Microalgae are among the most promising of non-food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light-emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two-phase strategy with an initial nutrient-sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18: 2), and methyl linolenate (C18: 3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011; 108: 2280-2287. (C) 2011 Wiley Periodicals, Inc.