Microbes Environ. Vol. 26, No. 1, 46–53, 2011
http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10159
Analytical Techniques for Quantifying 15N/14N of Nitrate, Nitrite, Total
Dissolved Nitrogen and Ammonium in Environmental Samples Using a
Gas Chromatograph Equipped with a Quadrupole Mass Spectrometer
KAZUO ISOBE1, YUICHI SUWA2, JUNKO IKUTANI3, MEGUMI KUROIWA3, TOMOKO MAKITA3, YU TAKEBAYASHI3,
MUNEOKI YOH3, SHIGETO OTSUKA1, KEISHI SENOO1, MASAYUKI OHMORI2, and KEISUKE KOBA3*
1Department
of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of
Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan; 2Department of Biological Sciences, Faculty of Science and
Engineering, Chuo University, 1–13–27 Kasuga, Bunkyo-ku, Tokyo 112–8551, Japan; and 3Graduate School of
Agriculture, Tokyo University of Agriculture and Technology, 3–5–8 Saiwaicho, Fuchu, Tokyo 183–8509, Japan
(Received August 18, 2010—Accepted October 23, 2010—Published online November 30, 2010)
The measurement of 15N concentrations in environmental samples requires sophisticated pretreatment devices and
expensive isotope-ratio mass spectrometry (IRMS). This report describes the use of a gas chromatograph equipped
with a quadrupole-type mass spectrometer (GC/MS) to measure 15N concentrations of ammonium, nitrate, nitrite,
and total dissolved nitrogen (TDN) in distilled water, a 2 M KCl solution and a 0.5 M K2SO4 solution. The system
measures nitrous oxide (N2O) that is ultimately converted from the target N compound, requiring no special apparatus
such as a purge-and-trap pretreatment device. It uses a denitrifier lacking N2O reductase, which produces N2O from
nitrate. Persulfate oxidation was applied to convert TDN to nitrate, while additional pretreatment with ammonia
diffusion was required for ammonium prior to the persulfate oxidation. Up to 100 samples can be measured daily using
the system. We can generally run 15N measurements with only 1–10 mL of sample for each chemical species of N,
a volume 1/10–1/100 times smaller than the amount necessary for conventional methods. Our method is useful for
measuring 15N with GC/MS, offering greater convenience than IRMS.
Key words: 15N, GC/MS, denitrifier, soil extract
The great interest in nitrogen (N) cycling in aquatic and
terrestrial ecosystems has been stimulating the development
of techniques to study microbial processes. For a better
understanding of N cycling, the linkages among microbial
composition, their ecological function, and N dynamics in
ecosystems need to be clarified. For this, analytical techniques based upon the principles of molecular biology or
geochemistry for the study of N cycling must be improved
greatly (39). The nitrogen isotope 15N has been frequently
used (20) to estimate gross rates of N transformation such as
ammonia oxidation (4, 6, 15), anaerobic ammonia oxidation
(2, 12, 22, 38), and denitrification (1, 5, 18, 37) in environmental and laboratory (incubated) samples. The high turnover of N caused by high biological demand indicates that
the net changes in the concentration of the target N compound cannot provide an accurate measure of the production
or consumption rate because production and consumption of
the target N compound occur simultaneously. Consequently,
to elucidate the N cycle and to identify microorganisms
which might carry out a component of the reaction in N
transformation, measurement of the gross rate of the target
process is necessary. Then, addition of 15N and tracing the
added 15N in the pool of the target compound is crucial to
separate the production and consumption of the target N
compound and thereby calculate its gross production or
consumption rate.
* Corresponding author. E-mail: [email protected];
Tel: +81–42–367–5951; Fax: +81–42–367–5951.
