Nitrogen isotope analysis of aqueous ammonium and nitrate by membrane inlet isotope ratio mass spectrometry (MIRMS) at natural abundance levels.

RATIONALE: Existing methods for the measurement of the 15 N/14 N isotopic composition of ammonium and nitrate are either only suitable for labelled samples or require considerable sample preparation efforts (or both). Our goal was to modify an existing analytical approach to allow for natural abundance precision levels. METHODS: Published reaction protocols were used to convert ammonium into N2 by NaOBr and nitrate into N2 O by TiCl3 . A membrane inlet system was developed and coupled to an isotope ratio mass spectrometer to allow precise determination of the analytes. RESULTS: Concentrations of ≥35 µmol/L N for both ammonium or nitrate could be analysed for δ15 N values with precisions of better than 0.9 mUr. While ammonium analyses exhibited a small concentration dependency and an offset of 2.7 mUr at high ammonium concentrations irrespective of the standard isotopic composition, nitrate analysis showed no offset but a blank contribution visible at very low concentrations. CONCLUSIONS: The presented method is capable of fast measurement of δ15 N values in ammonium and nitrate from aqueous samples with reasonable accuracy at natural abundance levels. It will thus facilitate the application of isotopic methods to studies of nitrogen cycling in ecosystems.

Development and verification of a novel isotopic N2 O measurement technique for discrete static chamber samples using cavity ring-down spectroscopy.

RATIONALE: N2 O isotopomers are a useful tool to study soil N cycling processes. The reliability of such measurements requires a consistent set of international N2 O isotope reference materials to improve inter-laboratory and inter-instrument comparability and avoid reporting inaccurate results. All these are the more important given the role of N2 O in anthropogenic climate change and the pressing need to develop our understanding of soil N cycling and N2 O emission to mitigate such emissions. Cavity ring-down spectroscopy (CRDS) could potentially overcome resource requirements and technical challenges, making N2 O isotopomer measurements more feasible and less expensive than previous approaches (e.g., gas chromatography [GC] and isotope ratio mass spectrometry [IRMS]). METHODS: A combined laser spectrometer and small sample isotope module (CRDS & SSIM) method enabled N2 O concentration, δ15 Nbulk , δ15 Nα , δ15 Nß and site preference (SP) measurements of sample volumes <20 mL, such as static chamber samples. Sample dilution and isotopic mixing as well as N2 O concentration dependence were corrected numerically. A two-point calibration procedure normalised δ values to the international isotope-ratio scales. The CRDS & SSIM repeatability was determined using a reference gas (Ref Gas). CRDS & SSIM concentration measurements were compared with those obtained by GC, and the isotope ratio measurements from two different mass spectrometers were compared. RESULTS: The repeatability (mean ± 1σ; n = 10) of the CRDS & SSIM measurements of the Ref Gas was 710.64 ppb (± 8.64), 2.82‰ (± 0.91), 5.41‰ (± 2.00), 0.23‰ (± 0.22) and 5.18‰ (± 2.18) for N2 O concentration, δ15 Nbulk , δ15 Nα , δ15 Nß and SP, respectively. The CRDS & SSIM concentration measurements were strongly correlated with GC (r = 0.99), and they were more precise than those obtained using GC except when the N2 O concentrations exceeded the specified operating range. Normalising CRDS & SSIM δ values to the international isotope-ratio scales using isotopic N2 O standards (AK1 and Mix1) produced accurate results when the samples were bracketed within the range of the δ values of the standards. The CRDS & SSIM δ15 Nbulk and SP precision was approximately one order of magnitude less than the typical IRMS precision. CONCLUSIONS: CRDS & SSIM is a promising approach that enables N2 O concentrations and isotope ratios to be measured by CRDS for samples <20 mL. The CRDS & SSIM repeatability makes this approach suitable for N2 O "isotopomer mapping" to distinguish dominant source pathways, such as nitrification and denitrification, and requires less extensive lab resources than the traditionally used GC/IRMS. Current study limitations highlighted potential improvements for future users of this approach to consider, such as automation and physical removal of interfering trace gases before sample analysis.

Nitrous oxide effluxes from plants as a potentially important source to the atmosphere.

