Research Article
Received: 25 January 2013
Revised: 31 March 2013
Accepted: 1 April 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6595
Isotopomer and isotopologue signatures of N2O produced in
alpine ecosystems on the Qinghai–Tibetan Plateau
Tomomichi Kato1,2*, Sakae Toyoda3, Naohiro Yoshida3,4,5, Yanhong Tang6
and Eitaro Wada1
1
Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, Yokohama, Kanagawa
236-0001, Japan
2
Laboratoire des Sciences du Climat et de l’Environnement, IPSL, CEA-CNRS-UVSQ, Orme des Merisiers, 91191 Gif sur
Yvette, France
3
Department of Environmental Science and Technology, Tokyo Institute of Technology, Yokohama 226-8502, Japan
4
Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, Yokohama 226-8502, Japan
5
Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan
6
National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8569, Japan
RATIONALE: Static-chamber flux measurements have suggested that one of the world’s largest grasslands, the QinghaiTibetan Plateau (QTP), is a potential source of nitrous oxide (N2O), a major greenhouse gas. However, production and
consumption pathways of N2O have not been identified by in situ field measurements.
METHODS: Ratios of N2O isotopomers (14N15N16O and 15N14N16O) and an isotopologue (14N14N18O) with respect to
14 14 16
N N O in the atmosphere, static chambers, and soils were measured by gas chromatography and mass spectrometry
in the summer of 2005 and the following winter of 2006 at three typical alpine ecosystems: alpine meadow, alpine shrub,
and alpine wetland, on the QTP, China.
RESULTS: Site preference (SP) values of soil-emitted N2O were estimated as 33.7% and 30.1% for alpine meadow and
shrub, respectively, suggesting larger contributions by fungal denitrification, than by bacterial denitrification and
nitrifier-denitrification, to N2O production. Statistical analysis of the relationship between SP and d15Nbulk values
indicated that in alpine meadow, shrub, and wetland sites fungal denitrification contributed 40.7%, 40.0%, and 23.2%
to gross N2O production and the produced N2O was reduced by 87.6%, 82.9%, and 92.7%, respectively.
CONCLUSIONS: The combined measurements of N2O concentration, flux, and isotopomeric signatures provide a robust
estimation of N2O circulation dynamics in alpine ecosystems on the QTP, which would contribute to the development of
ecosystem nitrogen cycle model. Copyright © 2013 John Wiley & Sons, Ltd.
Nitrous oxide (N2O) is a major greenhouse gas influencing
global climate change, and thus the mechanisms of its
production and consumption processes are of great interest.
Natural ecosystems are a major source of N2O, which is
produced through soil microbial activities of nitrification and
denitrification. These processes occur naturally and can be
enhanced in soils and waters that are enriched in nitrogencontaining species. In nitrification, N2O is produced as a byproduct in hydroxylamine oxidation to nitrite, as follows:
NH3 ! NH2 OH ! N2 O
(1)
In denitrification, N2O is produced as an intermediate by
bacterial/fungal denitrification:
NO
3 ! NO2 ! NO ! N2 O ! N2
(2)
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
NH3 ! NO
2 ! N2 O
(3)
Although many field studies have examined the temporal
and spatial variations in N2O fluxes between terrestrial
ecosystems and the atmosphere, the processes of production
and consumption of N2O gas before its release from soil
layers have received less attention. Therefore, much
uncertainty remains in our understanding of soil N2O and
its potential response to present and future climatic
variations, as well as to anthropogenic disturbances altering
natural and agricultural ecosystems at unprecedented scales,
both in rate and geographical extent.[1]
Stable isotope techniques can provide much information
regarding the attribution of N2O to different microbial
processes of production and consumption, and physical
processes of transportation between soil and the
atmosphere.[2–4] Isotopomer analysis has recently been used
to further refine the isotopic fingerprint of N2O. In contrast
Copyright © 2013 John Wiley & Sons, Ltd.
1517
* Correspondence to: T. Kato, Laboratoire des Sciences du
Climat et de l’Environnement, IPSL, CEA-CNRS-UVSQ,
Bât. 712, Orme des Merisiers, 91191 Gif sur Yvette. France.
E-mail: [email protected]
In addition to the above two processes, N2O can be
produced from nitrite through nitrification, which is often
termed nitrifier-denitrification:
T. Kato et al.
1518
to d18O and average d15N (d15Nbulk) values, the difference
between central and peripheral 15N enrichment (d15Na –
d15Nb = 15N site preference: SP) is considered to be
independent of the isotopic signature of the precursor[5] and
thus supplies process information even if isotopic signatures
of additional N species are lacking. However, there are still
relatively few in situ field measurements available for
comprehending the natural abundance of N2O isotopic and
isotopomeric values.
The Qinghai-Tibetan Plateau (hereafter referred to as
QTP) is one of the largest grasslands (~2.0 106 km2) in
the world, with high elevations (more than 4000 m above
sea level (a.s.l.)).[6] Alpine grasslands, which are
characterized by low temperature and plentiful sunlight,
might be a net carbon sink because of the lower
decomposition rate of organic matter and relatively
favorable photosynthetic conditions compared with high
latitude cold ecosystems, such as tundra or taiga forest.