Despite the importance of determining gross N transformation rates, the analytical procedures for 15N measurements
are still labor-intensive. They require sophisticated instruments and large samples. In general, the 15N/14N ratio is
measured using isotope-ratio mass spectrometry (IRMS)
after conversion of the target N compound such as ammonium (NH4+), nitrate (NO3−), nitrite (NO2−), or total dissolved
N (TDN) to a gaseous N-species (normally N2) using an
elemental analyzer connected with an IRMS system (EAIRMS). The high background concentration of N2 in the
atmosphere precludes measurement of 15N with a small
amount of N (e.g. <60 µg-N) by EA-IRMS. Moreover, specially modified analytical methods are necessary, especially
when soil and sediment samples are examined, because
exchangeable N compounds must be extracted from the soil
using near-saturated salt solutions such as 2 M KCl and 0.5
M K2SO4 solutions (19). Consequently, high-salinity soil or
sediment extracts must be analyzed.
Here we present a novel method of determining the 15N/
14N in biologically available N compounds such as NH +,
4
NO3−, NO2− and TDN. Using several chemical and microbial
pretreatments, each N compound is ultimately converted to
nitrous oxide (N2O), whose quantification cannot be affected
by atmospheric gases because its presence in air is negligible. The basic concept behind this method is to measure
15N O converted from NO −, NO −, NH +, and TDN in several
2
3
2
4
sample matrices (2 M KCl, 0.5 M K2SO4, and fresh water)
precisely using GC/MS (Fig. 1). Consequently, the amount
of sample used for an analysis can be reduced to 1/100 of
Analytical Techniques for 15N with GC/MS
47
were used. Chemicals were purchased from Wako Pure Chemical
Industries (Osaka, Japan) unless otherwise specified. KCl, NaCl,
MgO, and K2SO4 were each combusted at 450°C for 4 h before use
to reduce N contamination.
Diffusion method to recover NH4+
We recovered NH4+ from the 2 M KCl solution, distilled water
and freshwater as (NH4)2SO4 according to processes described by
Sigman et al. (31), Holmes et al. (16) and Koba et al. (21). We
used a “Teflon envelope” to capture ammonia gas produced during
diffusion (21) that sandwiched the acidified glass fiber filter (GF/D,
1 cm diameter, muffled at 450°C for 4 h; Whatman Int., Maidstone,
UK) with PTFE tape. A 10-mL aliquot of the standard solution
was placed in a high-density polyethylene bottle (NL2114–0002,
Nalgene; Thermo Fisher Scientific, Yokohama, Japan). Then 0.5 g
of combusted NaCl was added to an aliquot of the freshwater
standard, although no NaCl was added to the 2 M KCl extract. After
a PTFE envelope was placed in a bottle, 0.03 g of combusted
MgO was added, and the bottle was immediately closed firmly and
incubated at room temperature for 14 days. At the end of the
incubation period, the PTFE envelop was removed with tweezers,
washed with distilled water, and placed in a scintillation vial (03–
337–23A; Thermo Fisher Scientific). Each vial was placed in a
desiccator with silica gel and concentrated H2SO4 in an open container for 3 days to remove any trace of ammonia. The vials were
later removed from the desiccators and sealed with caps for storage.
Fig. 1. Flowchart showing the analytical procedures for different N
forms modified from (21). When the salinity of the sample is low, NaCl
must be added before the ammonia diffusion process and reduction of
NO2− to N2O by azide.
that required for a conventional N2 analysis. Moreover, our
system has several other advantages over conventional analytical procedures: (a) it uses a gas chromatograph equipped
with a quadrupole-type mass spectrometer (GC/MS), which
is much less expensive and more readily available in microbiology laboratories than EA-IRMS is; (b) it has no gas
concentration step using chemical traps and cryofocusing
units; (c) a simple headspace technique is applied instead of
a sophisticated purge-and-trap technique; and (d) it enables
the simultaneous measurement of 15N/14N ratios and NO3−
and TDN concentrations.
Materials and Methods
Solutions and range of standards
We evaluated the applicability of our analytical system to standard solutions, N compounds dissolved in distilled water, a 2 M
KCl solution and a 0.5 M K2SO4 solution. Therefore, we prepared
standard solutions for calculating the amounts of 15N in freshwater
samples, soil extracts and soil microbial biomass. Generally, a 2 M
KCl solution is used to extract soil N (19), although 0.5 M K2SO4 is
used to measure soil microbial biomass N (7). Therefore, we
prepared separate standard solutions with distilled water, 2 M KCl,
and 0.5 M K2SO4 as the respective proxies for freshwater samples,
soil extracts, and soil extracts for soil microbial biomass. Measurements not only of blanks but also standard solutions with different
15N atom% values as well as different concentrations were done.
