WO2016181802A1 - 構成型1,4-ジオキサン分解菌 - Google Patents
構成型1,4-ジオキサン分解菌 Download PDFInfo
- Publication number
- WO2016181802A1 WO2016181802A1 PCT/JP2016/062871 JP2016062871W WO2016181802A1 WO 2016181802 A1 WO2016181802 A1 WO 2016181802A1 JP 2016062871 W JP2016062871 W JP 2016062871W WO 2016181802 A1 WO2016181802 A1 WO 2016181802A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dioxane
- strain
- concentration
- constitutive
- cyclic ether
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
- C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
- C07C43/04—Saturated ethers
- C07C43/13—Saturated ethers containing hydroxy or O-metal groups
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present invention relates to a constitutive 1,4-dioxane degrading bacterium.
- 1,4-dioxane is a cyclic ether represented by the following formula (1).
- 1,4-Dioxane is excellent in compatibility with water and organic solvents, and is mainly used as a reaction solvent for organic synthesis.
- 1,4-dioxane The amount of 1,4-dioxane produced and imported in Japan in FY2010 was about 4500 t / year, and it is estimated that about 300 t / year was released into the environment. Since 1,4-dioxane is water-soluble, when it is released into the water environment, it diffuses over a wide area. Moreover, since all of volatile property, adsorptivity to solid, photodegradability, hydrolyzability, and biodegradability are low, removal from water is difficult. Since 1,4-dioxane has acute toxicity and chronic toxicity, and carcinogenicity has been pointed out, there is a concern that pollution of the water environment by 1,4-dioxane may adversely affect humans, animals and plants. . Therefore, in Japan, 1,4-dioxane is regulated by tap water quality standards (0.05 mg / L or less), environmental standards (0.05 mg / L or less) and wastewater standards (0.5 mg / L or less). ing.
- Non-patent Document 1 industrial wastewater containing 1,4-dioxane contains cyclic ethers such as 1,3-dioxolane and 2-methyl-1,3-dioxolane in addition to 1,4-dioxane. It has been reported that In particular, 1,3-dioxolane has been confirmed to have toxicity such as acute toxicity, and contaminated water containing 1,3-dioxolane must be appropriately treated.
- 1,4-dioxane is treated with ozone by adding hydrogen peroxide (O 3 / H 2 O 2 ), ozone treatment under ultraviolet irradiation (O 3 / UV), under irradiation with radiation or ultrasonic waves.
- hydrogen peroxide O 3 / H 2 O 2
- ozone treatment under ultraviolet irradiation (O 3 / UV)
- UV ultraviolet irradiation
- Non-patent document 2 reports that the treatment efficiency of 1,4-dioxane by the accelerated oxidation method decreases when an organic substance other than 1,4-dioxane is present.
- Patent Document 1 and Non-Patent Document 3 describe 1,4-dioxane degrading bacteria that are 1,4-dioxane-decomposing bacteria. Dioxane treatment has been proposed.
- a 1,4-dioxane-degrading bacterium is a bacterium that decomposes 1,4-dioxane as a single carbon source (assimilating bacterium) and a bacterium that can degrade 1,4-dioxane in the presence of a specific substrate such as tetrahydrofuran.
- the assimilating bacteria are further divided into inducible and constitutive types depending on whether or not 1,4-dioxane degrading enzyme is induced.
- inducible 1,4-dioxane-degrading bacteria produce and secrete degrading enzymes due to the presence of inducers such as 1,4-dioxane.
- inducers such as 1,4-dioxane.
- constitutive 1,4-dioxane-degrading bacteria always produce degrading enzymes, so they can be used immediately for 1,4-dioxane treatment without acclimatization.
- Non-Patent Document 3 the maximum specific decomposition rate of 1,4-dioxane-decomposing bacteria of the constituent-type 1,4-dioxane-degrading bacteria is lower than that of derivative 1,4-dioxane-degrading bacteria. There's a problem. It is also known whether 1,4-dioxane degrading bacteria disclosed in Patent Document 1, Non-Patent Documents 3 and 4 can degrade other cyclic ethers in the presence of 1,4-dioxane. Absent.
- Patent Document 2 by the inventors of the present application proposes a method for culturing 1,4-dioxane-degrading bacteria that increases 1,4-dioxane-degrading bacteria using a medium containing diethylene glycol. Since 1,4-dioxane-degrading bacteria are excellent in the ability to use diethylene glycol as a carbon source, by using a medium containing diethylene glycol, sterilization treatment can be carried out under conditions where other microorganisms inhabit. It can proliferate preferentially.
- JP 2008-306939 A Japanese Patent No. 5877918
- a constitutive 1,4-dioxane degrading bacterium which is the N23 strain deposited under accession number NITE BP-02032. 2.1. A suspension containing the constituent type 1,4-dioxane degrading bacterium described in 1. 3.1. 1. The constitutive 1,4-dioxane-degrading bacterium described in 1. A method for treating cyclic ether in water, comprising using the suspension described in 1. 4.1. 1. The constitutive 1,4-dioxane-degrading bacterium described in 1. A method for treating cyclic ether in soil, comprising using the suspension described in 1. 5. 2.
- the cyclic ether is one or more of 1,4-dioxane, 1,3-dioxolane, 2-methyl-1,3-dioxolane, and tetrahydrofuran. Or 4.
- a method for culturing a constitutive 1,4-dioxane-degrading bacterium which is the N23 strain deposited under the characteristic accession number NITE BP-02032. 8. N23 strain deposited under accession number NITE BP-02032 characterized by culturing using a medium containing one or more of 1,4-dioxane, ethylene glycol, diethylene glycol and 1,4-butanediol A method for culturing a constitutive type 1,4-dioxane degrading bacterium.
- the N23 strain is a constitutive 1,4-dioxane-degrading bacterium and always produces a degrading enzyme.
- the N23 strain has the highest 1,4-dioxane maximum specific decomposition rate among the constitutive-type degrading bacteria reported so far, and is excellent in dioxane decomposing ability.
- the N23 strain can degrade 1,4-dioxane to an extremely low concentration of 0.017 mg / L or less, and can process a high concentration of 1,4-dioxane of about 5200 mg / L.
- 1,4-dioxane In addition to 1,4-dioxane, it is excellent in the ability to treat cyclic ethers such as 1,3-dioxolane, 2-methyl-1,3-dioxolane, tetrahydrofuran, etc., and can treat a plurality of cyclic ethers simultaneously. You can also.
- cyclic ethers such as 1,3-dioxolane, 2-methyl-1,3-dioxolane, tetrahydrofuran, etc.
