APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1988, p. 1662-1667 Vol. 54, No. 70099-2240/88/071662-06$02.00/0Copyright © 1988, American Society for Microbiology

Enhancement of Butanol Formation by Clostridium acetobutylicumin the Presence of Decanol-Oleyl Alcohol Mixed Extractants

PATRICK J. EVANS AND HENRY Y. WANG*

Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109

Received 23 November 1987/Accepted 30 March 1988

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end productinhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutyli-cum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed tocircumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present ina mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated theinhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractantthat contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlledconditions. The production of acetone and acetoin was also increased, even though these two products were notextractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementationof glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement ofbutanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was notobserved unless an organic phase was present to extract butanol. A mechanism for the effects of decanol onproduct formation is proposed.

Batch fermentation by Clostridium acetobutylicum ischaracterized by two phases. During the first phase, oracidogenesis, C. acetobutylicum grows and produces acetateand butyrate from glucose. These acids attain their maximalconcentrations and are consumed in the second phase,which is known as solventogenesis. The acids are reduced,and neutral solvents including butanol, acetone, ethanol, andacetoin are produced. Butanol is generally produced inconcentrations of no greater than 12 g/liter (17, 23). Thislimitation is thought to be due to the toxicity of butanol to C.acetobutylicum (9, 14, 17).One technique for increasing butanol production is genetic

alteration of C. acetobutylicum to make it more tolerant tobutanol. A number of attempts have been made in this regardwith varying degrees of success (9, 16, 17). How muchimprovement can be accomplished is questionable though,because low-molecular-weight compounds such as butanolmay not have specific sites of toxicity (2). Butanol maylocalize in the plasma membrane and disrupt a number ofphysiological processes including membrane permeability,solute transport, maintenance of the proton motive force,and conformation and activity of intrinsic membrane pro-teins.Another technique is alteration of the environment rather

than the cell. The toxic or inhibitory product can be removedsimultaneously from the broth as it is formed. This techniqueis known as extractive fermentation. One approach is to addto the broth an immiscible organic liquid that has a highdistribution coefficient for butanol. A general problem inher-ent with liquid extractants is that liquids with high butanoldistribution coefficients are toxic, and nontoxic liquids havelow distribution coefficients (5). Toxicity is defined here asthe inhibition of growth of C. acetobutylicuim. Circumven-tion of extractant toxicity has resulted in the use of nontoxicextractants that have the highest distribution coefficients orthe use of membranes to separate toxic organic liquids fromthe broth (7, 8, 13, 19, 22).

* Corresponding author.

In a previous report (5) we described the effects of mixedextractants, namely, mixtures of toxic and nontoxic coex-tractants, on the growth of C. acetobutylicum. The combi-nation of toxic decanol and nontoxic oleyl alcohol, whichhave butanol distribution coefficients of 6.2 and 3.2, respec-tively, was chosen as a model system, designated as deca-nol-oleyl alcohol (DOA). Up to 40% (vol/vol) decanol inoleyl alcohol is nontoxic to growth. At greater concentra-tions of decanol, toxicity is attributable to dissolved decanolin the broth (i.e., a solute effect) rather than a surface effectattributable to the aqueous-organic interface; therefore, thetoxicity ofDOA is independent of the relative volumes of theorganic and aqueous phases. The purpose of this study wasto investigate the effects ofDOA on product formation by C.acetobutylicium.

MATERIALS AND METHODS

Microorganism and culture conditions. C. acetobutylicumATCC 4259 was sporulated and preserved as a spore sus-pension in a 5% (wt/vol) cooked corn meal medium (J. C.Leung, Ph.D. thesis, Massachusetts Institute of Technology,Cambridge, Mass., 1982). Inocula were prepared by adding0.2 ml of spore suspension to 10 ml of CAB medium (15) with5 g of glucose per ml and 0.1 ml of 2.5% (wt/vol)Na2S 9H20 in Bellco (Bellco Glass, Inc., Vineland, N.J.)anaerobic tubes with butyl bungs and aluminum crimps.CAB medium contains the following, in grams per liter ofdistilled water: yeast extract (Difco Laboratories, Detroit,Mich.), 4; tryptone (Difco), 1; KH2PO4, 0.7; K2HPO4, 0.7;DL-asparagine, 0.5; MgSO4 7H20, 0.1; MnSO4- H20, 0.1;NaCl, 0.1; FeSO4 7H20, 0.015; and resazurin, 0.002. ThepH was adjusted to 5.4 prior to autoclaving. Glucose wasautoclaved separately and added later. The spore suspensionwas heat shocked at 80°C for 2 min and incubated at 34°C.This culture was used to inoculate (5% [vol/vol]) 50 ml ofCAB medium with 60 to 70 g of glucose per liter and no Na2Sin a 100-ml serum bottle. This culture was incubated over-night and then used as inocula for the experiments. All media

