Unveiling novel pathways and key contributors in the nitrogen cycle: Validation of enrichment and taxonomic characterization of oxygenic denitrifying microorganisms in environmental samples

Microorganisms play a crucial role in both the nitrogen cycle and greenhouse gas emissions. A recent discovery has unveiled a new denitrification pathway called oxygenic denitrification, entailing the enzymatic reduction of nitrite to nitric oxide (NO) by a putative nitric oxide dismutase (nod) enzyme. In this study, the presence of the nod gene was detected and subsequently enriched in anaerobic-activated sludge, farmland soil, and paddy soil samples. After 150 days, the enriched samples exhibited significant denitrification, and concomitant oxygen production. The removal efficiency of nitrite ranged from 64.6 % to 79.0 %, while the oxygen production rate was between 15.4 mu L/min and 18.6 mu L/min when exposed to a sole nitrogen source of 80 mg/L sodium nitrite. Additionally, batch experiments and kinetic analyses revealed the intricate pathways and underlying mechanisms governing the oxygenic denitrification reaction by using CARBOXY-PTIO, 18O-labelled water, and acetylene to unravel the intricacies of the reaction. The quantitative polymerase chain reaction (qPCR) results indicated a significant surge in the abundance of nod genes, escalating from 7.59 to 10.12-fold. Moreover, analysis of 16S ribosomal DNA (rDNA) amplicons revealed Proteobacteria as the dominant phylum and Thauera as the main genus, with the presumed affiliation. In this study, a new nitrogen conversion pathway, oxygenic denitrification, was discovered in environmental samples. This process provides the possibility for the control of nitrous oxide in the treatment of nitrogenous wastewater.