JPH1190484A - Purification method of polluted medium by biological decomposition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To decompose/purify aromatic compounds and/or organochlorine compounds in a polluted medium efficiently and stably by a method in which the medium polluted with aromatic compounds and/or organochlorine compounds is contacted with a JMI and added with ethanol as a carbon source. SOLUTION: In a biological decomposition purification method of a polluted medium, a medium polluted with aromatic compounds and/or organochlorine compounds is contacted with a JMI (FERMBP-5352) and added with ethanol as a carbon source to decompose these pollutants contained in the medium. The decomposition treatment can be done by a process in which the pollutants in an aqueous medium, in soil, or in a gas phase are brought into contact with a JMI stock. The method of the contact is not restricted if the decomposition activity of the JMI stock is not hindered. Various methods including a batch method, a semi-continuous method, a continuous method can be implemented.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the biodegradation of volatile organic chlorine compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) released into the environment, and aromatic compounds such as phenol, toluene and cresol. Regarding the processing method. An environmental restoration method particularly useful for purifying aqueous media such as wastewater and wastewater containing volatile organochlorine compounds and aromatic compounds, repairing soil contaminated thereby, and purifying air contaminated by volatile organochlorine compounds. About.

[0002]

2. Description of the Related Art In recent years, environmental pollution by volatile organic chlorine compounds which are harmful to living organisms and are hardly decomposable has become a serious problem. In particular, the contamination of volatile organic chlorine compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene (DCE) has spread to a considerable extent in the soil of the paper and pulp industry and precision machinery-related industries in Japan and overseas. It has been reported that many cases were actually detected by environmental surveys. It is said that those volatile organic chlorine compounds remaining in soil are dissolved in groundwater by rainwater or the like and spread throughout the surrounding area. Such compounds are suspected of carcinogenicity and are very stable in the environment. In particular, the contamination of groundwater used as a drinking water source is regarded as a major social problem.

[0003] Therefore, purification of an aqueous medium such as contaminated groundwater, soil, and the accompanying gas phase by removing and decomposing volatile organic chlorine compounds is an important issue from the viewpoint of environmental conservation. The technology required for purification is being developed.

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

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

For example, TCE degrading bacteria include Welc
ia alkenophilasero 5 (USP
4877736, ATCC 53570), Welc
hia alkenophila sero 33 (U
SP 4877736, ATCC 53571), Me
thylcysis sp. strain M (A
gric. Biol. Chem. , 53, 2903 (1
989), Biosci. Biotech. Bioch
em. , 56,486 (1992), 56,736
(1992)), Methylosinus tric
hose OB3b (Am. Chem. So
c. Natl. Meet. Dev. Environ. M
microbiol. , 29, 365 (1989), Ap
pl, Environ. Microbiol. , 55,
3155 (1989), Appl. Biochem. B
iotechnol. , 28,877 (1991), JP-A-02-92274, JP-A-03-29297.
No. 0), Methylomonas sp. MM2
(Appl. Environ. Microbiol.,
57, 236 (1991)), Alcaligenes.
denitrificans ssp. xylos
oxidans JE75 (Arch.microbi)
ol. , 154, 410 (1990)), Alcali.
genes eutrophus JMP134 (Ap
pl. Environ. Microbiol. , 56,
1179 (1990)), Alcaligenes
trophos FERM-13761 (Japanese Unexamined Patent Publication No.
-123976), Pseudomonas a
eruginosa JI104 (Japanese Unexamined Patent Publication No. 07-236)
895), Mycobacterium vac
cae JOB5 (J. Gen. Microbio
l. , 82, 163 (1974)), Appl. Env
iron. Microbiol. , 54, 2960 (1
989), ATCC 29678), Pseudomon
as putida BH (Sewerage Association Journal, 24, 27
(1987)), Pseudomonas sp. st
line G4 (Appl. Environ. Micr
obiol. , 52, 383 (1986), 53, 9
49 (1987), 54, 951 (1989), 5
6,279 (1990) and 57,193 (199)
1), USP 4,925,802, ATCC 53617,
This bacterium was initially Pseudomonas cepacia
Pseudomonas sp.
Was changed to ), Pseudomonas mend
ocina KR-1 (Bio / Technol.,
7, 282 (1989)), Pseudomonas
putida F1 (Appl. Environ. Mi)
crobiol. , 54, 1703 (1988)), 54, 2578 (1988)), Pseudomona
s fluorescens PFL12 (Appl.
Environ. Microbiol. , 54, 257
8 (1988)), Pseudomonas puti
da KWI-9 (JP-A-06-70753),
(Pseudomonas cepasia KK01
(Japanese Patent Application Laid-Open No. Hei 06-22769), Nitrosom
onaseuropaea (Appl. Enviro)
n. Microbiol. , 56, 1169 (199
0)), Lactobacillus vital
issp. nov (Int. J. Syst. Bacte)
riol. , 39, 368 (1989), ATCC 4
9540), Nocardia corallina
B-276 (Japanese Patent Application Laid-Open No. 08-70881, FERM
BP-5124, ATCC 31338).

Here, when these decomposing bacteria are used for actual environmental purification treatment, a problem arises. In order to decompose TCE, aromatic compounds and methane are used as decomposition inducing substances. And so on.

