Kinetic study of immobilized lipases in biodiesel synthesis from waste cooking oil within ultrasonic system and its biodiesel performance

Two commercial immobilized lipases, Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RMIM) were evaluated with molar ratio of WCO to methyl acetate ranging from 1:3 to 1:12 under ultrasonic conditions at 45℃, 1.8 wt% enzyme loading and 5kHz ultrasonic frequency. As a result, CALB demonstrated the biodiesel yield of 81.2% at a 1:12 molar ratio, whereas RMIM attained a maximum yield of 16.6% at 1:3. The superior performance of CALB was attributed to its non-specific catalytic activity, enabling triglyceride conversion at all positions, in contrast to the sn-1,3 positional specificity of RMIM. The formation of triacetin as a by-product was confirmed by FTIR analysis, with a characteristic absorption peak at 1238.90 cm-1. Based on its superior catalytic performance at the optimum 1:12 WCO-to-methyl acetate molar ratio, CALB was selected for kinetic evaluation. The enzymatic transesterification kinetics, involving two substrates, were described using a Ping Pong Bi-Bi mechanism with competitive inhibition by methyl acetate, and kinetic parameters were estimated via non-linear regression using Polymath 6.10 software. The fittings results were as follows: Vmax = 0.000026M/min; KmTG = 0.0050M; KmA = 0.178M and KiA = 0.0057M. As a result, the model was fitted with enzyme kinetic data from the experiment and good correlations were achieved. The produced biodiesel was blended with Industrial Fuel (B7) and Commercial Diesel Fuel (B10) to form B20a and B20b blends respectively, each containing 20% biodiesel by volume. Diesel engine testing showed a reduction in engine run time with increasing biodiesel content. Exhaust emission analysis revealed a 2.13% reduction in NOx for B20a, while a slight 1.48% increase was observed for B20b. Notably, CO₂ emissions decreased significantly, by 13.9% for B20a and 20.9% for B20b, although CO emissions increased for both blends. The observed reductions in NOx and CO₂ demonstrate the potential of biodiesel derived from enzymatic transesterification of WCO as a more sustainable and lower-carbon alternative fuel. These findings support Malaysia’s biofuel policy promoting higher biodiesel blends and reinforce the environmental viability of WCO-based biodiesel.