Utilization and Transport of Acetic Acid in Dekkera anomala and Their Implications on the Survival of the Yeast in Acidic Environments

Transcription

1 96 Journal of Food Protection, Vol. 63, No. 1, 2000, Pages Copyright, International Association for Food Protection Utilization and Transport of Acetic Acid in Dekkera anomala and Their Implications on the Survival of the Yeast in Acidic Environments HERNÂNI GERÓS, FERNANDA CÁSSIO, AND CECÍLIA LEÃO* Centro de Ciências do Ambiente, Departamento de Biologia, Universidade do Minho, Braga, Portugal MS 99-92: Received 9 April 1999/Accepted 6 August 1999 ABSTRACT The yeast Dekkera anomala IGC 5153 exhibited a restricted ability to use weak acids as the only carbon and energy sources. Of the monocarboxylic, dicarboxylic, and tricarboxylic acids tested, only acetic acid was used in such a way. The cells were able to grow at acetic acid concentrations of 0.1 to 3% (vol/vol) over a ph range of 3.5 to 5.5, and the specific growth rates decreased exponentially with the increase of the undissociated acetic acid concentration in the culture medium. Transport assays carried out in cells that exhibited higher specific growth rates showed the presence of an acetate-proton symport associated with a simple diffusion component of the undissociated acetic acid, the weight of the latter increasing with the undissociated acid concentration in the culture media. The acetate carrier was shared by propionic, formic, and sorbic acids and was inducible and repressed by glucose and concentrations of undissociated acetic acid in the culture medium above 0.3% (vol/vol). In undissociated acetic acid repression conditions, the lowest values for the yeast specific growth rates were obtained, and the simple diffusion of the undissociated acid was the only mechanism involved in the acetic acid uptake by the cells. The results will be discussed in terms of the high tolerance of D. anomala to the acidic stress conditions present in wine. Yeasts that belong to the genus Dekkera/Brettanomyces are well known for their capacity to spoil alcoholic beverages, producing obnoxious flavors and odors (10, 11). Particularly in the wine industry, these yeasts are responsible for important economic losses because of their capacity to survive in wine and induce such detrimental effects on its organoleptical qualities (9). They are rarely found during the alcoholic fermentation of grape, probably due to their low growth rate, which makes them unable to compete with Saccharomyces cerevisiae. However, they are often found in bottled wines because of their higher tolerance to the stress environment of the wine. The identification and understanding of the physiological and biochemical properties, which make these yeasts so peculiar, are of most importance under either a fundamental or applied point of view. However, the scientific basis for this behavior is poorly understood, and the few data available focus mainly on the inhibition of alcoholic fermentation under anaerobic conditions (1, 16) and on the role of oxygen on acetic acid production in wine making (6). The aim of the present work was to contribute to the elucidation of the mechanisms underlying the survival of Dekkera anomala in acidic environments, such as the glucose and acetic acid present in vinification. In particular, the transport of acetic acid across the plasma membrane and its role in the use of the acid will be studied. MATERIALS AND METHODS Microorganism and growth conditions. D. anomala IGC 5153 was maintained on a medium containing glucose (2%, wt/ * Author for correspondence. Tel: ; Fax: ; cleao@bio.uminho.pt. vol), peptone (1%, wt/vol), yeast extract (0.5%, wt/vol), and agar (2%, wt/vol). The yeast was cultivated in a mechanically agitated fermenter (Minifermenter-System, Biolab, B. Braun Melsungen AG) with a working volume of 1 liter under ph and temperature (26 C) control. It used a mineral medium with vitamins (15) supplemented with the carbon sources at the concentrations and ph values indicated in the results. Growth was monitored by measuring the optical density at 640 nm (OD 640 ) (Spectronic 21, Bausch & Lomb). The specific growth rate ( max ) of the cultures was estimated from the plot of the logarithm of OD values versus time, since the slope of the linear part of the curve was a measure of the max. Transport assays. Cells from the midexponential growth phase (OD 640, 0.4 to 0.6) were harvested, centrifuged, washed twice with ice-cold distilled water, and suspended in distilled water at a final concentration of about 40 mg of dry weight per ml. For estimating the uptake rates of labeled acids, 10 l of yeast suspension was mixed with 30 l of 0.1 M potassium phosphate buffer in 10-ml conical tubes at the desired ph value. After 2 min of incubation at 26 C in a water bath, the reaction was started by the addition of 