Vol. 164, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 173-1800021-9193/85/100173-08$02.00/0Copyright © 1985, American Society for Microbiology

Mechanism of Ethanol Inhibition of Fermentation inZymomonas mobilis CP4t

YEHIA A. OSMAN AND LONNIE 0. INGRAM*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 15 May 1985/Accepted 23 July 1985

Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugarconversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can beattributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanoldecreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essentialcofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage ofintermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanolon fermentation in Zymomonas mobilis.

Zymomonas mobilis is an obligately fermentative bacte-rium that produces ethanol and carbon dioxide as principalfermentation products (42). This organism is of considerableinterest for commercial production of ethanol and has beenreported to be capable of sugar conversion rates severalfoldthose of Saccharomyces cerevisiae under optimal conditions(20, 33).Renewed interest in ethanol as a biofuel, octane enhancer,

and chemical feedstock has led to numerous investigationsinto methods for increasing rates and yields of fermentationby both S. cerevisiae (1, 2, 30, 37, 43) and Z. mobilis (3, 24,25, 34). One of the problems associated with large-scaleethanol production is the decrease in the rate of substrateconversion observed during ethanol accumulation in themedium (1, 19, 25, 26). Despite centuries of experience in theart of fermentation (5, 14), the mechanism of inhibition offermentation by ethanol has not been established. Currenthypotheses concerning this mechanism have proposed feed-back inhibition or inactivation of enzymes which are in-volved in alcohol production (15, 19, 24, 25, 27). However,these hypotheses are not fully supported by in vitro studiesof enzyme activity and stability (24, 25).

In this study, we developed a different approach to inves-tigate the mechanism for ethanol inhibition of fermentationin Z. mobilis. This approach involves treatment of cellssuspended in buffer or growth medium as if they wereenzymes. Glucose consumption was monitored as a measureof fermentation. Our results support the hypothesis that theprincipal action of ethanol which results in a decreased rateof fermentation is to increase membrane leakage, reducingthe intracellular concentrations of cofactors and coenzymesessential for the activity of enzymes involved in glycolysisand alcohol production.

MATERIALS AND METHODSOrganism and growth conditions. The organism used in

these studies, Z. mobilis CP4, was generously supplied by A.Ben-Bassat of Cetus Corporation (Berkeley, Calif.). Thisorganism was cultivated in the medium described bySkotnicki et al. (39) with various levels of glucose. Cells weremaintained on medium solidified with 1.5% agar. Growth was

* Corresponding author.t Publication no. 6397 of the Florida Agricultural Experiment

Station, University of Florida, Gainesville.

monitored by measuring optical density at 550 nm with a B&LSpectronic 70 spectrophotometer with 10-mm round cuvettes(disposable 10-mm culture tubes). For ethanol inhibitionstudies, cells were harvested in exponential phase (opticaldensity at 550 nm, 0.7; approximately 4 x 108 cells per ml; 175,ug of cell protein per ml).

Analytical methods. Glucose was measured by the glucoseoxidase assay (32) supplied by the Sigma Chemical Co. (St.Louis, Mo.). Magnesium was measured with the 60-SecondMagnesium reagent system, supplied by the American Mon-itor Corporation (Indianapolis, Ind.) and designed for clini-cal determinations. Nucleotides were estimated by measur-ing A260. Spectra did not contain shoulders corresponding toproteins at 280 nm and did not contain measurable amountsof protein. Cell protein was measured by the procedure ofLowry et al. (22) as described by Layne (21). Tritium, 32p,and 14C were measured with a model 8000 scintillationcounter (Beckman Instruments, Inc., Fullerton, Calif.) withScinti Verse II as a cocktail (Fisher Scientific Co., Pitts-burgh, Pa.).

