JPH09271749A - Biological restoration method of polluted soil by microorganism using suitable solute - Google Patents

Abstract

PROBLEM TO BE SOLVED: To apply a culture medium of high concentration to a wide range of areas to be restored and a lot of soil to be restored to obtain good restoration effect by bringing a culture medium for microorganisms and a suitable solute, together with microorganisms, into contact with polluted soil. SOLUTION: A culture medium for microorganisms and a suitable solute, together with microorganisms, are brought into contact with polluted soil polluted by a pollutant which is at least one of aromatic compounds such as phenol and organic chlorine compounds such as trichloroethylene to decompose the pollutant. The suitable solute has action of accelerating the adaptation of microorganisms having decomposition activity of aromatic compounds or organic chlorine compounds to a high concentration tissue culture medium and of improving the decomposition activity, and as such solute, for example, proline, betaine or the like is used. As the microorganisms, those in which an oxygenase which is an oxidizing enzyme appears, for example, those belonging to a corynebacterium genus are used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for biodegrading aromatic compounds and / or organic chlorine compounds in soil.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to organic chlorine compounds, which are harmful to living organisms and hardly decomposed, has become a serious problem. In particular, tetrachlorethylene (PC
E), trichlorethylene (TCE), dichloroethylene (DCE) and other organochlorine compounds are considered to have spread to a considerable extent, and many cases actually detected in environmental surveys have been reported. It is said that these organic chlorine compounds, which remain in the soil, dissolve in groundwater due to rainwater etc. and spread throughout the surrounding area. Since such compounds are suspected to have carcinogenicity and reproductive toxicity and are very stable in the environment, contamination of groundwater by these compounds, especially groundwater used as a source of drinking water, is large. It is considered a social problem.

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 the techniques necessary for purification have been developed. For example, adsorption treatment with activated carbon and decomposition treatment with light and heat have been studied, but they are not always practical in terms of cost and operability.

On the other hand, in recent years, decomposition of microbial compounds such as TCE, which is stable in the environment, has been reported.
Research is being started toward its practical application. 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 the labor and cost for maintenance can be reduced. And so on. For example, as a report of TCE-degrading bacteria, Welchi has been reported to be isolated by a microorganism capable of degrading organochlorine compounds.
a alkenophila sero 5 (US Pat. No. 4,877,736, ATCC5
3570), Welchia alkenophila sero 33 (US Pat. No. 4,873).
7,736, ATCC53571), Methylocystis sp. Strain M
[Agric. Biol. Chem., 53, 2903 (1989), Biosci. Bi
otech.Biochem., 56, 486 (1992), 56, 736 (199)
2)], Methylosinus trichosprium OB3b [Am. Chem. So
c. Natl. Meet. Dev. Environ. Microbiol., 29, 365
(1989), Appl. Environ. Microbiol., 55, 3155 (198
9), Appl. Biochem. Biotechnol., 28, 877 (1991), JP-A-02-92274, JP-A-03-292970], Methy
lomonas sp. MM2 [Appl. Environ. Microbiol., 57.,
236 (1991)], Alcaligenesdenitrificans ssp. Xyloso
xidans JE75 [Arch. microbiol., 154, 410 (199
0)], Alcaligenes eutrophus JMP134 [Appl. Enviro
n. Microbiol., 56, 1179 (1990)], Alcaligenes eutr
ophus KS01 (JP 07-123976), Mycobacteriu
m vaccae JOB5 [J. Gen. Microbiol., 82, 163 (197
4)], Appl. Environ. Microbiol., 54, 2960 (1989), A
TCC29678], Pseudomonas putida BH [Sewer Association Magazine,
24, 27 (1987)], Acinetobactor sp. Strain G4 [App
L. Environ. Microbiol., 52, 383 (1986), 53, 949.
(1987), 54, 951 (1989), 56, 279 (1990), 57, 1
93 (1991), U.S. Pat.No. 4,925,802; ATCC53617; the strain was originally classified as Pseudomonas cepacia.
inetobactor sp.), Pseudomonas mendoci
na KR-1 [Bio / Technol., 7, 282 (1989)], Pseudomon
as putida F1 [Appl. Environ. Microbiol., 54. 1703
(1988), ibid. 54, 2578 (1988)], Pseudomonas fluoresc.
ens PFL12 [Appl. Environ. Microbiol., 54, 2578 (1
988)], Pseudomonas fluorescens NRRL-B-18296 (U.S. Pat. No. 4,853,334), Pseudomonas putida KW1-9 (JP 06-70753 A), Pseudomonas cepacia KK01 (JP HEI)
06-227769), Nitrosomonas europaea [Appl. E
nviron. Microbiol., 56, 1169 (1990)], Lactobacill
us vaginalis sp. nov [Int. J. Syst. Bacteriol., 3
9, 368 (1989); ATCC49540] and the like are known.

