In situ transesterification of oleaginous microbes with short chain alcohol has been developed as a renewable process for the production of biodiesel. Dry biomass is often a requisite for the process to avoid the adverse effect of water on the productivity. As a consequence, large amount of energy consumption is required for prior biomass drying. In this study, the wet biomass of Rhodotorula glutinis, an oleaginous yeast, was used directly in in situ transesterification without biomass drying. The reaction conditions were optimized for the production of fatty acid methyl esters (FAME) and the effects of adding different surfactants were also studied. The highest FAME yield of 110% was achieved with a methanol loading of 1:100 at 90 degrees C for 8 h as catalyzed by 0.36 M H2SO4, and the FAME content was 97%, which meets the 96.5% specified in both European biodiesel standards and Taiwanese biodiesel standards. The addition of 50 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate (3-DMAPS, a zwitterionic surfactant) improved the FAME yield from 69% to 83%, which was obtained with a low methanol loading of 1:10 at 90 degrees C for 10 h. Hence, the production of FAME with wet biomass under optimized reaction conditions was as effective as that with the dry form. This clearly indicates that using wet R. glutinis as the feedstock is feasible for the production of biodiesel by in situ transesterification. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.