- the N23 strain can be used to treat cyclic ether in water or soil. Since the N23 strain, which is a constitutive 1,4-dioxane-degrading bacterium, can immediately exhibit a high cyclic ether treatment capacity without being habituated, it is possible to construct a simple and high-contamination treatment process. Since the N23 strain is excellent in the ability to use diethylene glycol as a carbon source, in the presence of diethylene glycol, the cell mass of the N23 strain can be maintained high even if other microorganisms are present. Therefore, when the cyclic ether treatment is performed in the presence of diethylene glycol, the cyclic ether treatment ability can be kept high. Further, in the presence of diethylene glycol, the cyclic ether treatment ability can be stably kept high even if the cyclic ether concentration varies.
- the N23 strain grows preferentially even in the presence of microorganisms that do not have 1,4-dioxane resolution.
- the N23 strain can be cultivated easily and in large quantities, supplying a large amount of cells necessary for the treatment of contaminated water and soil contaminated with cyclic ether. can do. Further, since no sterilization equipment or chemicals are required, the N23 strain can be cultured at a very low cost.
- the N23 strain has an excellent ability to use 1,4-dioxane, glyoxylic acid, glycolic acid, ethylene glycol, diethylene glycol, 1,4-butanediol, 1-butanol, tetrahydrofuran, glucose, acetic acid as a carbon source, By using a medium containing one or more of these, the growth rate of the N23 strain can be increased. Further, the N23 strain is superior in ability to use 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as a carbon source, compared to a microorganism having no 1,4-dioxane resolution.
- the N23 strain can be cultured without sterilizing other microorganisms. it can.
- FIG. 6 The figure which shows the time-dependent change of the cyclic ether density
- FIG. 7 The figure which shows the microbial cell density
- FIG. 8 The figure which shows the microbial cell density
- FIG. 8 The figure which shows the residual organic carbon density
- 1,4-Dioxane-degrading bacteria exist in nature, and sludge collected from water or soil contaminated with 1,4-dioxane is cultured in a medium containing only 1,4-dioxane as a carbon source. Can be screened. As described above, 1,4-dioxane-degrading bacteria are roughly classified into two types, assimilating bacteria and co-metabolizing bacteria, and assimilating bacteria are further divided into an induction type and a constitutive type.
- N23 strain The constitutive 1,4-dioxane degrading bacterium of the present invention (hereinafter referred to as N23 strain) was isolated from 1,4-dioxane-contaminated groundwater.
- the N23 strain is under the accession number NITE BP-02032.
- NPMD Patent Microorganism Depositary Center
- An SEM image of the N23 strain is shown in FIG.
- the N23 strain is positive for Gram staining and positive for catalase reaction.
- the N23 strain is a constitutive 1,4-dioxane-degrading bacterium and always produces a degrading enzyme.
- constitutive 1,4-dioxane-degrading bacteria show a lower maximum specific degradation rate of 1,4-dioxane compared to induced 1,4-dioxane-degrading bacteria, but N23 strain is a constituent type that has been reported so far.
- 1,4-dioxane degrading bacteria it has the highest 1,4-dioxane maximum specific degradation rate, and its value is equal to or higher than that of the induced 1,4-dioxane degrading bacteria.
- the N23 strain can degrade 1,4-dioxane to an extremely low concentration of 0.017 mg / L or less, and can treat 1,4-dioxane at a high concentration of about 5200 mg / L.
- the N23 strain does not need to be acclimatized with 1,4-dioxane or the like, has a high 1,4-dioxane maximum specific decomposition rate, and can decompose 1,4-dioxane to a very low concentration. Since 1,4-dioxane at a concentration can be treated, it can be suitably used for biological treatment of 1,4-dioxane.
- the N23 strain can efficiently decompose not only 1,4-dioxane but also cyclic ethers such as 1,3-dioxolane, 2-methyl-1,3-dioxolane, and tetrahydrofuran.
- cyclic ethers such as 1,3-dioxolane, 2-methyl-1,3-dioxolane, and tetrahydrofuran.
- a plurality of cyclic ethers can also be treated simultaneously. Therefore, the N23 strain can be suitably used for biological treatment of cyclic ethers such as 1,4-dioxane, 1,3-dioxolane, 2-methyl-1,3-dioxolane, and tetrahydrofuran.
- 16S rDNA of N23 strain was subjected to PCR amplification using 8F (5′-AGAGTTTGATCCTGGCTCAG-3 ′) and U1492R (5′-GGTTACCTTGTACTACACT-3 ′) as primers, and the resulting amplified product was subjected to sequence analysis. .
- the partial base sequence of 16S rDNA of N23 strain is shown in FIG. 2 and SEQ ID NO: 1 in the sequence listing.
- N23 strain was referred to as Pseudonocardia strainoxydans strain K1 (hereinafter referred to as K1 strain).
- K1 strain Pseudonocardia strainoxydans strain K1 (hereinafter referred to as K1 strain).
- the homology was as high as 99%.
- the K1 strain is a co-metabolite that degrades 1,4-dioxane by co-metabolism (S. Mahendra and L. Alvarez-Cohen: Kinetics of 1,4-dioxane biodegradation by monooxygenase-expressing bacteria, Environ. Sci. Technol., 40 (17) , pp 5435-5442, 2006.).
- the N23 strain is an assimilating bacterium that decomposes 1,4-dioxane as a single carbon source. Therefore, the N23 strain shows a high homology of 99% with the K1 strain, but is clearly a different species from the K1 strain.
- a phylogenetic tree prepared based on the 16S rDNA base sequence is shown in FIG.
- Examples of the medium for culturing the N23 strain include a liquid medium and a solid medium.
- the medium is not particularly limited as long as it can culture the N23 strain, and a known medium such as MGY medium or CGY medium can be used.
- a liquid medium In order to culture the N23 strain in a large amount, it is preferable to use a liquid medium, and it is further preferable to perform continuous culture while taking out the culture solution containing the same amount of the N23 strain as the liquid medium is supplied while supplying the liquid medium. preferable.
- inorganic substances and organic substances can be added. Since the activity amount of microorganisms is limited by the smallest factor among necessary factors such as nutrients, growth can be promoted by adding insufficient nutrients.
- inorganic substance to be added K 2 HPO 4, and the like (NH 4) 2 SO 4, MgSO 4 ⁇ 7H 2 O, FeCl 3, CaCl 2, NaCl.
- the organic substance to be added is not particularly limited, but corn steep liquor, casamino acid, yeast extract, peptone and the like are preferable.
- the N23 strain has an excellent ability to use 1,4-dioxane, glyoxylic acid, glycolic acid, ethylene glycol, diethylene glycol, 1,4-butanediol, 1-butanol, tetrahydrofuran, glucose, and acetic acid as a carbon source.
- Growth of N23 strain by using a medium containing one or more of 1,4-dioxane, glyoxylic acid, glycolic acid, ethylene glycol, diethylene glycol, 1,4-butanediol, 1-butanol, tetrahydrofuran, glucose, acetic acid Speed can be increased.
- the total concentration of the above compounds in the medium is not particularly limited, but is preferably 1.0 ⁇ 10 ⁇ 8 wt% or more and 10.0 wt% or less.