Alpine grassland soils store an estimated 33.52 Pg C in
organic form, accounting for approximately 2.5% of the
global soil carbon pool.[7] Several field measurements of
N2O flux have been conducted on the QTP. Kato et al.[8]
examined the spatial variation in N2O fluxes and reported
that some sites acted as sinks and others as sources of
atmospheric N2O. They also showed several relationships
between the N2O flux and environmental factors, including
an exponentially negative relationship with the C/N ratio
of surface soil in drier ecosystems and a negative correlation
with soil pH in wetlands. Lin et al.[9] showed that yak dung
and urea caused slightly higher emission of N2O in an alpine
meadow. Considering that finding, the QTP may be a
potential N2O source because of livestock raising and could
have a substantial impact on the global N2O budget.
Complicated interactions between microbes, nutrients, and
physical and chemical conditions lead to variations in the
relative intensity of nitrification, denitrification, and N2O
reduction. Previous studies have mainly examined the
contributions of each production and consumption process
by indirect methods, such as the relationship between net
ecosystem N2O flux and environmental factors. However,
not only quantitative but also qualitative approaches,
such as stable isotope analysis, are needed for better
understanding of the mechanisms of N2O production,
reduction, and transportation in field settings of the QTP.
The emerging ecosystem modeling approach to clarify
the behavior of green house gases on continental and
global scales also demands more in situ information about
the N2O production and consumption pathways for the
parameterization and validation of the model. To reduce
the uncertainties in the global N2O budget estimation for
better future climate projection, therefore, we have to
clarify the mechanisms involved in the N2O cycle in the
vast QTP alpine ecosystems both qualitatively and
quantitatively. Here we conducted the in situ field
measurement of isotopic signature of N2O in three typical
alpine ecosystems in northeastern QTP in the summer of
2005 and the following winter of 2006. We sampled the
gas in the atmosphere and soil layers, and soil-emitted
gas in static closed chambers at the soil surface, and
examined the temporal and vertical changes in the isotopic
signature of N2O. Considering that the net N2O flux is
controlled by N2O production and reduction occurring in
wileyonlinelibrary.com/journal/rcm
the aerobic/anaerobic part of soil layers, we raise several
questions that would provide a better understanding of the
mechanisms of N2O circulation in the QTP alpine ecosystems:
1. Does the N2O pathway show clear diurnal and seasonal
changes?
2. What microbial types are the most likely end members and
how much is the fractional contribution of each end
member for gross N2O production?
3. How much is the gross N2O production reduced by
oxidation in soil?
EXPERIMENTAL
Site description
The isotopic signatures and fluxes of N2O were measured in
daytime in summer 2005 and winter 2006 at three typical QTP
ecosystems: alpine meadow, alpine shrub, and alpine wetland.
The sites were located within approximately a 2-km radius of
the Haibei Alpine Meadow Ecosystem Research Station,
Northwest Plateau Institute of Biology, Chinese Academy
of Science, China (37 29’–45’N, 10112’–23’E; 3250 m a.s.l.).
The annual average temperature and precipitation for
1981–2000 were 1.7 C and 561 mm, respectively. In the
alpine meadow, the soil is clay loam of Mat Cry-gelic
Cambisol. In the alpine shrub, the soil is silty clay loam of
Mol-Cryic Cambisol.[10] The wetland is characterized by a
mixed
hummock-hollow
terrain
with
hummocks
representing 40%, hollows 55%, and other features 5% of
the landscape. The study sites are not closed by fence and
are grazed by yaks and sheep every winter.
The three perennial sedges Kobresia humilis, K. pygmaea, and
K. tibetica dominated the plant community at the alpine
meadow site.[6] A Rosaceous shrub, Potentilla fruticosa, and
the above three sedges dominated at the alpine shrub site,[11]
and K. tibetica, Carex pamirensis, Hippuris vulgaris, and Blysmus
sinocompressus dominated at the wetland.[12] In the wetland,
the soil surface under plant canopies was largely covered by
moss (Distichium inclinatum [Hedw.], Bruch and Schimp.,
Brachythecium spp., and Encalypta spp.), and free water was
partially covered by Potamogeton pectinatus L.[13] Vascular
plants start to grow in May and die out in October.[6]
Gas sampling
Gas samples for isotopic analysis were collected from one of the
static flux chambers after sampling for the flux measurement
through a capillary tube to avoid a sudden pressure change
inside the chamber. We sampled four times on a single day
(10:00, 14:00, 18:00, and 24:00 at Beijing Standard Time [BST])
to investigate the diurnal cycle of N2O flux and isotopic
signature. In summer, samples were taken on 21 July at the
alpine meadow, 23 July at the alpine shrub, and 25 July 2005
at the alpine wetland. In winter, samples were taken on
4 March at the alpine meadow, 6 March at the alpine shrub,
and 8 March 2006 at the alpine wetland. In addition, N2O gas
was collected from the atmosphere at 2.2 m height and from
the soil layer through a capillary tube and cylindrical soilgas-phase probes made of stainless steel (GL Science. Inc.,
Tokyo, Japan) installed individually at depths of 10, 30, and
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
N2O isotopomers in alpine ecosystems
70 cm at 14:00 and 24:00 (BST) in summer and winter at the
alpine meadow, 30 cm at 14:00 in summer at the alpine shrub,
and at no depth at the alpine wetland. The gas was stored in
an evacuated 500-mL container made of glass or stainless
steel. For soil gas, 10-, 30-, and 70-cm depths were chosen
for sampling; no soil gas was sampled in the wetland.
