Folia Microbiol.37 (1), 43-46 (1992)
Effect of Growth Rate on Ethanol Tolerance of Saccharomyces cerevisiae (~. NOVOTN~, M. FLIEGER,J. PANOS and L. DOLET~kLOV,~
Institute of Microbiology, CzechoslovakAcademy of Sciences, 142 20 Prague4 ReceivedMay 20, 1991 ABSTRACT. A5,7-Sterol-accumulatingSaccharomycescerevisiaecells growing in chemostatat a specificgrowth rate of 0,075/h exhibited higher ethanol tolerance measured as ethanol-induceddeath and anaerobic growth inhibition than the cells growing at 0.2/h, the differencebeing dependent on the carbon-to-nitrogenmolar proportion in the medium, The observed difference in sensitivityto ethanol of anaerobic growth between the slowlyand rapidly-growingcellswas completelyreversed as a result of a block in sterol synthesis causing a negligible synthesis of A5'7-sterols.Two physiologicalparameters, budding frequencyand membrane composition, evidenlly affected ethanol lolerance. Differencesbetween the A5'7-sterol-synthesizingand deficient strains documented a profound effect of the qualityof the slerol present on the physiologicalslate of the cell. Ethanol tolerance of a microorganism primarily depends on the composition of its cellular membranes (Ingrain and Buttke 1984; Casey and lngledew 1986). The presence of proper sterols and unsaturated fatty acids was found to be essential (Thomas et aL 1978; Watson 1982). Protection against the toxic effect of ethanol can also be ensured by addition of various complex compounds into the growth medium: proteolipids, soy flour, peptone, etc. (Casey and Ingledew 1986). Saccharomyces cerevisiae belongs to microorganisms that are highly resistant to ethanol, probably as a result of their evolutionary adaptation (Ingrain and Buttke 1984). Casey and lngledew (1986) reported the values of the minimum "threshold" concentrations of ethanol required before an inhibition of qrowth was observed and those completely suppressing growth to be 2.5-7.8 and 8.5-14.0 % (V/V), respectively, for Saccharomyces yeasts. The minimum levels of ethanol required to inhibit viability in stationary phase suspensions of sake, ale, lager and baker's yeasts were found to be 18, 18, 13 and 15 % (V/V), respectively (Kalmokoff and Ingledew 1985). Sensitivity to ethanol depends on the growth phase. Early-stationary-phase cells showed greater resistance to ethanol-induced death than exponential-phase ones (Kalmokoff and Ingledew 1985). As to the inhibition of growth, the exponentially growing cells can sometimes be less sensitive than the nongrowing ones (Thomas and Rose 1979) and, thus, contradictory results exist whether ethanol is more toxic or not to the stationary than to the exponentially growing cells (ef. Casey and Ingledew 1986). The purpose of our work was to examine the effect of growth rate in the chemostat on the sensitivity of the cells to ethanol measured by ethanol-induced death and the inhibition of their subsequent anaerobic growth. Comparison of the strains synthesizing various levels of AS'7-sterols was expected to indicate a possible role of the sterols involved.
MATERIALS
AND METHODS
Microorganisms. The As,7-sterol-synthesizing Saccharomyces cerevisiae strain FL100 (trpl ura3) and the sterol mutant 7011 (A8 "* A7 isomerization lesion, isogenic with FL100; erg2 trpl ura3) were used. Growth media. The strains were maintained on wort agar slopes. A two-fold concentrated Olson-Johnson (1949) synthetic medium containing glucose at 40 g / L (molar proportions of the carbon-to-ammonia-nitrogen sources: 109 or 5) was used. The chemostat cultures were grown in a 2-L fermenter at 30 ~ 10 Hz agitation frequency, 1 volume of air per rain, pH kept at 4.5- 5.0 with NaOH and H2SO4. Dilution rates used: 0.075 or 0.2/h. Ethanol tolerance. Ethanol-induced death was estimated by the method of Lee et al. (1981). Anaerobic growth in the Olson-Johnson (1949) medium containing 1 % (W/V) glucose in the presence of 0 to 12.2 % (V/V) exogenous ethanol was measured turbidimetrically at 540 nm. Before being used to measure ethanol tolerance, the chemostat cells were harvested and washed with saline solution.
44
(~. NOVOTNY et al.
