Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

δ O(N O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N O.

The precision and accuracy of this method were comparable with or better than previously reported for similar measurements. The proposed method allows for the analysis of all quantities within one run, thus reducing the measurement and sample preparation time as well as increasing the reliability of the results.

Abstract. The prediction of nitrous oxide (N2O) and of dinitrogen (N2) emissions formed by biotic denitrification in soil is notoriously difficult due to challenges in capturing co-occurring processes at microscopic scales. N2O production and reduction depend on the spatial extent of anoxic conditions in soil, which in turn are a function of oxygen (O2) supply through diffusion and O2 demand by respiration in the presence of an alternative electron acceptor (e.g. nitrate). This study aimed to explore controlling factors of complete denitrification in terms of N2O and (N2O + N2) fluxes in repacked soils by taking micro-environmental conditions directly into account. This was achieved by measuring microscale oxygen saturation and estimating the anaerobic soil volume fraction (ansvf) based on internal air distribution measured with X-ray computed tomography (X-ray CT). O2 supply and demand were explored systemically in a full factorial design with soil organic matter (SOM; 1.2 % and 4.5 %), aggregate size (2–4 and 4–8 mm), and water saturation (70 %, 83 %, and 95 % water-holding capacity, WHC) as factors. CO2 and N2O emissions were monitored with gas chromatography. The 15N gas flux method was used to estimate the N2O reduction to N2. N gas emissions could only be predicted well when explanatory variables for O2 demand and O2 supply were considered jointly. Combining CO2 emission and ansvf as proxies for O2 demand and supply resulted in 83 % explained variability in (N2O + N2) emissions and together with the denitrification product ratio [N2O / (N2O + N2)] (pr) 81 % in N2O emissions. O2 concentration measured by microsensors was a poor predictor due to the variability in O2 over small distances combined with the small measurement volume of the microsensors. The substitution of predictors by independent, readily available proxies for O2 demand (SOM) and O2 supply (diffusivity) reduced the predictive power considerably (60 % and 66 % for N2O and (N2O+N2) fluxes, respectively). The new approach of using X-ray CT imaging analysis to directly quantify soil structure in terms of ansvf in combination with N2O and (N2O + N2) flux measurements opens up new perspectives to estimate complete denitrification in soil. This will also contribute to improving N2O flux models and can help to develop mitigation strategies for N2O fluxes and improve N use efficiency.

This is the first report on oxygen exchange with water during fungal denitrification. The exchange appears to be within the range previously reported for bacterial denitrification. This adds to the difficulty of differentiating N2O producing processes based on the origin of N2O-O. However, the large oxygen exchange repeatedly observed for bacteria and now also fungi could lead to less variability in the δ(18)O values of N2O from soils, which could facilitate the assessment of the extent of N2O reduction.