The global budget for nitrous oxide (N2 O), an important greenhouse gas and probably dominant ozone-depleting substance emitted in the 21st century, is far from being fully understood. Cycling of N2 O in terrestrial ecosystems has traditionally exclusively focused on gas exchange between the soil surface (nitrification-denitrification processes) and the atmosphere. Terrestrial vegetation has not been considered in the global budget so far, even though plants are known to release N2 O. Here, we report the N2 O emission rates of 32 plant species from 22 different families measured under controlled laboratory conditions. Furthermore, the first isotopocule values (δ15 N, δ18 O and δ15 Nsp ) of N2 O emitted from plants were determined. A robust relationship established between N2 O emission and CO2 respiration rates, which did not alter significantly over a broad range of changing environmental conditions, was used to quantify plant-derived emissions on an ecosystem scale. Stable isotope measurements (δ15 N, δ18 O and δ15 Nsp ) of N2 O emitted by plants clearly show that the dual isotopocule fingerprint of plant-derived N2 O differs from that of currently known microbial or chemical processes. Our work suggests that vegetation is a natural source of N2 O in the environment with a large fraction released by a hitherto unrecognized process.

Improvement of the 15 N gas flux method for in situ measurement of soil denitrification and its product stoichiometry.

RATIONALE: Field measurement of denitrification in agricultural ecosystems using the 15 N gas flux method has been limited by poor sensitivity because current isotope ratio mass spectrometry is not precise enough to detect low 15 N2 fluxes in the presence of a high atmospheric N2 background. For laboratory studies, detection limits are improved by incubating soils in closed systems and under N2 -depleted atmospheres. METHODS: We developed a new procedure to conduct the 15 N gas flux method suitable for field application using an artificially N2 -depleted atmosphere to improve the detection limit at the given precision of mass spectrometry. Laboratory experiments with and without 15 N-labelling and using different flushing strategies were conducted to develop a suitable field method. Subsequently, this method was tested in the field and results were compared with those obtained from the conventional 15 N gas flux method. RESULTS: Results of the two methods were in close agreement showing that the denitrification rates determined were not biased by the flushing procedure. Best sensitivity for N2 + N2 O fluxes was 10 ppb, which was 80-fold better than that of the reference method. Further improvement can be achieved by lowering the N2 background concentration below the values established in the present study. CONCLUSIONS: In view of this progress in sensitivity, the new method will be suitable to measure denitrification dynamics in the field beyond peak events.

Improved isotopic model based on 15 N tracing and Rayleigh-type isotope fractionation for simulating differential sources of N2 O emissions in a clay grassland soil.

RATIONALE: Isotopic signatures of N2 O can help distinguish between two sources (fertiliser N or endogenous soil N) of N2 O emissions. The contribution of each source to N2 O emissions after N-application is difficult to determine. Here, isotopologue signatures of emitted N2 O are used in an improved isotopic model based on Rayleigh-type equations. METHODS: The effects of a partial (33% of surface area, treatment 1c) or total (100% of surface area, treatment 3c) dispersal of N and C on gaseous emissions from denitrification were measured in a laboratory incubation system (DENIS) allowing simultaneous measurements of NO, N2 O, N2 and CO2 over a 12-day incubation period. To determine the source of N2 O emissions those results were combined with both the isotope ratio mass spectrometry analysis of the isotopocules of emitted N2 O and those from the 15 N-tracing technique. RESULTS: The spatial dispersal of N and C significantly affected the quantity, but not the timing, of gas fluxes. Cumulative emissions are larger for treatment 3c than treatment 1c. The 15 N-enrichment analysis shows that initially ~70% of the emitted N2 O derived from the applied amendment followed by a constant decrease. The decrease in contribution of the fertiliser N-pool after an initial increase is sooner and larger for treatment 1c. The Rayleigh-type model applied to N2 O isotopocules data (δ15 Nbulk -N2 O values) shows poor agreement with the measurements for the original one-pool model for treatment 1c; the two-pool models gives better results when using a third-order polynomial equation. In contrast, in treatment 3c little difference is observed between the two modelling approaches. CONCLUSIONS: The importance of N2 O emissions from different N-pools in soil for the interpretation of N2 O isotopocules data was demonstrated using a Rayleigh-type model. Earlier statements concerning exponential increase in native soil nitrate pool activity highlighted in previous studies should be replaced with a polynomial increase with dependency on both N-pool sizes.

