Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibitedgrowth of Saccharomyces cerevisiae both in minimal medium andin media which contained supplements, such as yeast extract,corn steep powder, or a mixture of amino acids. However, theyeast grew when the pH of the medium containing acetic acidor lactic acid was adjusted to 4.5, even though the medium stillcontained the undissociated form of either acid at a concentrationof 102 mM. The results indicated that the buffer pair formedwhen the pH was adjusted to 4.5 stabilized the pH of the mediumby sequestering protons and by lessening the negative impactof the pH drop on yeast growth, and it also decreased the differencebetween the extracellular and intracellular pH values ( ${Delta}$ pH),the driving force for the intracellular accumulation of acid.Increasing the undissociated acetic acid concentration at pH4.5 to 163 mM by raising the concentration of the total acidto 267 mM did not increase inhibition. It is suggested thatthis may be the direct result of decreased acidification ofthe cytosol because of the intracellular buffering by the bufferpair formed from the acid already accumulated. At a concentrationof 102 mM undissociated acetic acid, the yeast grew to highercell density at pH 3.0 than at pH 4.5, suggesting that it isthe total concentration of acetic acid (104 mM at pH 3.0 and167 mM at pH 4.5) that determines the extent of growth inhibition,not the concentration of undissociated acid alone.

INTRODUCTION

Top

Introduction

The yeast Saccharomyces cerevisiae under aerobic conditionscan use short-chain organic acids, such as acetic acid and lacticacid, as carbon sources. The process involves induction of certainanabolic pathways, enzymes, and specific transport mechanisms(2, 4, 5, 6, 17). If glucose is available in the growth medium,these pathways and permeases are repressed. Glucose-repressedyeast cells are unable to take up the anions of these acids(6), but the undissociated acids diffuse freely into the cells.Once inside, these acids dissociate because of the higher intracellularpH and cause acidification of the cytoplasm. Generally, eucaryoticcells maintain their intracellular pH within a narrow rangedespite wide variations that may occur in the extracellularpH (7). Under fermentation conditions, the intracellular pHof S. cerevisiae is usually maintained between 5.5 and 5.75when the external pH is 3.0 (9) or between 5.9 and 6.75 whenthe external pH is varied between 6.0 and 10.0 (8). To maintainthe intracellular pH within a physiological range optimum formetabolism, the cells pump out protons at the expense of metabolicenergy (ATP). Increased diversion of energy (ATP) to pump outprotons results in decreased molar growth yield with respectto glucose (Y_glucose) (19, 24). It has also been reported thatthe Y_ATP (grams of biomass produced per mole of ATP generated)decreased from 14 to 4 when the concentration of acetic acidwas increased from 0 to 170 mM (19). As the gap between theextracellular pH and the intracellular pH widens, greater stressis placed on the cells and more energy is expended to maintainthe intracellular pH within the range that permits growth andsurvival of the yeast.

Growth of the yeast is faster and the biomass produced is greaterin media containing complex ingredients than is seen in minimalmedium (15). Improved growth in complex media has been thoughtto be entirely due to the availability of greater amounts ofnutrients, although it is now recognized that ingredients ofcomplex media also play nonnutritional roles in promoting thegrowth and survival of yeast (1, 12, 22). Therefore, it is reasonableto expect that complex ingredients in media will continue tostimulate yeast growth, albeit to a lesser degree, even in thepresence of organic acids, such as acetic acid or lactic acid.In other work, it was suggested that components such as yeastextract in yeast extract-peptone-dextrose broth offer some protectionagainst stress conditions (15). No systematic study, however,has been carried out to prove whether complex-medium ingredientscan protect yeast against the inhibitory effects of organicacids.

