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Denitrification enzyme activity and potential of subsoils under grazed grasslands assayed by membrane inlet mass spectrometer
Authors:MI Khalil  KG Richards
Institution:a Teagasc Environment Research Centre, Johnstown Castle, Wexford, Dublin, Ireland
b School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland
c Climate Change Research Programme, Environmental Protection Agency, Johnstown Castle Estate, Wexford, Ireland
Abstract:The importance of subsoil denitrification on the fate of agriculturally derived nitrate (NO3) leached to groundwater is crucial for budgeting N in an ecosystem and for identifying areas where the risk of excess NO3 is reduced. However, the high atmospheric background of di-nitrogen (N2) causes difficulties in assessing denitrification enzyme activity (DEA) and denitrification potential (DP) in soils directly. Here, we apply Membrane Inlet Mass Spectrometry (MIMS) technique to investigate indirectly DEA and DP in soils by measuring N2/Ar ratio changes in headspace water over soil. Soils were collected from 0-10, 15-25 and 60-70 cm depths of a grazed ryegrass and grass-clover. The samples were amended with helium-flushed deionized water containing ranges of NO3 and carbon (glucose-C) and were incubated for six hours in the dark at 21 °C. The peaks for N2/Ar ratio, declined with increasing soil depth, indicating a reduced substrate requirements to initiate DEA en-masse (15-30 mg NO3-N alone or with 60-120 mg glucose-C, kg−1 soil). The dissolved N2O concentrations were very small (0.004-0.269 μg N kg−1 soil) but responded well to the added N and C, showing a reduction in DEA with soil depth. In three separate studies, only subsoils were incubated for 3 days at 12 °C with 20-30 mg NO3-N ± 40-60 mg glucose-C, kg−1 soil. Denitrification capacity (DC, NO3 only treatment) was not statistically different to the control (no amendment) within a land use (0.03-0.05 vs. 0.07-0.22 mg N kg−1 soil d−1), the highest being in ryegrass subsoils receiving groundwater. The DP was significantly (P < 0.0001) higher in subsoils under ryegrass than under grass-clover (0.50-0.71 vs. 1.15 mg N kg−1 soil d−1). The rates of DP (NO3 + glucose-C) increased significantly (P < 0.0001) in unsaturated and saturated subsoils (0.92 and 2.19 mg N kg−1 soil d−1, respectively) of grass-clover, due to the higher reductive state resulting from the 10 day pre-incubation. Available C accelerated denitrification in soils and superseded the temporary elevation in oxidative state due to NO3 addition. The substrates load differences between the land uses regulated the degree of denitrification rates. Results suggest that both dissolved N2O measured by gas chromatography and N2/Ar ratio measured by MIMS to indirectly determine DEA, and the latter to quantify total DC/DP in soils can be used. However, interference of oxygen in the MIMS system should be considered if available C is added or is naturally elevated in soil or groundwater.
Keywords:Denitrification  Denitrification Enzyme Activity  Subsoils  Grazed grassland
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