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Methane production in relation with temperature, substrate and soil depth in Zoige wetlands on Tibetan Plateau
Acta Ecologica Sinica 31 (2011) 121–125
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Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Methane production in relation with temperature, substrate and soil depth in Zoige wetlands on Tibetan Plateau Jianqing Tian a,b,1, Huai Chen c,d, Yanfen Wang b,⇑, Xiaoqi Zhou b a
Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China c College of Forestry, Northwest Agriculture and Forest University, Yanglin 712100, PR China d Institute of Environment Sciences, Quebec University at Montreal, Montreal, Canada C3H3P8 b
a r t i c l e
i n f o
Article history: Received 21 November 2010 Revised 11 January 2011 Accepted 27 January 2011
Keywords: Tibetan Plateau Archea Methanogenesis Greenhouse gases Peatland Carbon biogeochemistry
a b s t r a c t To understand the effect of substrate and temperature on methane production in high frigid wetlands, soil samples along the soil profile were collected from Zoige alpine wetlands, a ‘‘hotspot’’ of methane emission on Qinghai-Tibetan Plateau. Our result showed that temperature had a positive influence on methane production. However, acetate accumulation was negative related with temperature. We found that supplemental methanol and acetate strongly stimulated methane production. Such result indicated that methanol and acetate are major substrates for methane production in Zoige wetlands. Moreover, we also found that methane production rates decreased with soil depths due to the decrease of available substrate in deeper soil. Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Among all natural methane (CH4) sources, natural wetland is regarded as the single largest source which may contribute 10%–30% to the global budget [1]. Among them, wetlands in cold areas, such as northern wetlands and tundra wetlands, contribute a large proportion [2]. Recently, scientists have been trying to get insight into the methanogensis in cold environments [3,4]. Major attentions are paid to the effect of substrate and temperature on CH4 production, the psychrophiles that may implement the bioconversion of biomass into CH4 and key environmental factors that are likely to have influence on CH4 emission. CH4 production depends on the quantity and quality of substrates. Because the amount and decomposability of soil organic matters determine the supply of substrate for methanogens [5]. On the micro-scale, the distribution of fresh organic matter is the dominant factor explaining the spatial variation in CH4 production [6]. Furthermore, methanogenesis is controlled more by the availability and composition of the substrate than by the amount of methanogens [7]. Hence, substrate availability is a predominant ⇑ Corresponding author. Address: 19(Jia), YuQuan Road, Shijingshan, Beijing 100049, PR China. Tel.: +86 10 88256066, fax: +86 10 88256715. E-mail addresses: [email protected] (J. Tian), [email protected] (Y. Wang). 1 Tel.: +86 10 64807512; fax: +86 10 64807505.
constraint for CH4 production under nature condition rather than abiotic factors [8]. Temperature is one of the most important abiotic factor controlling CH4 production [9,10]. Previous studies observed that CH4 fluxes can be described by a single linear equation with a factor of temperature. In particular, CH4 production rate depends greatly on the temperature under a physiological temperature range of methanogens in a low temperature environment [3,4,11,12]. Studies on CH4 emission in Qinghai-Tibetan Plateau (QTP) suggested that Zoige wetland is a major centre for CH4 generation in the plateau [13]. However, little is yet known about the effect of temperature and the substrate quality on CH4 production. Therefore, the objectives of this study are aimed to discuss how CH4 production response to temperature and substrate. 2. Material and Methods 2.1. Site description The Zoige alpine wetland is located on the northeastern edge of the QTP which belongs to the cold Qinghai-Tibetan climatic zone. Alpine lakes and peatlands are well developed. Mean annual temperature is about 0.6 °C–1.2 °C, and mean annual precipitation is approximately 560–860 mm. The soil pH is about 6.8–7.2. The total C of soil and the depth of standing water table are 179.17 g kg 1and 8.6 cm, respectively [14].
