Advances in Heterotrophic Nitrification–Aerobic Denitrification: Physiological Characteristics, Environmental Responses, and Nitrogen Metabolic Pathways

Heterotrophic nitrification–aerobic denitrification (HNAD) bacteria enable nitrogen removal under aerobic conditions and have attracted increasing attention in wastewater treatment. Although numerous studies have enriched HNAD strains and explored their nitrogen removal performance, the environmental responses and metabolic mechanisms underlying nitrogen transformation remain insufficiently evaluated. In this review, the ecological distribution and physiological characteristics of HNAD bacteria are summarized, followed by a systematic elaboration of the effects of nitrogen sources, carbon sources, C/N ratio, and environmental factors (e.g., temperature, pH, dissolved oxygen and salinity) on nitrogen removal and transformation performance. In addition, nitrogen depletion from the aqueous phase is likely to reflect both conversion to gaseous nitrogen products and assimilatory uptake into biomass, which may account for a non-negligible proportion of the apparent nitrogen removal in some strains. Accordingly, the nitrogen transformation mechanisms, encompassing nitrite/nitrate-mediated pathways, proposed hydroxylamine-associated pathways, and nitrogen assimilation, are critically examined, with attention to the strength and limitations of the available evidence. Furthermore, the regulatory roles of intracellular redox balance and electron allocation in mediating the interplay among nitrification, denitrification, and cellular respiration are addressed. Finally, the key challenges and future prospects of HNAD research and applications are highlighted. This review provides mechanistic insights into the metabolic versatility of HNAD bacteria and supports the development and evaluation of nitrogen removal strategies in advanced biological nitrogen removal systems.