Enhancement of dark fermentation H₂ production by the sustained provision of soluble Fe²⁺: Importance of electron reallocation and metabolic network regulation

Understanding how iron supplementation regulates metabolic pathways and electron flow is essential for optimizing dark fermentation hydrogen production. Here, the effects of Fe⁰, FeCl₂·4H₂O, and Fe(OH)₂ supplementation during glucose fermentation by Thermoanaerobacterium thermosaccharolyticum W16 were compared. Although the three iron sources were supplied at the same total iron concentration, only FeCl₂ delivered an immediate and sustained supply of soluble Fe²⁺, yielding the highest total hydrogen of 58.87 mL, a 29.87 % higher than that of the control. This FeCl₂-driven Fe²⁺ supply substantially enhanced intracellular reducing power, accelerated Fe-S cluster assembly, and improved [FeFe] hydrogenase activity and electron transport system function, thereby promoting the redirection of electron flow toward H₂ production and supporting acetate formation and hydrogen accumulation. Integrated analysis of electron distribution, NAD(P)H levels, and metabolite profiles revealed a distinct intracellular redox phenotype induced by FeCl₂ supplementation. This was characterized by a more reduced intracellular environment, heightened acidogenesis, and the coordinated remodeling of central carbon and amino acid metabolism. These findings establish FeCl₂ as a key regulator of microbial electron flow and metabolic network architecture during dark fermentation, supporting the design of iron supplementation strategies to improve biological hydrogen production.