JP2003154382A - Method for efficiently restoring environment or medium polluted with organic compound in presence of glutathione by microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enhancing decomposition efficiency at the time of decomposition treatment of an organic compound being a pollutant by microorganisms and a method for restoring a polluted environment or medium using the same. SOLUTION: At the time of restoration of the environment or medium by the decomposition of the organic compound polluting the environment or medium by microorganisms, glutathione is allowed to be present to enhance restoration efficiency.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the efficiency of remediation in the purification and remediation of groundwater contaminated with organic compounds, water such as rivers and lakes, environment such as soil and air, and media such as wastewater using microorganisms. And a repairing method using the same.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to volatile organic chlorine compounds which are harmful to living organisms and hardly decomposed has become a serious problem. In particular, contamination by volatile organic chlorine compounds such as tetrachlorethylene (PCE), trichlorethylene (TCE), and dichloroethylene (DCE) has spread to a considerable extent in the soil of paper and pulp industries and precision machinery-related industrial areas in Japan and overseas. It is believed that there are many cases, and many cases have been reported that were actually detected in environmental surveys. It is said that these volatile organic chlorine compounds that remain in the soil dissolve in groundwater due to rainwater etc. and spread throughout the surrounding area. Since such compounds are suspected to have carcinogenicity and reproductive toxicity and are extremely stable in the environment, the pollution of groundwater used as a water source for drinking water is a major social problem.

From the above, purification of an aqueous medium such as contaminated groundwater, soil and its surrounding gas phase by removing and decomposing volatile organic chlorine compounds is an important issue from the viewpoint of environmental conservation. The necessary technology is being developed.

For example, adsorption treatment with activated carbon and decomposition treatment with light or heat have been studied, but it is not always practical in terms of cost and operability.

On the other hand, in recent years, decomposition of volatile organochlorine compounds such as TCE, which is stable in the environment, by microorganisms has been reported in recent years, and studies for their practical use have begun. That is, in biodegradation treatment using microorganisms, it is possible to decompose volatile organochlorine compounds into harmless substances by selecting the microorganisms to be used, basically no special chemicals are required, and labor and cost required for maintenance. There are advantages such as reduction.

[0006] As an example of the TCE-degrading bacterium, Welch was isolated as a microorganism isolated with a microorganism capable of decomposing volatile organic chlorine compounds.
ia alkenophila sero 5 (USP 4877736, ATCC 53570),
Welchia alkenophila sero 33 (USP 4877736, ATCC 5357
1), Methylocystis sp. Strain M (Agric. Biol. Che
m., 53, 2903 (1989), Biosci. Biotech. Biochem., 5
6, 486 (1992), 56, 736 (1992), Methylosinus tri
chosprium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Env
iron.- Microbiol., 29, 365 (1989), Appl. Environ.M
icrobiol., 55, 3155 (1989), Appl. Biochem. Biotech
nol., 28, 877 (1991), JP-A-02-92274, JP-A
03-292970), Methylomonas sp.MM2 (Appl. Envi
ron. Microbiol., 57, 236 (1991)), Alcaligenes deni
trificansssp.xylosoxidans JE75 (Arch. microbiol.,
154, 410 (1990)), Alcaligeneseutrophus JMP134 (A
ppl. Environ. Microbiol., 56, 1179 (1990)), Alcal
igenes eutrophus FERM-13761 (JP 07-123976), Pseudomonas aeruginosa JI 104 (JP 07-23689)
5), Mycobacterium vaccae JOB5 (J. Gen. Micr
obiol., 82, 163 (1974), Appl. Environ. Microbiol.,
54, 2960 (1989), ATCC 29678), Pseudomonas putida
BH (Sewer Society Journal, 24, 27 (1987)), Acinetobactor s
p. strain G4 (Appl. Environ. Microbiol., 52, 383
(1986), 53, 949 (1987), 54, 951 (1989), 56,
279 (1990), 57, 193 (1991), USP 4925802, ATCC 53
617, this fungus was initially classified as Pseudomonas cepacia, but was changed to Pseudomonas sp.), Pseudomonas me
ndocina KR-1 (Bio / Technol., 7,282 (1989)), Pseudom
onas putida F1 (Appl. Environ. Microbiol., 54, 1703
(1988), ibid. 54, 2578 (1988)), Pseudomonas fluorescen
s PFL12 (Appl. Environ. Microbiol., 54, 2578 (198
8)), Pseudomonas putida KWI-9 (JP 06-70753A), Pseudomonas cepacia KK01 (JP 06-227769A), Nitrosomonas europaea (Appl. Environ. Micr)
obiol., 56, 1169 (1990)), Nocardia corallina B-276
(Japanese Patent Laid-Open No. 08-70881, FERM BP-5124, ATCC 31338)
Etc.

