The influence of medium constituents on the production of biosurfactants by Candida tropicalis cultivated in waste frying oil was investigated according to a fractional factorial 25‐1 design. The combined effect of the C/Ninorganic, C/Fe, C/Mg, and C/P ratios and yeast extract on surface tension reduction, biosurfactant yield, emulsification activity, and biomass were studied. The highest biosurfactant yield was reached when low C/Mg and low C/P ratio variables were combined, while the cell growth was favored by increasing the nitrogen concentration. The highest surface tension net decrease, on the other hand, was observed at low yeast extract concentration, low C/Fe, and high C/P ratios. Emulsification indices against lubrication and automobile waste oil of approximately 65 to 95% were observed. The crude biosurfactant produced in the medium—formulated with 2% waste frying oil, 0.067% NH4Cl, 0.025% MgSO4·7H2O, 0.067% KH2PO4, and 0.0026% FeCl3·6H2O—removed approximately 78 to 97% of the petroleum and motor oil adsorbed in sand samples.

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