JP4428620B2 - Methane oxidizing bacteria holding carrier manufacturing method, manufacturing apparatus, and methane generation suppression method - Google Patents

Description

The present invention relates to a method for manufacturing a responsible body for holding the methane oxidation decomposing bacteria is one of the greenhouse gases, a manufacturing apparatus and methane production inhibition method. In particular, the method of producing methane-oxidizing bacteria retaining support consists in adhere and propagate methane oxidation decomposing bacteria in multiple porous carrier, for the preparation apparatus and methane production inhibition method.

Methane (CH ₄ ) is a greenhouse gas that causes global warming, but it is present in the atmosphere at about 1.7 ppm, and its main source is not only natural wetlands and oceans, but also agricultural land such as paddy fields, waste It is considered to be a disposal site, ruminant, biomass burning. Methane is about 21 times the warming potential per molecule of carbon dioxide, and a slight increase in concentration is said to have a significant effect on global warming. For this reason, according to the Kyoto Protocol adopted at the 3rd Conference of the Parties to the United Nations Framework Convention on Climate Change (COP3), it is a numerical value that methane will reduce total emissions in terms of carbon dioxide by at least 5 wt% with a base year of 1990. It is set as a target, and countermeasures for major emission sources such as industry and agriculture / forestry are required.

  Techniques for suppressing methane generation are being studied mainly in paddy fields, and methods for applying rice straw, which is a raw material for methane, and water management such as middle drying are proposed. In addition, methane is adsorbed on porous agents such as activated carbon and charcoal (Patent Document 1), and iron-containing materials such as converter slag and used disposable body warmers that are industrial byproducts of the steel industry are applied to paddy fields to release methane. Has been proposed (Non-patent Document 1). However, in order to control methane generation by applying it to large-area sources such as wetlands and final waste disposal sites and composting facilities where high concentrations of methane are generated, it is possible to obtain sufficient effects in the medium to long term. There are problems with iron-containing materials that cannot be used or remain, and few practical techniques are known.

  On the other hand, in recent years, environmental remediation technology (bioremediation) using the resolution of microorganisms has attracted attention and has been put to practical use in pollutants such as petroleum, agricultural chemicals, and heavy metals. In addition, efforts have been made for empirical research on harmful chemical substances such as trichlorethylene and PCB, and it has been reported that microorganisms can be used to remove these pollutants. However, in the past, it was almost always to use a single bacterium, and since the microorganisms were sprayed directly onto pollution sources such as soil, they were preyed by many predators existing in the environment and had a sufficient purification effect. Is often not obtained. For this reason, the porous material is made to adsorb and propagate useful bacteria (Patent Document 2), and further accumulates microorganisms capable of decomposing pollutants from soil contaminated with pollutants such as agricultural chemicals. (Patent Document 3).

However, the prior art using these microorganisms is for liquid substances and cannot be applied to gaseous pollutants such as methane. For methane, only methods for separating and identifying microorganisms that oxidize and decompose have been reported (Non-patent Document 1), and these microorganisms are used to suppress the generation of methane from the natural environment and artificial facilities. There is nothing known to do.
JP 2001-17859 A Japanese Patent Laid-Open No. 8-252086 JP 11-318435 A Nutrient Cycling in Agroecosystems, Vol.64, pp.193-201 (2002) Appl. Environ. Microbiol., Vol. 65 (11), pp. 4887-4897 (1999)

Therefore, the problem of the present invention can be applied to an environment where it is difficult to suppress the generation of methane, such as a wide range of methane generation sources or high-concentration methane generation. manufacturing method and a manufacturing apparatus and methane production inhibition method of capital material capable of inhibiting is to provide.

The present inventors have, when intensive studies for solving the above problems, found that getting the methane adhere and propagate the oxidation decomposing bacteria on a porous support is held by the carrier, completion of the present invention I let you.