Assay of fermentation rate. Fermentation rate was mea-sured by determining glucose consumption by using wholecells suspended in buffer or growth medium. Preliminaryexperiments were done to determine the pH optimum (pH6.0) in sodium phosphate, sodium citrate, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid hydro-chloride buffers. Phosphate buffer was chosen for the assaysystem because it is a component of the growth medium andsupported high rates of fermentation. Growth conditionswere optimized (30°C; unshaken tube cultures containing0.2% glucose; harvesting in exponential growth immediatelybefore glucose exhaustion; optical density at 550 nm, 0.65 to0.70) to obtain reproducible and highly active fermentingcells. Cells were harvested and washed twice in either 50mM sodium phosphate buffer (pH 6.0) or growth mediumlacking glucose by using an unrefrigerated centrifuge (8,000x g for 5 min). Standard assay conditions were growthmedium or 50 mM sodium phosphate buffer (pH 6), 40 ,g ofcell protein per ml, and 0.6 mM glucose. Ethanol, nucleo-tides, yeast extracts, and other salts were also present invarious experiments. Assays were carried out at 30°C andwere initiated by adding glucose. Samples were removed at10-min intervals over a 40-min period and inactivated byboiling for 2 min. Screw-capped tubes with teflon liners wereused when high concentrations of ethanol were present.

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174 OSMAN AND INGRAM

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FIG. 1. Effects of added ethanol on fermentative activity. Sym-bols: 0, cells assayed in 50 mM sodium phosphate buffer (pH 6.0)containing ethanol; 0, cells exposed to various concentrations ofethanol in buffer at 30°C for 10 min, washed, and assayed in bufferlacking ethanol; A, cells exposed to ethanol in buffer, washed, andassayed in buffer supplemented with 5 mM magnesium sulfatelacking ethanol; *, cells assayed in growth medium containingethanol; 0, cells exposed to ethanol in growth medium at 30°C for 10min, washed, and assayed in growth medium lacking ethanol.

Residual glucose was measured by the glucose oxidaseassay. Linearity of this assay system was confirmed forprotein concentration and time. Typical activities obtainedwere 15 to 16 i,mol of glucose consumed per h per mg of cellprotein in buffer and 18 to 20 ,umol of glucose consumed perh per mg of cell protein in growth medium.

Determination of intracellular ethanol concentration. Intra-cellular concentrations of ethanol were determined forwashed cells suspended in buffer alone, in buffer containing1.7 M ethanol (8%, wt/vol; 10-min exposure), and in cellswhich had previously been exposed to 1.7 M ethanol for 10min, centrifuged, and suspended in buffer lacking ethanol. Inthese determinations, cell pellets were suspended in 4 ml ofbuffer per g of wet weight. Samples (1 ml) in Microfuge(Beckman) tubes were mixed with 10 RI of [14C]sorbitol (8.3,uCi/ml, 50 ,uCi/mmol) dissolved in tritiated water (198,uCi/ml) and allowed to equilibrate for 10 min at roomtemperature. Samples (50 RId each) were removed from thesesuspensions for counting. Cell suspensions were then cen-trifuged in an Eppendorf microcentrifuge for 2 min, andsamples were removed from the supernatants for counting.The 14C-tritium ratio of the suspension divided by that of thecell-free supernatant represents the fraction of aqueousvolume surrounding the cells (intercellular volume). Theintracellular volume is equal to one minus the intercellularvolume. Ethanol was measured in the suspension and in thesupernatant with a 560 gas chromatograph (Tracor Instru-ments, Austin, Tex.) equipped with an electronic integratoras described by Goel and Pamment (12). Intracellularethanol was calculated by the formula: ethanol concentra-tion in the suspension = (ethanol concentration in thesupernatant x fractional intercellular volume) + (ethanolconcentration within the cell x fractional intracellular vol-ume). All radioactive compounds were purchased from theAmersham Corp. (Arlington Heights, Ill.).

Fractionation of yeast extract. Two methods were used tofractionate yeast extract. In the first, 5 g of yeast extract was

dissolved in 500 ml of distilled water and treated three times

with 100 g of Norit A activated charcoal (Matheson, Cole-man & Bell, East Rutherford, N.J.) for 1 h each with gentleagitation. Charcoal was removed by centrifugation (8,000 xg). The second procedure involved ashing 5 g of yeastextract at 500°C for 48 h followed by dissolving the inorganicresidue in distilled water.

