acetobutylicum Intracellular ph and Cellular Distribution of

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1 APPLED AND ENVRONMENTAL MCROBOLOGY, June 1986, p Vol. 51, No /86/ $02.00/0 Copyright 1986, American Society for Microbiology nfluence of External ph and Fermentation Products on Clostridium acetobutylicum ntracellular ph and Cellular Distribution of Fermentation Products L HUANG, CECL W. FORSBERG,* AND L. N. GBBNS Department of Microbiology, University of Guelph, Guelph, Ontario NG 2WJ, Canada Received 16 December 1985/Accepted 7 March 1986 Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at ph 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at ph 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. ph 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at ph 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular ph, while butanol had relatively little effect until the external concentration reached 150 mm. Acetone had no effect at concentrations up to 200 mm. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic conditions and thus tend to lower the intracellular ph. The high intracellular butyrate concentration presumably leads to induction of solvent production, thereby circumventing a decrease in the intracellular ph great enough to be deleterious to the cell. Acetone-butanol fermentation by Clostridium acetobutylicum has been characterized as a biphasic batch-culture fermentation (14, 16, 18). The first phase is characterized by rapid growth and by the formation of acetic and butyric acids which are excreted into the medium, thereby lowering the medium ph. The second phase commences after the ph of the medium has fallen below approximately 5.0. During this period, butanol and acetone become the major fermentation products. The fatty acids, previously accumulated in the medium, pass through the cell membrane in their undissociated form (10) and are converted to solvents. At the end of the fermentation, the metabolic activity ceases primarily because the concentrations of the solvents have reached toxic levels (13). t has been shown that environmental changes that occur during the fermentation trigger solventogenesis (1, 6, 12, 13, 17). Of the major fermentation products, fatty acids, especially butyrate, were found to have a significant influence on the initiation of solventogenesis, and a critical level of undissociated butyric acid was reported to coincide with the onset of solvent formation (12). Butanol, on the other hand, was found to be the primary toxic substance in the acetone-butanol fermentation, and it inhibited cell growth by 50% when the concentration in the culture medium reached 150 mm (4, 13). However, in both cases, the concentration of the fermentation products within the cells was unknown. Furthermore, in recent studies, the intracellular ph of C. acetobutylicum cells grown in a chemostat was shown to decrease with decreasing external ph during acetogenic fermentation and then become stabilized after solventogenesis was initiated (9). t appears that the cells can maintain a relatively high intracellular ph under acidic growth conditions in a chemostat by redirecting metabolism from organic acid synthesis to solvent synthesis. Both acidic and neutral fermentation products have been shown to interfere with membranerelated functions such as energy generation which are re- * Corresponding author sponsible for generation of the transmembrane ph gradient (2, 5, 7, 13, 19). t was therefore of interest to determine the intracellular concentration of fermentation products and to investigate the effect of fermentation products on the intracellular ph of the cells. n this communication we present information on the distribution of the fermentation products across the cytoplasmic membrane of C. acetobutylicum grown at various ph values and on the sensitivity of the transmembrane ph gradient of the cells to these end products. The objective of the study was to determine the intracellular concentrations of fermentation products in cells and to assess the influence of these products on the maintenance of intracellular ph, since this information will be essential to understand the regulatory mechanism governing solvent production. MATERALS AND METHODS Organism and growth conditions. The bacterium used in this study was C. acetobutylicum ATCC 824. t was grown in a phosphate-limited chemostat at ph values ranging from 4.5 to 6.5. Cells grown at ph 6.5 carried out an acetogenic fermentation with acetate and butyrate being the major fermentation products, while at ph 4.5, butanol and acetone were the major fermentation product (9). The medium composition and the growth conditions have been described previously (9). The concentrations of fermentation products were measured by gas chromatography as described elsewhere (9). Reagents. [14C]benzoic acid (18.8 mci/mmol), [14C]butyric acid (13.4 mci/mmol), ['4C]butanol (1.0 mci/mmol), and [3H]raffinose (7.8 Ci/mmol) were purchased from New England Nuclear Corp. of Canada, Lachine, Quebec. Other materials were of reagent grade or the highest grade available. Metabolism of butyrate and butanol by C. acetobutylicum. The ability of cells to transform ['4C]butyric acid and [14C]butanol was tested as follows. Duplicate samples (2 ml) of culture from the chemostat operating at ph 4.5 were

