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Because it is a by-product of bio-diesel industry, glycerol is expected to be available in a large quantity with low costs.To valorize the raw glycerol derived from biodiesel processing, the oleaginous yeast R.toruloides Y4 was applied to convert glycerol into microbial lipid by using a two-staged batch fermentation process.The 250 mL flask fermentation system containing 50 g/L raw glycerol exhibited a lipid productivity of 1.28 g/(L·d) with average lipid concentrations around 6.40 g/L and lipid yield of 0.128 (g lipid of per g glycerol).Compared with refined glycerol, the crude glycerol led to significantly higher biomass concentration and lipid yield.The highest cell mass concentration was 16.6 g/L with intracellular lipid content of 39%, of which the content of saturated fatty acids was over 60%.Such microbial lipid may be an alternative for cocoa butter.Further studies were conducted to investigate the effects of 7 representative operational parameters on the performance of the yeast culture.The results indicated that crude glycerol was directly converted to triacylglycerols, and that salt and methanol promoted the accumulation of cell mass and lipid.