JPH09267083A - Biologically restoring and purifying method for contaminated soil - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively execute the biologically restoring purification of contami nated soil by keeping the temp. of the contaminated soil at the temp. lower than a growth temp. or a decomposition optimum temp. of a decomposition microorganism thereby controlling the level and duration of a decomposition activity of the decomposition microorganism. SOLUTION: The temp. of the contaminated soil is kept at the temp. lower than the growth temp. or the decomposition optimum temp. of the decomposition microorganism to control the level and the duration of the decomposition activity of the decomposition microorganism. The temp. of the contaminated soil is kept, for example, at the temp. range of 5-10 deg.C lower than the optimum suitable temp. Any method is good if the method does not prevent the activity of the decomposition microorganism as a cooling method. The microorganism is not confined if it decomposes an arom. compd. and an org. chlorine compd. as the decomposition microorganism. An initial proliferation speed and decomposition of trichloroethylene(TCE) is excellent at 30 deg.C, but the number of bacteria are reduced with the lapse of time, and as a result, TCE is well decomposed at 15 deg.C than 22 deg.C being the optimum suitable temp. of J 1 stock.

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, microbial decomposition of organochlorine compounds such as TCE, which is stable in the environment, has been reported in recent years, and studies for its 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 organochlorine compound, for example, as a TCE-degrading microorganism, Welchia alkenophila sero 5 (USP 4877736, ATC
C5370), Welchia alkenophila sero 33 (USP4877736, A
TCC53571), Methylocystis sp. Strain M [Agric. Bio
l. 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], Methylomonassp. 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), Pesudomonas 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), USP 4925802, ATCC53617, which was originally classified as Pseudomonas cepacia, but Acinetoba
ctor sp.], Pseudomonas mendocina KR-1
[Bio / Technol., 7, 282 (1989), Pesudomonas putida
F1 (Appl. Environ. Microbiol., 54., 1703 (1988),
54, 2578 (1988)], Pesudomonas fluorescens PFL12
(Appl. Environ. Microbiol., 54, 2578 (1988)), Pesu
domonas fluorescens NRRL-B-18296 (USP 4853334), Pe
sudomonas putida KWl-9 (Japanese Patent Laid-Open No. 06-70753), Ps
eudomonas cepacia KK01 (Japanese Patent Laid-Open No. 06-227769), N
itrosomonas europaea (Appl. Environ. Microbiol., 5
6, 1169 (1990)), Lactobacillus vaginalis sp. Nov.
(Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC495
40) etc. are known.

Recently, attempts have been made to use these degrading microorganisms to actually repair soils contaminated with aromatic compounds and / or organochlorine compounds.

However, in the natural environment of the soil, naturally, there are various factors peculiar to the soil such as pH, water content, temperature, oxygen content, which are different from those in the laboratory culture system. These factors make it difficult to optimize the growth of degrading microorganisms and the expression of degrading activity, and as a result, many problems still remain in 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 said that the temperature in soil in a temperate zone such as Japan is generally constant at about 15 ° C throughout the year, but when the wall surface is exposed due to a part near the surface or a fault. It is easily affected by the outside temperature, and the temperature varies greatly depending on the season. Also, on the earth,
Although there are cold regions and the temperature in the soil is stable to some extent, there are considerable variations in 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, pH, water content, and water content in decomposition of chlorophenol in soil using microorganisms belonging to the genus Rhodocox and Mycobacterium.
It is claimed to improve the bioactive function of microorganisms by adjusting the temperature along with the redox potential, nutrition, aeration and the like. Specifically, for example, the soil temperature is maintained at 20 ° C. to 35 ° C., which is the biological activity temperature of the microorganism, by composting to generate heat or by heating with an external heating means.

[0011] In DE3720833A1 as well, in order to treat the contaminated soil with microorganisms, hot air is introduced into the soil to increase the temperature in the soil and maintain the activity of the microorganisms.

