JPH11341978A - New fungus having dioxin decomposing ability and decomposition of dioxin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a dioxins-decomposing fungus having excellent dioxin decomposing and removing efficiency and to provide a method for decomposing dioxins. SOLUTION: This fungus is Fusarium avenaceum (Corda Fries) Saccardo 65 strain having dioxins-decomposing ability, Fusarium avenaceum (Corda Fries) Saccardo PC×65 strain having dioxins-decomposing ability, Peniophora 267 strain having dioxins-decomposing ability or Peniophora PC×267 strain having dioxins-decomposing ability. The method for decomposing dioxin comprises using the fungus or a fungus belonging to the family Coeticiaceae.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism capable of decomposing dioxin and a method for decomposing dioxin.

[0002]

2. Description of the Related Art In recent years, environmental pollution by dioxins has become a problem. In particular, in the field of incineration of waste, etc., dioxin contained in the exhaust gas is released to the field, causing dioxin contamination of the soil, or dioxin mixed in incineration ash remains unchanged. By being transported to the final disposal site for incinerated ash, it causes dioxin contamination of the surrounding soil and groundwater.

[0003] Dioxin, which is a kind of organochlorine compound, is a general term for a compound having a skeleton structure in which two benzene rings are linked by one or two oxygen atoms (dibenzofuran or dibenzo-p-dioxin). By substituting chlorine atoms for these skeletons, there are theoretically 135 and 75 isomers respectively [Paper and Pulp Technical Journal 45 (8), p887, 1991].
Year〕. It is said that dioxin is generally highly toxic, chemically stable, not easily decomposed, and has a high fat solubility, and therefore easily accumulates in living tissues. However, its toxicity varies greatly depending on the chlorine substitution position and the number of substituted chlorines, and some dioxins have extremely low toxicity. Among dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8
-TCDD) is the most toxic (ALPHA, No.
5, 2, 1991).

[0004] Known techniques for decomposing and removing such dioxins include a complete combustion method (melt solidification method), a thermal decomposition method (heat dechlorination method), a pelletizing firing method, a photolysis method, and various chemical methods. A decomposition method and a supercritical water treatment method have been reported. However, it is said that it is difficult to apply the above-mentioned known technology to dioxin treatment in contaminated soil or in the vicinity of a final disposal site of incinerated ash due to its small amount and widespread dispersion.

As described above, a technique utilizing microorganisms (bioremediation technique) has been reported as a method for decomposing dioxin which is minutely and widely dispersed in the environment. For example, Valli et al. (1) Phanelocaete chrysosporium (Ph), a kind of wood-rot fungi,
anerochaete chrysosporiu
m: hereinafter, abbreviated as PC bacterium) to decompose and remove 2,7-dichlorodibenzo-p-dioxin (hereinafter, abbreviated as 2,7-DCDD), which is a kind of dioxin; and 2) It was shown that enzymes involved in lignin degradation produced by the PC bacterium, ie, lignin peroxidase (LiP) and manganese peroxidase (MnP) are involved in the degradation of 2,7-DCDD [J. Bacteriology, 174
(7), p2131, 1992].

A bacterium capable of producing an enzyme involved in lignin degradation, that is, a dioxin-degrading bacterium, is a dye Remazol Brilliant Blue R (Remaz).
olbrilliant blue R), and has the property of decolorizing three types of bacteria (563) from the field.
Strain, V1 strain, and V2 strain).
Species and three species of wood rot fungi having lignin decomposability [Coriolus versicola (Coriolus versic)
color), Fusarium Solani
solani) and PC bacteria]
Examination of the CDD resolution revealed that all of the six bacteria had 2
It is reported to have a 7-DCDD resolution [Paper and Pulp Technical Journal, 50 (12), p122, 1996].
Year〕.

[0007]

However, the dioxin-degrading ability of the dioxin-degrading bacteria reported so far has not reached a sufficient level. There has been a demand for a bacterium having a higher dioxin decomposition / removal efficiency than known dioxin-degrading bacteria. Therefore, an object of the present invention is to provide a novel dioxin degrading bacterium having excellent dioxin decomposing and removing efficiency, and to further provide a dioxin decomposing method.