10 l of an aqueous solution of [U- 14 C]acetic acid or [2-14 C]propionic acid with about 5,000 dpm nmol 1, adjusted to the ph of the assay and at the desired concentration. The sampling times for the labeled acids were 0, 5, and 10 s, periods during which the uptake was linear. The reaction was stopped by dilution with 4 ml of ice-cold water, and the mixtures were immediately filtered through GF/C filters (Whatman, Inc, Clifton, N.J.). The filters were washed with 8 ml of ice-cold water, and the radioactivity was counted in the scintillation fluid OptiPhase HiSafe II (LKB Scintillation Products). The radioactivity was measured with a Packard Tri-Carb 2200 CA liquid scintillation counter (Packard Instruments Co., Inc., Rockville, Md.). Results were corrected for nonspecific 14 C adsorption

2 J. Food Prot., Vol. 63, No. 1 TRANSPORT OF ACETIC ACID IN D. ANOMALA 97 by diluting the cells with 4 ml of ice-cold distilled water before the addition of labeled acid. Inhibition by nonlabeled acids was assayed by simultaneous addition of the labeled and nonlabeled substrate. To estimate the initial rates of proton uptake on addition of carboxylic acids, a standard ph meter (PHM 82 Radiometer A/S, Copenhagen, Denmark) connected to a recorder (Kipp and Zonen) was used. The ph electrode was immersed in a water-jacketed chamber with magnetic stirring. A total of 4.5 ml of 10 mm potassium phosphate and 0.5 ml of yeast suspension were added to the chamber. The ph was adjusted to the desired value, and a baseline was obtained. The desired amount of carboxylic acid (adjusted to the experimental ph value) was added, and the subsequent alkalinization curve was monitored. The slope of the initial part of the ph trace was used to calculate the initial rates of proton uptake. Calibration was performed with HCl. Accumulation studies were performed using a method already described (5). Acetic acid grown cells (20 l, 40 mg of dry weight per ml) were added to 60 l of 0.1 M potassium phosphate buffer at the desired ph. The reaction was started by the addition of 20 l of labeled acetic acid or propionic acid, previously adjusted to the desired ph value. At appropriate times, 10- l aliquots were taken from the reaction mixture into 4 ml of ice-cold water and filtered immediately through Whatman GF/C membranes. The filters were washed with 8 ml of ice-cold water, and the radioactivity was counted as indicated above. The intracellular concentrations of acetic acid or propionic acid were estimated as the ratio between the intracellular and extracellular acid concentration, expressed as the anionic form of the acid and using the intracellular volume obtained as indicated below. Intracellular volume. The intracellular water volume was measured as previously described by De la Peña et al. (8) and Rottenberg (12). Values of l and l (mean SE, n 3) of intracellular water per mg of yeast dry weight for glucose- and acetic acid grown cells, respectively, were obtained. Estimation of glucose and acetic acid concentrations. Glucose and acetic acid concentrations in the cultures were estimated by high-pressure liquid chromatography after the cells had been separated by centrifugation. The apparatus used was a Gilson chromatograph with a Merck Polyspher OA KC (catalog no ) column maintained at 85 C, with N H 2 SO 4 as the mobile phase at a flow rate of 0.5 ml min 1. Calculation of the kinetic parameters. The application of a computer-assisted nonlinear regression analysis (GraphPad software, San Diego, Calif.) to the data of the initial uptake rates of the labeled carboxylic acids, as well as of the initial uptake rates of proton disappearance in cell suspensions, allowed us to determine the best fitting transport kinetics and the kinetic parameters. Calculation of the concentrations of carboxylic acid forms. Concentrations of the dissociated or undissociated forms of carboxylic acids were calculated by the use of the Henderson- Hasselbach equation with the pk a values of 4.76 for acetic acid and 4.87 for propionic acid (7). Chemicals. Radioactively labeled acids were [U- 14 C]acetic acid, sodium salt (CFA229; Amersham) and [2-14 C]propionic acid, sodium salt (CFA88; Amersham). All other chemicals were reagent grade and obtained from commercial sources. FIGURE 1. Growth of the yeast D. anomala IGC 5153 in media with acetic acid. (a) Values of max exhibited by the cells grown with different acetic acid concentrations at ph 3.5 (#), 4.0 ( ), 5.0 ( ), and 5.5 ( ). (b) Values of max plotted against the undissociated acetic acid concentration present in the culture medium in the conditions described in (a). RESULTS AND DISCUSSION Assessing acetic acid and other weak carboxylic acids as carbon and energy sources by D. anomala. The strain IGC 5153 of D. anomala was tested for its ability to use acetic, lactic, propionic, formic, succinic, malic, citric, sorbic, and