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This study focused on the valorization of the date syrup obtained from low-quality date fruits to be used as a low-cost alternative medium for producing single cell oil (SCO) by Rhodotorula glutinis PTCC5256, which could further be converted into biodiesel. The higher C/N ratio of date syrup (C/N 70) led to restricting the formation of cell biomass and enhancing the biosynthesis of SCO. The maximal cell biomass and lipid productivities were obtained 72 mg/L/h and 17 mg/L/h by C/N ratios of 20 and 70, respectively. Although the obtained biodiesel met the international standards for cold filter plugging point (4.92 °C), iodine value (87.22 g I2/100 g oil), cetane number (52.26), higher heating value (40.19 MJ/kg), cloud point (6.29 °C), pour point (0.00 °C), density (878 kg/m3), kinematic viscosity (4.30 mm2/s) and oxidation stability (7.87 h), its weak cold-flow properties might limit its application in cold areas in comparison with diesel fuel.