Achieving mesophilic-level methane production in low-temperature anaerobic digestion by zero-valent iron-driven...

Anaerobic digestion (AD) at low temperature is attractive for reducing the energy demand of sludge treatment, but methane production is strongly constrained by slow microbial kinetics and inefficient interspecies electron transfer. This study demonstrates that zero-valent iron (ZVI) exhibited a temperature-dependent shift in its dominant electron-transfer function. ZVI was applied to the AD of waste activated sludge across 20-35℃ at dosages of 0-20 g/L. Methane yield enhancement increased from 10.7% at 35℃ to 136.4% at 20℃, and the ZVI-amended reactor at 20℃ produced more methane than the unamended mesophilic control. The optimal ZVI dosage increased 60-fold as temperature decreased. A stoichiometric calculation based on endpoint dissolved Fe²⁺ yielded a theoretical CH₄ equivalent of 2.7 mL CH₄/g VS at 20 °C, corresponding to <5% of the observed methane increment. Stage-specific kinetic rates changed little at 35℃, whereas acidogenesis and hydrogenotrophic methanogenesis increased at 20 °C. Paired metagenomic and metatranscriptomic analyses further supported a temperature-dependent electron-transfer reconfiguration. At 35℃, ZVI primarily promoted H₂-associated hydrogenotrophic methanogenesis, whereas at 20℃ it promoted ZVI-assisted conductive electron exchange together with enhanced formate-associated electron transfer, as supported by increased activity of electroactive microorganisms, enrichment of formate-utilizing methanogens (Methanobacterium formicicum), and upregulation of formate-related genes including fdo and fdh. These findings establish a conceptual framework demonstrating that the electron-transfer function of ZVI is not a fixed property but dynamically regulated by environmental conditions, providing new insights into the adaptive roles of conductive materials for low-temperature anaerobic sludge digestion with reduced dependence on digester heating.