In the absence of good buffering, the pH of a growth mediumis lowered when organic acids are added. The inhibitory effectof low pH on yeast growth is compounded by the presence of organicacids in the medium. First, lowering the pH increases the concentrationof undissociated acid and thus enhances the inhibitory effectfor a given (total) amount of acid. Second, since accumulationof organic acids is a function of the difference between theextracellular and the intracellular pHs ( ${Delta}$ pH) (2, 14), greaterinhibition would be expected as the pH is decreased. It is notclear how much of the growth inhibition observed is due to lowpH and how much is due to the level of undissociated acid. Inthis study, we report that the inhibition of yeast growth byacetic acid and lactic acid is a function of the pH and thebuffering capacity of the medium and of the total amount ofthe organic acid added.

MATERIALS AND METHODS

Top

Materials and Methods

Yeast.
An industrial strain of S. cerevisiae (obtained from AlltechBiotechnology Center, Nicholasville, Ky.) was used throughoutthe study. This strain is widely used for fuel alcohol productionand is available as active dry yeast (superstart yeast). However,we obtained a slant culture of the yeast and ensured its purityby selection on yeast-peptone-dextrose agar. The yeast was keptat -70°C as a suspension in 20% glycerol.

Chemicals and growth media.
Common chemicals were of reagent grade and were purchased locally.Yeast extract (AYE 2200; Gilette Foods Inc., Union, N.J.), cornsteep powder (Marcor Development Corp., Hackensack, N.J.), andamino acids (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada)were used as nutrient supplements for yeast cultivation.

The minimal medium described by Wickerham (25) was used withminor modifications. The modifications included raising theconcentrations of glucose and ammonium sulfate to 556 and 10mmol per liter, respectively. The effect of nutrient supplementationon yeast growth was studied by adding yeast extract (10 g perliter), corn steep powder (10 g per liter), or a mixture of18 amino acids. The following amino acids were included in themixture: aspartic acid, glutamic acid, glycine, alanine, methionine,proline, arginine, cysteine, valine, threonine, leucine, isoleucine,tryptophan, phenylalanine, lysine, histidine, serine, and tyrosine.Concentrated solutions of these ingredients were sterilizedseparately by autoclaving them at 121°C for 15 min, andeach was added to sterile minimal medium to give a final concentrationof 1.8 mM. Where necessary, heat-sterilized solutions of aceticacid or lactic acid were added to sterile media to give thefinal concentrations described in the text.

Growth conditions.
The yeast was grown by either batch or continuous cultivation.In the batch method, 100-ml quantities of medium in sidearm250-ml Erlenmeyer flasks (Klett flasks) were inoculated with0.5 ml of broth culture. The inoculum was grown for 24 h at30°C in the minimal medium described above. The inoculatedflasks were incubated at 30°C with shaking (200 rpm). Growthwas followed turbidometrically using a Klett-Summerson colorimeterfitted with a red filter. In some cases, the pH of the sterilemedium before inoculation was aseptically adjusted to 4.5 with2 M KOH.

Continuous cultivation was carried out at 30°C and at adilution rate of 0.15 h^-1. For this, a 1-liter Omni-culturefermentor (The Virtis Company Inc., Gardiner, N.Y.) was convertedto operate in a continuous mode. The growth medium was pumpedinto the fermentor with a variable-speed peristaltic pump (modelPiper-31; Fred A. Dungey Inc., Agincourt, Ontario, Canada).The yeast was grown in 600 ml of the minimal medium (the workingvolume) with or without added acetic acid. The pH of the culturewas continuously monitored with a pH controller (model 169136;The Virtis Company Inc.) and maintained at predetermined valuesby automatic addition of 2 M KOH or 2 M HCl.

