Nitrogen Isotope Fractionation Research Articles

Using the natural abundance of nitrogen isotopes to identify cattle with greater efficiency in protein-limiting diets

The difficulty in selecting cattle for higher feed and nitrogen use efficiency (NUE) is an important factor contributing to poor growth and reproductive performance in dry-tropics rangelands. Therefore, the objectives were to examine the cattle variation in retaining nitrogen in a protein-deficient diet and the natural abundance of stable isotopes in body tissues as a practical alternative for the detection of more efficient cattle. In experiment 1, feed efficiency parameters were determined in 89 Brahman steers fed a protein-limiting diet for 70 days, followed by 7 days in metabolism crates for total collection of urine and faeces and calculation of nitrogen retention and NUE. The diet-animal fractionation of nitrogen isotopes (Δ15N) was quantified in tail hair and plasma proteins using isotope-ratio MS. There was a large variation in growth performance, feed efficiency and nitrogen losses among steers. Quantifying Δ15N in tail hair (Δ15Ntail hair) resulted in stronger correlations with feed efficiency and nitrogen metabolism parameters than when quantified in plasma proteins. Δ15Ntail hair was positively correlated with nitrogen losses in urine (r = 0.31, P < 0.01) and faeces (r = 0.25, P = 0.04), leading to a negative correlation with NUE (r = −0.40, P < 0.01). The group of steers with lower Δ15Ntail hair had greater feed efficiency, lower nitrogen losses, and greater NUE. In experiment 2, for evaluation of isotope fraction as a predictor of reproductive performance, 630 Brahman-crossed cows were classified for reproductive performance for 2 years. From this group, 25 cows with poor reproductive performance and 25 cows with good reproductive performance were selected. Tail hair representing 7 months of growth were segmented and analysed for carbon (δ13C) and nitrogen (δ15N) isotope enrichment. Reproductive performance was not associated with diet selection, as there was no difference in tail hair δ13C between groups. However, more productive cows had lower (P < 0.05) tail hair δ15N during the dry season, indicating differences in N metabolism and possibly lower N losses. In addition, cows with better reproductive performance and, therefore, greater nutrient demands, had similar body condition scores and a tendency (P = 0.09) for higher live weight at the end of the trial. In conclusion, the findings of the present study confirm that nitrogen isotope fractionation in tail hair can be used as a predictor of nitrogen losses, NUE, and reproductive performance of Brahman cattle on low-protein diets.

Density functional theory calculations of nitrogen and oxygen equilibrium isotope fractionations in NO3−–NO2−–H2O aqueous system reveal inverse kinetic isotope effects during nitrite oxidation

Nitrate and nitrite play key roles in the nitrogen cycle on Earth's surface. The isotope fractionation during nitrate reduction on enzymatic level involves multiple steps, including transfer of free NO3− to the activate site on nitrate reductase and NO2− equilibration with ambient water. However, the isotope fractionation factors of 15N and 18O among NO3−, NO2−, and H2O molecules in aqueous phases are poorly constrained. It strongly impedes the understanding of the involved processes and using stable isotopes to quantitatively examine the biogeochemical nitrogen cycle. In this contribution, we employ the density functional theory method with the Urey-Bigeleisen-Goeppert-Mayer model to predict the nitrogen and oxygen equilibrium isotope fractionation factors of NO3−, NO2−, and H2O molecules in gaseous and aqueous phases. Our calculation results show that the solvent effect has a large influence on equilibrium isotope fractionation for oxygen in water (+10.1‰ between liquid and vapor water at 25 °C), which is different to the little solvent effects on both oxygen and nitrogen in nitrate and nitrite. The calculated temperature-dependent equilibrium isotope fractionations between nitrogen of NO3− and NO2−, and oxygen of H2O and NO2− are consistent with previous laboratory experiments. Our results confirm that the oxygen equilibrium isotope fractionations between NO3− and NO2− should be +9.4‰ at 25 °C. Integrating the new results and previously reported kinetic isotope effects of nitrate reduction, we demonstrate inverse kinetic isotope effects for both nitrogen (+15.4‰) and oxygen (+5.2‰) during nitrite oxidation, which falls in the range of previous experiments. The new results enable us to use both nitrogen and oxygen isotopes as a bonded isotope tool to quantitatively assess the nitrogen cycle in low-temperature environments.

