JP6098918B2 - Organochlorine compound degrading bacteria capable of degrading low chlorinated PCBs, organochlorine compound decomposing material, method for producing the same, and purification apparatus for contaminated environment - Google Patents

Description

  The present invention relates to a novel organochlorine compound-degrading bacterium containing a degrading bacterium belonging to the genus Sphingomonas having the ability to degrade low-chlorinated PCBs or HCHs, and an organochlorine compound-decomposing material using this degrading bacterium and its production The present invention relates to a method and a purification apparatus for a polluted environment.

  PCBs sold under trade names such as Kanechlor, for example, low chlorinated PCBs represented by Kanechlor KC-300 (trade name) (Cl number 2 to 4) are highly concentrated in insulating oil in waste capacitors and transformers. In other words, treatment using chemical treatment methods (dechlorination decomposition method, etc.) is performed. However, this treatment method is expensive and the treatment has hardly progressed. In addition, this residual organic pollutant is not easily decomposed when released into the environment such as soil, and is likely to accumulate in the living body through a food chain or the like. Therefore, despite being harmful to human health and ecosystems, there is no effective degrading means if it is mixed in the soil even though it is in a trace amount.

  On the other hand, the organochlorine insecticide hexachlorocyclohexane (HCHs) mainly contains four isomers (α-form, β-form, γ-form, delta-form) in a ratio of 8: 1: 2: 1. It is an organic pollutant. In Japan, its use was banned in 1970 due to its strong toxicity and bioaccumulation, and its indegradability. Currently, HCHs are target agricultural chemicals for buried agricultural chemical treatment business, and in 2009, three isomers excluding δ were designated as persistent organic pollutants (POPs). Among the isomers, α and γ isomers have been aerobically decomposed and mineralized bacteria, and metabolic pathways and degrading enzyme genes have been clarified. However, no assimilating bacteria have been found that completely dechlorinate (mineralize) the chemically most stable β-form and the next stable delta-form.

  Regarding the purification methods for soil and groundwater contaminated with these pollutants, biostimulation methods that supply nutrients, oxygen, etc. to the contaminated site and propagate and activate indigenous degrading bacteria to purify, and are specified from outside In-situ bioremediation utilizing biological functions (especially resolution) of microorganisms, such as a bioaugmentation method for introducing microorganisms having the above functions, is expected, and bioremediation techniques relating to the degradation of simazine are disclosed in JP-A-2005. Japanese Patent Application Laid-Open No. 11-318435 (Patent Document 2) and the like disclose a bioremediation technique related to PCNB degradation in Japanese Patent Application Laid-Open No. 27536 / Patent Document 1 (Patent Document 1).

Japanese Patent Laying-Open No. 2005-27536 JP 11-318435 A

Thus, although PCBs or HCHs are designated as persistent organic pollutants, an effective decomposition technique has not been established.
Then, this invention is made | formed for the purpose of providing the bioremediation technique which decomposes | disassembles PCBs or HCHs effectively.

  In order to achieve the above object, some or all of the bacteriological properties of the Sphingomonas sp. TSK-1 strain having low chlorinated PCBs deposited under the receipt number: FERM AP-22222 (Accession number TBD) Provided is an organochlorine compound-degrading bacterium having properties.

  Receipt number: FERM AP-22222, an organochlorine compound-degrading bacterium having a part or all of the mycological properties of Sphingomonas sp. Since it is a newly discovered strain that degrades chlorinated PCBs, it can be expected to effectively purify contaminated environments represented by contaminated soil containing persistent low-chlorinated PCBs. Here, low chlorinated PCBs refers to PCBs having a chlorine number of 4 or less, preferably 2 to 4 among PCBs isomers having 1 to 10 chlorine atoms.

Also, the mycological properties of the Sphingomonas sp. TSK-1 strain having α-HCH, β-HCH, γ-HCH and δ-HCH resolution deposited under the receipt number: FERM AP-22222 (Accession number TBD) An organochlorine compound-degrading bacterium having a part or all of the properties of the present invention is provided.
A part or all of the bacteriological properties of the Sphingomonas sp. TSK-1 strain having α-HCH, β-HCH, γ-HCH and δ-HCH resolution deposited as FERM AP-22222 Since the organochlorine compound-degrading bacterium has a newly discovered strain that degrades not only α-HCH and γ-HCH but also β-HCH and δ-HCH, it is difficult to degrade β-HCH and δ. -It can be expected to effectively purify contaminated environments represented by contaminated soil containing HCH.

Organochlorine compound decomposing material comprising an organochlorine compound-degrading bacterium having a part or all of the mycological properties of Sphingomonas sp. TSK-1 strain in a porous material such as a wood carbonized material I will provide a.
Organochlorine compound-degrading bacteria having part or all of the mycological properties of Sphingomonas sp. Strain TSK-1 are contained in a porous material such as a wood carbonized material, so that these degrading bacteria can be retained stably. And can be grown. Moreover, since the porous material can be handled as a carrier containing organochlorine compound-decomposing bacteria, it is easy to handle organochlorine compound-degrading bacteria.