[0008] For example, aromatic compounds such as phenol and toluene are very excellent decomposition inducers, but their toxicity is high, so there is a problem that they are released into the environment. Methane is also an effective decomposition inducer. However, since it is a flammable gas, introducing and controlling it in the environment involves great risks and difficulties. That is, the purification treatment method using these degrading bacteria is not suitable as an actual purification treatment method in that a chemical substance harmful or dangerous to the induction of degradation must be used.

In order to solve these problems, Nelson et al. Have developed a method using tryptophan as a substance for inducing the decomposition of an organic chlorine compound (Japanese Patent Laid-Open No. Hei 4-502277). However, the fundamental mechanism of degradation, in which degradation is induced by a degradation-inducing substance, is similar to that of other degrading bacteria. That is, since TCE-degrading enzyme is an inducing enzyme, the enzyme activity once induced is usually a few. It is maintained for only about an hour, after which it is necessary to re-add the degradation inducer. Furthermore, there is no solution for the essential problem that the efficiency of TCE degradation is significantly reduced because TCE degradation is competitively inhibited by the presence of a degradation inducer. That is, introduction of the decomposition inducing substance into the environment has become practical, but it is still difficult to provide a simple and efficient purification method at low cost.

Therefore, at present, a plasmid incorporating a DNA fragment containing a gene region encoding such an oxygenase or hydroxylase as a TCE-degrading enzyme is introduced into a host bacterium, and a harmless degradation-inducing substance is used. Even if it does not exist, TC
Attempts have been made to develop E-degrading activity.
Pseudomonas mendocina KR-1 (JP-A-2-503866) and Pseudomonas putida KWI-9 (JP-A-6-1056) include DNA fragment-derived strains.
No. 91) and Pseudomonas putida BH (3rd Lecture Meeting on Research on Groundwater and Soil Pollution and Its Prevention Measures, 213 (1994)).

However, these purification methods using recombinant strains require IPTG (isopropylthiogalactopyranoside), which is a very expensive substance, as an inducer, or have a sufficient stability of the plasmid to the host strain. However, there are various problems such as unstable activity and expression during subculture. In addition, the release of recombinant strains into the environment is unavoidable, even on the basis of public acceptance.

[0012] In order to solve these problems, Shields et al. Have proposed Pseudomonas cepacia which has a TCE resolution without requiring a decomposition inducer (in this case, phenol or toluene) (changed to Pseudomonas species in the deposit with the ATCC). A mutant strain of G4 strain was obtained by introducing a mutation using a transposon (Appl. En.
viron. Microbiol. , 58, 3977
(1992), World Patent WO 92/19738).

[0013] However, the purification method using the mutant G4 strain is not only insufficient in terms of TCE degradation activity, but also has a problem in the stability of the mutation because the mutation is introduced using a transposon. Contains. In addition, since the transposon itself contains an aminoglycoside antibiotic resistance gene, when released into the environment, the epidemiological adverse effect of horizontal transmission to other bacteria becomes a problem, and it is still unsuitable as an actual purification method .

Here, the microorganism JM1 (special purpose) is used as a microorganism which can be used for decomposing aromatic compounds and / or organochlorine compounds without using a decomposition-inducing substance, and which satisfies practical conditions and has sufficient resolution. Kaihei 8-294
387).

[0015]

SUMMARY OF THE INVENTION However, JM1
When a strain is actually used for environmental purification treatment, not only does the degrading bacterium have a sufficient resolution but also satisfies various practical conditions, but also a variety of environmental conditions at the actual treatment site. Therefore, it is extremely important to provide a purification method capable of stably and efficiently exhibiting the decomposing ability of decomposing bacteria.

For example, assuming the treatment of waste liquid or soil containing TCE, the temperature, p, etc. of waste liquid or soil as well as the basic TCE resolution of degrading bacteria
There is a demand for a purification method capable of stably maintaining a high decomposition activity even under a variety of environmental conditions in which H, oxygen concentration, contained components, and the like are greatly different.

As described above, it is more preferable that the biological purification method be one that can be applied in a wide range of applications or one that can be used in a wide variety of forms. The ability of the JM1 strain to degrade aromatic compounds and / or organochlorine compounds can be varied. There is a strong demand for a purification method that is more practically advantageous than conventionally known purification methods that can be stably and efficiently exerted even under various environmental conditions.

Accordingly, an object of the present invention is to efficiently and stably decompose aromatic compounds and / or organic chlorine compounds by expressing the decomposing activity of the JM1 strain under various conditions stably and with high activity. A method for purification treatment, in particular, efficiently and stably removing aromatic compounds and / or organochlorine compounds contained in various environments such as aqueous media such as wastewater, wastewater and groundwater, in soil, and in the gas phase. An object of the present invention is to provide a method of performing a decomposition purification treatment.

[0019]

The above object is achieved by the present invention described below.

That is, the present inventors have found that ethanol is effective against the JM1 strain as a carbon source capable of stably increasing the decomposition activity under various conditions, and it has been found that aromatic compounds and / or aromatic compounds are effective. By contacting the JM1 strain (FERM BP-5352) with a medium (aqueous medium, soil, gas phase, etc.) contaminated with an organochlorine compound and adding ethanol as a carbon source, aromatic compounds contained in the contaminated medium And / or a method for efficiently and stably decomposing and purifying organochlorine compounds has been invented.