- the lower limit of the total concentration is more preferably 0.1 wt% or more, more preferably 0.5 wt% or more, and most preferably 1.0 wt% or more.
- the upper limit value of the total concentration is more preferably 9.0 wt% or less, further preferably 8.0 wt% or less, and most preferably 7.0 wt% or less.
- the total amount of the above-mentioned compounds is preferably 60 wt% or more, more preferably 80 wt% or more, further preferably 95 wt% or more with respect to the total amount of organic compounds in the medium, 99 Most preferably, it is 9 wt% or more.
- the N23 strain is superior in ability to use 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as a carbon source, compared to a microorganism having no 1,4-dioxane resolution. Therefore, it is more preferable that the medium contains one or more of 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as the main carbon source.
- the total amount of 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol is preferably 60 wt% or more, more preferably 80 wt% or more with respect to the total amount of organic compounds in the medium. 95 wt% or more is more preferable, and 99.9 wt% or more is most preferable.
- Microorganisms that do not have 1,4-dioxane degradability are inferior in their ability to use 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as carbon sources compared to the N23 strain.
- the N23 strain and a microorganism having no 1,4-dioxane resolution are cultured using the medium containing the N23 strain, the N23 strain proliferates preferentially. That is, when the N23 strain is cultured using a medium containing one or more of 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as a main carbon source, the N23 strain can be obtained without prior sterilization treatment.
- a culture method that requires sterilization is difficult to sterilize to every corner of the apparatus, and thus it is difficult to perform culture using a large-capacity apparatus.
- culturing of the N23 strain using a medium containing one or more of 1,4-dioxane, ethylene glycol, diethylene glycol, and 1,4-butanediol as a main carbon source does not require sterilization treatment. It can be cultured, and a large amount of bacterial cells required for the treatment of contaminated water and soil contaminated with cyclic ether can be supplied.
- the N23 strain of the present invention can be used to treat cyclic ether contained in contaminated water such as factory effluent, general sewage and groundwater, or contaminated soil such as industrial waste treatment plants, factories and illegal dumping sites. Since the N23 strain is a constitutive 1,4-dioxane-degrading bacterium and always produces a degrading enzyme, the cyclic ether treatment can be started immediately.
- the N23 strain may be a cell separated by filtration from a culture solution, a cryopreserved cell, an L-dried cell, a freeze-dried cell, an immobilization carrier in which the N23 strain is immobilized on a resin, a culture solution, It can be used for the cyclic ether treatment in any form such as a suspension containing N23 strain such as the concentrated solution. Since the cyclic ether can be treated simply by bringing the N23 strain into contact with the treatment object contaminated with the cyclic ether, a simple and high-contamination treatment process can be constructed.
- N23 strain can treat cyclic ether in contaminated water.
- the method of the cyclic ether treatment in the contaminated water using the N23 strain is not particularly limited.
- the N23 strain is immobilized in the aeration tank, the immobilized carrier, the suspension, etc.
- the cyclic ether in the contaminated water can be treated simply by adding in the form of Since the biological treatment of cyclic ether can be carried out simply by adding the N23 strain to the aeration tank, the equipment used in the conventional standard activated sludge method can be utilized almost as it is.
- the N23 strain can be easily cultured using a commercially available apparatus by using a medium containing diethylene glycol, the above-described continuous culture is performed at the contaminated water treatment site, and the culture solution containing the N23 strain is used as the contaminated water. Can be continuously injected.
- Diethylene glycol may be added to the contaminated water treated with the N23 strain. At this time, it is preferable to inject so that the concentration of diethylene glycol in the contaminated water is 1.0 ⁇ 10 ⁇ 8 wt% or more and 10.0 wt% or less.
- the lower limit value of the diethylene glycol concentration is more preferably 0.1 wt% or more, more preferably 0.5 wt% or more, and most preferably 1.0 wt% or more.
- the upper limit value of the diethylene glycol concentration is more preferably 6.0 wt% or less, further preferably 3.0 wt% or less, and most preferably 2.0 wt% or less.
- N23 strain can treat cyclic ether in contaminated soil. At this time, it is preferable to add so that the diethylene glycol concentration in the contaminated soil is 0.1 wt% or more and 10 wt% or less.
- the lower limit of the diethylene glycol concentration is more preferably 0.5 wt% or more, more preferably 1 wt% or more, and most preferably 2 wt% or more.
- the upper limit of the diethylene glycol concentration is more preferably 8 wt% or less, further preferably 7 wt% or less, and most preferably 5 wt% or less.
- the method of the cyclic ether treatment in the contaminated soil using the N23 strain is not particularly limited, and a method such as adding the N23 strain to the contaminated soil, mixing and stirring, and injecting a suspension containing the N23 strain into the contaminated soil. Can be mentioned. Since steps such as on-site plant construction, soil excavation, detoxification, and backfilling are unnecessary, it is preferable to inject a suspension containing the N23 strain into the soil. In general, since nutrients are insufficient in soil, it is preferable to inject a carbon source, inorganic salts, and the like into contaminated soil, and it is more preferable to inject diethylene glycol as a carbon source. By adding diethylene glycol to the soil, the treatment of cyclic ether in the soil can be accelerated.
- Example 1 "Determining the partial base sequence of 16S rDNA of N23 strain" N23 strain
- stock was cultured for 7 days (28 degreeC, 120 rpm) using the CGY liquid culture medium (casitone 5g / L, glycerol 5g / L, yeast extract 1g / L). The culture was collected by centrifugation at 10,000 ⁇ g, 4 ° C. for 3 minutes, and washed twice with 0.9% physiological saline.
- the obtained amplification product was electrophoresed on a 2% agarose gel. Thereafter, the target band was cut out, purified using MinElute Gel Extraction Kit (QUIAGEN), and sequence analysis was performed on the resulting amplified product.
- the partial base sequence of 16S rDNA of the N23 strain is as shown in FIG.
- Example 2 “Investigation of degradation characteristics of 1,23-dioxane of N23 strain” 100 mL of CGY medium (casitone 5 g / L, glycerin 5 g / L, yeast extract 1 g / L) was added to an Erlenmeyer flask with a 300 mL capacity baffle, and sterilized by autoclaving (121 ° C., 15 minutes). Thereafter, 1,4-dioxane was added so as to be 500 mg / L, and then one platinum loop of the N23 strain was inoculated, followed by rotary shaking culture (28 ° C., 120 rpm) for 7 days (pre-culture). .
- the cells were transferred to a CGY medium containing 500 mg / L 1,4-dioxane and cultured under the same conditions (pre-culture).
- the culture solution obtained in the preculture was collected and collected by centrifugation, and an inorganic salt medium (composition: 1 g / L K 2 HPO 4 , 1 g / L (NH 4 ) 2 SO 4 , 50 mg / L NaCl, 200 mg) / L MgSO 4 ⁇ 7H 2 O, 10 mg / L FeCl 3 , 50 mg / L CaCl 2 , pH: 7.3) was added to wash the cells.