1872-2032/$ - see front matter Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chnaes.2011.01.002
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J. Tian et al. / Acta Ecologica Sinica 31 (2011) 121–125
2.2. Incubation methods Soil samples were collected from a 75 75 m sampling square submerged wetland with the soil depth of 10 cm, 20 cm and 30 cm on 20th August, 2005. For each depth, three replicates were taken and put into three separate sterile 100 ml vials, flushed with nitrogen (N2) sealed with butyl rubber stopper, kept in a cool box, then immediately taken back to laboratory for preparation. Incubation experiment was set up to measure CH4 potential production of the soil. The slurries were made by the soil samples and modified medium (1:1, V/V). The modified medium [15] contained the waterlogged from the study site. Approximately 10 ml slurry was placed in 25 ml screw-capped tubes sealed with butyl rubber stoppers, with a gas phase of N2. All of them were done in the anaerobic glove box under the N2 atmosphere. Every triplicate was cultivated for six months at 6 °C, 15 °C and 30 °C. During the five month’s incubation time, gas samples (30 ll) were taken from the headspace with gas-tight syringes and analyzed by gas chromatography twice in the first month and once every month afterward. The liquid samples were taken and stored at –20 °C for further analysis. Twenty-fold diluted samples (5% v/v suspension) were used to examine the ability of the peat methanogens to produce CH4 with hydrogen:carbon dioxide (H2:CO2) (4:1), acetate, trimethylamine (TMA) and methanol. The diluted samples were put into the screw-capped tubes containing medium supplemented with 40 mM each of substrate. Triplicates of each treatment were incubated at 30 °C, and CH4 concentration was measured weekly. Before analysis, all the test tubes were thoroughly shaken to equilibrate gases between the slurries and headspace. To obtain more evidence for the existence of specific psychrophilic methanogenic communities, the wetland slurries (soil:medium; 1:1) pre-incubated at 6 °C for six months were diluted to 20-fold (soil: medium; 1:20) by medium with substrates (i.e. H2/ CO2, methanol, acetate, TMA) incubation at 15 °C and 30 °C, respectively. Triplicates of each treatment were set up, and CH4 concentration was measured weekly. CH4 and acetate concentrations were measured with a gas chromatograph (Shimadzu GC–14B) equipped with a Flame Ionization Detector and Nitrogen used as carrier gas at a flow rate of 50 ml min 1. For CH4 concentration measurement, column, injector, and detector temperatures were 50 °C, 80 °C and 100 °C, respectively, while acetate was measured by 230 °C, 250 °C and 280 °C, respectively.
Table 1 CH4 production rates at different incubation temperatures and soil depths. Temperature °C
6 15 30
CH4 production rates (lmol g
1
[fresh soil] d
1
)
30 cm
20 cm
10 cm
0.12 ± 0.03 0.21 ± 0.05 1.36 ± 0.02
0.12 ± 0.01 0.36 ± 0.04 1.65 ± 0.07
0.12 ± 0.01 0.49 ± 0.06 1.75 ± 0.02
(Table 1). At 6 °C, all the three layers had similar CH4 production potential and the rates of methane production was 0.12 lmol g 1 [fresh soil] d 1. The Q10 value showed that substrate (soil samples from different layers) have stronger influence on CH4 production than temperature at lower temperature treatment (data not shown). Q10 values varied with soil depth, which may be partly due to the components of substrates soil contains [8]. CH4 production was significantly related with acetate accumulation at lower temperature, such as 15 °C and 6 °C (Fig. 1A-D). Acetogenesis predominated on methanogenesis at 15 °C and below during the first month of incubation, and up to 14 mM acetate accumulated during this period. After that, the acetate accumulation was suspended by lack of substrate. At 6 °C, the accumulated acetate was not utilized even after five month of incubation. When incubated at 30 °C, there were no obvious acetate accumulation and CH4 production lagging. CH4 potential production reached the peak in the first month. 