BACKGROUND: Microalgae biodiesel has attracted considerable attention, however, low lipid content has significantly restricted its development. Many research studies have demonstrated that nutrient deficiency is an important factor stimulating lipid accumulation in algal cells. This present study investigated the combined effects of nitrogen, phosphorus, and carbon on algal lipid accumulation by Chlorella vulgaris in mixotrophic culture. RESULTS: Phosphorus at 0.2 g L-1 concentration inhibited Chlorella vulgaris growth but did not influence lipid accumulation. High concentration of NaNO3 (3.75 g L-1) inhibited both algal growth and lipid accumulation. Nitrogen sufficiency can stimulate lipid accumulation. Glucose limitation restricted both biomass production and lipid accumulation, resulting in lipid content decrease even under nitrogen sufficiency. CO2 was unable to satisfy the carbon demand for lipid accumulation when glucose was exhausted in mixotrophic culture. The initial C:N ratio of culture medium played an important role in algal lipid accumulation. Lipid content was maintained at 7.6-11.3% when the initial C: N of culture was less than 50, while it increased significantly to 23.9% with initial C: N of culture increased to 92.7. CONCLUSION: Superior lipid accumulation requires limiting nitrogen concentration in the medium solution. High initial C: N ratio greater than 86 was found to be beneficial for algal lipid accumulation. (C) 2014 Society of Chemical Industry