Nitrite accumulation and impairment of N2O reduction explains contrasting soil denitrification phenotypes

Soil denitrification produces the potent greenhouse gas and ozone depleter nitrous oxide (N2O). We previously linked soil N2O emission potential (N2O/N2O + N2) to a continuum of denitrification phenotypes in 20 pasture soils but were unable to determine their proximal cause. At one end of the continuum, soils carried out completely concurrent production and reduction of N2O (concurrent phenotype) while at the other end soils delayed N2O reduction until almost all added nitrate (NO3−) was accumulated as N2O (sequential phenotype). In an unsealed environment, the later phenotype is predicted to emit most added N as N2O. Here we tested the role of delayed N2O reductase synthesis and nitrite (NO2−) based impairment of N2O reduction as determinants of delayed N2O reduction in soils with sequential phenotypes. Nitric oxide (NO), N2O and N2 accumulation were measured in response to added NO3−, NO2− and N2O in anoxic batch incubations of fresh or pre-incubated soil using automated gas chromatography. Successive NO3− additions drove 6 of 7 soils towards increasingly concurrent N2O production/reduction, suggesting delayed production of N2O reductase may be the cause of delayed N2O reduction in initially sequentially denitrifying soils. NO2− addition (2 mM NO3− + 1 mM NO2− vs. 3 mM NO3− control) to sequentially and concurrently denitrifying soil demonstrated that NO2− impairs N2O reduction, even when N2O reductase was fully induced (pre-incubated soils, 1 mM NO2− + 42 μmol N2O vs. 42 μmol N2O control). Further, 2 pieces of evidence suggest that differences in NO2− accumulation are a probable cause of the previously observed denitrification phenotypes. 1) 48 fold higher NO2− accumulation in the sequential vs. concurrent soil in response to 3 mM NO3− addition. 2) the concurrent timing of NO2− depletion/N2O reduction in the sequential soil. However, these observations should be confirmed in a greater number of soils. Inhibition of N2O reductase by NO is proposed as a potential proximal cause of impaired early N2O reduction which could link separate effectors (NO2−, carbon, successive NO3− additions) to a common inhibitory mechanism.