Chlorella pyrenoidosa was grown in mixotrophic condition in presence of sodium acetate and glycerol. Nearly, six fold enhancement in biomass productivity and a remarkable 32 fold increment in lipid productivity were recorded in cultures grown with sodium acetate (10 g m(-3)) in comparison to autotrophic culture. With glycerol (0.5% by volume fraction), the biomass productivity and lipid productivity were three times and twenty times higher as compared to control. Glycerol proved to be more beneficial in lipid accumulation produces lipid content of 17.3%, which is about seven times higher to that of the control. While, lipid content with sodium acetate was 13.5%, more than five times of control. Oil samples collected from C. pyrenoidosa were converted into fatty acid methyl ester (FAME) through acid based transesterification and characterised by GC-MS. The fatty acid profiles of mixotrophic cultures showed its suitability for biofuel production than autotrophic cultures. The content of palmitic acid (C-16) and oleic acid (C-18) (indicator of biodiesel quality) is much higher in mixotrophic cultures than autotrophic cultures. C. pyrenoidosa, has shown significant improvement in growth and quantity as well as quality of lipid with acetate and glycerol. This provides a better way to produce biofuel at reduced overall cost since these substrates can be obtained as waste byproducts of some processes like bio-hydrogen production and biodiesel production. (C) 2013 Elsevier Ltd. All rights reserved.