[0008]

That is, the present invention provides:
(1) Fusarium avenaceum (corda: fries) sacchard having dioxin resolution [Fusari
um avenaceum (Corda: Fries)
Saccardo] 65 strains [hereinafter simply referred to as "65 strains"
(2) Fusarium avenaceum (corda: fryings) sacchard having dioxin resolution [Fusarium avenaceum (C
orda: Fries) Saccardo] PC × 65
Strain (hereinafter sometimes simply referred to as “PC × 65 strain”), (3) Kawatake (P.
eniphora) [hereinafter referred to simply as “2
67 strains], and (4) Kawatake mushroom (Peniophora) having dioxin decomposability
Of the genus PC × 267 (hereinafter sometimes simply referred to as “PC × 267 strain”).

[0009] The present invention also relates to a method for decomposing dioxin, comprising using one or more of the above microorganisms. Furthermore, the present invention relates to
The present invention also relates to a method for decomposing dioxin, which comprises using a microorganism belonging to the genus Titicaceae (particularly, a microorganism belonging to the genus Kawai (Peniophora)).

In the present specification, “dioxin” is a polychlorodibenzo-p-dioxin, which is a tricyclic compound having a structure in which two benzene rings are condensed via two oxygen atoms.
zo-p-dioxin), and two benzene rings are 1
Polychlorodibenzofuran, which is a tricyclic compound having a structure condensed via two oxygen atoms.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The 65 strains according to the present invention belong to the Fusarium avenaceum (corda: fries) sacchard species belonging to the genus Fusarium which is an incomplete bacterium, and have a higher dioxin degrading ability than conventionally known dioxin degrading bacteria. And newly isolated from the decay material / soil by the present inventors. The 267 strain according to the present invention is a basidiomycete Coeticiaea
e) A strain belonging to the genus Kawai (Peniophora) belonging to the family and having a higher dioxin degrading ability than conventionally known dioxin-degrading bacteria.
Freshly isolated from soil. In addition, among microorganisms belonging to Asteraceae (especially microorganisms belonging to the genus Kawatake),
The present inventors have found for the first time that a microorganism having a dioxin decomposability exists.

In addition, the PC × 65 strain according to the present invention is the same as the above-mentioned 65 strain and a known PC (Phaneroc).
Haete chrysosporium), which is a strain newly created by cell fusion with the bacterium, and belongs to the Fusarium avenaceum (Korda: Fries) sacchard species as in the case of the above-mentioned 65 strains, but is less than the parental cells of the above 65 strains and PC bacteria. It has better dioxin resolution.
Furthermore, the PC × 267 strain according to the present invention is a strain newly created by the present inventors by cell fusion of the above-mentioned 267 strain with a known PC bacterium, and belongs to the genus Kawatake like the 267 strain, but the parent It has an even better dioxin degradability than the 267 strain and PC strain, which are cells.

The 65 strains and 267 strains of the present invention can be prepared by a known method [eg, Paper Pulp and Paper Technical Association, 50 (12), p1
22, the following method disclosed in 1996]]. The method comprises:
A bacterium capable of producing an enzyme involved in lignin degradation, that is, a dioxin-degrading bacterium, is a dye Remazol brilliant blue R (Remazol brilli).
ant blue R) has the property of decolorizing. That is, the decay material and / or soil collected from the field are suspended in distilled water, and the suspension is further diluted to obtain a sample for screening. The sample is applied on an agar medium containing the dye Remazol Brilliant Blue R, and cultured at an appropriate temperature (for example, about 25 ° C.). The bacteria from which the pigment has been decolorized are separated by a plate smearing method and stored as a slant. The dioxin decomposability of the obtained microorganism is measured, for example, by using 2,7-dichlorodibenzo-p-dioxin (hereinafter, referred to as a kind of dioxin).
2,7-DCDD) to obtain the microorganism of the present invention.