2 VOL. 51, ,-10 -e 6jF a External FG. 1. Distribution of butyrate across the cytoplasmic membrane of C. acetobutylicum grown in a phosphate-limited chemostat maintained at various ph values (solid line). The dashed line represents the internal-to-external butyrate concentration ratios calculated from the ApH values, determined previously with ['4C]benzoic acid (9). incubated with [14C]butyric acid (36.3,uM, 13.4 mci/mmol) at 35 C with shaking. After 10 min of incubation, one sample was centrifuged at 34,800 x g for 10 min at room temperature to reproduce the procedure used in the actual determination of butyrate distribution (see below), heat treated at 80 C for 30 min, and centrifuged again. After 2 h of incubation, another sample was treated in the same manner. The supernatant fluid from each of the two samples was treated with Amberlite R-120 cation-exchange resin and filtered through a 0.45-,um-pore-size Metricel filter. The components in each sample were separated with a high-pressure liquid chromatograph (Waters Associates, nc., Milford, Mass.) equipped with a model ERC-7510 refractive index detector (Erma Optical Works, Ltd., Tokyo, Japan) and a model 730 data module (Waters Associates). Separation was achieved on a Bio-Rad Aminex ion-exclusion HPX-87H column (300 by 7.8 mm) fitted with a cation-exchange microguard precolumn (Bio-Rad Laboratories, Richmond, Calif.). The column temperature was maintained at 35 C. Degassed H2SO4 (0.01 N) was used as the solvent at a flow rate of 0.6 ml/min. Samples (10 Rd) were loaded onto the column, and fractions were collected with a LKB model 2112 Redirac fraction collector (Fisher Scientific Ltd., Toronto, Ontario, Canada). A sample of each fraction was mixed with ACS scintillation fluid (Amersham Canada Ltd., Oakville, Ontario, Canada), and the radioactivity was counted. The metabolism of [14C]butanol by the cells were examined in the same way except that the cells were incubated with [14C]butanol (3.04 mm, 1.0 mci/mmol) at 35 C for 1 h. Determination of distribution of fermentation products across the cell membrane of C. acetobutylicum. Two radioactively labeled compounds, [14C]butyric acid and [14C]butanol, were used to estimate the distribution of butyrate and butanol across the cytoplasmic membrane of cells. Samples (10 ml) of the culture withdrawn from the chemostat operating at selected ph values between 4.5 and 6.5 were dispensed anaerobically into centrifuge tubes, to which either [14C]butyric acid or [14C]butanol had been added to ph NTRACELLULAR ph OF C. ACETOBUTYLCUM 1231 give a final concentration of 4.46,uM (13.4 mci/mmol) or 0.25 mm (1.0 mci/mmol), respectively. [3H]raffinose was also included in the samples (2,uM, mci/mmol) to determine the extracellular space in the cell pellet. After incubation for 10 min at 35 C, the samples were centrifuged at 34,800 x g for 10 min at room temperature. The pellet and the supernatant fluid samples were processed and the radioactivity in these samples was counted as described previously for the measurement of the transmembrane ph gradient of the cells (9). From the total water content, determined gravimetrically, and the extracellular space in the cell pellet, the intracellular and extracellular radioactivity was obtained. The distribution of butyrate and butanol could then be estimated. Distribution of acetate and butyrate across the cell membrane was also determined by measuring the concentrations of these acids in a concentrated cell suspension from the chemostat culture with and without disintegration of the cells. Duplicate samples (100 ml) of the culture (1 g of dry cells per liter) were withdrawn from the chemostat operating at ph 4.5, 5.2, or 6.5, centrifuged anaerobically, and concentrated 100-fold by resuspending the pellet in the culture supernatant (Si) to a total volume of 1 ml. The cell suspension and the removed supernatant samples were heated in sealed tubes at 100 C in a steamer. After 