A novel 15N tracer approach for the quantification of N2 and N2O emissions from soil incubations in a completely automated laboratory set up

The microbial mediated production of nitrous oxide (N2O) and its reduction to dinitrogen (N2) via denitrification represents a loss of nitrogen (N) from fertilised agro-ecosystems to the atmosphere. Although denitrification has received great interest by biogeochemists in the last decades, the magnitude of N2lossesand related N2:N2O ratios from soils still are largely unknown due to methodical constraints. We present a novel 15N tracer approach, based on a previous developed tracer method to study denitrification in pure bacterial cultures which was modified for the use on soil incubations in a completely automated laboratory set up. The method uses a background air in the incubation vessels that is replaced with a helium-oxygen gas mixture with a 50-fold reduced N2 background (2 % v/v). This method allows for a direct and sensitive quantification of the N2 and N2O emissions from the soil with isotope-ratio mass spectrometry after 15N labelling of denitrification N substrates and minimises the sensitivity to the intrusion of atmospheric N2 at the same time. The incubation set up was used to determine the influence of different soil moisture levels on N2 and N2O emissions from a sub-tropical pasture soil in Queensland/Australia. The soil was labelled with an equivalent of 50 μg-N per gram dry soil by broadcast application of KNO3solution (4 at.% 15N) and incubated for 3 days at 80% and 100% water filled pore space (WFPS), respectively. The headspace of the incubation vessel was sampled automatically over 12hrs each day and 3 samples (0, 6, and 12 hrs after incubation start) of headspace gas analysed for N2 and N2O with an isotope-ratio mass spectrometer (DELTA V Plus, Thermo Fisher Scientific, Bremen, Germany(. In addition, the soil was analysed for 15N NO3- and NH4+ using the 15N diffusion method, which enabled us to obtain a complete N balance. The method proved to be highly sensitive for N2 and N2O emissions detecting N2O emissions ranging from 20 to 627 μN kg-1soil-1hr-1and N2 emissions ranging from 4.2 to 43 μN kg-1soil-1hr-1for the different treatments. The main end-product of denitrification was N2O for both water contents with N2 accounting for 9% and 13% of the total denitrification losses at 80% and 100%WFPS, respectively. Between 95-100% of the added 15N fertiliser could be recovered. Gross nitrification over the 3 days amounted to 8.6 μN g-1 soil-1 and 4.7 μN g-1 soil-1, denitrification to 4.1 μN g-1 soil-1 and 11.8 μN g-1 soil-1at 80% and 100%WFPS, respectively. The results confirm that the tested method allows for a direct and highly sensitive detection of N2 and N2O fluxes from soils and hence offers a sensitive tool to study denitrification and N turnover in terrestrial agro-ecosystems.