Denitrifier Method Research Articles

Analytical improvements and assessment of long‐term performance of the oxidation–denitrifier method

The analysis of the nitrogen (N) isotopic composition of organic matter bound to fossil biomineral structures (BB‐δ15N) using the oxidation–denitrifier (O–D) method provides a novel tool to study past changes in N cycling processes.MethodsWe report a set of methodological improvements to the O–D method, including (a) a method for sealing the reaction vials in which the oxidation of organic N to NO3− takes place, (b) a recipe for bypassing the pH adjustment step before the bacterial conversion of NO3− to N2O, and (c) a method for storing recrystallized dipotassium peroxodisulfate (K2S2O8) under Ar atmosphere.ResultsThe new sealing method eliminates the occasional contamination and vial breakage that occurred previously while increasing sample throughput. The protocol for bypassing pH adjustment does not affect BB‐δ15N, and it significantly reduces the processing time. Storage of K2S2O8 reagent under Ar atmosphere produces stable oxidation blanks over more than 3.5 years. We report analytical blanks, accuracy, and precision for this methodology from eight users over the course of ~3.5 years of analyses at the Max Planck Institute for Chemistry. Our method produces analytical blanks characterized by low N content (0.30 ± 0.13 nmol N, 1σ, n = 195) and stable δ15N (−2.20 ± 3.13‰, n = 195). The analysis of reference amino acid standards USGS 40 and USGS 65 indicates an overall accuracy of −0.23 ± 0.35‰ (1σ, n = 891). The analysis of in‐house fossil standards gives similar analytical precision (1σ) across a range of BB‐δ15N values and biominerals: zooxanthellate coral standard PO‐1 (6.08 ± 0.21‰, n = 267), azooxanthellate coral standard LO‐1 (10.20 ± 0.28‰, n = 258), foraminifera standard MF‐1 (5.92 ± 0.28‰, n = 243), and tooth enamel AG‐Lox (4.06 ± 0.49‰, n = 78).ConclusionsThe methodological improvements significantly increase sample throughput without compromising analytical precision or accuracy down to 1 nmol of N.

Discussion on the need for correction during isotopic analysis of nitrogen by the denitrifier method.

The nitrogen and oxygen isotopes of NO3 - are effectively used to trace the main nitrogen sources and migration processes in the atmosphere, water and soil. NO3 - can be converted into N2O by the bacterial denitrification method, which is an advanced method with high sensitivity. However, due to the existence of a small but inevitable blank during the whole experimental process, the N isotopic signal of N2O produced by denitrification superimposes on that of the N blank. Currently, the standard curve correction method is used to correct measured nitrogen isotope results to mitigate blank interference. It has been reported that high variability of the nitrogen isotope results have been produced by the denitrifier method by conducting an interlaboratory comparison of denitrifier methods and other methods on standards and environmental samples, and to reduce this problem, the nitrogen isotope calibration process with a standard curve is examined in depth in this paper, which uses PreCon-GC-IRMS to determine the nitrogen isotopes in N2O. We demonstrate for the first time that reliable results can be obtained without correction for samples with nitrogen isotope composition ranging from -9.9 to 19.5‰, which covers the natural sample range. This study establishes the double test approach for the bacterial denitrification method, ensuring the accuracy and long-term stability of different batches of nitrogen isotope results.

Influence of sample volume on nitrate N and O isotope ratio analyses with the denitrifier method.