[0007] However, in particular, when a degrading bacterium of a chlorinated ethylene compound such as TCE or DCE is used in an actual environmental purification treatment, in order to decompose these compounds, a decomposition inducer (index It means that there is a case where a chemical substance such as an aromatic compound or methane is required as a user.

[0008] For example, aromatic compounds such as phenol and toluene are very excellent inducers, but their toxicity is high, and their release into the environment must be carried out under some regulations, and similarly, they are also excellent inducers. Methane, which is a substance, is a flammable gas and is dangerous to handle.
Special measures are needed to control the diffusion into the environment.

One method for solving such a problem is disclosed in Japanese Patent Laid-Open No. 4-502277. in this way,
Tryptophan, which is one of the aromatic amino acids, is used as a microbial decomposition inducer of chlorinated ethylene to avoid the toxicity and danger problems of the above inducers. Further, according to JP-A-7-171548, using lignocellulose, which is a natural substance extracted from herbaceous plants, as an inducer,
It avoids the above problems.

From the viewpoint of improving degrading microorganisms, a plasmid into which a DNA fragment containing a gene region encoding a degrading enzyme oxygenase or hydroxylase is incorporated is introduced into a host bacterium, or a harmless inducer is used. Attempts have been made to constitutively express degrading activity even under the condition that no inducer is present. DN
Pseudomonas mendocina KR is a strain derived from the A fragment.
-1 (Japanese Patent Laid-Open No. 2-503866), Pseudomonas Putida K
W1-9 (JP-A-6-105691), Pseudomonas putida
BH (Groundwater / Soil Contamination and 3rd Lecture Meeting on Preventive Measures, 213 (1994)), etc.

Another solution is the international publication number WO 92/19.
It is disclosed in Japanese Patent No. 738. According to this report, a mutant strain that decomposes a chlorinated ethylene compound without using an inducer such as phenol or toluene has been obtained by mutation using a transposon.

Yet another solution is US Pat. No. 5543.
No. 317 and Japanese Patent Application Laid-Open No. 8-294387.
According to this report, a mutant strain that decomposes a chlorinated ethylene compound or an aromatic compound without using an inducer such as phenol or toluene has been obtained by treating it with nitrosoguanidine as a mutation source.

[0013]

As described above, the problems concerning the inducer in the microbial decomposition treatment of chlorinated ethylene compounds and aromatic compounds are being circumvented. Is becoming more and more practical.

Further, in the environmental remediation treatment of organic compounds by the decomposition of microorganisms, there are cases where long-term purification is required, and for that purpose, it is necessary to maintain the decomposition activity of microorganisms for a long period of time. In the technology, the degrading activity of these microorganisms is often deactivated in a very short period of time.

Further, in the environmental remediation treatment utilizing the decomposition of organic compounds by microorganisms, it takes time and cost to cultivate the microorganisms, and it is essential to effectively utilize the target ability of the microorganisms once cultivated. Is.

