KR20110006962A - Novel methane-oxidizing bacterium, cupriavidus sp. mbt 14 and method for removing methane and volatile organic compounds using the same - Google Patents

Abstract

PURPOSE: A novel methane-oxidizing bacteria, Cupriavidus sp., and a method for reducing methane and volatile organic compound using the same are provided to effectively decompose methane. CONSTITUTION: A novel Cupriavidus sp. MBT 14KCTC 11518BP is isolated and identified from a landfill. A composition for reducing methane and volatile organic compound contains Cupriavidus sp. MBT14 KCTC 11518BP. A bio-cover for reducing methane and volatile organic compound released from the earth contains the composition for reducing methane and volatile organic compound.

Description

Novel methane-oxidizing bacterium, Cupriavidus sp. Novel methane-oxidizing bacterium genus and simultaneous reduction of methane and volatile organic compounds using the same MBT 14 and method for removing methane and volatile organic compounds using the same}

The present invention relates to a novel methane-oxidizing bacillus genus Cupriaviders and a method for simultaneously reducing methane and volatile organic compounds using the same. At the same time, it can be disassembled effectively.

Methane is the second most important greenhouse gas after carbon dioxide, accounting for 18% of the total radiative forcing (IPCC, The Physical Science Basis.Working Group I Contribution to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (eds) Solomon S. et al . (Cambridge University Press, Cambridge, 2007; Dunfield et al., Nature 450, 879-882, 2007.) Since methane concentrations in the atmosphere have more than doubled since the industrial age, many studies have shown that The focus is on identifying sources and sinks or finding microbes associated with them (Bodelier et al ., FEMS Microbiol. Ecol . 52, 163-174, 2005). (global carbon cycle) were concentrated to investigate the distribution and variety of the habitat of the methane oxidizing bacteria that plays an important role (evoked, Bodelier et al., FEMS Microbiol. Ecol. 52, 163-174, 2005) in. typical methane generation There is as a landfill, methane generated by the landfill, so about 35% of the methane generated artificially in the United States and is estimated to account for 5 to 10% of global methane produced in the atmosphere (IPCC, 2001; Stern et al . , Waste Manage . 27, 1248-1258, 2007).

Landfills emit not only methane but also various volatile organic compounds such as chlorine-based hydrocarbons and aromatics (VOCs, Rettenberger et al ., Landfill gas compounds.In Landfilling of waste: biogas; Christensen, TH, Cossu, R., Stegmann, R., Eds. E & FN Spoon: London, pp. 51-58, 1996), volatile organic compounds destroy the ozone layer and contribute to global warming (Wallington et al., Environ. Sci. Technol . 28, 320A-326A, 1994). Representative volatile organic compound emissions from landfills Trichlorethylene (TCE), tetrachlorethylene (PCE), benzene (benzene), toluene (toluene) such as, 200 or more substances each year and is discharged approximately 10 ¹² kg (Allen et al, Environ. Sci. Tech., 31 (4), 1054-1061, 1997; Scheutz et al., Waste Manage . 28, 1892-1908, 2008).

Two important methods of removing methane from the atmosphere are the reaction with OH radicals in the troposphere (490 ± 85 Tg · y ⁻¹ ) and the oxidation of soil microorganisms estimated to be about 30 ± 15 Tg · y ⁻¹ (IPCC , The Science of Climate Change (eds Houghton, JT et al .), Cambridge University Press, Cambridge. 1996). Microbial oxidation by methane-oxidizing bacteria plays an important role in the reduction of methane from landfills (Scheutz and Kjeldsen, J. Air & Waste Manage.Assoc. 55, 878-885, 2005). Based on laboratory research, it is estimated to be between 10% and 50%, and in field studies, 10% to 100% of methane released annually can be oxidized (Scheutz and Kjeldsen, J. Air & Waste Manage.Assoc. 55 , 878-885, 2005). Although methane's natural savings in landfill soils have been concentrated in relation to landfill contributions to global warming, no technology has been developed to simultaneously remove volatile organic compounds released simultaneously with methane (Scheutz and Kjeldsen, J. Air & Waste). Manage.Assoc . 55, 878-885, 2005).

Therefore, it is necessary to develop a technology capable of simultaneously removing methane and volatile organic compounds.

It is an object of the present invention to isolate and identify new methane-oxidizing bacteria in landfills that can decompose methane and volatile organic compounds.