Analyses of the isotope ratios of nitrogen (15 N/14 N) and oxygen (18 O/16 O) in nitrate (NO3 - ) with the denitrifier method require relatively high sample volumes at low concentrations (≤1 μM) to afford sufficient analyte for mass spectrometry, resulting in isotopic offsets compared to more concentrated samples of the same isotopic composition. To uncover the origins of isotopic offsets, we analyzed the N and O isotope ratios of NO3 - reference materials spanning concentrations of 0.5-20 μM. We substantiated the incidence of volume-dependent isotopic offsets, then investigated whether they resulted from (a) incomplete sample recovery during N2 O sparging, (b) blanks - bacterial, atmospheric, or in reference material solutions - and (c) oxygen atom exchange with water during the bacterial conversion of NO3 - to N2 O. Larger sample volumes resulted in modest offsets in δ15 N, but substantial offsets in δ18 O. N2 O recovery from sparging was less complete at higher volumes, resulting in decreases in δ15 N and δ18 O due to associated isotope fractionation. Blanks increased detectably with volume, whereas oxygen atom exchange with water remained constant within batch analyses, being sensitive to neither sample volume nor salinity. The sizeable offsets in δ18 O with volume are only partially explained by the factors considered in our analysis. Our observations argue for bracketing of NO3 - samples with reference materials that emulate sample volumes (concentrations) to achieve improved measurement accuracy and foster inter-comparability.

Influence of δ18 O of water on measurements of δ18 O of nitrite and nitrate.

Oxygen isotope ratio measurements of NO2 - and NO3 - by the azide method and denitrifier method are sensitive to the δ18 O value of the sample water. However, the influence of δ18 OH2O on those measurements has not been quantitatively evaluated and documented so far. Therefore, we investigated the influence of δ18 OH2O of a sample on the δ18 O analysis of NO2 - and NO3 - . We prepared NO2 - and NO3 - standards (with known δ18 ONO2- and δ18 ONO3- values) dissolved in waters having different δ18 OH2O values (δ18 OH2O = -12.6, 25.9, 56.7, and 110.1‰). Nitrite and nitrate were converted into N2 O using the azide method and the denitrifier method, respectively. The isotope ratios of the generated N2 O were measured with a Sercon purge-and-trap gas chromatography/isotope ratio mass spectrometry (PT-GC/IRMS) system. The measured δ18 O values of the produced N2 O were plotted against known δ18 ONO2- and δ18 ONO3- values to evaluate the influence of exchange of an oxygen atom with H2 O during the conversion of NO2 - into N2 O and NO3 - into N2 O, respectively. The degree of oxygen isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0% in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δ18 OH2O value of water than the denitrifier method. Thus, the intercept of the standard calibration curve must be corrected to account for differences in δ18 OH2O . Abiotic NO2 -H2 O equilibrium isotope effect experiments yielded a rate constant of (1.13 ± 007) × 10-2 (h-1 ) and an equilibrium isotope effect of 11.9 ± 0.1‰ under the condition of pH = 7.5, 30°C, and 2.5% salinity. Oxygen isotope ratio measurements of NO2 - by the azide method are highly sensitive to δ18 OH2O as a result of significant oxygen isotope exchange between NO2 - and H2 O. Therefore, to obtain the most accurate measurements water with the same δ18 OH2O value as that of the sample must be used to make the NO2 - and NO3 - standards.

Further optimisation of the denitrifier method for the rapid 15 N and 18 O analysis of nitrate in natural water samples.

This study aims to develop a simplified denitrifier method for the δ15 N and δ18 O analysis of nitrate (NO3 - ) in natural water samples combining the method of Zhu et al (Sci Total Environ. 2018; 633: 1370-1378) and the original denitrifier method of Sigman et al (Anal Chem. 2001; 73: 4145-4153). Unlike in the aforementioned methods, the aerobic cultivation was performed without the addition or removal of nitrate in the liquid medium. We remove the nitrate contained in the nutrient medium as N2 O in the gas phase by an additional purging step after incubation overnight before the water sample is injected. This eliminates the need for another preparation step, thus saving working time. The δ15 N and δ18 O values of dissolved NO3 - were determined using a Delta V Plus isotope ratio mass spectrometer coupled to a GasBench II sample preparation device that included a denitrification kit. After optimising the influencing factors (i.e., purging gas, purging time, and type of crimp seals), the method yielded high accuracy and precision (standard deviations were generally ≤0.7‰ for δ18 O values and ≤0.3‰ for δ15 N values), confirming the suitability of this procedure. Finally, the potential applicability of the method was demonstrated by measuring the isotopic composition of NO3 - in natural water samples. The denitrifier method for converting NO3 - into N2 O for isotope analysis was optimised. This allowed the sample preparation time to be further reduced. The complete working time for sample preparation, including all steps, takes 10 min per vial if 60 vials are prepared in one run. The water samples are ready for isotope analysis on the fourth day after preparation has started. Isotope measurements can be performed up to 14 days after preparation.

