Ethanol tolerance in the yeast Saccharomyces

The effect of ethanol on yeast growth and fermentation was studied in Saccharomyces cerevisiae NCYC 479 (a commercial sake yeast) and Saccharomyces uvarum 5D-cyc (a laboratory haploid strain). The effect of ethanol on growth was similar in both strains, complex kinetics were exhibited since ethanol affected both growth rate and viability. Ethanol was less inhibitory towards fermentation than towards growth. Fermentation was more ethanol tolerant in the sake yeast than in the laboratory strain. The optimum temperature for fermentation by S.uvarum 5D-cyc was higher than that for its growth. Growth inhibition by ethanol was least at temperatures 5°C and 10°C below the growth optimum (35°C). Fermentation became more resistant to ethanol inhibition with increasing temperature. S.uvarum 5D-cyc was characterized in a glucose-limited chemostat. Yield, maintenance and KS constants were determined. Derivation of an inhibitor constant for growth suggested that at substrate concentrations of 50 gl -1 and 135 gl -1 glucose autogenously produced ethanol caused a deviation from substrate saturation kinetics. This was manifested by the measurement of µ max by washout as 0.41 h- 1 whereas D Crit was found to be 0.28 h-1. The effect of oxygen on cell and ethanol formation by S.uvarum 5D-cyc (a grande strain) and by a cytoplasmic petite mutant derived from it was studied. The optimum oxygen level in the input gas flow for cell and ethanol formation by the grande was 0.17%. At no oxygen level did the yield or productivity of ethanol by the petite exceed that obtainable with the grande. Mutants of S.uvarum 5D-cyc with increased tolerance to ethanol were isolated by a continuous selection technique which allows the culture itself to determine the intensity of selection via a feedback control circuit. This system permitted the selection of mutants which displayed a range of phenotypes, all of the mutants were viable in 120 gl-1 ethanol.