APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 110-114 Vol. 54, No. 10099-2240/88/010110-05$02.00/0Copyright © 1988, American Society for Microbiology

Intracellular Ethanol Accumulation in Saccharomyces cerevisiaeduring Fermentation

TONY D'AMORE,* CHANDRA J. PANCHAL, AND GRAHAM G. STEWART

Production Research Department, Labatt Brewing Company Limited, 150 Simcoe Street, London,Ontario, Canada N6A 4M3

Received 22 June 1987/Accepted 23 September 1987

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stagesof fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanolconcentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio ofintracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, theintracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing theosmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrientsupplementation of the medium increased the extent of growth and fermentation, resulting in complete glucoseutilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrientlimitation is a major factor responsible for the decreased growth and fermentation activities observed in yeastcells at higher osmotic pressures.

The intracellular accumulation of ethanol in Saccha-romyces cerevisiae cells and its potential effects on growthand fermentation have been topics of controversy for thepast several years (5a). It was initially reported by Nagoda-withana and Steinkraus (16) that when ethanol was added toS. cerevisiae cultures it was less toxic to the yeast cells thanwas ethanol produced by the yeast cells themselves. Thereasons proposed for this phenomenon are the build-up oftoxic by-products (21), the depletion of nutrients (5, 8), andthe intracellular accumulation of ethanol during fermentation(1, 16, 17). Various groups have reported that intracellularaccumulation of ethanol occurs during fermentation (1, 16,17, 19). These findings have recently been confirmed invarious laboratories (14, 15, 18, 20). On the other hand, otherreports suggest that the intracellular concentrations of etha-nol in fermenting suspensions of yeast cells are less than orequal to those in the extracellular environment (6, 7, 10).The discrepancy in the results from the various studies is

primarily due to the controversy surrounding the accuracy ofthe techniques used to measure intracellular ethanol concen-tration (6, 7). For example, washing of cells prior to mea-surement of intracellular ethanol concentration, assumptionof a constant intracellular water volume, and initial harvest-ing and cell suspension steps all introduce errors whichaffect the calculated intracellular ethanol concentration.Recently, Dombek and Ingram (7) have developed a tech-nique for the determination of intracellular ethanol concen-tration based on the exclusion of [14C]sorbitol to estimateaqueous cell volume. Similarly, Guijarro and Lagunas (10)have used tritiated water and L-[U-14C]glucose to estimateaqueous cell volume. These methods avoided many of thetechnical problems encountered in other studies. Their re-sults suggest that the intracellular concentration of ethanol infermenting suspensions of S. cerevisiae cells is less than orequal to that in the extracellular environment and that it doesnot increase to high levels even during the most active stagesof fermentation. By using these methods, we have recentlyshown that there is an increase in intracellular ethanolconcentration in S. cerevisiae cells during the early stages of

* Corresponding author.

fermentation of wort with increasing carbohydrate concen-tration (T. D'Amore, C. J. Panchal, and G. G. Stewart, J.Inst. Brew., in press). However, no effect on growth orfermentation rates was observed, indicating that the increasein intracellular ethanol concentration did not affect fermen-tation.

In this communication, we report on the determination ofthe intracellular ethanol concentration in an ale-brewingstrain of S. cerevisiae throughout fermentation and on theeffect of increasing osmotic pressure and nutrient supple-mentation. The results obtained support our previous obser-vations in this laboratory that intracellular accumulation ofethanol occurs during the early stages of fermentation (19).Although intracellular ethanol accumulation is more evidentat higher osmotic pressures, it is the limitation of nutrientswhich results in the decreased growth and fermentationactivities of the cells.

MATERIALS AND METHODS

Chemicals. D-[U-'4C]sorbitol (304 mCi/mmol) and tritiatedwater (5 mCi/ml) were obtained from Amersham CanadaLtd. (Oakville, Ontario, Canada). All other chemicals wereobtained from commercial sources and were of the highestavailable purity.