[0005]

Recently, attempts have been made to actually repair soil contaminated with aromatic compounds or organic chlorine compounds by using these degrading bacteria, but in many cases In order to grow the degrading bacteria in the soil, which is an oligotrophic environment, and to maintain the degrading activity, a medium containing nutrients such as carbon source, nitrogen source and phosphorus source is simultaneously injected into the contaminated soil together with the degrading bacteria. There is a need to.

In order to effectively repair a contaminated soil in the widest possible area without increasing the arrangement density of the injection site by such a simultaneous injection of nutrients, the medium itself as well as the degrading bacteria within the area to be repaired at a certain concentration. It is desirable to inject into. However, when the area to be repaired is expanded, the diffusion distance of the medium from the injection position becomes longer, and as the diffusion distance becomes longer, the concentration of the medium decreases due to the water originally present in the environment. Therefore, in order to maintain a sufficient medium concentration even in the region away from the injection position, it is necessary to increase the medium concentration at the time of injection. In this case, although the optimum medium concentration is obtained at a point some distance from the injection point into the soil, there is no choice but to reach a considerably high concentration state in excess of the optimum value near the injection point.
Also, the original salt concentration of the contaminated soil itself may be high due to salt damage or desertification.

However, when the medium components are present in a high concentration state exceeding the optimum value, there is a problem that the growth and activity of microorganisms are suppressed or inhibited.

That is, many microorganisms have optimum ranges of nutrients (medium composition), pH, temperature, etc. which constitute the environment in which the microorganisms are placed. The salt concentration is a typical example, and it is known that the growth ability and enzyme activity of microorganisms are reduced or inhibited in an environment of salt concentration outside the optimum concentration range. For example, due to the high salt concentration of the medium,
When the osmotic pressure of the medium is higher than the osmotic pressure of the cells, water is leached from the cells into the medium, the cytoplasmic volume is reduced, and membrane damage or the like occurs. In this case, in Gram-positive bacteria, the cell membrane is detached from the cell wall, that is, cytoplasmic separation occurs,
In Gram-negative bacteria, the cell wall retracts along with the cell membrane. These are described in "Microbiology", First Edition, 5th Edition (R.
Y. Suetani, J. et al. L. Ingram, M.A. L. Willies, P. R. Painter co-author, Baifukan, p187-18
9) and "Skillful Lifestyle of Bacteria Living in Special Environments" (Future Biological Science Series 27, Riki Motomoto, Kyoritsu Shuppan, p25-37).

An object of the present invention is to apply a high-concentration medium to a wider area to be repaired or a larger amount of soil to be repaired in a method for biologically repairing soil contaminated with aromatic compounds and / or organic chlorine compounds. Another object of the present invention is to provide a method capable of obtaining a good repair effect.

[0010]

The method for biologically repairing a contaminated soil by a microorganism according to the present invention is to decompose the pollutant into a contaminated soil contaminated with at least one pollutant of an aromatic compound and an organic chlorine compound. In the method for biologically remediating a contaminated soil by decomposing the pollutant by contacting a possible microorganism, the medium for the microorganism and a compatible solute are contacted with the microorganism in the contaminated soil. It is characterized by

Compatible sol used in the present invention
utes) are substances that are drawing attention as being involved in the adaptation function of microorganisms to high salt concentration environments. Usually, the regulation function of adaptation to high salt concentration environment in microorganisms is as follows. First, increase (accumulation) of amino acids centering on glutamic acid and potassium ion (K ⁺ ) as its counter ion.
Can be seen. However, since excessive accumulation of potassium ions adversely affects intracellular metabolic activity,
As a measure for the next step, the metabolic system involving the accumulation of substances that do not adversely affect the metabolic function in cells is activated, and the accumulation of such substances becomes observed. Such accumulating substances are called compatible solutes, and zwitterionic type proline compounds and betaine compounds have been found as general examples thereof, and some polyols such as glycerol, trehalose, and sucrose can be exemplified. . These are "the skillful lifestyle of bacteria living in a special environment."
(Future Biological Science Series 27, Riki Motomoto, Kyoritsu Shuppan, p80-90) and "Diversity of Microbial Function" edited by Teruhiko Beppu, Academic Publishing Center, p427-436.

[0012] The researches on the compatible solute in the prior art mainly relate to the relationship between the salt concentration and the compatible substance and various metabolic functions related thereto, mainly on Escherichia coli and Salmonella, cyanobacteria, sulfur bacterium, green bacterium. Aeruginosa,
It is done only in limited microorganisms such as Rhizobium and Brevibacterium. That is, the present situation is that no study has been made on the effect of degrading harmful substances by adding compatible solutes to the medium.