It is worth noting that 2 M KCl and 0.5 M K2SO4 introduce a
considerable amount of N. We typically prepared several standard
solutions for 15N with 0.37, 10.3, 20.2, 49.8, and 99.3 15N atom%,
and concentrations of 0–100 or 1000 µM-N in either 2 M KCl, 0.5
M K2SO4, or distilled water.
To prepare the standard solutions for 15N, 15N-labeled compounds
(NH4Cl, NaNO2, KNO3 and glycine, 99.3 15N atom%; Isotec, USA)
Persulfate oxidation of NH4+ and TDN to form NO3−
The PTFE envelope entrapping the (NH4)2SO4 was carefully
opened using cleaned tweezers. The glass fiber filter placed in
the envelope was transferred to a 14-mL borosilicate glass test
tube (71–063–006; Iwaki, AGC Techno Glass, Chiba, Japan) with
a Teflon-lined screw cap (71–028–004; Iwaki). Next, 2 mL of distilled water was pipetted into the tube, which was then capped and
shaken with a reciprocal shaker for 10 min at a rate of about 2
cycles/s to dissolve (NH4)2SO4 captured on the GF/D.
Total dissolved nitrogen, defined as the sum of dissolved organic
N (DON), NH4+, NO2− and NO3−, or (NH4)2SO4 collected using
the diffusion method, was converted to NO3− using the persulfateoxidation method (24, 33). Persulfate-oxidizing reagent (POR) was
prepared daily by dissolving 5 g of K2S2O8 and 3 g of boric acid
in 100 mL of 1.52% NaOH. The TDN standard solution of either 1
mL of 2 M KCl (or 0.5 M K2SO4), or 10 mL of distilled water was
pipetted to 14 mL of borosilicate glass test tubes with Teflon-lined
caps. Immediately after 1 mL of POR was added to the tube containing the TDN standard solution or captured (NH4)2SO4, the screw
cap was closed tightly. Then the tubes were autoclaved for 1 h at
121°C and preserved in a refrigerator until further analysis using the
denitrifier method.
Conversion of NO3− to N2O using the denitrifier method
Our method largely depends on a previously described approach
designated “the denitrifier method” (9, 10, 14, 32). The denitrifier
method uses a pure culture of a denitrifying bacterium lacking N2O
reductase, so that the strain produces N2O as the final product from
dissimilatory reduction of NO3−. Pseudomonas chlororaphis subsp.
aureofaciens ATCC 13985T, a denitrifying bacterium lacking the
capacity to reduce N2O (9), was cultivated in a 120-mL glass vial
(0501-09, vial No. 8, Maruemu, Osaka, Japan) containing 100 mL
of sterile Tryptic Soy Broth (Difco Laboratories, Detroit, USA)
amended with 10 mM KNO3, 7.5 mM NH4Cl, and 36 mM KH2PO4.
Cultures grown for 6–10 days were concentrated six-fold by centrifugation and then purged with pure He for 2–3 h to eliminate all dissolved N2O. The concentrated culture was transferred to vials, the
size and volume which depended on the solution’s N-concentration.
Typically, a 2-mL culture was dispensed in a 20-mL vial (Autosampler Vial, 20-CV; Chromacol, Welwyn Garden City, UK). The
vials were crimp-sealed with Teflon-backed silicone septa (20-ACCBT3; Chromacol) and purged for 30 min with ultrapure He to
48
eliminate all O2 and N2O. The standard solutions were then added at
a constant volume using a disposable syringe and needle. The vials
were incubated overnight to allow the complete conversion of NO3−
to N2O. The reaction was terminated by adding of 0.3 mL of 6 M
NaOH, which also acts to expel CO2 in the vial. A silicon sealant
(KE-42-T; Shin-Etsu Chemical, Tokyo, Japan) was pasted onto the
surface of the septum to prevent leakage of N2O from the vials. The
volume of the solutions should be constant to calibrate the blank because the 2 M KCl and 0.5 M K2SO4 solutions invariably contain N.