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ENHANCEMENT OF BUTANOL FORMATION BY C. ACETOBUTYLICUM 1663

and organic liquids were prepared anaerobically by themethod of Baronofsky et al. (2).

Fermentations were performed in either 50-ml serumbottles or a fermentor (Multigen; New Brunswick ScientificCo., Inc., Edison, N.J.). The 50-ml serum bottles thatcontained 10 ml of CAB medium with 60 to 70 g of glucoseper liter and 10 ml of various DOA mixtures were incubatedat 34°C overnight prior to inoculation, in order to equilibratedecanol concentrations in the organic and aqueous phases.Decanol was obtained from Aldrich Chemical Co., Inc.(Chicago, Ill.), and oleyl alcohol was obtained from EastmanKodak Co. (Rochester, N.Y.). The bottles were then inoc-ulated (5% [vol/vol]) and incubated without shaking for 48 h.Fermentations in a 1-liter fermentor (Multigen) contained300 ml of CAB medium with 60 to 70 g of glucose per literand 300 ml of various DOA mixtures, unless otherwisenoted. The extractant and medium, without glucose, weresterilized together. Sterile nitrogen that was passed througha catalyst (BASF R3-11; Chemical Dynamics, South Plain-field, N.J.) at 175°C to remove traces of oxygen was bubbledcontinuously into the organic phase at 100 ml/min to main-tain anaerobic conditions. Dual turbine impellers operated at100 rpm mixed each phase separately but did not disrupt theliquid-liquid interface. The fermentor was maintained underthese conditions and at 34°C overnight prior to inoculation(5% [vol/vol]). The pH was controlled at 4.5 + 0.1 with 0.5N NaOH and 0.5 N H2SO4, unless otherwise indicated. Thefermentation with butyrate addition was completed simi-larly, but 3.5 ml of butyric acid was added during solvento-genesis (29.5 h).

Analytical methods. The product concentrations in eachliquid phase were measured by gas chromatography. Aque-ous samples were centrifuged to remove cells, and thesupernatants were acidified with 10% (vol/vol) 10 N H3PO4to protonate the acids prior to analysis. A 10% AT1000 onChromosorb W-AW (80/100) column (2 m by 2 mm) on a gaschromatograph (5840A; Hewlett-Packard Co., Palo Alto,Calif.) with an autosampler (7672A; Hewlett-Packard) wasused. The conditions for the aqueous analysis were asfollows: injector temperature, 120°C; flame ionization detec-tor temperature, 250°C; column temperature, 70 to 235°C at30°C/min; flow rate, 30 ml of nitrogen per min. Acetone,butanol, acetoin, acetate, and butyrate were quantitated byan external standard method. Ethanol was not assayedbecause it was present as an impurity in the decanol. Theconditions for the organic phase analysis were as follows:injector temperature, 220°C; flame ionization detector tem-perature, 250°C; column temperature, 70 to 130°C at30°C/min, held at 130°C for 4.5 min, and then 130 to 250°C at30°C/min and then held at 250°C; flow rate, 30 ml of nitrogenper min. Acetoin and acetate were not quantitated in theorganic phase because they did not distribute significantlyinto this phase.Glucose in the aqueous supernatants was measured by the

colorimetric method of Somogyi (20).

RESULTS

Product formation without pH control. Final product con-centrations for fermentations in serum bottles with equalvolumes of broth and extractant are presented in Fig. 1. Anincrease in acetoin and butanol production was observed onthe addition of oleyl alcohol (decanol volume fraction, 0.0)compared with the control. The addition of decanol to theorganic phase inhibited butanol production as well as that ofacetone and acetoin. On the contrary, net butyrate produc-

tion was enhanced on the addition of oleyl alcohol, and thenet production of acetate and butyrate was enhanced as upto 20 to 30% (vol/vol) decanol was added to oleyl alcohol.Glucose consumption decreased from 170 to 18 mM as thedecanol volume fraction was increased from 0.0 to 0.3.