The organochlorine compound decomposing material further includes a degrading bacterium having part or all of the mycological properties of Nocardioides sp. PD653 strain having β-HCH resolution deposited internationally as FERM BP-10405. It can be used as an organic chlorine compound decomposition material.
Degrading bacteria having part or all of the mycological properties of Nocardioides sp. PD653 strain is converted into degrading bacteria having part or all of the mycological properties of Sphingomonas sp. In addition, since it is further included, it is possible to effectively decompose β-isomers that are difficult to decompose in HCHs. Therefore, it is an organochlorine-based compound decomposition material that is extremely excellent in resolution of HCHs.

Degradation having some or all of the mycological properties of Sphingomonas sp. TSK-1 strain versus degrading bacteria having some or all of the mycological properties of Nocardioides sp. PD653 It can be set as the organochlorine compound decomposition material which has a microbe in the ratio of 2-6 times.
Degrading bacteria having some or all of the mycological properties of Sphingomonas sp. TSK-1 strain versus degrading bacteria having some or all of the mycological properties of Nocardioides sp. PD653 In the porous material, the decomposition bacteria having the properties of the PD653 strain and the decomposition bacteria having the properties of the TSK-1 strain are contained in the porous material. It can coexist and HCHs can be effectively decomposed by the respective decomposing bacteria. If the proportion of the TSK-1 strain is less than twice, the growth of the TSK-1 strain may be inferior and the decomposition of δ-HCH may be insufficient. Further, when the proportion of the TSK-1 strain is more than 5 times, the growth of the PD653 strain is inferior, and β-HCH may be insufficiently decomposed. Such a ratio is more preferably more than 3 times and 6 times or less.

  This degradation material may contain pyruvic acid or a salt thereof and methionine as a substrate that becomes a carbon source or nitrogen source of the degradation bacteria together with the degradation bacteria. Since at least one of pyruvic acid or a salt thereof and methionine are included as a substrate serving as a carbon source or nitrogen source, the growth of decomposing bacteria in the porous material can be improved. Therefore, the resolution of low chlorinated PCBs or HCHs can be increased.

  Furthermore, the contaminated environment purification apparatus containing such decomposing bacteria and decomposition materials is provided. Since it contains decomposing bacteria and decomposition materials capable of degrading low chlorinated PCBs or HCHs, it is possible to purify contaminated soil and contaminated water.

Furthermore, regarding a method for producing an organochlorine compound decomposing material in which a degrading bacterium capable of degrading a predetermined organochlorine compound is accumulated in a porous material, a substrate serving as a nitrogen source or carbon source of the decomposing bacterium, and the decomposing bacterium A method for producing an organochlorine-based compound decomposing material, comprising immersing a culture solution containing a lysate in the porous material and supporting a decomposing bacterium and a substrate in the porous material.
A substrate containing a nitrogen source or a carbon source of a decomposing bacterium capable of degrading a predetermined organochlorine compound and a culture solution containing the decomposing bacterium is immersed in the porous material, so that the decomposing bacterium and the substrate are contained in the porous material. Therefore, the decomposing bacteria and the substrate can be efficiently contained in the porous material. Therefore, it is possible to easily produce a degradation material containing a degrading bacterium and a substrate.

  The predetermined organic compound is low chlorinated PCBs or HCHs, and the degrading bacterium has a part or all of the bacteriological properties of Sphingomonas sp. TSK-1 strain or the bacterium possessed by Nocardioides sp. PD653 strain If it is a degrading bacterium having some or all of the chemical properties, an organochlorine compound decomposing material capable of effectively degrading low chlorinated PCBs or HCH can be obtained.

According to the organochlorine compound decomposing bacteria, the organochlorine compound decomposing material, and the contaminated environment purification apparatus of the present invention, it is possible to easily purify contaminated environments such as soil contaminated with low chlorinated PCBs and HCHs.
Further, according to the method for producing an organic chlorine compound decomposing material in which the degrading bacteria capable of decomposing the predetermined organic chlorine compound of the present invention are accumulated in the porous material, the organic chlorine compound decomposing material and the purification apparatus for contaminated environment are provided. Easy to manufacture.

