A combined immobilization system for high-solids cellulosic ethanol production by simultaneous saccharification and fermentation

A novel combined immobilization technology was introduced in this work to improve the efficiency and stability of a high-solids biocatalysis system. First, a nano biocatalyst based on commercial cellulase immobilized onto the Fe3O4@SiO2-APTES nanoparticles was synthesized utilizing glutaraldehyde as a chemical crosslinking agent with a final immobilization efficiency of 93.0 %. A decrease in the Vmax and Km values indicates that the nanoparticle-immobilized cellulase enzyme has an increased binding affinity for the cellulose substrate. Next, a recombinant yeast strain was constructed via yeast cell-surface immobilization with laccase and versatile peroxidase for synergistic lignin-degradation. Functional accessibility was detected by using flow cytometry and immunofluorescence microscopy. Finally, the cooperative biocatalysis of nanoparticle-immobilized cellulase and cell-surface immobilized S. cerevisiae strain for SSF were investigated. Fed-batch operation of the solids at a 30.0 % (DW, w/w) final substrate loading was implemented to manage the instantaneous concentration level of inhibitors and enhance the saccharification of undetoxified biomass. The combined immobilization produced a maximum ethanol titer of 79.5 ± 4.3 g/L and a theoretical ethanol yield of 88.2 % with a low cellulase loading of 10 FPU/g cellulose. This work presented a promising green pathway for achieving efficient cellulosic ethanol production, highlighting potential industrial applications and contributing to manufacturing optimization.