Another object of the present invention is to provide a method of simultaneously reducing methane and volatile organic compounds using the novel methane oxidizing bacteria.

Still another object of the present invention is to provide a biocover capable of simultaneously removing methane and volatile organic compounds using the novel methane oxidizing bacteria and a system for simultaneously reducing methane and volatile organic compounds including the same.

Still another object of the present invention is to provide a method for simultaneously reducing methane and volatile organic compounds using the system.

In order to achieve the above object, the present invention provides Cupriavidus sp. MBT14 KCTC 11518BP.

The present invention also provides a composition for simultaneously reducing methane and volatile organic compounds including Cupriavidus sp. MBT14 KCTC 11518BP.

The present invention also provides a method for simultaneously reducing methane and volatile organic compounds comprising the step of decomposing methane and volatile organic compounds in the composition for simultaneously reducing methane and volatile organic compounds.

The present invention also provides a biocover for simultaneously reducing methane and volatile organic compounds emanating from the cover layer or surface of a landfill containing the composition for simultaneously reducing methane and volatile organic compounds of the present invention.

The present invention also provides a bio-active layer on the cover layer or the surface of the landfill waste, the bio-active layer in the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds emitted from the cover layer or surface,

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

It provides a system for simultaneously reducing methane and volatile organic compounds comprising a ventilation layer surrounding the biocover layer.

The present invention also provides a bio-active layer on the cover layer or the surface of the landfill waste, the bio-active layer in the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds emitted from the cover layer or surface,

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

It provides a simultaneous reduction system of methane and volatile organic compounds, characterized in that it comprises a ventilation layer laminated under the biocover layer.

The present invention also provides a method for simultaneously reducing methane and volatile organic compounds, comprising the step of decomposing methane and volatile organic compounds by injecting a sample into the system for simultaneously reducing methane and volatile organic compounds.

Cupriavidus sp. MBT14 , a novel methane-oxidizing bacterium of the present invention, can simultaneously decompose methane and volatile organic compounds.

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

The present invention relates to Cupriavidus sp. MBT14 KCTC 11518BP.

The microorganism of the present invention is a novel methane oxidizing bacterium having excellent resolution of methane and volatile organic compounds, sealing a culture bottle in which a landfill soil and an enrichment medium, for example, NMS (nitrate mineral salts) medium are added, and methane and Cupriavidus sp. MBT14 , a novel methane-oxidizing bacterium with excellent resolution of methane and volatile organic compounds, by injecting volatile organic compounds and continuously supplying nitrogen and phosphorus during incubation, and then using concentrated culture medium as inoculum. Was identified and deposited on June 3, 2009 at Korea Institute of Bioscience and Biotechnology, and received the deposit number KCTC 11518BP.

The present invention also relates to a composition for simultaneously reducing methane and volatile organic compounds comprising Cupriavidus sp. MBT14 KCTC 11518BP.

The composition of the present invention may use the novel Cupriavidus sp. MBT14 KCTC 11518BP of the present invention as methane oxidizing bacteria to simultaneously reduce methane and volatile organic compounds.

In addition, the composition for simultaneously reducing the methane and volatile organic compounds of the present invention may further include soil or earthworm fecal soil as a filler for the composition of the culture environment of the bacterial culture.

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.

In addition, the earthworm fecal soil is an earthworm fecal soil produced by supplying the sewage sludge generated in the sewage treatment process as the earthworm food, using impurities having a particle size of 0.2 to 2 mm after the natural fermentation / drying process for at least 6 months. It is desirable to.

PH of the soil and fecal soil may be 5 to 7, water content of 1 to 40%, organic content of 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. When in the above content range, the simultaneous reduction efficiency of methane and volatile organic compounds is high.

The present invention also relates to a method for simultaneously reducing methane and volatile organic compounds comprising the step of decomposing methane and volatile organic compounds in the composition for simultaneously reducing methane and volatile organic compounds.

Cupriavidus sp. MBT14 KCTC 11518BP strain of the present invention is excellent in resolution for methane and volatile organic compounds, unlike the prior art that decomposes methane or volatile organic compounds using oxygen separately from the oxygen production agent Without addition, it can effectively decompose methane and volatile organic compounds simultaneously.

The present invention also relates to a biocover for simultaneously reducing methane and volatile organic compounds emanating from the cover layer or surface of a waste landfill, comprising the composition for simultaneously reducing methane and volatile organic compounds of the present invention.