Further, the PC × 65 strain and PC ×
The 267 strain is, for example, a 65 or 267 strain isolated by the above method and a PC having dioxin degradability.
It can be obtained by cell fusion with bacteria according to a conventional method. For example, the PC × 65 strain of the present invention is a fused bacterium obtained by preparing a protoplast from each of the 65 strains and a known PC strain (IFO 31249) by a cell fusion method using polyethylene glycol. In the same manner, the PC × 267 strain of the present invention can also be used for the 267 strain of the present invention and a known PC strain (IFO 31249).
And a fused bacterium obtained from

The 65 strains, 267 strains, PC × 65 strain, and PC × 267 strains according to the present invention have been deposited at the Research Institute of Biotechnology, Industrial Technology Institute of Japan from March 6, 1998. The accession number of the 65 strains is FERMP-
The accession number of the 267 strain is FERM
P-16689, and the accession number of the PC × 65 strain is F
ERM P-16690 and PC × 267
The accession number of the strain is FERM P-16691.

The dioxin decomposing method according to the present invention can be carried out using one or more of 65, PC × 65, 267 or PC × 267 strains according to the present invention. Alternatively, the method for decomposing dioxin according to the present invention comprises a microorganism belonging to the family Asteraceae (particularly,
It can be carried out using one or more microorganisms belonging to the genus Kawatake, preferably microorganisms belonging to the family Asteraceae having dioxin decomposability (in particular, microorganisms belonging to the genus Kawatake having dioxin decomposability). Other than using the above-mentioned microorganism, a known dioxin decomposition method using a microorganism can be used as it is.

That is, dioxin contaminants (eg, incinerated ash) can be detoxified by utilizing the microorganisms described above. In order to carry out this detoxification method, for example, it is conceivable to add the microorganism of the present invention to incinerated ash to decompose dioxin in the incinerated ash. In addition, the microorganisms can be used to detoxify the exhaust gas from the incinerator. To carry out this detoxification method, for example, it is conceivable to dispose a carrier carrying the above-mentioned microorganisms in the flow path of the exhaust gas and decompose dioxin in the exhaust gas.

Furthermore, dioxin-contaminated soil can be rendered harmless by using the above microorganisms. To carry out this detoxification method, for example,
Alternatively, spray the substance carried on the carrier onto the contaminated soil,
Decomposition of dioxin in soil can be considered.

[0019]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

[Isolation example] << Isolation and identification of 65 strains and 267 strains >> (1) Isolation process Decay material and soil are collected from the field and sterilized distilled water (10 ml).
In a few rounds, a few loops of the decay material and / or soil were suspended (ie, 2-3 loops per loop). Further, a diluted solution obtained by diluting the suspension in a range of 1,000 to 10,000 times was used as a sample for screening. As an agar medium plate for screening, Czapek-D was used in the lower layer.
ox medium (10 ml) was overlaid with 1% dye (Remaz
ol brilliant blue R) and 5ppm
An agar medium plate prepared by overlaying a malt-agar medium (8 ml) containing menomiles was used. The composition of the Czapek-Dox medium is described in the literature [for example, “Basic knowledge of illustrated applied microorganisms” by Takazo Nishiyama (1992).
2 years) Ohmsha, p153], specifically, sucrose (10 g), KH ₂ PO ₄ (1 g), M
_{gSO 4 · 7H 2 O (0.5g} ), KCl (0.5g),
NaNO ₃ (2.0 g), FeSO ₄ .7H ₂ O (0.0
1g) and agar (15g) in distilled water (1000ml)
, The pH was adjusted to 5.6, and the mixture was autoclaved. The screening sample (1 ml) was applied on the agar medium plate and cultured at 25 ° C. The bacteria from which the pigment had been decolorized were separated by a plate smearing method and stored as slants. According to the method described in the column of the resolution evaluation described below, when the 2,7-DCDD resolution was evaluated for various bacteria stored as slants,
Two strains having excellent 2,7-DCDD resolution were obtained, and were named 65 strains and 267 strains, respectively.

(2) Identification of 65 strains The 65 strains were inoculated on potato dextrose agar plate medium (PDA) and potato sucrose agar plate medium (PSA) and cultured at 25 ° C. for 4 to 7 days. After the culture, the color tone of the colonies, the conidial formation structure, and the like were observed for the grown mold. The morphological characteristics of the colonies are shown in Table 1 below together with the culture medium.

