WO2011007907A1 - Novel methane oxidizing bacteria, methylocystis microorganism and method for reducing methane using same - Google Patents

Abstract

The present invention relates to novel methane oxidizing bacteria, Methylocystis microorganism and a method for reducing methane using the same. More specifically, the novel Methylocystis microorganism has good degradability for methane or other volatile organic compounds and can more effectively degrade methane under biocover or a reduced system using an earthworm-cast as filler material.

Description

Novel Microorganisms of Methyloxidation Bacteria

The present invention relates to a novel microorganism of the genus methane oxidizing bacteria methyloscis and to a method for reducing methane using the same. More specifically, the novel microorganism of the genus methyloscistis excellent in resolution for methane or volatile organic compounds is earthworm as a filler. Methane can be more effectively decomposed in methane abatement biocovers or abatement systems that use fecal soil.

Representative greenhouse gases that cause global climate change include carbon dioxide (CO ₂ ), methane (CH ₄ ), nitrous oxide (N ₂ O), and F-gases (HFCs, PFCs, SF ₆ ). Since the Industrial Revolution, global human greenhouse gas emissions have risen rapidly due to human activities. Methane, the second-highest greenhouse gas contributing to carbon dioxide, is a colorless, odorless greenhouse gas that is explosive if it contains 5-15% of air and is a harmful gas that causes respiratory distress after long-term exposure to 0.5% methane. In particular, the residence time of methane is 12 ± 3 years, which is relatively short compared to other greenhouse gases. Therefore, GWP is 20 times higher than that of carbon dioxide. Methane is characterized not only by the agriculture and fossil fuel use sectors, but also by other sources of waste treatment, unlike other greenhouse gases. The amount of methane from landfills and waste is estimated to be around 35-73 Tg per year. Methane is produced by methane-producing bacteria in the anaerobic decomposition of organic matter, and the amount of methane produced by these biological activities is estimated to be about 70-80% of the total generation. Methane removal is largely eliminated by reaction with OH radicals in the troposphere (CH ₄ + OH-> CH ₃ + H ₂ O) and with chlorine in the stratosphere (CH ₄ + Cl-> CH ₃ + + HCl). . Also, in aerobic environments, methane is finally oxidized to carbon dioxide by methane nutrients as the only carbon and energy source, which is the main extinction mechanism of methane.

On the other hand, some landfills, which are the major sources of methane, are equipped with gas collection wells that can capture biogas. Generally, about 40 to 60% of the biogas produced can be recovered. It is reported that up to 90% of biogas can be recovered if synthetic resin covers are installed in the landfill. Biogas containing methane discharged from landfills can be recovered, recycled for energy, or burned, or oxidized and removed using cover soil microorganisms. Combustion of biogas as an energy source by burning biogas is an ideal way to reduce greenhouse gases and secure renewable energy.However, methane content of biogas is over 30% and biogas flux is 50 m ³ h. Only applicable when ^-1 . For landfill biogas collecting Jung installed, installation cost and operation cost required to recycling the methane gas corresponding to CO ₂ in terms of tone of the first tone as an energy source is take US $ 3.1. The chemical oxidation method of oxidizing methane gas at 600-800 ° C. in a packed column using a catalyst is a method applicable when the concentration of methane gas is low at 0.1-1%. In addition, a method of converting methane into methanol to be recycled is also being studied. To prevent the risk of explosion in landfills, biogas is simply burned off without energy recovery. This simple incineration method is also applicable to landfills equipped with gas collection wells, when the concentration of methane in biogas is at least 20% and biogas flux is 10-15 m ³ h ^-1 . In addition, there is a problem that secondary pollutants such as dioxins are generated during incineration of biogas. In Korea as well as in developed countries, there are still many landfills without gas collection facilities, and many landfills with low biogas emissions exist due to the lapse of time or the size of landfills.

In the case of landfills without gas collection facilities, landfills with low biogas emissions due to the lapse of time or small landfills, researches using biological methods such as biofilters or biocovers to minimize methane emissions, greenhouse gases Has been very active in recent years. The biological method uses methane oxidizing bacterium activity to remove and methane oxidize under aerobic conditions, and biofilters or biocovers have been developed that utilize various materials such as soil, compost, and peat as fillers.

Therefore, the performance of biofilters and biocovers evaluated in the field application is known to vary greatly in methane removal efficiency according to landfill characteristics, filler types and environmental conditions. Therefore, it is essential to select effective methane oxidizing bacteria and fillers for methane removal. will be.

It is an object of the present invention to isolate and identify new methanogenic bacteria that can decompose methane in landfills.

Another object of the present invention is to provide a method for reducing methane by using the novel methane oxidizing bacteria.

Still another object of the present invention is to provide a biocover capable of removing methane using the novel methane oxidizing bacteria and a methane abatement system including the same.

Another object of the present invention is to provide a method of reducing methane using the system.

In order to achieve the above object, the present invention provides a Methylocystis sp. M6 KCTC 11519BP.

The present invention also provides a composition for reducing methane comprising Methylocystis sp. M6 KCTC 11519BP.

The present invention also provides a methane reduction method comprising the step of decomposing methane methane reduction composition of the present invention.

The present invention also provides a methane-reducing biocover emanating from the cover layer or surface of a landfill containing the methane-reducing composition of the present invention.

The present invention also provides a methane abatement system for biologically decomposing methane emitted from the cover layer or the ground surface by installing a bio active layer on the cover layer or the ground surface of the landfill.

A biocover layer in which at least one biocover of the present invention is laminated; And

It provides a methane reduction system comprising a ventilation layer surrounding the biocover layer.

The present invention also provides a methane abatement system for biologically decomposing methane emitted from the cover layer or the ground surface by installing a bio active layer on the cover layer or the ground surface of the landfill.

A biocover layer in which at least one biocover of the present invention is laminated; And

It provides a methane reduction system comprising a ventilation layer stacked on the lower bio cover layer.

The present invention also provides a methane abatement method comprising the step of decomposing methane by injecting a sample into the methane abatement system of the present invention.

The novel microorganism of the genus methylosciss of the present invention can be used in a biocover installed in a landfill cover or surface that is capable of biologically decomposing methane due to its excellent resolution for methane or volatile organic compounds. In addition, the present invention can further increase the methane oxidation efficiency by using earthworm fecal soil as a filler.