It is explanatory drawing which shows the molecular phylogenetic tree of TSK-1 stock | strain based on a base sequence. It is a graph which compares the growth degree of the TSK-1 strain | stump | stock in TSK culture medium and R2A culture medium. It is a graph which shows decomposition | disassembly of various low chlorinated PCBs by TSK-1 stock | strain in a TSK culture medium. It is a graph which shows Cl < ^- > density | concentration after various low-chlorination PCBs decomposition | disassembly by TSK-1 strain | stump | stock in TSK culture medium. It is a graph which shows KC-300 density | concentration after dechlorination decomposition | disassembly of KC-300 by TSK-1 strain | stump | stock in TSK culture medium. It is a graph which shows the chlorine ion density | concentration after the dechlorination decomposition | disassembly of KC-300 by the TSK-1 strain | stump | stock in a TSK culture medium. It is a graph which shows decomposition | disassembly of (alpha) -HCH by TSK-1 strain | stump | stock in an inorganic salt culture medium. It is a graph which shows decomposition | disassembly of (gamma) -HCH by TSK-1 strain | stump | stock in an inorganic salt culture medium. It is a graph which shows decomposition | disassembly of (beta) -HCH by TSK-1 strain | stump | stock in an inorganic salt culture medium. It is a graph which shows decomposition | disassembly of (delta) -HCH by TSK-1 strain | stump | stock in an inorganic salt culture medium. It is a graph which shows decomposition | disassembly of (alpha) -HCH by the TSK-1 strain | stump | stock in a TSK culture medium. It is a graph which shows decomposition | disassembly of (beta) -HCH by TSK-1 strain | stump | stock in TSK culture medium. It is a graph which shows decomposition | disassembly of (delta) -HCH by TSK-1 strain | stump | stock in a TSK culture medium. It is a graph which shows simultaneous decomposition | disassembly of 4 types of HCH by the TSK-1 strain | stump | stock in a TSK culture medium. It is a graph which shows the proliferation of the TSK-1 strain at the time of simultaneous decomposition | disassembly of 4 types of HCH by the TSK-1 strain in a TSK culture medium. It is a graph which shows decomposition | disassembly of 4 types HCH by 2 types complex system in a TSK culture medium. It is a graph which shows the change of turbidity in decomposition | disassembly of 4 types HCH by 2 types complex system in a TSK culture medium. It is a graph which shows decomposition | disassembly of 4 types HCH by 2 types complex system in contaminated soil.

  Techniques for decomposing low chlorinated PCBs or HCHs are described in detail below.

TSK-1 strain that degrades low chlorinated PCBs or HCHs :
In order to perform bioremediation, the presence of degrading bacteria (hereinafter referred to as “degrading bacteria”) for low-chlorinated PCBs or HCHs is indispensable. For obtaining the decomposing bacteria, a means applying the soil charcoal reflux method was used to obtain a decomposing material containing the decomposing bacteria.

20 g of γ-hexachlorocyclohexane (γ-HCH) and chlorothalonil (TPN) continuous soil sieved to 2 mm or less, and wood charcoal material A100 crushed and sieved to 4 to 6 mm (hardwood at 400 ° C to 600 ° C) Firing, mixing with ¹ g of specific surface area 95-120 m ^{2 /} g, pH 7.8) and filling the percolator as a reflux device (“degradable bacteria rapid accumulation device” manufactured by Fujiwara Seisakusho Co., Ltd.), the carbon source is γ-HCH ( 250 mL of an inorganic salt medium (5H) containing only 5 mg / L was refluxed and cultured at 25 ° C. in the dark for 3 weeks. Then, the concentration of γ-HCH and Cl ⁻ (γ-HCH metabolite) in the reflux solution over time was examined by GC-ECD and IC to confirm the state of accumulation of the decomposed bacteria.

The composition of the inorganic salt medium is as follows.
Each 1 L of medium contains 1.2 g of Na ₂ HPO ₄ · 12H ₂ O, 0.5 g of KH ₂ PO ₄ , 100 mg of NH ₄ NO ₃ , 10 mL of trace element, and here contains 5 mg of the above-mentioned γ-HCH, pH Is 6.8.

After washing, the autoclave-sterilized A100 was filled with 7.5 g in another percolator of the same type as the above-described percolator. To this, 0.25 g of A100 in which accumulation of degrading bacteria progressed in the above culture was inoculated, and as described above, 250 mL of an inorganic salt medium (5H) in which the carbon source was only γ-HCH (5 mg / L) was refluxed. The cells were cultured at 25 ° C. in the dark for 3 weeks.
Then, A100 in which degrading bacteria were accumulated was taken out from the percolator, ground, and diluted to 10 ⁻⁴ with a phosphate buffer, inoculated into an inorganic salt medium (5H), cultured, and the decomposing activity was confirmed.

Further, this culture solution was serially diluted to 10 ⁻⁷ times by a limiting dilution method to increase the density of degrading bacteria, and a culture solution with a 10 ⁻⁷ dilution having a degrading activity was obtained. The culture broth was smeared on an R2A agar medium, sprayed with 5% γ-HCH-containing ether, cultured, and the degrading bacteria were isolated and identified from the formed clear zone. The reason why the R2A agar medium was used in place of the inorganic salt medium was to increase the growth of degrading bacteria.