Cupriavidus sp. MBT14 KCTC 11518BP culture of the present invention is put into the biocover for the simultaneous reduction of methane and volatile organic compounds and they can be effectively reduced by contacting the biocover and methane or volatile organic compounds.

Biocover of the present invention

Biomedia layer containing methane oxidizing bacteria; And

An upper protective sheet and a lower support sheet for maintaining the biomedia layer in a mat shape;

It may include a coupling means for coupling the upper protective sheet, the bio-media layer and the lower support sheet to each other.

The biomedia layer is a methane oxidizing bacterium capable of biologically decomposing methane and volatile organic compounds, and may include Cupriavidus sp. MBT14 KCTC 11518BP of the present invention.

In addition, the biomedia layer may further include a conventional methane oxidizing bacteria to increase the decomposition efficiency of methane and volatile organic compounds. 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.

The present invention also provides a bio-active layer on the cover layer or the surface of the landfill waste, the bio-active layer in the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds emitted from the cover layer or surface,

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

It relates to a system for simultaneously reducing methane and volatile organic compounds comprising a ventilation layer surrounding the biocover layer.

The biocover layer contains methane oxidizing bacteria and can simultaneously biologically decompose methane and volatile organic compounds.

The methane oxidizing bacteria may include the novel Cupriavidus sp. MBT14 KCTC 11518BP of the present invention.

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

In addition, the biocover layer may further include soil and earthworm fecal soil to increase the oxidation efficiency of methane and volatile organic compounds. Details of the soil and the earthworm fecal soil 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 bio-active layer on the cover layer or the surface of the landfill waste, the bio-active layer in the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds emitted from the cover layer or surface,

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

It relates to a simultaneous reduction system of methane and volatile organic compounds, characterized in that it comprises a ventilation layer laminated under the biocover layer.

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

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 method for simultaneously reducing methane and volatile organic compounds comprising the step of decomposing methane and volatile organic compounds by injecting a sample into the system for simultaneously reducing methane and volatile organic compounds.

When methane gas or volatile organic compounds are injected into the system for simultaneously reducing methane and volatile organic compounds of the present invention, they can be effectively decomposed by the methane oxidizing bacteria of the present invention.

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 Securing Degraded Bacteria Consortium for Methane and Volatile Organic Compounds

Soil was harvested from landfill to obtain methane and BT co-decomposed thickening broth. The soil collection site is Gapyeong, Gyeonggi-do, and is an active landfill where landfill gas continues to be discharged. The collected soil was transported in an ice box and stored at 4 ° C.

Thickening culture was performed to obtain a mixed strain capable of co-decomposing methane and BT. To obtain a microbial enrichment broth, 8 g of wet landfill soil and 20 mL of nitrate mineral salts (NMS) medium were added to a 600 mL serum bottle and sealed with a rubber stopper. In a sealed serum bottle, 5% (v / v) of methane was injected as the only carbon source, 111.9 and 93.6 μmole of benzene and toluene, respectively, and the serum bottle was incubated at 30 ° C. and 180 rpm. Once 100% of the injected methane, benzene and toluene were removed, the rubber bottle was opened to allow air saturation for 1 hour. After air saturation, the rubber stopper was closed and methane, benzene, and toluene were 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 confirm that methane, benzene and toluene are degraded during the thickening, 0.3 mL of each serum bottle was collected from the headspace of the serum bottle. 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.

Methane, Benzene and Toluene Decomposition Thickening Solution (Consortium) 100% decomposed 5% methane within 65 hours, benzene and toluene completely decomposed within 17 hours and 13 hours, respectively (Fig. 1). The decomposition rates of methane, benzene, and toluene in the concentrated culture medium were 4.83 ± 0.39 μmole g-dry soil ^-1 · h ^-1 for methane and 2.71 ± 0.53 μmole g-dry soil ^-1 · h ^-1 for methane. Experimental results confirm that this consortium decomposes methane, benzene and toluene effectively.

To determine the microbial community of methane, benzene and toluene degrading microbial consortium (enriched culture medium), DNA was extracted from the initial soil and enriched culture medium. About 0.5 g of wet soil was extracted using BIO101 kit for microbial community analysis of initial soil. In the concentrated culture solution, the methane, benzene, and toluene were completely decomposed, and 1 mL of the culture solution was centrifuged at 14,000 × g for 5 minutes to extract DNA from the precipitate. DNA extraction was performed according to the BIO101 kit method and all samples were extracted two times.

