- Email: [email protected]
Energetics of the effect of acetic acid on growth of Saccharomyces cerevisiae
FEMS Microbiology Letters 184 (2000) 69^72
www.fems-microbiology.org
Energetics of the e¡ect of acetic acid on growth of Saccharomyces cerevisiae M. Elisa Pampulha b
a;b
, Maria C. Loureiro-Dias
a;b;
*
a Laboratory of Microbiology, Gulbenkian Institute of Science, Ap. 14, 2781 Oeiras Codex, Portugal Departamento de Botaªnica e Engenharia Biolo¨gica, Instituto Superior de Agronomia, 1399 Lisbon Codex, Portugal
Received 11 October 1999; accepted 14 January 2000
Abstract In batch cultures of a respiratory deficient mutant of Saccharomyces cerevisiae the maximum specific growth rate and the yield coefficient decreased, but the specific glucose consumption rate increased, in the presence of acetic acid. The ATP yield decreased from approximately 14 to 4 g biomass (mol ATP)31 when the concentration of acetic acid increased from 0 to 170 mM. Intracellular acidification was much weaker than previously reported for non-adapted cells. A linear relation was obtained between the ATP specific production rate and the uptake rate of acetic acid, suggesting that about 1 mol ATP is consumed per mol of acetic acid diffusing into the cells. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Acetic acid; ATP yield; Saccharomyces cerevisiae
1. Introduction Under acidic conditions, undissociated weak acids may di¡use through the plasma membrane reaching the more alkaline environment of the cellular cytoplasm where dissociation occurs. As a result H and the anion, less soluble in the lipids, accumulate in the cytoplasm, disturbing the metabolism of the cell. The behavior of microorganisms in the presence of weak acids has received special attention because many food preservatives belong to this category of compounds. We have previously shown that the inhibition of fermentation and the intracellular acidi¢cation induced by acetic acid in fermenting cells of Saccharomyces cerevisiae can be attributed to the undissociated form of the acid [1] and that the inhibition of fermentation by acetic acid can probably be explained by decreased activity of hexokinase, phosphofructokinase and enolase, while glucose transport is certainly not affected [2]. There is a general agreement in the literature that at sublethal concentrations weak acids inhibit growth of microorganisms by reducing the growth yield [3^6]. The decrease in growth has been attributed to a diversion of ATP
* Corresponding author. Tel. : +351 (1) 3638161; Fax: +351 (1) 3635031; E-mail: [email protected]
from anabolic processes to the pumping of H through the plasma ATPase [6]. To evaluate the extent of the diversion of ATP from anabolic processes, the ATP yield, yATP (g biomass (mol ATP)31 ), is a useful parameter which was shown to be reduced in S. cerevisiae in the presence of several weak acids in glucose-limited continuous culture under anaerobiosis [4]. Our objectives in this study were (1) to evaluate the e¡ect of acetic acid on growth and energetic parameters of S. cerevisiae in batch cultures in the exponential phase, under glucose saturation, (2) to ¢nd clues on the extent of adaptation, when the results are compared with those obtained in non-adapted cells, and (3) to establish a relationship between the ATP consumption and the amount of acetic acid di¡using into the cells. 2. Materials and methods 2.1. Yeast strain and culture conditions A respiratory de¢cient mutant of S. cerevisiae (IGC 3507-III) was used. Inocula were prepared from 24 h old slants on YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar). Cells were grown in a mineral medium [7] supplemented with vitamins, 2% (w/v) glucose and acetic acid at several concentrations. In each
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 2 2 - 7
FEMSLE 9264 10-2-00
70
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
experiment, 600 ml of medium in 1-l conical £asks with magnetic stirring, was used. The temperature was kept at 27³C in a thermostated water bath. The pH was monitored and kept at 4.5 by the use of a pH-stat (Radiometer), connected to a combined glass electrode (Sho«tt Gerate). The system controlled the addition of 1 M KOH to the culture vessel. Growth was followed by optical density (OD) at 640 nm and dry weight. At pH 4.5, 64% of acetic acid is in the undissociated form (pK = 4.76). 2.2. Yield factors Yield factors were estimated for glucose, ethanol and glycerol during the exponential growth. Samples were quickly centrifuged in an Eppendorf bench centrifuge and concentrations of glucose, ethanol and glycerol in the supernatants were evaluated by enzymatic tests (Boehringer, Mannheim). Yield factors were calculated from the biomass and products formed and glucose consumed in the same time intervals. For concentrations of acetic acid above 80 mM, inhibition of the auxiliary enzymes present in the kits was observed, for all pH values tested. This was taken into consideration by diluting in all cases the samples of supernatants, prior to the determination of concentrations. Since the work was performed with a respiratory de¢cient mutant, all ATP comes from fermentation and the yATP (g biomass (mol ATP)31 ) is easily calculated. The amount of ATP produced is equivalent to that of ethanol formed minus that of glycerol, since one ATP is consumed during the metabolism of glucose into glycerol. The yATP is derived by dividing the biomass formed during a time interval by the di¡erence of ethanol and glycerol formed in the same time interval: yATP biomass= ethanol3glycerol 2.3. Intracellular pH The classical method described by Rottenberg [8] was applied, utilizing [3-14 C]propionic acid as an intracellular probe.