In this method, ethanol can also be supplied as a gaseous vapor, which has the advantage that a wide range of carbon sources can be supplied easily and effectively in an actual purification treatment.

[0022]

Embodiments of the present invention will be described below in detail.

First, the medium used for culturing microorganisms basically contains all of the carbon source, nitrogen source, inorganic salts, and special required substances (vitamin, amino acid, nucleic acid base, etc.) necessary for growth. It is preferable to include.

Generally, from the viewpoint of growing microorganisms such as bacteria in a short time, yeast extract, meat extract, peptone, casamino acid, CSL (Corn stee) are used.
epliquor), SVP (Soluble veg)
A natural medium such as an E. coli protein, malt extract, and waste beet sugar, and a complete nutrient medium such as a 2xYT medium or an LB medium combining them are effective, and are often used as a growth medium for pure culture. Due to its properties, such a complete nutrient medium contains a wide variety of carbon sources such as amino acids, saccharides, and organic acids.

On the other hand, when the growth substrate is single or limited, the number of microorganisms that can efficiently assimilate the growth substrate is limited as compared with the case of a completely vegetative natural medium. Such a medium is called a selection medium, and is used, for example, when accumulating and screening microorganisms for a certain purpose from the environment where various microorganisms are present.

The present inventors have used these complete nutrient media or selective media under various conditions to grow the JM1 strain and evaluated its degradation activity. The present inventors have found that there is a medium component suitable for stably expressing the decomposition activity and maintaining the high decomposition activity.

That is, it has been found that it is effective to use ethanol as a carbon source as a medium component that enables the JM1 strain to stably express and maintain high-degrading activity. As a result, efficient and stable decomposition and purification of aromatic compounds and / or organochlorine compounds in the environment by the JM1 strain was made possible.

Furthermore, ethanol can be supplied as gaseous vapor, which has the advantage that the carbon source can be easily and effectively supplied to a wide range in the actual purification treatment.

The basic inorganic salt medium used for culturing the strain JM1 used in the present invention is not particularly limited as long as it contains components necessary for the growth of the microorganism. It is possible to culture with M9 medium supplemented with ethanol.

An example of the composition of the M9 medium is shown below (1 L of medium).
Medium; pH 7.0).

Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g

The culture can be performed under aerobic conditions,
Liquid culture or solid culture may be used. The culture temperature is desirably about 15 ° C to 30 ° C. For the pre-culture, other carbon sources can be used, and for example, malic acid or a salt thereof may be used.

In the present invention, the decomposition treatment of the aromatic compound and / or the organochlorine compound is carried out by mixing the aromatic compound and / or the organochlorine compound in the aqueous medium, in the soil or in the gas phase with the JM.
It can be carried out by contacting one strain.
The contact between the JM1 strain and the aromatic compound and / or the organochlorine compound can be carried out by any method as long as the JM1 strain can exhibit a decomposition activity, and various methods such as a batch method, a semi-continuous method, and a continuous method can be used. The method can be performed using The microorganism can be used in a semi-fixed state or by immobilization on a suitable carrier. The object to be treated such as waste liquid, soil, gas phase, etc. may be subjected to various treatments as necessary.

In the present invention, the decomposition treatment of the aromatic compound and / or the organochlorine compound in the aqueous medium is performed by bringing the aromatic compound and / or the organochlorine compound present in the aqueous medium into contact with the decomposing microorganism. Can be. The main modes of use are described below, but the present invention is not limited to these modes and can be used for purification treatment of any aqueous medium for contamination with aromatic compounds and / or organochlorine compounds.

For example, the simplest method is to directly introduce a decomposing microorganism into an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound. In this case, it is necessary to adjust the pH, salt concentration, temperature, contaminant concentration and the like of the aqueous medium, but the decomposing microorganisms maintain their decomposing activity unless they are extremely acidic or alkaline and have a high salt concentration.

As another application, a culture tank is provided to culture degrading microorganisms, and an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the culture tank at a predetermined flow rate to decompose the microorganism. The form which makes it do. The introduction and drainage of the aqueous medium may be carried out continuously, but it is also possible to carry out the treatment intermittently or batchwise according to the treatment capacity. Such control may be optimized by controlling the system according to the concentration of the aromatic compound and / or the organic chlorine compound.

Further, the decomposed microorganisms are made to adhere to a carrier, for example, soil particles and the like, filled in a reaction layer, and an aromatic compound and / or an organic chlorine compound-contaminated aqueous medium is introduced into the reaction tank to perform a decomposition treatment. There is a form. In this case, the carrier to be used is not limited to soil particles, but any carrier can be used. However, a carrier that has excellent ability to retain microorganisms and does not impair air permeability is more desirable. For example, various materials commonly used in bioreactors conventionally used in the pharmaceutical industry, the food industry, wastewater treatment systems, and the like can be used as a material that provides a habitat for microorganisms. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid And gel-like carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

In the present invention, the decomposition treatment of the aromatic compound and / or the organochlorine compound in the soil can be performed by contacting the aromatic compound and / or the organochlorine compound present in the soil with the decomposing microorganism. . The main use forms are described below, but the present strain is not limited to these forms, and can be used for purification treatment of contamination with aromatic compounds and / or organochlorine compounds in any soil.

