The oleaginous alga Chlorella protothecoides accumulates lipid in its biomass when grown in nitrogen-restricted conditions. To assess the relationship between nitrogen provision and lipid accumulation and to determine the contribution of photosynthesis in mixotrophic growth, C protothecoides was grown in mixo- and heterotrophic nitrogen-limited continuous flow cultures. Lipid content increased with decreasing C/N, while biomass yield on glucose was not affected. Continuous production of high lipid levels (57% of biomass) was possible at high C/N (87-94). However, the lipid production rate (2.48 g L-1 d(-1)) was higher at D = 0.84 d(-1) with C/N 37 than at D = 0.44 d(-1) and C/N 87 even though the lipid content of the biomass was lower (38%). Photosynthesis contributed to biomass and lipid production in mixotrophic conditions, resulting in 13-38% reduction in CO2 production compared with heterotrophic cultures, demonstrating that photo- and heterotrophic growth occurred simultaneously in the same population. (C) 2013 Elsevier Ltd. All rights reserved.