The bacterial community structure of methane, benzene and toluene degrading bacterial consortium was analyzed using PCR-denaturing gradient gel electrophoresis (DGGE) technique. First, the primer pairs used to amplify methane degrading 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 ').

In addition, primer pairs used to detect benzene and toluene degrading bacteria were TMOAfGC (5'- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G CGA AAC CGG CTT YAC CAA YAT G-3 ') and TMOAr (5'- ACC GGG ATA TTT YTC TTC SAG CCA-3 ').

PCT amplification conditions were performed 40 times of initial denaturation at 96 ° C. for 5 minutes, denatured 96 ° C. for 1 minute, annealing 58 ° C. for 1 minute, amplification 72 ° C. for 1 minute in the first step, and 72 ° C. at the last step. Experiment was 5 minutes. 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. After the DGGE experiment, the band was cut from the gel, 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, the DNA of the supernatant was amplified: The primer used for the amplification was a primer except for the GC portion of the A189fGC primer for the detection of methane-degrading bacteria, and TMOAfGC for the detection of benzene and toluene-degrading bacteria. Primer except for GC part in primer was used. The nucleotide sequence of the amplified DNA was analyzed and identified.

As shown in Figure 2, after the PCR using the A189fGC and mb661r primer that can detect the gene (pmmo) of methane monooxygenase involved in methane oxidation, the clone obtained after analysis by DGGE was confirmed that was identified by analyzing the nucleotide sequence, in seutiseu when methyl (Methylocystis sp.), Sinners in (Methylosinus sp.) of methyl, and the methane-oxidizing bacteria such as micro emptying album (Methylomicrobium album) dominant with methyl (FIG. 2).

In addition, PCR was performed using TMOAfGC and TMOAr primers capable of detecting monooxygenase coding genes involved in benzene and toluene oxidation, followed by DGGE analysis. ( Pseudomonas sp.), Cupriavidus sp. And Ralstonia sp. (Genus Ralstonia) was found to be dominant species (Fig. 3).

Example 2 Isolation and Identification of Novel Methane Oxidizing Bacteria Having Degradability Against Methane and Volatile Organic Compounds

The experiment was carried out using the concentrated culture medium as an inoculum to separate pure bacteria that decompose methane, benzene and toluene. 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 in two serum bottles containing 4 mL NMS medium according to their shape and color, and sealed with a rubber stopper, 5% methane and 56 and 47 μmole of benzene and toluene were injected. After injection, headspace concentrations were analyzed over time to determine the resolution of methane, benzene, and toluene. The analysis method is the same as above.

DNA was extracted using the same method as above to identify pure bacteria capable of decomposing methane, benzene and toluene. 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 are as described above, and identified by analyzing the nucleotide sequence of the amplified sample.

Strain MBT14, which can simultaneously decompose methane and BT, was isolated, and was subjected to 16S rDNA partial sequencing, and belonged to the genus Cupriavidus sp. The base sequence results of the MBT14 strain are shown in Table 1 and the phylogenetic tree results similar to the MBT14 strain are shown in FIG. 4.

Phylogenetic analysis showed that Cupriavidus sp. MBT14 was associated with other Cupriavidus and Alcaligenes and Ralstonia . The similarity with species was high (FIG. 4). These results were also observed in the concentrated culture broth, but the benzene and toluene degrading bacterial populations of the concentrated culture broth showed that the similarity with Cupriavidus and Ralstonia species was high (FIG. 3). These results indicate that Cupriavidus sp. MBT14 strain plays an important role in the decomposition of methane, benzene and toluene.

In addition, as shown in Figure 6, the cupriavidus sp. ( Cupriavidus sp.) MBT14 methane, benzene and toluene degradation characteristics of the MBT14 strain was found to decompose 5% methane completely in 6 days, 112 μmole of BT 100% degradation in 3 days. The methane decomposition rate of MBT14 was 4.3 mmole · L ⁻¹ · h ⁻¹ , and the BT degradation rates were 79.2 and 92.4 μmole · L ⁻¹ · h ⁻¹ , respectively. Therefore, Cupriavidus sp. MBT 14, which has high resolution for methane and volatile organic compounds, was deposited with the Korea Institute of Biotechnology and Biotechnology Center on June 3, 2009 and received accession number KCTC 11518BP.