Quantifying N2O reduction to N2 during denitrification in soils via isotopic mapping approach: Model evaluation and uncertainty analysis.

The last step of denitrification, i.e. the reduction of N2O to N2, has been intensively studied in the laboratory to understand the denitrification process, predict nitrogen fertiliser losses, and to establish mitigation strategies for N2O. However, assessing N2 production via denitrification at large spatial scales is still not possible due to lack of reliable quantitative approaches. Here, we present a novel numerical "mapping approach" model using the δ15Nsp/δ18O slope that has been proposed to potentially be used to indirectly quantify N2O reduction to N2 at field or larger spatial scales. We evaluate the model using data obtained from seven independent soil incubation studies conducted under a He-O2 atmosphere. Furthermore, we analyse the contribution of different parameters to the uncertainty of the model. The model performance strongly differed between studies and incubation conditions. Re-evaluation of the previous data set demonstrated that using soils-specific instead of default endmember values could largely improve model performance. Since the uncertainty of modelled N2O reduction was relatively high, further improvements to estimate model parameters to obtain more precise estimations remain an on-going matter, e.g. by determination of soil-specific isotope fractionation factors and isotopocule endmember values of N2O production processes using controlled laboratory incubations. The applicability of the mapping approach model is promising with an increasing availability of real-time and field based analysis of N2O isotope signatures.

NO Reduction to N2O Improves Nitrate 15N Abundance Analysis by Membrane Inlet Quadrupole Mass Spectrometry.

Earlier an automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the 15N abundances and concentrations of nitrate, nitrite and ammonium of aqueous solutions without any sample preparation. Here we describe an alternative analytical protocol to convert NO3- to N2O instead of NO before measurement. This is advantageous because NO strongly interacts with surfaces, requires long purge times, and still shows considerable carryover between samples, all of which is avoided when N2O is used as analyte. The sensitivity of the measurement of NO3- as N2O is comparable to the earlier measurements with NO as analyte.

Estimating N2 O processes during grassland renewal and grassland conversion to maize cropping using N2 O isotopocules.

RATIONALE: Enhanced nitrous oxide (N2 O) emissions can occur following grassland break-up for renewal or conversion to maize cropping, but knowledge about N2 O production pathways and N2 O reduction to N2 is very limited. A promising tool to address this is the combination of mass spectrometric analysis of N2 O isotopocules and an enhanced approach for data interpretation. METHODS: The isotopocule mapping approach was applied to field data using a δ15 NspN2O and δ18 ON2O map to simultaneously determine N2 O production pathways contribution and N2 O reduction for the first time. Based on the isotopic composition of N2 O produced and literature values for specific N2 O pathways, it was possible to distinguish: (i) heterotrophic bacterial denitrification and/or nitrifier denitrification and (ii) nitrification and/or fungal denitrification and the contribution of N2 O reduction. RESULTS: The isotopic composition of soil-emitted N2 O largely resembled the known end-member values for bacterial denitrification. The isotopocule mapping approach indicated different effects of N2 O reduction on the isotopic composition of soil-emitted N2 O for the two soils under study. Differing N2 O production pathways in different seasons were not observed, but management events and soil conditions had a significant impact on pathway contribution and N2 O reduction. N2 O reduction data were compared with a parallel 15 N-labelling experiment. CONCLUSIONS: The field application of the isotopocule mapping approach opens up new prospects for studying N2 O production and consumption of N2 O in soil simultaneously based on mass spectrometric analysis of natural abundance N2 O. However, further studies are needed in order to properly validate the isotopocule mapping approach.

Preliminary assessment of stable nitrogen and oxygen isotopic composition of USGS51 and USGS52 nitrous oxide reference gases and perspectives on calibration needs.

