AbstractEight hundreds and eighty nine yeast strains were isolated from soils and wastes of palm oil mill and biodiesel plant in southern region of Thailand using glucose or glycerol as carbon source and at acidic condition (pH 4.0) or neutral condition (pH 6.0) with 0.0001% chloramphenicol. By applying Sudan Black B tests, 23 strains were identified as potential lipid producer or oleaginous yeast. The lipid contents of these 23 strains were compared in crude glycerol based medium. It was found that BY4-523 accumulated highest lipid content up to 53.28% while JU4-57 grew fastest and gave comparable high lipid content 41.50%. They were identified as Kodamaea ohmeri and Trichosporonoides spathulata, respectively. Among organic nitrogen sources tested, a mixture of yeast extract and peptone (1:1) gave the best biomass (17.05g/L for T. spathulata and 11.1g/L for K. ohmeri) and the maximum lipid production (10.43g/L for T. spathulata and 4.53g/L for K. ohmeri). In the view point of economic strategy, the cheaper inorganic nitrogen sources were also tested. Among inorganic nitrogen sources tested, ammonium sulfate was selected as a suitable nitrogen source. It gave the best biomass (9.17g/L for T. spathulata and 10.45g/L for K. ohmeri) and the maximum lipid production (3.85g/L for T. spathulata and 3.17g/L for K. ohmeri). The results showed that the newly isolated yeasts could grow and accumulate high lipid content in crude glycerol based medium supplemented with only ammonium sulfate. The optimal medium composition for both strains was 0.5% ammonium sulfate and 10% crude glycerol (C/N ratio of 17). Under this condition, the maximum biomass of 10.40g/L and lipid production of 4.45g/L were achieved for T. spathulata. Similarly, K. ohmeri also reached the maximum biomass of 10.50g/L and lipid production of 3.22g/L.