JP6701973B2 - 1,4-dioxane decomposition activity improver, composition for 1,4-dioxane decomposition treatment and method for producing the same, and method for culturing 1,4-dioxane-decomposing bacteria and 1,4-dioxane decomposition treatment method - Google Patents

Description

  The present invention relates to a 1,4-dioxane decomposition activity improver, a composition for 1,4-dioxane decomposition treatment, a method for producing the same, a method for culturing 1,4-dioxane-decomposing bacteria and a method for 1,4-dioxane decomposition treatment. .. More specifically, it relates to a 1,4-dioxane decomposition activity improver containing a cyclic ester compound.

  1,4-dioxane was added to the WHO environmental standards in 2002 as having a potential carcinogenic effect (Group 2B: suspected carcinogenic to humans). 1,4-dioxane is contained in wastewater from business establishments such as chemical products industry such as photosensitive resin production, petrochemical industry such as ethylene oxide and ethylene glycol production, and chemical fiber industry such as polyester production. It is said that 1,4-dioxane is difficult to decompose and is difficult to remove when it is released into the environment. Therefore, development of a method capable of decomposing 1,4-dioxane contained in wastewater easily and at low cost is desired.

  Patent Document 1 is a wastewater treatment method for decomposing and removing 1,4-dioxane in wastewater, in which 1,4-dioxane-degrading bacteria are added to the wastewater to decompose and remove 1,4-dioxane in the wastewater. A wastewater treatment method is described (see claim 14). The document discloses that, as a method for efficiently culturing 1,4-dioxane-decomposing bacteria having an extremely low growth rate, "a seed sludge containing 1,4-dioxane-decomposing bacteria, and only 1,4-dioxane as an organic substance are used. After culturing in an inorganic medium containing the above, an isolation step of forming a colony of the 1,4-dioxane-decomposing bacterium on an agar medium and isolating the colony, and a colony of the isolated 1,4-dioxane-decomposing bacterium in the above 1 , A culturing step of culturing in an organic medium containing an organic substance other than 4-dioxane, and a method for culturing a 1,4-dioxane-degrading bacterium" are disclosed (see claim 1). In this method, in the isolation step, the 1,4-dioxane-decomposing bacterium is preferentially grown so as not to be contaminated with other microorganisms, and in the culturing step, the isolated 1,4-dioxane-decomposing bacterium is rapidly grown. It is intended to proliferate.

  The inorganic medium used in the isolation step disclosed in Patent Document 1 is used by adding 1,4-dioxane (see, for example, paragraph 0105 and Table 1).

JP, 2008-306939, A

  Conventionally, in order to obtain 1,4-dioxane-degrading bacteria in a state of having 1,4-dioxane-degrading activity, culture is performed in the presence of 1,4-dioxane itself in order to induce an enzyme that catalyzes the activity. There was a need. Also in the test of Patent Document 1, when 1,4-dioxane was added to the organic medium and culturing was performed (see Test 2 in Table 4), the removal rate of 1,4-dioxane was relatively high at 48%. high. However, in the case of culturing in an organic medium in the absence of 1,4-dioxane, which is an embodiment of the invention according to the document (test 3 in Table 4), the removal rate is 25%. Stays in. This is because even if the growth of the 1,4-dioxane-decomposing bacterium is achieved at high speed in the culture in the organic medium, in the absence of the 1,4-dioxane, the 1,4-dioxane-decomposing bacterium is sufficiently decomposed in the grown bacterium. It shows that the activity is not induced.

  When 1,4-dioxane is used for inducing 1,4-dioxane-degrading activity, 1,4-dioxane may be released into the environment from the medium of 1,4-dioxane-degrading bacterium which is an aerobic microorganism. is there. For this reason, equipment investment is required to separate and decompose 1,4-dioxane released from the medium.

  Therefore, the main object of the present invention is to provide a technique for culturing 1,4-dioxane-degrading bacteria excellent in 1,4-dioxane-degrading activity without using 1,4-dioxane.