RATIONALE: Despite a long history and growing interest in isotopic analyses of N2 O, there is a lack of isotopically characterized N2 O isotopic reference materials (standards) to enable normalization and reporting of isotope-delta values. Here we report the isotopic characterization of two pure N2 O gas reference materials, USGS51 and USGS52, which are now available for laboratory calibration (https://isotopes.usgs.gov/lab/referencematerials.html). METHODS: A total of 400 sealed borosilicate glass tubes of each N2 O reference gas were prepared from a single gas filling of a high vacuum line. We demonstrated isotopic homogeneity via dual-inlet isotope-ratio mass spectrometry. Isotopic analyses of these reference materials were obtained from eight laboratories to evaluate interlaboratory variation and provide preliminary isotopic characterization of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and site preference (SP ) values. RESULTS: The isotopic homogeneity of both USGS51 and USGS52 was demonstrated by one-sigma standard deviations associated with the determinations of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and SP values of 0.12 mUr or better. The one-sigma standard deviations of SP measurements of USGS51 and USGS52 reported by eight laboratories participating in the interlaboratory comparison were 1.27 and 1.78 mUr, respectively. CONCLUSIONS: 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.

Measuring 15N Abundance and Concentration of Aqueous Nitrate, Nitrite, and Ammonium by Membrane Inlet Quadrupole Mass Spectrometry.

An automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the 15N abundances and concentrations of nitrate, nitrite, and ammonium in aqueous solutions without any sample preparation. The minimum N concentration for an accurate determination of the 15N abundance is 7 µmol/L for nitrite and nitrate, with a relative standard deviation (RSD) of repeated measurements of <1%, and 70 µmol/L with an RSD < 0.4% in the case of ammonium. The SPIN-MIMS system provides a wide dynamic range (up to 3500 µmol/L) for all three N species for both isotope abundance and concentration measurements. The comparison of parallel measurements of 15N-labeled NH4+ and NO3- from soil extracts with the denitrifier method and the SPIN-MIMS system shows a good agreement between both methods.

Use of oxygen isotopes to differentiate between nitrous oxide produced by fungi or bacteria during denitrification.

RATIONALE: Fungal denitrifiers can contribute substantially to N2 O emissions from arable soil and show a distinct site preference for N2 O (SP(N2 O)). This study sought to identify another process-specific isotopic tool to improve precise identification of N2 O of fungal origin by mass spectrometric analysis of the N2 O produced. METHODS: Three pure bacterial and three fungal species were incubated under denitrifying conditions in treatments with natural abundance and stable isotope labelling to analyse the N2 O produced. Combining different applications of isotope ratio mass spectrometry enabled us to estimate the oxygen (O) exchange accelerated by denitrifying enzymes and the ongoing microbial pathway in parallel. This experimental set-up allowed the determination of δ18 O(N2 O) values and isotopic fractionation of O, as well as SP(N2 O) values, as a perspective to differentiate between microbial denitrifiers. RESULTS: Oxygen exchange during N2 O production was lower for bacteria than for fungi, differed between species, and depended also on incubation time. Apparent O isotopic fractionation during denitrification was in a similar range for bacteria and fungi, but application of the fractionation model indicated that different enzymes in bacteria and fungi were responsible for O exchange. This difference was associated with different isotopic fractionation for bacteria and fungi. CONCLUSIONS: δ18 O(N2 O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N2 O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N2 O.

Deep ploughing increases agricultural soil organic matter stocks.

Subsoils play an important role within the global C cycle, since they have high soil organic carbon (SOC) storage capacity due to generally low SOC concentrations. However, measures for enhancing SOC storage commonly focus on topsoils. This study assessed the long-term storage and stability of SOC in topsoils buried in arable subsoils by deep ploughing, a globally applied method for breaking up hard pans and improving soil structure to optimize crop growing conditions. One effect of deep ploughing is translocation of SOC formed near the surface into the subsoil, with concomitant mixing of SOC-poor subsoil material into the 'new' topsoil. Deep-ploughed croplands represent unique long-term in situ incubations of SOC-rich material in subsoils. In this study, we sampled five loamy and five sandy soils that were ploughed to 55-90 cm depth 35-50 years ago. Adjacent, similarly managed but conventionally ploughed subplots were sampled as reference. The deep-ploughed soils contained on average 42 ± 13% more SOC than the reference subplots. On average, 45 years after deep ploughing, the 'new' topsoil still contained 15% less SOC than the reference topsoil, indicating long-term SOC accumulation potential in the topsoil. In vitro incubation experiments on the buried sandy soils revealed 63 ± 6% lower potential SOC mineralisation rates and also 67 ± 2% lower SOC mineralisation per unit SOC in the buried topsoils than in the reference topsoils. Wider C/N ratio in the buried sandy topsoils than in the reference topsoils indicates that deep ploughing preserved SOC. The SOC mineralisation per unit SOC in the buried loamy topsoils was not significantly different from that in the reference topsoils. However, 56 ± 4% of the initial SOC was preserved in the buried topsoils. It can be concluded that deep ploughing contributes to SOC sequestration by enlarging the storage space for SOC-rich material.