Yeast strain. The yeast strain used in this study was thepolyploid S. cerevisiae ale-brewing strain 3001 from theLabatt Brewing Company, Ltd., culture collection.Growth medium. The yeast cells were subcultured in PYN

medium, which consisted of the following (in grams, alldissolved in 1 liter of distilled water and adjusted to pH 5.6):peptone, 3.5; yeast extract, 3.0; KH2PO4, 2.0; (NH4)2SO4,1.0; MgSO4 7H2O, 1.0; glucose, 100. Fermentations wereconducted in PYN medium containing 100 or 200 g ofglucose per liter (10 and 20% glucose, respectively) and werecarried out at 30°C in a BioFlow model 30 bench topfermentor (New Brunswick Scientific Co., Inc., Edison,N.J.) containing 1.3 liters of medium and with constantagitation of 150 rpm and constant aeration. In nutrientsupplementation experiments, PYN medium containingtwice the amounts of components (2x PYN) was used. The

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INTRACELLULAR ACCUMULATION OF ETHANOL 111

inoculum used in all studies was 3.5 g (wet weight) of cellsper liter.

Cell sampling and determination of ethanol concentration.At specified times during fermentation, 10-ml portions of cellsuspension were withdrawn. Intracellular ethanol concentra-tion was determined by using a modification of the perchloricacid extraction method described previously (19; D'Amoreet al., in press). In this procedure, 1-ml aliquots of cellsuspension were initially placed into Eppendorf tubes, intriplicate. A 0.1-ml sample from each suspension was with-drawn and added directly to a sample vial containing 0.1 mlof ice-cold 0.58 M perchloric acid. The remaining suspen-sions were immediately centrifuged at 10,000 x g for 1 minin a microcentrifuge. A 0.1-ml sample from each resultingsupernatant was added directly to a sample vial containing0.1 ml of ice-cold 0.58 M perchloric acid. To each pellet wasalso added 0.1 ml of ice-cold 0.58 M perchloric acid toliberate the intracellular ethanol. All samples were incubatedat 4°C for 12 h. Perchlorate ions were precipitated by theaddition of 0.4 ml of 4 M KOH and incubation at roomtemperature for 15 min. The precipitates were removed bycentrifugation at 10,000 x g for 5 min. The extracts werequickly frozen for subsequent ethanol concentration deter-mination. As a control, known volumes of ethanol weresubjected to perchloric acid treatment. Of the remaining cellsuspension, 5 ml was immediately centrifuged at 10,000 x gfor 10 min, and the supernatant was frozen for subsequentanalysis.

Determination of intracellular aqueous volume. The yeastcell intracellular aqueous volume was determined by twomethods. The first method was based on the proceduresdeveloped by Dombek and Ingram (7) and Guijarro andLagunas (10). The total aqueous volume of packed cells(interstitial volume plus true cellular volume) was measuredwith tritiated water (3H20). The interstitial volume of packedcells was measured with [14C]sorbitol. To a 1-ml sample ofcell suspension were added 20 ,ul of [14C]sorbitol and 10 RI of3H20 at final concentrations of 60.8 nCi/ml and 0.5 ,Ci/ml,respectively. The suspension was incubated at room temper-ature for 5 min, and 0.1-ml samples were pipetted directlyinto 10 ml of scintillation fluid. The remaining suspensionwas centrifuged, and 0.1-ml samples of the supematant werepipetted directly into 10 ml of scintillation fluid. The cellpellet was solubilized by the addition of 1.0 ml of 1% TritonX-100 and incubated at 30°C for 15 min. A 0.1-ml sample ofthis suspension was then pipetted directly into 10 ml ofscintillation fluid. The intracellular ethanol concentrationwas computed on the basis of the aqueous cell volume,which was calculated by subtracting the interstitial volume(sorbitol-excluded volume) from the total aqueous volume,as described elsewhere (7, 10).

In the second method, the total aqueous volume in thepellet was determined from the difference in wet and dryweights as described elsewhere (15). The interstitial volumeof the pellet was determined with [14C]sorbitol as describedabove. The aqueous cell volume was calculated by subtract-ing the interstitial volume from the total aqueous volume.Both methods yielded similar results.

Biomass. Cell population densities during fermentationwere determined by withdrawing 5 ml of suspension atvarious times, washing the portion twice with distilled water,and weighing the pellet after drying the portion at 100°C for4 h.

Analytical methods. Ethanol concentration was deter-mined by using a Carle AGC Series 100 gas chromatographoperating at 180°C having an 8-ft-long column (1 ft = 30.48

cm) packed with Poropak Q (81,000 mesh) and a Hewlett-Packard model 3380A integrator. Glucose and glycerol con-centrations were determined by using a Spectra-Physicsmodel SP8100 high-performance liquid chromatograph in-corporating a Bio-Rad oligosaccharide column (AminexHPX-42A) measuring 300 by 7.8 mm, a Micromeritics model771 refractive index detector, and a Spectra-Physics modelSP4270 computing integrator.

