APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1986, p. 1230-1234 Vol. 51, No. 60099-2240/86/061230-05$02.00/0Copyright © 1986, American Society for Microbiology

Influence of External pH and Fermentation Products on Clostridiumacetobutylicum Intracellular pH and Cellular Distribution of

Fermentation ProductsLI HUANG, CECIL W. FORSBERG,* AND L. N. GIBBINS

Department of Microbiology, University of Guelph, Guelph, Ontario NIG 2WJ, Canada

Received 16 December 1985/Accepted 7 March 1986

Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture main-tained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grownat pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than therespective 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 andbutyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively littleeffect 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 andthus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads toinduction of solvent production, thereby circumventing a decrease in the intracellular pH great enough to bedeleterious to the cell.

Acetone-butanol fermentation by Clostridium acetobu-tylicum has been characterized as a biphasic batch-culturefermentation (14, 16, 18). The first phase is characterized byrapid growth and by the formation of acetic and butyric acidswhich are excreted into the medium, thereby lowering themedium pH. The second phase commences after the pH ofthe medium has fallen below approximately 5.0. During thisperiod, butanol and acetone become the major fermentationproducts. The fatty acids, previously accumulated in themedium, pass through the cell membrane in their undissoci-ated form (10) and are converted to solvents. At the end ofthe fermentation, the metabolic activity ceases primarilybecause the concentrations of the solvents have reachedtoxic levels (13). It has been shown that environmentalchanges that occur during the fermentation triggersolventogenesis (1, 6, 12, 13, 17). Of the major fermentationproducts, fatty acids, especially butyrate, were found tohave a significant influence on the initiation ofsolventogenesis, and a critical level of undissociated butyricacid was reported to coincide with the onset of solventformation (12). Butanol, on the other hand, was found to bethe primary toxic substance in the acetone-butanol fermen-tation, and it inhibited cell growth by 50% when the concen-tration in the culture medium reached 150 mM (4, 13).However, in both cases, the concentration of the fermenta-tion products within the cells was unknown. Furthermore, inrecent studies, the intracellular pH of C. acetobutylicumcells grown in a chemostat was shown to decrease withdecreasing external pH during acetogenic fermentation andthen become stabilized after solventogenesis was initiated(9). It appears that the cells can maintain a relatively highintracellular pH under acidic growth conditions in a chemo-stat by redirecting metabolism from organic acid synthesis tosolvent synthesis. Both acidic and neutral fermentationproducts have been shown to interfere with membrane-related functions such as energy generation which are re-

* Corresponding author.

sponsible for generation of the transmembrane pH gradient(2, 5, 7, 13, 19). It was therefore of interest to determine theintracellular concentration of fermentation products and toinvestigate the effect of fermentation products on the intra-cellular pH of the cells.

In this communication we present information on thedistribution of the fermentation products across the cyto-plasmic membrane of C. acetobutylicum grown at variouspH values and on the sensitivity of the transmembrane pHgradient of the cells to these end products. The objective ofthe study was to determine the intracellular concentrationsof fermentation products in cells and to assess the influenceof these products on the maintenance of intracellular pH,since this information will be essential to understand theregulatory mechanism governing solvent production.

MATERIALS AND METHODS

Organism and growth conditions. The bacterium used in thisstudy was C. acetobutylicum ATCC 824. It was grown in aphosphate-limited chemostat at pH values ranging from 4.5 to6.5. Cells grown at pH 6.5 carried out an acetogenicfermentation with acetate and butyrate being the majorfermentation products, while at pH 4.5, butanol and acetonewere the major fermentation product (9). The mediumcomposition and the growth conditions have been describedpreviously (9). The concentrations of fermentation productswere measured by gas chromatography as describedelsewhere (9).

