Construction of lignin humic-like biochar composites for intensifying thermophilic biohydrogen production from lignocellulosic residues.

Dark fermentative hydrogen production from lignocellulosic residues is often limited by inefficient electron distribution and competing metabolic pathways. In this study, a lignin-derived humic substance-biochar (LHS-BC) was developed to enhance thermophilic hydrogen production from lignocellulosic residues. Among the tested materials, the lignin-derived humic-like substance obtained via alkaline oxidative humification and subsequently combined with biochar (HSH@BC) exhibited the best performance, increasing hydrogen production (mL/L) by 29.76% compared with the control. Kinetic analysis showed that HSH@BC significantly increased hydrogen production potential (1113.98 mL/L) and reduced lag time. The composite promoted cellulose degradation, enhanced cellulase and hydrogenase activities, and increased intracellular NAD+/NADH levels. Metabolic analysis revealed a shift from ethanol-type fermentation to acetate-butyrate pathways, leading to higher hydrogen yield. Electrochemical characterization suggests that cytochrome c may be involved in electron exchange with the quinone functional groups in LHS-BC. PICRUSt-based functional prediction suggested potential enrichment of central metabolic pathways, including glycolysis, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). Overall, LHS-BC improved hydrogen production by simultaneously regulating microbial community structure, metabolic pathways, and electron transfer processes, providing an effective strategy for thermophilic biohydrogen production from lignocellulosic biomass.