Oxygenation of methane by methane-grown Pseudomonas methanica and Methanomonas methanooxidans

1. Experimental conditions have been found in which small amounts of methanol (approximately 2.5mm) accumulated when washed cell suspensions of methane-grown Pseudomonas methanica and Methanomonas methanooxidans were incubated with methane+oxygen mixtures in Warburg flasks. 2. The methanol formed could be separated completely from water by fractional distillation through glass helices followed by gas chromatography using 20% polyethylene glycol 400 on a Celite 545 support. 3. By using ¹⁸O-enriched oxygen gas the abundance of ¹⁸O in the methanol formed from oxidation of methane was measured with a Perkin–Elmer 270 combined gas chromatograph/mass spectrometer. The results showed that the oxygen in methanol was derived exclusively from gaseous oxygen in both micro-organisms. 4. Control experiments using [¹⁸O]water in incubation mixtures confirmed that there was negligible incorporation of the oxygen atom from water into methanol.     

Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

Heme-Dependent Rubber Oxygenase RoxA of Xanthomonas sp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of ¹⁶O₂, ¹⁸O₂, H₂¹⁶O, and H₂¹⁸O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of ¹⁸O-compounds. Incorporation of one ¹⁸O atom in ODTD was found if the cleavage reaction was performed in the presence of ¹⁸O₂ and H₂¹⁶O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H₂¹⁸O in the presence of ¹⁶O₂ indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of ¹⁸O₂, H₂¹⁶O, and alcohol dehydrogenase/NADH, incorporation of two atoms of ¹⁸O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Spontaneous Hydrolysis and Dehydration of Dehydroascorbic Acid in Aqueous Solution

The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in¹⁸O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H₂¹⁸O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H₂¹⁸O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H₂¹⁸O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

New Type of Oxygenase Involved in the Metabolism of Propane and Isobutane

Nocardia paraffinicum (Rhodococcus rhodochrous), a hydrocarbon-degrading microorganism, was used in a study of propane and isobutane metabolism. The bacterium was able to utilize propane or isobutane as a sole source of carbon, and oxygen was found to be essential for its metabolism. Gas chromatographic analysis showed that n-propanol was the major compound recovered from the metabolism of propane by resting cells, although trace amounts of isopropanol and acetone were detected. When a mixture of propane and isobutane was used, drastic inhibition (72 to 88%) of hydrocarbon utilization by resting cells occurred. The ratio of hydrocarbon to oxygen consumed was found to be approximately 2:1 during the metabolism of propane or isobutane by resting cells when these substrates were provided individually to the organism. Gas chromatographic-mass spectrometric analysis of products formed from ¹⁸O₂ confirmed that the initial oxidative step in the metabolism of these substrates involved molecular oxygen. The proportion of the alcohol containing ¹⁸O was the same as that of ¹⁸O₂ in the gas mixture. Only a negligible amount of ¹⁸O was detected in the alcohol when H₂¹⁸O was incorporated into the system. The observed 2:1 ratio of hydrocarbon to oxygen consumption suggests that the oxygenase in N. paraffinicum, unlike the conventional mono- or dioxygenases, requires two hydrocarbon-binding sites for each of the oxygen-binding sites and is therefore an intermolecular dioxygenase. The newly described oxygenase, which catalyzes the reaction of two molecules of propane with one molecule of oxygen to yield two molecules of a C₃ alcohol, is proposed as the initial oxidation step of the hydrocarbon substrate.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Methane oxidation by methanotrophs and its effects on the phosphate flux over the sediment-water interface in a eutrophic lake

The effect of methane oxidation in aerobic sediment on oxygen consumption and phosphate flux was investigated in diffusion chambers. The diffusion chambers consisted of two compartments separated by a Teflon membrane. In the upper chamber a thin sediment layer was present and the lower chamber was continuously flushed with gas. The hydrophobic membrane allowed for diffusion of gases from the lower chamber through the sediment layer toward the headspace of the upper chamber. In experiments with a methane oxidation rate of 9.8 mmol m^–2 day^–1, the oxygen consumption rate increased by a factor of two compared with controls without methane oxidation (8.6 vs 17.7 mmol m^–2 day^–1). Methane oxidation significantly decreased oxygen penetration depth (2.5–4.0 vs 1.0–2.0 mm). However, despite the shrinkage of the oxidized microlayer, no differences were found in phosphate flux across the sediment water interface. Batch experiments with standard additions of methane revealed that the growth of methanotrophic bacteria contributes to the phosphate uptake of aerobic sediment. From the batch experiments a molar ratio of carbon to phosphate of 45 mol:mol was calculated for the growth of methanotrophs. Results suggest that a decrease in chemical phosphate adsorption caused by a decrease in the oxygen penetration depth could be compensated for entirely by the growth of methanotrophic bacteria. Send offprint requests to: A.J.C. Sinke     