Reagents. [14C]benzoic acid (18.8 mCi/mmol), [14C]butyricacid (13.4 mCi/mmol), ['4C]butanol (1.0 mCi/mmol), and[3H]raffinose (7.8 Ci/mmol) were purchased from New Eng-land Nuclear Corp. of Canada, Lachine, Quebec. Othermaterials were of reagent grade or the highest grade avail-able.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

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External pHFIG. 1. Distribution of butyrate across the cytoplasmic mem-

brane of C. acetobutylicum grown in a phosphate-limited chemostatmaintained at various pH values (solid line). The dashed linerepresents the internal-to-external butyrate concentration ratioscalculated 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 samplewas centrifuged at 34,800 x g for 10 min at room tempera-ture to reproduce the procedure used in the actual determi-nation of butyrate distribution (see below), heat treated at80°C for 30 min, and centrifuged again. After 2 h of incuba-tion, another sample was treated in the same manner. Thesupernatant fluid from each of the two samples was treatedwith Amberlite IR-120 cation-exchange resin and filteredthrough a 0.45-,um-pore-size Metricel filter. The componentsin each sample were separated with a high-pressure liquidchromatograph (Waters Associates, Inc., Milford, Mass.)equipped with a model ERC-7510 refractive index detector(Erma Optical Works, Ltd., Tokyo, Japan) and a model 730data module (Waters Associates). Separation was achievedon a Bio-Rad Aminex ion-exclusion HPX-87H column (300by 7.8 mm) fitted with a cation-exchange microguardprecolumn (Bio-Rad Laboratories, Richmond, Calif.). Thecolumn temperature was maintained at 35°C. DegassedH2SO4 (0.01 N) was used as the solvent at a flow rate of 0.6ml/min. Samples (10 Rd) were loaded onto the column, andfractions were collected with a LKB model 2112 Rediracfraction collector (Fisher Scientific Ltd., Toronto, Ontario,Canada). A sample of each fraction was mixed with ACSscintillation fluid (Amersham Canada Ltd., Oakville,Ontario, Canada), and the radioactivity was counted.The metabolism of [14C]butanol by the cells were exam-

ined in the same way except that the cells were incubatedwith [14C]butanol (3.04 mM, 1.0 mCi/mmol) at 35°C for 1 h.

Determination of distribution of fermentation productsacross the cell membrane of C. acetobutylicum. Two radioac-tively labeled compounds, [14C]butyric acid and [14C]bu-tanol, were used to estimate the distribution of butyrate andbutanol across the cytoplasmic membrane of cells. Samples(10 ml) of the culture withdrawn from the chemostat oper-ating at selected pH values between 4.5 and 6.5 weredispensed anaerobically into centrifuge tubes, to whicheither [14C]butyric acid or [14C]butanol had been added to

give a final concentration of 4.46 ,uM (13.4 mCi/mmol) or0.25 mM (1.0 mCi/mmol), respectively. [3H]raffinose wasalso included in the samples (2 ,uM, 124.6 mCi/mmol) todetermine the extracellular space in the cell pellet. Afterincubation for 10 min at 35°C, the samples were centrifugedat 34,800 x g for 10 min at room temperature. The pellet andthe supernatant fluid samples were processed and the radio-activity in these samples was counted as described previ-ously for the measurement of the transmembrane pH gradi-ent of the cells (9). From the total water content, determinedgravimetrically, and the extracellular space in the cell pellet,the intracellular and extracellular radioactivity was ob-tained. The distribution of butyrate and butanol could thenbe estimated.

Distribution of acetate and butyrate across the cell mem-brane was also determined by measuring the concentrationsof these acids in a concentrated cell suspension from thechemostat culture with and without disintegration of thecells. Duplicate samples (100 ml) of the culture (1 g of drycells per liter) were withdrawn from the chemostat operatingat pH 4.5, 5.2, or 6.5, centrifuged anaerobically, and con-centrated 100-fold by resuspending the pellet in the culturesupernatant (Si) to a total volume of 1 ml. The cell suspen-sion and the removed supernatant samples were heated insealed tubes at 100°C in a steamer. After 25 min of heattreatment, the samples were cooled and then centrifuged at4°C. The supernatant fluid samples (S2) from the cell sus-pension samples, together with duplicate Si samples, wereanalyzed for fermentation products by gas chromatography.The intracellular concentrations of fatty acids were calcu-lated from the gas chromatography results, using the intra-cellular space values obtained previously from the intracel-lular pH determination (9).

