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Aerobic and Anaerobic Nonmicrobial Methane Emissions from Plant Material Zhi-Ping Wang,†,* Zong-Qiang Xie,† Bao-Cai Zhang,‡ Long-Yu Hou,† Yi-Hua Zhou,‡ Ling-Hao Li,† and Xing-Guo Han†,§ †
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing 100093, China ‡ State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China § Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China ABSTRACT: Methane (CH4) may be generated via microbial and nonmicrobial mechanisms. Nonmicrobial CH4 is also ubiquitous in nature, such as in biomass burning, the Earth's crust, plants, and animals. Relative to microbial CH4, nonmicrobial CH4 is less understood. Using fresh (living) and dried (dead) leaves and commercial structural compounds (dead) of plants, a series of laboratory experiments have been conducted to investigate CH4 emissions under aerobic and anaerobic conditions. CH4 emissions from fresh leaves incubated at ambient temperatures were nonmicrobial and enhanced by anaerobic conditions. CH4 emissions from dried leaves incubated at rising temperature ruled out a microbialmediated formation pathway and were plant-species-dependent with three categories of response to oxygen levels: enhanced by aerobic conditions, similar under aerobic and anaerobic conditions, and enhanced by anaerobic conditions. CH4 emissions in plant structural compounds may help to fully understand nonmicrobial CH4 formation in plant leaves. Experiments of reactive oxygen species (ROS) generator and scavengers indicate that ROS had a significant role in nonmicrobial CH4 formation in plant material under aerobic and anaerobic conditions. However, the detailed mechanisms of the ROS were uncertain.
1. INTRODUCTION Methane (CH4) is an important trace gas, contributing to global warming and atmospheric redox chemistry. The change in atmospheric CH4 concentrations from 715 nL 3 L1 in 1750 to 1774 nL 3 L1 in 2005 figures out an average radiative forcing of 0.48 W 3 m2, ranking CH4 as the second most important anthropogenic greenhouse gas after CO2.1 CH4 has been traditionally considered an end product of organic matter degradation by microbes. The microbes are a limited group of obligate prokaryotes called methanogens that thrive under anaerobic conditions.2,3 Microbial CH4 has been widely studied in the past decades and understood profoundly.2 Nonmicrobial CH4 is also widespread in nature, such as in biomass burning,4,5 the Earth's crust,6 plants,7 and animals.8,9 However, it has been less understood. Nonmicrobial CH4 emissions by plants and its global strength still remain controversial.10 Previous studies described plant CH4 emissions as aerobic since plant tissues/compounds were incubated under aerobic conditions.7,1113 However, recent studies indicated that nonmicrobial CH4 was also generated in plant leaves when they were incubated under anaerobic conditions.10,14 Earlier studies demonstrated hypoxia-induced generation of nonmicrobial CH4 in mitochondria and eukaryotic cells of animals.8,9 Thus, we would propose that it might be better to use aerobic and r 2011 American Chemical Society
anaerobic nonmicrobial CH4 that are defined as those from organisms, including plants and animals, when incubated under aerobic and anaerobic conditions, respectively. In this study, we postulated that nonmicrobial CH4 formation in plant material may occur under both aerobic and anaerobic conditions. To test this hypothesis, we concentrated on a comparison of nonmicrobial CH4 emissions from fresh and dried leaves of plants between aerobic and anaerobic conditions. Several structural compounds of plants such as pectin, lignin, and cellulose were examined to aid a better understanding of the effect of oxygen levels on the emissions from plant leaves. Furthermore, experiments of reactive oxygen species (ROS) generators and scavengers were conducted to investigate the potential role of ROS in nonmicrobial CH4 formation in plant material.