Conversion of NO2− to N2O using the azide method
The azide method (25) was used to convert NO2− directly to
N2O. Into a 20-mL glass vial, 5 mL of a 2 M sodium azide solution
was pipetted; the vial was then tightly capped with a Teflon-backed
silicone septum and crimp-sealed with aluminum closure. The vial
was transferred into a ventilated chemical hood, a short needle was
used to puncture the septum, and 5 mL of a 20% acetic acid solution
was injected via a syringe into the vial. The azide/acetic acid buffer
in the vial was purged using ultrapure He for at least 30 min in the
hood to remove N2O. A volume of NO2− standard solution was
pipetted into the sample vial (typically, 5 mL of sample in a 20-mL
vial). When the matrix of the standard solutions was freshwater,
combusted NaCl was added to a final concentration of 0.5 M. The
sample vial was crimp-sealed with a Teflon-backed septum, and
transferred into the hood. The purged azide buffer was injected via a
syringe into the sample vial in the hood. The amount of the azide
buffer injected depended on the amount of the standard solution:
the ratio of sample volume to buffer volume should be maintained
constant. According to McIlvin and Altabet (25), 0.3 mL of azide
buffer was added to 5 mL of sample solution. The vial was shaken
vigorously for several seconds and left for 30 min in the hood for
the complete conversion of NO2− into N2O. Finally, a 6 M NaOH
solution in the same amount as that of the azide buffer, was injected
into the sample vial via a syringe to terminate the reaction. All vials
were kept in the hood because toxicity of HN3 was expected as long
as the pH of the mixed solution in the vial was low.
ISOBE et al.
Concentration and 15N atom% of N2O measured by GC/MS
The concentrations and 15N atom% values of N2O were measured
using GC/MS with a modified injection port. We used a GC/MS
system (GCMS-QP2010 Plus; Shimadzu, Kyoto, Japan) with a
CP-PoraPLOT Q-HT column (25 m×0.32 mm, Varian, USA). The
detector worked with an electronic impact (EI) ionization source
and a quadrupole analyzer. The sample gas (50 µL) was injected
via a sample loop connected with an eight-port valve with purge
housing (Valco Instruments, Houston, USA) into the column at
50°C with a split ratio of 30. Ultrapure He was used as a carrier
gas with a flow rate of 2.0 mL min−1. For MS detection, the general
configuration was as follows: ionization energy, 70 eV; detection
voltage, 1.2 kV; and interface temperature, 280°C. Mass spectra
were obtained in the selected ion monitoring mode.
The headspace of the sample vial (250 µL) was removed by
a gas-tight syringe (Series A-2, VICI; Valco Instruments) and
injected into the GC/MS system via a sample loop (50 µL). It
took about 15 min to analyze several samples. Consequently, 100 or
more analyses were possible in a day. Figure 2 portrays two chromatograms obtained with our analytical system. It is crucial for a
precise measurement of N2O to remove CO2 because CO2 and N2O
have the same molecular weight. A comparison of m/z 44 (Fig. 2A)
and m/z 30 (Fig. 2B) enabled us to check the separation of N2O
from CO2 (also see Fig. 3). About 30% of N2O was determined as
NO (m/z 30 or 31) and m/z 30 and 31 were not produced from
CO2 (27). Therefore, the monitoring of m/z 30 and 31 enables us to
trace CO2 on the chromatograph (Fig. 3). The tiny peak at ca. 5.4
min was CO2 with no signal for m/z 30 or 31 (Fig. 3). This CO2
peak is caused by air contamination and CO2 in the vial that is not
trapped by NaOH. Removal of this CO2 was not possible using our
analytical procedures. Although it is quite difficult to eliminate the
CO2, good separation of CO2 from N2O (at 5.5 min) was established
using our analytical procedures.
Fig. 2. Typical chromatograms. The upper two graphs show chromatograms with an 15N atom% value of 0.37 and different concentrations (0,
100, 200, 500, and 1000 µM) scanning m/z 44 (A) and m/z 30 (B). The lower three graphs show chromatograms with a constant N concentration of
100 µM with different 15N atom% values (0.37, 10.3, 20.2, 49.8, and 99.3) scanning m/z 44 (C), m/z 45 (D) and m/z 46 (E).