Kinetic isotope effects of C and N indicate different transformation mechanisms between atzA- and trzN-harboring strains in dechlorination of atrazine.

Compound-specific stable isotope analysis provides an alternative method to insight into the biotransformation mechanisms of diffuse organic pollutants in the environment, e.g., the endocrine disruptor herbicide atrazine. Biotic hydrolysis process catalyzed by chlorohydrolase AtzA and TrzN plays an important role in the detoxification of atrazine, while the catalytic mechanism of AtzA is still speculative. To investigate the catalytic mechanism of AtzA and answer whether both enzymes catalyze hydrolytic dechlorination of atrazine by the same mechanism, in this study, apparent kinetic isotope effects (AKIE) for carbon and nitrogen were observed by three atzA-harboring bacterial isolates and their membrane-free extracts. The AKIEs obtained from atzA-harboring bacterial isolates (AKIEC = 1.021 ± 0.010, AKIEN = 0.992 ± 0.003) were statistically different from that of trzN-harboring strains (AKIEC = 1.040 ± 0.006, AKIEN = 0.983 ± 0.006), confirming the different activation mechanisms of atrazine preceding to nucleophilic aromatic substitution of Cl atom in actual enzymatic reaction catalyzed by AtzA and TrzN, despite the limitation of variable dual-element isotope plots. The lower degree of normal carbon and inverse nitrogen isotope fractionation observed from atzA-harboring strains, suggesting AtzA catalyzing hydrolytic dechlorination of atrazine by coordination of Cl and one aromatic N to the Fe2+ drawing electron density from carbon-chlorine bond that facilitating the nucleophilic attack, rather than in TrzN case that protonation of aromatic N increasing nucleophilic substitution of Cl atom. This study suggests considering the potential influences of phylogenetic diversity of bacterial isolates and evolution of enzymes on the applications of CSIA method in future study.

Isotope effects at the origin of life: Fingerprints of the Strecker synthesis

Strecker synthesis creates α-amino acids from prebiotically plausible substrates (cyanide, ammonia, and aldehydes) and is widely hypothesized to be a key mechanism in the chemistry that led to life on Earth and on other planets. To better constrain the synthetic environments and precursors of abiotic α-amino acids, and to determine unique signatures of abiogenic amino acids, we measured the molecular-averaged and site-specific carbon and nitrogen isotope effects for the Strecker synthesis of alanine. The reaction steps of the Strecker synthesis can be divided into two groups: an initial series of reversible amination and nitrile-addition reactions (‘equilibration’) and a second series of irreversible hydrolysis reactions (‘hydrolysis’). The equilibration of cyanide, acetaldehyde, and ammonia with the intermediate, α-aminopropionitrile (α-APN), has a measured 55.1‰ equilibrium nitrogen isotope effect between the 15N-rich amine nitrogen in α-aminopropionitrile and the 15N-poor ammonia and a 20.0‰ equilibrium carbon isotope effect between the 13C-poor C-2 site in α-aminopropionitrile and the 13C-rich carbonyl carbon in acetaldehyde. The first irreversible hydrolysis step is inferred to have an up to 10‰ normal carbon fractionation (i.e., faster for 12C, slower for 13C) for the whole molecule, but it also has one or more side reactions that deplete the reactive α-APN reservoir by up to 15‰. The second hydrolysis step has a 15.4‰ normal kinetic isotope effect on the amide (C-1) site of alaninamide, which becomes the carboxyl site of alanine. Other α-amino acids will likely experience similar nitrogen isotope fractionations between ammonia and their amine sites, and similar carbon isotope fractionations between the carbonyl carbon in reactant aldehydes or ketones and the intermediate α-aminonitrile, and between cyanide and the carboxyl site. Therefore, these isotope effects allow us to predict the carbon and nitrogen isotopic contents and intramolecular structures of α-amino acids formed by Strecker synthesis based on their substrates’ isotopic compositions, or to infer the isotopic compositions of substrates from which amino acids formed, for example in the case of the amino-acid-rich carbonaceous chondrites. The site-specific C and N isotopic compositions of amino acids formed by Strecker chemistry contrast with those typical of terrestrial biosynthetic amino acids, so these data also provide a means of discriminating between biogenic and abiogenic α-amino acids.