In order to solve the above problems, the present invention provides the following [1] to [29].
[1] A 1,4-dioxane decomposition activity improver, which is used for a bacterium having a 1,4-dioxane decomposition activity, which contains a cyclic ester compound.
[2] The cyclic ester compound is selected from the group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone. [1] 1,4-dioxane decomposition activity improver of any one or more of
[3] The 1,4-dioxane decomposition activity improver of [1] or [2], which does not contain 1,4-dioxane.
[4] [1] to [3], wherein the bacterium belongs to one or more genera selected from the group consisting of Pseudonocardia, Rhodococcus, and Mycobacterium. 1,4-dioxane decomposition activity improver.
[5] The above-mentioned fungus is Pseudonocardia dioxanovorans, Pseudonocardia benzenivorus, Pseudonocardia benzencoorca, Pseudonoca 1,4-dioxane degradation of any one of [1] to [3] belonging to one or more species selected from the group consisting of Rhodococcus aterivorans, Rhodococcus aetherivorans and Mycobacterium vaccae Activity enhancer.
[6] The above-mentioned fungus is Pseudonocardia dioxanovorans CB1190, Pseudonocardia benzenica lucifera codica serracodicacondactica, and Pseudonocardia vulcoa serracodica anticontica (Pseudonocardia varica serracodica sd. (Rhodococcus ruber) 219, Rhodococcus aetherivorans JCM14343, Mycobacterium vaccae (Mycobacterium spp.), and Mycobacterium sp. The 1,4-dioxane decomposition activity improver according to any one of [1] to [3].

[7] A method for culturing a bacterium having 1,4-dioxane degrading activity, which comprises:
A method for culturing a bacterium having 1,4-dioxane-degrading activity, which comprises a step of culturing the bacterium in a medium containing a 1,4-dioxane-degrading activity improving agent containing a cyclic ester compound.
[8] The cyclic ester compound is selected from the group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone. [7] The method of culturing [7], which comprises one or more of the following:
[9] The culture method according to [7] or [8], wherein the cyclic ester compound contained in the 1,4-dioxane decomposition activity improver is present in the medium at 0.001 to 0.15 mol/L.
[10] The culture method according to any one of [7] to [9], wherein the 1,4-dioxane decomposition activity improver does not contain 1,4-dioxane.
[11] The fungus belongs to one or more genera selected from the group consisting of Pseudonocardia genus, Rhodococcus genus and Mycobacterium genus, [7] to [10] Any of the culture methods of.
[12] The above-mentioned fungus is Pseudonocardia dioxanovorans, Pseudonocardia benzenivorcans, Pseudonocardia benzencoorca, Pseudonoca The method for culturing according to any one of [7] to [10], which belongs to one or more species selected from the group consisting of Rhodococcus aetherivorans and Mycobacterium vaccae.
[13] The above-mentioned fungus is Pseudonocardia dioxanovorans CB1190, Pseudonocardia benzenica lucifera codica serracodica sunderacondica Pentacodica serracodica sd. (Rhodococcus ruber) 219, Rhodococcus aetherivorans JCM14343, Mycobacterium vaccae (Mycobacterium spp.), and Mycobacterium sp. The culture method according to any one of [7] to [10].

[14] A method for decomposing 1,4-dioxane, which comprises a step of contacting 1,4-dioxane with a bacterium obtained by the culturing method according to any one of [7] to [13], , 4-dioxane decomposition treatment method.

[15] A method for producing a composition for 1,4-dioxane decomposition treatment, which comprises a bacterium having 1,4-dioxane decomposition activity,
A method for producing a composition for 1,4-dioxane decomposition treatment, comprising a step of culturing a bacterium in a medium containing a 1,4-dioxane decomposition activity improver containing a cyclic ester compound.
[16] The cyclic ester compound is selected from the group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone. [15] The method for producing [15], which is one or more of the following.
[17] The production method of [15] or [16], wherein the cyclic ester compound contained in the 1,4-dioxane decomposition activity improver is present in the medium at 0.001 to 0.15 mol/L.
[18] The production method of any of [15] to [17], wherein the 1,4-dioxane decomposition activity improver does not contain 1,4-dioxane.
[19] The above-mentioned bacterium belongs to one or more genera selected from the group consisting of Pseudonocardia genus, Rhodococcus genus and Mycobacterium genus, [15] to [18] One of the manufacturing method of.
[20] The fungus is Pseudonocardia dioxanovorans, Pseudonocardia benzenivorcans (Pseudonocardia benzencoorca), Pseudonocardia benzenicoruca var. The method according to any one of [15] to [18], which belongs to one or more species selected from the group consisting of Rhodococcus aetherivorans and Mycobacterium vaccae.
[21] The above-mentioned fungus is Pseudonocardia dioxanovorans CB1190, Pseudonocardia benzenica lucifera codica serracodica sunderacondica Pentaeudocorcus var. (Rhodococcus ruber) 219, Rhodococcus aetherivorans JCM14343, Mycobacterium vaccae (Mycobacterium spp.), and Mycobacterium sp. The manufacturing method according to any one of [15] to [18].