Accordingly, the present invention consists of a porous carrier a mixture of culture medium containing bacteria with methane oxidizing ability, then by attaching the bacteria by circulating air containing methane porous, breeds The present invention relates to a method for producing a carrier for holding methane oxidizing bacteria. The culture medium may be absorbed by the porous carrier before mixing the culture medium containing bacteria having methane oxidizing ability with the porous carrier. In the present invention, the oxidation and oxidative decomposition of methane mean that methane is decomposed into carbon dioxide and water. Therefore, bacteria that oxidize and decompose methane and methane-oxidizing bacteria mean the same group of bacteria.

  Further, the present invention comprises a double container of an inner container and an outer container, the inner container having an inlet and an outlet, and introducing the methane-containing air into the inlet and introducing the exhaust into the inner container. A blowing means for discharging from the mouth, and the outer container forms a flow path for returning air containing methane discharged from the inner container to the inlet, between the inner container and methane in the inner container. A carrier for supporting methane-oxidizing bacteria, comprising a porous carrier sprayed with a nutrient solution containing oxidizing bacteria and forming a circulating flow of air containing methane passing through the porous carrier in the inner container Relates to the device.

The invention further a carrier which holds a methanotrophic bacterium obtained by the production method of methanotrophic bacteria hold-back carrier above mentioned method of inhibiting methane production which consists in spraying or disposed.

1. Methane-oxidizing bacteria Bacteria that oxidize and decompose methane are microorganisms called methane-oxidizing bacteria or methane-utilizing bacteria, and are widely used in land and lakes, rivers, pastures, and broad-leaved forest soils and water. It is known that In the present invention, methane can be used as a carbon source, and is not particularly limited as long as it is a bacterium that can finally decompose methane into carbon dioxide and water. Such microorganisms are described in Non-Patent Document 2, for example Methylococcus sp, Methylopsphaera genus Methylomonas sp, Methylomicrobium genus Methylobacter genus Methylosinus genus include Methylocystis sp and Methanotrophic bacteria of the genus, preferably Hyphomicro bi um methylovorum, Hyphomicro bi um facilis, Hyphomicro bi um sp. belonging to these same clusters. Strains isolated as S4, T2-06 strain, T2-07 strain, T2-17 strain classified as type II methane-oxidizing bacteria, and bacteria closely related to these and having methane-oxidizing ability, etc. It is.

2. Acquiring Methane Oxidizing Bacteria These methane oxidizing bacteria are widely inhabited in the soil and water of wetlands, lakes and rivers, paddy fields, upland fields, pastures, broadleaf forests, and there are few types compared to land. Inhabiting has been confirmed even in the submarine mud. Methane-oxidizing bacteria can be obtained by collecting soil from paddy fields with high methane generation, lake wetlands, riverside wetlands in agricultural waterways, etc., and culturing them using methane as the sole carbon source. Therefore, it is preferable to select a methane-oxidizing bacterium having a higher methane-oxidizing ability or a methane-oxidizing bacterium obtained from the soil of the application site.

  The methane-oxidizing bacterium can be selected by using methane consumption and carbon dioxide generation as indicators because the methane-oxidizing bacterium decomposes methane into carbon dioxide and water. Methane oxidizing bacteria can be cultured according to the method for separating chloroethylene and chloroethane-degrading bacteria by Yagi et al. (National Institute for Environmental Studies Special Research Report SR-31-2000, 2000). Specifically, it can be carried out as follows. Put an inorganic salt liquid medium suitable for cultivation of methane-oxidizing bacteria into the gas vial, add more soil, and seal the vial with. Next, after partially extracting air from the head space of the gas vial, methane is added as a carbon source. This is cultured with shaking in a constant temperature incubator at 30 ° C. After culturing for a certain period, air is collected from the head space, and the methane concentration is measured with a gas chromatograph. About what the reduction | decrease of methane density | concentration was recognized, the liquid culture medium is added to the gas vial containing a liquid culture medium, and a 2nd shake culture is performed. In this culture, a methane-oxidizing bacterium having a large decrease in methane concentration is selected. Further, the methane-oxidizing bacterium having a large decrease in methane concentration is selected by performing planting in a new liquid medium and performing a third enrichment culture.

