Mixotrophic growth of microalgae to boost lipid production is currently under active investigation. Such a process could be of practical importance if a cheap source of organic carbon, such as waste glycerol from biodiesel production, could be used. Several previous studies have already demonstrated that this carbon source can be used by different indigenous strains of microalgae. In this study it is shown that different nitrogen limitation strategies can be applied to further increase lipid production during growth with glycerol. In one strategy, cultures were grown in nitrogen replete medium and then resuspended in nitrogen free medium. In a second strategy, cultures were grown with different initial concentrations of nitrate. Lipid production by the two microalgal strains used, Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), was shown to be boosted by strategies of nitrogen limitation, but they responded differently to how nitrogen limitation was imposed. (C) 2016 Elsevier Ltd. All rights reserved.