Hydrothermal acid (combined with autoclaving and nitric acid) pretreatment was applied to Nannochloropsis salina as a cost-effective yet efficient way of lipid extraction from wet biomass. The optimal conditions for this pretreatment were determined using a statistical approach, and the roles of nitric acid were also determined. The maximum lipid yield (predicted: 24.6%; experimental: 24.4%) was obtained using 0.57% nitric acid at 120 degrees C for 30 min through response surface methodology. A relatively lower lipid yield (18.4%) was obtained using 2% nitric acid; however, chlorophyll and unsaturated fatty acids, both of which adversely affect the refinery and oxidative stability of biodiesel, were found to be not co-extracted. Considering its comparable extractability even from wet biomass and ability to reduce chlorophyll and unsaturated fatty acids, the hydrothermal nitric acid pretreatment can serve as one direct and promising route of extracting microalgae oil. (C) 2014 Elsevier Ltd. All rights reserved.