Promotion of biological H (Bio-H) production by the nitrogen-fixing anaerobic microbial consortia using humin, a solid-phase humic substance.

Dark fermentative biological hydrogen (Bio-H) production is expected to be a clean and sustainable H production technology, and the technologies have been studied to increase in the product yield as index. This study achieved high product yields of Bio-H using nitrogen-fixing consortia under nitrogen-deficient conditions with glucose or mannitol as substrate and humin as the extracellular electron mediator: 4.12 mol-H/mol-glucose and 3.12 mol-H/mol-mannitol. The high Bio-H production was observed under the conditions where both nitrogenase and hydrogenase were active in the presence of humin. Nitrogenase activity was confirmed by acetylene reduction activity and hydrogenase activity by Bio-H production under nitrogenase-inhibiting conditions with NHNO. [Fe-Fe] hydrogenase detected by a specific PCR and acetate, butyrate, formate, lactate, and pyruvate produced as by-products suggested the involvement of both pyruvate-ferredoxin-oxidoreductase and pyruvate formate lyase pathways in Bio-H production. Humin promoted the Bio-H production beyond the capacity of the consortium, which had reached saturation with the optimum concentrations of glucose and mannitol. Carbon balance suggested the concurrent H consumption by hydrogenotrophic methanogenesis and acetogenesis. Bio-H production of the washed and starved consortium with reduced humin under conditions with or without NHNO suggests that humin promoted hydrogenase and nitrogenase activity by donating extracellular electrons. Clostridium and Ruminococcus in the consortia were considered major hydrogen producers. Thus, this study demonstrated the outstanding potential of nitrogen-fixing consortia under nitrogen-deficient conditions with humin as an extracellular electron mediator for dark fermentative Bio-H production with high yields.