Flux measurement of N2O
A polyvinyl chloride (PVC) cylinder chamber (200 mm in
diameter by 200 mm high) was used to measure the fluxes.
Four chambers were used in summer and two were used in
winter. In both seasons, the chambers were set in 5 m 5 m
rectangle grids spaced at 5-m intervals. They were buried at
approximately 70-mm depth vertically at least 24 h before
sampling. At every sampling time, the PVC lids were put
on the chamber tops and sealed tightly by vinyl tape. The
air inside the chambers was sampled in 10-mL glass vials
for N2O gas at 1, 10, 20, 30, and 60 min in summer, and at
1, 10, 30, 60, and 90 min in winter. The flux was calculated
as a linear slope of the concentration evolution over the time
courses. The concentrations of N2O in the air samples were
measured by gas chromatographs (models GC-7A and GC-14B;
Shimadzu, Kyoto, Japan) equipped with a (63Ni)-electroncapture detector. From each vial, 2 mL of air was withdrawn
using a gas-tight syringe, and injected into a gas chromatograph
and purged with pure N2 gas at a flow rate of 40 mL min–1.
The samples were directly injected from sample vials into the
gas chromatograph with a gas-tight syringe (A-2 type gas
syringes; VICI Precision Sampling, Baton Rouge, LA, USA).
On-site measurements of environmental factors
Soil temperature and water temperature (in case of an openwater site) at 5 cm around the chambers were measured by
thermometers. The average values of soil-water content at 0
and 12 cm depths were determined by time domain
reflectometry (TDR) sensors (CS-620; Campbell Sci. Inc.,
Logan, UT, USA) only in summer. In wetlands, the soil
oxidation–reduction potential (ORP) was measured using a
glass electrode. After chamber gas sampling, 5-cm-deep soil
cores were collected. Using these cores, the soil air-filled space
was measured with a soil three-phase meter (model DIK-1130;
Daiki, Tokyo, Japan). The water-filled-pore space (WFPS) was
also determined by dividing the soil-water-filled space, as
evaporated soil water from oven-dried soil, by the porosity,
which is calculated as a sum of the air-filled space and the
evaporated soil water from oven-dried soil. Above-ground
plant mass was collected by mowing. Below-ground plant
mass and mineral soil were separately collected by sieving
the soil samples collected with a core sampler from 0- to
30-cm depths. Each sample was then weighed before and
after oven drying at around 80 C for 48 h. The total carbon
and nitrogen contents of plant bodies and of sieved and
decarboxylated soil samples were measured with an NC
analyzer (Sumigraph NC-900; Sumika Chemical Analysis
Service Ltd., Osaka, Japan).
Measurement of isotopomers and an isotopologue of N2O
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
d15 Ni ¼ 15 Risample =15 Rstd 1Þ 1000ð%Þði ¼ a; b; or bulkÞ (4)
d18 O ¼
18
Rsample =18 Rstd 1 1000ð%Þ
(5)
where 15Ra and 15Rb represent the 15N/14N ratios at the center
and end sites of N atoms, respectively, and 15Rbulk and 18R
denote the isotope ratios for average 15N/14N and 18O/16O,
respectively. The subscripts ’sample’ and ’std’, respectively,
indicate the isotope ratios for the sample and the standard,
with the standard being atmospheric N2 for N and Vienna
standard mean ocean water (V-SMOW) for O. We also define
the 15N site preference (hereafter SP) as an illustrative
parameter of the intramolecular distribution of 15N:
15
N site preference ðSPÞ ¼ d15 Na d15 Nb
(6)
The N2O concentration was obtained simultaneously with
the isotopomer and isotopologue ratios from the peak area
of the major ions (masses 44 and 30 in the molecular ion
and fragment ion analysis, respectively) measured in the
sample and the reference gas (the synthetic air), and the
volume of the processed sample and reference.
Definition of enrichment factors
The relationships between isotope ratios and concentrations
of substrate or accumulated products are expressed by the
Rayleigh equation:
dS ðtÞ ¼ dS ðt ¼ 0Þ þ eln f
(7)
where dS(t) and dS(t = 0) are the d values of substrate S at
times t and t = 0, respectively, e is an enrichment factor,
and f equals the remaining fraction of substrate S at time
t ([S][t]/[S][t = 0]). Equation (7) holds only if e is constant
during the progress of the reaction.[17] In this study, e was
used for analysis as an enrichment factor only when the
uptake of flux by soil occurred in the alpine meadow and
alpine shrub sites.
