NO-driven Anammox systems contribute to nitrogen removal via altering nitrogen transformation pathways and microbial interactions

Conventional Anammox systems using NO₂ − -N as an electron acceptor consistently face challenges of incomplete nitrogen removal due to NO₃ − -N production. Nitric oxide, a key intermediate in nitrogen cycling, presents a promising alternative electron acceptor for Anammox bacteria (AnAOB). To explore its feasibility, this study investigates the effects of NO on nitrogen transformations, microbial community dynamics and metabolic regulation within Anammox systems. Results revealed that NO elicited varied responses among nitrogen-transforming bacteria. Specifically, NO stimulation increased the relative abundance of AnAOB by 20.1 %–40.2 %, while suppressing nitrite-oxidizing bacteria (NOB) and denitrifiers (DNB). Consequently, the NO-dependent Anammox pathway contributed 15.4 % of total nitrogen removal and the NO₂ − -based pathway improved by 49.4 % according to isotope tracing. This shift was attributed to the ability of AnAOB to moderately uptake and efficiently utilize NO, maintaining intracellular NO levels 13.6 %–45.6 % lower than other bacteria, thereby avoiding oxidative stress and enhancing core metabolism. Additionally, NO facilitated ecological niche differentiation, strengthening interspecies cooperation mediated by metabolites, particularly between AnAOB and DNB ( Planctomycetia bacterium , Cyanobacteria bacterium CYA , etc.). Notably, NO supplementation increased NH 4 + -N removal by 1.5–1.9 times, with ΔNO₃ − -N/ΔNH₄ + -N decreasing by 45.2 %–54.8 %. These findings underscore the potential of NO-enhanced Anammox systems for advanced nitrogen removal in wastewater treatment.