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JOURNALOF FERMENTATION AND BIOENGINEERINGVol. 85, No. 2, 209-212. 1998

Effect of Acetic and Butyric Acids on the Stability of Solvent andSpore Formation by Clostridium acetobutylicum ATCC 824during Repeated SubculturingOMAR ASSOBHEI, ABDELLAH EL KANOUNI,* MOHAMMED ISMAILI, MOHAMMED LOUTFI,

AND HENRI PETITDEMANGE2Lab. Microbiologic, FacuM des Sciences Ben Msik, B.P. .7955, Casablanca, M~rocco,~ and Lab. Chimie Biologique I,UniversitP de Nancy I, B.P. 239, 54506 Vandceuvre-Les Nancy, Cedex, Fran&

Received 28 July 1997/Accepted 27 October 1997We studied the effect of serial transfers on solventogenesis capacity and spore-forming ability by Clostridiumacetobutylicum ATCC 824 in two culture media. In reinforced clostridial medium (RCM), an abrupt decreaseof the ability to ferment glucose and produce solvent occurred after the 10th to 12th transfer and spore for-mation failed after the 25th transfer. When 1.5 g/Zof acetic and butyric acids were added to the RCM, however,the capacity to produce a normal level of solvents was not lost, whereas spore-forming ability was lost after the23rd transfer. The shift from solventogenesis to acidogenesis did not occur in chemically defined medium(CDM) even by the 40th transfer. The dramatic decrease of solventogenesis in the degenerate culture was coupledwith acetate/butyrate CoA transferase and acetoacetate decarboxylase deficient activities.[Key words: solventogenesis, Clostridium acetobutylicum, fermentation, spore formation, degeneration]

It is known that during repeated subculturing of batchculture (l-3) or during continuous fermentation (4-8) ofsaccharolytic clostridia, solvent formation declines withtime. From an industrial point of view, the most promis-ing results concern the significant increase in productiv-ity obtained with continuous cultures (9). At the presentstage of development, the problem of stability and lon-gevity of the solventogenesis in Clostridium acetobutyli-cum may be solved. Significant effects have been reportedregarding the pH of the acetic and butyric acids onthe induction of acetone and butanol production (lo-12). The relation between spore and solvent formationwas extensively investigated (13, 14) and spore-formingdeficient solventogenic strains were reported (14). Recent-ly, it has been shown that the loss of solventogenesis isdue to a defect in the region of the chromosome encod-ing the solvent genes (15). The aim of our investigationwas to elucidate the culture conditions that allow rapidand spontaneous degeneration of C. acetobutylicum andto determine the relationships between the level of totalacid concentration and solventogenesis stability duringsubculturing, and between the loss of solventogenesisand spore forming ability.

MATERIALS AND METHODSMicroorganism Clostridium acetobutylicum ATCC824 was maintained in reinforced clostridial medium(RCM, Oxoid Ltd., Basingstoke, UK) at 35C for 5 d fol-lowed by storage at 4C. For inoculum preparation, thisstored culture was transferred to fresh RCM. After heatshocking at 80C for 45 min, the culture was incubatedat 35C.Media The following media were used. The chemi-cally defined medium (CDM) (16) consisted of (per literof distilled water): glucose, 6Og; KH2P04, 0.5 g; K2HP04.3Hz0, 0.5 g; MgS04.7Hz0, 0.2g; MnS04.Hz0, 10mg;

* Corresponding author.

FeS04. 7H20, 10 mg; NaCI, 10 mg; ammonium acetate,2.2 mg; p-amino-benzoic acid, 1 mg; biotin, 0.01 mg andCaCOr, 3 g (buffer agent). The RCM was supplementedwith glucose in log/l. Acetic and butyric acids weresterilized by filtration and added to the RCM as potassiumsalt. The pH of the media was adjusted to 6.8.Serial-transfer technique In the first experiment, C.acetobutylicum was maintained by growth in a Hungatetube (18 x 150mm) containing 10ml of RCM. Every12 h, 1 ml of culture was transferred to 9 ml of freshRCM allowing maintenance of growth in low-acidogenicconditions, 5 ml was transferred to 45 ml of fresh CDMin order to study the fermentation profile (after 72 h)and 1 ml was transferred to 9 ml of fresh RCM in orderto test spore formation which was induced by growthoccurrence at 35C after heat shock (45 min at SOC). Inthe second experiment, every 24 h, 5 ml of culture in a45 ml CDM tube was used to inoculate the next CDMtube, permitting the strain to be kept in acidogenic condi-tions. The residual volume of the culture was used tostudy the fermentation profile (after 72 h). At the sametime, analysis of the end-products was performed ineach culture at the moment of each transfer.Analysis Cell concentration was estimated by drycell weight using a correlation between OD at 600 nmand dry cell weight (1 OD =0.3 g/l). After 72 h of cul-ture in CDM, analyses were made on the supernatantsamples. The concentration of residual glucose was deter-mined by the method of Miller et al. (17). The end-product concentration was determined by injectingacidified supernatant into an Intersmat ICC 121 FL gaschromatograph equipped with a flame ionization detec-tor. The isobutanol was used as an internal standard.Separation took place in a 2 m long glass column packedwith Porapack Q.l00/120 mesh. N2 was used as carriergas. Injector and detector temperatures were 220C andthe column temperature was programmed to be between175C and 225C. The analyse of the chromatographicdata were carried out by an Intersmat ICR IB Integrator.