For example, the simplest method is to directly introduce a decomposing microorganism into soil contaminated with an aromatic compound and / or an organic chlorine compound.
As a method of introduction, not only a method of spraying on the soil surface but also a method of introducing from a well inserted into the ground in the case of treatment in a relatively deep stratum. Further, when pressure is applied by air, water, or the like, the decomposing microorganisms are spread over a wide range, which is more effective. In this case, it is necessary to adjust the conditions in the soil to be suitable for the decomposing microorganism,
Degradation microorganisms are more rapidly proliferated in the presence of a carrier such as soil particles, and in that sense, the condition of being in soil is advantageous.

Further, the decomposed microorganisms are adhered to a carrier, and this is filled in a reaction vessel, and the reaction vessel is filled with an aromatic compound and / or
Alternatively, a form in which soil contaminated with an organic chlorine compound is mainly introduced into an aquifer to perform decomposition treatment may be mentioned. The form of the reaction tank is desirably one that can cover a wide range in soil, such as a fence or a film. In this case, any carrier can be used, but it is more preferable that the carrier has an excellent ability to retain microorganisms and does not impair air permeability. For example, various materials commonly used in bioreactors conventionally used in the pharmaceutical industry, the food industry, wastewater treatment systems, and the like can be used as a material that provides a habitat for microorganisms. More specifically, porous glass, ceramics, metal oxides, activated carbon,
Inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan,
Gel-like carriers such as agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester and the like can be mentioned. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

The decomposition treatment of the aromatic compound and / or the organochlorine compound in the gas phase in the present invention is performed by bringing the aromatic compound and / or the organochlorine compound present in the gas phase into contact with the JM1 strain. Can be. The main use forms are described below, but the present strain is not limited to these forms and can be used for purification treatment of gas phase contamination by any aromatic compound and / or organochlorine compound in the gas phase. .

For example, there is a form in which a decomposing microorganism is cultured in a culture tank, and a gas contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the culture tank at a predetermined flow rate to decompose the microorganism. There is no limitation on the method of introducing the gas, but it is more preferable that the introduction of the gas stirs the culture solution to promote aeration. The introduction and exhaust of the gas may be performed continuously, but it is also possible to perform the processing intermittently or batchwise according to the processing capacity. Such control may be optimized by performing system control in accordance with the concentration of the organic chlorine compound.

As another application form, decomposed microorganisms are attached to a carrier, for example, soil particles and the like, and this is filled in a reaction layer, and contaminated gas due to an aromatic compound and / or an organic chlorine compound is filled in the reaction tank. Introducing a decomposition process. In this case, the carrier to be used is not limited to soil particles, but any carrier can be used. However, a carrier that has excellent ability to retain microorganisms and does not impair air permeability is more desirable. For example, as a material that provides a space for microorganisms to live, various microorganism carriers commonly used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid And gel-like carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

For purifying the pollutant gas, the microorganism may be introduced after preliminarily being filled with a substance to be a carrier, or pre-culture may be performed. In order to perform the decomposition reaction more efficiently, the above-described carbon source, water content ratio, oxygen concentration, and the like are preferably maintained at desired conditions. Further, the ratio of the amount of water to the amount of the carrier in the reaction tank may be appropriately selected from the growth of the microorganism and the air permeability. The form of the reaction tank may be appropriately selected depending on the amount of the gas to be treated, the concentration, and the like.However, it is good to consider that contact between the gas and the microorganisms held in the carrier is promoted, for example, a column, a tube, a tank, A box-shaped thing can be used. Further, a unit having such a shape may be unitized with an exhaust duct, a filter, or the like, or some units may be connected in accordance with the capacity.

The contaminated gas may be adsorbed on the carrier material at first, and there are rare cases where the effect of utilizing microorganisms is not well observed. However, after a certain period of time, the contaminant adhering to the carrier material is decomposed, In addition, it is said that the resorption of the contaminant on the surface of the material in which the contaminant is decomposed regenerates the adsorptivity to the carrier material. In this way, a constant decomposition can always be expected without saturating the decontamination ability.

The method of the present invention can be applied to waste water treatment, soil treatment, and air treatment in both closed and open systems. The microorganisms may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0047]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Here, the microorganism JM1 strain used in the present invention is a specific microorganism (an international deposit number based on the Budapest Treaty: FERM BP) which can degrade aromatic compounds and chlorinated ethylene compounds by using a decomposition inducer.
-5102) is obtained by mutating by a mutation operation using a mutagen, and is a mutant strain capable of decomposing an organic compound without using a decomposition inducing substance. This mutant strain is designated as JM1 strain (FERM BP-5352 / Identification for identification: Corynebacterium sp. Strain JM1 under the Budapest Treaty on the International Recognition of Deposit of Microorganisms under Patent Proceedings). sp.JM1))
Has been deposited. Although this strain was initially indicated as "Corynebacterium sp. JM1 strain" as belonging to the genus Corynebacterium, it was confirmed by later examination that the strain does not belong to the genus Corynebacterium. Therefore, the identification display was changed to "JM1 strain".

