Enhanced methanogenesis of wastewater anaerobic digestion by nanoscale zero-valent iron: Mechanism on intracellular energy conservation and amino acid metabolism.

Nanoscale zero-valent iron (nZVI)-mediated anaerobic digestion commonly focuses on electron transfer between syntrophic bacteria, neglecting intracellular energy conservation strategies and amino acid metabolism. In this study, FH dehydrogenase abundance increased by 5.1 %, 27.0 %, and 31.5 % at 10 mM, mM， 30 mM, and 50 mM nZVI dosing, respectively, enabling an efficient transmembrane proton-coupled electron transfer mode. Electron bifurcation (EB) enzymes involved in methanogenesis responded differently to nZVI, with HdrABC initially increasing at 10 mM and decreasing at 30 mM and 50 mM, while MvhADG-HdrABC was completely down-regulated. Metabolomics further demonstrated that nZVI reduced riboflavin and flavin mononucleotide content, which is detrimental to the EB. Instead, an alternative measure to maintain electron flow and energy conservation under high nZVI exposure is high expression of ndh and F-type or V/A-type ATPase genes. Additionally, enhancing C-unit carrier expression through amino acid metabolism regulation emerged as a key strategy. This study provides new perspectives on nZVI-mediated anaerobic digestion.