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm
1519
The relative abundances of isotopomers (14N15N16O and
15 14 16
N N O) and an isotopologue (14N14N18O) with respect to
14 14 16
N N O in the gas samples were determined in the
laboratory at Tokyo Institute of Technology (Yokohama, Japan)
using an on-line analytical system described elsewhere.[14,15]
Briefly, an aliquot of gas sample containing 1–5 nmol of N2O
was manometrically measured and N2O was cryogenically
concentrated and separated from H2O and CO2 using chemical
adsorbents. After further purification by gas chromatography,
the N2O was injected into an isotope-ratio mass spectrometer
(MAT 252; Thermo Fisher Scientific K. K., Yokohama, Japan).
Site-specific N-isotope analysis in N2O was conducted using
ion detectors that had been modified for mass analysis of the
N2O fragment ions (NO+), which contain the N atoms in the
center positions of the N2O molecules. Bulk (average) N- and
O-isotope ratios were determined from the molecular ions.[16]
Isotopic calibration was carried out by measuring a synthetic
air standard containing 349 nL L–1 of N2O (Japan Fine Products
Co., Ltd., Kawasaki, Japan), which had been previously
calibrated against international standards for atmospheric N2
and standard mean ocean water.[16] The notation of the
isotopomer or isotopologue ratios is shown below. The
measurement precision was typically better than 0.1% for
d15Nbulk and d18O values, and better than 0.5% for d15Na and
d15Nb values:
T. Kato et al.
Calculation of the isotopic signature of N2O emitted from
the soil
When N2O is emitted from the soil, the collected N2O in the flux
chambers is a mixture of atmospheric and soil-derived N2O.
The isotopic signatures of the soil-emitted N2O (dsoil values)
were calculated from those of the chamber gas samples
(dchamber values) and the ambient air samples (dair values)
assuming two-component mixing. We conducted sampling
only once during each measurement time; therefore, it was
difficult to obtain the stable dsoil values from the slope of the
regression line for dchamber values versus inverse Cchamber
(Keeling plot). Thus, the dsoil value was calculated using the
following mass balance equation:
dchamber Cchamber ¼ dair Cair þ dsoil Csoil
Fractional composition of N2O production and
consumption pathways estimated by SP and d15Nbulk
Assuming that N2O is produced by two sources and that the
mixed N2O is also consumed by N2O reduction, x, the fraction
of N2O produced by one of two sources, and Fr, the progress
of N2O reduction, are estimated by SP and d15Nbulk by Monte
Carlo calculation. Details of this calculation are explained in
the Auxiliary Material of the report by Toyoda et al.[15]
In SP–d15N space, the following equation defines a line,
which makes it possible to deduce the SP and d15N values
of the produced N2O, and the relative contributions from
the two sources, by tracing the observed values back
along the line until it intersects with the mixing line defined
by the two sources:
SPsoil fxSPnit þ ð1 xÞSPdenit g
(9)
1520
where soil, nit, and denit denote soil, nitrification, and
denitrification, respectively, e(SP)red and e(15Nbulk)red are
the fractions of reduction of SP and 15Nbulk, and C is the
N2O concentration.
We obtained x and Fr, approximate measures of the degree
of N2O reduction, and their uncertainties by Monte Carlo
calculation as follows. First, the e and SP values for N2O
production and consumption processes were calculated using
random numbers that obey a normal distribution, with the
average and 1s values obtained from published results
(’best estimates’ in Table 3, Toyoda et al.[15]). For e(SP)/e
(15Nbulk), we used the average values of e(SP)/e(15Nbulk) in
each experimental condition or the literature, instead of
the independent average of e(SP) and e(15Nbulk) shown in
Table 3 of Toyoda et al.[15] The isotopic signature of N2O
produced by the two sources was then calculated from the
observed isotope ratios in substrates (ammonium and nitrate;
if not available, the d15N value of nitrate was calculated from
the d15N value of ammonium and the enrichment factor for
wileyonlinelibrary.com/journal/rcm
x ¼ ðSPintersect SPdenit Þ=ðSPdenit SPnit Þand
(10)
k ¼ ½SPsoil þ fxSPnit þ ð1 xÞSPdenit g=eðSPÞred
(11)
where SPintersect is the SP value of the calculated intersect, and
k is a positive number. If N2O production follows sequentially
with the reduction, k = ln(C/C0) and therefore the degree of
N2O reduction, Fr, is calculated as follows:
Fr ¼ 1 C=C0 ¼ 1 expðkÞ
(12)
(8)
where C is the N2O concentration and Csoil = Cchamber Cair.
The dsoil values obtained from small Csoil values (<10 ppbv)
were not used for further data analysis because in such cases
dchamber was equal to dair within the precision of the analysis
and the error propagated in the calculation of dsoil was large.
