JPH09267085A - Biologically restoring and purifying method for contaminated soil - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce not only the decomposition efficiency of a contaminated substance but also an equipment and the cost required to soil restoration by using a decomposition microorganism precultured in a range within a specified temp. difference with a previously monitored contaminated soil as the decomposition microorganism to be brought into contact with the contaminated soil. SOLUTION: The decomposition microorganism to be brought into contact with the contaminated soil is the decomposition microorganism precultured at a range within 5 deg.C temp. difference between the previously monitored contaminated soil. The effective proliferation of a decomposed matter and activity are obtained without especially increasing the temp. of the soil since a temp shift at series of growth process including from the preculture of the decomposition microorganism having the decomposition ability of the contaminated substance to the growth in the soil affects largely to its decomposition ability, and moreover, the decomposition microorganism cultured by the preculture at which the temp. difference with the soil is within 5 deg.C is charged to the contaminated soil, thereby not only the decomposition efficiency of the contaminated substance but also the reducing of the equipment and the cost required for the soil restoration are possible.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biological remediation and purification method effective for remediating soil contaminated with at least one of aromatic compounds and organic chlorine compounds.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to organic chlorine compounds, which are harmful to living bodies and hardly decomposed, has become a major issue. Especially, tetrachlorethylene (PCE) is found in the soil of high-tech industrial areas such as IC factories in Japan and overseas.
It is considered that the contamination by chlorinated aliphatic hydrocarbon compounds such as trichlorethylene (TCE) and dichloroethylene (DCE) has spread to a considerable extent, and many cases actually detected by environmental surveys have been reported. There is.
It is said that those organic chlorine compounds remaining in the soil are dissolved in groundwater by rainwater or the like and spread throughout the surrounding area. Since such compounds are suspected to have carcinogenicity and reproductive toxicity and are extremely stable in the environment, pollution of groundwater used as a water source for drinking water is a major social issue.

From the above, the purification and restoration of soil by removing and decomposing organic chlorine compounds is an important issue from the viewpoint of environmental protection, and various technical incentives necessary for purification have been made. For example, adsorption treatment with activated carbon and decomposition treatment with light and heat have been studied, but these methods are not always practical in terms of cost and operability.

On the other hand, in recent years, decomposition of organic chlorine 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 the biodegradation treatment using microorganisms, it is possible to decompose organic chlorine compounds into harmless substances by selecting the microorganisms to be used, basically no special chemicals are required, and maintenance labor and cost are reduced. There are advantages such as what can be done.

As a report isolated from a microorganism capable of degrading an organic chlorine compound, for example, as a TCE-degrading bacterium, Welchia alkenophila sero 5 (USP 4877736, ATCC5
370), Welchia alkenophila sero 33 (USP4877736, ATC
C53571), Methylocystis sp. Strain M [Agric. Biol.
Chem., 53, 2903 (1989), Biosci. Biotech. Bioche
m., 56, 486 (1992), ibid. 56,736 (1992)), Methylosinus
trichosprium OB3b (Am. Chem. Soc. Natl. Meet. Dev.
Environ. Microbiol., 29, 365 (1989), Appl. Enviro
n. Microbiol., 55, 3155 (1989), Appl. Biochem. Bio
technol., 28, 877 (1991), JP 02-92274 A, JP 03-292970 A], Methylomonas sp.MM2 (Appl.
Environ. Microbiol., 57, 236 (1991)), Alcaligenes
denitrificans ssp. xylosoxidans JE75 (Arch. microb
iol., 154, 410 (1990)), Alcaligenes eutrophus JMP1
34 (Appl. Environ. Microbiol., 56, 1179 (1990)), Al
caligenes eutrophus KSO1 (JP 07-123976), Mycobacterium vaccae JOB5 (J. Gen. Microbio
l., 82, 163 (1974), Appl. Environ. Microbiol., 54,
2960 (1989), ATCC29678), Pseudomonas putida BH [Sewer Association Journal, 24, 27 (1987), Acinetobactor sp. Strain
G4 (Appl. Environ. Microbiol., 52, 383 (1986), 5
3,949 (1987), 54,951 (1989), 56,279 (1990),
57,193 (1991), USP4925802, ATCC53617,
It was classified as Pseudomonas cepacia, but Acinetobact
or sp.], Pseudomonas mendocina KR-1
[Bio / Technol., 7, 282 (1989), Pseudomonas putida
F1 (Appl. Environ. Microbiol., 54., 1703 (1988), same
54,2578 (1988)], Pseudomonas fluorescens PFL12 (A
ppl. Environ. Microbiol., 54, 2578 (1988)), Pseudo
monas fluorescens NRRL-B-18296 (USP 4853334), Pseu
domonas putida KWl-9 (Japanese Patent Laid-Open No. 06-70753), Pseu
domonas cepacia KK01 (Japanese Patent Laid-Open No. 06-227769), Nit
rosomonas europaea (Appl. Environ. Microbiol., 56,
1169 (1990)), Lactobacillus vaginalis sp. Nov (In
t. J. Syst. Bacteriol., 39, 368 (1989), ATCC49540)
Etc. are known.