Denitrifier Method for Nitrite Removal in Electrochemical Analysis of the Electron Accepting Capacity of Humic Substances.

Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox battery, however, remains poorly understood. Mediated electrochemical analysis is an emerging tool to determine the redox properties (i.e., electron donating capacity (EDC), electron accepting capacity (EAC), and redox state) of HS. However, the presence of nitrite (NO2-), a central intermediate of the N-cycle, interferes with the electrochemical determination of the EAC. To eliminate this interference, we developed a bioassay to remove nitrite in HS samples using the denitrifying bacterium Pseudomonas nitroreducens. Cell suspensions of P.nitroreducens completely removed NO2- at various concentrations (1, 2, and 5 mM) from humic acid samples (1 g HA/L) of different redox states. As P.nitroreducens is not able to exchange electrons with dissolved humic acids, the procedure allows an accurate and reliable determination of the EAC of humic acid samples. The proposed method thus opens new perspectives in biogeochemistry to study interactions between HSs and N cycling.

Factors influencing the accuracy of the denitrifier method for determining the oxygen isotopic composition of nitrate.

The denitrifier method is widely used as a novel pretreatment method for the determination of nitrogen and oxygen isotope ratios as it can provide quantitative and high-sensitivity measurements. Nevertheless, the method is limited by relatively low measurement accuracy for δ18O. In this study, we analyzed the factors influencing the accuracy of δ18O determination, and then systematically investigated the effects of dissolved oxygen concentrations and nitrate sample sizes on estimates of the δ15N and δ18O of nitrate reference materials. The δ18O contraction ratio was used to represent the relationship between the measured difference and true difference between two reference materials. We obtained the following main results: (1) a gas-liquid ratio of 3:10 (v/v) in ordinary triangular flasks and a shaking speed of 120 r/min produced an optimal range (1.9 to 2.6 mg/L) in the concentration of dissolved oxygen for accurately determining δ18O, and (2) the δ18O contraction ratio decreased as nitrate sample size decreased within a certain range (1.0 to 0.1 μmol). Our results suggested that δ18O contraction is influenced mainly by dissolved oxygen concentrations in pure culture, and provided a model for improving the accuracy of oxygen isotope analysis.

Controlled induction of denitrification in Pseudomonas aureofaciens: A simplified denitrifier method for dual isotope analysis in NO3−

Dual isotope signatures (δ15N and δ18O) of nitrate (NO3−) are invaluable to constrain nitrogen transformation processes in the biosphere. Biological conversion to nitrous oxide (N2O) by denitrifiers lacking the gene coding for N2O reductase has become the method of choice for isolating NO3− from natural samples for 15N and 18O analysis (“denitrifier method”). The success of the method depends on the ability of the culture to rapidly and quantitatively convert NO3− to N2O with as little as possible contamination from non-sample NO3− or interference with water 18O. We studied oxic-anoxic transition in Pseudomonas chlororaphis ss. aureofaciens grown oxically in complex medium. When letting the culture turn anoxic starting with ample O2 and mM NO3−, nitric oxide accumulated to toxic levels, impairing denitrification. With initially low O2 availability and μM NO3−, efficient conversion to N2O depended on the presence of mM ammonium. At higher cell densities, conversion efficiencies decreased, suggesting that the ability to induce balanced denitrification is growth dependent. Our results show that P. aureofaciens can induce denitrification successfully in the presence of μM NO3−, making anoxic pre-culturing with extraneous NO3− obsolete. We devise a simplified denitrifier method, which is fast and robust and does not require concentration of cells or removal of extraneous NO3− or N2O. Background NO3− in the medium can be removed as N2 by anoxic pre-incubation with P. denitrificans. Repeatability at natural abundance ranged from 0.1–0.3‰ for δ15N and 0.2–0.7‰ for δ18O. We successfully tested the method for various sample matrices including low pH (pH=4) and high salinity (0.25 - 1M KCl). Analyzing NO3− isotopes in the presence of highly 15N-labelled NH4+ or amino acids showed no cross-contamination. We conclude that the simplified denitrifier method is well suited for analyzing dual NO3− isotopes in a wide range of natural samples.

