Use of oxygen isotopes to differentiate between nitrous oxide produced by fungi or bacteria during denitrification

Fungal denitrifiers can contribute substantially to N₂ O emissions from arable soil and show a distinct site preference for N₂ O (SP(N₂ O)). This study sought to identify another process-specific isotopic tool to improve precise identification of N₂ O of fungal origin by mass spectrometric analysis of the N₂ O produced.

Three pure bacterial and three fungal species were incubated under denitrifying conditions in treatments with natural abundance and stable isotope labelling to analyse the N₂ O 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 δ¹⁸ O(N₂ O) values and isotopic fractionation of O, as well as SP(N₂ O) values, as a perspective to differentiate between microbial denitrifiers.

Oxygen exchange during N₂ O 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.

δ¹⁸ O(N₂ O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N₂ O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N₂ O.