The morphological characteristics of this degrading bacterium are Neisseria gonorrhoeae (0.7-0.8 × 1.5-5.0 μm), and the culture property is possible at 30 ° C. on R2A agar medium, yellow and glossy “+ ”, Pigment production“ + ”, presence / absence of surface growth“ − ”in R2A liquid medium 30 ° C., presence / absence of“ + ”in medium turbidity, growth state“ − ”in gelatin puncture medium 30 ° C., gelatin liquefaction“ -", Litmus milk coagulated at 30 ° C"-", liquefied"-".
Physiological properties include Gram stainability "-", nitrate reduction "-", denitrification "-", MR test "-", VP test "-", indole production "-", hydrogen sulfide production "- ”, Starch hydrolysis“-”, citric acid utilization (Koser)“-”, citric acid utilization (Christensen)“-”, inorganic nitrogen source utilization (nitrate)“-”, inorganic nitrogen source utilization ( Ammonium salt) "-", urease activity "-", catalase "-", oxidase "+", growth range is pH 5 "-", pH 6 "+ w", pH 7 to 9.5 "+" The ranges are 15 ° C. “+ w”, 20 ° C. “+”, 25 ° C. “+”, 37 ° C. “−”, anaerobic growth “-”, OF test (oxidation / fermentation) “-/-”. It is.

  In the acid production test medium composition shown below, for acid production / gas production from saccharides, L-arabinose “− / −”, D-glucose “− / −”, D-furanose “− / −”, Maltose "-/-", lactose "-/-", D-sorbitol "-/-", inositol "-/-", D-xylose "-/-", D-mannose "-/-", D- Galactose “− / −”, saccharose “− / −”, trehalose “− / −”, D-mannitol “− / −”, and glycerin “− / −”.

The acid production test medium composition is Yeast Extract 0.5 g, Bacto Proteose Peptone No. 3 0.5 g, Bacto Casamino Acid 0.5 g, Soluble Starch 0.5 g, Sodium Pyruvate 0.3 g, K ₂ HPO ₄ 0.3 g, MgSO ₄ .7H ₂ O 0.05 g, agar 0.02 g, ultrapure water 1000 mL, 0.2% Phenol Red 0.05 mL, each sugar 10 g, and pH is 8.0.
Other physiological properties are [beta] -galactosidase activity "-", arginine dihydrolase "-", lysine decarboxylase activity "-", tryptophan deaminase activity "-", gelatinase activity "-".

  “+” Represents “positive”, “−” represents “negative”, and “w” represents “weak reaction”. In addition, each test is conducted at the NCIMB Laboratory in the UK and 1) BARROW, (GI) and FELTHAM, (RKA): Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University Press 2) Toshikazu Sakazaki, Goro Yoshizaki, Kanji Miki: New Bacteriology and Culture Laboratory (2nd edition). 1988, published by Kyundai 3) Takeshi Hasegawa, edited and classified by microorganisms (below). And a bacterial identification kit AP120E, (bioMerieux, France).

The obtained degrading bacteria were assigned as Sphingomonas sp. TSK-1 (also referred to as “TSK-1 strain”). The 16S rRNA base sequence (1482 bases) is shown in the sequence listing with the base sequence of SEQ ID NO: 1. The TSK-1 strain will be registered at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-11-1 Higashi, Tsukuba City, Ibaraki Prefecture 305-8566, receipt number: FERM AP-22222, date of deposit: March 1, 2012 ( The deposit number is undecided. A molecular phylogenetic tree of this strain based on this base sequence is shown in FIG. As can be seen from this phylogenetic tree, it is systematically different from existing degrading bacteria.

PD653 strain that degrades β-HCH :
The PD653 strain is a degrading bacterium that has been internationally deposited as FERM BP-10405 at the National Institute of Advanced Industrial Science and Technology (AIST) as Nocardioides sp. PD653 ( Nocardioides sp. PD653). There is also a description in the publication.

The mycological properties of the PD653 strain are as follows. Morphological features are Neisseria gonorrhoeae (0.7-0.8 × 1.0-1.2μm), no sporulation, pale yellow, round, semi-lens-like ridged state, smooth all edges, opaque, butter-like Colonies having a consistency of The culture property is aerobic culture for 3 to 7 days at 30 ° C. in an R2A agar medium. There is no change in colony morphology due to mutation, culture conditions, or physiological conditions. Motility "-", Gram staining "-". Physiological properties are catalase “+”, oxidase “−”, acid / gas production (glucose): “− / −”, O / F test (glucose) “− / −”, GC content: 70.8% is there. The notations “+” and “−” are the same as above.
Molecular phylogenetic analysis based on the partial base sequence of 16S rRNA of PD653 strain was carried out, and continuous 1487 bases were determined from the base sequence of 16S rRNA of PD653 strain. Show.

  It is known that the PD653 strain alone has a resolution for PCNB and also has a resolution for HCB alone, but it is known that it has the resolution for each HCH. It was not done. However, considering that HCB and β-HCH are similar in three-dimensional structure in that all Cl groups are in the equatorial position, the degradation performance of β-HCH by the PD653 strain was examined.

