JP2000167577A - Restoration of environment contaminated with organic compound by microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently restore environment such as underground water, waste water or air contaminated with an org. compd. such as a chloroethylene compd. or an aromatic compd. by microorganisms. SOLUTION: Microorganisms capable of decomposing an org. compd. being a contamination source are cultured in the presence of a propagating substrate and an energy source, and the cltured microorganisms are converted to a microorganism soln. of a resting state free from the propagating substrate and the energy source to be divided into two fractions and the energy source is added to the respective fractions corresponding to a period decomposing the org. compd. to be brought into contact with the org. compd.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying and repairing an environment such as liquid, soil, and air contaminated with an organic compound using microorganisms.

[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 soil in 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 the soil dissolve in rainwater and the like, mix into groundwater, and spread throughout the surrounding area. Such compounds are suspected of carcinogenicity and reproductive toxicity, and are very stable in the environment. Therefore, in particular, the pollution of groundwater used as a water source for drinking water is regarded as a major social problem.

[0003] Therefore, purification by removing and decomposing volatile organic chlorine compounds in the aqueous medium such as contaminated groundwater, soil, and the surrounding gas phase is an important issue from the viewpoint of environmental conservation. Necessary technologies are 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 studies for practical use have begun. In other words, in biodegradation treatment using microorganisms, it is possible to decompose volatile organic chlorine compounds to harmless substances by selecting microorganisms to be used, basically there is no need for special chemicals, and labor and cost for maintenance are reduced. There are advantages such as reduction. Such advantages have been a problem in the past so-called BETX (benzene, ethylbenzene,
The present invention can also be applied to the purification treatment of contamination by aromatic compounds represented by toluene, xylene), phenol and cresol.

Examples of microorganisms having the ability to decompose volatile organochlorine compounds include, for example, TCE degrading bacteria such as Welch
ia alkenophila sero 5 (USP 4877736, ATCC 53570),
Welchia alkenophila sero 33 (USP 4877736, ATCC 5357
1), Methylocystis sp. Strain M (Agric. Biol.
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,
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-A-07-123976), Pseudomonas aeruginosa JI104 (JP-A-07-23689)
No. 5), Mycobacterium vaccae JOB5 (J. Gen. Micr
obiol., 82, 163 (1974), Appl. Environ. Microbiol.,
54, 2960 (1989), ATCC 29678), Pseudomonas putida
BH (Sewer Association of Japan, 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), 54, 2578 (1988)), Pseudomonas fluorescen
s PFL12 (Appl.Environ.Microbiol., 54, 2578 (198
8)), Pseudomonas putida KWI-9 (JP-A-06-70753), Pseudomonas cepacia KK01 (JP-A-06-227769), Nitrosomonaseuropaea (Appl. Environ. Micro)
biol., 56, 1169 (1990)), Nocardia corallina B-276
(JP 08-70881 A, FERM BP-5124, ATCC 31338)
Etc. However, especially when using bacteria that decompose chlorinated ethylene compounds such as TCE and DCE in actual environmental purification treatments, it is important to use those decomposers (inducers) to decompose those compounds. This means that chemical substances such as aromatic compounds and methane are required.

[0007] For example, aromatic compounds such as phenol and toluene are very good inducers, but their toxicity is high, and their release into the environment must be carried out according to some regulations. Methane, which is an induced substance, is a flammable gas, and its control in the environment is very dangerous and complicated.

One method for solving such a problem is disclosed in JP-A-4-502277. in this way,
Tryptophan, one of aromatic amino acids, is used as a biodegradation inducer for chlorinated ethylene to avoid the toxicity and danger of the inducer. In addition,
According to 7-171548, the above problem is avoided by using lignocellulose, a natural substance extracted from herbaceous plants, as an inducer.