3. Porous carrier In the present invention, the porous carrier is not particularly limited as long as it can hold the above-mentioned methane-oxidizing bacteria, that is, can adsorb and grow methane-oxidizing bacteria. Porous ores such as perlite, pumice, and zeolite are exemplified, and charcoal and activated carbon are preferable. When the porous carrier of the present invention is applied to the environment, it is required to effectively use wooden waste such as dam driftwood and building waste, thinned wood, etc. as part of the construction of a recycling society. You may use the charcoal which made these woody waste and the thinning material as a raw material. In this case, a porous material can be obtained, for example, by chipping these woods to a grain size of 1 to 100 mm, preferably about 5 to 10 mm, and carbonizing usually at 300 to 1000 ° C., preferably 400 ° C. or more. . When the porous carrier of the present invention is applied to ruminants such as cattle, it is preferable to use a porous carrier such as charcoal or zeolite that does not affect the living body.

4). Porous carrier holding methane-oxidizing bacteria When producing a porous carrier on which methane-oxidizing bacteria propagate, from the viewpoint of practical use such as cost, the smallest possible amount of methane-oxidizing bacteria is efficiently attached to the entire porous body. It is preferable to breed. Therefore, a porous carrier chip is formed, and a nutrient solution containing methane-oxidizing bacteria is added to the upper part thereof, and air containing methane is circulated to promote adhesion and propagation to charcoal. The method will be described taking charcoal as an example of the porous carrier.
(1) Spray an inorganic salt medium on charcoal and fully moisten it to a moisture content of about 80%.
(2) Put charcoal (1) into a sealable container and add a culture solution containing methane-oxidizing bacteria from the top.
(3) The inside air is evacuated with a vacuum pump, and a mixed gas having a methane / air ratio of 1: 9 is injected.
(4) Operate a fan and circulate air containing methane.
(5) The container is kept at a temperature of about 30 ° C.
In addition, in the propagation of methane oxidizing bacteria, an environment in which the initial methane concentration is 5 to 45 vol%, the ambient temperature is 20 to 40 ° C., and the oxygen concentration is maintained at least 5 vol%, preferably 8 vol% or more is suitable. It is not restricted to the above-mentioned conditions.

  After the above operation is completed, the internal methane concentration and carbon dioxide are measured over time, and the air circulation is stopped when the methane concentration falls below the detection limit. Here, by using the charcoal taken out from the container, it is possible to confirm the adherence and breeding state of methane-oxidizing bacteria and the ability to remove methane by the following methods.

  First, the charcoal taken out from the container is thoroughly mixed, and a certain amount is randomly picked up and sealed in a gas vial. Next, after partially extracting air, methane is injected instead, and the gas vial is left in a thermostat at 30 ° C. It can be confirmed by sampling at intervals of 24 hours and measuring the concentrations of methane and carbon dioxide with a gas chromatograph. For example, if the desired ability to remove methane is not obtained, the methane-degrading ability of the methane-oxidizing bacterium used is confirmed at the time of separation, so that the state of methane-oxidizing bacteria adherence and breeding may be insufficient. Conceivable.

5). Suppression of methane generation The porous carrier holding the bacteria having the ability to oxidize methane of the present invention is placed, spread or buried in a paddy field, wetland, waste disposal site or composting facility. When buried, the methane-oxidizing bacterium is an aerobic bacterium, so it is necessary to make the inside of the carrier aerobic. The amount to be arranged, sprayed or buried varies depending on the conditions under which methane generation should be suppressed and the methane oxidation / decomposition of the bacteria, but is, for example, about 100 to 50 kg, preferably about ² to 25 kg per 1 m ² . For example, in the case of paddy fields and wetlands, a sufficient methane suppression effect can be obtained even at about 100 g / m ² , but a layer of about 1 to 20 cm, preferably about 5 to 10 cm, is formed in a waste disposal site. The required amount increases accordingly.

