Background/Question/Methods Nitrous oxide (N2O), a potent greenhouse gas that contributes amply to climate change, is emitted mostly from soils by a suite of microbial metabolic pathways that are nontrivial to identify, and subsequently, to manage. Using enriched or natural abundance stable isotope methods has aided in identifying microbial sources of N2O, but each approach has limitations. Amending soils with isotopically labelled nitrogen (N) and measuring the emitted enriched N2O is robust, but reveals only nitrification vs. denitrification. Conversely, measuring natural abundance intramolecular site preference (SP) from emitted N2O is less categorical, but reveals multiple N2O-generating pathways. In this study, we paired natural abundance and enriched methods on the same soils and hypothesized this pairing would improve our confidence identifying microbial sources of N2O. Our laboratory experiment incubated soils from a corn agroecosystem and a subalpine forest, at 50 to 95% soil saturation, and under paired enriched and natural abundance conditions. Enriched soils were amended with 99 AP excess 15NO3- or 15NH4+, and all soils were incubated for 28 hr. The isotopic composition of microbially-emitted N2O was measured using a laser-based N2O isotopic analyzer to characterize the enriched and natural abundance signatures. Results/Conclusions Our pairing of natural abundance and enriched methods for disentangling microbial sources of N2O yielded three key findings. First, isotopic signatures from enriched and natural abundance N2O generally agreed in interpretation. SP decreased in natural abundance soils as moisture increased (R2=0.26; p=0.01), indicating a transition from nitrification to denitrification. Concomitantly, wetter enriched soils primarily emitted 15N2O from 15NO3--amended soils compared to 15NH4+-amended soils (p<0.001), also indicating denitrification. Second, paired methodologies removed ambiguity in dry agricultural soils. The SP in this soil had an atypically high value of 65‰, which suggested nitrification. However, the enriched assay revealed >87% of N2O came from 15NO3- as compared to 15NH4+ (p<0.001), illustrating that nitrification was minor. Perhaps DNRA and denitrification occurred simultaneously in anoxic microsites, giving rise to uncharacteristically enriched SP and the high 15NO3- use in the enriched assay. Finally, we identified a persistent position-specific enrichment pattern in the enriched assay. Surprisingly, more than 50% of the 15N enrichment appeared in the β-position (p<0.001), far in excess of expectations from SP-level fractionation. These counter-intuitive enrichment patterns raise the possibility of previously unrecognized biochemical or biogeochemical transformations in these soils.