[0006] Recently, attempts have been made to use these degrading bacteria to actually repair soil contaminated with aromatic compounds and / or organic chlorine compounds.

However, in the soil which is a natural environment, it is natural that p, which is different from the ideal culture system in the laboratory,
There are various factors peculiar to soil such as H, water content, temperature and oxygen content, and these factors make it difficult to optimize the growth of degrading microorganisms and expression of degrading activity. As a result, many problems remain for soil restoration by degrading microorganisms.

Among them, the temperature condition is very important because it is directly involved in the growth of the degrading microorganism and the expression of the degrading activity. It is generally said that the temperature in soil in a temperate zone such as Japan is about 15 ° C. throughout the year, which is fairly low as a general microorganism culture temperature.

[0009] On the other hand, attempts have been made so far to increase the efficiency of soil restoration by degrading microorganisms by controlling the temperature in the soil.

In Japanese Patent Publication No. Hei 4-503528, in the decomposition of chlorophenols in soil using microorganisms belonging to the genus Rhodococcus and the genus Mycobacterium, the temperature along with pH, moisture, redox potential, nutrition, aeration, etc. It is disclosed that the bioactive function of microorganisms is improved by adjusting Specifically, for example, composting that generates heat or heating the soil by an external heating means maintains the soil temperature at 20 ° C. to 35 ° C., which is the biological activity temperature of microorganisms.

DE 3720833A1 also discloses a method for maintaining the activity of the microorganism by increasing the temperature in the soil (about 15 ° C. to 50 ° C.) by introducing warm air into the soil to treat the contaminated soil. ing.

In addition, in the bioreactor treatment of contaminated soil (DE3921066C2) and the groundwater pumping treatment (Japanese Patent Laid-Open No. 57-209692), a method of increasing the activity of the degrading microorganisms is employed by increasing the temperature.

As described above, in the conventional technique, means for raising the temperature of the environment (soil temperature) has been adopted in order to enhance the activity of the degrading microorganisms.

[0014]

However, the above-mentioned method for controlling the temperature of soil has a problem that a large amount of energy and special equipment are required to raise the soil temperature, which is a problem in terms of economical efficiency. It was something that had.

An object of the present invention is to provide not only the efficiency of decomposing pollutants, but also the equipment and cost required for soil restoration, and an economically efficient biological restoration and purification method for contaminated soil. Especially.

[0016]

The method for biological remediation and purification of contaminated soil according to the present invention provides a degrading microorganism capable of decomposing the compound in contaminated soil contaminated with at least one of an aromatic compound and an organic chlorine compound. In the method for repairing and purifying the contaminated soil by contacting and decomposing the compound, the degrading microorganisms that come into contact with the contaminated soil are pre-cultured within a temperature difference of 5 ° C. or less with the previously monitored contaminated soil. It is characterized by being a microorganism.

