JP5598646B2 - Cyclic diene compound decomposing bacteria and cyclic diene compound decomposing agent, contaminated environment purification apparatus, cyclic diene compound decomposing method, and pollutant purifying method - Google Patents

Description

  The present invention relates to cyclic diene pesticides, particularly degrading bacteria classified as filamentous fungi having the ability to degrade at least one compound selected from aldrin, dieldrin, heptachlor, heptachlor epoxide, and the like, and more particularly Mucor racemosus spp. It relates to degrading bacteria belonging to. The present invention also relates to a cyclic diene compound decomposing agent using the decomposing bacteria, a purification apparatus for contaminated environments, a method for decomposing cyclic diene compounds, and a method for purifying contaminated soil.

Six types of organochlorine pesticides whose production and use were prohibited in the mid 1970s by the POPs Convention adopted in 2001 (DDT, aldrin (see Fig. 1), dieldrin (see Fig. 2), endrin, heptachlor (see Fig. 3) ), 12 substances including hexachlorobenzene) have been designated as POPs, and each country has decided to promote appropriate management and treatment of these pesticides within an international framework.
In Japan, after the ban on production and use, these agricultural chemicals are buried in containers by agricultural chemical manufacturers, agricultural cooperatives, etc., and then the embedded agricultural chemicals are excavated and recovered, and physicochemical methods (high temperature incineration, supercritical water, etc.) Decomposed and detoxified by oxidation method, vacuum heating method). However, it has been pointed out that containers may be damaged due to long-term embedding, and the surrounding soil and groundwater may be contaminated at low concentrations. On the other hand, in Tokyo and Yamagata prefectures, dieldrin exceeding the residue standard was detected from cucumbers, and these were because the drin agent applied about 30 years ago remained at low sub-ppm levels in farmland soil. Conceivable.

  With regard to a purification method for soil and groundwater contaminated extensively with such low-concentration organic chemicals, in-situ bioremediation utilizing biological functions (particularly resolution) of microorganisms and the like is expected. Such bioremediation includes biostimulation methods in which nutrients, oxygen, etc. are supplied to the contaminated site to grow and activate native degrading bacteria for purification, and bioorganisms that introduce microorganisms with specific functions from outside. There is a known shading method.

  However, bioremediation technology for soil contaminated with cyclic diene pesticides designated by POPs is stagnant. The cause of the stagnation may be that it is difficult to isolate microorganisms with high resolution for these compounds. In fact, Aldrin is relatively easily oxidized (epoxidized) by microorganisms to become dieldrin, but the half-life of dieldrin in soil is stable at 7 years or more, and there are almost no microorganisms that can degrade dieldrin in soil. Conceivable.

Although some studies on dieldrin degradation have been reported, the degree of degradation is limited. For example, Matsumura and Boush the filamentous fungi Trichoderma viride (Trichoderma viride) (Non-Patent Documents 1 and 2), also, Anderson is Mucor Aruterunansu (Mucor alternans) (Non-Patent Document 3) to degrade dieldrin However, there is only about 20% degradation. Wedermeyer reports that Aerobacter aerogenes converts dieldrin to aldrindiol, but does not show dechlorination (Non-patent Document 4). Furthermore, although recently Burke by Matsumoto et al. Burkholderia (Burkholderia) genus and Cupriavidus (Cupriavidus) spp reported to degrade dieldrin, the degree of decomposition is as much as 50% (Non-Patent Document 5), There are no reports of dechlorination of dieldrin. Chucko et al. Examined the degradation of dieldrin using Mucor ramanianus , but no degradation was observed (Non-patent Document 6).

  Even if cyclic diene compounds such as dieldrin are decomposed unless dechlorinated, toxicity may remain, so dechlorination is desirable for detoxification. However, with regard to the reported cyclic diene compound-degrading bacteria, most of the bacteria whose metabolism is stopped by epoxidizing the left half of the molecule (hydrocarbon ring portion). When a decomposition test was conducted using a compound labeled with C in the common skeleton, the mineralization rate was 5% or less for most degrading bacteria, so the chlorine substituent was attacked and dechlorinated. It is thought that there are almost no decomposable bacteria. That is, at present, there is no degradable bacterium that can be used in bioremediation techniques including biostimulation methods and bioaugmentation methods for soil contaminated with cyclic diene compounds.

