Dissolved nitrate isotope analysis (15N & 18O) by “denitrifier
method”
Isotope Science Laboratory – Applied Geochemistry Group
Geoscience Department
University of Calgary
Overview:
A method used to determine the N and O isotopic composition of dissolved nitrate in
water. The ‘denitrifier method’ is based on bacterial reduction of nitrate (NO3-) to nitrous
oxide (N2O) via a bacterium that lacks nitrous oxide reductase, so further reduction to
N2 does not occur. This enables simultaneous determination of both δ15N and δ18O
values of the sample NO3- by measuring δ15N and δ18O values of the produced N2O.
The bacterial strain Pseudomonas aureofaciens (ATCC# 13985) is used to reduce NO3to N2O.
Extraction:
The system is comprised of a home made, 24 vial autosampler interfaced to a HP 6890
gas chromatogram with PreCon® device interfaced to a Finnigan Mat Delta+XL mass
spectrometer.
Briefly, the bacteria must be grown in a special tryptic soy broth prepared 1 week in
advance. After 7 days of growth, the bacteria will have exhausted the nutrients in the
broth and be of sufficient population for use. The bacteria are then harvested and
subdivided into 24 separate sample vials. The vials are flushed with inert N 2 (UHP 4.8)
for ~5 hours to lower the atmospheric blank before aliquots of sample are injected. The
target amount of NO3- depends on the sensitivity of the mass spec, but is generally in
the range of 20 to 50 nmole [NO3-]. After ~16 hours the bacteria are lysed by injecting
0.1-0.2 mL of 10 N NaOH and the vials mounted in a 24 slot autosampler for analysis.
The instrumental technique is briefly as follows: the headspace of the vial is flushed
with Helium (UHP 5.0) at a flow rate of 20 ml/min. The N2O + carrier then pass through
a series of traps (alcohol slush Nafion® (PermaPure p/n MD 070)  Ascarite) to
remove excess moisture and CO2 before being cryofocussed by the PreCon device.
The N2O once released from the PreCon passes through a GC (HP Plot-U, 30m x
320m @ room temperature) to separate N2O from any remaining CO2, then into the
open split interface (located in a GCC-III® device) which ‘leaks’ the gas into the mass
spectrometer. The 15N and δ18O values are calculated by the instrument software
(ISODAT 2.63).
Data correction:
Ion currents of masses 44, 45 and 46 are measured simultaneously and the
15/14Nitrogen and 18/16Oxygen ratios of the sample are compared to that of a workingreference N2O gas (4.8, ‘Semiconductor Process Gas’, Praxair Air, Canada). Raw data
is corrected to the International N2 and VSMOW scales by normalization with reference
materials analyzed in the same sequence as the samples (the “IT” principal described in
Werner & Brand, 2001).
Reference materials:
Identifier
15N (‰)
18O (‰)
IAEA NO3
USGS 34
USGS 35
+4.69 ± 0.2
-1.80 ± 0.2
+2.70 ± 0.2
+25.6 ± 0.6
-27.9 ± 0.6
+57.5 ± 0.8
Accuracy and Precision:
Accuracy and precision of 15N determinations based on a long term record of analyses
of ISL in-house reference (ISL-KNO3) is 0.3 permil (n=108)
Accuracy and precision of 18O determinations based on a long term record of analyses
of ISL in-house reference (ISL-KNO3) is 0.7 permil (n=109)
Misc. comments:
Dissolved nitrate samples must be submitted with [NO3-] concentration data.
References:
Bohlke, J.K., Mroczkowski, S.J. and T.B. Coplen, 2003, Oxygen isotopes in nitrate: new
reference materials for 18O: 17O: 16O measurements and observations on nitratewater equilibration, Rapid Commun.Mass Spectrom. 2003; 17; 1835-1846
Casciotti, K.L., D.M. Sigman, M. Galanter Hastings, J.K. Böhlke, and A. Hilkert, 2002,
Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater
using the denitrifier method. An. Chem. 74: 4905-4912.
Rock, L. , and Ellert, B., 2007, Nitrogen-15 and Oxygen-18 Natural Abundance of
Potassium Chloride Extractable Soil Nitrate Using the Denitrifier Method, Soil Science
Society of America Journal, vol. 71 (2), 355-361.
Sigman, D.M., K.L. Casciotti, M. Andreani, C. Barford, M. Galanter, and J.K. Böhlke.
2001. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and
freshwater. Anal. Chem. 73: 4145-4153.
Silva, S.R., C. Kendall, D.H. Wilkison, A.C. Ziegler, C.C.Y. Chang, and R.J. Avanzino.
2000. A new method for collection of nitrate from fresh water and the analysis of
nitrogen and oxygen isotope ratios. J. Hydrol. 228: 22-36.
R.A. Werner and W.A. Brand, Referencing strategies and techniques in stable isotope
ratio analysis, Rapid Communications in Mass Spectrometry, 2001: 15: 501-519