According to the present invention, a temperature shift (temperature gap) in a series of growth processes including pre-culture of a degrading microorganism having the ability to decompose pollutants and growth in soil has a great influence on the resolution. When applying degrading microorganisms for repair and purification of contaminated soil, rather than raising the temperature of the soil to a relatively high optimum temperature at which the degrading microorganisms actively grow or exhibit high activity, By administering the degrading microorganisms cultivated by pre-culturing with a temperature difference of 5 ° C or less to the contaminated soil, sufficient and efficient growth and activity of the degrading microorganisms can be achieved without increasing the soil temperature by an external heating means. It was made based on a new finding by the present inventors that it can be obtained in soil.

According to the present invention, not only the efficiency of decomposing pollutants but also the equipment and cost required for soil restoration can be reduced, and an economically efficient biological restoration and purification method for contaminated soil is provided. It becomes possible.

For example, in the above-mentioned conventional example, a degrading microorganism previously cultivated at a normal culturing temperature (generally around 25 ° C. to 37 ° C.) is introduced into the soil. However, in such a case, there is already a temperature difference (gap) between the medium and the soil at the time of introduction, and the temperature difference adversely affects the growth and activity of the degrading microorganism. That is, the initial growth and activity of the degrading microorganisms are insufficient due to the damage due to the temperature difference, while the growth and activation of the degrading microorganisms are achieved by the microorganisms originally present in the soil (indigenous microorganisms). Oxygen and nutrients introduced for the purpose are consumed,
As a result, sufficient decomposition activity may not be exhibited. In addition, when the temperature in the soil is raised from 25 ° C to around 30 ° C before the introduction of the degrading microorganisms, the optimum growth temperature is close to that of the degrading microorganisms and activates the action of the indigenous microorganisms that compete with the degrading microorganisms. It becomes a factor and this method is not a good idea.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is particularly applicable to a case where the temperature in soil is higher than the lower limit temperature at which the degrading microorganism used grows and is 20 ° C. or less, that is, the SoD determined by the US Department of Agriculture.
fl Taxi in il Taxonomy
id, mesic, thermic (the)
rmic) Effective in soil.

In the present invention, for example, the temperature is 15 ° C.
When microorganisms with a normal cultivation temperature of about 25 ° C are added to the soil, the pre-cultivation conditions are adjusted appropriately so that the cultivation temperature is 10 ° C.
Pre-culture is performed so that the temperature is between about -20 ° C.

At this time, it is preferable to set the conditions that can secure the same number of bacteria as in the case of culturing at 25 ° C. In this case, it takes longer to grow to the target number of bacteria than in the case of culturing at 25 ° C. However, there is no problem because the activity itself is usually improved with the growth. Then, when the microorganisms cultivated at the set temperature are introduced into the soil, the gap with the soil temperature becomes smaller than that when cultivated at 25 ° C,
Only that much grows smoothly.

Once the number of bacteria of about 10 ⁷ cells / g soil is secured before the growth of the indigenous microorganisms becomes active, the growth of the indigenous microorganisms is suppressed to some extent, and the rapid and continuous expression of degrading activity is achieved. Done.

The degrading microorganism used in the present invention is not particularly limited as long as it can grow at the temperature of the introduced soil and can decompose an aromatic compound and / or an organic chlorine compound. For example, those expressing an oxygenase and having a decomposing activity of at least one of these organic compounds can be preferably used. Corynebacterium (Coryneba) is one of such microorganisms.
genus Cterium, Pseudomonas
onas), Acinetobacter (Acinetoba)
genus Cter, Alcaligenes
s) genus, Vibrio genus, Nocardia (N)
genus ocardia), Bacillus
Genus Lactobacillus
Genus, Achromobacter
Genus, Arthrobacter
Genus, Micrococcus genus, Mycobacterium genus,
Methylosinus genus, Methylomonas genus, Belchia (W
genus elchia, Methylocistis (Methyloc)
genus ytisis, Nitrosomom
onas), Saccharomyces
ces genus, Candida genus, and Torulopsis genus, and one or more of them can be appropriately selected and used according to the type of pollutant to be decomposed.