- an inorganic salt medium composition: 1 g / L K 2 HPO 4 , 1 g / L (NH 4 ) 2 SO 4 , 50 mg / L NaCl, 200 mg
- L MgSO 4 ⁇ 7H 2 O 10 mg / L FeCl 3 , 50 mg / L CaCl 2 , pH: 7.3
- FIG. 4 shows the time course of 1,4-dioxane concentration.
- the N23 strain is a 1,4-dioxane degrading bacterium capable of degrading 1,4-dioxane to an extremely low concentration range.
- FIG. 5 shows the relationship between initial 1,4-dioxane concentration and cell yield.
- the N23 strain can treat contaminated water containing 1,4-dioxane at a high concentration. From the above results, it was shown that the N23 strain is an assimilating bacterium capable of growing using 1,4-dioxane as a single carbon source. From the results of Example 1, the N23 strain was presumed to be Pseudonocardia persistentoxydans strain K1 and a genus fungus, but the N23 strain is an assimilating bacterium and the K1 strain is a co-metabolite, so the N23 strain and the K1 strain are different. It is a fungus.
- Example 4 "Inductivity investigation of 1,4-dioxane degrading enzyme of N23 strain" In the preculture described in Example 2, a degradation test was performed using the inoculated liquid prepared in a system in which 1,4-dioxane was added (induction system) and a system in which 1,4-dioxane was not added (non-induction system).
- 1,4-dioxane-degrading bacteria N23 strain and Pseudonocardia dioxanivorans CB1190 (hereinafter referred to as CB1190 strain), which is a known inductive 1,4-dioxane-degrading bacterium, were used.
- the CB1190 strain was purchased from ATCC (ATCC 55486).
- an inoculum was prepared so that the protein concentration at the start of the test was 10 mg / L.
- sampling was performed as appropriate, and the 1,4-dioxane concentration was measured using a headspace GC / MS.
- FIG. 6 (a) shows the time course of 1,4-dioxane concentration in the system using N23 strain and FIG. 6 (b) using CB1190 strain.
- the graph shows the time course of 1,4-dioxane concentration from the average value, and divides the slope (1,4-dioxane degradation per hour) by the amount of protein at the start of the experiment to determine the specific degradation rate. Asked.
- the specific decomposition rate was calculated by carrying out an experimental system to which the N23 strain was not added and subtracting the reduction amount of 1,4-dioxane due to volatilization. In accordance with the method shown in Example 2, the initial protein concentration was 128 to 206 mg / L. Moreover, it measured similarly about CB1190 strain.
- FIG. 7 shows the specific decomposition rate relative to the initial 1,4-dioxane concentration. It has been shown that it follows a typical Monod equation that increases proportionally with the concentration of the substrate at low concentrations, but reaches a peak when the substrate concentration increases to some extent and approaches the maximum specific decomposition rate. . From this analysis result, when the maximum specific degradation rate in each strain was determined, the maximum specific degradation rate in the CB1190 strain was 0.051 mg-1,4-dioxane / mg-protein ⁇ h. On the other hand, the maximum specific degradation rate of the N23 strain was 0.216 mg-1,4-dioxane / mg-protein ⁇ h, which was about 4.2 times that of the CB1190 strain.
- Non-Patent Document 3 Kiyo et al. Conducted an investigation of the maximum specific degradation rate of 1,4-dioxane degrading bacteria.
- Table 1 shows the results of Example 5, the strains described in Non-Patent Document 3 and their types, and the maximum specific decomposition rate.
- the N23 strain had a maximum specific degradation rate that was 2.3 to 4.2 times higher than the constitutive 1,4-dioxane degrading bacteria reported so far. Further, the value was equal to or higher than that of the induced 1,4-dioxane degrading bacterium.
- the N23 strain was confirmed to be a strain having a high maximum specific degradation rate as compared with the constitutive type 1,4-dioxane degrading bacteria reported so far.
- Example 6 "Investigation of degradation of 1,3-dioxolane and 2-methyl-1,3-dioxolane by N23 strain" An experiment was conducted to confirm whether the N23 strain, which is a constitutive 1,4-dioxane-degrading bacterium, can degrade 1,3-dioxolane and 2-methyl-1,3-dioxolane.
- FIG. 8 shows the change with time of each cyclic ether concentration. It was confirmed that all cyclic ether concentrations decreased immediately after the start of the experiment. That is, it was confirmed that the N23 strain can simultaneously decompose 1,4-dioxane, 1,3-dioxolane and 2-methyl-1,3-dioxolane, and can be used for the treatment of plural kinds of cyclic ethers.
- Example 7 “Verification of N23 strain growth by carbon source” N23 strain
- an inorganic salt medium composition: 1 g / L K 2 HPO 4 , 1 g / L (NH 4 ) 2 SO 4 , 50 mg / L NaCl, 200 mg / L MgSO 4 .7H 2 O, 100 mL of 10 mg / L FeCl 3 , 50 mg / L CaCl 2 , pH: 7.3
- an inorganic salt medium composition: 1 g / L K 2 HPO 4 , 1 g / L (NH 4 ) 2 SO 4 , 50 mg / L NaCl, 200 mg / L MgSO 4 .7H 2 O, 100 mL of 10 mg / L FeCl 3 , 50 mg / L CaCl 2 , pH: 7.3
- the carbon source 1,4-dioxane, diethylene glycol, glucose, and lactic acid were used.
- the culture was terminated on the 4th day from the start of the test, and the bacterial cells in the solution were collected by filtration through suction filtration, dried at 105 ° C. overnight, and then measured for the weight of the bacterial cells at the end of the culture.
- the bacterial cell concentration (mg / L) was determined.
- FIG. 9 shows the cell concentration at the end of the culture for each carbon source.
- the growth of the N23 strain was confirmed in all carbon sources, and the cell concentration after 4 days in culture was highest, especially in the system cultured with glucose.
- other microorganisms can also be used as growth substrates for glucose, it is difficult to preferentially grow the N23 strain using glucose as a carbon source in an environment where other microorganisms exist.
- diethylene glycol can specifically grow 1,4-dioxane-degrading bacteria, as reported in Patent Document 2 above. Since the N23 strain can use diethylene glycol as a carbon source, the N23 strain can be preferentially grown over other microorganisms using diethylene glycol.
- Example 8 “Confirmation of growth potential of N23 strain due to different carbon sources 2”
- the N23 strain was cultured for 2 weeks using MGY medium (Mal Extract: 10 g / L, Glucose: 4 g / L, Yeast Extract: 4 g / L, pH 7.3).
- the culture solution was collected by centrifugation at 10,000 ⁇ g, 4 ° C. for 3 minutes, and collected with an inorganic salt medium (composition: 1 g / L K 2 HPO 4 , 1 g / L (NH 4 ) 2 SO 4 , 50 mg / L).