1 is a comparison of the methane oxidation rate according to the earthworm feces mixing ratio, (a) the initial, (b) shows the activity.

Figure 2 shows the methane oxidation characteristics of soil and feces mixed soil.

Figure 3 shows the effect of water content (a) and temperature (b) on the rate of methane oxidation.

Figure 4 is a comparison of the bacterial community characteristics of paddy soil, fecal soil and mixed soil, (a) general bacteria, (b) methane oxide bacteria.

Figure 5 shows the phylogenetic relationship of pmoA -clone and pmoA -DGGE band clone of fecal soil samples.

Figure 6 shows the methane decomposition of the landfill thickening broth, symbol ●, experimental group ○, control.

Figure 7 shows a phylogenetic tree of clones obtained from landfill thickening broth.

Figure 8 shows a phylogenetic tree of Methylocystis sp. M6 strain of the present invention.

Figure 9 shows the methane decomposition characteristics of Methylocystis sp. M6 of the present invention.

10 shows a laboratory scale biocover and a laboratory scale biocover installation drawing.

FIG. 11 compares the methane concentration emitted from the biocover surface (40% CH ₄ + 60% CO ₂ (v / v) mixed gas flow rate: 10 mL / min).

12 shows changes in O ₂ , N ₂ , CH ₄ and CO ₂ concentrations at each height of the biocover (40% CH ₄ + 60% CO ₂ (v / v) mixed gas flow rate: 10 mL / min).

FIG. 13 compares the methane concentration emitted from the biocover surface (40% CH ₄ + 60% CO ₂ (v / v) mixed gas flow rate: 20 mL / min).

FIG. 14 shows changes in O ₂ , N ₂ , CH ₄ and CO ₂ concentrations at each height of the biocover (40% CH ₄ + 60% CO ₂ (v / v) mixed gas flow rate: 10 mL / min).

15 shows a methane or volatile organic compound reduction system including the biocover of the present invention for installation in a landfill.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention relates to Methylocystis sp. M6 KCTC 11519BP.

The microorganism of the present invention is a novel methane oxidizing bacterium having excellent resolution of methane or volatile organic compounds, sealing a culture bottle containing landfill soil and enriched culture medium, for example, NMS (nitrate mineral salts) medium, and methane as a carbon source. Injected with nitrogen and phosphorus continuously during incubation, isolates and identified a new methane-oxidizing bacterium, Methylocystis sp. On 3rd, he was deposited with the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC 11519BP. Methylocystis sp. M6 strain is excellent in the resolution of volatile organic compounds in addition to methane.

The present invention also relates to a composition for reducing methane comprising Methylocystis sp. M6 KCTC 11519BP.

The composition of the present invention can use the novel Methylocystis sp. M6 KCTC 11519BP of the present invention as methane oxidizing bacteria to remove methane.

In addition, the methane reducing composition of the present invention may be used alone or both of the soil, earthworm fecal soil, etc. as a filler for the culture environment composition of the bacterial culture.

The earthworm fecal soil is an earthworm fecal soil produced by supplying the sewage sludge generated in the sewage treatment process to earthworm food. After the natural fermentation / drying process for more than 6 months, impurities may be removed and the particle size may be 0.2 to 2 mm. .

In addition, the soil may be paddy / field soil, forest soil, or wetland soil, but is not particularly limited thereto.

The soil can be collected at a depth of 100 to 200 cm from the surface layer, and then sieved up to 3 mm to remove large particles.

The soil and earthworm fecal soil pH is 5 to 7, the water content is 1 to 40%, the organic content may be used as 1 to 40%, but is not particularly limited thereto.

In addition, the soil and earthworm fecal soil is preferably mixed in a ratio of 50: 50 to 90: 10 to weight. It is because the reduction efficiency of methane is high when it is in the said content range.

The present invention also relates to a methane reduction method comprising the step of decomposing methane methane reduction composition of the present invention.

The methane reducing composition of the present invention can effectively decompose these using methane as a carbon source.

The present invention also relates to a methane-reducing biocover emanating from the cover layer or surface of a landfill containing the methane-reducing composition of the present invention.

When the methane reducing composition of the present invention is put in a methane reducing biocover and the biocover is contacted with methane, methane may be effectively decomposed.

The biocover of the present invention may include a biomedia layer containing the composition for reducing methane of the present invention.

The biomedia layer may include Methylocystis sp. M6 KCTC 11519BP as a methane oxidizing bacterium capable of biologically decomposing methane.

In addition, the biomedia layer may further include a conventional methane oxidizing bacteria in order to increase the decomposition efficiency of methane. In the methane is methyl by oxidation bacteria Pseudomonas genus (Methylomonas), methyl micro emptying in (Methylomicrobium), methyl bakteo in (Methylobacter), methyl local dumsok (Methylocaldum), waves with methyl acceleration (Methylophaga), Sar with methyl or when a Methylosarcina , Methylothermus , Methylohalobius , Methylosphaera , Methylocystis , Methylocella , Methyl rokap sasok (Methylocapsa), may be used alone or two or more kinds in Sinners (Methylosinus), or methyl Rhodococcus genus (Methylococcus) such as methyl.

In addition, the biomedia layer may include soil or earthworm fecal soil as a filler. The type of the soil, the manufacturing method of earthworm fecal soil is as described above.

In addition, the biomedia layer may further include an oxygen generating agent to supply the oxygen required by the methane oxide bacteria to reduce the thickness of the bio cover.

The oxygen generating agent may be used alone or two or more of magnesium peroxide, calcium peroxide, sodium percarbonate and the like.

The present invention also provides a methane abatement system for biologically decomposing methane emitted from the cover layer or the ground surface by installing a bio active layer on the cover layer or the ground surface of the landfill.

A biocover layer in which at least one biocover of the present invention is laminated; And

It relates to a methane abatement system comprising a ventilation layer surrounding the biocover layer.

The biocover layer contains methane oxidizing bacteria and earthworm fecal soil, which can biologically decompose methane.

The methane oxidizing bacteria may include the novel Methylocystis sp. M6 KCTC 11519BP of the present invention.

The methane oxidizing bacteria may further comprise the above-described conventional methane oxidizing bacteria.

In addition, the biocover layer may use soil or earthworm fecal soil as a filler to increase the oxidation efficiency of methane. Details of the soil or earthworm feces are as described above.