[0009] In terms of improvement of degrading microorganisms, a plasmid into which a DNA fragment containing a gene region coding for a degrading enzyme, oxygenase or hydroxylase, is introduced into a host bacterium, and the plasmid is introduced with a harmless inducer or an inducer. Attempts have been made to constitutively express a degrading activity even under conditions where no is present. Examples of DNA fragment-derived strains include Pseudomonas mendocina KR-1 (JP-A-2-503866) and Pseudomonas putida KW1-9.
(Japanese Unexamined Patent Publication No. 6-105691), Pseudomonas putida BH
(The 3rd Lecture Meeting on Groundwater and Soil Pollution and Its Prevention Measures, 213 (1994)).

[0010] Another solution is disclosed in WO 92/19738. According to the present report, mutants that degrade chlorinated ethylene compounds without using inducers such as phenol and toluene were obtained by mutation using transposons.

Yet another solution is disclosed in US Pat. No. 5,543,317.
And JP-A-8-294387. According to the present report, mutant strains that degrade chlorinated ethylene compounds and aromatic compounds without using inducers such as phenol and toluene were obtained by treating them with nitrosoguanidine, a mutagen.

As described above, the problem relating to the inducer in the microbial decomposition treatment of a chlorinated ethylene compound or an aromatic compound is being avoided, and the technology of environmental restoration treatment by the decomposition of microorganisms can be sufficiently practically used. It is becoming something. However, in the case of environmental restoration treatment of organic compounds by the decomposition of microorganisms, long-term purification may be required. For this purpose, it is necessary to maintain the decomposition activity of microorganisms for a long period of time. There is the fact that the microbial degradation activity of the microorganisms is deactivated in a very short time.

[0013] In the environmental restoration treatment of an organic compound by decomposing microorganisms, it takes time and cost to culture the microorganisms, and it is essential to maximize the intended ability of the microorganisms once cultured.

The cause of the phenomenon that the decomposing activity of the microorganism is deactivated in a very short period of time is considered to be the autolysis of the decomposing enzyme in the cells, the toxicity of the decomposed intermediate, the lack of energy required for decomposing, and the like. Among them, the lack of energy is a very important issue in the sense that the microbial degrading activity of organic compounds is lost.

In order to solve such a problem of energy shortage, in Japanese Patent Application Laid-Open No. 1-160479, etc., formaldehyde is used as a method for restoring the activity of methane monooxygenase of methane assimilating bacteria. This is a one-time event, and it cannot be said that it shows a sufficient effect in terms of maintaining the decomposition activity.

[0016]

SUMMARY OF THE INVENTION An object of the present invention is to decompose and purify the environment, such as groundwater, wastewater, soil and air, contaminated by organic compounds such as chlorinated ethylene compounds and aromatic compounds using microorganisms. An object of the present invention is to provide a method for effectively and stably repairing an environment contaminated with an organic compound by effectively utilizing the intended resolution of these microorganisms during treatment.

[0017]

When the organic compound to be decomposed is present in a sufficient amount, the treatment effect can be achieved by initially providing an energy source in an amount sufficient for the microorganism to exhibit sufficient decomposition activity. . However, at actual contamination sites,
Organic compounds at very low concentrations may be treated continuously or intermittently, in which case the microorganisms may be contacted with an energy source that provides sufficient degrading activity at the beginning. An activity higher than that of an enzyme capable of decomposing an organic compound is expressed, and as a result, the enzyme is wasted, and the period for maintaining the activity is shortened as a whole.
In addition, the energy source itself may be toxic to the microorganism, and it was a double-edged sword to give a single high concentration (that is, an amount in which the microorganism exerts sufficient decomposition activity). From the above viewpoints, the inventor has reached the following invention.

That is, the method of the present invention provides a method for repairing an environment contaminated by an organic compound using a microorganism having the ability to decompose the organic compound, wherein (1) culturing a microorganism capable of decomposing the organic compound, (2) dividing the microorganism solution from which the growth substrate and energy source have been removed into two or more fractions,
(3) The method is characterized in that an energy source is added to each fraction according to the timing of decomposing the organic compound, and the fraction is brought into contact with the organic compound.

However, in the case of the first fraction, the culture solution can be used as it is when it is used for the degradation purification treatment immediately after the culture.

