Nitrogen Isotope Effects Research Articles

Kinetic nitrogen isotope effects of 18 amino acids degradation during burning processes

Understanding the nitrogen isotopic variations of individual amino acids (AAs) is essential for utilizing the nitrogen isotope values of individual amino acids (δ15N-AA) as source indicators to identify proteinaceous matter originating from biomass combustion processes. However, the nitrogen isotope effects (ε) associated with the degradation of individual amino acids during combustion processes have not been previously explored. In this study, we measured the nitrogen isotope values of residual free amino acids -following a series of controlled combustion experiments at temperatures of 160–240 °C and durations of 2 min to 8 h, as described in Part 1. δ15N values of proline, aspartate, alanine, valine, glycine, leucine, and isoleucine are more positive than their initial δ15N values after prolonged combustion. Variations in δ15N values of the most AAs conform to the Rayleigh fractionation during combustion and their nitrogen isotope effects (ε) are greatly impacted by their respective combustion degradation pathways. This is the first time the ε values associated with the degradation pathways of AAs during combustion have been characterized. Only the ε values associated with Pathway 1 (dehydration to form dipeptide) and 2 (simultaneous deamination and decarboxylation) are found to be significant and temperature-dependent, ranging from + 2.9 to 6.4‰ and + 0.9‰ to + 3.8‰, respectively. Conversely, ε values associated with other pathways are minor. This improves the current understanding on the degradation mechanisms of protein nitrogen during biomass burning.

Changes in isotope fractionation during nitrate assimilation by marine eukaryotic and prokaryotic algae under different pH and CO2 conditions

AbstractThe impact of environmental factors on nitrogen (N) and oxygen (O) isotope effects during algal nitrate assimilation causes uncertainty in the field application of sedimentary N isotope records and nitrate isotopes to understand the marine nitrogen cycle. Ocean acidification is predicted to change nitrogen cycling including nitrate assimilation, but how N and O isotope effects during algal nitrate assimilation vary in response to changes in seawater pH and partial pressure CO2 (pCO2) remains unknown. We measured N and O isotope effects during nitrate assimilation and physiological states of the marine diatom Thalassiosira weissflogii and Synechococcus under different pH (8.1 or 7.8) and pCO2 (400 or 800 μatm) conditions. Low pH and/or high pCO2 equally decreased N and O isotope effects during nitrate assimilation by diatoms possibly due to reducing cellular nitrate efflux/uptake ratio and decreased isotope effects for nitrate uptake, whereas they did not affect those by Synechococcus with low intracellular nitrate concentration and limited nitrate efflux. Our results provide compelling experimental evidence showing different changes in N and O isotope effects during nitrate assimilation by marine eukaryotic and prokaryotic phytoplankton at low pH and/or high pCO2. These findings suggest new insight into environmental controls on variability in the isotope effect during algal nitrate assimilation, and have implications for improving a predictive understanding of N and O isotope tools in acidified oceans.

Nitrous oxide production from biological and chemodenitrification by Fe(II) in estuarine and coastal sediments

Although biological processes are assumed the main sources of nitrous oxide (N2O), it has been increasingly highlighted that abiotic mechanisms are also at play. Here we investigated abiotic and biological N2O production from nitrate and nitrite reduction by Fe(II) in estuarine sediments through a series of anoxic incubations. N2O concentrations increased and then decreased with prolonged incubation time in biological treatment, which was attributed to biological reduction. N2O from chemodenitrification was not mediated by nitrate reduction, but instead was generated from nitrite reduction. Electrons transfer of Fe(II) oxidation decreased by 44.5% from abiotic to biological processes, reflecting that abiotic nitrite reduction was mainly responsible for Fe(II) oxidation. Pseudo-first-order rate constants of Fe(II) were higher in abiotic than biotic processes, and the constants of nitrite were higher in biotic than abiotic processes. 18ɛ:15ɛ ratios of nitrate and nitrite reduction were higher in abiotic than biological processes, suggesting that abiotic process represented a stronger oxygen isotope effect relative to nitrogen isotope effect than biological process. Site preference (SP) of N2O was higher in biological than abiotic processes and Fe(II) addition increased N2O production with accompanying high SP. Based on the reaction stoichiometry and electron transfer balance, chemodenitrification contributed to 20.2–38.4% of N2O production, and 61.6–79.8% of N2O was produced from biological denitrification. These result suggest that biological and chemodenitrification coupled with Fe(II) oxidation can produce N2O, which further mediate the kinetics of N2O production and have wide implications for coupled biogeochemical reactions between nitrogen and iron in global aquatic ecosystems.

