Low-Temperature Anammox Supported by Zero-Valent Iron (ZVI): Microbial and Physicochemical Changes during Treatment of Synthetic and Municipal Wastewater

The anaerobic ammonium oxidation (anammox) process offers a sustainable and energy-efficient alternative for nitrogen removal in wastewater treatment, but its performance at low temperatures remains a major challenge. This study investigated the role of zero-valent iron (ZVI) in enhancing anammox activity across a temperature range of 10-30 °C using both synthetic and municipal wastewater (MWW). Short-term batch tests demonstrated that low-dose ZVI (5-10 mg/L) stimulated specific anammox activity (SAA) particularly at 13-20 °C, while ZVI increasing concentration (1-10 mg/L) enhanced the enzymatic activity of HDH and decreased NIR activity, as well as modulated oxidative stress (ROS and GSH balance). In contrast, the long-term operation of the anammox process in sequencing batch reactors (SBR) showed that while ZVI (5 mg/L) improved SAA and microbial stability under synthetic conditions at 13 °C in compared to control (without ZVI), these benefits diminished once real municipal wastewater was introduced, most likely due to biomass stress and organic load. Metataxonomic analysis confirmed that ZVI selectively promoted genera such as Candidatus Brocadia, Denitratisoma, Micavibrionales_unclassified, while reducing overall microbial diversity. These results indicate that low-dose ZVI can temporarily enhance anammox resilience at suboptimal temperatures. However, its long-term application in MWW requires further optimization to mitigate potential inhibitory effects and iron passivation.