Effects of ethanol on leakage. Ethanol-induced leakage ofmagnesium, nucleotides, and protein was determined bysuspending cells (800 ,ug of protein per ml) in either 50 mMsodium phosphate buffer (pH 6.0) or growth medium con-taining a series of ethanol concentrations but lacking glu-cose. Cells were incubated for 30 min at 30°C and pelleted bycentrifugation for 2 min in an Eppendorf microcentrifuge,and the supernatants were analyzed. Magnesium was mea-sured as indicated above. Nucleotide leakage was measuredin two ways. In buffer, nucleotide concentration was mea-sured as A260. In both buffer and growth medium, cells wereprelabeled for five generations in complex medium bygrowth with [32P]phosphate (80 ,Ci added per 300-ml culturecontaining 0.7 mM added monobasic potassium phosphate);cells were washed and suspended to measure leakage. Afterremoval of cells by centrifugation, perchloric acid (finalconcentration, 10%) was added to the supernatant, and thesamples were centrifuged a second time to eliminate nucleicacids before removing 0. 1-ml samples for nucleotide mea-surement. Protein leakage was measured by using cellsprelabeled for five generations in complex growth mediumsupplemented with tritiated leucine (10 ,uCi; specific activity,72 Ci/mmol; added to 300-ml broth culture). Cells werewashed and suspended in various concentrations of ethanolto determine the influence of ethanol on protein leakage intothe supernatants. Leakage values for protein and magnesiumare expressed as a percentage of release into the supernatantby vortex-mixing suspensions for 30 s with chloroform (0.2ml per 2 ml of suspension). This treatment released 70% ofthe total cellular protein and 80% of the total magnesium.Nucleotide values are expressed as A260 and as a percentageof the total nucleotides released by 10% perchloric acid at0°C during a 30-min period.

RESULTSEffects of ethanol addition on fermentation rates. Addition

of ethanol to Z. mobilis cells caused dose-dependent inhibi-tion of fermentation in both buffer and growth medium (Fig.1). However, fermentation was much more sensitive toinhibition by low concentrations of ethanol in buffer than ingrowth medium. Inhibition of 50% of activity was caused by1.1 M ethanol (5%, wt/vol) in buffer and 2.2 M ethanol (10%,wt/vol) in growth medium. Fermentation was completelyinhibited by the highest concentration of ethanol tested, 4.4M (20%, wt/vol), in both buffer and growth medium.

Ethanol removal by washing. We determined intracellularconcentrations of ethanol in cells as controls for experimentsinvolving ethanol addition and removal. Intracellular ethanolconcentration was measured in washed cells suspended inbuffer, in cells suspended in buffer containing 1.7 M ethanol(8%, wt/vol), and in cells exposed to 1.7 M ethanol immedi-ately after pelleting and suspending in fresh buffer lackingethanol. The initial ethanol concentration was less than 0.01M. The intracellular ethanol concentration was 1.5 M (stan-dard deviation, 0.2) for cells suspended in buffer containing1.7 M ethanol and 0.02 M (standard deviation, 0.01) ethanolin cells exposed to ethanol and suspended in fresh bufferlacking ethanol. These results indicated that added ethanolwas rapidly equilibrated within Z. mobilis cells and wasefficiently removed by washing.

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ETHANOL INHIBITION OF FERMENTATION 175

Reversibility of inhibition of fermentation by ethanol wasexamined after ethanol removal by pelleting cells and sus-pending them in fresh medium. Removal of ethanol fromcells suspended in buffer did not cause an increase infermentative activity (Fig. 1). Removal of ethanol from cellsexposed to ethanol in growth medium resulted in partialrecovery of fermentative activity in all but the' highestethanol concentrations. These results indicated that expo-sure to ethanol in buffer and, to a lesser extent, in growthmedium resulted in persistent damage to fermentative activ-ity.The effects of ethanol exposure time on the extent of

inhibition of fermentation were examined with cells sus-pended in buffer and in growth medium (Fig. 2). Fermenta-tive activity decreased more rapidly in buffer than in growthmedium. Under both conditions, loss of fermentative activ-ity with time was biphasic, with a rapid loss during the initial10 min of exposure, followed by a slower rate of declineduring the subsequent 30-tnin period examined.

