Mechanistic insights into in situ N₂O fluxes in a German crop rotation integrating chamber measurements, isotopocule signatures, and functional nitrogen cycling genes

Nitrous oxide (N₂O) is one of the most relevant anthropogenic greenhouse gases, mainly produced in agricultural soils. Understanding the mechanisms responsible for N₂O production and consumption is crucial for developing N₂O mitigation strategies in croplands but field studies combining N₂O flux measurements with isotopocule signatures and metagenomic analysis are very rare.

With the aim to clarify how isotopic signatures and functional genes can help explaining N₂O fluxes in a cropland, we conducted a two years study, from February 2023 to January 2025, at the Reinshof experimental farm of the University of Göttingen in Germany (51.49° N, 9.93° E). The crop sequence was sugar beet, winter wheat and winter barley.

We measured N₂O fluxes with eight manually-operated static chambers and gas chromatography and additionally collected samples for analysis of N₂O isotopocule signatures. We applied FRAME, a three-dimensional model based on ¹⁵N site preference, bulk ¹⁵N and ¹⁸O isotopic signatures to distinguish between the source processes (bacterial denitrification, nitrifier denitrification, fungal denitrification, nitrification) and to estimate the reduction of N₂O to N₂.

We regularly monitored soil water content, mineral nitrogen (N_min) and dissolved organic carbon (DOC). Additionally, every four months we collected soil samples for real-time quantitative polymerase chain reaction (qPCR)-based quantification of bacterial and fungal DNA, as well as functional genes involved in nitrification (ammonia-oxidizing archaea and bacteria amoA genes) and denitrification (nirK, nirS, and nosZ clade I and II).

We observed mean N₂O fluxes of 19.8 µg N₂O-N m^-2 h^-1. Individual chamber measurements ranged from -26.7 to 573.1 µg N₂O-N m^-2 h^-1. The spatial variability between chambers within one day showed a high coefficient of variation of 123%. N₂O fluxes increased after fertilization, rewetting and harvest while highest fluxes occurred after a freeze-thawing event. Cumulative fluxes showed that 0.97% of applied fertilizer N was emitted as N₂O-N. We observed a significant positive effect of soil moisture on N₂O fluxes, no significant effect of N_min and a significant negative effect of DOC.

Preliminary results showed that bacterial denitrification was the dominant process responsible for N₂O production. As N₂O fluxes increased, the proportion of bacterial denitrification increased while the proportion of nitrification decreased. Following freeze-thawing, there was more bacterial denitrification than after the fertilization events and very little fungal denitrification. Overall, the residual N₂O fraction of 45.9 ± 15.0% suggested extensive nitrogen loss as N₂ via denitrification. The dominance of nosZ over nirK and nirS further implied substantial conversion of nitrogen to N₂.

In addition, bacterial DNA was more abundant than fungal DNA, and denitrifiers were more abundant than nitrifiers. No clear differences in processes or gene copy numbers were observed between chambers. Seasonally, gene copy numbers of most functional genes were higher during the growing season and lower in winter, consistent with higher N₂O fluxes during the growing season and lower fluxes in winter.

In future analysis, we will show how soil characteristics, isotopic signatures, and functional genes jointly shape the spatial and temporal variation of the measured N₂O fluxes.