[0025] Suitable strains include, for example, J1 strain (F
ERM BP-5102), JM1 strain (FERM BP
-5352), Pseudomonas sp. TL1 strain; FERM.
P-14726), Alcaligenes Spices TL
2 strains (Alcaligenes sp. TL2; FER
MP-14642) and the like.

"FERM P" or "FERM"
The strain with the deposit number indicated by "BP" has been deposited at the Ministry of International Trade and Industry, the Agency of Industrial Science and Technology, and the Institute of Biotechnology and Industrial Technology (1-3 East, Tsukuba City, Ibaraki Prefecture) and has the BP number. Is an international deposit under the Budapest Treaty. The dates of these deposits are as follows. J1; FERM BP-5102; Deposit Date: May 25, 1994 (transferred from FERMP-14332 to International Deposit on May 17, 1995) JM1; FERM BP-5352; Deposit Date: 1995
January 10, (from FERMP-14727 to 1995 January
Transferred to international deposit on February 22) TL1; FERM P-14726; Deposit date: 1995
January 10, TL2; FERM P-14642; Deposit date: Heisei 6
November 15, 2013 JM1 strain was obtained by mutating J1 strain that oxidizes aromatic compounds and organic chlorine compounds with oxygenase, and is a mutant strain that constitutively expresses oxygenase without inducer. The mycological properties are as follows. Bacteriological characteristics of J1 strain and JM1 strain; Gram stainability and morphology: Growth in each medium of Gram-negative bacilli 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: fermentation glucose positive sugar: negative sucrose: negative raffinose: negative galactose: Negative Maltose: Negative Urease: Positive Esculin hydrolysis (β-glucosidase): Positive Nitrate reduction: Negative Indole production: Negative Glucose acidification: Negative Arginine dihydrolase: Negative Gelatin hydrolysis (protease): Negative β-galactosidase: Negative Assimilation of: glucose Negative L-arabinose: Negative D-mannose: 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 Quen Sodium acid: positive Phenyl acetate: negative Therefore, at the time of deposit, these were tentatively classified as Corynebacterium sp., But thereafter, the identification labels of the deposited strains were changed to J1 and JM1 strains.

The medium used for culturing the microorganism used in the present invention may be any medium as long as the microorganism can grow, such as LB medium and 2 × YT medium, as long as the microorganism can grow. For example, it is also possible to culture with M9 medium added with a carbon source.

The composition of the M9 medium (in 1 liter of medium: pH 7.0) 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 The culture can be carried out under aerobic conditions and may be liquid culture or solid culture. .

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the soil in the present invention can be carried out by bringing the decomposing microorganism into contact with the aromatic compound and / or the organic chlorine compound existing in the soil.

A typical example of the method of applying the degrading microorganisms to soil will be described below. For example, the simplest method is a method of directly introducing the degrading microorganism into soil contaminated with an aromatic compound and / or an organic chlorine compound. As a method of introduction, in addition to the method of spraying on the soil surface, in the case of treatment in a relatively deep formation,
There is a method of introducing from a well inserted in the ground. Furthermore, when pressure is applied by air, water, etc., the decomposing microorganisms spread over a wide area, which is more effective. In this case, it is preferable to adjust various conditions in the soil to be suitable for the degrading microorganisms, and the degrading microorganisms can be grown faster in the presence of a carrier such as soil particles. Is.