Intracellular pH of cells suspended in anaerobic citrate-phosphate buffer. Culture fluid was withdrawn from thechemostat operating at either pH 4.5 or 6.5. The cells wereharvested by centrifugation at 13,200 x g for 10 min at roomtemperature, washed with anaerobic citrate-phosphatebuffer (pH 4.5 or 6.5) supplemented with 20 mM glucose, andconcentrated fourfold by resuspending the pellets in S ml ofthe same buffer of various pH values (pH 3.0 to 6.5). Afterincubation for 10 min at 35°C, the intracellular pH of thesamples was measured by using [14C]benzoic acid as theprobe as described previously (9). [3H]raffinose was used inthis experiment to permit determination of the extracellularspace (9).

Effects of fermentation products on intracellular pH of thecells. The anaerobic pellet of the cells collected from 200 mlof culture grown in the chemostat operating at pH 4.5 waswashed 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 thecytoplasmic membrane of C. acetobutylicum ATCC 824 grown ina phosphate-limited chemostat maintained at various pH valuesa

Acetate Butyrate

put[Acetatelin: [acetate]ou, pHinb [Butyratelin: [butyrate]out pHinb4.5 13.2 6.0 7.1 5.85.2 4.2 5.9 4.6 6.06.5 2.2 6.8 2.2 6.8a Intracellular 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 at35°C were taken to be 4.76 and 4.84, respectively.

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TABLE 2. Effect of the pH of a phosphate-limited culture of C. acetobutylicum ATCC 824 on the intracellular concentrations of acetateand butyrate

[Acetate]in (mM) [Acetate],,, (mM) [Butyrateli, (mM) [ButyrateI0u, (mM)pH0ut Calculateda Observedb observedb Calculated' Observedb observedb4.5 134 134 10 147 (86)C 95 135.0 158 179 (167)5.2 150 37 169 375.5 211 228 (204)6.0 157 190 (244)6.5 95 110 50 124 (132) 143 65

a The calculated intracellular concentrations of acetate and butyrate were derived from the ApH determined previously with ['4C]benzoic acid as a probe, thePKa 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 intocentrifuge tubes, to which one of the following compoundswas added to final concentrations indicated in the corre-sponding figures: 5 M acetic acid (pH was adjusted to 4.5with 10 M NaOH); 5 M butyric acid (pH was adjusted to 4.5with 10 M NaOH); acetone; and n-butanol. After incubationfor 10 min at 35°C, the intracellular pH values of the sampleswere measured by using [14C]benzoic acid as describedpreviously (9).

RESULTSMetabolism of butyrate and butanol by the cells. The use of

[14C]butyric acid and [14C]butanol to determine the distribu-tion of butyrate and butanol across the cell membranerequires that no significant transformation of the radioactivecompound occur during the experiment. In this study,[14C]butyric acid was added to the cells taken from thechemostat at pH 4.5. It was found that less than 3% of theadded [14C]butyrate was metabolized (mostly into[14C]butanol) during the 10-min incubation period, althoughprolonged incubation (2 h) resulted in approximately 25%conversion to butanol. Therefore, the error due to thetransformation of [14C]butyrate during the incubation period(10 min) used in these experiments was considered to benegligible.

[14C]butanol conversion to butyrate by the cells wasinvestigated in a similar way. The added [14C]butanol was

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FIG. 2. Intracellular pH of nongrowing cells in citrate-phosphatebuffer at various pH values. Growing culture was harvested from thechemostat operating at either pH 4.5 (0) or 6.5 (0), washed, andsuspended in anaerobic citrate-phosphate buffer (20 mM glucose) ofselected pH values. After incubation at 35°C, the intracellular pH ofthe samples was measured with ['4C]benzoic acid.

almost unchanged after 1 h of incubation, suggesting that thereaction(s) leading to butanol formation was actually irre-versible under the conditions used in this experiment.