Analysis of nitrite oxidation process and nitrification performance by nitrogen and oxygen isotope fractionation effect

The N and O isotope fractionation effects in NO2−-N oxidation and nitrification performance of an activated sludge system treating municipal wastewater are unknown. The nitrifying sludge was cultured under different temperature (33 ± 1 °C, 25 ± 1 °C,and 18 ± 1 °C) and dissolved oxygen (DO: 0.5–1 mg/L, 3–4 mg/L, and 7–8 mg/L). The inverse kinetic isotope effects of N and O (15εNO2 and 18εNO2) were −0.62‰ to −7.08‰ and −0.87‰ to −1.68‰ in the process of NO2−-N oxidation, respectively. 15εNO3 gradually increased with increasing of temperature (15εNO3–33°C (14.49‰) > 15εNO3–25°C (10.43‰) > 15εNO3–18°C (7.3‰)), while the 15εNO3:18εNO3 was maintained at 1.02–5.32. The increase of temperature improved the nitrification activity, which promoted the fractionation effect, but the change of DO did not highlight this difference. The exchange of NO2−-N and H2O (XNOB) was 32.5 ± 1.5%, and the kinetic isotope effect of H2O participating in the reaction (18εk, H2O, 2) was 22.57 ± 1.79‰, indicating that H2O was involved in the NO2−-N oxidation rather than DO. In summary, the elevated temperature enhanced the fractionation effect of NO2−-N oxidation. This study provides a new perspective to reveal the mechanism of NO2−-N oxidation, optimize the process of nitrogen removal from wastewater and further control water eutrophication.

Carbon and Nitrogen Isotope Fractionation During Uv-A and Uv-C Photo-Transformation of 3-Nitro-1,2,4-Triazol-5-One (Nto)

Carbon and Nitrogen Isotope Fractionation During Uv-A and Uv-C Photo-Transformation of 3-Nitro-1,2,4-Triazol-5-One (Nto)

Nitrogen isotope fractionation during magma ocean degassing: tracing the composition of early Earth’s atmosphere

Nitrogen isotope fractionation during magma ocean degassing: tracing the composition of early Earth’s atmosphere

Nitrogen Kinetic Isotope Effects of Nitrification by the Complete Ammonia Oxidizer Nitrospira inopinata.

ABSTRACTAnalysis of nitrogen isotope fractionation effects is useful for tracing biogeochemical nitrogen cycle processes. Nitrification can cause large nitrogen isotope effects through the enzymatic oxidation of ammonia (NH3) via nitrite (NO2−) to nitrate (NO3−) (15εNH4+→NO2- and 15εNO2-→NO3-). The isotope effects of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and of nitrite-oxidizing bacteria (NOB) have been analyzed previously. Here, we studied the nitrogen isotope effects of the complete ammonia oxidizer (comammox) Nitrospira inopinata that oxidizes NH3 to NO3−. At high ammonium (NH4+) availability (1 mM) and pH between 6.5 and 8.5, its 15εNH4+→NO2- ranged from −33.1 to −27.1‰ based on substrate consumption (residual substrate isotopic composition) and −35.5 to −31.2‰ based on product formation (cumulative product isotopic composition), while the 15εNO2-→NO3- ranged from 6.5 to 11.1‰ based on substrate consumption. These values resemble isotope effects of AOB and AOA and of NOB in the genus Nitrospira, suggesting the absence of fundamental mechanistic differences between key enzymes for ammonia and nitrite oxidation in comammox and canonical nitrifiers. However, ambient pH and initial NH4+ concentrations influenced the isotope effects in N. inopinata. The 15εNH4+→NO2- based on product formation was smaller at pH 6.5 (−31.2‰) compared to pH 7.5 (−35.5‰) and pH 8.5 (−34.9‰), while 15εNO2-→NO3- was smaller at pH 8.5 (6.5‰) compared to pH 7.5 (8.8‰) and pH 6.5 (11.1‰). Isotopic fractionation via 15εNH4+→NO2- and 15εNO2-→NO3- was smaller at 0.1 mM NH4+ compared to 0.5 to 1.0 mM NH4+. Environmental factors, such as pH and NH4+ availability, therefore need to be considered when using isotope effects in 15N isotope fractionation models of nitrification.IMPORTANCE Nitrification is an important nitrogen cycle process in terrestrial and aquatic environments. The discovery of comammox has changed the view that canonical AOA, AOB, and NOB are the only chemolithoautotrophic organisms catalyzing nitrification. However, the contribution of comammox to nitrification in environmental and technical systems is far from being completely understood. This study revealed that, despite a phylogenetically distinct enzymatic repertoire for ammonia oxidation, nitrogen isotope effects of 15εNH4+→NO2- and 15εNO2-→NO3- in comammox do not differ significantly from those of canonical nitrifiers. Thus, nitrogen isotope effects are not suitable indicators to decipher the contribution of comammox to nitrification in environmental samples. Moreover, this is the first systematic study showing that the ambient pH and NH4+ concentration influence the isotope effects of nitrifiers. Hence, these key parameters should be considered in comparative analyses of isotope effects of nitrifiers across different growth conditions and environmental samples.