The mycological properties of the JM1 strain are as follows. Gram stainability and morphology: Gram-negative bacilli Growth in each medium BHIA: Good growth MacConkey: Viable Colony color: Cream Optimal temperature: 25 ° C> 30 ° C> 35 ° C Motility: negative (semi-fluid medium) TSI ( slant / butt): alkali / alkali, H ₂ S (-) oxidase: positive (weak) catalase: positive sugar fermentation glucose: negative sucrose: negative raffinose: negative galactose: negative maltose: negative urease: negative esculin hydrolysis ( β-glucosidase): positive Nitrate reduction: negative Indole productivity: negative Glucose acidification: negative Arginine dihydrolase: negative Gelatin hydrolysis (protease): negative β-galactosidase: negative Assimilation of each compound Glucose: negative L-arabinose: negative D-Man North: negative D-mannitol: negative N-acetyl-D-glucosamine: negative Maltose: negative potassium gluconate: negative n-capric acid: positive adipic acid: negative dl-malic acid: positive sodium citrate: positive phenyl acetate: negative

Example 1 (Decomposition of phenol by JM1 strain in sand system (30 ° C.)) A decomposition treatment was carried out in a system in which 60 g of Sawara sand was added to a 67.5 ml vial bottle. First, 6 ml of M9 medium containing 0.2% of various nutrients (this example: ethanol, comparison: sodium lactate, yeast extract) was prepared, and inoculated with 60 μl (1/100 amount) of the pre-cultured JM1 strain. did. Here, the preculture was performed using M9 containing 2.0% sodium malate.
Using the medium, shaking culture was performed at 15 ° C. for 3 days. Further, phenol was added and mixed to a final concentration of 300 ppm as a substance to be decomposed, and added to the sand in the vial bottle. After the addition, the mixture was capped with a silicon stopper, and allowed to stand still at 30 ° C., and the decrease in phenol was measured. The measurement was performed by liquid chromatography (detection: spectrophotometer),
The residual ratio with respect to the initial concentration after time was shown. Table 1 shows the results. The superiority of ethanol as a carbon source in phenol degradation in soil is apparent.

[0051]

[Table 1]

Example 2 (Decomposition of phenol by JM1 strain in a sand system (15 ° C.)) A decomposition treatment was carried out in a system in which 60 g of Sawara sand was added to a 67.5 ml vial. First, 6 ml of M9 medium containing 0.2% of various nutrients (this example: ethanol, comparison: sodium lactate, yeast extract) was prepared, and 60 μl (1/100 amount) of the pre-cultured JM1 strain was inoculated thereto. did. Here, the preculture was performed using M9 containing 2.0% sodium malate.
Using the medium, shaking culture was performed at 15 ° C. for 3 days. Further, phenol was added and mixed so as to have a final concentration of 300 ppm as a substance to be decomposed, and added to the sand in the vial bottle. After the addition, the mixture was sealed with a silicon stopper and incubated at 15 ° C. for static culture to measure the decrease in phenol. The measurement was carried out by liquid chromatography (detection: spectrophotometer),
The residual ratio with respect to the initial concentration after time was shown. Table 2 shows the results. The superiority of ethanol as a carbon source in phenol degradation in soil is apparent.

[0053]

[Table 2]

Example 3 (Decomposition of TCE by JM1 strain in sand system (30 ° C.)) A decomposition treatment was carried out in a system in which 60 g of Sawara sand was added to a 67.5 ml vial bottle. First, 6 ml of M9 medium containing 0.2% of various nutrients (this example: ethanol, comparison: sodium lactate, yeast extract) was prepared, and inoculated with 60 μl (1/100 amount) of the pre-cultured JM1 strain. did. Here, the preculture was performed using M9 containing 2.0% sodium malate.
Using the medium, shaking culture was performed at 15 ° C. for 3 days.

The culture solution inoculated with the bacteria was added to the sand in the vial, sealed with a butyl rubber stopper and an aluminum cap, and air containing TCE was added thereto to adjust the TCE concentration in the final solution to 1%.
The culture was allowed to stand still at 30 ° C. at 0 ppm, and the TCE resolution was evaluated.

The amount of TCE was determined 48 hours later by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 3 shows the results. The superiority of ethanol as a carbon source in TCE degradation in soil is apparent.

[0057]

[Table 3]

Example 4 (Decomposition of TCE by JM1 strain in a sand system (15 ° C.)) A decomposition treatment was carried out in a system in which 60 g of Sawara sand was added to a 67.5 ml vial bottle. First, 6 ml of M9 medium containing 0.2% of various nutrients (this example: ethanol, comparison: sodium lactate, yeast extract) was prepared, and inoculated with 60 μl (1/100 amount) of the pre-cultured JM1 strain. did. Here, the preculture was performed using M9 containing 2.0% sodium malate.
Using the medium, shaking culture was performed at 15 ° C. for 3 days.

The culture solution inoculated with the bacteria was added to the sand in the vial, sealed with a butyl rubber stopper and an aluminum cap, and air containing TCE was added thereto to reduce the TCE concentration in the final solution to 1 l.
It was set to 0 ppm, static culture was performed at 15 ° C., and the TCE resolution was evaluated.

