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Climate change due to global warming has led to the expansion of deciduous forests. Increased product yields from hardwood provide an opportunity for the exploitation of biomass resources, such as bioethanol. In this study, the enzymatic hydrolysis of various hardwoods pretreated using the hydrogen peroxide acetic acid (HPAC) method was evaluated. Glucose and xylose were fermented sequentially to improve the bioethanol production from hardwood. The HPAC pretreatment significantly reduced lignin and improved the hydrolysis efficiency compared with no treatment. The enhanced hydrolysis efficiency contributed to an increase in the bioethanol productivity, which was similar to that of simultaneous saccharification and fermentation, by reducing the processing time of separate hydrolysis and fermentation. The sequential fermentation of glucose and xylose via Saccharomyces cerevisiae and Pichia stipitis, respectively, improved ethanol production by approximately 12% over conventional fermentation used with glucose alone. The results suggest that sequential fermentation can improve bioethanol production from hardwood. (C) 2019 Elsevier Ltd. All rights reserved.