RESULTSEffect of glucose concentration on growth and fermentation.

It is well documented that increases in osmotic pressure arecorrelated with decreases in yeast growth and fermentation(2, 11, 12). In the present study, the effects of 10% and 20%glucose (13 and 29 atm, respectively [1 atm = 101.29 kPa])on S. cerevisiae growth and fermentation were investigated.Figure 1 shows that increasing the sugar concentrationresults in a decrease in the theoretically expected yield ofethanol. For example, utilization of 10% glucose would beexpected to result in production of 4.8% (wt/vol) ethanol.Figure 1A shows that 4.6% (wt/vol) ethanol was obtainedfrom the fermentation of 10% glucose. At a glucose concen-tration of 20%, however, this strain was unable to fullyferment the substrate under the conditions used (Fig. 1B). Inthis case, with the utilization of approximately 68% of theglucose, 6.6% (wtlvol) ethanol should have been produced.However, only 5.6% (wt/vol) ethanol was obtained. It hasbeen previously proposed that this phenomenon could beattributed to intracellular accumulations of ethanol, particu-larly at higher osmotic pressures (16, 19). However, Fig. 1also shows that complete fermentation of 20% glucose couldbe achieved by supplementing the media with 2x PYN,indicating that nutrient limitation is responsible for thedecrease in fermentation activity in 20% glucose. Table 1also shows that increasing the sugar concentration results ina decrease in cell growth as represented by the biomass.However, nutrient supplementation of the medium resultedin a twofold increase in biomass by the end of fermentation,supporting the increase in ethanol production observed.

It was also observed that an increase in osmotic pressureresulted in an increase in the rate of glycerol production(Table 2). Increases in glycerol concentration have beenshown to be a response of the yeast cells to osmotic stress (3,13). It should be noted that glycerol production from 20%glucose was not affected by nutrient supplementation.

Yeast intracellular aqueous volume. In order to calculatethe intracellular concentration of ethanol in yeast cells

A B

T150

11004

0 12 24 60 n 0 12 24 36 46 60F hndn Unw(hou) FS imUtoUm. (houm)

FIG. 1. Effect of glucose concentration on fermentation by the S.cerevisiae ale-brewing strain 3001. (A) Ethanol production; (B)glucose utilization. Symbols indicate cells grown on 10% glucose(0), 20% glucose (A), and 20% glucose with 2x PYN (0).

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during fermentation, the intracellular aqueous volume mustfirst be determined. Table 3 shows the results of suchdeterminations in S. cerevisiae during the fermentation of10% and 20% glucose by using two methods (7, 10). In one

method, the total aqueous volume of packed cells (interstitialvolume plus true cellular volume) was measured with triti-ated water. In the other method, the total aqueous volumewas determined from the difference in wet and dry weights.In both methods, the interstitial aqueous volume was deter-mined with ['4C]sorbitol and the aqueous cell volumes were

calculated by subtracting the interstitial aqueous volumefrom the total aqueous volume. Under our experimentalconditions, both methods yielded similar results.

In agreement with the results of other studies (1, 6, 10;D'Amore et al., in press), the intracellular aqueous volumewas found to decrease as the age of the culture increased.This was found to occur at both glucose concentrations.Interestingly, it was found that with 20% glucose the intra-cellular aqueous volume decreased by about 50% after 48 hwith nutrient supplementation, as compared with normalgrowth conditions, indicating increased cell growth withnutrient supplementation. Thus, a change in intracellularaqueous volume is an important factor to consider whendetermining the intracellular concentration of ethanol.

Distribution of intracellular and extracellular ethanol con-

centrations. To determine whether yeast cells accumulateethanol during the course of fermentation, the intracellularand extracellular concentrations of ethanol were examinedat different stages of growth. Furthermore, two concentra-tions of glucose in the medium were used to observe anyeffects due to increasing osmotic pressure. Under the testconditions used, the results obtained showed that ethanoldoes accumulate in the cells during the early stages offermentation (Table 4). The highest ratio of intracellular toextracellular ethanol was obtained at 3 h of fermentation,and this value decreased during fermentation until the intra-cellular and extracellular concentrations of ethanol were

similar (Table 5). Furthermore, the results indicate thatunder the higher osmotic pressure (higher glucose concen-

trations), the ratio of intracellular to extracellular ethanolwas greater than at the lower osmotic pressure. The perch-loric acid treatment method used to determine the intracel-lular ethanol concentration did not affect ethanol concentra-tion values because full recoveries of known ethanolstandards were obtained (data not shown). Table 4 alsoshows that nutrient supplementation of 20% glucose mediumresulted in an increase in ethanol production, although no

significant effect on the intracellular and extracellular etha-nol distribution during fermentation was observed (Table 5).