Further, a degrading microorganism is attached to a carrier, which is filled in a reaction tank, and this reaction tank is introduced mainly into an aquifer of a soil contaminated with an aromatic compound and / or an organic chlorine compound to decompose it. Processing can also be performed. The form of the reaction tank is preferably fence-shaped or film-shaped so that it can cover a wide range in soil. In this case, any carrier can be used, but it is more preferable that the carrier has 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, the wastewater treatment system, and the like can be used as a material that provides a living space for microorganisms. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carriers such as anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. , Polyacrylamide, carrageenan, agaro
And gel-like carriers such as gelatin and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester and the like. As natural products, cellulose, cotton, hemp, paper, and other cellulose-based products, and wood powder, bark, and lignin-based products can also be used.

The method of the present invention can be applied to soil remediation in both closed and open systems. It should be noted that the microorganism may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0034]

【Example】

Example 1 (Phenol and TCE by growth of J1 strain in soil)
Of J1 strain on M9 agar medium containing 0.2% by weight of yeast extract is inoculated into 200 ml of a liquid medium of the same composition prepared in a 500 ml Sakaro flask.
Shaking culture was carried out at 25 ° C. for 24 hours. 200 μl of the obtained culture solution (including proliferating cells) was added to 100 ml of two liquid media having the same composition, one at 22 ° C. and the other at 15 ° C.
When shaking culture was carried out at 0 ° C and the number of bacteria was monitored by plate count, the time required to reach 10 ⁸ cells / ml was about 18 hours in the system at 22 ° C and about 3 in the system at 15 ° C.
It was 6 hours. Then, repeat the same operation,
The culture solution (including the proliferating cells) at the time of culturing for about 18 hours in the system of ° C and about 36 hours in the system of 15 ° C was used for the next operation.

Yeast extract 0.2% by weight, phenol 40
1m of M9 medium containing 0ppm and 100ppm of TCE
1 was poured into a 15-mL Teflon inner vial equipped with an inner lid, and 4 g of air-dried Sahara passed sand was added to prepare a large number. Next, 10 μl each of the J1 strain culture solution obtained as described above was inoculated into individual vials, completely sealed, and then statically cultured at 15 ° C., which is close to the actual temperature in the soil, to determine the number of bacteria. , Phenol and TCE concentrations were measured daily.

For the measurement of the number of bacteria, 5 ml of sterilized water was added to the soil sample in the vial, and the supernatant obtained by stirring and extracting for 1 minute was serially diluted to obtain M9 containing 0.2% by weight of yeast extract.
It was applied to an agar medium and the plate count method was used. To distinguish between indigenous bacteria and J1 strain colonies,
When cultivated at 5 ° C, the colonies of the J1 strain became recognizable in about 2 days, whereas the indigenous bacteria required 3 days or more. The colonies of the J1 strain decomposed phenol to give a yellow substance. I went out by using. Also, the correspondence between the plate count and the number of bacteria by FCM was confirmed in advance. As a method for quantifying phenol, 5 ml of sterilized water was added to a soil sample in a vial, and phenol in a supernatant obtained by stirring and extracting for 1 minute was detected by a JIS method using aminoantipyrine (JIS K010).
2-1993, 28.1). Then, the TEC concentration was determined by adding 5 ml of n-hexane to the soil sample in the vial, stirring and extracting for 3 minutes, diluting 10 times depending on the case, and quantifying by gas chromatography (ECD detector).

Regarding phenol and TCE, sand that had been sterilized in the same experimental system was used as a control, and quantitative determination was also performed in a system in which the J1 strain was not added to determine the residual ratio with respect to the concentration of the control. The results are shown in FIG. 1 (change in bacterial count when precultured at 22 ° C.), FIG. 2 (change in bacterial count when precultured at 15 ° C.), FIG. 3 (change in phenol concentration) and FIG.
(Change of TCE concentration) is shown respectively.

