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.