TABLE 1. Effect of glucose concentration on biomass

Biomass (mg [dry wt] of cells/ml) after growth in:Fermentation

time (h)a 10% Glucose 20% Glucose +2x

PYN

0 1.0 ±0.1 1.0 0.2 1.0 0.13 2.9 0.5 1.3 ±0.2 0.9 0.26 4.7 0.7 2.7 0.4 1.0 0.212 6.7 1.1 4.4 0.8 2.4 0.424 8.9 1.5 4.7 0.8 4.6 0.248 8.0 1.4 4.3 0.6 7.3 0.872 7.7 1.0 4.1 0.4 8.7 1.0

a 5. cerevisiae 3001 was grown in 10% and 20% glucose in PYN medium orin 20% glucose plus 2x PYN medium at 30°C. After incubation for theindicated times, the biomass was determined as described in Materials andMethods. The results are the average of at least three trials.

TABLE 2. Effect of glucose concentration on glycerol production

Glycerol concn (g/liter) after growth in:Fermentation

time (h)a 10% Glucose 20% Glucose 20% Glucose+ 2x PYN

03 0.5 ± 0.1 0.2 ± 0.1 0.2 ± 0.16 0.8 ± 0.2 1.0 ± 0.2 0.9 ± 0.2

12 1.4 ± 0.2 2.8 ± 0.5 2.6 ± 0.424 3.3 ± 0.2 7.1 ± 0.8 6.6 ± 0.648 3.4 ± 0.2 9.0 ± 1.0 8.3 ± 0.672 3.4 ± 0.2 10.1 ± 1.1 9.4 ± 0.4

a S. cerevisiae 3001 was grown in 10% and 20% glucose in PYN medium orin 20% glucose plus 2x PYN medium. After incubation for the indicatedtimes, glycerol concentrations were determined as described in Materials andMethods. The results are the average of at least three trials.

These results, which are consistent with the initial accumu-lation of intracellular ethanol and subsequent diffusionthrough the yeast cell membrane, are in agreement withprevious results from this laboratory (19; D'Amore et al., inpress) and from other laboratories (14, 15, 18, 20).

DISCUSSION

The intracellular accumulation of ethanol in yeast cells hasbeen the topic of considerable controversy for the pastseveral years. It has been proposed by various groups thatethanol does accumulate in yeast cells during fermentationand that this accumulation may be a factor contributing tothe toxic effects of ethanol (1, 14, 16, 17, 19). On the otherhand, other reports suggest that the intracellular concentra-tion of ethanol in fermenting suspensions of yeast cells is lessthan or equal to that in the extracellular environment (6, 7,10). The differences in the results from the various studieshave been attributed to inaccuracies in the techniques usedto measure intracellular ethanol concentration. For example,washing of cells prior to measurement of intracellular etha-nol concentration, assumption of a constant intracellularwater volume throughout fermentation, and errors arisingduring the initial harvesting and cell suspension steps com-mon to all methods (such as continued fermentation aftersampling) all introduce errors which can affect the calculatedintracellular ethanol concentration (6, 7). In the presentinvestigation, the method of Dombek and Ingram (7) wasused for the determination of intracellular ethanol concen-tration. This method avoided many of the technical problemsencountered in other studies, such as the difficulty in wash-ing the cells or breaking them open.The data presented in this communication clearly indicate

that there is an accumulation of ethanol in the yeast cells at3 h of fermentation (Table 4). This finding is in agreementwith previous results from this laboratory (19; D'Amore etal., in press). As fermentation proceeds, the intracellular andextracellular ethanol concentrations become similar. Theseresul'ts contradict those of other studies which have indi-cated that ethanol does not accumulate during any stage offermentation (6, 7, 10). It should be noted that, in theseprevious studies, the earliest time point for the determina-tion of intracellular ethanol concentration was taken after 12h of fermentation. The results presented here indicate thatthere is an accumulation of ethanol in the yeast cells duringthe early stages of fermentation (as early as 3 h) and that theethanol subsequently diffuses out through the yeast cellmembrane so that by 12 h of fermentation the intracellularand extracellular concentrations of ethanol are similar. This

