Saccharomyces spp. are widely used for ethanologenic
fermentations, however yeast metabolic rate and
viability decrease as ethanol accumulates during fermentation,
compromising ethanol yield. Improving ethanol tolerance
in yeast should, therefore, reduce the impact of
ethanol toxicity on fermentation performance. The purpose
of the current work was to generate and characterise ethanol-
tolerant yeast mutants by subjecting mutagenised and
non-mutagenised populations of Saccharomyces cerevisiae
W303-1A to adaptive evolution using ethanol stress as a
selection pressure. Mutants CM1 (chemically mutagenised)
and SM1 (spontaneous) had increased acclimation and
growth rates when cultivated in sub-lethal ethanol concentrations,
and their survivability in lethal ethanol concentrations
was considerably improved compared with the parent
strain. The mutants utilised glucose at a higher rate than the
parent in the presence of ethanol and an initial glucose concentration
of 20 g l¡1. At a glucose concentration of
100 g l¡1, SM1 had the highest glucose utilisation rate in

the presence or absence of ethanol. The mutants produced
substantially more glycerol than the parent and, although
acetate was only detectable in ethanol-stressed cultures,
both mutants produced more acetate than the parent. It is
suggested that the increased ethanol tolerance of the
mutants is due to their elevated glycerol production rates
and the potential of this to increase the ratio of oxidised and
reduced forms of nicotinamide adenine dinucleotide
(NAD+/NADH) in an ethanol-compromised cell, stimulating
glycolytic activity.