Sustainable, substrate-specific catalysis: Green Fe3O4 nanocatalysts drive hydrogen production and metabolic redirection in thermophilic xylose fermentation

The efficient production of biohydrogen (BioH 2 ) relies on the effective use of the hemicellulose component from lignocellulosic biomass. This study investigated the efficiency of iron oxide nanocatalysts (Fe 3 O 4 NCs) in catalyzing substrate-specific thermophilic dark fermentation (55 °C) of xylose and glucose with acclimated industrial waste inoculum. The addition of 300 mg L −1 Fe 3 O 4 NCs increased hydrogen (H 2 ) production and metabolite yields for xylose fermentation over glucose fermentation. Specifically, the NCs increased the hydrogen molar yield (HMY) from 1.41 to 2.2 mol H 2 mol −1 xylose, indicating that the Fe 3 O 4 NCs exhibit enhanced performance and metabolic stability when compared to glucose over three cycles of fermentation. Brunauer-Emmett-Teller (BET) analyses revealed the mesoporous nature of Fe 3 O 4 NCs, with an average surface area of 18.356 m 2 g −1 and a pore diameter range of 10-20 nm. Mechanistically, Fe 3 O 4 NCs act as thermodynamic catalysts by lowering the Gibbs free energy barrier (ΔG°)’ to facilitate the rate-limiting conversion of pyruvate to acetyl-CoA, thus diverting metabolic flux from competing pathways leading to ethanol and enhancing production of H 2 -rich acetate and butyrate. Additionally, electrochemical analyses demonstrated that the NCs increase the secretion of flavin-based electron shuttles, thereby enhancing the extracellular electron transfer (EET). This work provides mechanistic insights into the sustainable, substrate-specific catalytic properties of Fe 3 O 4 NCs in thermophilic xylose fermentation, offering a robust strategy for enhanced BioH 2 production. • Thermodynamic catalysis by Fe 3 O 4 NCs was confirmed by lowered (ΔG)’ for the rate limiting step. • Fe 3 O 4 NCs showed substrate-specific enhancement, increasing H 2 yield from xylose greater than glucose. • Fe 3 O 4 NCs resulted in enhanced flavin-mediated extracellular electron transfer (EET). • Fe 3 O 4 NCs sustained superior H 2 yields across three fermentation cycles. • The lowered (ΔG)’ redirects metabolic flux, away from ethanol formation to H 2 production.