Acetate-driven reorganization of Fe(III) mineral interfaces suppresses Anammox bacteria-associated Feammox

Fe(III)-coupled anaerobic ammonium oxidation (Feammox) couples NH₄⁺-N oxidation to Fe(III) reduction, yet the involvement of anaerobic ammonium-oxidizing bacteria (AnAOB) in complex enrichment systems and the mechanism underlying organic inhibition remain unclear. Here, using a Feammox-acclimated anaerobic ammonium oxidation (Anammox) enrichment exposed to different acetate loadings, we combined continuous-flow operation, isotope tracing, microscopic characterization, and integrated metagenomic and metatranscriptomic analyses to clarify the functional attribution of AnAOB-associated Feammox and the mechanism underlying its inhibition. Although acetate was readily consumed, NH₄⁺-N removal decreased from 100% to 72.1 ± 9.5% and 61.2 ± 9.3%, respectively. ¹⁵NH₄⁺-N tracing, Raman spectroscopy, and inhibitor assays supported a close association between AnAOB and Fe(III)-dependent NH₄⁺-N conversion. 16S ribosomal ribonucleic acid (rRNA) gene sequencing further indicated the dominance of Candidatus (Ca.) Brocadia under Feammox conditions, and metagenome-assembled genome (MAG)-resolved multi-omics resolved three Brocadia-like candidate Feammox-AnAOB populations, namely MAG288, MAG18, and MAG274. Among them, MAG288 showed nearly 100% average nucleotide identity to the extracellular electron transfer (EET)-capable Ca. Brocadia sp. BROELEC01, supporting a BROELEC01-like genetic basis for EET. Mechanistically, acetate-associated Feammox inhibition was not explained by depletion of the bulk Fe(III) inventory. Acetate exposure coincided with heterotrophic expansion and increased coverage of Fe(III) mineral surfaces by filament-like structures. Together with the redox signals associated with extracellular polymeric substances (EPS) and MAG-resolved transcriptional responses, these observations suggest that acetate may impair access of candidate Feammox-associated AnAOB to solid-phase Fe(III). Potential trophic restructuring may have further reinforced this interface-limitation response. Collectively, acetate-driven Fe(III) interface reorganization limits AnAOB-associated Feammox.