Analytical Techniques for 15N with GC/MS
49
1 h, filtered by a glass-fiber filter (GF/F, Whatman; muffled at
450°C for 4 h) and stored at 4°C until further measurements. Ten
milliliters of the filtered 2 M KCl solution was used for measurements of NH4+, NO3− and TDN. Concentrations and 15N atom%
values of DON, NH4+ and NO3− were determined as for the soil
extracts above.
Results and Discussion
15
Fig. 3. Closeup of the chromatogram (20.2 15N atom% in Figs. 2C–
2E). The tiny peak at ca. 5.4 min was CO2 with no signal for m/z 30 or
31, and the large peak at ca. 5.5 min was N2O. This peak is caused by
air contamination and CO2 in the vial that is not trapped by NaOH.
N analysis of NO3−, TDN and NH4+ in distilled water
Figure 4 depicts calibration curves for concentrations and
15N atom% of NO − (Figs. 4A and 4D), TDN (Figs. 4B and 4E),
3
and NH4+ (Figs. 4C and 4F) in distilled water. We examined
configurations of two types (Fig. 4A) with different ratios of
solution and vial size to determine the applicability of our
simple headspace method. A smaller volume of headspace
raised the N2O concentration in the headspace; consequently,
the amount of N2O introduced into the GC/MS system in a
50-µL sample loop can be increased. With both configurations, we were able to obtain good regression lines between
NO3− and GC/MS peak area (m/z 44+45+46; Fig. 4A). The
For calculation of the 15N content, the equation proposed by
Stevens et al. (34, 35) was applied:
15
N atom% in N2O=100×(45R+2×46R−17R−2×18R)/(2+2×45R+2×46R),
—(eq. 1)
where 45R and 46R are the ratios of measured peak areas (45R=peak
area of m/z 45/peak area of m/z 44; 46R=peak area of m/z 46/peak
area of m/z 44, respectively), and 17R and 18R are the 17O/16O
(379.9×10−6) (23) and 18O/16O (2005.2×10−6) (3), respectively (see
Kaiser et al. (17) for details about 17R and 18R).
N-tracer experiment to determine the incorporation of NO3− and
NH4+ into DON
We used water samples from a temperate lake in Japan, Lake
Kizaki, to study the fate of NO3− under anoxic conditions. The
anoxic lake water collected from a depth of 22 m in Sep. 2009, was
incubated in 120-mL vials either with 15NH4Cl (99.3 15N atom%) or
with Na15NO3− (99.3 15N atom%). One milliliter of tracer solution
(6 mM 15NH4Cl and 12 mM Na15NO3−) was added to a 120-mL
vial to make a final concentration of ca. 50 µM for NH4+ and 100
µM for NO3−, and the vial was incubated for 2 h at 6.8°C. Vials
without 15N were incubated in parallel as a control experiment.
After incubation for 2 h, the sample was filtered using a syringe
filter (Dismic 25CS, 0.45 µM; Advantec, Tokyo, Japan); 10 mL
each of the filtered solution was used for measurements of NH4+,
NO3− and TDN. Concentrations and 15N atom% values of TDN and
NO3− were determined as described above. Ammonium concentrations were determined colorimetrically (19). Concentrations and 15N
atom% values of DON were calculated using the mass balance
between TDN and (NH4++NO3−).