According to the present invention, when such a compatible solute is added to a medium having a high concentration composition, the contaminant degrading bacteria can be adapted to a medium having a high concentration composition, and at the same time, contaminants, particularly aromatic compounds and organic chlorine compounds It was completed based on the new finding that the effect of improving the decomposition activity can be obtained.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of aromatic compounds to be decomposed in the present invention include phenol, toluene and cresol, and examples of organic chlorine compounds include trichloroethylene and dichloroethylene. .

Compatible solutes used in the method of the present invention include:
Among compatible solutes found in various microorganisms,
Any substance can be used without limitation as long as it has an action of promoting adaptation to a high-concentration composition medium of a microorganism having a decomposition activity of an aromatic compound or an organic chlorine compound and improving the decomposition activity thereof. Specific examples include proline, betaine (glycine betaine), glycerol, mannitol, trehalose, sucrose, ectoine, carnitine, choline, N.
-Carbamoyl-L-glutamine 1-amide, glucosyl glycerol, N-acetyl-β-lysine, galactosyl glycerol, and the like.
More than one species can be used. Among these, proline and betaine (glycine betaine) are particularly preferable.

The compatible solute is applied to the contaminated soil together with the microorganism for decomposing the pollutant and the culture medium thereof, and these are introduced individually or in a mixture of two or more thereof into the contaminated soil. The concentration of the compatible solute at least when the compatible solute and the medium are mixed and used can be appropriately selected within a range in which a desired effect is obtained, but for example, the upper limit thereof is 2.0% by weight, preferably 1. It is desirable that the lower limit is 0% by weight and the lower limit is 0.02% by weight, preferably 0.1% by weight.

The medium composition is selected according to the contaminant-decomposing bacteria to be used, and the composition amount can be appropriately selected according to the diffusion distance from the injection position, the amount of contaminated soil to be repaired, etc. It is possible to use a high-concentration composition of about 2 to 10 times, more preferably about 2 to 5 times the composition concentration usually used for culturing a microorganism for decomposing pollutants. Speaking of the salt concentration, for example, when the Na ⁺ (sodium ion) concentration is about 0.2 to 1.0 mol, more preferably about 0.2 to 0.5 mol, the effect can be more suitably exhibited.

As a microorganism for decomposing pollutants which can be used in the present invention, any microorganism can be used without limitation as long as it can decompose an aromatic compound or an organic chlorine compound to be decomposed, but among them, an oxygenase is used. Expressing microorganisms are preferred. For example, Corynebacterium (Co
rynebacterium), Pseudomonas
Genus, Acinetobacter genus, Alcaligenes genus, Vibrio genus, Nocardia genus, Bacillus genus, Lactobacillus genus, Achromobacter genus, Achromobacter genus, Arthrobacter (Arthrobacte
r) genus, Micrococcus genus, Mycobacterium genus, Methylosinas (Methylo)
sinus) genus, Methylomonas genus, Welchia genus, Methylocystis
Examples thereof include microorganisms belonging to the genera Nitrosomonas, Saccharomyces, Candida, and Torulopsis, and one or more of these microorganisms can be used.

As these microorganisms, those which can be obtained according to known literatures or from various depository institutions can be used.

For example, J1 strain (FERM BP-510
2), JM1 strain (FERM BP-5352), Pseudomonas sp. TL1 strain (Pseudomonas sp. TL)
1: FERM P-14726), Pseudomonas alcaligenes KB2 strain (Pseudomonas alcaligenes KB
2: FERM BP-5354), Pseudomonas cepacia KK01: FER
M BP-4235), Alcaligenes Species T
L2 strain (Alcaligenes sp. TL2: FERM P-146
42) and Vibrio species KB1 strain (Vibrio sp.
KB1: FERM P-14643) and the like. These strains are collected and isolated from soil or the like, and have an advantage that they can exhibit good TCE decomposing ability even in a poor environment such as soil. Strains having these FERM numbers have been deposited with the Ministry of International Trade and Industry, the Agency of Industrial Science and Technology, and the Institute of Biotechnology and Industrial Technology, and those with BP numbers are international deposits under the Budapest Treaty. The dates of these deposits are shown below. J1 stock: deposit date, May 25, 1994 (transferred from FERM P-14332 on May 17, 1995) JM stock: deposit date, January 10, 1995 (January 1, 1995
Transferred from FERMP-14727 on February 22) Pseudomonas cepacia KK01 strain: Deposit date, March 11, 1992 (FERM P- on March 9, 1993)
(Transferred from 12869) Pseudomonas species TL1 strain: deposit date, January 10, 1995 Pseudomonas alkaligenes KB2 strain: deposit date, November 15, 1994 (F December 25, 1995)
Transferred from ERM P-14644) Alcaligenes Species TL2 strain: deposit date, November 15, 1994 Vibrio Species KB1 strain: deposit date, November 15, 1994 JM1 strain is an aromatic compound and an organochlorine compound. Was obtained by mutating the J1 strain that oxidizes oxygenase with oxygenase, and is a mutant strain that constitutively expresses oxygenase without an inducer. The mycological properties of these 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.

