JP2003153686A - New microorganism and method for degrading persistent organochlorine compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a new microorganism having an excellent degrading ability for persistent aromatic compounds, especially chlorinated dioxins without requiring new other carbon sources as a nutrient source and a method for industrially and advantageously degrading the persistent aromatic compounds using the new miroorganism. SOLUTION: This NK32 strain of the genus Corynebacterium, especially a new strain has a degrading ability for the persistent aromatic compounds.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism having excellent decomposability of a persistent organochlorine compound, particularly dioxins, and a method for decomposing the persistent organochlorine compound using the microorganism.

[0002]

2. Description of the Related Art Persistent halogenated aromatic compounds, especially dioxins, exhibit extremely high toxicity and teratogenicity to the human body, and because of their hydrophobic nature, they are bioconcentrated by the food chain. In recent years, these dioxins are unintentionally produced during the production process of chemical substances and the combustion process of waste,
Global environmental pollution that diffuses through incinerated ash and leachate from waste disposal sites is a major social problem. Although dioxins can be decomposed by heat treatment or chemical treatment, they are present in the environment, especially in soil or bottom mud of rivers, at low concentrations, and when the treatment is performed by the above method. Is inefficient and economically burdensome.

In order to solve such a problem, a method of decomposing diiochicin chloride using a microorganism has been considered,
There have been reports on some microorganisms having dioxin decomposing ability such as Pseudomonas, Sphingomonas and Terrabactor. However, the conventionally known decomposition products of dioxin chlorides have the following problems. Since co-metabolism is involved as a decomposition mechanism, it is necessary to supply another carbon source as its nutritional source. Poor resolution of highly chlorinated dioxins.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object of the present invention is not to require another new carbon source as a nutritional source, and in particular, a persistent aromatic compound. It is an object of the present invention to provide a novel microorganism excellent in decomposing ability of dioxins chloride and an industrially advantageous decomposition method of a hardly decomposable aromatic compound using the same.

[0005]

[Means for Solving the Problems] The inventors of the present invention searched for new microorganisms effective for solving the above problems in soil and activated sludge of industrial wastewater treatment plants all over the country, and found that Corynebacterium sp. It was found that the specific strains of the above were effective.

[0006] That is, according to the present invention, firstly, a Corynebacterium NK32 strain is provided. Secondly, the strain according to the first aspect is provided, which has the ability to decompose persistent aromatic compounds. Thirdly, there is provided the strain according to the second, wherein the hardly decomposable aromatic compound is dioxins. Fourth, there is provided the strain according to the third, wherein the dioxins are chlorinated dioxins. Fifth, there is provided a method for decomposing a hardly-decomposable aromatic compound, which comprises using the strains according to the first to fourth aspects. Sixth, the decomposition method according to the fifth is characterized in that the hardly decomposable aromatic compound is a dioxin. Seventh, there is provided the decomposition method according to the sixth, wherein the dioxins are chlorinated dioxins.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The novel microorganism of the present invention can be obtained by separating it from nature by the following steps. Soil nationwide 30 locations, samples were taken approximately 50 points, such as activated sludge industrial waste water treatment plant, first, minimal medium 20ml test tube (composition: 1000 ml per _{4.1 g Na 2 HPO 4 · 2H} 2 0; 0.4
g KH ₂ PO ₄ ; 0.5 g (NH ₄ ) ₂ SO ₄ ; 0.1 g MgCl ₂・ 6H ₂ O; 5
_{0 mg Ca (NO 3) 2} · 4H 2 O; 20 mg Fe (NH 4) 2 - citrate, 0.1m
la trace element solution without EDTA) and dibenzofuran at a final concentration of 0.2 mM, add about 0.1 g of the sample and stir. Incubate this at 30 ° C for about 1 week, observe the change in the color and turbidity of the culture medium in a test tube, collect 20 μl from the changed culture medium, and inoculate it into the medium of the above composition. Then, this operation was repeated 5 times to search for a bacterium that decomposes and assimilates dibenzofuran. The bacteria extracted by culturing are single colonyed on a minimal agar medium containing 0.2 mM dibenzofuran. This single bacterium is cultured in a liquid minimal medium containing 0.1 mM unchlorinated dioxin for about 7 days at 30 ° C. with shaking. Decomposition of dioxin from these strains
The strain obtained as having assimilation ability is the novel microorganism of the present invention and is named Corynebacterium urealyticum NK32. This strain was isolated from a sample (soil) collected in Ibaraki prefecture, and has been deposited at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM P-18501.