The cause of the phenomenon that the degrading activity of microorganisms is inactivated in a very short period of time is considered to be self-digestion of degrading enzymes in cells, toxicity due to degrading intermediate products, lack of energy required for degrading, etc. However, among these, the problem of toxicity due to decomposition intermediates is a very important issue in the sense that the microbial decomposition activity of organic compounds disappears.

An object of the present invention is to provide a method for improving the decomposition efficiency when an organic compound as a pollutant is decomposed by a microorganism and a method for repairing a contaminated environment or a medium using the method.

[0018]

[Means for Solving the Problems] Therefore, as a result of diligent research on the method of solving the problem of reduction in the decomposition activity due to toxicity due to decomposition intermediates, the present inventors have found that when a microorganism is brought into contact with an organic compound to decompose it. In addition, it was found that the coagulation of reduced glutathione in the decomposition system makes the decomposition process more efficient. The present invention has been made based on such findings.

The method for improving the efficiency of repairing a polluted environment or medium of the present invention is a method for efficiently repairing an environment or medium polluted with an organic compound using a microorganism capable of degrading the organic compound. When the compound is brought into contact with the organic compound to cause decomposition, the decomposition is made efficient by coexisting reduced glutathione in the decomposition system.

The method of repairing a contaminated environment or medium according to the present invention is characterized by using the above-described efficiency improving method.

[0021] According to the present invention, it is possible to provide a method capable of efficiently decomposing polluted organic compounds in the environment or various media without using an inducer which requires careful handling.

[0022]

DETAILED DESCRIPTION OF THE INVENTION Glutathione is glutamic acid,
It is a peptide consisting of cysteine and glycine (L-γ-glutamyl-L-cysteinylglycine). Various physiological activities have been found in glutathione, and its function is γ-
It is believed to be based on the glutamyl group and the SH group of cysteine residues. Glutathione has a reducing ability and causes other molecules (small molecules, enzymes, proteins) to newly generate SH groups,
It has the function of maintaining the SH group. Some enzyme proteins form mixed disulfides with glutathione, which may regulate enzyme activity. Further, it also plays a very important role in terms of biological defense such as reduction and detoxification of peroxide generated by oxidative stress, conjugation and excretion of various foreign compounds. Another important function of glutathione is to maintain the redox system. In vivo
95% or more usually exists in a reduced form (GSH). Oxidized type (GSSG)
The redox reaction with and is coupled to the pentose phosphate cycle and is performed by glutathione reductase and glutathione oxidase.

The effective concentration when giving such glutathione is 0.1 mM to 10 mM, and more preferably 0.5 mM.
It is about 5 to 5 mM, but it varies slightly depending on the type of microorganism used.

Examples of pollutants to be purified include chlorinated ethylene compounds such as trichlorethylene (TCE) and dichloroethylene (DCE), and the method of the present invention is applicable to the environment and various media polluted with such pollutants. Can be suitably applied to.

The polluted environment or medium may be in the form of an aqueous liquid, a solid, a gas or a mixture of two or more thereof. Examples of the form of the aqueous liquid include groundwater, water in the environment such as rivers and lakes, various industrial processes, wastewater generated in waste treatment, and water media such as wastewater. Examples of the mixture of the aqueous liquid, the solid and the gas include soil. Examples of the gas include air, exhaust gas or waste gas generated in various industrial processes, waste treatment, and the like. It should be noted that the medium to be treated also includes those that are discharged from various processes and reused in other processes.

