Combining the ¹⁵N Gas Flux Method and N₂O Isotopocule Data for the Determination of Soil Microbial N₂O Sources

Rationale: The analysis of natural abundance isotopes in biogenic N₂O molecules provides valuable insights into the nature of their precursors and their role in biogeochemical cycles. However, current methodologies (for example, the isotopocule map approach) face limitations, as they only enable the estimation of combined contributions from multiple processes at once rather than discriminating individual sources. This study aimed to overcome this challenge by developing a novel methodology for the partitioning of N₂O sources in soil, combining natural abundance isotopes and the use of a ¹⁵N tracer (¹⁵N Gas Flux method) in parallel incubations.

Methods: Laboratory incubations of an agricultural soil were conducted to optimize denitrification conditions through increased moisture and nitrate amendments, using nitrate that was either ¹⁵N-labeled or unlabeled. A new linear system combined with Monte Carlo simulation was developed to determine N₂O source contributions, and the subsequent results were compared with FRAME, a Bayesian statistical model for stable isotope analysis.

Results: Our new methodology identified bacterial denitrification as the dominant process (87.6%), followed by fungal denitrification (9.4%), nitrification (1.5%), and nitrifier denitrification (1.6%). Comparisons with FRAME showed good agreement, although FRAME estimated slightly lower bacterial denitrification (80%) and higher nitrifier-denitrification (9%) contributions.

Conclusions: This approach provides an improved framework for accurately partitioning N₂O sources, enhancing understanding of nitrogen cycling in agroecosystems, and supporting broader environmental applications.