[0021]

Table 1 Item Medium Morphological growth rate of colony (25 ° C, 4 days) Diameter of PDA colony 30-36mm PSA diameter of 36-38mm Growth rate (20 ° C, 4 days) PDA colony diameter of 34-38mm PSA colony 38-40 mm in diameter of the surface of the settlement PDA Grayish red PSA Grayish ruby Color of the backside of the settlement PDA Grayish red PSA Grayish red

In addition, the tissue of the colony is wool-like, and no sclerotium is formed, but sporodkia is formed. The conidium-forming cells are single or branched ampule-shaped phialides, and are mono-phialide-type. The size of the conidial cells was 10-16 μm × 2-4 μm.
The large conidia are colorless, curved, crescent-shaped or spindle-shaped, and have 1 to 3 partitions (mainly 3 partitions). 3
The size of the conidia of the partition is 18-34 μm × 3-4 μm
It is. Small conidia are colorless and elliptical, have no or one partition, are formed in a viscous mass at the tip of phialide, and have a size of 10 to 16 μm × 2 to 3 μ.
m. In addition, chlamydospores are not formed.

From the above results, 65 strains were found to be Fusarium avenaceum (coder: Fries) sacchard belonging to the genus Fusarium of the imperfect fungus [F.
usarium avenaceum (Corda: F
ries) Saccardo] species. Fusarium genus is a genus of imperfect fungi, and the colonies are white, yellow, brown, pink, red, or purple, etc., and the form is spindle-shaped or sickle-shaped large large with one to several partitions from phialide. It is characterized by forming two types of conidia and pear-shaped or spindle-shaped small conidia. Some species form only large conidia. Generally called red mold, it is widely distributed around the world and is isolated from soil, cereals, foods, and the like. Fusarium avenaceum (F. avenaceum) grows rapidly, the colony exhibits a red or white color, and the conidial cells are of the monophyllide type. Widely distributed around the world, plant fruits, seeds,
There are examples of isolation from leaves and branches of dead plants, and also known as phytopathogenic fungi (spot rot).

(3) Identification of 267 strain The 267 strain was inoculated on a 2% malt extract agar plate medium (2% MEA) and a phyton yeast extract agar plate medium (PYE) and cultured at 25 ° C. for 4 to 13 days. In addition, the color tone and the organization of the colonies, the conidial formation structure, etc. were observed for the grown mold, and the enzyme activity test was performed. The morphological characteristics of the colonies are shown in Table 2 below together with the medium, and the results of the enzyme activity tests are shown in Table 3 below. In Table 3, "+" means that enzyme activity was observed.

[0025]

[Table 2] Item Medium Morphological growth rate of colonies (25 ° C., 7 days) 2% MEA Colony diameter of 87 mm or more PYE Colony diameter of 87 mm or more Color tone of colony surface 2% MEA colorless PYE White Color tone of colony back surface 2% MEA colorless PYE white

[0026]

[Table 3]

In addition, the tissue of the colony was felt-like, and the formation of a clamp connection and the formation of segmented conidia were observed. It should be noted that budding conidia are hardly formed, and chlamydospores are not formed. No fruiting bodies or complete generations were observed. From the above results, 267 strains were found to be of the genus Kawai (Peniophor).
a).

[0028]

[Preparation Example 1] << Preparation of PC × 65 strain and PC × 267 strain >> (1) Preparation of protoplasts 65 strains (0.5 strain) cultured in a glucose / peptone medium
g) after washing with 0.6M MgSO ₄ solution,
0.6M MgSO ₄ solution containing 2.5% cellulase, 1% nobozyme and 0.5% chitinase (2.5 ml)
Suspended in water. After reacting at 35 ° C. for 3 hours, centrifugation (3000 rpm, 10 minutes) is carried out.
65 strains of protoplasts were obtained. 1.5 × 10 ⁶ protoplasts were obtained per 1 g of the live 65 bacterial strains.

