Lipid was produced by Rhodosporidiobolus fluvialis DMKU-SP314 from a mixture of low-cost substrates: sugarcane top hydrolysate (STH) and biodiesel-derived crude glycerol. The optimized lipid production medium for shaking flask cultivation contained STH supplemented with 59 g/L crude glycerol, 0.21 g/L soybean powder, 0.9 g/L (NH4)(2)SO4, 0.4 g/L KH2PO4, 2.0 g/L MgSO4 center dot 7H(2)O and pH 6.1 (C/N ratio 60), while the optimal cultivation temperature was 28 degrees C. Batch cultivation in a 2 L stirred-tank fermenter at an agitation speed of 300 rpm and an aeration rate of 2 vvm achieved the maximum lipid quantity of 18.2 g/L, which was 75.0% of dry biomass (24.3 g/L biomass), after 240 h of cultivation. These represent a 5.4-fold and 1.7-fold improvement in the lipid concentration and lipid content, respectively, compared to non-optimized shaking flask cultivation. Under optimal conditions, the lipid produced had a high monounsaturated fatty acid content (36.6% of oleic acid), which is suitable for biodiesel production. Thus, R. fluvialis DMKU-SP314 is capable of producing large quantities of lipid from low-cost raw materials in a process that can be readily scaled up for industrial production.