Corn fiber hydrolysate was used as feedstock for microbial lipids production using the oleaginous Mortierella isabellina M2. First, we evaluated the effects of varying the HCl concentration, temperature, time and substrate concentration on corn fiber hydrolysis, then the effects of varying the concentrations of sugars, the nitrogen source, and culture time on lipid accumulation by M. isabellina M2. Under optimized corn fiber hydrolysis conditions, the sugars yield reached 83%. The microbial lipid content reached up to 50% when glucose, xylose, mannose and arabinose were used as single carbon sources, and when the corn fiber hydrolysate was used, the optimum sugar concentration, nitrogen source and cultivation time (for batch cultivation under otherwise constant conditions) were 60 g/l, 2 g/l (NH4)(2)SO4, and 6 days, respectively. In validation experiments under this set of culture conditions the biomass, lipid content and lipid yield reached 17.5 g/l, 50.5% and 8.84 g/l, respectively. Results of GC/MS analysis indicate that the fatty acid composition of the lipid was similar to that of vegetable oil, thus it is a promising material for biodiesel production. (C) 2012 Elsevier Ltd. All rights reserved.