Organochlorine compound decomposition materials :
In order to effectively decompose organic pollutants such as low chlorinated PCBs and HCHs in contaminated environments such as soil and water, organochlorine-based compound-degrading bacteria are present in soil and water at high density for a long period of time. There is a need. Therefore, it is preferable to carry | support to the porous material like the wood carbonization material which becomes a living place of an organochlorine compound decomposing bacteria with the substrate used as the bait | organism of an organochlorine compound decomposing bacteria. Among the substrates, the carbon source is preferably pyruvic acid or a salt thereof such as sodium pyruvate. As the nitrogen source, methionine, tyrosine and the like are preferable, but methionine is more preferable.
The carrier which is a dwelling of degrading bacteria specific surface area as found in wood carbonization material or the like is 50m 2 ^/ g~600m 2 ^{/ g} porous material is preferable. This is because the assimilation material is easily adsorbed in a form in which the degrading bacteria can easily be used, and the degrading bacteria can be stably accumulated on the carrier.
In addition, since the porous material increases the content of degrading bacteria, it is preferable that pores having a size of 2 μm to 50 μm, more preferably 5 μm to 20 μm are 10% or more by volume ratio.

Purification method of pollutants using organochlorine compound decomposition materials :
There are the following methods for purifying substances contaminated with low chlorinated PCBs and HCHs using organochlorine compound decomposition materials.
For removal of low chlorinated PCBs and HCHs in contaminated soil, organochlorine compound decomposition materials are buried in the contaminated soil and mixed. By burying in the soil, low chlorinated PCBs and HCHs contained in the soil are decomposed by the decomposing bacteria. According to this method, it is possible to avoid low chlorinated PCBs and HCHs in the soil from being mixed into the groundwater, and it is possible to prevent groundwater contamination.
As an application of this technology, mixing into surface and lower soils where low chlorinated PCBs and HCHs exist, mixing into lower soils on the green surface of golf courses, mixing into lower soils at industrial waste disposal sites, in factories, etc. For example, it can be mixed into the lower layer soil of the organic waste liquid storage, and such applications can treat low chlorinated PCBs and HCHs.

Pollution environment purification equipment :
Low-chlorinated PCBs and HCHs can be easily used as bioreactors if an organic chlorinated compound decomposing material is packed in an air-permeable housing, and an accumulation layer containing the organic chlorinated compound decomposing material in the soil is held in the housing. It can be set as the purification apparatus (organochlorine compound decomposition removal apparatus) of the polluted environment which decomposes and removes.
The soil can be purified by mixing soil contaminated with HCHs into the apparatus and refluxing the water. Moreover, this contaminated water can be purified by refluxing water contaminated with HCHs to the apparatus. In addition, it can handle similarly about low chlorinated PCBs instead of HCHs.
Alternatively, by installing this device in a part of waterways such as domestic drainage channels, paddy field agricultural drainage channels, golf course drainage channels, etc., the low-chlorinated PCBs and HCHs dissolved and dispersed in water can be decomposed and removed. It can be used as a pollution environment purification device for purifying a pollution environment.

<Experimental example 1>
[Preparation of TSK medium: Fig. 2]
A TSK medium, which is a semi-synthetic medium in which the TSK-1 strain grows and has a low Cl ⁻ concentration and can be dechlorinated by the TSK-1 strain, was developed.
The composition of TSK medium is 1.0 g of peptone CE90M, 0.25 g of yeast extract BSP-B, 0.5 g of methionine, 0.6 g of sodium pyruvate, 0.08 g of K ₂ HPO ₄ in 1 L of the TSK medium. It contains 0.05 g of MgSO ₄ .7H ₂ O and has a pH of 7.2 to 7.4.

The TSK medium is obtained by changing Proteose Peptone No.3, Bacto Yeast Extract and casamino acid in R2A (Difco) liquid medium suitable for culturing TSK-1 strain into peptone CE90M, yeast extract BSP-B and methionine. The Cl ^- concentration of the solution was reduced from about 68.1 mg / L to about 0.69 mg / L to 1/100, soluble starch and glucose were excluded, methionine was added, and sodium pyruvate was doubled. They succeeded in changing the carbon source to accelerate the growth compared to the R2A liquid medium (FIG. 2).

  The nutrient source was changed in this way because the TSK-1 strain does not have the ability to use soluble starch and glucose as a carbon source, and can use pyruvic acid as another nutrient source. This is because it has been found that increasing the amount improves growth. As for methionine, the nitrogen source was examined from 20 kinds of amino acids, and it was found that methionine was the most excellent nitrogen source, and that the combination of pyruvic acid and methionine had a good effect on growth. .

<Experimental example 2>
[Degradation test of low chlorinated PCBs by TSK-1 strain: FIGS. 3 to 4]
A low chlorinated PCBs degradation test was performed using TSK medium.
First, as low chlorinated PCBs, 2,4′-dichlorobiphenyl (IUPAC # = # 8), 2,2 ′, 5-trichlorobiphenyl (IUPAC # = # 18), 2,4 ′, 5-trichlorobiphenyl ( IUPAC # = # 31), 2 ′, 3,4-trichlorobiphenyl (IUPAC # = # 33), 2,2 ′, 3,5′-tetrachlorobiphenyl (IUPAC # = # 44), 2,2 ′, Contains 1000 ppm each of seven low-chlorinated PCBs mixed with 5,5′-tetrachlorobiphenyl (IUPAC # = # 52) and 2,3 ′, 4,4′-tetrachlorobiphenyl (IUPAC # = # 66) An acetone solution A and an acetone solution B containing 1000 ppm of 2,4,4′-trichlorobiphenyl (IUPAC # = # 28) as low-chlorinated PCBs were prepared.