Distribution of fermentation products across the cell mem-brane. The distribution of butyrate across the cytoplasmicmembrane of cells withdrawn from the chemostat main-tained at various pH values was examined with ['4C]butyricacid as a probe. The butyrate concentration within the cellswas higher than that in the medium, and the internal-to-external concentration gradient increased as the external pHwas lowered (Fig. 1). This was confirmed by direct determi-nation and, furthermore, extended to include acetate (Table1). We have reported that C. acetobutylicum ATCC 824grown under conditions identical to those used in this studymaintained an internal-alkaline transmembrane pH gradientwhich increased with decreasing external pH (9). Sincebutyric acid, a weak acid, can passively diffuse across thecell membrane in its undissociated form (10), the distributionof this acid is dependent upon the pH values on both sides ofthe cell membrane. As demonstrated in Fig. 1 and Table 2, atpH values above 5.0, the concentration gradient of butyratedetermined in this experiment is in good agreement with thatcalculated from the transmembrane pH gradient of the cellsobtained previously when benzoate was used as the probe(9). However, the intracellular butyrate level was lower thanexpected at pH 4.5 at which solventogenesis is favored. Incontrast to butyrate, the intracellular concentration of ace-tate calculated from the transmembrane pH gradient corre-sponded very closely to the experimentally determinedconcentration. The intracellular concentrations of acetateand butyrate were the highest at a culture pH of approxi-mately 5.5

Butanol distribution across the membrane of the cellsgrown at pH 4.5 was determined with [14C]butanol. Theinternal-to-external concentration ratio for butanol was cal-culated to be approximately 1.30, indicating that the intra-cellular and extracellular butanol concentrations were simi-lar.

Intracellular pH of cells suspended in anaerobic citrate-phosphate buffer. These experiments were performed toestablish assay conditions under which the transmembranepH gradient of the cells could be generated and studied in theabsence of the fermentation products. It was found that cellsharvested from the chemostat operating at either pH 4.5 or6.5 and then washed and resuspended in anaerobic citrate-phosphate buffer at various pH values were able to generatea transmembrane pH gradient upon the addition of glucose(20 mM), and a steady-state level of the pH gradient could bereached within 20 min of incubation at 35°C. Two differentintracellular pH profiles were observed (Fig. 2). Although

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FIG. 3. Effect of acetate (A) and butyrate (B) on intracellular pHof the cells of C. acetobutylicum ATCC 824. A sample of the culturegrown at pH 4.5 was centrifuged, and the cell pellet was washed andsuspended in anaerobic citrate-phosphate buffer (pH 4.5) supple-mented with 20 mM glucose. Samples (5 ml) of the suspension weredispensed into centrifuge tubes, to which acetic or butyric acid(neutralized to pH 4.5 with 5 N NaOH) was added to give finalconcentrations ranging from 0 to 70 mM. After incubation for 10 minat 350C, the intracellular pH of the samples was measured with[14C]benzoic acid.

the nongrowing cells of both origins maintained a highintracellular pH within a certain external pH range, thesolvent-producing cells exhibited a higher intracellular pHthan the acid-producing cells. The cells from the chemostatoperating at pH 4.5 were able to keep the intracellular pHabove 6.0 even when the external pH was as low as 3.5. Adrop in intracellular pH occurred when the external pH wasfurther decreased. In contrast, the intracellular pH of thecells originating from the chemostat set at pH 6.5 declinedrapidly as the external pH was lowered and fell to 6.0 at anexternal pH of 4.5. A marked drop in intracellular pH cameafter the external pH fell below 4.0.

Effect of fermentation products on transmembrane pHgradient of cells suspended in anaerobic citrate-phosphatebuffer. The intracellular pH of nongrowing cells suspendedin the anaerobic citrate-phosphate buffer (pH 4.5) supple-mented with 20 mM glucose was measured after incubationwith each of the four major acetone-butanol fermentationproducts at various concentrations. It can be seen in Fig. 3that the addition of either acetic or butyric acid to the cellsuspension markedly lowered the intracellular pH, indicat-ing the uncoupling effect of both acids. The drop in intracel-lular pH was found to be linearly related with the fatty acidconcentration within the range of concentrations tested.

Butanol slightly decreased the intracellular pH of the cellsat concentrations below 150 mM (Fig. 4). However, whenbutanol was added to the cell suspension to give a concen-tration above 150 mM, the transmembrane pH gradientcollapsed and the intracellular pH dropped dramatically.Acetone, on the other hand, did not affect the intracellularpH even at concentrations much higher than that encoun-tered in the normal acetone-butanol fermentation (Fig. 4)(18).

