A direct method to determine gross N2O reduction potential: Downscaling soil mass to constrain the reduction hotspots

N₂O reduction is a key process controlling and mitigating the highly heterogeneous N₂O emission from soils. We developed a new method (¹⁵N₂O reduction tracing: ¹⁵N₂O RT) to quantify potential gross reduction rates of N₂O by incubating only a few grams of soil samples, spiking single-labeled ¹⁵N₂O tracer as the direct substrate for N₂O reduction, and analyzing its direct product (the ¹⁵N/¹⁴N ratio of N₂). In the present study, we proved two concepts of the method: (a) direct determination of N₂O reduction potential and (b) downscaling of soil mass to identify N₂O reduction hotspots. First, the mass balance of ¹⁵N between N₂O consumption and N₂ production was confirmed (recovery rate: 107% ± 10%) using pure cultures of complete denitrifying bacteria. Second, we applied our method to soil profiles at a secondary forest (O, 0–5 cm A1, 5–20 cm A2 horizons), no-tillage agricultural plot (O, A1, A2), and conventional tillage plot (0–20 cm Ap horizon). Their N₂O reduction potentials under a controlled soil water potential (−1 kPa) and 0.1% ¹⁵N₂O air varied across orders of magnitude: higher in the shallower, carbon-rich horizons (O–A1). Our method allowed the direct comparison between the N₂O reductions and copy numbers of nosZ (the functional gene responsible for N₂O reduction), which revealed no clear relationship across the studied samples. Instead, the variation in N₂O reduction potential co-varied with the soil total carbon (C) content, C/N ratio, and 16S rRNA gene copy number, suggesting C substrate control on the N₂O reduction. By further reducing the required soil mass, the current method may help disentangle N₂O production and reduction hotspots at a macroaggregate scale (approximately > 2 mm diameter) to clarify mechanisms behind the heterogeneous N₂O dynamics in soils.

Keywords: nitrous oxide, isotopic labeling, incubation, N₂O reducers, soil profile scale