Boron and Nitrogen Isotope Effects on Hexagonal Boron Nitride Properties.

The unique physical, mechanical, chemical, optical, and electronic properties of hexagonal boron nitride (hBN) make it a promising 2D material for electronic, optoelectronic, nanophotonic, and quantum devices. Here, the changes in hBN's properties induced by isotopic purification in both boron and nitrogen are reported. Previous studies on isotopically pure hBN have focused on purifying the boron isotope concentration in hBN from its natural concentration (≈20 at% 10 B, 80 at% 11 B) while using naturally abundant nitrogen (99.6 at% 14 N, 0.4 at% 15 N), that is, almost pure 14 N. In this study, the class of isotopically purified hBN crystals to 15 N is extended. Crystals in the four configurations, namely h10 B14 N, h11 B14 N, h10 B15 N, and h11 B15 N, are grown by the metal flux method using boron and nitrogen single isotope (> 99%) enriched sources, with nickel plus chromium as the solvent. In-depth Raman and photoluminescence spectroscopies demonstrate the high quality of the monoisotopic hBN crystals with vibrational and optical properties of the 15 N-purified crystals at the state-of-the-art of currently available 14 N-purified hBN. The growth of high-quality h10 B14 N, h11 B14 N, h10 B15 N, and h11 B15 N opens exciting perspectives for thermal conductivity control in heat management, as well as for advanced functionalities in quantum technologies.

Nitrogen isotope effects on boron vacancy quantum sensors in hexagonal boron nitride

There has been growing interest in studying hexagonal boron nitride (hBN) for quantum technologies. Here, we investigate nitrogen isotope effects on boron vacancy (V B) defects, one of the candidates for quantum sensors, in 15N isotopically enriched hBN synthesized using a metathesis reaction. The Raman shifts are scaled with the reduced mass, consistent with previous work on boron isotope enrichment. We obtain nitrogen isotopic composition-dependent magnetic resonance spectra of V B defects and determine the magnitude of the hyperfine interaction parameter of 15N spin to be 64 MHz. Our investigation provides a design policy for hBNs for quantum sensing.

The essential bioactive role of nickel in the oceans: Evidence from nickel isotopes

The role of nickel (Ni) in ocean biogeochemical cycles is both under-studied and controversial. Strong correlations between Ni and organic carbon in modern and ancient marine sediments suggest a prominent biogeochemical role over a substantial portion of Earth history. Addition of Ni to culturing and seawater incubation experiments produces strong responses in terms of cell growth, particularly of nitrogen-fixing organisms. But the implied limiting role for phytoplankton growth is inconsistent with observations in the real ocean, specifically that photic zone Ni concentrations never descend to the very low values that characterise other bioactive, and often bio-limiting, metals like iron. These two observations can be reconciled if a large portion of the total dissolved Ni present in open-ocean surface waters is not bio-available on short timescales. Here we present new Ni concentration and stable isotope data from the GEOVIDE transect in the North Atlantic. We interpret these new data in the light of the growing database for Ni stable isotopes in the modern ocean, with implications for the biogeochemical importance of Ni.In the new North Atlantic dataset, the lowest Ni concentrations (1.8-2.6 nmol/L) and highest δ60Ni (up to +1.67‰) are associated with low nitrate, south of the subarctic front (SAF). By contrast, stations at latitudes north of the SAF, with higher surface nitrate, show very subdued variation in Ni concentrations throughout the entire depth of the water column (3.6±0.3 nmol/L, mean and 2SD), and no variation in δ60Ni beyond the narrow global deep-ocean range (+1.33±0.13 ‰). These North Atlantic Ni isotope data also show relationships with nitrogen isotope effects, observed in the same samples, that are suggestive of a link between Ni utilisation, isotope fractionation and nitrogen fixation.The global dataset, including the new data presented here, reveals a biogeochemical divide with Ni isotope fractionation only occurring in low latitude surface waters. A simple observationally constrained three-dimensional model of Ni cycling suggests that the creation of this isotopically heavy, Ni-poor, end-member, together with the physical circulation and remineralisation at depth, can explain the global Ni-δ60Ni systematics. Taken together, these findings hint at Ni-N co-limitation in the modern ocean. We advocate for more extensive and detailed culturing/incubation studies of this neglected metal in order to elucidate its potentially crucial biogeochemical role.

