A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater

Sigman, Daniel M.; Casciotti, Karen L.; Andréani, M.; Barford, Carol; Galanter, M.; Böhlke, John K.

doi:10.1021/ac010088e

Cited by 1,673 publications

(1,332 citation statements)

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“…Since N is balanced during denitrification (2NO 3 -→ 2NO 2 -→2NO → N 2 O), the isotopes of the N molecules within NO 3 -are more likely to suffer from fractionation due to incomplete conversion into N 2 O. 14 NO 3 -requires less kinetic energy during the denitrification reaction and would be expected to produce a final product with an isotopic signature less enriched in 15 N if the entire pool was not denitrified. Only one in six O atoms is conserved during denitrification by P. aureofaciens, suggesting any fractionation of isotopologues of O in NO 3 -may be buffered by O from the sample solution.…”

Section: Methods For Correcting For the Kcl Matrixmentioning

confidence: 99%

“…If enriched isotopic tracers are going to be used in conjunction with KCl extracts, as in 15 N tracer studies, it will be imperative to determine the effects of KCl on higher δ 15 N values.…”

Section: Methods For Correcting For the Kcl Matrixmentioning

confidence: 99%

“…[14,15] The bacteria P. aureofaciens (ATCC# 13985) lack nitrous oxide reductase activity and therefore produce N 2 O gas as a final product of denitrification which allows the isotopic ratios of N and O to be simultaneously determined. Briefly, the bacteria were grown in a modified soy broth solution for 7-10 days, concentrated, and sealed in a 20 mL headspace vial and sparged with helium gas for 2 h. Samples were then added to the reaction vials, which were inverted overnight to allow for complete conversion of NO 3 -while minimizing N 2 O loss.…”

Section: Experimental Sample Analysismentioning

confidence: 99%

“…[12,13] Pseudomonas aureofaciens was initially used in seawater to study nitrogen cycling in marine ecosystems, [14,15] suggesting that high salt concentrations should not affect the method. Standard reagent grade KCl has a label purity of 99-100.5% KCl.…”

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

Bell

Sickman

2014

Rapid Commun. Mass Spectrom.

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RATIONALE: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 (NO 3 -) by isotope ratio mass spectrometry. We discovered that N 2 O 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. METHODS: 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. RESULTS: The amount of N 2 O 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 NO 3 -in the KCl reagents. However, we also noted reduced conversion of NO 3 -into N 2 O as the KCl molarity increased, suggesting there is a physiological response of P. aureofaciens to KCl. CONCLUSIONS: There is a small amount of NO 3 -present in crystalline KCl, which can bias isotopic measurement of NO 3 -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 NO 3 -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. Copyright © 2014 John Wiley & Sons, Ltd.Mass spectrometry of the stable isotopes of oxygen (O) and nitrogen (N) of nitrate (NO 3 -) through bacterial denitrification has advanced our understanding of reactive nitrogen in terrestrial, freshwater and marine ecosystems. [1][2][3][4] Anthropogenic and naturally produced NO 3 -have distinct isotopic signatures of both δ 15 N and δ 18 O. [5] Mixing models based on these values and background NO 3 -concentrations can help identify the sources and transformation of nitrogen within a local system. [4,6,7] Recent studies have analyzed potassium chloride (KCl) solutions containing NO 3 -extracted from soil samples using the denitrifying bacteria Pseudomonas chlororaphis ssp. aureofaciens (P. aureofaciens) without identifying any isotopic artifacts, [7][8][9] although earlier work by Stark and Hart [10] measured NO 3 -contamination by 2 M KCl while performing diffusion digests. While conducting similar analyses on KCl-extracted soils in southern California, we noted an increase in N 2 O production in blanks prepared in KCl and worrying shifts in th...

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Section: Methods For Correcting For the Kcl Matrixmentioning

confidence: 99%

Section: Methods For Correcting For the Kcl Matrixmentioning

confidence: 99%

Section: Experimental Sample Analysismentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Bell

Sickman

2014

Rapid Commun. Mass Spectrom.