[0020]

Here, "energy" and "energy source" in the present invention are as follows. In other words, in microorganisms that decompose organic compounds such as aromatic compounds and chlorinated aliphatic compounds such as TCE using an oxidase called oxygenase, the process of introducing molecular oxygen into the organic compounds to form epoxides and hydroxyl groups is decomposed. This is the first stage. At this time, NADH which is a coenzyme
(Nicotinamide-adenine dinucleotide reduced form) or NADPH (nicotinamide-adenine dinucleotide phosphate reduced form) is required. These reduced nicotinamide nucleotides become oxidized in the electron transfer system in the microorganism, and the electrons released at this time are finally received by molecular oxygen to form epoxides and hydroxyl groups, and NADH or NADPH respectively. It becomes oxidized NAD + or NADP +. In order to reduce these oxidized nicotinamide nucleotides,
For this purpose, a substrate is required, usually using an alcohol, an aldehyde or a carboxylic acid as a substrate, and reduced by an enzyme called dehydrogenase.
PH. In the present invention, NADH or NA in this case is used.
DPH is defined as "energy" and the substrate alcohol, aldehyde or carboxylic acid as "energy source".

The energy source depends on the dehydrogenase system of the microorganism used, but usually, methanol, formaldehyde, formic acid and the like are used.

Japanese Patent Application Laid-Open No. 9-10794 discloses a method for introducing a microorganism capable of decomposing a contaminant into a medium in a method for purifying the medium contaminated with the contaminant; A carbon source capable of selectively improving at least one of the decomposition activity of the microorganism with respect to competitor microorganisms originally present in the environment where the contaminant is present, and contacting the microorganism with the microorganism twice or more times. And a step of selectively decomposing this contaminant. This publication intends to selectively grow only a target microorganism by contacting a carbon source such as an organic acid a plurality of times during the growth of the microorganism.

On the other hand, in the present invention, the microorganisms are not normally grown on an energy source, and the microorganisms can be brought into contact with the microorganisms at the stage when the carbon source of the microorganism culture medium is depleted or after the carbon source is removed. Required. Further, when a carbon source such as an organic acid shown in JP-A-9-10794 is given as a source of energy for the purpose of the present invention, microorganisms are multiplied, and energy is multiplied. It is not enough to realize the idea of the invention.

As a specific method of the present invention, the microorganism to be used is cultured and used for purification treatment when the growth is almost completed. At this time, the carbon source (sugar, organic acid, If hydrocarbons are almost gone, use the microbial culture solution as it is.If a sufficient amount of carbon source remains, collect the microbial cells by centrifugation, filtration, etc., and resuspend in a medium without carbon source. Turbid, resting state of microorganisms
And The solution containing the microorganism thus prepared (hereinafter referred to as a microorganism solution) is divided into two or more containers, and an energy source is sequentially added every time the solution is used for the purification treatment.

For example, when the groundwater contaminated by the organic compound flows out sequentially, the microorganisms once cultured are prepared and the microorganism solution as described above is prepared, and the amount of the groundwater that can be treated by the microorganisms in the container is increased. At the time of collection, first the energy source is added to the first container for purification treatment, and then when the amount of groundwater that can be treated by microorganisms in the second container is collected, the energy source is added to the first container. Is added to perform a purification treatment, and this operation is sequentially repeated. Alternatively, the prepared cells may be immobilized on an immobilization carrier, and an energy source may be sequentially added to the immobilized carrier and introduced into groundwater for treatment. According to such a method, it is possible to carry out efficient treatment by maximally utilizing the ability of decomposing microorganisms without repeating time-consuming and costly cultivation of microorganisms according to the speed at which groundwater flows out.

When purifying soil contaminated with an organic compound, a method of excavating the soil, throwing it into a microorganism solution, and treating the soil is considered. In this case, since the excavation takes time, if it is put into a vessel containing a microbial solution to which an energy source is sequentially added according to the time, and then processed, efficient processing utilizing the decomposing ability of the microorganisms to the utmost effective can be achieved. It becomes possible. Further, when purifying soil contaminated with the organic compound, there is a method in which a well is dug in the soil and a microorganism solution is injected from the well to directly purify the soil. In this case, soil contamination is often widespread, in which case many wells are dug and microbial solutions are sequentially injected. By using the method of adding and injecting the microorganism, efficient treatment can be performed by effectively utilizing the decomposition ability of the microorganism.