Effects of Seasonal Thermal Stratification on Nitrogen Transformation and Diffusion at the Sediment-Water Interface in a Deep Canyon Artificial Reservoir of Wujiang River Basin

Watershed-scale nitrogen pollution in aquatic systems has become a worldwide concern due to its continuous impact on water quality deterioration, while the knowledge of key influencing factors dominating nitrogen transportation and transformation at the sediment-water interface (SWI) remains limited, especially in impounded rivers with an artificial reservoir. Hence, for a better understanding of the effects of thermal stratification on nitrogen transformation, we investigated the nitrogen species and isotopes in the sediment of a deep reservoir in Southwest China. Our results confirmed a significant difference in nitrogen species and isotopic composition in sediment between those in the thermal stratification period and non-thermal stratification period and indicated that the sediment biogeochemical process and transportation were clearly linked to the variations in water temperature and dissolved oxygen dominated by the process of thermal stratification. Significant seasonal differences in NH4+-N and NO3−-N in pore water of the upper layer (0–19 cm) revealed that nitrification exhausted NH4+ in the non-stratified period (NSP), and a potential low mineralization rate appeared when compared with those in the stratified period (SP). Seasonal differences in nitrogen species and isotope fractionation of δ15N-PON (about 2.3‰ in SP) in the upper layer sediment indicated a higher anaerobic mineralization rate of organic matter in SP than that in NSP. The diffusion fluxes of NH4+-N at SWI were 9.48 and 15.66 mg·m−2·d−1 in NSP and SP, respectively, and annual NH4+-N diffusion accounted for 21.8% of total storage in the reservoir. This study demonstrated that the nitrogen cycling processes, especially nitrification, denitrification, and mineralization, have been largely altered along with the changes in dissolved oxygen and that the diffusion of nitrogen species varied with the presence of the oxygen. The results contribute to the future study of watershed nitrogen budget evaluation and suggest that the endogenous nitrogen released from the sediment-water interface should be emphasized when aiming to fulfil water management policies in deep reservoirs.

Trophic Enrichment Factors of Carbon and Nitrogen Isotopic Ratios (Δ13C and Δ15N) in Four Marine Ciliates.

Understanding the magnitude and causes of isotopic fractionation between organisms and their dietary resources is crucial for gaining knowledge on stable isotope ecology. However, little is known regarding the diet-tissue fractionation values of marine ciliates, which play a critical role in the reconstruction of microbial food webs. In the present study, we conducted experiments on two benthic (Pseudokeronopsis pararubra and Protocruzia labiata) and two pelagic (Strombidium sulcatum and Uronemella filificum) marine ciliates, where they were fed with isotopically constant foods (Chaetoceros calcitrans and Isochrysis galbana) under laboratory culture conditions to determine their carbon and nitrogen isotopic fractionation values (Δ13C and Δ15N). The stable isotope values (δ13C and δ15N) of ciliates for all experiments rapidly increased after the initial feeding, with half-lives ranging from 6.1 to 23.0h for δ13C and from 3.1 to 24.9h for δ15N. The Δ13C and Δ15N for all ciliates represented significantly positive enrichments, with overall mean fractionations of 0.6±0.2 and 1.2±0.4, respectively. Irrespective of the dietary type, both Δ13C and Δ15N were very similar for the same ciliate species. These results suggest that Δ13C and Δ15N for marine ciliates are similar to those found in common marine organisms with very little food-dependent variation. Overall, quantifying the specific isotopic fractionation of marine ciliates is expected to provide fundamental information on the trophic transfer of carbon, nitrogen, and energy flow through the microbial pathway in marine ecosystems.

