Urea-NaHCO₃ co-activated biochar enhances biohydrogen production via microbial community restructuring and metabolic...

Biochar enhances dark fermentative biohydrogen production (BHP), yet conventional biochar is limited by low porosity and few active sites. While nitrogen doping and chemical activation can individually upgrade biochar, the synergistic effect of urea doping combined with sodium bicarbonate (NaHCO₃) activation, and its consequence for intracellular metabolic networks, remains unclear. Herein, material characterization, 16S rRNA sequencing, and non-targeted metabolomics were integrated to elucidate how urea-doped NaHCO₃-activated rice-straw biochar (UBC-A) enhances cellulolytic BHP. UBC-A achieved the highest hydrogen production of 192.52 mL g^-1, representing a 6.6-fold (561.35% relative improvement) of the control; the hydrogen production lag period was shortened to 13.93 h, and the energy conversion efficiency was 14.19%. UBC-A exhibited enhanced graphitization and hierarchical porous structure. Microbiome analysis revealed selective enrichment of hydrogen-producing taxa (Clostridia, Thermoanaerobacterium) and cellulolytic microbes, alongside suppression of competitors. Metabolomics identified 113 significantly differential metabolites (P < 0.05), revealing system-wide metabolic rewiring centered on three interconnected hubs: (i) L-glutamate-driven TCA cycle activation and GABA-mediated acid stress alleviation; (ii) 2-hydroxyglutarate as a novel indicator of enhanced NADH regeneration capacity; and (iii) glycerophospholipid-mediated membrane restructuring facilitating extracellular electron transfer. Correlation analysis established significant associations between these hydrogen producers and key upregulated metabolites, indicating that UBC-A optimizes BHP by synchronizing community assembly with metabolic pathway redirection. These findings advance a structure-microbiome-metabolism framework for agricultural-waste valorization and biohydrogen industrialization.