Combining low level labelling with 15N and 15N site preference to distinguish N2O production by nitrification and fungal denitrification

Isotopocules of the greenhouse gas nitrous oxide (N₂O), i.e. δ¹⁸O, average δ¹⁵N (δ¹⁵N^bulk), and ¹⁵N site preference (SP) values were used to distinguish between N₂O production pathways in soil. However, as many N₂O production pathways coexist and N₂O can be reduced to N₂, it is not possible to distinguish pathways based only on the natural abundance of N₂O. This applies especially to nitrification and fungal denitrification, where the specific high SP values overlap. Combining ¹⁵N tracer approaches and natural abundance approaches (especially using SP values) could serve to disentangle such pathways, but with the disadvantage that both approaches have to be carried out as parallel experiments.

With this contribution, we present an experimental concept based on the theory, that low level labelling with ¹⁵N of N₂O precursors may allow both, a clear distinction of nitrate or ammonium (NO₃^- or NH₄⁺, respectively) derived N₂O fluxes by ¹⁵N tracing, and the use of SP values of N₂O as additional constraint. This could potentially expand possibilities to evaluate and validate current natural abundance isotopocule mapping approaches.

We will present first results of three experiments to investigate the impact of low labelled precursors on SP values of N₂O produced. Each experiment included treatments with unlabeled and low labelled ¹⁵N precursors to test if low labelling with ¹⁵N affects N₂O isotopocules. In one incubation experiment (i) various levels of ¹⁵N labelling of NO₃^- (between 0.6 and 5 at% ¹⁵N) were used for incubations with Pseudomonas aureofaciens. In another experiment (ii) two pure bacterial (P. aureofaciens and Paracoccus denitrificans) and one pure fungal culture (Fusarium oxysporum) known to be capable of reducing NO₃^- or NO₂^-, respectively, were used. In all experiments, isotopocules of N₂O were unaffected by N₂O reduction as this reduction step could be excluded with selected species. To further investigate isotopocules of N₂O affected by co-occuring processes as well as N₂O reduction a third incubation experiment with two repacked soils was conducted. For this approach, nitrification and/or denitrification was induced by applying NH₄SO₄ and KNO₃ as N₂O precursors, either unlabeled in one treatment or with ¹⁵N labelled KNO₃ (max. 1.1 at% ¹⁵N) in another treatment, both under dry (40% water filled pore space (WFPS)) or wet (80% WFPS) soil conditions.

Based on the results presented, we will be able to give an outlook whether this method can be used to distinguish between nitrification and fungal denitrification.