Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method

Casciotti, Karen L.; Sigman, Daniel M.; Hastings, Meredith G.; Böhlke, John K.; Hilkert, Andreas

doi:10.1021/ac020113w

Cited by 1,312 publications

(1,339 citation statements)

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“…Water samples and soil extracts were analyzed for ammonium (NH 4 + ) and NO 3 − concentrations, as well as for N and O isotopes of NO 3 − . The denitrifier method (33,34) was used to determine δ 15 N and δ 18 O of NO 3 − in all samples, excluding stream waters collected from JFL forests in 2012 and soil extracts from JFL and DHS forest soil profiles collected in 2013, which were determined by the chemical conversion method (35). The Δ 17 O of NO 3 − of selected precipitation and stream water samples was determined by combining bacterial reduction (i.e., denitrifier method) and the thermal decomposition method (36).…”

Section: Methodsmentioning

confidence: 99%

Microbial denitrification dominates nitrate losses from forest ecosystems

Fang

Koba

Makabe

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

188

124

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Denitrification removes fixed nitrogen (N) from the biosphere, thereby restricting the availability of this key limiting nutrient for terrestrial plant productivity. This microbially driven process has been exceedingly difficult to measure, however, given the large background of nitrogen gas (N 2 ) in the atmosphere and vexing scaling issues associated with heterogeneous soil systems. Here, we use natural abundance of N and oxygen isotopes in nitrate (NO 3 − ) to examine dentrification rates across six forest sites in southern China and central Japan, which span temperate to tropical climates, as well as various stand ages and N deposition regimes. Our multiple stable isotope approach across soil to watershed scales shows that traditional techniques underestimate terrestrial denitrification fluxes by up to 98%, with annual losses of 5.6-30.1 kg of N per hectare via this gaseous pathway. These N export fluxes are up to sixfold higher than NO 3 − leaching, pointing to widespread dominance of denitrification in removing NO 3 − from forest ecosystems across a range of conditions. Further, we report that the loss of NO 3 − to denitrification decreased in comparison to leaching pathways in sites with the highest rates of anthropogenic N deposition.denitrification | nitrate isotopes | nitrogen cycling | forested watersheds

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Section: Methodsmentioning

confidence: 99%

Microbial denitrification dominates nitrate losses from forest ecosystems

Fang

Koba

Makabe

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

188

124

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“…[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%

“…These reference standards were chosen because they represent the two endmembers of our isotope calibration curve and they do not contain any excess 17 O isotope, which can introduce error into δ 15 N measurements of NO 3 -. [14] The reference samples were prepared in DIW as well as in a 1 M KCl solution made with Fisher-Reagent KCl, and then precisely mixed to create a gradient of isotope working standards in both DIW and KCl matrices. All standards were diluted to a concentration of 1 μg NO 3 -mL -1 (molarity held constant) and were injected into helium-sparged P. aureofaciens vials in 3 mL aliquots.…”

Section: Experiments 4: Effect Of Kcl Molarity On Isotope Fractionationmentioning

confidence: 99%

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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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“…N 2 O is stripped from the sample vial using helium as carrier gas, purified, and analyzed for its N and O isotopic composition. The determination of the 18 O/ 16 O of nitrate requires the correction for fractionation during O atom removal and exchange with O atoms from water during the reduction of nitrate to nitrous oxide [Casciotti et al, 2002]. O isotope exchange was always <3% (for P. aureofaciens).…”

Section: Nitrate Isotope Analysesmentioning

confidence: 99%

Origin of the deep Bering Sea nitrate deficit: Constraints from the nitrogen and oxygen isotopic composition of water column nitrate and benthic nitrate fluxes

Lehmann¹,

Sigman

McCorkle

et al. 2005

Global Biogeochemical Cycles

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[1] On the basis of the normalization to phosphate, a significant amount of nitrate is missing from the deep Bering Sea (BS). Benthic denitrification has been suggested previously to be the dominant cause for the BS nitrate deficit. We measured water column nitrate 15 N/ 14 N and 18 O/ 16 O as integrative tracers of microbial denitrification, together with pore water-derived benthic nitrate fluxes in the deep BS basin, in order to gain new constraints on the mechanism of fixed nitrogen loss in the BS. The lack of any nitrate isotope enrichment into the deep part of the BS supports the benthic denitrification hypothesis. On the basis of the nitrate deficit in the water column with respect to the adjacent North Pacific and a radiocarbon-derived ventilation age of $50 years, we calculate an average deep BS (>2000 m water depth) sedimentary denitrification rate of $230 mmol N m À2 d À1 (or 1.27 Tg N yr À1 ), more than 3 times higher than high-end estimates of the average global sedimentary denitrification rate for the same depth interval. Pore water-derived estimates of benthic denitrification were variable, and uncertainties in estimates were large. A very high denitrification rate measured from the base of the steep northern slope of the basin suggests that the elevated average sedimentary denitrification rate of the deep Bering calculated from the nitrate deficit is driven by organic matter supply to the base of the continental slope, owing to a combination of high primary productivity in the surface waters along the shelf break and efficient down-slope sediment focusing along the steep continental slopes that characterize the BS.

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Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method

Cited by 1,312 publications

References 30 publications

Microbial denitrification dominates nitrate losses from forest ecosystems

Microbial denitrification dominates nitrate losses from forest ecosystems

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

Origin of the deep Bering Sea nitrate deficit: Constraints from the nitrogen and oxygen isotopic composition of water column nitrate and benthic nitrate fluxes

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