DISCUSSIONIt has been found that the level of fatty acids in the culture

medium of C. acetobutylicum for transition from ace-

togenesis to solventogenesis is dependent upon the externalpH and that this level is higher when the solvent productionis initiated at a higher pH value (12). In the present study, thedistribution of the acidic fermentation products across thecytoplasmic membrane of C. acetobutylicum grown in achemostat at various pH values was examined to provide abasis for further consideration of the "fatty acid effect."Both acetate and butyrate were concentrated by growingcells at pH values of 6.5 and lower, as would be expectedfrom the transmembrane pH gradient determined previously(9), although the intracellular butyrate concentration waslower than expected in solvent-producing cells. This appar-ent anomoly could be attributed either to the vigoroustransformation of butyrate to butanol within the cells or to animbalance in the metabolic pathway for butyrate synthesis.However, at present there is insufficient information to reacha definitive conclusion.Based on the intracellular pH values determined from

[14C]benzoate distribution, the pKa values of acetate andbutyrate, and the extracellular concentrations of these acids,the calculated intracellular concentrations were estimated tobe the greatest at a pH value of 5.5. The calculated intracel-lular concentration for butyrate based on [14C]butyrate dis-tribution was highest at pH 6.0. These data demonstrate thatthe butyrate and acetate concentrations within the cells arehighest at or just above the pH at which solvent productionwas shown to be induced (9). This coincidence supports thecontention that the high intracellular concentration of fattyacids leads to the induction of solvent production (8, 12).However, there is no evidence to support the suggestion thatundissociated fatty acids alone serve as the inducing agent.These data do not permit an evaluation of the relativeimportance of acetate and butyrate in the induction ofsolvent synthesis.When cells are fermenting sugars, their ability to maintain

an intracellular pH value near neutrality is subject to thestress imposed by the fermentation products. The extent towhich cells can cope with the stress is significant, since anyvariation in intracellular pH will lead to the modification ofthe cellular functions (11, 15). In the absence of fermentation

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FIG. 4. Effect of butanol (A) and acetone (B) on intracellular pHof the cells of C. acetobutylicum ATCC 824. The procedure de-scribed in the legend to Fig. 3 was followed, except that eitherbutanol or acetone was added instead of the fatty acids.

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products, the nongrowing cells suspended in buffer wereable to maintain a high intracellular pH even at low externalpH, and such ability was more pronounced in the cellsoriginating from the chemostat set at pH 4.5 than in the cellsfrom the chemostat set at pH 6.5. The enhanced ability ofsolvent-producing cells to generate a transmembrane pHgradient is presumably due to the adapted cells having agreater capacity to convert fatty acids to solvents. Theaddition of either acetate or butyrate to the cells suspendedin citrate-phosphate buffer at pH 4.5 resulted in a markeddrop in the intracellular pH of the cells. This effect increasedwith the concentration of the added acid within the testedrange. It appears that even a relatively low concentration ofthe fatty acid would be a considerable load on the ApH-generating system of the cells at low external pH. The effectof the fatty acid on the transmembrane pH gradient has beenattributed to its ability to diffuse across the cell membrane inits undissociated form in response to the difference in pHbetween the two sides (2, 5, 10). Butanol showed a differenteffect on the intracellular pH of solvent-producing cells.When added at concentrations below 150 mM, butanol onlyslightly decreased the intracellular pH. However, at concen-trations over 150 mM, it caused a dramatic drop in intracel-lular pH. This observation has also been reported from otherlaboratories (3). It has been observed that 150 mM butanolcaused 50% inhibition of growth (13). Clearly, the growthinhibition and the fall in intracellular pH caused by butanolare related. Butanol has been shown to increase lipid fluidityand to inhibit some membrane-related functions, such asATPase activity and nutrient transport systems, in C.acetobutylicum (13, 19). Thus, the failure to maintain a highintracellular pH may be caused by damage to the protonpump or to the shortage of energy (3). Acetone, on the otherhand, displayed little effect on the intracellular pH even at aconcentration which was approximately three times as highas the maximum acetone level encountered in culture fil-trates from the acetone-butanol fermentation (18). This isconsistent with the finding that acetone did not inhibit thegrowth of C. acetobutylicum at concentrations of less than500 mM (4).The results obtained on the influence of acetate, butyrate,

butanol, and acetone were for cells suspended in anaerobiccitrate-phosphate buffer with glucose as a readily availablecarbon source. This allowed the separate assessment of theeffect of each major fermentation product on the intracellularpH, which would not have been possible in growth medium.As a result of this approach, the data cannot be directlyapplied to growing cultures since a more complicated arrayof components influences the response of cells to the fer-mentation 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.