2. MATERIALS AND METHODS 2.1. Plant Species Collection. A total of nine plant species were collected from the Xilin River basin in the Inner Mongolia12 Received: June 14, 2011 Accepted: September 30, 2011 Revised: September 27, 2011 Published: September 30, 2011 9531
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Environmental Science & Technology and the Beijing Botanical Garden (39°590 2400 N, 116°120 3600 E; 66 m above sea level) throughout the year 2010. The plants were of distinctive morphotypes, including five wood, one shrub, and three herb species. For the purpose of clarity, plant species abbreviations are used in figures: AF (Artemisia frigida), LG (Larix gmelinii), LS (Lactuca sativa), MS (Medicago sativa), PB (Populus beijingensis), PT (Pinus tabulaeformis), QW (Quercus wutaishanica), RP (Robinia pseudoacacia), and SO (Spinacia oleracea). 2.2. Sample Preparation. Fresh leaves were detached to limit transpiration as a potential microbial CH4 source. The leaves were collected in plastic bags and transported to the laboratory within 15 min. They were immediately washed in deionized water and air-dried for about 0.5 h prior to commencement of measurements. Dried leaves were obtained by oven-drying fresh leaves at 40 °C to constant mass. Plant structural compounds were commercial citrus pectin (CAS no. 9000-69-5), lignin (CAS no. 8068-05-1), and cellulose (CAS no. 9004-34-6). They were obtained from SigmaAldrich Chemical Co., Shanghai, China. 2.3. Chemical Addition. Fenton reagent was used to generate • OH,15 whereas 1,4-diazabicyclo[2.2.2]octane (DABCO, C6H12N2), potassium iodide (KI), and D-mannitol [C6H8(OH)6] were reported to be scavengers of 1O2, H2O2, and •OH, respectively.16,17 In ROS generator experiments, fresh or dried leaves were impregnated consecutively by 2 mL of 20 mM Na2EDTA 3 2H2O (disodium ethylenediaminetetraacetate dihydrate) and 2 mL of 20 mM FeSO4 3 7H2O, sealed in gastight serum bottles, and flushed with CH4-free compressed oxygen or nitrogen. Immediately afterward, 2 mL of deionized water (equivalent to 0% H2O2), 2 mL of 1% H2O2, or 2 mL of 2% H2O2 were added via syringe into the samples. Plant structural compounds were also treated as above but with 1 mL of chemical solutions. In ROS scavenger experiments, dried leaves were soaked in 0, 5, or 50 mM DABCO, KI, or D-mannitol solutions for about 5 h, removed, and then air-dried for a few days. The air-dried leaves containing the chemical were used as samples. 2.4. Laboratory Incubation. CH4 emissions were examined from fresh and dried leaves and structural compounds of plants in closed-bottle laboratory incubations in the dark. For each plant sample, a few grams of prepared plant material was placed in a 120-mL serum bottle. Parallel blanks were employed to determine whether background CH4 concentrations in serum bottles changed in the absence of plant material. If blanks had undetectable change in CH4 concentrations, they were usually omitted in figures for the purpose of clarity. A flushing method was used to establish aerobic and anaerobic conditions.14 In brief, the bottles were immediately sealed with butyl rubber stoppers (diameter 20 mm) and flushed for 15 min with CH4-free compressed oxygen (O2), nitrogen (N2), hydrogen (H2), or helium (He) by use of “inletoutlet” needles inserted through the stoppers at a rate of 400 mL 3 min1, respectively. To avoid plant structural compound being flushed out of the bottle, a piece of glass microfiber filter (Whatman GF/A, diameter 12.5 cm) was used to separate a slow flushing (200 mL 3 min1) from the sample. Before usage, glass microfiber filters were baked for a few hours in an oven at 200 °C to remove possible organic contaminants. Initial CH4 concentrations were measured immediately prior to incubations. Fresh leaves were incubated at ambient temperatures. At the end of each experiment, their dry matter was determined by oven-drying at 40 °C to constant mass. To increase the signal-tonoise ratio, dried leaves and structural compounds of plants were incubated at rising temperature, unless stated otherwise.
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Figure 1. CH4 emissions from (a) fresh leaves at ambient temperatures of 2324 °C for 3 h and (b) dried leaves at rising temperature of 70 °C for 1 h under oxygen, hydrogen, nitrogen, and helium conditions.