- 1,4-dioxane, glyoxylic acid, glycolic acid, glyoxal, ethylene glycol, diethylene glycol, triethylene glycol, 1,4-butanediol, 1-butanol, phenol, tetrahydrofuran, glucose and acetic acid were used.
- the solid content in the culture solution was collected using glass fiber filter paper (GF / B, Whatman, diameter 47 mm), dried at 105 ° C., and the cell concentration was measured. Further, the total organic carbon concentration remaining in the solution recovered by filtration was measured with a TOC meter. For comparison, a blank system to which no carbon source was added was also tested. The bacterial cell concentration is shown in FIG. 10, and the residual organic carbon concentration is shown in FIG.
- the cell concentration after 7 days of culture was greatly increased except for experiments using glyoxal, triethylene glycol and phenol.
- 1,4-dioxane, 1,4-butanediol, 1-butanol and glucose showed high cell concentrations.
- the residual organic carbon concentration showed a low value in an experimental system in which clear cell growth could be confirmed.
- the N23 strain can be grown using 1,4-dioxane, glyoxylic acid, glycolic acid, ethylene glycol, diethylene glycol, 1,4-butanediol, 1-butanol, tetrahydrofuran, glucose and acetic acid as carbon sources. It has been shown. Therefore, the N23 strain can be efficiently cultured by using these carbon sources.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Hydrology & Water Resources (AREA)
- Water Supply & Treatment (AREA)
- Biodiversity & Conservation Biology (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Emergency Management (AREA)
- Business, Economics & Management (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
Description
2.1.に記載の構成型1,4-ジオキサン分解菌を含む懸濁液。
3.1.に記載の構成型1,4-ジオキサン分解菌、または2.に記載の懸濁液を用いることを特徴とする水中の環状エーテル処理方法。
4.1.に記載の構成型1,4-ジオキサン分解菌、または2.に記載の懸濁液を用いることを特徴とする土壌中の環状エーテル処理方法。
5.前記環状エーテルが、1,4-ジオキサン、1,3-ジオキソラン、2-メチル-1,3-ジオキソラン、テトラヒドロフランの1種以上であることを特徴とする3.または4.に記載の環状エーテル処理方法。
6.ジエチレングリコールの存在下で行うことを特徴とする3.~5.のいずれかに記載の環状エーテル処理方法。
7.1,4-ジオキサン、グリオキシル酸、グリコール酸、エチレングリコール、ジエチレングリコール、1,4-ブタンジオール、1-ブタノール、テトラヒドロフラン、グルコース、酢酸の1種以上を含有する培地を用いて培養することを特徴とする受託番号NITE BP-02032として寄託されたN23株である構成型1,4-ジオキサン分解菌の培養方法。
8.1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールの1種以上を含有する培地を用いて培養することを特徴とする受託番号NITE BP-02032として寄託されたN23株である構成型1,4-ジオキサン分解菌の培養方法。
さらに、N23株は、1,4-ジオキサン分解能を有さない微生物と比較して、1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールを炭素源として利用する能力に優れているため、1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールの1種以上を主たる炭素源として含む培地を用いることにより、他の微生物を滅菌することなくN23株を培養することができる。
1,4-ジオキサン分解菌は自然界に存在しており、1,4-ジオキサンで汚染された水中や土壌中から採取した汚泥等を、炭素源として1,4-ジオキサンのみを含む培地で培養することでスクリーニングすることができる。上記したように、1,4-ジオキサン分解菌は、資化菌と共代謝菌の2種類に大別され、資化菌はさらに誘導型と構成型とに分けられる。
, pp 5435-5442, 2006.)。それに対し、N23株は1,4-ジオキサンを単一炭素源として分解する資化菌である。よって、N23株は、K1株と99%と高い相同性を示すが、K1株とは明らかに別種の菌である。16S rDNA塩基配列に基づいて作成した系統樹を図3に示す。
それに対し、1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールの1種以上を主たる炭素源として含む培地を用いたN23株の培養は滅菌処理が不要なため、大規模スケールで培養することができ、環状エーテルで汚染された汚染水や汚染土壌等の処理に必要とされる大量の菌体を供給することができる。1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールの1種以上を主たる炭素源として含む培地を用いると、他の微生物による汚染(コンタミネーション)が起こらないため、N23株を容易に培養することができる。また、滅菌のための設備や薬品が不要なため、非常に低コストで培養することができる。
「N23株の16S rDNAの部分塩基配列決定」
N23株をCGY液体培地(カシトン5g/L、グリセリン5g/L、酵母エキス1g/L)を用いて7日間培養した(28℃、120rpm)。この培養液を、10000×g、4℃、3分間遠心分離して集菌し、0.9%生理食塩水を用いて2回洗浄した。得られた洗浄菌体から、「西郷薫、佐野弓子共訳:分子生物学実験プロトコールI細菌ゲノムDNAの調整 basic protocol 細菌ゲノムDNAの少量調整(ミニレップ), pp36-37, 丸善株式会社, 1997.」の記載に準じてDNAを抽出し、16S rDNAのPCR増幅を行った。プライマーとして、8F(5’-AGAGTTTGATCCTGGCTCAG-3’)とU1492R(5’-GGTTACCTTGTTACGACTT-3’)を用いた。PCR増幅は、94℃で10分間保持した後、変性(94℃、1分)、アニーリング(58℃、1分)、伸長(72℃、2分)を35サイクル行い、最後に10分間、72℃で保持した。
得られた増幅産物を、2%アガロースゲルで電気泳動を行った。その後、標的のバンドを切り出し、MinElute Gel Extraction Kit(QUIAGEN)を用いて精製を行い、得られた生成増幅産物に対してシーケンス解析を実施した。N23株の16S rDNAの部分塩基配列は図2に示した通りである。
「N23株の1,4-ジオキサンの分解特性調査」
300mL容量のバッフル付の三角フラスコにCGY培地(カシトン5g/L、グリセリン5g/L、酵母エキス1g/L)を100mL添加し、オートクレーブにて滅菌処理(121℃、15分)を行った。その後、500mg/Lになるように1,4-ジオキサンを添加してから、N23株を一白金耳量植菌し、回転振盪培養(28℃、120rpm)を7日間行った(前々培養)。