When purifying soil contaminated with a highly volatile organic compound such as TCE, there is a method of digging a well in the soil and sucking air from the well to treat the air. Also, in the case of groundwater contamination, there is a method of aerating water and treating the air. In many cases, the concentration of organic compounds contained in the air is uneven,
It is efficient to carry out the treatment when the contamination is high. In this case as well, the concentration of organic compounds in the air is monitored online in advance, and when a gas with a high concentration is discharged to a certain extent, it is treated sequentially with a microbial solution to which an energy source has been added to maximize the ability to decompose microorganisms. Efficient and efficient processing is possible.

The microorganism used in the present invention may be any microorganism as long as it can decompose the target organic compound. The combination of the microorganism and an efficient energy source and the optimum concentration thereof are evaluated in advance. There is a need. For example, the J1 strain (FERM strain) used in the examples of the present invention.
Formaldehyde and methanol are particularly effective against BP-5102) and JM1 strain (FERM BP-5352). The concentration of formaldehyde used is 0.1 mM to 5 mM in total,
More preferably, the concentration is 0.5 mM to 2 mM, and the concentration of methanol used is 0.01% to 0.5% (v / v), more preferably 0.05% to 0.2%, and an effect appears when the concentration is lower than that. Difficult and high concentrations have toxic effects on the microbial cells themselves. Sodium formate is particularly effective against the KK01 strain (FERM BP-4235). The concentration of sodium formate used is preferably 0.5 mM to 20 mM in total, more preferably 1 mM to 5 mM. If the concentration is lower than this, the effect is hardly exhibited, and even if the concentration is high, the effect is not increased.

Pollutants to be purified include chlorinated ethylene compounds such as trichlorethylene (TCE) and dichloroethylene (DCE), and benzene, toluene, phenol,
And aromatic compounds such as cresol, and the method of the present invention provides a liquid contaminated with these contaminants;
It can be used for both soil and air purification.

Pseudomonas cepacia strain KK01 (an international deposit number based on the Budapest Treaty: FERM BP-4235), which is one of the microorganisms used in the present invention, is an aromatic compound such as phenol, cresol, etc. A strain that has been announced as a bacterium that degrades
Japanese Patent Application Laid-Open Publication No. H11-157, discloses that an aromatic compound such as phenol is used as a derivative to decompose an organic chlorine compound such as TCE. The strain of the genus Pseudomonas cepacia has now been taxonomically changed to Burkholderia cepacia.

One of the microorganisms used in the present invention, strain J1 (an international deposit number based on the Budapest Treaty: FERM BP-51)
02) is a strain disclosed in Japanese Patent Application Laid-Open No. 8-66182, which discloses that an aromatic compound such as phenol is used as a derivative to decompose an organic chlorine compound such as TCE.

The JM1 strain (an international deposit number based on the Budapest Treaty: FERM BP-53) which is one of the microorganisms used in the present invention
52) is a mutant obtained by mutating the J1 strain by a mutation operation using a mutagen and capable of decomposing an organochlorine compound such as TCE without using an inducer. No. 8-294387. Although this strain was initially indicated as belonging to the genus Corynebacterium, "Corynebacterium sp. JM1 strain" was displayed, but it was confirmed by later examination that the strain was "not belonging to the genus Corynebacterium". As a result, the display of the identification
M1 strain ".

The medium used for culturing these microorganisms is basically a carbon source, a nitrogen source, a phosphorus source,
What is necessary is just to contain inorganic salts etc. The basic inorganic salt medium is not particularly limited as long as it contains components necessary for growth of the strain, and for example, a basal salt medium such as M9 medium or MSB medium is used.

The composition of the 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 (in 1 liter of medium; pH 7.0) Cultivation can be performed under aerobic conditions. Culture or solid culture may be used. The culture temperature is preferably from 15 ° C to 30 ° C.