Fig. 1. E¡ect of acetic acid on the growth parameters of S. cerevisiae growing in mineral medium at pH 4.5. Maximum speci¢c growth rate, Wmax (F); yield coe¤cient, y (E); glucose speci¢c consumption rate, Qgluc (a).
was on the elongation of the lag phase, which was very short in its absence and increased to about 5 days when the concentration was 170 mM. During the exponential phase, samples were taken to assess biomass and glucose concentration. The e¡ects on the maximum speci¢c growth rate, Wmax h31 , and on the yield coe¤cient, y (g biomass (mol glucose)31 ), are presented in Fig. 1. Acetic acid decreased both parameters, the e¡ect being more pronounced on y. We derived values for the speci¢c consumption rate of glucose, Qgluc = Wmax /y (mmol glucose (g biomass)31 h31 ), which are also shown in Fig. 1: acetic acid stimulated the consumption of glucose. Although growth was slower, the cells acquired the ability to utilize glucose at a higher rate. The increase in Qglu was about 50% when the concentration of acetic acid was 170 mM. 3.2. E¡ect of acetic acid on energy metabolism In the same samples as above, ethanol and glycerol were also measured. For each concentration of acetic acid the values of speci¢c production rate for ethanol, Qet , and glycerol, Qgly , were calculated. The results are shown in
3. Results 3.1. E¡ect of acetic acid on growth parameters To perform this study we chose a respiratory de¢cient mutant of S. cerevisiae. This strain does not metabolize acetic acid and moreover the metabolism of glucose is fermentative, allowing the amount of ATP produced to be calculated from the production of ethanol and glycerol. Experiments were performed without previous adaptation of the inoculum to acetic acid. In all cases the pH was kept constant at 4.5. The ¢rst obvious e¡ect of the acid
Fig. 2. E¡ect of acetic acid on the speci¢c production rates, Q, of ethanol (F) and of glycerol (a) in S. cerevisiae at pH 4.5.
FEMSLE 9264 10-2-00
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
71
while in the second case the cells contacted with acetic acid just before the measurement of intracellular pH. In this case 120 mM was the highest concentration of acetic acid at which fermentation activity could be detected. These results indicate that adaptation to the acid involves a dramatic increase of intracellular pH, occurring during the lag phase. 4. Discussion
Fig. 3. E¡ect of acetic acid on the ATP yield in S. cerevisiae at pH 4.5.
