Dual sludge system driven NDFO-Feammox coupling: Optimization of the iron cycling network for sustainable and...

This research addresses the significant reliance on carbon sources and high energy consumption in traditional biological nitrogen removal methods. It introduces sponge iron (SI) as a source of iron to drive the coupled nitrate-dependent Fe(II) oxidation (NDFO) and Fe(III)-mediated ammonium oxidation (Feammox) processes using activated sludge and anaerobic ammonium oxidation (Anammox) sludges. The findings indicate that supplementing with SI significantly enhances the nitrogen removal capacity of the system. The system achieved up to 87.1 ± 3.2 % removal of NH₄⁺-N and 91.8 ± 2.7 % removal of NO₃⁻-N. A dual-constraint mathematical model confirms that the NDFO pathway provides at least 66.1 % of the Fe(III) required for Feammox metabolism. This lays the theoretical foundation for constructing a sustainable, autotrophic iron-nitrogen cycling system under anaerobic conditions. To elucidate the mechanisms underlying the coupled nitrogen removal, batch kinetic experiments and metagenomic analyses were conducted. These analyses identified the dominance of Candidatus Brocadia in Feammox metabolism. It was also noted that Anammox genes were enriched with the addition of SI. Combined with the experimental observation of in situ Anammox activity restoration, this suggests a high degree of metabolic overlap between the Anammox and Feammox pathways. Unexpected metabolic couplings were also revealed. In particular, divergent trends in the relative abundances of gene clusters encoding Nar and Nir triggered partial NDFO metabolism, driving Candidatus Brocadia to engage in co-metabolism of Feammox and Anammox. This process diversifies nitrogen escape pathways. Further investigations showed that sustained NDFO metabolism benefited from the synergistic effects of extracellular iron oxidation, Feammox-mediated iron reduction, and iron regulatory networks. Additionally, Feammox metabolism established an iron turnover mechanism through iron storage, subsequently providing positive feedback to Feammox metabolism for high energy gain.