[0012] In addition, also in the bioreactor treatment of contaminated soil (DE3921066C2) and the groundwater pumping treatment (JP-A-57-209692), the method of increasing the activity of degrading microorganisms by increasing the soil temperature is used.

As described above, the conventional temperature control in the method for purifying contaminated soil is to raise the temperature of the soil exclusively to enhance the activity of the degrading microorganisms.

[0014]

However, when an aromatic compound or an organic chlorine compound present in soil is subjected to microbial decomposition treatment at the location in the soil, as in the above-mentioned conventional example,
Regarding the culture obtained in the liquid culture experiment in the laboratory,
Alternatively, it is not always effective to apply the optimum temperature for degrading activity to soil as it is.

For example, the temperature in soil is 25 ° C. to 30 ° C.
When raised before and after, the action of the indigenous microorganisms that compete with the degrading microorganisms is also activated, and there is a high possibility that the growth and activity of the degrading microorganisms cannot be effectively exerted.

Thus, although the temperature of the soil to be repaired and purified is an important factor in biological treatment, the conventional method of controlling the temperature of the soil still has problems to be improved. It was a thing.

An object of the present invention is to provide a method for efficiently carrying out biological remediation and purification of contaminated soil from the viewpoint of temperature control of contaminated soil.

[0018]

The method for biological remediation and purification of contaminated soil according to the present invention comprises contacting soil contaminated with at least one of an aromatic compound and an organic chlorine compound with a degrading microorganism capable of decomposing the compound. In the method for repairing and purifying the contaminated soil by decomposing the compound, the temperature of the contaminated soil is maintained at a temperature lower than the optimum temperature for the growth and decomposition of the degrading microorganisms, thereby degrading the degrading activity of the degrading microorganisms. It is characterized by adjusting the size and duration.

According to the method of the present invention, the temperature of the soil to be treated is maintained in accordance with the optimum temperature for the growth or decomposition activity of the degrading microorganisms capable of degrading pollutants to be introduced into the contaminated soil, thereby degrading the degrading microorganisms. It regulates the growth activity and the decomposition activity of pollutants to achieve more effective repair and purification.

The effect of the present invention can be obtained by maintaining the temperature of the contaminated soil at a temperature lower than the optimum temperature in the temperature range of 5 to 10 ° C., for example.

It is considered that maintaining the temperature of the contaminated soil in the method of the present invention has various actions. For example, the degrading activity of the degrading microorganisms introduced into the contaminated soil is at a practically sufficient level and for a longer period of time. You can list the points to be demonstrated. That is, when the soil is subjected to a temperature condition in which the initial growth rate and the decomposition activity are highest in the liquid culture system (optimum temperature; usually around 30 ° C., which is higher than the soil temperature), the degrading microorganisms introduced thereinto. Has a peak of decomposition activity that exceeds the amount of pollutants that can be decomposed in the early stage after introduction, but at the stage where pollutants gradually elute from soil particles, the peak of decomposition activity has already been reached. Since it is too low, it may result in the fact that sufficient decomposition activity is not obtained. On the other hand, in the method of the present invention, the temperature of the contaminated soil is adjusted so that the degrading activity of the degrading microorganisms is maintained at a practically sufficient level, and for a longer period of time. Eluted pollutants can also be effectively decomposed.