benzoic acids as the only carbon and energy sources. From all these substrates, only acetic acid was metabolizable by the yeast, and the max values were dependent on the acid concentration and on the ph of the culture medium. The results are shown in Figure 1. For each growth ph value tested (3.5 to 5.5), there was an acetic acid concentration associated with the maximum value for max above which it decreased exponentially, the effect being accentuated with the decrease of the ph of the medium (Fig. 1a). When max values correspondent to the different experimental growth conditions were plotted against the acid concentration but expressed as the undissociated form, it was observed that max decreased with the increase of the undissociated acid concentration in the medium (Fig. 1b). An exponential relation was again obtained, expressed as follows: x 0 e k i [x x min] max max (1) where x and 0 max max are the maximal specific growth rates at external concentrations x and 0 of undissociated acetic acid, x min is the minimum inhibitory concentration of undissociated acid, and k i is the exponential growth inhibition constant. Otherwise, when cells grown with 0.5% of acetic acid

3 98 GERÓS ET AL. J. Food Prot., Vol. 63, No. 1 FIGURE 3. (a) Accumulation of labeled acetic acid at ph 5.0 ( ) by cells of D. anomala IGC 5153 grown in 0.5% of acetic acid, ph 5.5. The initial acetic acid extracellular concentration was 4 mm. At the time indicated by the arrow, additions were as follows: cold acetic acid to a final concentration of 0.2 M ( ), cold propionic acid to a final concentration of 0.2 M ( ), and CCCP to a final concentration of 0.05 mm ( ). A total of 0.05 mm of CCCP ( ) was added to the reaction mixture before addition of labeled acid. (b) Dependence of the accumulation ratio of labeled acetic and propionic acids on the extracellular ph in cells grown as indicated in (a). FIGURE 2. Initial uptake rates of labeled acetic acid at ph 5.0 by cells of D. anomala IGC 5153 grown in 0.5% of acetic acid, ph 5.5. Insert: Eadie-Hofstee plot of the initial uptake rates. at ph 5.5 (undissociated acid of about 0.08%) were transferred into a medium with the same total acetic acid concentration but at ph 3.5 (undissociated acid of about 0.47%), a biphasic growth curve was observed (not shown) and the max values estimated for each exponential growth phase were in accordance with those presented in Figure 1. Altogether, the results suggested that the yeast growth was mainly conditioned by the ph once it determines the relative amount of each form of the acetic acid present in the medium, because the undissociated form rather than the anion appeared to be associated with the negative effects observed on growth. From equation 1 a k i value of about 27 liter mol 1 was estimated for the exponential growth inhibition constant induced by the undissociated acid. To further clarify the growth kinetics reported above, the transport of acetic acid across the plasma membrane, the first step of its metabolism, was measured in cells grown under different conditions. The following sections deal with the results obtained on this subject. Higher growth rates are associated with the presence of a monocarboxylate-proton symport for the transport of acetic acid. The initial uptake rates of radiolabeled acetic acid at ph 5.0, over a concentration range of 0.1 to 4 mm, were measured in cells displaying the highest growth rates (concentration of the undissociated acetic acid in culture medium below 0.3%, vol/vol). Figure 2 depicts representative results of these experiments in cells grown with 0.5% of acetic acid, ph 5.5. The uptake followed a Michaelis-Menten kinetics, and the application of a computer-assisted nonlinear regression analysis to the data suggested the involvement of a mediated transport system associated with a simple diffusion component. The values estimated for the kinetic parameters were as follows: K m, mm of total acetic acid; V max, nmol s 1 mg 1 of dry weight; and for the diffusion constant of the undissociated acetic acid, k d, l s 1 mg 1 of dry weight. Search for possible competitive inhibitors of the acetic acid transport was carried out by measuring the initial uptake rates of labeled acetic acid in the presence of other carboxylic acids. Formic, propionic, and sorbic acids acted as competitive inhibitors of acetic acid transport, whereas lactic, pyruvic, citric, and succinic acids had no significant effect on the acid transport (not shown). The addition of acetic, formic, propionic, or sorbic acids to weak buffered cell suspensions promoted a transient alkalinization of the extracellular media, and the initial velocities of the proton disappearance also followed Michaelis-Menten kinetics (not shown). None of the other acids induced such alkalinization of the extracellular medium. The