Repair of fermentative activity during incubation afterethanol removal. We examined the ability of Z. mobilis cellsexposed to 1.1 M ethanol (30°C for 10 min) in buffer to regainfermentative activity after ethanol removal (Fig. 3). Thefermentative activity of ethanol-damaged cells which hadbeen suspended'in buffer continued to decline in the absenceof ethanol. In contrast, the fermentative activity of ethanol-damaged cells which had been suspended in growth mediumshowed progressive improvement with time, reaching 70%of original activity after 2 h. Cell mass was monitoredspectrophotometrically during the incubation period and didnot increase. Thus, the improvement in fermentative activityobserved in growth medium appears to result from repair ofethanol damage rather than cell growth.The various components of growth medium were tested

individually in buffer for their ability to facilitate repair ofethanol damage to fermentation (Table 1). Growth mediumcontained potassium phosphate, glucose, and yeast extract.

100

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TIME OF EXPOSURE (min)FIG. 2. Time dependence of ethanol exposure on inhibition of

fermentation. Cells were suspended in ethanol solutions at 30°C.Samples were removed at various times, centrifuged, suspended inthe absence of ethanol, and assayed for fermentative activity.Symbols: 0, cells exposed to 1.1 M ethanol (5%, wt/vol) in 50 mMsodium phosphate buffer (pH 6.0) and assayed in buffer lackingethanol; 0, cells exposed to 2.2 M ethanol (10%, wt/vol) in growthmedium and assayed in growth medium lacking ethanol.

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TIME (h)FIG. 3. Recovery of cells from ethanol damage. Cells were

exposed to 1.1 M ethanol (5%, wt/vol) in 50 mM sodium phosphatebuffer (pH 6) for 10 min at 30°C, centrifuged, and suspended in eitherbuffer (pH 6.0) or growth medium. These suspensions of ethanol-damage cells were incubated for 2 h at 30°C. Samples were removedat 30-min intervals, centrifuged, and suspended in buffer for mea-surement of fermentative activity. Optical density at 550 nm wasmonitored as a measure of growth during incubation and did notincrease in buffer or growth medium during the 2-h incubationperiod. Symbols: 0, cells incubated in growth medium; 0, cellsincubated in buffer.

Of these, only yeast extract was required for partial recoveryof fermentation activity. Unfottunately, yeast extract is avery complex nutrient and contains appreciable amounts ofthe other'medium components as well.

Yeast extract was fractionated in two ways: (i) by treat-ment with charcoal to remove nucleotides, vitamins, and avariety of hydrophobic organic compounds and (ii) by ash-ing, which destroyed all but the inorganic components. Bothof these preparations were as effective as whole yeastextract (Table 1), indicating that only the inorganic compo-

TABLE 1. Effects of growth medium components on recovery offermentation in ethanol-damaged cells

Fermentative activityCells and treatment ptrnol/mg of % of

protein per h control

Control cells 15.0 100

Ethanol-damaged cells assayed in:aBuffer 5.2 35Complete medium 11.0 73Buffer + glucose (2 g/liter) 7.6 51Buffer + KCl (2 g/liter) 7.8 52Buffer + yeast extract (10 g/liter) 10.8 72Buffer + charcoal-treatedb yeast extract 10.5 70Buffer + ashedb yeast extract 11.1 74

aCells were exposed to 1.1 M ethanol (5%, wt/vol) in 50 mM sodiumphosphate buffer (pHI 6.0) for 10 min. Ethanol was removed by centrifugationand suspension in buffer. Cell suspensions were then incubated for 2 h at 30Cto allow repair of ethanol damage, centrifuged, and assayed for fermentativeactivity in buffer.

b Charcoal-treated and ashed yeast extracts were added at a concentrationequivalent to that of whole yeast extract.

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176 OSMAN AND INGRAM

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MAGNESIUM CONC. (mM)FIG. 4. Effects of added magnesium sulfate on inhibition of

fermentation by ethanol. Cells were exposed to ethanol either in 50mM sodium phosphate buffer (pH 6.0) or in growth medium for 10min at 30°C and assayed in the presence of ethanol or centrifugedafter ethanol exposure and assayed in the absence of ethanol.Various concentrations of magnesium sulfate were added to theassay solutions. A. Buffer with 2.2 M ethanol (10%, wt/vol); B.

