 to 15 g . l - l (i.e., 0.027 tool). Fig. 1 shows the effect of butyric acid on biomass formation and a c e t o n e - b u t a n o l productions. The cell concentration for this mutant strain is higher in the presence o f 66 g . l - i o f glucose (Fig. 1A, curve 3) than with a 50 g,1- 1 glucose concentration (Fig. 1A, curve 1). In both cases, butyric acid involves a slight inhibition o f biomass formation (curves 2 and 4). Acetone and butanol productions are increased in the presence of butyric acid (curves 2 and 4, Fig. IB, 1C). Fig. 2 shows the evolution o f specific rates of butanol and acetone formations versus time. This evolution explains the variations o f cellular behavior due to a precise change in fermentation medium. It can__be noted that the addition of butyric acid markedly results in an increase of specific butanol and acetone formation rates (Fig. 2A and B, curves 2 and 4). Using a fermentation carried out with 50 g.lof glucose, the specific butanol formation rate grows very slowly and reaches a maximum value of 0.125 h -I (Fig. 2A, curve 1). This value remains

Table I. Effect of butyric acid on glucose fermentation by Clostridium acetobutylicurn mutant strain 77. Exp. Glucose Butyric added acid a (g.i- l) (g.l- I,h- 1) 1

50

0

2 3 4

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Solvents formed

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Glucose fermented (g.l-I)

3.5 2.7 3.8 3.3

4.0 3.0 5.0 4

2.2 3.5 1.5 4

47 47 61 51

Acetone Butanol Ethanol Total (g.l- l) solvent 2.9 4.6 3.4 4.6

10.2 12.6 13 15

1.0

1.0 2.4 1.8

14.1 18.2 18.8 21.4

,Butyric acid is continuously fed to the medium as described under Materials and Methods.

G. Matta-EI-Ammouri et al.

112

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Fig. 1. Influence of butyric acid on glucose fermentation by C. acetobutylicum strain 77. IA. Kinetics of biomass formation in 50 and 66 g-l- I glucose fermentations without (respectively curves l and 3) and with the addition of 0.36 g.l- Uh- ' of butyric acid for 34 h (respectively, curves 2 and 4). lB. Kinetics of acetone and butanol productions in a 50 g.l - ~glucose fermentation without (curve 1) and with the addition of 0.36 g . l - ' - h - J of butyric acid, for 34 h (curve 2). IC. Kinetics of acetone and butanol productions in a 66 g-l-' glucose fermentation without (curve 3) and with the addition of 0.36 g.l-I.h-i of butyric acid for 34 h (curve 4).

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Fig. 2. Influence of butyric acid on the specific rates of butanol formation (A) and of acetone formation (B) in glucose fermentation by C. acetobutylicum mutant strain 77. Curves 1,2: fermentations with 50 g.I- i of glucose, without (curve 1) and with the addition of 0.36 g-l-I.h-i of butyric acid for 34 h (curve 2). Curves 3,4: fermentations with 66 g.1-~ of glucose, without (curve 3) and with the addition of 0.36 g.I-~.h-i of butyric acid for 34 h (curve 4).

weaker than the maximum value (0.175 h-1) observed with a fermentation realized with 66 g.l- i of glucose (Fig. 2A, curve 3). A high glucose concentration allows a better conversion of sugar to butanol. In the presence of butyric acid, the specific butanol formation rate rapidly reaches a maximum

value of 0.225 h - t which is the highest value obtained by the mutant strain (Fig. 2A, curve 4). Fig. 2B (curves 2 and 4) shows that butyric acid raises the specific acetone formation rates. Using fermentations carried out with 50 and 66 g4- i of glucose without added butyric acid, the maximal specific acetone formation rates remained quite similar: 0.04 h -I (Fig. 2B, curves 1 and 3). With added butyric acid, these rates increase to 0.07 h -t and 0.06 h - l , respectively (Fig. 2B, curves 2 and 4). These experiments demonstrate that butyric acid accelerates cellular metabolism in the solventogenetic phase and that its action is not specific, since it increases both acetone and butanol formations.

Effect o f acetic and butyric acids on acetate and butyrate kinase activities and on acetate and butyrate CoA transferase activities The last steps of acetic and butyric acid formations are catalyzed by acetate and butyrate kinases, whereas acetate and butyrate coerazyme A transferase activities are involved in acetoacetate and butyryl-CoA or acetyl-CoA formations.

