Abstract As atmospheric CO2 is rising alarmingly, focused research on microalgal based bio-mitigation of CO2 has become relevant. With the aim of single-objective maximization, response surface methodology (RSM) study was employed through central composite design (CCD) for the phototropic cultivation of Nannochloropsis sp. using Flue gas and synthetic fertilizer (NPK-10:26:26). Influence of crucial external factors such as light, salinity, carbon content and nutrients were investigated along with their interactions on three objectives such as Biomass, Lipid, and carbon bio-fixation rate. Using fertilizer assisted RSM optimized media, an enhancement of 43, 31, and 79% was observed in three responses over standard f/2 medium. Variation in carbon content (32%) and C/N ratio (100%) was observed, that validated diversion of metabolism under stress. The multi-objective optimization was carried out by genetic algorithm. A carbon supply strategy was employed that enhances biomass growth. Carbon sequestration was calculated first time precisely. Light intensity was concluded as a dominant external inducer to carbon-sequestration and lipid accumulation. Carbon sequestration rate was revealed to enhance in conditions that favor both good growths and induce stress. Single and multi-objective optimization data in this study can help in commercial decision making required in large scale-cultivations for biotechnological gains.