Stable nitrogen isotope composition of NOx of biomass burning in China

Owing to the local characteristics of stable nitrogen isotopes in nitrogen oxides (δ15N-NOx) emitted from biomass burning, the lack of data on δ15N-NOx values associated with biomass burning in China limits the use of this parameter in identifying and quantifying the sources of atmospheric nitrate (NO3-) and NOx. The results showed that the δ15N-NOx values of open burning and rural cooking stoves in China ranged from -3.7‰ to 3.1‰ and -11.9‰ to 1.5‰, respectively. The δ15N values of nine biomass fuel sources (δ15N-biomass) ranged from 0.1‰ to 4.1‰. Significant linear relationships between the δ15N-biomass values and δ15N-NOx values of open burning (δ15N-NOx = 1.1δ15N-biomass - 2.7; r2 = 0.63; p < 0.05) and rural cooking stoves (δ15N-NOx = 1.7δ15N-biomass - 9.8; r2 = 0.72; p < 0.01) suggested that the variations in δ15N-NOx values from biomass burning were mainly controlled by the biomass fuel source. The isotopic fractionation of nitrogen during the biomass burning process might have led to the higher δ15N-NOx values from open burning in comparison to rural cooking stoves. By combining the δ15N-NOx values of biomass burning with biomass burning emission inventory data, a model for calculating the δ15N-NOx values of biomass burning in different regions of China was established, and the estimated δ15N-NOx value of biomass burning at the national scale was -0.8 ± 1.2‰. But the limited δ15N-biomass values increase the uncertainty of model in national scale.

Interpretation of Nitrogen Isotope Profiles in Petroleum Systems: A Review

Bulk sedimentary nitrogen isotope profiles are often used as proxies for depositional redox conditions, nitrogen cycling, and nutrient uptake in modern and ancient marine systems. The general preference in terms of analysis is that the sediments measured have undergone minimal thermal alteration, as post-depositional processes might have altered the initial δ15N signal, thus complicating the interpretation of these records. Although not a traditional proxy for petroleum evaluation purposes, recently the use of nitrogen isotopes in petroleum systems has been investigated as potential proxies to reconstruct paleoenvironmental conditions such as redox, and for organic matter evaluation. In this paper we review the use of nitrogen isotope data in petroleum systems, their interpretations, and factors that may complicate their use as proxies. We review the evidence for nitrogen isotopic fractionation during diagenesis, catagenesis, and fluid migration as determined by lab experiments, and how these might impact interpretation of δ15N data in petroleum systems. We also analyze the use and interpretation of δ15N data from petroleum-producing reservoir units, including unconventional reservoirs and lacustrine basins. Lastly, we discuss potential applications for nitrogen isotopes in petroleum systems with regards to their use as both geochemical proxies and as tools to evaluate petroleum reservoirs.

Nitrogen loss and isotopic fractionation during granulite-facies metamorphism in the lower crust (Ivrea Zone, NW Italy)

