JOURNAL
OF FERMENTATION AND BIOENGINEERINGVol. 85, No. 2, 209-212. 1998
Effect of Acetic and Butyric Acids on the Stability of Solvent and Spore Formation by Clostridium acetobutylicum ATCC 824
during Repeated Subculturing
OMAR ASSOBHEI,’ ABDELLAH EL KANOUNI,‘* MOHAMMED ISMAILI,’ MOHAMMED LOUTFI,’
AND
HENRI PETITDEMANGE2
Lab. Microbiologic, FacuM des Sciences Ben M’sik, 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 1997
We studied the effect of serial transfers on solventogenesis capacity and spore-forming ability by Clostridium acetobutylicum ATCC 824 in two culture media. In reinforced clostridial medium (RCM), an abrupt decrease of 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 the 23rd 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 coupled with 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 batch culture (l-3) or during continuous fermentation (4-8) of saccharolytic clostridia, solvent formation declines with time. 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 present stage of development, the problem of stability and lon- gevity of the solventogenesis in Clostridium acetobutyli- cum may be solved. Significant effects have been reported regarding the pH of the acetic and butyric acids on the induction of acetone and butanol production (lo- 12). The relation between spore and solvent formation was extensively investigated (13, 14) and spore-forming deficient solventogenic strains were reported (14). Recent- ly, it has been shown that the loss of solventogenesis is due to a defect in the region of the chromosome encod- ing the solvent genes (15). The aim of our investigation was to elucidate the culture conditions that allow rapid and spontaneous degeneration of C. acetobutylicum and to determine the relationships between the level of total acid concentration and solventogenesis stability during subculturing, and between the loss of solventogenesis and spore forming ability.
MATERIALS AND METHODS
Microorganism Clostridium acetobutylicum ATCC 824 was maintained in reinforced clostridial medium (RCM, Oxoid Ltd., Basingstoke, UK) at 35°C for 5 d fol- lowed by storage at 4°C. For inoculum preparation, this stored culture was transferred to fresh RCM. After heat shocking at 80°C for 45 min, the culture was incubated at 35°C.
Media The following media were used. The chemi- cally defined medium (CDM) (16) consisted of (per liter of 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 and CaCOr, 3 g (buffer agent). The RCM was supplemented with glucose in log/l. Acetic and butyric acids were sterilized by filtration and added to the RCM as potassium salt. 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 Hungate tube (18 x 150mm) containing 10ml of RCM. Every 12 h, 1 ml of culture was transferred to 9 ml of fresh RCM allowing maintenance of growth in low-acidogenic conditions, 5 ml was transferred to 45 ml of fresh CDM in order to study the fermentation profile (after 72 h) and 1 ml was transferred to 9 ml of fresh RCM in order to test spore formation which was induced by growth occurrence at 35°C after heat shock (45 min at SO’C). In the second experiment, every 24 h, 5 ml of culture in a 45 ml CDM tube was used to inoculate the next CDM tube, permitting the strain to be kept in acidogenic condi- tions. The residual volume of the culture was used to study the fermentation profile (after 72 h). At the same time, analysis of the end-products was performed in each culture at the moment of each transfer.
Analysis Cell concentration was estimated by dry cell weight using a correlation between OD at 600 nm and dry cell weight (1 OD =0.3 g/l). After 72 h of cul- ture in CDM, analyses were made on the supernatant samples. The concentration of residual glucose was deter- mined by the method of Miller et al. (17). The end- product concentration was determined by injecting acidified supernatant into an Intersmat ICC 121 FL gas chromatograph 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 packed with Porapack Q.l00/120 mesh. N2 was used as carrier gas. Injector and detector temperatures were 220°C and the column temperature was programmed to be between
175°C and 225°C. The analyse of the chromatographic data were carried out by an Intersmat ICR IB Integrator.
209
210
ASSOBHEI ET AL. J.
FERMENT. BIOENG..Enzyme activities After 24 h of fermentation in CDM, cells were separated by centrifugation (2O,OOOxg, 20 min) at 4°C. The pellet was suspended in a degassed Tris-HCl (0.01 M, pH 7) buffer and disrupted by sonica- tion at 4°C for 4min at 20,000 cycles. Cell-free extracts were obtained by centrifugation (20,0OO~g, 1 h) at 4°C.
Acetate and butyrate kinase activities were performed as described by Twarog and Wolfe (18), CoA transferase activity was performed according to the method of Andersch et al. (19) and acetoacetate decarboxylase activity was determined in a Warburg manometer as described by Westheimer (20). Proteins were measured by the method of Lowry et al. (21) using bovine serum albumin as the standard.
RESULTS AND DISCUSSION
Effect of serial transfers in RCM medium with and without 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 (Fig 1A). Whereas during the first 10 transfers, glucose consumption was maintained at high levels (50-52g/t) and solvents were accumulated at high concentrations (15 g/Z). Spore formation was not affected by this metabolism shift. However, after the 25th transfer, the culture failed to form spores and after shock heating, no growth occurred at all.
