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Stable carbon isotopic evidence of methane consumption andproduction in three alpine ecosystems on the QinghaieTibetanPlateau

Tomomichi Kato a,b,*, Keita Yamada c, Yanhong Tang d, Naohiro Yoshida c,e, f, Eitaro Wada a

aResearch Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, Yokohama, Kanagawa 236-0001, Japanb Laboratoire des Sciences du Climat et de l’Environnement, IPSL, CEA-CNRS-UVSQ, Orme des Merisiers, F-91191 Gif sur Yvette, FrancecDepartment of Environmental Chemistry and Engineering, Tokyo Institute of Technology, Yokohama 226-8502, JapandNational Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, JapaneDepartment of Environmental Science and Technology, Tokyo Institute of Technology, Yokohama 226-8502, Japanf Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo, 152-8551, Japan

h i g h l i g h t s

� Stable carbon isotope ratio in soil CH4 was examined in alpine ecosystems in China.� Alpine meadow and shrub showed different fractionation factors in methane oxidation.� In alpine wetland, acetate fermentation was major contributor on methane production.� Hummocks showed plant-mediated transport of CH4 from deep soil to the atmosphere.� In hollows, fractional oxidation rate on methane production was estimated at 7e36%.

a r t i c l e i n f o

Article history:Received 5 December 2012Received in revised form4 April 2013Accepted 3 May 2013

Keywords:Acetate fermentationCO2/H2 reductionFractionation factorPlant-mediated CH4 transport

a b s t r a c t

To understand the mechanisms of soil CH4 consumption and production in alpine ecosystems, we forthe first time examined the stable carbon isotope ratio of CH4 (d13CeCH4) at three major grasslandvegetation types, alpine meadow, alpine shrub, and alpine wetland, on the QinghaieTibetan Plateau.

The alpine meadow and shrub showed net CH4 absorption in their vertical profiles of CH4 concentrationin summer, but a difference in their processes. Whereas the alpine shrub was dominated by CH4 con-sumption in its soil profile, CH4 production in the alpine meadow could slightly cancel consumed CH4 inshallow soil from�0.3 to�0.1 m. This potential CH4 production can be attributed to the relatively wet soiltype of that ecosystem, which might allowmethanogenesis to act in moist soil lumps in the shallow layer.

The alpine wetland differed in methane production, consumption, and transport pathways betweenhummock and hollow plots. In summer, both plots were enriched in 13CeCH4 in dissolved CH4 in soil porewater, suggesting that CH4 production was conducted mainly by acetate fermentation. In autumn, CH4

productionwas shifted toward CO2/H2 reduction. Furthermore, in hummocks, plant-mediated transport ofCH4 by vascular plants appeared to perform passive CH4 flow fromdeep soil to atmosphere, which allowedthe produced CH4 to bypass the oxidation zone in shallow soil. In hollows, however, CH4 produced inshallow soil was subject to simultaneous oxidation. The fractional oxidation rate on gross CH4 productionin hollows was estimated by simple mass balance model at 7e17% in summer and 35e36% in autumn.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Methane (CH4), a major greenhouse gas (GHG), is exchangedbetween terrestrial ecosystems and the atmosphere through a va-riety of biological, physical, and chemical processes. However, hugeuncertainties still remain in our understanding of processes in theconsumption and production of methane in terrestrial ecosystems(IPCC, 2007).

* Corresponding author. Laboratoire des Sciences du Climat et de l’Envir-onnement(LSCE), IPSL, CEA-CNRS-UVSQ, Bât. 712, Orme des Merisiers, F-91191 Gifsur Yvette, France. Tel.: þ33 169 08 41 54; fax: þ33 169 08 77 16.

E-mail address: tomomichi.kato@lsce.ipsl.fr (T. Kato).

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The QinghaieTibetan Plateau (QTP) is one of the largest grass-lands (w2.0 � 106 km2) at high elevation (>4000 m a.s.l.) in theworld (Li and Zhou, 1998). Alpine grasslands, which are charac-terized by low temperature and plentiful sunlight, could be a netcarbon sink because of the lower decomposition rate of organicmatter and relatively favorable photosynthetic conditionscompared with higher-latitude cold ecosystems, such as tundra ortaiga forest. Alpine grassland soils store 33.5 pg C in organic form,accounting for approximately 2.5% of the global soil carbon pool(Wang et al., 2002). Many field measurements of CH4 flux havebeen conducted in the QTP. Kato et al. (2011) examined spatialvariation of CH4 fluxes and reported that both of their sites acted assinks and sources of atmospheric CH4 and also showed severalrelationships of CH4 flux with environmental factors over spatialvariation, including positive and negative relationships betweenCH4 flux and soil biomass to a 5 cm depth in nine grassland andseven wetland sites, respectively. Other studies have shown thatspatial variations in CH4 are related to vegetation zonations inswamps (Hirota et al., 2004) and have also indicated that yak dungand urea cause slight reductions in CH4 uptake in alpine meadows,suggesting positive impacts of grazing on regional GHG emissions(Lin et al., 2009). Several recent studies have also reported that netCH4 flux had apparent temporal changes: diurnal change in alpinewetland (Chen et al., 2010) and seasonal change in alpine meadow(Cao et al., 2008). Especially, Chen et al. (2010) showed that CH4

emission from Zoigewetland changed diurnally associatedwith thechange in redox potential of surface soil, and moreover, themaximum CH4 emission in the afternoon could be related tochanges in the fraction of CH4 oxidation and production by plantgas transport mechanism. As much as usual seasonal variation,thus, diurnal change should be examined to know the constrainingfactors for understanding the mechanism of CH4 circulation and todesign the sampling strategy for estimating daily average CH4budget, not only in alpine wetland, but also in other drier alpineecosystems: alpine meadow and alpine shrub. However, these CH4flux studies have discussed the mechanisms of CH4 consumptionand production pathways, mainly by the indirect relationship be-tween net ecosystem CH4 flux and environmental factors. Moredirect measurements, including isotopic information, should betaken for additional analysis of these mechanisms in alpine eco-systems on the QTP, particularly in-situ field experiments.

