Application of the <sup>15</sup>N gas-flux method for measuring in situ N<sub>2</sub> and N<sub>2</sub>O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique

Abstract

Abstract. Soil denitrification is considered the most un-constrained process in the global N cycle due to uncertain in situ N2 flux measurements, particularly in natural and semi-natural terrestrial ecosystems. 15N tracer approaches can provide in situ measurements of both N2 and N2O simultaneously, but their use has been limited to fertilized agro-ecosystems due to the need for large 15N additions in order to detect 15N2 production against the high atmospheric N2. For 15N–N2 analyses, we have used an “in-house” laboratory designed and manufactured N2 preparation instrument which can be interfaced to any commercial continuous flow isotope ratio mass spectrometer (CF-IRMS). The N2 prep unit has gas purification steps and a copper-based reduction furnace, and allows the analysis of small gas injection volumes (4 µL) for 15N–N2 analysis. For the analysis of N2O, an automated Tracegas Preconcentrator (Isoprime Ltd) coupled to an IRMS was used to measure the 15N–N2O (4 mL gas injection volume). Consequently, the coefficient of variation for the determination of isotope ratios for N2 in air and in standard N2O (0.5 ppm) was better than 0.5 %. The 15N gas-flux method was adapted for application in natural and semi-natural land use types (peatlands, forests, and grasslands) by lowering the 15N tracer application rate to 0.04–0.5 kg 15N ha−1. The minimum detectable flux rates were 4 µg N m−2 h−1 and 0.2 ng N m−2 h−1 for the N2 and N2O fluxes respectively. Total denitrification rates measured by the acetylene inhibition technique in the same land use types correlated (r = 0.58) with the denitrification rates measured under the 15N gas-flux method, but were underestimated by a factor of 4, and this was partially attributed to the incomplete inhibition of N2O reduction to N2, under a relatively high soil moisture content, and/or the catalytic NO decomposition in the presence of acetylene. Even though relatively robust for in situ denitrification measurements, methodological uncertainties still exist in the estimation of N2 and N2O fluxes with the 15N gas-flux method due to issues related to non-homogenous distribution of the added tracer and subsoil gas diffusion using open-bottom chambers, particularly during longer incubation duration. Despite these uncertainties, the 15N gas-flux method constitutes a more reliable field technique for large-scale quantification of N2 and N2O fluxes in natural terrestrial ecosystems, thus significantly improving our ability to constrain ecosystem N budgets.

Improvement of the 15N gas flux method for in situ measurement of soil denitrification and its product stoichiometry

“…In the past, comparison of different methods to determine denitrification processes had always been complicated by the difficulty in keeping the soil conditions for the compared methods identical. 13,[29][30][31][32] This also applied to our study because we did not have the resources to conduct 15 NGF+ and conventional 15 NGF at exactly the same time. Figure 4), we showed that subsoil diffusion was indeed relevant in our setup.…”

“…Underestimation of denitrification rates can occur because a certain fraction of the 15 Nlabelled denitrification products is not emitted from the soil surface, but diffuses to the subsoil. 13 When applying 15 NGF in the laboratory, the sensitivity can be augmented by incubation under an N 2 -depleted atmosphere in a closed system 14,15 or in an open system where the soil headspace is continuously flushed at a constant rate with an artificial N 2 -depleted atmosphere. 16 Direct measurement of N 2 fluxes using the He/O 2 method is not subject to the problems associated with 15 N-based methods, 17 but the need for sophisticated gastight incubation systems limits its use to laboratory incubations only and low N 2 fluxes are difficult to detect due to the difficulty in completely removing atmospheric N 2 .…”

Denitrification Rate and Its Potential to Predict Biogenic N2O Field Emissions in a Mediterranean Maize-Cropped Soil in Southern Italy

“…− production via nitrification, the possible slow diffusion of C 2 H 2 into fine textured soils and rapid decomposition of C 2 H 2 [44,[47][48][49]. Nonetheless, beyond this limitation of the method, the AIT has been effectively and widely used and recommended (also in recent pertinent literature) to compare sites and experimental treatments (in both terrestrial and aquatic environments) and to evaluate controlling factors [44,46,[50][51][52].…”

Underestimation of denitrification rates from field application of the <sup>15</sup>N gas flux method and its correction by gas diffusion modelling

“…Closing incubation vessels for a limited time and estimating denitrification from headspace concentration (e.g. Meyer et al, 2010;Siegel et al, 1982) inevitably lead to underestimation of denitrification rates. Experimental evidence for this underestimation was obtained by destroying pore structure at final sampling to homogenize headspace and pore space (Harter et al, 2016).…”

“…There are three principles to overcome the latter problem: (i) adding NO − 3 that is highly enriched in 15 N and monitoring 15 N-labelled denitrification products ( 15 N gas flux method; e.g. Siegel et al, 1982), (ii) adding acetylene to block N 2 O reductase quantitatively and estimating total denitrification from N 2 O production (acetylene inhibition technique, e.g. Felber et al, 2012), and (iii) measuring denitrification gases during incubation of soils in the absence of atmospheric N 2 using gas-tight containers and artificial helium-oxygen atmosphere (HeO 2 method; Scholefield et al, 1997;Butterbach-Bahl et al, 2002).…”