The amount of TCE was determined after 72 hours by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 4 shows the results. The superiority of ethanol as a carbon source in TCE degradation in soil is apparent.

[0061]

[Table 4]

Example 5 (Decomposition of DCE by JM1 strain in sand system (30 ° C.)) The substances to be decomposed in the medium were cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1, 2
DCE in the same manner as in Example 3 except that each of dichloroethylene (trans-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) was 10 ppm.
Was measured. Table 5 shows the results after 48 hours. Cis-1,2-DCE in soil, trans-
In all of the decomposition of 1,2-DCE and 1,1-DCE, the superiority of ethanol as a carbon source is apparent.

[0063]

[Table 5]

Example 6 (Decomposition of DCE by JM1 strain in sand system (15 ° C.)) The substance to be decomposed in the medium was cis-1,2-DCE, tr
ans-1,2-DCE and 1,1-DCE each 1
The decrease in DCE was measured in the same manner as in Example 4 except that 0 ppm was used. Table 6 shows the results after 72 hours. Cis-1,2-DCE in soil, trans-
The superiority of ethanol as a carbon source is apparent for all 1,2-DCE and 1,1-DCE decomposition.

[0065]

[Table 6]

Example 7 (Soil extract mixed liquid culture system (30
Decomposition of TCE by JM1 strain at 0 ° C.) The decomposition treatment was performed in a system in which 20 ml of a liquid medium mixed with a soil extract was added to a 67.5 ml vial.

The soil extract was applied to 30 g of Sawara Sanda for 30 m.
1 M9 medium was added and vortexed for 30 seconds to suspend the soil, and the liquid phase portion was separated and prepared. Various nutrients (this example:
Ethanol, comparison: sodium lactate, yeast extract) was added, and 200 μl (1/10) of the pre-cultured JM1 strain was added thereto.
0 volume). Here, the preculture was performed by shaking culture at 15 ° C. for 3 days using an M9 medium containing 2.0% sodium malate.

The culture solution inoculated with the bacteria was added to a vial, and then sealed with a butyl rubber stopper and an aluminum cap.
TCE concentration in the final solution is 10 pp by adding air containing CE.
m, shaking culture was performed at 30 ° C., and the TCE resolution was evaluated.

The amount of TCE was quantified after 48 hours by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 7 shows the results. The superiority of ethanol as a carbon source for TCE degradation in soil extract mixtures is apparent.

[0070]

[Table 7]

Example 8 (Soil extract mixed liquid culture system (15
Decomposition of TCE by JM1 strain at 0 ° C.) The decomposition treatment was performed in a system in which 20 ml of a liquid medium mixed with a soil extract was added to a 67.5 ml vial.

The soil extract was applied to 30 g of Sawara sand by 30 m.
1 M9 medium was added and vortexed for 30 seconds to suspend the soil, and the liquid phase portion was separated and prepared. Various nutrients (this example:
Ethanol, comparison: sodium lactate, yeast extract) was added, and 200 μl (1/10) of the pre-cultured JM1 strain was added thereto.
0 volume). Here, the preculture was performed by shaking culture at 15 ° C. for 3 days using an M9 medium containing 2.0% sodium malate.

The culture solution inoculated with the bacteria was added to a vial, and then sealed with a butyl rubber stopper and an aluminum cap.
TCE concentration in the final solution is 10 pp by adding air containing CE.
m, shaking culture was performed at 15 ° C., and the TCE resolution was evaluated.

The amount of TCE was determined by gas chromatography (detection: FID detector) after 72 hours by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 8 shows the results. The superiority of ethanol as a carbon source for TCE degradation in soil extract mixtures is apparent.

[0075]

[Table 8]

Example 9 (JM1 in a polluted water system (30 ° C.)
Decomposition of TCE by strain) Decomposition treatment was performed in a system in which 20 ml of contaminated water was added to a 67.5 ml vial. 2 ml of 10xM9 in 20 ml of water collected from a very polluted reservoir in Atsugi, Kanagawa
A culture medium was added to obtain the polluted water used in this example. Various nutrients (this example:
Ethanol, comparison: sodium lactate, yeast extract) was added, and 200 μl (1/10) of the pre-cultured JM1 strain was added thereto.
0 volume). Here, the preculture was performed by shaking culture at 15 ° C. for 3 days using an M9 medium containing 2.0% sodium malate.

After the culture solution inoculated with the bacteria was added to the vial, it was sealed with a butyl rubber stopper and an aluminum cap.
TCE concentration in the final solution is 10 pp by adding air containing CE.
m, shaking culture was performed at 30 ° C., and the TCE resolution was evaluated.

The amount of TCE was quantified 48 hours later by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 9 shows the results. The superiority of ethanol as a carbon source in TCE degradation in polluted water is apparent.

[0079]

[Table 9]

Example 10 (JM in polluted water system (15 ° C.)
Decomposition of TCE by One Strain) The decomposition treatment was performed in a system in which 20 ml of contaminated water was added to a 67.5 ml vial. 2 ml of 10xM9 in 20 ml of water collected from a very polluted reservoir in Atsugi, Kanagawa
A culture medium was added to obtain the polluted water used in this example. Various nutrients (this example:
Ethanol, comparison: sodium lactate, yeast extract) was added, and 200 μl (1/10) of the pre-cultured JM1 strain was added thereto.
0 volume). Here, the pre-culture was performed by shaking culture at 15 ° C. for 3 days using an M9 medium containing 2.0% sodium malate.

