For the environmentally sustainable development, the recovery of resource or energy from wastes has attracted worldwide attention in recent years. The wastewater produced from distillery is more difficult to treat than the domestic wastewater due to the high organic loads. This study explores the possibility of developing a process, which could produce microbial lipid convertible to biodiesel by recovering nutrients from non-sterile wastewater while simultaneously removing part of organic matters through cultivating oleaginous microorganisms. Especially, it is intended to further optimize the initial cell density and the usage efficiency of seed culture medium. The lipid productivity by oleaginous yeast Rhodosporidium toruloides was studied using wastewater from rice wine distillery, at 30ºC under different initial cell densities (2 x 10⁷, 1 x 10⁷, 0.8 x 10⁷, and 0.5 x 10⁷ cells/mL) and incubation periods (2-5 days). Chemical oxygen demand (COD) removal efficiency, cell yield, and lipid yield were analyzed and the potential for bioenergy recovery from wastewater was assessed. The seed culture medium was found reusable for the second time without the addition of extra nutrient and possible for the third time with the addition of glucose.