Growth medium with 2.2 Methanol (10%, wt/vol). Symbols: 0, cellsin the continuous presence of ethanol; 0, cells exposed to ethanoland assayed after ethanol removal.

nents of yeast extract were required for the repair of ethanoldamage to fermentation.

Restoration of fermentAtive activity in ethanol-damagedcells by addition of divalent metals. The cellular requirementsfor fermentation in Z. mobilis are relatively simple: sugarsubstrate, functional enzymes (glycolytic enzymes, pyruvatedecarboxylase, and alcohol dehydrogenase), coenzymes(NAD/NADH; ATP/ADP), thiamin pyrophosphate cofactors(magnesium, calcium [?], potassium 1?], and phosphate), andappropriate pH and temperature. In addition, fermentationrequires a semipermeable membrane which retains all essen-tial metabolites and enzymes while permitting the entry ofsubstrate by facilitated diffusion (6) and exit of end products.In comparing the requirements for fermentation with theresults obtained with fractionated yeast extract, it becameclear that only inorganic cofactors could be supplied by ash..Phosphate was abundant in the buffer used, and addition ofpotassium chloride did not support appreciable repair, leav-ing magnesium and calcium as possible inorganic compo-nents of yeast extract which are required for repair ofethanol damage to fermentation.The effects of added magnesium sulfate on fermentation in

buffer and growth medium are shown in Fig. 4. With washedcells that had been exposed to 2.2 M ethanol in growthmedium or buffer, fermentative activity was completelyrestored by addition of 0.5 and 2.0 mM magnesium sulfate,respectively. Fermentative activity was partially restored byaddition of magnesium sulfate in the presence of 2.2 Methanol, with optimal concentrations of 2 mM for cellsassayed in growth medium and 5 mM for cells assayed inbuffer. Concentrations of magnesium sulfate above 2 mMcaused a progressive reduction in fermentation rate.We also examined divalent metal ion specificity for im-

provement of fermentative activity in the presence ofethanol and in cells which had been washed after exposure toethanol. Comparisons were made with the addition of 0.5,1.0, and 2.0 mM chlorides or sulfates or both in buffer (1.1 Methanol) and in growth medium (2.2 M ethanol). Magnesiumwas most effective, followed closely by manganese. Zincprovided a slight improvement, whereas calcium and cobaltwere not helpful.To further examine the significance of divalent metals

such as magnesium as an explanation for differences in theethanol sensitivity of fermentation in buffer and growthmedium, we reexamined the inhibition of fermentation byethanol in buffer supplemented with magnesium sulfate (Fig.1). Cells suspended in buffer containing 5 mM magnesiumsulfate exhibited decreased sensitivity to inhibition of fer-mentation by ethanol, equivalent to that of cells suspendedin growth medium. Further, the addition of 10 mM EDTA togrowth medium increased the extent of inhibition of fermen-tation by 2.2 M ethanol (10%, wt/vol) from 45% to over 60%in comparison with control cells in the absence of ethanol.Subsequent analysis of growth medium revealed that itcontained approximately 0.35 mM magnesium ions.

Ethanol-induced leakage. The effects of added ethanol oncellular leakage are shown in Fig. 5. Cells were exposed toethanol and centrifuged, and the supernatants were exam-ined to determine the extent of leakage. The 100lo valuesobtained corresponded to 1.4 ,umol of magnesium per mg ofcell protein, 5.4 ,umol of nucleotide (ADP equivalents basedon A260) per mg of cell protein, and 70%o of total cellularprotein. Increasing concentrations of ethanol caused a dose-dependent (though not linear) increase in the leakage ofnucleotides and magnesium. Magnesium leakage was moresensitive to the addition of ethanol than was nucleotideleakage. The addition of 1.1 M ethanol in buffer and 2.8 Methanol (12.7%, wt/vol) in growth medium resulted in theleakage of 50% of the magnesium. The leakage of 50% of thecellular nucleotides was caused by 2.4 M ethanol (11%,wt/vol) in buffer and 2.8 M ethanol in growth medium.Appreciable leakage of cellular proteins was not observed,even in the presence of 4.4 M ethanol.