25 min of heat treatment, the samples were cooled and then centrifuged at 4 C. The supernatant fluid samples (S2) from the cell suspension samples, together with duplicate Si samples, were analyzed for fermentation products by gas chromatography. The intracellular concentrations of fatty acids were calculated from the gas chromatography results, using the intracellular space values obtained previously from the intracellular ph determination (9). ntracellular ph of cells suspended in anaerobic citratephosphate buffer. Culture fluid was withdrawn from the chemostat operating at either ph 4.5 or 6.5. The cells were harvested by centrifugation at 13,200 x g for 10 min at room temperature, washed with anaerobic citrate-phosphate buffer (ph 4.5 or 6.5) supplemented with 20 mm glucose, and concentrated fourfold by resuspending the pellets in S ml of the same buffer of various ph values (ph 3.0 to 6.5). After incubation for 10 min at 35 C, the intracellular ph of the samples was measured by using [14C]benzoic acid as the probe as described previously (9). [3H]raffinose was used in this experiment to permit determination of the extracellular space (9). Effects of fermentation products on intracellular ph of the cells. The anaerobic pellet of the cells collected from 200 ml of culture grown in the chemostat operating at ph 4.5 was washed with 100 ml of the anaerobic citrate-phosphate buffer (ph 4.5) containing 20 mm glucose and resuspended in 55 ml TABLE 1. Distribution of acetate and butyrate across the cytoplasmic membrane of C. acetobutylicum ATCC 824 grown in a phosphate-limited chemostat maintained at various ph valuesa Acetate Butyrate put[acetatelin: [acetate]ou, phinb [Butyratelin: [butyrate]out phinb a ntracellular concentrations of acetate and butyrate were calculated from direct determinations of these compounds and the intracellular space. bpphjn values were calculated from the distribution data of the two fatty acids as previously described (9). pka values for acetic and butyric acids at 35 C were taken to be 4.76 and 4.84, respectively.

3 o< TABLE 2. Effect of the ph of a phosphate-limited culture of C. acetobutylicum ATCC 824 on the intracellular concentrations of acetate and butyrate [Acetate]in (mm) [Acetate],,, (mm) [Butyrateli, (mm) [Butyrate0u, (mm) ph0ut Calculateda Observedb observedb Calculated' Observedb observedb (86)C (167) (204) (244) (132) a The calculated intracellular concentrations of acetate and butyrate were derived from the ApH determined previously with ['4C]benzoic acid as a probe, the PKa values, and the extracellular concentrations of the respective acids. bthe observed intracellular and extracellular concentrations were obtained by direct determinations, as outlined in the Materials and Methods. C The intracellular butyrate concentration (in parentheses) was calculated from the distribution ratio for ['4C]butyrate and extracellular butyrate concentration. of the same buffer. Samples (5 ml) were dispensed into almost unchanged after 1 h of incubation, suggesting that the centrifuge tubes, to which one of the following compounds reaction(s) leading to butanol formation was actually irreversible under the conditions used in this experiment. was added to final concentrations indicated in the corresponding figures: 5 M acetic acid (ph was adjusted to 4.5 Distribution of fermentation products across the cell membrane. The distribution of butyrate across the cytoplasmic with 10 M NaOH); 5 M butyric acid (ph was adjusted to 4.5 with 10 M NaOH); acetone; and n-butanol. After incubation membrane of cells withdrawn from the chemostat maintained at various ph values was examined with ['4C]butyric for 10 min at 35 C, the intracellular ph values of the samples were measured by using [14C]benzoic acid as described acid as a probe. The butyrate concentration within the cells previously (9). was higher than that in the medium, and the internal-toexternal concentration gradient increased as the external ph RESULTS was lowered (Fig. 1). This was confirmed by direct determination and, furthermore, extended to include acetate (Table Metabolism of butyrate and butanol by the cells. The use of [14C]butyric acid and [14C]butanol to determine the distribution of butyrate and butanol across the cell membrane 1). We have reported that C. acetobutylicum ATCC 824 grown under conditions identical to those used in this study requires that no significant transformation of the radioactive maintained an internal-alkaline transmembrane ph gradient compound occur during the experiment. n this study, which increased with decreasing external ph (9). Since [14C]butyric acid was