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.

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.

Isotope fractionation (δ13C, δ15N) and microbial community response in degradation of petroleum hydrocarbons by biostimulation in contaminated soil.

This study investigated the isotope effects of δ13C and δ15N and microbial response during biodegradation of hydrocarbons by biostimulation with nitrate or compost in the petroleum-contaminated soil. Compost and KNO3 amendments promoted the total petroleum hydrocarbon (TPH) removal accompanied by a significant increase of Actinobacteria and Firmicutes phyla. Soil alpha diversity decreased after 90 days of biostimulation. An inverse significant carbon isotope effect (εc = 16.6 ± 0.8‰) and strong significant nitrogen isotope effect (εN = -24.20 ± 9.54‰) were shown by the KNO3 supplementation. For compost amendment, significant carbon and nitrogen isotope effect were εc = 38.8 ± 1.1‰ and εN = -79.49 ± 16.41‰, respectively. A clear difference of the carbon and nitrogen stable isotope fractionation was evident by KNO3 or compost amendment, which indicated that the mechanisms of petroleum degradation by adding compost or KNO3 may be different.

Nitrogen isotope effects can be used to diagnose N transformations in wastewater anammox systems

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, − 16 to − 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19–32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems.

Predicting nitrogen and oxygen kinetic isotope effects of nitrate reduction by periplasmic dissimilatory nitrate reductase

Kinetic isotope effect (KIE) reveals the transition state structure of an elementary reaction and can be predicted by quantum chemical calculation. Density Functional Theory calculation of an enzymatic reaction with large numbers of atoms is computationally prohibitive, especially if explicit solvent effect is considered. Cutoff method, which simplifies an entire molecule to a cluster around a target position, can simplify position-specific equilibrium isotope effect calculation for a large organic molecule. It should also be applicable to KIE calculation of an enzymatic reaction and allow us to introduce explicit solvent molecules to the system. If this treatment is feasible and trustable, it will provide an efficient method to estimate a number of KIEs produced by enzyme reactions. Obviously, its robustness must be tested. Here, using NO3− reduction by the active site of periplasmic dissimilatory nitrate reductase (Nap) in Rhodobacter sphaeroides as an example, we built 17 models to test the influence of cutoff size, implicit, and explicit solvent effects on KIE calculation. The results show that to estimate the KIE value of an enzymatic reaction accurately and efficiently, we can first simplify the reaction model to a cutoff model with 3 proximal bonds to the active position. Then, incorporating implicit-plus-explicit solvent models can simulate a reaction environment more realistically, which is necessary for accuracy. Our calculated lnKIE values for nitrate Nap reduction at 25 °C are −32.4 ± 1.8‰ for 15N and −20.9 ± 0.4 ‰ for 18O, respectively, with a ln18KIE/ln15KIE ratio of 0.65 ± 0.05. Although additional reservoir-transport processes need to be considered, our calculation results are consistent with calibrated isotope effects from laboratory experiments, suggesting that the transition state we calculated depicts the general reaction mechanism of NO3− reduction by Nap.

Isotope Fractionation (δ13C, δ15N) in the Microbial Degradation of Bromoxynil by Aerobic and Anaerobic Soil Enrichment Cultures.

