The airlift bioreactor had superior performance on the growth of R. glutinis as compared to the agitation tank. Furthermore, the operation of fed-batch by feeding crude glycerol could enhance cell growth as compared to simple batch cultivation. The exponential feeding criteria could avoid the high concentration of crude glycerol accumulated, and led to the enhancement of cell growth rate. Nevertheless, the conventional simple pulse feeding criteria achieved the highest total lipids and β-carotene productivity. Therefore, from the perspective of collecting the maximum total lipids for biodiesel production, the fed-batch operation by pulse feeding is suggested as a suitable criterion for the cultivation of R. glutinis with crude glycerol as the carbon source.