Advancement of the Acetylene Inhibition Technique Using Time Series Analysis on Air-Dried Floodplain Soils to Quantify Denitrification Potential

Denitrification in floodplain soils is one key process that determines the buffering capacity of riparian zones in terms of diffuse nitrate pollution. One widely used approach to measure the denitrification potential is the acetylene inhibition technique that requires fresh soil samples. We conducted experiments with air-dried soils using a time series analysis to determine the optimal rewetting period. Thus, air-dried soil samples from six different floodplain areas in Germany were rewetted for 1 to 13days to 100% water-filled pore space. We analyzed nitrogen accumulated as N2O in the top of anaerobic flasks with and without acetylene by gas chromatography after four hours of incubation. We observed an overall optimal rewetting of at least seven days for complete denitrification. We also saw the strong influence of pH and field capacity on the denitrification product ratio; in soils with pH < 7, we hardly assumed complete denitrification, whereas the treatments with pH > 7 achieved stable values after seven days of rewetting. This advanced method provides the opportunity to carry out campaigns with large soil sample sizes on the landscape scale, as samples can be stored dry until measurements are taken.

Application of the ¹⁵N gas-flux method for measuring in situ N₂ and N₂O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique

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.

Application of the ¹⁵N-Gas Flux method for measuring in situ N₂ and N₂O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique

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 fertilised 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, 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 Pre-concentrator (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. For our chamber design (volume / surface = 8:1) and a 20 h incubation period, 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. The N2 flux ranged between 2.4 and 416.6 μg N m−2 h−1, and the grassland soils showed on average 3 and 14 times higher denitrification rates than the woodland and organic soils respectively. The N2O flux was on average 20 to 200 times lower than the N2 flux, while the denitrification product ratio (N2O/N2 + N2O) was low, ranging between 0.03 and 13 %. Total denitrification rates measured by the acetylene inhibition technique under the same field conditions 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 attributed to the incomplete inhibition of N2O reduction to N2 under relatively high soil moisture content. The results show that the 15N Gas-Flux method can be used for quantifying N2 and N2O production rates in natural terrestrial ecosystems, thus significantly improving our ability to constrain ecosystem N budgets.

Acetylene inhibition for measuring denitrification rates in activated sludge

A method of determining the actual and potential denitrification rates in activated sludge samples by measuring accumulation of nitrous oxide after inhibition by acetylene was adapted and applied.Rates obtained with the acetylene inhibition technique were compared with those calculated through mass balances of total nitrogen from process data as well as those measured as nitrate utilisation.The acetylene inhibition technique was a reliable method for determination of denitrifying activity.The influence of acetate, ethanol, methanol and nitrate additions was examined.Acetate gave the best response upon denitrification followed by ethanol and methanol.The bacteria in the activated sludge could not denitrify at full capacity due to limitations in the supply of electron donors.The actual rates measured with the acetylene inhibition technique corresponded to the rates obtained from process data.The potential rates, determined with additions of acetate and nitrate in excess (240 mg l−1 and 28 mg N l−1), were 3–7 times higher than the actual rates and the rates calculated from process data.There is, thus, a potential for a more rapid denitrification rate.The potential denitrification rate measured with the acetylene inhibition technique was equivalent to the nitrate utilisation rate.