Matsumura & Boush (1967), Science Vol. 156, 959-961 Matsumura & Boush (1968), JEE Vol. 61, 610-612 Anderson JPE, Lichtenstein EP, Whittingham WF (1970), JEE Vol. 63, 1595-1599 Wedemeyer G (1967), AEM Vol. 16, 661-662 Matsumoto E, Kawanaka Y, Yun S-J, Oyaizu H (2008), AMB, 1095-1103 Chacko CI, Lockwood JL, Zabik M (1966) Science Vol. 154, 893-895

  Therefore, the present invention is capable of decomposing compounds containing cyclic diene pesticides such as aldrin, dieldrin, heptachlor, heptachlor epoxide (see FIG. 4), and decomposing these compounds using the decomposing bacteria. The purpose is to provide technology.

That is, the present invention provides a cyclic diene compound decomposing agent comprising a fungus of the genus Mucor racemosus having the resolution of a cyclic diene compound in order to achieve the above object.
The cyclic diene compound includes a compound having a 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure (see FIG. 5), and more specifically, an aldrin. , Dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, and at least one compound selected from the group consisting of endosulfan sulfate.
Such a cyclic diene compound is preferably dechlorinated and decomposed. The toxicity of the cyclic diene compound can be reduced by dechlorination.
Moreover, it is preferable to decompose 50% or more of the cyclic diene-based compound, more preferably 60% or more, 80% or more, or 90% or more. If it can be decomposed by 50% or more, its toxicity can be weakened. If it can be decomposed by 60% or more, it is more effective. If it can be decomposed by 80% or more, the residue of the substance can be almost eliminated.
Furthermore, the cyclic diene compound decomposing agent preferably has a resolution of DDT (dichlorodiphenyltrichloroethane) or DDE (dichlorodiphenyldichloroethylene). DDT is a pesticide designated as POPs, and DDE is a main metabolite generated by DDT being decomposed in soil and the like, and remains in agricultural land for a long time. If the cyclic diene compound decomposing agent also has DDT or DDE resolution, it is possible to more efficiently purify the environment such as contaminated soil and contaminated water.

The cyclic diene compound decomposing agent comprises a filamentous fungus of the genus Mucor racemosus. More specifically, it can contain a filamentous fungus of the genus Mucor racemosus having the base sequence represented by SEQ ID NO: 1 in the ITS (Internal transcribed spacer) region. It may also comprise at least one strain selected from the group consisting of M. racemosus DDF (Accession No. FERM P-21775), IFO 6745, and NBRC 4581. it can.
The cyclic diene compound-decomposing bacterium is a filamentous fungus of the genus Mucor racemosus, and a more detailed example is a filamentous fungus of the genus Mucor racemosus having the base sequence represented by SEQ ID NO: 1 in the ITS region, Since it is at least one strain selected from the group consisting of Mosas DDF strain, IFO 6745 strain, and NBRC 4581 strain, it can degrade 90% or more of dieldrin, heptachlor, heptachlor epoxide, and at least 50 cyclic diene compounds. % Can be decomposed. Also, dieldrin can be dechlorinated and decomposed.

The cyclic diene compound-decomposing agent can be obtained by growing the above-mentioned cyclic diene compound-decomposing bacterium on a substrate such as bran or rice bran serving as the feed, and including the substrate. Since the cyclic diene compound-decomposing bacterium includes the substrate on which the cyclic diene compound-decomposing bacterium has been grown, the cyclic diene compound-decomposing bacterium can be grown at a high density for a long period of time.
Moreover, it can be set as the cyclic | annular diene type compound decomposition | disassembly agent formed by mixing porous materials, such as a wood carbonization raw material, in such a substrate. Furthermore, since the porous material is mixed, the cyclic diene compound-decomposing bacteria are more excellently retained, and the cyclic diene compound can be stably decomposed. This cyclic diene compound decomposing agent can be used in bioremediation technology and can purify soil and water contaminated with the cyclic diene compound.

