This study aimed to optimize the growth conditions of the microalgaeAuxenochlorella protothecoidesto maximize the biomass and lipid content. The pH values and acetate concentrations of a tris-acetate-phosphate medium were varied for a photoheterotrophically grown culture ofA. protothecoides. Approximately 23% of the microalgal biomass consisted of saturated fatty acids (SFAs). The highest lipid content (47.8%) was achieved with a medium containing 17 mM acetate at pH 6.2, and a medium containing 50 mM acetate at pH 5.8 resulted in the highest SFA content (43.7%). Surface response methodology was used to analyze the effects of two independent variables, pH (5.8-7.2) and acetate (0-35 mM), on lipid and biomass content. Four quadratic models, using the Box-Wilson central composite design, were proposed to correlate the optimal pH and acetate concentrations that would achieve the maximum biomass and lipid content. The study demonstrated that medium containing 24.4 mM acetate at pH 5.96 resulted in the highest lipid content (47.8%), and resulted in the highest biomass (1537 mg/L). Microalgae growth under these optimized conditions can enhance biomass and lipid productivity and can provide suitable conditions for high-quality biodiesel.