We also incubated soil collected from the Uryu Experimental
Forest of Hokkaido University with 15N to determine the fate of
added NH4+ and NO3−. We collected surface mineral soil (0–10 cm)
at five random spots in the representative plots (20×20 m). The soil
was well mixed and sieved (4 mm) to remove coarse roots and
gravel. Seven grams of the sieved soil was put in a centrifuge tube
(50 mL, Corning Int’l., Tokyo, Japan), 1 mL of 5 mM Na15NO3
(99.8 15N atom%) or 15NH4Cl (99.7 15N atom%) was injected into
the tube, and the soil was incubated at 26°C. After a 2-h incubation,
the samples were extracted with 35 mL of 2 M KCl. The 2 M KCl
extract was prepared by shaking the soil with the KCl solution for
15
Fig. 4. Calibration curves for concentrations and peak area (m/z
44+45+46; A, B, C) with 15N atom% of 0.37, and for measured and
true 15N atom% values (D, E, F, with a concentration of 100 µM) for
different N forms (A and D for NO3−, B and E for TDN and C and F
for NH4+) dissolved in distilled water. The measured 15N atom%
was obtained with peak areas of m/z 44, 45, and 46 and eq. 1. For
NO3−, different amounts of solution were applied (2 mL sample in 10
mL vial: cross, and 10 mL sample in 20 mL vial: open circle), resulting
in different slopes and intercepts (A), although the calibration curves
for 15N content were quite similar (D). For NH4+, two sets of data with
the diffusion process conducted on different days (October 1, 2009 as
crosses and October 8, 2009 as open circles) are shown for comparison.
ISOBE et al.
50
linearity was maintained over a wide concentration range
(1–150 µM; Fig. 4A), which can accommodate most samples
from natural environments such as eutrophic lakes and rivers. The wide dynamic range of GC/MS and steady conversion of NO3− to N2O by denitrifying bacteria reduce the
laboratory work greatly because steps such as the dilution of
samples and changing of analytical configurations according
to the concentration range are not necessary. Despite the
pretreatment processes involved in TDN and NH4+ measurements (Fig. 1), good regression curves were obtained (Figs.
4B and 4C). For the 15N analysis, good calibration curves
were obtained for all N forms dissolved in distilled water
(Figs. 4D–4F). Even with different sample/headspace ratios
(Fig. 4D) and different dates (Fig. 4F), the calibration curves
were quite similar, which indicates the robustness of our
analytical procedures.
15
N analysis of NO3−, TDN and NH4+ in 2 M KCl extract
Figure 5 shows the calibration curves for concentrations
and 15N atom% of NO3− (Figs. 5A and 5D), TDN (Figs. 5B
and 5E), and NH4+ (Figs. 5C and 5E) in 2 M KCl. We
confirmed that the monitoring of m/z 44+45+46 and m/z
30+31 each produced a good regression curve (Figs. 5A–
5C), suggesting that the separation of CO2 from N2O worked
well in our system (Fig. 3). Because the peak area was
smaller for m/z 30+31 than m/z 44+45+46, it is not necessary
to use the peak area of m/z 30+31. When we expanded
the concentration range to 1000 µM (Figs. 5A and 5B),
the linearity remained sufficient, as indicated by the high
correlation coefficient (R2>0.999).
For the 15N analysis, good calibration curves were
obtained for all N forms (Figs. 5D–5F). Ammonium in 2 M
KCl is expected to be the most difficult to quantify, especially when it is at a low concentration and low 15N/14N ratio
because several pretreatment processes were needed to convert NH4+ to N2O (Fig. 1). Despite this, the regression curves
for NH4+ with a lower 15N ratio (15N atom%, 0.37 to 10) were
acceptable (inset in Fig. 5F). Using this analytical configuration, we usually run 15N measurements with less than 20 mL
of soil extract to measure gross mineralization and nitrification rates in forest soils. The necessary sample volume is
reduced to one-tenth of that needed for the conventional
combination of diffusion methods and EA-IRMS because
this new method uses a denitrifier and GC/MS, which is
advantageous for ecological studies because they often
require the investigation of numerous samples.
15
N and concentration of TDN in 0.5 M K2SO4
The regression curves for concentrations (Fig. 6A) and 15N
atom% (Fig. 6B) of TDN were also good with 0.5 M K2SO4,
which is commonly used to measure soil microbial biomass
with chloroform-fumigated and unfumigated soil samples
(7, 8). The regression lines for concentrations with m/z
44+45+46 and m/z 30+31 each produced an acceptable
correlation (R2>0.999), demonstrating that m/z 44+45+46
is useful. For 15N contents (Fig. 6B), the high correlation
coefficient (R2>0.999) confirms the applicability of our
analytical procedures. We confirmed that our method
worked not only for TDN extracted with 0.5 M K2SO4 but
also for NO3− and NH4+ in 0.5 M K2SO4 (data not shown).