[22] A method for decomposing 1,4-dioxane, which comprises a composition for decomposing 1,4-dioxane obtained by the production method according to any one of [15] to [21], and 1,4-dioxane. A method for decomposing 1,4-dioxane, which comprises a step of contacting

[23] A composition for decomposing 1,4-dioxane, which comprises a bacterium having a 1,4-dioxane decomposing activity and a cyclic ester compound.
[24] The cyclic ester compound is selected from the group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone. The composition for decomposing 1,4-dioxane according to [23], which is any one or more of the following.
[25] The composition for decomposing 1,4-dioxane according to [23] or [24], which does not contain 1,4-dioxane.
[26] The fungus belongs to one or more genera selected from the group consisting of Pseudonocardia, Rhodococcus, and Mycobacterium, [23] to [25] A composition for decomposing 1,4-dioxane according to any one of 1.
[27] The above-mentioned fungus is Pseudonocardia dioxanovorans, Pseudonocardia benzenivorus, Pseudonocardia benzencoorca, Pseudonoca Degradation of 1,4-dioxane of any one of [23] to [25] belonging to one or more species selected from the group consisting of Rhodococcus aterivorans, Mycobacterium vaccae Treatment composition.
[28] The above-mentioned fungus is Pseudonocardia dioxanovolans CB1190, Pseudonocardia benzenica lucifera codica serracodica scond ancedonicus (Pseudonocardia benzica serracodactica), and Pseudonocardia benzica serracodica sunderacondica Pd. (Rhodococcus ruber) 219, Rhodococcus aetherivorans JCM14343, Mycobacterium vaccae (Mycobacterium spp.), and Mycobacterium sp. The composition for 1,4-dioxane decomposition treatment according to any one of [23] to [25].

[29] A method for decomposing 1,4-dioxane, which comprises a step of bringing the composition for decomposing 1,4-dioxane of any one of [23] to [28] into contact with 1,4-dioxane. A method for decomposing 1,4-dioxane, which comprises:

  The present invention provides a technique for culturing 1,4-dioxane-degrading bacteria excellent in 1,4-dioxane-degrading activity without using 1,4-dioxane.

  Hereinafter, a suitable mode for carrying out the present invention will be described. The embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention should not be construed narrowly.

(1,4-dioxane decomposition activity improver)
The 1,4-dioxane decomposition activity improver according to the present invention is used for a bacterium having 1,4-dioxane decomposition activity and contains a cyclic ester compound.

  The cyclic ester is a compound represented by the following formula (1), (2) or (3).

(In the formulas (1) to (3), R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ represent hydrogen or a linear or branched alkyl group having 1 to 4 carbon atoms, They may be the same or different from each other.The alkyl group is a hydrocarbon group remaining after removing one hydrogen atom from a saturated or unsaturated hydrocarbon, and n represents an integer of 1 to 3. )

The cyclic ester is preferably a compound represented by the above formula (1). In the cyclic ester, more preferably, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms. The cyclic ester is more preferably 1,4-dioxan-2-one in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms and n=1.

  As the cyclic ester, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone are also preferable.

  The 1,4-dioxane decomposition activity improver according to the present invention may be one that contains one or more of the above cyclic esters, and other components are not particularly limited. However, it is preferable that the 1,4-dioxane decomposition activity improver according to the present invention does not contain 1,4-dioxane. Here, "does not contain 1,4-dioxane" means that the 1,4-dioxane decomposition activity improver does not substantially contain 1,4-dioxane, and specifically, It means that 1,4-dioxane is present at 0.05 ppm or less in the 4-dioxane decomposition activity improver.

(Method of culturing 1,4-dioxane-degrading bacterium)
The method for culturing a bacterium having 1,4-dioxane-degrading activity according to the present invention is characterized by including a procedure of culturing the bacterium in the presence of the above-mentioned 1,4-dioxane-degrading activity enhancer.