Analysis.
Glucose, ethanol, acetic acid, and lactic acid were measuredby high-performance liquid chromatography using a Waters (Milford,Mass.) chromatographic system. Supernatant portions of culturesobtained by centrifugation (10,300 x g; 15 min) were filteredthrough a Millipore membrane (0.22-µm pore size) and dilutedwith distilled water, and a sample (5 µl) was injectedinto an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules,Calif.) maintained at 40°C. Deionized water (Milli-Q) containingsulfuric acid (5 mM) was used as the eluant. The elution ratewas 0.7 ml per min, and boric acid was used as the internalstandard. The separated components were detected with a Watersdifferential refractometer (model 410) and quantified with aMillennium³² Chromatography Manager computer program suppliedby Waters Corp.

RESULTS

Top

Results

Yeast growth without pH adjustment.
The yeast grew to only a limited extent in the control minimalmedium (Fig. 1). On supplementation of the medium with yeastextract, corn steep powder, or the amino acid mixture, growthimproved considerably. The growth response to corn steep powderwas identical to that shown for yeast extract and thereforeis not included in Fig. 1. The results seemed to indicate thatthe poor growth in the minimal medium was the result of nutritionaldeficiency. Although the supplements used here are excellentsources of nutrients, the buffering provided by these ingredients(see "Buffering capacity of growth medium" below) played animportant role in stimulating yeast growth. The yeast failedto grow when acetic acid (167 mM) was incorporated into themedium (Fig. 1). None of the three nutrient supplements alleviatedthe growth inhibition caused by acetic acid. Lactic acid ata concentration of 548 mmol per liter inhibited yeast growthin a similar fashion (data not shown). As will be shown below,the amounts of acetic and lactic acid added to the medium weresuch that, at pH 4.5, they would provide identical concentrations(102 mM) of undissociated acid.

View larger version (22K):
[in this window]
[in a new window]
FIG. 1. Growth of S. cerevisiae in minimal medium with or without nutrient supplements and in the presence or absence of 167 mM acetic acid. ${circ}$ , control; •, 10 g of yeast extract per liter; ${blacksquare}$ , mixture of 18 amino acids, each at a final concentration of 1.8 mM; ${triangleup}$ , 10 g of yeast extract plus 167 mmol of acetic acid per liter; ${blacktriangleup}$ , 10 g of corn steep powder plus 167 mmol of acetic acid per liter; ${triangledown}$ , amino acid mixture plus 167 mmol of acetic acid per liter. (Note that the solid triangles are almost hidden by the open triangles.)

The addition of acetic acid and lactic acid lowered the pH ofthe minimal medium to 2.76 and 2.12, respectively (Table 1).If the medium contained nutrient supplements, such as yeastextract, corn steep powder, or a mixture of amino acids, thepH did not decrease to the same extent on addition of the acids.Growth inhibition was, however, still observed. The final pHvalues of media which contained either acetic acid or lacticacid were slightly elevated. This may be related to the factthat there was no yeast growth in these media (Fig. 1), andsome loss of acids may have occurred through evaporation duringthe 12 days of incubation or through lysis of the yeast cellsused for inoculation.

View this table:
[in this window]
[in a new window]
TABLE 1. Effects of various additives and initial pH values of media on final pH at the end of incubation with S. cerevisiae

Yeast growth with pH adjustment.
The initial pH of the medium and the presence or absence ofeither of the two acids seemed to determine whether the yeastgrew or failed to grow. As shown in Table 1 and Fig. 1, theyeast grew if the initial pH of the medium was 3.2 or higherand if the medium did not contain either acetic acid or lacticacid. To study the effect of the initial pH on yeast growth,the pH of each medium listed in Table 1 was adjusted asepticallyto 4.5, and then the medium was inoculated with a 24-h yeastculture prepared in minimal medium. Once again, the yeast grewto only a limited extent in the minimal medium (Fig. 2A). Thenutrient supplements stimulated yeast growth. The addition ofyeast extract reduced the lag period and increased the productionof biomass. The unexpected observation was that if the initialpH of the medium was adjusted to 4.5, the yeast grew to a greaterextent in the presence of 167 mM acetic acid or 548 mM lacticacid than in the absence of either acid. The lag phase in eachcase, however, was extended by about 6 h by these acids. Similarextended lag periods have been reported by Pampulha and Loureiro-Dias(19). Growth of the yeast in yeast extract-supplemented mediumwas marginally reduced by the presence of acetic acid or lacticacid in the medium. Similar results were obtained when cornsteep powder was used as the nutrient supplement (Fig. 2B).Yeast growth in the medium supplemented with a mixture of aminoacids was about the same as that in the medium that containedlactic acid (Fig. 2).