培養後、500mg/Lの1,4-ジオキサンを含んだCGY培地に植え継ぎ、同様の条件にて培養を行った(前培養)。
前培養にて得られた培養液を遠心分離によって集菌・回収し、無機塩培地(組成:1g/L K2HPO4、1g/L (NH4)2SO4、50mg/L NaCl、200mg/L MgSO4・7H2O、10mg/L FeCl3、50mg/L CaCl2、pH:7.3)を加えて、菌体の洗浄を行った。
100mL無機塩培地に植菌液を添加した後、1.25mg/Lになるように1,4-ジオキサンを添加し、28℃、120rpmにて回転振盪培養を行った(n=3)。なお、事前に植菌液におけるタンパク質濃度をPR.Meyersらの方法に準じて測定し(PR. Meyers, WR. Bourn, LM. Steyn, PD. van Helden, AD. Beyers, and GD. Brown: Novel method for rapid measurement of growth of mycobacteria in detergent-free media, J Clin Microbiol., 36(9), pp.2752-2754, 1998)、試験開始時のタンパク質濃度を30mg/Lになるように調製した。培養は12時間行い、2時間ごとに溶液中の1,4-ジオキサン濃度をヘッドスペースガスクロマトグラフ質量分析計(島津製作所:GC/MS-QP2010 PLUS、TURBOMATRIX HS40 以下、ヘッドスペースGC/MCという。)にて測定した。また、比較としてN23株を添加していない実験系も実施した。
「1,4-ジオキサンによるN23株の増殖性確認」
実施例2で作製したN23株の植菌液1mLを、無機塩培地19mLに添加した後、所定の濃度になるように1,4-ジオキサンを添加し、28℃、120rpmにて回転振盪培養を行った(n=3)。培養は6時間行い、培養前後における1,4-ジオキサン濃度をヘッドスペースGC/MSにて測定するとともに、実施例2で示した方法に準じてタンパク質濃度を測定した。1,4-ジオキサンの分解量に対する増加したタンパク質量の割合を細胞収率として算出した。図5に、初期の1,4-ジオキサン濃度と細胞収率の関係を示す。
以上の結果から、N23株は1,4-ジオキサンを単一炭素源として増殖できる資化菌であることが示された。実施例1の結果から、N23株は、Pseudonocardia tetrahydrofuranoxydans strain K1と類菌種と推定されたが、N23株は資化菌、K1株は共代謝菌であるため、N23株とK1株とは異なる菌である。
「N23株の1,4-ジオキサン分解酵素の誘導性調査」
実施例2に記載の前培養において1,4-ジオキサンを添加する系(誘導系)と、添加しない系(非誘導系)で、作成した植菌液を用いて分解試験を実施した。1,4-ジオキサン分解菌として、N23株と、公知の誘導型1,4-ジオキサン分解菌であるPseudonocardia dioxanivorans CB1190(以下、CB1190株という。)を用いた。なお、CB1190株は、米国ATCCから購入した(ATCC 55486)。
「N23株の1,4-ジオキサン最大比分解速度測定」
実施例2の手順で作成した植菌液を用いて分解試験を行った。19mL無機塩培地に植菌液1mL添加した後、所定濃度になるように1,4-ジオキサンを添加し、28℃、120rmpにて回転振盪培養を8時間行った。実験は、初期の1,4-ジオキサン濃度を11、107、258、564及び1136mg/Lとし、各系においてn=2にて実施した。実験期間中には1時間もしくは2時間ごとにサンプリングを行い、1,4-ジオキサン濃度をヘッドスペースGC/MSにて測定した。それらの平均値から1,4-ジオキサン濃度の経時変化をグラフ化し、それぞれの傾き(時間あたりの1,4-ジオキサン分解量)に対して実験開始時のタンパク質量で割ることにより比分解速度を求めた。なお、比分解速度は、N23株を添加していない実験系を実施し、揮発による1,4-ジオキサンの減少量を差し引いて算出した。実施例2で示した方法に準じ、初期のタンパク質濃度は、128~206mg/Lとした。また、CB1190株に対しても同様に測定した。
基質が低濃度のときは濃度に比例的に増加するが、基質濃度がある程度まで高くなると頭打ちとなり、最大比分解速度に漸近していくような、典型的なMonod式に従うことが明らかになった。この解析結果から、各菌株における最大比分解速度を求めたところ、CB1190株における最大比分解速度は、0.051mg-1,4-dioxane/mg-protein・hを示した。一方で、N23株の最大比分解速度は、0.216mg-1,4-dioxane/mg-protein・hを示し、CB1190株のおよそ4.2倍の値であった。また、上記非特許文献3において、清らは、1,4-ジオキサン分解菌の最大比分解速度の調査を実施している。表1に、実施例5の結果、及び上記非特許文献3に記載されている各菌株とそのタイプ、及び最大比分解速度を示す。N23株は、これまでに報告されている構成型1,4-ジオキサン分解菌よりも2.3~4.2倍も高い最大比分解速度を有していた。また、その値は誘導型1,4-ジオキサン分解菌と同等以上であった。N23株は、これまで報告されている構成型の1,4-ジオキサン分解菌と比較して高い最大比分解速度を有する菌株であることが確かめられた。
「N23株による1,3-ジオキソラン及び2-メチル-1,3-ジオキソランの分解調査」
構成型1,4-ジオキサン分解菌であるN23株が、1,3-ジオキソランと2-メチル-1,3-ジオキソランとを分解可能であるか確認する実験を行った。分解試験では、19mL無機塩培地に植菌液1mLを添加した後、所定の濃度になるように1,4-ジオキサン、1,3-ジオキソラン及び2-メチル-1,3-ジオキソランの3種の環状エーテルを添加した(n=3)。実験期間中には、適宜サンプリングを行い、ヘッドスペースGC/MSを用いて1,4-ジオキサン、1,3-ジオキソラン、2-メチル-1,3-ジオキソランの濃度を測定した。なお、実施例2で示した方法に準じて、試験開始時のタンパク質濃度がおよそ90mg/Lになるように事前に植菌液を調製した。
「炭素源の違いによるN23株の増殖性確認」
N23株をCGY液体培地(カシトン5g/L、グリセリン5g/L、酵母エキス1g/L)を用いて7日間培養した(28℃、120rpm)。この培養液を、10000×g、4℃、3分間遠心分離して集菌し、0.9%生理食塩水を用いて2回洗浄した。その後、0.9%生理食塩水を用いて、所定濃度になるように菌体を懸濁して植菌液を作成した。
「炭素源の違いによるN23株の増殖性確認2」
N23株を、MGY培地(Malt Extract:10g/L、グルコース:4g/L、Yeast Extract:4g/L、pH 7.3)を用いて2週間培養した。この培養液を、10000×g、4℃、3分間遠心分離して集菌し、無機塩培地(組成:1g/L K2HPO4、1g/L (NH4)2SO4、50mg/L NaCl、200mg/L MgSO4・7H2O、10mg/L FeCl3、50mg/L CaCl2、pH:7.3)を用いて二回洗浄した。
50ml容量のバイアル瓶に、炭素源を100mg-C/L含む無機塩培地を20mL添加した後、N23株を50mg-cell/Lになるように添加し、28℃、120rpmにて、回転振盪培養を行った(n=3)。炭素源としては、1,4-ジオキサン、グリオキシル酸、グリコール酸、グリオキサール、エチレングリコール、ジエチレングリコール、トリエチレングリコール、1,4-ブタンジオール、1-ブタノール、フェノール、テトラヒドロフラン、グルコース及び酢酸を用いた。
7日間培養後、ガラス繊維ろ紙 (GF/B, Whatman, 直径 47mm)を用いて、培養液中の固形分を回収し、105℃にて乾燥させて菌体濃度を測定した。また、ろ過によって回収した溶液に残存する全有機炭素濃度をTOC計により測定した。なお、比較として炭素源を添加しないブランク系も同様に試験を行った。菌体濃度を図10に、残存有機炭素濃度を図11に示す。
Claims (8)
- 受託番号NITE BP-02032として寄託されたN23株である構成型1,4-ジオキサン分解菌。
- 請求項1に記載の構成型1,4-ジオキサン分解菌を含む懸濁液。
- 請求項1に記載の構成型1,4-ジオキサン分解菌、または請求項2に記載の懸濁液を用いることを特徴とする水中の環状エーテル処理方法。
- 請求項1に記載の構成型1,4-ジオキサン分解菌、または請求項2に記載の懸濁液を用いることを特徴とする土壌中の環状エーテル処理方法。
- 前記環状エーテルが、1,4-ジオキサン、1,3-ジオキソラン、2-メチル-1,3-ジオキソラン、テトラヒドロフランの1種以上であることを特徴とする請求項3または4に記載の環状エーテル処理方法。
- ジエチレングリコールの存在下で行うことを特徴とする請求項3~5のいずれかに記載の環状エーテル処理方法。