Vol. 37
Various analyses. The sterol content was determined by HPLC (Rodriguez and Parks 1985), fatty acid content by GC. The conditions of the fatty acid analysis were as follows: a gas chromatograph Perkin-Elmer 900; column - stainless steel, 1800 • 2.7 mm, packed with 2 % EGSS-X, 0.2 % NEPSS, 0.2 % EGA on Gas Chrom P 100/120 mesh; column temperature 170 ~ injection-block temperature 200 ~ FID temperature 195 ~ FN2 = 30 mL/min; injection volume 1.0/zL; methylation carried out with 10 % BF3/MeOH. Ethanol concentration in the medium was determined according to Williams and Reese (1950). R E S U L T S A N D DISCUSSION A large body of data on yeast ethanol tolerance has accumulated whose major part consists of the results from industrial ethanol fermentations that represent complex and dynamic systems including numerous biological and nutritional parameters (Casey and Ingledew 1986; Jones 1989). Therefore, experimental approaches minimizing the number of transient factors are of much importance to define the real significance of various physiological changes making up a higher ethanol tolerance of the microbial cell. In our broader study trying to specify the role of sterols in ethanol tolerance under strictly defined conditions in the chemostat, we observed that the actual value of specific growth rate could significantly affect the sensitivity to the toxic effect of ethanol. We measured the effect on cell viability and on the anaerobic growth and analyzed it in detail.
I
I
too cv
% O
SO
10
2O
o
1 lo
2o
Irt OH, % Fig. 1. Effect of growth rate in a chemostat on cell viability (CV, %) after 6 h at different concentrations of exogenous ethanol (EtOH, %). S. cerevisiae strains FL100 (AS,7-sterol-synthesizing, circles) and 7011 (sterol mutant, triangles) were grown in chemostat at a growth rate of 0.075/h (open symbols) or 0.2/h (closed symbols). Growth medium had C/N ratio equal to 109
(left) or 5 (r/g/u). If S. cerevisiae was grown in the chemostat in a medium having a carbon-to-nitrogen (C/N) molar ratio of 109, a significant difference in the sensitivity to ethanol-induced death was observed between cells growing at specific growth rates/z of 0.075 and 0.2/h (Fig. 1 left). The more rapidly growing cells of both the as,7-sterol-synthesizing and deficient strains exhibited a greater sensitivity to ethanol. The difference was less distinct if the C:N ratio was 5 (Fig. 1 right). All the cells were growing in the chemostat under a permanent stress of about 5 to 18 g of endogenous ethanol per L medium, the sterol content of the cells being in the range of 3 to 10 mg As,7-sterol per g dry matter in the as,7-sterolsynthesizing strain. An increase of specific growth rate from 0.075 to 0.2/h reduced the AS,7-sterol con-
E T H A N O L T O L E R A N C E O F S. cerevisiae
1992
45
tent by about one half. Detailed information on the sterol and fatty acid composition of the cells grown under these conditions is published elsewhere (Novotn3~et al. 1992). The observed difference in the sensitivity to ethanol-induced death can be explained by a higher proportion of budding cells in the rapidly growing population (Vranfi 1990) since these cells were found to be more sensitive to ethanol throughout fermentation than nonbudding ones (Lee et al. 1981) and, in the case of FL100, also by a higher content of As,7-sterols in the biomass grown at 0.075/h (Lafon-Lafourcade and Ribereau-Gayon 1984; Novotn~ et al. 1992). Our results show that the concentration of about 15 % (V/V) ethanol (cf. Kalmokoff and Ingledew 1985) at which ethanol-induced death of many Saccharomyces strains usually starts to manifest can decrease to less than 10 % just by imposing a higher growth rate. This behavior may have important technological implications. I
I
5
10
lOO
Vmax %
SO
I
-0
I
0
10
5 EtOH,%
Fig. 2. Effect of growth rate in a chemostat on anaerobic growth (I/max, %) at various concentrations of exogenous ethanol (EtOH, %). S. cerevisiae strains FL100 (AS,7-sterol-synthesizing, circles and 7011 (sterol mutant, triangles) were grown in chemostat at a growth rate of 0.075/h (open symbols) or 0.2/h (closed symbols). Growth medium had C / N ratio equal to 109 (left) or 5
(right).