In addition, the thickness of the biocover layer is preferably 50 to 500 mm. When the thickness is less than 50 mm, the time for contact between the methane oxidizing bacteria and methane gas is short so that the oxidation does not occur sufficiently that the methane gas is not converted to carbon dioxide. When the thickness exceeds 500 mm, oxygen diffused in the atmosphere cannot diffuse to the bottom of the biocover layer and thus cannot establish aerobic conditions.

The biocover layer is surrounded by a ventilation layer for supplying oxygen. The component constituting the ventilation layer is not particularly limited as long as it has a particle size capable of supplying oxygen. For example, it may consist of sand or gravel. In addition, the ventilation layer may be provided with at least one vent pipe for supplying air. Air may be injected into the blower through the vent pipe.

The present invention also provides a methane abatement system for biologically decomposing methane emitted from the cover layer or the ground surface by installing a bio active layer on the cover layer or the ground surface of the landfill.

A biocover layer in which at least one biocover of the present invention is laminated; And

It relates to a methane abatement system comprising a ventilation layer stacked below the biocover layer.

In the methane reduction system of the present invention, a ventilation layer capable of supplying oxygen between the cover layer and the biocover layer may be stacked.

Details of the biocover layer are as described above.

The component constituting the ventilation layer is not particularly limited as long as it has a particle size capable of supplying oxygen. For example, it may consist of sand or gravel. In addition, the ventilation layer may be provided with at least one vent pipe for supplying air. Air may be injected into the blower through the vent pipe.

The present invention also relates to a methane abatement method comprising the step of decomposing methane by injecting a sample into the methane abatement system of the present invention.

When the methane gas is injected into the methane abatement system of the present invention, the methane oxidizing bacteria of the present invention and the earthworm fecal soil as a filler can be effectively decomposed.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example 1 Methane Reduction Effect of Earthworm Fecal Soil

The soil used in this example was paddy soil of Gyeonggi-do, which was collected after digging more than about 30 cm from the surface layer, and removing the large particles by striking the collected soil with a 2 mm sieve. Earthworm fecal soil was also collected from sewage treatment facility in Seoul. An earthworm fecal soil produced by supplying sewage sludge from sewage treatment to earthworm food, and using average particle size of 0.2-2 mm after natural fermentation / drying process for more than 6 months and impurities removed and particle size selection process It was.

Soil and fecal soil pHs were 6.47 ± 0.08 and 5.23 ± 0.13, respectively, and the water contents were 2.27 ± 0.11%, 38.09 ± 1.39%, and organic matter contents were 1.71 ± 0.24% and 37.41 ± 1.21%, respectively.

Experimental Example 1 Effect of Soil and Earthworm Fecal Mix Ratio on Methane Oxidation Rate

In order to investigate the difference of methane removal rate according to the difference of soil and fecal soil mix ratio, paddy soil and fecal soil were treated with 10: 0 (w / w), 1: 9, 2: 8, 3: 7, 4: 6 and 5: 5. After mixing to the conditions, distilled water was added so that the water content of the mixed soil was 25%. 50 g of each mixed soil was added to 600 mL-serum bottles, sealed with butyl rubber, and injected with methane gas at a final concentration of 5% (50,000 ppmv). When the methane concentration of the upper portion of the serum bottle was periodically analyzed while stationary incubation at 25 ° C., the methane gas having the same concentration as above was re-injected. Methane gas re-injection was performed 5-10 times in the same way, and the rate of methane oxidation following re-injection was determined.

0.5 mL of gas from the headspace of each serum bottle was collected using a gas-tight syringe (Agilent), and then CH ₄ concentration analysis was performed using gas chromatography (Agilent 6890 plus, USA) equipped with a flame ionization detector (FID). It was. Analytical conditions were HP-1 column (30 m × 0.320 mm × 0.25 μm), carrier gas (H ₂ 35 mL / min, air 300 mL / min), make-up gas (N ₂ 30 mL / min), split ratio 12: 1, injection unit temperature 250 ° C, detection unit temperature 250 ° C, oven temperature 100 ° C (2.5 minutes hold). Gas chromatography, Agilent 7890A, USA (GC) equipped with TCD (Thermal conductivity detector) was used for oxygen and carbon dioxide concentration analysis, including CH ₄ . Analytical conditions were Molesive 5A column (30 m × 0.53 mm × 25 μm), carrier gas (He 20 mL / min), make-up gas (He 3 mL / min) split ratio 2: 1, inlet temperature 200 ° C, The detection unit temperature was performed at 250 ° C., oven temperature 50 ° C. (3 minutes hold), and 30 ° C./min at 250 ° C. (2 minutes hold).

Methane removal capacity (oxidation rate) according to the mixing ratio of soil and fecal soil is shown in FIG. 1.

As shown in Fig. 1, the methane oxidation rate of only non-mixed bunbyeonto soil was ^{1.5 ㎍ · g-day soil -1} · h -1 in an initial state, after the concentrated culture 4.9 1.5 ㎍ · g-day soil - ^It was improved to ¹ · h ^-1 .

When fecal soil is mixed with soil at a ratio of 10, 20, 30, 40, 50% (w / w), the methane oxidation capacity is 8.6-25.1 ㎍ g-day soil ^-1 h ^-1 (6.1-17.9 g ^-3 h ^-1 ). This shows that satisfactory methane oxidation can be obtained even if the ratio of earthworm fecal soil is reduced to 10%.

The filter bed is a place where biofilm containing methane oxidizing bacteria is cultured, so it needs enough space for growth of microorganisms, requires high water content, meets physical / chemical / biological properties, and the lower the cost, the better. Therefore, fecal soil was sufficiently satisfied as a filter bed, and it was confirmed that it can be used as a material for biocover and biofilter because it obtains good methane oxidation ability even with a small amount of mixing.

Experimental Example 2 Effect of Temperature and Water Content on Methane Oxidation Capacity

To investigate the effects of temperature and water content on methane oxidation rate in soils mixed with 5: 5 of paddy soils and earthworm feces, mixed soils in 13 600 mL-serum bottles (paddy soil: fecal soil = 5: 5, After 100 g of w / w) was added, methane gas was added to 5%. When the methane concentration was lowered to 200 ppmv or less while incubated at 25 ° C. under constant temperature, the process of reinjecting methane was performed five times. The mixed soil reaching the steady state was removed from each serum bottle and mixed uniformly (about 1300 g in total), and then naturally dried for 3 days until the moisture content of the mixed soil became 15% or less. 50 g each of the naturally dried mixed soil was dispensed into 600 mL-serum bottles.