Example 2 Biocover Performance Evaluation Using Novel Methane Oxidized Bacteria

Laboratory-level biocovers simulated from landfills were inoculated with microbial consortiums predominantly Cupperidus sp. MBT 14 to examine the biodegradation profiles of benzene / toluene as malignant volatile organic compounds with methane.

Forest soil and earthworm fecal soil were collected, filtered through a 2 mm sieve, and mixed at a ratio of 75:25 (w / w), respectively, and filled in the biocover, wherein the microbial consortium culture solution of Example 1 was 300 mL (2.1 × 10 ^8). cells / mL) were mixed with soil.

The biocover was built in two stages with a diameter of 200 mm and a height of 500 mm, with a column of 300 mm at the top, which simulates the atmosphere directly in contact with the landfill soil. At the bottom of the column, a methane / carbon dioxide (40% / 60%) mixed gas was injected at a rate of 10 mL / min, and benzene / toluene was volatilized with an methane gas at about 200 ppmv. Flow was supplied at a concentration (FIG. 5). At the top of the column, air was injected at 1.45 atm, 200 mL / min, simulating the condition of the atmosphere in contact with landfill soil. Gas profiles were collected 300 μl from the sampling port using gas tight syringes for each column height, and were equipped with gas chromatography (Agilent 7890A, 6890N USA) equipped with a capillary column (30 m × 530 μm × 50 μm). Analysis was carried out using a thermal conductivity detector. The analysis temperature of methane, oxygen, and nitrogen gas was 50 degreeC of oven, 200 degreeC of injection parts, and 250 degreeC of detection parts. In the case of carbon dioxide, it was oven 280 degreeC, injection | pouring part 200 degreeC, and detection part 250 degreeC. Benzene and toluene are identical to the benzene and toluene analysis conditions in the concentrated culture broth.

7 and 8 show the removal characteristics of methane, benzene, and toluene of a biocover made of soil mixed with a forest soil inoculated with a microorganism consortium obtained by enrichment culture at a ratio of 75:25 (w / w). The removal efficiency of methane was about 99% or more, and almost 100% of benzene and toluene were removed, so that methane, benzene and toluene emitted from the biocover surface were hardly detected (FIG. 7). That is, it was confirmed that the bio cover can remove methane and volatile organic compounds at the same time.

Meanwhile, methane was mainly removed from the top of the biocover, while benzene and toluene were removed from the bottom (Fig. 8).

Example 3 Investigation of the Resolution of Other Pollutants of Cupriavidus sp.

In order to investigate the availability of other hydrocarbon compounds of Cupriavidus sp. MBT14 of the present invention having excellent resolution of methane, a total of 14 materials were selected to investigate the resolution or growth capacity of MBT14 for these materials.

The MBT14 strain to be used for the substrate test was 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. However, M6 strain for methanol, ethanol, acetone and diesel (Diesel) degradation confirmation for measuring the value of OD ₆₀₀ was dispensed in 20 mL in 600 mL-serum bottle, 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 OD600nm value, and the resolution of the remaining 10 materials was analyzed by measuring the concentration of the substrate, with 50 μl gas tight syringe. 30 μl was collected from the headspace of the serum bottle 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 Table 2, MBT14 strain degraded ethylbenzene, m -xylene, p -xylene, o -xylene and methanol.

Figure 1 shows the methane, benzene and toluene decomposition results of the landfill thickening liquid, ●, experimental group ○, control ▲, benzene experimental group ■, benzene control △, toluene experimental group □, toluene control.

2 shows a phylogenetic tree of clones associated with methane oxidation in landfill consortium bacteria.

3 shows a phylogenetic tree of clones associated with methane and toluene oxidation among consortium bacteria.

Figure 4 shows a phylogenetic tree of Cupriavidus sp. MBT14 of the present invention decomposing methane and volatile organic compounds.

5 is an installation diagram of a laboratory scale biocover using Cupriavidus sp. MBT14 of the present invention.

Figure 6 shows the methane and toluene decomposition characteristics of Cupriavidus sp. MBT14 of the present invention, methane, experimental group ▲, benzene experimental group △, toluene experimental group.

Figure 7 shows the concentration of methane, benzene and toluene discharged from the biocover surface of the present invention.

8 is a profile of the concentration of methane, benzene and toluene by the biocover height of the present invention.