added to the cells taken from the butyric acid, a weak acid, can passively diffuse across the chemostat at ph 4.5. t was found that less than 3% of the cell membrane in its undissociated form (10), the distribution added [14C]butyrate was metabolized (mostly into of this acid is dependent upon the ph values on both sides of [14C]butanol) during the 10-min incubation period, although the cell membrane. As demonstrated in Fig. 1 and Table 2, at prolonged incubation (2 h) resulted in approximately 25% ph values above 5.0, the concentration gradient of butyrate conversion to butanol. Therefore, the error due to the determined in this experiment is in good agreement with that transformation of [14C]butyrate during the incubation period calculated from the transmembrane ph gradient of the cells (10 min) used in these experiments was considered to be obtained previously when benzoate was used as the probe negligible. (9). However, the intracellular butyrate level was lower than [14C]butanol conversion to butyrate by the cells was expected at ph 4.5 at which solventogenesis is favored. n investigated in a similar way. The added [14C]butanol was contrast to butyrate, the intracellular concentration of acetate calculated from the transmembrane ph gradient corresponded very closely to the experimentally determined 0 concentration. The intracellular concentrations of acetate 7.01 and butyrate were the highest at a culture ph of approxi- 'i 6.51 C 60[ 0o/0,s // 4.JD ~ ~ ~ ~ ~ ~ ~ 1 < a) c 5.5[ 8 0/; 5.01 L_ External ph FG. 2. ntracellular ph of nongrowing cells in citrate-phosphate buffer at various ph values. Growing culture was harvested from the chemostat operating at either ph 4.5 (0) or 6.5 (0), washed, and suspended in anaerobic citrate-phosphate buffer (20 mm glucose) of selected ph values. After incubation at 35 C, the intracellular ph of the samples was measured with ['4C]benzoic acid. mately 5.5 Butanol distribution across the membrane of the cells grown at ph 4.5 was determined with [14C]butanol. The internal-to-external concentration ratio for butanol was calculated to be approximately 1.30, indicating that the intracellular and extracellular butanol concentrations were similar İntracellular ph of cells suspended in anaerobic citratephosphate buffer. These experiments were performed to establish assay conditions under which the transmembrane ph gradient of the cells could be generated and studied in the absence of the fermentation products. t was found that cells harvested from the chemostat operating at either ph 4.5 or 6.5 and then washed and resuspended in anaerobic citratephosphate buffer at various ph values were able to generate a transmembrane ph gradient upon the addition of glucose (20 mm), and a steady-state level of the ph gradient could be reached within 20 min of incubation at 35 C. Two different intracellular ph profiles were observed (Fig. 2). Although 1232 HUANG ET AL. APPL. ENVRON. MCROBOL.

4 VOL. 51, 1986 A 6.5~ k 5.5k B Concn.(mM) FG. 3. Effect of acetate (A) and butyrate (B) on intracellular ph of the cells of C. acetobutylicum ATCC 824. A sample of the culture grown at ph 4.5 was centrifuged, and the cell pellet was washed and suspended in anaerobic citrate-phosphate buffer (ph 4.5) supplemented with 20 mm glucose. Samples (5 ml) of the suspension were dispensed into centrifuge tubes, to which acetic or butyric acid (neutralized to ph 4.5 with 5 N NaOH) was added to give final concentrations ranging from 0 to 70 mm. After incubation for 10 min at 350C, the intracellular ph of the samples was measured with [14C]benzoic acid. the nongrowing cells of both origins maintained a high intracellular ph within a certain external ph range, the solvent-producing cells exhibited a higher intracellular ph than the acid-producing cells. The cells from the chemostat operating at ph 4.5 were able to keep the intracellular ph above even when the external ph was as low as 3.5. A drop in intracellular ph occurred when the external ph was further decreased. n contrast, the intracellular ph of the cells originating from the chemostat set at ph 6.5 declined rapidly as the external ph was lowered and fell to at an external ph of 4.5. A marked drop in intracellular ph came after the external ph fell below 4.0. Effect of fermentation products on transmembrane ph gradient of cells suspended in anaerobic citrate-phosphate buffer. The intracellular ph of nongrowing cells suspended in the anaerobic citrate-phosphate buffer (ph 4.5) supplemented with 20 mm glucose was measured after incubation with each of the four major acetone-butanol fermentation products at various concentrations. t