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Ruminant urine patches on grazed grassland are a significant source of agricultural nitrous oxide (N 2 O) emissions. Of the many biotic and abiotic N 2 O production mechanisms initiated following urine-urea deposition, codenitrification resulting in the formation of hybrid N 2 O, is one of the least understood. Codenitrification forms hybrid N 2 O via biotic N-nitrosation, co-metabolising organic and inorganic N compounds (N substrates) to produce N 2 O. The objective of this study was to assess the relative significance of different N substrates on codenitrification and to determine the contributions of fungi and bacteria to codenitrification. 15 N-labelled ammonium, hydroxylamine (NH 2 OH) and two amino acids (phenylalanine or glycine) were applied, separately, to sieved soil mesocosms eight days after a simulated urine event, in the absence or presence of bacterial and fungal inhibitors. Soil chemical variables and N 2 O fluxes were monitored and the codenitrified N 2 O fluxes determined. Fungal inhibition decreased N 2 O fluxes by ca. 40% for both amino acid treatments, while bacterial inhibition only decreased the N 2 O flux of the glycine treatment, by 14%. Hydroxylamine (NH 2 OH) generated the highest N 2 O fluxes which declined with either fungal or bacterial inhibition alone, while combined inhibition resulted in a 60% decrease in the N 2 O flux. All the N substrates examined participated to some extent in codenitrification. Trends for codenitrification under the NH 2 OH substrate treatment followed those of total N 2 O fluxes (85.7% of total N 2 O flux). Codenitrification fluxes under non-NH 2 OH substrate treatments (0.7-1.2% of total N 2 O flux) were two orders of magnitude lower, and significant decreases in these treatments only occurred with fungal inhibition in the amino acid substrate treatments. These results demonstrate that in situ studies are required to better understand the dynamics of codenitrification substrates in grazed pasture soils and the associated role that fungi have with respect to codenitrification.

Intensively managed agricultural pastures contribute to N 2 O and N 2 fluxes resulting in detrimental environmental outcomes and poor N use efficiency, respectively. Besides nitrification, nitrifier-denitrification and heterotrophic denitrification, alternative pathways such as codenitrification also contribute to emissions under ruminant urine-affected soil. However, information on codenitrification is sparse. The objectives of this experiment were to assess the effects of soil moisture and soil inorganic-N dynamics on the relative contributions of codenitrification and denitrification (heterotrophic denitrification) to the N 2 O and N 2 fluxes under a simulated ruminant urine event. Repacked soil cores were treated with 15 N enriched urea and maintained at near saturation (-1 kPa) or field capacity (-10 kPa). Soil inorganic-N, pH, dissolved organic carbon, N 2 O and N 2 fluxes were measured over 63 days. Fluxes of N 2 , attributable to codenitrification, were at a maximum when soil nitrite (NO 2 - ) concentrations were elevated. Cumulative codenitrification was higher (P = 0.043) at -1 kPa. However, the ratio of codenitrification to denitrification did not differ significantly with soil moisture, 25.5 ± 15.8 and 12.9 ± 4.8% (stdev) at -1 and -10 kPa, respectively. Elevated soil NO 2 - concentrations are shown to contribute to codenitrification, particularly at -1 kPa.

Soil plays a key role in the global carbon (C) cycle. Most current assessments of SOC stocks and the guidelines given by Intergovernmental Panel on Climate Change (IPCC) focus on the top 30 cm of soil. Our research shows that, when considering only total quantities, most of the SOC stocks are found in this top layer. However, not all forms of SOC are equally valuable as long-term stable stores of carbon: the majority of SOC is available for mineralisation and can potentially be re-emitted to the atmosphere. SOC associated with micro-aggregates and silt plus clay fractions is more stable and therefore represents a long-term carbon store. Our research shows that most of this stable carbon is located at depths below 30 cm (42% of subsoil SOC is located in microaggregates and silt and clay, compared to 16% in the topsoil), specifically in soils that are subject to clay illuviation. This has implications for land management decisions in temperate grassland regions, defining the trade-offs between primary productivity and C emissions in clay-illuviated soils, as a result of drainage. Therefore, climate smart land management should consider the balance between SOC stabilisation in topsoils for productivity versus sequestration in subsoils for climate mitigation.

Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N 2 O and N 2 emissions. Soil pH regulates the reduction of N 2 O to N 2 , however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N 2 O emission ratio (N 2 O/(NO + N 2 O + N 2 )) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N 2 O emission ratio and community changes. Soil pH was negatively associated with N 2 O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir &nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N 2 O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N 2 O emission ratio through more efficient conversion of N 2 O to N 2 .

Pasture-based livestock systems are often associated with losses of reactive forms of nitrogen (N) to the environment. Research has focused on losses to air and water due to the health, economic and environmental impacts of reactive N. Di-nitrogen (N2) emissions are still poorly characterized, both in terms of the processes involved and their magnitude, due to financial and methodological constraints. Relatively few studies have focused on quantifying N2 losses in vivo and fewer still have examined the relative contribution of the different N2 emission processes, particularly in grazed pastures. We used a combination of a high (15)N isotopic enrichment of applied N with a high precision of determination of (15)N isotopic enrichment by isotope-ratio mass spectrometry to measure N2 emissions in the field. We report that 55.8 g N m(-2) (95%, CI 38 to 77 g m(-2)) was emitted as N2 by the process of co-denitrification in pastoral soils over 123 days following urine deposition (100 g N m(-2)), compared to only 1.1 g N m(-2) (0.4 to 2.8 g m(-2)) from denitrification. This study provides strong evidence for co-denitrification as a major N2 production pathway, which has significant implications for understanding the N budgets of pastoral ecosystems.