In addition to these, for example, Welchia alkeno
phila sero 5 (US Pat. No. 4,877,736, ATCC53570),
Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736
No., ATCC53571), Methylocystis sp. Strain M [Agric.
Biol. Chem., 53, 2903 (1989), Biosci. Biotech. B
iochem., 56, 486 (1992), ibid. 56, 736 (1992)], Methy
losinus trichosprium OB3b [Am. Chem. Soc. Natl. M
eet. Dev. Environ. Microbiol., 29, 365 (1989), App
l. Environ. Microbiol., 55, 3155 (1989), Appl. Bio
Chem. Biotechnol., 28, 877 (1991), JP-A-02-92274
No. JP-A 03-292970], Methylomonas sp.
MM2 [Appl. Environ. Microbiol., 57., 236 (199
1)], Alcaligenes denitrificans ssp. Xylosoxidans
JE75 [Arch. Microbiol., 154, 410 (1990)], Alcali
genes eutrophus JMP134 [Appl. Environ. Microbio
l., 56, 1179 (1990)], Alcaligenes eutrophus KS01
(JP 07-123976), Mycobacterium vaccae JO
B5 [J. Gen. Microbiol., 82, 163 (1974)], Appl. En
viron. Microbiol., 54, 2960 (1989), ATCC29678], P
seudomonas putida BH [Sewer Association, 24, 27 (198
7)], Acinetobactor sp. Strain G4 [Appl. Environ.
Microbiol., 52, 383 (1986), 53, 949 (1987), 5
4, 951 (1989), 56, 279 (1990), 57, 193 (199)
1), U.S. Pat.No. 4,925,802; ATCC53617;
Although classified as Pseudomonas cepacia, Acinetobac
tor sp.), Pseudomonas mendocina KR-1
[Bio / Technol., 7,282 (1989)], Pseudomonas putida
F1 [Appl. Environ. Microbiol., 54.1703 (1988),
54, 2578 (1988)], pseudomonas fluorescens PFL12
[Appl. Environ. Microbiol., 54, 2578 (1988)], P
seudomonas fluorescens NRRL-B-18296 (U.S. Pat.
53,334), Pseudomonas putida KW1-9 (Japanese Patent Laid-Open No. 06-7075
3), Nitrosomonas europaea [Appl. Environ. M
icrobiol., 56, 1169 (1990)], Lactobacillus vagina
lis sp. nov [Int. J. Syst. Bacteriol., 39, 368 (19
89); ATCC49540] and other known TCE-degrading strains can be used.

Microorganisms for decomposing pollutants can be cultivated in an appropriate medium, and the resulting microbial cells can be used for contact treatment of contaminated soil. The culture medium for preparing the proliferating cells may be selected according to the microorganism used, and various culture forms such as liquid culture and solid culture can be selected. Further, culture conditions such as temperature, aeration, and stirring are appropriately selected according to the microorganism to be used. Usually, the culture temperature is about 15 to 30 ° C.

The soil remediation of the present invention is carried out by contacting the pollutant degrading microorganism, its medium and compatible solute with the contaminated soil. The simplest method of bringing them into contact is, for example, a method of directly introducing these components into the contaminated soil. As described above, the introduction of these components into the soil is carried out by individually introducing the microorganisms for decomposing pollutants, the culture medium and the compatible solute, and mixing two of these three components in advance, A method of introducing separately from the remaining components, or a method of previously mixing and introducing these three components can be used. As the aqueous medium when the microorganisms are introduced separately from the other components, for example, the medium used for the preparation of the growing bacterial cells can be used.

As the introduction method, there are a method of spraying on the soil surface and a method of using a well inserted in the ground in the case of treatment in a relatively deep formation. It is more effective to apply pressure with air or the like when introducing into the well, because these components can be permeated in a wide range. In this case, it is necessary to adjust the various conditions in the soil to be suitable for the degrading microorganisms, but the presence of soil particles or the like provides an environment suitable for the growth of the introduced microorganisms and often accelerates the growth. Therefore, the condition of being in soil is convenient. It is also possible to use a method in which a suitable carrier capable of giving a better growth environment to the microorganism is sprayed in the soil, or a method in which the microorganism is carried on the carrier and introduced into the soil.