The Ivrea Zone, exposed in the Southern Alps, affords an examination of nitrogen mobilization and storage in deep continental crust, in rocks spanning the amphibolite to granulite facies transition and that experienced multiple devolatilization reactions and partial melting. Such information is important for considerations of whole-Earth N cycling, as continental crust is thought to contain up to ~15% of global N, much of it organic in origin and thus tied to ancient-Earth biogeochemical processes.In these rocks, N is redistributed among silicate phases during devolatilization and both H2O-saturated and dehydration melting, showing particular affinity for potassic phases such as biotite, muscovite and alkali feldspar. In metasedimentary rocks, N concentrations decrease strongly with increasing grade, from the Kinzigite Zone (amphibolite facies) with 29 to 181 μg/g to the Stronalite Zone (granulite facies) containing only 10 to 15 μg/g. The decrease in N is intermediate in magnitude to that of Cs, showing somewhat greater loss, and that of Rb that shows smaller degrees of loss. The loss of N with increasing grade is due to breakdown of biotite, which is the main mineral host of N. It also reflects a strong preference of N for the fluid during dehydration and a moderate preference of N for the melt during partial melting. Accompanying this loss of N is a shift by up to 9‰ in δ15N towards lower values. The direction of this shift contrasts with what would be expected for loss of N into fluids speciated as either N2 or NH3 and likely reflects complex isotope fractionation effects during equilibration between melt, fluid, and rock, perhaps during evolving redox conditions. The lowest δ15N values for metabasaltic rocks in this suite are near that of the mantle (−5‰) and the array of values across the traverse could represent initial protolith heterogeneities and superimposed effects of magmatic degassing of the protolith. The leucosomes broadly overlap in N concentrations and δ15N values with their host rocks and lack any systematic relationships with metamorphic grade or type of host lithology.As has been noted in previous work, estimation of the size and isotopic composition of the N reservoir in continental crust is complicated its great lithologic heterogeneity and by the paucity of data for metamorphic rocks representing greater depths. Superimposed on the lithologic heterogeneity associated with varying protoliths is the ability of highly devolatilized and partially melted deep crustal rocks to store N over long time periods. We used our new data of deep crustal rocks from the Ivrea Zone, combined with published N data and based on lithological proportions in the crust, to provide a first estimate for the N content in the middle continental crust of 56 ± 4 μg/g. For the lower continental crust, we estimate a N content of 26 ± 4 μg/g N but note that the data base of granulite-facies samples is still small. Calculation of the total continental crust N content yields 74 ± 4 μg/g, which is in between previous estimates of 56, 60 and 88 μg/g N. Finally, we estimate the N isotopic composition of the bulk continental crust as δ15N = 4.3 ± 0.5.

Nitrogen removal performance of sulfur autotrophic denitrification under different S2O32− additions using isotopic fractionation of nitrogen and oxygen

The dual isotope fractionation of nitrogen (N) and oxygen (O) is an effective way to track the transformation of NO3−-N in biological denitrification process. The Sulfur autotrophic denitrification combined with the different concentrations of S2O32− was investigated using the dual isotope fractionation of nitrogen (N) and oxygen (O) to reveal the nitrogen removal mechanism of the activated sludge. Based on successful autotrophic denitrification incubation, the modified Logistic model responded to the short-term effects of S2O32− addition on NO3−-N removal and SO42− generation. Under the S2O32− addition of 0.5, 1, 2 and 4 times of the incubation stage (49.29 mg/L–394.32 mg/L), the fractionation effect of N in NO3−-N (15εNO3) decreased from 8.74 ± 1.81‰ to 2.08 ± 0.06‰, and the fractionation effect of O in NO3−-N (18εNO3) declined from 11.34 ± 0.46‰ to 5.48 ± 0.46‰. The 15εNO3/18εNO3 was maintained at 0.46–0.94, indicating a negative correlation between addition amount and isotope effect, and the addition of high concentrations of S2O32− was not suitable for system stabilization. Moreover, the 18O-labeled H2O (δ18OH2O) tests significantly proved the presence of O exchange between NO2−-N/NO3−-N and H2O (67%/97%) during the nitrogen removal process, while the reoxidation of NO2−-N was explored in the autotrophic denitrification. The kinetic models coupled with isotope fractionation effectively revealed the nitrogen removal characteristics in the autotrophic denitrification systems, and verified the difference between the activated sludge-based wastewater treatment process and the natural ecosystem.

86 Using nitrogen isotopic fractionation to estimate in vitro ammonia emissions from dairy cattle manure

86 Using nitrogen isotopic fractionation to estimate in vitro ammonia emissions from dairy cattle manure

Enzyme-Specific Coupling of Oxygen and Nitrogen Isotope Fractionation of the Nap and Nar Nitrate Reductases.

Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR is catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases and fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O), which is reflected in residual environmental nitrate pools. Data on the relationship between the pattern in oxygen vs nitrogen isotope fractionation (18ε/15ε) suggests that systematic differences exist between marine and terrestrial ecosystems that are not fully understood. We examined the 18ε/15ε of nitrate-reducing microorganisms that encode Nar, Nap, or both enzymes, as well as gene deletion mutants of Nar and Nap to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of 18ε/15ε fractionation ratios of all examined nitrate reductases forms two distinct peaks centered around an 18ε/15ε proportionality of 0.55 (Nap) and 0.91 (Nar), with the notable exception of the Bacillus Nar reductases, which cluster isotopically with the Nap reductases. Our findings may explain differences in 18ε/15ε fractionation between marine and terrestrial systems and challenge current knowledge about Nar 18ε/15ε signatures.

Combining dual isotopes and a Bayesian isotope mixing model to quantify the nitrate sources of precipitation in Ningbo, East China.

Nitrate (NO3-) is becoming a significant contributor to acid deposition in many cities in China. Based on the chemical compositions and stable isotopes of NO3- in precipitation (δ15N-NO3- and δ18O-NO3-), the NO3- sources and their formation pathways were determined to aid in reducing NOx emissions in Ningbo, an important port city. The acid rain frequency in Ningbo was 67%, and the mean SO42-/NO3- ratio was 1.07. The δ18O-NO3- (49.5‰-82.8‰) and δ15N-NO3- values (-4.3‰-2.7‰) both varied seasonally, with high values during the cold season and low values during the warm season. The seasonal variations in the δ18O-NO3- values were mainly controlled by the NO3- formation pathways, following the OH· pathway during the warm season and N2O5 pathway during the cold season. The Monte Carlo simulation results indicated that the contributions of the OH· pathway ranged from 28.3% to 75.4%, with the remainder contributed by the N2O5 pathway. The improved Bayesian model incorporating nitrogen (N) isotopic fractionation (Ԑ=4‰) indicated that mobile sources, including ship emissions (35.0%)>coal combustion (26.0%)>biomass burning (20.0%)>soil emissions (19.0%), were the major sources of NOx emissions in Ningbo. The results indicate that the influence of isotopic fractionation on source apportionment must be considered in a Bayesian model.

Experimental investigation of formation and decomposition of roaldite in ammonia atmosphere at 300–700 °C and associated nitrogen isotope fractionations

Roaldite (Fe4N) is one of the few nitride minerals found in meteorites. Their nitrogen (N) isotopic signatures carry important information for understanding the early N cycle in the proto-solar nebula. However, the lack of knowledge on the N isotopic effects from nitride formation to its survival from frictional heating during landing impedes the interpretation and application of N isotope compositions of nitride minerals in meteorites. Here, we carried out laboratory experiments under a recently proposed roaldite forming condition, i.e., NH3 (as starting N source) reacting with metallic Fe at medium temperatures. We observed Fe4N formation over a large range of temperatures from 300 °C to 700 °C. The formation of Fe4N was associated with equilibrium N isotope fractionations with αFe4N-NH3 values of 0.9907 (±0.0004) at 300 °C and 0.9936 (±0.0004) at 500 °C, respectively. In the experimental pressure conditions (initial PNH3 = 3.9–6.4 bar, PTotal < 8.3 bar), the formed Fe4N remained stable at 300 °C, but was unstable and quickly decomposed to Fe and N2 at 500 °C and 700 °C. The decomposition of Fe4N was associated with large kinetic isotope fractionations with αN2-Fe4N values of 0.9811 (±0.0009) at 500 °C and 0.9839 (±0.0011) at 700 °C, respectively. Our experimental results suggest that roaldite formed from NH3 can carry an isotopic signature very close to that of its source, but partial decomposition (if there is any) can easily shift its N isotope composition for several tens of per mil, and in extreme cases, to >300‰. Thus, great caution is needed when using N isotope composition of roaldite (and probably other nitride minerals as well) to trace source information.

Oxidation and sources of atmospheric NOx during winter in Beijing based on δ18O-δ15N space of particulate nitrate.