Influence of Lumbricus terrestris and Folsomia candida on N2 O formation pathways in two different soils - with particular focus on N2 emissions.

RATIONALE: The gaseous N losses mediated by soil denitrifiers are generally inferred by measuring N2 O fluxes, but should include associated N2 emissions, which may be affected by abiotic soil characteristics and biotic interactions. Soil fauna, particularly anecic earthworms and euedaphic collembola, alter the activity of denitrifiers, creating hotspots for denitrification. These soil fauna are abundant in perennial agroecosystems intended to contribute to more sustainable production of bioenergy. METHODS: Two microcosm experiments were designed to evaluate gaseous N emissions from a silty loam and a sandy soil, both provided with litter from the bioenergy crop Silphium perfoliatum (cup-plant) and inoculated with an anecic earthworm (Lumbricus terrestris), which was added alone or together with an euedaphic collembola (Folsomia candida). In experiment 1, litter-derived N flux was determined by adding 15 N-labelled litter, followed by mass spectrometric analysis of N2 and N2 O isotopologues. In experiment 2, the δ18 O values and 15 N site preference of N2 O were determined by isotope ratio mass spectrometry to reveal underlying N2 O formation pathways. RESULTS: Lumbricus terrestris significantly increased litter-derived N2 emissions in the loamy soil, from 174.5 to 1019.3 µg N2 -N kg-1 soil, but not in the sandy soil (non-significant change from 944.7 to 1054.7 µg N2 -N kg-1 soil). Earthworm feeding on plant litter resulted in elevated N2 O emissions in both soils, derived mainly from turnover of the soil mineral N pool during denitrification. Folsomia candida did not affect N losses but showed a tendency to redirect N2 O formation pathways from fungal to bacterial denitrification. The N2 O/(N2  + N2 O) product ratio was predominantly affected by abiotic soil characteristics (loamy soil: 0.14, sandy soil: 0.26). CONCLUSIONS: When feeding on S. perfoliatum litter, the anecic L. terrestris, but not the euedaphic F. candida, has the potential to cause substantial N losses. Biotic interactions between the species are not influential, but abiotic soil characteristics have an effect. The coarse-textured sandy soil had lower gaseous N losses attributable to anecic earthworms. Copyright © 2016 John Wiley & Sons, Ltd.

Automated system measuring triple oxygen and nitrogen isotope ratios in nitrate using the bacterial method and N2 O decomposition by microwave discharge.

RATIONALE: Triple oxygen and nitrogen isotope ratios in nitrate are powerful tools for assessing atmospheric nitrate formation pathways and their contribution to ecosystems. N2 O decomposition using microwave-induced plasma (MIP) has been used only for measurements of oxygen isotopes to date, but it is also possible to measure nitrogen isotopes during the same analytical run. METHODS: The main improvements to a previous system are (i) an automated distribution system of nitrate to the bacterial medium, (ii) N2 O separation by gas chromatography before N2 O decomposition using the MIP, (iii) use of a corundum tube for microwave discharge, and (iv) development of an automated system for isotopic measurements. Three nitrate standards with sample sizes of 60, 80, 100, and 120 nmol were measured to investigate the sample size dependence of the isotope measurements. RESULTS: The δ17 O, δ18 O, and Δ17 O values increased with increasing sample size, although the δ15 N value showed no significant size dependency. Different calibration slopes and intercepts were obtained with different sample amounts. The slopes and intercepts for the regression lines in different sample amounts were dependent on sample size, indicating that the extent of oxygen exchange is also dependent on sample size. The sample-size-dependent slopes and intercepts were fitted using natural log (ln) regression curves, and the slopes and intercepts can be estimated to apply to any sample size corrections. When using 100 nmol samples, the standard deviations of residuals from the regression lines for this system were 0.5‰, 0.3‰, and 0.1‰, respectively, for the δ18 O, Δ17 O, and δ15 N values, results that are not inferior to those from other systems using gold tube or gold wire. CONCLUSIONS: An automated system was developed to measure triple oxygen and nitrogen isotopes in nitrate using N2 O decomposition by MIP. This system enables us to measure both triple oxygen and nitrogen isotopes in nitrate with comparable precision and sample throughput (23 min per sample on average), and minimal manual treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of methods to determine triple oxygen isotope composition of N2O.