Stable isotope techniques have provided much information onthe attribution of CH4 to different microbial processes of con-sumption and production, as well as physical and chemical pro-cesses of transportation between the soil and atmosphere (e.g.,Chanton, 2005; Conrad, 2005). Nakagawa et al. (2002) measuredd13C in emitted CH4 fromwater surface in grasses in eastern Siberiaand proposed the fractional contribution of methane productionprocesses between acetate fermentation and CO2 reduction.Moreover, d13CeCH4 was used as a tracer in conjunction with theapplication of the inhibitor methyl fluoride (CH3F), which stoppedacetate fermentation, in Italian rice field soils (Krüger et al., 2002)and Finnish boreal peatland (Galand et al., 2010). Thus, d13CeCH4 isa useful proxy in determining the relative contribution of CH4production and consumption processes. However, compared to thelarge number of laboratory studies, in-situ field measurements ofd13CeCH4 are still quite limited.

To better understand the mechanisms of CH4 circulation inalpine ecosystems on the QTP, we examined the carbon isotopicsignature of the CH4 exchanged between the atmosphere and thesoil and the vertical profile of the carbon isotopic signature of CH4in the soil in three typical QTP ecosystems: alpine meadow, alpineshrub, and alpine wetland, at Haibei station of the Chinese Acad-emy of Sciences in different seasons. Focusing on temporal andbiome-specific variation of CH4 pathways, and assumed the

dominating contribution to net CH4 exchange with atmosphere byCH4 consumption in alpine dryer land, i.e. alpine meadow andalpine shrub, and that by CH4 emission in alpine wetland as pre-viously shown in Kato et al. (2011), here we arise several questionsparticularly as follows.

1. Does the CH4 pathway show clear diurnal and seasonal changesin alpine dryer land and in alpine wetland, respectively?

2. Is net CH4 uptake by ecosystem from atmosphere driven onlyby methanotroph in alpine dryer land?

3. Is net CH4 emission from ecosystem to atmosphere driven bysame pathway between geomorphologically typical two plots:hummocks and hollows, in alpine wetland?

2. Material and methods

2.1. Overview

d13CeCH4 was measured in three different biomes, alpinemeadow, alpine shrub, and alpine wetland, near the Haibei AlpineMeadow Ecosystem Research Station, Northwest Plateau Instituteof Biology, Chinese Academy of Science (37�29e450N, 101�12e230E;3250m a.s.l.). Diurnal courses of CH4 fluxweremeasured using fourchambers placed within a 5 � 5 rectangular grid in summer andtwo static chambers with 5-m distance in summer and winter,respectively. To measure d13CeCH4, just after the CH4 flux mea-surements, an air sample was collected from one static chamberand also from atmosphere at a height of 2.2 m. We used thechambers to measure the diurnal cycle of flux and d13CeCH4 at10:00, 14:00, 18:00, and 24:00 (Beijing Standard Time, BST) on 21July at the alpine meadow, 23 July at the alpine shrub, and 25 July2005 at the alpinewetland in summer, and on 4March at the alpinemeadow, 6 March at alpine shrub, and 8 March 2006 at the alpinewetland in winter.

Soil pore gas was sampled for CH4 concentration and d13CeCH4measurements at 14:00 and 24:00 (BST) in the alpine meadow atthe same time we measured chamber flux in summer and winterand at 14:00 in alpine shrub in summer only. At the alpine wetland,additional samplings were conducted to measure d13CeCH4 in CH4efflux and dissolved CH4 in the pore water of the soil layer simul-taneously both in hummocks and hollows at 14:00 (BST) on 22, 24,and 26 July 2007 and 20 and 24 October 2007.

2.2. Site description

The three measurement sites, an alpine meadow, alpine shrub,and wetland, were located within about a 2 km radius of the Haibeistation. The annual average temperature and precipitation for1981e2000 were �1.7 �C and 561 mm, respectively. The alpinemeadow soil is clay loam of Mat Cry-gelic Cambisols, whereas thealpine shrub soil is silty clay loam of Mol-Cryic Cambisols (Li et al.,2001). The wetland is characterized by mixed hummockehollowterrain, with hummocks representing 40%, hollows representing55%, and other features, 5% of the landscape. The study sites weregrazed by yaks and sheep every winter.