Further, a microorganism for decomposing pollutants is supported on a suitable carrier, and the reaction tank is filled with a medium and a compatible solute, and the reaction tank is placed in a region to be repaired in soil, for example, in an aquifer. It is possible to carry out the decomposition treatment by introducing into. This reaction tank can be composed of various materials such as metal and plastic. For example, a fence-shaped or film-shaped structure in which a carrier supporting microorganisms is sandwiched between two sheets is It is desirable in that it can cover a wide range. The outer wall of this reaction tank has a structure that allows water permeability and air permeability, and for example, a sheet-like or film-like net material having a mesh of a size such that the filled carrier does not flow out to the outside, or a large number of holes. A sheet-shaped or film-shaped wall material or the like in which the space is opened can be used.

Even in the method using this reaction tank, after introducing the reaction tank into the soil, in the diffusion process of the medium or degrading bacteria from the inside of the reaction tank into the soil, or when ground water etc. Since the inflow causes the dilution of the medium, it is possible to maintain a good decomposition treatment against this dilution for a longer period by preliminarily making the medium a high concentration composition.

On the other hand, it is also possible to use a method in which a mixed solution based on an aqueous medium containing a microorganism for decomposing pollutants, its medium and a compatible solute is prepared and treated with contaminated soil. Also in this method, the microorganism can be immobilized on a suitable carrier before use. If the medium used at that time is made to have a high-concentration composition against the dilution due to the addition of contaminated soil, a better decomposition treatment can be performed.

In the above various methods, the carrier for supporting the microorganism may be any one as long as it has an excellent ability to retain the microorganism and does not impair the air permeability when introduced into the soil,
For example, various microbial carriers that have been widely used in bioreactors and the like conventionally used in the pharmaceutical industry, food industry, wastewater treatment system, etc. can be used as a material for providing a habitat space for microorganisms. As a specific example,
Porous glass, ceramics, metal oxide, activated carbon, kaolinite, bentonite, zeolite, silica gel,
Alumina, particulate carrier made of inorganic material such as anthrite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, carrier made of gelled substance such as agarose, gelatin, ion-exchangeable cellulose, ion Exchange resin, cellulose derivative, carrier made of glutaraldehyde, polyacrylic acid, polyurethane, polyester, etc., carrier made of natural cellulosic material such as cotton, hemp, paper, carrier made of natural lignin material such as wood flour, bark, etc. And one or more of these can be used.

The method of the present invention can be applied to both closed system and open system soil remediation, and in addition to a medium component for growth and a compatible solute, various compounds capable of further improving decomposition efficiency can be further used. You can also

[0030]

The present invention will be described in more detail with reference to the following examples. In the following examples, all “%” represent “% by weight”, and M9 medium (pH per liter,
The composition of 7.0) is as follows. Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g Water: balance Example 1 [Phenol decomposition treatment in soil in high salt concentration medium of J1 strain] Effect of L-Proline on Brown Forest Soil]
A J1 strain colony grown on M9 agar medium containing 0.2% yeast extract was inoculated into 200 ml of M9 medium containing 0.1% pyruvic acid and 0.02% yeast extract in a 500 ml Sakaguchi flask. Shaking culture was performed at 25 ° C. for 24 hours to obtain a preculture liquid.

A 5-fold and 2-fold concentration of M9 medium containing 600 ppm of phenol and 2% pyruvic acid was prepared, neutralized with sodium hydroxide, and a portion of L-proline had a concentration of 0.1%. So added. Next, 1 ml of each of these 4 types of medium was injected into a 15 ml volume sterilized tube, and 4 g of brown forest soil collected from Atsugi City, Kanagawa Prefecture was added thereto, and 1 of the above preculture liquid was added.
After inoculating 0 μl, it was cotton plugged. A large number of similar products were prepared, and these were statically cultured at 15 ° C., which is close to the actual temperature in soil, and the phenol concentration was measured daily.

As a method for quantifying phenol, 5 ml of sterilized water was added to a tube, and the phenol in the supernatant obtained by stirring and extracting for 1 minute was detected by the JIS method using aminoantipyrine (JIS K0102-199).
3, 28.1). The results expressed as the residual rate (%) with respect to the initial concentration are shown in FIG.

Example 2 [Effect of L-proline on TCE (trichloroethylene) decomposition treatment in soil in high salt medium of J1 strain (brown forest soil)] 50 ppm TCE, 300 ppm phenol as a TCE decomposition inducer, and A 5-fold and 2-fold concentration M9 medium containing 0.1% pyruvic acid was prepared. A large number of 1 ml of each of these media was poured into a screw vial bottle with a Teflon inner lid having a volume of 15 ml, and 4 g of brown forest soil similar to that of Example 1 was added to all the bottles. Next, for each of these,
Pre-culture was carried out by adding L-proline in an amount similar to that of Example 1 such that the amount of L-proline contained in each of the 9 concentrations was 0.1% and the amount of L-proline not contained was the same. 0.1 ml of the liquid was inoculated, then completely sealed, and statically cultivated at 15 ° C., which is close to the actual temperature in soil.