View larger version (20K):
[in this window]
[in a new window]
FIG. 2. Effect of adjusting pH to 4.5 on the growth of S. cerevisiae in media with or without 10 g of yeast extract per liter, acetic acid (167 mM), or lactic acid (548 mM) (A) and with or without a mixture of 18 amino acids (each at a final concentration of 1.8 mM), 10 g of corn steep powder per liter, acetic acid (167 mM), or lactic acid (548 mM) (B). ${circ}$ , control; •, yeast extract; ${square}$ , acetic acid; ${blacksquare}$ , lactic acid; ${triangleup}$ , yeast extract plus acetic acid; ${blacktriangleup}$ , yeast extract plus lactic acid; ${blacklozenge}$ , mixture of amino acids; ${lozenge}$ , mixture of amino acids plus acetic acid; ${triangledown}$ , mixture of amino acids plus lactic acid; ${triangleright}$ , corn steep powder; ${blacktriangleright}$ , corn steep powder plus acetic acid; ${boxtimes}$ , corn steep powder plus lactic acid.

High-performance liquid chromatography analysis confirmed thatthe yeast used neither acetic acid nor lactic acid as a carbonsource while glucose was still in the medium. However, within48 h, 95 to 98% of the glucose was consumed in all media whichcontained supplements (results not shown). Moreover, as longas glucose remained in the medium, the yeast did not producedetectable quantities of acetic acid. This is in agreement withthe observation that production of acetic acid by S. cerevisiaeoccurs only after the exhaustion of glucose from the medium(23), although other workers have reported "consecutive" productionand reassimilation of acetate in media containing low concentrationsof glucose (<0.35%) (20). In the present study, only 38%of the glucose was used in media which did not contain supplements.A greater proportion of glucose was converted to ethanol ifthe medium contained acetic acid and if the pH was adjustedto 4.5. This increased conversion was independent of the presenceof nutrient supplements in the medium. On average, 19.8% moreethanol was produced when acetic acid was present in the mediumthan when it was absent (Fig. 3). The average conversion efficiencyof glucose to ethanol in the absence of acetic acid was 72.6%,while in its presence the efficiency increased to 87%. It appearsthat in the presence of acetic acid, a greater proportion ofglucose was diverted for the production of energy (ATP), andthis resulted in increasing the ethanol yield. Lactic acid didnot have similar stimulatory effects on the conversion of glucoseto ethanol. This suggested that the mechanisms by which thesetwo acids inhibit yeast growth may be different, as reportedby Narendranath et al. (16).

View larger version (62K):
[in this window]
[in a new window]
FIG. 3. Effects of various additions to minimal medium on ethanol production by S. cerevisiae. The minimal medium contained 100 g of glucose per liter. AA, 18 amino acids (each at a final concentration of 1.8 mM); LA, lactic acid (548 mM); AcA, acetic acid (167 mM); CSP, corn steep powder (10 g per liter); YE, yeast extract (10 g per liter).

