Stable isotopic analyses of soil-emitted N₂O (δ¹⁵N^bulk, δ¹⁸O and δ¹⁵N^sp = ¹⁵N site preference within the linear N₂O molecule) may help to quantify N₂O reduction to N₂, an important but rarely quantified process in the soil nitrogen cycle. The N₂O residual fraction (remaining unreduced N₂O, rN₂O) can be theoretically calculated from the measured isotopic enrichment of the residual N₂O. However, various N₂O-producing pathways may also influence the N₂O isotopic signatures, and hence complicate the application of this isotopic fractionation approach. Here this approach was tested based on laboratory soil incubations with two different soil types, applying two reference methods for quantification of rN₂O: helium incubation with direct measurement of N₂ flux and the ¹⁵N gas flux method. This allowed a comparison of the measured rN₂O values with the ones calculated based on isotopic enrichment of residual N₂O. The results indicate that the performance of the N₂O isotopic fractionation approach is related to the accompanying N₂O and N₂ source processes and the most critical is the determination of the initial isotopic signature of N₂O before reduction (δ₀). We show that δ₀ can be well determined experimentally if stable in time and then successfully applied for determination of rN₂O based on δ¹⁵N^sp values. Much more problematic to deal with are temporal changes of δ₀ values leading to failure of the approach based on δ¹⁵N^sp values only. For this case, we propose here a dual N₂O isotopocule mapping approach, where calculations are based on the relation between δ¹⁸O and δ¹⁵N^sp values. This allows for the simultaneous estimation of the N₂O-producing pathways' contribution and the rN₂O value.

The approaches based on natural abundance N₂O stable isotopes are often applied for the estimation of mixing proportions between various N₂O-producing pathways as well as for estimation of the extent of N₂O reduction to N₂. But such applications are associated with numerous uncertainties; hence, their limited accuracy needs to be considered. Here we present the first systematic validation of these methods for laboratory and field studies by applying the ¹⁵N gas-flux method as the reference approach. Besides applying dual-isotope plots for interpretation of N₂O isotopic data, for the first time we propose a three dimensional N₂O isotopocule model based on Bayesian statistics to estimate the N₂O mixing proportions and reduction extent based simultaneously on three N₂O isotopic signatures (δ¹⁵N, δ¹⁵N ^SP, and δ¹⁸O). Determination of the mixing proportions of individual pathways with N₂O isotopic approaches often appears imprecise, mainly due to imperfect isotopic separation of the particular pathways. Nevertheless, the estimation of N₂O reduction is much more robust, when applying an optimal calculation strategy, typically reaching an accuracy of N₂O residual fraction determination of about 0.15.

Soil moisture strongly affects the balance between nitrification, denitrification and N₂O reduction and therefore the nitrogen (N) efficiency and N losses in agricultural systems. In rice systems, there is a need to improve alternative water management practices, which are designed to save water and reduce methane emissions but may increase N₂O and decrease nitrogen use efficiency. In a field experiment with three water management treatments, we measured N₂O isotope ratios of emitted and pore air N₂O (δ¹⁵N, δ¹⁸O and site preference, SP) over the course of 6 weeks in the early rice growing season. Isotope ratio measurements were coupled with simultaneous measurements of pore water ${\mathrm{NO}}_{\mathrm{3}}^{-}$ , ${\mathrm{NH}}_{\mathrm{4}}^{+}$ , dissolved organic carbon (DOC), water-filled pore space (WFPS) and soil redox potential (Eh) at three soil depths. We then used the relationship between SP × δ¹⁸O-N₂O and SP × δ¹⁵N-N₂O in simple two end-member mixing models to evaluate the contribution of nitrification, denitrification and fungal denitrification to total N₂O emissions and to estimate N₂O reduction rates. N₂O emissions were higher in a dry-seeded + alternate wetting and drying (DS-AWD) treatment relative to water-seeded + alternate wetting and drying (WS-AWD) and water-seeded + conventional flooding (WS-FLD) treatments. In the DS-AWD treatment the highest emissions were associated with a high contribution from denitrification and a decrease in N₂O reduction, while in the WS treatments, the highest emissions occurred when contributions from denitrification/nitrifier denitrification and nitrification/fungal denitrification were more equal. Modeled denitrification rates appeared to be tightly linked to nitrification and ${\mathrm{NO}}_{\mathrm{3}}^{-}$ availability in all treatments; thus, water management affected the rate of denitrification and N₂O reduction by controlling the substrate availability for each process (${\mathrm{NO}}_{\mathrm{3}}^{-}$ and N₂O), likely through changes in mineralization and nitrification rates. Our model estimates of mean N₂O reduction rates match well those observed in ¹⁵N fertilizer labeling studies in rice systems and show promise for the use of dual isotope ratio mixing models to estimate N₂ losses.