¼ eðSPÞred =eð15 Nbulk Þred½d15 N bulk soil
xd15 N bulk nit þ ð1 xÞd15 N bulk denit nitrification) and the randomly determined parameters. Next,
the intersection of the fractionation and source-mixing lines
was obtained and x and k were calculated as follows:
In some cases, the intersection could not be obtained
mathematically or was outside the mixing line, and thus
was discarded. The calculation was repeated until 1000
results were obtained.
This method can be applied for the combination of two sources
that have apparently distant SP values, for example, four
combinations: fungal denitrification or hydroxylamine oxidation
(nitrification) vs. denitrification or nitrifier-denitrification.
RESULTS
Diurnal change in environmental factors, N2O gas flux, and
N2O isotopic signature
The soil-surface temperature reached its diurnal maximum at
14:00 in summer and 18:00 in winter and reached its
minimum at 24:00 in summer and 6:00 in winter (Fig. 1(a)).
The soil water was relatively constant diurnally in the alpine
meadow and shrub, but decreased considerably at 24:00 in
the alpine wetland (Fig. 1(b)).
The N2O gas fluxes fluctuated temporally and
occasionally changed their signs in the range of 9.24 to
20.27 mg N2O m–2 h–1 (Fig. 2). The daily averaged fluxes for the
single chamber that was used for isotopic measurements were
positive, with values of 0.81, 3.01, and 3.35 mg N2O m–2 h–1
for alpine meadow, alpine shrub, and alpine wetland,
respectively, in summer. In winter, these values were 1.30,
8.29, and 0.26 mg N2O m–2 h–1 for alpine meadow, alpine shrub,
and alpine wetland, respectively. The daily averaged fluxes for
all four chamber-averaged values were similar to values for the
single chamber except for the wetland, which had a negative
value of 1.05 mg N2O m–2 h–1 in summer. The amplitude of
temporal fluctuations was much smaller in winter than in
summer for alpine meadow and wetland.
Isotopomers and an isotopologue in soil-emitted N2O (dsoil
values) showed large diurnal changes in summer and winter
for alpine meadow, and in winter for alpine wetland (Fig. 3).
However, they showed relatively stable values in summer
and winter for alpine shrub and in summer for alpine
wetland (Fig. 3).
Vertical profile of N2O isotopomers and isotopologue
At the alpine meadow site, the N2O concentration in the soil
gas was similar to that in the atmosphere at 0.1 and 0.3 m
below the surface, but it increased to 2273 ppb at 0.7 m in
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
N2O isotopomers in alpine ecosystems
1
a
Soil water content (m3 m-3)
Soil surface temperature (°C)
25
20
15
Alpine Meadow (Summer)
Alpine Shrub (Summer)
Alpine Wetland (Summer)
Alpine Meadow (Winter)
Alpine Shrub (Winter)
Alpine Wetland (Winter)
10
5
0
-5
-10
6
12
18
Alpine Meadow (Summer)
Alpine Shrub (Summer)
Alpine Wetland (Summer)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
6
24
b
0.9
6
12
Hour (Beijing Standard Time)
18
6
24
Hour (Beijing Standard Time)
Figure 1. Diurnal variations in (a) soil surface temperature and (b) volumetric soil water
content in surface soil. Data points are averaged values of four chambers in summer and
two chambers in winter.
25
a
20
b
c
15
10
5
N2O flux ( g N2O m-2 h-1)
0
-5
-10
Average of surrounding chambers (n=4)
-15
Single chamber for isotope sampling (n=1)
-20
-25
d
20
15
e
f
10
5
0
-5
-10
-15
-20
-25
6
12
18
24
6
12
18
24
6
12
18
24
30
Hour (Beijing Standard Time)
Figure 2. Diurnal variations in N2O emissions at the alpine meadow (a, d), alpine shrub (b, f), and
alpine wetland (c, e) sites in summer 2005 (a, b, c) and winter 2006 (d, e, f). Filled circles show the
averaged N2O emissions of four chambers in summer and two chambers in winter including the
chamber for isotopomer sampling. Open squares show N2O emission from the chamber, which is
used for isotopomer sampling.
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
showed nearly the same values (38.6% and 39.3%,
respectively), which were slightly lower than the value for
the atmosphere (41.9%).
DISCUSSION
Diurnal change in N2O flux and the isotopomers and
isotopologue
The observed N2O fluxes showed diurnal variations in
several cases (Fig. 2). At the alpine shrub site, the N2O flux
was higher at 14:00 and 18:00 than at 10:00 and 24:00 in the
winter, corresponding with the variation in soil-surface
temperature (Fig. 1). This might suggest that the microbial
Copyright © 2013 John Wiley & Sons, Ltd.
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1521
summer (Fig. 4). The ratios of the isotopomers or the
isotopologue of N2O in the shallower soil layers were also
similar to those in the atmosphere and showed distinct values
at 0.7 m in summer (d15Nbulk = 7.9%, d15Na = 7.3%, d15Nb =
23.2%, d18O = 47.7%, SP = 30.5%). In contrast, such
increases or decreases in concentration and d values at the
deep soil layer were not observed in winter.