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TABLE 3. Intracellular aqueous volumes of packed S. cerevisiae cells during fermentation

Intracellular aqueous vol (pl1/mg [dry wtl of cells)b after growth in:Fermentation 10% Glucose 20% Glucose 20% Glucose + 2x PYN

Method 1 Method 2 Method 1 Method 2 Method 1 Method 2

3 4.0 ± 0.3 3.8 ± 0.4 7.5 ± 0.8 7.7 ± 0.8 8.0 ± 0.6 8.2 ± 0.86 3.2 ± 0.1 2.9 ± 0.1 5.9 ± 0.4 5.8 ± 0.8 7.0 ± 0.4 6.8 ± 0.612 3.1 ± 0.1 2.7 ± 0.2 5.8 ± 0.4 5.3 ± 0.4 4.1 ± 0.4 4.4 ± 4.424 3.2 ± 0.2 2.6 ± 0.1 5.6 ± 0.2 5.1 ± 0.2 3.5 ± 0.2 3.7 ± 0.4

a S. cerevisiae 3001 was grown in 10% and 20% glucose in PYN medium and in 20% glucose plus 2x PYN medium at 30°C.b Determined by wet weight - dry weight differences of packed yeast cells (method 1) or by the distribution of tritiated water (method 2) as described in

Materials and Methods. The results are the average of at least three trials.

possibility may explain why these investigators did notobserve an intracellular accumulation of ethanol. In concur-rence with this possibility, Legmann and Margalith (14)observed an intracellular accumulation of ethanol in yeastcells after 5 h of fermentation. Therefore, the discrepancy inthe results in determining intracellular ethanol concentrationmay be related not only to the techniques used to measure

intracellular ethanol concentration but also to the assay

times selected. Furthermore, the yeast strain used in thepresent study is polyploid, and the cells are larger than thoseof strains used in other studies. Therefore, strain variabilitymay also explain the differences in the results.

In agreement with previous studies (1, 6, 10; D'Amore etal., in press), the results also indicate that the intracellularaqueous volume decreases as the age of the culture increases(Table 3). This relationship is a consequence of the cellsgetting smaller with culture age due to increased cell density,as well as of the build-up of glycerol (Table 2), which reducesthe aqueous volume. This decrease in intracellular aqueousvolume with fermentation time is an important factor toconsider when determining intracellular concentrations ofethanol. The intracellular accumulation of ethanol observedat 3 h of fermentation is complicated by possible errorsarising in the determination of ethanol concentration as theresult of the low number of cells and the low ethanolconcentrations. The method that we used, though, providesthe least opportunity for error. The assay is very fast; lessthan 1 min is required from the time that the sample iswithdrawn to perchloric acid extraction of both cell suspen-sion and supernatant. Furthermore, these results supportrecent observations which indicate that by the end of fer-mentation the intracellular and extracellular ethanol concen-trations are similar. In addition, full recovery of knownstandard volumes of ethanol were obtained by the use of thismethod (data not shown). The reason for the initial build-up

of ethanol in the cells is not immediately certain. In order forproduced ethanol to diffuse out of the cells, there must be aninitial gradient which favors the exit of ethanol. On thisbasis, it would be expected that in the initial stages offermentation there is a build-up of ethanol which subse-quently diffuses out of the cells as fermentation continues.An increase in intracellular ethanol concentration was

observed in response to an increase in osmotic pressure(Table 4). This finding is in agreement with our previousobservations in which sorbitol or total carbohydrate in wortwas used to increase the osmotic pressure (19; D'Amore etal., in press). Increases in osmotic pressure have beenshown to cause decreases in yeast cell growth and fermen-tation (2, 11, 12) as well as an increase in glycerol productionto counteract the effect of the higher osmotic pressure. Insupport of this observation, increasing the glucose concen-tration from 10% to 20% in our study caused a decrease inyeast cell growth and in the theoretical ethanol yield (Table1, Fig. 1) and an increase in glycerol production (Table 2). Infact, this strain was unable to fully ferment the 20% glucose.One could speculate, therefore, that the increase in intracel-lular ethanol concentration at the higher glucose concentra-tion may contribute to the decreased growth and fermenta-tion activities. We have recently demonstrated that there isan increase in the ratio of intracellular to extracellularethanol concentration in S. cerevisiae cells with increasingcarbohydrate concentration in wort (D'Amore et al., inpress). However, no effect on growth or fermentation rateswas observed with increasing intracellular ethanol concen-tration in this case. Therefore, other factors, such as nutri-tional requirements and accumulation of toxic by-products(such as octanoic and decanoic acids) at higher osmoticpressures, may have a contributing role (21). The datapresented in Fig. 1 and Table 1 demonstrate that nutrientsupplementation of the medium results in a twofold increase