Reductions in the final pH were associated with theaddition of oleyl alcohol to the broth and of decanol to oleylalcohol (Fig. 1, inset). This reduction was probably due tothe accumulation of acetate which, unlike butyrate, was notextractable. Butyric acid had a distribution coefficient ofapproximately 2.3 in oleyl alcohol and 2.6 in a mixture of20% decanol in oleyl alcohol at pH 4.5. The distributioncoefficient of acetate was less than 0.2 in oleyl alcohol at pH4.5.

Effect of pH control. Fermentations were completed in afermentor with and without pH control to ascertain the effectof pH on solvent production. The pH was maintained at 4.5with pH control. In the absence of extractant (i.e., thecontrol fermentation), different amounts of butyrate wereproduced with and without pH control, namely, 44 and 21mM, respectively; but in both cases butyrate was completelyconsumed during the solventogenic phase (data not shown).Equal amounts of butanol were produced at equal rates inboth cases (data not shown). Data for fermentations with andwithout pH control in the presence of 20% decanol in oleylalcohol are given in Fig. 2. Consumption of butyrate wasobserved only when the pH was controlled. Furthermore,butanol production was greater in terms of rate and amountwhen the pH was controlled.

Solvent formation with pH control. Results for fermenta-tions with the pH maintained at 4.5 are shown in Fig. 3 to 6.Figure 3 shows the total amounts of solvents that wereproduced. A slight increase in butanol production wasobserved on the addition of oleyl alcohol. The addition ofdecanol up to 30% caused a much greater increase in butanolproduction. The maximal rate of butanol production alsoincreased from 6.1 mmol/liter- h in the control to 7.6mmol/liter* h in 20% decanol in oleyl alcohol. More decanol(40%) caused a reduction in butanol formation. Acetone andacetoin production were also affected by decanol in a similarmanner. These products were not extracted significantly intothe organic phase.The enhancement of butanol formation did not appear to

be solely due to the alleviation of its end product toxicity. Onthe addition of oleyl alcohol, the aqueous concentration ofbutanol was reduced from 87 to 25 mM, but the totalconcentration increased only from 87 to 96 mM. Totalbutanol increased from 96 to 150 mM on the addition of 20%decanol, and the aqueous butanol concentration increasedrather than decreased, from 25 to 33 mM.

Supplementation of glucose and butyrate. Table 1 showsthat the addition of DOA with 20% decanol (case 2) causedalmost complete utilization of glucose relative to that of thecontrol (case 1). When the initial glucose concentration wasincreased (case 3), no significant increases in butanol pro-duction or in the maximal total butyrate concentration wereobserved, even though glucose was in excess and an in-crease in glucose consumption was observed. The finalaqueous butyrate concentrations were low in all three ofthese fermentations. The addition of butyrate in the middleof the solventogenic phase, in addition to the presence ofexcess glucose (case 4), resulted in an increase in butanolproduction. Butyrate was not depleted, and increased con-sumption of butyrate and glucose was observed. Cells lysedat the end of the fermentation in case 4, and no spores wereevident in the broth.

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90.0

80.0

70.0

':i" 60.0

50.00°. 40.0

30.0

20.0

10.0-

0.0-econtrol

i I TI0.0 0.1 0.2 0.3

Decanol volume fraction0.4

FIG. 1. Final product concentrations for fermentations in serum bottles. The concentrations represent the total amount of product presentin the aqueous and organic phases based on the aqueous phase volume. The control had no organic phase. The abscissa is the volume fractionof decanol in the organic phase. Symbols: 0, acetone; A, acetoin; O, butanol; *, acetate; E, butyrate. (Inset) Final pH of the fermentations.