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Enzyme activities After 24 h of fermentation inCDM, cells were separated by centrifugation (2O,OOOxg,20 min) at 4C. The pellet was suspended in a degassedTris-HCl (0.01 M, pH 7) buffer and disrupted by sonica-tion at 4C for 4min at 20,000 cycles. Cell-free extractswere obtained by centrifugation (20,0OO~g, 1 h) at 4C.Acetate and butyrate kinase activities were performedas described by Twarog and Wolfe (18), CoA transferaseactivity was performed according to the method ofAndersch et al. (19) and acetoacetate decarboxylase activitywas determined in a Warburg manometer as describedby Westheimer (20). Proteins were measured by themethod of Lowry et al. (21) using bovine serum albuminas the standard.

RESULTS AND DISCUSSIONEffect of serial transfers in RCM medium with andwithout acids Repeated serial subculturing of C.acetobutylicum has been maintained on the RCM con-

taining 1% glucose. After 10 to 12 successive transfers,the cells abruptly lost their ability to use up 20 g/l of glu-cose and to accumulate a normal level of solvents (Fig1A). Whereas during the first 10 transfers, glucoseconsumption was maintained at high levels (50-52g/t)and solvents were accumulated at high concentrations(15 g/Z). Spore formation was not affected by thismetabolism shift. However, after the 25th transfer, theculture failed to form spores and after shock heating, nogrowth occurred at all.It has been shown that acetic and butyric acids inducethe solventogenesis (10-12) and that the production ofbutanol begins when 3-4 g/l of acids are accumulated inthe medium. The data from the analysis of the solventsand acids at the time of various transfers (Table 1) indi-cated that in RCM, the level of acids was found to be2.4g/l lower than that required for solventogenesisinduction. On the basis of these data, 1.5 g of acetic acidand 1.5 g of butyric acid were added to the RCM. Underthese experimental conditions, solvent production wasmaintained at high levels (15-16 g/l) and glucose utiliza-tion was stable in spite of transfers (Fig 1B). Sporulatingability ceased after the 23rd transfer on RCM sup-plemented with acetic and butyric acids.Effects of transfers during the acidic phase in CDMmedium C. acetobutylicum showed the biphasic pat-tern of the acetone-butanol fermentation when it wasgrown in batch culture in CDM with a glucose concentra-tion of 60 g/l. Successive subculturing was carried out atapproximately 24 h intervals. At transfer time, the acidlevel was approximately 7 g/l (Table l), which is morethan the required concentration for solventogenesis onsetand explains the stability of the solvent formation andTABLE 1. Culture conditions of C. acetobutylicum at the time ofvarious serial transfers (values are the average of 40 transfers)

Medium Acids added(g/0Time oftransfer Acids Solvent(h) (g/0 (g/0 pH

0 12 2.4

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VOL. 85, 1998 SOLVENTOGENESIS STABILITY IN C. ACETOBUTYLICUM 211

04 I I I I I I I I I I I, I I I I II72 4 6 8 1 0 1 2 1 4 1 6 1 6 2 0 2 2 2 4 2 6 2 6 3 0 3 2 3 4 3 6 3 6 40

Transfer numberFIG. 2. Effects of transfers during the acidic phase on the fermen-

tation. C. acetobutylicum was maintained on the CDM and after 24 hat transfer times, CDM was inoculated to study the fermentation.

mids containing solvent formation genes, Stim-Herndonet al. (15) have shown that normal solventogenesis canbe restored in mutant degenerate strains as well as inspontaneous degenerate strains. Enzymatic investigationshows that the CoA transferase and acetoacetate decar-boxylase specific activities, are drastically decreased,whereas the acetate and butyrate kinase activities areincreased. The serial subculturing experiments described inthis paper lead to a rapid production of nonsolventogen-ic culture in spite of the inoculum size used (10%). Ourresults showed that a high concentration of acids permitsthe stabilization of the solventogenic ability of theculture so that degenerative changes did not occur after 40transfers. However, a shift to acid production occurredafter 10 to 12 volume changes when the acid concentra-tion was below 5 g/l. During the two experimental condi-tions, the lost of the spore-forming ability of the cultureoccured independently of the solventogenic capacityand spore formation was able to be maintained even ifsolventogenesis was lost. On the basis of these results, wesuggest that there is a relation between the presence ofacids in the growth medium and the activity of solvento-genie strains of C. acetobutylicum. This could be due tothe magnitude of the carbon flux generated by acid addi-tion: one mole of butyrate or two moles of acetatemetabolized permit one mole of glucose to breakdownthrough the glycolytic pathway.It is known that during continuous fermentation,

TABLE 2. Enzyme activities of C. acetobutylicum after 24 h ofculture in CDM

Soecific activities ofCOAT B.K. A.K. A.A.D.

Normal culturea 1.2 3 7 600Degenerate c&u& 0.004 6.5 13.5 20COAT, Acetate/butyrate CoA transferase; B.K., butyrate kinase;

A.K., acetate kinase; A.A.D., acetoacetate decarboxylase. Specificactivities of COAT., B.K. and A.K. are in pmol/min/mg protein.Specific activities of A.A.D. are in pmol CO*/min/g dry cell.

a Culture from the 12th transfer in RCM with acid addition.b Culture from the 12th transfer in RCM without acid addition.

solvent formation activity declines with time. Thus, on thebasis of our experiments, procedures that ensure asufficiently high total concentration of acids (acetic andbutyric) must be employed in stock culture in order tomaintain the solventogenesis of C. acetobutykum.

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