TABLE 4. Distribution of intracellular and extracellular ethanol concentrations in S. cerevisiae during fermentation

% (wt/vol) Ethanolb after growth in:Fermentation 10% Glucose 20W Glucose 20% Glucose + 2x PYN

Int Ext Int Ext Int Ext

3 1.6 ± 0.2 0.19 ± 0.05 2.03 ± 0.4 0.09 ± 0.04 2.49 ± 0.6 0.12 + 0.046 0.92 ± 0.2 0.56 ± 0.08 1.15 ± 0.2 0.41 + 0.05 1.68 ± 0.6 0.38 + 0.0812 1.78 ± 0.1 1.66 ± 1.15 1.87 ± 0.2 1.78 ± 0.1 3.02 ± 0.4 2.69 ± 0.224 4.14 ± 0.1 4.30 ± 0.23 3.58 + 0.1 3.95 ± 0.1 8.1 ± 0.8 6.8 ± 0.648 ND ND ND ND 10.2 ± 0.8 10.8 ± 0.6

a S. cerevisiae 3001 was grown in 10% and 20% glucose in PYN medium or 20% glucose plus 2x PYN medium at 30°C.b After incubation for the indicated times, the intracellular (Int) and extracellular (Ext) ethanol concentrations were determined as described in Materials and

Methods. Intracellular ethanol concentration was determined by using an average intracellular aqueous volume from Table 3 for each time period. The resultsare the average of at least three trials. ND, Not determined.

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TABLE 5. Ratios of intracellular to extracellularethanol concentrationa

Int ethanol concn/ext ethanol concn after growth onb:Fermentation

time (h) 10% Glucose 20% Glucose 20% Glucose+ 2 x PYN

3 8.42 22.56 20.86 1.64 2.80 4.4

12 1.07 1.05 1.124 0.96 0.91 1.248 ND ND 0.94

a The ratios of intracellular (Int) to extracellular (Ext) ethanol concentrationwere determined from the data in Table 4.

b ND, Not determined.

in growth and in complete fermentation of 20% glucose.Under these conditions, no effect on the distribution ofintracellular or extracellular concentration was observed(Table 4). This observation provides strong evidence thatnutrient limitation, not necessarily the intracellular accumu-lation of ethanol or of by-products, is responsible for theobserved decreases in growth and fermentation activities ofyeast cells at higher osmotic pressures. In support of thiscontention, it has recently been shown that nutrient limita-tion is the major factor responsible for the decline in fermen-tative activity during the early stages of fermentation inyeast cells (8). Furthermore, magnesium has been identifiedas the active component responsible for relieving ethanoltoxicity during yeast fermentation (9). We are currently inthe process of identifying the active component(s) in thePYN medium.

In conclusion, the results presented in this communicationindicate that there is an intracellular accumulation of ethanolduring the early stages of yeast fermentation. As fermenta-tion proceeds, the intracellular and extracellular ethanolconcentrations become similar. In addition, increases inosmotic pressure are associated with increased intracellularaccumulation of ethanol. However, it was observed thatnutrient limitation, not increased intracellular accumulationof ethanol, is responsible for the decreases in growth andfertnentation activities of yeast cells at higher osmotic pres-sures.

ACKNOWLEDGMENTS

We thank I. Russell, C. A. Bilinski, Y. Haj-Ahmad, and R. Jonesfor helpful discussions in the preparation of this manuscript.

T.D. is the recipient of an Industrial Research Fellowship fromthe Natural Sciences and Engineering Research Council of Canadafor which gratitude is expressed.

LITERATURE CITED1. Beaven, M. J., C. Charpentier, and A. H. Rose. 1982. Production

and tolerance of ethanol in relation to phospholipid fatty-acylcomposition in Saccharomyces cerevisiae NCYC 431. J. Gen.Microbiol. 128:1447-1455.