2.5. Extraction of Structural Compounds in Plant Cell Wall. Structural compounds were extracted from dried leaves of selected plants. The dried leaves were extracted with 70% ethanol and chloroform/methanol (1:1 v/v) to prepare alcohol-insoluble residues (AIRs) of cell walls.18 For the measurement of pectin content, AIRs were extracted by 1% ammonium oxalate (w/v), and then soluble pectin was precipitated and trapped on a crucible to weigh. To determine the contents of uronic acids, AIRs were destarched and methanolyzed in 1 M methanolic hydrochloric acid. The trimethylsilyl derivatives were generated with Trisil reagent and finally analyzed by Agilent GC 7900/ 5975C MS with a DB-1 column.19 The content of lignin was determined as described previously.19 In brief, AIRs were treated with 72% 1 N sulfuric acid to remove polysaccharides, and then the insoluble lignin was trapped and weighed after being thoroughly dried. To determine the content of crystalline cellulose, AIRs were destarched with amylase and then hydrolyzed in Updegraff reagent (8:1:2 v/v/v in acetic acid/nitric acid/ water) at 100 °C for 30 min. After centrifuge collection and washing, the cellulose was hydrolyzed and used for anthrone assay.18 2.6. CH4 Concentration Measurement. CH4 concentrations in the headspace of serum bottles were analyzed at various time intervals by use of a Hewlett-Packard 5890 series II gas chromatograph. The GC running conditions were described previously.14 A 5-mL gas sample was withdrawn from a 120-mL serum bottle by syringe and immediately replaced by 5 mL of CH4-free compressed oxygen or nitrogen to maintain headspace pressure. 2.7. Statistical Analysis. Emission rate was calculated by CH4 accumulation over time and recorded as nanograms per gram dry weight per hour. Value is mean ( 1 standard deviation (n = 3 in Figures 1 and 46 and n = 4 in Figures 2 and 3). Statistical analysis was performed by use of a Statistical Analysis System program.20 Duncan’s multiple range test was employed to compare 9532
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Figure 2. CH4 emissions from fresh leaves of plants at ambient temperatures of 2223 °C under (a) a cycle of 01 h aerobic (100% O2), 23 h anaerobic (0% O2), and 45 h aerobic conditions and (b) a cycle of 01 h anaerobic, 23 h aerobic, and 45 h anaerobic conditions. Between incubation periods, the samples were appropriately flushed again.
the variation in CH4 emission rates among treatments at P < 0.05. One-way analysis of variance was used to evaluate statistical difference in CH4 emission rates between aerobic and anaerobic conditions. The different letters indicated significant differences (P < 0.05) in each group of treatments. If statistically significant differences were easily self-explanatory, the different letters were omitted for the purpose of clarity.
3. RESULTS AND DISCUSSION 3.1. Aerobic and Anaerobic Incubation Conditions. CH4 emissions from fresh and dried leaves of plants had significant differences (P < 0.05) between the treatments of O2 and the other gases, with the exception of those from dried leaves of A. frigida. For all plant species investigated, however, the emissions had no significant differences (P > 0.05) in the treatments of H2, N2, and He (Figure 1). H2 is an available growth substrate for a large diversity of anaerobic bacteria, notably obligate methanogens; CH4 generation by anaerobic bacteria using H2 as substrate contributes approximately 1050% to total CH4.2 In this study if H2 would provide a substrate for CH4 generation, the emission in H2 treatment should be higher than those in the treatments of N2 and He. Accordingly, H2 did not serve as a substrate for CH4 generation during incubation periods. On the other hand, temporal kinetic experiments showed that no significant microbial CH4 was generated over a few hours of incubation, since methanogens need adequate time to multiply.10 These indicate that CH4 emitted from the leaves was indeed nonmicrobial. O2 provided
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aerobic conditions while H2, N2, and He provided anaerobic conditions during these short-term incubations. 