The present invention also provides a purification apparatus for a contaminated environment containing the cyclic diene compound decomposing agent or the cyclic diene compound decomposing bacteria.
According to the polluted environment purification apparatus containing the cyclic diene compound decomposing agent or the cyclic diene compound decomposing bacteria, soil and water contaminated with the cyclic diene compound can be purified.

Furthermore, the present invention is directed to aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, endosulfan sulfate using a cyclic diene compound decomposing agent, a cyclic diene compound degrading bacterium, and a purification apparatus for contaminated environment containing them. A ring that decomposes a cyclic diene compound having a 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure, exemplified by at least one compound selected from the group consisting of A method for decomposing a diene compound is provided.
A cyclic diene compound decomposing agent, a cyclic diene compound degrading bacterium, and a contaminated environment purification device containing them are brought into contact with the cyclic diene compound to decompose the cyclic diene compound, and cyclic such as soil and groundwater An environment contaminated with diene compounds can be purified.

In addition, the present invention provides a method for purifying pollutants by inoculating a soil contaminated with a cyclic diene compound with a cyclic diene compound degrading bacterium or a cyclic diene compound decomposing agent. Contaminated soil can be purified by inoculating a soil contaminated with a cyclic diene compound with a cyclic diene compound degrading bacterium or a cyclic diene compound decomposing agent.
Moreover, the purification | cleaning method of the pollutant which inoculates the cucurbitaceae plant contaminated with the cyclic diene type compound and the cyclic diene type compound decomposing agent or the cyclic diene type compound decomposing agent is provided. By inoculating and contacting a cucurbitaceae plant contaminated with a cyclic diene compound with a cyclic diene compound degrading bacterium or a cyclic diene compound decomposing agent, fertilizer using the cucurbitaceae plant as a raw material is decomposed. It can also be manufactured.

  According to the cyclic diene compound decomposing bacteria and the cyclic diene compound decomposing agent of the present invention, 1,2,3, such as aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, endosulfan sulfate, which are cyclic diene pesticides. A compound having a 4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure can be decomposed.

  Furthermore, according to the decomposition method of the cyclic diene compound and the purification method of contaminated soil of the present invention, the cyclic diene compound contained in the environment such as contaminated soil and contaminated water is decomposed to purify the contaminated environment. Can do.

It is a chemical formula of aldrin. It is the chemical formula of dieldrin. It is the chemical formula of heptachlor. It is a chemical formula of heptachlor epoxide. It is a chemical formula showing 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure. It is explanatory drawing which shows the molecular phylogenetic tree based on the base sequence of Mucor racemosus DDF strain. It is a graph which shows dieldrin degradation by Mucor racemosus DDF strain. It is a graph which shows dieldrin decomposition | disassembly by three strains which belong to a racemosus species. It is a graph which shows heptachlor epoxide decomposition | disassembly by Mucor racemosus DDF strain | stump | stock. It is a graph which shows decomposition | disassembly of various cyclic diene type compounds by Mucor racemosus DDF strain | stump | stock. It is a graph which shows DDT degradation by Mucor racemosus DDF strain | stump | stock. It is a graph which shows DDE degradation by Mucor racemosus DDF stock | strain.

  The cyclic diene compound-decomposing bacterium and cyclic diene compound-decomposing agent of the present invention, a polluted environment purification apparatus using them, a cyclic diene-based compound degradation method, and a purification method for the environment such as contaminated soil are described in detail below. To do.

Cyclic diene compound-degrading bacteria :
The present inventors have found that mucor fungus isolated from endosulfan continuous soil has the ability of dieldrin, a cyclic diene pesticide. By the way, endosulfan is not designated as POPs. However, because the properties and characteristics of endrins designated as POPs are similar, the endorphan continuous soil contains 1,2,3,4,7,7-hexachlorobicyclo shown in Fig. 5 such as endrin and dieldrin. [2.2.1] Based on the idea that there may be inhabiting degrading bacteria capable of degrading cyclic diene compounds having a hepta-2-ene structure. As soil inhabiting desired decomposing bacteria, in addition to endosulfan continuous soil, soil using cyclic diene compounds can be considered.