Fig. 2. Increasing the concentration of acetic acid resulted in an increase of Qet and a decrease of Qgly . With the values of production of biomass, ethanol and glycerol the e¡ect of acetic acid on the ATP yield was evaluated. The results are shown in Fig. 3. The presence of 170 mM acetic acid reduced the yATP to 30% of the value obtained in its absence. 3.3. E¡ect of acetic acid on intracellular acidi¢cation The intracellular pH of cells in the mid-exponential phase was measured with [14 C]propionic acid. The value did not change signi¢cantly during the exponential phase for each concentration of acetic acid. The results are shown in Fig. 4 as closed symbols. In Fig. 4 as open symbols, we present the values previously obtained for the intracellular pH of cells of the same strain grown in the absence of acetic acid and incubated in 50 mM phosphate bu¡er pH 4.5 with 2% (w/v) glucose [1]. In the ¢rst case the cells were grown in the presence of acetic acid (after a rather long lag phase for higher concentrations),
Fig. 4. E¡ect of acetic acid on the intracellular pH of S. cerevisiae during exponential growth in the presence of the acid (F) at pH 4.5. Data representing the e¡ect of acetic acid on the intracellular pH of nonadapted fermenting cells at the same pH are also represented (a) (from [1]).
As expected, the presence of acetic acid a¡ected growth of the respiratory de¢cient mutant of S. cerevisiae. Growth was a¡ected, ¢rst, by an increase of the duration of the lag phase. During this period an increase in intracellular pH does certainly occur since a signi¢cant di¡erence in intracellular pH was observed between cells non-adapted and adapted to acetic acid, as Fig. 4 documents. This result agrees with previous works that report that during the lag phase activation of the ATPase and active glycolytic £ux take place before growth starts [9,10]. The hypothesis that acetic acid might have been metabolized was discarded in our work by the observation (by high performance liquid chromatography) that the concentration of the acid in the fresh medium did not vary during the assay (not shown). It was previously reported that weak acids increase the glycolytic £ux in yeasts in continuous culture under glucose limitation [5,6]. It is worth noting that in this work, in batch cultures, this result was also observed, although the yeast was growing at saturating concentrations of glucose. This indicates an improvement of glycolytic activity, even when the cytoplasmic pH was rather acidic. This observation is in apparent con£ict with previous reports that show that weak acids inhibit fermentation [1], and, in particular, acetic acid inhibits hexokinase, phosphofructokinase and enolase in yeast cell free extracts [2]. We checked in the present work that these activities (measured as in [2] in cell extracts in the absence of the acid) were not enhanced by the presence of acetic acid in the growth medium (results not shown). Conceivably glycolysis is activated by the low-
Fig. 5. Relation between the uptake rate of acetic acid during growth of S. cerevisiae and the ATP speci¢c production rate.
FEMSLE 9264 10-2-00
72
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
er level of ATP, as was previously observed in resting cells in the presence of acetic acid [2]. Comparing the values of 13 and 4 g biomass (mol ATP)31 obtained for yATP , respectively, in the absence and in the presence of 170 mM acetic acid, it is valid to state that most ATP is diverted for energy purposes in the presence of high concentrations of the acid. We con¢rmed that metabolic energy was diverted from anabolism when acetic acid was present in batch culture. However, a question remains: how much ATP is diverted in response to acetic acid entering the cells? To answer this question it is necessary to calculate how much acid is entering the cells. We made the following assumption: the acid permeates into the cell in the undissociated form by simple di¡usion but if there were no growth a steady state would be reached in which the net £ux of acid into the cells would be zero. Since the biomass is growing, there is a constant driving force inwards created by an e¡ect of intracellular dilution due to an increase of biomass volume. Thus, the amount of acid entering the whole biomass is the same as if the biomass were formed of two fractions: the existing biomass, x, in which the acid is in equilibrium, and the biomass formed per unit of time, Wx, in which the intracellular concentration of the acid is zero. Thus, the in£ux rate of acetic acid, X (mmol (g biomass)31 h31 ), should be:
X K D Uundissociated acetic acidU W where KD is the di¡usion coe¤cient of acetic acid. To calculate X we used KD = 0.117 Wl s31 (mg dry weight)31 derived for glucose grown S. cerevisiae at pH 4.5, from [11]. We calculated the speci¢c production rate of ATP, QATP = W/yATP (mmol (g biomass)31 h31 ). Fig. 5 relates QATP with the in£ux rate of acetic acid. A linear relationship was obtained with a slope of 1.3. This suggests that in S. cerevisiae about 1 mol of ATP is spent per mol of acetic acid entering the cells. This ATP can be used to pump out 1 mol of H , keeping the intracellular pH at a constant value.