It has been shown that acetic and butyric acids induce the solventogenesis (10-12) and that the production of butanol begins when 3-4 g/l of acids are accumulated in the medium. The data from the analysis of the solvents and acids at the time of various transfers (Table 1) indi- cated that in RCM, the level of acids was found to be 2.4g/l lower than that required for solventogenesis induction. On the basis of these data, 1.5 g of acetic acid and 1.5 g of butyric acid were added to the RCM. Under these experimental conditions, solvent production was maintained at high levels (15-16 g/l) and glucose utiliza- tion was stable in spite of transfers (Fig 1B). Sporulating ability ceased after the 23rd transfer on RCM sup- plemented with acetic and butyric acids.
Effects of transfers during the acidic phase in CDM medium C. acetobutylicum showed the biphasic pat- tern of the acetone-butanol fermentation when it was grown in batch culture in CDM with a glucose concentra- tion of 60 g/l. Successive subculturing was carried out at approximately 24 h intervals. At transfer time, the acid level was approximately 7 g/l (Table l), which is more than the required concentration for solventogenesis onset and explains the stability of the solvent formation and TABLE 1. Culture conditions of C. acetobutylicum at the time of
various serial transfers (values are the average of 40 transfers)
Medium Acids added (g/0
Time of
transfer Acids Solvent
(h) (g/0 (g/0 pH
0
12 2.4 <l 4.8
RCM
1.5 acetic + 1.5 butyric 12 5 <2 4.8
CDM 0 24
RCM, Reinforced clostridial medium.
CDM, Chemically defined medium.
I 12 4.8
glucose utilization in the course of serial transfers (Fig 2).
Enzyme activities Enzymatic investigations are illus- trated in Table 2. The results show that acetate and butyrate kinase specific activities were two times higher in the degenerate culture than in the normal one. At same time, the specific activities of CoA transferase and the acetoacetate decarboxylase were strongly altered in the degenerate culture.
In the present study we describe a repeated transfer of C. acetobutylicum to fresh medium which leads to a rapid degenerated culture. After 10 to 12 transfers, the culture did not exhibit normal solventogenesis: acids were accumulated at the expense of solvent production.
Attempt to reverse this degeneration of solventogenesis by further acid addition was unsuccessful. Using plas-
50
0
2 4 6 8 10 12 14 16 16 20 22 24 26 28 30 32 34 36 38 40
Transfer number
20 1 (6)
81 I I I I I I I I I I I I I
2 4 6 6 10 12 14 16 16 20 22 24 26 28 30 32 34 36 38 40
Transfer number
FIG. 1. Glucose fermentation and product formation by C.
aeetobutylicum during repeated subculturing. In A, tubes of CDM
were inoculated from repeated serial subculturing of C. acetobutyli-
cum maintained in RCM. In B, the experimental conditions were the
same as in A but 1.5 g/l of acetic and 1.5 g/l of butyric acids were
added to the RCM. Symbols: 0, residual glucose; A, solvents; 0,
acids.
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 II7 2 4 6 8 10 12 14 16 16 20 22 24 26 26 30 32 34 36 36 40Transfer number
FIG.
2.
Effects of transfers during the acidic phase on the fermen- tation. C.acetobutylicum
was maintained on the CDM and after 24 h at transfer times, CDM was inoculated to study the fermentation.mids containing solvent formation genes, Stim-Herndon et al. (15) have shown that normal solventogenesis can be restored in mutant degenerate strains as well as in spontaneous degenerate strains. Enzymatic investigation shows that the CoA transferase and acetoacetate decar- boxylase specific activities, are drastically decreased, whereas the acetate and butyrate kinase activities are increased. The serial subculturing experiments described in this paper lead to a rapid production of nonsolventogen- ic culture in spite of the inoculum size used (10%). Our results showed that a high concentration of acids permits the stabilization of the solventogenic ability of the culture so that degenerative changes did not occur after 40 transfers. However, a shift to acid production occurred after 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 culture occured independently of the solventogenic capacity and spore formation was able to be maintained even if solventogenesis was lost. On the basis of these results, we suggest that there is a relation between the presence of acids in the growth medium and the activity of solvento- genie strains of C. acetobutylicum. This could be due to the magnitude of the carbon flux generated by acid addi- tion: one mole of butyrate or two moles of acetate metabolized permit one mole of glucose to breakdown through the glycolytic pathway.
It is known that during continuous fermentation,
TABLE 2. Enzyme activities of C.
acetobutylicum
after 24 h of culture in CDMSoecific activities of
COAT B.K. A.K. A.A.D.
Normal culturea 1.2 3 7 600
Degenerate c&u& 0.004 6.5 13.5 20
COAT, Acetate/butyrate CoA transferase; B.K., butyrate kinase;
A.K., acetate kinase; A.A.D., acetoacetate decarboxylase. Specific activities 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 the basis of our experiments, procedures that ensure a sufficiently high total concentration of acids (acetic and butyric) must be employed in stock culture in order to maintain the solventogenesis of C. acetobutykum.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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