According to the method and apparatus for producing a carrier for holding methane-oxidizing bacteria of the present invention , a small amount of methane-oxidizing bacteria can be efficiently attached to and propagated on the entire charcoal or other porous carrier, and the methane-oxidizing bacteria can be produced at low cost. The holding carrier can be mass-produced. The methane-oxidizing bacterium holding carrier can efficiently suppress methane generation from a waste final disposal site, a wetland, a paddy field, etc. over a wide range of methane generation sources at low cost. Furthermore, natural materials such as charcoal, especially timber waste such as dam driftwood and building waste, and charcoal made from thinned wood can be used as a porous material, so the burden on the environment is small.

  The present invention will be described with reference to the following examples, but the present invention is not limited to the following examples.

1. Collection and culture of methane-oxidizing bacteria The methane-oxidizing bacteria group in this example is obtained from the paddy field, the Teganuma wetland, the soil of riverside wetlands of agricultural waterways (about 10 cm x 10 cm from the black surface to a depth of about 5 cm) by the following method. Harvested and enriched cultures were selected to have high methane decomposition ability.
A 30 mL gas vial was charged with the inorganic salt liquid medium for methane oxidizing bacteria shown in Table 1, 0.1 g of soil was further added, the butyl rubber stopper was sealed with an aluminum seal, and 10 mL of methane was added as a carbon source. Each gas vial was cultured with shaking (rotation speed: 120 rpm) in a constant-temperature incubator at 30 ° C. One week later, 1 mL of the liquid medium was collected, added to a gas vial containing 10 mL of a new liquid medium, and methane was added again to perform shaking culture. This operation was repeated to perform a fourth enrichment culture. FIG. 1 shows the methane decomposition ability of the accumulated methane-oxidizing bacteria group. In the methane removal ability of these methane-oxidizing bacteria, the methane concentration decreased to the detection limit or less on the third day from the start of the experiment in Teganuma Wetland A ward, and the paddy ward, irrigation wetlands A, B ward, Compared with the fact that the methane concentration was reduced by only about 50%, a high removal effect was exhibited. Similarly, in the carbon dioxide concentration, Teganuma Wetland A Ward showed higher concentration than other Wards.

2. Attachment and breeding of methane-oxidizing bacteria using charcoal Using the methane-oxidizing bacteria in the Aga Teganuma wetland, which had a high removal ability among the five types of methane-oxidizing bacteria obtained in Example 1, Attempt to adhere and breed. As charcoal, charcoal made from chips of dam driftwood (ratio of hardwood and conifer 7: 3) was used. Table 2 shows the specific surface area of charcoal. The charcoal used in the experiment was screened to a particle size of 5 mm to 10 mm with a sieve, washed thoroughly with distilled water, and then dried at 100 ° C. for 24 hours. As an inoculation source for the charcoal adhesion / reproduction experiment, 1 mL of a liquid medium in which methane-oxidizing bacteria were accumulated was placed in 10 mL of a new liquid medium, and 3 mL of methane was added to grow for 1 week. The viable cell count (expressed in cfu) in the prepared nutrient solution was estimated to be 0.14 × 10 ¹⁰ cfu MPN / mL.

  When preparing charcoal in which methane-oxidizing bacteria propagate, it is desirable to efficiently attach and propagate as little methane-oxidizing bacteria as possible to the entire charcoal from the viewpoint of practical use. Therefore, it is considered that charcoal is chip-shaped, and a nutrient solution containing methane-oxidizing bacteria is added to the top, and air containing methane is circulated to promote adhesion and propagation to charcoal. A holding carrier manufacturing apparatus (FIG. 2) was prototyped.