And the said acetone solution A was added to TSK culture medium so that the final concentration of each PCBs might be set to 5 ppm. Similarly, acetone solution B was added to another TSK medium so that the final concentration of PCBs was 5 ppm. Then, the TSK medium to which the acetone solution A was added and the TSK medium to which the acetone solution B was added were respectively experimented in the following procedure.
The TSK medium containing PCBs contains TSK-1 strain culture solution (composition is phosphate buffer containing 1.2 g of Na ₂ HPO ₄ · 12H ₂ O and 0.5 g of KH ₂ PO ₄ per liter of the culture solution). 1 mL of OD ₆₀₀ = 1.0 including TSK-1 strain) was added to 10 mL of the medium. As a control, 1 mL of phosphate buffer was added.

Then, it was cultured with shaking in a 50 mL Erlenmeyer flask with a stopper at 180 rpm and 30 ° C. for 10 days. One week later, 22 mL of hexane was added, shaken for 10 minutes, the hexane layer was diluted 10-fold, and each PCBs concentration was analyzed by GC-ECD. The result is shown in FIG. Further, the Cl ⁻ concentration was measured by ion chromatography, and the result is shown in FIG.

From the PCB (2,4,4′-trichlorobiphenyl) degradation test of # 28 in acetone solution B, from FIG. 3, # 28 was degraded by 17% (0.95 mg / L) in 10 days of culture. From the results, it was found that 0.39 mg / L of chlorine ions was produced. Since the molecular weight of # 28 PCB was 257 and the proportion of chlorine was 41.4%, 3 chlorine was eliminated. Therefore, it was found that # 28 was completely dechlorinated.
Also, from the seven PCBs decomposition tests with acetone solution A, about 25% to 30% for PCBs with 4 chlorines, about 15% to 20% for PCBs with 3 chlorines, about 10% for PCBs with 2 chlorines It turns out that it has decomposed. Thereby, it turned out that PCBs with many chlorine numbers are easier to decompose | disassemble.

<Experimental example 3>
[Degradation test of KC-300 (trade name) by TSK-1 strain: FIGS. 5 to 6]
A degradation test of KC-300 (trade name) was performed using TSK medium. KC-300 is a mixture of various PCBs sold as Kanechlor KC-300 (trade name), and is a substance in which about 96% of low-chlorinated PCBs containing 2 to 4 chlorine atoms in the molecule accounted for.
An acetone solution containing 1000 ppm of this KC-300 was prepared, and an experiment similar to that of Example 12 was performed except that this acetone solution was used instead of the acetone solution A used in Example 12 above. The results are shown in FIGS.
5 and 6, it was found that 0.64 mg / L of chlorine ions was generated and KC-300 was decomposed by 26%.

<Experimental example 4>
[Α-HCH degradation test with TSK-1 strain using inorganic salt medium: Fig. 7]
20 mL of inorganic salt medium containing α-HCH, 17.2 μM (5 mg / L) as a sole carbon source (composition is 1.2 g Na ₂ HPO ₄ · 12H ₂ O and 0 KH ₂ PO ₄ per liter of medium) 0.5 g, NH ₄ NO ₃ 100 mg, Trace element 10 mL, and here α-HCH 5 mg, pH is 6.8) in TSK-1 strain culture medium (composition is Na per 1 L culture medium) Inoculate 1 mL of phosphate buffer containing 1.2 g of ₂ HPO ₄ · 12H ₂ O and 0.5 g of KH ₂ PO ₄ (including TSK-1 strain and OD ₆₀₀ = 2.04) at 30 ° C. and 180 rpm Cultured for 10 days. On the 4th, 7th, and 10th days, 20 mL of hexane was added for total extraction, and the α-HCH concentration of the hexane layer was measured by GC-ECD, and the Cl ⁻ concentration of the aqueous layer was measured by ion chromatography. At the same time, the turbidity (OD ₆₀₀ ) of the medium was also measured.

As a result of the degradation test of α-HCH using the inorganic salt medium, it was found that α-HCH disappeared and was decomposed 100% after 10 days of culture, as shown in FIG. Further, since 4 equivalents of Cl ^- was detected, it was found that the TSK-1 strain eliminated 4 chlorine of α-HCH.

<Experimental example 5>
[Γ-HCH degradation test by TSK-1 strain using inorganic salt medium: Fig. 8]
In the inorganic salt medium of Experimental Example 4, a γ-HCH decomposition test was performed in the same manner as in Experimental Example 4 except that γ-HCH was used instead of α-HCH. The results are shown in FIG.
As a result of the degradation test of γ-HCH using an inorganic salt medium, 6 equivalents of Cl ⁻ was detected in 10 days of culture. Therefore, the TSK-1 strain must completely dechlorinate γ-HCH and degrade 100%. I understood.

