A direct method to determine gross N<sub>2</sub>O reduction potential: Downscaling soil mass to constrain the reduction hotspots

Nitrous oxide (N2O) reduction is a key process controlling and mitigating the highly heterogeneous N2O emission from soils. We here developed a new method (15N2O reduction tracing) 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). First, the mass balance between 15N2O consumption and 15N2 production was confirmed (recovery rate: 107% ± 10%) using pure cultures of complete denitrifying bacteria. Second, we tested our method’s applicability 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 to identify N2O reduction hotspots at smaller scales and clarify mechanisms behind the heterogenous N2O dynamics in soils.