Cultivation of bacterial cells collected from endosulfan continuous soil can be performed by contacting with a material containing a cyclic diene compound under conditions where these bacterial cells can grow. Examples of the conditions under which these cells can grow include PDA medium, Martin medium, and modified Czapec medium, which will be described later. Examples of medium other than these mediums include a medium containing an energy source such as glucose.
Examples of the material containing a cyclic diene compound include agricultural chemicals containing a cyclic diene compound, soil and groundwater containing a cyclic diene compound, and the like.
Isolation of cyclic diene-based compound-degrading bacteria is carried out by smearing a suspension obtained by diluting endosulfan continuous soil, etc. on Martin medium, etc., distinguishing the color and form of the colonies that have appeared, and further subjecting them to degradation tests, respectively. To identify.

  Cyclic diene compound-degrading bacteria can be decomposed bacteria with enhanced resolution against cyclic diene compounds obtained using genetic recombination technology, or newly-produced degrading bacteria. Degradable bacteria obtained from soil are preferred. This is because the degrading bacteria obtained from the cyclic diene compound compound soil are the degrading bacteria that inhabit the soil, and there are fewer safety problems than the degrading bacteria obtained by using the genetic recombination technology.

  Such a degrading bacterium is a fungus belonging to the genus Mucor having a resolution for cyclic diene compounds, more specifically, a degrading bacterium belonging to the genus Mucor racemosus. The degrading bacteria belonging to the Mucor racemosus species include filamentous fungi belonging to the Mucor racemosus species isolated from endosulfan continuous soil. Examples of strains belonging to the genus Mucor racemosus include Mucor racemosus DDF strain, which is a novel strain isolated from endosulfan continuous soil by the inventors, and IFO 6745 strain and NBRC 4581 strain obtained from strain storage organizations.

Mucor racemosus DDF strain showed 100% homology (471/471) in the base sequence of its ITS region by BLAST search compared to the mucor racemosus species (accession number: FJ228217).
The base sequence (5′-3 ′) in the ITS region of this Mucor racemosus DDF strain is shown in the sequence listing as SEQ ID NO: 1. Mucor racemosus DDF strain is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number “FERM P-21775”. A molecular phylogenetic tree using the base sequence of this strain is shown in FIG.
The base sequence (5′-3 ′) in the ITS region of the IFO 6745 strain is shown in the sequence listing as SEQ ID NO: 2. Furthermore, the base sequence (5′-3 ′) in the ITS region of the NBRC4581 strain is shown in the sequence listing as SEQ ID NO: 3.

Mucor racemosus DDF strain has the following mycological characteristics.
(1) Morphological properties: In addition to the basic hyphae having a branch in the liquid medium and on the agar medium, aerial mycelium develops to form many sporangia. The spore spore is circular and contains dozens of spores. Moreover, a bowl-shaped column axis can be confirmed at the tip of the spore handle.
(2) Culture characteristics; Growth is faster in various nutrient agar media as the mycelium covers 9 cm dishes in 3 days. Although no soluble pigment is secreted, when a spore is formed in the aerial hyphae, it becomes light yellowish. Moreover, it is recognized that many zygospores are formed and become dark black with long-term culture for one month or longer.

Also, the mycological characteristics of the IFO 6745 strain are the same as those of the Mucor racemosus DDF strain and are as follows.
(1) Morphological properties: In addition to the basic hyphae having a branch in the liquid medium and on the agar medium, aerial mycelium develops to form many sporangia. The spore spore is circular and contains dozens of spores. Moreover, a bowl-shaped column axis can be confirmed at the tip of the spore handle.
(2) Culture characteristics; Growth is faster in various nutrient agar media as the mycelium covers 9 cm dishes in 3 days. Although no soluble pigment is secreted, when a spore is formed in the aerial hyphae, it becomes light yellowish.

Further, the mycological characteristics of the NBRC4581 strain are the same as those of the Mucor racemosus DDF strain and are as follows.
(1) Morphological properties: In addition to the basic hyphae having a branch in the liquid medium and on the agar medium, aerial mycelium develops to form many sporangia. The spore spore is circular and contains dozens of spores. Moreover, a bowl-shaped column axis can be confirmed at the tip of the spore handle.
(2) Culture characteristics; Growth is faster in various nutrient agar media as the mycelium covers 9 cm dishes in 3 days. Although no soluble pigment is secreted, when a spore is formed in the aerial hyphae, it becomes light yellowish.