3.2. Supplemental experiments To test the effect of substrate on CH4 production, supplemental experiments were set up. Comparing to control, all substrates can stimulate CH4 production with different rates: methanol >TMA>acetate>H2/CO2 (Fig. 2). Both methanol and TMA can be used to produce CH4 at the same time. H2/CO2 was hardly used as substrate by hydrogenotrophic methanogens in this area (Fig. 2). When samples were pre-incubated at 6 °C, methanogens used all the supplemental substrates except H2/CO2 to form CH4 very quickly from the second day of the 30 °C incubation, the seventh day of the 15 °C incubation with a short-lag phase. The final CH4 concentration with methanol supplemented as substrate at 15 °C was higher than that at 30 °C (Fig. 3). 4. Discussion
2.3. Statistics analysis Within one month, CH4 concentration increased linearly at all temperature tested. The rates of CH4 production and the growth rates of methanogens were calculated by linear regression analysis and the accumulation by the difference between the final and initial gas concentrations. One-way analysis of variance (ANOVA) was employed to determine the effect of temperature and soil depth on methane production. ANOVA and regression analysis were performed using SPSS 12.0 software (SPSS Inc., USA). 3. Results 3.1. CH4 production during incubation The experiment was performed with slurries of fresh soil samples that were incubated at three temperatures. Measurement of CH4 potential production in incubated peat samples revealed highest CH4 production in the upper waterlogged peat layer at 30 °C and 15 °C, and a decrease of rates with depth and temperature
4.1. Temperature effects Supposing a linearity between CH4 production in situ and in laboratory, CH4 production in laboratory can be used to reflect production in situ [10]. Hence, under the laboratory incubation conditions, it is not only possible but also convenient to understand the relationship between temperature and CH4 production when the other regulating factors are controlled. In this study, CH4 production can be detected at all temperatures with different rates. The average rates at all the depth showed that CH4 production rates were approximately 2–3 times and 7–10 times greater at 30 °C than that at 15 °C and 6 °C, respectively. Hence, CH4 production rates strongly depended on temperature. Besides methane, acetate was accumulated in fresh soil of Zoige wetland during first month at 15 °C and 6 °C. During the subsequent months, acetate was transformed into CH4 at 15 °C; while the acetate kept untransformed at 6 °C. The explanation was that 15–20 °C was a threshold for end products formation in lowtemperature environments like tundra permafrost soil, lake sediments and paddy soil [11,16,17].
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J. Tian et al. / Acta Ecologica Sinica 31 (2011) 121–125
B
20
16
20
15ºC
30cm 20cm 10cm
Acetate concentration (mmol/L)
CH4 concentration (mmol/L)
A
12
8
4
30cm 15ºC 20cm 10cm
16
12
8
4
0
0 0
30
60
90
120
150
0
30
60
Days
D
20
16
120
150
20
6ºC
30cm 20cm 10cm
Acetate concentration (mmol/L)
CH4 concentration (mmol/L)
C
90
Days
12
8
4
6ºC
30cm 20cm 10cm
16
12
8
4
0
0 0
30
60
90
120
150
0
30
Days
60
90
120
150
Days
Fig. 1. Formation CH4 and acetate in two times diluted samples not supplemented with substrate at different temperatures (6 °C, 15 °C) and depths (10 cm, 20 cm and 30 cm).
0.6
CH4 concentration (mmol/L)
CH4 rates umol L-1 d-1
2.0
0.4
0.2
30ºC 15ºC
1.6
1.2
0.8
0.4
0.0 0.0
0
H2/CO2
TMA
Acetate
Methanol
CK
Fig. 2. CH4 production rates from four different substrates at 30 °C (TMA = trimethyaine, CK = without adding substrate). The rates were calculated by linear regression analysis.
10
20
30
40
50
Days Fig. 3. CH4 final concentration produced from methanol at 30 °C and 15 °C after pre-incubation at 6 °C. The circle and square symbol represented 15 °C and 30 °C, respectively.
