| Summary: | Microbial lipids are able to produce from various raw materials including lignocellulosic biomass by the effective oleaginous microorganisms using different cultivation processes. This study aimed to enhance microbial lipid production from the low-cost substrates namely sugarcane top hydrolysate and crude glycerol by <i>Rhodosporidiobolus fluvialis</i> DMKU-SP314, using two-stage fed-batch cultivation with different feeding strategies in a 3 L stirred-tank fermenter. The effect of two feeding strategies of 147.5 g/L crude glycerol solution was evaluated including pulse feeding at different starting time points (48, 24, and 72 h after initiation of batch operation) and constant feeding at different dilution rates (0.012, 0.020, and 0.033 h<sup>−1</sup>). The maximum lipid concentration of 23.6 g/L and cell mass of 38.5 g/L were achieved when constant feeding was performed at the dilution rate of 0.012 h<sup>−1</sup> after 48 h of batch operation, which represented 1.24-fold and 1.27-fold improvements in the lipid and cell mass concentration, respectively. Whereas, batch cultivation provided 19.1 g/L of lipids and 30.3 g/L of cell mass. The overall lipid productivity increased to 98.4 mg/L/d in the two-stage fed-batch cultivation. This demonstrated that the two-stage fed-batch cultivation with constant feeding strategy has the possibility to apply for large-scale production of lipids by yeast.
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