2. Beuchat, L. R. 1983. Influence of water activity on growth,metabolic activities and survival of yeasts and molds. J. Food

Protect. 46:135-141.3. Brown, A. D. 1978. Compatible solutes and extreme water stress

in eukaryotic micro-organisms. Adv. Microb. Physiol. 17:181-242.

4. Casey, G. P., and W. M. Ingledew. 1985. Reevaluation ofalcohol synthesis and tolerance in brewer's yeast. Master Brew.Assoc. Am. (Tech. Quart.) 22:133-141.

5. Casey, G. P., C. A. Magnus, and W. M. Ingledew. 1984.High-gravity brewing: effects of nutrition on yeast composition,fermentative ability, and alcohol production. AppI. Environ.Microbiol. 48:639-646.

5a.D'Amore, T., and G. G. Stewart. 1987. Ethanol tolerance ofyeast. Enzyme Microb. Technol. 9:322-330.

6. Dasari, G., F. Roddick, M. A. Connor, and N. B. Pamment.1983. Factors affecting the estimation of intracellular ethanolconcentrations. Biotechnol. Lett. 5:715-720.

7. Dombek, K. M., and L. 0. Ingram. 1986. Determination of theintracellular concentration of ethanol in Saccharomyces cerevi-siae during fermentation. Appl. Environ. Microbiol. 51:197-200.

8. Dombek, K. M., and L. 0. Ingram. 1986. Nutrient limitation asa basis for the apparent toxicity of low levels of ethanol duringfermentation. J. Ind. Microbiol. 1:219-225.

9. Dombek, K. M., and L. 0. Ingram. 1986. Magnesium limitationand its role in apparent toxicity of ethanol during yeast fermen-tation. Appl. Environ. Microbiol. 52:975-981.

10. Guijarro, J. M., and R. Lagunas. 1984. Saccharomyces cerevi-siae does not accumulate ethanol against a concentration gradi-ent. J. Bacteriol. 160:874-878.

11. Hahn-Hagerdal, B., M. Larsson, and B. Mattison. 1982. Shift inmetabolism towards ethanol production in Saccharomyces cere-visiae using alterations of the physical-chemical micro-environ-ment. Biotechnol. Bioeng. Symp. 12:199-202.

12. Jones, R. P., N. Pamment, and P. F. Greenfield. 1981. Alcoholfermentation by yeasts: the effect of environmental and othervariables. Process Biochem. 16:42-49.

13. Kenyon, C. P., A. P. Bernard, and H. J. J. van Vuuren. 1986.Water relations of ethanol fermentation by Saccharomycescerevisiae: glycerol production under solute stress. EnzymeMicrob. Technol. 8:461-464.

14. Legmann, R., and P. Margalith. 1986. Ethanol formation byhybrid yeasts. Appl. Microbiol. Biotechnol. 23:198-202.

15. Loureiro, V., and H. G. Ferrera. 1983. On the intracellularaccumulation of ethanol in yeast. Biotechnol. Bioeng. 25:2263-2269.

16. Nagodawithana, T. W., and K. H. Steinkraus. 1976. Influence ofthe rate of ethanol production and accumulation on the viabilityof Saccharomyces cerevisiae in "rapid fermentation." Appl.Environ. Microbiol. 31:158-162.

17. Navarro, J. M., and G. Durand. 1978. Fermentation alcoolique:influence de la temperature sur l'accumulation d'alcool dans lescellules de levure. Ann. Microbiol. (Paris) 129B.215-224.

18. Novak, M., P. Strehaiano, and M. Mareno. 1981. Alcoholicfermentation: on the inhibitory effect of ethanol. Biotechnol.Bioeng. 23:201-211.

19. Panchal, C. J., and G. G. Stewart. 1980. The effect of osmoticpressure on the production and excretion of ethanol and glyc-erol by a brewing yeast strain. J. Inst. Brew. 86:207-210.

20. Strehaiano, P., and G. Goma. 1983. Effects of initial substrateconcentration on two wine yeasts: relation between glucosesensitivity and ethanol inhibition. Am. J. Enol. Vitic. 34:1-5.

21. Viegas, C. A., I. Sa-Correia, and J. M. Novais. 1985. Synergisticinhibition of the growth of Saccharomyces cerevisiae by ethanoland octanoic or decanoic acids. Biotechnol. Lett. 7:611-614.

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