The amount of TCE was determined by adding 5 ml of n-hexane to the bottle and stirring and extracting for 3 minutes, diluting the supernatant 10 times, and quantifying by gas chromatography (ECD detector). The decrease in TCE was measured daily. As a control, TCE was also quantified in a system in which the J1 strain was not added in the same experimental system, and the residual rate (%) with respect to the TCE amount of the control was determined. The results are shown in FIG.

Example 3 [Effect of L-proline on DCE (dichloroethylene) decomposition treatment in soil in high-salt concentration medium of J1 strain (brown forest soil)] The substance to be decomposed was replaced by TEC and ci.
s-1,2-dichloroethylene (cis-1,2-DCE), t
trans-1,2-dichloroethylene (trans-1,2-DC
E) and 1,1-dichloroethylene (1,1-DCE) were individually used, and the decrease in DCE was measured in the same manner as in Example 2 except that the concentration of each was 30 ppm. Table 1 shows the residual rate (%) of each DCE on the 7th day of culture.

[0036]

[Table 1] Example 4 [Effect of betaine on phenol and TCE degradation treatment in soil in high salt medium of J1 strain (brown forest soil)] Instead of using 0.1% L-proline as a compatible solute, 0.2 % Examples 1 and 2 except using% betaine
Similarly, the effect of betaine on the phenol and TCE decomposition treatment in soil under the high salt concentration medium condition of strain J1 was confirmed. Table 2 shows the residual ratio (%) of phenol and TCE on the 5th day of culture.

[0037]

[Table 2] Example 5 [Effect of L-proline on Phenol Decomposition Treatment in Soil in High Salt Concentration Medium of KK01 Strain (Brown Forest Soil)] Pseudomonas cepacia grown on M9 agar medium containing 0.2% of yeast extract. KK01 strain (FERM B
P-4235) colonies were inoculated into 200 ml of M9 medium containing 0.2% of sodium glutamate and 0.02% of yeast extract in a 500 ml Sakaguchi flask, and the temperature was 30 ° C.
Culture was carried out with shaking for 18 hours to obtain a preculture liquid.

A 5-fold and 2-fold concentration of M9 medium (pH 7) containing 800 ppm of phenol and 1% sodium glutamate was prepared, and L-proline was added to a part of the medium to a concentration of 0.2%. Next, these 4
Inject 1 ml each of the seed medium into a 15 ml sterile tube, add 4 g of brown forest soil collected from Atsugi City, Kanagawa Prefecture, and further add 10 μ of the above preculture liquid.
l was inoculated and then cotton plugged. Prepare a lot of similar things,
These were statically cultured at 20 ° C., and the phenol concentration was measured daily.

As a method for quantifying phenol, 5 ml of sterilized water was added to a tube, and the phenol in the supernatant extracted by stirring for 1 minute was detected by the JIS method using aminoantipyrine (JIS K0102-199).
3, 28.1). The results expressed as the residual ratio (%) with respect to the initial concentration are shown in FIG.

Example 6 [Effect of L-Proline on Cresol and Toluene Decomposition Treatment in Soil in High Salt Concentration Medium of KK01 Strain (Brown Forest Soil)] Decomposition targets were o-cresol and m-
Effect of L-proline on cresol and toluene decomposition treatment in soil under high salt concentration medium condition of strain KK01 in the same manner as in Example 5 except that cresol, p-cresol (600 ppm each) and toluene (100 ppm) were changed. It was confirmed. When toluene was used, a Teflon sheet was used as the inner lid of the tube, and the tube was tightly sealed so that gas did not leak.

The amount of each cresol was determined by the JIS method using p-hydrazinobenzenesulfonic acid (JI
SK0102-1993, 28.2), and the amount of toluene was determined by a method of measuring toluene in a gas sampled from the head space of the tube by gas chromatography. Table 3 shows the residual rate (%) of each aromatic compound on the 4th day of culture.

[0042]

[Table 3] Example 7 [TCE in soil in high salt medium of strain KK01]
Effect of L-proline on degradation (brown forest soil)]
A 5-fold concentration and 2-fold concentration M9 medium containing 50 ppm of TCE, 300 ppm of phenol as a TCE decomposition inducer, and 1% sodium glutamate was prepared. A large number of 1 ml of each of these media was poured into a screw vial bottle with a Teflon inner lid having a volume of 15 ml, and 4 g of brown forest soil similar to that of Example 1 was added to all the bottles. Next, for each of these M9 concentrations, L-proline was added so that the content of L-proline was 0.2% and the content of L-proline was not the same.
In each bottle, 0.1% of the preculture liquid prepared in the same manner as in Example 1 was used.
After inoculation with 1 ml, the cells were completely sealed and statically cultured at 20 ° C.