Bromoxynil is an increasingly applied nitrile herbicide. Under aerobic conditions, hydration, nitrilation, or hydroxylation of the nitrile group commonly occurs, whereas under anaerobic conditions reductive dehalogenation is common. This work studied the isotope effects associated with these processes by soil cultures. The aerobic soil enrichment culture presented a significant increase in Stenotrophomonas, Pseudomonas, Chryseobacterium, Achromobacter, Azospirillum, and Arcticibacter, and degradation products indicated that nitrile hydratase was the dominant degradation route. The anaerobic culture was dominated by Proteobacteria and Firmicutes phyla with a significant increase in Dethiosulfatibacter, and degradation products indicated reductive debromination as a major degradation route. Distinct dual-isotope trends (δ13C, δ15N) were determined for the two routes: a strong inverse nitrogen isotope effect (εN = 10.56 ± 0.36‰) and an insignificant carbon isotope effect (εC = 0.37 ± 0.36‰) for the aerobic process versus a negligible effect for both elements in the anaerobic process. These trends differ from formerly reported trends for the photodegradation of bromoxynil and enable one to distinguish between the processes in the field.

Isotope analysis method for the herbicide bromoxynil and its application to study photo-degradation processes

Bromoxynil is an increasingly applied nitrile herbicide used for post-emergent control of annual broadleaved weeds. Compound-specific isotope analysis (CSIA) of the compound is of interest for studying its environmental fate, yet is challenging following its polar nature. We present a CSIA method for bromoxynil that includes offline thin-layer chromatography purification followed by an elemental analyzer isotope ratio mass spectrometer (EA-IRMS). This method was shown to be accurate and precise for δ13C and δ15N analysis of the compound (standard deviation of replicate standards <0.5‰). The method was applied to photodegraded samples, either radiated under laboratory condition with a UV lamp, or exposed to sunlight under environmental conditions. Dominating degradation products were similar in both cases. Nevertheless, isotope effects differed, presenting a strong inverse carbon isotope effect (εC = 4.74 ± 0.82‰) and a weak inverse nitrogen isotope effect (εN = 0.76 ± 0.12‰) for the laboratory experiment, and an insignificant carbon isotope effect (εC = 0.34 ± 0.44‰) and a normal nitrogen isotope effect (εN = −3.70 ± 0.30‰) for the natural conditions experiment. The differences in δ13C vs. δ15N enrichment trends suggest different mechanism for the two processes. Finally, the obtained dual isotope trend for natural conditions provide the basis for studying the dominance of photodegradation as a degradation route in the environment.

Inconsistent carbon and nitrogen isotope fractionation in the biotransformation of atrazine by Ensifer sp. CX-T and Sinorihizobium sp. K

Compound-specific stable isotope analysis (CSIA) has been used to quantify and identify biodegradation of organic pesticides in the environment. In this work, the kinetic isotope fractionation of carbon and nitrogen was investigated during rapid atrazine degradation by two bacterial isolates, CX-T (Ensifer sp.) and K (Sinorihizobium sp.), under aerobic conditions. Significant isotope fractionation (εcarbon = -2.40 ± 0.07‰, εnitrogen = 1.30 ± 0.13‰, strain CX-T; εcarbon = -1.40 ± 0.07‰, εnitrogen = 2.5 ± 0.5‰, strain K) was observed for both isolates. The same pattern of normal carbon and inverse nitrogen isotope effects were obtained, confirming the same reaction mechanisms of atrazine. The calculated, apparent kinetic isotope effects of strain CX-T (AKIEcarbon = 1.020; AKIEnitrogen = 0.994) and strain K (AKIEcarbon = 1.011; AKIEnitrogen = 0.988) were observed, implying that carbon-chlorine bonds weakened and additional bonds appeared on the triazine ring nitrogen at the transition state. However, isotope enrichment factors and dual isotope slopes (Δ = δ15N/δ13C) of both isolates (Δ = -0.55 ± 0.05, strain CX-T; Δ = -1.07 ± 0.14, strain K) were, nevertheless, inconsistent even for the same compound and same transformation pathway.