Collection Method for Isotopic Analysis of Gaseous Nitrous Acid.

The sources and chemistry of gaseous nitrous acid (HONO) in the environment are of great interest. HONO is a major source of atmospheric hydroxyl radical (OH), which impacts air quality and climate. HONO is also a major indoor pollutant that threatens human health. However, the large uncertainty of HONO sources and chemistry hinders an accurate prediction of the OH budget. Isotopic analysis of HONO may provide a tool for tracking the sources and chemistry of HONO. In this study, a modified annular denuder system (ADS) was developed to quantitatively capture HONO for offline nitrogen and oxygen isotopic analysis (δ15N and δ18O) using the denitrifier method. The ADS method was tested using laboratory generated HONO (400 ppbv to 1 ppmv) and validated by parallel HONO collection with a standard, basic impinger (BI) method. The ADS system shows complete capture of HONO without isotopic fractionation. The uncertainty (1σ) based on repeated measurements across the entire analytical procedure is 0.6‰ for δ15N and 0.5‰ for δ18O. The ADS method was also tested in roadside collections of ambient HONO (0.4-1.3 ppbv) for isotopic analysis and was found to be robust for low concentration collections over 3 and 12 h collection times. In order to ensure ability to use this method in the laboratory and in the field, storage conditions for the collected HONO samples were tested and samples can be stored with consistent δ15N and δ18O for 60 days. This method enables future work to utilize the isotopic composition of HONO for studying HONO chemical formation pathways, as well as atmospheric sources and chemistry.

Isotope analysis of ammonium and nitrate: A review on measured methods and their application

In the past several decades, a variety of methods have been developed for measuring the isotopic composition of ammonium (δ15N) and nitrate (δ15N and δ18O). This review summarized the advantages and disadvantages of these methods. Nowadays, the most popular method for measu-ring δ15N of ammonium is the combined hypobromite (BrO-) and hydroxylamine (NH2OH) me-thod, while for δ15N and δ18O of nitrate is the denitrifier method and the sodium azide (NaN3) me-thod. These methods convert NH4+ or NO3- into nitrous oxide (N2O) and measure its isotopic compositions, with higher analytical precision because of the lower background concentration of atmospheric N2O. Accordingly, these methods are suitable for the samples with lower N concentration, and normally require 10-60 nmol N. The development of new methods for measuring N isotopic composition has greatly stimulated the studies in nitrogen cycling worldwide.

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.

Novel Method for Nitrogen Isotopic Analysis of Soil-Emitted Nitric Oxide.

The global inventory of NOx (NOx = NO + NO2) emissions is poorly constrained, with a large portion of the uncertainty attributed to soil NO emissions that result from soil abiotic and microbial processes. While natural abundance stable N isotopes (δ15N) in various soil N-containing compounds have proven to be a robust tracer of soil N cycling, soil δ15N-NO is rarely quantified due to the measurement difficulties. Here, we present a new method that collects soil-emitted NO through NO conversion to NO2 in excess ozone (O3) and subsequent NO2 collection in a 20% triethanolamine (TEA) solution as nitrite and nitrate for δ15N analysis using the denitrifier method. The precision and accuracy of the method were quantified through repeated collection of an analytical NO tank and intercalibration with a modified EPA NOx collection method. The results show that the efficiency of NO conversion to NO2 and subsequent NO2 collection in the TEA solution is >98% under a variety of controlled conditions. The method precision (1σ) and accuracy across the entire analytical procedure are ±1.1‰. We report the first analyses of soil δ15N-NO (-59.8‰ to -23.4‰) from wetting-induced NO pulses at both laboratory and field scales that have important implications for understanding soil NO dynamics.