results suggested that the acetic, propionic, formic, and sorbic acids shared the same carrier and that a proton symport could be involved on their transport through the plasma membrane. In agreement with this hypothesis, it was observed that the transport of labeled acetic acid (Fig. 3) or its nonmetabolizable analogue propionic acid (not shown) along time, at ph 5.0, was accumulative. Cold acetic or propionic acids added at the steady state promoted a rapid efflux of the radiolabeled acetic acid. The addition of 50 M m-chlorophenylhydrazone (CCCP) to the mixture after the acid had been accumulated promoted their rapid efflux, and the accumulation was prevented by the presence of the ionophore (Fig. 3a). Furthermore, when the initial uptake rates of labeled acetic acid (0.1 to 4 mm) were measured in the presence of 50 M CCCP, an inhibition of about 60% was observed, supporting the hypothesis that a proton symport mechanism was involved in the transport (4). The uptake of labeled acetic acid in cells grown with 0.5% of acetic acid, ph 5.5, was also assayed at different extracellular ph values. The transport kinetic best fitting the experimental initial uptake rates at ph 3.0 to 6.0 was determined by GraphPad software. The results showed that in all conditions a mediated trans-

4 J. Food Prot., Vol. 63, No. 1 TRANSPORT OF ACETIC ACID IN D. ANOMALA 99 TABLE 1. Kinetic parameters (K m and V max ) and diffusion constants (k d ) for the transport of labeled acetic acid as a function of extracellular ph (ph ext ), in D. anomala IGC 5153 grown in a medium with acetic acid (0.5%, vol/vol; ph 5.5) a K m (mm) for: ph ext Total acid Anion c acid d Undissociated V max (nmol s 1 mg 1 dry wt) k d b ( l s 1 mg 1 dry wt) a Values are means standard error; n 3. b For undissociated acid. c Hypothesis of transport of anionic form. d Hypothesis of transport of undissociated acid. port system associated with a component of simple diffusion of the undissociated acid was present (Table 1). As shown in this table, the variation of the K m values with the extracellular ph, expressed as the concentration of undissociated or dissociated acid, was similar, which did not allow us to distinguish clearly between the two forms of the acid as the substrate for the carrier. Nevertheless, the accumulation ratios estimated as either [acetate] in /[acetate] out or [propionate] in /[propionate] out over the same ph range revealed to be a linear function of the extracellular ph (Fig. 3b), suggesting the presence of an electroneutral proton symport for the anionic form of the acid, with a stoichiometry of 1:1. Accordingly, when the uptake of labeled acetic acid was measured in the presence of 10 mm of the ionophore tetraphenlylphosphonium, which short-circuits the transmembranar electrical potential, no inhibitory effect was detected on the acid transport (not shown). The presence of the acetate-proton symport described above was also assayed in cells cultivated with other acetic acid concentrations and at different ph values. Table 2 summarizes the results obtained. On the whole, these experimental data suggested that acetic acid grown cells of D. anomala IGC 5153, under conditions that allowed higher max (undissociated acid present in the medium at concentrations below 0.3%), were able to produce an electroneutral monocarboxylate proton symport. Additionally, a component of simple diffusion of the undissociated acetic acid was measured, the weight of which increased with the increase of the undissociated acid concentration in the culture medium. In regard to the dependence on energy, substrate specificity, and Michaelis-Menten constants, the acetate carrier behaved similarly to those carriers identified in acetic acid grown cells of the yeasts S. cerevisiae (2), Torulaspora delbrueckii (3), and Zygosaccharomyces bailii (13). Lowest growth rates are associated with simple diffusion of the undissociated acid. As shown in Table 2, cells exhibiting the lowest growth rates (concentration of the undissociated acetic acid in culture medium above 0.3%, vol/vol) did not display activity for the acetate-proton symport. Figure 4 presents typical results of these experiments in cells grown with 0.5% of total acetic acid at ph TABLE 2. Kinetic parameters (K m and V max ) and diffusion constants (k d ) for the transport of labeled acetic acid at ph 5.0 in cells of D. anomala IGC 5153 grown in acetic acid as a carbon and energy source in the experimental conditions presented in Figure 1 Undissociated acetic acid in the culture medium (%, vol/vol) Kinetic parameters K m (mm) V max (nmol s 1 mg 1 dry wt) k d a ( l s 1 mg 1 dry wt) a For undissociated acid. b, not measurable b FIGURE 4. Initial uptake rates of labeled acetic acid at ph 5.0 by cells of D. anomala IGC 5153 grown in 0.5% of acetic acid at ph 3.5. Insert: Eadie-Hofstee plot of the data.