  If the medium used for the culture contains a cyclic ester compound (or a 1,4-dioxane-decomposing activity enhancer containing the same), it has been conventionally used for culturing 1,4-dioxane-degrading bacteria. A known medium can be used without particular limitation.

  The medium contains compounds other than cyclic ester compounds, which are necessary for the growth and growth of 1,4-dioxane-degrading bacteria. Examples of medium components for growing and growing 1,4-dioxane-degrading bacteria include monosaccharides such as glucose and fructose as carbon sources; disaccharides such as lactose, sucrose and maltose; oligosaccharides; polysaccharides such as starch. Etc. are organic nitrogen sources such as yeast extract as nitrogen sources; free amino acids such as arginine, glutamic acid and glutamine; inorganic nitrogen sources such as ammonium chloride; water-soluble vitamins such as vitamin C and vitamin B12 as vitamin sources; vitamin A; Examples of fat-soluble vitamins such as vitamin E and mineral sources include salts such as phosphate ion, magnesium ion, manganese ion, iron ion, cobalt ion and zinc ion, or hydrates thereof.

In the culturing method according to the present invention, by culturing 1,4-dioxane-decomposing bacteria in a medium containing a cyclic ester compound, high 1,4-dioxane decomposition can be achieved without adding 1,4-dioxane to the medium. A bacterium having activity can be obtained. In order to induce sufficient 1,4-dioxane-degrading activity, the concentration of the cyclic ester compound in the medium is 0.001 to 0.15 mol/L, preferably 0.005 to 0.1 mol/L, more preferably 0. It is set to 0.01 to 0.05 mol/L.
Further, in order to induce sufficient 1,4-dioxane-degrading activity, the amount of the cyclic ester compound added to the medium is 0.1 to 15 ml/L, preferably 0.5 to 10 ml/L, and more preferably 1. It is set to 0 to 5 ml/L.

  The 1,4-dioxane-degrading bacterium is not particularly limited as long as it is a bacterium capable of expressing 1,4-dioxane-degrading activity. It is known that 1,4-dioxane-degrading bacteria are generally present in activated sludge, and the bacteria isolated from activated sludge can also be used in the present invention. In addition, 1,4-dioxane-degrading bacteria are treated with ultraviolet rays, NTG (N-methyl-N'-nitro-N-nitrosoguanidine), methylmethanesulfonic acid, etc. to modify the gene to decompose 1,4-dioxane. A mutant bacterium with improved activity may be used. Furthermore, even if the wild type is a bacterium that does not have 1,4-dioxane degrading ability, it is possible to obtain a recombinant bacterium to which 1,4-dioxane degrading ability is imparted by using a normal genetic engineering method, In the present invention, such a recombinant bacterium can also be used.

Examples of 1,4-dioxane-degrading bacteria include the following bacteria.
(1) Pseudonocardia genus. In particular, Pseudonocardia dioxanivorans, Pseudonocardia benzenivorans and Pseudonocardia antarctica. More specifically, Pseudonocardia dioxanovolans CB1190, Pseudonocardia benzenivorans B5, Pseudonocardia antarctica DVS 5a1.

(2) Rhodococcus genus. In particular, Rhodococcus ruber and Rhodococcus aetherivorans. More specifically, Rhodococcus rubber 219 and Rhodococcus atheerivorans JCM14343.

(3) Mycobacterium genus. Especially Mycobacterium vaccae. More specifically, Mycobacterium sp. PH-06.

  Among these bacteria, Pseudonocardia dioxanovorans CB1190 and Rhodococcus ateribolance JCM14343 are particularly preferable because they are excellent in the resolution of 1,4-dioxane. Pseudonocardia dioxanovorance CB1190 is available from Japan Collection of Microorganisms of RIKEN BioResource Center (JCM13855). Further, the DSM strain is available from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. ATCC strains are available from the American Type Culture Collection.

  The culture can be carried out by inoculating the above-mentioned medium, which has been appropriately sterilized, with the bacteria, and may be carried out under the conventionally known conditions. The seed amount of the bacteria in the medium is, for example, 0.1 to 10 (v/v)%, preferably 0.2 to 5 (v/v)%, more preferably 0.2 to 1 (v/v)%. It should be about. The culture conditions are aerobically cultivated at a pH of 5.0 to 9.0, preferably 6.0 to 8.0, and a temperature of 25 to 40°C, preferably 28 to 32°C. The culturing time may be appropriately set depending on the amount of the culturing solution and the desired number of bacterial cells.