Furthermore, the method of the present invention is effective against volatile pollutants such as TCE and DCE. Highly volatile compounds such as TCE and DCE exist in soil in a three-phase medium of gas / liquid / solid. That is, the volatile pollutants are present in the air in the soil in a gaseous state, dissolved in the water containing the soil, and sorbed on the surface and inside of the soil particles themselves. Among them, the microorganisms can be efficiently decomposed when they are dissolved in the water content of the soil to form an aqueous solution. In other words, the portion dissolved in the water phase is first decomposed, and the concentration equilibrium causes the portion in the gas phase and solid phase to move into water, dissolve, and be decomposed. Go ahead. However, the dissolution of volatile pollutants from the gas phase to the water phase occurs almost instantaneously, whereas the rate at which the sorption once inside the soil particles dissolves in the water phase is It is much slower than dissolution and can take days to weeks depending on the type of contaminant and the period of contamination. In such a situation, if the soil temperature is raised too high, contaminants in the liquid phase that can be used by the degrading microorganisms may move into the gas phase, which may cause a problem of lowering the decomposition efficiency. On the other hand, in the method of the present invention, the temperature of the contaminated soil is adjusted to a low temperature range to prevent volatile pollutants from escaping from the liquid phase to the gas phase, and more effectively decompose the pollutants. It can be performed.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is particularly effective when the temperature in soil is 20 ° C. or higher. This corresponds to hyperthermic soil according to US Department of Agriculture's Soil Taxonomy. Further, it is also effective when the soil temperature rises due to the combined use of physical soil treatment or the treatment such as air circulation for the activation of microorganisms.

As described above, the temperature inside the soil in Japan is generally constant at about 15 ° C. throughout the year, but various additives such as microorganisms, nutrient solutions and oxygen are introduced, gas and The temperature may rise during forced circulation of the liquid. In preparation for such a case, a temperature sensor is previously installed in the soil, and when the temperature reaches 20 ° C or higher, the soil temperature is lowered to, for example, about 15 ° C. Also, when the original soil temperature is 20 ° C. or higher in the tropical region, the soil temperature is similarly lowered.

As the cooling method, a method of directly supplying cooling air to the soil, or an indirect introduction method of performing heat exchange on the conduit wall through a conduit or the like (in this case, air such as ammonia gas in the case of a closed system) Other gas may be used) method,
A method of directly spraying cooling water to the soil, a method of passing heat through the conduit wall through a conduit, etc., and indirectly cooling (in this case, various refrigerants and organic solvents may be used if it is a closed system), water Examples of such a method include introducing salts into the soil that can undergo an endothermic reaction when dissolved in the soil, and as a result, the temperature in the soil is lowered to the target temperature, which does not hinder the activity of the degrading microorganisms. Any method can be used.

The degrading microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of degrading 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. Examples of such microorganisms include Corynebacterium (Coryn).
genus Pseudomonas (Pseu)
genus domonas, Acinetobacter (Acinet)
genus, Alcalig
genus, Vibrio genus, Nocardia genus, Bacillus
s) genus, Lactobacillus
s) genus, Achromobacte
r) genus, Arthrobacter
r) genus, Micrococcus
Genus Mycobacterium
m) genus, Methylosinus
Genus, Methylomonas genus, Welchia genus, Methylocistis (Met)
genus hylochystis, Nitrozomonas (Nit
genus rosomonas, Saccharomyces
aromyces) genus, Candida genus,
Examples thereof include microorganisms belonging to the genus Torulopsis, and one or more of these can be appropriately selected and used according to the type of contaminant to be decomposed.

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 in a Mg medium to which a carbon source is added. Below Mg
The composition of the medium (in 1 liter; pH 7.0) is shown.

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 culture temperature is preferably about 15 ° C to 30 ° C.

Decomposition treatment of aromatic compounds and / or organic chlorine compounds in soil in the present invention can be carried out by contacting the aromatic compounds and / or organic chlorine compounds present in soil with the decomposing microorganisms. .

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 a degrading microorganism into soil contaminated with an aromatic compound and / or an organic chlorine compound. The method of introduction includes not only the method of spraying on the soil surface, but also the method of introduction from a well inserted into the ground in the case of treatment in a relatively deep formation. 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 the conditions in the soil to be suitable for degrading microorganisms,
The degrading microorganisms are allowed to grow faster in the presence of a carrier such as soil particles, and in that sense, the condition of being in soil is convenient.