As the microorganism used in the present invention, a microorganism that has the ability to decompose an organic compound to be decomposed and whose decomposition efficiency is improved in the presence of glutathione is used. Examples of such microorganisms include Pseudomonas cepacia KK01 strain, JM1 strain, Methylocystis sp. Strain M strain (Agric. Bio
l. Chem., 53, 2903 (1989), Biosci. Biotech. Bioche
m., 56, 486 (1992), 56, 736 (1992)), Methylosinus trichospr strain OB3b (Methylosinus trichospr)
ium OB3b) (Am. Chem. Soc. Natl. Meet. Dev. Enviro
n.-Microbiol., 29, 365 (1989), Appl. Environ.Micro
biol., 55, 3155 (1989), Appl. Biochem. Biotechno
L., 28, 877 (1991), JP 02-92274 A, JP 03
-292970), Mycobacterium vaccae JOB5 strain (J.Gen. Microbiol.,
82, 163 (1974), Appl. Environ. Microbiol., 54, 296
0 (1989), ATCC 29678), Pseudomonas putida BH strain (Sewer Association, 24, 27 (1
987)), Acinetobacter species G4 strain (Acinetobac
tor sp. strain G4) (Appl. Environ. Microbiol., 5
2, 383 (1986), 53, 949 (1987), 54, 951 (198)
9), 56, 279 (1990), 57, 193 (1991), USP 492580
2, ATCC 53617), Pseudomonas mendocina KR-1 strain (P
seudomonas mendocina KR-1) (Bio / Technol., 7, 282
(1989)), Pseudomonas pu
tida F1) (Appl. Environ. Microbiol., 54, 1703 (198
8), ibid. 54, 2578 (1988)) and the like.

Pseudomonas cepacia strain KK01 (International Deposition number under the Budapest Treaty number: FERM BP-4235), which is one of the microorganisms used in the embodiment of the present invention, is disclosed in Japanese Examined Patent Publication No. 8-24589 as phenol, cresol and the like. It is a strain that has been publicly announced as a bacterium that decomposes aromatic compounds, and JP-A-6-296711 discloses that an aromatic compound such as phenol is used as an inducer to decompose organic chlorine compounds such as TCE. In addition, Pseudomonas
The strains of the genus Sepacia are currently taxonomically classified as Burkholderia
It has been changed to Sepacia.

JM1 strain (an international deposit number under the Budapest Treaty number: FERM BP-5352), which is one of the microorganisms used in the embodiment of the present invention, is obtained by mutating the J1 strain by a mutation operation using a mutagen. Japanese Patent Laid-Open No. 8-294387 discloses that the obtained mutant strain is capable of degrading an organochlorine compound such as TCE without using an inducer. Initially, this strain was designated as "Corynebacterium species JM1 strain" as belonging to the genus Corynebacterium, but it was confirmed that the strain was "not belonging to the genus Corynebacterium" in the later examination. Therefore, the identification display was changed to "JM1 share".

The medium used for culturing microorganisms is basically a carbon source necessary for obtaining growth and decomposition activity,
It may contain a nitrogen source, a phosphorus source, an inorganic salt and the like. The basic inorganic salt medium is not particularly limited as long as it contains components necessary for the growth of microorganisms, and for example, basal salt medium such as M9 medium and MSB medium can be used.

The composition of M9 medium is shown below.

Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g balance: water (in 1 liter of medium; pH 7.0) Cultivation can be carried out under aerobic conditions. It may be liquid culture or solid culture. The culture temperature is preferably 15 to 30 ° C.

The method according to the present invention comprises a step of bringing a microorganism having a decomposing activity for an organic compound as a pollutant into contact with an object to be purified and repaired in the presence of glutatin. Microorganisms can be used in a state of being supported on a carrier as needed, and as the carrier, those used in various steps utilizing reactions using microorganisms in the synthetic chemical industry, pharmaceutical industry, food industry, etc. Can be used publicly. In addition, when the microorganisms supported on the carrier are put into the environment such as soil, those which have no influence on the environment and those which can be naturally eliminated after the purification / restoration treatment can be used.

Examples of such carriers include porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, and other inorganic particulate carriers, starch, agar, chitin, and the like. Examples thereof include gel carriers such as chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, agarose and gelatin, ion exchangeable cellulose, ion exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. Also as a natural product. Cellulose-based materials such as cotton, linen and paper, and lignin-based materials such as wood flour and bark can also be used.

Examples of the organic compound include chlorinated aliphatic compounds such as trichloroethylene and dichloroethylene.

