The isotopic composition of nitrous oxide (N2O) provides useful information for evaluating N2O sources and budgets. Due to the co‐occurrence of multiple N2O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N2O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope‐ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N2O isotopes, while LAS is more suitable for in situ/inline studies and offers advantages for site‐specific analyses. When reviewing the link between the N2O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot‐to‐global scales, mixing of N2O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi‐quantitative attribution of co‐occurring N2O production and reduction processes. More recently, process‐based N2O isotopic models have been developed for natural abundance and 15N‐tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter‐laboratory comparisons, further exploration of N2O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N2O isotope community will continue to advance our understanding of N2O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.

Abstract. For the past two decades, the measurement of nitrous oxide (N2O) isotopocules – isotopically substituted molecules 14N15N16O, 15N14N16O and 14N14N18O of the main isotopic species 14N14N16O – has been a promising technique for understanding N2O production and consumption pathways. The coupling of non-cryogenic and tuneable light sources with different detection schemes, such as direct absorption quantum cascade laser absorption spectroscopy (QCLAS), cavity ring-down spectroscopy (CRDS) and off-axis integrated cavity output spectroscopy (OA-ICOS), has enabled the production of commercially available and field-deployable N2O isotopic analyzers. In contrast to traditional isotope-ratio mass spectrometry (IRMS), these instruments are inherently selective for position-specific 15N substitution and provide real-time data, with minimal or no sample pretreatment, which is highly attractive for process studies. Here, we compared the performance of N2O isotope laser spectrometers with the three most common detection schemes: OA-ICOS (N2OIA-30e-EP, ABB – Los Gatos Research Inc.), CRDS (G5131-i, Picarro Inc.) and QCLAS (dual QCLAS and preconcentration, trace gas extractor (TREX)-mini QCLAS, Aerodyne Research Inc.). For each instrument, the precision, drift and repeatability of N2O mole fraction [N2O] and isotope data were tested. The analyzers were then characterized for their dependence on [N2O], gas matrix composition (O2, Ar) and spectral interferences caused by H2O, CO2, CH4 and CO to develop analyzer-specific correction functions. Subsequently, a simulated two-end-member mixing experiment was used to compare the accuracy and repeatability of corrected and calibrated isotope measurements that could be acquired using the different laser spectrometers. Our results show that N2O isotope laser spectrometer performance is governed by an interplay between instrumental precision, drift, matrix effects and spectral interferences. To retrieve compatible and accurate results, it is necessary to include appropriate reference materials following the identical treatment (IT) principle during every measurement. Remaining differences between sample and reference gas compositions have to be corrected by applying analyzer-specific correction algorithms. These matrix and trace gas correction equations vary considerably according to N2O mole fraction, complicating the procedure further. Thus, researchers should strive to minimize differences in composition between sample and reference gases. In closing, we provide a calibration workflow to guide researchers in the operation of N2O isotope laser spectrometers in order to acquire accurate N2O isotope analyses. We anticipate that this workflow will assist in applications where matrix and trace gas compositions vary considerably (e.g., laboratory incubations, N2O liberated from wastewater or groundwater), as well as extend to future analyzer models and instruments focusing on isotopic species of other molecules.

The agreement of isotope-delta values obtained in the interlaboratory comparison was not sufficient to provide reliable accurate isotope measurement values for USGS51 and USGS52. We propose that provisional values for the isotopic composition of USGS51 and USGS52 determined at the Tokyo Institute of Technology can be adopted for normalizing and reporting sample data until further refinements are achieved through additional calibration efforts.

Abstract. Soil moisture strongly affects the balance between nitrification, denitrification and N2O reduction and therefore the nitrogen (N) efficiency and N losses in agricultural systems. In rice systems, there is a need to improve alternative water management practices, which are designed to save water and reduce methane emissions but may increase N2O and decrease nitrogen use efficiency. In a field experiment with three water management treatments, we measured N2O isotope ratios of emitted and pore air N2O (δ15N, δ18O and site preference, SP) over the course of 6 weeks in the early rice growing season. Isotope ratio measurements were coupled with simultaneous measurements of pore water NO3-, NH4+, dissolved organic carbon (DOC), water-filled pore space (WFPS) and soil redox potential (Eh) at three soil depths. We then used the relationship between SP × δ18O-N2O and SP × δ15N-N2O in simple two end-member mixing models to evaluate the contribution of nitrification, denitrification and fungal denitrification to total N2O emissions and to estimate N2O reduction rates. N2O emissions were higher in a dry-seeded + alternate wetting and drying (DS-AWD) treatment relative to water-seeded + alternate wetting and drying (WS-AWD) and water-seeded + conventional flooding (WS-FLD) treatments. In the DS-AWD treatment the highest emissions were associated with a high contribution from denitrification and a decrease in N2O reduction, while in the WS treatments, the highest emissions occurred when contributions from denitrification/nitrifier denitrification and nitrification/fungal denitrification were more equal. Modeled denitrification rates appeared to be tightly linked to nitrification and NO3- availability in all treatments; thus, water management affected the rate of denitrification and N2O reduction by controlling the substrate availability for each process (NO3- and N2O), likely through changes in mineralization and nitrification rates. Our model estimates of mean N2O reduction rates match well those observed in 15N fertilizer labeling studies in rice systems and show promise for the use of dual isotope ratio mixing models to estimate N2 losses.