Acid formation and consumption with pH control. Figure 4shows that the profiles of the final total acid concentrationswere, in a sense, mirror images of the solvent profiles (cf.Fig. 3). The final concentrations of the acids were not themaximal concentrations attained during the course of thefermentation because of acid consumption during solvento-genesis. Acid consumption (i.e., the final concentrationssubtracted from the maximal concentrations) is shown inFig. 5. The maximal acid concentration profiles (Fig. 4),which reflect acid production, are relatively flat comparedwith the consumption profiles (Fig. 5), indicating that theaddition of DOA to the fermentation broth affected acidconsumption during solventogenesis rather than acid pro-duction during acidogenesis. Acid consumption was minimalin the presence of 0 to 10% decanol and 40% decanol. Aconcentration of 20 to 30% decanol in oleyl alcohol rein-stated acid consumption to levels observed in the control.The maximal aqueous concentration of butyric acid wasrelatively independent of the volume fraction of decanol; itvaried between 19 and 26 mM. The control had a maximalaqueous butyric acid concentration of 44 mM.The effects of altered acid consumption were manifested

in the yields of acids and solvents and glucose consumption(Fig. 6). Greater acid yields and lower solvent yields wereobserved in the presence of 0 to 10% decanol and 40%decanol. The addition of DOA containing up to 30% decanolincreased glucose consumption relative to that of the con-trol. Decanol at 40% was inhibitory in terms of glucoseconsumption.

Effect of product extraction. Experiments were performedto determine whether product extraction was required forthe enhancement of butanol production or whether the

presence of dissolved decanol in the broth was the solecause. Figure 7 shows butanol production in the presence ofdifferent relative volumes of broth and extractant; the or-ganic phase was composed of 20% decanol in oleyl alcohol,and the control had no organic phase. Butanol productionwith a phase volume ratio (organic to aqueous) of 1:100 was

160.0

140.0- /

0~~~~~~~~~~~120.0 ;

100.0- /.2-E 80.0- 0

0.0 *21

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0rime (hr)

FIG. 2. Butyrate (O, *) and butanol (0, 0) formed in fermen-tations without pH control starting at pH 5.1 (O, 0) or with the pHcontrolled at 4.5 (-, *). Equal volumes of medium and DOAextractant with a decanol volume fraction of 0.2 were present. Theconcentrations are total amounts produced based on the aqueousvolume.

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ENHANCEMENT OF BUTANOL FORMATION BY C. ACETOBUTYLICUM 1665

-

%oo

0-._

L

(U

C

0

2C

0.1 0.2 tDecanol volume fraction

0.4

FIG. 3. Concentration of solvents produced in fermentationswith the pH controlled at 4.5. The concentrations are total amountsproduced based on the aqueous volume. Symbols: A, acetoin; 0,

acetone; O, aqueous butanol; *, total butanol.

no better than that in the control. In fact, the production ratewas slightly inhibited by decanol. On the other hand, butanolproduction was enhanced in the presence of equal volumesof broth and extractant. The final aqueous concentration ofbutanol in this case was 33 mM. This was less than 86 mM ata ratio of 1:100, in which the total and aqueous concentra-tions were approximately equal.A volume ratio of 1:100 effectively rendered product

extraction negligible. It distributed butanol as 4% in theorganic phase and 96% in the aqueous phase at equilibrium.Since thermodynamic phase equilibrium is dependent onlyon the chemical potentials of the particular components andnot on the volumes of the phases (4), decanol was present inthe culture medium to perturb C. acetobutylicum at a

concentration that was independent of the phase volumeratio. Thus, the data for the 1:100 ratio represent the effectsof dissolved decanol in the broth, and the data for the 1:1ratio represent the effects of dissolved decanol and productextraction.

DISCUSSION

Growth of C. acetobutylicum is not severely inhibited byDOA mixed extractants that contain up to 40% decanol.Approximately 50% decanol in oleyl alcohol, however, iscompletely inhibitory to growth (5). The same results werenot observed for product formation. Since the addition ofdecanol to oleyl alcohol increases the butanol distributioncoefficient of the mixed extractant, butanol production was

0.0 0.1 0.2Decanol volume fraction

FIG. 4. Maximal and final total concentrations of acids producedin fermentations with the pH controlled at 4.5. Symbols: 0, maximalacetate; 0, maximal butyrate; *, final acetate; *, final butyrate.