N2O and CH4 fluxes in undisturbed and burned holm oak, scots pine and pyrenean oak forests in central Spain

We investigated N₂O and CH₄ fluxes from soils of Quercus ilex, Quercus pyrenaica and Pinus sylvestris stands located in the surrounding area of Madrid (Spain). The fluxes were measured for 18?months from both mature stands and post fire stands using the static chamber technique. Simultaneously with gas fluxes, soil temperature, soil water content, soil C and soil N were measured in the stands. Nitrous oxide fluxes ranged from ?11.43 to 8.34?μg N₂O–N?m^?2?h^?1 in Q.ilex, ?7.74 to 13.52?μg N₂O–N?m^?2?h^?1 in Q. pyrenaica and ?28.17 to 21.89?μg N₂O–N?m^?2?h^?1 in P. sylvestris. Fluxes of CH₄ ranged from ?8.12 to 4.11?μg CH₄–C?m^?2?h^?1 in Q.ilex, ?7.74 to 3.0?μg CH₄–C m^?2?h^?1 in Q. pyrenaica and ?24.46 to 6.07?μg CH₄–C?m^?2?h^?1 in P. sylvestris. Seasonal differences were detected; N₂O fluxes being higher in wet months whereas N₂O fluxes declined in dry months. Net consumption of N₂O was related to low N availability, high soil C contents, high soil temperatures and low moisture content. Fire decreased N₂O fluxes in spring. N₂O emissions were closely correlated with previous day’s rainfall and soil moisture. Our ecosystems generally were a sink for methane in the dry season and a source of CH₄ during wet months. The available water in the soil influenced the observed seasonal trend. The burned sites showed higher CH₄ oxidation rates in Q. ilex, and lower rates in P. sylvestris. Overall, the data suggest that fire alters both N₂O and CH₄ fluxes. However, the magnitude of such variation depends on the site, soil characteristics and seasonal climatic conditions.     

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen

Methanosarcina barkeri was cultured on methanol, H₂-CO₂, and acetate, and the ¹³C/¹²C ratios of the substrates and the methane produced from them were determined. The discrimination against ¹³C in methane relative to substrate decreased in the order methanol > CO₂ > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO₂. These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher ¹³C/¹²C ratios than those from environments where other substrates predominate.     

Controllable vesicles based on unconventional cyclodextrin inclusion complexes

Vesicles were assembled from an unconventional inclusion complex between β-cyclodextrin (βCD), and N,N′-bis(ferrocenylmethylene)diaminohexane (1). The vesicles formed in water and in a mixed solvent (water/methanol) were observed by transmission electron microscopy. The peculiar inclusion effects of 1·βCD were characterized by UV and cyclic voltammetry. The structure of the complex was characterized by ¹H- and 2D ROESY NMR spectroscopies. The size of the vesicles in water, methanol, and in mixtures of water and methanol was investigated by dynamic light scattering. The vesicles disappeared upon addition of an oxidizing agent. The structures of the inclusion complex and the vesicles formed via the complex are discussed according to the experimental data.     

Effects of salt water immersion caused by a tsunami on δ<Superscript>13</Superscript>C and δ<Superscript>18</Superscript>O values of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> tree-ring cellulose

Pinus thunbergii trees growing on Pacific coastal sand dunes in Japan were immersed by the tsunami that followed the Great East Japan Earthquake on 11 March 2011. Even trees that survived direct physical damage began to die during the following summer, probably because of the physiological stress of salt water immersion. The objectives of this study were to analyze the relationship between the carbon and oxygen isotope values (δ¹³C and δ¹⁸O, respectively) of P. thunbergii tree-ring cellulose and the effects of salt water immersion caused by the tsunami. Pinus thunbergii trees were sampled in Yamamoto, Miyagi Prefecture, and in Misawa, Aomori Prefecture. Each tree-ring that formed between 2008 and 2012 was sliced into four to eight equal subdivisions, and the isotope values were analyzed at a high time-scale resolution. Tree rings that were immersed in seawater from the tsunami had higher δ¹³C values in the earlywood that formed in the spring following the tsunami than those formed prior to the disaster. This increase in δ¹³C values was likely caused by osmotic stress from root immersion in salt water. We did not observe a clear change in tree-ring δ¹⁸O values after the tsunami. This lack of variance might have resulted from the post-photosynthetic exchange of carbonyl oxygens with non-¹⁸O-enriched xylem water.     

Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (−)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used ¹⁸O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under ¹⁸O₂, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H₂¹⁸O. Pisatin synthesized by pea pods in the presence of ¹⁸O₂ or H₂¹⁸O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H₂O and not from O₂. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.     

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, δ¹⁸O cellulose = 0.48 δ¹⁸O water + 24.1%‰. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of ¹⁸O in the form of O₂ bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O₂ of normal ¹⁸O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.     

Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen

Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% ¹⁸O₂ and 80% N₂ indicates that one atom of ¹⁸O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing ¹⁸O₂ indicates that one atom of ¹⁸O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

     

Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis

Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 (¹⁸O). We used the relationship between the amount of ¹⁸O in the water and in the cellulose that was synthesized to determine the number and ¹⁸O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis.     

Physiological control of leaf methane emission from wetland plants

The transport of methane from the rhizosphere to the atmosphere takes place in the intercellular spaces and stomata of wetland plants, and foliar gas exchange is one of the critical steps of the transport process. The objectives of our research were to investigate: (i) variation in foliar gas exchange among four common wetland plant species (i.e., Peltandra virginica L., Orontium aquaticum L., Juncus effusus L., and Taxodium distichum L.), (ii) the role of key environmental factors (i.e., light, temperature, and carbon dioxide concentration) in controlling foliar methane emission, and (iii) physiological mechanisms underlying the variation in methane emission due to species and the environment. Experiments were conducted in an instantaneous, flow-through gas-exchange system that operated on a mass balance approach and concurrently measured foliar fluxes of methane, water vapor, and carbon dioxide. The chamber system allowed for the control of light, temperature, humidity, and carbon dioxide concentration. Diel patterns of methane emission varied among species, with daylight emissions from P. virginica and O. aquaticum 2–4 times those of J. effusus and T. distichum in saturating light. Foliar methane emission from P. virginica (1.80 μmol m⁻² s⁻¹) under ambient daylight conditions was an order of magnitude higher than that of the other three species (∼0.20 μmol m⁻² s⁻¹). As leaf temperature was increased by 10 °C, methane emission increased by a factor of 1.5–2.2, and the temperature effect was independent of stomatal conductance. When data were pooled among the four species, varying the light and carbon dioxide concentrations in a stepwise manner produced changes in foliar methane emission that were associated with stomatal conductance (r² = 0.52). To scale our observations to other wetland plant species, a stepwise multiple regression model is offered that incorporates stomatal conductance and net carbon dioxide assimilation to estimate instantaneous methane emission from foliar surfaces. The model indicates that changes in stomatal conductance affect methane emission three times more than equivalent changes in net carbon dioxide assimilation.     

鄱阳湖苔草湿地甲烷释放特征

2009年5月-2010年4月在鄱阳湖南矶湿地国家级自然保护区选择以灰化苔草为建群种的洲滩,设置土壤-植物系统(TC)、剪除植物地上部分 (TJ)2个试验处理,利用密闭箱-气相色谱法测定了鄱阳湖典型苔草湿地的甲烷(CH₄)释放通量。结果表明:1)TC、TJ 2个试验处理CH₄释放速率变化范围分别为-0.094-17.75 mg · m^-2 · h^-1、-0.122-19.16 mg · m^-2 · h^-1,均表现出明显的季节变化规律;2)地表未淹水期间,剪草处理CH₄释放显著高于非剪草处理(t=2.69, P<0.05);地表淹水达到15 cm后,剪草处理CH₄释放明显低于非剪草处理。3)土壤5 cm温度、土壤水分与2处理非淹水期间CH₄释放速率均呈显著正相关,是非淹水期间CH₄通量变化的主要控制因子,2因子能够共同解释非淹水期苔草湿地65%-74%的CH₄通量变异;4)试验期间,苔草湿地CH₄释放量约为12.77 gC/m²,相当于同期土壤有机质分解碳排放量的4%,甲烷释放的碳消耗不足苔草湿地年NPP的1%。