LITERATURE CITED1. Bahl, H., W. Andersch, K. Braun, and G. Gottschalk. 1982.

Effect of pH and butyrate concentration on the production ofacetone and butanol by Clostridium acetobutylicum grown incontinuous culture. Eur. J. Appl. Microbiol. Biotechnol. 14:17-20.

2. Baronofsky, J. J., W. J. A. Schreurs, and E. R. Kashket. 1984.Uncoupling by acetic acid limits growth of and acetogenesis byClostridium thermoaceticum. Appl. Environ. Microbiol. 48:1134-1139.

3. Bowles, L. K., and W. L. Ellefson. 1985. Effects of butanol onClostridium acetobutylicum. Appl. Environ. Microbiol.50:1165-1170.

4. Costa, J. M. 1981. Solvent toxicity in the acetone-butanolfermentation. Proc. Annu. Biochem. Eng. Symp. 11:83-90.

5. Freese, K., and B. C. Levin. 1978. Action mechanisms ofpreservatives and antiseptics. Dev. Ind. Microbiol. 19:207-227.

6. Gottschal, J. C., and J. G. Morris. 1981. The induction ofacetone and butanol production in cultures of Clostridiumacetobutylicum by elevated concentrations of acetate and butyr-ate. FEMS Microbiol. Lett. 12:385-389.

7. Herrero, A. A. 1983. End-product inhibition in anaerobic fer-mentations. Trends Biotechnol. 1:49-53.

8. Holt, R. A., G. M. Stephens, and J. G. Morris. 1984. Productionof solvents by Clostridium acetobutylicumn cultures maintainedat neutral pH. Appl. Environ. Microbiol. 48:1166-1170.

9. Huang, L., L. N. Gibbins, and C. W. Forsberg. 1985.Transmembrane pH gradient and membrane potential in Clos-tridium acetobutylicum during growth under acetogenic andsolventogenic conditions. Appl. Environ. Microbiol. 50:1043-1047.

10. Kell, D. B., M. W. Peck, G. Rodger, and J. G. Morris. 1981. Onthe permeability to weak acids and bases of the cytoplasmicmembrane of Clostridium pasteurianum. Biochem. Biophys.Res. Commun. 99:81-88.

11. Konings, W. N., and H. VeldkaMpp. 1983. Energy transductionand solute transport mechanisms in relation to environmentsoccupied by microorganisms. Symp. Soc. Gen. Microbiol.34:153-186.

12. Monot, F., J. M. Engasser, and H. Petitdemange. 1984. Influenceof pH and undissociated butyric acid on the production ofacetone and butanol in batch cultures of Clostridiumacetobutylicum. Appl. Microbiol. Biotechnol. 19:422-426.

13. Moreira, A. R., D. C. Ulmner, and J. C. Linder. 1981. Butanoltoxicity in the butylic fermentation. Biotechnol. Bioeng. Symp.11:567-579.

14. Ross, D. 1961. The acetone-butanol fermentation. Prog. Ind.Microbiol. 3:73-90.

15. Schuldiner, S., and E. Padan. 1982. How does Escherichia coliregulate internal pH?, p. 65-73. In A. N. Martonosi (ed.),Membranes and transport, vol. 1. Plenum Publishing Corp.,New York.

16. Spivey, M. J. 1978. The acetone/butanol/ethanol fermentation.Process Biochem. 13(11):2-4, 25.

17. Thauer, R. K., and J. G. Morris. 1984. Metabolism ofchemotrophic anaerobes: old views and new aspects. Symp.Soc. Gen. Microbiol. 36:123-168.

18. Volesky, B., A. Mulchandani, and J. Williams. 1981. Biochem-ical production of industrial solvents (acetone-butanol-ethanol)from renewable resources. Ann. N.Y. Acad. Sci. 369:205-218.

19. Vollherbst-Schneck, K., J. A. Sands, and B. S. Montenecourt.1984. Effect of butanol on lipid composition and fluidity ofClostridium acetobutylicum ATCC 824. AppI. Environ. Micro-biol. 47:193-194.

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