Improved performance of a packed-bed reactor for biodiesel production through whole-cell biocatalysis employing a high-lipase-expression system

► A packed-bed reactor system was developed using recombinant fungal whole-cells. ► We used immobilized Aspergillus oryzae employing a high-lipase-expression system. ► Optimization enabled the achievement of a methyl ester content above 96%. ► The batch methanolysis could be repeated for 5 cycles. To improve enzymatic biodiesel production, we developed a packed-bed reactor (PBR) system using recombinant cells, into which a strong enolase promoter and 5′ untranslated region were introduced. Aspergillus oryzae expressing Fusarium heterosporum lipase was immobilized within biomass support particles (BSPs) during cultivation and used directly as a whole-cell biocatalyst. BSP-immobilized A. oryzae carrying three copies of the expression cassette showed a higher performance than previously developed cell systems, resulting in 87.5% conversion after 10 passes in PBR. The performance was also affected by operational factors including residence time and methanol feeding. After optimization, the PBR system attained a final methyl ester content of 96.1% with a residence time of 140min per pass and stepwise addition of 4.25molar equivalents of methanol to oil for 6 passes. Moreover, lipase activity was maintained for 5 batch cycles. Therefore, the developed PBR employing a high-lipase-expression system is considered useful for improving enzymatic biodiesel production.