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4.2. Substrates influenced CH4 production Theoretically, about two thirds of CH4 is produced from acetogenic methanogenesis [22]. Our incubation experiments with soil samples from alpine ecosystem provided evidence that CH4 produced from acetate and methanol is much more than that from H2/CO2. Jiang et al. [23] reported that methanol-derived methane could account for a large portion in Eleocharis valleculosa soil in the same peatlands. In situ, methanol is emitted from the flowering plants [24–26] and plant decay [27]. Pectin of dead plant material alone can produced 800 Tg y 1 methanol, and the large part is going to dissolve in soil water and utilized as microorganism substrate [24]. 90%–95% of Zoige Plateau was covered with plant. Because of long-term waterlogged conditions, such wetlands are able to release much methanol, which may supply substrate for methanogens. Furthermore, phylogenetic tree revealed that most of sequences were affiliated with methylotrophic and acetoclastic methanogens [28], which indirectly support most of CH4 origination from acetate or/and methanol. 4.3. Soil depth influenced CH4 production In anoxic environment with constant thermal conditions, the potential CH4 production depends on substrate availability [18]. In Zoige wetlands, the maximum CH4 production rate took place within the upper 10 cm and the rate then decreased with soil depth at all incubation temperatures (Fig. 2). This is consistent with previous studies [19–21]. Decreased CH4 potential production with depth may be associated with different substrate availability at varied depth. As the fresh organic matter is a dominant factor for the spatial variation in methane production [6]. Shallow layer is younger and contains more labile and easily mineralized carbon sources from newly deposited organic matter [5], which will lead to higher methanogens activity and the overcoming of methanogens in such layer. Additionally, deeper peat deposits in natural wetlands showed limited production rates due to unfavorable component accumulations for microbiological activity, such as ligins, and phenolic or humic substrates. Another explanation is possible, that other micro-organisms in deeper layer out-competed methanogens and resulted in low CH4 potential production and high acetate accumulation. These results, acetate accumulation increased with soil depth suggested that the capability of methanogens competing with other acetogenic bacteria at deeper layer was weaker than in the upper layer at low temperature.
5. Conclusion The activity of methanogensis strongly depended on temperature, substrate and soil depth which indicated both are important influence factors for CH4 production. Methanogensis occurred at a widely temperature range even at low temperature (6 °C), it is consistent with its cold environment. Notably, methanol could serve as the more important methanogenic precursors than expected in Zoige wetland. In future, the contribution of methanol-derived methane should be paid more attention. To some extent, global warming might result in more CH4 emission from wetlands on the QTP due to rising temperature and more substrates from the plants. Acknowledgements This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (kzcx2-yw-418-03).
Two anonymous reviewers were thanked for their detailed evaluation and suggestion on our manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chnaes.2011.01.002. References [1] IPCC. Summary for Policymakers, in: Climate Change 2007. The Physical Science Basis, Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. [2] R.J. Cicerone, R.S. Oremland, Biogeochemical aspects of atmospheric methane, Global Biogeochemical Cycles 24 (1988) 299–327. [3] M. Metje, P. Frenzel, Effect of temperature on anaerobic ethanol oxidation and methanogenesis in acidic peat from a northern wetland, Applied and Environmental Microbiology 71 (2005) 8191–8200. [4] M. Metje, P. Frenzel, Methanogenesis and methanogenic pathways in a peat from subarctic permafrost, Environmental Microbiology 6 (2007) 954–964. [5] D.W. Valentine, E.A. Holland, D.S. Schimel, Ecosystem and physiological controls over methane production in northern wetlandsM, Journal of Geophysical Research 99 (1994) 1563–1571. [6] G. Wachinger, S. Fiedler, K. Zepp, et al., Variability of soil methane production on the micro-scale: spatial association with hot spots of organic material and Archaeal populations, Soil Biology and Biochemistry 32 (2000) 1121–1130. [7] C. Joulian, B. Ollivier, H.