Thermostable CaCO₃-Immobilized Bacillus subtilis Lipase for Sustainable Biodiesel Production from Waste Cooking Oil

Due to the increasing demand for green processes in renewable energy production, the extracellular Bacillus subtilis B-1-4 lipase was used as a biocatalyst for producing biodiesel from waste cooking oil. Response surface methodology was employed for the optimization of enzyme production. Lipase activity was modeled with a quadratic function of four factors that primarily influence the culture medium. Thanks to this model, an optimal lipase activity of 1.7 ± 0.082 U/mL was achieved with the best culture medium composition: 16 g/L of tryptone, 15 g/L of yeast extract, 15 g/L of NaCl, and a 0.15 initial optical density at 600 nm (OD600 nm). The maximal lipase activity was measured at 45 °C and pH 8, using para-nitrophenyl palmitate as a substrate. The enzyme maintained above 94% and 99% of its initial activity at temperatures ranging from 40 to 50 °C and at pH 8, respectively. Moreover, it exhibited a higher residual activity than other Bacillus lipases in the presence of organic solvents. Residual activities of 86.7% and 90.2% were measured in the presence of isopropanol and ethanol, respectively. The lipase was immobilized by adsorption onto CaCO3 powder. FT-IR and SEM were used to characterize the surface-modified support. After immobilization, a lipase activity of 7.1 U/mg of CaCO3 was obtained. Under the optimized conditions, the highest biodiesel yield of 71% was obtained through the transesterification of waste cooking oil using the CaCO3-immobilized Bacillus subtilis lipase. This research reveals a method for the utilization of waste cooking oil for biodiesel production using an efficient immobilized thermostable lipase, providing environmental and economic security.