  The above-mentioned culture (main culture) can be performed after pre-culture, if necessary. The method of pre-culture is not particularly limited, for example, glucose, carbon sources such as fructose, nitrogen sources such as yeast extract, arginine, glutamic acid, free amino acids such as glutamine, vitamins, in a medium containing inorganic salts, It is preferable to control the pH at 6.0 to 8.0 and the temperature at 28 to 32° C. and culture aerobically.

(Method for producing composition for decomposing 1,4-dioxane)
The method for producing a 1,4-dioxane decomposition treatment composition containing a bacterium having 1,4-dioxane decomposition activity according to the present invention is a bacterium in the presence of a 1,4-dioxane decomposition activity improver containing a cyclic ester compound. The method is characterized by including the step of culturing (i.e., the above-mentioned culture method).

  Since the 1,4-dioxane-decomposing bacterium obtained by the culture method according to the present invention has a high 1,4-dioxane-decomposing activity, the composition containing the bacterium is decomposed by 1,4-dioxane and removed. Can be effectively used for.

  The composition for 1,4-dioxane decomposition treatment obtained by the method for producing a composition for 1,4-dioxane decomposition treatment according to the present invention contains a cyclic ester compound derived from a culturing step of a bacterium, Characterized by the absence of dioxane.

(Method for decomposing 1,4-dioxane)
The method for decomposing 1,4-dioxane according to the present invention is a composition for decomposing 1,4-dioxane obtained by the above-mentioned culture method or 1,4-dioxane-decomposing bacterium, And a step of contacting with 4-dioxane.

  The 1,4-dioxane to be treated is used in the wastewater from chemical industry such as photosensitive resin production, petrochemical industry such as ethylene oxide and ethylene glycol production, and chemical fiber industry such as polyester production. It may be included.

  By contacting 1,4-dioxane-decomposing bacteria or 1,4-dioxane-decomposing composition with 1,4-dioxane (or wastewater containing the same) in an appropriate reaction solution, 1,4-dioxane Can be disassembled. Specifically, the bacterium obtained by the above culture method, is added to the drainage as a composition of the culture solution as it is, or added to the drainage as the cells separated by centrifuging the culture solution, Alternatively, the culture solution is centrifuged to dry the separated bacterial cells, which are added as dry bacterial cells in the waste water, and then stirred as necessary to accelerate the decomposition reaction of 1,4-dioxane. Alternatively, by fixing or immobilizing the bacterium or the composition on the carrier and immersing the carrier in the waste water, or by arranging the carrier at a position in the flow path of the waste water which can be contacted with the waste water, 1,4-dioxane A decomposition reaction occurs. As a method of fixing or immobilizing the bacterium or the composition, known methods that have been conventionally used are particularly limited as long as they are methods of fixing or immobilizing 1,4-dioxane-degrading bacteria so as not to flow out from the carrier. It can be used without being used. Specific fixing or immobilizing methods include, for example, an adherent biofilm method using a carrier to which a bacterium or a composition adheres to form a biofilm, and a carrier for culturing a bacterium by mixing the carrier and a medium. Culturing method, carrier-binding method of binding bacteria or composition to water-insoluble carrier, method of encapsulating bacteria or composition in pores of the carrier under reduced pressure, and cross-linking into cells with a reagent having two or more functional groups Immobilization method, entrapment immobilization method for confining bacteria or composition in the inside of polymer gel or film, covalent bond method, physical adsorption method, ionic bond method and biochemical specific A carrier binding method and the like which are classified as a binding method and the like can be mentioned.

  INDUSTRIAL APPLICABILITY The 1,4-dioxane-degrading bacterium and the composition for 1,4-dioxane-degrading treatment containing the same according to the present invention have high 1,4-dioxane-degrading activity, and therefore have a higher efficiency in draining water than conventional techniques. It is possible to decompose and remove 1,4-dioxane contained therein.

<Example 1: Culture of 1,4-dioxane-degrading bacterium>
(1) Preculture Pseudonocardia dioxanivorans CB1190 which had been cryopreserved was thawed in a hot water bath at 37° C., and inoculated into the preculture medium shown below at 0.26 (v/v)%, 30° C., stirring number 230 rpm. The cells were shake-cultured for 5 days.