can be seen in Fig. 3 that the addition of either acetic or butyric acid to the cell suspension markedly lowered the intracellular ph, indicating the uncoupling effect of both acids. The drop in intracellular ph was found to be linearly related with the fatty acid concentration within the range of concentrations tested. Butanol slightly decreased the intracellular ph of the cells at concentrations below 150 mm (Fig. 4). However, when butanol was added to the cell suspension to give a concentration above 150 mm, the transmembrane ph gradient collapsed and the intracellular ph dropped dramatically. Acetone, on the other hand, did not affect the intracellular ph even at concentrations much higher than that encountered in the normal acetone-butanol fermentation (Fig. 4) (18). NTRACELLULAR ph OF C. ACETOBUTYLCUM 1233 DSCUSSON t has been found that the level of fatty acids in the culture medium of C. acetobutylicum for transition from acetogenesis to solventogenesis is dependent upon the external ph and that this level is higher when the solvent production is initiated at a higher ph value (12). n the present study, the distribution of the acidic fermentation products across the cytoplasmic membrane of C. acetobutylicum grown in a chemostat at various ph values was examined to provide a basis for further consideration of the "fatty acid effect." Both acetate and butyrate were concentrated by growing cells at ph values of 6.5 and lower, as would be expected from the transmembrane ph gradient determined previously (9), although the intracellular butyrate concentration was lower than expected in solvent-producing cells. This apparent anomoly could be attributed either to the vigorous transformation of butyrate to butanol within the cells or to an imbalance in the metabolic pathway for butyrate synthesis. However, at present there is insufficient information to reach a definitive conclusion. Based on the intracellular ph values determined from [14C]benzoate distribution, the pka values of acetate and butyrate, and the extracellular concentrations of these acids, the calculated intracellular concentrations were estimated to be the greatest at a ph value of 5.5. The calculated intracellular concentration for butyrate based on [14C]butyrate distribution was highest at ph. These data demonstrate that the butyrate and acetate concentrations within the cells are highest at or just above the ph at which solvent production was shown to be induced (9). This coincidence supports the contention that the high intracellular concentration of fatty acids leads to the induction of solvent production (8, 12). However, there is no evidence to support the suggestion that undissociated fatty acids alone serve as the inducing agent. These data do not permit an evaluation of the relative importance of acetate and butyrate in the induction of solvent synthesis. When cells are fermenting sugars, their ability to maintain an intracellular ph value near neutrality is subject to the stress imposed by the fermentation products. The extent to which cells can cope with the stress is significant, since any variation in intracellular ph will lead to the modification of the cellular functions (11, 15). n the absence of fermentation -n C C S B 6.5 * -A 5.5 r OO Concn.(mM) FG. 4. Effect of butanol (A) and acetone (B) on intracellular ph of the cells of C. acetobutylicum ATCC 824. The procedure described in the legend to Fig. 3 was followed, except that either butanol or acetone was added instead of the fatty acids.

5 1234 HUANG ET AL. products, the nongrowing cells suspended in buffer were able to maintain a high intracellular ph even at low external ph, and such ability was more pronounced in the cells originating from the chemostat set at ph 4.5 than in the cells from the chemostat set at ph 6.5. The enhanced ability of solvent-producing cells to generate a transmembrane ph gradient is presumably due to the adapted cells having a greater capacity to convert fatty acids to solvents. The addition of either acetate or butyrate to the cells suspended in citrate-phosphate buffer at ph 4.5 resulted in a marked drop in the intracellular ph of the cells. This effect increased with the concentration of the added acid within the tested range. t appears that even a relatively low concentration of the fatty acid would be a considerable load on the ApHgenerating system of the cells at low external ph. The effect of the fatty acid on the transmembrane ph gradient has been attributed to its ability to diffuse across the cell membrane in its undissociated form in response to the difference in ph between the two sides (2, 5, 10). Butanol