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“…이를 극복하기 위해서 질소의 안정동위원소 추 적자( 15 N)를 이용하여 각 과정을 측정하는 실험방법이 발달하였다 (Dugdale and Goering, 1967;Blackburn, 1979;Caperon et al, 1979;Lin et al, 2011 (Sigman, 2001;Brandes et al, 2007). 최근 들어 해양환경에서 DNRA (dissimilatory nitrate reduction to ammonium) 나 ANAMMOX (anaerobic ammonium oxidation)와 같은 질소변환 과정이 새롭게 발견되거나 그 중요성이 재평가 받음으로써 암모 늄 안정동위원소 측정의 필요성은 점차 증가하고 있다 (Brandes et al, 2007;Thamdrup, 2012).…”

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Stable Isotope Measurement of Ammonium Using HPLC-RTS (high performance liquid chromatography-retention time shift)

Lee

Gardner³

2013

The Sea

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Despite the usefulness of nitrogen isotope tracer experiments in nitrogen cycling studies, there are not such many measurement data mainly due to the difficulties in analytical methods. Although Gardner et al. (1996) developed a relatively simple and accurate method that can measure ammonium isotope using HPLC and used it widely in various N dynamics studies, the technique was not adopted to other laboratories. An HPLC-RTS system using updated HPLC pumps that can perform the same measurements as that of Gardner et al. (1996) was built. The result of standard sample showed linear increase of RTS with the 15 N proportions. Centroid retention times calculated with Matlab ® program enhanced the linearity of the response. In a sea water incubation experiment spiked with 15 NH 4 + , the uptake and regeneration of ammonium could be separately estimated using the temporal change of 15 N/ 14 N.

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Evidence of parallel denitrification and nitrite oxidation in the ODZ of the Arabian Sea from paired stable isotopes of nitrate and nitrite

Gaye

Nagel

Dähnke³

et al. 2013

Global Biogeochemical Cycles

102

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[1] The Arabian Sea is a major oceanic nitrogen sink, and its oxygen-deficient zone (ODZ) extends from 150 m to 1200 m water depth. To identify the dominant transformation processes of reactive nitrogen and to quantify the amounts of nitrogen turned over in the different reactions of the nitrogen cycle, we use paired data on stable isotope ratios of nitrogen and oxygen in nitrate and nitrite measured at four near-coastal and five open ocean stations in the Arabian Sea. We find significant nitrate reduction and denitrification between 100 m and 400 m in the open Arabian Sea, which are most intense in the eastern and northern part of the basin, and estimate that about 50% of initial nitrate is being reduced either to dinitrogen gas (denitrification) or to nitrite (nitrate reduction) in the core zone of denitrification. Nitrite accumulates in concentrations above 4 μM in the water column of the eastern and northern Arabian Sea. Large differences in isotopic ratios of nitrate and nitrite and a decoupling of their stable nitrogen and oxygen isotopes can be explained by the reoxidation of nitrite. The observed decoupling of the paired isotopes may be due to the exchange of oxygen of nitrite with oxygen from ambient water. In agreement with model estimates from the literature, about 25% of the nitrate initially reduced to nitrite is returned to the nitrate pool by nitrification in the upper and lower denitrification layer while 40% is denitrified.Citation: Gaye, B., B. Nagel, K. Da¨hnke, T. Rixen, and K.-C. Emeis (2013), Evidence of parallel denitrification and nitrite oxidation in the ODZ of the Arabian Sea from paired stable isotopes of nitrate and nitrite, Global Biogeochem. Cycles, 27,

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A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater

Cited by 1,673 publications

References 31 publications

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

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

Stable Isotope Measurement of Ammonium Using HPLC-RTS (high performance liquid chromatography-retention time shift)

Evidence of parallel denitrification and nitrite oxidation in the ODZ of the Arabian Sea from paired stable isotopes of nitrate and nitrite

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