65 cultured in a glucose-peptone medium
In place of the strain, a 267 strain cultured in a glucose-peptone medium was used, and as enzymes, 2% cellulase and 1% chitinase were used instead of 2% cellulase, 1% nobozyme, and 0.5% chitinase. The above procedure was repeated, except that the protoplasts of 267 strains were obtained. 1 g of 267 strains in a viable state
1.3 × 10 ⁶ protoplasts were obtained. In addition, a PC strain cultured in a glucose-peptone medium was used instead of the 65 strain cultured in a glucose-peptone medium, and 2% cellulase was used as an enzyme.
Protoplasts of PC strain were obtained by repeating the above procedure, except that 2% nobozyme and 0.2% chitinase were used. 1 g of viable PC bacteria per gram.
0 × 10 ⁶ protoplasts were obtained. The PC
The strain was obtained from the Institute of Fermentation (Institute F)
ermentation Osaka, Japan; I
FO) (IFO number = 31249; ATC)
C number = 34541).

(2) Cell fusion Protoplasts of 65 strains (1.0 × 10⁶PC)
Protoplast of the strain (1.0 × 10⁶) And it
0.6M-MgSO4 each_FourSuspended in a solution (2.5 ml)
After that, 65 strain protoplast solution (1 ml) and PC bacteria
Mix with protoplast solution (1 ml) and further add 30 ml
% Polyethylene glycol solution [0.1 M glycine-N
aOH buffer (pH 8.5) and 50 mM CaCl_Two
(2 ml). Incubate at 30 ° C for 30 minutes
After centrifugation (3000 rpm, 10 minutes),
gave. After removing the supernatant, the precipitate was washed with 0.6M-Mg.
SO _FourBy suspending in a solution (5 ml), the fused bacteria
Obtained. About the obtained various bacteria,
The 2,7-DCDD resolution was evaluated according to the method described in the column.
As a result, the strain 1 having excellent 2,7-DCDD resolution was obtained.
The seed was obtained and was named PC × 65 strain.

Instead of protoplasts of 65 strains, 2
By repeating the above procedure except that 67 strains of protoplasts were used, one strain having excellent 2,7-DCDD resolution could be obtained and named PC × 267 strain.

(3) Identification of PC × 65 strain For the PC × 65 strain, the identification procedure for the 65 strains described in the above Isolation Example 1 (2) was repeated to obtain P.
The C × 65 strain was identified. The morphological characteristics of the colonies are shown in Table 4 below together with the culture medium.

[0033]

Table 4 Item Medium Morphological growth rate of colony (25 ° C, 4 days) Diameter of PDA colony 44-49mm PSA diameter of colony 49-52mm Growth rate (20 ° C, 4 days) PDA colony diameter 40-42mm PSA colony 40-41 mm in diameter of the surface of the settlement PDA Grayish red PSA Grayish red Color of the backside of the settlement PDA Grayish red PSA Grayish red

The tissue of the colony is wool-like, and no sclerotium is formed, but sporodkia is formed. The conidium-forming cells are single or branched ampule-shaped phialides, and are mono-phialide-type. The size of the conidial cells was 10 to 19 μm × 2 to 4 μm.
The large conidia are colorless, half-moon-shaped or spindle-shaped, and have 2 to 5 partition walls. The size of the large conidia of the three partitions is 24-37 μm × 3-4 μm, the size of the large conidia of the four partitions is 33-47 μm × 3-4 μm, and the size of the large conidia of the five partitions The size is 38-54μm ×
3 to 4 μm. Small conidia are colorless and elliptical, have no or one partition, are formed in a viscous mass at the tip of phialide, and have a size of 8 to 17 μm
× 2 to 3 μm. In addition, chlamydospores are not formed.
From the above results, the PC × 65 strain, like the 65 strain, also has Fusarium avenaceum belonging to the incomplete fungus genus Fusarium (coder: Fries).
Sacchard [Fusarium avenaceum]
(Corda: Fries) Saccardo] species. (4) Identification of PC x 267 strain The PC x 267 strain was identified by repeating the identification procedure for the 267 strain described in Isolation Example 1 (3) above. The morphological characteristics of the colonies are shown in Table 5 below together with the medium, and the results of the enzyme activity tests are shown in Table 6 below. In Table 6, "+" means that enzyme activity was observed.

