Biomass and lipid accumulation of heterotrophic microalgae Chlorella protothecoides by supplying mixed waste substrate of brewer fermentation and crude glycerol were investigated. The biomass concentrations of the old and the new C. protothecoides strains on day 6 reached 14.07 and 12.73 g/L, respectively, which were comparable to those in basal medium with supplement of glucose and yeast extract (BM-GY) (14.47 g/L for old strains and 11.43 g/L for new strains) (P > 0.05). Approximately 81.5% of total organic carbon and 65.1% of total nitrogen in the mixed waste were effectively removed. The accumulated lipid productivities of the old and the new C. protothecoides strains in BM-GY were 2.07 and 1.61 g/L/day, respectively, whereas in the mixed waste, lipid productivities could reach 2.12 and 1.81 g/L/day, respectively. Our result highlights a new approach of mixing carbon-rich and nitrogen-rich wastes as economical and practical alternative substrates for biofuel production. (C) 2014 Elsevier Ltd. All rights reserved.