Occurrence and 15N-quantification of simultaneous nitrification and denitrification in N-fertilised soils incubated under oxygen-limiting conditions

Nitrification and denitrification are known to co-occur in soils, but the effect of fertilisation history on N2O fluxes and the relative source partitioning of the N2O has not been thoroughly investigated. In this study, we therefore combined a high-tech 15N stable isotope tracing technique with quantitative PCR (qPCR) to explore the relative contributions of nitrification and denitrification to N2O production by a sandy-loam Eutric Cambisol soil treated repeatedly with ammonium sulfate [(NH4)2SO4] or potassium nitrate (KNO3) for 3 years prior. Both soils (historically (NH4)2SO4 and historically KNO3 treated) were amended separately with (15NH4)2SO4 and K15NO3 and incubated at 80% water filled pore space for 30 days. Soil N2O emissions, NH4+ and NO3− concentrations and their corresponding 15N-enrichments were determined. The effect of N addition on N transformation rates was also calculated. The total abundance of nitrifiers was estimated by qPCR of the amoA gene from bacteria and archaea, and that of denitrifiers by using the nirK, nirS, norB and nosZI genes as molecular targets. In the historically (NH4)2SO4-treated soil, 49.0–58.0% of the N2O emitted originated from nitrification and 42.0–51.0% from denitrification during incubation. The production of N2O was accompanied by a decrease in soil NH4+ concentrations and a parallel increase in the concentration of soil NO3−. In addition, the abundance of the bacterial and archaeal amoA gene increased during the incubation. Conversely, in the soil historically treated with KNO3, the 15N isotopic analyses showed that denitrification contributed 84.0–99.0% of the total N2O produced. Decreases in soil NO3− concentrations paralleled the increase in 15N enrichment of N2O and the abundance of the nirK, nirS, norB and nosZI genes. The results also showed that values of 15N2 enrichment were significantly higher in the KNO3-treated soil, which is in line with the higher abundance of the nosZI gene. Calculation of the N transformation rates indicated that autotrophic nitrification and denitrification were responsible for N2O production in the historically (NH4)2SO4-treated soil and that denitrification was the most important N2O source in the soil treated with KNO3. We conclude that N-fertilisation history, and not simply soil oxygen availability, affect the relative contributions of nitrification and denitrification to soil N2O emissions. Indeed, here we have shown that nitrification can be an important N2O source pro