Plant communities at the fluxmeasurement sites are dominatedmainly by three major perennial sedges, Kobresia humilis, Kobresiapygmaea, and Kobresia tibetica in the alpine meadow (Li and Zhou,1998); a rosaceous shrub, Potentilla fruticosa, and the above threesedges in the alpine shrub (Zhao et al., 2006); and four species(K. tibetica, Carex pamirensis, Hippuris vulgaris, Blysmus sinocom-pressus) in the wetland (Zhao et al., 2010). In the wetland, the soilsurface is covered largely by hollows, and under plant canopies,hummocks are partially covered with moss (Distichium inclinatum

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(Hedw.) Bruch & Schimp., Brachythecium spp., and Encalypta spp.).Vascular plants begin growth in May and die out in October (Li andZhou, 1998).

2.3. d13CeCH4 measurement

2.3.1. SamplingChamber air was collected for isotopic measurement immedi-

ately after CH4 gas flux sampling (see section 2.4) through capillarytubes to avoid a sudden pressure change inside the static fluxchamber. An atmospheric air sample was also collected at 2.2 mheight and from the soil layer via capillary tubes and cylindricalstainless-steel soil gas-phase probes (GL Science Inc., Tokyo, Japan)at depths of�0.1,�0.3, and�0.7 m at alpine meadow and �0.1 and�0.3 m at alpine shrub. Pore water was sampled for the isotopicmeasurement of dissolved CH4 at depths of �0.1, �0.2, �0.3, �0.4,and�0.6m fromhummocks and�0.1,�0.2,�0.3,�0.5, and�0.7mfrom the alpine wetland. Water was also sampled at a nearbystream flowing into the river from the wetland. Additionally, in thealpine wetland, emerging gas bubbles were collected occasionallyon the same day as the flux measurement by placing invertedplastic cups at the surface of small open water spaces around thehollow chambers. The gas was stored in a pre-evacuated glass orstainless-steel bottle with a volume of 10 mL, 200 mL, or 500 mL,depending on the CH4 concentration.

2.3.2. Procedure of d13CeCH4 measurementd13CeCH4 and CH4 concentrations were measured using an on-

lineCH4 extraction systemconnected to a gas chromatography (GC)-combustion interface (ThermoQuest GC/C Interface; ThermoFinni-gan, Bremen, Germany) and a FinniganMAT252mass spectrometer(Finnigan MAT, Bremen, Germany). The GC-combustion interfaceconsisted of a HewlettePackard model 6890 gas chromatographequipped with a split/splitless injector and a capillary column (HP-PLOT Q, 30 m � 0.32 mm i.d., 20-mm film; HewlettePackard, PaloAlto, CA, USA), a combustion furnace (Al2O3 ceramic tube packedwith CuO, NiO, and Pt wires, 320 mm� 0.5 mm i.d.), a Nafion dryer,and an open split. The principle of operation is described in Yamadaet al. (2003). In this system, CH4 is extracted from air samples andintroduced online into the gas chromatograph,where it is purified at40 �C and 0.8 mL min�1 column flow (ultrapure helium), and thentransferred into the combustion furnace (960 �C) to convert CH4 intoCO2. CO2 is introduced into the mass spectrometer via the Nafiondryer and open split interface. d13CeCH4 is calibrated againstreference CO2 gas introduced before and after the sample gas.Quantification of CH4 is achieved by comparing the intensity of themass 44 peak to a CH4 standard mixture (102.60 ppmv CH4 in N2;TaiyoToyo Sanso Co. Ltd., Osaka, Japan). d13CeCH4 is calculated fromthe equation

d ¼�Rsample=Rstandard � 1

�� 1000ð&Þ (1)

where R denotes the isotope ratio (13C/12C) of the sample andstandard. The value of d13CeCH4 is relative to the isotope ratio ofthe international standard Vienna Peedee Belemnite (VPDB;Coplen,1995; Gonfiantini et al., 1995). The precision of repeated analyses(1s) was �0.3& for d13C and �0.03 ppmv for CH4 concentration(Yamada et al., 2003).

2.3.3. Definition of the fractionation factorTo calculate the fractionation factor in CH4 consumption by

alpine dryer land’s soil, the following formulas are applied. WhenCH4 is uptaken by the ecosystem, 13C enrichment in residual CH4can be written as

dchamber ¼ dair þ 3lnðCair=CchamberÞ (2)

where dchamber and dair are d13CeCH4 for the chamber gas andambient air samples, respectively, 3is the enrichment factor, andCchamber and Cair are the CH4 concentrations in the chamber air andambient air samples, respectively. The isotope fractionation factor,asoil, the bulk fractionation effect by the soil, is defined as

asoil ¼ 1000=ð1000þ 3Þ (3)

2.3.4. Kinetic isotope effect of microbial oxidationThe kinetic isotope effect bymicrobial oxidation of CH4 in alpine

dryer land is calculated in the following formulas as proposed inboreal forest soils by Reeburgh et al. (1997). Assuming onlyoxidation and diffusion of methane within the soil and zero CH4concentration at the bottom of the soil, then aox, the fractionationfactor in methane oxidation, could be calculated by the followingequation:

aox ¼ 1:0195*�1=

�1þ lnððdCzþ1000Þ=ðdC0þ1000ÞÞ

ln�½CH4�z=½CH4�0

��2

(4)

where dCz and dCo are d13CeCH4 at depths of z and 0 m, respec-tively, and [CH4]z and [CH4]0 are CH4 concentrations at depths of zand 0 m, respectively.

We can also estimate aox from asoil using chamber flux-derivedd13CeCH4 in the following equation (Snover and Quay, 2000):

asoil ¼�adiff*aox

�0:5(5)

where adiff, the fractionation effect by molecular diffusion, is givenas 1.0195 (Snover and Quay, 2000).