This apparatus for producing a carrier for holding methane-oxidizing bacteria comprises a double container of an inner container 1 and an outer container 2, and the inner container 1 is provided with an inlet 3 and an outlet 4 and air 5 containing methane at the inlet 3 is provided. A blowing means 6 is provided for introducing into the inner container 1 and discharging from the exhaust port 4, and the outer container 2 circulates air 5 containing methane discharged from the inner container 1 to the inlet 3. The flow path 7 is formed, the porous carrier 9 in which the nutrient solution 8 containing methane oxidizing bacteria is dispersed is accommodated in the inner container 1, and the air 5 containing methane that passes through the porous carrier 9 in the inner container 1. A circulation flow is formed. Here, the size of the inner container 1 is 100 mm (W) × 100 mm (D) × 200 mm (H), and a 12 V sirocco fan as the blowing means 6 is attached to the lid portion that closes the inlet 3. A structure is provided in which four exhaust ports 4 each having a 5 mm ² hole are provided in the lower part of 1. The inner container 1 is housed in a vacuum polycarbonate desiccator (capacity 2.4 L) as the outer container 2 to form a double container, and a flow that can form a circulating flow of air containing methane between the desiccator 2 and the inner container 1. Form roads and spaces. Using this methane-oxidizing bacteria-retaining carrier production apparatus, an experiment was performed according to the following procedure.
(1) Spray an inorganic salt medium on charcoal and fully moisten it to a moisture content of about 80%.
(2) 20 g of charcoal chips as the porous carrier 9 are put into the inner container 1, and 1 mL of nutrient solution 8 containing methane oxidizing bacteria is added dropwise from above.
(3) Place the inner container 1 in the desiccator 2.
(4) The air in the desiccator 2 is extracted with a vacuum pump (not shown), and a mixed gas having a methane / air ratio of 1: 9 is injected.
(5) The sirocco fan 6 is operated, and the air 5 containing methane is sent into the inner container 1.
(6) The desiccator 2 is placed in a thermostat (not shown) at 30 ° C.

  After the above operation is completed, the gas in the desiccator 2 is extracted from the sampling port 10, the methane concentration and the carbon dioxide concentration are measured over time, and the sirocco fan 6 is stopped when the methane concentration falls below the detection limit. The circulation of the air 5 was stopped, and the inner container 1 was taken out from the desiccator 2. The result of examining the methane removal capability using the charcoal 9 taken out from the inner container 1 is shown in FIG. 1 g of charcoal (porous carrier) 9 was randomly collected from the container 1 to prepare 5 samples. And the air and charcoal containing 3 mL of methane were put into a 30 mL gas vial bottle, and after standing, it was left still in a 30 ° C. thermostat. Methane and carbon dioxide concentrations were measured over time with a gas chromatograph. As a result, it was found that in any charcoal, the methane concentration rapidly decreased from the start of the experiment, and became 1% or less on the second day and below the detection limit on the seventh day. The carbon dioxide concentration also reached about 4% on the 2nd day and 5% on the 7th day as the methane concentration decreased. From this, it was confirmed that the methane injected into the gas vial was removed by the methane oxidizing bacteria attached to and propagated on the charcoal. Furthermore, it was confirmed that methane-oxidizing bacteria propagated in the pores of charcoal by taking pictures of decomposed charcoal with an SEM electron microscope (FIG. 4). The moisture content of charcoal was about 80% at the start of the experiment, but decreased to 40% at the end of the experiment, and it became easy to handle because it was dried by air circulation.

3. Evaluation of Methane Oxidation / Resolution The results of examining the effect of removing methane by changing the weight of charcoal obtained in Example 2 are shown in FIG. The air containing 3 mL of methane and charcoal with different weights were placed in a 30 mL gas vial, sealed, and then placed in a 30 ° C. incubator. The methane concentration was measured with a gas chromatograph, and when it was no longer detected, the operation of adding air containing methane was continued until no decrease in the concentration was observed. As a result, it was found that the amount of methane removed by charcoal increased in proportion to the weight of charcoal. That is, it was shown that cracked charcoal has a methane removal capacity of about 6.1 mg / g. Since the amount of methane removed per gram by weight of charcoal alone was about 4.7 × 10 ⁻⁴ mg / g, the ability to remove charcoal increased by about 13,000 times due to the propagation of methane oxidizing bacteria.

