Visible light-driven modulation of hydrogen biosynthesis pathways boosts photocatalytic hydrogen production in Klebsiella sp.-GO-NiFe2O4 biohybrid systems.

Photocatalytic biohydrogen production using semiconductor nanomaterials coupled with microbial cells has recently led to a boom in solar hydrogen production. However, current photo-biohybrid platforms are suboptimal for achieving a synergistic enhancement of photocatalytic hydrogen production, and their nanoparticles (NPs) distribution and electron transfer mechanisms are poorly understood. In this study, green-synthesized graphene oxide-doped nickel ferrite (GO-NiFe2O4) NPs were combined with Klebsiella sp. (Ka) and its engineered strain (Ka-GL) to construct Ka/NPs and Ka-GL/NPs biohybrid systems. Both biohybrid systems efficiently produced hydrogen driven by visible-light irradiation, with cumulative hydrogen productions reaching 533.1 ± 8.3 and 644.1 ± 10.8 mmol/L at 120 h of reaction, respectively, which were significantly higher than those of their respective strains and NPs. Further results demonstrated that GO-NiFe2O4 NPs distributed extracellularly and intracellularly efficiently excited the photo-generated electrons and transported them into the bacterial cell, further activating hydrogenase and formate-hydrogen lyase to produce hydrogen gas. In particular, Ka-GL/NPs exhibited better photoelectrochemical performance, higher expression levels of iron and nickel transport genes as well as electron transfer and transport genes, and enhanced hydrogenase and formate-hydrogen lyase activities and related gene expression, reducing potential, and cell viability. This proved that the biohybrid system constructed using engineered strain overexpressing hydrogenase subunits (Ka-GL) can more effectively enhance the efficiency of photocatalytic hydrogen production. This study provides novel biohybrid systems for highly efficient photocatalytic hydrogen production and offers insights into the NPs distribution and electron transfer mechanism for improving hydrogenase and formate-hydrogen lyase catalyzed hydrogen synthesis pathways.