Novel nitric oxide dismutase drives effective NO degradation in oxygenic denitrification.

Nitrogenous wastewater is a major environmental problem. Denitrification refers to the sequential reduction of nitrate (NO3-) and nitrite (NO2-) to nitric oxide (NO) and nitrous oxide (N2O) to form N2. Oxygenic denitrification could transform NO into N2 and O2 via NO dismutase (Nod), an unusual O2-forming process to reduce N2O emission during nitrogen removal. After confirming the presence of the nod gene in three samples including anaerobically-activated sludge, farmland soil, and paddy soil, the targeted enrichment experiment was conducted. The abundance of key denitrification genes rose by 4.6-13 fold, including NO2- reductase (nir), N2O reductase (nos), NO reductase (nor), and nod. Principal coordinate and α-diversity analyses revealed the progressive enrichment of oxygenic denitrifiers by eliminating irrelevant microorganisms. Focusing on paddy soil with optimal O2-generating capacity, novel nod genes were identified. Specifically, recombinant-engineered bacteria containing the nod genes were constructed to assess their NO dismutation activity. Results demonstrated that these recombinant strains exhibited excellent NO degradation efficiency at 72.6-85.4%, indicating superior dismutation performance. Apparent enzymatic activity assays using the crude Nod enzyme extract from the representative recombinant strain SD52149 showed Michaelis-Menten-type saturation behavior. These findings provide critical insights into the application of oxygenic denitrification for reducing N2O emissions in nitrogenous wastewater treatment and enhancing microbial remediation strategies.