The result of examining the methane removal rate by charcoal holding methane oxidizing bacteria is shown in FIG. Air containing 2 mL of methane and 1 g of charcoal were placed in a 20 mL test tube, sealed, and then placed in a thermostat at 30 ° C. The methane concentration was measured over time with a gas chromatograph. As a result, the methane concentration at the start of the experiment was about 2.5 mg CH ₄ (volume ratio: 15%), but after 6 hours it was 75% of the initial concentration, after 15 hours it was about 40%, and after 26 hours. It was found that it was almost below the detection limit. Since the methane concentration decreased in inverse proportion to the time, the removal rate of methane by charcoal holding methane oxidizing bacteria was calculated to be 0.091 mg CH ₄ / hr / g. Moreover, the methane removal rate in the different temperature conditions of the charcoal which hold | maintains methane oxidation bacteria is shown in FIG. On the seventh day from the start of the experiment, the methane removal rate was about 5% at a temperature of 5 ° C., and the removal rate increased with an increase in temperature, about 30% at 10 ° C. and about 50% at 15 ° C. Since the methane removal rate on the 7th day increased compared to the 5th day from the start of the experiment, it is considered that the methane oxidizing bacteria are active. On the other hand, when the temperature was 20 ° C. to 40 ° C., the methane removal rate reached 100% on the fifth day, and all the injected methane was removed. However, when the temperature was set to 50 ° C., the removal rate of methane was small and hardly removed. From the above, it was considered that methane-oxidizing bacteria propagated on charcoal were considered to have a suitable growth temperature of 20 to 40 ° C., the ability to remove methane was lowered at 15 ° C. or lower, and the ability to decompose was lost at 50 ° C. or higher. In addition, the experiment regarding the methane removal of the methane-oxidizing bacterium breeding charcoal under different temperature conditions was carried out by putting air containing 3 mL of methane and 1 g of charcoal into a 20 mL test tube, sealing it, then 5, 10, 15, 20, 30, 40. , Left in a 50 ° C. incubator. The methane concentration was measured with a gas chromatograph on the 5th and 7th days, and the reduction rate relative to the initial concentration was taken as the removal rate.

4). Identification of methane-oxidizing bacteria group In order to identify the species of methane-oxidizing bacteria group that adhered to and propagated on charcoal, this charcoal was ground, and genomic DNA was extracted from those cultured in the liquid medium shown in Table 1, using this as a template. PCR was performed to amplify the 16S rRNA gene region. As the primers, primers 27F (5′-GAGTTTGATCMTGGCTCA-3 ′; SEQ ID NO: 1) and 1544R (5′-AGAAAGGAGGTGATCCAGCC-3 ′; SEQ ID NO: 2), which are highly conserved throughout bacteria, were used. After confirming amplification of the target region by agarose gel electrophoresis, this PCR product was cloned. Using pGEMT-easy (Promega) as a vector, TA cloning was performed by a conventional method and introduced into E. coli by electroporation.

The emerged colony is M13 primer set present in the vector M13 forward primer: 5'-GTTTTCCCAGTCACGACGTT-3 '(SEQ ID NO: 3)
M13 rear primer: 5′-GGAAACAGCTATGACCATGA-3 ′; (SEQ ID NO: 4)
Amplification was performed directly, and the insert was confirmed. For those for which amplification of the target product was confirmed, sequencing was performed using the M13 forward primer, three samples Met16, 21, and 31 considered to be methane-oxidizing bacteria were selected, and a sequence covering the entire length was performed. . Assembly is performed based on the obtained sequence data, the full-length sequence is determined, homology search and phylogenetic tree generation are performed using the base sequence of 16S rRNA gene, and the fungus is prepared according to the method of Non-Patent Document 2. Species estimation was performed. The homology search used a BLAST search against the GenBank database. ClustalX and Treeview were used for multiple alignment and generation of phylogenetic trees.

