JP6695612B2 - Method for producing microbial preparation for treating organic solvent and method for treating organic solvent - Google Patents

Description

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

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

In particular, when considering bioprocesses, microorganisms 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 show good growth for microorganisms required for purification of oil-polluted environment due to oil spill accident, etc. while decomposing these in the presence of high concentration of hardly volatile compounds, not only resolution but also coexistence It is required that the anti-volatile compound has high resistance to toxicity.
In order to analyze these properties, first, the organic solvent resistance of the microorganism is required as a cut point, and in particular, in the bioprocess, growth in the presence of a high-concentration organic solvent is required.

The present inventors have found that the Rhodococcus rhodochrous S-2 strain is a high-concentration petroleum-resistant petroleum-degrading bacterium (see, for example, Non-Patent Document 1), and as a result of further studies on its petroleum resistance. , It has been clarified that the extracellular polysaccharide (hereinafter referred to as "EPS") produced by the same bacterium is deeply involved in the resistance of a non-volatile organic solvent such as a long-chain alkane.
Furthermore, when the colony morphology and solvent resistance of Rhodococcus were examined, rough type bacteria with low EPS production had high affinity for the solvent and consequently solvent sensitivity, while mucoid type bacteria with high EPS production were found. Showed that there was a high correlation between colony morphology and organic solvent resistance in the same bacterium.
Further, EPS has been shown to confer solvent resistance to solvent-sensitive rough bacterium, and from these facts, the formation of mucoid type colony is one index for considering the solvent resistance of the genus bacterium. It was found.

  The Rhodococcus erythropolis PR4 strain (hereinafter sometimes referred to as “PR4 strain”) decomposes pristane (2,6,10,14-tetramethyl-pentadecane), which is a kind of branched alkane. It is a strain isolated as a bacterium (see, for example, Non-Patent Document 2), and is a strain that changes its colony morphology based on EPS production from rough type to mucoid type to rough type with the progress of culture. . It is also known that this strain shows resistance to a hardly volatile organic solvent.

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

  In the culture / alkane bilayer culture system, the PR4 strain has two different localizations depending on the carbon number of the alkane to be added, that is, "adsorption type" that is adsorbed on the alkane particle surface and "transfer type" that is transferred into the alkane particle. The strain is shown (for example, see Non-Patent Document 3), and is expected as a host for bioremediation and bioprocess.

  The present inventors have revealed that the PR4 strain can be transferred to tetradecane, pentadecane, hexadecane, pristane, squalane, etc. 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) Preservation, maintenance and transfer of bacteria while maintaining expression of degrading enzymes. There are some problems such as measures for easy handling, 3) measures for increasing the affinity of the medium / alkane after administration of the preparation to the entire bilayer culture system.

  Generally, the emulsification technology in a water / oil mixed system can be classified into two methods, a chemical method using a surfactant or an emulsion stabilizer and a physical method using mechanical energy. In the former chemical method, the surfactant and the like are substances that are difficult to decompose, and if they flow out to the river or the sea, they remain and may cause environmental pollution.

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

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

That is, the present invention includes the following aspects.
[1] and Rhodococcus erythropolis PR4 strain was mixed with an alkane having 13 or more carbon atoms, and a medium, wherein the performed Rhodococcus erythropolis PR4 strain sonicated in viable conditions, before Symbol Rhodococcus erythropolis comprises as engineering oil droplets of the alkanes PR4 strain was transferred to obtain a dispersed organic solvent treatment microbial agent in the medium, the manufacturing method of the microorganism preparation for organic solvent treatment.
[ 2 ] The method for producing a microbial preparation for organic solvent treatment according to [1], wherein the alkane has 13 or more and 19 or less carbon atoms.
[ 3 ] The microbial preparation for organic solvent treatment according to [1] or [2] , wherein the oil droplets have an average particle size of 1 μm or more and 100 μm or less.
[ 4 ] A method for treating an organic solvent , which uses the microbial preparation for treating an organic solvent obtained by the method for producing a microbial preparation for treating an organic solvent according to any one of [1] to [ 3 ].

  According to the present invention, it is possible to provide a microbial preparation for treatment with an organic solvent, which can be stored at room temperature and which is produced by an emulsification technique with a low environmental load and has a high organic solvent decomposing ability.

It is the image which image | photographed the organic solvent process microbe preparation manufactured using various organic solvents (C12, C15, C16, C19) in Example 1 with the phase contrast microscope. 1 is an image photographed by a phase contrast microscope after double-staining the organic solvent-treated microorganism preparation produced using C16 or C19 as an organic solvent in Example 1 with a DAPI reagent and a CTC reagent. The average particle size and the average number of transitions immediately after ultrasonic treatment in the organic solvent-treated microbial preparation produced using C16 or C19 as the organic solvent in Example 1 are visually measured using a phase contrast microscope. It is a graph. Using a phase-contrast microscope, the average particle diameter and the average number of transitions on the 1st to 7th days after ultrasonic treatment with the organic solvent-treated microbial preparation produced using C16 or C19 as the organic solvent in Example 1 were used. 5 is a graph showing the results of visual measurement by 5 is an image of a preparation for an organic solvent-treated microorganism produced by using C19 as an organic solvent and MM medium as an aqueous solvent in Example 2 by a phase contrast microscope. 6 is an image of a preparation for an organic solvent-treated microorganism produced by using C19 as an organic solvent and an NP medium as an aqueous solvent in Example 3, taken by a phase contrast microscope. (A) C16 as an organic solvent in Example 4, an organic solvent-treated microorganism preparation produced using MM medium as an aqueous solvent, and a control micelle produced using C16 as an organic solvent and MM medium as an aqueous solvent. It is the image which image | photographed with the phase contrast microscope. (B) C16 as an organic solvent in Example 4, an organic-solvent-treated microorganism preparation produced by using MM medium as an aqueous solvent, and a control micelle produced by using C16 as an organic solvent and MM medium as an aqueous solvent. Is a graph showing the particle size measured by a laser diffraction type particle size distribution measuring device. GC-MS analysis (SIM conditions) was performed on various residual components other than C16 of the weathered-crude oil (w-oil) after the treatment under each decomposition condition (Patterns 1 to 3) in Test Example 1 with Pattern 4 as a control. It is a graph which shows a result. Results of GC-MS analysis (SIM conditions) of the residual component (C16) of the weathered-crude oil (w-oil) after the treatment under each decomposition condition (Patterns 1 to 3) in Test Example 1 with the 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 with an organic solvent, which comprises an organic solvent-degrading bacterium that is micelle-encapsulated with an organic solvent.

  The microbial preparation for treating with an organic solvent of the present embodiment can be stored at room temperature and has a low environmental load. Furthermore, the microbial preparation for organic solvent treatment of the present embodiment has a high organic solvent decomposing ability and can be used for environmental purification or substance production.

<Organic solvent>
In the present embodiment, the organic solvent used for encapsulation of micelles is not particularly limited and may be, for example, a solid at room temperature and atmospheric pressure, or a liquid at room temperature and atmospheric pressure. Good. When a solid substance is used at room temperature and atmospheric pressure, the presence of an organic solvent that is liquid at ordinary temperature and atmospheric pressure can dissolve the organic solvent that is solid at ordinary temperature and atmospheric pressure to form a liquid. Among them, from the viewpoint of storage stability of micelles, the organic solvent used for encapsulating micelles in the present embodiment is preferably a liquid at room temperature and atmospheric pressure.

In the present specification, the “normal temperature” means a temperature at which it is not cooled or heated, that is, a normal temperature, and examples thereof include a temperature of 15 to 25 ° C.
Further, in the present specification, "normal pressure" means a pressure when neither depressurization nor pressurization is specifically performed, that is, a pressure equal to atmospheric pressure, and for example, about 1 atm (1 atm, 101, 325 Pa) or the like. Can be mentioned.

In addition, in the present embodiment, the organic solvent used for encapsulating the micelle preferably contains an alkane having 13 or more carbon atoms. With alkanes having 12 or less carbon atoms, it is usually difficult to transfer organic solvent-degrading bacteria into an organic solvent. Therefore, as the organic solvent used for encapsulating the micelle, a hydrocarbon having 12 or less carbon atoms or another hydrophobic substance may be contained in addition to the alkane having 13 or more carbon atoms. It may be 100% occupied. When the alkane having 12 or less carbon atoms is contained, 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. It is more preferable that the content is 60% (v / v) or more.
Above all, it is preferable that the organic solvent used for encapsulating micelles is 100% occupied by alkanes having 13 or more carbon atoms.

  Therefore, as an organic solvent which is a liquid at normal temperature and pressure and in which an organic solvent-decomposing bacterium can be transferred into an organic solvent, for example, a linear, branched or cyclic alkane having 13 to 16 carbon atoms, etc. 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 chain alkane having 13 to 16 carbon atoms include, for example, 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), 3,3-Diethyl-4,5,5 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), farnesane (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) , 2,4,6-trimethyltridecane (C16), 3-methylpentadecane (C16), 6-pentylundecane (C16), 4,6-diethyldodecane (C16), 2,2,4,4,6,6. Examples thereof include, but are not limited to, 6,7-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) and 3-cyclohexyl-4-. Examples include methyl heptane (C14), cyclopentadecane (C15), cyclohexadecane (C16), and the like, but are not limited thereto.

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, examples of the halogen atom substituting the hydrogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
When the organic solvent used for encapsulating micelles is a linear, branched or cyclic alkane having 13 or more and 16 or less carbon atoms, the organic solvent-degrading bacterium can be transferred into the organic solvent. Furthermore, since the organic solvent-degrading bacterium can decompose and metabolize the organic solvent itself used for encapsulating micelles, the organic solvent used for encapsulating micelles does not remain and the environmental load can be reduced. ..

<Organic solvent degrading bacteria>
In the present specification, the term "organic solvent-degrading bacterium" means that an organic solvent can be decomposed, can grow in an organic solvent, and is not adsorbed on the surface of the organic solvent but is in a transferred state, that is, an organic solvent. It means a bacterium existing in a state of being submerged in a solvent. The organic solvent decomposing bacterium is not particularly limited, and examples thereof include bacteria belonging to the genus Rhodococcus.
As bacteria belonging to the genus Rhodococcus, more specifically, for example, Rhodococcus Australis (Rhodococcus australis) ATCC35215 strain, Rhodococcus coprophilus (Rhodococcus coprophilus) ATCC29080 strain, Rhodococcus coprophilus JCM3200 strain, Rhodococcus erythropolis (Rhodococcus erythropolis) ATCC27854 Strains, Rhodococcus erythropolis ATCC47072 strains, Rhodococcus erythropolis DSM1069 strains, Rhodococcus erythropolis JCM3201 strains, Rhodococcus erythropolis PR4 strains (hereinafter sometimes referred to as "PR4 strains"), Rhodococcus globellus (Rucorus vulcorus vulcolus rus vulcorus rubous). ) ATCC14346 strain, Rhodococcus globellus ATCC15076 strain, Rhodococcus globellus ATCC21292 strain, Rhodococcus globellus ATCC25669 strain, Rhodococcus globellus ATCC25688 strain, Rhodococcus globellus ATCC3110 strain, Rhodococcus roscodocolis octocoloscodochroscoccus 48 globinus ATCC25688 strain Rhodococcus Opakasu (Rhodococcus opacus) ATCC170391 strain, Rhodococcus Opakasu ATCC51881 strain, Rhodococcus Opakasu ATCC51882 strain, Rhodococcus Opakasu JCM9703 strain, Rhodococcus Perukoratasu (Rhodococcus percolatus) JCM10087 strain, Rhodococcus Rodoni (Rhodococcus rhodnii) ATCC35071 strain, Rhodococcus Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC9808, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodochrous ATCC9808 Rhodococcus rhodochrous ATCC184 strain, Rhodococcus rhodochrous ATCC17041 strain, Rhodococcus S. rhodochrous ATCC 19150 strain, Rhodococcus rhodochrous ATCC 12674 strain, Rhodococcus rhodochrous JCM2156 strain, Rhodococcus rhodochrous JCM2157 strain, Rhodococcus rhodochrous R-1 strain, Rhodococcus rhodochrous R-2 strain, Rhodococcus S. rhodochrous S. Rhodococcus louver (Rhodococcus ruber) IFO15591 strain, Rhodococcus species PG7-2 strain, Rhodococcus species JCM3376 strain, Rhodococcus sp. Examples include, but are not limited to:
Among them, the organic solvent decomposing bacterium used in the present embodiment is preferably a bacterium belonging to the genus Rhodococcus erythropolis, and 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 degrading and metabolizing an organic solvent.

<Micelle>
In the microbial preparation for treating an organic solvent 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 size of the micelle is within the above range, at least one organic solvent-decomposing bacterium can be included, and the micelle can be stably dispersed.

  The number of organic solvent-decomposing bacteria to be enclosed in the micelle is preferably at least 1 or more, more preferably 1 or more and 10 or less, and 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-decomposing bacterium enclosed in the micelle is within the above range, the micelle can be stably dispersed in the state in which the organic solvent-decomposing bacterium is enclosed, and the organic solvent can be further efficiently dispersed. Can be disassembled.

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

When IB2 liquid medium is used as the medium, the IB2 liquid medium contains a yeast extract in order to transfer the organic solvent-decomposing bacteria from the aqueous solvent or the organic solvent used for encapsulating micelles to the organic solvent to be treated. Preferably. By including the yeast extract in the IB2 liquid medium, the organic solvent-degrading bacterium can be smoothly transferred from the aqueous solvent or the organic solvent used for encapsulating micelles to the organic solvent to be treated.
The amount of the yeast extract added to the aqueous solvent is preferably 0.05% (w / w) or more, and 0.5% (w), based on the total amount of the aqueous solvent (100%). / W) or more and 5% (w / w) or less, more preferably about 1% (w / w).

  In the present specification, "migrating into an organic solvent" means that an organic solvent-degrading bacterium existing in an aqueous solvent is an aqueous solvent, or an organic solvent used for encapsulating micelles, and an organic substance to be treated. It means to be adsorbed or transferred to a solvent.

In the microbial preparation for treating an organic solvent of the present embodiment, it is usually difficult for an organic solvent-decomposing bacterium to transfer to an alkane having 12 or less carbon atoms, but by limiting the concentration of the inorganic salt added to the aqueous solvent, Decomposing 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 further preferably 0.9 nM or less. Examples of the inorganic salt include magnesium salts, and more specifically, magnesium chloride, magnesium sulfate, and magnesium nitrate.

  The method for producing the microbial preparation for treating with an organic solvent of the present embodiment will be shown in << Method of producing microbial preparation for treating with organic solvent >> described below.

  Regarding the storage conditions of the organic solvent treatment microbial preparation of the present embodiment, the organic solvent-decomposing bacteria should be stored at a temperature higher than the temperature at which the organic solvent used for encapsulating micelles or the aqueous solvent solidifies at a temperature at which the organic solvent-degrading bacteria do not die. For example, it may be stored at room temperature, for example, may be stored at a temperature higher than room temperature or a temperature below room temperature, for example, stored in refrigeration (4 ° C or lower) or frozen (-20 ° C or lower). You may. Above all, it is preferable to store the micelles at room temperature from the viewpoint of stability.

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

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

  In the present specification, "treatment of an organic solvent" means adding an organic solvent that can be decomposed and metabolized by an organic solvent-degrading bacterium to the above-mentioned microbial preparation for treating an 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 it can decompose and metabolize an organic solvent-degrading bacterium. Examples of the organic solvent to be treated include linear, branched or cyclic alkanes having 6 or more carbon atoms. When the organic solvent-degrading bacterium is transformed, it can grow even a linear, branched, or cyclic alkane having 6 to 12 carbon atoms and is adsorbed at least by these low-carbon alkanes. Therefore, the low carbon number alkane can be metabolized and decomposed.
Further, the upper limit of the carbon number of the alkane is not limited, for example, even if it is a solid at room temperature and atmospheric pressure, it is a solid at room temperature and atmospheric pressure due to the presence of an alkane that is liquid at room temperature and atmospheric pressure. It is sufficient if the alkane can be dissolved and made into a liquid.
Further, the linear, branched or cyclic alkane having 6 or more carbon atoms may be contained in one kind or two kinds or more.

  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 Examples thereof include (C25) and n-triacontane (C30), but are not limited thereto.

Examples of the branched chain alkane having 6 or more carbon atoms include, but are not limited to, the followings.
Carbon number 6: 2-methylpentane, 2,3-dimethylbutane, 3-methylpentane, 3-methylpentane, dimethylbutane, 2,2-dimethylbutane Carbon number 7: 3-methylhexane, 3,3-dimethylpentane , 2,2,3-Trimethylbutane, 2,3-Dimethylpentane, 3-Ethylpentane, 2-Methylhexane, 2,2-Dimethylpentane, 2,4-Dimethylpentane C8: (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 C10: 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-tetramethyi Ruhexane, 2,2,5-trimethylheptane, 4-methylnonane, 2,4,5-trimethylheptane, 2,4,6-trimethylheptane, 3,4-diethylhexane C11: 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 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- Propylnonane, 2-methylundecane, 3,4-dimethyldecane, 2,2,7,7-tetramethyloctane, 2,4,5,7-tetramethyloctane, 2,2-dimethyldecane, 2,2,2. 4,4,6-Pentamethylheptane, 2,2-dibutylbutane C13: 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 C14: 2-methyltridecane, 7-methyltridecane, 3,8-diethyldecane, (6R, 7S) -6,7-dimethyldodecane, 3,3,4,4-tetraethyl Hexane, 2,2,3,3,4,5,5-octamethylhexane, 5-butyldecane, 2,5-dimethyl-3,4-diisopropylhexane, 2,2,3,3,5,6 ， -Heptamethylheptane, 3-tert-butyl-2,2,5,5-tetramethylhexane C15: 4-methyltetradecane, 2,7,10-trimethyldodecane, 7-methyltetradecane, 6-propyldodecane, Farnesane, [R, (−)]-3-methyltetradecane, 2,6-dimethyl-3,5-diisopropylheptane, 5-butylundecane, 5-pentyldecane C16: (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 Chillundecan, 4,6-diethyldodecane, 2,2,4,4,6,6,7-heptamethylnonane, 2,2,4,4,6,8,8-heptamethylnonane 17: 4 carbon atoms , 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 C18: 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 Carbon number 19: 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 C20: phytane, 2-methyl nonadecane, 3-methyl nonadecane, 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 Methylhexadecane, 5-butylhexadecane 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, hasian, 2-methyltetracosane, 2,2,8,8-tetramethyl-5,5-bis (3,3-dimethyl) Butyl) nonane, 2,6,10,14,18-pentamethylicosane, carbon number 30: squalane, 2 10-dimethyloctacosane, 7-methylnonacosane, 11-nonylhenicosan, (R) -3-methylnonacosane, 8,12-dimethyloctacosane, 3-methylnonacosane, 9-octyldocosane, 7,12- Dihexyl octadecane, Rizan, 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-tetrane. Methylcyclohexane (C10), 1-methyl-2,4-diethylcyclopentane (C10), 1,5-dimethylcyclooctane (C10), cycloundegane (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-cyclohexyl-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), cembran (C20), cycloicosane (C20), tetradecylcyclohexane (C20), cyclopentacosane (C25), (1- Nonyldecyl) cyclohexane (C25), 9- (3-cyclopentylpropyl) heptadecane (C25), cyclotriacontane (C30), icosamethylcyclodecane (C30), pentacosylcyclopentane (C30), and the like. Not limited to.

<Use>
The treatment method of this embodiment can be used for treating a contaminated environment containing an organic solvent. Examples of the contaminated environment treatment include treatment of domestic wastewater and treatment of industrial water.
Moreover, the processing method of the present embodiment can be used for the 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 the present embodiment, the above-mentioned organic solvent treatment microbial preparation does not contain an emulsifier such as a surfactant, and the organic solvent contained in the organic solvent treatment microbial preparation itself is also decomposed by the organic solvolytic bacterium. Also, since it is metabolizable, the environmental load can be reduced.

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

<< Method for producing microbial preparation for organic solvent treatment >>
In one embodiment, the present invention is a mixture of an organic solvent-degrading bacterium, an organic solvent, and a medium, sonicated under conditions in which the organic solvent-degrading bacterium can grow, and encapsulated in micelles by the organic solvent. Also provided is a method for producing a microbial preparation for treating an organic solvent, which comprises a micelle encapsulation step for obtaining the organic solvent-degrading bacterium.

  According to the production method of the present embodiment, a microbial preparation for treating an organic solvent, which can be stored at room temperature, has a small environmental load, and has a high organic solvent decomposing ability, can be easily obtained.

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

Examples of the organic solvent-decomposing bacterium used in the present embodiment include the same ones as exemplified in the above <<<< organic solvent treatment microorganism preparation >>>>. Among them, the organic solvent degrading bacterium used in the present embodiment is preferably a bacterium belonging to the genus Rhodococcus erythropolis, and 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 degrading and metabolizing an organic solvent.

  In the present embodiment, the organic solvent is preferably an organic solvent that is liquid at room temperature and atmospheric pressure and is capable of transferring organic solvent-degrading bacteria into the organic solvent. For example, a linear chain having 13 to 16 carbon atoms Examples thereof include linear, branched, or cyclic alkanes. Examples of the linear, branched or cyclic alkane having 13 or more and 16 or less carbon atoms include the same as those exemplified in the above <<<< microbial preparation for treating with organic solvent >>.

  Examples of the medium used in the present embodiment include the same as those exemplified in the above <<<< microbial preparation for treating with organic solvent >>. Among them, the medium used in the present embodiment is preferably IB2 liquid medium, MM medium or NP medium.

In the present 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-degrading bacterium can be easily encapsulated in micelles.
The concentration of the organic solvent-decomposing bacterium in the mixed solution may be appropriately adjusted depending on the type of the organic solvent-decomposing bacterium, the number of cells to be encapsulated in each prepared micelle, the condition of ultrasonic treatment, and the like. it can. For example, 1 mL of the organic solvent and 10 mL of the medium may be mixed so that the organic solvent-degrading bacterium is 1.0 × 10 ⁷ cells / mL.

In the present specification, the “condition capable of growing” means a condition in which the organic solvent-degrading bacterium is not killed or the microbial cell is not damaged and the growth is not hindered by ultrasonication. Examples of the ultrasonic treatment under the “growing condition” include, for example, an ultrasonic cleaner containing 4 L of water (Azuwan USD-4R), and an organic solvent-degrading bacterium 1.0 × 10 ⁷ cells / mL, Conditions such as 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 room temperature and normal pressure at 40 kHz for 15 minutes can be mentioned. 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 a mixed solution of an organic solvent-degrading bacterium, an organic solvent, and a medium, or via a container containing a mixed solution of an organic solvent-degrading bacterium, an organic solvent, and a medium. It may be done indirectly. Among them, the organic solvent-degrading bacteria are not killed, or the cells are not damaged, and the conditions are such that the growth is not hindered. Therefore, the organic solvent-degrading bacteria, the organic solvent, and a container containing a mixed solution of the medium are used. It is preferably performed indirectly.
The ultrasonic generator to be used is not particularly limited, and examples thereof include a standing wave (washer type) ultrasonic generator in which the vibrator is mounted on a flat plate (vibration plate), and a cylindrical shape at the tip of the vibrator. The horn type (homogenizer type) ultrasonic wave generation device in which the horn of FIG. Above all, it is preferable to use the standing wave type (washer type) ultrasonic generator because the organic solvent decomposing bacteria are not killed or the bacterial cells are not damaged and the growth is not disturbed.

  Regarding the produced microbial preparation for treating with an organic solvent, the number of cells of the organic solvent-decomposing bacterium enclosed in the micelle can be confirmed by, for example, a method using a phase contrast microscope or the like. The average particle size of the micelles can be confirmed by, for example, a method of visually measuring using a phase contrast microscope or the like, a method of measuring using a laser diffraction type particle size distribution measuring device, or the like.

Regarding the storage conditions of the produced microbial preparation for treating with an organic solvent, if it is stored at a temperature at which the organic solvent-degrading bacterium does not die and is higher than the temperature at which the organic solvent or aqueous solvent used for encapsulating micelles solidifies. Well, for example, it may be stored at room temperature, for example, may be stored at a temperature higher than room temperature or a temperature below room temperature, for example, refrigerated (4 ° C or less) or frozen (-20 ° C or less). May be. In addition, when stored by refrigeration (4 ° C or lower) or frozen (-20 ° C or lower), even if the organic solvent solidifies to break the micelle and separate into the organic solvent phase and the medium phase, the above-mentioned By performing the <micelle encapsulation step>, the organic solvent-degrading bacterium can be encapsulated in micelles.
Among them, it is preferable to store the micelles at room temperature because the micelles can be stably stored without the need for ultrasonic treatment again.

  Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[material]
1. Strains Used Rhodococcus erythropolis PR4 strain (hereinafter, sometimes referred to as “PR4 strain”) was used in the following examples.

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

2-2. IB2 liquid medium 5 g of glucose (manufactured by Wako Pure Chemical Industries, Ltd.) in 5 mL of milli-Q water, 5 g of yeast extract (Becton, manufactured by Dickinson and company), 0.09 g of MgCl ₂ .6H ₂ O (Wako Pure Chemical Industries, Ltd.) Made), 0.05 g CaCl ₂ .2H ₂ O (made by Wako Pure Chemical Industries, Ltd.), 0.05 g NaCl (made by Wako Pure Chemical Industries, Ltd.), 0.01 g FeCl ₂ .6H ₂ O (Wako Pure Chemical Industries, Ltd.) (Manufactured by Kogyo Co., Ltd.) and 0.25 g of (NH ₄ ) ₂ SO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.) were added. Then, after adjusting to pH 7.2, it was autoclaved at 121 ° C. for 15 minutes and cooled.

2-3. MM medium 0.09 g of MgCl ₂ .6H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.), 0.05 g of CaCl ₂ .2H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.), 0.05 g of NaCl in 500 mL of Milli-Q water. (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 (Wako Pure Chemical Industries, Ltd.) was added. Then, it autoclaved at 121 degreeC 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. Then, it autoclaved at 121 degreeC for 15 minutes, and cooled.

3. Reagents used In order to confirm whether the PR4 strain is alive or dead, DAPI (4 ′, 6-diamidino-2-phenylinodole, dihydrochloride) reagent (manufactured by Wako Pure Chemical Industries, Ltd.) and CTC (5-Cyano-2,3-ditoylyl-2H). -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.)
Pristane (hereinafter sometimes referred to as "C19") (manufactured by ACROSS)

Further, 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 adjusted to 100 mL. Then, it autoclaved at 121 degreeC for 15 minutes, and cooled.

3-2. CaCl ₂ solution To MilliQ water, 10 g of CaCl ₂ .2H ₂ O (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the volume was raised to 100 mL. Then, it autoclaved at 121 degreeC for 15 minutes, and cooled.

3-3. NaCl solution To Milli-Q water, 10 g of NaCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added to make up to 100 mL. Then, it autoclaved at 121 degreeC 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 MilliQ water, and the volume was adjusted to 100 mL. Then, it autoclaved at 121 degreeC 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. Then, it autoclaved at 121 degreeC for 15 minute (s), and cooled.

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

(2) Encapsulation of PR4 strain into micelles Subsequently, a test tube which had been subjected to dry heat sterilization in advance at 180 ° C. for 30 minutes was prepared, and 10 mL of IB2 liquid medium was added thereto using an autopipetter 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 liquid of the PR4 strain prepared in (1) was added using Pipetman P-200, and this was cultivated with shaking at 28 ° C. and 110 rpm for 1 day. The cell concentration after culture was 1.0 × 10 ⁷ cells / mL. Subsequently, ultrasonic treatment was performed at 40 kHz for 15 minutes at room temperature and atmospheric pressure using an ultrasonic cleaner (USD-4R, manufactured by AS ONE) containing 4 L of water. The ultrasonic treatment was performed so 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 ultrasonic treatment was sampled. The sampled solution was observed using a phase contrast microscope. For observation, using a Pipetman P-20, 8 μL of each solution was dropped on the slide, a cover glass was placed, and one drop of immersion oil was dropped on a stage of a phase contrast microscope (Olympus). And the objective lens was magnified 100 times. The results are shown in Figure 1.

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 the IB2 liquid medium was used as the aqueous solvent, the localization became metastatic and the PR4 strain could be encapsulated in micelles. Further, it was confirmed that the PR4 strain was transferred to about 1 to 5 cells in each micelle.

(3) Confirmation of Influence on PR4 Strain by Ultrasonic Treatment Further, the organic solvent-treated microorganism preparation produced using C16 or C19 as an organic solvent was subjected to double staining with DAPI and CTC.
Double staining was performed as follows. Using 100 μL of each organic solvent-treated microorganism preparation produced using C16 or C19 as an organic solvent, 0.5 μL of Enhancing reagent B solution (manufactured by Dojindo Co., Ltd.) and 1.5 μL of CTC reagent were used using Pipetteman P-2. Each of them was added and thoroughly stirred by vortex. Then, the cells were cultured for 15 minutes in THERMO MINDER 50 mini (manufactured by TAITEC) set at 37 ° C. Subsequently, 1 μL each of DAPI reagent was added using Pipetman P-2, thoroughly stirred by vortex, and left at room temperature for 15 minutes. Then, the sample was observed using the phase contrast microscope. The DAPI reagent is a nuclear staining reagent used as counterstain, and the CTC reagent is a viable cell selective fluorescent staining reagent. The results are shown in Figure 2.

From FIG. 2, red fluorescence due to CTC staining was detected for the organic solvent-treated microorganism preparation produced using C16 or C19 as the organic solvent. Further, although detailed data are not shown, when the number of colonies of PR4 strains with and without sonication was compared, it was found that PR4 strains subjected to sonication (conditions described above) were not sonicated. It was confirmed that they formed colonies of the same degree as the strain. From this, it was confirmed that sonication did not affect the colony forming ability.
From the above, it was confirmed that the PR4 strain had respiratory activity even after ultrasonic treatment. That is, it was confirmed that the PR4 strain was not killed by the ultrasonic treatment under the above conditions.

(4) Measurement of average particle size of micelles and average number of transitions of PR4 strain In addition, a phase-contrast microscope was used for an organic solvent-treated microbial preparation (4 samples each) produced using C16 or C19 as an organic solvent. The measurement was carried out visually three times independently, and the average particle size of the micelles and the average number of transitions were calculated. Results are shown in FIG.

From FIG. 3, with respect to the organic-solvent-treated microorganism preparation produced using C16 as the organic solvent, no significant difference was observed in the average particle size and the average number of transitions among the four samples. Further, when the average value of the average particle size and the average number of dislocations was obtained using four samples, the average particle size was 12.27 μm and the average number of dislocations was 3.27.
Regarding the organic solvent-treated microorganism preparation produced using C19 as the organic solvent, similar to the case of C16, no significant difference was observed in the average particle size and the average number of transitions among the four samples. Further, when the average value of the average particle size and the average number of dislocations was determined using four samples, the average particle size was 13.26 μm and the average number of dislocations was 4.23.
From the above, it was confirmed that by using an organic solvent-degrading bacterium whose localization is a transfer type, cells of about 1 to 5 cells can be encapsulated into micelles regardless of the type of organic solvent.

(5) Examination of stability of micelle Further, the stability of micelle was examined for the organic solvent-treated microorganism preparation produced by using C16 or C19 as the organic solvent used in (4). As a method, an organic solvent-treated microorganism preparation produced by using C16 or C19 as an organic solvent, which was shake-cultured at 28 ° C. or statically cultured at room temperature, was used on the first day, the third day, and the fifth day after ultrasonic treatment. Whether or not there was stability was examined by sampling on the 7th and 7th days and visually observing the average particle size and the average number of transitions with a phase contrast microscope. The results are shown in Fig. 4.

From FIG. 4, the average particle size and the average number of transfer of the organic solvent-treated microorganism preparation produced using C16 as the organic solvent did not change significantly even after the number of days of culture passed. When the stability was compared using two culture methods, shake culture and static culture, the static culture was more stable in both the average particle size and the average number of transfers. From this, it was speculated that shaking had an effect such as binding of micelles in the vicinity.
Regarding the organic solvent-treated microorganism preparation produced using C19 as the organic solvent, the average particle size and the average number of transfers did not change significantly even after the number of culture days, as in the case of C16. In C19, as in the case of C16, when the stability was compared using two culture methods of shaking culture and static culture, the static culture showed more stable average particle size and average number of transfers. Was there. From this, it was speculated that shaking had an effect such as binding of micelles in the vicinity.
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] Production of a microbial preparation for treating an organic solvent using MM medium as an aqueous solvent (1) Washing of bacterial cells The preculture liquid prepared in (1) of Example 1 was put into a 2.0 mL Eppendorf pipette P Using -1000, 500 μL was added. Then, centrifugation was performed at 10,000 g and 4 ° C. for 10 minutes to remove the supernatant. Subsequently, 500 μL of sterilized Milli-Q water was added, thoroughly stirred, and again centrifuged at 10,000 g at 4 ° C. for 10 minutes. This operation was repeated 4 times in total, and after suspending in 500 μL of sterile Milli-Q water, it was used in the subsequent step (2).

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

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

[Example 3] Production of microbial preparation for treatment with organic solvent using NP medium as aqueous solvent (1) Washing of bacterial cells Using the same method as in (1) of Example 2, the bacterial cells in the preculture liquid were washed. It was washed, suspended in 500 μL of sterile Milli-Q water, and then used in (2).

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

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

[Example 4] Analysis of particle size of microbial preparation for organic solvent treatment using laser diffraction particle size distribution analyzer (1) Washing of cells Using the same method as (1) in Example 2, preculture liquid The microbial cells therein were washed, suspended in 500 μL of sterile Milli-Q water, and then used in (2).

(2) Micelle encapsulation of PR4 strain C16 was used as an organic solvent, and sonication was performed in the same manner as in (2) of Example 1 except that MM medium was used instead of IB2 liquid medium. In addition, control micelles were prepared by performing ultrasonic treatment without containing the PR4 strain. Subsequently, 100 μL of the solution after ultrasonic treatment was sampled. The sampled solution was observed using a phase contrast microscope. The results are shown in Fig. 7 (A).

  From FIG. 7 (A), it was confirmed that the PR4 strain was micelle-encapsulated in the organic solvent-treated microbial preparation.

(3) Analysis of particle size Further, the particle size of the microbial preparation for organic solvent treatment and the control micelle prepared in (2) were 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. 7 (B).

  From FIG. 7B, 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 organic solvent treatment microbial preparation, 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] Degradation test of weathered-crude oil (w-oil) using a microbial preparation for treating with organic solvent (1) Degradation conditions C16 was used as an organic solvent, and MM medium was used as an aqueous solvent. Moreover, 1 mg / mL of weathered-crude oil (w-oil) was used as a decomposition target. The w-oil is a heat-treated product of Arabian light crude oil, and was assumed to be a non-volatile oil remaining in a contaminated environment. The initial cell amount was 1.0 × 10 ⁶ cells / mL. In addition, samples under the following four decomposition conditions were prepared and cultured with shaking at 28 ° C for 48 hours.
Pattern 1: PR4 strain and C16 were separately charged to MM medium and not sonicated Pattern 2: Pre-sonicated PR4 strain and MM medium and pre-sonicated C16 and MM medium Mixture (ultrasonic conditions are the same as (2) of Example 1)
Pattern 3: C16 as an organic solvent, sonicated by the same method as in (2) of Example 1 except that MM medium was used instead of the IB2 liquid medium. Pattern 4: C16 as an organic solvent. Was sonicated using MM medium instead of IB2 liquid medium, containing no PR4 strain and using the same method as (2) of Example 1.

(2) Analysis of residual components using GC-MS From the culture solution of each sample after the decomposition treatment in (1), residual components were extracted using chloroform, and GC-MS (manufactured by Shimadzu Corporation) was used. Analysis was performed. In addition, for each sample, since C16 contains much more than other alkanes in the w-oil, it cannot be shown with the same strength. Therefore, components other than C16 and C16 are separately measured, SIM analysis was performed. In each case, the pattern 4 was used as a control (the residual amount of each residual component was 100%) and expressed as a relative residual rate. The results are shown in FIGS. 8 and 9.

  From FIG. 8, in comparison with the decomposition conditions of pattern 1 and pattern 2 for various components other than C16 in w-oil, decomposition of all alkanes proceeded and the relative residual ratio decreased under the decomposition conditions of pattern 3. Was confirmed. Further, in Pattern 2, since the ultrasonic treatment was performed without containing C16, it was speculated that the PR4 strain was damaged and the decomposition rate was decreased.

  Further, from FIG. 9, it was confirmed that, with respect to the C16 derived from the organic solvent and w-oil, the decomposition progressed and the relative residual rate decreased under the decomposition conditions of Pattern 3 as compared with the decomposition conditions of Pattern 1 and Pattern 2. It was Therefore, the microbial preparation for treating with an organic solvent of the present invention can decompose the organic solvent contained in the preparation and does not remain, so it is presumed that the environmental load is small.

  From the above, it became clear that the microbial preparation for treating with an organic solvent of the present invention can be stored at room temperature, has a small environmental load, and has a high organic solvent decomposing ability.

  According to the present invention, it is possible to provide a microbial preparation for treatment with an organic solvent, which can be stored at room temperature and which is produced by an emulsification technique with a low environmental load and has a high organic solvent decomposing ability. Further, by using the microbial preparation for treating an organic solvent of the present invention, it can be used for environmental purification or substance production.

Claims (4)

And Rhodococcus erythropolis PR4 strain, and alkanes having 13 or more carbon atoms, a mixture of a medium, the performed Rhodococcus erythropolis PR4 strain sonicated in viable condition, the previous SL Rhodococcus erythropolis PR4 strain translocated oil droplets of said alkane comprises as engineering to obtain a dispersed organic solvent treatment microbial agent in the medium, the manufacturing method of the microorganism preparation for organic solvent treatment. The method for producing a microbial preparation for treating an organic solvent according to claim 1, wherein the alkane has 13 or more and 19 or less carbon atoms. The method for producing a microbial preparation for treating an organic solvent according to claim 1 or 2 , wherein the average particle size of the oil droplets is 1 µm or more and 100 µm or less. An organic solvent treatment microbial agent obtained by the production method of the organic solvent treatment for microbial formulation according to any one of claims 1 to 3 the method of treating organic solvents.