In order to investigate the effect of the water content on the methane decomposition ability, distilled water was added and sealed after adding 15, 20, 25, 30, 35 and 40% water content of the mixed soil of the serum bottle containing 50 g of the mixed soil. Methane gas was injected (final concentration: 50,000 ppmv) and methane concentration was periodically analyzed at the top of the serum bottle while static culture at 25 ° C. When the methane concentration was lowered below 200 ppmv, methane gas was reinjected to the same concentration. Re-injection was performed 5 times in this way, and the methane oxidation rate was calculated by calculating the methane concentration decrease over time. The experiment was performed by preparing two sets of serum bottles for each moisture content condition.

On the other hand, to investigate the effect of temperature on the methane oxidation capacity, distilled water was added so that the moisture content of the mixed soil of the serum bottle containing 50 g of mixed soil was 25%, and then methane gas was injected after closing (final concentration: 50,000 ppmv). ). The methane degradation rate was measured while each serum bottle was incubated at 15, 20, 25, 30, 35 and 40 ° C. Experiments were performed by preparing two sets of serum bottles for each temperature condition. In the temperature effect experiment, methane was reinjected 5 times in the same manner as the water content effect experiment, and the average value was calculated by calculating the methane decomposition rate at each injection.

Serum disease experiments were conducted to investigate the methane oxidation characteristics of paddy soil and fecal mixed soil in detail. First, in order to investigate whether methane is removed by physical / chemical reaction in the mixed soil, the methane was removed by sterilizing the mixed soil, and the removal of methane by physical adsorption or chemical reaction was not observed (not shown).

In addition, as shown in FIG. 2, when the methane removal characteristics of the mixed soil were examined, methane was removed after about two days of lag period when the first methane was injected, but the methane was removed without a lag period from the second methane reinjection. Was observed (FIG. 2A). This result suggests that fecal soil contains more microorganisms involved in methane decomposition than paddy soil. Fecal soil is therefore considered one of the best inoculum in methane removal system.

In addition, oxygen concentration and carbon dioxide concentration also increased with the decrease of the concentration of methane, but no increase of the carbon dioxide concentration was observed when no methane was injected (FIG. 2B). This means that methane was decomposed by methane oxidizing bacteria and released into carbon dioxide. Methane oxidizing bacteria follow an energy metabolic pathway that completely oxidizes CH ₄ to CO ₂ or a metabolic pathway that transforms them into cellular components: CH ₄ + 2O ₂ Biomass + CO ₂ + 2H ₂ O, ie CH ₄ oxidation By using HCHO, a product, for the synthesis of cellular materials, theoretically, the amount of CO ₂ produced per molecule of CH ₄ is less than one molecule. Therefore, although CH ₄ is oxidized to CO ₂ by methane oxidizing bacteria, some of them can reduce the total amount of greenhouse gases by metabolism. In this Experimental Example, the ratio of CO ₂ generation to CH ₄ consumption at 25% humidity and 25 ° C was 0.46, and 0.97 when the water content was 40%. Therefore, it was confirmed that not all CH ₄ is oxidized to the same concentration of CO ₂ , which shows the possibility of engineering the fact that the total amount of global warming gas can be reduced in terms of prevention of global warming. Giving.

3A shows the effect of methane oxidation ability according to the change in water content using a mixed soil having a mixing ratio of soil and earthworm fecal soil by 5: 5 concentration. The rate of methanation at 25% water content was 559.5 ㎍ · g-dry soil ^-1 · h ^-1, and at 57% -78% water content at 25% water content above or below 25% water content. Value was shown. However, the mixed soil of earthworm fecal soil and paddy soil effectively removed methane in a wide range of water content of 15-40%. In general, considering that the water content of the landfill is widely distributed from 7 to 45%, it can be seen that the earthworm feces mixed soil is suitable as a biocover (biocover) material.

In addition, as a result of examining the effect of water content on the methane oxidation, the methane removal capacity when the water content of earthworm fecal mixed soil is 15-40% is more than 4.4 ~ 7.8 times that of landfill soil, which is much higher than other reports. see.

The effect of temperature on the rate of methane oxidation is shown in Figure 3b. The optimum temperature for methane oxidation was 25 ℃ and the rate of methane oxidation was 504.8 ㎍ · g-dry soil ^-1 · h ^-1 after enrichment. As the temperature was lowered below 25 ° C., the methane oxidation capacity decreased drastically and at 15 ° C. decreased to about 26% of 25 ° C. However, the methane oxidation rate in the range of 20 ~ 40 ℃ excluding 25 ℃ 129 ~ 380 ㎍ · g-dry soil ^-1 · h ^-1 was able to obtain excellent methane oxidation capacity in a relatively wide temperature range.

As a result, methane oxidizing bacteria can be regarded as mesophilic microorganisms. There is no significant difference in seasonal temperature changes in the landfill, but the temperature changes due to the direct contact with air in the 10–30 cm deep soil layer, which is expected to have the most active methane oxidizing bacteria. Since the uppermost temperature of the cover layer in winter keeps about 15 ° C on average, it can be said that the biocover landfill can be applied.

In this experiment, the high oxidation capacity was shown in the range of 15 ~ 40 ℃ effective methane oxidation capacity.

Experimental Example 3 Analysis of Microbial Community

(Total DNA extraction)

DNA was extracted from fecal soil (RES), paddy soil (RPS), and mixed soil (EEPS, earthworm cast: paddy = 5: 5) using Fast DNA SPIN for Soil Kit (MP Biomedical). The extracted DNA was stored at -20 ° C until used as PCR material.