Pre-culture medium:
Glucose 10g/L
Yeast extract (manufactured by Oriental Yeast Co., Ltd.) 3g/L
Potassium dihydrogen phosphate 0.5g/L
Magnesium sulfate heptahydrate 0.5 g/L
Sodium glutamate 10g/L
Iron (II) sulfate heptahydrate 10 mg/L
pH 6.0

(2) Main culture The culture solution after the pre-culture was inoculated into the main culture medium shown below at 0.2 (v/v)% and shake-cultured at 30° C. and 230 rpm for 5 days with shaking.

Main culture medium:
Glucose 10g/L
Yeast extract (manufactured by Oriental Yeast Co., Ltd.) 3g/L
Potassium dihydrogen phosphate 0.5g/L
Magnesium sulfate heptahydrate 0.5 g/L
Sodium glutamate 10g/L
Iron (II) sulfate heptahydrate 10 mg/L
Manganese (II) sulfate pentahydrate 10 mg/L
Zinc sulfate heptahydrate 10 mg/L
Cobalt (II) chloride hexahydrate 10 mg/L
1,4-dioxan-2-one 1.0 ml/L
pH 6.0

<Example 2: Culture of 1,4-dioxane-degrading bacterium>
Culture was carried out in the same manner as in Example 1 except that 2.0 ml/L of 1,4-dioxan-2-one was added to the main culture medium.

<Example 3: Culture of 1,4-dioxane-degrading bacterium>
Culture was carried out in the same manner as in Example 1 except that 1.0 ml/L of 1,4-dioxane-2,5-dione was added to the main culture medium.

<Example 4: Culture of 1,4-dioxane-degrading bacterium>
Culture was performed in the same manner as in Example 1 except that 0.3 ml/L of γ-valerolactone was added to the main culture medium.

<Example 5: Culture of 1,4-dioxane-degrading bacterium>
Culture was performed in the same manner as in Example 1 except that 0.3 ml/L of δ-valerolactone was added to the main culture medium.

<Example 6: Culture of 1,4-dioxane-degrading bacterium>
Culture was performed in the same manner as in Example 1 except that 0.3 ml/L of γ-butyrolactone was added to the main culture medium.

<Comparative Example 1: Culture of 1,4-dioxane-degrading bacterium>
Culture was carried out in the same manner as in Example 1 except that 1,4-dioxan-2-one was removed from the main culture medium.

<Comparative Example 2: Culture of 1,4-dioxane-degrading bacterium>
Culturing was carried out in the same manner as in Example 1 except that 1,4-dioxan-2-one was removed from the main culture medium and 1.0 ml/L of 1,4-dioxane was added.

<Test Example 1: Evaluation of 1,4-dioxane decomposition activity>
The culture medium obtained in the main culture of Examples 1 to 6 and Comparative Examples 1 and 2 was centrifuged to separate the cells, and the cells were washed twice with 0.05M phosphate buffer. The collected bacterial cells were resuspended in 0.05M phosphate buffer so that the bacterial concentration was 1.0 g/L. 1,4-dioxane was added to the suspension at 10 mg/L to prepare a reaction solution, which was shaken at 30° C. and a stirring rate of 230 rpm for 2 hours.

  The concentration of 1,4-dioxane in the reaction solution before and after shaking for 2 hours was measured by the headspace gas chromatography mass spectrometry. A 1,4-dioxane standard solution (1 mg/ml methanol solution, Wako Pure Chemical Industries, Ltd.) was appropriately diluted and used as the standard solution, and a calibration curve was prepared by the absolute calibration curve method.

(1) Equipment used Equipment: Agilent GC/MS 6890/5973N
Headspace sampler: Hewlett-Packard headspace autosampler HP7694
(2) Analysis conditions Headspace sampler Syringe size: 2.5 ml
Injection capacity: 2.0ml
Oven: 80℃ 20 minutes Syringe temperature: 120℃
Gas Chromatography/Mass Spectrometer Column: Frontier Lab UltraALLOY-5 30m×0.25mm I.D. Film thickness: 0.25μm
Inlet temperature: 180℃
Oven temperature: 35℃(5min), then 10℃/min-200℃(5min)
Gas flow rate: 2.0 ml/min
Split ratio: 1:10
Sample amount: 0.5 ml
Ionization method: EI
Detector mode: SIM (58, 88)
Quantitative ion: 88

  From the concentration of 1,4-dioxane before and after shaking for 2 hours, the removal rate of 1,4-dioxane by 1,4-dioxane-degrading bacteria was calculated. The results are shown in "Table 1".