At the alpine shrub site, the N2O concentration showed a
monotonic increase from the atmosphere (333 ppb) to 0.3 m
below the soil surface (5483 ppb; sampling was conducted
only in summer). On the other hand, the isotopic signature
showed a corresponding increase (SP, from 19.3 to 32.1%)
or decrease (d15Nbulk values, from 7.9 to 17.6%; d15Na
values from 17.6 to 1.5%; d15Nb values from 1.7 to
33.6%), although d18O in the soil gas at 0.1 and 0.3 m
T. Kato et al.
Alpine Meadow
80
Summer
Winter
15Nbulk
(‰)
60
Alpine Shrub
Alpine Wetland
80
80
60
60
40
40
40
20
20
20
0
0
6
18
24
30 6
-20
12
18
24
0
30 6
-20
-40
-40
-40
-60
-60
-60
-80
80
-80
80
-80
80
60
60
60
40
40
40
(‰)
12
20
20
20
15N
-20
0
6
-20
0
12
18
24
(‰)
18
24
0
30 6
-20
-40
-40
-60
-60
-60
-80
-80
-80
60
60
60
30
30
30
0
6
-30
15N
12
-40
12
18
24
0
30 6
-30
12
18
24
0
30 6
-30
-60
-60
-90
-90
-90
-120
-120
-120
160
160
160
120
120
120
80
80
80
40
40
40
18O
(‰)
-60
0
30 6
-40
-40
0
30 6
-40
-80
160
-80
-80
160
160
120
120
120
80
80
80
40
40
40
0
0
0
6
SP (‰)
30 6
-20
6
12
12
18
18
-40
24
24
30 6
-40
12
12
18
18
24
24
0
30 6
-40
-80
-80
-80
-120
-120
-120
12
18
24
30
12
18
24
30
12
18
24
30
12
18
24
30
12
18
24
30
Hour (Beijing Standard Time)
Figure 3. Diurnal variations in isotopes and isotopomers from soil-emitted N2O gas at the alpine
meadow, alpine shrub, and alpine wetland sites in summer 2005 (filled circles ●) and winter 2006
(open squares □). Error bars show propagated error calculated from 1s of the isotopomer ratio
and concentration analysis.
1522
activity that produces N2O depends on temperature. In other
cases, however, the N2O fluxes were not correlated with
surface temperature or soil moisture.
The ratios of the N2O isotopomers or isotopologue also
showed temporal variations with larger amplitudes especially
at the alpine meadow site and alpine shrub site (Fig. 3).
wileyonlinelibrary.com/journal/rcm
However, attention should be paid to the uncertainty of the
data because the amplitude of flux is basically small, as shown
in Fig. 2 and mentioned in previous reports of studies at the
same sites.[8] Therefore, the calculated d values for soilemitted N2O have large uncertainty as represented by the
large error bars in their diurnal courses in Fig. 3. At the alpine
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
N2O isotopomers in alpine ecosystems
Alpine Meadow
Alpine Shrub
Height (m)
2
2
1.5
1.5
1
Alpine Meadow - Summer
0.5
1000
0
2000
3000
4000
00
-0.5
-0.5
-1
-1
ppb
2.5
2.5
Height (m)
N
2
2
1.5
1.5
1
1
0.5
0.5
0
-5
0
-0.5
-20 -15 -10
‰
N
1000 2000 3000 4000 5000 6000
ppb
15 bulk
15
Alpine Shrub - Summer
1
0.5
Alpine Meadow - Winter
0
5
10
-1
‰
Height (m)
2
2
1.5
1
1
0.5
0.5
0
5
10
15
0
-5
20
-0.5
-1
0
5
-1
‰
Height (m)
-30
Height (m)
20
‰
2.5
2
2
1.5
1.5
1
1
0.5
0.5
0
-10
0
-0.5
-20
18O 2.5
10
-40
-30
0
-10
0
-0.5
-20
-1
‰
10
-1
2.5
2
2
1.5
1.5
1
1
0.5
0.5
36
40
44
48
52
0
-1
44
48
52
2
2
1.5
1
1
0.5
0.5
15
20
25
‰
2.5
1.5
-1
40
-1
‰
2.5
0
10
-0.5
36
-0.5
-0.5
Height (m)
15
-1
10
‰
2.5
SP
10
-0.5
15N
-40
5
2.5
1.5
0
-5
0
-5
0
-0.5
-20 -15 -10
2.5
0
that no diurnal changes in N2O isotopic signatures could be
detected in soil-emitted N2O in this chamber-based approach
partly due to the very low N2O flux.
2.5
2.5
N2O conc.
30
35
40
0
10
15
20
25
30
35
40
-0.5
‰
-1
‰
Figure 4. Vertical profile of the N2O concentration and
isotopic signature in the atmosphere (2.2 m) and the soil
layers (0.1, –0.3, and 0.7 m) at the alpine meadow and
alpine shrub sites in summer 2005 (filled circles ●) and winter
2006 (open squares □).