Further, the degrading microorganisms are attached to a carrier, which is filled in a reaction tank, and this reaction tank is introduced mainly into the aquifer of soil contaminated with aromatic compounds and / or organic chlorine compounds. It can also be decomposed. The form of the reaction tank is preferably fence-shaped or film-shaped so that it can cover a wide range in soil. The carrier used in this case is
Any material can be used, but it is more preferable that it has an excellent ability to retain microorganisms and does not impair the 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, shikagel, alumina, inorganic particulate carriers such as anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. Gel 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, cellulose-based products such as cotton, linen and paper, and lignin-based products such as wood flour and bark can be used.

The method of the present invention can be applied to soil purification 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.

[0035]

EXAMPLE First, the apparatus used in the experiments of Examples 1 to 8 is shown in FIG. 1, and the explanation of the apparatus and the outline of the experimental method will be described below.

A stainless steel pot 1 having an inner diameter of about 32 cm and a height of about 31 cm is filled with gravel 2 having a diameter of about 1 cm up to 4 cm, and fine sand having a water content ratio of about 11% (Sahara thread;
2mm) 3 is packed into a thickness of about 25cm (about 20mm)
Liter), the upper layer was sealed with a Teflon sheet 4, and an injection pipe 5 and an exhaust pipe 6 for feeding gas, culture solution and the like were provided. Further, air was circulated by connecting a Teflon tube 7 provided with a temperature control device 9 and a pump 8 on the way to the injection pipe 5 and the exhaust pipe 6. The temperature of the fine sand in the pot is the temperature sensor 10
Then, the temperature was adjusted to the target temperature by adjusting the temperature control device 9. The number of bacteria and the concentration of each compound were measured by collecting fine sand around point A.

Example 1 (Growth of J1 strain in soil, phenol decomposition and TEC
Effect of gas cooling on decomposition treatment) 50 colonies of the J1 strain on M9 agar medium containing 0.1% by weight of yeast extract were
200 ml of the same composition medium in a 0 ml volume Sakaguchi flask was inoculated and shake culture was carried out at 22 ° C. for 24 hours.

In a thermostatic chamber at 15 ° C., 200 ml / mi of saturated TCE vapor was introduced through the injection pipe 5 of the apparatus shown in FIG.
n. After flowing for 10 minutes (while the exhaust pipe 6 is open), the injection pipe 5 and the exhaust pipe 6 were sealed and left to stand. After one week, the J1 strain cultured as described above in M9 medium containing 500 ppm of phenol and 0.2% by weight of yeast extract was added with 10 ⁶ cells.
5 liter of the bacterial solution was added to 300 ls / m
Injection from the injection pipe 5 at a rate of ml / min (during which the exhaust pipe 6 is opened), then the temperature of the thermostatic chamber is set to 30 ° C.
By connecting the Teflon tube 7 to the exhaust pipe 6 and circulating cooled air, 30 ° C. (simply circulate only air),
Conditions of 22 ° C (optimum temperature of J1 strain) and 15 ° C were prepared (monitored by the temperature of exhaust gas), and static culture was performed. Three samples of each system were prepared, and after 1 day, 2 days, and 4 days, fine sand around the point A was collected, and the number of bacteria, phenol concentration and TE
The C concentration was measured.

The number of bacteria was measured by adding 10 g of sample to 10 m of sterilized water.
l, and the mixture was stirred and extracted for 30 seconds, and the extract was serially diluted to give 0.1% by weight of yeast extract and 100 pp of phenol.
Colony counting was performed by culturing on M9 agar medium containing m. As a method for quantifying phenol, 10 ml of sterilized water was added to 10 g of a sample, and phenol in a supernatant obtained by stirring and extracting for 1 minute was detected by a JIS method using aminoantipyrine (JIS K102).
-1993, 28.1). The concentration of TCE was measured by adding 10 ml of n-hexane to 10 g of the sample and extracting TCE in the supernatant extracted by stirring for 3 minutes by glass chromatography using an ECD detector. As a control experiment, sterilized fine sand was used, only the medium was injected, and a system under the same conditions other than the above was performed at the same time. Phenol and TCE were shown as percentages when the values were 100. The results are shown in Fig. 2 (number of bacteria), Fig. 3 (phenol) and Fig. 4 (TCE).

