The recovery of resources and energy from wastes has been drawing worldwide attention for an environmentally sound and sustainable development. Studies have been focusing on the production of microbial lipid from sterile wastewater and its further conversion to biodiesel while simultaneously removing part of organic matter via cultivation of oleaginous microorganisms. An enhancement of the initial cell density has shown this process applicable to real distillery wastewater but lots of waste seed culture medium was generated while preparing high cell density seed culture. Current study explores the possibility to reuse the spent seed culture medium (YPD medium) to save resource and cost. Compared to the fresh YPD medium producing 2.8 x 10 ¹¹ yeast cells/L (average), the 2 nd and 3 nd time reused media produced 2.1 x 10 ¹¹ and 1.3 x 10 ¹¹ cells/L, respectively, without any extra addition of carbon source. Results suggest the seed culture medium for oleaginous yeast strain could successfully be reused for the 2 nd time with similar lipid productivity without further addition of nutrients. The lipid productivity simultaneously with the wastewater treatment for real distillery wastewater and the cost reduction analysis were assessed using cells from the reused media and compared with the fresh medium.