Compared with the system pre-cultured at 22 ° C., which is the optimum temperature of J1 strain, pre-culture at 15 ° C. which is the same as the soil temperature allows the J1 strain to grow more rapidly and the growth of indigenous microorganisms. Suppressed In addition, along with it, decomposition of phenol, TC
For the decomposition of E, it was better to carry out pre-culture at 15 ° C.

Example 2 (Cresol Degradation Treatment with J1 Strain) O-cresol, m-instead of phenol and TCE as decomposition targets
The same experiment as in Example 1 was performed except that cresol (both was 300 ppm) was used to examine the effect of the gap between the preculture temperature and the soil temperature on the cresol decomposition of the J1 strain in the soil. The quantification of each cresol was carried out by the same extraction method as for phenol, and the detection method by the JIS method using p-hydrazinobenzenesulfonic acid (JIS K0102-1993, 28.
2). Table 1 shows the survival rate of each cresol on the 6th day of culture.

[0040]

[Table 1] Degradation of each cresol was better when pre-cultured at 15 ° C., which is the same as the soil temperature.

Example 3 (Phenol and TC by growth of TL1 strain in soil)
E-degradation treatment) Colony of TL1 strain on M9 agar medium containing 0.2% by weight of yeast extract was inoculated into 200 ml of the same composition medium in a 500 ml Sakaro flask, and at 24 ° C at 24 ° C.
Shaking culture was performed for hours.

20 of the obtained culture solution (including proliferating cells)
0 μl was added to each 100 m of two liquid media of the same composition.
Shake culture at 25 ° C for one and 15 ° C for the other.
Performed and monitored the number of bacteria by plate count,
It showed that the growth rate was very slow at 15 ° C.
Then, shaking culture was performed again at 18 ° C. As a result, 10
⁸Time to reach cells / ml is 25 ° C
It took about 22 hours and about 40 hours for the system at 18 ° C.

Then, the same operation was repeated, using the culture solution at the time of about 22 hours in the system of 25 ° C. and about 40 hours in the system of 18 ° C., the phenol concentration was 200 ppm, TC
The effect of the temperature difference between the preculture and the soil was examined in the same manner as in Example 1 except that the E concentration was 40 ppm. The obtained results are shown in FIG. 5 (change in the number of bacteria when precultured at 25 ° C.),
Fig. 6 (change in the number of bacteria when precultured at 18 ° C), Fig. 7
(Change of phenol) and FIG. 8 (Change of TCE) are shown respectively.

Compared to the system in which the TL1 strain was pre-cultured at 25 ° C. which is the optimum temperature, the pre-culture at 18 ° C. which is close to the soil temperature allows the TL1 strain to grow more rapidly and the growth of indigenous microorganisms. Suppressed Also, along with it, the soil temperature is close to 1
The pre-culture at 8 ° C. was better in the degradation of phenol and TCE of the TL1 strain.

Example 4 (Cresol Degradation Treatment with TL1 Strain) p-cresol (20
0 ppm), except that the same experiment as in Example 3 was performed.
The effect of temperature difference between pre-culture and soil on cresol degradation in soil with L1 strain was investigated. The quantification of cresol was carried out by the same extraction method as for phenol, and the detection method by JIS method using p-hydrazinobenzenesulfonic acid (JIS K010
2-1993, 28.2). Table 2 shows the survival rate on day 6 of culture.

[0046]

[Table 2] Regarding the decomposition of cresol, it was better to carry out the pre-culture at 18 ° C which is close to the soil temperature.

Example 5 (Phenol and TCE decomposition treatment of soil of TL2 strain) T on M9 agar medium containing 0.2% by weight of yeast extract.
A L2 strain colony was inoculated into 200 ml of the same composition medium prepared in a 500 ml Sakaro flask, and the inoculum was kept at 25 ° C. for 24 hours.
Shaking culture was performed for hours. 200 μl of the obtained culture solution (including proliferating cells) was added to 100 ml of two liquid media of the same composition.
When one was shake-cultured at 25 ° C. and the other at 18 ° C., and the number of bacteria was monitored by plate count, it was shown that the growth rate was extremely slow at 18 ° C. When shaking culture was performed at 20 ° C., 10 ⁸
The time required to reach cells / ml was about 36 hours in the system at 25 ° C and about 48 hours in the system at 20 ° C.