5 100 GERÓS ET AL. J. Food Prot., Vol. 63, No. 1 FIGURE 6. Diauxic growth of D. anomala IGC 5153 at ph 5.5 in a mineral medium with vitamins, glucose (0.12%, wt/vol), and acetic acid (0.6%, vol/vol). FIGURE 5. ph dependence of the acetic acid diffusion constants by cells of D. anomala IGC 5153 grown in 2% of glucose ( ) and 0.5% of acetic acid at ph 3.5 ( ) (high concentration of undissociated acetic acid, repressed conditions). Diffusion constants for labeled acetic acid reported by Casal et al. (2) for S. cerevisiae ( ) and by Casal and Leão (3) for T. delbrueckii (#) As can be seen in these cells, the plot of the initial velocities of labeled acetic acid uptake at ph 5.0, over the concentration range tested in derepressed grown cells, was linear. These results were in good agreement with the involvement of a simple diffusion mechanism in the transport of the acetic acid in cells growing with higher undissociated acetic acid concentrations. We must emphasize that, in these experimental conditions, the lack of the symporter cannot be attributed to the insufficient amount of the anionic form of acetic acid available for growth. In fact, cells grown with identical quantity of the anionic form of acetic acid but with lower concentration of uncharged acid (e.g., 0.1% of total acetic acid at ph 3.5) were able to show activity for the acetate-proton symport. As mentioned above, the shift of the ph to 3.5 in cells growing with 0.5% acetic acid at ph 5.5 (presence of the acetate-proton symport) promoted a change in the max, and the max values estimated for each exponential phase are in accordance with those presented in Figure 1. The results from the transport assays in cells obtained from the second growth phase showed that simple diffusion of the undissociated acid was the only mechanism involved in the acetic acid uptake by the cells. These data reinforced that the increase of the undissociated acid concentration in the culture medium was associated with a decrease in the max and the loss of activity for the acetate carrier. Furthermore, it appeared that the inhibitory effect of acetic acid in growth was due to the higher rate of acid transport that can be reached by simple diffusion as compared with acetate-proton symport (Table 2). Transport of acetic acid in cells grown with other carbon sources. To further understand the mechanism involved in the regulation of the acetate-proton symport, cells were cultivated in media with other carbon sources. Similar to those observed in cells grown in concentrations of the undissociated acetic acid above 0.3%, glucose-, fructoseand ethanol-grown cells did not display activity for the acetate-proton symport, measured as the uptake of labeled acid or proton uptake. However, these cells were still permeable to the acid that crossed the plasma membrane by simple diffusion. For each type of cell, the values of the acetic acid diffusion constant for the undissociated acid, at ph 5.0, expressed as l s 1 mg 1 of dry weight, were about 0.30 for glucose-grown cells, 0.48 for fructose-grown cells, 0.65 for ethanol-grown cells, and 0.45 for repressed undissociated acetic acid grown cells. Furthermore, either in glucose-grown or repressed acetic acid grown cells, the values for the diffusion constants were identical and increased about threefold with the increase of the extracellular ph from 3.0 to 6.0 (Fig. 5). As shown in this figure, a similar dependence of the acetic acid diffusion constants on the extracellular ph has been found in other yeast species, namely in S. cerevisiae (2) and T. delbrueckii (3). Growth of the yeast in a medium containing a mixture of glucose and acetic acid at ph 5.0 was diauxic (Fig. 6), since the acetate-proton symport was only detectable after the glucose had been exhausted from the culture medium. These results supported that in D. anomala IGC 5153 the acetate carrier was not only inducible but also subject to glucose repression. In conclusion, the results presented herein showed that D. anomala IGC 5153 exhibited a narrow capacity for using weak acids as the only carbon and energy sources, since acetic acid was the only one metabolized by the yeast. Similar behavior has been described for the yeast Z. bailii, another dangerous species frequently found in spoiled food and beverages (13). In addition, the growth of D. anomala in medium with acetic acid occurred at relatively high total acid concentration and over a broad ph range, including ph 3.5. Specifically in wine, this property of D. anomala may be associated with its presence at the end of the vinification process, although such environmental conditions are too severe to allow the survival of S. cerevisiae. According to our results, the main difference between these species was that D. anomala was able to grow at low ph values and in the presence of high concentrations of acetic acid in the culture medium, whereas S. cerevisiae was not, at least up to 1 week (results not shown). Particularly in dry white wines in which there is no residual sugar, the