Buffering capacity of growth medium.
Stimulation of yeast growth by acetic and lactic acids at pH4.5 appeared most likely to be through the buffering actionof these acids. The final pH values of the fermented media thatcontained these acids did not drop to the same extent as inthe controls (Table 1). In such cases, the final pH remainedat 4.3 or higher. The final pHs of the cultures that receivednutrient supplements, but not the acids, also decreased to relativelylow values. Considering the amount of biomass produced in thepresence of these nutrients, the yeast would have pumped largequantities of protons into the medium. Nutrient supplementslikely sequestered some of these protons (buffering) beforethe pH became too low for the yeast to grow. This was verifiedby measuring the buffering capacities of the media. A 50-mlaliquot of each medium was titrated with 0.1 M NaOH or 0.1 MHCl, and the buffering capacity was calculated. Buffering capacityis defined here as the number of milliequivalents of NaOH orHCl required to shift the pH of 1 liter of the medium by 1 unit.As shown in Fig. 4, the minimal medium demonstrated very poorbuffering in the pH range that was optimal for yeast growth.The buffering capacity of the minimal medium at pH 2.8 was 2.14meq of NaOH, while that of the medium with amino acids was 3.73meq (1.74 times more buffering capacity than that shown by thecontrol). This increased buffering by amino acids was sufficientto stimulate growth significantly.

View larger version (26K):
[in this window]
[in a new window]
FIG. 4. Buffering capacities of minimal medium (control) and minimal medium with a mixture of 18 amino acids (each at a final concentration of 1.8 mM), with yeast extract (10 g per liter), or with corn steep powder (10 g per liter).

A reference point of pH 2.8 was chosen to compare the bufferingcapacities because below this value yeast growth begins to declinedrastically. Yeast extract and corn steep powder, each at aconcentration of 10 g per liter, increased the buffering capacityof the medium at pH 2.8 to 13.9 and 16.6 meq of NaOH, respectively(Fig. 4). The greatly improved growth with these nutrients inthe medium (Fig. 1) is most likely related to these increasedbuffering capacities.

To test whether the reduced growth of the yeast in minimal mediumwas caused by the rapid lowering of the pH (lack of buffering),the yeast was grown under pH control. The pH of the batch cultureduring growth was continuously monitored and was controlledat 4.6 by automatic addition of 1 M KOH. The yeast grew to agreater extent (2.3-fold) in the minimal medium with pH controlthan in medium without it (Fig. 5). This suggested that it wasthe low pH itself and not the exhaustion of nutrients or theaccumulation of toxic compounds that reduced the growth of theyeast. This was further confirmed by another approach. The yeastwas grown in minimal medium without pH control until the culturewas well into the stationary phase (96 h), and then the pH ofthe culture was adjusted to 4.6 aseptically with 1 M KOH. Theculture was further incubated. As shown in Fig. 5, the culturegrew to a much higher cell density during the second phase ofgrowth. This suggested that the original medium was not deficientin nutrients and that toxic metabolic products did not accumulateto force the culture to enter the stationary phase. The solereason for the reduced growth during the primary phase (no pHcontrol) appeared to be the rapid drop in pH. The lack of asimilar lowering of pH during the second phase (after the pHof the stationary-phase culture was adjusted to 4.6) was unexpected.As shown in Fig. 5, the pH during the second phase of growthdid not decrease as fast as it did during the first phase. Inaddition, greater amounts of biomass were produced during thesecond phase. The improved growth and reduced drop in pH seemagain to be related to the improved buffering provided by theculture. Since the original medium itself did not have appreciablebuffering, the resistance to change in pH during the secondphase must have been caused by some substance produced by theyeast during the first phase of growth and excreted into themedium. To test this, the yeast was grown in minimal mediumwithout pH control for 96 h until the culture was well intothe stationary phase. The culture was filtered through membranefilters (pore diameter, 0.45 µm). Fifty milliliters ofthe filtrate was titrated with 0.1 M NaOH, and the bufferingcapacity was calculated. The culture filtrate had greater bufferingcapacity at all pH values than did the original minimal medium(Fig. 6). The pH of the culture had decreased to 2.32 duringyeast growth, and at this pH both the culture filtrate and theminimal medium had relatively strong buffering. The culturefiltrate contained a substance, or substances, which had a higherbuffering capacity than did the minimal medium at pH valuesbetween 3.2 and 5.0. At present, the nature of this substanceor substances is not known. Once the pH of the yeast culture(grown without pH control) was adjusted to a value in this range,the yeast was able to grow to a higher cell density. The reduceddecrease in pH during the second phase of growth (Fig. 5) alsoseems to be related to the presence of the buffering substance(s)in the medium.

