BACKGROUND Oleaginous yeast Naganishia albida is well known for its ability to utilize a variety of carbon sources, including lignocellulosic hydrolysates, complex sugars and fatty acids. In this study, crude glycerol derived from the biodiesel industry and onion waste hydrolysate (OWH) were used as potential substrates for microbial lipid production. RESULTS Both substrates provided sufficient nutrition to support cell growth and lipid production as sole carbon source. Enhanced lipid production and cell growth rate were observed with supplementation of yeast extract as nitrogen source. Co-fermentation of OWH and crude glycerol led to improved biomass as well as lipid production. The highest lipid content of 34.0% (w/w) with 21.1 g L-1 of dry cell weight was obtained after 168 h of fed-batch fermentation with intermittent feeding of crude glycerol and OWH. The lipid concentration was approximately 7 times higher than using mineral medium with crude glycerol as sole carbon source, where a lipid content of 12.6% (w/w) and a maximum biomass of 8.32 g L-1 were obtained at 168 h. CONCLUSION Optimization of glycerol and OWH feeding could enhance lipid accumulation in N. albida. This study suggests that the co-fermentation of agricultural and industrial waste as substrates can provide a competitive route for single cell oil production. (c) 2019 Society of Chemical Industry