6 J. Food Prot., Vol. 63, No. 1 TRANSPORT OF ACETIC ACID IN D. ANOMALA 101 capacity of the yeasts to grow will be limited to the use of respirable substrates and by the amount of dissolved oxygen. A previously published study reported that the wine spoiler yeast Z. bailii is also able to use acetic acid alone or simultaneously with glucose at low ph values such as 3.5 (14). According to the authors, this behavior may be correlated with the fact that, under these growth conditions, an acetate carrier is operational and both the membrane transport and the intracellular metabolic flux of the acid are regulated in such a way that the intracellular free acetic acid is maintained at levels below which negative effects may occur. The results presented in this work indicated that in D. anomala the acetate carrier was subject to glucose repression. Thus, it appeared that the strategy used by this species for surviving in acidic environments is different from that of Z. bailii. Under these conditions, D. anomala exhibited the highest values for the acetic acid diffusion constants associated with the lowest max. The yeast would be able to survive as long as the rates of the acid uptake were less or equal to its catabolic rate. Higher values of acid uptake rates due to an enhanced diffusion process may lead to toxic intracellular acid concentrations. The survival of D. anomala could be achieved by the existence of a pump for promoting the efflux of acetic acid from the inside of the cell. This fact will certainly be correlated with the high-energy expenses for cell maintenance, which, in turn, will account for the observed low max. Studies for elucidating this hypothesis are now under development. ACKNOWLEDGMENT This study was supported by a research grant (contract PBIC/C/BIO/ 2189/95) from Junta Nacional de Investigação Científica e Tecnológica, Lisbon, Portugal. REFERENCES 1. Carrascosa, J. M., M. D. Viguera, I. Núñes de Castro, and W. A. Scheffers Metabolism of acetaldehyde and Custers effect in the yeast Brettanomyces abstinens. Antonie van Leeuwenhoek 47: Casal, M., H. Cardoso, and C. Leão Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae. Microbiology 142: Casal, M., and C. Leão Utilization of short-chain monocarboxylic acids by the yeast Torulaspora delbrueckii: specificity of the transport systems and their regulation. Biochim. Biophys. Acta 1267: Cássio, F., M. Côrte-Real, and C. Leão Quantitative analysis of proton movements associated with the uptake of weak carboxylic acids: the yeast Candida utilis as a model. Biochim. Biophys. Acta 1153: Cássio, F., and C. Leão Low and high-affinity transport systems for citric acid in the yeast Candida utilis. Appl. Environ. Microbiol. 57: Ciani, M., and L. Ferraro Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking. J. Sci. Food Agric. 75: Dawson, R. M. C., D. C. Elliot, H. E. William, and K. M. Jones Data for biochemical research. Clarendon Press, Oxford. 8. De la Peña, P., F. Barros, S. Gascon, P. S. Lazo, and S. Ramos Effect of yeast killer toxin on sensitive cells of Saccharomyces cerevisiae. J. Biol. Chem. 256: Fugelsang, K. C., M. M. Osborn, and C. J. Muller Brettanomyces and Dekkera: implications in wine making, chapter 7, p In B. H. Gump (ed.), Beer and wine production, analysis, characterization and technological advances. American Chemical Society, Washington, D.C. 10. Heresztyn, T Formation of substituted tetrahydropyridines by species of Brettanomyces and Lactobacillus isolated from mousy wines. Am. J. Enol. Vitic. 37: Heresztyn, T Metabolism of volatile phenolic compounds from hidroxycinnamic acids by Brettanomyces yeasts. Arch. Microbiol. 146: Rottenberg, H The measurement of membrane potential and ph in cells, organelles and vesicles. Methods Enzymol. 55: Sousa, M. J., L. Miranda, M. Côrte-Real, and C. Leão Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistence of the yeast to acidic environments. Appl. Environ. Microbiol. 62: Sousa, M. J., F. Rodrigues, M. Côrte-Real, and C. Leão Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii. Microbiology 144: Van Uden, N Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. Microbiol. 58: Wijsman, M. R., J. P. Van Dijken, B. H. A. Van Kleeff, and W. A. Scheffers Inhibition of fermentation and growth in batch cultures of yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie van Leeuwenhoek 50: