JP2017201962A - Microbial preparation for organic solvent treatment, method for producing microbial preparation for organic solvent treatment, and method for treating organic solvent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel microbial preparation for organic solvent treatment having high organic solvent decomposing ability, which can be stored at room temperature and prepared by an emulsification technique having less environmental burden.SOLUTION: According to the present invention, there is provided a microbial preparation for organic solvent treatment, the preparation comprising an organic solvent-degrading bacterium encapsulated in micelle with an organic solvent. According to the present invention, there is also provided a method for producing a microbial preparation for organic solvent treatment, the method comprising a micelle encapsulation step in which an organic solvent-degrading bacterium, an organic solvent, and a culture medium are mixed and the mixture is then subjected to an ultrasonic treatment under the conditions that the organic solvent-degrading bacterium can grow to obtain the organic solvent-decomposing bacterium encapsulated in the micelle with the organic solvent.SELECTED DRAWING: None

Description

  The present invention relates to a microorganism preparation for treating an organic solvent, a method for producing a microorganism preparation for treating an organic solvent, and a method for treating an organic solvent.

Rhodococcus bacteria, which are organic solvent-degrading bacteria, are a kind of gram-positive bacteria and coryneform bacteria having a high G + C content that are commonly found in soil and the ocean. Rhodococcus bacteria have the ability to decompose and assimilate many persistent compounds including petroleum hydrocarbons and polychlorinated biphenyls (PCB), in addition to acrylamide and useful enzymes, or It is known that there are many producing bacteria such as functional biopolymers including extracellular polysaccharides.
Therefore, it is positioned as an industrially important fungus group, and is expected to be applied to environmental purification or material production by a bioprocess capable of reducing energy and reducing the environmental load.

In particular, when considering a bioprocess, microorganisms that are expected to be applied are required to have properties such as good growth and active metabolic activity in a special environment containing an organic solvent. In addition, in order to demonstrate good growth while decomposing these microorganisms in the presence of highly-concentrated volatile compounds, microorganisms necessary for the purification of the oil-contaminated environment due to an oil spill accident, etc. not only have the ability to coexist, but also coexist. Therefore, it is required to have high resistance to toxicity of the hardly volatile compound.
In order to analyze these properties, first, the organic solvent resistance of microorganisms is required as a starting point, and growth in the presence of a high concentration organic solvent is required particularly in a bioprocess.

The present inventors have found that Rhodococcus rhodochrous strain S-2 is a high-concentration oil-resistant oil-degrading bacterium (see, for example, Non-Patent Document 1), and the results of investigations into its oil resistance. It has been clarified that an extracellular polysaccharide (hereinafter referred to as “EPS”) produced by the same bacterium is deeply involved in the resistance of volatile organic solvents such as long-chain alkanes.
Further, when the colony morphology and solvent resistance of Rhodococcus bacteria were examined, rough type bacteria having a small amount of EPS production have high affinity for the solvent and consequently are solvent sensitive, while mucoid type bacteria having a large amount of EPS production. Showed resistance, it was clarified that there is a high correlation between colony morphology and organic solvent resistance in bacteria belonging to the same genus.
EPS has also been shown to impart solvent resistance to solvent-sensitive rough bacteria, and from these, the formation of mucoid colonies is one index for considering the solvent resistance of the genus bacteria. It was found.

  Rhodococcus erythropolis PR4 strain (hereinafter sometimes referred to as “PR4 strain”) is a degradation of pristane (2,6,10,14-tetramethyl-pentadecane) which is a kind of branched chain alkane. It is a strain isolated as a fungus (for example, see Non-Patent Document 2), and changes its own colony form based on the production of EPS from rough type to mucoid type to rough type with the progress of culture. . In addition, it is known that this strain is resistant to hardly volatile organic solvents.

  In general, when an organic solvent is treated with bacteria, it is carried out in a two-layer culture system of an organic solvent and an aqueous solvent containing medium components. However, since bacteria added in an aqueous solvent can only react at the interface between an organic solvent and an aqueous solvent, it is necessary to improve the lipophilicity of bacterial cells for application to environmental purification or substance production by bioprocess. It is necessary to improve the processing efficiency by transferring to alkane.

  The PR4 strain has two different localities in the medium / alkane bilayer culture system, depending on the carbon number of the added alkane, “adsorption type” adsorbed on the surface of the alkane particle and “transition type” transferred into the alkane particle. This strain is shown (for example, see Non-Patent Document 3) and is expected as a host for bioremediation and bioprocesses.

  The present inventors have revealed that the PR4 strain can be transferred to tetradecane, pentadecane, hexadecane, pristane, squalane and the like having 14 or more carbon atoms to metabolize and decompose these alkanes.

Iwabuchi, N. et al., “Relationships between Colony Morphotypes and Oil Tolerance in Rhodococcus rhodochrous”, Applied Environmental Microbiology, Vol. 66, No. 11, p5073-5077, 2000. Komukai-Nakamura, S. et al., “Construction of bacterial consortia which degrade Arabian light crude oil”, J. Ferment. Bioeng., Vol. 82, p 570-574, 1996. Iwabuchi, N. et al., “Role of Interfacial Tensions in the Translocation of Rhodococcus erythropolis during Growth in a Two Phase Culture”, System Environmental Science & Technology, vol. 43, p8290-8294, 2009.

  In developing microbial preparations using Rhodococcus bacteria, 1) measures for inducing degrading enzymes without pre-culture, 2) storage, maintenance and transfer of bacteria while maintaining the expression of degrading enzymes There are several problems such as measures for ease of use, and 3) measures for increasing the affinity of the medium / alkane after administration of the preparation to the entire bilayer culture system.

  In general, emulsification techniques in a water / oil mixed system can be classified into two methods: a chemical method using a surfactant and an emulsion stabilizer and a physical method using mechanical energy. In the former chemical method, a surfactant or the like is a hardly decomposable substance, and if it flows out into a river or the sea, it may remain and cause environmental pollution.

  The present invention has been made in view of the above circumstances, and provides a novel microbial preparation for organic solvent treatment having high organic solvent resolution, which can be stored at room temperature and is produced by an emulsification technique with low environmental impact. .

  The present inventors have found that a culture medium / alkane bilayer culture system containing an organic solvent-degrading bacterium is micellized by sonication, and completed the present invention.

That is, the present invention includes the following aspects.
[1] A microorganism preparation for organic solvent treatment, comprising an organic solvent-degrading bacterium encapsulated in micelles with an organic solvent.
[2] The microorganism preparation for organic solvent treatment according to [1], wherein the organic solvent is a liquid at normal temperature and normal pressure.
[3] The microorganism preparation for organic solvent treatment according to [1] or [2], wherein the organic solvent is an alkane having 13 to 16 carbon atoms.
[4] The microorganism preparation for organic solvent treatment according to any one of [1] to [3], wherein the organic solvent-degrading bacterium is a bacterium belonging to the genus Rhodococcus.
[5] The microorganism preparation for organic solvent treatment according to any one of [1] to [4], wherein the micelle has an average particle size of 1 μm to 100 μm.
[6] A method for treating an organic solvent, wherein the microorganism preparation for treating an organic solvent according to any one of [1] to [5] is used.
[7] The organic solvent-degrading bacterium mixed with micelles in the organic solvent by mixing an organic solvent-degrading bacterium, an organic solvent, and a medium, performing sonication under conditions that allow the organic solvent-decomposing bacterium to grow. A method for producing a microbial preparation for treating an organic solvent, comprising a step of encapsulating micelles.
[8] The method for producing a microorganism preparation for organic solvent treatment according to [7], wherein the organic solvent is a liquid at normal temperature and normal pressure.
[9] The method for producing a microorganism preparation for organic solvent treatment according to [7] or [8], wherein the organic solvent is an alkane having 13 to 16 carbon atoms.
[10] The method for producing a microorganism preparation for organic solvent treatment according to any one of [7] to [9], wherein the organic solvent-degrading bacterium is a bacterium belonging to the genus Rhodococcus.

  ADVANTAGE OF THE INVENTION According to this invention, the microbial formulation for organic solvent processing which can be preserve | saved at normal temperature and was produced with the emulsification technique with little environmental impact and which has the high organic solvent resolution | decomposability can be provided.

It is the image which image | photographed the formulation for organic-solvent treatment microorganisms manufactured using the various organic solvents (C12, C15, C16, C19) in Example 1 with the phase-contrast microscope. It is the image which carried out the double dyeing | staining using the DAPI reagent and the CTC reagent, and image | photographed with the phase-contrast microscope the preparation for organic-solvent process microorganisms manufactured using C16 or C19 as an organic solvent in Example 1. The average particle diameter and the average number of transition immediately after ultrasonication in the formulation for organic solvent treatment microorganisms manufactured using C16 or C19 as an organic solvent in Example 1 are shown by visual observation using a phase contrast microscope. It is a graph. Using a phase contrast microscope, the average particle diameter and the average number of transitions from day 1 to day 7 after sonication in the preparation for organic solvent-treated microorganisms produced using C16 or C19 as the organic solvent in Example 1 were used. It is a graph which shows the result measured visually. It is the image which image | photographed the formulation for organic-solvent treatment microorganisms manufactured using C19 as an organic solvent in Example 2, and MM culture medium as an aqueous solvent with a phase contrast microscope. It is the image which image | photographed the formulation for organic-solvent treatment microorganisms manufactured using C19 as an organic solvent in Example 3, and NP culture medium as an aqueous solvent with a phase contrast microscope. (A) A preparation for an organic solvent-treated microorganism produced using C16 as an organic solvent and an MM medium as an aqueous solvent in Example 4, and a control micelle produced using C16 as an organic solvent and an MM medium as an aqueous solvent Is an image taken with a phase contrast microscope. (B) A preparation for organic solvent-treated microorganisms produced using C16 as an organic solvent and an MM medium as an aqueous solvent in Example 4, and a control micelle produced using MM medium as an aqueous solvent and C16 as an organic solvent; Is a graph showing the particle diameter measured by a laser diffraction particle size distribution measuring device. Various residual components other than C16 of the weathered-crude oil (w-oil) after treatment under each decomposition condition (Patterns 1 to 3) in Test Example 1 were subjected to GC-MS analysis (SIM conditions) using Pattern 4 as a control. It is a graph which shows a result. Result of GC-MS analysis (SIM conditions) of residual component (C16) of weathered-crude oil (w-oil) after treatment under each decomposition condition (patterns 1 to 3) in Test Example 1 using pattern 4 as a control It is a graph which shows.

<< Microbial preparation for organic solvent treatment >>
In one embodiment, the present invention provides a microbial preparation for treating an organic solvent containing an organic solvent-degrading bacterium encapsulated in micelles with an organic solvent.

  The microorganism preparation for organic solvent treatment of the present embodiment can be stored at room temperature and has a low environmental load. Furthermore, the microorganism preparation for organic solvent treatment of the present embodiment has high organic solvent resolving power and can be used for environmental purification or substance production.

<Organic solvent>
In this embodiment, the organic solvent used for micelle encapsulation is not particularly limited, and may be, for example, a solid at normal temperature and normal pressure, or a liquid at normal temperature and normal pressure. Also good. In the case of using a solid that is solid at normal temperature and normal pressure, the organic solvent that is solid at normal temperature and normal pressure may be dissolved to form a liquid by the presence of the organic solvent that is liquid at normal temperature and normal pressure. Among these, from the viewpoint of micelle storage stability, the organic solvent used for micelle encapsulation in the present embodiment is preferably a liquid at normal temperature and normal pressure.

In this specification, “normal temperature” means a temperature that is not cooled or heated, that is, a normal temperature, and examples thereof include a temperature of 15 to 25 ° C.
In the present specification, “normal pressure” means a pressure when neither depressurizing nor pressurizing, that is, a pressure equal to atmospheric pressure, for example, about 1 atm (1 atm, 101, 325 Pa) or the like. Can be mentioned.

Moreover, in this embodiment, it is preferable that the organic solvent used for micelle encapsulation contains an alkane having 13 or more carbon atoms. In the case of alkanes having 12 or less carbon atoms, it is usually difficult to transfer organic solvent-degrading bacteria into the organic solvent. Therefore, the organic solvent used for encapsulating the micelles may contain hydrocarbons having 12 or less carbon atoms or other hydrophobic substances in addition to alkanes having 13 or more carbon atoms, and alkanes having 13 or more carbon atoms. 100% may be occupied. In the case of containing an alkane having 12 or less carbon atoms, the alkane having 13 or more carbon atoms is preferably contained in an amount of 20% (v / v) or more, and 40% (v / v) or more, based on the total amount of the organic solvent. More preferably, it is more preferably 60% (v / v) or more.
Especially, as an organic solvent used for micelle encapsulation, it is preferable that 100% is occupied by alkanes having 13 or more carbon atoms.

  Therefore, examples of the organic solvent that is liquid at normal temperature and normal pressure and in which the organic solvent-degrading bacteria can be transferred into the organic solvent include linear, branched, or cyclic alkanes having 13 to 16 carbon atoms. Is mentioned.

  Examples of the linear alkane having 13 to 16 carbon atoms include n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), and n-hexadecane (C16).

  Examples of the branched alkane having 13 to 16 carbon atoms include 2,2,7-trimethyldecane (C13), 2,6,8-trimethyldecane (C13), and 2,4,6-trimethyldecane. (C13), 3,5-dimethylundecane (C13), 4,7-dimethylundecane (C13), 2,5,5-trimethyldecane (C13), 5,7-dimethylundecane (C13), 2,8- Dimethylundecane (C13), 4,8-dimethylundecane (C13), 2,3-dimethylundecane (C13), 2,2,9-trimethyldecane (C13), 5-methyl-5-propylnonane (C13), 2,5,6-trimethyldecane (C13), [R, (−)]-3-methyldodecane (C13), 3,3,5-trimethyldecane (C13), 3, -Diethyl-4,5,5-trimethyloctane (C13), 4,4-dipropylheptane (C13), 2,2,3,3-tetramethylnonane (C13), 2,4-dimethyl-3,3 -Diisopropylpentane (C13), 2-methyltridecane (C14), 7-methyltridecane (C14), 3,8-diethyldecane (C14), (6R, 7S) -6,7-dimethyldodecane (C14) 3,3,4,4-tetraethylhexane (C14), 2,2,3,3,4,4,5,5-octamethylhexane (C14), 5-butyldecane (C14), 2,5-dimethyl -3,4-diisopropylhexane (C14), 2,2,3,3,5,6,6-heptamethylheptane (C14), 3-tert-butyl-2,2,5,5-tetramethylhexane (C14), 4-methyltetradecane (C15), 2,7,10-trimethyldodecane (C15), 7-methyltetradecane (C15), 6-propyldodecane (C15), farnesan (C15), [R, (- ]]-3-Methyltetradecane (C15), 2,6-dimethyl-3,5-diisopropylheptane (C15), 5-butylundecane (C15), 5-pentyldecane (C15), (R) -5-ethyl -5-propylundecane (C16), 2,2,4,4,5,5,7,7-octamethyloctane (C16), 3,5,9-trimethyltridecane (C16), 7-propyltridecane (C16), 5,8-diethyldodecane (C16), 4-methylpentadecane (C16), 3,3,6,6-tetraethyloctane (C16), , 4,6-trimethyltridecane (C16), 3-methylpentadecane (C16), 6-pentylundecane (C16), 4,6-diethyldodecane (C16), 2,2,4,4,6,6, Examples include, but are not limited to, 7-heptamethylnonane (C16), 2,2,4,4,6,8,8-heptamethylnonane (C16), and the like.

  Examples of the cyclic alkane having 13 to 16 carbon atoms include cyclotridecane (C13), cyclotetradecane (C14), 1,1,3,5-tetramethylcyclohexane (C14), 3-cyclohexyl-4- Examples include, but are not limited to, methylheptane (C14), cyclopentadecane (C15), cyclohexadecane (C16), and the like.

These linear, branched or cyclic alkanes may be used alone or in combination of two or more. Furthermore, one or more hydrogen atoms of these linear, branched or cyclic alkanes may be substituted with a halogen atom or a hydroxyl group. Here, as a halogen atom which substitutes a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom etc. are mentioned, for example.
When the organic solvent used for micelle encapsulation is a linear, branched or cyclic alkane having 13 to 16 carbon atoms, the organic solvent-degrading bacterium can be transferred into the organic solvent. Furthermore, since the organic solvent decomposing bacteria can also decompose and metabolize the organic solvent itself used for the micelle encapsulation, the organic solvent used for the micelle encapsulation does not remain, and the environmental load can be reduced. .

<Organic solvent degrading bacteria>
In the present specification, the “organic solvent-degrading bacterium” means an organic solvent that can be decomposed and grown in the organic solvent, and is not adsorbed on the surface of the organic solvent, but in a transferred state, ie, organic. It means a bacterium that exists in a solvent. The organic solvent-degrading bacterium is not particularly limited, and examples thereof include bacteria belonging to the genus Rhodococcus.
Specific examples of the bacterium belonging to the genus Rhodococcus include, for example, Rhodococcus australis ATCC35215 strain, Rhodococcus coprofilus ATCC29080 strain, Rhodococcus cotrophodoc3 strain, Rhodococcus coprophyrus d3 Rhodococcus erythropolis ATCC 47072 strain, Rhodococcus erythropolis DSM1069 strain, Rhodococcus erythropolis JCM3201 strain, Rhodococcus erythropolis PR4 strain (hereinafter sometimes referred to as "PR4 strain"), Rhodococcus globelras (ccho) s globerulus) ATCC 14346 strain, Rhodococcus globerras ATCC15076 strain, Rhodococcus globerras ATCC21292 strain, Rhodococcus globerras ATCC25669 strain, Rhodococcus globerlas ATCC25688 strain, Rhodococcus globerlas ATCC3110 strain, Rhodococcus globerlas ATCC3110 strain, Rhodococcus opacus ATCC 170391 strain, Rhodococcus opacus ATCC51881 strain, Rhodococcus opacus ATCC 51882 strain, Rhodococcus opacus JCM9703 strain, Rhodococcus percolatus c us percolatus) JCM10087 strain, Rhodococcus Rodoni (Rhodococcus rhodnii) ATCC35071 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) ATCC271 strain, Rhodococcus rhodochrous ATCC999 strain, Rhodococcus rhodochrous ATCC4001 strain, Rhodococcus rhodochrous ATCC13808 strain, Rhodococcus rhodochrous ATCC14348 strain, Rhodococcus rhodochrous ATCC 14349, Rhodococcus rhodochrous ATCC 15905, Rhodococcus rhodochlaus ATCC 15906, Rhodococcus rhodochrous ATCC 184, Rhodococcus rhodochrous ATCC 17041, Rhodococcus rhodochrac US ATCC 19150, Rhodococcus rhodochrous ATCC 12274, Rhodococcus rhodochlaus JCM2156, Rhodococcus rhodochlaus JCM2157, Rhodococcus rhodochlaus R-1, Rhodococcus rhodochrous R-2, Rhodococcus rhodochrous S-1 -2 strain, Rhodococcus ruber IFO15591 strain, Rhodococcus sp. PG7-2 strain, Rhodococcus sp. JCM3376 strain, Rhodococcus sp. However, it is not limited to these.
Among them, the organic solvent-degrading bacterium used in the present embodiment is preferably a bacterium belonging to the genus Rhodococcus erythropolis, more preferably a PR4 strain.
The organic solvent-degrading bacterium in the present embodiment may be a naturally derived strain, or a transformant that maintains or improves the property of decomposing and metabolizing the organic solvent.

<Micelle>
In the microorganism preparation for organic solvent treatment of the present embodiment, the organic solvent is dispersed as micelles, and the organic solvent-degrading bacteria are enclosed in the micelles.
The average particle size of the micelle is preferably 1 μm or more and 100 μm or less, more preferably 1 μm or more and 50 μm or less, and further preferably 5 μm or more and 30 μm or less. When the average particle diameter of the micelle is in the above range, at least one organic solvent-degrading bacterium can be included and can be stably dispersed as a micelle.

  In addition, the number of cells of the organic solvent-degrading bacterium sealed in the micelle is preferably at least 1 or more, more preferably 1 or more and 10 or less, further preferably 2 or more and 8 or less, It is particularly preferably 3 or more and 6 or less, and most preferably 4 or more and 5 or less. Since the number of cells of the organic solvent-degrading bacteria enclosed in the micelle is in the above range, the micelles can be stably dispersed in a state where the organic solvent-degrading bacteria are enclosed, and the organic solvent can be more efficiently dispersed. Can be disassembled.

<Aqueous solvent>
The aqueous solvent used in the microorganism preparation for organic solvent treatment of the present embodiment is not particularly limited as long as it is a medium capable of culturing organic solvent-degrading bacteria. The medium may be any medium that is generally used for culturing bacteria. For example, IB2 liquid medium, YG liquid medium, LB medium, marine broth, nutritive broth, tryptosoy broth, MM medium, NP Examples include a medium. Among them, it is preferable to use IB2 liquid medium, MM medium, or NP medium as the medium.

When an IB2 liquid medium is used as a medium, an organic solvent-degrading bacterium contains an yeast extract in the IB2 liquid medium in order to shift from an aqueous solvent or an organic solvent used for micelle encapsulation to an organic solvent to be treated. It is preferable. By including the yeast extract in the IB2 liquid medium, the organic solvent degrading bacteria can smoothly shift from the aqueous solvent or the organic solvent used for the micelle encapsulation to the organic solvent to be treated.
The addition amount of the yeast extract in the aqueous solvent is preferably 0.05% (w / w) or more, based on the total amount of the aqueous solvent (100%), 0.5% (w / W) or more and 5% (w / w) or less, more preferably about 1% (w / w).

  In this specification, “transition into an organic solvent” means that an organic solvent-degrading bacterium that was present in an aqueous solvent is an organic solvent to be treated from an aqueous solvent or an organic solvent used for micelle encapsulation. It means adsorbing or transferring to a solvent.

In the microbial preparation for organic solvent treatment of the present embodiment, it is usually difficult for an organic solvent-degrading bacterium to transfer to an alkane having 12 or less carbon atoms, but by limiting the concentration of inorganic salt added to the aqueous solvent, Degradable bacteria can be transferred to alkanes having 12 or less carbon atoms.
The concentration of the inorganic salt in the aqueous solvent is preferably 88.5 nM or less, more preferably 8.9 nM or less, and even more preferably 0.9 nM or less. Examples of the inorganic salt include a magnesium salt, and more specifically include, for example, magnesium chloride, magnesium sulfate, and magnesium nitrate.

  The method for producing a microbial preparation for organic solvent treatment of the present embodiment will be described in << Method for producing microbial preparation for organic solvent treatment >> described later.

  With respect to the storage conditions of the microorganism preparation for organic solvent treatment of the present embodiment, the organic solvent-degrading bacteria are not killed, and are stored at a temperature higher than the temperature at which the organic solvent or aqueous solvent used for the micelle encapsulation is solidified. For example, it may be stored at room temperature, for example, it may be stored at a temperature higher than room temperature or lower than room temperature, for example, refrigerated (4 ° C. or lower) or frozen (−20 ° C. or lower). May be. Especially, it is preferable to preserve | save at normal temperature from a stability viewpoint of a micelle.

<< Method of treating organic solvent >>
In one embodiment, the present invention provides a method for treating an organic solvent using the above-mentioned microorganism preparation for treating an organic solvent.

  According to the treatment method of the present embodiment, an organic solvent can be treated easily and efficiently.

  In the present specification, “treatment of organic solvent” means that an organic solvent that can be decomposed and metabolized by an organic solvent-degrading bacterium is added to the above-mentioned microbial preparation for treating organic solvent.

<Organic solvent to be treated>
In the present embodiment, the organic solvent to be treated using the above-mentioned microbial preparation for treating an organic solvent is not particularly limited as long as the organic solvent-degrading bacterium can be decomposed and metabolized. Examples of the organic solvent to be treated include linear, branched or cyclic alkanes having 6 or more carbon atoms. When an organic solvent-degrading bacterium is transformed, it can grow even in a linear, branched or cyclic alkane having 6 to 12 carbon atoms, and at least adsorbs to these low-carbon alkanes. Therefore, the low carbon number alkane can be metabolized and decomposed.
Furthermore, the upper limit of the carbon number of the alkane is not limited, for example, even if it is solid at normal temperature and normal pressure, it is solid at normal temperature and normal pressure due to the presence of alkane that is liquid at normal temperature and normal pressure. It is sufficient that the alkane can be dissolved to form a liquid.
One or two or more linear, branched or cyclic alkanes having 6 or more carbon atoms may be included.

  Examples of the linear alkane having 6 or more carbon atoms include n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n- Tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), n-octadecane (C18), n-nonadecane (C19), n-icosane (C20), n-pentacosane (C25), n-triacontane (C30), etc. are mentioned, It is not limited to these.

Examples of the branched chain alkane having 6 or more carbon atoms include, but are not limited to, the following.
6 carbon atoms: 2-methylpentane, 2,3-dimethylbutane, 3-methylpentane, 3-methylpentane, dimethylbutane, 2,2-dimethylbutane 7 carbon atoms: 3-methylhexane, 3,3-dimethylpentane 2,2,3-trimethylbutane, 2,3-dimethylpentane, 3-ethylpentane, 2-methylhexane, 2,2-dimethylpentane, 2,4-dimethylpentane Carbon number 8: (3R, 4S)- 3,4-dimethylhexane, 2,2,3,3-tetramethylbutane, (S) -3-methylheptane, 3,4-dimethylhexane, 3-methylheptane, 3-ethylhexane, 3-methyl-3 -Ethylpentane, 2-methyl-3-ethylpentane, 2,3,3-trimethylpentane, isooctane, 2-methylheptane, 4-methylheptane 2,3,4-trimethylpentane, 2,2,3-trimethylpentane, 2,4-dimethylhexane, 2,3-dimethylhexane, carbon number 9: 2,2,4,4-tetramethylpentane, 3-ethyl Heptane, 3,3,4-trimethylhexane, 2,2-dimethylheptane, 2,4-dimethylheptane, 3-ethyl-2,3-dimethylpentane, 2,3,4-trimethylhexane, 2,2,3 , 4-tetramethylpentane, 4-ethylheptane, 3,5-dimethylheptane, carbon number 10: 2,4-dimethyl-3-isopropylpentane, 2,3,5-trimethylheptane, 2,2-dimethyloctane, 2 , 2,4,5-tetramethylhexane, 5-ethyl-2-methylheptane, 2-methyl-3,3-diethylpentane, 2,3,3,5-tetramethyl Ruhexane, 2,2,5-trimethylheptane, 4-methylnonane, 2,4,5-trimethylheptane, 2,4,6-trimethylheptane, 3,4-diethylhexane Carbon number 11: 4-isopropyloctane, 3, 6-dimethylnonane, 2,2,6,6-tetramethylheptane, 3,4-diethylheptane, 2,6-dimethylnonane, 2,3,7-trimethyloctane, 2,4,6-trimethyloctane, 3 , 3,5,5-tetramethylheptane, 4-ethyl-4-methyloctane, 3,5-diethylheptane, 2,3,6-trimethyloctane, 2,2,6-trimethyloctane, 2-methyldecane, 2 , 2,3,3,4,4-hexamethylpentane, 3,3-diethylheptane, 2,5,6-trimethyloctane, carbon number 12: 3,7- Methyldecane, 5,6-dimethyldecane, 5-methylundecane, 2,2,4,6,6-pentamethylheptane, 4-ethyldecane, 2,3,5-trimethylnonane, 2,9-dimethyldecane, 5- Propyl nonane, 2-methylundecane, 3,4-dimethyldecane, 2,2,7,7-tetramethyloctane, 2,4,5,7-tetramethyloctane, 2,2-dimethyldecane, 2,2, 4,4,6-pentamethylheptane, 2,2-dibutylbutane Carbon number 13: 2,2,7-trimethyldecane, 2,6,8-trimethyldecane, 2,4,6-trimethyldecane, 3,5 -Dimethylundecane, 4,7-dimethylundecane, 2,5,5-trimethyldecane, 5,7-dimethylundecane, 2,8-dimethylundecane, 4,8-dimethyl Ndecane, 2,3-dimethylundecane, 2,2,9-trimethyldecane, 5-methyl-5-propylnonane, 2,5,6-trimethyldecane, [R, (−)]-3-methyldodecane, 3 , 3,5-trimethyldecane, 3,3-diethyl-4,5,5-trimethyloctane, 4,4-dipropylheptane, 2,2,3,3-tetramethylnonane, 2,4-dimethyl-3 , 3-Diisopropylpentane Carbon number 14: 2-methyltridecane, 7-methyltridecane, 3,8-diethyldecane, (6R, 7S) -6,7-dimethyldodecane, 3,3,4,4-tetraethyl Hexane, 2,2,3,3,4,4,5,5-octamethylhexane, 5-butyldecane, 2,5-dimethyl-3,4-diisopropylhexane, 2,2,3,3,5,6 , -Heptamethylheptane, 3-tert-butyl-2,2,5,5-tetramethylhexane 15: 4-methyltetradecane, 2,7,10-trimethyldodecane, 7-methyltetradecane, 6-propyldodecane, Farnesan, [R, (−)]-3-methyltetradecane, 2,6-dimethyl-3,5-diisopropylheptane, 5-butylundecane, 5-pentyldecane Carbon number 16: (R) -5-ethyl-5 -Propyl undecane, 2,2,4,4,5,5,7,7-octamethyloctane, 3,5,9-trimethyltridecane, 7-propyltridecane, 5,8-diethyldodecane, 4-methyl Pentadecane, 3,3,6,6-tetraethyloctane, 2,4,6-trimethyltridecane, 3-methylpentadecane, 6-pen Cylundecane, 4,6-diethyldodecane, 2,2,4,4,6,6,7-heptamethylnonane, 2,2,4,4,6,8,8-heptamethylnonane 17: 4 , 6,8,10-tetramethyltridecane, 5,9-dimethylpentadecane, 2,5-dimethylpentadecane, (S) -3-methylhexadecane, 5,5-dibutylnonane, 4,4-dipropylundecane, 6 -Pentyldodecane, 2,6,10-trimethyltetradecane, 2,2,4,4-tetramethyl-3,3-di-tert-butylpentane, 2-methylhexadecane 18 carbon atoms: 2-methylheptadecane, 3 -Methylheptadecane, 2,3,4,5,6,7,8,9-octamethyldecane, 7,9-dimethylhexadecane, 4,9-dipropyldodecane, 2,2,5 5-tetramethyl-3,4-di-tert-butylhexane, 4-methylheptadecane, 8-methylheptadecane, 2,2,4,9,11,11-hexamethyldodecane, 7-methylheptadecane, 4,5,6,7-tetraethyldecane, 7-butyltetradecane 19 carbon atoms: pristane, 2,6-dimethylheptadecane, 3-methyloctadecane, 3,3-dimethylheptadecane, (7R, 11S) -7, 11-dimethylheptadecane, 5,9-dimethylheptadecane, 5-methyloctadecane, 2,6,10,13-tetramethylpentadecane, 7-hexyltridecane, 5,5,7,7-tetraethylundecane, 2- Methyl octadecane 20 carbon atoms: phytane, 2-methylnonadecane, 3-methylnonadecane, 8-tert-buty Ruhexadecane, 4-methylnonadecane, 7,11-dimethyloctadecane, 2,6-dimethyloctadecane, 9-methylnonadecane, 4-propylheptadecane, 3-methyl-3-ethylheptadecane, 2,6,11,15-tetra Methyl hexadecane, 5-butylhexadecane Carbon number 25: 9-octylheptadecane, 10-hexylnonadecane, 2,6,10,15,19-pentamethylicosane, 2,6,10,14,19-pentamethyl Icosan, 7,7-dihexyltridecane, 9- (2-ethylhexyl) heptadecane, hacyan, 2-methyltetracosane, 2,2,8,8-tetramethyl-5,5-bis (3,3-dimethyl) Butyl) nonane, 2,6,10,14,18-pentamethylicosane 30 carbon atoms: squalane, 2 10-dimethyloctacosane, 7-methylnonacosane, 11-nonylhenicosan, (R) -3-methylnonacosane, 8,12-dimethyloctacosane, 3-methylnonacosane, 9-octyldocosan, 7,12- Dihexyl octadecane, lysane, 2,6-dimethyloctacosane

  Examples of the cyclic alkane having 6 or more carbon atoms include cyclohexane (C6), cycloheptane (C7), cyclooctane (C8), cyclononane (C9), cyclodecane (C10), 1,1,3,3-tetra Methylcyclohexane (C10), 1-methyl-2,4-diethylcyclopentane (C10), 1,5-dimethylcyclooctane (C10), cycloundecane (C11), cyclododecane (C12), 1-ethyl-2 -Pentylcyclopentane (C12), 1-methylcycloundecane (C12), butylcyclooctane (C12), cyclotridecane (C13), cyclotetradecane (C14), 1,1,3,5-tetramethylcyclohexane (C14) ), 1,2,4,5-tetraethylcyclohexane (C14), 3-cycl Hexyl-4-methylheptane (C14), 1,1,2-trimethylcycloundecane (C14), isopropylcycloundecane (C14), cyclopentadecane (C15), cyclohexadecane (C16), (1-propylheptyl) cyclohexane ( C16), 1-butylcyclododecane (C16), methylcyclopentadecane (C16), cycloheptadecane (C17), cyclooctadecane (C18), (1-pentylheptyl) cyclohexane (C18), dodecamethylcyclohexane (C18), Cyclononadecane (C19), Sembran (C20), cycloicosane (C20), tetradecylcyclohexane (C20), cyclopentacosane (C25), (1-nonyldecyl) cyclohexane (C25), 9- (3-cycl Pentyl propyl) heptadecane (C25), cycloalkyl triacontyl Tan (C30), equalization Sa methyl tricyclodecane (C30), pen octopus sill cyclopentane (C30) and the like, but are not limited to.

<Uses>
The treatment method of this embodiment can be used for treating a contaminated environment containing an organic solvent. Examples of contaminated environment treatment include domestic wastewater treatment or industrial water treatment.
Moreover, the processing method of this embodiment can be used for production of a substance. Examples of the production of the substance include production of a linear, branched or cyclic alkane having a small molecular weight using a linear, branched or cyclic alkane having a large molecular weight as a substrate.

  In the treatment method of this embodiment, the above-mentioned microbial preparation for treating an organic solvent does not contain an emulsifier such as a surfactant, and the organic solvent contained in the microbial preparation for treating an organic solvent itself is also decomposed by an organic solvent-degrading bacterium. In addition, since it can be metabolized, the environmental load can be reduced.

  In the treatment method of the present embodiment, the above-mentioned microorganism preparation for treating an organic solvent used has a large gradient of the decomposition rate of the organic solvent, and the initial velocity rapidly increases. That is, the above-mentioned microorganism preparation for treating an organic solvent used has a high resolution of the organic solvent and a high degradation rate. Therefore, for example, when applied to the purification of sewage, usually the sewage contained in the septic tank or the like is replaced in about 2 to 3 days. However, according to the treatment method of this embodiment, the above-described organic solvent treatment is performed from the start of purification. Microbial preparations for use have a high affinity for organic solvents and have a high degradation rate, so they can efficiently decompose and metabolize target organic solvents and purify wastewater within a limited period of time. .

<< Method for producing microbial preparation for organic solvent treatment >>
In one embodiment, the present invention comprises mixing an organic solvent-degrading bacterium, an organic solvent, and a medium, performing sonication under conditions that allow the organic solvent-degrading bacterium to grow, and encapsulating micelles with the organic solvent. In addition, a method for producing a microbial preparation for treating an organic solvent, comprising a step of encapsulating micelles for obtaining the organic solvent degrading bacterium is provided.

  According to the production method of the present embodiment, a microorganism preparation for organic solvent treatment that can be stored at room temperature, has a low environmental load, and has a high organic solvent resolution can be easily obtained.

<Micelle encapsulation process>
First, an organic solvent-decomposing bacterium, an organic solvent, and a medium are mixed, and sonication is performed under conditions that allow the organic solvent-decomposing bacterium to grow.

Examples of the organic solvent-degrading bacterium used in the present embodiment include the same as those exemplified in the above <<<< microorganism preparation for organic solvent treatment >>. Among them, the organic solvent-degrading bacterium used in the present embodiment is preferably a bacterium belonging to the genus Rhodococcus erythropolis, more preferably a PR4 strain.
The organic solvent-degrading bacterium in the present embodiment may be a naturally derived strain, or a transformant that maintains or improves the property of decomposing and metabolizing the organic solvent.

  In the present embodiment, the organic solvent is preferably an organic solvent that is liquid at normal temperature and normal pressure, and in which the organic solvent-degrading bacteria can be transferred into the organic solvent, for example, a straight chain having 13 to 16 carbon atoms. , Branched or cyclic alkanes and the like. Examples of the linear, branched, or cyclic alkane having 13 to 16 carbon atoms are the same as those exemplified above in <<<< microorganism preparation for organic solvent treatment >>.

  Examples of the medium used in the present embodiment include the same media as those exemplified above in <<<< Microorganism preparation for organic solvent treatment >>. Especially, as a culture medium used in this embodiment, it is preferable that they are IB2 liquid culture medium, MM culture medium, or NP culture medium.

In this embodiment, the mixing ratio of the organic solvent and the medium may be, for example, about 1: 1 to 1:20. By mixing the organic solvent in an amount smaller than that of the medium, the organic solvent-decomposing bacteria can be easily encapsulated in micelles.
The concentration of the organic solvent-decomposing bacteria in the mixed solution can be adjusted as appropriate according to the type of organic solvent-degrading bacteria, the number of cells to be encapsulated per produced micelle, the conditions of ultrasonic treatment, and the like. it can. For example, what is necessary is just to mix so that an organic-solvent decomposing microbe may be 1.0 * 10 < ⁷ > cells / mL with respect to 1 mL of organic solvents and 10 mL of culture media.

In the present specification, “a condition under which growth is possible” means a condition under which the organic solvent-degrading bacteria are not killed or the cells are not damaged and the growth is not hindered by ultrasonic treatment. As the ultrasonic treatment under “growth conditions”, for example, using an ultrasonic cleaning machine (As One USD-4R) containing 4 L of water, organic solvent-degrading bacteria 1.0 × 10 ⁷ cells / mL, Examples include a condition of treating a container such as a test tube containing 11 mL of a mixed solution of 1 mL of an organic solvent and 10 mL of a medium at 40 kHz for 15 minutes at room temperature and normal pressure. At this time, the liquid level of the mixed solution in the container is preferably about 1.5 cm to 2.0 cm below the water level of the ultrasonic cleaner.
The ultrasonic treatment may be performed directly on the mixed solution of the organic solvent-decomposing bacterium, the organic solvent, and the medium, and through a container containing the mixed solution of the organic solvent-degrading bacterium, organic solvent, and the medium. It may be done indirectly. Among them, the organic solvent-degrading bacteria are not killed, or the cells are not damaged, and the growth is not hindered, so through a container containing a mixed solution of organic solvent-degrading bacteria, organic solvent, and medium. It is preferable to carry out indirectly.
The ultrasonic generator to be used is not particularly limited, for example, a standing wave type (cleaner type) ultrasonic generator in which the vibrator is mounted on a flat plate (vibrating plate), and a cylindrical shape at the tip of the vibrator A horn type (homogenizer type) ultrasonic generator to which the horn is attached is exemplified. Among them, it is preferable to use a standing wave type (washer type) ultrasonic generator because the organic solvent degrading bacteria are not killed or the cells are not damaged and the growth is not hindered.

  With respect to the produced microorganism preparation for treating an organic solvent, the number of cells of the organic solvent-degrading bacterium encapsulated in the micelle can be confirmed, for example, by a method using a phase contrast microscope or the like. Moreover, the average particle diameter of the micelle can be confirmed by, for example, a method of measuring visually using a phase contrast microscope or the like, or a method of measuring using a laser diffraction particle size distribution measuring device or the like.

As for the storage conditions of the prepared organic preparation for organic solvent treatment, if the organic solvent-degrading bacteria are not killed and stored at a temperature higher than the temperature at which the organic solvent or aqueous solvent used for micelle encapsulation is solidified Well, for example, it may be stored at room temperature, for example, it may be stored at a temperature higher or lower than room temperature, for example, refrigerated (4 ° C. or lower) or frozen (−20 ° C. or lower). May be. Moreover, when preserve | saved by refrigeration (4 degrees C or less) or freezing (-20 degrees C or less) etc., even if a micelle breaks by the organic solvent solidifying and isolate | separates into an organic solvent phase and a culture medium phase, it is again above-mentioned. By performing the <micelle encapsulation step>, the organic solvent degrading bacteria can be encapsulated.
Among them, it is preferable to store at normal temperature because micelles can be stably stored without the need for ultrasonic treatment again.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[material]
1. In the following Examples, Rhodococcus erythropolis PR4 strain (hereinafter sometimes referred to as “PR4 strain”) was used.

2. Medium used In the following examples, the method for preparing the medium used is as follows.
2-1. IB2 agar milli-Q water 800mL to 8g of glucose (manufactured by Wako Pure Chemical Industries, Ltd.), 8g of yeast the Extract (Becton, manufactured by Dickinson and company _{Ltd.), MgCl 2 · 6H 2 O} ( Wako Pure Chemical Industries, Ltd. of 0.16g Manufactured), 0.08 g CaCl ₂ · 2H ₂ O (manufactured by Wako Pure Chemical Industries), 0.08 g NaCl (manufactured by Wako Pure Chemical Industries), 0.016 g FeCl ₂ · 6H ₂ O (Wako Pure Chemical) Kogyo Co., Ltd.) and 0.4 g of (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries, Ltd.) were added. Subsequently, 12 g of agar (manufactured by Wako Pure Chemical Industries, Ltd.) was added after adjusting to pH 7.2. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

2-2. IB2 liquid medium Milli-Q water 500mL to 5g of glucose (manufactured by Wako Pure Chemical Industries, Ltd.), yeast the Extract of 5g (Becton, manufactured by Dickinson and company _{Ltd.), MgCl 2 · 6H 2 O} ( Wako Pure Chemical Industries, Ltd. of 0.09g Ltd.), manufactured by CaCl ₂ · _{2H 2} O (Wako pure Chemical Industries, Ltd. of 0.05 g), manufactured by NaCl (Wako pure Chemical Industries, Inc. _{0.05g), FeCl 2 · 6H 2} O in 0.01 g (Wako pure Chemical 0.25 g of (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries) was added. Subsequently, after adjusting to pH 7.2, the mixture was autoclaved at 121 ° C. for 15 minutes and cooled.

2-3. MM medium 0.09 g MgCl ₂ · 6H ₂ O (manufactured by Wako Pure Chemical Industries), 0.05 g CaCl ₂ · 2H ₂ O (manufactured by Wako Pure Chemical Industries), 0.05 g NaCl (Manufactured by Wako Pure Chemical Industries, Ltd.), 0.01 g of FeCl ₂ .4H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 g of (NH ₄ ) ₂ SO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.), 25 g of K ₂ HPO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.) was added. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

2-4. NP medium 0.25 g of (NH ₄ ) ₂ SO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.25 g of K ₂ HPO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.) were added to 500 mL of milli-Q water. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

3. Reagent used In order to confirm the life and death of the PR4 strain, DAPI (4 ′, 6-diamidino-2-phenyllinole, dihydrochloride) reagent (manufactured by Wako Pure Chemical Industries, Ltd.) and CTC (5-Cyano-2, 3-diitolyl-2H) were used. -Tetrazolium chloride) reagent (manufactured by Dojindo) was used.

Moreover, the organic solvent used in the following examples is as follows.
n-dodecane (hereinafter sometimes referred to as “C12”) (manufactured by Wako Pure Chemical Industries, Ltd.)
n-Tetradecane (hereinafter sometimes referred to as “C14”) (manufactured by Wako Pure Chemical Industries, Ltd.)
n-Pentadecane (hereinafter sometimes referred to as “C15”) (manufactured by Wako Pure Chemical Industries, Ltd.)
n-Hexadecane (hereinafter sometimes referred to as “C16”) (manufactured by Wako Pure Chemical Industries, Ltd.)
Pristan (hereinafter sometimes referred to as “C19”) (manufactured by ACROSS)

Various inorganic salts added to the medium were prepared as follows.
3-1. MgCl ₂ solution 18.4 g of MgCl ₂ .6H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.) was added to MilliQ water, and the volume was increased to 100 mL. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

3-2. CaCl ₂ solution 10 g CaCl ₂ .2H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.) was added to Milli-Q water, and the volume was increased to 100 mL. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

3-3. NaCl solution 10 g NaCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added to Milli-Q water, and the volume was increased to 100 mL. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

3-4. FeCl ₂ solution 1.7 g of FeCl ₂ .4H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.) was added to Milli-Q water, and the volume was increased to 100 mL. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

3-5. _(NH4) 2 SO ₄ solution milli-Q water to 50g of the _{(NH 4)} 2 SO ₄ (Wako Pure Chemical Industries, Ltd.) was added and filled up to 100 mL. Subsequently, it was autoclaved at 121 ° C. for 15 minutes and cooled.

[Example 1] Manufacture of organic solvent-treated microorganism preparations using various organic solvents (C12, C15, C16, C19) (1) Preparation of PR4 strain pre-culture solution Dry heat sterilization is performed at 180 ° C for 30 minutes in advance. A test tube was prepared, and 5 mL of IB2 liquid medium was added thereto using an auto pipettor and a 10 mL measuring pipette. Subsequently, the PR4 strain was scraped off from the IB2 agar medium with a disposable, and inoculated. Subsequently, a test tube inoculated with these PR4 strains was shake-cultured at 28 ° C. and 110 rpm for 2 to 3 days, and used as a preculture solution.

(2) Encapsulation of PR4 strain micelle Subsequently, a test tube preliminarily sterilized by dry heat at 180 ° C. for 30 minutes was prepared, and 10 mL of IB2 liquid medium was added thereto using an autopipettor and a 10 mL measuring pipette. Subsequently, 1 mL each of C12, C15, C16, and C19 was added using Pipetteman P-1000. Subsequently, 100 μL of the preculture solution of PR4 strain prepared in (1) was added using Pipetteman P-200, and this was cultured with shaking at 28 ° C. and 110 rpm for 1 day. The cell density after the culture was 1.0 × 10 ⁷ cells / mL. Subsequently, ultrasonic treatment was performed at 40 kHz for 15 minutes at room temperature and normal pressure using an ultrasonic cleaning machine (USD-4R, manufactured by ASONE) containing 4 L of water. The ultrasonic treatment was performed such that the liquid level of the solution in the test tube was about 1.5 cm to 2.0 cm below the water level of the ultrasonic cleaner. Subsequently, 100 μL of the solution after sonication was sampled. The sampled solution was observed using a phase contrast microscope. For observation, 8 μL each of the solution was dropped onto a preparation using a Pipetman P-20, a cover glass was placed, and one drop of immersion oil was dropped on the stage of a phase contrast microscope (Olympus). And the magnification of the objective lens was set to 100 times. The results are shown in FIG.

From FIG. 1, when C12 is used as the organic solvent and IB2 liquid medium is used as the aqueous solvent, the localization is adsorption type, and the PR4 strain is adsorbed on the surface of the organic solvent, and the PR4 strain is encapsulated in micelles. I couldn't.
On the other hand, when C15, C16 or C19 was used as the organic solvent and IB2 liquid medium was used as the aqueous solvent, the localization was transferred and the PR4 strain could be encapsulated in micelles. In each micelle, the state where the PR4 strain was transferred by about 1 to 5 cells was confirmed.

(3) Confirmation of influence on strain PR4 by ultrasonic treatment Further, double staining with DAPI and CTC was performed on the preparation for organic solvent-treated microorganisms produced using C16 or C19 as the organic solvent.
Double staining was performed as follows. Pipetteman P-2 is used to add 100 μL each of the preparations for organic solvent-treated microorganisms prepared using C16 or C19 as the organic solvent, and 0.5 μL of Enhancing Reagent B solution (manufactured by Dojindo Co., Ltd.) and 1.5 μL of CTC reagent. It added one by one, and it fully stirred by vortex. Then, it culture | cultivated for 15 minutes by THERMO MINDER 50mini (made by TAITEC) set to 37 degreeC. Subsequently, 1 μL of DAPI reagent was added using Pipetman P-2, and the mixture was sufficiently stirred by vortexing, and then allowed to stand at room temperature for 15 minutes. Subsequently, the sample was observed using a phase contrast microscope. The DAPI reagent is a nuclear staining reagent used for counterstaining, and the CTC reagent is a viable cell selective fluorescent staining reagent. The results are shown in FIG.

From FIG. 2, red fluorescence by CTC staining was detected for the organic solvent-treated microorganism preparation produced using C16 or C19 as the organic solvent. Moreover, although detailed data are not shown, when the number of colonies of the PR4 strain with and without sonication was compared, the PR4 strain subjected to sonication (the above conditions) was not subjected to sonication. It was confirmed that the same colony as the strain was formed. This confirmed that there was no effect of sonication on colony forming ability.
From the above, it was confirmed that the PR4 strain had respiratory activity even when sonication was performed. That is, it was confirmed that the PR4 strain was not killed by sonication under the above-mentioned conditions.

(4) Measurement of average particle size of micelles and average number of transitions of PR4 strain For organic solvent-treated microorganism preparations (4 samples each) produced using C16 or C19 as an organic solvent, using a phase contrast microscope Visual measurement was performed three times independently, and the average particle size and average number of transitions of micelles were calculated. The results are shown in FIG.

From FIG. 3, regarding the preparation for organic solvent-treated microorganisms produced using C16 as the organic solvent, there was no significant difference in average particle diameter and average number of transitions among the four samples. Moreover, when the average value of an average particle diameter and an average number of transitions was calculated | required using four samples, they were an average particle diameter of 12.27 micrometers and an average number of transitions of 3.27.
Moreover, about the formulation for organic-solvent treatment microorganisms manufactured using C19 as an organic solvent, the big difference was not looked at by the average particle diameter and the average number of transitions among four samples like C16. Moreover, when the average value of an average particle diameter and an average number of transitions was calculated | required using four samples, they were an average particle diameter of 13.26 micrometers and an average number of transitions of 4.23.
From the above, it was confirmed that by using an organic solvent degrading bacterium whose localization is transferable, micelles of about 1 to 5 cells can be encapsulated regardless of the type of organic solvent.

(5) Examination of micelle stability Furthermore, the stability of micelles was examined for organic solvent-treated microorganism preparations produced using C16 or C19 as the organic solvent used in (4). As a method, a preparation for organic solvent-treated microorganisms produced using C16 or C19 as an organic solvent that has been shaken at 28 ° C. or statically cultured at room temperature is used. Sampling was performed on day 7 and day 7, and the presence or absence of stability was examined by measuring the average particle diameter and the average number of transitions over time by visual observation using a phase contrast microscope. The results are shown in FIG.

From FIG. 4, the average particle diameter and the average number of transitions of the preparation for organic solvent-treated microorganisms produced using C16 as the organic solvent did not change greatly even when the number of culture days passed. When the stability was compared using two culture methods, shaking culture and static culture, the static culture was more stable in terms of both average particle diameter and average number of metastases. From this, it was inferred that there was an influence such as the coupling of nearby micelles by shaking.
Moreover, about the formulation for organic-solvent treatment microorganisms manufactured using C19 as an organic solvent, the average particle diameter and the average number of transition did not change a lot even if the culture | cultivation days passed like C16. In C19, as in the case of C16, when the stability was compared using two culture methods, shaking culture and stationary culture, stationary culture was more stable in terms of both average particle size and average number of metastases. It was. From this, it was inferred that there was an influence such as the coupling of nearby micelles by shaking.
From the above, it was confirmed that the organic solvent-treated microorganism preparation produced using C16 or C19 as the organic solvent can be stored at room temperature for about one week.

[Example 2] Manufacture of microorganism preparation for organic solvent treatment using MM medium as aqueous solvent (1) Bacterial cell washing The preculture solution prepared in (1) of Example 1 was put into a Pipetteman P in a 2.0 mL Eppendorf tube. 500 μL was added using -1000. Subsequently, centrifugation was performed at 10,000 g and 4 ° C. for 10 minutes, and the supernatant was removed. Subsequently, 500 μL of sterilized milli-Q water was added, the mixture was sufficiently stirred, and centrifuged again at 10000 g and 4 ° C. for 10 minutes. This operation was repeated 4 times in total, suspended in 500 μL of sterilized milli-Q water, and used in the subsequent (2).

(2) Encapsulation of micelle of PR4 strain Ultrasonication was performed using the same method as in (2) of Example 1 except that C19 was used as the organic solvent and MM medium was used instead of IB2 liquid medium. Subsequently, 100 μL of the solution after sonication was sampled. The sampled solution was observed using a phase contrast microscope. The results are shown in FIG.

  From FIG. 5, it was confirmed that the PR4 strain was encapsulated in the micelle. From this, it was confirmed that by using an organic solvent-degrading bacterium whose localization is transferable, about 1 to 5 cells can be encapsulated in micelles regardless of the type of medium.

[Example 3] Manufacture of microorganism preparation for organic solvent treatment using NP medium as aqueous solvent (1) Bacterial cell washing Using the same method as in (2) of Example 2, the cells in the precultured solution After washing and suspending in 500 μL of sterile MilliQ water, it was used in the subsequent step (2).

(2) Encapsulation of micelle of PR4 strain Ultrasonication was performed using the same method as in (2) of Example 1 except that C19 was used as the organic solvent and MM medium was used instead of IB2 liquid medium. Subsequently, 100 μL of the solution after sonication was sampled. The sampled solution was observed using a phase contrast microscope. The results are shown in FIG.

  From FIG. 6, it was confirmed that the PR4 strain was encapsulated in the micelle. From this, it was confirmed that by using an organic solvent-degrading bacterium whose localization is transferable, about 1 to 5 cells can be encapsulated in micelles regardless of the type of medium.

[Example 4] Analysis of particle size of microorganism preparation for organic solvent treatment using laser diffraction particle size distribution analyzer (1) Cell washing Using the same method as (1) of Example 2, pre-culture solution The bacterial cells inside were washed and suspended in 500 μL of sterilized milli-Q water, and then used in (2).

(2) Micelle encapsulation of PR4 strain Ultrasonication was performed using the same method as in (2) of Example 1 except that C16 was used as the organic solvent and MM medium was used instead of IB2 liquid medium. Further, ultrasonic treatment was carried out without containing PR4 strain to produce control micelles. Subsequently, 100 μL of the solution after sonication was sampled. The sampled solution was observed using a phase contrast microscope. The results are shown in FIG.

  From FIG. 7 (A), it was confirmed that PR4 strain was encapsulated in micelles in the microorganism preparation for organic solvent treatment.

(3) Particle size analysis Further, for the microbial preparation for organic solvent treatment and the control micelle prepared in (2), the particle size was measured using a Shimadzu laser diffraction particle size distribution analyzer (SALD-2300, manufactured by Shimadzu Corporation). It was measured. The results are shown in FIG.

  From FIG. 7 (B), in the control micelle, the particle size was distributed in the range of 0.7 to 3 μm, and the average particle size was 1.58 ± 0.07 μm. On the other hand, in the microorganism preparation for organic solvent treatment, the particle size was distributed in the range of 0.9 to 400 μm, and the average particle size was 23.40 ± 3.76 μm.

[Test Example 1] Weathered-crude oil (w-oil) degradation test using a microorganism preparation for organic solvent treatment (1) Degradation conditions C16 was used as an organic solvent, and MM medium was used as an aqueous solvent. Moreover, 1 mg / mL weathered-crude oil (w-oil) was used as a decomposition target. “w-oil” is a heat-treated product of Arabian light crude oil, and was used assuming a non-volatile oil remaining in a contaminated environment. The initial cell amount was 1.0 × 10 ⁶ cells / mL. In addition, samples having the following four decomposition conditions were prepared, and cultured with shaking at 28 ° C. for 48 hours.
Pattern 1: PR4 strain and C16 are separately added to MM medium and not subjected to ultrasonic treatment. Pattern 2: PR4 strain and MM medium pre-sonicated, and C16 and MM medium pre-sonicated. Mixed (ultrasonic conditions are the same as (2) of Example 1)
Pattern 3: Sonicated using the same method as in (2) of Example 1 except that C16 was used as the organic solvent and MM medium was used instead of the IB2 liquid medium. Pattern 4: C16 as the organic solvent Was sonicated using the same method as (2) of Example 1 using MM medium instead of IB2 liquid medium and not containing PR4 strain

(2) Analysis of residual components using GC-MS From the culture solution of each sample after the decomposition treatment in (1), the residual components were extracted using chloroform, and GC-MS (manufactured by Shimadzu Corporation) was used. Analysis was performed. In addition, about each sample, since C16 contains very much compared with the other alkane in w-oil, since it cannot show with the same intensity | strength, it measures separately components other than C16 and C16, SIM analysis was performed. In any case, the pattern 4 was used as a control (remaining amounts of various remaining components were set to 100%) and expressed as a relative remaining rate. The results are shown in FIGS.

  From FIG. 8, regarding the various components other than C16 in w-oil, compared to the decomposition conditions of pattern 1 and pattern 2, in the decomposition conditions of pattern 3, decomposition of all alkanes progressed and the relative residual ratio decreased. Was confirmed. In Pattern 2, since the ultrasonic treatment was performed without including C16, it was speculated that the PR4 strain was damaged and the degradation rate was lowered.

  Further, from FIG. 9, it was confirmed that C16 derived from the organic solvent and w-oil was decomposed and decreased in the relative residual ratio under the pattern 3 decomposition condition as compared with the pattern 1 and pattern 2 decomposition conditions. It was. Therefore, it was speculated that the organic solvent-treating microbial preparation of the present invention can decompose the organic solvent contained in the preparation and does not remain, and therefore has a low environmental load.

  From the above, it was revealed that the microorganism preparation for organic solvent treatment of the present invention can be stored at room temperature, has a low environmental load, and has a high organic solvent resolving power.

  ADVANTAGE OF THE INVENTION According to this invention, the microbial formulation for organic solvent processing which can be preserve | saved at normal temperature and was produced with the emulsification technique with little environmental impact and which has the high organic solvent resolution | decomposability can be provided. Furthermore, by using the microorganism preparation for organic solvent treatment of the present invention, it can be used for environmental purification or substance production.

Claims (10)

  A microorganism preparation for organic solvent treatment, comprising an organic solvent-degrading bacterium encapsulated in micelles with an organic solvent.   The microorganism preparation for organic solvent treatment according to claim 1, wherein the organic solvent is a liquid at normal temperature and normal pressure.   The microorganism preparation for organic solvent treatment according to claim 1 or 2, wherein the organic solvent is an alkane having 13 to 16 carbon atoms.   The microorganism preparation for organic solvent treatment according to any one of claims 1 to 3, wherein the organic solvent-degrading bacterium is a bacterium belonging to the genus Rhodococcus.   The microorganism preparation for organic solvent treatment according to any one of claims 1 to 4, wherein the micelle has an average particle size of 1 µm to 100 µm.   The organic solvent processing microbial formulation as described in any one of Claims 1-5 is used, The processing method of the organic solvent characterized by the above-mentioned.   A micelle obtained by mixing an organic solvent-decomposing bacterium, an organic solvent, and a medium, and performing sonication under conditions that allow the organic solvent-decomposing bacterium to grow, thereby obtaining the organic solvent-decomposing bacterium encapsulated in micelles with the organic solvent A method for producing a microorganism preparation for organic solvent treatment, comprising an encapsulation step.   The method for producing a microbial preparation for treating an organic solvent according to claim 7, wherein the organic solvent is liquid at normal temperature and normal pressure.   The method for producing a microorganism preparation for organic solvent treatment according to claim 7 or 8, wherein the organic solvent is an alkane having 13 to 16 carbon atoms.   The method for producing a microorganism preparation for organic solvent treatment according to any one of claims 7 to 9, wherein the organic solvent-degrading bacterium is a bacterium belonging to the genus Rhodococcus.