[0036]

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

Example 1 A colony of strain J1 on an agar M9 medium (containing 0.1% yeast extract and 200 ppm phenol) was cultured at 0.
M9 medium containing 1% yeast extract and 300ppm phenol
200 ml was inoculated and shake-cultured at 25 ° C. in a 500 ml shake flask. At 24 hours, which is the late stage of logarithmic growth, the cells were collected by centrifugation and resuspended in an equal volume of M9 medium without a carbon source. Ten bacterial suspensions prepared in this manner were injected into 10 ml 27.5 ml vials. Each of these five tubes was made into a series under the following conditions, and shaken at 30 ° C., and TCE was added one by one so that the final concentration (concentration in the reaction solution) was 5 ppm every 6 hours. A: Formaldehyde is added in advance to a final concentration of 2 mM for all five tubes. B: Final concentration of 2 mM every 6 hours, ie, immediately before adding TCE.
A system in which only M9 medium was added to five vials as blanks was prepared as blanks, and the degradation of TCE was evaluated. In the TCE decomposition experiment, after adding a prescribed amount of a saturated aqueous solution of TCE, completely sealed with a Teflon liner butyl rubber stopper and an aluminum cap, shaken at 30 ° C, and after 5 hours, 0.1 ml of gas phase in the vial bottle was shaken.
Was collected and TCE was measured by gas chromatography (Shimadzu Gas Chromatograph GC-14B; FID detector) by the headspace method. Table 1 shows the residual TCE concentration at each time.

[0038]

[Table 1] From these results, the effect of the present invention was shown in the J1 strain, a liquid TCE decomposition treatment using formaldehyde as an energy source.

Example 2 A colony of the JM1 strain on an agar M9 medium (containing 0.5% sodium glutamate) was inoculated into 200 ml of an M9 medium containing 0.5% sodium glutamate and 500 ml.
Shaking culture was performed at 25 ° C. in a shake flask. At 22 hours, the late phase of logarithmic growth, cells were collected by centrifugation,
Resuspended in an equal volume of M9 medium without carbon source. Using the bacterial suspension thus prepared, TCE degradation was evaluated in the same manner as in Example 1. Table 2 shows the residual TCE concentration at each time.

[0040]

[Table 2] From these results, the effect of the present invention was demonstrated in the decomposition treatment of liquid TCE using JM1 strain and formaldehyde as an energy source.

Example 3 In the same manner as in Example 2, the energy source to be supplied was methanol (final concentration: 0.1% (v / v)).
The degradation of TCE was evaluated. Table 3 shows the residual TCE concentration at each time.

[0042]

[Table 3] From these results, the effect of the present invention was shown in the JM1 strain, the decomposition treatment of liquid TCE using methanol as an energy source.

Example 4. The substances to be decomposed were cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,2-dichloroethylene, and 1,1-dichloroethylene (1,1-DCE), respectively. The effect of the present invention was evaluated in the same manner as in Example 2 except that 3.0 ppm was used. Table 4 (cis-1,2-DCE) and Table 5 (t
rans-1,2-DCE) and Table 6 (1,1-DCE).

[0044]

[Table 4]

[0045]

[Table 5]

[0046]

[Table 6] From these results, the effect of the present invention was shown in the JM1 strain, a liquid DCE decomposition treatment using formaldehyde as an energy source.

Example 5 The effect of the present invention was evaluated in the same manner as in Example 2 except that the substance to be decomposed was 40 ppm of toluene. Table 7 shows the results.

[0048]

[Table 7] From the results, the effect of the present invention was shown in the decomposition treatment of liquid toluene using formaldehyde as an energy source for JM1 strain. Example 6 10 ml of the bacterial suspension prepared in the same manner as in Example 2 was
Then, each of the mixture was poured into a 50 ml sterile tube, and phenol (50 ppm) and o-cresol (50 ppm) were prepared in the same manner as in Example 2.
The effect of the present invention on the decomposition of was evaluated. Quantification is in solution
0.1 ml was collected and phenol was analyzed by spectrophotometry using 4-aminoantipyrine (JIS K 0102-1993 28.1).
Cresol was measured by a spectrophotometric method using p-hydrazinobenzenesulfonic acid (JISK 0102-1993 28.2). The results are shown in Table 8 (phenol) and Table 9 (o-cresol).