  In the cells obtained in Comparative Example 1 in which main culture was performed in the absence of 1,4-dioxan-2-one, the removal rate of 1,4-dioxane was only 20%.

  When 1,4-dioxane was added to the main culture, the removal rate was 48% (Comparative Example 2). It was considered that this was because 1,4-dioxane induced the decomposition activity of 1,4-dioxane in the cells.

In contrast to the comparative example, in the bacterial cells obtained in Examples 1 and 2 in which the main culture was performed in the presence of 1,4-dioxan-2-one, the 1,4-dioxane removal rate was 100%. It was
In the case of Example 3 in which the main culture was performed in the presence of 1,4-dioxane-2,5-dione, the removal rate was 65%.
In the case of Example 4 in which the main culture was carried out in the presence of γ-valerolactone, the removal rate was 35%.
In the case of Example 5 in which the main culture was carried out in the presence of δ-valerolactone, the removal rate was 49%.
In the case of Example 6 in which the main culture was performed in the presence of γ-butyrolactone, the removal rate was 51%.
From these results, by culturing in the presence of a cyclic ester compound, without using 1,4-dioxane, having 1,4-dioxane decomposing activity equivalent to or better than the case of using it, It was revealed that 4-dioxane-degrading bacteria can be obtained.

Claims (12)

Any one or more cyclic ester compounds selected from the group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone . only contains 1,4 to cultivation of bacteria with dioxane degrading activity Te use Irare, compositions for enhancing the 1,4-dioxane degradation activity of the fungus. The composition of claim 1, which is free of 1,4-dioxane . A method for culturing a bacterium having 1,4-dioxane-degrading activity, comprising:
Look including the steps of culturing the microorganism in a medium containing a composition according to claim 1 or 2, having a non-existence higher than the bacteria cultured under 1,4-dioxane degradation activity of the composition A culture method for obtaining a bacterium. In the medium, before Symbol cyclic ester compound is present in 0.001~0.15mol / L, 3. The method of culturing according.   The culture method according to claim 3 or 4, wherein the bacterium belongs to one or more genera selected from the group consisting of Pseudonocardia, Rhodococcus, and Mycobacterium.   The fungus is Pseudonocardia dioxanivorans, Pseudonocardia benzeniborans, Pseudonocardia radicocord, Pseudonocordia, Pseudocona, The culture method according to claim 3 or 4, which belongs to at least one species selected from the group consisting of aterivorans (Rhodococcus aeterivorans) and Mycobacterium vaccae (Mycobacterium vaccae).   The fungus is Pseudonocardia dioxanivorans CB1190, Pseudonocardia benzenicorsa var. ) 219, Rhodococcus aeterivorans JCM14343, Mycobacterium vaccae, Mycobacterium sp. PH-06, and more than one group selected from the group consisting of 3 or more. Or the culturing method described in 4. A method for decomposing 1,4-dioxane, comprising:
A method for decomposing 1,4-dioxane, which comprises a step of bringing the bacterium obtained by the culturing method according to any one of claims 3 to 7 into contact with 1,4-dioxane. A method for producing a composition for 1,4-dioxane decomposition treatment, which comprises a bacterium having 1,4-dioxane decomposition activity,
Look including the step of culturing the bacteria in a culture medium comprising the composition of claim 1 or 2, the absence higher than the bacteria cultured under 1,4-dioxane degradation activity of the composition A method for producing a composition for decomposing 1,4-dioxane, which comprises a bacterium having the same . A method for decomposing 1,4-dioxane, comprising:
A method for decomposing 1,4-dioxane, which comprises a step of bringing the composition for decomposing 1,4-dioxane obtained by the production method according to claim 9 into contact with 1,4-dioxane. Bacteria having 1,4-dioxane degrading activity , and a group consisting of 1,4-dioxan-2-one, 1,4-dioxane-2,5-dione, γ-valerolactone, δ-valerolactone and γ-butyrolactone It includes any one or more of the cyclic ester compound selected from, and 1,4-dioxane contains no 1,4-dioxane decomposition treatment composition. A method for decomposing 1,4-dioxane, comprising:
A method for decomposing 1,4-dioxane, comprising a step of bringing the composition for decomposing 1,4-dioxane according to claim 11 into contact with 1,4-dioxane.