-U. Neue, et al., Microbiological aspects of methane emission by a ricefield soil from the Camargue (France): 1. Methanogenesis and related microflora, European Journal of Soil Biology 32 (1996) 61–70. [8] I. Bergman, B.H. Svensson, M. Nilsson, Regulation of methane production in a Swedish acid mire by pH, temperature and substrate, Soil Biology and Biochemistry 30 (1998) 729–741. [9] B.H. Svensson, Different temperature optima for methane formation when enrichments from acid peat are supplemented with acetate or hydrogen, Applied and Environmental Microbiology 48 (1984) 389–394. [10] P. Westermann, Temperature regulation of methanogenesis in wetlands, Chemosphere 26 (1993) 321–328. [11] A.N. Nozhevnikova, C. Holliger, A. Ammann, et al., Methanogenesis in sediments from deep lakes at different temperatures(2–70°C), Water Science and Technology 36 (1997) 57–64. [12] O.R. Kotsyurbenko, A.N. Nozhevnikova, T.I. Soloviova, et al., Methanogensis at low temperatures by microflora of tundra wetland soil, Antonie van Leeuwenhoek 69 (1996) 75–86. [13] H. Jin, J. Wu, G. Cheng, et al., Methane emissions from wetlands on the Qinghai–Tibet plateau, Chinese Science Bulletin 44 (1999) 2283–2286. [14] H. Chen, N. Wu, Y. Gao, et al., Spatial variations on methane emissions from Zoige alpine wetlands of Southwest China, Science of the Total Environment 407 (2009) 1097–1104. [15] A.J.B. Zehnder, K. Wuhrmann, Physiology of a methanobacterium strain, AZ Archives of Microbiology 111 (1977) 199–205. [16] A.N. Nozhevnikova, V. Nekrasova, A. Ammann, et al., Influence of temperature and high acetate concentrations on methanogenensis in lake sediment slurries, FEMS Microbiology Ecology 62 (2007) 336–344. [17] R. Conrad, F. Bak, H.J. Seitz, et al., Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment, FEMS Microbiology Letters 62 (1989) 285–293. [18] J.-L. Garcia, B.K.C. Patel, B. Ollivier, Taxonomic, phylogenetic, and ecological diversity of methanogenic archaea, Anaerobe 6 (2000) 205–226. [19] C. McKenzie, S. Schiff, R. Aravena, et al., Effect of temperature on production of CH4 and CO2 from peat in a natural and flooded boreal forest wetland, Climatic Change 40 (1998) 247–266. [20] P.E. Galand, S. Saarnio, H. Fritze, et al., Depth related diversity of methanogen Archaea in Finnish oligotrophic fen, FEMS Microbiology Ecology 42 (2002) 441–449. [21] B.A. Hales, C. Edwards, D.A. Ritchie, et al., Isolation and identification of methanogen-specific DNA from blanket bog feat by PCR amplification and sequence analysis, Applied and Environmental Microbiology 62 (1996) 668– 675. [22] R. Conrad, Control of microbial methane production in wetland rice fields, Nutrient Cycling in Agroecosystems 64 (2002) 59–69. [23] N. Jiang, Y. Wang, X. Dong, Methanol as the primary methanogenic and acetogenic precursor in the cold Zoige wetland at tibetan plateau, Microbial Ecology 60 (2010) 206–213. [24] I.E. Galbally, W. Kirstine, The production of methanol by flowering plants and the global cycle of methanol, Journal of Atmospheric Chemistry 43 (2002) 195–229. [25] Y. Fukui, P.V. Doskey, Air-surface exchange of nonmethane organic compounds at a grassland site: Seasonal variations and stressed emissions, Journal of Geophysical Research – Atmospheres 103 (1998) 13153–13168.
J. Tian et al. / Acta Ecologica Sinica 31 (2011) 121–125 [26] M. Nemecekmarshall, R.C. Macdonald, F.J. Franzen, et al., Methanol emission from leaves – enzymatic detection of gas-phase methanol and relation of methanol fluxes to stomatal conductance and leaf development, Plant Physiology 108 (1995) 1359–1368. [27] C. Warneke, T. Karl, H. Judmaier, et al., Acetone, methanol, and other partially oxidized volatile organic emissions from dead plant matter by abiological processes: Significance for atmospheric HOx chemistry, Global Biogeochemical Cycles 13 (1999) 9–17.
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[28] G. Zhang, J. Tian, N. Jiang, et al., Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC–I, Environmental Microbiology 10 (2008) 1850–1860. Jianqing Tian, 1981, Shanxi Province, Ph.D, Research Associate, mainly focused on microbial community structure in wetland.