- 1,4-ジオキサン、グリオキシル酸、グリコール酸、エチレングリコール、ジエチレングリコール、1,4-ブタンジオール、1-ブタノール、テトラヒドロフラン、グルコース、酢酸の1種以上を含有する培地を用いて培養することを特徴とする受託番号NITE BP-02032として寄託されたN23株である構成型1,4-ジオキサン分解菌の培養方法。
- 1,4-ジオキサン、エチレングリコール、ジエチレングリコール、1,4-ブタンジオールの1種以上を含有する培地を用いて培養することを特徴とする受託番号NITE BP-02032として寄託されたN23株である構成型1,4-ジオキサン分解菌の培養方法。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201680027167.4A CN107849520A (zh) | 2015-05-11 | 2016-04-25 | 组成型1,4‑二恶烷降解菌 |
CA2985866A CA2985866A1 (en) | 2015-05-11 | 2016-04-25 | Constitutive 1,4-dioxane-degrading bacteria |
US15/573,401 US10329631B2 (en) | 2015-05-11 | 2016-04-25 | Constitutive 1,4-dioxane-degrading bacteria |
KR1020177032492A KR102597380B1 (ko) | 2015-05-11 | 2016-04-25 | 구성형 1,4-디옥산 분해균 |
EP16792527.0A EP3296389B1 (en) | 2015-05-11 | 2016-04-25 | Constitutive 1,4-dioxane-degrading bacterium |
JP2016554710A JP6117450B1 (ja) | 2015-05-11 | 2016-04-25 | 構成型1,4−ジオキサン分解菌 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015-096577 | 2015-05-11 | ||
JP2015096577 | 2015-05-11 | ||
JP2015-205643 | 2015-10-19 | ||
JP2015205643 | 2015-10-19 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016181802A1 true WO2016181802A1 (ja) | 2016-11-17 |
Family
ID=57248898
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/062871 WO2016181802A1 (ja) | 2015-05-11 | 2016-04-25 | 構成型1,4-ジオキサン分解菌 |
Country Status (8)
Country | Link |
---|---|
US (1) | US10329631B2 (ja) |
EP (1) | EP3296389B1 (ja) |
JP (1) | JP6117450B1 (ja) |
KR (1) | KR102597380B1 (ja) |
CN (1) | CN107849520A (ja) |
CA (1) | CA2985866A1 (ja) |
TW (1) | TW201702374A (ja) |
WO (1) | WO2016181802A1 (ja) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017042097A (ja) * | 2015-08-26 | 2017-03-02 | 学校法人東京電機大学 | 環状エーテル構造を有する有機化合物の分解能を有する新規微生物とその使用 |
JP2018094455A (ja) * | 2016-12-08 | 2018-06-21 | 大成建設株式会社 | 環状エーテルの生分解処理方法 |
WO2018235743A1 (ja) * | 2017-06-19 | 2018-12-27 | 大成建設株式会社 | 有機化合物の生分解処理方法 |
JP2019000074A (ja) * | 2017-06-19 | 2019-01-10 | 大成建設株式会社 | 構成型1,4−ジオキサン分解菌n23株の培養方法 |
WO2019097921A1 (ja) * | 2017-11-17 | 2019-05-23 | 大成建設株式会社 | ジオキサン分解菌固定担体、生分解処理方法、および生分解処理装置 |
JP2020110788A (ja) * | 2018-07-03 | 2020-07-27 | 大成建設株式会社 | 有機塩素化合物の生分解処理方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7300242B2 (ja) * | 2017-11-07 | 2023-06-29 | 大成建設株式会社 | 汚染水処理方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100864448B1 (ko) * | 2007-02-20 | 2008-10-20 | 경상북도도지사 | 1,4-디옥산 분해능을 갖는 신규한 슈도노카디아 속gb7 균주 및/또는 이의 배양물, 및 이의 용도 |
JP5046183B2 (ja) | 2007-06-12 | 2012-10-10 | 株式会社日立プラントテクノロジー | 1,4−ジオキサン分解菌の培養及び馴養方法、1,4−ジオキサン分解菌固定化担体の製造方法、廃水処理方法及び装置 |
JP5802141B2 (ja) * | 2011-02-07 | 2015-10-28 | 株式会社日立製作所 | 1,4−ジオキサン含有廃水の処理方法及び処理装置 |
US20140154784A1 (en) * | 2012-12-03 | 2014-06-05 | Gregory van Buskirk | Method and Process for the Degradation of Cyclic Ethers in Ethoxylate-Containing Actives |
JP5877918B1 (ja) | 2014-10-09 | 2016-03-08 | 大成建設株式会社 | 1,4−ジオキサン分解菌の培養方法、培地、1,4−ジオキサン分解菌を利用する1,4−ジオキサン処理方法 |
-
2016
- 2016-04-25 CA CA2985866A patent/CA2985866A1/en not_active Abandoned
- 2016-04-25 EP EP16792527.0A patent/EP3296389B1/en active Active
- 2016-04-25 WO PCT/JP2016/062871 patent/WO2016181802A1/ja active Application Filing
- 2016-04-25 US US15/573,401 patent/US10329631B2/en active Active
- 2016-04-25 JP JP2016554710A patent/JP6117450B1/ja active Active
- 2016-04-25 CN CN201680027167.4A patent/CN107849520A/zh active Pending
- 2016-04-25 KR KR1020177032492A patent/KR102597380B1/ko active IP Right Grant
- 2016-05-04 TW TW105113823A patent/TW201702374A/zh unknown
Non-Patent Citations (5)
Title |
---|
KOHLWEYER, ULRIKE ET AL.: "Tetrahydrofuran degradation by a newly isolated culture of Pseudonocardia sp. strain Kl", FEMS MICROBIOL. LETT., vol. 186, no. 2, 2000, pages 301 - 306, XP027360401 * |
MAHENDRA, SHAILY ET AL.: "Kinetics of 1,4-Dioxane biodegradation by monooxygenase- expressing bacteria", ENVIRON. SCI. TECHNOL., vol. 40, no. 17, 2006, pages 5435 - 5442, XP055328570 * |
NORIFUMI YAMAMOTO ET AL.: "A Study of Biotreatment for Groundwater Contaminated with 1,4-Dioxane", REPORT OF TAISEI TECHNOLOGY CENTER, vol. 46, 2013, pages 1 - 4, XP055328568 * |
