A mixture of 5% (w/v) glucose, 4% (w/v) xylose and 5% (w/v) cellobiose was fermented into ethanol using non-recombinant yeasts. Two series of experiments were carried out: (1) sequential fermentation with Candida shehatae D45-6 and Saccharomyces cerevisiae (Cs-Sc), and (2) sequential fermentation with C. shehatae D45-6 and Brettanomyces bruxellensis (Cs-Bb). C shehatae D45-6 was initially used for glucose and xylose fermentation before adding highly ethanol-tolerant yeasts, either S. cerevisiae or B. bruxellensis, for cellobiose fermentation. For the sequential fermentation using S. cerevisiae, B-glucosidase was also included in the second step. In these two experiments, ethanol concentration reached 5.6-5.8% (w/v) and 99% sugar was consumed. Our results suggest that restricted glucose production from cellulose by saccharification could allow D45-6 to complete monosaccharide fermentation before the ethanol concentration exceeded its tolerance level. (C) 2012 Elsevier Ltd. All rights reserved.