Then, the same operation was repeated, and the culture solution at the time of culturing for about 36 hours in the system at 25 ° C. and about 48 hours in the system at 20 ° C. was used, and the phenol concentration of the medium was adjusted to 700.
The effect of the temperature difference between the preculture and the soil was examined in the same manner as in Example 1 except that the ppm was used. The results are shown in FIG. 9 (change in bacterial count when pre-cultured at 25 ° C.), FIG. 10 (change in bacterial count when pre-cultured at 20 ° C.), and FIG. 11 (change in phenol).
12 and FIG. 12 (change of TEC).

Compared with the system pre-cultured at 25 ° C., which is the optimum temperature of the TL2 strain, pre-culture at 20 ° C. which is closer to the soil temperature allows the TL2 strain to grow more rapidly and the growth of indigenous microorganisms. Was suppressed. Along with this, pre-culturing at 20 ° C., which is close to the soil temperature, was better in the degradation of phenol and TCE of the TL2 strain.

Example 6 (Growth of JM1 Strain in Soil and TCE Degradation Treatment) Colonies of the JM1 strain on an M9 agar medium containing 0.2% by weight of yeast extract were mixed with the same composition medium in a 500 ml Sakaro flask. 200 ml was inoculated and shaking culture was performed at 22 ° C. for 24 hours. 200 μl of the obtained culture solution (including proliferating cells)
Was added to 100 m of two liquid media of the same composition, one of which was 2
At 2 ℃ where, the other performs a shaking culture at 15 ° C., and monitored the number of bacteria in plate count, 10 ⁸ cell
The time required to reach s / ml was about 18 hours in the 22 ° C. system and about 36 hours in the 15 ° C. system. JM1 strain is J
It was a mutant strain of 1 strain and had a TCE decomposing ability under the condition that an inducer such as phenol was not present, and the change in growth due to this temperature was the same as that of the parent strain.

Further, by repeating the same operation as described above,
In the same manner as in Example 1, except that the culture solution at the time of culturing for about 18 hours in the system of 15 ° C. and for about 36 hours in the system of 15 ° C. was used and phenol was not used, the influence of the temperature difference between the pre-culture and the soil was used. I checked. The obtained results are shown in FIG. 13 (change in bacterial count when pre-cultured at 22 ° C.), FIG. 14 (change in bacterial count when pre-cultured at 15 ° C.), and FIG. 15 (change in TCE), respectively. .

Compared to the system pre-cultured at 22 ° C., which is the optimum temperature of JM1 strain, the pre-culture at 15 ° C. which is the same as the soil temperature allows the J1 strain to grow faster and suppresses the growth of soil microorganisms. Was done. Also, along with it, the same as the soil temperature, 15 ℃
The JM1 strain showed better TCE degradation when pre-cultured in (1).

Example 7 (DCE decomposition treatment with JM1 strain) The substance to be decomposed in soil was cis-1,2-dichloroethylene (cis-1,2).
-DCE), trans-1,2-dichloroengineered ethylene (trans-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) (50 ppm each), the same method as in Example 6. The decrease in DCE was measured by. Table 3 shows the survival rate of each DCE on the 6th day of culture.

[0054]

[Table 3] It was shown that decomposition of each DCE proceeded in the pre-culture system at 15 ° C.

[0055]

According to the method of the present invention, the temperature difference between the pre-culture and the contaminated soil to be repaired and purified is kept within 5 ° C., so that the growth of degrading microorganisms and the degrading activity due to these temperature differences can be prevented. By eliminating the influence, it becomes possible not only to decompose the soil contaminated with the aromatic compound and / or the organic chlorine compound but also to perform the biological decomposition / restoration treatment with good economical efficiency.

