Oxygen isotope exchange and isotopic fractionation during N2O production by denitrification Dominika Lewicka-Szczebak1 · Jan Kaiser2 · Reinhard Well1 1 Thünen Institute of Climate-Smart Agriculture, Germany, 2 Centre for Ocean and Atmospheric Sciences, School of Environmental Sciences, University of East Anglia, Norwich, UK Methods Introduction In previous experiments, an 18O-enriched tracer was applied to quantify the magnitude of oxygen exchange (1,2). Such an approach does not permit determining any oxygen isotope fractionation. Here, we applied two novel experimental approaches (FIG. 2): (i) waters of two distinct δ18O values within the natural range (–1.5 and –14.8 ‰ relative to VSMOW) were used for rewetting the incubated soils and nitrates of two distinct δ18O values (27 and 56‰ relative to VSMOW) were used for fertilizing the soils and the oxygen exchange was calculated from comparing the relative isotope ratio difference between N2O and H2O for these different treatments (2); (ii) soils were amended with natural NaNO3 Chile Saltpeter characterized by a high Δ17O (ca. 20‰) and with synthetic NaNO3 characterised by negative Δ17O (ca. -5‰) and the 17O excess of the N2O product was measured. Both approaches were applied simultaneously on the same soil samples, which allowed quantifying the oxygen isotope exchange with two independent methods at the same time. The N2O reduction was inhibited with acetylene method and δ18O values of the N2O produced were measured to determine the oxygen fractionation during N2O production. Stable isotopic analyses of N2O may help in quantification of soil denitrification processes and distinguishing various microbial pathways. Oxygen isotopes may be especially useful, in particular the 17O excess, Δ17O, to quantify any oxygen isotope exchange between soil water and intermediate products (NO2-, NO) (FIG.1). This significantly influences the δ18O values of the N2O produced. Previous studies showed this exchange to be nearly complete, up to 90% (1,2). However, there are very few studies on the associated oxygen isotopic fractionation and its potential coupling to the magnitude of oxygen isotope exchange with soil water (2,3). We hypothesize that for high oxygen exchange, the oxygen isotopic fractionation will show little variability providing consistent and characteristic δ18O values for N2O produced during denitrification. FIG.1 Oxygen isotopic fractionation during denitrification as a result of branching effects (εb) und exchange effects (εe) associated with the following enzymatic steps: nar, nir and nor. FIG. 2 Scheme of microcosm design Results & Discussion O isotope effect between N2O and H2O (Δδ18O (N2O-H2O)) is very stable. In contrast, the isotope effect between N2O and NO3- (Δδ18O (N2O-NO3-)) shows large variations (FIG. 3): Both methods showed nearly complete (93-100%) oxygen exchange with soil water for both soil types (sand and silt loam), water contents (50 to 80% WFPS), temperatures (8 and 22°C), and N2O production rates (from 0.25 to 12.41 mg d-1 kg-1). The method proposed by Snider et al.(2) was applied to estimate O-exchange with soil water and O isotopic fractionation based on the δ18O values of N2O, H2O and NO3- (FIG. 4): Very low Δ17O values were measured in N2O (TAB. 1), so the 17O excess of soil NO3- is not reflected in the produced N2O, hence the majority of O-atoms must have been exchanged with soil water. The magnitude of O-exchange was calculated as: δ18O (N2O) values show clear relations to δ18O of soil water, whereas δ18O of soil nitrates has only very minor influence. Hence, the O isotopic fractionation during denitrification should be considered in relation to soil water, rather than soil nitrates. Δ17O (N2O) show very consistent low values, which indicate that denitrification is rather not the source of N2O with significant 17O excess, as previously supposed (4,5). TAB.1 17O excess in soil nitrate and in N2O with calculated % of 17O excess remained in N2O when compared to NO3- and % of O-atoms exchanged with soil water experiment Δ17O (NO3-) Δ17O (N2O) remained Δ17O in % O-exchange in % 1 sand, 8°C 11.7 0.6 5.3 94.7 sand, 22°C 10.4 0.4 3.7 96.3 silt loam, 22°C 8.1 0.2 2.9 97.1 2 3.4 6.2 93.8 -4.5 -0.3 7.3 92.7 2.6 0.1 2.7 97.3 -4.8 -0.2 5.0 95.0 Conclusions Denitrification is associated with nearly complete O-exchange with soil water. δ18O values of produced N2O are almost independent of soil nitrate isotopic signature but in strict relation to soil water. The oxygen isotope effect Δδ18O (N2O-H2O), defined as the relative isotope ratio difference between product (N2O) and substrate (soil water), is quite stable, from 16 to 20 ‰. This confirms that δ18O values of N2O produced during denitrification may be useful in distinguishing pathways and product ratio determination, when δ18O of soil water is known. Moreover, the very low 17O excess found in N2O indicates that the hypothesis of soil denitrification contributing to the oxygen isotope excess of atmospheric N2O can be rejected (4,5). FIG.3 Isotope effects between produced N2O and soil water and nitrate, on the right δ18O values of the initial soil nitrate for different treatments FIG.4 Corelation between O isotopic signatures of N2O and soil water expressed in relation to soil nitrate, the equation of linear fit allows for estimation of O-exchange with soilwater and the associated isotopic fractionation Acknowledgements: This study was funded by the Deutsche Forschungsgemeinschaft. We thank Jens Dyckmanns for Δ17O analyses performed at the Centre for Stable Isotope Research and Analysis (University of Göttingen). References: 1. Kool, et al., 2009. The O-18 signature of biogenic nitrous oxide is determined by O exchange with water .Rapid Communication in Mass Spectrometry 23: 104-108 2. Snider, et al., 2013. A new mechanistic model of d18O-N2O formation by denitrification. Geochimica et Cosmochimica Acta 112: 102-115 3. Lewicka-Szczebak, et al., 2014. Experimental determinations of isotopic fractionation factors associated with N2O production and reduction during denitrification in soils .Geochimica et Cosmochimica Acta 134: 55-73 4. Kaiser, et al., 2004. Contribution of mass-dependent fractionation to the oxygen isotope anomaly of atmospheric nitrous oxide. Journal of Geophysical Research-Atmospheres 109 (D3): D03305 5. Michalski, et al., 2003. First measurements and modeling of Delta O-17 in atmospheric nitrate. Geophysical Research Letters 30 (16): 1870 Contact: dominika.lewicka-szczebak@ti.bund.de