Next, the bacteriological properties of the strain according to the present invention are shown below. A. Morphology V-form of bacillus Size (μm): width 1.0, length 1.5 to 3.0 B. Growth condition in medium (1) Growth temperature range: Optimal growth temperature range is 30 degrees C. Physiological and biochemical properties (1) Gram reaction: + (2) Motility:-(3) Aerobic growth: + (4) Anaerobic growth:-(5) Catalase reaction: + (6) Oxidase reaction: + (7 ) O / F test:-(8) Growth temperature: NT (9) Sulfate reduction:-(10) Pyrosinamidase: + (11) Pyrrolidonyl allyl amidase:-(12) Alkaline phosphatase:-(13) β- Glucuronidase:-(14) β-galactosidase: + (15) α-glucosidase:-(16) N-acetyl-β-glucosaminidase:-(17) Esculin (glucosidase): + (18) Urease: + (19) Gelatin Liquefaction:-(20) Fermentability of carbohydrates Glucose:-Mannitol:-Ribose:-Xylose:- Maltose: - lactose: - sucrose: - (21) Glycogen: - (22) urease hydrolyzable: + (23) Acid production from carbohydrates: +

The above-mentioned various properties are described by Berjay's manual.
8th Edition of Bergen's Manual of Determinative Bacteriology,
Based on the above properties, it was confirmed that this bacterium belongs to the genus Corynebacterium and corresponds to Corynebacterium urealyticum. However, this bacterium is different from known strains belonging to Corynebacterium urealyticum in yellow pigment production, β-galactosidase positive, esculin hydrolysis positive, and the like, and is considered to be a novel strain. The bacterium can be stored, for example, after growing it in LB medium, adding 750 μl of the bacterium to 350 μl of glycerin, stirring it well, and maintaining it at −80 ° C.

The novel microorganism according to the present invention exhibits excellent assimilation ability and decomposition ability with respect to a hardly decomposable aromatic compound.
The target hardly-decomposable aromatic compound includes all compounds having an aromatic ring, may be either a homocyclic ring or a heterocyclic ring, and may be substituted with a halogen, a hydroxyl group, a carboxyl group or the like. Good.

Examples of the monocyclic ring include benzene, benzene having a substituent such as chlorobenzene, benzoic acid, phenol, catechol, salicylic acid, indole and toluene. Examples of the heterocycle include dibenzothiophene; fluorene-9-one; xanthene; biphenyl; fluorene; benzophenone; xanthene-9-one; anthrone; naphthalene; dibenzosuberenone.

The hardly decomposable aromatic compounds to which the present invention is applied include dioxines represented by dibenzofuran, PCDDs (polychlorinated dibenzodioxins) and PCDFs (polychlorinated dibenzofurans).

In particular, the novel microorganisms according to the present invention, as will be shown in the Examples below, are in the environment with chlorinated dioxins represented by PCDDs (polychlorinated dibenzodioxins) and PCDFs (polychlorinated dibenzofurans). , Which is said to coexist with dibenzo-p-dioxin, is a highly toxic chlorinated dioxin, and is dioxins in which chlorine is substituted at any of positions 2, 3, 7 and 8. It has excellent resolution.

Therefore, the novel microorganism according to the present invention does not require any other new carbon source as a nutrient source as in the conventional case, and chlorine is substituted at any position of 2, 3, 7 and 8. Since dioxins can be decomposed efficiently, it can be said to be extremely useful industrially.

[0015]

EXAMPLES The properties of the novel microorganism according to the present invention will be described below based on the test results.

(1) Dioxin assimilation ability Dioxin assimilation ability was determined by the following test. The strain is inoculated into 20 ml of a liquid minimal medium containing 0.1 mM dibenzo-p-dioxin and shake-cultured at 30 ° C. culture
A culture solution is collected every 24 hours, and the turbidity of the culture solution is measured by a spectrophotometer [HP8452 manufactured by Hurret Packard], and the quality of the growth of the bacteria is observed to determine whether or not there is an assimilation ability. . As a control, Corynebacterium gl
The inoculated utamicum IAM 12435 was shake-cultured. The results of this test are shown in FIG. The strain Corynebacterium urealyticum according to the present invention (Corynebacterium ur
ealyticm) NK32 strain is a known Corynebacterium glutamic
Unlike um IAM 12435, it is recognized that it grows with dibenzofuran as a nutrient source, which shows that it has an excellent assimilation ability for dioxin.

