ORIGINAL PAPER Selection of thermotolerant Saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement Sornsiri Pattanakittivorakul . Noppon Lertwattanasakul . Mamoru Yamada . Savitree Limtong Received: 11 October 2018 / Accepted: 12 January 2019 © Springer Nature Switzerland AG 2019 Abstract A thermotolerant ethanol fermenting yeast strain is a key requirement for effective ethanol production at high temperature. This work aimed to select a thermotolerant yeast producing a high ethanol concentration from molasses and increasing its ethanol production by mutagenesis. Saccha- romyces cerevisiae DMKU 3-S087 was selected from 168 ethanol producing strains because it produced the highest ethanol concentration from molasses at 40 °C. Optimization of molasses broth composition was performed by the response surface method using Box–Behnken design. In molasses broth containing optimal total fermentable sugars (TFS) of 200 g/L and optimal (NH 4 ) 2 SO 4 of 1 g/L, with an initial pH of 5.5 by shaking flask cultivation at 40 °C ethanol, productivity and yield were 58.4 ± 0.24 g/L, 1.39 g/L/h and 0.29 g/g, respectively. Batch fermentation in a 5 L stirred-tank fermenter with 3 L optimized molasses broth adjusted to an initial pH of 5.5 and fermentation controlled at 40 °C and 300 rpm agitation resulted in 72.4 g/L ethanol, 1.21 g/L/h productivity and 0.36 g/g yield at 60 h. Strain DMKU 3-S087 improvement was performed by mutagenesis using ultraviolet radiation and ethyl methane sul- fonate (EMS). Six EMS mutants produced higher ethanol (65.2 ± 0.48–73.0 ± 0.54 g/L) in molasses broth containing 200 g/L TFS and 1 g/L (NH 4 ) 2 SO 4 by shake flask fermentation at 37 °C than the wild type (59.8 ± 0.25 g/L). Among these mutants, only mutant S087E100-265 produced higher ethanol (62.5 ± 0.26 g/L) than the wild type (59.5 ± 0.02 g/L) at 40 °C. In addition, mutant S087E100-265 showed better tolerance to high sugar concentration, furfural, hydroxymethylfurfural and acetic acid than the wild type. Keywords Ethanol fermentation · Thermotolerant yeast · Molasses · Mutagenesis · Ethyl methane sulfonate Introduction High temperature ethanol fermentation is required for effective ethanol production in tropical countries that have elevated temperatures throughout the year. Fermentation at high temperature has several advan- tages including rapid fermentation rate, reduction of S. Pattanakittivorakul · N. Lertwattanasakul · S. Limtong (&) Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand e-mail: fscistl@ku.ac.th M. Yamada Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan S. Limtong Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand 123 Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-019-01230-6