The mechanism of oxygen isotope fractionation during N₂O production by denitrification

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 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. We performed several soil incubation experiments. For the first time, ∆17 O isotope tracing was applied to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found bacterial denitrification to be typically associated with almost complete oxygen isotope exchange and a stable difference in δ18O between soil water and the produced N₂O of δ18O(N₂O / H₂O) = (17.5±1.2) ‰. However, some experimental setups yielded oxygen isotope exchange as low as 56 % and a higher δ18O(N₂O / H₂O) of up to 37‰. The extent of isotope exchange and δ18O(N₂O / H₂O) showed a very significant correlation (R2 = 0.70, p < 0.00001). We hypothesise 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 the isotope exchange occurs prior to the isotope branching and that the mechanism of 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 bacte- 5 rial and fungal denitrification, due to their different magnitudes of branching isotope effects