Contents lists available at ScienceDirect
Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Methane production in relation with temperature, substrate and soil depth in Zoige wetlands on Tibetan Plateau Jianqing Tian a,b,1, Huai Chen c,d, Yanfen Wang b,⇑, Xiaoqi Zhou b a
Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China c College of Forestry, Northwest Agriculture and Forest University, Yanglin 712100, PR China d Institute of Environment Sciences, Quebec University at Montreal, Montreal, Canada C3H3P8 b
a r t i c l e
i n f o
Article history: Received 21 November 2010 Revised 11 January 2011 Accepted 27 January 2011
Keywords: Tibetan Plateau Archea Methanogenesis Greenhouse gases Peatland Carbon biogeochemistry
a b s t r a c t To understand the effect of substrate and temperature on methane production in high frigid wetlands, soil samples along the soil profile were collected from Zoige alpine wetlands, a ‘‘hotspot’’ of methane emission on Qinghai-Tibetan Plateau. Our result showed that temperature had a positive influence on methane production. However, acetate accumulation was negative related with temperature. We found that supplemental methanol and acetate strongly stimulated methane production. Such result indicated that methanol and acetate are major substrates for methane production in Zoige wetlands. Moreover, we also found that methane production rates decreased with soil depths due to the decrease of available substrate in deeper soil. Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Among all natural methane (CH4) sources, natural wetland is regarded as the single largest source which may contribute 10%–30% to the global budget [1]. Among them, wetlands in cold areas, such as northern wetlands and tundra wetlands, contribute a large proportion [2]. Recently, scientists have been trying to get insight into the methanogensis in cold environments [3,4]. Major attentions are paid to the effect of substrate and temperature on CH4 production, the psychrophiles that may implement the bioconversion of biomass into CH4 and key environmental factors that are likely to have influence on CH4 emission. CH4 production depends on the quantity and quality of substrates. Because the amount and decomposability of soil organic matters determine the supply of substrate for methanogens [5]. On the micro-scale, the distribution of fresh organic matter is the dominant factor explaining the spatial variation in CH4 production [6]. Furthermore, methanogenesis is controlled more by the availability and composition of the substrate than by the amount of methanogens [7]. Hence, substrate availability is a predominant ⇑ Corresponding author. Address: 19(Jia), YuQuan Road, Shijingshan, Beijing 100049, PR China. Tel.: +86 10 88256066, fax: +86 10 88256715. E-mail addresses: [email protected] (J. Tian), [email protected] (Y. Wang). 1 Tel.: +86 10 64807512; fax: +86 10 64807505.
constraint for CH4 production under nature condition rather than abiotic factors [8]. Temperature is one of the most important abiotic factor controlling CH4 production [9,10]. Previous studies observed that CH4 fluxes can be described by a single linear equation with a factor of temperature. In particular, CH4 production rate depends greatly on the temperature under a physiological temperature range of methanogens in a low temperature environment [3,4,11,12]. Studies on CH4 emission in Qinghai-Tibetan Plateau (QTP) suggested that Zoige wetland is a major centre for CH4 generation in the plateau [13]. However, little is yet known about the effect of temperature and the substrate quality on CH4 production. Therefore, the objectives of this study are aimed to discuss how CH4 production response to temperature and substrate. 2. Material and Methods 2.1. Site description The Zoige alpine wetland is located on the northeastern edge of the QTP which belongs to the cold Qinghai-Tibetan climatic zone. Alpine lakes and peatlands are well developed. Mean annual temperature is about 0.6 °C–1.2 °C, and mean annual precipitation is approximately 560–860 mm. The soil pH is about 6.8–7.2. The total C of soil and the depth of standing water table are 179.17 g kg 1and 8.6 cm, respectively [14].