Consequently, this method is applicable to various N
solutions extracted with either 2 M KCl or 0.5 M K2SO4
using methods conventionally employed in soil science.
15
Fig. 5. Calibration curves for concentrations and peak area (m/z
44+45+46 or 30+31; A, B, C) with 15N atom% of 0.37, and for
measured and true 15N atom% values (D, E, F with a concentration
of 100 µM) for different N forms (A and D for NO3−, B and E for TDN
and C and F for NH4+) dissolved in 2 M KCl. Regression curves for
concentration (A, B and C) with peak area of m/z 44+45+46 (open
square) and m/z 30+31 (solid circle) each produced sufficient quality
(R2>0.999 in all cases). We checked 15N analysis with low 15N contents
(<10 15N atom%) for NH4+ (inset in F) using the most complicated
procedure (Fig. 1). Our analytical procedures showed a good relation
between measured and true 15N atom% values (inset in F).
N analysis of NO2− in 2 M KCl
We explored the possibility of using this method to measure 15NO2− because the complicated dynamics of NO2− in
soil, as revealed in recent studies (28, 29), requires the use of
an 15N tracer to properly elucidate the production and consumption of NO2−. The applicability of the “azide method”
(25) to soil extracts was confirmed with 15N standards. Our
analytical method works well for NO2− dissolved in 2 M KCl
(Fig. 7). It is worth noting that the N2O produced with this
azide method obtained one N atom from NO2−, whereas the
other N atom came from azide, resulting in a low slope of the
regression line, close to 0.5 (0.475).
Case study 1: Rapid incorporation of NO3− into DON in an
anoxic zone in Lake Kizaki
Table 1 presents results obtained from study with anoxic
water from Lake Kizaki. When 15NO3− was added to the
Analytical Techniques for 15N with GC/MS
51
Fig. 6. Calibration curves for concentrations and the peak area (m/z 44+45+46 or m/z 30+31) with 15N atom% of 0.37, and for the measured
and true 15N atom% values with a concentration of 100 µM for TDN in 0.5 M K2SO4. To produce the N2O to measure, 1 mL of 10 mL of the persulfate-oxidized solution (2 mL of POR plus 8 mL of standard solution) was fed to the denitrifier. Regression curves for concentration (A) with peak
area of m/z 44+45+46 (open squares) and m/z 30+31 (solid circles) produced sufficient quality (R2>0.999). For 15N contents (B), high correlation
(R2>0.999) confirmed the applicability of our analytical procedures.
ment, followed by nitrosation of NO2− with organic matter
(36). The occurrence of this rapid, abiotic immobilization
of NO3− has recently been discussed in soil biogeochemical
studies (11, 13).
Fig. 7. Calibration curve for 15N content for NO2− in 2 M KCl with a
constant concentration of 100 µM: 1 mL of the solution was reacted
with azide buffer in a 10-mL headspace vial.
anoxic water, a considerable amount of 15N was detected
as DON in 2 h (Table 1). A parallel experiment with 15NH4+
showed no incorporation of 15N into DON (Table 1), which
suggests that the quick incorporation of 15NO3− is not microbial assimilation because microbes generally prefer NH4+
to NO3−. This high 15N level and concentration of DON in
15NO −-added samples was maintained for 8 days (data not
3
shown), which also suggests that the rapid 15N incorporation
was triggered by the addition of 15NO3− at the beginning of
the incubation, and no further reaction occurred. This quick
incorporation of NO3− into DON is suspected to involve
the abiotic immobilization of NO3− driven by the partial
reduction of NO3− to NO2− in an anoxic, reduced environ-
Case study 2: Slow incorporation of 15N into DON in a
forest soil
Table 2 presents results obtained from study with forest
mineral soil (0–10 cm) collected from the Uryu Experimental
forest of Hokkaido University. Concentrations and 15N levels
of NH4+, NO3− and DON in 2 M KCl soil extract were measured with the method developed in this study. Contrary to
expectations of abiotic immobilization of NO3− or rapid
incorporation of 15NH4+ into DON, concentrations and 15N
levels did not change with 15NH4+ and 15NO3−, although rapid
nitrification was detected when 15NH4+ was added to the soil
(Table 2). The fact that there was no rapid incorporation of
15N into DON indicated the DON in this forest soil to be
recalcitrant and not involved in the rapid biological cycling
between microbes and available N.