Although the initial growth rate and the decomposition of phenol and TCE were excellent at 30 ° C., the number of bacteria decreased with the passage of time, and as a result, when the temperature was raised to 15 ° C., the liquid culture system of the J1 strain was used. Phenol and TCE were decomposed better than the optimum temperature of 22 ° C.

Example 2 (Effect of Gas Cooling on DEC Decomposition Treatment of Strain J1 in Soil) The substance to be decomposed in soil was replaced with ci instead of TCE.
s-1,2-dichloroethylene (cis-1,2-DC)
E), trans-1,2-dichloroethylene (tra
ns-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were used, and the decrease in DCE was measured by the same method as in Example 1. Table 1 shows the survival rate of each DCE on the 4th day of culture.

[0042]

[Table 1] It was shown that each DCE was most decomposed at 15 ° C.

Example 3 (Effect of gas cooling on cresol decomposition treatment of J1 strain in soil) Without introducing TCE, o-cresol and m-cresol (both 400 ppm) were used as decomposition targets instead of phenol. The same experiment as in Example 1 was carried out except that the effect of cooling air introduction on the cresol decomposition in soil of the J1 strain was evaluated. The quantification of each cresol was carried out by the same extraction method as for phenol, and the detection method by JIS method using p-hydrazinobenzenesulfonic acid (JIS K0102).
-1993, 28.2). Table 2 shows the survival rate of each cresol on the 4th day of culture.

[0044]

[Table 2] It was shown that each cresol decomposed most at 15 ° C.

Example 4 (Growth of TL1 strain in soil, phenol degradation and TC
Effect of gas cooling on E decomposition treatment) Yeast extract 0.1
Colonies of TL1 strain on M9 agar containing
200 ml of the same composition medium in a 500 ml Sakaguchi flask
Were cultivated with shaking and cultured at 25 ° C. for 24 hours.

The phenol concentration of the medium was 200 ppm, the temperature was 20 ° C., 25 ° C. (optimum growth temperature of the TL1 strain) and 3
In the same manner as in Example 1 except that the temperature was 0 ° C., the effect of temperature maintenance on the growth, phenol degradation, and TCE degradation of the TL1 strain was evaluated. The results are shown in Fig. 5 (number of bacteria), Fig. 6 (phenol) and Fig. 7 (TCE).

Although the initial growth rate and the decomposition of phenol and TCE were excellent at 30 ° C., the number of bacteria decreased with the passage of time, and as a result, when the temperature was raised to 20 ° C., the liquid culture system of the TL1 strain was used. Phenol and TCE decomposition was performed better than the optimum temperature of 25 ° C.

Example 5 (Effect of Gas Cooling on Cresol Decomposition Treatment of Strain TL1 in Soil) Without introducing TCE, p-cresol (300 pp) was used as a decomposition target instead of phenol.
The same experiment as in Example 4 was carried out except that m) was used to evaluate the effect of introducing cooling air on the cresol decomposition in soil of the TL1 strain. Cresol was quantified by the same extraction method as phenol using JIS using p-hydrazinobenzenesulfonic acid.
Detection method (JIS K102-1993, 28.
2). Table 3 shows the survival rate on the 4th day of culture.

[0049]

[Table 3] It was shown that cresol also decomposed most at 25 ° C.

Example 6 (Growth of Strain TL2 in Soil, Phenol Degradation and TC)
Effect of gas cooling on E decomposition treatment) Yeast extract 0.1
Colonies of TL2 strain on M9 agar containing
200 ml of the same composition medium in a 500 ml Sakaguchi flask
Were cultivated with shaking and cultured at 25 ° C. for 24 hours with shaking.