3.2. Nonmicrobial CH4 Emissions from Fresh Leaves. When fresh leaves of A. frigida, M. sativa, and Q. wutaishanica were alternately incubated under aerobic and anaerobic conditions, their CH4 emissions were significantly higher (P < 0.05) under anaerobic than aerobic conditions. However, CH4 emissions from fresh leaves of L. gmelinii, P. beijingensis, and P. tabulaeformis had no statistically significant difference from zero. Despite this, the emissions were enhanced by anaerobic conditions (Figure 2). Accordingly, it is concluded from results obtained from these experiments and previous studies in plants10,14 and animals8,9 that instant CH4 formation in living organisms is a nonmicrobial process enhanced by anaerobic conditions. Keppler et al.7 suggested a nonenzymatic process for CH4 formation in plants. Nisbet et al.21 did not find necessary biochemical pathways to synthesize CH4 in plants. Thus, enhanced CH4 formation under anaerobic conditions might be due to physiological activities of living organisms, such as a passive consequence of physiological processes. It is well-known that microbial CH4 is generated under anaerobic conditions.2 As a result, in nature both microbial and nonmicrobial CH4 should be simultaneously generated under anaerobic environment. In a number of field studies where significant CH4 emissions were observed, it has been reported that these were transmitted to the atmosphere by plants.2224 Such field emissions might now also include a contribution from nonmicrobial source. However, with current knowledge, it is difficult to distinguish between microbial and nonmicrobial CH4 generated in nature. 3.3. Nonmicrobial CH4 Emissions from Dried Leaves. When dried leaves of R. pseudoacacia and Q. wutaishanica were alternately incubated at ambient and rising temperatures, their CH4 emissions were repeatedly provoked by rising temperature (70 °C) but were undetectable at ambient temperatures (Figure 3a,b). Microbial CH4 emission was usually observed as a parabolic curve with respect to temperature; the emission peak corresponded to the most appropriate temperature of 2530 °C required by enzymatic metabolism of microbes.25 Accordingly, the emissions at rising temperature excluded microbial activity as the source. Nonmicrobial CH4 emissions from the dried leaves incubated at rising temperature had three categories of response to oxygen levels. Specifically, the emissions were enhanced by aerobic conditions in M. sativa, P. beijingensis, R. pseudoacacia, S. oleracea, and L. sativa (left of the left dashed line); similar under aerobic and anaerobic conditions in A. frigida and L. gmelinii (between the two dashed lines), and enhanced by anaerobic conditions in P. tabulaeformis and Q. wutaishanica (right of the right dashed line) (Figure 3c,d). Three categories of response were also reflected in nonmicrobial CH4 emissions between the treatments of O2 and the other gases (Figure 1b). When pectin and lignin were incubated at rising temperatures, their CH4 emissions were enhanced by aerobic conditions (Figure 4). This may be used to interpret that the emissions from dried leaves were enhanced by aerobic conditions. Commercial pectin or lignin incubated in serum bottles did not have an opportunity to react with other compounds like those in dried leaves. This might be one reason why the emissions from these pure structural compounds were much lower than those from dried leaves when based on structural compound equivalents (Figure 3c,d; Table 1). Previous studies used ultraviolet radiation as a trigger to drive nonmicrobial CH4 formation in terrestrial 9533
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Figure 3. CH 4 emissions from dried leaves of plants under (a, b) a cycle of ambient and rising temperatures and (c, d) a cycle of aerobic and anaerobic conditions. Dried leaves of (a) R. pseudoacacia and (b) Q. wutaishanica were alternately incubated at ambient temperature of 21 °C in the periods of 13 and 46 h and rising temperature of 70 °C in the periods of 01, 34, and 67 h. Dried leaves of plants were alternately incubated at rising temperature of 70 °C under (c) a cycle of 01 h aerobic, 23 h anaerobic, and 45 h aerobic conditions and (d) a cycle of 01 h anaerobic, 23 h aerobic, and 45 h anaerobic conditions. Between incubation periods, the samples were appropriately flushed again.
Figure 4. CH4 emissions from plant structural compounds at rising temperatures of (a) 70 °C and (b) 90 °C for 3 h under aerobic and anaerobic conditions.
plant tissues and compounds.2628 Rising temperature, such as heat wave in summer and biomass burning, has wide implications for terrestrial plants on the Earth's surface. CH4 emission was lower in pectin than lignin when rising temperature was a driver (Figure 4). This is inconsistent with results by Vigano et al.,27 where there was higher CH4 emission in pectin than lignin under ultraviolet irradiation. Accordingly, different structural compounds prefer to accept their distinctive drivers in nonmicrobial CH4 formation. In addition, almost no CH4 emission was observed in cellulose when incubated at rising temperatures (Figure 4). This may suggest that nonmicrobial CH4 formation was not derived from the cellulose of dried leaves at rising temperature.