[0035]

[Table 5] Item Medium Morphological growth rate of colonies (25 ° C., 7 days) 2% MEA Diameter of colony 87 mm or more PYE Diameter of colony 87 mm or more PYE white

[0036]

[Table 6]

In addition, the tissue of the colony was felt-like, and the formation of segmented conidia was observed. No clamp connection is formed, budding conidia are hardly formed, and chlamydospores are not formed. No fruiting bodies or complete generations were observed. From the above results, the PC × 267 strain was also similar to the 267 strain in the genus Kawatake (Peniop).
hora).

[0038]

[Reference] 65 strains, 267 strains, PC × 65
For the identification of the strain and the PC × 267 strain, the following literature was referred to. (1) Domsch, K .; H. , Gams, W.C. and
Anderson, Traute-Heidi: "C
Ompendium of Soil Fungi "
(1993) IHW-Verlag (2) Nelson, P .; E. FIG. , Toussoon,
T. A. and Marasas, W.M. F. O. : "F
usarium species An Illust
rated Manual for Identify
nation "(1983) The Pennsylv
ania State University Pre
ss (3) Booth, C.I. : "The genus Fu
sarium "(1971) CAB Common
weight Mylogical Institute
ute (4) Samson, R.A. A. , Hoekstra,
E. FIG. S. Frisvad, J. et al. C. and Filt
Enborg, O .; : "Introduction
Food-Borne Fungi "5th edi
Tion (1996) Centraalbureau
voor Schimmelcultures (5) Stalpers, J .; A. : “Studies
in Mycology No. 16 "(1978)
Centralalureau voor Skim
Melcultures (6) Stalpers, J .; A. : “Studies
in Mycology No. 24 "(1984)
Centralalureau voor Skim
Melcultures (7) Masakatsu Ichinohe: Antibacterial and antifungal, 18,399-406 (1
(990) (8) Rokuya Imaseki, Yoshio Otani, Tsuguo Hongo, Ed .: "Mt.

[0039]

[Evaluation of resolution] (1) Synthesis of dioxin 2,7-DCDD which is a kind of dioxin used for evaluation of resolution of dioxin was obtained from Advan. Chem. S
er. , 120, p126, 1973, according to the following procedure. That is, 1N methanolic KOH (29.5 ml) was added to 2-bromo-4-chlorophenol (5.19 g) and dissolved, and the mixture was allowed to stand for 2 hours. Then, benzene (2.5 m) was added to the concentrate obtained by concentrating the solution under reduced pressure.
l) and hexane (5 ml) were added. After removing the upper layer, the residue was dried under reduced pressure to obtain potassium salt of 2-bromo-4-chlorophenol (6.14 g). on the other hand,
0.5 g of zinc powder (23.0 g) was added to a solution of copper sulfate (pentahydrate) (73 g) dissolved in distilled water (280 ml) with stirring while blowing nitrogen. After the mixture was heated to 70 ° C., it was subjected to suction filtration. The residue was washed with 5% hydrochloric acid and then neutralized with distilled water to obtain a copper catalyst (15.84 g). The prepared catalyst is
Stored in refrigerator until use. The potassium salt of 2-bromo-4-chlorophenol prepared as described above (3.
66 g) and bis (2-ethoxyethyl) ether (5
ml), ethylene diacetate (0.05 ml), and the copper catalyst (20 mg) prepared as described above,
The mixture was heated and refluxed at 00 ° C. for 15 hours. After cooling, the product was extracted with chloroform. 1N chloroform extract
After extraction with an aqueous sodium hydroxide solution to remove the alkali-soluble portion, the extract was dried with sodium sulfate decahydrate.
Thereafter, the mixture was concentrated under reduced pressure to obtain a reaction product (4.45 g). This product was sublimated at 170 ° C. (under 25 mmHg) for 3 hours to obtain white crystals (3.18 g). The crystals were recrystallized from methanol to obtain 2,7-DCDD (2.95 g) as white crystals (melting point = 209).
２１１211 ° C .; yield = 39.2%).

