Culturing microalgae heterotrophically for producing lipid-based biofuels such as biodiesel and renewable hydrocarbons has attracted increasing attention due to the advantages of fast growth and high lipid yield under this growth mode without being subjected to light limitation. High cell density in the culture broth is desirable for reducing downstream processing costs. Oleaginous microalga Chlorella sorokiniana was investigated for high cell density culture with glucose as the carbon source. Best growth performance was obtained first with batch culture at pH 7.0 when ammonium was the nitrogen source. Then, two-stage fed-batch fermentation was conducted under the optimal conditions. The algal biomass grew linearly in the first stage with a productivity of 24.2 g L-1 d(-1), and the lipid content increased from 14.5% to 38.7% in the second stage. This fermentation strategy resulted in algal biomass and lipid concentrations of 103.8 g L-1 and 40.2 g L-1 respectively. Analysis of lipid and fatty acid profiles showed C. sorokiniana accumulated a large amount of neutral lipids (92.9% of total lipids), triacylglycerols (82.8% of neutral lipids), and high contents of palmitic, oleic and linoleic acids, which are ideal form of lipid for making biodiesel. These results suggest that heterotrophic culture of C sorokiniana holds great potential for lipid-based biofuel production. (c) 2013 Elsevier Ltd. All rights reserved.