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
Direct measurement of N2O in the soil gas revealed that the
summer vertical profile had significantly high N2O
concentrations and high or low isotopic signatures in the
deeper soil layers, 0.7 m and deeper in alpine meadow and
0.3 m and deeper in alpine shrub (Fig. 4). This suggests that
N2O is intensively produced in the deeper layers rather than
in the shallower layers, and that the produced N2O can be
transported to the shallower layers or atmosphere by
diffusion. Because the negative flux (i.e., atmosphere to soils)
was observed on several occasions (Fig. 2), consumption of
N2O also could have occurred in the shallow layers. The lack
of gradient in the concentration and isotopic signature in the
winter vertical profile of N2O at the alpine meadow site
indicates that there was no significant microbial activity in
N2O production.
In order to discuss the production and consumption
processes of N2O, we estimated the isotopic signature of the
soil N2O source using the relationship between d values
and the inversed concentration (i.e., Keeling plot; Fig. 5). In
summer, the d15Nbulk and SP values in the atmosphere,
soil-emitted gas (chambers), and soil layers show significant
correlation with the inverse concentration (determinant
coefficient [r2] >0.80), except for SP at the alpine wetland
site. This suggests that the observed N2O concentration
and isotopic signatures can be explained by mixing of two
components (two end-members): atmospheric N2O and
soil-produced N2O. The isotopic signature of the latter was
estimated from the y intercept of the regression line, and
values of 12.7, –17.6, and 2.4% for d15Nbulk, and 33.7,
30.1, and 23.1% for SP, were obtained at the alpine meadow,
shrub, and wetland sites, respectively.
Production and consumption processes of N2O in alpine
soils
The estimated values of SP and d15Nbulk of N2O produced in
the alpine soils are plotted in Fig. 6 together with literature
data. Our data have conspicuously high SP values, especially
for alpine meadow and shrub soils (33.7 and 30.1%,
respectively). These values are close to the SP values reported
for N2O produced by nitrification (oxidation of NH2OH,
about 33%, Sutka et al.[18–20]) or fungal denitrification (37%,
Sutka et al.[21]), rather than by bacterial denitrification or
nitrifier-denitrification (reduction of nitrite, about 0%, Sutka
et al.[18–20] and Toyoda et al.[14]). Therefore, assuming that
nitrification does not occur in deep soils that are not
cultivated nor fertilized and that N2O reduction is also not
occurring, the N2O in the alpine meadow and shrub soils
would be produced predominantly by fungal denitrification.
In the case of alpine wetland soil, the estimated SP value
(23.1%) could be explained by the combination of fungal
denitrification and bacterial denitrification because the latter
process can be activated under the anaerobic condition
induced by waterlogging and because nitrification is likely
to be suppressed under anaerobic conditions.
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm
1523
shrub site, the d values were almost constant both in the
summer and winter, probably indicating that the production
and consumption pathways changed little. Thus, we consider
Vertical profile of the N2O concentration, isotopomers, and
isotopologue
T. Kato et al.
Alpine Meadow
Alpine Shrub
Alpine Wetland
10
15Nbulk
(‰)
5
0
-5
y = -12.7 + 6574.2x
r 2 = 0.85
-10
-20
y = -17.6 + 8774.9x
r 2 = 0.94
Air
Chamber
Soil
-15
0
0.001
0.002
0.003
0.004
0.001
0.002
0.003
0.004
y = -2.4 + 3070.3x
r 2 = 0.87
0.001
0.002
0.003
0.004
35
y = 33.7 - 4621.2x
r 2 = 0.80
SP (‰)
30
y = 30.1 - 4139.4x
r 2 = 0.85
y = 23.1 - 1241.6x
r 2 = 0.87
25
20
15
0
0.001
0.002
0.003
0.004
0.001
0.002
0.003
0.004
0.001
0.002
0.003
0.004
1/[N2O] (ppb-1)
Figure 5. Relationship of d15Nbulk and SP values against the reciprocal of the N2O
concentration in the atmosphere (2.2 m; filled circles ●) and the soil gas emitted from the
surface (chamber, open squares □) and the soil layer (0.1, –0.3, and 0.7 m; filled
diamonds ◆) at the alpine meadow, alpine shrub, and alpine wetland sites in summer 2005.
