The continuous increase of nitrous oxide (N ₂ O) abundance in the atmosphere is a global concern. Multiple pathways of N ₂ O production occur in soil, but their significance and dependence on oxygen (O ₂ ) availability and nitrogen (N) fertilizer source are poorly understood. We examined N ₂ O and nitric oxide (NO) production under 21%, 3%, 1%, 0.5%, and 0% (vol/vol) O ₂ concentrations following urea or ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] additions in loam, clay loam, and sandy loam soils that also contained ample nitrate. The contribution of the ammonia (NH ₃ ) oxidation pathways (nitrifier nitrification, nitrifier denitrification, and nitrification-coupled denitrification) and heterotrophic denitrification (HD) to N ₂ O production was determined in 36-h incubations in microcosms by ¹⁵ N- ¹⁸ O isotope and NH ₃ oxidation inhibition (by 0.01% acetylene) methods. Nitrous oxide and NO production via NH ₃ oxidation pathways increased as O ₂ concentrations decreased from 21% to 0.5%. At low (0.5% and 3%) O ₂ concentrations, nitrifier denitrification contributed between 34% and 66%, and HD between 34% and 50% of total N ₂ O production. Heterotrophic denitrification was responsible for all N ₂ O production at 0% O ₂ . Nitrifier denitrification was the main source of N ₂ O production from ammonical fertilizer under low O ₂ concentrations with urea producing more N ₂ O than (NH ₄ ) ₂ SO ₄ additions. These findings challenge established thought attributing N ₂ O emissions from soils with high water content to HD due to presumably low O ₂ availability. Our results imply that management practices that increase soil aeration, e.g., reducing compaction and enhancing soil structure, together with careful selection of fertilizer sources and/or nitrification inhibitors, could decrease N ₂ O production in agricultural soils.

The use of organic fertilizers constitutes a sustainable strategy to recycle nutrients, increase soil carbon (C) stocks and mitigate climate change. Yet, this depends largely on balance between soil C sequestration and the emissions of the potent greenhouse gas nitrous oxide (N₂O). Organic fertilizers strongly influence the microbial processes leading to the release of N₂O. The magnitude and pattern of N₂O emissions are different from the emissions observed from inorganic fertilizers and difficult to predict, which hinders developing best management practices specific to organic fertilizers. Currently, we lack a comprehensive evaluation of the effects of OFs on the function and structure of the N cycling microbial communities. Focusing on animal manures, here we provide an overview of the effects of these organic fertilizers on the community structure and function of nitrifying and denitrifying microorganisms in upland soils. Unprocessed manure with high moisture, high available nitrogen (N) and C content can shift the structure of the microbial community, increasing the abundance and activity of nitrifying and denitrifying microorganisms. Processed manure, such as digestate, compost, vermicompost and biochar, can also stimulate nitrifying and denitrifying microorganisms, although the effects on the soil microbial community structure are different, and N₂O emissions are comparatively lower than raw manure. We propose a framework of best management practices to minimize the negative environmental impacts of organic fertilizers and maximize their benefits in improving soil health and sustaining food production systems. Long-term application of composted manure and the buildup of soil C stocks may contribute to N retention as microbial or stabilized organic N in the soil while increasing the abundance of denitrifying microorganisms and thus reduce the emissions of N₂O by favoring the completion of denitrification to produce dinitrogen gas. Future research using multi-omics approaches can be used to establish key biochemical pathways and microbial taxa responsible for N₂O production under organic fertilization.

Summary: Soil ecosystem represents the largest contributor to global nitrous oxide (N ₂ O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N ₂ O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the ¹⁵ N‐ ¹⁸ O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N ₂ O pathways including nitrification, nitrifier‐induced denitrification (nitrifier denitrification and nitrification‐coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier‐induced denitrification were the dominant pathways of N ₂ O production, and application of the nitrification inhibitor 3,4‐dimethylpyrazole phosphate (DMPP) significantly reduced the N ₂ O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia‐oxidizing bacteria (AOB) in the DMPP‐amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N ₂ O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N ₂ O production from nitrifier‐induced denitrification, a potential significant source of N ₂ O production in agricultural soils.