The phenol concentration in the medium was 700 ppm, the temperature was 20 ° C., 25 ° C. (optimum temperature for culturing TL2 strain) and 3
In the same manner as in Example 1 except that the temperature was 0 ° C., the effect of temperature maintenance on the growth, phenol degradation, and TCE degradation of the TL2 strain was evaluated. The results are shown in Fig. 8 (number of bacteria), Fig. 9 (phenol) and Fig. 10 (TCE).

Although the initial growth rate and the decomposition of phenol and TCE were excellent at 30 ° C., the number of bacteria decreased with the passage of time, and as a result, when the temperature was 20 ° C., the liquid culture system of the TL2 strain was used. Phenol and TCE decomposition was performed better than the optimum temperature of 25 ° C.

Example 7 (Effect of gas cooling on growth of JM1 strain in soil and TCE decomposition treatment) M9 containing 0.1% by weight of yeast extract
The JM1 strain colony on the agar medium was inoculated into 200 ml of the same composition medium in a 500 ml Sakaguchi flask, and the temperature was 22 ° C.
The culture was carried out for 24 hours with shaking.

The JM1 strain is a mutant strain of the J1 strain and is capable of degrading TCE under the condition that an inducer such as phenol is not present. Therefore, the same method as in Example 1 except that the phenol of the injection medium was removed. The effect of temperature maintenance on the proliferation and TCE degradation of JM1 strain was evaluated. FIG. 11 shows the results.
(Number of bacteria) and FIG. 12 (TCE) are shown.

Initial growth rate and TCE decomposition were 30 ° C.
Although it was excellent, the number of bacteria decreased over time,
As a result, when the temperature was set to 15 ° C, the number of cells and the TCE degrading activity were maintained well, and TCE degradation was performed better than 22 ° C, which is the optimum temperature in the liquid culture system of the JM1 strain.

Example 8 (Effect of gas cooling on DEC decomposition treatment of JM1 strain in soil) Degradation target substances in soil were cis-1,2-dichloroethylene (cis-1,2-DCE) and tran.
s-1,2-dichloroethylene (trans-1,2-
DCE) and 1,1-dichloroethylene (1,1-DC
The decrease in DCE was measured in the same manner as in Example 5 except that the difference was E). Table 4 shows the survival rate of each DCE on the 4th day of culture.

[0057]

[Table 4] It was shown that each DCE was most decomposed at 15 ° C.

The experiments in Examples 9 and 10 below
It performed with the apparatus shown in FIG. That is, a coiled stainless steel pipe 11 is put in a stainless pot 1 similar to the device of FIG. 1, gravel 2 and fine sand 3 are packed as in the device of FIG. 1, and sealed with a Teflon sheet 4, an injection pipe 5, and an exhaust pipe. The tube 6 and the temperature sensor 10 were embedded. The stainless steel pipe 11 is connected to the pump 8 and the temperature control device 9, and the inside thereof is filled with water. Water is circulated by the pump 8 and the temperature sensor 10
The temperature in the fine sand was monitored by and the target temperature was maintained by the temperature controller 9.

Example 9 (Effect of Gas Cooling on TEC Degradation Treatment in Soil of J1 Strain) An experiment was conducted in the same manner as in Example 1 using the apparatus shown in FIG. 13, and the phenol and TCE degradation of J1 strain was evaluated. did. Table 5 shows the survival rate on the 4th day of culture at each temperature.

[0060]

[Table 5] Similar to the case of Example 1, even in the liquid cooling system, both phenol and TCE showed the best decomposition when cooled to 15 ° C.

Example 10 (Effect of Gas Cooling on TEC Degradation Treatment in Soil of JM1 Strain) The same experiment as in Example 7 was conducted using the apparatus used in Example 9 to evaluate the TCE degradation of JM1 strain. Table 6 shows the survival rate on the 4th day of culture.

[0062]

[Table 6] As in the case of Example 1, the best TCE decomposition was observed in the liquid cooling system when cooled to 15 ° C.

