Redox-active biochar as insoluble electron acceptors for anaerobic oxidation of ammonium

Anaerobic ammonium oxidation (anammox) is a promising nitrogen removal process, but its reliance on nitrite presents operational challenges. Here, we demonstrated that redox-active pyrogenic biochar could serve as an insoluble electron acceptor for NH₄⁺ oxidation by anammox consortia. Through batch incubations, over 95 % total nitrogen removal was consistently achieved with biochar as the sole electron acceptor across two consecutive cycles, though at a slower rate (0.77-1.18 mmol-N·d^-1 g^-1 VSS) compared to conventional anammox reaction. Isotopic tracing confirmed the role of biochar as an electron sink, and electrochemical and spectroscopic analyses illustrated that biochar's electron-accepting capability was attributed to surface quinone-like and pyridinic N groups. High-throughput sequencing and metagenomics revealed the dominance of anammox species Candidatus Brocadia in biochar-driven NH₄⁺ oxidation, with upregulated genes for extracellular electron transfer (EET) associated with c-type cytochromes. Metabolic reconstruction further elucidated the hydroxylamine pathway in biochar-driven anammox, distinctively different from the canonical nitrite-dependent route. These findings underscored biochar geobatteries as an inexpensive, environmentally sustainable electron acceptor, circumventing nitrite supply limitations. This work advances the understanding of EET-mediated anammox, thereby providing the potential for developing energy-efficient nitrogen removal technologies.