Heterotrophic microalgae, Aurantiochytrium sp. KRS101 had a large amount of lipid (56.8% total lipids). The cells in the culture medium were easily ruptured due to thin cell wall of Aurantiochytrium sp., which facilitated in-situ fatty acid methyl esters (FAMEs) production directly from biomass. The harvested biomass had a high content of free fatty acids (FFAs), which was advantageous for glycerol-free FAMEs production. FAMEs were directly produced from Aurantiochytrium sp. KRS101 biomass (48.4% saponifiable lipids) using Novozyme 435-catalyzed in-situ esterification in dimethyl carbonate (DMC). DMC was used as a lipid extraction reagent, acyl acceptor and reaction medium. A 433.09 mg FAMEs/g biomass was obtained with 89.5% conversion under the optimal condition: DMC to biomass ratio of 5:1 (v/w) and enzyme to biomass ratio of 30% (w/w) at 50 degrees C for 12 h. Glycerol could not be detected in the produced FAMEs. (C) 2016 Elsevier Ltd. All rights reserved.