  Examples of cyclic diene compounds to be decomposed include compounds having a 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure. The compound is at least one compound selected from the group consisting of aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, and endosulfan sulfate.

  For the decomposition of the cyclic diene compound, it is preferable to decompose 90% or more of those compounds, and more preferably to dechlorinate. This is because the compound can be almost eliminated if it is decomposed by 90% or more, and the toxicity based on the compound can be weakened if it is dechlorinated.

Cyclic diene compound decomposition agent :
Cyclic diene compound-degrading bacteria are thought to be able to grow in general soil, but in order to effectively degrade cyclic diene-based compounds in soil, these degrading bacteria exist at a high density for a long period of time. It is preferable that there exists a decomposing bacteria with the substrate used as the bait of the decomposing bacteria. As the substrate, bran, rice bran, beer ground, coffee grounds, oil grounds and the like are preferable. Also, peanut shells, sawdust, kana waste, wood chips, bark, coconut husks, rice husks, etc. could be used as substrates. Such a substrate also functions as a carrier for holding the degrading bacteria.

That is, the cyclic diene compound-decomposing agent is obtained by separating the cyclic diene-based compound-degrading bacterium from the substrate and using only the degrading bacterium as the cyclic diene-based compound decomposing agent. Can be included with each substrate.
The cyclic diene compound decomposer containing a substrate produces a more effective cyclic diene compound decomposer by mixing a porous material with a large number of pores and a high adsorption coefficient, such as a wood carbonized material, into the substrate. can do. The porous material may also serve as a substrate.
The application to contaminated soil is to purify contaminated soil by introducing cyclic diene compound-decomposing bacteria grown with the substrate into the soil together with the substrate, rather than putting the substrate and cyclic diene compound-degrading bacteria into the soil separately. It is preferable to do.

Pollution environment purification equipment :
If an accumulation layer of a cyclic diene compound decomposing agent is formed by packing the cyclic diene compound decomposing agent in a gas-permeable casing, it can be easily used as a bioreactor for decomposing and removing the cyclic diene compound. .
It is also possible to purify the soil by mixing the soil contaminated with the cyclic diene compound into the apparatus and refluxing the water. Further, the contaminated water can be purified by refluxing the water contaminated with the cyclic diene compound into the apparatus.
Alternatively, the apparatus is provided in a part of a water channel such as a domestic drainage channel, an agricultural drainage channel in a paddy field, a drainage channel of a golf course, etc., so that the cyclic diene compound dissolved and dispersed in water can be decomposed and removed. It can be used as a polluted environment purification device for purifying the environment.

Purification method of pollutants using cyclic diene compound decomposition material :
There are the following methods for purifying a substance contaminated with a cyclic diene compound using a cyclic diene compound decomposer.
Regarding removal of the cyclic diene compound in the contaminated soil, a cyclic diene compound decomposing agent is embedded in the contaminated soil and mixed. By embedding in the soil, the cyclic diene compound already contained in the soil is decomposed by the degrading bacteria. According to this method, it is possible to avoid the cyclic diene compound in the soil from being mixed into the groundwater, and it is possible to prevent the contamination of the groundwater.
Applications of this technology include contamination of the surface and lower soils where cyclic diene compounds are present, contamination of lower green soils of golf courses, contamination of lower soils of industrial waste disposal sites, and organic waste liquids in factories, etc. For example, it can be mixed into the lower soil of the storage area, and cyclic diene compounds can be treated by such application.

Moreover, the cyclic diene compound in the cucurbitaceae plant can be decomposed and detoxified by inoculating the cucurbitaceae plant contaminated with the cyclic diene compound with the cyclic diene compound decomposing agent. The detoxified cucurbitaceae plant can be used as a fertilizer.
Cucurbitaceae plants include cucumbers, watermelons, pumpkins, loofahs and melons.

<Experimental example 1>
[Isolation of Mucor racemosus DDF strain (1); acquisition of candidate strains having dieldrin resolution]
0.5 g of endosulfan continuous soil was diluted with 4.5 mL of sterilized water to prepare a 10 ² , 10 ³ order soil dilution (soil suspension). The soil dilution solution (0.1 mL) thus obtained was smeared on Martin medium and cultured at 25 ° C. The color and morphology of the appearing filamentous fungal colonies were visually discriminated, and each one was transplanted to a newly prepared PDA medium and cultured at 25 ° C. In this way, 36 strains of filamentous fungi that were candidates for degrading bacteria having deldrin resolution were isolated.