After the culture solution inoculated with the bacteria was added to the vial, it was sealed with a butyl rubber stopper and an aluminum cap.
TCE concentration in the final solution is 10 pp by adding air containing CE.
m, shaking culture was performed at 15 ° C., and the TCE resolution was evaluated.

The amount of TCE was determined after 72 hours by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 10 shows the results.
Shown in The superiority of ethanol as a carbon source in TCE degradation in polluted water is apparent.

[0083]

[Table 10]

Example 11 (Decomposition of TCE in gas phase by soil aeration using JM1 strain) First, 0.2% of various nutrients (this example: ethanol,
Comparison: M9 medium 3 containing sodium lactate, yeast extract)
0 ml was prepared, and the pre-cultured JM1 strain was
1 (1/100 amount) was inoculated. Here, preculture was performed using M9 medium containing 2.0% sodium malate,
Shaking culture was performed at 5 ° C. for 3 days.

Brown forest soil was added to the surface of the culture solution inoculated with the fungi to the surface of the water, sealed with a silicon stopper, and allowed to stand at 20 ° C. for 24 hours, followed by decanting off the excess culture solution. The air aerated in a 100 ppm TCE solution was added to this at a flow rate of 60 ppm.
After flowing through the soil at a rate of 30 ml / min for 30 minutes, the mixture was completely sealed with a butyl rubber stopper and an aluminum seal, and then subjected to stationary culture at 20 ° C. The amount of TCE was determined by gas chromatography (detection: FID detector) by the headspace method.
The amount was measured. The results after 10 hours are shown in Table 11. The superiority of ethanol as a carbon source in TCE decomposition in the gas phase by soil aeration is apparent.

[0086]

[Table 11]

Example 12 (Decomposition of TCE in Gas Phase by Continuous Aeration of Soil Using JM1 Strain) First, 0.2% of various nutrients (this example: ethanol,
Comparison: M9 medium 3 containing sodium lactate, yeast extract)
0 μl was prepared, and the pre-cultured JM1 strain was
1 (1/100 amount) was inoculated. Here, preculture was performed using M9 medium containing 2.0% sodium malate,
Shaking culture was performed at 5 ° C. for 3 days.

Brown forest soil was added to the surface of the culture solution inoculated with the fungi to the surface of the water, sealed with a silicon stopper, and allowed to stand at 20 ° C. for 24 hours, and then the excess culture solution was decanted off. After completely sealing this with a butyl rubber stopper and aluminum seal,
The air aerated in a 100 ppm TCE solution was flowed at 0.
Static culture was performed at 20 ° C. while continuously flowing through the soil at 5 ml / min. The TCE amount is determined by the T
The amount of CE was determined by gas chromatography, and the amount of TCE was measured over time. The results are shown in FIG. In the TCE decomposition in the gas phase by continuous aeration of soil,
The superiority of ethanol as a carbon source is apparent.

Example 13 (TCE in sand system (30 ° C.) using JM1 strain cultured by supplying ethanol vapor)
Decomposition) A decomposition treatment was performed in a system in which 60 g of Sawara sand was added to a 67.5 ml vial. First, 6 ml of an M9 medium containing no carbon source was prepared, and 60 μl of the precultured JM1 strain was added thereto.
(1/100 amount). Here, the preculture is 2.0%
Using M9 medium containing sodium malate,
Shaking culture was performed for one day.

After adding the M9 medium inoculated with the bacteria to the sand in the vial, it was sealed with a butyl rubber stopper and an aluminum cap.
Air containing ethanol vapor was continuously added thereto for 10 minutes. As the air containing ethanol vapor, a 50% ethanol solution aerated at a flow rate of 60 ml / min was used. Here, a control without ethanol vapor was used as a control.

Air containing TCE was added thereto to adjust the TCE concentration in the final solution to 10 ppm, and the mixture was allowed to stand still at 30 ° C. to evaluate the TCE resolution.

The amount of TCE was quantified 48 hours later by gas chromatography (detection: FID detector) by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 12 shows the results.
Shown in Degradation of TCE was shown in the JM1 strain cultured by supplying ethanol vapor.

[0093]

[Table 12]

Example 14 (TCE in sand system (15 ° C.) using JM1 strain cultured by supplying ethanol vapor)
Decomposition) A decomposition treatment was performed in a system in which 60 g of Sawara sand was added to a 67.5 ml vial. First, 6 ml of an M9 medium containing no carbon source was prepared, and 60 μl of the precultured JM1 strain was added thereto.
(1/100 amount). Here, the preculture is 2.0%
Using M9 medium containing sodium malate,
Shaking culture was performed for one day.

After adding the M9 medium inoculated with the fungus to the sand in the vial, the mixture was sealed with a butyl rubber stopper and an aluminum cap.
Air containing ethanol vapor was continuously added thereto for 10 minutes. As the air containing ethanol vapor, a 50% ethanol solution aerated at a flow rate of 60 ml / min was used. Here, a control without ethanol vapor was used as a control.