(PCR amplification)

DNA extracted from RPS, RES, and EEPS was used to amplify 16S rRNA gene and pmoA gene, respectively. Approximately 200 bp of the 16S rRNA gene fragment was prepared using primers 341f (CCT ACG GGA GGC AGC AG) and 518r (ATT ACC GCG GCT GCT) with 40-bp GC clamp (CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G). Amplification via PCRdmf using GG). Fragments of the particulate methane monooxigenase (pMMO approximately 510 bp) encoded by the pmoA gene were identified by primers A189f (GGN GAC TGG GAC TTC TGG) and mb661r (CCG GMG) stained with 6-FAM (6-carboxyfluorescein). Amplification was by PCR using CAA CGT CYT TAC C). PCR was performed in 50-μl of reaction solution and the components were as follows: 100 ng DNA, 0.2 mg BSA, 0.4 mM each primer, 1 × Ex Taq buffer, 0.75 U Takara Ex Taq DNA polymerase (TaKaRa Bio Inc.) , 200 mM dNTP. PCR was performed using a ^{GeneAmp ® PCR system Model 2700 (PE} Applied Biosystems) that conditions were as follows: initial denaturation 95 ℃, 5 min; 35 cycles-denatured 95 ° C., 30 seconds, annealing 55 ° C. or 60 ° C., 30 seconds, amplification 72 ° C., 30 seconds; 72 ° C., 10 minutes in the last step. Each PCR product was identified on a 1% agarose gel and purified using a QIAquick PCR purification kit (Qiagen). In order to identify the dominant methane-oxidizing bacteria of each soil through DGGE and sequencing, the secondary pmoA- PCR was carried out under the same conditions as above, and the product was purified using the primary pmoA- PCR product as a material.

(DGGE analysis)

Purified 16S rRNA gene fragments (approximately 200 bp) were subjected to 8% polyacrylamide gel (40-60% gradient urea and deionized formamide) to analyze common microorganisms in RES, RPS and EEPS soils. Electrophoresis on. Approximately 6 mg of 16S rRNA gene fragments were used and 15 at 50 V in 1 × TAE buffer (10 mM Tris base, 20 mM sodium acetate, 1 mM EDTA) warmed to 60 ° C. using DCode ^™ system (BioRed). Electrophoresis was performed for hours. Gene fragments isolated on the gel were stained with SYBR GOLD solution (Invitrogen) (1/10000 in 0.5 × TAE buffer) and confirmed using UV-illuminator. Similarity between the DGGE band patterns of each soil sample was analyzed using GelCompa II software version 3.0 (Applied Maths, Inc.) and the relative frequency of each band was numerically obtained. As the microbial diversity index in DGGE band analysis using the numerical value, Shannon diversity Index ( H ) was calculated using the following formula.

H =-∑ P _i log P _i

P _i , the relative strength of each band with respect to the total band strength total.

The purified secondary pmoA- PCR product was electrophoresed on 6% polyacrylamide gel (40-80% gradient urea and deionized formamide). The conditions were electrophoresed for 15 hours at 100V in 60 ° C. 1 × TAE buffer. DNA fragments were extracted by freeze-thaw method only in dominant bands as band intensities, and extracted pmoA gene fragments were PCR amplified using primers A189f-GC and mb661r. The amplified pmoA gene fragment was purified using a QIAquick PCR purification kit, and the nucleotide sequence was determined by primer mb661r with BigDye v3.1 and ABI auto sequencer 3730XL DNA analyzer (Applied Biosystem).

(pmoA-based T-RFLP analysis)

6-FAM stained pmoA gene fragments derived from DNAs of RES, EPS, and EEPS were purified, and about 200 ng were digested with 37 U of each restriction enzyme Msp I and Hha I (BEAMS Biotechnology) overnight at 37 ° C. Terminal restriction fragments (T-RFs) were electrophoresed in denaturing polyacrylamide gels (6 M urea and 5% polyacrylamide) using an ABI 377 DNA auto sequencer. Only 50-500 bp of T-RFs were selected and analyzed using GENESCAN analytical software (ABI). The peak width of the selected fragment sizes was considered only those peaks with 50 relative fluorescent units (RFU) or more. Each fragment peak area was calculated from the sum of the total peak areas, and the principal component analysis (PCA) was performed using the software SPSS version 12.0K (SPSS Inc.).

(pmoA-based PCR cloning and sequencing)

Purification of the pmoA a gene fragment derived from EEPS DNA, which was cloned using the pGEM-T-Easy vector system ( Promega). As a cloning vector, E. coli DH5α was transformed using a cell-pulser electroporator (GIBCO BRL.), And only white colonies transformed in selection medium ( LacZ / X-gal, ampicillin) were selected. The selected colonies were suspended in 50 μl of distilled water, then boiled at 95 ° C. for 30 minutes, and centrifuged at 13,000 rpm for 10 minutes to obtain a supernatant. PCR was performed with primer A189f and mb661r using 1 μl of the supernatant containing the clone-vector, and the sequence of the amplified pmoA gene fragment was determined as primer A189f.

Phylogenetic Analysis

EEPS-pmoA Gene sequences In the GenBank databasepmoA Nucleic acid and amino acid sequences of genes were compared and identified by Blastn and Blastx search (www.ncbi.nlm.nih.gov/BLAST). Of samplepmoA Identified and compared with the gene sequencepmoA Gene sequencing the software BioEdit version 7.0.9.0. Alignment using the (BioEdit Sequence Alignment Editor), only sequencing sequences (> 450 bp) of the softwareMEGA It was applied to phylogeny generation using version 4 (Tamura et al., 2007). The phylogenetic tree includes the Neighbor-Joining method and the Substitution model; Calculations were made using bootstrap 1000 iterations using amino acid position correction.

(Nucleotide sequence identification number)

The insoluble methane monooxigenase ( pmoA ) base sequence obtained through this experiment was listed in the GenBank database with the following certification number: Certificate number: EEPS pmoA-DGGE band sequence, FJ775131 to FJ775133 EEPS pmoA-clone base Sequence, FJ775134 through FJ77513164.

In this experiment, methane enrichment of paddy soil (RPS), earthworm fecal soil (RES), and mixed soil (EEPS) was carried out, and the diversity of general microbial community was investigated by DGGE method using 16s rRNA gene amplicons amplified from them (Fig. 4). These diversity indices ( H ' ) were RPS = 3.32, RES = 3.18 and RESS = 3.35, respectively. The similarities of DGGE band cluster in general microorganisms were 71.43% for RPS and EEPS, 58.00% for RPS and RES, and 64.59% for RES and EEPS, respectively. In the PCA analysis using the relative diversity of DGGE bands, the general microbial community of EEPS showed a closer relationship with the general microbial community of RPS than RES (FIG. 4A). These results suggest that the general microbial community of EEPS is derived from the general microbial community of RPS rather than RES.