2.3.5. Calculation of d13CeCH4 in efflux from the soilThe CH4 collected in the flux chambers in alpine wetland was a

mixture of atmospheric and soil-derived CH4. d13CeCH4 for soil-emitted CH4 (dsoil) was calculated from chamber gas (dchamber)and ambient air (dair) samples, assuming two-component mixing.In our study, sampling was conducted only once during eachmeasurement period; therefore, obtaining stable dsoil from theslope of the regression line for dchamber versus inverse Cchamber(Keeling plot) was difficult. Hence, we calculated dsoil using thefollowing mass balance equation:

dchamber$Cchamber ¼ dair$Cair þ dsoil$Csoil (6)

where C is CH4 concentration, and Csoil ¼ Cchamber � Cair. dsoil valuesobtained from small Csoil values (<10 ppbv, corresponding to flux of<10 mg Nm�2 h�1) were not used for further data analysis, as inthese cases, dchamber was equal to dair within the precision of theanalysis, and the error propagated in the calculation of dsoil waslarge.

2.4. Chamber flux and environmental factor measurement

CH4 fluxes were measured using a 200-mm-diameter � 200-mm-long polyvinyl chloride (PVC) cylinder chamber (details arein Kato et al., 2011). At each sampling time, the PVC lids were tightlysealed to the top of the chambers with vinyl tape, and the inside airwas collected in 5-mL glass vials for CH4 gas at 1, 10, 20, 30, and60 min. The flux rates were calculated as a linear slope of con-centration evolution over the time courses. The concentrations ofCH4 were determined using a gas chromatograph equipped with aPorapak Q column (80/120 mesh) and a flame ionization detector

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(FID; GL 390B; GL Science, Tokyo, Japan), with pure helium (He) as acarrier gas. Injection, detection, and column-oven temperatureswere 120 �C, 150 �C, and 50 �C, respectively.

Environmental factors were measured within the gas samplingtime. The temperatures of the soil at 5 cm and open water sitesaround the chambers weremeasured using thermometers (CT-220;CUSTOM, Tokyo, Japan). Time domain reflectance (TDR) sensors(CS-620; Campbell Scientific Inc., Logan, UT, USA) were used todetect the averaged values of soil water content at 0e12 cm depth.In wetland sites, the water table height and soil oxidationereduc-tion potential (ORP) were collected. After chamber gas sampling, 5-cm-depth soil cores were collected, and the soil air filled space wasmeasured using a soil three-phase meter (Model DIK-1130; Daiki,Tokyo, Japan), and the water-filled pore space (WFPS) of the 5-cm-depth soil columns were determined by dividing the soil-water-filled space, as evaporated soil water from oven-dried soil, by theporosity, which was calculated as the sum of air-filled space andevaporated soil water from oven-dried soil. Aboveground plantbodies were collected by mowing. Soil samples were collected us-ing a core sampler for 0 to �5 cm depth, but belowground plantbiomass was not separated. Each sample was then weighed beforeand after oven-drying at around 80 �C for 48 h. The total carbon andnitrogen contents of plant bodies and of sieved and decarboxylatedsoil samples were measured using an NC analyzer (Sumigraph NC-900; Sumika Chemical Analysis Service Ltd., Osaka, Japan).

2.5. Measurement of d13C and d15N in plant and soil biomass

Dried plant and soil biomasses were placed in tin capsules afterprecise weighing, which totaled 0.1 and 4.0e5.0 mg for plantbiomass C and N, respectively, and w100 and w500 mg for soilbiomass C and N, respectively, and were then combusted at 1000 �Cin an Elemental Analyzer (Flash EA, 1112 Series; Thermo FisherScientific,Waltham,MA, USA). d13C and d15Nwere then determinedusing a Thermo Delta V Plus continuous-flow isotope ratio massspectrometer (Thermo Fisher Scientific) interfaced with theelemental analyzer using a Conflo III gas controller (Thermo FisherScientific). d15N is a relative value to the isotope ratio of the AirInternational Reference Standard (AIR).

3. Results

3.1. Alpine meadow and alpine shrub

3.1.1. Diurnal changes in isotope ratio and environmental factorsIn summer, CH4 fluxes fluctuated temporally, but showed

negative fluxes, except at 14:00 (BST) in the alpine shrub, with dailyaveraged fluxes of �0.022 � 0.014 in the alpine meadow, and�0.016 � 0.027 mg CH4 m�2 h�1 in the alpine shrub (Fig. 1b). Incontrast, in winter, CH4 fluxes were nearly neutral, as shown by thedaily average of �0.003 � 0.004 in the alpine meadow and0.002 � 0.005 mg CH4 m�2 h�1 in the alpine shrub (Fig. 1e).

The fractionation factor, asoil, showed large diurnal fluctuationsboth in summer and winter (Fig. 1a and d) due to the small dailyaverage of CH4 uptake by the ecosystem soil.

Soil surface temperature (Tsoil) showed a diurnal cycle, reachinga maximum at 14:00 or 18:00 and minimum at 24:00 or 6:00 insummer and winter (Fig. 1c and f). Soil water content (SWC)showed a constant value in summer (Fig. 1c).

