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This study demonstrates the feasibility of nitrite-free anaerobic ammonium oxidation (Anammox) in mixed microbial communities through extracellular electron transfer (EET) driven by anodic potential and conductive carrier. The results show a significant increase in nitrogen removal efficiency and stability of the process. The findings have important implications for the development of low-carbon nitrogen removal technologies.
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Anaerobic ammonium oxidation (Anammox) provides a low-carbon pathway for nitrogen removal, yet its reliance on nitrite (NO 2 - -N) constrains large-scale application. Emerging evidence indicates that Anammox bacteria (AnAOB) can oxidize ammonium (NH 4 + -N) through extracellular electron transfer (EET) without NO 2 - -N. However, the long-term stability of this process in mixed communities remains unresolved. Here, microbial electrolysis cells were operated for 260 days to investigate how anodic potential and conductive carrier regulate EET-dependent Anammox. A threshold potential of 0.4-0.6 V (vs. SHE) enabled NO 2 - -N-free NH 4 + -N removal of 103.61 ± 9.22 mg N·L -1 ·d -1 (approximately 2.5-fold higher than highly enriched communities) via a hydroxylamine oxidoreductase-mediated pathway. The conductive carrier increased electron flux 4.9-fold, enhanced protein secretion, and stabilized biofilms. High potential combined with conductive carrier enriched electroactive AnAOB (Candidatus Kuenenia, Candidatus Brocadia) and induced a shift from NO 2 - -N-dependent to EET-dependent metabolism. These findings demonstrate sustained long-term EET-dependent Anammox and inform scalable, carbon-free nitrogen removal.