[Brief description of drawings]

FIG. 1 is a diagram showing changes in the number of bacteria when precultured at 22 ° C. in Example 1.

FIG. 2 shows changes in the number of bacteria when precultured at 15 ° C. in Example 1.

FIG. 3 is a diagram showing changes in phenol concentration in Example 1.

FIG. 4 is a diagram showing changes in TCE concentration in Example 1.

FIG. 5 is a view showing changes in the number of bacteria when precultured at 25 ° C. in Example 3.

FIG. 6 shows changes in the number of bacteria when precultured at 18 ° C. in Example 3.

7 is a diagram showing changes in phenol in Example 3. FIG.

FIG. 8 is a diagram showing changes in TCE in Example 3.

FIG. 9 shows changes in the number of bacteria when precultured at 25 ° C. in Example 5.

FIG. 10 shows changes in the number of bacteria when precultured at 20 ° C. in Example 5.

FIG. 11 is a diagram showing changes in phenol in Example 5.

FIG. 12 is a diagram showing changes in TEC in Example 5.

FIG. 13 shows changes in the number of bacteria when precultured at 22 ° C. in Example 6.

FIG. 14 shows changes in the number of bacteria when precultured at 15 ° C. in Example 6.

FIG. 15 is a diagram showing changes in TCE in Example 6.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:05) (72) Inventor Shinya Kozaki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Within the corporation

Claims (11)

[Claims] 1. A contaminated soil contaminated with at least one of an aromatic compound and an organic chlorine compound is contacted with a degrading microorganism capable of decomposing the compound to decompose the compound, thereby repairing and purifying the contaminated soil. In the method, the decomposing microorganism that is brought into contact with the contaminated soil is a decomposing microorganism that has been pre-cultured in a temperature range of 5 ° C. or less with respect to the previously monitored contaminated soil. 2. The method for biological remediation and purification of contaminated soil according to claim 1, wherein the degrading microorganism is a microorganism expressing an oxygenase, which is an oxidase. 3. The microorganism expressing the oxygenase is Corynebacterium, Pseudomonas, Acinetobacter, Alcaligenes, Vibrio, Nocardia, Bacillus, Lactobacillus, Achromobacter, Arthrobacter, Micrococcus, Mycobacterium, Methylosynas, Methylomonas,
The biological remediation and purification method for contaminated soil according to claim 2, which is at least one kind of microorganisms belonging to the genus Berchia, the genus Methylocistis, the genus Nitrozomonas, the genus Saccharomyces, the genus Candida, and the genus Tollopsis. 4. The J1 strain (FERM BP) is used as the microorganism.
-5102), The method for biological remediation and purification of contaminated soil according to claim 2. 5. The JM1 strain (FERM B)
P-5352), The method for biological remediation and purification of contaminated soil according to claim 2. 6. The method for biological remediation and purification of contaminated soil according to claim 3, wherein the microorganism is Pseudomonas species TL1 strain (FERM P-14726). 7. The method for biological remediation and purification of contaminated soil according to claim 3, wherein the microorganism is Alcaligenes sp. TL2 strain (FERM P-14642). 8. The biological remediation of the contaminated soil according to claim 1, which is carried out by introducing an aqueous medium containing the decomposing microorganism into the contaminated soil and allowing the decomposing microorganism to grow in the soil. Purification method. 9. The method for biologically remediating and purifying contaminated soil according to claim 8, wherein the introduction of the microorganism into the soil is performed by pressure from an injection well provided in the soil. 10. The method for biological remediation and purification of contaminated soil according to claim 1, wherein the aromatic compound is at least one of phenol and cresol. 11. The method for biological remediation and purification of contaminated soil according to claim 1, wherein the organic chlorine compound is at least one of trichlorethylene and dichloroethylene.