Acetate kinase evolution Using a fermentation realized with 66 g.l-i of glucose, acetate kinase reaches its maximum value at the beginning of the fermentation. The specific acetate kinase activity is 8 U . m g - l of protein,

Effects o f butyric and acetic acids on acetone-butanol fermentation C ACETATE KINASE '; 8 i

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Fig. 3. Influence of acetic and butyric acid on the kinetics of acetone production (A), kinetics of acetate production (B), and on the specific activity of acetate kinase evolution (C) in glucose fermentation by C. acetobutylicum mutant strain 77. Curve 1 : fermentation with 66 g.l -~ of glucose. Curve 2: fermentation with 66 g.I -~ of glucose with acetic acid addition (1 g-I- t initially and 0.26 g.i- ~.h- ~ during the first 24 h then 0.37 g.l-~.h-~ during the following 24 h. Curve 3: fermentation with 66 g.I -~ of glucose with butyric acid addition (0.36 g.l-~.h -~ for 34 h).

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Fig. 4. Influence of acetic (A) and butyric (B) acids on the specific activities of acetate and butyrate CoA transferase in glucose fermentation by C. acetobutylicum strain 77. A and B, curves I, show the evolution of the specific activities versus time of coenzyme A transferase either with acetate or butyrate as the acceptor of coenzyme A. Assays were run with cells grown in 66 g.I-~ of glucose. A, curves 2, show the evolution of the specific activities versus time of coenzyme A transferase either with acetate or butyrate as the acceptor of coenzyme A. Assays were run on cells grown in 66 g.l-x of glucose supplemented with acetic acid (1 g.I- ~ initially and 0.26 g.I- ~.h- ~during the first 24 h, then 0.37 g.l-~.h - ' during the following 24 h. B, curves 3, show the evolution of the specific activities versus time of coenzyme A transferase either with acetate or butyrate as the acceptor of the coenzyme A. Assays were run with cells grown in 66 g.1- I of glucose supplemented with butyric acid addition (0.36 g.l-l.h -' for 34 h).

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Fig. 5. Influence of butyric and acetic acids on the kinetics of butanol production (A), of butyrate production (B), and on specific activity evolution of butyrate kinase, in glucose fermentation by C. acetobutylicum strain 77. Curves 1 : fermentation in 66 g.I-t of glucose. Curves 2: fermentation in 66 g-l-~ of glucose with acetic acid addition (1 g.I -~ initially, 0.26 g-I- I.h- i during the first 24 h then 0.37 g.l-~.h-t during the following 24 h). Curves 3: fermentation with 66 g-l-~ of glucose with butyric acid addition (0.36 g.I -I for 34 h.).

after 5 h of fermentation, then it decreases when solvents appear in the medium (Fig. 3C, curve 1). In this culture medium, with added acetic acid, the acetone concentration increases very markedly IlCio_ "~A_ e l l r v e ~_ w h i c h ig $_~ t i m e g h i ~ h a r t h a n

its usual concentration. At the same time, if acetic

acid is measured in the culture medium and if the initial concentration is subtracted, it can be noted that the cells excrete only a negligible acetic acid amount into the medium (Fig. 3B, curve 2). The decrease in acetic acid formation by the strain (Fig. 3B, curve 2) is accompagnied by a weak fall in acetate kinase specific activity (Fig. 3C, curve 2) at the beginning of the fermentation, and then by an early disappearance of this enzyme (Fig. 3C, curve 2). The increase in acetone concentration is correlated with a slight increase of the coenzyme A transferase specific activities (Fig. 4A and 4B, curves 2 and 3), when acetic (Fig. 4A) or butyric (Fig. 4B) acids are added to the medium. Using a fermentation carried out with 66 g.l-t of glucose and with added butyric acid, acetic acid (Fig. 3B, curve 3) and acetone (Fig. 3A, curve 3) concentrations are above the average ones. These results are in agreement with a slight increase in acetate kinase specific activity at the beginning of the fermentation, which rises from 8 to 10 U.mgof protein (Fig. 3C, curve 3).

