BackgroundThe use of microbial biomasses, such as fungal biomass, to catalyze the transesterification of triglycerides (TG) for biodiesel production provides a sustainable, economical alternative while still having the main advantages of expensive immobilized enzymes.ResultsBiomasses ofAspergillus flavusandRhizopus stoloniferawere used to catalyze the transesterification of TG in waste frying oil (WFO). Isopropanol as an acyl-acceptor reduced the catalytic capability of the biomasses, while methanol was the most potent acyl-acceptor with a final fatty acid methyl ester (FAME) concentration of 85.5 and 89.7%, w/w, forR. stoloniferandA. flavus, respectively. Different mixtures of the fungal biomasses were tested, and higher proportions ofA. flavusbiomass improved the mixture's catalytic capability.C. sorokinianacultivated in synthetic wastewater was used as feedstock to cultivateA. flavus.The biomass produced had the same catalytic capability as the biomass produced in the control culture medium. Response surface methodology (RSM) was adopted using central composite design (CCD) to optimize theA. flavusbiomass catalytic transesterification reaction, where temperature, methanol concentration, and biomass concentration were selected for optimization. The significance of the model was verified, and the suggested optimum reaction conditions were 25.5 °C, 250 RPM agitation with 14%, w/w, biomass, 3 mol/L methanol, and a reaction duration of 24 h. The suggested optimum conditions were tested to validate the model and a final FAME concentration of 95.53%. w/w was detected.ConclusionBiomasses cocktails might be a legitimate possibility to provide a cheaper technical solution for industrial applications than immobilized enzymes. The use of fungal biomass cultivated on the microalgae recovered from wastewater treatment for the catalysis of transesterification reaction provides an additional piece of the puzzle of biorefinery. Optimizing the transesterification reaction led to a valid prediction model with a final FAME concentration of 95.53%, w/w.