(2) Evaluation Method of Dioxin Degradability Evaluation of the dioxin decomposition efficiency of the four dioxin-degrading bacteria of the present invention was carried out in a liquid. In addition, PC bacteria and V2 strain were used as dioxin-decomposing bacteria for comparison. The V2 strain is one of the three dioxin-degrading bacteria (563) isolated from the field by Tachibana et al.
Strain, V1 strain, and V2 strain).
It is the strain having the highest DD decomposition removal efficiency [Paper and Pulp Technical Journal, 50 (12), p122, 1996].

Kirk et al. (Arch. Microbio)
1,117, p277, 1978), 20 mM sodium succinate, 2% glucose, and 1.2 mM ammonium tartrate as a nitrogen source were added to the Basal solution, and the medium adjusted to pH 4.5 was used for culture. Liquid medium. After the liquid medium was sterilized in an autoclave, the liquid medium (20 m
l) was dispensed, and one platinum loop of the test bacterium was inoculated therein, and the flask was double-capped and cultured statically at 25 ° C. Oxygen was blown into each culture flask once a day. After culturing for 6 days, 2,7-DC dissolved in N, N-dimethylformamide (DMF) (100 μl)
DD (1.26 mg) (2,7-DCDD addition concentration =
0.25 mM) or 2,7-DCDD (6.3 mg) dissolved in DMF (100 μl) (additional concentration of 2,7-DCDD = 1.25 mM). 2,7-DCDD
Was added, and the cells were further cultured for 15 days or 30 days. During the culture period, oxygen was blown once a day.

The combinations of 2,7-DCDD addition concentration, culture period, and test bacteria are shown below. That is, 2,7-
The conditions in which the DCDD addition concentration was 0.25 mM and the culture treatment period was 15 days were as follows: 65 strains (Example 1);
It carried out about PC * 65 strain (Example 2), PC * 267 strain (Example 3), PC strain (Comparative Example 1), and V2 strain (Comparative Example 2). For the condition where the 2,7-DCDD addition concentration is 0.25 mM and the culture treatment period is 30 days, 65 strains (Example 4), 267 strains (Example 5) and PC × 267 strains (Example 6) , PC strain (Comparative Example 3), and V2 strain (Comparative Example 4).
For the condition where the 2,7-DCDD addition concentration is 1.25 mM and the culture treatment period is 15 days, 267 strains (Example 7), PC × 65 strain (Example 8), PC × 267
It carried out about the strain (Example 9), the PC strain (Comparative Example 5), and the V2 strain (Comparative Example 6). As for the conditions in which the 2,7-DCDD addition concentration is 1.25 mM and the culture treatment period is 30 days, 65 strains (Example 10), 267 strains (Example 11), and PC × 65 strains (Example 12) , P
It carried out about C * 267 strain (Example 13), PC strain (Comparative Example 7), and V2 strain (Comparative Example 8). The experiment was performed twice for each strain, each concentration, and each culture period, and this was repeated twice, and the average value was used as data.

The dioxin decomposition efficiency was evaluated by the following method. That is, after culturing under the above conditions, the pH of the culture was adjusted to 2.0 with dilute hydrochloric acid, and ethyl acetate (40 ml) was added thereto, followed by vigorous stirring. Thereafter, the mixture was subjected to suction filtration, the culture filtrate was collected, and the ethyl acetate layer and the aqueous layer were separated. The aqueous layer was further extracted twice with ethyl acetate (each 40 m
l) This extract is combined with the ethyl acetate layer,
After washing with saturated saline (50 ml), sodium sulfate
After drying with 0-hydrate, the metabolite was obtained by concentration under reduced pressure. To this metabolite (1.0 mg), N, O-bis-trimethylsilylacetamide (40 μl), trimethylchlorosilane (20 μl), and pyridine (40 μl) were added, and the mixture was heated (80 ° C., 10 minutes) to give trimethylsilyl ( TMS) was used as a sample for gas chromatography (GC).

