Denitrification enzyme activity and potential of subsoils under grazed grasslands assayed by membrane inlet mass spectrometer

Abstract

The importance of subsoil denitrification on the fate of agriculturally derived nitrate (NO 3) leached to groundwater is crucial for budgeting N in an ecosystem and for identifying areas where the risk of excess NO 3 is reduced. However, the high atmospheric background of di-nitrogen (N 2) 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 N 2/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 NO 3 and carbon (glucose-C) and were incubated for six hours in the dark at 21 °C. The peaks for N 2/Ar ratio, declined with increasing soil depth, indicating a reduced substrate requirements to initiate DEA en-masse (15–30 mg NO 3-N alone or with 60–120 mg glucose-C, kg −1 soil). The dissolved N 2O 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 NO 3-N ± 40–60 mg glucose-C, kg −1 soil. Denitrification capacity (DC, NO 3 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 (NO 3 + 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 Available C accelerated denitrification in soils and superseded the temporary elevation in oxidative state due to NO 3 addition. The substrates load differences between the land uses regulated the degree of denitrification rates. Results suggest that both dissolved N 2O measured by gas chromatography and N 2/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.