Modified Method for Trapping and Analyzing 15N in NO Released from Soils.

15N isotope tracing is an effective and direct approach to investigate sources of nitric oxide (NO) formed in soils. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone, making it impossible to directly measure 15N in NO. Various wet-chemical methods for conversion of NO to nitrite (NO2-) and nitrate (NO3-) have been proposed for 15N analysis in high-concentration NO sources, such as combustion processes. In contrast, NO concentrations in the soil surface-near air are usually small (ppbv-range), posing major challenges to conversion efficiency and blank correction. Here, we present a modified method in which NO is oxidized quantitatively to NO2 by chromium trioxide (CrO3), before conversion to NO2- and NO3- in an alkaline hydrogen peroxide (H2O2) solution. A denitrifier method was used to reduce NO2- and NO3- in the trapping solution quantitatively to nitrous oxide (N2O) for subsequent 15N analysis. NO trapping efficiencies of >85% were obtained with 50 ppb NO in a 0.5 L min-1 air stream bubbling through a solution of 1.2 M H2O2 and 0.5 M NaOH. In a laboratory test with distinct 15NO abundances, the overall precision was 0.29‰ (δ-values) for natural abundance NO and 0.13 atom % for labeled NO, suggesting that our method can be used for both natural abundance studies and 15N labeling experiments. In a soil incubation experiment with 15NH4NO3, NH415NO3, or Na15NO2 amendments, we found distinct 15N abundances in NO, indicating that our method is well suited to investigate NO sources in soils.

Technical considerations for the use of passive samplers to quantify the isotopic composition of NOx and NO2 using the denitrifier method

The isotopic composition of nitrogen oxides (NOx = NO + NO2) hold potential to trace emissions sources, as well as chemistry in the atmosphere. Passive samplers that collect NOx and NO2 as nitrite could provide a simple and inexpensive means by which to collect samples in a variety of environments, and we report here on several methodological considerations when using Ogawa passive collectors of NOx and NO2 for concentration analysis via colorimetric methods and isotopic analysis via the denitrifier method. Using Ogawa samplers, NOx and NO2 were collected in Providence, RI, USA (41.8°N, 71.4°W) with exposure times of seven, fourteen, twenty-one, and twenty-eight days during summer and winter in an area dominated by vehicle emissions, particularly diesel delivery trucks. Average values were −9.7 ± 0.7‰ for δ15N-NOx and −8.3 ± 0.9‰ for δ15N-NO2 based on duplicate seven, fourteen, twenty-one, and twenty-eight day exposures (n = 8) in summertime, and −11.9 ± 0.7‰ for δ15N-NOx and −6.4 ± 1.4‰ for δ15N-NO2 based on seven, fourteen, twenty-one, and twenty-eight day exposures (n = 7) in wintertime. The oxygen isotopic composition (δ18O, Δ17O) was also determined with averages of: 36.3 ± 2.4‰ for δ18O-NOx (n = 8), 50.5 ± 3.2‰ for δ18O-NO2 (n = 8), 6.2 ± 0.8‰ for Δ17O-NOx and 10.8 ± 0.6‰ for Δ17O-NO2 (n = 4) in summer; and 38.7 ± 2.9‰ for δ18O-NOx and 47.4 ± 1.2‰ for δ18O-NO2 (n = 7), 7.7 ± 1.5‰ for Δ17O-NO2 (n = 4) and 5.3 ± 1.2‰ for Δ17O-NOx (n = 5), in winter. Our isotopic results differ from previous studies that utilized passive samplers and suggest specific nitrogen and oxygen isotopic signatures associated with vehicular emissions. To collect sufficient sample for multiple isotopic analyses (e.g., 10 nmol N for δ15N and δ18O; 50 nmol N for Δ17O) the exposure time for the samplers, even in urban areas, is at least days. This necessarily results in collection of a mixture of local emissions sources, as well as background air that can represent multiple NOx sources. Thus, passive samplers cannot be used to diagnose isotopic signatures unless only exposed to a single emissions source. The oxygen isotopic composition of nitrite (the collection analyte) results determined via the denitrifier method must be corrected in comparison to nitrate isotopic reference materials, resulting in an increase of ∼25‰. Even with this correction, the oxygen isotopic results are lower than what would be expected for ozone-dominated atmospheric chemistry in an urban area, and the oxygen isotopic results for NOx were consistently lower than that found for NO2. We suggest that the oxygen isotopic composition of NO and NO2 were modified upon capture and conversion to nitrite on the passive sampler pads, and does not directly reflect abundances in the atmosphere.

