We attempted to integrate lipase-catalyzed ethanolysis into fermentative bioethanol production. To produce bioethanol, ethanol fermentation from brown rice was conducted using a tetraploid Saccharomyces cerevisiae expressing alpha-amylase and glucoamylase. The resultant ethanol was distilled and separated into three fractions with different concentrations of water and fusel alcohols. In ethanolysis using the first fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst expressing Fusarium heterosporum lipase (r-FHL) afforded the highest ethyl ester content of 94.0% after 96 h. Owing to a high concentration of water in the bioethanol solutions, r-FHL, which works best in the presence of water when processing ethanolysis, was found to be more suitable for the integrative process than a commercial immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch ethanolysis, resulting in an ethyl ester content of more than 80% even after the fifth batch. Fusel alcohols such as 1-butanol and isobutyl alcohol are thought to decrease the lipase activity of r-FHL. Using this process, a high ethyl ester content was obtained by simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water concentration. The developed process model, therefore, would contribute to biodiesel production from only biomass-derived feedstocks. (c) 2012 Elsevier Inc. All rights reserved.