The activity of methanases for methanation in real soils is higher than that of the soluble methane monooxigenase (sMMO), which is encoded by the pmoA gene (particulate methane monooxigenase, pMMO). It is thought to be more broadly practical. In this experiment, the diversity of methane-oxidizing bacteria present in RPS, RES, and EEPS was investigated by T-RFs obtained from T-RFLP method amplified with pmoA -gene and treated with restriction enzymes Msp I and Hha I by PCA method. As a result, the methane oxidative bacterial community of EEPS was closer to the methane oxidative bacterial community of RES than RPS (FIG. 4B). This is an interesting finding, suggesting that the methane oxidative bacterial community of EEPS is derived from the methane oxidative bacterial community of RES rather than RPS. As mentioned above, on the contrary, it was suggested that the general microbial community of EEPS was derived from the general microbial community of RPS rather than RES. These results demonstrate that earthworm feces are a good source of methane oxidizing bacteria.

In addition, 31 pmoA clones were randomly selected from the pmoA gene clone library derived from DNA extracted from EEPS to determine and compare the base sequences. As a result of investigation using BLAST, all 31 belonged to Methylocystis (type II methanotrophs), and their phylogenetic tree is shown in FIG. 5. In addition, three dominant pmoA -DGGE bands (EEPS pmoA -DGGE band EBL1-band EBL3) with band (brightness) intensity in DGGE of the secondary pmoA- PCR product were used to identify methane oxidative bacterial dominant species of RPS, RES and EEPS Were selected and their sequencing was determined and compared.

Investigation using the BLAST, EEPS pmoA -DGGE EBL1 band and band EBL2 is methyl local dumsok (Methylocaldum sp.) In seutiseu (Methylocystis) when in the band intensity was high EEPS pmoA -DGGE band EBL3 is methyl the (type I methanotrophs) (type II) (not shown). In contrast, the EEPS pmoA- DGGE band EBL2, which appeared in RES and EEPS, did not appear in RPS. These results confirmed that earthworm fecal soil contributed more directly to methane oxidative bacterium concentration than general soil (paddy soil, RPS).

As shown in the phylogenetic tree (FIG. 5), the dominant methane oxidizing bacterium enriched in EEPS (pmoA-methanotrophs are the genusMethylocaldum sp.) (type I) and Methylosissis (Methylocystis sp.) (type II). Therefore, it can be seen that type I and type II methanogenic bacteria were involved in methane oxidation in EEPS.

In terms of thickening and dominant in EEPS, the seutiseu in (Methylocystis sp.) (Type II ) is methyl seutiseu in (Methylocystis) when a relative to the strength of the number of clones with pmoA pmoA -DGGE band belonging thereto when methyl Presumably the methane oxidizing bacteria.

On the other hand, Methylocystis ( Methylocystis ) is the result of the predominance of methane-oxidizing bacteria is interesting. This is because the EEPS methane oxidizing bacteria enrichment environment was an environment rich in oxygen, low concentrations of methane (5%) and nutrient rich (earthworm feces), which favored type I methane oxidizing bacteria. However, the results are not unexpected. Because the diversity of methane oxidizing bacteria is determined by a variety of environmental factors, rather than one environmental factor: methane concentration, oxygen availability, temperature, nitrogen source, soil material and water content, and so far The reported results show no evidence that type I and type II methanogenic bacteria can dominate independently of each other due to high and low methane concentrations.

Example 2 Methane Oxidation Consortium Culture from Landfill Soil

Soil was taken from the landfill to obtain a methane oxide consortium. The soil collection site is Gongju, Chungcheongnam-do, and is an active landfill where landfill gas is continuously discharged. The collected soil was transported in an ice box and stored at 4 ° C.

Thickening culture was performed as follows to obtain a methane oxide consortium. In a 600 mL serum bottle, 8 g of wet landfill soil and 20 mL of nitrate mineral salts (NMS) medium were added and sealed with a rubber stopper. In a sealed serum bottle, 5% (v / v) of methane was injected as the only carbon source, and the serum bottle was incubated at 30 ° C. and 180 rpm. Once 100% of the injected methane was removed, the rubber bottle was opened with a serum bottle for air saturation for 1 hour. After air saturation, the rubber stopper was closed and methane was injected at the same concentration. In order to prevent inhibition due to the depletion of nitrogen and phosphorus, 1 mL of nitrogen and phosphorus concentrates were added to each other when air saturation was performed.

The NMS medium composition is as follows (MgSO ₄ .7H ₂ O 1 g / L; CaCl ₂ · 2H ₂ O 0.295 g / L; KNO ₃ 1 g / L; KH ₂ PO ₄ 0.26 g / L; 0.41 g / L ). Nitrogen and phosphorus concentrates were referred to NMS medium composition, nitrogen was prepared by adding 2 g of KNO ₃ in 100 mL distilled water and 0.52 g of KH ₂ PO ₄ and 1.65 g of Na ₂ HPO ₄ · 12H ₂ O in 100 mL distilled water. .

In order to check whether methane is decomposed during the enrichment culture, 0.3 mL was collected from the headspace of the serum bottle using a 1 mL gas tight syringe. Sample gas was analyzed using a gas chromatography (Agilent 6850N, USA) -flame ionization detector equipped with a wax column (Supelco, 30 × 0.32 mm × 0.25 μm). The analysis temperature was 100 degreeC of oven, and 230 degreeC of injection part and a detection part.

As shown in FIG. 6, methane oxidized (decomposed) concentrated culture medium enriched using landfill soil was 100% decomposed 5% methane within 65 hours, and the methane decomposition rate of the concentrated culture medium was 4.95 ± 0.26 μmole g-dry. soil ^-1 h ^-1 . From the experimental results, it was confirmed that the thickening medium effectively decomposed methane.

To investigate the microbial community of methane oxide consortium, DNA was extracted from the initial soil and consortium. About 0.5 g of wet soil was extracted using BIO101 kit for microbial community analysis of initial soil. The methane oxide consortium sample was centrifuged at 14,000 × g for 5 min after the complete decomposition of methane to extract DNA from the precipitate. BIO101 kit was used as the soil. DNA extraction was performed according to the BIO101 kit method and all samples were extracted two times.

