Acetylene inhibition of N2O reduction in laboratory soil and groundwater denitrification assays: evaluation by 15N tracer and 15N site preference of N2O

Acetylene inhibition of N2O reduction in laboratory soil and groundwaterdenitrification assays: evaluation by 15N tracer and 15N site preference ofN2ODaniel Weymann (1), Reinhard Well (2), Dominika Lewicka-Szczebak (2,3), and Rohe Lena (2)(1) Forschungszentrum Juelich, Agrosphere Institute (IBG-3), Juelich, Germany (d.weymann@fz-juelich.de), (2)Thünen-Institute of Climate-Smart Agriculture, Braunschweig, Germany, (3) University of Wroclaw, PolandThe measurement of denitrification in soils and aquifers is still challenging and often enough associated withconsiderable experimental effort and high costs. Against this background, the acetylene inhibition technique (AIT)applied in laboratory soil and groundwater denitrification assays is by far the most effective approach. However,this method has been largely criticized, as it is susceptible to underestimate denitrification rates and adds anadditional carbon source to the substrates to be investigated.Here we provide evidence that the AIT is not necessarily an inappropriate approach to measure denitrification,that its reliability depends on the drivers governing the process, and that the 15N site preference of N2O (SP)may serve as a tool to assess this reliability. Two laboratory batch experiments were conducted, where sandyaquifer material and a peat soil were incubated as slurries. We established (i) a standard anaerobic treatment byadding KNO3 (10 mg N L-1), (ii) an oxygen treatment by adding KNO3 and O2 (5 mg L-1), and (iii) a glucosetreatment by adding KNO3 supplemented with glucose (200 mg C L-1). Both experiments were run under 10 %(v/v) acetylene atmosphere and as 15N tracer treatments using labeled K15NO3 (60 atom % 15N).In the case of the standard anaerobic treatments, we found a very good agreement of denitrification potentialobtained by the AIT and 15N tracer methods. SP of N2O of the AIT samples from this treatment ranged between-4.8 and 2.6 h which is indicative for N2O production during bacterial denitrification but not for N2O reductionto N2. In contrast, we observed substantial underestimation of denitrification by AIT for the glucose treatmentscompared to the 15N method, i.e. denitrification was underestimated by 36 % (sandy aquifer material) and 47 %(peat soil). SP of N2O of the AIT samples from this treatment ranged between 4.5 and 9.6 h which suggestsoccurrence of bacterial N2O reduction. In the case of the oxygen treatments, we observed a very good agreementof denitrification potential obtained by the AIT and 15N tracer methods for the aquifer material, but a significantunderestimation of 20 % in the AIT samples of the peat soil. The 15N site preference of N2O again mirrored thisand ranged between -1.2 and -3.5 h(aquifer material) and 5.5 and 11.0 (peat soil), respectively.We conclude that the AIT can act as a reliable method in laboratory soil and groundwater bacterial denitrificationassays, but our results suggest that this relies on substrate types and incubation conditions. Additional measurementsof SP have potential to assess AIT efficacy and can help to reduce parallel time-consuming and expensive15N tracer experiments