Effect of nucleotides plus magnesium addition on inhibitionof fermentation by 4.4 M ethanol. The damaging effects oflow ethanol concentrations were almost completely reversedby addition of magnesium ions, but this addition was insuf-ficient to prevent inhibition of fermentation at high ethanolconcentrations. Since both nucleotides and magnesium ionsare required for fermentation, and ethanol increased theleakage of both, we examined the effect of nucleotides addedwith magnesium on cells in which fermentation was com-pletely inhibited by 4.4 M ethanol. Addition of magnesiumsulfate alone resulted in the recovery Qf 20%o of the controlactivity. In combination with 0.2 mM NAD, a decrease inactivity was observed. In combination with 0.2 mM NAD,0.9 mM ATP, and 0.1 mM ADP, 30% of the control activitywas recovered. High ADP:ATP ratios were less effective.

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ETHANOL INHIBITION OF FERMENTATION 177

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FIG. 5. Effects of ethanol on cell leakage. Cells were exposed toethanol for 30 min at 30°C in either 50 mM phosphate buffer (pH 6.0)for growth medium and pelleted by centrifugation, and the extent ofleakage of mnagnesium ions (A), nucleotides (B), and proteins (C)was determined. Values are plotted as the percentage of constituentreleased by membrane disruption with chloroform (defined as100%). Symbols for panels A and C: 0, buffer; 0, growth medium.Symbols for panel B: O, A260 in buffer; 0, [32P]phosphate in buffer;0, [32P]phosphate in growth medium.

These results indicated that the enzymes of glycolysis werenot sufficiently inactive to prevent fermentation, even in thepresence of 4.4 M ethanol.

Relationship between leakage and the extent of inhibition offermentation. Figure 6 shows the relationship betweenethanol-induced leakage of magnesium and nucleotides andethanol-induced inhibition of fermentation. The relation-ships between the extent of leakage of magnesium and theextent of inhibition of fermentation in growth medium and inbuffer were very similar, although different concentrations ofethanol were required to cause an equivalent extent ofleakage. Leakage of up to 50% of cellular magnesium was

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age and that of ethanol-induced inhibition of fermentation. Theextent of inhibition of fermentation by various ethanol concentra-tions in either 50 mM sodium phosphate buffer (pH 6.0) or growthmedium is plotted against the extent of leakage of magnesium ions(A) and the extent of leakage of nucleotides (B) which occurred atthese ethanol concentrations. Symbols: 0, growth medium; 0,buffer.

accompanied by modest inhibition of fermentation (<20%).Further leakage resulted in a very rapid decline in the rate offermentation. Complete release of magnesium was accom-panied by complete inhibition of fermentation.

Relationships between nucleotide leakage and inhibitionof fermentation in growth medium and in buffer were alsosimilar. Even low levels of nucleotide leakage were accom-panied by a reduction in fermentative activity. Althoughdifferent concentrations of ethanol were required to causesimilar extents of nucleotide leakage in buffer and in growthmedium, both were accompanied by equal inhibition offermentation.

DISCUSSIONThe accumulation of ethanol as an end product of fermen-

tation is known to cause progressive inhibition in the rate ofsugar conversion to ethanol by Z. mobilis (3, 19) and byyeasts (1, 25-28). This phenomenon is particularly importantduring commercial ethanol production and increases thetime required to complete the conversion of sugar substrateto ethanol and limits the final concentration of ethanolachieved (16). Many studies have hypothesized that thisinhibition is due to direct action of ethanol on key enzymes

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178 OSMAN AND INGRAM

of glycolysis and ethanol production involving feedbackinhibition or enzyme inactivation (2, 15, 19, 24, 26). How-ever, in vitro studies of these enzymes in Z. mobilis and S.cerevisiae do not fully support this hypothesis (24).