To investigate the community structure of methane-degrading bacteria, PCR-denaturing gradient gel electrophoresis (DGGE) was used. Primer pairs used to amplify methane degradation bacteria were A189fGC (5'- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G GGN GAC TGG GAC TTC TGG-3 ') and mb661r (5'-CCG GMG CAA CGT CYT) TAC C-3 '). The amplification conditions were performed 40 times at the first stage of the initial denaturation at 96 ℃ 5 minutes at 96 ℃, denatured 96 ℃ 1 minute, annealing 58 ℃ 1 minute, amplification 72 ℃ 1 minute, and at the last stage 72 ℃ 5 minutes Experiment with. The amplified DNA concentrations were adjusted to 200-300 ng / μl and injected into 6% polyacrylamide gels. The urea change was 35-70% and operated for 17 hours at 60 ° C and 50V. The band was cut from the gel after the DGGE experiment, and DNA was extracted from the cut gel. To extract DNA from the gel, 30 μl of sterile water was added and frozen at -20 ° C. Then dissolved at 70 ℃ for 3 minutes and mixed well. After repeating the following three times amplified DNA of the supernatant, the primer used in the amplification except for the GC portion in the A189fGC primer. Amplification conditions are the same as above. The nucleotide sequence of the amplified DNA was analyzed to determine the community of methane-degrading bacteria in the concentrated culture medium.

Methane oxidative consortium was analyzed by PCR-DGGE, and the nucleotide sequences of the obtained clones were identified. As a result, Methylocystis sp., Methylosinus sp., And Methylmicrobiium alboom were identified. ( Methylomicrobium album), such as methane oxidized bacteria was confirmed that the dominant species (Fig. 7).

Example 3 Isolation and Identification of a Novel Methylocystis sp.

In order to isolate methane degrading bacteria, an experiment was carried out using a concentrated culture medium as an inoculum. 1 mL of the concentrated culture solution was added to 9 mL 0.9% NaCl solution and diluted using the serial dilution method. The diluted solution was plated in NMS agar medium, placed in a desiccator, and strain growth was observed while adding 20% methane and incubating at 30 ° C. The grown colonies were inoculated into a serum bottle containing 4 mL of NMS medium, blocked with a rubber stopper, and then injected with 5% methane. After injection, the concentration of methane in the headspace was analyzed over time to determine the methane resolution. Analysis method is the same as in Example 2.

DNA was extracted using the same method as Example 2 to identify methane-degraded pure bacteria. The extracted DNA was amplified using 27f (5'-AGA GTT TGA TCM TGG CTC AG-3 ') and 1492r (5'-TAC GGY TAC CTT GTT ACG AC-3'). Amplification conditions were the same as in Example 2, and the amplified samples were identified by sequencing.

To obtain pure bacteria capable of decomposing methane from the methane oxidation consortium, NMS agar medium was inoculated with a consortium and incubated in a desiccator containing 20% methane. Strain M6 capable of decomposing methane was isolated from the culture medium, and belonged to the genus Methylocystis sp. According to 16S rDNA partial sequencing. The nucleotide sequence results of the M6 strain are shown in Table 1, and the phylogenetic tree results similar to the M6 strain are shown in FIG. 8. The isolate was identified as Methylocystis sp. These results show that Methylocystis sp. M6 strains dominate in the thickening medium and play an important role in decomposing methane.

As a result of methane decomposition of Methylocystis sp. M6, M6 strain completely degraded 5% methane in 3 days. At this time, the decomposition rate of methane was 9.98 mmole-L ^-1 -h ^-1 (Fig. 9).

Therefore, Methylocystis sp. M6, which has high resolution against methane, was deposited on June 3, 2009 to the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC 11519BP.

[Revisions under Rule 26 26.10.2009]
Table 1

Example 4 Biocover Using New Strain

A laboratory scale biocover was fabricated and tested to investigate the CH ₄ oxidation characteristics by simulating the cover layer of landfill on a laboratory scale. The column used in this experiment consisted of three layers, consisting of two 20 cm diameter, 50 cm high charging sections, and a 20 cm diameter and 30 cm high ventilation section. The biocover is made of PVC material and a detailed configuration diagram of the device is shown in FIG. 10.

At the bottom of each charging section, a PVC perforated plate and a 20 cm diameter rubber filter were raised to a height of 50 cm to allow the CH ₄ / CO ₂ mixed gas to diffuse as uniformly as possible. Soil mixed with forest soil and earthworm fecal soil at 75:25 (w / w) was charged to two stages of filling. In addition, 300 mL (1.3 × 10 ⁸ cells / mL) of the microbial consortium culture solution developed in Example 3 was added to the filling part. As a control for verifying the mixing effect of earthworm fecal soil, a biocover filled with only forest soil without earthworm fecal soil was prepared.

The sample injection port installed a gas-tight valve every 10 cm and inserted a GC analysis septum at the outlet to prevent mixed gas leakage in the column. Air was continuously flowed at the top of the column at a flow rate of 200 mL / min to simulate the description of the atmospheric layer and the redox gradient of the landfill's natural soil layer. Synthetic landfill gas concentrations were 40% CH ₄ and 60% CO ₂ and fed at 10 and 20 mL / min from bottom to top. All experiments were run in a laboratory at 20 ± 5 ° C.

When a mixed gas of 40% CH ₄ and 60% CO ₂ (v / v) was supplied at a rate of 10 mL / min to a laboratory-scale biocover, the concentration of CH ₄ detected in the biocover surface layer over time was shown in FIG. 11. Shown in

In addition, the concentration changes of O ₂ , N ₂ , CH ₄ and CO ₂ at each height of the biocover when methane oxidation is observed stably are shown in FIG. 12.

The lower end of the biocover (-0.5 to -1 m) is anaerobic, and the CH ₄ is hardly diluted into the atmosphere because the air flowing from the upper vent is not sufficiently diffused to the lower end. In addition, because of the anaerobic environment with little concentration of O ₂ , the activity of methanetrophs (methanotrophs) is also considered to be insignificant. When the concentration of CH ₄ is above 1% (v / v) and the concentration of O ₂ is about 1% (v / v), the growth rate of type II methane oxidizing bacteria is higher than that of type I methane oxidizing bacteria. This is not to say that there is no CH ₄ oxidation.

