Pyrite-Mediated Rewiring of Redox Homeostasis and Metabolic Priority Enables Synergistic Dark Fermentation of Mixed Sugars

This study elucidates the mechanism by which natural pyrite enhances hydrogen production during the cofermentation of glucose and xylose in both synthetic mixed sugars and real lignocellulosic hydrolyzates. In synthetic mixed sugars, pyrite increased the maximum hydrogen production rate by 1.8–2.4-fold, shortened the xylose metabolic lag by 3–5 h, and elevated the hydrogen molar yield by up to 35%. Similar improvements were observed in lignocellulosic hydrolyzates, where pyrite facilitated earlier xylose utilization and increased the peak hydrogen production rate by over 60%. These enhancements corresponded to intensified EPS and flavin secretion-showing 30–70% higher fluorescence signals-which strengthened extracellular electron transfer, consistent with the markedly increased redox peak currents in cyclic voltammetry. Pyrite also buffered acidification by maintaining pH above 5.0 during critical fermentation stages and reshaped the microbial community toward Firmicutes-dominant hydrogenogenic consortia, with Clostridium spp. increasing by 20–40% in relative abundance. Integrating these biochemical, electrochemical, and microbial effects, pyrite established a more stable and electron-efficient fermentation environment, thereby enabling more effective cometabolism of mixed sugars. This work highlights pyrite as a simple and low-cost strategy to substantially improve hydrogen yields from lignocellulosic substrates.