expected to increase as the decanol volume fraction in-creased. Without pH control, the opposite was observed,along with an increase in the production of acetate andbutyrate.Three hypotheses were formulated in order to explain the

results. First, decanol may directly be inhibitory to solvent-ogenesis. If so, the acids would not have been consumedduring solventogenesis and would have accumulated asobserved. Because butyrate is extractable and the additionof oleyl alcohol can reduce its aqueous concentration, thesecond hypothesis is that butyrate was not available to thecells for the reduction to butanol. The third hypothesis isbased on the low distribution coefficient of acetic acid.Accumulation of acetate in the broth as the decanol volumefraction was increased may have inhibited the cells orlowered the pH of the broth and consequently inhibitedsolventogenesis.The third hypothesis is supported by the pH data, which

showed a decrease in the pH as the decanol volume fractionwas increased. However, these data do not invalidate theother hypotheses. Nevertheless, the third hypothesis, spe-cifically the pH aspect, appears to be correct, becausemaintenance of the pH at 4.5 reinstated butyrate consump-tion and butanol production.The significance of a reduction of 0.3 pH units (cf. Fig. 1,

inset) was explored. Gottwald and Gottschalk (6) havedemonstrated that C. acetobutylicum does not producesolvents if the intracellular pH is less than 5.5. Theseresearchers found that the pH gradient component (ApH) ofthe proton motive force ranged from 0.9 to 1.3. While these

TABLE 1. Effect of additional glucose and butyric acid on product regulation at pH 4.5

Concn (mM)

Case Conditions Final Initial Glucose Total Final Maximal Butyrateglucose glucose consumption butanol aqueous butyrate consumption

1 Control 160 390 230 87 2.7 44 412 20% DOA 38 390 350 150 5.5 59 423 20% DOA; glucose 180 640 460 160 4.9 48 224 20% DOA; glucose; 160 750 590 220 20 70

butyrate

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1666 EVANS AND WANG

i 35.0

c30.0

0

-8 25.0-

:320.0

10.0

5.0-

0.0*

control 0.0 0.1 0.2 0.3 0.4

Decanol volume fractionFIG. 5. Consumption of acids during solventogenesis for pH-

controlled fermentations. Consumption was defined as the final totalacid concentration subtracted from the maximal total acid concen-

tration attained during the course of the fermentation. Symbols: 0,

acetate; *, butyrate.

data are dependent on the culture conditions and the strain,they are useful as first approximations. Using the value of1.3 for ApH, the intracellular pH in the control (Fig. 1, inset)was 5.4. This is near the limiting value of 5.5 for solventproduction. If the ApH remained constant, a reduction of theextracellular pH would have caused a concomitant reductionof the intracellular pH. Thus, a reduction of the extracellularpH by only 0.3 units, which would, in turn, cause a reductionof the intracellular pH from 5.4 to 5.1, may have caused theinhibition of solventogenesis. Butanol has been shown todecrease the ApH in C. acetobutylicum (3, 6, 11, 21).Decanol may cause the same effect because linear alcoholsoften have similar effects, differing only in potency, inbiological systems (12). For example, membrane-bound AT-Pase activity, alanine uptake, and 3-0-methyl glucose up-

take in C. acetobutylicum have been shown to be similarlyinhibited by n-alkanols (18). Thus, the ApH may havedecreased as the decanol volume fraction increased, becauseof an increase in the aqueous decanol concentration (5), and

90.0-

80.0-

* 70.0-

o 60.0-

50.0-

- 40.0-

! 30.0-

20.0-

10.0

-380.0

-360.0

- 340.0

- 320.0 dC

-300.0 8

-280.0 8

-260.0 5

-240.0

control 0.0 0.1 0.2 0.3 0.4

Decanol volume fractionFIG. 6. Total acid (0) and solvent (A) yields (not including

ethanol) and glucose consumption (M) for pH-controlled fermenta-tions.

i~ 120.0-100.0-

8 40.0-;

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0Time (hr)

FIG. 7. Total butanol production in the presence of differentphase volume ratios of 20% decanol in oleyl alcohol and CABmedium. Time zero is the onset of solventogenesis for each fermen-tation. Symbols: 0, control with no organic phase; A, 1:100organic-aqueous phase volume ratio; 0, 1:1 organic-aqueous phasevolume ratio.

lowered the intracellular pH to a greater degree than theextracellular pH.When the pH was maintained at 4.5, the incorporation of

decanol into the extractant led to an increase in butanolproduction. This increase was not explicable in terms of thealleviation of end product inhibition of butanol. This is not tosay that the alleviation of end product inhibition has nosignificance in these experiments (indeed, the presence of anorganic phase was required for the enhancement of butanolformation), but rather, it is to say that it does not serve as amechanism for the increase.The increase in butanol formation was partially attribut-