The effect of the cells' history on the sensitivity of subsequent anaerobic growth to exogenous ethanol is documented in Fig. 2. Whereas the trend of a greater sensitivity of rapidly growing cells holds for the AS,7-sterol synthesizing strain (Fig. 2 left) including the fading-away difference at a C/N ratio of 5 (Fig. 2 right), contrasting behavior was observed with the aS,7-sterol-deficient mutant. The differences between slowly and rapidly growing cells became pronounced for ethanol concentrations excecding 6 % (V/V), the ethanol inhibition of growth of the two strains being in the range reported by Casey and Ingledew (1986) for Saccharomyces yeasts. The latter strain exhibited not only a negligible synthesis of AS,7-sterols but also a low unsaturated-to-saturated fatty acid (FA) ratio (0.2 to 0.3). The corresponding value for FL100 strain was 1.7-2.2. These results show that the observed differences in cell lipid composition may completely change the strain's behaviour under ethanol stress even though the range of sensitivity to the toxic compound remains unchanged. The increased inhibition of anaerobic growth by high concentrations of exogenous ethanol was often paralleled by a longer lag period before growth started. The results of our study show that not only growing cells may differ from resting ones in their sensitivity to ethanol (cf. Jones 1989) but also slowly and rapidly growing cells which stresses the importance of the physiological state of the population. Gilbert and Wright (1987) provided evidence that similar differences can be observed with carbon- and nitrogen-limited Escherichia coli cells with respect to their resistance to antibacterial phospholipid-combining agents. The manifestation of these differences is, however, dependent on the composition of the growth medium (Figs 1 and 2; Gilbert and Wright 1987). Our observations with strains differing in sterol synthesis as well as those with E. coli
46
~. N O V O T N ~ et al.
Vol. 37
suggest that the difference between slowly and rapidly growing cells in resistance to various toxic cffects probably depends on the composition of the cytoplasmic membrane. The authors thank Prof. F. Karst (Universit~ de Poitiers, France) for kindly providing the yeast strains. Skillcd tcchnical assistance of M. Vyletalov~i and J. Honzikovfi is gratefully acknowledged.
REFERENCES CASEV G.P., |NGLEDEWW.M.: Ethanol tolerance in yeasts. CRC Crit.Rev.Microbiol. 13,219- 279 (1986). GILaERT P., WRlo~rr N.E.: Interrelation between the physiological status of E. coli and its susceptibility towards inactivation by antimicrobial agents, poster at 9th lnternat. Symp. Continuous Cultivation, |lradec Kr~lov6, Book of Abstracts, p. 13 (1987). INORAM L.O., BUTrKE T.M.: Effect of alcohols on microorganisms. Adv.Microb.PhysioL 25, 254 - 300 (1984). JONES R.P.: Biological principles for the effects of ethanol. Enzyme Microb.Technol. 11, 130-153 (1989). KALMOKOFFM., INGLEDEWW.M.: Evaluation of ethanol tolerance in related Saccharornyces strains. J.Am.Soc.Brcw.Chem. 43, 189 - 201 (1985). LAFoN-LAFOURCADES., RIBEREAU-GAYONP.: Developments in the microbiology of wine production, pp. 1-45 in Progress in Industrial Microbiology (M.E. Bushell, Ed.). Elsevier, Amsterdam 1984. LEE S.S., ROBINSON F.M., WANG H.Y.: Rapid determination of yeast viability. BiotechnoLBioeng.Symp. 11, 641- 649 (1981). NOVOTN~' C., FLIEGER M., PANOS J., KARST F.: Effect of 5,7-unsaturated sterols on ethanot tolerance in S. cerevisiae. Biotechnol. AppLBiochem., in press (1992). OLSON P.It., JOrINSON M.J.: Factors producing high yeast yields in synthetic media. J.BacterioL 57, 235 -246 (1949). RODRIGUEZ R.J., PARKS L.W.: High-performance liquid chromatography of sterols: yeast sterols, pp. 37-50 in Methods in Enzymology, Vol. 111, part 3 (J.H. Law, H.C. RiUing, Eds). Academic Press, London 1985. THOMAS D.S., HOSSACK J.A., ROSE A.IL: Plasma-membrane lipid composition and ethanol tolerance in S. cerevisiae. Arch.MicrobioL 117, 239- 245 (1978). THOMAS D.S., ROSE A.H.: Inhibitory effect of ethanol on growth and solute accumulation by S. cerevisiae as affected by plasma membrane lipid composition. Arch.MicrobioL 122, 49-54 (1979). VRANA D.: Distribution of cells in different stages of the cell cycle and some age-related physiological characteristics in dependence on the dilution rate in chemostat cultures of C. utilis. Folia Microbiol. 35, 245 -250 (1990). WATSON K.: Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces. BiotechnoLLett. 4, 397-402 (1982). WILLL~MS M.P., REESE H.D.: Colorimetric determination of ethylalcohol. Anal.Chem. 22, 1556-1561 (1950).