[0063]

INDUSTRIAL APPLICABILITY The method of the present invention enables efficient biodegradation and repair of soil contaminated with aromatic compounds and / or organic chlorine compounds.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of an experimental apparatus used in Examples 1 to 8.

FIG. 2 is a diagram showing changes in the number of bacteria obtained in Example 1.

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

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

FIG. 5 is a diagram showing changes in the number of bacteria obtained in Example 4.

FIG. 6 is a diagram showing changes in phenol obtained in Example 4.

7 is a diagram showing changes in TCE obtained in Example 4. FIG.

FIG. 8 is a diagram showing changes in the number of bacteria obtained in Example 6.

9 is a diagram showing changes in phenol obtained in Example 6. FIG.

FIG. 10 is a diagram showing changes in TCE obtained in Example 6.

FIG. 11 is a diagram showing changes in the number of bacteria obtained in Example 7.

12 is a diagram showing changes in TCE obtained in Example 7. FIG.

FIG. 13 is a diagram showing an outline of an experimental apparatus used in Examples 9 and 10.

[Explanation of symbols]

 1 Stainless steel pot 2 Gravel layer 3 Fine sand (Sahara through sand) layer 4 Teflon sheet 5 Injection pipe 6 Exhaust pipe 7 Teflon tube 8 Pump 9 Temperature control device 10 Temperature sensor 11 Stainless pipe

─────────────────────────────────────────────────── ─── 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 (72) Inventor Chieko Mihara 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Yukitoshi Okubo 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (17)

[Claims] 1. A method for repairing and purifying the contaminated soil by contacting soil contaminated with at least one of an aromatic compound and an organic chlorine compound with a degrading microorganism capable of degrading the compound to decompose the compound. In, the temperature of the contaminated soil is maintained at a temperature lower than the optimum temperature for the growth and decomposition of the degrading microorganisms, and the magnitude and duration of the degrading activity of the degrading microorganisms are adjusted, whereby the organism of the contaminated soil is characterized. Repair purification method. 2. The method for biological remediation and purification of contaminated soil according to claim 1, wherein the temperature of the contaminated soil is maintained at a temperature lower than the optimum temperature by 5 to 10 ° C. 3. The biological remediation and purification method for contaminated soil according to claim 1, wherein the temperature of the contaminated soil is maintained within a range of 10 to 25 ° C. 4. The method for biological remediation and purification of contaminated soil according to claim 3, wherein the temperature of the contaminated soil is maintained in the range of 15 to 20 ° C. 5. The method for biological remediation and purification of contaminated soil according to claim 1, wherein the temperature of the contaminated soil is maintained by a gas or a liquid for heating or cooling introduced into the contaminated soil. 6. The method for biological remediation and purification of contaminated soil according to claim 5, wherein the gas is a gas containing oxygen or air. 7. The method for biological remediation and purification of contaminated soil according to claim 5, wherein the liquid is an aqueous medium. 8. The method according to claim 1, wherein the degrading microorganism is a microorganism that expresses oxygenase which is an oxidase. 9. 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 8, 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. 10. The J1 strain (FERM B
P-5102), The method for biological remediation and purification of contaminated soil according to claim 9. 11. The JM1 strain (FERM)
BP-5352), The method for biological remediation and purification of contaminated soil according to claim 9. 12. The method for biological remediation and purification of contaminated soil according to claim 9, wherein the microorganism is Pseudomonas species TL1 strain (FERM P-14726). 13. The method for biological remediation and purification of contaminated soil according to claim 9, wherein the microorganism is Alcaligenes species TL2 strain (FERM P-14642). 14. The biological remediation of a contaminated soil according to claim 1, which is carried out by introducing an aqueous medium containing the degrading microorganism into the contaminated soil and allowing the degrading microorganism to grow in the soil. Purification method. 15. The method for biologically remediating and purifying contaminated soil according to claim 14, wherein the introduction of the microorganism into the soil is performed by pressure from an injection well provided in the soil. 16. 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. 17. 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.