Beyond ammonia-oxidizing bacteria-centric paradigms: Geobacter-assisted anodic anaerobic ammonia oxidation

Anodic anaerobic ammonium oxidation (anodic anammox) presents a sustainable approach for nitrogen removal, yet its bioelectrochemical mechanisms remain unclear due to biofilm complexity and undefined roles of electroactive microorganisms (EAMs). This study reveals that nitrite (NO₂⁻) is the direct product of ammonia-oxidizing bacteria (AOB)-driven anodic anammox, with extracellular electron transfer (EET) mediated by indirect mechanisms via redox shuttles. Metagenomic analysis identified two ammonia oxidation pathways: (1) a novel short-range nitrification pathway (NH₄⁺ → NO₂⁻) governed by ncd2 genes, and (2) a traditional ammonia oxidation pathway (NH₄⁺ → NH₂OH) facilitated by amoABC. Intriguingly, Geobacter exhibited potential NH₂OH oxidation capability, bridging AOB activity and electrode respiration. Functional inhibition experiments demonstrated that EAMs-derived electron shuttles and reactive oxygen species (ROS) are critical for enhancing EET efficiency, with ROS serving as a key electron acceptor for AOB under anaerobic conditions. Spatial and metabolic synergy between EAMs and AOB-via substrate cross-feeding, cofactor provision, and electron transfer-was essential for maintaining biofilm stability. These findings challenge the conventional view of AOB-driven anodic anaerobic anammox mechanisms and provide new insights into sustainable nitrogen removal in engineered bioelectrochemical systems.