However, we cannot exclude the possibility of other
mechanisms of N2O production because high SP values are
also observed when N2O is partly reduced by denitrifying
50
Alpine Meadow (this study)
Alpine Shrub (this study)
Alpine Wetland (this study)
Crop (field)
Crop (incubation)
Grassland (field)
Grassland (incubation)
Forest (field)
Forest (incubation)
40
SP (‰)
30
20
10
-70
-60
-50
-40
-30
-20
-10
0
0
10
-10
15Nbulk
(‰)
1524
Figure 6. Correlation diagram of site preference (SP) and
d15Nbulk values at the three sites in summer 2005 and at various
sites from the literature. Literature values are averages of
multiple measurements at each site. Crop (field) consists of
values from Perez et al.,[28] Park et al.,[29] and Toyoda et al.;[15]
crop (incubation) consists of values from Well et al.;[30,31]
grassland (field) consists of values from Yamulki et al.,[32]
Opdyke et al.,[25] and Ostrom et al.;[33] grassland (incubation)
consists of values from Bol et al.[34] and Cardenas et al.;[35] forest
(field) consists of values from Well et al.[36] and Park et al.;[29]
and forest (incubation) consists of values from Perez et al.[37]
wileyonlinelibrary.com/journal/rcm
bacteria.[22] Hence, we further applied the computational
analysis of production and reduction of N2O based on
isotopic data as presented by Toyoda et al.[15]
Assuming that fungal denitrification and bacterial
denitrification are the major two end-members of N2O
sources in the alpine soils, the degree of N2O reduction (Fr)
and the relative fraction of fungal denitrification to the gross
N2O production (x) were estimated from SP and d15Nbulk
values of soil N2O (see the previous subsection). In addition,
the d15N value of nitrate in the soil (27 to approximately
11%, based on the d15N value of organic nitrogen measured
for soils between 0 and 0.1-m depth and the reported
enrichment factor for conversion of ammonium into nitrate)
was estimated using the procedure explained in the
Experimental section. Other input parameters required for
the calculation were taken from Toyoda et al.[15]: the SP values
of N2O produced by fungal and bacterial denitrifications
were 37.0 2.6% and 0.40 5.50%, respectively; the
15 bulk
N -enrichment factor e(15Nbulk) for N2O produced by
fungal or bacterial denitrification was 26 11%; the
e(15Nbulk) for N2O reduction was 9.8 6.0%; and the ratio
of e(SP) to e(15Nbulk) for N2O reduction was 0.86 0.36. The
averaged value of x obtained from 1000 iterations of Monte
Carlo calculations was 40.7%, 40.0%, and 23.2% for alpine
meadow, alpine shrub, and alpine wetland soils, respectively
(Table 1). The averaged Fr values were 87.6%, 82.9%, and
92.7% for alpine meadow, alpine shrub, and alpine wetland
soils, respectively (Table 1).
Alpine meadow and shrub showed moderate contributions
by fungal denitrification of 40.7% and 40.0%, respectively, to
total N2O production. Recent studies[23,24] have also shown
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
N2O isotopomers in alpine ecosystems
Table 1. Fractional contributions to the N2O production
pathway of two sources (fungal denitrification and
bacterial denitrification) and N2O reduction at the Haibei
station site in the summer of 2005
x (%; meanstandard
deviation; share of
fungal denitrification
Fr (%; mean
between the
standard
two production
deviation; fraction
pathways)
of N2O reduction)
Alpine meadow
Alpine shrub
Alpine wetland
40.7 22.6
40.0 21.3
23.2 17.2
87.6 14.8
82.9 18.5
92.7 10.0
ranging from 87.6 to 92.7%. This supported our observations
of the high N2O concentration in deeper soil and smaller N2O
flux at the soil surface. Thus, the combined measurement of
N2O concentration, N2O flux, and N2O isotopomeric signatures
could provide a robust estimation of N2O-circulation dynamics
in alpine ecosystems on the QTP. However, the N2O
isotopomeric measurements have high uncertainty due to the
lack of iterative evidence of temporal isotopic change,
especially in the diurnal cycle, and the lack of isotopic
measurement on N2O substrates. Further investigations are
needed to both qualitatively and quantitatively clarify the
interaction between production pathways and substrates, and
the reduction contribution.
Acknowledgements
that fungal denitrification was significant in N2O production
in alpine ecosystems. On the other hand, this statistical
analysis suggests that bacterial denitrification contributed
largely, by 59.3% and 60.0%, in alpine meadow and shrub,
respectively, similar to the relatively high contributions by
bacterial denitrification reported from N2O isotopomeric
measurement studies, such as in temperate agricultural soils
in Michigan, USA (61–92%[25]), and in temperate coniferous
forests in Japan (96.8%[26]). Finally, we can conclude that the
N2O emitted from the soil surface was conveyed by diffusion
from deeper soil layers, in which the N2O produced by both
fungal and bacterial denitrifications was greatly reduced.
The alpine wetland had a lower contribution of fungal
denitrification (23.2%) than the predominant contribution of
81% found in peat incubation in tropical wetland soil with
respiration-inhibiting antibiotics in Indonesia.[27] It can be
concluded that the N2O gas was produced predominantly by
bacterial denitrification, and also that the produced N2O gas
was strongly eliminated in the soil layers by N2O reduction.
CONCLUSIONS
Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526
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This study provided the isotopic signature of N2O produced in
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The N2O production in the soil appeared to be moderately
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This study was supported by JSPS Grant-in-Aid for Scientific
Research (Kiban-A, No. 19201006) from the Ministry of
Education, Science, Culture, Sports and Technology of Japan.
The authors thank Associate Prof. Dr K. Yamada and Dr A. Fujii,
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Department of Environmental Chemistry and Engineering,
Tokyo Institute of Technology, for providing help and
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