Metabolic enhancement of AnAOB via enzyme and cofactor regulation for nitrogen removal in wastewater: a critical review

Anaerobic ammonium oxidation (anammox) is a promising low-carbon and energy-efficient biological nitrogen removal technology, yet its application is constrained by the slow growth, limited metabolic flux, and poor environmental adaptability of anaerobic ammonium-oxidizing bacteria (AnAOB). Recent research has shifted from traditional macroscopic optimization toward micro-scale strategies centered on key enzymes and their regulatory networks. This review systematically examines the AnAOB metabolic framework along the enzyme–cofactor–signaling–application axis, covering the catalytic roles and rate-limiting features of core enzymes including Nitrite reductase (Nir), Hydrazine synthase (Hzs), Hydrazine oxidoreductase (Hzo), and auxiliary enzymes such as nitrate reductase (Nar) and hydroxylamine oxidoreductase (Hao), emphasizing their dependence on metal cofactors like iron and copper and the regulatory role of metal homeostasis in modulating electron transfer and energy coupling. It further explores endogenous signaling networks—including siderophore-mediated metal uptake, quorum sensing (QS), and cyclic nucleotide pathways—in coordinating metabolic activation, biofilm formation, and stress responses. Exogenous strategies involving iron-based materials, biochar, porous carriers, and redox mediators are assessed for their capacity to enhance metal bioavailability, facilitate electron transfer, and stabilize microenvironments, thereby boosting enzymatic activity and engineering performance. These micro-scale mechanisms are evaluated within integrated systems including mainstream anammox, partial nitritation/anammox (PN/A), Comammox–Anammox, and bioelectrochemical processes. Future efforts integrating multi-omics, materials design, and AI-based modeling will enable more precise regulation of AnAOB metabolism, advancing anammox from a nitrogen removal unit toward a carbon-driven platform for nitrogen transformation and resource recovery.