The isotopic composition of soil-derived N₂O can help differentiate between N₂O production pathways and estimate the fraction of N₂O reduced to N₂. Until now, δ¹⁸O of N₂O has been rarely used in the interpretation of N₂O isotopic signatures because of the rather complex oxygen isotope fractionations during N₂O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H₂O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N₂O production by soil denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the δ¹⁸O values of the produced N₂O. In our soil incubation experiments Δ¹⁷O isotope tracing was applied for the first time to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found that N₂O formation in static anoxic incubation experiments was typically associated with oxygen isotope exchange close to 100 % and a stable difference between the ¹⁸O ∕ ¹⁶O ratio of soil water and the N₂O product of δ¹⁸O(N₂O ∕ H₂O) = (17.5 ± 1.2) ‰. However, flow-through experiments gave lower oxygen isotope exchange down to 56 % and a higher δ¹⁸O(N₂O ∕ H₂O) of up to 37 ‰. The extent of isotope exchange and δ¹⁸O(N₂O ∕ H₂O) showed a significant correlation (R² = 0.70, p < 0.00001). We hypothesize that this observation was due to the contribution of N₂O from another production process, most probably fungal denitrification. An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification, isotope exchange occurs prior to isotope branching and that this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0 ± 0.9) ‰, in contrast to fungal denitrification where higher values of up to 30 ‰ have been reported previously. This suggests that δ¹⁸O might be used as a tracer for differentiation between bacterial and fungal denitrification, due to their different magnitudes of branching isotope effects.

Nitrogen is an essential nutrient in the environment that exists in multiple oxidation states in nature. Numerous microbial processes are involved in its transformation. Knowledge about very complex N cycling has been growing rapidly in recent years, with new information about associated isotope effects and about the microbes involved in particular processes. Furthermore, molecular methods that are able to detect and quantify particular processes are being developed, applied and combined with other analytical approaches, which opens up new opportunities to enhance understanding of nitrogen transformation pathways. This review presents a summary of the microbial nitrogen transformation, including the respective isotope effects of nitrogen and oxygen on different nitrogen-bearing compounds (including nitrates, nitrites, ammonia and nitrous oxide), and the microbiological characteristics of these processes. It is supplemented by an overview of molecular methods applied for detecting and quantifying the activity of particular enzymes involved in N transformation pathways. This summary should help in the planning and interpretation of complex research studies applying isotope analyses of different N compounds and combining microbiological and isotopic methods in tracking complex N cycling, and in the integration of these results in modelling approaches.

Nitrous oxide (N₂O) is of major importance as a greenhouse gas and precursor of ozone (O₃) destruction in the stratosphere mostly produced in soils. The soil-emitted N₂O is generally predominantly derived from denitrification and, to a smaller extent, nitrification, both processes controlled by environmental factors and their interactions, and are influenced by agricultural management. Soil water content expressed as water-filled pore space (WFPS) is a major controlling factor of emissions and its interaction with compaction, has not been studied at the micropore scale. A laboratory incubation was carried out at different saturation levels for a grassland soil and emissions of N₂O and N₂ were measured as well as the isotopocules of N₂O. We found that flux variability was larger in the less saturated soils probably due to nutrient distribution heterogeneity created from soil cracks and consequently nutrient hot spots. The results agreed with denitrification as the main source of fluxes at the highest saturations, but nitrification could have occurred at the lower saturation, even though moisture was still high (71 % WFSP). The isotopocules data indicated isotopic similarities in the wettest treatments vs. the two drier ones. The results agreed with previous findings where it is clear there are two N pools with different dynamics: added N producing intense denitrification vs. soil N resulting in less isotopic fractionation.