When the microorganisms are brought into contact with the environment or medium to be repaired, when they are aqueous liquids, a method of administering the microorganisms into the aqueous liquid to bring them into contact with each other, and supporting the microorganisms on a carrier, A method of bringing an aqueous liquid into contact with the carrier can be used. When a carrier is used, an aqueous medium is introduced into the reaction area where the carrier can be stored and brought into contact with them to decompose the contaminating organic compounds, and the aqueous liquid after the decomposition is discharged to the outside of the container. The method of doing is suitable. This treatment can be performed by a desired treatment method such as a batch method, a semi-continuous method or a continuous method.

The concentration of the polluted organic compound in the aqueous liquid introduced into the reaction zone can be selected depending on the decomposition activity of the microorganism. If it is necessary to dilute it, dilute it with water and introduce it into the reaction zone. In the case of a chlorinated aliphatic compound, it is preferable to set it in the range of 1 ppm to 100 ppm, for example.

Further, in order to make glutathione present in the reaction region, a method of dissolving a predetermined concentration of glutathione in a culture solution of the microorganism or a solution in which the microorganism is suspended and adding it to the reaction region can be used.

The concentration of glutatinone is 0.1 m
It is preferable to select from the range of M to 20 mM.

When the environment to be treated is soil, any one of the following methods or a combination of two or more of them can be suitably used for contacting the contaminated organic compound with the microorganism. (1) A method in which a liquid containing microorganisms is introduced into soil by providing a well or the like for introduction. (2) A method of introducing soil into a solution containing microorganisms. (3) A method of mixing a solution containing a microorganism and soil. (4) A method of bringing a carrier supporting microorganisms into contact with soil. (5) A method in which an organic compound is extracted from soil into water and the extract is brought into contact with microorganisms.

When the soil to be contacted with microorganisms is collected from the ground and treated, the concentration of polluted organic compounds contained in the collected soil can be selected according to the degrading activity of the microorganisms used, etc. If so, it can be used for contact with microorganisms after mixing with a suitable medium such as water and soil and diluting. In the case of chlorinated aliphatic compounds, for example,
It is preferable to set it in the range of 1 ppm to 50 ppm.

Furthermore, in order to make glutathione present in the place of contact with soil, glutathione is dissolved in a predetermined concentration in a culture solution of the microorganism or a solution in which the microorganism is suspended, and the glutathione is sprayed on the soil in the reaction area, or Methods such as digging a well and injecting it can be used.

When the object to be treated is air or gas, a method of introducing air or gas into a solution containing microorganisms, a method of bringing air or gas into contact with a carrier carrying microorganisms, a combination of these methods, and the like. Can be used.

As in the case of treating the above-mentioned aqueous liquid, air or gas is introduced into the reaction region in the container accommodating the carrier supporting the microorganisms to bring the microorganisms into contact with the air or gas, and A method in which an organic compound in a gas is decomposed and the air or the gas after the decomposition is discharged to the outside of the container can be suitably used.

The concentration of the polluted organic compound in the air or gas to be brought into contact with the microorganisms can be selected according to the decomposition activity of the microorganisms to be used, and if necessary, after diluting with an applicable gas such as air. It can be used for contact with microorganisms.

[0045]

The present invention will be described in more detail with reference to the following examples.

Reference Example 1 (Evaluation of Effect of Glutathione itself on TCE) In order to evaluate the effect of reduced glutathione (hereinafter referred to as GSH) on TCE, TCE in distilled water containing GSH and distilled water not containing GSH was evaluated. The concentrations were compared.

In a 27.5 ml vial, 10 ml of distilled water was placed, and GSH was added to a final concentration of 2 mM,
Make four bottles each with the same amount of distilled water added, and adjust the concentration in the vial to about 8, 17, 26, and 35 ppm.
A CE saturated aqueous solution was added, the container was sealed with a butyl rubber stopper with a Teflon liner and an aluminum seal, and the mixture was shaken at 30 ° C. for 27 hours, and the gas in the gas phase was measured by gas chromatography.