Figure 5. Effects of ROS generator, Fenton’s reagent, on CH4 emissions in (a) fresh leaves of P. tabulaeformis, (b) dried leaves of P. tabulaeformis and R. pseudoacacia, and (c) plant structural compounds. The samples were incubated at ambient temperatures. Treatments had aerobic and anaerobic conditions; 1% and 2% H2O2; PT and RP species; P (pectin) and L (lignin). Undetectable CH4 emissions in plant material infiltrated with deionized water as blanks (0% H2O2) and cellulose treatments were omitted for the purpose of clarity. 9534
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Cell wall compounds in dried leaves of three types of plants are listed in Table 1. AIRs were obtained via washing dried leaves with ethanol, in which polysaccharides are insoluble. Pectic polysaccharides mainly consist of sugar residues, methyl esters, and O-acetyl groups,28 while in sugar residues galactouronic acid (GalUA) and glucuronic acid (GlcUA) are major components.29 On average, the first type of plants with enhanced CH4 emissions under aerobic conditions had a higher proportion of AIRs to biomass when compared with the other two types. Again, the first type had higher pectin and lower lignin and cellulose contents in
Figure 6. Effect of ROS scavengers on CH4 emissions in dried leaves of (a) P. tabulaeformis and R. pseudoacacia and (b) P. tabulaeformis. The leaves were incubated at rising temperature of 70 °C for 1 h.
AIRs and higher GalUA and GlcUA contents in destarched AIRs. The values in pectin are incomparable with GalUA and GlcUA contents, since destarched AIRs are different from and much lower than AIRs. For dried leaves in each type of plant, however, contents of their structural compounds had large variabilities, for example with coefficients of variation (CV) of 128.1%, 65.5%, and 109.9% in the first, second, and third types of plants, respectively. Together with less plants examined, thus, it is uncertain that the response of nonmicrobial CH4 emissions to oxygen levels is classified by contents of the structural compounds. To profoundly understand the response, more plant species and structural compounds need to be investigated in the future. Previous studies found that methoxyl groups of plant pectin and/or lignin serve as precursors for nonmicrobial CH4 formation.2628 If precursors would restrictedly come from pectin and/or lignin, the result of enhanced nonmicrobial CH4 emissions by aerobic conditions in the structural compounds (Figure 4) cannot explain the emissions from dried leaves of the other two types of plants (Figure 3c,d). This indicates that more precursors should be responsible for nonmicrobial CH4 formation in dried leaves. The precursors might include oxidative and reductive categories that coexist in the dried leaves. Response of nonmicrobial CH4 formation to oxygen levels presumably depends upon a mixture of various categories of precursors. 3.4. Role of ROS in Nonmicrobial CH4 Formation in Plant Material. ROS are exceedingly reactive and short-lived.15,28 Previous studies did not directly monitor ROS.28,30 Because of difficulty in monitoring ROS, we used ROS generator and scavengers as done previously 28,30 to examine potential role of ROS in mechanisms of nonmicrobial CH4 formation in plant material. More CH4 was irritatingly emitted by ROS generator, H2O2, in all categories of plant material under anaerobic than aerobic
Table 1. Cell Wall Compounds in Dried Leaves of Plantsa pectin/AIRs
GalUA/D-AIRs
GlcUA/D-AIRs
lignin/AIRs
cellulose/AIRs
AIRs/biomass (%)
(μg 3 mg1)
(μg 3 mg1)
(μg 3 mg1)
(μg 3 mg1)
(μg 3 mg1)
M. sativa
72.6
18.9
78.5
9.4
122.5
73.2
P. beijingensis
80.1
57.9
78.8
3.6
330.6
72.4
R. pseudoacacia S. oleracea
66.1 72.9
7.4 5.0
63.5 48.0
6.3 ndb
446.4 58.6
67.1 49.4
L. sativa
68.0
152.2
79.2
nd
139.5
107.7
mean
71.9
48.3
69.6
6.4
219.5
73.9
SD
5.4
61.8
13.8
2.9
162.4
21.2
CV (%)
7.5
128.1
19.8
45.6
74.0
28.6
A. frigida
63.1
17.6
95.4
11.4
309.7
164.6
L. gmelinii mean
64.3 63.7
6.4 12.0
45.7 70.6
3.4 7.4
566.0 437.8
71.8 118.2
species
SD
0.9
7.9
35.2
5.6
181.2
65.6
CV (%)
1.4
65.5
49.8
76.0
41.4
55.5
P. tabulaeformis
62.2
0.6
25.3
1.5
450.3
228.9
Q. wutaishanica
67.1
5.1
82.4
2.9
312.1
125.6
mean
64.7
2.9
53.9
2.2
381.2
177.2
3.4 5.3
3.2 109.9
40.4 75.1
1.0 44.7
97.8 25.6
73.0 41.2
SD CV (%)
a Three groups of plant species showed distinctive responses in nonmicrobial CH4 emissions to aerobic and anaerobic conditions (see Figure 3c,d). AIRs are alcohol-insoluble residues of cell wall, while D-AIRs are destarched alcohol insoluble residues. b Content is under detection threshold.
