Evaluating the role of high N2O affinity complete denitrifiers and non-denitrifying N2O reducing bacteria in reducing N2O emissions in river

Freshwater rivers are hotspots of N2O greenhouse gas emissions. Dissolved organic carbon (DOC) is the dominant electron donor for microbial N2O reduction, which can reduce N2O emission through enriching high N2O affinity denitrifiers or enriching non-denitrifying N2O-reducing bacteria (N2ORB), but the primary regulatory pathway remains unclear. Here, field study indicated that high DOC concentration in rivers enhanced denitrification rate but reduced N2O flux by improving nosZ gene abundance. Then, four N2O-fed membrane aeration biofilm reactors inoculated with river sediments from river channel, estuary, adjacent lake, and a mixture were continuously performed for 360 days, including low, high, and mixed DOC stages. During enrichment stages, the (nirS+nirK)/nosZ ratio showed no significant difference, but the community structure of denitrifiers and N2ORB changed significantly (p < 0.05). In addition, N2ORB strains isolated from different enrichment stages positioned in different branches of the phylogenetic tree. N2ORB strains isolated during high DOC stage showed significant higher maximum N2O-reducing capability (V-max: 0.6 +/- 0.4 x10(-4) pmol h(-1) cell(-1)) and N2O affinity (a(0): 7.8 +/- 7.7 x1(0)(-12) L cell(-1) h(-1)) than strains isolated during low (V-max: 0.1 +/- 0.1 x10(-4) pmol h(-1) cell(-1), a(0): 0.7 +/- 0.4 x1(0)(-12) L cell(-1) h(-1)) and mixed DOC stages (V-max: 0.1 +/- 0.1 x10(-4) pmol h(-1) cell(-1), a(0): 0.9 +/- 0.9 x1(0)(-12) L cell(-1) h(-1)) (p < 0.05). Hence, under high DOC concentration conditions, the primary factor in reducing N2O emissions in rivers is the enrichment of complete denitrifiers with high N2O affinity, rather than non-denitrifying N2ORB.