able to an increase in butyrate consumption as the decanolvolume fraction was increased from 0.0 to 0.2. The additionof oleyl alcohol to the fermentation resulted in the extractionof butyrate and the subsequent decrease of its maximal,aqueous concentration. This decrease led to the alleviationof end product inhibition by butyrate, as evidenced by theincrease in glucose consumption. The decrease in the max-imal aqueous concentration of butyrate probably resulted ina decrease of the maximal intracellular concentration of theundissociated acid. This intracellular concentration mayhave a triggering role (1, 10, 21; M. Huesemann and E. T.Papoutsakis, presentation at the 194th national meeting ofthe American Chemical Society, New Orleans, La., 1987) intransforming the cells from the acidogenic state to thesolventogenic state. The decreased acid consumption in thepresence of 0 to 10% decanol in oleyl alcohol may haveresulted because solventogenesis was incompletely trig-gered. Other mechanisms are also conceivable. The de-creased consumption is not a kinetic effect resulting from adecreased rate in accordance with a decrease substrate (i.e.,aqueous butyrate) concentration, since the addition of 20 to30% decanol, which did not significantly affect the maximalaqueous butyrate concentration, did cause an increase inacid consumption. The intracellular, undissociated acid con-centration is proportional to the extracellular acid concen-tration (undissociated plus dissociated) because the externalpH is constant and the intracellular and extracellular undis-sociated acid concentrations are approximately equal (21).

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ENHANCEMENT OF BUTANOL FORMATION BY C. ACETOBUTYLICUM 1667

When the decanol volume fraction is increased, the aqueousdecanol concentration increases (5). Even though dissolveddecanol in the broth did not account solely for the enhance-ment of butanol formation, it may have substituted forbutyrate in triggering acid consumption. This hypothesis isthe subject of a forthcoming report.The reinstatement of acid consumption allowed an in-

crease in butanol formation, as well as an increase in acetoneand acetoin formation. This increase resulted in an addi-tional increase in glucose consumption which was attribut-able to the alleviation of the end product inhibition ofbutanol, the disposal of excess reducing equivalents createdby the production of acetate and butyrate, or both.No increase in solvent production was observed as the

decanol volume fraction was increased from 0.2 to 0.3. Thetoxicity of decanol was not found to be the explanation.Rather, the increase in butanol was limited by the depletionof glucose and butyrate. Excess glucose alone did not lead toan increase in butanol production because it did not causegreater production of butyrate; thus, butyrate itself becamelimiting during solventogenesis. When excess glucose andbutyrate were provided, the cells produced an increasedamount of butanol but did not sporulate and lysed at the endof the fermentation. The additive toxicity of acids, solvents,and decanol may have limited the production in this case.More decanol (40%) in oleyl alcohol caused a decrease in

glucose consumption and solvent formation and completelyinhibited acid consumption. Acid consumption was inhibitedprobably by the toxicity of decanol, since 50% decanol wasinhibitory to growth. This result is in contrast to the inhibi-tion in the presence of 0 to 10% decanol, which resulted fromthe fact that acid consumption was not triggered. Twopossible explanations exist for the decreased glucose con-sumption. One is that decanol directly inhibited glucosetransport or glycolysis. The second explanation is thatdecanol may have decreased the ApH and caused the intra-cellular pH to be too low to allow solvent production asdescribed earlier. The inability of C. ac etobutylicini toreduce the acids to solvents could have subsequently led toaccumulation of these acids, their uncoupling of the cell (2),and the inhibition of proton motive force-mediated glucosetransport.

ACKNOWLEDGMENT

We are grateful for financial and technical assistance from theMichigan Biotechnology Institute.

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Effect of pH and butyrate concentration on the production ofacetone and butanol by Clostridiiiin acetobutylichun 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 byClostridilin thermoaceticiin. Appl. Environ. Microbiol. 48:1134-1139.

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1165-1170.4. Denbigh, F. R. S. K. 1981. The principles of chemical equilib-

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15. Kim, B. H., P. Bellows, R. Datta, and J. G. Zeikus. 1984. Controlof carbon and electron flow in Clostriidiinm acetobutliculnfermentations: utilization of carbon monoxide to inhibit hydro-gen production and to enhance butanol yields. Appl. Environ.Microbiol. 48:764-770.

16. Largier, S. T., S. Long, J. D. Santangelo, D. T. Jones, and D. R.Woods. 1985. Immobilized Clostr-idiiumii acetobitlicumi P262mutants for solvent production. Appl. Environ. Microbiol.50:477-481.

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