Updates to instrumentation and protocols for isotopic analysis of nitrate by the denitrifier method.

The denitrifier method allows for highly sensitive measurement of the (15) N/(14) N (δ(15) N value) and (18) O/(16) O (δ(18) O value) of nitrate dissolved in natural waters and for highly sensitive δ(15) N measurement of other N forms (e.g., organic N) that can be converted into nitrate. Here, updates to instrumentation and protocols are described, and improvements in data quality are demonstrated. A 'heart cut' of the N2 O was implemented in the extraction system to (1) minimize introduction of contaminants into the mass spectrometer, reducing isotopic drift and (2) decrease the fraction of sample lost at the open split to improve sensitivity. Referencing protocols were updated, including a correction scheme for a weak dependence of nitrate δ(18) O values on nitrate concentration. Analyses of samples from the US GEOTRACES North Atlantic Program and of reference solutions from the same analysis batches were used to characterize performance. The drift is typically <0.1‰ for both δ(15) N and δ(18) O values. Within-batch and inter-batch replication yields 1 standard deviation (SD) of ≤0.06‰ for δ(15) N values and ≤0.14‰ for δ(18) O values down to 5 μM nitrate and ≤0.08‰ and ≤0.23‰ at 2 and 1 μM. The blank is typically 0.06 nmol N, 0.3% of the N in a 20 nmol N sample. Differences between reference materials in seawater are indistinguishable from reported differences for δ(15) N values, with a contraction for δ(18) O values of ≤5%. The new instrumentation and protocols yield nitrate isotopic data with external precision of ≤0.1‰ for large sample sets such as those derived from oceanographic sections. Further study should investigate the causes of (1) the weak dependence of nitrate δ(18) O values on nitrate concentration and (2) the inter-batch variation in the δ(18) O contraction (due mostly to oxygen atom exchange with water). Nevertheless, comprehensive correction schemes are in place for the measurement of both the δ(15) N and δ(18) O values of nitrate. Copyright © 2016 John Wiley & Sons, Ltd.

Characteristics of the $$\delta ^{15} N_{NO_3 } $$ δ 15 N N O 3 distribution and its drivers in the Changjiang River estuary and adjacent waters