1872-2032/$ - see front matter Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chnaes.2011.01.002
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J. Tian et al. / Acta Ecologica Sinica 31 (2011) 121–125
2.2. Incubation methods Soil samples were collected from a 75 75 m sampling square submerged wetland with the soil depth of 10 cm, 20 cm and 30 cm on 20th August, 2005. For each depth, three replicates were taken and put into three separate sterile 100 ml vials, flushed with nitrogen (N2) sealed with butyl rubber stopper, kept in a cool box, then immediately taken back to laboratory for preparation. Incubation experiment was set up to measure CH4 potential production of the soil. The slurries were made by the soil samples and modified medium (1:1, V/V). The modified medium [15] contained the waterlogged from the study site. Approximately 10 ml slurry was placed in 25 ml screw-capped tubes sealed with butyl rubber stoppers, with a gas phase of N2. All of them were done in the anaerobic glove box under the N2 atmosphere. Every triplicate was cultivated for six months at 6 °C, 15 °C and 30 °C. During the five month’s incubation time, gas samples (30 ll) were taken from the headspace with gas-tight syringes and analyzed by gas chromatography twice in the first month and once every month afterward. The liquid samples were taken and stored at –20 °C for further analysis. Twenty-fold diluted samples (5% v/v suspension) were used to examine the ability of the peat methanogens to produce CH4 with hydrogen:carbon dioxide (H2:CO2) (4:1), acetate, trimethylamine (TMA) and methanol. The diluted samples were put into the screw-capped tubes containing medium supplemented with 40 mM each of substrate. Triplicates of each treatment were incubated at 30 °C, and CH4 concentration was measured weekly. Before analysis, all the test tubes were thoroughly shaken to equilibrate gases between the slurries and headspace. To obtain more evidence for the existence of specific psychrophilic methanogenic communities, the wetland slurries (soil:medium; 1:1) pre-incubated at 6 °C for six months were diluted to 20-fold (soil: medium; 1:20) by medium with substrates (i.e. H2/ CO2, methanol, acetate, TMA) incubation at 15 °C and 30 °C, respectively. Triplicates of each treatment were set up, and CH4 concentration was measured weekly. CH4 and acetate concentrations were measured with a gas chromatograph (Shimadzu GC–14B) equipped with a Flame Ionization Detector and Nitrogen used as carrier gas at a flow rate of 50 ml min 1. For CH4 concentration measurement, column, injector, and detector temperatures were 50 °C, 80 °C and 100 °C, respectively, while acetate was measured by 230 °C, 250 °C and 280 °C, respectively.
Table 1 CH4 production rates at different incubation temperatures and soil depths. Temperature °C
6 15 30
CH4 production rates (lmol g
1
[fresh soil] d
1
)
30 cm
20 cm
10 cm
0.12 ± 0.03 0.21 ± 0.05 1.36 ± 0.02
0.12 ± 0.01 0.36 ± 0.04 1.65 ± 0.07
0.12 ± 0.01 0.49 ± 0.06 1.75 ± 0.02
(Table 1). At 6 °C, all the three layers had similar CH4 production potential and the rates of methane production was 0.12 lmol g 1 [fresh soil] d 1. The Q10 value showed that substrate (soil samples from different layers) have stronger influence on CH4 production than temperature at lower temperature treatment (data not shown). Q10 values varied with soil depth, which may be partly due to the components of substrates soil contains [8]. CH4 production was significantly related with acetate accumulation at lower temperature, such as 15 °C and 6 °C (Fig. 1A-D). Acetogenesis predominated on methanogenesis at 15 °C and below during the first month of incubation, and up to 14 mM acetate accumulated during this period. After that, the acetate accumulation was suspended by lack of substrate. At 6 °C, the accumulated acetate was not utilized even after five month of incubation. When incubated at 30 °C, there were no obvious acetate accumulation and CH4 production lagging. CH4 potential production reached the peak in the first month. 3.2. Supplemental experiments To test the effect of substrate on CH4 production, supplemental experiments were set up. Comparing to control, all substrates can stimulate CH4 production with different rates: methanol >TMA>acetate>H2/CO2 (Fig. 2). Both methanol and TMA can be used to produce CH4 at the same time. H2/CO2 was hardly used as substrate by hydrogenotrophic methanogens in this area (Fig. 2). When samples were pre-incubated at 6 °C, methanogens used all the supplemental substrates except H2/CO2 to form CH4 very quickly from the second day of the 30 °C incubation, the seventh day of the 15 °C incubation with a short-lag phase. The final CH4 concentration with methanol supplemented as substrate at 15 °C was higher than that at 30 °C (Fig. 3). 4. Discussion