View larger version (28K):
[in this window]
[in a new window]
FIG. 5. Growth of S. cerevisiae in minimal medium during batch cultivation with pH controlled at 4.6 and without pH control until the culture reached stationary phase (96 h), at which time the pH of the culture was adjusted to 4.6 but not controlled. Changes in pH during growth of the yeast in minimal medium without pH control (pH) are also shown.

View larger version (21K):
[in this window]
[in a new window]
FIG. 6. Buffering capacities of the minimal medium and a culture filtrate recovered after S. cerevisiae was grown in minimal medium without pH control until the culture was in stationary phase.

pH controlled growth in continuous culture.
Although the mechanism of the inhibition of yeast growth byorganic acids is well studied, not much is known about how thatinhibition is affected by the pH of the medium itself. The yeastgrew quite well at pH 4.5 and 4.0, but the growth declined graduallyas the pH was lowered below 4.0 (Fig. 7A). The greatest reductionin growth occurred when the pH was lowered to 2.0 from 2.5.The presence of 167 mM acetic acid in the medium reduced thegrowth of the yeast at all pH values, and there was no growthwhen the pH was maintained below 3.0. With acetic acid in themedium, the cell density attained at pH 4.5 was only about halfof that observed in the medium which did not contain the acid.

View larger version (23K):
[in this window]
[in a new window]
FIG. 7. Changes in growth (A) and ethanol productivity (B) at different pH values during continuous cultivation of S. cerevisiae at a dilution rate of 0.15 per h in minimal medium with and without (control) 167 mM (1%) acetic acid.

The volumetric productivity of ethanol in the absence of aceticacid in the medium was unchanged between pH 2.5 and 3.5, butat higher pH values, the productivity increased (Fig. 7B). Inthe presence of acetic acid, the productivity at pH 4.0 and4.5 was about the same as that observed in media which did notcontain the acid.

Undissociated acetic acid concentration and inhibition of yeast growth.
Since only undissociated acid diffuses into glucose-repressedcells, growth inhibition is a function of the concentrationof the undissociated acid in the medium. The pH of the mediumdirectly influences the concentration of undissociated acid.To study the effects of pH and the concentration of undissociatedacid on yeast growth, the yeast was grown at 30°C by continuousculture methods at a dilution rate of 0.15 h^-1 and with pH control.The pH was held at 4.5 or at 3.0. As shown in Table 2, whenthe total concentration of the acid was 167 mM, the calculated(using a pK_a of 4.7 for acetic acid) concentration of undissociatedacid was 102 mM at pH 4.5 and 164 mM at pH 3.0. The decreasedgrowth at pH 3.0 was, therefore, the direct result of low pHand an elevated concentration of the undissociated acid (Table2). However, when the concentration of the undissociated acidwas lowered to the same level as at pH 4.5 (102 mM) by decreasingthe total acid concentration to 104 mM, the yeast was not inhibitedat pH 3.0 to the same extent as it was at pH 4.5. The expectedresult was that at equal concentrations of undissociated acidin the medium, the yeast growth would be inhibited to a greaterextent at pH 3.0 than at pH 4.5. The only difference betweenthe treatments (other than the respective pH values) was inthe total concentration of the acid used. This suggests thatthe inhibition of yeast growth by acetic acid is not only afunction of the concentration of the undissociated acid butalso of the total concentration of the acid.