References [1] Pampulha, M.E. and Loureiro-Dias, M.C. (1989) Combined e¡ect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast. Appl. Microbiol. Biotechnol. 31, 547^550. [2] Pampulha, M.E. and Loureiro-Dias, M.C. (1990) Activity of glycolytic enzymes of Saccharomyces cerevisiae in the presence of acetic acid. Appl. Microbiol. Biotechnol. 34, 375^380. [3] Pons, M.N., Rajab, A. and Engasser, J.M. (1986) In£uence of acetate on growth kinetics and production control of Saccharomyces cerevisiae on glucose and ethanol. Appl. Microbiol. Biotechnol. 24, 193^ 198. [4] Verduyn, C., Postma, E., Sche¡ers, W.A. and van Dijken, J.P. (1990) Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136, 405^412. [5] Verduyn, C., Postma, E., Sche¡ers, W.A. and van Dijken, J.P. (1992) E¡ect of benzoic acid on metabolic £uxes in yeasts: a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501^517. [6] Warth, A.D. (1988) E¡ect of benzoic acid on growth yield of yeasts di¡ering in their resistance to preservatives. Appl. Environ. Microbiol. 54, 2091^2095. [7] van Uden, N. (1967) Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. Mikrobiol. 58, 155^168. [8] Rottenberg, H. (1979) Measurement of membrane potential and vpH in cells, organelles and vesicles. Methods Enzymol. 55, 547^569. [9] Holyoak, C.D., Stratford, M., McMullin, Z., Cole, M.B., Crimmins, K., Brown, A.J.P. and Coote, P.J. (1996) Activity of the plasma membrane H -ATPase and optimal glycolytic £ux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158^3164. [10] Viegas, C.A., Almeida, P.F., Cavaco, M. and Sa¨-Correia, I. (1998) The H -ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-supplemented medium accompanying the decrease in intracellular pH and cell viability. Appl. Environ. Microbiol. 64, 779^783. [11] Casal, M., Cardoso, H. and Lea¬o, C. (1996) Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae. Microbiology 142, 1385^1390.
FEMSLE 9264 10-2-00
www.fems-microbiology.org
Energetics of the e¡ect of acetic acid on growth of Saccharomyces cerevisiae M. Elisa Pampulha b
a;b
, Maria C. Loureiro-Dias
a;b;
*
a Laboratory of Microbiology, Gulbenkian Institute of Science, Ap. 14, 2781 Oeiras Codex, Portugal Departamento de Botaªnica e Engenharia Biolo¨gica, Instituto Superior de Agronomia, 1399 Lisbon Codex, Portugal
Received 11 October 1999; accepted 14 January 2000
Abstract In batch cultures of a respiratory deficient mutant of Saccharomyces cerevisiae the maximum specific growth rate and the yield coefficient decreased, but the specific glucose consumption rate increased, in the presence of acetic acid. The ATP yield decreased from approximately 14 to 4 g biomass (mol ATP)31 when the concentration of acetic acid increased from 0 to 170 mM. Intracellular acidification was much weaker than previously reported for non-adapted cells. A linear relation was obtained between the ATP specific production rate and the uptake rate of acetic acid, suggesting that about 1 mol ATP is consumed per mol of acetic acid diffusing into the cells. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Acetic acid; ATP yield; Saccharomyces cerevisiae
1. Introduction Under acidic conditions, undissociated weak acids may di¡use through the plasma membrane reaching the more alkaline environment of the cellular cytoplasm where dissociation occurs. As a result H and the anion, less soluble in the lipids, accumulate in the cytoplasm, disturbing the metabolism of the cell. The behavior of microorganisms in the presence of weak acids has received special attention because many food preservatives belong to this category of compounds. We have previously shown that the inhibition of fermentation and the intracellular acidi¢cation induced by acetic acid in fermenting cells of Saccharomyces cerevisiae can be attributed to the undissociated form of the acid [1] and that the inhibition of fermentation by acetic acid can probably be explained by decreased activity of hexokinase, phosphofructokinase and enolase, while glucose transport is certainly not affected [2]. There is a general agreement in the literature that at sublethal concentrations weak acids inhibit growth of microorganisms by reducing the growth yield [3^6]. The decrease in growth has been attributed to a diversion of ATP