Effect of Growth Rate on Ethanol Tolerance of Saccharomyces cerevisiae (~. NOVOTN~, M. FLIEGER,J. PANOS and L. DOLET~kLOV,~
Institute of Microbiology, CzechoslovakAcademy of Sciences, 142 20 Prague4 ReceivedMay 20, 1991 ABSTRACT. A5,7-Sterol-accumulatingSaccharomycescerevisiaecells growing in chemostatat a specificgrowth rate of 0,075/h exhibited higher ethanol tolerance measured as ethanol-induceddeath and anaerobic growth inhibition than the cells growing at 0.2/h, the differencebeing dependent on the carbon-to-nitrogenmolar proportion in the medium, The observed difference in sensitivityto ethanol of anaerobic growth between the slowlyand rapidly-growingcellswas completelyreversed as a result of a block in sterol synthesis causing a negligible synthesis of A5'7-sterols.Two physiologicalparameters, budding frequencyand membrane composition, evidenlly affected ethanol lolerance. Differencesbetween the A5'7-sterol-synthesizingand deficient strains documented a profound effect of the qualityof the slerol present on the physiologicalslate of the cell. Ethanol tolerance of a microorganism primarily depends on the composition of its cellular membranes (Ingrain and Buttke 1984; Casey and lngledew 1986). The presence of proper sterols and unsaturated fatty acids was found to be essential (Thomas et aL 1978; Watson 1982). Protection against the toxic effect of ethanol can also be ensured by addition of various complex compounds into the growth medium: proteolipids, soy flour, peptone, etc. (Casey and Ingledew 1986). Saccharomyces cerevisiae belongs to microorganisms that are highly resistant to ethanol, probably as a result of their evolutionary adaptation (Ingrain and Buttke 1984). Casey and lngledew (1986) reported the values of the minimum "threshold" concentrations of ethanol required before an inhibition of qrowth was observed and those completely suppressing growth to be 2.5-7.8 and 8.5-14.0 % (V/V), respectively, for Saccharomyces yeasts. The minimum levels of ethanol required to inhibit viability in stationary phase suspensions of sake, ale, lager and baker's yeasts were found to be 18, 18, 13 and 15 % (V/V), respectively (Kalmokoff and Ingledew 1985). Sensitivity to ethanol depends on the growth phase. Early-stationary-phase cells showed greater resistance to ethanol-induced death than exponential-phase ones (Kalmokoff and Ingledew 1985). As to the inhibition of growth, the exponentially growing cells can sometimes be less sensitive than the nongrowing ones (Thomas and Rose 1979) and, thus, contradictory results exist whether ethanol is more toxic or not to the stationary than to the exponentially growing cells (ef. Casey and Ingledew 1986). The purpose of our work was to examine the effect of growth rate in the chemostat on the sensitivity of the cells to ethanol measured by ethanol-induced death and the inhibition of their subsequent anaerobic growth. Comparison of the strains synthesizing various levels of AS'7-sterols was expected to indicate a possible role of the sterols involved.
MATERIALS
AND METHODS
Microorganisms. The As,7-sterol-synthesizing Saccharomyces cerevisiae strain FL100 (trpl ura3) and the sterol mutant 7011 (A8 "* A7 isomerization lesion, isogenic with FL100; erg2 trpl ura3) were used. Growth media. The strains were maintained on wort agar slopes. A two-fold concentrated Olson-Johnson (1949) synthetic medium containing glucose at 40 g / L (molar proportions of the carbon-to-ammonia-nitrogen sources: 109 or 5) was used. The chemostat cultures were grown in a 2-L fermenter at 30 ~ 10 Hz agitation frequency, 1 volume of air per rain, pH kept at 4.5- 5.0 with NaOH and H2SO4. Dilution rates used: 0.075 or 0.2/h. Ethanol tolerance. Ethanol-induced death was estimated by the method of Lee et al. (1981). Anaerobic growth in the Olson-Johnson (1949) medium containing 1 % (W/V) glucose in the presence of 0 to 12.2 % (V/V) exogenous ethanol was measured turbidimetrically at 540 nm. Before being used to measure ethanol tolerance, the chemostat cells were harvested and washed with saline solution.
44
(~. NOVOTNY et al.
