Zero-valent iron regulates metabolic pathways to simultaneously enhance biohydrogen production and H2/CO2 ratio in thermophilic straw hydrolysate fermentation.

Deep strata anaerobic fermentation of crop straw hydrolysate for bio-hydrogen (H2) production mitigates open-field burning pollution and supports net-negative carbon emissions by substituting and converting geologically sequestered CO2 into CH4. However, the thermophilic conditions in deep strata differ significantly from traditional mesophilic systems for biohydrogen fermentation. Crucially, optimizing subsequent methanogenesis of pre-stored CO2 requires enhancing H2 yield and H2/CO2 ratio simultaneously. This study demonstrates that zero-valent iron (ZVI) can enhance the biohydrogen during straw hydrolysate fermentation at 55 °C. Experimental results indicate that, compared to the control, the reactor with a ZVI dosage of 1 g/L exhibited a 24.53% increase in cumulative hydrogen yield, accompanied by a 26.90% elevation in the H2/CO2 ratio, thereby achieving simultaneous improvements in both hydrogen yield and purity. Mechanistic investigation reveal that ZVI maintains appropriate reductive conditions and faciliates interspecies electron transfer efficiency within the fermentation system. Moreover, molecular ecological network analysis indicates that ZVI promotes the formation of syntrophic microbial networks. KEGG-based functional predictions further suggest that key enzyme genes associated with glycolysis and butyrate-type fermentation were upregulated, whereas the expression of genes participating in non-hydrogen-producing pathways was suppressed. Furthermore, acetyl-CoA metabolism was shifted toward the acetate pathway during butyrate-type fermentation, favoring elevated hydrogen production while suppressing CO2 co-generation. This study establishes a conceptual and experimental foundation for achieving net-negative carbon emissions via straw-derived biohydrogen in deep strata, although significant engineering challenges remain.