In this study, we conducted investigations in the Changjiang (Yangtze) River estuary and adjacent waters (CREAW) in June and November of 2014. We collected water samples from different depths to analyze the nitrogen isotopic compositions of nitrate, nutrient concentrations (including inorganic N, P, and Si), and other physical and biological parameters, along with the vertical distribution and seasonal variations of these parameters. The compositions of nitrogen isotope in nitrate were measured with the denitrifier method. Results show that the Changjiang River diluted water (CDW) was the main factor affecting the shallow waters (above 10 m) of the CREAW, and CDW tended to influence the northern areas in June and the southern areas in November. \(\delta ^{15} N_{NO_3 } \) values in CDW ranged from 3.21‰–3.55‰. In contrast, the deep waters (below 30 m) were affected by the subsurface water of the Kuroshio Current, which intruded into the waters near 31°N in June. The \(\delta ^{15} N_{NO_3 } \) values of these waters were 6.03‰–7.6‰, slightly higher than the values of the Kuroshio Current. Nitrate assimilation by phytoplankton in the shallow waters of the study area varied seasonally. Because of the favorable temperature and nutrient conditions in June, abundant phytoplankton growth resulted in harmful algae blooms (HABs). Therefore, nitrate assimilation was strong in June and weak in November. The \(\delta ^{15} N_{NO_3 } \) fractionations caused by assimilation of phytoplankton were 4.57‰ and 4.41‰ in the shallow waters in June and November, respectively. These results are consistent with previous laboratory cultures and in situ investigations. Nitrification processes were observed in some deep waters of the study area, and they were more apparent in November than in June. The fractionation values of nitrification ranged from 24‰–25‰, which agrees with results for Nitrosospira tenuis reported by previous studies.

Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate.

The dynamics of nitrate (NO−3), a major nitrogen (N) source for natural plants, has been studied mostly through experimental N addition, enzymatic assay, isotope labeling, and genetic expression. However, artificial N supply may not reasonably reflect the N strategies in natural plants because NO−3 uptake and reduction may vary with external N availability. Due to abrupt application and short operation time, field N addition, and isotopic labeling hinder the elucidation of in situ NO−3-use mechanisms. The concentration and natural isotopes of tissue NO−3 can offer insights into the plant NO−3 sources and dynamics in a natural context. Furthermore, they facilitate the exploration of plant NO−3 utilization and its interaction with N pollution and ecosystem N cycles without disturbing the N pools. The present study was conducted to review the application of the denitrifier method for concentration and isotope analyses of NO−3 in plants. Moreover, this study highlights the utility and advantages of these parameters in interpreting NO−3 sources and dynamics in natural plants. We summarize the major sources and reduction processes of NO−3 in plants, and discuss the implications of NO−3 concentration in plant tissues based on existing data. Particular emphasis was laid on the regulation of soil NO−3 and plant ecophysiological functions in interspecific and intra-plant NO−3 variations. We introduce N and O isotope systematics of NO−3 in plants and discuss the principles and feasibilities of using isotopic enrichment and fractionation factors; the correlation between concentration and isotopes (N and O isotopes: δ18O and Δ17O); and isotope mass-balance calculations to constrain sources and reduction of NO−3 in possible scenarios for natural plants are deliberated. Finally, we offer a preliminary framework of intraplant δ18O-NO−3 variation, and summarize the uncertainties in using tissue NO−3 parameters to interpret plant NO−3 utilization.

Correcting for background nitrate contamination in KCl-extracted samples during isotopic analysis of oxygen and nitrogen by the denitrifier method

Previous research has shown that the denitrifying bacteria Pseudomonas chlororaphis ssp. aureofaciens (P. aureofaciens) can be used to measure the δ(15)N and δ(18)O values of extracted soil nitrate (NO3(-)) by isotope ratio mass spectrometry. We discovered that N2O production from reference blanks made in 1 M KCl increased relative to blanks made of deionized water (DIW). Further investigation showed that isotopic standards made in KCl yielded δ(15)N and δ(18)O values different from the standards prepared in DIW. Three grades of crystalline KCl were dissolved in DIW to create solutions of increasing molarity (0.1 M to 2 M), which were added to P. aureofaciens broth and measured as blanks. Reference standards USGS-32, USGS-34, and USGS-35 were then dissolved in a range of KCl concentrations to measure isotopic responses to changing KCl molarity. Reference blanks and standards created in DIW were analyzed as controls to measure the impact of KCl on the δ(15)N and δ(18)O values. The amount of N2O in the KCl blanks increased linearly with increasing molarity, but at different rates for each KCl grade. The isotopic values of the reference standards measured in KCl were systematically different from those measured in DIW, suggesting contamination by background NO3(-) in the KCl reagents. However, we also noted reduced conversion of NO3(-) into N2O as the KCl molarity increased, suggesting there is a physiological response of P. aureofaciens to KCl. There is a small amount of NO3(-) present in crystalline KCl, which can bias isotopic measurement of NO3(-) at low sample concentrations. This can be minimized by making standards and blanks in the same KCl as is used in samples, diluting all samples and standards to the appropriate NO3(-) concentration using matched KCl solutions, and adding samples and standards to the broth at a constant volume to standardize the KCl molarity in the reaction vial.