* Corresponding author. Tel. : +351 (1) 3638161; Fax: +351 (1) 3635031; E-mail: [email protected]
from anabolic processes to the pumping of H through the plasma ATPase [6]. To evaluate the extent of the diversion of ATP from anabolic processes, the ATP yield, yATP (g biomass (mol ATP)31 ), is a useful parameter which was shown to be reduced in S. cerevisiae in the presence of several weak acids in glucose-limited continuous culture under anaerobiosis [4]. Our objectives in this study were (1) to evaluate the e¡ect of acetic acid on growth and energetic parameters of S. cerevisiae in batch cultures in the exponential phase, under glucose saturation, (2) to ¢nd clues on the extent of adaptation, when the results are compared with those obtained in non-adapted cells, and (3) to establish a relationship between the ATP consumption and the amount of acetic acid di¡using into the cells. 2. Materials and methods 2.1. Yeast strain and culture conditions A respiratory de¢cient mutant of S. cerevisiae (IGC 3507-III) was used. Inocula were prepared from 24 h old slants on YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar). Cells were grown in a mineral medium [7] supplemented with vitamins, 2% (w/v) glucose and acetic acid at several concentrations. In each
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 2 2 - 7
FEMSLE 9264 10-2-00
70
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
experiment, 600 ml of medium in 1-l conical £asks with magnetic stirring, was used. The temperature was kept at 27³C in a thermostated water bath. The pH was monitored and kept at 4.5 by the use of a pH-stat (Radiometer), connected to a combined glass electrode (Sho«tt Gerate). The system controlled the addition of 1 M KOH to the culture vessel. Growth was followed by optical density (OD) at 640 nm and dry weight. At pH 4.5, 64% of acetic acid is in the undissociated form (pK = 4.76). 2.2. Yield factors Yield factors were estimated for glucose, ethanol and glycerol during the exponential growth. Samples were quickly centrifuged in an Eppendorf bench centrifuge and concentrations of glucose, ethanol and glycerol in the supernatants were evaluated by enzymatic tests (Boehringer, Mannheim). Yield factors were calculated from the biomass and products formed and glucose consumed in the same time intervals. For concentrations of acetic acid above 80 mM, inhibition of the auxiliary enzymes present in the kits was observed, for all pH values tested. This was taken into consideration by diluting in all cases the samples of supernatants, prior to the determination of concentrations. Since the work was performed with a respiratory de¢cient mutant, all ATP comes from fermentation and the yATP (g biomass (mol ATP)31 ) is easily calculated. The amount of ATP produced is equivalent to that of ethanol formed minus that of glycerol, since one ATP is consumed during the metabolism of glucose into glycerol. The yATP is derived by dividing the biomass formed during a time interval by the di¡erence of ethanol and glycerol formed in the same time interval: yATP biomass= ethanol3glycerol 2.3. Intracellular pH The classical method described by Rottenberg [8] was applied, utilizing [3-14 C]propionic acid as an intracellular probe.
Fig. 1. E¡ect of acetic acid on the growth parameters of S. cerevisiae growing in mineral medium at pH 4.5. Maximum speci¢c growth rate, Wmax (F); yield coe¤cient, y (E); glucose speci¢c consumption rate, Qgluc (a).