<110> Ewha University-Industry Collaboration Foundation <120> Novel methane-oxidizing bacterium, Cupriavidus sp. MBT 14 and          method for removing methane and volatile organic compounds using          the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1373 <212> DNA <213> Cupriavidus sp. MBT14 <220> <221> gene (222) (1) .. (1373) <223> 16S rDNA from Cupriavidus sp. MBT14 <400> 1 caggctcaga tgaacgctgg cggcatgcct tacacatgca agtcgaacgg cagcacgggc 60 ttcggcctgg tggcgagtgg cgaacgggtg agtaatacat cggaacgtgc cctgtagtgg 120 gggataacta gtcgaaagat tagctaatac cgcatacgac ctgagggtga aagcggggga 180 ccggtaacgg cctcgcgcta caggagcggc cgatttttgt ttagttagtt tgggggggta 240 aggcccttcc caggggacgg accggtagct ggtgggagag gacgatcagc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtggg gaattttgga caatgggggc 360 aaccctgatc cagcaatgcc gcgtgtgtga agaaggcctt cgggttgtaa agcacttttg 420 tccggaaaga aatggctctg gttaataccc ggggtcgatg acggtaccgg aagaataagc 480 accggctaac tacgtgccag cagccgcggt aatacgtagg gtgcgagcgt taatcggaat 540 tactgggcgt aaagcgtgcg caggcggttt tgtaagacag gcgtgaaatc cccgagctca 600 acttgggaat ggcgcttgtg actgcaaggc tagagtatgt cagagggggg tagaattcca 660 cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg atggcgaagg cagccccctg 720 ggacgtcact gacgctcatg cacgaaagcg tggggagcaa acaggattag ataccctggt 780 agtccacgcc ctaaacgatg tcaactagtt gttggggatt catttcttca gtaacgtagc 840 taacgcgtga agttgaccgc ctggggagta cggtcgcaag attaaaactc aaaggaattg 900 acggggaccc gcacaagcgg tggatgatgt ggattaattc gatgcaacgc gaaaaacctt 960 acctaccctt gacatgccac taacgaagca gagatgcatt aggtgcccga aagggaaagt 1020 ggacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgtc tctagttgct acgaaagggc actctagaga gactgccggt 1140 gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttatg ggtagggctt 1200 cacacgtcat acaatggtgc gtacagaggg ttgccaaccc gcgaggggga gctaatccca 1260 gaaaacgcat cgtagtccgg atcgtagtct gcaactcgac tacgtgaagc tggaatcgct 1320 agtaatcgcg gatcagcatg ccgcggtgaa tacgtttccc gggtcttgta cac 1373  

Claims (10)

Cupriavidus sp. MBT14 KCTC 11518BP. Cupriavidus sp. MBT14 KCTC 11518BP comprising a composition for the simultaneous reduction of methane and volatile organic compounds. The method of claim 2, A composition for simultaneously reducing methane and volatile organic compounds further comprising at least one selected from the group consisting of soil and earthworm fecal soil. The method of claim 3, Soil and earthworm fecal soil composition for the simultaneous reduction of methane and volatile organic compounds are mixed in a ratio of 50:50 to 90:10 by weight. A method for simultaneously reducing methane and volatile organic compounds comprising the step of simultaneously decomposing methane and volatile organic compounds in the composition for simultaneously reducing methane and volatile organic compounds. A biocover for simultaneously reducing methane and volatile organic compounds emitted from a cover layer or surface of a waste landfill, comprising the composition for simultaneously reducing methane and volatile organic compounds according to claim 2. 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 , methyl Sinners in (Methylosinus) and methyl Rhodococcus genus (Methylococcus) bio cover for the simultaneous reduction of the methane and volatile organic compounds further include one or more methane-oxidizing bacteria selected from the group consisting of. In the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds 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 Simultaneous reduction of methane and volatile organic compounds comprising a ventilation layer surrounding the biocover layer. In the simultaneous reduction system of methane and volatile organic compounds to biologically decompose methane and volatile organic compounds 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 Simultaneous reduction system of methane and volatile organic compounds, characterized in that it comprises a ventilation layer laminated under the biocover layer. A method of simultaneously reducing methane and volatile organic compounds, comprising injecting a sample into a system for simultaneously reducing methane and volatile organic compounds according to claim 8 or 9.

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