Previous studies showed that lipids could be produced from non-sterile distillery wastewater using high initial cell density of oleaginous yeast Rhodosporidium toruloides while lots of spent seed culture medium was generated during the seed culture preparation. This study explores the possibility to reuse the spent seed culture medium while saving resource and cost. The cells produced from the 2nd time reused medium without extra nutrient addition showed similar performances to the fresh medium in lipid production (biomass 7.42 +/- 0.31 g/L, lipid yield 2.74 +/- 0.42 g/L, and lipid content 36.90 +/- 4.36%) and removal efficiencies for organics and nutrients (chemical oxygen demand, total nitrogen, and total phosphorus removal of 86.47 +/- 1.40%, 50.73 +/- 6.15%, and 74.36 +/- 3.39%, respectively), in non-sterile distillery and domestic mixed wastewater after two-day cultivation. The 3rd time reused medium showed relatively low removal efficiencies for organics and nutrients as well as poor lipid production at the early culture stage, resulting in the reutilization of 3rd time spent medium not recommended even with 50% nutrient supplementation. Around 30% reduction in the material cost for the medium preparation could be achieved by the spent medium reutilization. (C) 2016 Elsevier Ltd. All rights reserved.