The results are shown in FIG. The TCE concentration was converted from the obtained area value of the gas chromatography as the concentration when all the TCEs were dissolved in the aqueous solution in the vial and shown (the same applies to the following examples).

From this result, it was found that TCE was not directly affected by GSH.

Example 1 (Effect of GSH on TCE Degradation of JM1 Strain (Liquid System)) Colonies of JM1 strain on agar M9 medium (containing 0.5% sodium glutamate) were added to 200 ml of liquid M9 medium containing 0.5% sodium glutamate. Inoculate and in a 500 ml shake flask, 25
Shaking culture was performed at ° C. At 20 hours, which is the latter stage of logarithmic growth, the cells were collected by centrifugation and resuspended in M9 medium containing no equivalent amount of carbon source.

10 ml aliquots of the bacterial suspension thus prepared
Two bottles were prepared by injecting into a 27.5 ml vial. GSH was added to one of these so that the final concentration was 2 mM, and the same amount of sterile distilled water was added to the other. To these vials and a total of three vials containing only 10 ml of M9 medium as a blank, add TCE saturated aqueous solution so that the concentration in the vial is about 12 ppm, and use a butyl rubber stopper with a Teflon liner and an aluminum seal. It was sealed.

These vials were shaken at 30 ° C., and the TCE of the gas phase portion was measured with time by gas chromatography. When the value became 10% or less of the initial concentration, it was once opened, and about 90% of the initial TCE saturated aqueous solution was added, and the container was resealed (the blank remains in the initial state). By repeating such operations, the effect of GSH on the repeated decomposition of TCE was evaluated.

Formaldehyde was added as an energy source for the third time, the fifth time, and the seventh time so that the final concentration was 1 mM, for a total of three times.

The results are shown in FIG. The effect of glutathione was clearly seen.

Further, as a result of carrying out the same experiment by changing the concentration of glutathione, the effect was observed from 0.5 mM to 5 mM,
The effect of 2 mM was most prominent.

Example 2 (Effect of GSH on DCE decomposition of JM1 strain (liquid system)) cis-1,2-dichloroethylene (cis-1,2-DCE), tra
The effects on the degradation of the JM1 strain were evaluated when ns-1,2-dichloroethylene and 1,1-dichloroethylene (1,1-DCE) were each 5.0 ppm and GSH was added at 2 mM. As an experimental system, basically the same as in Example 2, the first time (after 1 hour), the second
(1.5 hours later, total 2.5 hours later), 3rd time (2 hours later, total 4.5)
After 4 hours (after 2.5 hours, after a total of 7 hours), 5th time (after 3 hours, after a total of 10 hours), 6th time (after 12 hours, after a total of 22 hours)
Each concentration was measured. Formaldehyde as an energy source was added to the final concentration of 1 mM at the first, third and fifth times.

The results are shown in Table 1 (cis-1,2-DCE) and Table 2 (trans-1,2-DCE).
DCE) and Table 3 (1,1-DCE).

[0058]

[Table 1]

[0059]

[Table 2]

[0060]

[Table 3] From this result, GSH in the decomposition treatment of DCE of JM1 strain liquid system
The effect of was shown.

Example 3 (Example 4. Effect of GSH on TCE degradation of KK01 strain (liquid system)) Colonies of KK01 strain on agar M9 medium (containing 0.1% yeast extract and 2 mM phenol) were treated with a liquid of the same composition. M9 medium
200 ml was inoculated and shake culture was performed at 30 ° C. in a 500 ml shake flask.

At 24 hours, which is the latter stage of logarithmic growth, the bacterial cells were collected by centrifugation and resuspended in M9 medium containing no equal amount of carbon source.

10 ml each of the bacterial suspension thus prepared
Two bottles were prepared by injecting into a 27.5 ml vial. GSH was added to one of these so that the final concentration was 1 mM, and the same amount of sterile distilled water was added to the other. To these vials and a total of three vials containing only 10 ml of M9 medium as a blank, add TCE saturated aqueous solution so that the concentration in the vial is about 12 ppm, and use a butyl rubber stopper with a Teflon liner and an aluminum seal. It was sealed.