was on the elongation of the lag phase, which was very short in its absence and increased to about 5 days when the concentration was 170 mM. During the exponential phase, samples were taken to assess biomass and glucose concentration. The e¡ects on the maximum speci¢c growth rate, Wmax h31 , and on the yield coe¤cient, y (g biomass (mol glucose)31 ), are presented in Fig. 1. Acetic acid decreased both parameters, the e¡ect being more pronounced on y. We derived values for the speci¢c consumption rate of glucose, Qgluc = Wmax /y (mmol glucose (g biomass)31 h31 ), which are also shown in Fig. 1: acetic acid stimulated the consumption of glucose. Although growth was slower, the cells acquired the ability to utilize glucose at a higher rate. The increase in Qglu was about 50% when the concentration of acetic acid was 170 mM. 3.2. E¡ect of acetic acid on energy metabolism In the same samples as above, ethanol and glycerol were also measured. For each concentration of acetic acid the values of speci¢c production rate for ethanol, Qet , and glycerol, Qgly , were calculated. The results are shown in
3. Results 3.1. E¡ect of acetic acid on growth parameters To perform this study we chose a respiratory de¢cient mutant of S. cerevisiae. This strain does not metabolize acetic acid and moreover the metabolism of glucose is fermentative, allowing the amount of ATP produced to be calculated from the production of ethanol and glycerol. Experiments were performed without previous adaptation of the inoculum to acetic acid. In all cases the pH was kept constant at 4.5. The ¢rst obvious e¡ect of the acid
Fig. 2. E¡ect of acetic acid on the speci¢c production rates, Q, of ethanol (F) and of glycerol (a) in S. cerevisiae at pH 4.5.
FEMSLE 9264 10-2-00
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
71
while in the second case the cells contacted with acetic acid just before the measurement of intracellular pH. In this case 120 mM was the highest concentration of acetic acid at which fermentation activity could be detected. These results indicate that adaptation to the acid involves a dramatic increase of intracellular pH, occurring during the lag phase. 4. Discussion
Fig. 3. E¡ect of acetic acid on the ATP yield in S. cerevisiae at pH 4.5.
Fig. 2. Increasing the concentration of acetic acid resulted in an increase of Qet and a decrease of Qgly . With the values of production of biomass, ethanol and glycerol the e¡ect of acetic acid on the ATP yield was evaluated. The results are shown in Fig. 3. The presence of 170 mM acetic acid reduced the yATP to 30% of the value obtained in its absence. 3.3. E¡ect of acetic acid on intracellular acidi¢cation The intracellular pH of cells in the mid-exponential phase was measured with [14 C]propionic acid. The value did not change signi¢cantly during the exponential phase for each concentration of acetic acid. The results are shown in Fig. 4 as closed symbols. In Fig. 4 as open symbols, we present the values previously obtained for the intracellular pH of cells of the same strain grown in the absence of acetic acid and incubated in 50 mM phosphate bu¡er pH 4.5 with 2% (w/v) glucose [1]. In the ¢rst case the cells were grown in the presence of acetic acid (after a rather long lag phase for higher concentrations),
Fig. 4. E¡ect of acetic acid on the intracellular pH of S. cerevisiae during exponential growth in the presence of the acid (F) at pH 4.5. Data representing the e¡ect of acetic acid on the intracellular pH of nonadapted fermenting cells at the same pH are also represented (a) (from [1]).
As expected, the presence of acetic acid a¡ected growth of the respiratory de¢cient mutant of S. cerevisiae. Growth was a¡ected, ¢rst, by an increase of the duration of the lag phase. During this period an increase in intracellular pH does certainly occur since a signi¢cant di¡erence in intracellular pH was observed between cells non-adapted and adapted to acetic acid, as Fig. 4 documents. This result agrees with previous works that report that during the lag phase activation of the ATPase and active glycolytic £ux take place before growth starts [9,10]. The hypothesis that acetic acid might have been metabolized was discarded in our work by the observation (by high performance liquid chromatography) that the concentration of the acid in the fresh medium did not vary during the assay (not shown). It was previously reported that weak acids increase the glycolytic £ux in yeasts in continuous culture under glucose limitation [5,6]. It is worth noting that in this work, in batch cultures, this result was also observed, although the yeast was growing at saturating concentrations of glucose. This indicates an improvement of glycolytic activity, even when the cytoplasmic pH was rather acidic. This observation is in apparent con£ict with previous reports that show that weak acids inhibit fermentation [1], and, in particular, acetic acid inhibits hexokinase, phosphofructokinase and enolase in yeast cell free extracts [2]. We checked in the present work that these activities (measured as in [2] in cell extracts in the absence of the acid) were not enhanced by the presence of acetic acid in the growth medium (results not shown). Conceivably glycolysis is activated by the low-
Fig. 5. Relation between the uptake rate of acetic acid during growth of S. cerevisiae and the ATP speci¢c production rate.
