J Ind Microbiol Biotechnol (2010) 37:139–149 DOI 10.1007/s10295-009-0655-3 123 ORIGINAL PAPER Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae Dragana Stanley · Sarah Fraser · Paul J. Chambers · Peter Rogers · Grant A. Stanley Received: 31 March 2009 / Accepted: 21 October 2009 / Published online: 10 November 2009  Society for Industrial Microbiology 2009 Abstract Saccharomyces spp. are widely used for etha- nologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermenta- tion, compromising ethanol yield. Improving ethanol toler- ance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise etha- nol-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 concen- trations, and their survivability in lethal ethanol concentra- tions 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 con- centration 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. Keywords Saccharomyces · Ethanol · Stress · Mutant · NADH Introduction Microbial production of ethanol has become increasingly important due to renewed interest in its use as a biofuel. Many new ethanol plants are being built to increase supply, and researchers are investigating ways of increasing etha- nol output [3]. One approach that can be used to achieve this end is improvement of the microbial strains used in fer- mentation [25]. A considerable amount of research to date has focussed on improving the ethanol tolerance of ethanol- producing organisms, in the belief that such improvement will consequently lead to higher ethanol productivities and yields [10, 29]. The use of genetic engineering to improve the ethanol tolerance of Saccharomyces cerevisiae is limited by our lack of knowledge, and the complexity, of the ethanol- stress-related mechanisms that inhibit cell performance [16]. It is perhaps not surprising that research on ethanol tolerance mechanisms in S. cerevisiae has mostly involved isolating ethanol-sensitive mutants to identify genes that are necessary for growth under ethanol stress [34]. While this has provided some insight into ethanol tolerance per se, the mechanisms associated with ethanol sensitivity do not necessarily translate into eVective strategies for improving D. Stanley · S. Fraser · G. A. Stanley (&) School of Engineering and Science, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia e-mail: grant.stanley@vu.edu.au P. J. Chambers The Australian Wine Research Institute, PO Box 197, Glen Osmond, SA 5064, Australia P. Rogers Foster’s Group Ltd, 4 Southampton Crescent, Abbotsford, VIC 3067, Australia