These vials were shaken at 30 ° C., and the TCE of the gas phase portion was measured by gas chromatography after a certain period of time. After that, open the cap once and keep the same level as the initial one.
After adding a saturated aqueous solution of TCE, the bottle was sealed again (the blank remains in the initial state). By repeating such operations, the effect of GSH on the repeated decomposition of TCE was evaluated. The measurement time is as follows. 1st time: 1 hour later, 2nd time: 1 hour later (total 2 hours later), 3rd time: 1.5 hours later (total 3.5 hours later), 4th time: 1.5
After 5 hours (total 5 hours), 5th time: 1.5 hours (total 6.5 hours), 6th time: 2 hours (total 8.5 hours), 7th time: 2 hours
(Total 10.5 hours later), 8th time: 2 hours later (total 12.5 hours later),
9th time: 10 hours later (total 22.5 hours later). The results are shown in Table 4.

[0065]

[Table 4] From this result, GSH in the decomposition treatment of TCE of KK01 strain liquid system
The effect of was shown. Moreover, as a result of performing the same experiment by changing the concentration of glutathione, the effect was observed from 0.5 mM to 1 mM, but the inhibition was observed conversely beyond that.

Example 5 (Effect of GSH on TCE decomposition of JM1 strain (soil system)) 50 g of Sahara through sand (water content ratio 13%, unsterilized) in 15 68 ml vials.
Then, a saturated aqueous solution of TCE was added so that the concentration in the soil would be 5 ppm, and the container was sealed and left standing at 15 ° C for 3 days to prepare three pseudo-TCE-contaminated soil samples. Then, 5 ml of the bacterial suspension of JM1 strain prepared with the same method as in Example 2 (with or without GSH2mM added) was injected into each vial bottle in an amount of 5 ml, and the other was blanked with M9. 5 ml of the medium alone was added to evaluate the effect of GSH on the purification of TCE-contaminated soil. The static suspension and the decomposition experiment were carried out at 20 ° C, which is close to the actual soil temperature.

The TCE decomposition experiment was conducted in such a manner that, after obtaining a bacterial suspension, the concentration when TCE was completely dissolved in the liquid part of the soil was 10 ppm.
A saturated aqueous solution of TCE was added to the mixture so that it was completely sealed with a Teflon liner butyl rubber stopper and an aluminum cap. 20 ° C
After standing still, the TCE of the gas phase part was measured by gas chromatography after a certain period of time. Then open the cap and TCE
The same operation was repeated by adding gas and resealing.
Each measurement time is 1st: 2 hours later, 2nd: 3 hours later (total 5 hours)
Hours), 3rd time: 4 hours later (total 9 hours later), 4th time: 5 hours later (total 14 hours later), 5th time: 10 hours later (total 24 hours later). In addition, formaldehyde as an energy source
The final concentration of 1 mM was added to the 1st, 3rd, and 5th times. The results are shown in Table 5.

[0068]

[Table 5] From these results, the effect of GSH was shown in the decomposition treatment of soil-based TCE using JM1 strain.

Example 6 (Example 7. Effect of GSH on TCE degradation of JM1 strain (gas phase)) Porous cellulose carrier (average pore size of about 500 mm,
Sterilized by autoclave (about 5 mm in diameter) and cooled to room temperature or lower were filled in 3 vials similar to those in Example 2.

15 ml of the JM1 strain bacterial suspension (GSH2 mM added system, non-added system) prepared as in Example 2 was injected into a vial bottle, and every 4 hours, TCE-contaminated air (prepared by a permeator, A gas concentration of 100 ppm) was added to the vial with a 1 ml gas tight syringe to evaluate the effect of the present invention in cleaning contaminated air. The decomposition experiment was performed at 25 ° C. In addition, formaldehyde as an energy source, the first, third,
At the 5th addition, each final concentration was 1 mM. The results are shown in Table 6.
Shown in. The concentration is a gas concentration.

