Enhanced Nitrogen Removal in Constructed Wetlands with Multivalent Manganese Oxides: Mechanisms Underlying Ammonium Oxidation Processes

The ammonium (NH4+-N) removal efficiency in constructed wetlands (CWs) is often limited by insufficient oxygen. In this study, an extract of Eucalyptus robusta Smith leaves was used to prepare multivalent manganese oxides (MVMOs) as substrates, which were used to drive manganese oxide (MnOx) reduction coupled to anaerobic NH4+-N oxidation (Mnammox). To investigate the effects and mechanisms of MVMOs on ammonia removal, four laboratory-scale CWs (0%/5%/15%/25% volume ratios of MVMOs) were set up and operated as continuous systems. The results showed that compared to controlled C-CW (0% MVMOs), Mn25-CW (25% MVMOs) improved the average NH4+-N removal efficiency from 24.31% to 80.51%. Furthermore, N2O emissions were reduced by 81.12% for Mn25-CW. Isotopic tracer incubations provided direct evidence of Mnammox occurrence in Mn-CWs, contributing to 18.05–43.64% of NH4+-N removal, primarily through the N2-producing pathway (73.54–90.37%). Notably, batch experiments indicated that Mn(III) played a predominant role in Mnammox. Finally, microbial analysis revealed the highest abundance of the nitrifying bacteria Nitrospira and Mn-cycling bacteria Pseudomonas, Geobacter, Anaeromyxobacter, Geothrix and Novosphingobium in Mn25-CW, corresponding to its superior NH4+-N removal efficiency. The enhancement of NH4+ oxidation, first to hydroxylamine and then to nitrite, in Mn25-CW was attributed to the upregulation of ammonia monooxygenase genes (amoABC and hao). This study enhanced our understanding of Mnammox and provided further support for the use of manganese oxide substrates in CWs for efficient NH4+-N removal.