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Environmental Science & Technology conditions (Figure 5). The emissions showed logarithmic kinetics with respect to time from fresh leaves of P. tabulaeformis and linear kinetics from dried leaves of P. tabulaeformis and R. pseudoacacia. The linear kinetics may be interpreted as due to a large precursor reservoir that continuously served CH4 formation. The emissions also had logarithmic kinetics in pectin but were almost undetectable in lignin. CH4 emissions were significantly (P < 0.05) constrained by the ROS scavengers immerged in dried leaves when incubated at rising temperature (Figure 6). KI showed increasing inhibitory effect on the emissions from dried leaves of P. tabulaeformis and R. pseudoacacia. DABCO and D-mannitol, even at their low concentration treatments, had significant inhibition on the emissions from dried leaves of P. tabulaeformis. The differences in inhibitory magnitude may be due to KI, DABCO, and Dmannitol acting as distinctive scavengers of H2O2, 1O2, and •OH, respectively.16,17 Similar/identical change trends in nonmicrobial CH4 emissions were observed between aerobic and anaerobic conditions (Figures 5 and 6). This indicates that ROS were involved in nonmicrobial CH4 formation in plant material while other factors could be responsible for the differences in emission rates between aerobic and anaerobic conditions. However, it is necessary to mention that the detailed mechanisms of the ROS were unclear. Less nonmicrobial CH4 emissions were observed in pectin than lignin at rising temperatures (Figure 4), whereas more emissions were stimulated by ROS generator in pectin than lignin at ambient temperatures (Figure 5c). Previous studies suggested that nonmicrobial CH4 is generated via pyrogenic and thermogenic reactions in biomass burning4,5 and within the Earth's crust.6 Thus, free radicals and pyrogenic and thermogenic reactions might together be responsible for nonmicrobial CH4 formation in dried leaves when incubated at rising temperature. Plant tissues naturally generate certain ROS during growth via the Fenton or HaberWeiss reactions (see ref 28). On the other hand, in nature plants are frequently subjected to various forms of environmental stress such as extreme weather, solar UV radiation, soilwater deficit and flooding, hypoxia and hyperoxia, wounding, herbicides, and pathogens.10 These environmental stress factors stimulate ROS generation in plant cells.31 Thus, the ROS’ role in nonmicrobial CH4 formation simulated in laboratory conditions may be extended to natural situations. Previous studies indicated that microbial CH4 formation in soils may occur under aerobic conditions.32,33 This is inconsistent with the traditionally held view that microbial CH4 is generated under anaerobic conditions. This study confirms that nonmicrobial CH4 formation in plant material occurred under both aerobic and anaerobic conditions. Accordingly, it is clearly shown that CH4 formation, regardless of via microbial or nonmicrobial mechanisms, does not completely depend upon oxygen levels. Conrad2 suggested that aerobic microbial CH4 may be generated via the coincidence of electron donors and electron acceptors. When electron donor availability coincides with electron acceptors in a medium involved by microorganisms, sequential reduction occurs largely to produce microbial CH4 under aerobic conditions. The electron coincidence might provide a basic point for understanding microbial and nonmicrobial CH4 formation under aerobic and anaerobic conditions. Whether nonmicrobial CH4 formation is also realized via the coincidence of electron donors and electron acceptors in a plant medium needs further work to test.