The composition of the aforementioned Martin medium is shown below.
Martin medium (per liter)
Peptone: 5g
KH ₂ PO ₄ : 1 g
MgSO ₄ · 7H ₂ O: 0.5g
Glucose: 10g
Rose Bengal: 0.033g
Agar: 20g
Chloramphenicol: 0.25g
Ultra pure water: the rest

The reason why the transplant destination medium is a PDA medium instead of the Martin medium is that the Martin medium contains a mycelial elongation inhibitor, whereas the PDA medium does not contain an elongation inhibitor. It is.
The composition of the PDA medium is shown below.
PDA medium (per liter)
Potato leachate powder: 4.0g
Glucose: 20.0g
Agar: 15.0g
Ultra pure: the rest

<Experimental example 2>
[Isolation of Mucor racemosus DDF strain (2); identification of dieldrin-degrading bacteria Mucor racemosus DDF strain]
Among the 36 strains obtained from each of the PDA media, a degrading bacterium having dieldrin-degrading ability was identified.
Each of the above 36 strains was inoculated into the following modified zapek medium, pre-cultured in the dark at 25 ° C. for 1 week, added with dieldrin to 5 mg / L, and further cultured for 2 weeks. After completion of the culture, 15 mL of acetonitrile was added and the cells were homogenized. After centrifugation at 3000 rpm for 10 minutes, the supernatant was transferred into hexane, the dieldrin concentration was measured by GC / ECD, and the chloride ion concentration in the culture filtrate was measured by ion chromatography. As a result, degradation of dieldrin was observed in one of the 36 strains.
The base sequence (ITS region) of this strain showed 100% homology with the base sequence of the genus Mucor racemosus. The nucleotide sequence (5′-3 ′) of the ITS region of Mucor racemosus DDF strain is shown in the sequence listing as SEQ ID NO: 1. This new strain is deposited under the accession number “FERM P-21775” as “Mucor racemosus DDF” at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

The reason why the PDA medium was inoculated in place of the modified Czapek medium was that the PDA medium is a medium based on potato exudate and natural ingredients, and therefore the components and their concentrations cannot be changed and are not suitable for the degradation test. . Therefore, a degradation test was performed using a modified Czapek medium that is a synthetic medium.
The composition of the modified Czapek medium is as follows.
Modified zapek medium (per liter)
Glucose: 10g
MgSO ₄ · 7H ₂ O: 0.5g
NaNO ₃ : 2g
K ₂ HPO ₄ : 1 g
FeSO ₄ · _7H 2 O: 0.01g
Yeast: 0.5g
Ultra pure water: the rest

<Experimental example 3: FIG. 7>
[Degradin degradation test by Mucor racemosus DDF strain]
10 mL of the above modified zapek medium was placed in a 100 mL Erlenmeyer flask with a lid, and autoclaved at 121 ° C. for 10 minutes. On the other hand, Mucor racemosus DDF strain previously cultured in a petri dish was punched out with a cork borer having a diameter of 6 mm, and one of the disks was placed in this modified zapek medium. At this time, a dieldrin solution dissolved in acetone was added to 5 mg / L. And it culture | cultivated for 10 days at the dark place 25 degreeC. The culture solution was sampled over time, the dieldrin concentration was determined by GC / ECD, and the chloride ion concentration was determined by ion chromatography. In addition, homogenized and centrifuged cells at the time of measurement of dieldrin were suction filtered and dried with a dryer at 70 ° C., and the dry cell weight was determined. The result is shown in the line graph of FIG.

In the graph of FIG. 7, the left vertical axis represents the chloride ion concentration and dieldrin concentration (μM) in the culture solution, the right vertical axis represents the dry cell weight (g), and the horizontal axis represents the culture time (days). .
As shown in FIG. 7, 59% of dieldrin was degraded on the 7th day of culture, almost 100% of dieldrin disappeared after 10 days of culture, and 25 μM of chloride ion was detected. This suggested that 2 chlorines were desorbed from 1 molecule of dieldrin.