3.1.2. Vertical profiles of CH4 concentration and d13CeCH4

In summer, low values of CH4 concentration in the alpinemeadow at a depth of �0.7 m were 1.50e1.60 ppm (Fig. 2c), andthose of d13CeCH4 ranged from �48.2& to �46.9& (Fig. 2a). Incontrast, high values were recorded at different depths beneath the

ground surface: �0.1 m for d13CeCH4 at 14:00 and at �0.3 m forCH4 concentration (Fig. 2a and c). In winter, the vertical profiles ofCH4 concentration and d13CeCH4 from the atmosphere to soil var-ied much less.

Summer CH4 concentrationwas greatly reduced at depth, with aminimum at �0.3 m in the alpine shrub (Fig. 2d).

3.2. Alpine wetland

3.2.1. Diurnal changes in soil-emitted d13CeCH4 and environmentalfactors

Soil-emitted d13CeCH4 (dsoil13 CeCH4) varied diurnally from�60.6& to �54.6& in summer (Fig. 3a). In winter, dsoil13 CeCH4 in-creases were associated with decreased CH4 flux (Fig. 3c).

In summer, CH4 fluxes showed large emissions, with a dailyaveraged flux of 11.84 � 12.20 mg CH4 m�2 h�1 (Fig. 3a). However,in winter, CH4 fluxes were small, averaging 0.0206 � 0.0134 mgCH4 m�2 h�1 daily (Fig. 3c).

Soil surface temperature showed a diurnal cycle (Fig. 3b and d).Soil water content decreased greatly at 24:00 in summer (Fig. 3b).

3.2.2. Vertical profile in d13CeCH4 and environmental factors inhummocks and hollows

d13CeCH4 dissolved in soil pore water (dDIS13 CeCH4) graduallydecreased in deeper soil in both hummocks and hollows (Fig. 4aand b), although it fluctuated temporally at �0.1 m depth inhummocks. dDIS13 CeCH4wasmore depleted in October than in July inhollows. High values of dissolved CH4 were found in the zone from�0.7 to�0.3 m in hummocks and�0.3 to�0.0 m in hollows (Fig. 4cand d), although these values fluctuated temporally.

Environmental factors highlighted the contrasts in vertical pro-files between the two sites and among seasons (Fig. 5). Tsoildecreased gradually to near zero along the depth profile in July, butwas relatively constant in value toward deeper soils at around 3 �Ce5 �C in October at both sites. The frozen soil layer was deeper than�0.7m in hummocks and�0.6m in hollows in July, but disappearedinOctober. ORP showed stable negative values at less than�0.3m inhummocks and�0.1m in hollows.MaximumpHoccurred at�0.1min hummocks, but was relatively constant in value in hollows alongthe depth profile. Hummocks showed gradual changes in d13C andd15N along the depth. Hollows were higher in soil carbon contentand nitrogen and C/N ratio but registered relatively lower values ofd13C and d15N from �0.1 to �0.3 m than in deeper soil. The watertable was nearly constant throughout measurements at both sites,occurring at�0.40 to�0.36m in hummocks and�0.04 to�0.015min hollows from the ground surface.

4. Discussion

The pathways of CH4 exchanged between soil and atmosphereare discussed here separately for alpine dryer land, i.e. alpinemeadow and alpine shrub in 4.1, and for alpine wetland, i.e. hum-mocks and hollows, in 4.2. Former part focuses on the fractionationfactor in CH4 consumption by soil, assuming the domination by soiloxidation of atmospheric CH4 in alpine dryer land. Latter part fo-cuses on the fractionation effect by CH4 production, consumptionand transport, given that soil-produced methane dominates theCH4 emitted from soil surface to atmosphere in alpine wetland.

4.1. CH4 production and consumption in alpine meadow and alpineshrub

CH4 fluxes showed diurnal variations of their mean values, butwith large standard deviation among chambers in summer (Fig. 1).asoil also showed a large amplitude in temporal variations in alpine

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meadow (Fig. 1a), but relatively constant values in alpine shrub,ranging from 0.9850 to 1.0130 in summer, probably indicating thatconsumption and production pathways did not change very muchdespite of large change in net CH4 flux. Nevertheless, the reliabilityof the data should be considered especially inwinter. We think thatobtaining large enough CH4 fluxes to yield sufficient accuracy incarbon isotopic signatures of CH4 flux estimated from chamber-derived CH4 collected within the short time period of this studywas difficult.

In summer, the vertical profile of CH4 concentration in thealpine meadow showed the lowest CH4 concentration at �0.7 mdepth (Fig. 2c). CH4 consumption occurred in deeper soil, and CH4could be transported from upper soil to lower soil by diffusion. Onthe other hand, the highest value of CH4 concentration occurred at�0.3 m, implying that a slight CH4 production occurred around thatdepth to compensate for the consumed CH4 in the soil (Fig. 2c). Incontrast, in winter, no apparent vertical profile was found in CH4concentration and d13CeCH4 in the alpine meadow, demonstratingno significant activity in consuming CH4, which was confirmed bythe nearly neutral CH4 flux (Fig. 1e).

