Microbial nitrogen metabolic pathways in biological wastewater treatment: mechanisms, interactions, and engineering perspectives

Biological nitrogen removal in wastewater treatment has long been described as a two-step process, in which ammonia is oxidized aerobically to nitrate and nitrate is reduced anoxically to dinitrogen gas. Over the past 2 decades, this picture has been extended by the recognition of (1) complete ammonia oxidation (comammox), (2) anaerobic ammonium oxidation (anammox), (3) heterotrophic nitrification–aerobic denitrification (HNAD), and (4) dissimilatory nitrate reduction to ammonium (DNRA). Earlier reviews have mostly treated these pathways one at a time. Three questions have received less attention: how the pathways interact within a shared community, how operating conditions shift nitrogen among them, and how the contribution of each pathway can be demonstrated in a working reactor. This review addresses all three. Organizing the pathways by nitrogen oxidation state shows that ammonium, nitrite, and nitrate are the points where they compete and exchange intermediates. Cooperative and competitive relationships are examined together with the spatial structure of flocs, biofilms, and granules. These include the nitrite handoff in partial nitritation–anammox, the dual role of DNRA, which competes with anammox for nitrite but can also return ammonium to it, and the division of denitrification steps among different organisms. Dissolved oxygen and organic carbon are then used to define a conceptual niche space for the main guilds. Its boundaries shift with pH, temperature, solids retention time, reactor configuration, and salinity, so no single threshold, such as a fixed carbon-to-nitrate ratio, reliably predicts which pathway will dominate. A guild-level summary links each group to its preferred operating conditions, main limitations, and optimization strategies. The review also sets out four levels of evidence for a pathway: metabolic potential (genes), expression (transcripts and proteins), pathway-specific flux, and contribution to reactor-scale nitrogen fate. Most molecular methods remain correlative, and attributing removal to a pathway requires isotope tracing, gaseous-product measurement, and nitrogen mass balances, ideally linked to the responsible organisms. Remaining priorities include measuring pathway-specific rates in operating reactors, explaining when an operating condition redirects nitrogen from one pathway to another, and understanding how microbial interactions withstand and recover from disturbance.