<Experimental Example 4; FIG. 8>
[Deldrin degradation test of strains other than Mucor racemosus DDF strain belonging to racemosas species]
Using the following two strains belonging to the genus Mucor racemosus purchased from the National Institute for Biotechnology and Genetic Resources (NBRC), the strain preservation organization (mucor race shown in Experimental Example 3 using the IFO 6745 strain and the NBRC 4581 strain) The same dieldrin degradation test was performed with the Mothus DDF strain, and the results are shown in the bar graph of Fig. 8 together with the degradation test of the Mucor racemosus DDF strain.

FIG. 8 shows that the initial dose of dieldrin of 15 μM was not detected in the 10 day culture except for the control. In the control, chloride ions were not detected from the beginning, but in all three strains belonging to the genus Mucor racemosus, chloride ions that were not initially detected in 10 days of culture were detected. Yes. In addition, the control added dead cells instead of inoculating live cells.
That is, 90% or more of dieldrin was degraded on the 10th day of culture in the IFO6745 and NBRC4581 strains as well as the Mucor racemosus DDF strain. However, there was a difference in the chlorine ion concentration produced for each degrading bacterium, suggesting that there was a difference in the degree of dechlorination.

<Experimental Example 5: FIG. 9>
[Decomposition test of heptachlor epoxide using Mucor racemosus DDF strain]
In Example 3, a heptachlor epoxide degradation test was performed on the Mucor racemosus DDF strain by performing the same operation as in Example 3 except that the procedure was performed by adding 13 μM of heptachlor epoxide solution instead of adding 15 μM of the dieldrin solution. Went. The result is shown in FIG.
FIG. 9 shows that the initial 13 μM heptachlor epoxide was reduced to 0.8 μM in the 10-day culture except for the control. Moreover, it shows that chloride ions were not detected even after 10 days of culture.
From this result, it was confirmed that the Mucor racemosus DDF strain also decomposes 90% or more of heptachlor epoxide. However, it could not be confirmed until the dechlorination of heptachlor epoxide.

<Experimental example 6: FIG. 10>
[Decomposition test of various cyclic diene pesticides (other than dieldrin and heptachlor epoxide) by Mucor racemosus DDF strain]
In Experimental Example 3 and Experimental Example 5, instead of the operation of adding 15 μM of dieldrin solution and 13 μM of heptachlor epoxide solution, the operation of adding predetermined amounts of heptachlor solution, α-endosulfan solution, β-endosulfan solution, and endosulfan sulfate solution, respectively. Except for culturing for 10 to 14 days, the same procedure as in Experimental Example 3 and Experimental Example 5 was carried out to decompose heptachlor, α-endosulfan, β-endosulfan and endosulfan sulfate for Mucor racemosus DDF strain. A test was conducted. The result is shown in FIG.
In FIG. 10, the initial heptachlor was 13.3 μM and heptachlor epoxide was 13.0 μM, but in 10 days of culture, heptachlor was 0.75 μM and heptachlor epoxide was 0.84 μM, both decreased to 1 μM or less. The initial α-endosulfan 16.5 μM, β-endosulfan 16.3 μM, and endosulfan sulfate 17.1 μM decreased to 2.52 μM, 6.58 μM, and 8.21 μM, respectively, after 14 days of culture. Show. In the control, dead cells were added instead of inoculating live cells.
That is, as shown in FIG. 10, Mucor racemosus DDF strain decomposes 90% or more of heptachlor or heptachlor epoxide, 80% or more of α-endosulfan, 60% or more of β-endosulfan, and 50 of endosulfan sulfate. % Can be confirmed to decompose.

<Experimental example 7: FIG. 11>
[Decomposition test of DDT by Mucor racemosus DDF strain]
In Example 3, a DDT degradation test was performed on the Mucor racemosus DDF strain in the same manner as in Example 3 except that DDT was added at 12.8 μM in place of the procedure with addition of the dieldrin solution 15 μM. went. The result is shown in FIG.
FIG. 11 shows that the initial DDT of 12.8 μM became 1.76 μM after 10 days of culture. In the control, dead cells were added instead of inoculating live cells. In this experimental example, the DDT concentration also decreased in the control because the part of the initially added DDT was adsorbed by dead cells and could not be recovered.
As shown in FIG. 11, the addition of the Mucor racemosus DDF strain decreased the DDT concentration compared to the control, confirming that the Mucor racemosus DDF strain degraded 45.9% of the DDT. .
Moreover, DDE was not detected by the DDT decomposition | disassembly after culture | cultivation for 10 days by this experiment, and dechlorination was able to be confirmed.

