Lipids extracted from microalgae have been considered as a potential source for the production of biodiesel. Enhancement of lipid has the limitations of low biomass productivity. So, the main objective of the present study was to deduce suitable conditions for the improvement of biomass production followed by enhancement of lipid content. After optimization, a strategy for two stage cultivation was utilized where high lipid content was obtained with a high biomass concentration. Optimization of biomass production of Chlorella minutissima MCC 5 was carried out under different intensities of light, temperatures, concentrations of nitrate and phosphate using Taguchi model. A suitable synergy of the four parameters yielded maximum biomass (1.93 g L-1) in airlift reactor. Temperature was found to be relatively effective than other parameters for higher biomass production. Activation energy for the cell growth was determined (47.95 kJ mol(-1)). Among the various (photo, thermal, nitrate and phosphate) stress conditions studied, nitrate limitation (1 mM) was found to be suitable for the enhancement of lipid resulting highest yield (48.26% w/w). Two stage cultivation of the microalgae yielded a maximum lipid content of 46% w/w with a biomass concentration of 2.2 g L-1. Additionally, FAME analysis exhibited significant increase of oleic add in the biodiesel. So, C. minutissima MCC 5 cultivated under nitrate stress could be a possible feedstock for biodiesel production. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.