Conclusion
The results demonstrated the applicability of our analytical
method to several matrix solutions (Figs. 4–7). The method
enables the measurement of tiny N samples with low N concentrations such as 10 mL of 20 µM-N because of the low
risk of contamination by N2O from the atmosphere. Its
advantages are partly based on the use of GC/MS system,
which is much less expensive and much more commonly
Table 1. Incorporation of 15N from NH4+ and NO3− into DON in an anoxic zone in Lake Kizaki
Treatment
Control (no addition)
15NO − addition
3
15NH + addition
4
NH4+
µM-N
4.5 (0.3)
4.6 (0.1)
55.5 (0.7)
15N
NO3−
atom% excess
n.d.
n.d.
90.8 (1.1)
Data are shown as the mean (s.e.) (n=5)
n.d.=not detected
All samples were incubated for 2 hrs with/without the tracer.
15N atom% excess=15N atom%
measured−0.37
µM-N
1.5 (0.1)
107.6 (0.8)
0.1 (0.0)
15N
DON
atom% excess
n.d.
94.7 (0.2)
n.d.
µM-N
6.7 (0.6)
14.2 (0.7)
5.7 (0.5)
15N
atom% excess
n.d
81.0 (1.6)
n.d.
ISOBE et al.
52
Table 2.
Treatment
15NO −
3
15NH +
4
addition
addition
The fate of 15NH4+ and 15NO3− in a forest soil
NH4+
µmol-N g
soil−1
0.43 (0.06)
1.45 (0.04)
15N
NO3−
atom% excess
µmol-N g
n.d.
57.8 (4.5)
soil−1
1.14 (0.07)
0.09 (0.03)
15N
DON
atom% excess
89.2 (3.2)
2.7 (1.0)
µmol-N g
soil−1
3.93 (0.31)
3.53 (0.37)
15N
atom% excess
n.d.
n.d.
Data are shown as the mean (s.e.) (n=4)
n.d.=not detected
All samples were incubated for 2 hrs after the addition of the tracer.
15N atom% excess=15N atom%
measured−0.37
used among microbiologists than is IRMS. Furthermore, GC/
MS system enables the simultaneous monitoring of many
mass numbers (m/z), which helps analysts to identify problems related to analytical procedures. Moreover, the wide
dynamic range of GC/MS system (Figs. 4–6; 0.5–1000 µM
and 0.37–99 15N atom %) can greatly reduce the laboratory
work necessary to measure environmental samples with high
variations in concentration and 15N atom%. Our method can
be easily modified with an auto-sampler to provide more
high-throughput measurements because it obviates a purgeand-trap system. Finally, most procedures with our analytical
method (Fig. 1) have been proven applicable for 18O natural
abundance measurements (9, 21, 25). Moreover, more
methods to convert the target compound to N2O exist, all of
which can be improved for 15N and 18O measurements; some
examples are hydroxylamine to N2O (30) and H218O to N218O
(26). Consequently, our analytical method can be modified
for additional 15N and 18O measurements of other compounds
by virtue of the use of N2O as the target analytical gas.
Acknowledgements
This work was supported by grants-in-aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Nos. 19658060, 20780113, 19380078, 22248016
and 19201004), the Chuo University Joint Research Grant and the
Mitsui & Co., Ltd. Environment Fund (R08-C108). K. K. was also
supported by the Program to Create an Independent Research Environment for Young Researchers from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. K.I. was supported
by a Grant-in-aid for JSPS fellows from the Japan Society for the Promotion of Science. We appreciate Hideaki Shibata and Masamichi
Yamamoto providing us the soil and lake water samples. We
thank Ikuo Yoshinaga, Teruki Amano, Takao Yamagishi and Chie
Katsuyama for fruitful discussions on 15N measurements.
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