The determination of both stable nitrogen (δ15N-NO3-) and stable oxygen (δ18O-NO3-) isotopic signatures of nitrate in PM2.5 has shown potential for an approach of assessing the sources and oxidation pathways of atmospheric NOx (NO+NO2). In the present study, daily PM2.5 samples were collected in the megacity of Beijing, China during the winter of 2017-2018, and this new approach was used to reveal the origin and oxidation pathways of atmospheric NOx. Specifically, the potential of field δ15N-NO3- signatures for determining the NOx oxidation chemistry was explored. Positive correlations between δ18O-NO3- and δ15N-NO3- were observed (with R2 between 0.51 and 0.66, p<0.01), and the underlying environmental significance was discussed. The results showed that the pathway-specific contributions to NO3- formation were approximately 45.3% from the OH pathway, 46.5% from N2O5 hydrolysis, and 8.2% from the NO3+HC channel based on the δ18O-δ15N space of NO3-. The overall nitrogen isotopic fractionation factor (εN) from NOx to NO3- on a daily scale, under winter conditions, was approximately+16.1‰±1.8‰ (consistent with previous reports). Two independent approaches were used to simulate the daily and monthly ambient NOx mixtures (δ15N-NOx), respectively. Results indicated that the monthly mean values of δ15N-NOx compared well based on the two approaches, with values of-5.5‰ ± 2.6‰,-2.7‰ ± 1.9‰, and-3.2‰ ± 2.2‰ for November, December, and January (2017-2018), respectively. The uncertainty was in the order of 5%, 5‰ and 5.2‰ for the pathway-specific contributions, the εN, and δ15N-NOx, respectively. Results also indicated that vehicular exhaust was the key contributor to the wintertime atmospheric NOx in Beijing (2017-2018). Our advanced isotopic perspective will support the future assessment of the origin and oxidation of urban atmospheric NOx.

Nitrogen isotopic fractionations during nitric oxide production in an agricultural soil

Abstract. Nitric oxide (NO) emissions from agricultural soils play a critical role in atmospheric chemistry and represent an important pathway for loss of reactive nitrogen (N) to the environment. With recent methodological advances, there is growing interest in the natural-abundance N isotopic composition (δ15N) of soil-emitted NO and its utility in providing mechanistic information on soil NO dynamics. However, interpretation of soil δ15N-NO measurements has been impeded by the lack of constraints on the isotopic fractionations associated with NO production and consumption in relevant microbial and chemical reactions. In this study, anoxic (0 % O2), oxic (20 % O2), and hypoxic (0.5 % O2) incubations of an agricultural soil were conducted to quantify the net N isotope effects (15η) for NO production in denitrification, nitrification, and abiotic reactions of nitrite (NO2-) using a newly developed δ15N-NO analysis method. A sodium nitrate (NO3-) containing mass-independent oxygen-17 excess (quantified by a Δ17O notation) and three ammonium (NH4+) fertilizers spanning a δ15N gradient were used in soil incubations to help illuminate the reaction complexity underlying NO yields and δ15N dynamics in a heterogeneous soil environment. We found strong evidence for the prominent role of NO2- re-oxidation under anoxic conditions in controlling the apparent 15η for NO production from NO3- in denitrification (i.e., 49 ‰ to 60 ‰). These results highlight the importance of an under-recognized mechanism for the reversible enzyme NO2- oxidoreductase to control the N isotope distribution between the denitrification products. Through a Δ17O-based modeling of co-occurring denitrification and NO2- re-oxidation, the 15η for NO2- reduction to NO and NO reduction to nitrous oxide (N2O) were constrained to be 15 ‰ to 22 ‰ and −8 ‰ to 2 ‰, respectively. Production of NO in the oxic and hypoxic incubations was contributed by both NH4+ oxidation and NO3- consumption, with both processes having a significantly higher NO yield under O2 stress. Under both oxic and hypoxic conditions, NO production from NH4+ oxidation proceeded with a large 15η (i.e., 55 ‰ to 84 ‰) possibly due to expression of multiple enzyme-level isotopic fractionations during NH4+ oxidation to NO2- that involves NO as either a metabolic byproduct or an obligatory intermediate for NO2- production. Adding NO2- to sterilized soil triggered substantial NO production, with a relatively small 15η (19 ‰). Applying the estimated 15η values to a previous δ15N measurement of in situ soil NOx emission (NOx=NO+NO2) provided promising evidence for the potential of δ15N-NO measurements in revealing NO production pathways. Based on the observational and modeling constraints obtained in this study, we suggest that simultaneous δ15N-NO and δ15N-N2O measurements can lead to unprecedented insights into the sources of and processes controlling NO and N2O emissions from agricultural soils.