With the growing energy crisis and environmental pollution worldwide, the environmental benefits of biodiesel have made it a more attractive product. However, the production of biodiesel feedstock can only satisfy a small fraction of the demand for transportation fuels, and the cost of this feedstock is the main obstacle to the commercialization of biodiesel. The marine microalgae Schizochytrium is a promising producer of biodiesel feedstock because of its short life cycle, high cell density, high lipid content and high palmitic acid (PA) content, all of which can improve the quality of biodiesel. In this study, a sequential optimization of the culture medium, based on response surface methodology (RSM), was employed to increase the PA production in Schizochytrium and lower the process cost. As a result, the PA production by Schizochytrium sp. S056 increased from 14.69 +/- 0.62 g/L to 25.95 +/- 0.11 g/L using the optimal conditions. In addition, the quality analysis of the crude lipid showed that the lipid produced by the microbial process is a promising feedstock for biodiesel production. (C) 2015 Elsevier Ltd. All rights reserved.