aox was estimated by equation (4) from the vertical profile ofCH4 concentration and d13CeCH4 in the soil. In the alpinemeadow, the shallow layer from �0.3 to �0.1 m showed potentialCH4 production, and Equation (4) is not supposed to derive aox.On the other hand, we calculated aox in summer as 1.0070 and1.0048 at 14:00 and 24:00, respectively, in the �0.7 to �0.3 mlayer, and 1.0187 at 14:00 in the soil layer from �0.3 to �0.1 m inthe alpine shrub (Table 1). The difference in aox between alpinemeadow and shrub can be attributed to the variation in meth-anotrophic microbial compositions between the two ecosystems.In the alpine meadow, aox was within the range of that reportedin several culture studies: 1.005e1.031 (Barker and Fritz, 1981)and 1.007e1.027 (Zyakun et al., 1988) and similar to that in landfillcover soils: 1.008 � 0.003 (Bergamaschi and Harris, 1995) and1.008 � 0.004 (Bergamaschi et al., 1998). In the alpine shrub, aoxwas similar to that in grassland and forest soils: 1.022e1.025measured in situ in Alaskan boreal forest soil (Reeburgh et al.,1997) and 1.0121e1.0183 in native grassland and temperate for-est soil in Washington State, USA (Snover and Quay, 2000). Thoseimply that there could be a similarity in the methane pathways of

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Fig. 1. Diurnal changes in (a and d) the enrichment factor ( 3), (b and e) CH4 flux, and (c and f) soil surface temperature and soil water content in the alpine meadow and alpine shrubat Haibei station. Left panels show the results in (aec) summer, and right panels show (def) winter results. Error bars show standard deviation among chambers.

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Fig. 3. Diurnal changes in (a and c) soil-emitted d13CeCH4 and CH4 flux, and (b and d) soil surface temperature and soil water content in the alpine wetland at Haibei station. Leftpanels show the results in (a and b) summer, and right panels show them in (c and d) winter. Error bars show standard deviation among chambers.

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CH4

concentration (ppmv)

CH4

concentration (ppmv)

b d

ca

Heig

ht (m

)H

eig

ht (m

)

Air

AirShrub

Fig. 2. Vertical profile of (a and b) d13CeCH4 and (c and d) CH4 concentration in the soil in the (a and c) alpine meadow and (b and d) alpine shrub. Filled symbols show summer dataon 21 July and 23 July 2005 at the alpine meadow and shrub, respectively. Open symbol shows winter data on 4 March 2006 at the alpine meadow.

T. Kato et al. / Atmospheric Environment 77 (2013) 338e347 343

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both alpine meadow and shrub with those in corresponding typesof soil in quoted studies.

4.2. CH4 production and consumption in alpine wetland

4.2.1. Diurnal change in CH4 productionIn summer, dsoil13 CeCH4 fluctuated temporally, suggesting that

methane production pathways changed temporally, but did notshow apparent periodical diurnal changes along with temporalchanges in environmental factors: CH4 flux, Tsoil, and SWC (Fig. 3aand b). Methane production and consumption, thus, are likelycontrolled by other environmental conditions (e.g., pH and ORP)below the surface layer.

In winter, dsoil13 CeCH4 and CH4 flux showed similar temporalchanges in the opposite direction (Fig. 3c). This contrast suggeststhat the magnitude of CH4 flux is controlled by methane oxidation,which reduces net CH4 efflux and enriches d13CeCH4 in the soilsimultaneously. However, the CH4 source is questionable, as the soillayers were frozen (Fig. 3d), and soil microbes should not have beenso active. Hence, we assumed that the emitted CH4 gas originatedpartly from trapped CH4 produced before the soil was frozen.

4.2.2. CH4 production, consumption, and transport in hummocksLow ORP and high dissolved CH4 concentration indicated that

methane production occurred largely in soil deeper than �0.3 m(Fig. 5a). Moreover, the dDIS

13 CeCH4 in soil pore water, ranging from�61.3& to�53.9& (Fig. 4a), suggested that methanewas producedmore by acetate fermentation (reported as �60& to �27&;Chanton, 2005) than by CO2/H2 reduction (reported as �83.6& to

�61.5&; Nakagawa et al., 2002). Slightly lower dDIS13 CeCH4 values inOctober at soil below �0.3 m (Fig. 4a) implied that the fractionalrate of the methane production pathway shifted toward CO2/H2reduction rather than acetate fermentation. This possible shiftcould attribute to a seasonal change in the quality and quantity ofsubstrate supply. As discussed in spatial variation of dDIS

13 CeCH4values in soil pore water in Canadian peatland (Bellisario et al.,1999), when fresh organic material is utilized in sites with highplant productivity, the acetate fermentation pathway is thought todominate over CO2/H2 reduction. In opposite, less productive plantcommunity tends to use CO2/H2 reduction pathway. Assumed thatfresh organic material from vascular plant was greatly reduced dueto the death of aboveground biomass in October, the CH4 produc-tion pathways can be shifted toward CO2/H2 reduction, which couldalso be partly affected by lower Tsoil and ORP in October (Fig. 5a).

