Background: Microalgae, due to its well-recognized advantages have gained renewed interest as potentially good feedstock for biodiesel. Production of fatty acid methyl esters (FAMEs) as a type of biodiesel was carried out from Chlorella vulgaris bio-oil. Biodiesel was produced in the presence of nano-biocatalysts composed of immobilized lipase on functionalized superparamagnetic few-layer graphene oxide via a transesterification reaction. A hybrid of few-layer graphene oxide and Fe 3 O 4 (MGO) was prepared and characterized. The MGO was functionalized with 3-aminopropyl triethoxysilane (MGO-AP) as well as with a couple of AP and glutaraldehyde (MGO-AP-GA). The Rhizopus oryzae lipase (ROL) was immobilized on MGO and MGO-AP using electrostatic interactions as well as on MGO-AP-GA using covalent bonding. The supports, MGO, MGO-AP, and MGO-AP-GA, as well as nano-biocatalyst, ROL/MGO, ROL/ MGO-AP, and ROL/MGO-AP-GA, were characterized using FESEM, VSM, FTIR, and XRD. The few-layer graphene oxide was characterized using AFM and the surface charge of supports was evaluated with the zeta potential technique. The nano-biocatalysts assay was performed with an evaluation of kinetic parameters, loading capacity, relative activity, time-course thermal stability, and storage stability. Biodiesel production was carried out in the presence of nanobiocatalysts and their reusability was evaluated in 5 cycles of transesterification reaction. Results: The AFM analysis confirmed the few-layer structure of graphene oxide and VSM also confirmed that all supports were superparamagnetic. The maximum loading of ROL (70.2%) was related to MGO-AP-GA. The highest biodiesel conversion of 71.19% achieved in the presence of ROL/MGO-AP-GA. Furthermore, this nano-biocatalyst could maintain 58.77% of its catalytic performance after 5 cycles of the transesterification reaction and was the best catalyst in the case of reusability. Conclusions: In this study, the synthesized nano-biocatalyst based on bare and functionalized magnetic graphene oxide was applied and optimized in the process of converting microalgae bio-oil to biodiesel for the first time and compared with bare lipase immobilized on magnetic nanoparticles. Results showed that the loading capacity, kinetic parameters, thermal stability, and storage stability improved by the functionalization of MGO. The biocatalysts, which were prepared via covalent bonding immobilization of enzyme generally, showed better characteristics.

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