The amount of TCE was determined by adding 5 ml of n-hexane to a bottle, stirring and extracting for 3 minutes, diluting the supernatant 10 times, and quantifying by gas chromatography (ECD detector). The decrease in TCE was measured daily. As a control, TCE was also quantified in the same experimental system without the addition of the KK01 strain.
The residual rate (%) with respect to the amount was determined. FIG. 4 shows the results.

Example 8 [Effect of betaine on phenol and TCE degradation treatment in soil in high salt medium of strain KK01 (brown forest soil)] As a compatible solute, 0.2% L-proline was used instead of 0.2% L-proline. The effects of betaine on phenol and TCE decomposition treatment in soil under high salt concentration medium conditions of strain KK01 were confirmed in the same manner as in Examples 5 and 7 except that 5% betaine was used. Table 4 shows the residual rate (%) of phenol and TCE on the 4th day of culture.

[0045]

[Table 4] Example 9 [Effect of L-proline on TCE and DCE decomposition treatment in soil in high salt medium of JM1 strain (fine sand)] Cultured on M9 agar medium containing 1.0% sodium malate The obtained JM1 strain colony was inoculated into 200 ml of a liquid medium of the same composition in a Sakaguchi flask having a volume of 500 ml, and shake culture was performed at 22 ° C. for 20 hours to obtain a preculture liquid.

TCE (50 ppm) as a decomposition target,
Decomposition treatment was performed using cis-1,2-DCE, trans-1,2-DCE, and 1,1-DCE (30 ppm each) individually.

That is, the decomposition target with a predetermined concentration and 2.0
% And double concentration M9 containing 10% sodium malate
A medium was prepared. A large number of 1 ml of each of these media was poured into a screw vial bottle with a Teflon inner lid having a capacity of 15 ml, and 5 g of "Sahara's continuous sand" was added to all the bottles. Next, with respect to these, L-proline was added to each of the M9 concentrations so that the amount of L-proline was 0.1% and the amount of L-proline was not, and the pre-culture was carried out in each bottle. Inoculate with 0.1 ml of the liquid and then completely seal it.
The culture was allowed to stand at 0 ° C.

The amount of TCE was determined by adding 5 ml of n-hexane to the bottle, stirring and extracting for 3 minutes, and diluting the supernatant 10 times if necessary, and quantifying by gas chromatography (ECD detector). The decrease in TCE was measured daily. As a control, TCE was quantified in a system in which the JM1 strain was not added in the same experimental system, and the residual rate (%) with respect to the TCE amount of the control was determined. Table 5 shows the residual rate (%) on the 5th day of culture.

[0049]

[Table 5] Example 10 [Effect of betaine on TCE and DCE decomposition treatment in soil in high salt medium of JM1 strain (fine sand soil)]
As a compatible solute, 0.3% instead of 0.1% L-proline
% JM1 in the same manner as in Example 9 except that% betaine was used.
TCE and D in soil under high salt medium conditions
The effect of betaine on CE degradation treatment was confirmed. Table 6 shows the residual rate on day 7 of culture.

[0050]

[Table 6] Example 11 [Effect of L-proline on TCE degrading treatment in soil under high salt concentration medium condition by other degrading bacteria (loam soil)] As a degrading bacterium to be used, any aromatic compound assimilating TCE degrading bacterium Pseudomonas species TL1 strain, Alcaligenes species TL2
The following operations were performed using the strains, Vibrio species KB1 strain, and Pseudomonas alcaligenes KB2 strain, respectively.

First, as a preculture, yeast extract 0.2%
Inoculate 200 ml of liquid medium of the same composition in a 500 ml Sakaguchi flask with a colony on M9 agar medium containing
Shaking culture was performed at 22 ° C. for 36 hours.

Next, the TCE (50 pp
m) and phenol (200 p as an inducer of TCE decomposition)
The M9 medium containing 0.2% yeast extract containing pm) and 5 times concentration and 2 times concentration was prepared. 1 ml of each of these two types of medium was poured into a screw vial bottle with a Teflon inner lid having a volume of 15 ml, and 4 g of loam soil collected from Ibaraki Prefecture was added to prepare a large number. Next, for each of these M9 concentrations, L-proline was added so that 0.2% of L-proline was contained and the same number was not contained. Furthermore, 0.1 ml of the above-mentioned preculture liquid of the microorganism was inoculated into each vial, completely sealed, and statically cultured at 20 ° C.