114

G. Matta-EI-Ammouri et al. Glucose I

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Fig. 6. Proposed scheme for glucose fermentation by C. acetobutylicum. Solid arrows indicate product formations from glucose and open arrows the pathways of acetic and butyric acids used as co-suhstrates. I : glycolysis; II: NAD(P)H ferredoxin oxidoreductases; lII: hydrogenase; IV: pyruvate ferredoxin oxidoreductase; V: phosphate acetyl transferase; VI: acetate kinase; VII: coenzyme A transferase; VIII: pathway of ethanol formation; IX: acetyI-CoA acetyl transferase; X: pathway of butyryl CoA formation; XI: phosphate butyryl transferase; XII: butyrate kinase; XIII: pathway of butanol formation; XIV: acetoacetate decarboxylase.

Butyrate kinase evolution Using a fermentation carried out with 66 g.l- 1 of glucose, butyrate kinase activity is at its maximum (3 U . m g - l of protein) after 15 h (Fig. 5C, curve 1). With added acetic acid, butyrate kinase specific activity decreases slightly and disappears earlier than with a usual fermentation (Fig. 5C, curve 2). With butyric acid added to the culture medium, butyrate kinase specific activity decreases by about 33°70. The specific activity falls from 3 to 2 U-mg -I of protein (Fig. 5C, curve 3). The butyrate kinase decrease is correlated with a drop in butyric acid excretion (Fig. 5B, curve 3). Indeed, Fig. 5, shows that butyric acid secreted into the culture medium by the mutant strain falls from 1.5 to 0.3 g.l-1 corresponding to a decrease of 80°70.

Discussion It has been previously demonstrated [8] that a continuous flow of acetic acid into the medium favored the production of acetone by C. acetobutylicum without any change in butanol formation. The present investigation clearly shows that a continuous butyric acid addition to the medium results in an increase of both acetone and butanol productions, particularly when 50 g.l- l of glucose were fermented (Fig. 1 and Table I). Incorporation of acetic acid by coenzyme A transferase explained the increase in the specific rate of acetone biosynthesis [8]. In the same way, the incorporation of the butyric acid by the coenzyme A transferase also explained the increase of acetone

Effects o f butyric and acetic acids on acetone-butanol fermentation

and butanol: each time that the coenzyme A transferase produces 1 mol of acetoacetic acid, 1 mol of butyryl-CoA is formed which is the substrate of butyraldehyde and butanol dehydrogenases (Fig. 6). The measured specific activities of the acetate and butyrate kinases, as well as that of the coenzyme A transferase corroborate preceeding reports [15, 18, 19] and explain the re-metabolization of acetic and butyric acids by the eoenzyme A transferase (Fig. 6). The specific activities of this enzyme remain stable throughout the acetone butanol formation (i.e., during the re-metabolization of the acids) (Fig. 4), whereas acetate and butyrate kinases markedly decrease during the solvent phase (Figs. 3 and 5). The mechanism of acetic and butyric acid pathways proposed (Fig. 6) demonstrates clearly the correlation between the acetone production and the reutilization by the cells of the acids which are produced during the first phase of the fermentation. These acids act as inducers of the solvent phase [4, 7, 20] and as co-substrates. In this report, we show the regulation of the acetate and butyrate productions: acetic acid addition nearly inhibits its own formation but has no effect on butyric acid biosynthesis, and butyric acid addition results in an inhibition of its own formation without affecting acetate biosynthesis (Figs. 3 and 5). These results emphasize the crucial importance of intracellular regulations and explain that according to the culture conditions, butanol and acetone production varies. The control of acetate production by the cells is the key point of acetone biosynthesis which is not coupled to butanol formation [8], whereas a high butyrate production increases both acetone and butanol formations. From our results, it appears that the acetone/ butanol final ratio depends upon the ratio of acetic/ butyric acid formed at the end of the acidic phase and which are reutilized for solvent production. Acknowledgements This research was supported by a grant from the Centre National de la Recherche Scientifique (CNRS-PIRSEM). The authors wish to thank Mr. G. Raval for excellent technical assistance.

115

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70-77 17 Martin J.R., Petitdemange H., Marczak R. & Gay R. (1982) Coiioque Soc Fr. Microbiol. I.F.P., RueilMalmaison, 123-137 18 Hartmanis M.G.N. & Gatenbeck S. (1984) Appl. Environ. MicrobioL 47, 1277-1283 19 Hartmanis M.G.N., Klason T. & Gatenbeck S. (1984) Appl. MicrobioL BiotechnoL 20, 66-71 20 Ballongue J., Amine J., Masion E., Petitdemange H. & Gay R. (1985) FEMS MicrobioL Lett 29, 273-277