<Experimental example 6>
[Β-HCH degradation test by TSK-1 strain using inorganic salt medium: Fig. 9]
In the inorganic salt medium of Experimental Example 4, a β-HCH decomposition test was performed in the same manner as in Experimental Example 4 except that β-HCH was used instead of α-HCH. The results are shown in FIG.
As a result of the degradation test of β-HCH using an inorganic salt medium, β-HCH was degraded in 10 days of culture and 1 equivalent of Cl ⁻ was detected. Further, β-HCH disappeared. As a result, from the TSK-1 strain inoculated into an inorganic salt medium containing β-HCH as the sole carbon source, β-HCH was dechlorinated and decomposed 100%, but until complete dechlorination occurred. I found out that

<Experimental example 7>
[Δ-HCH degradation test by TSK-1 strain using inorganic salt medium: Fig. 10]
In the inorganic salt medium of Experimental Example 4, a δ-HCH decomposition test was performed in the same manner as in Experimental Example 4 except that δ-HCH was used instead of α-HCH. The remaining hexane layer was concentrated 100 times and then measured by GC-MS to analyze the metabolite of δ-HCH. The results are shown in FIG.
As a result of the degradation test of δ-HCH using an inorganic salt medium, δ-HCH was degraded by 80% after 10 days of culture, and 2 equivalents of Cl ^- was detected. Analysis of metabolites revealed pentachlorocyclohexanol and tetrachlorocyclohexenol.
As a result, from the TSK-1 strain inoculated in an inorganic salt medium containing δ-HCH as the sole carbon source, it was found that δ-HCH was dechlorinated but not completely dechlorinated. .

<Experimental Example 8>
[Α-HCH degradation test with TSK-1 strain using TSK medium: FIG. 11]
An α-HCH degradation test was performed using the TSK medium. 30 mL of TSK medium is mixed with α-HCH, 13.8 μM (4 mg / L), inoculated with 1.5 mL of the same TSK-1 culture solution (OD ₆₀₀ = 1.00) as in Experimental Example 4, at 30 ° C. in the dark, Cultured with shaking at 180 rpm for 10 days. Sampling was performed on 4th, 7th and 10th days (destructive analysis), and α-HCH concentration, Cl ⁻ concentration and turbidity (OD ₆₀₀ ) of the medium were measured. More specifically, 30 mL of hexane was added to the medium for total extraction, the α-HCH concentration was measured by GC-ECD from the hexane layer, and the Cl ⁻ concentration was measured by ion chromatography from the aqueous layer. The result is shown in FIG.
As a result of the degradation test of α-HCH using TSK medium, it was found that 6 equivalents of Cl ⁻ was detected after 10 days of culture, and α-HCH was completely dechlorinated to decompose 100%.

<Experimental Example 9>
[Β-HCH degradation test by TSK-1 strain using TSK medium: Fig. 12]
A β-HCH degradation test was performed in the same manner as in Experimental Example 8 except that β-HCH was used instead of α-HCH in the TSK medium of Experimental Example 8. The results are shown in FIG.
As a result of the degradation test of β-HCH using TSK medium, it was found that β-HCH was decomposed by 90% with the growth of degrading bacteria after 12 days of culture, causing 2-chlorine elimination.

<Experimental example 10>
[Δ-HCH degradation test with TSK-1 strain using TSK medium: Fig. 13]
Using δ-HCH instead of α-HCH in the TSK medium of Experimental Example 8 and changing the turbidity with the same culture solution as the TSK-1 strain culture solution of Experimental Example 8 (OD ₆₀₀ = 0.05) A δ-HCH decomposition test was conducted in the same manner as in Experimental Example 8 except that inoculation was performed. The results are shown in FIG.
As a result of the degradation test of δ-HCH using the TSK medium, it was found that 6 equivalents of Cl ⁻ was detected after 10 days of culture, and that the TSK-1 strain could completely decompose δ-HCH and decompose 100%. .
Further, from the comparison with the result of the δ-HCH decomposition test, it was found that the decomposition rate was higher for α-HCH than for δ-HCH.

<Experimental example 11>
[Four kinds of HCH degradation tests by TSK-1 strain using TSK medium: FIGS. 14 and 15]
Experimental Example 8 except that in addition to 4 mg / L of α-HCH in the TSK medium of Experimental Example 8, β-HCH was added to 2 mg / L, γ-HCH was added to 4 mg / L, and δ-HCH was added to 4 mg / L. In the same manner as above, the decomposition test of 4 types of HCH of α-form, β-form, γ-form, and delta-form was conducted. The results are shown in FIG. 14 and FIG.
In 10 days of culture, α-form, γ-form and δ-form were degraded 100%, β-form was degraded 91%, and the dechlorination rate was 53.0%. Moreover, it was found that the TSK-1 strain decomposes 4 types of HCH with growth.