(2) Assimilation capacity of aromatic compounds The assimilation capacity of aromatic compounds was determined by the following test. Inoculate the strain in a minimal liquid medium containing 0.2 mM of aromatic compounds and incubate at 30 ° C for about 72 hours. After culturing for 72 hours, the quality of the growth of the bacterium is observed and the presence or absence of assimilation ability is judged. The results of this test are shown in Table 1,
The strain Corynebacterium urealyticm NK32 strain according to the present invention is a monocyclic aromatic compound such as toluene, benzene or phenol, together with a dicyclic thiophene, naphthalene, a bicyclic aromatic compound such as biphenyl, etc. Growth was recognized with the nutrient source. Therefore, it has the ability to assimilate a wide range of compounds,
It can be seen that it has a better assimilation ability of aromatic compounds than other strains already known.

[0018]

[Table 1]

(3) Dioxin Chloride Decomposition Test This test was conducted to investigate the decomposability of various dioxin isomers, and the procedure is as follows. One platinum loop of the bacterium of the present invention (Corynebacterium urealyticm NK32) is collected from a petri dish and added to a culture flask (capacity 100 ml) containing 50 ml of a minimal medium. 7 at 30 ° C
Incubate with shaking (120 rpm) for 2 hours. This culture solution is 6,000rp
Centrifuge at m for 15 minutes, collect the cells, and wash 3 times with phosphate buffer (pH 7.5). And 5 ml of minimal medium
Add 1 ppm of dioxin chloride to a culture flask (capacity 50 ml) containing and add the above cells. This is shake-cultured at 30 ° C. for 7 days.

After culturing, 5 ml of ethyl acetate is added to the culture broth to extract dioxin chloride. Of the obtained extract, 1 ml is concentrated by jetting nitrogen gas. Dioxin chloride in this solution was analyzed for gas chromatograph mass spectrometer (GC-MS) [Hewlett-Packard HP58
90, HP5971A]. A column DB5 with an inner diameter of 0.25 mm and a length of 30 m was used as the column. Temperature rising condition is 8
The temperature is raised from 0 to 280 ° C. in 10 minutes. The flow rate of helium gas is 0.8 ml / min. A commercially available standard product can be used to identify and quantify the decomposition components of dioxin chloride. The decomposition rate in this test was calculated by the following formula, and the results of the decomposition test are shown in Table 2.

[0021]

[Formula 1] Decomposition rate (%) = (Detected chlorinated dioxin amount /
Added dioxin amount) x 100

[0022]

[Table 2]

Generally, there are 75 kinds of isomers depending on the number and position of chlorine substitution in dioxin chloride, and the highly toxic one is 2,3,7,8, especially di-, It is a tritetra form, and it is said that the ability of microorganisms to decompose dioxin decreases as the number of chlorine in the dioxin molecule increases, but this bacterium has a chlorine substitution number (mono substitution) of 1 or more. It can be seen that even in the case of a plurality of (poly-substituted), that is, a highly toxic dioxin, its decomposition ability is improved. Further, it is understood that it also has a decomposability with respect to trichlorination.

[0024]

As described above, the novel microorganism according to the present invention has the ability to assimilate dioxin and many kinds of aromatic compounds and also has a high ability to decompose dioxin chloride. It can be grown and maintained in contaminated water or contaminated soil without adding any carbon source. Therefore, it can be used for decomposing and treating dioxin chloride that pollutes river water, sewage, etc., and for cleaning soil and the like contaminated with dioxin chloride. Further, this novel microorganism has degradability with respect to high chloride, for example, dichlorinated chloride having a degree of trichlorination, and can be used for treating contaminated water and contaminated soil containing high chloride, and further has toxicity. It can be used for decomposing dioxin chloride with chlorine substituted at the 2,3,7,8 position.

[Brief description of drawings]

[Fig. 1] Corynebacterium ureaticum that grows with dioxin as a nutrient source
alyticm) NK32 strain growth curve graph.

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) C07D 319/24 C12R 1:15 // (C12N 1/20 B09B 3/00 ZABE C12R 1:15)

Claims (7)

[Claims] 1. A Corynebacterium NK32 strain. 2. The strain according to claim 1, which has the ability to decompose persistent aromatic compounds. 3. The strain according to claim 2, wherein the hardly decomposable aromatic compound is dioxins. 4. The strain according to claim 3, wherein the dioxins are chlorinated dioxins. 5. A method for decomposing a hardly decomposable aromatic compound, which comprises using the strain according to any one of claims 1 to 4. 6. The decomposition method according to claim 5, wherein the hardly decomposable aromatic compounds are dioxins. 7. The decomposition method according to claim 6, wherein the dioxins are chlorinated dioxins.