Barley hull hydrolysate was used as the fermentation feedstock of Trichosporon cutaneum CTM-30125 yeast strain for production of microbial lipid as the potential raw material of biodiesel. An effective hydrolysis process based on the combination of dilute acid hydrolysis and enzymatic saccharification was developed to maximize recovery of total sugars. On synthetic media, CTM-30125 can assimilate glucose and xylose simultaneously and accumulated intracellular lipid up to 43.6% (w/w). However, when grown on acid barley hull hydrolysate (ABHH) the lipid content was low (16.6% w/w) and a detoxification step was required. Overliming by Ca(OH)(2) treatment and adsorption by activated charcoal improved the fermentability of ABHH significantly by removing the inhibitors and reducing the nitrogen content. The solid fraction resulting from the acid treatment of the barley hull was treated with hydrolytic enzyme complex produced by the hyper-cellulolytic fungal strain Trichoderrna reesei Rut C-30. Enzyme production and hydrolytic conditions were optimized to enhance the saccharification. The structural changes in native and treated raw material were monitored using FTIR spectra. Detoxified ABHH and enzymatic hydrolysate were successively fermented by the CTM-30125 strain in fed-batch bioreactor without addition of external nitrogen source. A total biomass of 17.5 g/L with a lipid content of 38.2% (cor-responding to a lipid yield of 6.7 g/L) could be achieved after 168 h of culture. The relative fatty acid composition indicates the presence of increased levels of saturated (41.2%) and monounsaturated (52.5%) fatty acids in the oil, making it ideal for biodiesel production. (C) 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.