One major obstacle of simultaneous saccharification and fermentation (SSF) is that enzymatic hydrolysis in SSF is conducted at non-optimal temperature. To break through this bottleneck, thermotolerant Saccharomyces cerevisiae strains mbc 1-4 were screened by spot-assay from respiration-deficient mutants induced by ethidium bromide. When four strains placed in liquid medium, the strains exhibited significant increased growth and fermentability at 42 degrees C in comparison to the host Consequently, S. cerevisiae mbc 2 was selected to be the most suitable for bioethanol production at 42 degrees C. Miscanthus was pretreated with 1.5 M NaOH at 150 degrees C for 30 min in 1 L-vessel. As a result, 42.6% of solids containing 77.9% cellulose were recovered. Next, SSF was conducted with pretreated Miscanthus containing 3% glucan to estimate the fermentability of S. cerevisiae mbc 2 at 42 degrees C. In results, Ethanol concentration and theoretical ethanol yield by S. cerevisiae mbc 2 in 48 h were 15.3 g/L and 90.1%, respectively, whereas those by the control Were 8.3 g/L and 49.3%, respectively. In addition, SSF with increased substrate concentration upto 9% glucan resulted in 86.3% and 82.2% of theoretical ethanol yield, respectively. Thus, our approach for SSF with high solid loading using thermotolerant S. cerevisiae mbc 2 contributes to process and microbial strain development for bioethanol production. (C) 2015 Elsevier Ltd. All rights reserved.