Rationale: Fungal denitrifiers can contribute substantially to N2O emissions from arable soil
and show a distinct site preference for N2O (SP(N2O)). This study sought to identify another process‐specific isotopic tool to improve precise identification of N2O of fungal origin by mass spectrometric analysis of the N2O produced. Methods: Three pure bacterial and three fungal species were incubated under denitrifying conditions in treatments with natural abundance and stable isotope labelling to analyse the N2O produced. Combining different applications of isotope ratio mass spectrometry enabled us to estimate the oxygen (O) exchange accelerated by denitrifying enzymes and the ongoing microbial pathway in parallel. This experimental set‐up allowed the determination of ~d18O(N2O) values and isotopic fractionation of O, as well as SP(N2O) values, as a perspective to differentiate between microbial denitrifiers. Results: Oxygen exchange during N2O production was lower for bacteria than for fungi, differed between species, and depended also on incubation time. Apparent O isotopic fractionation during denitrification was in a similar range for bacteria and fungi, but application of the fractionation model indicated that different enzymes in bacteria and fungi were responsible for O exchange. This difference was associated with different isotopic fractionation for bacteria and fungi. Conclusions: ~d18O(N2O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N2O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N2O.
