Biodiesel production from microalgae is generally conducted through light dependent growth. Though promising and technically feasible, it is logistically difficult to produce a high-density biomass of microalgae due to poor light penetration. Light dependency complicates cultivation process, increases cost and reduces production of desired products. Heterotrophic culture is an attractive alternative to this challenge. However, lipid accumulation and total lipid productivity remain low, increasing downstream processing cost. The current study avoids these issues by growing microalgae, Chlorella kessleri, in nutrient limited heterotrophic cultures supplemented with exogenous pure sugars or lignocellulose hydrolysate from Big Blue Stem. Lignocellulosic material is a readily available renewable source of carbon. Its economic feasibility makes it an attractive carbon source. In addition, we have explored strategies to increase lipid content of cells in this research. Growth of microalgae in stressful environment under low nitrogen conditions causes algal cells to synthesize more lipids. We studied effects of phosphorus and iron limitation on lipid accumulation. The supplemented sugar promotes biomass while phosphorus and iron limitation stresses cell altering its metabolism and generating high lipid densities within cell. Cell counting method and chlorophyll measurements measured heterotrophic growth of microalgae at different concentrations of phosphorus (0 mM-5 mM) and iron (0 μM-20 μM) each day until stationary phase was reached. Furthermore, Nile Red staining measured lipid content of cells at stationary phase. A modified Bligh and Dyer lipid extraction method was adapted to extract lipids from microalgae. When phosphorus was limited to 95%, it resulted in 24.07 mg L-1 and 26.93 mg L-1 total lipid concentration in microalgae grown heterotrophically with sucrose and Big Blue Stem hydrolysate respectively. Therefore, this novel approach allows for flexible production of renewable fuels within existing infrastructure.