Abstract
Achieving high-purity biohydrogen (Bio-H₂) production necessitates the suppression of hydrogenotrophic methanogens, as their activity can impede hydrogen yield. Various inoculum pretreatments have been employed to suppress methane-producing microorganisms; however, these methods can negatively impact the enzymatic activity of hydrogen-producing microorganisms, thereby reducing hydrogen production. To address this challenge, this research investigates a novel approach to enhance Bio-H₂ production by activating microbial enzymes using magnetite Fe₃O4-doped carbonized nanoparticles (NPs) derived from vegetable leaves (VLCFe₃O4-NPs) within a coupled dark fermentation-microbial Electrohydrogenesis system. Characterization results revealed that VLCFe₃O4-NPs exhibited cubic and spherical morphologies, with a small diameter of 1 ± 100 nm and a mean crystallite size of 38.1 nm, indicating high purity. Fermentation tests investigated the impact of different nanoparticle dosages on Bio-H₂ generation, hydrogenase gene expression (Fe-Fe and Ni-Fe), and microbial biodiversity. Bio-H₂ production significantly improved with 500 mg/L VLCFe₃O4-NPs, yielding 1.2-fold more than the control group, while even a low dose of 25 mg/L resulted in a 0.22-fold increase. Relative gene expression analysis using qPCR and the 2-ΔΔCT method demonstrated a 30-fold increase in Cbei 1773 (Fe-Fe hydrogenase) and a 23-fold increase in hucL (Ni-Fe hydrogenase) gene expression, along with an increase in 16S rDNA. Additionally, the abundance of biohydrogen-producing bacteria, Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_11, increased by 14.3% and 11.1%, respectively, compared to 4.9% and 3.9% in the control group. This research indicates that VLCFe₃O4-NPs offer an eco-friendly solution for boosting biohydrogen production within microbial electrohydrogenesis cells with dark fermentation systems, thereby supporting sustainable bioenergy generation. One-sentence summary: Green carbonized nanoparticles Fe3O4-doped have been shown to turn on the genes of bacteria (Fe-Fe and Ne-Fe) and increase the biodiversity of microbes, both of which are important for biohydrogen production.