SALES, M. CHRISTOPHER ET AL.: "Genome sequence of the 1,4-Dioxiane-degrading pseudonocardia dioxanivorans strain CB1190", J. BACTERIOL., vol. 193, no. 17, 2011, pages 4549 - 4550, XP055328569 * |
SEI, KAZUNARI ET AL.: "Isolation and characterization of bacterial strains that have high ability to degrade 1,4-dioxane as a sole carbon and energy source", BIODEGRADATION, vol. 24, no. 5, 2013, pages 665 - 674, XP055328562 * |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017042097A (ja) * | 2015-08-26 | 2017-03-02 | 学校法人東京電機大学 | 環状エーテル構造を有する有機化合物の分解能を有する新規微生物とその使用 |
JP2018094455A (ja) * | 2016-12-08 | 2018-06-21 | 大成建設株式会社 | 環状エーテルの生分解処理方法 |
JP7017323B2 (ja) | 2017-06-19 | 2022-02-08 | 大成建設株式会社 | 構成型1,4-ジオキサン分解菌n23株の培養方法 |
JP2019000074A (ja) * | 2017-06-19 | 2019-01-10 | 大成建設株式会社 | 構成型1,4−ジオキサン分解菌n23株の培養方法 |
JP2019000831A (ja) * | 2017-06-19 | 2019-01-10 | 大成建設株式会社 | 有機化合物の生分解処理方法 |
EP3643686A4 (en) * | 2017-06-19 | 2021-03-24 | Taisei Corporation | ORGANIC COMPOUND BIODEGRADATION TREATMENT PROCESS |
WO2018235743A1 (ja) * | 2017-06-19 | 2018-12-27 | 大成建設株式会社 | 有機化合物の生分解処理方法 |
JP7053173B2 (ja) | 2017-06-19 | 2022-04-12 | 大成建設株式会社 | 有機化合物の生分解処理方法 |
US11306013B2 (en) | 2017-06-19 | 2022-04-19 | Taisei Corporation | Biodegradation treatment method for organic compounds |
WO2019097921A1 (ja) * | 2017-11-17 | 2019-05-23 | 大成建設株式会社 | ジオキサン分解菌固定担体、生分解処理方法、および生分解処理装置 |
JPWO2019097921A1 (ja) * | 2017-11-17 | 2020-12-17 | 大成建設株式会社 | ジオキサン分解菌固定担体、生分解処理方法、および生分解処理装置 |
JP7219225B2 (ja) | 2017-11-17 | 2023-02-07 | 大成建設株式会社 | ジオキサン分解菌固定担体、生分解処理方法、および生分解処理装置 |
US11618699B2 (en) | 2017-11-17 | 2023-04-04 | Taisei Corporation | Dioxane-degrading bacteria-immobilized carrier, biodegradation treatment method, and biodegradation treatment apparatus |
JP2020110788A (ja) * | 2018-07-03 | 2020-07-27 | 大成建設株式会社 | 有機塩素化合物の生分解処理方法 |
JP7190980B2 (ja) | 2018-07-03 | 2022-12-16 | 大成建設株式会社 | 有機塩素化合物の生分解処理方法 |
Also Published As
Publication number | Publication date |
---|---|
US10329631B2 (en) | 2019-06-25 |
EP3296389B1 (en) | 2019-10-30 |
JP6117450B1 (ja) | 2017-04-19 |
KR20180016984A (ko) | 2018-02-20 |
US20180135141A1 (en) | 2018-05-17 |
EP3296389A4 (en) | 2018-10-17 |
JPWO2016181802A1 (ja) | 2017-05-25 |
EP3296389A1 (en) | 2018-03-21 |
CN107849520A (zh) | 2018-03-27 |
KR102597380B1 (ko) | 2023-11-01 |
CA2985866A1 (en) | 2016-11-17 |
TW201702374A (zh) | 2017-01-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6117450B1 (ja) | 構成型1,4−ジオキサン分解菌 | |
JP7017323B2 (ja) | 構成型1,4-ジオキサン分解菌n23株の培養方法 | |
JP6664708B2 (ja) | 1,4−ジオキサン分解菌を利用する1,4−ジオキサン処理方法 | |
Jin et al. | Biodegradation of the benzo [a] pyrene-contaminated sediment of the Jiaozhou Bay wetland using Pseudomonas sp. immobilization | |
Liang et al. | Aerobic biodegradation of diethyl phthalate by Acinetobacter sp. JDC-16 isolated from river sludge | |
JP6835311B2 (ja) | 環状エーテルの生分解処理方法 | |
JP4707251B2 (ja) | 活性汚泥及び排水処理方法 | |
CN107586751B (zh) | 一株二恶烷降解菌d2及其应用 | |
JP4416975B2 (ja) | 芳香族化合物分解活性細菌及びその製造法 | |
JP7053173B2 (ja) | 有機化合物の生分解処理方法 | |
JP7300242B2 (ja) | 汚染水処理方法 | |
CN104946563A (zh) | 一株好氧降解吲哚的伯克霍尔德菌及其应用 | |
JP4670425B2 (ja) | 新規分解菌及びそれを用いた有機化合物の分解処理方法 | |
JP2005270970A (ja) | テトラクロロエチレンの分解方法および脱塩素微生物 | |
JP4578879B2 (ja) | ビスフェノールa分解細菌およびその用途 | |
KR102627006B1 (ko) | 디옥산 분해균 고정 담체, 생분해 처리방법 및 생분해 처리장치 | |
JP2006061055A (ja) | ビスフェノールaの分解微生物および該微生物を用いるビスフェノールaの分解方法 | |
JP2001128665A (ja) | ベンゾフェノンの分解法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
ENP | Entry into the national phase |
Ref document number: 2016554710 Country of ref document: JP Kind code of ref document: A |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16792527 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 20177032492 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2985866 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15573401 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2016792527 Country of ref document: EP |