Influence of mixotrophic mode and its transition to various trophic modes under stress conditions was assessed during two stage cultivation of Chlorella pyrenoidosa. Significant lipid productivity was triggered under low light intensity, glucose + bicarbonate supplementation and nitrogen starvation. The association between biomass and lipid productivity, fatty acid composition during mixotrophic transition was critically evaluated. Biomass in growth phase (GP) and stress phase (SP) was 6.14 g/l and 5.14 g/l, respectively, in mixotrophic mode. Higher lipid productivity of 284 g/kg and 154.3 g/kg of neutral lipids was achieved in SP in mixotrophic-mixotrophic (MM) and mixotrophic-heterotrophic (MH) modes, respectively. Stress conditions resulted in high unsaturated fatty acid methyl esters in MH mode. In addition, neutral lipid content was 58% in MH and 52% in MM, that can be attributed to carbon source that is supplemented even in stress phase. Exploring such novel strategies can generate sustainable avenues for biodiesel production.