Vol. 37
Various analyses. The sterol content was determined by HPLC (Rodriguez and Parks 1985), fatty acid content by GC. The conditions of the fatty acid analysis were as follows: a gas chromatograph Perkin-Elmer 900; column - stainless steel, 1800 • 2.7 mm, packed with 2 % EGSS-X, 0.2 % NEPSS, 0.2 % EGA on Gas Chrom P 100/120 mesh; column temperature 170 ~ injection-block temperature 200 ~ FID temperature 195 ~ FN2 = 30 mL/min; injection volume 1.0/zL; methylation carried out with 10 % BF3/MeOH. Ethanol concentration in the medium was determined according to Williams and Reese (1950). R E S U L T S A N D DISCUSSION A large body of data on yeast ethanol tolerance has accumulated whose major part consists of the results from industrial ethanol fermentations that represent complex and dynamic systems including numerous biological and nutritional parameters (Casey and Ingledew 1986; Jones 1989). Therefore, experimental approaches minimizing the number of transient factors are of much importance to define the real significance of various physiological changes making up a higher ethanol tolerance of the microbial cell. In our broader study trying to specify the role of sterols in ethanol tolerance under strictly defined conditions in the chemostat, we observed that the actual value of specific growth rate could significantly affect the sensitivity to the toxic effect of ethanol. We measured the effect on cell viability and on the anaerobic growth and analyzed it in detail.
I
I
too cv
% O
SO
10
2O
o
1 lo
2o
Irt OH, % Fig. 1. Effect of growth rate in a chemostat on cell viability (CV, %) after 6 h at different concentrations of exogenous ethanol (EtOH, %). S. cerevisiae strains FL100 (AS,7-sterol-synthesizing, circles) and 7011 (sterol mutant, triangles) were grown in chemostat at a growth rate of 0.075/h (open symbols) or 0.2/h (closed symbols). Growth medium had C/N ratio equal to 109
(left) or 5 (r/g/u). If S. cerevisiae was grown in the chemostat in a medium having a carbon-to-nitrogen (C/N) molar ratio of 109, a significant difference in the sensitivity to ethanol-induced death was observed between cells growing at specific growth rates/z of 0.075 and 0.2/h (Fig. 1 left). The more rapidly growing cells of both the as,7-sterol-synthesizing and deficient strains exhibited a greater sensitivity to ethanol. The difference was less distinct if the C:N ratio was 5 (Fig. 1 right). All the cells were growing in the chemostat under a permanent stress of about 5 to 18 g of endogenous ethanol per L medium, the sterol content of the cells being in the range of 3 to 10 mg As,7-sterol per g dry matter in the as,7-sterolsynthesizing strain. An increase of specific growth rate from 0.075 to 0.2/h reduced the AS,7-sterol con-
E T H A N O L T O L E R A N C E O F S. cerevisiae
1992
45
tent by about one half. Detailed information on the sterol and fatty acid composition of the cells grown under these conditions is published elsewhere (Novotn3~et al. 1992). The observed difference in the sensitivity to ethanol-induced death can be explained by a higher proportion of budding cells in the rapidly growing population (Vranfi 1990) since these cells were found to be more sensitive to ethanol throughout fermentation than nonbudding ones (Lee et al. 1981) and, in the case of FL100, also by a higher content of As,7-sterols in the biomass grown at 0.075/h (Lafon-Lafourcade and Ribereau-Gayon 1984; Novotn~ et al. 1992). Our results show that the concentration of about 15 % (V/V) ethanol (cf. Kalmokoff and Ingledew 1985) at which ethanol-induced death of many Saccharomyces strains usually starts to manifest can decrease to less than 10 % just by imposing a higher growth rate. This behavior may have important technological implications. I
I
5
10
lOO
Vmax %
SO
I
-0
I
0
10
5 EtOH,%
Fig. 2. Effect of growth rate in a chemostat on anaerobic growth (I/max, %) at various concentrations of exogenous ethanol (EtOH, %). S. cerevisiae strains FL100 (AS,7-sterol-synthesizing, circles and 7011 (sterol mutant, triangles) were grown in chemostat at a growth rate of 0.075/h (open symbols) or 0.2/h (closed symbols). Growth medium had C / N ratio equal to 109 (left) or 5
(right).