Ethanol caused dose-dependent inhibition of fermentationin vivo with measurable inhibition at concentrations as lowas 0.4 M (2%, wt/vol), whereas concentrations over 3.3 M(15%, wt/vol) are required to inhibit enzymes in vitro, andeven higher concentrations are required to inactivate theseenzymes (24, 25). One possible explanation which has beenproposed to reconcile these differences is that the actualintracellular concentration of ethanol during active fermen-tation is severalfold higher than in the medium (24-26).However, our measurements and those of Laudrin andGoma (19) indicate that Z. mobilis is freely permeable toethanol and that intracellular concentrations of ethanol in Z.mobilis do not reach the high levels which are required toinactivate key enzymes in vitro (24). Certainly, other aspectsof the intracellular environmnent could increase the sensitiv-ity of these enzymes to ethanol in vivo, and this possibility isnot readily eliminated.The results of our investigations indicate that ethanol

causes inhibition of enzymes involved in alcohol productionas previously proposed (16, 25) but provide evidence for anovel, indirect mech4nism of action by ethanol. Inhibition offermentation by ethanol appears to result from increasedleakage through the plasma membrane, allowing loss ofcofactors and coenzymes. This conclusion is supported bythe lack of complete reversibility of inhibition by ethanolremoval, even after exposure to low concentrations ofethanol (less than 5%, wt/vol in some cases), by ethanol-induced leakage of magnesium ions and nucleotides, by amagnesium ion requirement for repair of ethanol-induceddamage to fermentation (after removal of ethanol), and bythe ability of added magnesium and added nucleotides toincrease the rate of fermentation in the presence of ethanol.The extent of leakage of magnesium ions and nucleotidescorrelated well with the extent of inhibition of fermentationunder two different experimental assay conditions in whichthe sensitivity of fermentation to ethanol inhibition was verydifferent, i.e., cells suspended in phosphate buffer and cellssuspended in growth medium.

It is likely that leakage of magnesium ions (small mole-cules) and nucleotides (molecules of intermediate size) isonly symptomatic of a more generalized increase in mem-brane permeability, which may include many other ions,cofactors, and metabolites, such as intermediates ofglycolysis. The ability of magnesium to restore activity in thepresence of low concentrations of ethanol suggests thatmagnesium ion leakage is the dominant effect under theseconditions and is consistent with the leakage of magnesiumions induced by low ethanol concentrations. A variety ofenzymes in Entner-Doudoroff glycolysis require magnesiumas a cofactor, including glucokinase, glucose-6-phosphatedehydrogenase, phosphoglycerate kinase, and enolase (4,11, 24, 35, 40, 41). The metal ion specificity for the protec-tion and repair of ethanol damage to fermentation is similarto the metal ion specificity for these enzymes, i.e., magne-sium and manganese supporting activity and the inability ofcalcium to substitute. Thus, it is not surprising that leakageof magnesium ions results in inhibition of fermentation.Unlike S. cerevisiae, alcohol dehydrogenase in Z. mobilisdoes not appear to require magnesium (15, 45). Other metalcofactors which may be needed for fermentation includecalcium, which is reported to activate pyruvatedecarboxylase in Z. mobilis (15), and potassium for pyruvate

kinase (40). However, the addition of neither calcium nor ofpotassium reduced ethanol inhibition of fermentation.

Higher concentrations of ethanol were required to induceleakage of nucleotides than were required for magnesiumleakage, which is consistent with a requirement for moreextensive membrane damage to allow leakage of these largermolecules. Magnesium alone only partially relieved inhibi-tion of fermentation under these conditions. At very highconcentrations of ethanol (4.4 M), at which fermentation wascompletely inhibited, 30% of the fermentative activity ofcells could be restored by addition of nucleotides withmagnesium ions. It is unlikely that fermentative activity canbe completely restored in the presence of such high concen-trations of ethanol, owing to probable diffusion of interme-diary metabolites out of and into the cell and owing to apossible reduction in the catalytic rates of enzymes involvedin glycolysis and alcohol production (24). However, theseresults do demonstrate that complete inhibition of fermenta-tion by 4.4 M ethanol (20%, wt/vol) does not result fromfeedback inhibition or enzyme denaturation, even at thesehigh concentrations of ethanol.