2.3. Statistics analysis Within one month, CH4 concentration increased linearly at all temperature tested. The rates of CH4 production and the growth rates of methanogens were calculated by linear regression analysis and the accumulation by the difference between the final and initial gas concentrations. One-way analysis of variance (ANOVA) was employed to determine the effect of temperature and soil depth on methane production. ANOVA and regression analysis were performed using SPSS 12.0 software (SPSS Inc., USA). 3. Results 3.1. CH4 production during incubation The experiment was performed with slurries of fresh soil samples that were incubated at three temperatures. Measurement of CH4 potential production in incubated peat samples revealed highest CH4 production in the upper waterlogged peat layer at 30 °C and 15 °C, and a decrease of rates with depth and temperature
4.1. Temperature effects Supposing a linearity between CH4 production in situ and in laboratory, CH4 production in laboratory can be used to reflect production in situ [10]. Hence, under the laboratory incubation conditions, it is not only possible but also convenient to understand the relationship between temperature and CH4 production when the other regulating factors are controlled. In this study, CH4 production can be detected at all temperatures with different rates. The average rates at all the depth showed that CH4 production rates were approximately 2–3 times and 7–10 times greater at 30 °C than that at 15 °C and 6 °C, respectively. Hence, CH4 production rates strongly depended on temperature. Besides methane, acetate was accumulated in fresh soil of Zoige wetland during first month at 15 °C and 6 °C. During the subsequent months, acetate was transformed into CH4 at 15 °C; while the acetate kept untransformed at 6 °C. The explanation was that 15–20 °C was a threshold for end products formation in lowtemperature environments like tundra permafrost soil, lake sediments and paddy soil [11,16,17].
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4.2. Substrates influenced CH4 production Theoretically, about two thirds of CH4 is produced from acetogenic methanogenesis [22]. Our incubation experiments with soil samples from alpine ecosystem provided evidence that CH4 produced from acetate and methanol is much more than that from H2/CO2. Jiang et al. [23] reported that methanol-derived methane could account for a large portion in Eleocharis valleculosa soil in the same peatlands. In situ, methanol is emitted from the flowering plants [24–26] and plant decay [27]. Pectin of dead plant material alone can produced 800 Tg y 1 methanol, and the large part is going to dissolve in soil water and utilized as microorganism substrate [24]. 90%–95% of Zoige Plateau was covered with plant. Because of long-term waterlogged conditions, such wetlands are able to release much methanol, which may supply substrate for methanogens. Furthermore, phylogenetic tree revealed that most of sequences were affiliated with methylotrophic and acetoclastic methanogens [28], which indirectly support most of CH4 origination from acetate or/and methanol. 4.3. Soil depth influenced CH4 production In anoxic environment with constant thermal conditions, the potential CH4 production depends on substrate availability [18]. In Zoige wetlands, the maximum CH4 production rate took place within the upper 10 cm and the rate then decreased with soil depth at all incubation temperatures (Fig. 2). This is consistent with previous studies [19–21]. Decreased CH4 potential production with depth may be associated with different substrate availability at varied depth. As the fresh organic matter is a dominant factor for the spatial variation in methane production [6]. Shallow layer is younger and contains more labile and easily mineralized carbon sources from newly deposited organic matter [5], which will lead to higher methanogens activity and the overcoming of methanogens in such layer. Additionally, deeper peat deposits in natural wetlands showed limited production rates due to unfavorable component accumulations for microbiological activity, such as ligins, and phenolic or humic substrates. Another explanation is possible, that other micro-organisms in deeper layer out-competed methanogens and resulted in low CH4 potential production and high acetate accumulation. These results, acetate accumulation increased with soil depth suggested that the capability of methanogens competing with other acetogenic bacteria at deeper layer was weaker than in the upper layer at low temperature.
5. Conclusion The activity of methanogensis strongly depended on temperature, substrate and soil depth which indicated both are important influence factors for CH4 production. Methanogensis occurred at a widely temperature range even at low temperature (6 °C), it is consistent with its cold environment. Notably, methanol could serve as the more important methanogenic precursors than expected in Zoige wetland. In future, the contribution of methanol-derived methane should be paid more attention. To some extent, global warming might result in more CH4 emission from wetlands on the QTP due to rising temperature and more substrates from the plants. Acknowledgements This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (kzcx2-yw-418-03).
Two anonymous reviewers were thanked for their detailed evaluation and suggestion on our manuscript.
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