This sample was injected into GC, and 2,7-DCDD was quantified from a calibration curve prepared in advance.
The conditions of GC are as follows. (1) Column: TC-1 (0.25 mm × 30 m) (2) Temperature: Hold at 100 ° C. for 1 minute, then raise to 295 ° C. at 10 ° C./minute, hold at 295 ° C. for 20 minutes (3) Detection (4) Injector temperature: 250 ° C (5) Detector temperature: 250 ° C (6) Carrier gas: helium (3 kg / cm ² ),
Air (1.1 kg / cm ² ), split ratio: 1: 3
0

(3) Results The degradation and removal rate of 2,7-DCDD was
-Addition amount of DCDD and 2,7-DC in metabolite after culture
It was determined from the amount of DD. Tables 7 to 10 show the results of the experiment.

[0046]

Table 7 (2,7-DCDD added concentration: 0.25 mM, culture treatment period: 15 days) Degradation removal rate (%) used Example 1 65 strain 90.8 Example 2 PC × 65 strain 98. 6 Example 3 PC × 267 strain 94.7 Comparative example 1 PC strain 90.1 Comparative example 2 V2 strain 84.8

[0047]

[Table 8] (2,7-DCDD added concentration: 0.25 mM, culture treatment period: 30 days) Degradation and removal rate (%) used strain Example 4 65 strain 94.8 Example 5 267 strain 89.1 Example 6 PC × 267 strain 99.8 Comparative Example 3 PC strain 78.4 Comparative Example 4 V2 strain 81.6

[0048]

[Table 9] (Concentration of 2,7-DCDD added: 1.25 mM, culture treatment period: 15 days) Degradation and removal rate (%) used Example 7 267 strain 68.2 Example 8 PC × 65 strain 96. 7 Example 9 PC × 267 strain 79.7 Comparative example 5 PC strain 59.9 Comparative example 6 V2 strain 62.3

[0049]

[Table 10] (2,7-DCDD added concentration: 1.25 mM, culture treatment period: 30 days) Degradation removal rate (%) used Example 10 65 strain 82.9 Example 11 267 strain 75.2 Performed Example 12 PC × 65 strain 99.1 Example 13 PC × 267 strain 85.5 Comparative Example 7 PC strain 67.5 Comparative Example 8 V2 strain 70.1

[0050]

The microorganism of the present invention can decompose and remove dioxin with high efficiency, so that it can be used for purification of soil contaminated with dioxin and final disposal sites of incinerated ash, for example.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 15/02 B09B 3/00 ZABZ // (C12N 1/15 C12N 15/00 B C12R 1: 645) (C12N 1/14 C12R 1: 645) (C12N 1/14 C12R 1:77) (72) Inventor Kazuki Ito 3-5-7 Tarumi, Matsuyama-shi, Ehime Pref. Faculty of Agriculture, Ehime University Forest Resource Utilization Laboratory (72) Inventor Yamauchi Yoko 3-5-7 Tarumi, Matsuyama City, Ehime Prefecture Ehime University Faculty of Agriculture, Department of Bioresources, Forest Resource Utilization Research Laboratory (72) Inventor Tetsuya Sasaki 1-1-2 Marunouchi, Chiyoda-ku, Tokyo Nihon Kokan Co., Ltd. (72 ) Inventor Kazuyoshi Suzuki 1-2-2 Marunouchi, Chiyoda-ku, Tokyo Inside Nihon Kokan Co., Ltd. (72) Inventor Takaaki Kondo 1-chome Marunouchi, Chiyoda-ku, Tokyo No. 1-2 in Nihon Kokan Co., Ltd.

Claims (7)

[Claims] 1. A 65 strain of Fusarium avenaceum (Korda: Fries) Sacchard having dioxin decomposability. 2. Fusarium avenaceum (corda: Fries) sacchard PC × with dioxin resolution
65 strains. 3. A strain of Kawatake 267 having dioxin degradability. 4. A PC × 267 strain of the genus Kawatake having dioxin decomposability. 5. A method for decomposing dioxin, comprising using the microorganism according to any one of claims 1 to 4. 6. A method for decomposing dioxin, comprising using a microorganism belonging to the family Asteraceae. 7. The method for decomposing dioxin according to claim 6, wherein the microorganism belonging to the family Asteraceae is a microorganism belonging to the genus Kawatake.