Abstract. Atmospheric nitrous oxide (N2O) levels have been continuously growing since preindustrial times. Mitigation requires information about sources and sinks on the regional and global scales. Isotopic composition of N2O in the atmosphere could contribute valuable constraints. However, isotopic records of N2O in the unpolluted atmosphere remain too scarce for large-scale N2O models. Here, we report the results of discrete air samples collected weekly to biweekly over a 5-year period at the high-altitude research station Jungfraujoch, located in central Switzerland. High-precision N2O isotopic measurements were made using a recently developed preconcentration and laser spectroscopy technique. The measurements of discrete samples were accompanied by in situ continuous measurements of N2O mixing ratios. Our results indicate a pronounced seasonal pattern with minimum N2O mixing ratios in late summer, associated with a maximum in δ15Nbulk and a minimum in intramolecular 15N site preference (δ15NSP). This pattern is most likely due to stratosphere–troposphere exchange (STE), which delivers N2O-depleted but 15N-enriched air from the stratosphere into the troposphere. Variability in δ15NSP induced by changes in STE may be masked by biogeochemical N2O production processes in late summer, which are possibly dominated by a low-δ15NSP pathway of N2O production (denitrification), providing an explanation for the observed seasonality of δ15NSP. Footprint analyses and atmospheric transport simulations of N2O for Jungfraujoch suggest that regional emissions from the planetary boundary layer contribute to seasonal variations of atmospheric N2O isotopic composition at Jungfraujoch, albeit more clearly for δ15NSP and δ18O than for δ15Nbulk. With the time series of 5 years, we obtained a significant interannual trend for δ15Nbulk after deseasonalization (-0.052±0.012 ‰ a−1), indicating that the atmospheric N2O increase is due to isotopically depleted N2O sources. We estimated the average isotopic signature of anthropogenic N2O sources with a two-box model to be -8.6±0.6 ‰ for δ15Nbulk, 34.8±3 ‰ for δ18O and 10.7±4 ‰ for δ15NSP. Our study demonstrates that seasonal variation of N2O isotopic composition in the background atmosphere is important when determining interannual trends. More frequent, high-precision and interlaboratory-compatible measurements of atmospheric N2O isotopocules, especially for δ15NSP, are needed to better constrain anthropogenic N2O sources and thus the contribution of biogeochemical processes to N2O growth on the global scale.

Nitrous oxide (N 2 O) is the primary atmospheric constituent involved in stratospheric ozone depletion and contributes strongly to changes in the climate system through a positive radiative forcing mechanism. The atmospheric abundance of N 2 O has increased from 270 ppb (parts per billion, 10 −9 mole mole −1 ) during the pre-industrial era to approx. 330 ppb in 2018. Even though it is well known that microbial processes in agricultural and natural soils are the major N 2 O source, the contribution of specific soil processes is still uncertain. The relative abundance of N 2 O isotopocules ( 14 N 14 N 16 N, 14 N 15 N 16 O, 15 N 14 N 16 O, and 14 N 14 N 18 O) carries process-specific information and thus can be used to trace production and consumption pathways. While isotope ratio mass spectroscopy (IRMS) was traditionally used for high-precision measurement of the isotopic composition of N 2 O, quantum cascade laser absorption spectroscopy (QCLAS) has been put forward as a complementary technique with the potential for on-site analysis. In recent years, pre-concentration combined with QCLAS has been presented as a technique to resolve subtle changes in ambient N 2 O isotopic composition.From the end of May until the beginning of August 2016, we investigated N 2 O emissions from an intensively managed grassland at the study site Fendt in southern Germany. In total, 612 measurements of ambient N 2 O were taken by com-bining pre-concentration with QCLAS analyses, yielding δ 15 N α , δ 15 N β , δ 18 O, and N 2 O concentration with a temporal resolution of approximately 1 h and precisions of 0.46 ‰, 0.36 ‰, 0.59 ‰, and 1.24 ppb, respectively. Soil δ 15 N-NO − 3 values and concentrations of NO − 3 and NH + 4 were measured to further constrain possible N 2 O-emitting source processes. Furthermore, the concentration footprint area of measured N 2 O was determined with a Lagrangian particle dispersion model (FLEXPART-COSMO) using local wind and turbulence observations. These simulations indicated that nighttime concentration observations were largely sensitive to local fluxes. While bacterial denitrification and nitrifier denitrification were identified as the primary N 2 O-emitting processes, N 2 O reduction to N 2 largely dictated the isotopic composition of measured N 2 O. Fungal denitrification and nitrification-derived N 2 O accounted for 34 %-42 % of total N 2 O emissions and had a clear effect on the measured isotopic source signatures. This study presents the suitability of on-site N 2 O isotopocule analysis for disentangling source and sink processes in situ and found that at the Fendt site bacterial denitrification or nitrifier denitrification is the major source for N 2 O, while N 2 O reduction acted as a major sink for soil-produced N 2 O.