Enrichment of dDIS13 CeCH4 in surface soil associated with

increasing ORP (Fig. 5; i.e., becoming oxidative) suggested that theCH4 produced in deeper soil was consumed by methanotrophs inshallower soil. Methane oxidation could also be supported by thecontrast between the very low CH4 concentration at �0.1 m andvery high CH4 concentration at �0.7 to �0.3 m (Fig. 4c). Thus, thispossible methane oxidation will greatly explain the far smaller CH4

efflux in hummocks than in hollows (Fig. 6).Except for 24 October, dsoil13 CeCH4 (Fig. 6) was more depleted

than dDIS13 CeCH4 in shallow soil at �0.1 and �0.2 m by 8.8&e22.9&

(Fig. 4a), similar to results reported in a Minnesota bog (Chasaret al., 2000). This suggests that the aerenchyma of vascular plantsconducts passive CH4 transport from soil deeper than �0.2 mdirectly to the atmosphere through air spaces in the plant body

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ig

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)

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Fig. 4. Vertical profile of (a and b) d13CeCH4 and (c and d) CH4 concentration of dissolved methane in pore water in the soil at (a and c) hummocks and (b and d) hollows in thealpine wetland at Haibei station. Mean values and standard deviations (error bars) are shown for bubbles among multiple samplings. Identical values are indicated in the streambetween hummocks and hollows. Gray bars indicate the range of the water table during the measurements.

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before enrichment by methanotrophs in shallow soil, as onedominant sedge species at this study site has been reported toperform plant-mediated transport of methane gas in severalstudies (Whiting and Chanton, 1992; Kelker and Chanton, 1997).This idea is also consistent with our finding of slightly moreenriched dsoil

13 CeCH4 in October than in July (Fig. 4a). Assuming thatplant-mediated transport decreased due to vascular plant senes-cence in October, the blocked CH4 would rise through the shallowsoil, where methanotrophs enriched dDIS

13 CeCH4, leading to theenriched dsoil

13 CeCH4 captured by the static chamber at the groundsurface. This plant-mediated transport is a very important issue inGHG budgets in alpine wetlands that are grazed by livestock, asgrazing would provoke larger mass-flow of methane outgassing, asproposed by Hirota et al. (2005).

4.2.3. CH4 production, consumption, and transport in hollowsThe vertical profile showed relatively constant dDIS13 CeCH4 values

in July in hollows (Fig. 4b), suggesting that the pathway in methaneproduction was possibly kept constant by the straight vertical

profiles of ORP and pH, except for positive ORP at 0 m (Fig. 5b). Incontrast, the vertical profile of dissolved CH4 concentration varied,indicating relatively smaller values below �0.4 m, and thus lessproduction of CH4 (Fig. 4d). This was probably due to a low sub-strate supply for methanogenesis induced by the low decomposi-tion rate of soil organic biomass associated with lower Tsoil andlower C and N content below �0.4 m (Fig. 5b). In July, enricheddDIS13 CeCH4 (�60.5& to �56.5& at all depths) revealed thatmethane production was mainly due to acetate fermentation(Fig. 4b) as it was in hummocks. On the other hand, in October,dDIS13 CeCH4 was depleted to between �72.9& and �65.8% below�0.4 m, and dDIS

13 CeCH4 in water bubbles was also more depleted at�72.4& (Fig. 4b). Those low dDIS

13 CeCH4 values indicated that themethane production pathway could be dominated by CO2/H2

reduction rather than by acetate fermentation. This shift in themethane production pathway in October was apparently also dueto the change in quality and quantity of the substrate supply fromroot exudation due to the death of vascular plants in October, aswell as by lower Tsoil in shallower soil (Fig. 5b), similar to the sit-uation in hummocks.

We measured heavier dsoil13 CeCH4 than dDIS13 CeCH4 in surface soil

pore water (�0.1 m) by 1.4&e6.6&, reflecting the effect ofmethane oxidation on produced CH4. Chasar et al. (2000) reportedmethane oxidation in shallow water in a Minnesota bog to be arelative fraction of 0.17e0.74. We applied the simple mass balanceequation to estimate the fraction of methane oxidized by meth-anotrophs, f, as proposed by Chasar et al. (2000):

f ¼ ðdsoilC� dDISCÞ=½ðaox � 1Þ*1000� (7)

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N/C)%( N)%( CHp)C

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N/C)%( N)%( CHp)C

15N

(‰)

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15N

(‰)

Fig. 5. Vertical profile of environmental factors: soil temperature (Tsoil; �C), oxidationeredox potential (ORP; mV), soil pH, soil organic carbon content (%), nitrogen content (%), C/Nratio, soil organic d13C (&), and soil organic d15N (&) in the alpine wetland. Upper panels show the results in (a) hummocks and lower panels show them in (b) hollows.

Table 1Fractionation factor in methane oxidation (aox) in the soil at alpine meadow andshrub in summer of 2005.

Interval depth Alpine meadow Alpine shrub

21 July2005 (14 h)

21 July2005 (24 h)

23 July2005 (14 h)

�0.1 to �0.3 m n.d. n.d. 1.0187�0.3 to �0.7 m 1.0070 1.0048 n.d.

n.d. shows no data.

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where aox is valued at 1.018 as the median value of the reportedrange of 1.005e1.031 (Barker and Fritz, 1981;Zyakun et al., 1988;Bergamaschi and Harris, 1995; Reeburgh et al., 1997; Bergamaschiet al., 1998; Snover and Quay, 2000). Then, f was calculated as0.07, 0.14, and 0.17 on 22, 24, and 26 July, respectively, and at 0.35and 0.36 on 22 and 24 October, respectively. The enhancedmethane oxidation ratio in October should have contributed partlyto the decrease in CH4 efflux in October compared to that in July(Fig. 6b). Recently, methane oxidation within shallow water hasbeen reported to be attributed to symbiotic methanotrophic bac-teria in brown-moss cover in Siberian tundra (Liebner et al., 2011),as reported previously in a laboratory study of peat-moss from ninepeatlands (Kip et al., 2010). Hollow sites in this study were coveredlargely with mosses, which was potentially influenced by the pro-posed symbiotic methanotrophic bacteria.