RATIONALE: The oxygen isotope anomaly, Δ(17) O, of N2 O and nitrate is useful to elucidate nitrogen oxide dynamics. A comparison of different methods for Δ(17) O measurement was performed. METHODS: For Δ(17) O measurements, N2 O was converted into O2 and N2 using microwave-induced plasma in a quartz or corundum tube reactor, respectively, or conversion was carried out in a gold wire oven. In each case, isotope ratios were measured by isotope ratio mass spectrometry. RESULTS: All the tested methods showed acceptable precision (coefficient of variation <2.4 % at 160 nmol N2 O) with high sample size but the sample size dependence was lowest when using microwave-induced plasma in a corundum tube reactor. CONCLUSIONS: The use of microwave-induced plasma in a corundum tube yields best results for Δ(17) O measurement on N2 O gas samples.

Isotope fractionation factors controlling isotopocule signatures of soil-emitted N2O produced by denitrification processes of various rates.

RATIONALE: This study aimed (i) to determine the isotopic fractionation factors associated with N2O production and reduction during soil denitrification and (ii) to help specify the factors controlling the magnitude of the isotope effects. For the first time the isotope effects of denitrification were determined in an experiment under oxic atmosphere and using a novel approach where N2O production and reduction occurred simultaneously. METHODS: Soil incubations were performed under a He/O2 atmosphere and the denitrification product ratio [N2O/(N2 + N2O)] was determined by direct measurement of N2 and N2O fluxes. N2O isotopocules were analyzed by mass spectrometry to determine δ(18)O, δ(15)N and (15)N site preference within the linear N2O molecule (SP). An isotopic model was applied for the simultaneous determination of net isotope effects (η) of both N2O production and reduction, taking into account emissions from two distinct soil pools. RESULTS: A clear relationship was observed between (15)N and (18)O isotope effects during N2O production and denitrification rates. For N2O reduction, diverse isotope effects were observed for the two distinct soil pools characterized by different product ratios. For moderate product ratios (from 0.1 to 1.0) the range of isotope effects given by previous studies was confirmed and refined, whereas for very low product ratios (below 0.1) the net isotope effects were much smaller. CONCLUSIONS: The fractionation factors associated with denitrification, determined under oxic incubation, are similar to the factors previously determined under anoxic conditions, hence potentially applicable for field studies. However, it was shown that the η(18)O/η(15)N ratios, previously accepted as typical for N2O reduction processes (i.e., higher than 2), are not valid for all conditions.

Fungal oxygen exchange between denitrification intermediates and water.

