An effective cell disruption method, including alkaline pretreatment and subsequent enzymatic treatment, was established to break cell walls and extract lipid from Nannochloropsis sp. A synergistic effect was found between alkaline pretreatment and enzymatic treatment. The combination of commercialize enzymes (cellulase, protease, lysozyme, and pectinase) achieved higher lipid yield compared with a single enzyme application. With the compromise between economic feasibility and lipid yield, the optimum reaction conditions were obtained with alkaline pretreatment at pH 10.5 at 110 degrees C for 4 h, and subsequent enzymatic treatment at pH 4 at 50 degrees C for 30 min with the dosage of each enzyme at 200 IU/g. As high as 90.0% of lipid was extracted under optimal conditions from Nannochloropsis sp. (C) 2016 Elsevier Ltd. All rights reserved.