The amount of TCE was determined by adding 5 ml of n-hexane to the bottle, stirring and extracting for 3 minutes, diluting the supernatant 10 times, and quantifying by gas chromatography (ECD detector). The decrease in TCE was measured daily. As a control, TCE was also quantified in a system in which the strain was not added in the same experimental system, and the residual ratio (%) to the TCE amount of the control was determined. Table 7 shows the residual rate on the 4th day of culture.
Shown in

[0054]

[Table 7] Example 12 [Effect of betaine on TCE degradation treatment in soil under high salt concentration medium condition with other degrading bacteria (loam soil)] As a compatible solute, 0.5% betaine was used instead of 0.2% L-proline. In the same manner as in Example 11 except that TCE in the soil under high salt concentration medium conditions was used.
The effect of betaine on the degradation process was confirmed. Table 8 shows the residual rate on day 7 of culture.

[0055]

[Table 8]

[0056]

EFFECTS OF THE INVENTION The present invention enables efficient biodegradation / restoration of soil contaminated with aromatic compounds and / or organic chlorine compounds.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of L-proline on the phenol degradation of strain J1.

FIG. 2 shows the effect of L-proline on TCE degradation of J1 strain.

FIG. 3 shows the effect of L-proline on the phenol degradation of strain KK01.

FIG. 4 shows the effect of L-proline on TCE degradation of strain KK01.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinya Furusaki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (17)

[Claims] 1. The polluted soil is decomposed by contacting a polluted soil polluted with at least one pollutant of an aromatic compound and an organic chlorine compound with a microorganism capable of degrading the pollutant, thereby contaminating the polluted soil. A method for biologically repairing contaminated soil, comprising: contacting a contaminated soil with a medium for the microorganism and a compatible solute together with the microorganism. 2. The method for bioremediating a contaminated soil according to claim 1, wherein the compatible solute is proline or betaine. 3. The method for biologically repairing contaminated soil according to claim 2, wherein the microorganism expresses an oxygenase, which is an oxidase. 4. The microorganism expressing the oxygenase is Corynebacterium, Pseudomonas, Acinetobacter, Alcaligenes, Vibrio, Nocardia, Bacillus, Lactobacillus, Achromobacter, Arthrobacter, Micrococcus, Mycobacterium, Methylosynas, Methylomonas,
The method for biologically remediating a contaminated soil according to claim 3, which is a microorganism belonging to any one of the genus Berchia, the genus Methylocistis, the genus Nitrozomonas, the genus Saccharomyces, the genus Candida, and the genus Tollopsis, and one or more of these microorganisms is used. 5. The method according to claim 4, wherein the microorganism is one or more selected from the group consisting of Corynebacterium species, Pseudomonas species, Pseudomonas alcaligenes, Pseudomonas cepacia, Alcaligenes species and Vibrio species. A method for biologically repairing contaminated soil as described. 6. The J1 strain (FERM BP)
-5102), JM1 strain (FERM BP-535)
2), Pseudomonas species TL1 strain (FERM
P-14726), Pseudomonas alcaligenes KB2 strain (FERM BP-5354), Pseudomonas cepacia KK01 strain (FERM BP-423).
5), Alcaligenes Species TL2 strain (FERM
P-14642) and Vibrio species KB1 strain (FERM P-14643), which is one or more selected from the group consisting of the following. 7. The method for biologically repairing contaminated soil according to claim 1, wherein a mixture of the medium and the compatible solute is used in a mixed manner. 8. The method for bioremediating contaminated soil according to claim 7, wherein the concentration of the compatible solute in the mixture is in the range of 0.01% to 5.0%. 9. The method of bioremediating contaminated soil according to claim 8, wherein the concentration of said compatible solute in said mixture is in the range of 0.1% to 1.0%. 10. The method for biologically repairing contaminated soil according to claim 9, wherein the salt concentration of the medium is in the range of 0.2 to 1.0 molar in terms of Na ⁺ (sodium ion) concentration. 11. The method for biologically repairing contaminated soil according to claim 9, wherein the salt concentration of the medium is in the range of 0.2 to 0.5 mol in terms of Na ⁺ (sodium ion) concentration. 12. The contact between the contaminated soil and the microorganism is carried out by introducing the microorganism, the medium and the compatible solute into the contaminated soil, and growing the microorganism in the contaminated soil. Item 12. A biological repair method for contaminated soil according to any one of Items 1 to 11. 13. Introducing the microorganism into a contaminated soil,
The method for bioremediating a contaminated soil according to claim 12, wherein the method is performed by pressure from an injection well. 14. The method according to claim 1, wherein the contaminated soil is contacted with the microorganism by introducing the contaminated soil into a liquid phase containing the microorganism, the medium and the compatible solute. Biological remediation method for contaminated soil. 15. The method for biologically repairing contaminated soil according to claim 1, wherein the carrier supporting the microorganism is brought into contact with the contaminated soil. 16. The method for bioremediating a contaminated soil according to claim 1, wherein the aromatic compound is one or more compounds selected from phenol, toluene and cresol. 17. The method for biologically repairing contaminated soil according to claim 1, wherein the organic chlorine compound is at least one of trichlorethylene and dichloroethylene.