FEMSLE 9264 10-2-00
72
M.E. Pampulha, M.C. Loureiro-Dias / FEMS Microbiology Letters 184 (2000) 69^72
er level of ATP, as was previously observed in resting cells in the presence of acetic acid [2]. Comparing the values of 13 and 4 g biomass (mol ATP)31 obtained for yATP , respectively, in the absence and in the presence of 170 mM acetic acid, it is valid to state that most ATP is diverted for energy purposes in the presence of high concentrations of the acid. We con¢rmed that metabolic energy was diverted from anabolism when acetic acid was present in batch culture. However, a question remains: how much ATP is diverted in response to acetic acid entering the cells? To answer this question it is necessary to calculate how much acid is entering the cells. We made the following assumption: the acid permeates into the cell in the undissociated form by simple di¡usion but if there were no growth a steady state would be reached in which the net £ux of acid into the cells would be zero. Since the biomass is growing, there is a constant driving force inwards created by an e¡ect of intracellular dilution due to an increase of biomass volume. Thus, the amount of acid entering the whole biomass is the same as if the biomass were formed of two fractions: the existing biomass, x, in which the acid is in equilibrium, and the biomass formed per unit of time, Wx, in which the intracellular concentration of the acid is zero. Thus, the in£ux rate of acetic acid, X (mmol (g biomass)31 h31 ), should be:
X K D Uundissociated acetic acidU W where KD is the di¡usion coe¤cient of acetic acid. To calculate X we used KD = 0.117 Wl s31 (mg dry weight)31 derived for glucose grown S. cerevisiae at pH 4.5, from [11]. We calculated the speci¢c production rate of ATP, QATP = W/yATP (mmol (g biomass)31 h31 ). Fig. 5 relates QATP with the in£ux rate of acetic acid. A linear relationship was obtained with a slope of 1.3. This suggests that in S. cerevisiae about 1 mol of ATP is spent per mol of acetic acid entering the cells. This ATP can be used to pump out 1 mol of H , keeping the intracellular pH at a constant value.
References [1] Pampulha, M.E. and Loureiro-Dias, M.C. (1989) Combined e¡ect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast. Appl. Microbiol. Biotechnol. 31, 547^550. [2] Pampulha, M.E. and Loureiro-Dias, M.C. (1990) Activity of glycolytic enzymes of Saccharomyces cerevisiae in the presence of acetic acid. Appl. Microbiol. Biotechnol. 34, 375^380. [3] Pons, M.N., Rajab, A. and Engasser, J.M. (1986) In£uence of acetate on growth kinetics and production control of Saccharomyces cerevisiae on glucose and ethanol. Appl. Microbiol. Biotechnol. 24, 193^ 198. [4] Verduyn, C., Postma, E., Sche¡ers, W.A. and van Dijken, J.P. (1990) Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136, 405^412. [5] Verduyn, C., Postma, E., Sche¡ers, W.A. and van Dijken, J.P. (1992) E¡ect of benzoic acid on metabolic £uxes in yeasts: a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501^517. [6] Warth, A.D. (1988) E¡ect of benzoic acid on growth yield of yeasts di¡ering in their resistance to preservatives. Appl. Environ. Microbiol. 54, 2091^2095. [7] van Uden, N. (1967) Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. Mikrobiol. 58, 155^168. [8] Rottenberg, H. (1979) Measurement of membrane potential and vpH in cells, organelles and vesicles. Methods Enzymol. 55, 547^569. [9] Holyoak, C.D., Stratford, M., McMullin, Z., Cole, M.B., Crimmins, K., Brown, A.J.P. and Coote, P.J. (1996) Activity of the plasma membrane H -ATPase and optimal glycolytic £ux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158^3164. [10] Viegas, C.A., Almeida, P.F., Cavaco, M. and Sa¨-Correia, I. (1998) The H -ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-supplemented medium accompanying the decrease in intracellular pH and cell viability. Appl. Environ. Microbiol. 64, 779^783. [11] Casal, M., Cardoso, H. and Lea¬o, C. (1996) Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae. Microbiology 142, 1385^1390.
FEMSLE 9264 10-2-00