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’ ACKNOWLEDGMENT We greatly thank two anonymous referees and editors for their constructive comments that improved the paper. We are also very grateful to Frank Keppler and John T. G. Hamilton for their helpful comments. This research was supported by the general program of the National Natural Science Foundation of China (30970518), the Key Project of National Natural Science Foundation of China (30830026), and funding from the State Key Laboratory of Vegetation and Environmental Change (2011zyts07). ’ REFERENCES (1) Forster, P.; et al. Changes in Atmospheric Constituents and in Radiative Forcing. In Climate Change 2007: The Physical Science Basis; Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change; Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K. B., Tignor, M., Miller, H. L., Eds.; Cambridge University Press: Cambridge, U.K. and New York, 2007; pp 129234. (2) Conrad, R. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). Microbiol. Rev. 1996, 60, 609–640. (3) Conrad, R. Quantification of methanogenic pathways using stable carbon isotopic signatures: a review and a proposal. Org. Geochem. 2005, 36, 739–752. (4) Crutzen, P. J.; Andreae, M. O. Biomass burning in the tropics: impact on atmospheric chemistry and biogeochemical cycles. Science 1990, 250, 1669–1678. (5) Andreae, M. O.; Merlet, P. Emission of trace gases and aerosols from biomass burning. Global Biogeochem. Cycles 2001, 15, 955–966. (6) Etiope, G.; Klusman, R. W. Geologic emissions of methane to the atmosphere. Chemosphere 2002, 49, 777–789. (7) Keppler, F.; Hamilton, J. T. G.; Brass, M.; R€ockmann, T. Methane emissions from terrestrial plants under aerobic conditions. Nature 2006, 439, 187–191. (8) Ghyczy, M.; Torday, C.; Boros, M. Simultaneous generation of methane, carbon dioxide, and carbon monoxide from choline and ascorbic acid: a defensive mechanism against reductive stress? FASEB J. 2003, 17, 1124–1126. (9) Ghyczy, M.; Torday, C.; Kaszaki, J.; Szabo, A.; Cz obel, M.; Boros, M. Hypoxia-induced generation of methane in mitochondria and eukaryotic cells - An alternative approach to methanogenesis. Cell. Physiol. Biochem. 2008, 21, 251–258. (10) Wang, Z. P.; Keppler, F.; Greule, M.; Hamilton, J. T. Nonmicrobial methane emissions from fresh leaves: effects of physical wounding and anoxia. Atmos. Environ. 2011, 45, 4915–4921. (11) Ferretti, D. F.; Miller, J. B.; White, J. W. C.; Lassey, K. R.; Lowe, D. C.; Etheridge, D. M. Stable isotopes provide revised global limits of aerobic methane emissions from plants. Atmos. Chem. Phys. 2007, 7, 237–241. (12) Wang, Z. P.; Han, X. G.; Wang, G. G.; Song, Y.; Gulledge, J. Aerobic methane emission from plants in the Inner Mongolia steppe. Environ. Sci. Technol. 2008, 42, 62–68. (13) Bruhn, D.; Mikkelsen, T. N.; Øbro, J.; Willats, W. G. T.; Ambus, P. Effects of temperature, ultraviolet radiation and pectin methyl esterase on aerobic methane release from plant material. Plant Biol. 2009, 11 (Suppl. 1), 43–48. (14) Wang, Z. P.; Gulledge, J.; Zheng, J. Q.; Liu, W.; Li, L. H.; Han, X. G. Physical injury stimulates aerobic methane emissions from terrestrial plants. Biogeosciences 2009, 6, 615–621. 9536
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Environmental Science & Technology
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dx.doi.org/10.1021/es2020132 |Environ. Sci. Technol. 2011, 45, 9531–9537
