Carbon-Restricted Anoxic Zone as an Overlooked Anammox Hotspot in Municipal Wastewater Treatment Plants

The anoxic zone serves as the core functional unit in municipal wastewater treatment plants (MWWTPs). Unfortunately, in most cases, the downstream range of the anoxic zone is severely lacking in available organic carbon and thus contributes little to the removal of nutrients. This undesirable range is termed the "carbon-restricted anoxic zone", representing an insurmountable drawback for traditional MWWTPs. This study uncovers a previously overlooked role for the carbon-restricted anoxic zone: a hotspot for anaerobic ammonium oxidation (anammox). In a continuous-flow pilot-scale plant treating municipal wastewater (55 m³/d), virgin biocarriers were introduced into the carbon-restricted anoxic zone (downstream 25% of the anoxic zone with BOD₅ of 5.9 ± 2.3 mg/L). During the 517-day monitoring, anammox bacteria highly self-enriched within the biofilms, with absolute and relative abundance reaching up to (9.4 ± 0.1) × 10⁹ copies/g-VSS and 6.17% (Candidatus Brocadia), respectively. ¹⁵N isotopic tracing confirmed that anammox overwhelmingly dominated nitrogen metabolism, responsible for 92.5% of nitrogen removal. Following this upgrade, the contribution ratio of the carbon-restricted anoxic zone to total nitrogen removal increased from 9.2 ± 4.1% to 19.2 ± 4.2% (P < 0.001), while its N₂O emission flux decreased by 84.5% (P < 0.001). These findings challenge stereotypes about the carbon-restricted anoxic zone and highlight the multiple environmental implications of this newfound anammox hotspot.