[0049]

[Table 8]

[0050]

[Table 9] From the results, the effect of the present invention was demonstrated in the JM1 strain, a liquid phenol and cresol decomposition treatment using formaldehyde as an energy source.

Example 7 Colonies on the agar M9 medium (containing 0.1% yeast extract and 200 ppm phenol) of the KK01 strain were
M9 containing 0.05% yeast extract and 400 ppm phenol
The medium was inoculated into 200 ml of the medium, and shake-cultured at 30 ° C. in a 500 ml shake flask. At 22 hours, which is the late stage of logarithmic growth, the cells were collected by centrifugation, and resuspended in an equal volume of M9 medium containing no carbon source. The bacterial suspension prepared in this way was
10 bottles each of which was poured into a 27.5 ml vial bottle were prepared. Each of these five is made into a series under the following conditions, and 30
TCE was added one by one so that the final concentration was 5 ppm every 8 hours by shaking at ° C. A: Add sodium formate in advance to a final concentration of 4 mM for all five tubes B: Final concentration of 4 mM every 8 hours, ie immediately before adding TCE
As a blank, a system in which only M9 medium was added to five vials as blanks was further prepared as a blank, and the degradation of TCE was evaluated in the same manner as in Example 1. Table 10 shows the residual TCE concentration at each time.

[0052]

[Table 10] From these results, the effect of the present invention was shown in the liquid TCE decomposition treatment using sodium formate as an energy source for KK01 strain.

Example 8. Sawara sand (13% water content, not sterilized)
50g into 15 68ml vials, the concentration in soil is 5pp
m, add a saturated aqueous solution of TCE, stopper tightly, and add
After standing for a day, a simulated TCE-contaminated soil sample was prepared. Thereafter, the bacterial suspension of the JM1 strain prepared as in Example 2 was divided into a series under the following conditions, and 5 ml was injected into each vial every 7 hours, and the suspension was used for purification of contaminated soil. The effects of the invention were evaluated. In addition, the stationary and decomposition experiments of the bacterial suspension were performed at 20 ° C. A: Formaldehyde is added in advance to a final concentration of 2 mM. B: Every 7 hours, that is, 1 to the bacterial suspension added to the vial.
Formaldehyde was added so that the final concentration was 2 mM each, and a system in which only the M9 medium was added to five vials as blanks was also prepared, and the degradation of TCE was evaluated. In the TCE decomposition experiment, after adding a specified amount of the bacterial suspension, the cell was completely sealed with a Teflon liner butyl rubber stopper and an aluminum cap, and then cooled to 20 ° C.
After 6 hours, collect 10 g of soil in the vial
in 3 ml of normal hexane and vigorously stirred for 3 minutes,
The TCE of the normal hexane phase was measured by gas chromatography (Shimadzu Gas Chromatograph GC-14B; ECD detector). Table 11 shows the residual TCE concentration at each time.

[0054]

[Table 11] From these results, the effect of the present invention was demonstrated in the decomposition treatment of soil-based TCE using the JM1 strain.

Example 9 A porous cellulose carrier (average pore size: about 500 mm, diameter: about 5 mm) was sterilized in an autoclave, cooled to room temperature or lower, and packed in the same vial as in Example 2. The bacterial suspension of the JM1 strain prepared as in Example 2 was divided into a series under the following conditions, and 15 ml was injected into a vial. TCE-contaminated air (prepared with a permeator, gas (Concentration: 100 ppm) was added to the vial with a 1 ml gas-tight syringe to evaluate the effect of the present invention on purification of contaminated air. The decomposition experiment was performed at 25 ° C. A: Formaldehyde is added to a final concentration of 2 mM in advance. B: Every 6 hours, that is, to each sample immediately before adding TCE-contaminated air, one formaldehyde is added to a final concentration of 2 mM. Five additional vials as blanks In addition, a system to which only the M9 medium was added was prepared, and the degradation of TCE was evaluated. The TCE amount was measured in the same manner as in Example 2. Table 12 shows the results. The concentration is a gas concentration.

