Molecular techniques and stable isotope ratios at natural abundance give complementary inferences about N2O production pathways in an agricultural soil following a rainfall event

Abstract

The abatement of anthropogenic nitrous oxide (N2O) emissions depends on sound management of manure nitrogen (N) and an ability to track and predict manure-derived microbial N2O production in soils. The objective of this study was to investigate the utility of applying a novel combination of micrometeorological, stable isotope, and molecular methods to determine the short-term dynamics of N2O production processes in soil. Nitrous oxide emissions were continuously monitored in two drought-stressed agricultural fields treated with liquid dairy manure applied either in the fall or spring over an N2O emission event triggered by a heavy rainfall. In situ δ15N–N2O and δ18O–N2O measurements were used in conjunction with abundance (DNA) and potential activity (cDNA) measurements of key microbial N cycling gene and transcript (mRNA) targets to evaluate if these two techniques provided similar inferences about N2O soil processes occurring over the emission event. Soil gas was sampled at 4 depths along a profile from 10 to 50 cm below the surface and a multi-layer diffusion model was used to calculate the vertical N2O fluxes, the change in storage, and the net production of N2O in each layer.The rainfall event triggered N2O production in both fields at all depths, but the uppermost soil layer was the main source of N2O emissions throughout. Soil concentrations and surface emissions of N2O increased rapidly reaching a maximum 4 d following rainfall and were accompanied by a significant increase in the abundance of both nitrifier and denitrifier genes and total bacterial and archaeal genes and transcripts. A similar increase in the transcription of functional genes was not detected, but relatively high levels of archaeal ammonia monooxygenase (crenamoA) and bacterial nitrite reductase (nirS) transcripts were present in both fields throughout the study, indicating nitrification and denitrification were occurring concomitantly. High levels of H2O–NOx oxygen exchange at our study site precluded the use of δ18O–N2O values to separate different sources. Prior to the rain, both fields had very small amounts of soil ammonium and the stable nitrogen isotope characterization of N2O in the dry soil implicated denitrification as the main source of N2O rather than nitrifier-denitrification or hydroxylamine oxidation. Following the rain event, the δ15N–N2O values showed a change in production process as nitrifier-denitrification became a more dominant N2O source. By pairing stable isotope measurements with molecular analyses we were able to verify that these two approaches provide similar inferences about soil N2O production processes during an emission event in manure-applied fields. Although significantly higher N2O flux and N2O accumulation in the soil profile were observed for the field receiving manure in the spring, differences in isotope signal and molecular analysis between fields were not observed. To our knowledge this is the first time that isotope and molecular techniques have been used to study processes resulting in N2O production in manure-amended soils in situ. Whereas stable isotopes were useful for directly tracking the pathways of N2O production, molecular analyses revealed the status of the N cycling communities before, during and after the emission event. This information helped explain the observed differences in emissions between fields, and it helped to support our isotope-based conclusions.