<Experimental example 12>
[Decomposition test of 4 types of HCH by 2 types (TSK-1 strain + PD653 strain) composite system: FIG. 16, FIG. 17]
Using the above TSK medium, α-, β-, γ-, and δ-HCH degradation tests were performed. 20 mL of TSK medium contains 5 mg / L of α-HCH, 2 mg / L of β-HCH, 5 mg / L of γ-HCH, and 5 mg / L of δ-HCH, and the ratio of TSK-1 degrading bacteria to PD653 degrading bacteria is Inoculated with 1.0 mL of the same turbidity of the same culture solution as in Experimental Example 8 so that the ratio was 3: 1 (the composition was PD2 in the R2A medium and OD ₆₀₀ = 0.52), The culture was shaken at 30 ° C. in the dark at 180 rpm for 14 days. Sampling was performed on days 4, 7, and 14 (destructive analysis), and each HCH concentration, Cl ⁻ concentration, and turbidity (OD ₆₀₀ ) of the medium were measured. More specifically, 30 mL of hexane was added to the medium for total extraction, the δ-HCH concentration was measured by GC-ECD from the hexane layer, and the Cl ⁻ concentration was measured by ion chromatography from the aqueous layer. Each HCH concentration and Cl ⁻ concentration are shown in FIG. 16, and turbidity is shown in FIG.

  As a result of the degradation test of 4 types of HCH by the 2 types of complex system, it was found that α-HCH was decomposed 100%, β-HCH 100%, and γ-HCH 100%. δ-HCH was decomposed by 90%, and the dechlorination rate was 65%. This result is thought to be because the growth of the TSK-1 strain was somewhat inhibited because the PD653 strain had a faster growth rate than the TSK-1 strain.

<Experimental example 13>
[Degradation and removal test of HCHs actual contaminated soil by 2 types (TSK-1 strain + PD653 strain) composite system: FIG. 18]
Decomposing bacteria-containing culture solution in which the above-mentioned culture solution containing TSK-1 degrading bacteria (OD ₆₀₀ = 0.6) and the above-mentioned culture solution containing PD653-degrading bacteria (OD ₆₀₀ = 1.0) are mixed at a ratio of 6: 1. 30 mL was immersed in 20 g of a cleaned and sterilized wood carbonized material (coconut shell charcoal CC150; specific surface area 150 m ² / g, pH 7.8, particle size 0.5 mm to 4.0 mm) overnight. Then, this wood charcoal material 1.5g (corresponding to 5% of dry matter equivalent to dry matter) mixed with decomposed bacteria is mixed with 30g of HCHs contaminated soil (equivalent to dry soil) to adjust the soil moisture to 30%. Then, static culture was performed at 25 ° C. in the dark for 4 weeks. During this period, stirring and moisture adjustment were performed every week. Then, 10 g of contaminated soil mixed with a wood carbonized material was extracted with ASE (trademark) in the second week of culture, and after purification, each HCH was analyzed with GC-ECD. The result is shown in FIG.

  From FIG. 18, it was found that all of the four types of HCH decreased by 15% or more (18.5% for β-HCH and 34.6% for δ-HCH). In particular, it was found that γ-HCH was 76.1% and α-HCH was 67.1%, exceeding 50%.

Claims (8)

Decomposition of novel organochlorine compounds belonging to Sphingomonas sp. TSK-1 strain having the resolution of low chlorinated PCBs deposited under the accession number: FERM P-22222, α-HCH, β-HCH, γ-HCH and δ-HCH Fungus. Accession number: A novel organochlorine compound-degrading bacterium belonging to Sphingomonas sp. TSK-1 strain having α-HCH, β-HCH, γ-HCH and δ-HCH resolution deposited as FERM P-22222. An organochlorine compound decomposing material comprising the decomposing bacterium according to claim 1 or 2 in a porous material such as a wood carbonized material. The organochlorine-based compound decomposing material according to claim 3, further comprising a degrading bacterium belonging to Nocardioides sp. PD653 strain having β-HCH resolution deposited internationally as FERM BP-10405. The organochlorine according to claim 4, which has a degrading bacterium according to claim 1 or 2 in a ratio of 2 to 6 times that of the degrading bacterium belonging to Nocardioides sp. PD653 strain according to claim 4. Material decomposition materials. The substrate as a carbon source or nitrogen source of the degrading bacterium according to claim 1 or 2, comprising at least one of pyruvic acid or a salt thereof and methionine, according to any one of claims 3 to 5. Organochlorine compound decomposition material. An apparatus for purifying a polluted environment comprising the decomposing bacterium according to claim 1 or 2, or the organochlorine compound decomposing material according to any one of claims 3 to 6. In the method for producing an organic chlorine compound decomposing material in which a degrading bacterium capable of decomposing an organic chlorine compound is accumulated in a porous material,
The organochlorine compound is at least one of low chlorinated PCBs or HCHs;
The degrading bacterium is a first decomposing bacterium, accession number: FERM P-22222 deposited as Sphingomonas sp. TSK-1 strain, and the second degrading bacterium Nocardioides sp. PD653 strain. It is belong degrading bacteria,
A culture solution containing a substrate serving as a carbon source or a nitrogen source of the first decomposing bacterium, the first decomposing bacterium, and the second decomposing bacterium is immersed in the porous material, and the first A method for producing an organochlorine compound-decomposing material, comprising supporting a decomposing bacterium, the second decomposing bacterium, and the substrate in a porous material.

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