The effect of the cells' history on the sensitivity of subsequent anaerobic growth to exogenous ethanol is documented in Fig. 2. Whereas the trend of a greater sensitivity of rapidly growing cells holds for the AS,7-sterol synthesizing strain (Fig. 2 left) including the fading-away difference at a C/N ratio of 5 (Fig. 2 right), contrasting behavior was observed with the aS,7-sterol-deficient mutant. The differences between slowly and rapidly growing cells became pronounced for ethanol concentrations excecding 6 % (V/V), the ethanol inhibition of growth of the two strains being in the range reported by Casey and Ingledew (1986) for Saccharomyces yeasts. The latter strain exhibited not only a negligible synthesis of AS,7-sterols but also a low unsaturated-to-saturated fatty acid (FA) ratio (0.2 to 0.3). The corresponding value for FL100 strain was 1.7-2.2. These results show that the observed differences in cell lipid composition may completely change the strain's behaviour under ethanol stress even though the range of sensitivity to the toxic compound remains unchanged. The increased inhibition of anaerobic growth by high concentrations of exogenous ethanol was often paralleled by a longer lag period before growth started. The results of our study show that not only growing cells may differ from resting ones in their sensitivity to ethanol (cf. Jones 1989) but also slowly and rapidly growing cells which stresses the importance of the physiological state of the population. Gilbert and Wright (1987) provided evidence that similar differences can be observed with carbon- and nitrogen-limited Escherichia coli cells with respect to their resistance to antibacterial phospholipid-combining agents. The manifestation of these differences is, however, dependent on the composition of the growth medium (Figs 1 and 2; Gilbert and Wright 1987). Our observations with strains differing in sterol synthesis as well as those with E. coli
46
~. N O V O T N ~ et al.
Vol. 37
suggest that the difference between slowly and rapidly growing cells in resistance to various toxic cffects probably depends on the composition of the cytoplasmic membrane. The authors thank Prof. F. Karst (Universit~ de Poitiers, France) for kindly providing the yeast strains. Skillcd tcchnical assistance of M. Vyletalov~i and J. Honzikovfi is gratefully acknowledged.
REFERENCES CASEV G.P., |NGLEDEWW.M.: Ethanol tolerance in yeasts. CRC Crit.Rev.Microbiol. 13,219- 279 (1986). GILaERT P., WRlo~rr N.E.: Interrelation between the physiological status of E. coli and its susceptibility towards inactivation by antimicrobial agents, poster at 9th lnternat. Symp. Continuous Cultivation, |lradec Kr~lov6, Book of Abstracts, p. 13 (1987). INORAM L.O., BUTrKE T.M.: Effect of alcohols on microorganisms. Adv.Microb.PhysioL 25, 254 - 300 (1984). JONES R.P.: Biological principles for the effects of ethanol. Enzyme Microb.Technol. 11, 130-153 (1989). KALMOKOFFM., INGLEDEWW.M.: Evaluation of ethanol tolerance in related Saccharornyces strains. J.Am.Soc.Brcw.Chem. 43, 189 - 201 (1985). LAFoN-LAFOURCADES., RIBEREAU-GAYONP.: Developments in the microbiology of wine production, pp. 1-45 in Progress in Industrial Microbiology (M.E. Bushell, Ed.). Elsevier, Amsterdam 1984. LEE S.S., ROBINSON F.M., WANG H.Y.: Rapid determination of yeast viability. BiotechnoLBioeng.Symp. 11, 641- 649 (1981). NOVOTN~' C., FLIEGER M., PANOS J., KARST F.: Effect of 5,7-unsaturated sterols on ethanot tolerance in S. cerevisiae. Biotechnol. AppLBiochem., in press (1992). OLSON P.It., JOrINSON M.J.: Factors producing high yeast yields in synthetic media. J.BacterioL 57, 235 -246 (1949). RODRIGUEZ R.J., PARKS L.W.: High-performance liquid chromatography of sterols: yeast sterols, pp. 37-50 in Methods in Enzymology, Vol. 111, part 3 (J.H. Law, H.C. RiUing, Eds). Academic Press, London 1985. THOMAS D.S., HOSSACK J.A., ROSE A.IL: Plasma-membrane lipid composition and ethanol tolerance in S. cerevisiae. Arch.MicrobioL 117, 239- 245 (1978). THOMAS D.S., ROSE A.H.: Inhibitory effect of ethanol on growth and solute accumulation by S. cerevisiae as affected by plasma membrane lipid composition. Arch.MicrobioL 122, 49-54 (1979). VRANA D.: Distribution of cells in different stages of the cell cycle and some age-related physiological characteristics in dependence on the dilution rate in chemostat cultures of C. utilis. Folia Microbiol. 35, 245 -250 (1990). WATSON K.: Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces. BiotechnoLLett. 4, 397-402 (1982). WILLL~MS M.P., REESE H.D.: Colorimetric determination of ethylalcohol. Anal.Chem. 22, 1556-1561 (1950).