View this table:
[in this window]
[in a new window]
TABLE 2. Growth of S. cerevisiae during continuous cultivation (D = 0.15 h^-1) in minimal medium at two pH values and at two different concentrations of added acetic acid

DISCUSSION

Top

Discussion

The results clearly show that the poor growth of S. cerevisiaein minimal medium was the result of a very rapid decrease ofpH to values below 2.5, and it did not appear to be due to nutritionalinsufficiency. This was indicated by the fact that the yeastgrew to much higher cell density in the same minimal mediumif the pH was maintained at 2.5 or higher. The observed improvementof yeast growth in media supplemented with yeast extract, cornsteep powder, or a mixture of amino acids also appeared to bedue to maintaining the pH (buffering) above certain criticalvalues below which growth was drastically reduced. It is possiblethat the greatly improved growth and fermentation performanceseen on incorporation of soy flour (26) or other "yeast foods"(10) is due more to buffering than to yeast nutrition. The abilityof the yeast to completely ferment wheat mashes in spite oftheir low free amino nitrogen content may also be related tobuffering—in this case provided by soluble and insolublewheat proteins (21). Apparently, the critical pH at which growthinhibition occurs is increased if carboxylic acids, such asacetic acid or lactic acid, are added to the medium (Fig. 7).

Growth of the yeast was completely inhibited if 167 mM aceticacid or 548 mM lactic acid was incorporated into the minimalmedium. Neither the nutrients contained in yeast extract orcorn steep powder nor the buffering these preparations providedwas sufficient to protect the yeast against inhibition by theweak acids. This lack of protection may be related to the relativelyhigh concentrations of the acids used. Inhibition of yeast growthby weak acids in media containing glucose is a function of theconcentration of the undissociated acid (2, 5), which in turnis a function of the concentration of total acid (dissociatedplus undissociated) and the pH of the medium. Acidificationof cytoplasm, the primary cause of growth inhibition by weakacids, increases (i) with increasing concentrations of undissociatedacid (18), (ii) through increased permeation of the acids athigher pH (3), and (iii) in proportion to the amount of ethanolin the culture (3).

The inhibition of yeast growth by acetic acid and lactic acidwas alleviated considerably if the pH of the medium after additionof the acids was adjusted to 4.5. When the pH was adjusted,the yeast grew to a greater extent than those observed in minimalmedia which did not contain either of these acids (controls).This was unexpected. The likely reason for this improved growthwas that in each case, the buffer pair formed by poising themedium at pH 4.5 buffered the medium and prevented the pH fromdropping to inhibitory levels (Table 1). Such buffering wasnot available in the controls because the acids were not addedto them. The yeast also grew in complex media containing theseacids if the pH was adjusted to 4.5, although the maximum biomassproduced was less than that observed in the medium that didnot contain the acids. The total concentrations of acetic andlactic acids were such that the concentration of both undissociatedacids at pH 4.5 was 102 mM. Even at this concentration, inhibitionof yeast growth was not as severe as expected, although it isreported that diffusion of undissociated acetic and lactic acidsinto yeast cells increases with increasing pH and with increasingconcentrations of undissociated acid (2, 3, 5). Inhibition ofgrowth at pH 4.5 did not increase when the concentration ofundissociated acetic acid was raised to 164 mM by increasingthe total concentration of the acid to 267 mM (1.6% [wt/vol]).It has been reported by Pampulha and Loureiro-Dias (18) thatthe decrease in internal pH with increasing concentration oftotal acid in the external medium was less at high than at lowpH values. Thus, the reduced inhibition of growth at increasedtotal acetic acid concentration at pH 4.5 (Table 2) may be relatedto a decreased acidification of cytoplasm. We suggest that thebuffer pair formed internally resisted the decrease in pH (acidificationof cytosol) and that this allowed the cells to survive and grow.