Previous studies with a variety of systems have shownthat ethanol increases membrane leakage (6a, 8, 16, 23, 31).There are three basic ways in which ethanol would beexpected to decrease the effectiveness of the plasma mem-brane as a hydrophobic barrier: (i) by altering the colligativeproperties of the environment, (ii) by directly interactingwith the membrane, and (iii) by altering the dielectric prop-erties of the environment. First, the addition of ethanol to anaqueous milieu is known to alter water structure and de-crease the strength of hydrophobic interactions (46). Thisdecreased strength of hydrophobic interactions would tendto decrease the extent to which acyl chains participate inhydrophobic interactions which form the hydrophobic coreof the membrane and to increase the extent of incursion ofpolar molecules into the two membrane surfaces. Highconcentrations of ethanol would be expected to actuallysolubilize some of the membrane components by increasingthe ability of the milieu to accommodate hydrophobic func-tional groups (29, 44). In addition, high molar concentrationsof ethanol would be expected to replace water to someextent as a hydrogen bonding partner and in solvation shells(hydration shells which include ethanol). Second, ethanol isan amphipathic molecule and is known to partition into thehydrophobic region of the membrane (7, 13, 36, 38). Sinceethanol is much more polar than the hydrocarbon membranecore, its presence would tend to increase the average polar-ity of this environment and reduce its effectiveness as ahydrophobic barrier. Partitioning of ethanol into the mem-brane is also known to increase bulk membrane fluidity;increased fluidization per se has been shown to increasemembrane leakage (31). Finally, the presence of ethanol atrelatively high molar concentrations alters the dielectricproperties of an aqueous milieu, strengthening coulombicinteractions and shifting the equilibrium of charged ionicspecies toward neutral, conjugated forms (9, 18). In general,membranes are more permeable toward these neutral spe-cies (10). It is not possible to predict the biological conse-quences of these changes in coulombic interactions onmembranes. However such changes may alter the packingarrangement and allow segregation of mermbrane compo-nents such that discontinuities are produced whichpermeabilize cells, allowing leakage of small molecules butretention of larger molecules. Such an effect is consistentwith the release of cellular magnesium and nucleotidesobserved with 4.4 M ethanol in the absence of significant

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ETHANOL INHIBITION OF FERMENTATION 179

protein leakage. This type of global change in membraneorganization is also consistent with continued loss of fermen-tative activity in ethanol-treated cells during subsequentincubation in buffer after ethanol removal.Our initial experiments indicated that ethanol was more

inhibitory for fermentation in phosphate buffer than ingrowth medium, even though phosphate buffer was a com-

ponent of growth medium. The basis of this difference insensitivity appears to be the presence of magnesium ions inthe yeast extract equivalent to 35 ,umol per g. Addition ofmagnesium to phosphate buffer decreased ethanol inhibitionof fermentation to a level equivalent to that of growthmedium. Addition of EDTA to fermentation assays contain-ing growth medium increased the extent of ethanol inhibi-tion, providing further evidence for the importance of diva-lent metals in the growth medium as the basis for thereduction in ethanol damage.Microorganisms are known to actively transport magne-

sium (17). An active transport system would be expected topartially counter increased leakage of magnesium across theplasma membrane when appreciable quantities of magne-

sium ions are available in the surrounding milieu. Similarly,increasing the extracellular concentration of magnesium byaddition would be expected to decrease the problem ofproviding sufficient intracellular magnesium for use as an

enzyme cofactor. It is also likely that active magnesiumtransport is responsible for the repair of ethanol damage tofermentation which occurs during incubation in fresh growthmedium lacking ethanol. Thus, the presence of extracellularmagnesium in yeast extract is sufficient to explain theobserved reduction in the potency of ethanol as an inhibitorof fermentation in growth medium compared with buffer.

ACKNOWLEDGMENTSWe thank J. E. Gander and J. F. Preston for their discussions and

suggestions in the preparation of the manuscript.This work was supported in part by a grant from the National

Science Foundation (DMB 8204928), by the Egyptian Ministry ofHigher Education, which provided support for Y.A.O., and by theFlorida Agricultural Experiment Station.

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