In contrast to hummocks, dsoil13 CeCH4 was more enriched thandDIS13 CeCH4 (Fig. 6) in shallow soil at�0.1 and�0.2 m in the range of�1.4& to �7.8& (Fig. 4b). The mostly smaller coverage of vascularplants in hollows than in hummocks did not seem to conduct sig-nificant passive CH4 transport from deeper soil directly to theatmosphere.

5. Conclusions

Our field investigation depicted the temporal and biome-specific changes in carbon isotopic signatures in CH4 flux, soilgaseous CH4, and dissolved CH4 in soil pore water in three alpineecosystems on the QTP for the first time.

Diurnal fluctuation in asoil or soil-emitted dsoil13 CeCH4 was shown

in all three ecosystems. Although identifying the controlling factoron them only from soil-emitted dsoil

13 CeCH4 measurements wasdifficult due to poor correlation between isotopic signatures andenvironmental factors, alpine meadow showed a large diurnalchange in the pathway of CH4 exchange in summer, but alpineshrub described a relatively constant composition of the pathwayin summer. On the other hand, diurnal change in soil-emitteddsoil13 CeCH4 in alpine wetland in winter implied the contributionbymethane oxidation. Moreover, the seasonality in vertical profilesof isotopic ratios and environmental factors in alpine wetlandyielded many insights about seasonal changes of CH4 consumptionand production pathways. CH4 production appeared to be

conducted mainly by acetate fermentation in summer at both plots,as indicated by the enrichment of 13CeCH4 in dissolved CH4 in soil,but at different soil depths: �0.7 to �0.4 m in hummocks and �0.4to 0 m in hollows, respectively. In autumn, those 13CeCH4 valueswere more depleted, and shifted toward CO2/H2 reduction, slightlyin the hummocks and significantly in the hollows.

Although the alpine meadow and shrub showed similar mag-nitudes of net CH4 absorption in chamber flux measurements insummer, their pathways appeared to differ. Whereas alpine shrubindicated domination by CH4 consumption in its soil profile, alpinemeadow showed that slight CH4 production could occur, whichcanceled consumed CH4 in shallow soil from �0.3 to �0.1 m. Thispotential CH4 production can be attributed to the relatively wet soilof that ecosystem, which might allow methanogenesis to occur inmoist soil lumps in the shallow layer. The difference in aoxs esti-mates,1.0187 in alpine shrub and 1.0048e1.0070 in alpinemeadow,suggests the variation in methanotrophic microbial compositionsbetween the two sites.

The alpine wetland was a clear example of large spatial hetero-geneity in methane production, consumption, and transport path-ways between hummock and hollow plots, which led to contrastingsmall and large CH4 effluxes, respectively, from the ecosystem to theatmosphere. In addition to the difference in CH4 productionpathway mentioned above, the consumption and transportationindicated clear differences as follows. In hummocks, the transportand consumption of part of the produced CH4 was apparentlytransported directly fromdeep soil to the atmosphere by the passiveCH4 flow induced by the aerenchyma of vascular plants, but most ofthe produced CH4was oxidized in shallow soil. This plant-mediatedtransport, which allowed the produced CH4 to bypass the oxidationzone in shallow soil, would support a larger d value in soil emitted13CeCH4 than that in dissolved 13CeCH4 in shallow soil pore waterbetween�0.1 and�0.2 m in the range of 8.8&e22.9&. In contrast,in hollows, the produced CH4 in shallow soil was also subject tosimultaneous oxidation. A simple mass balance model suggestedthat gross CH4 production was reduced by 7%e36% on its way torelease into the atmosphere through soil pore water.

Those findings contribute to the understanding of globalmethane circulation, especially through improving terrestrialbiosphere models. Nevertheless, further investigation should beconducted using carbon and hydrogen isotopic signatures in the

wolloH )bkcommuH )a

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Fig. 6. Temporal changes in soil-emitted dsoil13 CeCH4, CH4 efflux, and CO2 efflux in (a) hummocks and (b) hollows in July and October 2007. Values are means with standard deviation

of the data from four chambers.

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substrates (e.g., dissolved organic carbon, acetate acid, and dis-solved CO2 gas), which were not measured in this study, to describethe mechanisms more precisely.

Acknowledgments

This study was supported by JSPS Grant-in-Aid for ScientificResearch (Kiban-A, No. 19201006) from the Ministry of Education,Science, Culture, Sports and Technology, Japan, and also by aresearch project on climate change and its impact on alpine eco-systems from Ministry of the Environment, Japan. The authorsthank Prof. S. Toyoda, Department of Environmental Science andTechnology, Tokyo Institute of Technology, and Dr. N. Suzuki andother members in Prof. N. Yoshida’s laboratory in the Departmentof Environmental Chemistry and Engineering, Tokyo Institute ofTechnology, for providing help and instruments for isotopic mea-surement of CH4. The authors also thank Prof. M. Hirota, Universityof Tsukuba, and Prof. S. Mariko, Hosei University, for providing theinstruments and advice to measure CH4 gas concentrations.

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