[0056]

[Table 12] From these results, the effect of the present invention was demonstrated in the decomposition treatment of gas phase TCE using the JM1 strain.

[0057]

As described above in detail, according to the present invention, an efficient biodegradation purification treatment of liquid, soil or air contaminated with an organic compound such as an aromatic compound or a chlorinated ethylene compound can be performed. It becomes possible.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/20 C12N 1/20 F 1/21 1/21 // B09B 3/00 ZAB B09B 3/00 ZABZ (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo F-term within Canon Inc. DD31

Claims (29)

[Claims] Claims: 1. A method for repairing an environment contaminated by an organic compound using a microorganism capable of decomposing the organic compound, the method comprising: (1) culturing a microorganism capable of decomposing the organic compound by adding a growth substrate and an energy source; (2) Dividing the microorganism after cultivation into two or more fractions as a dormant microbial solution without a growth substrate and energy source, and (3) depending on the time at which the organic compound is decomposed, the energy source in each fraction. And repairing the organic compound with the organic compound. 2. The method according to claim 1, wherein said energy source is an alcohol, aldehyde or carboxylic acid. 3. The method according to claim 2, wherein said alcohol is methanol. 4. The method according to claim 2, wherein said aldehyde is formaldehyde. 5. The method according to claim 2, wherein the carboxylic acid is formic acid or a salt thereof. 6. The method according to claim 1, wherein the organic compound is a chlorinated aliphatic compound. 7. The method according to claim 6, wherein the chlorinated aliphatic compound is trichlorethylene or dichloroethylene. 8. The method according to claim 1, wherein the organic compound is an aromatic compound. 9. The method according to claim 9, wherein the aromatic compound is benzene, toluene,
9. The method according to claim 8, which is phenol or cresol. 10. The method according to claim 1, wherein the environment contaminated by the organic compound is a liquid. 11. The method according to claim 10, wherein the liquid is groundwater or waste liquid. 12. The method according to claim 10, wherein the organic compound is decomposed by bringing the liquid into contact with a carrier supporting the microorganism. 13. The method according to claim 12, wherein the carrier supporting the microorganism is contained in a container, and the liquid is introduced from one end of the container and discharged from the other end. 14. The method according to claim 1, wherein the environment contaminated by the organic compound is soil. 15. The method according to claim 14, further comprising a step of introducing a liquid containing the microorganism into the soil. 16. The method according to claim 15, wherein the microorganism is introduced into the soil from an injection well provided in the soil. 17. The method according to claim 14, wherein the soil is introduced into a solution containing the microorganism. 18. The method according to claim 14, wherein the soil containing the microorganism is mixed with the soil. 19. The method according to claim 14, wherein the soil is brought into contact with a carrier carrying the microorganism. 20. The method according to claim 1, wherein the environment contaminated by the organic compound is air. 21. The method according to claim 20, wherein the air is introduced into a solution containing the microorganism. 22. The method according to claim 20, wherein the air is brought into contact with a carrier supporting the microorganism. 23. The method according to claim 22, wherein the carrier carrying the microorganism is contained in a container, and the air is introduced from one end of the container and discharged from the other end. 24. The method according to claim 1, wherein the microorganism is a microorganism obtained from a natural environment. 25. The method according to claim 24, wherein the microorganism is strain J1 (FERM BP-5102). 26. The method according to claim 26, wherein the microorganism is Pseudomonas cepacia.
The method according to claim 24, which is a strain KK01 (FERM BP-4235). 27. The method according to claim 1, wherein the microorganism is a microorganism obtained by a mutation operation. 28. The method according to claim 27, wherein the microorganism is a JM1 strain (FERM BP-5352). 29. The method according to claim 1, wherein the microorganism is a microorganism obtained by a genetic recombination operation.