Ab initio study of nitrogen and position-specific oxygen kinetic isotope effects in the NO + O3 reaction

Ab initio calculations have been carried out to investigate nitrogen (k15/k14) and position-specific oxygen (k17/k16O & k18/k16) kinetic isotope effects (KIEs) for the reaction between NO and O3 using CCSD(T)/6-31G(d) and CCSD(T)/6-311G(d) derived frequencies in the complete Bigeleisen equations. Isotopic enrichment factors are calculated to be -6.7‰, -1.3‰, -44.7‰, -14.1‰, and -0.3‰ at 298 K for the reactions involving the 15N16O, 14N18O, 18O16O16O, 16O18O16O, and 16O16O18O isotopologues relative to the 14N16O and 16O3 isotopologues, respectively (CCSD(T)/6-311G(d)). Using our oxygen position-specific KIEs, a kinetic model was constructed using Kintecus, which estimates the overall isotopic enrichment factors associated with unreacted O3 and the oxygen transferred to NO2 to be -19.6‰ and -22.8‰, respectively, (CCSD(T)/6-311G(d)) which tends to be in agreement with previously reported experimental data. While this result may be fortuitous, this agreement suggests that our model is capturing the most important features of the underlying physics of the KIE associated with this reaction (i.e., shifts in zero-point energies). The calculated KIEs will useful in future NOx isotopic modeling studies aimed at understanding the processes responsible for the observed tropospheric isotopic variations of NOx as well as for tropospheric nitrate.

13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects

A large number of flavoproteins catalyze the oxidation of amines. Because of the importance of these enzymes in metabolism, their mechanisms have previously been studied using deuterium, nitrogen, and solvent isotope effects. While these results have been valuable for computational studies to distinguish among proposed mechanisms, a measure of the change at the reacting carbon has been lacking. We describe here the measurement of a 13C kinetic isotope effect for a representative amine oxidase, polyamine oxidase. The isotope effect was determined by analysis of the isotopic composition of the unlabeled substrate, N, N’-dibenzyl-1,4-diaminopropane, to obtain a pH-independent value of 1.025. The availability of a 13C isotope effect for flavoprotein-catalyzed amine oxidation provides the first measure of the change in bond order at the carbon involved in this carbon-hydrogen bond cleavage and will be of value to understanding the transition state structure for this class of enzymes.

Aerobic respiration along isopycnals leads to overestimation of the isotope effect of denitrification in the ocean water column

The nitrogen (N) isotopes provide an integrative geochemical tool for constraining the fixed N budget of the ocean. However, N isotope budgeting requires a robust estimate for the organism-scale nitrogen isotope effect of denitrification, in particular as it occurs in water column denitrification zones (εwcd). Ocean field data interpreted with the Rayleigh model have typically yielded estimates for εwcd of between 20 and 30‰. However, recent findings have raised questions about this value. In particular, culture experiments can produce a substantially lower isotope effect (∼13‰) under conditions mimicking those of ocean suboxic zones. In an effort to better understand prior field estimates of εwcd, we use a geochemical multi-box model to investigate the combined effects of denitrification, aerobic respiration, and isopycnal exchange on the δ15N of nitrate. In the context of this admittedly simplistic model, we consider three isopycnals extending from the Southern Ocean to the Eastern Tropical North Pacific (ETNP). We show that the data from the ETNP suboxic zone can be reproduced with an εwcd of 13‰, given a rate of aerobic respiration consistent with the nutrient data on these isopycnals and a plausible range in the δ15N of the sinking flux being remineralized. We discuss the limitations of our analysis, additional considerations, as well as possible data-based tests for the proposal of a lower εwcd than previously estimated. All else held constant, a lower εwcd would imply a lower global ocean rate of denitrification that is more similar to the estimated rate of N input to the global ocean, providing a major impetus for further investigation.

Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream.

Ammonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ(15)NNO2- and δ(18)ONO2-, respectively) are geochemical tracers for evaluating the sources and the in situ rate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilic Thaumarchaeota populations composed almost entirely of "Candidatus Nitrosocaldus." The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ(18)O value of nitrite produced from ammonia oxidation varied with the δ(18)O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilic Thaumarchaeota could be estimated using δ(18)ONO2- in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments. Because ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilic Thaumarchaeota and elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

Relating Carbon and Nitrogen Isotope Effects to Reaction Mechanisms during Aerobic or Anaerobic Degradation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Pure Bacterial Cultures.

Kinetic isotopic fractionation of carbon and nitrogen during RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) biodegradation was investigated with pure bacterial cultures under aerobic and anaerobic conditions. Relatively large bulk enrichments in (15)N were observed during biodegradation of RDX via anaerobic ring cleavage (ε(15)N = -12.7‰ ± 0.8‰) and anaerobic nitro reduction (ε(15)N = -9.9‰ ± 0.7‰), in comparison to smaller effects during biodegradation via aerobic denitration (ε(15)N = -2.4‰ ± 0.2‰). (13)C enrichment was negligible during aerobic RDX biodegradation (ε(13)C = -0.8‰ ± 0.5‰) but larger during anaerobic degradation (ε(13)C = -4.0‰ ± 0.8‰), with modest variability among genera. Dual-isotope ε(13)C/ε(15)N analyses indicated that the three biodegradation pathways could be distinguished isotopically from each other and from abiotic degradation mechanisms. Compared to the initial RDX bulk δ(15)N value of +9‰, δ(15)N values of the NO2 (-) released from RDX ranged from -7‰ to +2‰ during aerobic biodegradation and from -42‰ to -24‰ during anaerobic biodegradation. Numerical reaction models indicated that N isotope effects of NO2 (-) production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε(15)N-NO2 (-) values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO2 (-) formation associated with anaerobic ring cleavage. These results indicate the potential for isotopic analysis of residual RDX for the differentiation of degradation pathways and indicate that further efforts to examine the isotopic composition of potential RDX degradation products (e.g., NOx) in the environment are warranted. This work provides the first systematic evaluation of the isotopic fractionation of carbon and nitrogen in the organic explosive RDX during degradation by different pathways. It also provides data on the isotopic effects observed in the nitrite produced during RDX biodegradation. Both of these results could lead to better understanding of the fate of RDX in the environment and help improve monitoring and remediation technologies.

Isotope fractionation and isotope decoupling during anammox and denitrification in marine sediments

To evaluate the relation of isotope fractionation during sedimentary nitrate reduction with sediment reactivity, we measured nitrate and nitrite reduction rates and corresponding isotope changes in marine sediments in the Skagerrak. Our sampling sites encompassed a gradient of reactivity, oxygen consumption, and manganese concentration. Anammox was the main N2-production pathway at the deepest site. For this site, we calculated the intrinsic isotope effect of nitrite consumption by anammox in marine sediments, and found that it is ∼ −20‰, in accordance with culture studies. Denitrification was dominant at shallower sites, which confirms previous studies from whole core incubations. The N-isotope effect of denitrification, 15ε, ranged from −12‰ to −16‰. Oxygen isotopes of nitrate were more variable, and the ratio of 18ε/15ε, was highly variable in all sediments we investigated. At all stations, the oxygen isotope effect was (partly or entirely) decoupled from the nitrogen isotope effect. In denitrification-dominated sediments, this decoupling of oxygen and nitrogen isotopes appeared to be driven by nitrite reoxidation in anoxic sediment incubations, either due to enzymatic reversibility of the respiratory nitrate reductase Nar, or to reversibility on the community level. In anammox-dominated sediments, this effect was also evident in N-isotope changes, likely due to net nitrate production and isotope exchange that is promoted by anammox. These findings suggest that the ratio of 18ε/15ε in marine environments is more flexible than previously assumed, because enzymatic or community-driven isotope exchange can alter both N and O isotopes, deviating from standard Rayleigh-type fractionation.