Abstract. 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.