RATIONALE: Fungi can contribute greatly to N2O production from denitrification. Therefore, it is important to quantify the isotopic signature of fungal N2O. The isotopic composition of N2O can be used to identify and analyze the processes of N2O production and N2O reduction. In contrast to bacteria, information about the oxygen exchange between denitrification intermediates and water during fungal denitrification is lacking, impeding the explanatory power of stable isotope methods. METHODS: Six fungal species were anaerobically incubated with the electron acceptors nitrate or nitrite and (18)O-labeled water to determine the oxygen exchange between denitrification intermediates and water. After seven days of incubation, gas samples were analyzed for N2O isotopologues by isotope ratio mass spectrometry. RESULTS: All the fungal species produced N2O. N2O production was greater when nitrite was the sole electron acceptor (129 to 6558 nmol N2O g dw(-1) h(-1)) than when nitrate was the electron acceptor (6 to 47 nmol N2O g dw(-1) h(-1)). Oxygen exchange was complete with nitrate as electron acceptor in one of five fungi and with nitrite in two of six fungi. Oxygen exchange of the other fungi varied (41 to 89% with nitrite and 11 to 61% with nitrate). CONCLUSIONS: This is the first report on oxygen exchange with water during fungal denitrification. The exchange appears to be within the range previously reported for bacterial denitrification. This adds to the difficulty of differentiating N2O producing processes based on the origin of N2O-O. However, the large oxygen exchange repeatedly observed for bacteria and now also fungi could lead to less variability in the δ(18)O values of N2O from soils, which could facilitate the assessment of the extent of N2O reduction.

Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification--a pure culture study.

RATIONALE: The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS: To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under denitrifying conditions, with either NO3(-) or NO2(-) as the electron acceptor, and measured the isotopic signature (δ(18)O, δ(15)Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS: Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO2(-) only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of (15)Nbulk were associated with larger N2O production. The δ(18)O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS: Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

Interlaboratory assessment of nitrous oxide isotopomer analysis by isotope ratio mass spectrometry and laser spectroscopy: current status and perspectives.

RATIONALE: In recent years, research and applications of the N2O site-specific nitrogen isotope composition have advanced, reflecting awareness of the contribution of N2O to the anthropogenic greenhouse effect, and leading to significant progress in instrument development. Further dissemination of N2O isotopomer analysis, however, is hampered by a lack of internationally agreed gaseous N2O reference materials and an uncertain compatibility of different laboratories and analytical techniques. METHODS: In a first comparison approach, eleven laboratories were each provided with N2O at tropospheric mole fractions (target gas T) and two reference gases (REF1 and REF2). The laboratories analysed all gases, applying their specific analytical routines. Compatibility of laboratories was assessed based on N2O isotopocule data for T, REF1 and REF2. Results for T were then standardised using REF1 and REF2 to evaluate the potential of N2O reference materials for improving compatibility between laboratories. RESULTS: Compatibility between laboratories depended on the analytical technique: isotope ratio mass spectrometry (IRMS) results showed better compatibility for δ(15)N values, while the performance of laser spectroscopy was superior with respect to N2O site preference. This comparison, however, is restricted by the small number of participating laboratories applying laser spectroscopy. Offset and two-point calibration correction of the N2O isotopomer data significantly improved the consistency of position-dependent nitrogen isotope data while the effect on δ(15)N values was only minor. CONCLUSIONS: The study reveals that for future research on N2O isotopocules, standardisation against N2O reference material is essential to improve interlaboratory compatibility. For atmospheric monitoring activities, we suggest N2O in whole air as a unifying scale anchor.

Isotopologue ratios of N2O and N2 measurements underpin the importance of denitrification in differently N-loaded riparian alder forests.

Known as biogeochemical hotspots in landscapes, riparian buffer zones exhibit considerable potential concerning mitigation of groundwater contaminants such as nitrate, but may in return enhance the risk for indirect N2O emission. Here we aim to assess and to compare two riparian gray alder forests in terms of gaseous N2O and N2 fluxes and dissolved N2O, N2, and NO3(-) in the near-surface groundwater. We further determine for the first time isotopologue ratios of N2O dissolved in the riparian groundwater in order to support our assumption that it mainly originated from denitrification. The study sites, both situated in Estonia, northeastern Europe, receive contrasting N loads from adjacent uphill arable land. Whereas N2O emissions were rather small at both sites, average gaseous N2-to-N2O ratios inferred from closed-chamber measurements and He-O laboratory incubations were almost four times smaller for the heavily loaded site. In contrast, groundwater parameters were less variable among sites and between landscape positions. Campaign-based average (15)N site preferences of N2O (SP) in riparian groundwater ranged between 11 and 44 ‰. Besides the strong prevalence of N2 emission over N2O fluxes and the correlation pattern between isotopologue and water quality data, this comparatively large range highlights the importance of denitrification and N2O reduction in both riparian gray alder stands.