[0071]

[Table 6] From this result, in the decomposition treatment of gas phase TCE using JM1 strain,
The effect of GSH was shown.

[0072]

According to one embodiment of the present invention, an efficient biodegradation and purification treatment of liquid, soil, and air contaminated with an organic compound such as a chlorinated ethylene compound becomes possible.

[Brief description of drawings]

FIG. 1 is a diagram showing test results for the effect of GSH on TCE.

FIG. 2 is a diagram showing test results for the effect of glutathione on the microbial degradation of TCE.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/00 B09B 3/00 ZABE 4D040 (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon In-house F-term (reference) 4B065 AA41X BD25 BD39 CA56 4D002 AA21 BA17 CA06 DA59 4D003 AA01 EA21 EA22 EA23 EA24 EA25 FA06 4D004 AA41 AB06 AC07 CA15 CA18 CA19 CC07 CC08 CC15 4D027 CA01 4D040 DD03 DD11

Claims (21)

[Claims] 1. A method for efficiently repairing an environment or medium contaminated with an organic compound using a microorganism capable of degrading the organic compound, wherein the microorganism is brought into contact with the organic compound for decomposition. In addition, a method for improving the efficiency of contaminated environment or medium restoration, characterized in that the degradation is made efficient by coexisting reduced glutathione in the degradation system. 2. The method of claim 1, wherein the organic compound is a chlorinated aliphatic compound. 3. The efficiency improving method according to claim 2, wherein the chlorinated aliphatic compound is trichloroethylene or dichloroethylene. 4. The efficiency improving method according to claim 1, wherein the environment or medium polluted by the organic compound is an aqueous liquid. 5. The efficiency improving method according to claim 4, wherein the aqueous liquid is groundwater, river water, or lake water. 6. The efficiency improving method according to claim 4, wherein the aqueous liquid is waste water. 7. The method for improving efficiency according to claim 1, wherein the microorganism is supported on a carrier. 8. The aqueous liquid is introduced into a container accommodating a carrier supporting the microorganisms to bring the microorganisms into contact with the aqueous liquid to decompose organic compounds in the aqueous liquid,
The efficiency improving method according to claim 7, wherein the aqueous liquid that has undergone the decomposition treatment is discharged to the outside of the container. 9. The efficiency improving method according to claim 1, wherein the environment is soil. 10. The efficiency improving method according to claim 9, further comprising the step of introducing a liquid containing the microorganism into the soil. 11. The method according to claim 10, wherein the introduction of the microorganisms into the soil is performed from an injection well provided in the soil. 12. The efficiency improving method according to claim 9, wherein the soil is introduced into a solution containing the microorganism. 13. The method according to claim 9, wherein the solution containing the microorganism is mixed with the soil. 14. The method according to claim 9, wherein the carrier supporting the microorganism is brought into contact with the soil. 15. The environment according to claim 1, wherein the environment is air.
The efficiency method described in. 16. The method according to claim 15, wherein the air is introduced into a solution containing the microorganism. 17. The efficiency improving method according to claim 15, wherein the air is brought into contact with a carrier supporting the microorganisms. 18. The air is introduced into a container accommodating a carrier supporting the microorganisms to bring the microorganisms into contact with the air to decompose organic compounds in the air, and the air after the decomposition treatment. 18. The efficiency improving method according to claim 17, wherein the gas is discharged to the outside of the container. 19. The microorganism is a JM1 strain (FERM BP-5352).
The efficiency improving method according to any one of claims 1 to 18, wherein 20. The efficiency-improving method according to claim 1, wherein the microorganism is Pseudomonas cepacia KK01 strain (FERM BP-4235). 21. A method for repairing a contaminated environment or a medium, which uses the efficiency improving method according to any one of claims 1 to 21.