<Experimental Example 8: FIG. 12>
[Decomposition test of DDE by Mucor racemosus DDF strain]
In Example 3, the DDE degradation test was performed on the Mucor racemosus DDF strain in the same manner as in Example 3 except that the culture was performed by adding 15.7 μM of DDE instead of the operation of adding 15 μM of the dieldrin solution. went. The result is shown in FIG.
FIG. 12 shows that the initial DDE of 15.7 μM became 3.28 μM after 10 days of culture. In the control, dead cells were added instead of inoculating live cells. Even in this experimental example, the DDE concentration decreased in the control because the part of the initially added DDE was adsorbed to dead cells and could not be recovered.
As shown in FIG. 12, the addition of the Mucor racemosus DDF strain decreased the DDE concentration compared to the control, confirming that the Mucor racemosus DDF strain degraded 51.6% of the DDE. .

Claims (14)

Cyclic diene compounds comprising filamentous fungi of Mucor racemosus species with resolution of cyclic diene compounds having 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure Decomposing agent. The cyclic diene compound decomposing agent according to claim 1 , wherein the cyclic diene compound is at least one compound selected from the group consisting of aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, and endosulfan sulfate. The cyclic diene compound decomposing agent according to claim 1 or 2, wherein dechlorination decomposition is performed on the cyclic diene compound. The cyclic diene compound decomposing agent according to any one of claims 1 to 3 , wherein 50% or more of the cyclic diene compound can be decomposed. The cyclic diene compound decomposing agent according to any one of claims 1 to 4, which has a resolution of DDT and DDE. Mucor Rasemosasu species filamentous fungus, Mucor racemate mode Blu DDF strain (accession number FERM P-21775), claims 1 to 5 is at least one strain selected from the group consisting of IFO6745 strain, and NBRC4581 strain The cyclic diene compound decomposing agent according to any one of the above. The cyclic diene compound degradation according to any one of claims 1 to 6 , comprising the filamentous fungus grown on a substrate such as bran or rice bran serving as a bait for the fungus of the genus Mucor racemosus. Agent. The cyclic diene compound decomposing agent according to claim 7, wherein a porous material such as a wood carbonized material is further mixed with the substrate. In Mucor racemosus DDF strain (accession number FERM P-21775) having the resolution of cyclic diene compounds having 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure A certain cyclic diene compound-degrading bacterium. The cyclic diene compounds aldrin, dieldrin, heptachlor, heptachlor epoxide, endrin, endosulfan, cyclic diene compound-decomposing bacterium according to claim 9, wherein at least one compound selected from the group consisting of Endosulfan sulfate. The cyclic diene compound-decomposing bacterium according to claim 9 or 10, which has a resolution of DDT and DDE. A purification apparatus for a polluted environment comprising the cyclic diene compound decomposing agent according to any one of claims 1 to 8 or the cyclic diene compound decomposing bacterium according to any one of claims 9 to 11 . The cyclic diene compound decomposing agent according to any one of claims 1 to 8, the cyclic diene compound decomposing bacterium according to any one of claims 9 to 11 , or the purification of a contaminated environment according to claim 12. A method for decomposing a cyclic diene compound comprising decomposing a cyclic diene compound having a 1,2,3,4,7,7-hexachlorobicyclo [2.2.1] hept-2-ene structure using an apparatus. The cyclic diene compound-decomposing agent according to any one of claims 1 to 8 , or the cyclic diene compound-decomposing bacterium according to any one of claims 9 to 11 , wherein 1 , 2, 3, 4, 7, 7-hexachlorobicyclo [2.2.1] cyclic diene compound having a hepta-2-ene structure, or a method for purifying pollutants inoculated in soil or cucurbitaceae plants contaminated with DDT or DDE.