On‐line triple oxygen isotope analysis of nitrous oxide using decomposition by microwave discharge

The oxygen isotope anomaly, Δ(17)O, of N2O and nitrate is useful to elucidate nitrogen oxide dynamics. The previously developed method for Δ(17)O measurement presents difficulty in maintaining optimal conditions of the gold tube for thermal decomposition of N2O to O2 and the Δ(17)O value is also sample size dependent. Trace amounts (5-40 nmol) of N2O were decomposed quantitatively to O2 in a quartz tube by microwave discharge. The O2 was purified using gas chromatography. Triple oxygen isotopes were measured using isotope ratio mass spectrometry. Each step was connected online and was applied to the analysis of nitrate in precipitation samples collected in Yokohama, Japan. Precision (1σ) of Δ(17)O analysis was better than 0.26‰ when more than 20 nmol of N2O with a small Δ(17)O value (approx. 1‰) was measured. It was better than 0.76‰ when more than 60 nmol of nitrate was converted into N2O using the denitrifier method and then measured on the developed system. The obtained Δ(17)O values in precipitation samples (14.5-26.4‰) agreed with findings from previous studies. A novel on-line analytical method was developed to measure the triple oxygen isotopes of N2O using microwave discharge to decompose N2O. This easy-to-use method is free from conditioning of reaction devices, and is applicable to molecules other than N2O such as NO and NO2.

Characterizing the isotopic composition of atmospheric ammonia emission sources using passive samplers and a combined oxidation‐bacterial denitrifier approach

Ammonia (NH3) emissions are a substantial source of nitrogen pollution to sensitive terrestrial, aquatic, and marine ecosystems and dependable quantification of NH3 sources is of growing importance due to recently observed increases in ammonium (NH4(+)) deposition rates. While determination of the nitrogen isotopic composition of NH3 (δ(15)N-NH3) can aid in the quantification of NH3 emission sources, existing methods have precluded a comprehensive assessment of δ(15)N-NH3 values from major emission sources. We report an approach for the δ(15)N-NH4(+) analysis of low concentration NH4(+) samples that couples the bromate oxidation of NH4(+) to NO2(-) and the microbial denitrifier method for δ(15)N-NO2(-) analysis. This approach reduces the required sample mass by 50-fold relative to standard elemental analysis (EA) procedures, is capable of high throughput, and eliminates toxic chemicals used in a prior method for the analysis of low concentration samples. Using this approach, we report a comprehensive inventory of δ(15)N-NH3 values from major emission sources (including livestock operations, marine sources, vehicles, fertilized cornfields) collected using passive sampling devices. The δ(15)N-NH4(+) analysis approach developed has a standard deviation of ±0.7‰ and was used to analyze passively collected NH3 emissions with a wide range of ambient NH3 concentrations (0.2 to 165.6 µg/m(3)). The δ(15)N-NH3 values reveal that the NH3 emitted from volatilized livestock waste and fertilizer has relatively low δ(15)N values (-56 to -23‰), allowing it to be differentiated from NH3 emitted from fossil fuel sources that are characterized by relatively high δ(15)N values (-15 to +2‰). The isotopic source signatures presented in this emission inventory can be used as an additional tool in identifying NH3 emission sources and tracing their transport across localized landscapes and regions. The insight into the transport of NH3 emissions provided by isotopic investigation is an important step in devising strategies to reduce future NH3 emissions, a mounting concern for air quality scientists, epidemiologists, and policy-makers.