showed a different effect on the intracellular ph of solvent-producing cells. When added at concentrations below 150 mm, butanol only slightly decreased the intracellular ph. However, at concentrations over 150 mm, it caused a dramatic drop in intracellular ph. This observation has also been reported from other laboratories (3). t has been observed that 150 mm butanol caused 50% inhibition of growth (13). Clearly, the growth inhibition and the fall in intracellular ph caused by butanol are related. Butanol has been shown to increase lipid fluidity and to inhibit some membrane-related functions, such as ATPase activity and nutrient transport systems, in C. acetobutylicum (13, 19). Thus, the failure to maintain a high intracellular ph may be caused by damage to the proton pump or to the shortage of energy (3). Acetone, on the other hand, displayed little effect on the intracellular ph even at a concentration which was approximately three times as high as the maximum acetone level encountered in culture filtrates from the acetone-butanol fermentation (18). This is consistent with the finding that acetone did not inhibit the growth of C. acetobutylicum at concentrations of less than 500 mm (4). The results obtained on the influence of acetate, butyrate, butanol, and acetone were for cells suspended in anaerobic citrate-phosphate buffer with glucose as a readily available carbon source. This allowed the separate assessment of the effect of each major fermentation product on the intracellular ph, which would not have been possible in growth medium. As a result of this approach, the data cannot be directly applied to growing cultures since a more complicated array of components influences the response of cells to the fermentation products. ACKNOWLEDGMENTS Appreciation is expressed to Donna Eby for typing the manuscript. This work was supported by research grants from the National Sciences and Engineering Research Council of Canada to C.W.F. and L.N.G. APPL. ENVRON. MCROBOL. LTERATURE CTED 1. Bahl, H., W. Andersch, K. Braun, and G. Gottschalk Effect of ph and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture. Eur. J. Appl. Microbiol. Biotechnol. 14: Baronofsky, J. J., W. J. A. Schreurs, and E. R. Kashket Uncoupling by acetic acid limits growth of and acetogenesis by Clostridium thermoaceticum. Appl. Environ. Microbiol. 48: Bowles, L. K., and W. L. Ellefson Effects of butanol on Clostridium acetobutylicum. Appl. Environ. Microbiol. 50: Costa, J. M Solvent toxicity in the acetone-butanol fermentation. Proc. Annu. Biochem. Eng. Symp. 11: Freese, K., and B. C. Levin Action mechanisms of preservatives and antiseptics. Dev. nd. Microbiol. 19: Gottschal, J. C., and J. G. Morris The induction of acetone and butanol production in cultures of Clostridium acetobutylicum by elevated concentrations of acetate and butyrate. FEMS Microbiol. Lett. 12: Herrero, A. A End-product inhibition in anaerobic fermentations. Trends Biotechnol. 1: Holt, R. A., G. M. Stephens, and J. G. Morris Production of solvents by Clostridium acetobutylicumn cultures maintained at neutral ph. Appl. Environ. Microbiol. 48: Huang, L., L. N. Gibbins, and C. W. Forsberg Transmembrane ph gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions. Appl. Environ. Microbiol. 50: Kell, D. B., M. W. Peck, G. Rodger, and J. G. Morris On the permeability to weak acids and bases of the cytoplasmic membrane of Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 99: Konings, W. N., and H. VeldkaMpp Energy transduction and solute transport mechanisms in relation to environments occupied by microorganisms. Symp. Soc. Gen. Microbiol. 34: Monot, F., J. M. Engasser, and H. Petitdemange nfluence of ph and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 19: Moreira, A. R., D. C. Ulmner, and J. C. Linder Butanol toxicity in the butylic fermentation. Biotechnol. Bioeng. Symp. 11: Ross, D The acetone-butanol fermentation. Prog. nd. Microbiol. 3: Schuldiner, S., and E. Padan How does Escherichia coli regulate internal ph?, p n A. N. Martonosi (ed.), Membranes and transport, vol. 1. Plenum Publishing Corp., New York. 16. Spivey, M. J The acetone/butanol/ethanol fermentation. Process Biochem. 13(11):2-4, Thauer, R. K., and J. G. Morris Metabolism of chemotrophic anaerobes: old views and new aspects. Symp. Soc. Gen. Microbiol. 36: Volesky, B., A. Mulchandani, and J. Williams Biochemical production of industrial solvents (acetone-butanol-ethanol) from renewable resources. Ann. N.Y. Acad. Sci. 369: Vollherbst-Schneck, K., J. A. Sands, and B. S. Montenecourt Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. App. Environ. Microbiol. 47: