Nano zero-valent iron-mediated synergism chemical reduction and biological degradation of azo dye: The multiple regulation roles towards electron transfer and stress responses

Azo dyes in textile wastewater present treatment challenges due to their structural complexity and resistance to biodegradation. While anaerobic digestion offers potential for azo dye degradation, its efficiency is limited by slow reaction kinetics and electron transfer constraints. This study demonstrated that nano zero-valent iron (nZVI) overcame these limitations through synergistic chemical and biological mechanisms. Using reactive orange 16 (RO16) as a representative azo dye, our results demonstrated that at the optimal concentration of 0.5 g/L, nZVI enhanced both RO16 degradation (98.4 % removal efficiency) and methane production (18.2 % increase) through chemical-biological synergy. First, nZVI rapidly cleaved azo bonds through chemical reduction, generating more biodegradable intermediates (2-phenylamine-5-nitrobenzenesulfonic acid and 4-vinylbenzenesulfonamide), thereby providing favorable substrates for subsequent microbial metabolism. Meanwhile, nZVI elevated the electron transfer system activity by up to 200 % and stimulated the production of humic acid-like substances, which functioned as electron shuttles to enhance direct interspecies electron transfer (DIET). At the community level, nZVI induced the enrichment of electroactive Georgenia (for dye degradation) by 99.9 % and DIET-capable methanogens by 55.8 %. Further gene expression analysis confirmed that nZVI upregulated critical functional genes, including those involved in acidogenic (phbB, 1.6-fold) and methanogenic (hmd, 1.7-fold) pathways, along with ABC transport genes (wzt, 1.2-fold) that potentially enhanced heme-dependent azoreductase production (hemH, 1.5-fold). Most remarkably, nZVI upregulated key electron transfer-related genes, particularly cytochrome oxidases (coxAC, 2.6-fold) and quinone biosynthesis (ubiC, 5.1-fold), thereby establishing a continuous conversion pathway from RO16 to methane. However, the beneficial effects of nZVI exhibited a concentration threshold, as elevated dose led to physical obstruction, reactive oxygen species overproduction (catE, 1.6-fold), and severe motility suppression, ultimately inhibiting methanogenesis. These insights advance fundamental understanding of bio-nano interactions in anaerobic environments and provide practical guidelines for implementing nZVI-enhanced treatment of refractory industrial wastewater.