As a result of the above, Met16 is, Hyphomicro bi um methylovorum, in a strain belonging to the same cluster as the Hyphomicro bi um facilis, Hyphomicro bi um sp. A strain that was systematically very close to the strain isolated as S4 was identified. Furthermore, the Met21 strain and the Met31 strain have high homology in the sequence data classified as type II methane-oxidizing bacteria [Methanotroph], the Met21 strain is closely related to the T2-06 strain, and the Met31 strain is the T2- It was judged to be closely related to the 07 strain and the T2-17 strain.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited to this, and various modifications can be made without departing from the scope of the present invention. For example, the methane oxidant holding carrier production apparatus exemplified in FIG. 2 has a structure suitable for experiments for convenience, but is not limited to this in industrial implementation. That is, since it is necessary to accurately measure the methane concentration in the experiment, the outer container 2 uses a desiccator. However, the outer container 2 is not necessarily a desiccator, and may be a sealed container that does not leak methane-containing air. However, the temperature for breeding does not need to be kept constant at 30 ° C. using a thermostat or the like, and if it is in the range of 20 ° C. to 40 ° C., it may fluctuate between them or be controlled at a constant temperature. Can be propagated efficiently. In addition, if the oxygen concentration of air containing methane is at least about 5 vol%, it can be propagated and desirably maintained at 8 vol% or more. However, since air usually contains 24 vol% oxygen, 5 The oxygen concentration can be maintained at 8 vol% or more even in the circulation use in a sealed state including methane in the range of ˜45 vol%. Furthermore, the size of the inner container 1 and the positions, sizes, number, and the like of the introduction port 3 and the exhaust port 4 are not limited to those of the illustrated embodiment, and are appropriately selected according to the production scale of the methane oxidizing bacteria holding carrier. In the present embodiment, a sirocco fan is used as the air blowing means 6, but the present invention is not limited to this, and any means can be used as long as it can secure an air blowing amount suitable for the propagation of methane oxidizing bacteria.

It is a graph which shows the methane decomposition ability of the methane oxidizing bacteria group obtained from the soil collected from the paddy field, Teganuma wetland, and the riverside wetland of the agricultural canal. (A) is the change of methane concentration, (B) is the carbon dioxide concentration. The change of each is shown. It is the schematic of the apparatus for making methane oxidation bacteria group adhere and propagate to charcoal. It is a graph which shows the methane decomposition | disassembly ability of the charcoal which adhered and propagated the methane oxidation bacteria group, (A) shows the change of methane concentration, (B) shows the change of carbon dioxide concentration, respectively. It is a SEM photograph in which methane-oxidizing bacteria propagate in the pores of charcoal. It is a graph which shows the relationship between the weight of decomposition | disassembly charcoal, and the amount of methane decomposition | disassembly. It is a graph showing the decomposition rate of methane by cracked charcoal. It is a graph which shows the methane decomposition ability of cracked charcoal under different temperature conditions.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Inner container 2 Outer container 3 Inlet port 4 Exhaust port 5 Air containing methane 6 Blowing means 7 Reflux path 8 Nutrient solution containing methane oxidizing bacteria 9 Porous carrier

Claims (6)

A culture solution containing bacteria capable of oxidizing methane is sprayed or added to the porous carrier and mixed, and then air containing methane is circulated through the porous carrier to attach the bacteria to the porous material. A method for producing a carrier for holding methane-oxidizing bacteria , which comprises allowing the microorganism to propagate . The method according to claim 1, wherein the culture solution is absorbed by the porous carrier before the culture solution containing the bacterium capable of oxidizing methane is dispersed or added to the porous carrier and mixed. The method according to claim 1, wherein the porous carrier is selected from charcoal, activated carbon, perlite, pumice or zeolite . The method according to claim 3, wherein the porous carrier is charcoal obtained by chipping a wood material into a particle size of about 1 to 100 mm and carbonizing at 300 ° C. to 1000 ° C. An air blowing means comprising a double container of an inner container and an outer container, the inner container having an inlet and an outlet, and introducing air containing methane into the inlet and discharging the air from the outlet The outer container forms a flow path for returning air containing methane discharged from the inner container to the introduction port between the outer container and the nutrient solution containing methane oxidizing bacteria in the inner container An apparatus for producing a carrier for holding methane-oxidizing bacteria , wherein a circulating flow of air containing methane passing through the porous carrier in the inner container is formed . A method for inhibiting methane generation, comprising spraying or arranging a methane oxidant holding carrier produced by the method for producing a methane oxidant holding carrier according to any one of claims 1 to 4 on a methane generation source .