Both biocovers oxidized most of CH ₄ at a height of 0–0.3 m, an aerobic state formed by diffusion of air. On the biocover surface filled with paddy soil only, 60,000 ppmv of CH ₄ was released without decomposition and showed about 85% methanation rate. However, about 5,000 ppmv of CH ₄ was emitted from the biocover surface (75% soil + 25% earthworm fecal soil) in which paddy soil and earthworm fecal soil were mixed. However, the methane emissions increased due to the deepening of the channeling phenomenon after the 12th day in the biocover, but the methane oxidation efficiency was recovered after recharging all the fillers.

The density of 100% soil and biocover mixed with soil and fecal soil were 0.83 and 0.76, respectively, and the organic contents were 13.6 ± 1.23% and 23.09 ± 0.60%, respectively. From these results, it was found that the density of the filter bed was lowered when the earthworm fecal soil was mixed, so that the air was diffused smoothly, and the growth and activity of methane oxidizing bacteria was increased because the organic matter was richer than the forest soil. Therefore, when applying the cover material mixed with the earthworm fecal soil, it was confirmed that the thinning of the final cover layer and the high methane oxidation rate could be derived.

When a mixed gas of 40% CH ₄ and 60% CO ₂ (v / v) was supplied at a rate of 20 mL / min to a laboratory-scale biocover, the concentration of CH ₄ detected in the biocover surface layer over time was shown in FIG. 13. Shown in

In addition, the concentration changes of O ₂ , N ₂ , CH ₄ and CO ₂ at each height of the biocover when methane oxidation is observed stably are shown in FIG. 14.

Example 5 Degradation of Contaminants of a Novel Methylocystis sp. M6

In order to investigate the availability of other hydrocarbon compounds of Methylocystis sp. M6, which has a high resolution of methane, a total of 14 materials were selected to investigate the resolution or growth capacity of M6.

Methylocystis sp. M6 strains to be used for substrate testing were inoculated in NMS medium after mass cultivation in R2A medium and washing. This was dispensed in 4 mL portions of 120 mL serum bottles, and 1 μl of each substance was injected after closing with a rubber stopper. M6 strains for methanol, ethanol, acetone and diesel decomposition for measuring the OD ₆₀₀ value were divided into 600 mL-serum bottles by 20 mL, and 10 μl of each material was injected after blocking with a rubber stopper. The growth capacity using methanol, ethanol, acetone and diesel was analyzed by measuring the OD ₆₀₀ value, and the resolution of the remaining 10 materials was analyzed by measuring the concentration of the substrate, using 50 μl gas tight syringe. 30 μl was collected from the headspace of the serum bottle, and a gas chromatography (Agilent 6850N, USA) -flame ionization detector equipped with a wax column (Supelco, 30 × 0.32 mm × 0.25 μm) was used. The analysis temperature was 100 degreeC of oven, and 230 degreeC of injection part and a detection part.

As shown in Table 2, Methylocystis sp. M6 strain was able to decompose m -xylene, p -xylene, methanol and ethanol.

TABLE 2

Example 6 Methane Reduction System

The methane reduction system using the biocover according to the present invention was prepared as follows.

The biocover material was used in the ratio of 75:25 to weight ratio of soil and earthworm stool, and Methylocystis sp. M6 was used as methane oxidizing bacterium.

In addition, in order to supply oxygen required by the methane oxidizing bacterium, the biocover layer may be prepared by mixing magnesium peroxide (MgO ₂ ) and calcium peroxide (CaO ₂ ), which are oxygen generating agents.

15A is a cross-sectional view showing an example of installation of a biocover installed on the cover layer or the ground surface of the landfill according to the present invention. The methane abatement facility using the biocover consists of a landfill layer, a cover layer, a biocover layer, and a planting layer. Installed and configured to supply air to the blower.

15B is a cross-sectional view showing an example of installation of another biocover installed on the cover layer or the ground surface of the landfill according to the present invention. Methane abatement facility using biocover consists of landfill layer, cover layer, sand and gravel layer, biocover layer, and plant layer, and supplies air to sand and gravel layer between cover layer and biocover layer to supply oxygen to biocover layer. Can be installed and configured to supply air to the blower.

The present invention can biologically decompose methane using a novel strain having excellent degradability for methane or volatile organic compounds, and thus can be used in biocovers installed on landfill cover layers or ground surfaces.

Claims (12)

1. Methylocystis sp. M6 KCTC 11519BP.
2. Methylocystis sp. M6 KCTC 11519BP comprising a composition for reducing methane.
3. The method of claim 2,

   Methane reducing composition further comprises one or more selected from the group consisting of soil and earthworm fecal soil.
4. The method of claim 3,

   Soil and earthworm fecal soil methane reduction composition is mixed in a ratio of 50:50 to 90:10 by weight.
5. Methane reduction method comprising the step of decomposing methane methane reduction composition of claim 2.
6. A methane-reducing biocover emanating from the cover layer or surface of a landfill containing the composition for reducing methane of claim 2.
7. The method of claim 6,

   Sar as to Pseudomonas genus (Methylomonas), in Micro-away with methyl (Methylomicrobium), bakteo in (Methylobacter) methyl, methyl local dumsok (Methylocaldum), wave acceleration as methyl (Methylophaga), methyl or when in (Methylosarcina), methyl Methylothermus , Methylohalobius , Methylosphaera , Methylocystis , Methylocella , Methylocapsa , Methylosinus ( Methylosinus ) and Methylococcus ( Methylococcus ) Methane reduction biocover further comprising one or more methane oxidizing bacteria selected from the group consisting of.
8. The method of claim 6,

   Methane reduction biocover further comprising an oxygen generator.
9. The method of claim 8,

   Oxygen generator is one or more selected from the group consisting of magnesium peroxide, calcium peroxide and sodium percarbonate methane reduction biocover.
10. In the methane reduction system for biologically decomposing methane emitted from the cover layer or surface by installing a bio active layer on the cover layer or surface of the landfill, the bio active layer is

    A biocover layer comprising one or more biocovers of claim 6 stacked thereon; And

    And a ventilation layer surrounding the biocover layer.
11. In the methane reduction system for biologically decomposing methane emitted from the cover layer or surface by installing a bio active layer on the cover layer or surface of the landfill, the bio active layer is

    A biocover layer comprising one or more biocovers of claim 6 stacked thereon; And

    Methane reduction system comprising a ventilation layer stacked below the biocover layer.
12. A method of methane abatement comprising the step of decomposing methane by injecting a sample into the methane abatement system of claim 10.

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