Raising the external pH has a number of effects on yeast metabolism.First, an external pH closer to the intracellular pH placesless stress on cells, and less energy is wasted in maintainingthe internal pH within a range optimal for growth. Second, ifthe media contain organic acids, raising the pH to a highervalue would reduce the concentration of undissociated acid fora given amount of total acid and thus decrease its inhibitoryeffect on yeast growth. It has been suggested, however, thatpassive diffusion of undissociated acetic acid or lactic acidacross the plasma membrane of S. cerevisiae is subject to opposinginfluences and that with increasing pH, the permeability ofundissociated acids increases while their concentrations decrease(2, 5). Third, although undissociated acids enter cells by passivediffusion, their accumulation inside cells is still a functionof the difference between external and internal pH values ( ${Delta}$ pH)(2, 14). One reason for the reduced inhibition by weak acidsat higher pH values may be the smaller ${Delta}$ pH. Fourth, the bufferpairs formed from organic acids when the pH is adjusted protectthe yeast against a rapid decrease in pH. This explains whythe acidification of cytoplasm with increasing concentrationsof acetic acid is much less in adapted cells than in nonadaptedcells (19). Adaptation may involve building up a sufficientconcentration of the acid to create a buffer pair internallyto resist pH change. It has been reported that anions formedthrough dissociation of carboxylic acids are retained withinthe cells (18) while protons are ejected through an energy (ATP)-coupledreaction involving plasma membrane ATPase. Potassium ions aretaken up by yeast cells in exchange for hydrogen ions to maintainionic stasis (11, 13). Depending on the ${Delta}$ pH and the externalconcentration of the acid, the molarity of the buffer pair insidethe cells at equilibrium can be very large (16), and this wouldenhance the internal buffering capacity. Reduced inhibitionby acetic acid may therefore be related to maintenance of intracellularpH by internal buffering at a level that is close to optimumfor growth. Less than expected growth inhibition at low totalacetic acid concentration (in spite of the low pH) may be attributedto the depletion of undissociated acid or to a reduction inits concentration through its continued diffusion into yeastcells and subsequent dissociation inside the cells. As protonsformed through dissociation of the acid are pumped out, moreof the undissociated acid from the medium diffuses into thecells. This process will continue until the undissociated acidin the medium is exhausted or until the cells can no longerpump out protons and thereby prevent acidification of the cytoplasm.As more and more undissociated acid from the medium diffusesinto the cells, more of the dissociated acid external to thecells is converted to undissociated acid to reestablish theequilibrium. These observations can be summarized as follows.First, the amount of undissociated acid that diffuses into cellsand its equilibrium concentration inside the cells are functionsof the external concentration of undissociated acid and thepH of the medium. Second, the equilibrium between the externaland internal concentrations of undissociated acid will be reestablishedif the undissociated acid inside the cells dissociates as aresult of higher intracellular pH. If the dissociation continues,more and more undissociated acid will diffuse into the cells,and the amount taken up will be a function of the external concentrationof the undissociated acid, which is, in turn, a function ofthe total acid concentration and the pH. The implication isthat the magnitude of growth inhibition is a function of thetotal concentration of the acid even though only undissociatedacid diffuses into the cells. Because of the dynamic equilibriumbetween undissociated and dissociated forms both outside andinside the cells and the passage of undissociated acids acrossthe membrane, continued removal of one component of the equilibriumreaction (protons in this case) would shift the reaction tofurther dissociation, and the removal of undissociated acidfrom the medium would continue.

ACKNOWLEDGMENTS

We thank the Natural Sciences and Engineering Research Councilof Canada, the Chippewa Valley Ethanol Company, the Corn PlusCo-Operative, and Alltech Inc. for research funds which in partsupported this work.

FOOTNOTES

* Corresponding author. Mailing address: Department of Applied Microbiology and Food Science, Agriculture 3E08, University of Saskatchewan, 51 Campus Dr., Saskatoon, Saskatchewan, Canada S7N 5A8. Phone: (306) 966-5028. Fax: (306) 966-8898. E-mail: ingledew{at}sask.usask.ca.

REFERENCES

Top