Air containing TCE was added thereto to adjust the TCE concentration in the final solution to 10 ppm, and static culture was carried out at 15 ° C. to evaluate the TCE resolution.

The amount of TCE was determined by gas chromatography (detection: FID detector) after 72 hours by the headspace method. As a control, JCE was measured in a similar system.
Quantification of the amount of TCE in the system without adding the M1 strain was also performed.
The residual ratio with respect to the TCE amount of the control was determined. Table 13 shows the results.
Shown in Degradation of TCE was shown in the JM1 strain cultured by supplying ethanol vapor.

[0098]

[Table 13]

Example 15 (DCE in sand system (30 ° C.) using JM1 strain cultured by supplying ethanol vapor)
Of cis-1,2-DCE, trans-
1,2-DCE and 1,1-DCE each at 10 ppm
The decrease in DCE was measured in the same manner as in Example 13 except for the above. Table 14 shows the results after 48 hours. In the JM1 strain cultured by supplying ethanol vapor,
Decomposition of DCE was shown.

[0100]

[Table 14]

Example 16 (DCE in sand system (15 ° C.) using JM1 strain cultured by supplying ethanol vapor)
Of cis-1,2-DCE, trans-
1,2-DCE and 1,1-DCE each at 10 ppm
The decrease in DCE was measured in the same manner as in Example 14 except for the above. Table 15 shows the results after 48 hours. In the JM1 strain cultured by supplying ethanol vapor,
Decomposition of DCE was shown.

[0102]

[Table 15]

[0103]

As is apparent from the above description, according to the present invention, a contaminated medium under a variety of environmental conditions such as an aqueous medium containing an aromatic compound and / or an organochlorine compound, soil, and a gaseous phase. Even so, a practically efficient and stable biodegradation and purification treatment can be performed.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results over time of TCE decomposition in the gas phase by continuous aeration of soil.

────────────────────────────────────────────────── ─── Continued on front page (51) Int.Cl. ⁶ Identification symbol FI C02F 1/58 B01D 53/34 120D 3/00 134E C12N 1/00 B09B 3/00 ZABE (72) Inventor Shinya Furusaki Ota, Tokyo 3-30-2 Shimomaruko-ku, Canon Inc.

Claims (16)

[Claims] A medium JM1 (FERM BP-53) is contaminated with an aromatic compound and / or an organic chlorine compound.
52) A method for biodegrading and purifying a contaminated medium, which comprises contacting and contacting with ethanol to decompose an aromatic compound and / or an organic chlorine compound contained in the contaminated medium. 2. The method according to claim 1, wherein ethanol used as a carbon source is supplied as ethanol vapor. 3. The method for biodegrading and purifying a contaminated medium according to claim 1, wherein the contaminated medium is an aqueous medium. 4. The method for biodegrading and purifying a contaminated medium according to claim 3, wherein the contacting is carried out by bringing the microorganisms carried on the carrier into contact with an aqueous medium containing an aromatic compound and / or an organic chlorine compound. 5. The contact is carried out by containing a carrier carrying microorganisms in a container, introducing an aqueous medium containing an aromatic compound and / or an organic chlorine compound from one of the containers, and discharging the aqueous medium from the other. Item 6. A method for biodegrading and purifying contaminated media according to Item 4. 6. The method for biodegrading and purifying a contaminated medium according to claim 1, wherein the contaminated medium is soil. 7. The method according to claim 6, wherein the decomposition purification is carried out by introducing an aqueous medium containing microorganisms into the contaminated soil and providing the carbon source and / or oxygen to grow the microorganisms in the soil. Biodegradation purification method for polluted media. 8. The method of introducing an aqueous medium containing microorganisms into soil by pressure from an injection well provided in the soil.
The biodegradation purification method of the contaminated medium according to the above. 9. The method according to claim 6, wherein the contacting is carried out by introducing soil containing an aromatic compound and / or an organic chlorine compound into a liquid phase containing microorganisms. 10. The contacting is performed by contacting a microorganism supported on a carrier with a microorganism.
The method for biodegrading and purifying a contaminated medium according to claim 6, wherein the method is carried out by contacting the soil with an aromatic compound and / or an organic chlorine compound. 11. The method according to claim 1, wherein the contaminating medium is air.
The biodegradation purification method of the contaminated medium according to the above. 12. The method according to claim 11, wherein the contacting is performed by introducing air containing an aromatic compound and / or an organic chlorine compound into a liquid phase containing microorganisms. 13. The contacting is carried out by contacting a microorganism supported on a carrier with a microorganism.
The method for biodegrading and purifying a contaminated medium according to claim 11, wherein the method is performed by contacting with air containing an aromatic compound and / or an organic chlorine compound. 14. The contact is carried out by accommodating a carrier carrying microorganisms in a container, introducing air containing an aromatic compound and / or an organic chlorine compound from one of the containers, and discharging the air from the other. The biodegradation purification method of the contaminated medium according to the above. 15. The method according to claim 1, wherein the aromatic compound is at least one of phenol, toluene and cresol.
4. The method for biodegrading and purifying a contaminated medium according to any one of 4 above. 16. The method for biodegrading and purifying a contaminated medium according to claim 1, wherein the organic chlorine compound is at least one of trichloroethylene and dichloroethylene.