This study investigated the cecal microbiota and serum metabolite profile of chickens fed with plant essential oils (PEO) or virginiamycin (VIRG) using high-throughput 16S rRNA gene sequencing and untargeted metabolomics approach. the main aim of this work was to explore the biochemical mechanisms involved in the improved growth performance of antibiotics and their alternatives in animal production. The results showed that both PEO and VIRG treatment significantly increased the relative abundance of phyla Bacteroidetes and decreased the abundance of phyla firmicutes and genus of Lactobacillus in cecal microbiota of chickens. compared to the control group (ct group), the relative abundance of genus of Alistipes, unclassified Rikenellaceae, Roseburia, and Anaeroplasma was enriched in the peo group; that of genus Bacteroides, Lachnospiraceae, and unclassified Enterobacteriaceae was enriched in the cecal microbiota of the ViRG group. Untargeted metabolomics analyses revealed that the PEO treatment modified 102 metabolites and 3 KEGG pathways (primary bile acid biosynthesis and phenylalanine metabolism) in the cecal microbiota, and 81 metabolites and relevant KEGG pathways (fructose and mannose metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid.) in the serum of the chicken. Compared to the CT group, VIRG treatment group differed 217 metabolites and 10 KEGG pathways in cecal contents and 142 metabolites and 7 KEGG pathways in serum of chickens. pearson's correlation analysis showed that phyla Bacteroidetes and genus of Bacteroides, Alistipes, and unclassified Rikenellaceae (in the VIRG and PE group) were positively correlated with many lipid metabolites. However, phyla firmicutes and genera Lactobacillus (higher in the ct group) were negatively correlated with the lipid and thymine metabolism, and positively correlated with hydroxyisocaproic acid, cytosine, and taurine. this study shows that dietary supplementation with peo and VIRG altered the composition and metabolism profile of the cecal microbiota, modified the serum metabolism profile. The intestinal microbiota, the population of microorganisms that inhabit the intestine, plays an important role in the intestinal morphology, immunity, nutrient digestion and absorption, and host health 1-3. Many studies have demonstrated that intestinal microbiota participates in many metabolic pathways, such as lipid metabolism and amino acid synthesis 4,5. The mechanism by which PEO promote growth of may be alter gut microflora and hence improved absorption of nutrients 6 , increase absorption of micronutrients in the small intestine 7 and reducing the deleterious effects of the microbial metabolites 8-10. There are many alternatives to antibiotics, such as acidifiers, probiotics, oligosaccharides, and plant extracts, which play a growth promoting role by regulating gut microbes in pig and poultry 11. Plant essential oils (PEO), which can be extracted from plants by steam distillation, extrusion, or solvent extraction 12,13 , serve as alternatives for antibi...

In this study, the effects of plant extracts (PEs) and virginiamycin (VIRG) on broiler growth performance, as well as on host intestinal microbiota composition and function were investigated. A total of 288 one-day-old male Cobb broiler chickens were randomly divided into four treatment groups (with six replicates per group). The duodenal, ileal, and cecal content of six broilers per treatment group after 14 and 28 days of treatment were sampled. This material was used for high-throughput Illumina sequencing of the V3–V4 region of the 16S rRNA gene. The results showed that chickens fed 400 mg/kg plant extracts (HPE group) had significantly higher average body weights at day 28 as compared to the control group (CT; P < 0.05), and lower feed-to-meat ratios over days 15–42 ( P < 0.01). Within the HPE group at day 14, the relative abundances of two bacterial phyla and 10 bacterial genera increased significantly in the ileal microbiota, and the relative abundance of three bacterial phyla and four bacterial genera decreased. The relative abundance of the genus Lactobacillus in the cecal microbiota decreased from 21.48% (CT group) to 8.41% (fed 200 mg/kg PEs; LPE group), 4.2% (HPE group), and 6.58% (fed 30 mg/kg virginiamycin; VIRG group) after 28 days. In contrast, Faecalibacterium and unclassified Rikenellaceae increased in abundance in the HPE group (from 18 to 28.46% and from 10.83 to 27.63%, respectively), while Bacteroides (36.7%) and Lachnospiraceae increased in abundance in the VIRG group. PICRUSt function analysis showed that the ileal microbiota of the PE treatment groups were more enriched in genes related to the meolism of cofactors and vitamins. In addition, the cecal microbiotas of the LPE and HPE groups were enriched in genes predicted to encode enzymes within 15 and 20 pathways, respectively. These pathways included protein digestion and absorption, amino acid metabolism, lipid biosynthesis, lipopolysaccharide biosynthesis, the citrate cycle (TCA cycle), and lipoic acid metabolism. Similarly, the VIRG group was enriched in 55 metabolic pathways (17 in the duodenum, 18 in the ileum, and 20 in the cecum) on day 28 ( P < 0.05). Thus, the results indicated that the observed increase in broiler growth performance after PE or VIRG supplementation might be attributed to an improvement in intestinal microbial composition and metabolic function.