JP5303176B2 - New aquatic rhizosphere microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism having cleaning ability of aromatic organic contaminants, especially a microorganism useful for cleaning an organic contaminant in the environment by phytoremediation. <P>SOLUTION: The microorganism belonging to the genus Acinetobacter has degrading ability to a hydroxy group-containing monocyclic aromatic compound or a polycyclic aromatic compound to produce a hydroxy group on an aromatic ring by the cleavage reaction of an aromatic ring. An environmental contaminant such as phenol is cleaned by using the microorganism absolutely harmless to the nature. Particularly, the biologicals efficiently and extremely inexpensively treat in the natural environment, environmental contaminants such as phenol leaked to the environment, especially lakes, marshes or rivers without recovering contaminated water etc. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention has a resolution for a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is generated on an aromatic ring by the cleavage reaction of the aromatic ring, and attaches to duckweed roots. Acinetobacter genus microorganism having the ability, and contamination by monocyclic aromatic compound having a hydroxyl group or polycyclic aromatic compound in which a hydroxyl group is generated on the aromatic ring by an aromatic ring cleavage reaction The present invention relates to a method for purifying collected water or soil.

  Phenol and naphthalene are aromatic compounds important as raw materials for producing synthetic resins, dyes, explosives, plasticizers, and surfactants, as well as used as disinfectants, preservatives, insecticides, and dye intermediates. All of these are highly toxic to living organisms, and outflow from factories and the like may cause serious environmental pollution.

  Therefore, for example, the phenol emission standard is set at 5 mg / L by the Sewerage Law and Water Pollution Control Law, etc., and as a method of treating wastewater containing phenolic compounds generated in the manufacturing process of the chemical industry, the incineration method, distillation Recovery methods, activated sludge methods, etc. are being implemented.

  The above treatment method is a method developed mainly for the purpose of being carried out inside a facility such as a factory where wastewater is generated. Therefore, it is practically impossible to treat environmental pollution sources such as phenol that have leaked into the open environment such as soil, rivers, and lakes outside the facility by the above method.

  Since it is not easy to physically or chemically recover or decompose these sources that have leaked into the environment, biological purification technologies using plants and microorganisms are It is used as a removal method. In particular, a technique for purifying environmental pollutants utilizing the ability of plants to absorb and metabolize organic compounds is generally called phytoremediation. This technique is particularly excellent for removal of nitrogen and phosphorus that plants use as their own nutrient sources, and removal of heavy metals using absorption and accumulation in plants.

  The greatest merit of environmental purification technology using plants is the economic efficiency and environmental compatibility using solar energy, and plants such as water hyacinth and reed are widely used as passive systems. However, the recovery and removal reaction of environmental pollutants such as organic compounds by plants generally takes a long time, so it cannot be said that it is efficient.

  As a method for solving this problem, there has been proposed a method for improving the efficiency of phytoremediation by combining microorganisms that can coexist with plants and have an environmental purification effect. A typical example is an environmental purification technique called rhizomediation, which utilizes the symbiotic relationship between microorganisms and plants around the roots of plants.

  For example, there is an example in which crude oil around the root of the indigenous plant is removed by a combination of an indigenous plant and a crude oil-degrading microorganism in a crude oil-contaminated desert (Non-Patent Document 1).

In addition, regarding the rhizosphere microorganisms of aquatic plants, it has been reported that the resolution of aromatic compounds is improved in the presence of plants and rhizosphere microorganisms (Non-patent Document 2).
Samir Radwan et al., Nature, 1995, 376, 6538-302. Toyama et al. Biosci. Bioeng. 2006, Vol. 101, No. 4, pages 346-353

  An object of the present invention is to provide a microorganism having a purification ability of aromatic organic pollutants, particularly a microorganism that can be used for purification of a contaminated environment by phytoremediation.

  The present inventors isolated microorganisms capable of degrading phenol and naphthalene from duckweed rhizosphere, and biologically degrading and removing phenol and naphthalene, in particular, degrading and removing pollutants by phytoremediation or the pollutants. It was found that it can be used to purify the environment polluted by, and the following inventions have been completed.

(1) Acinetobacter microorganisms having a resolution for a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction.

(2) The microorganism of the genus Acinetobacter according to (1), which does not show resolution against benzene.

(3) The Acinetobacter genus microorganism according to (1) or (2), which is a rhizosphere microorganism of an aquatic plant.

(4) The Acinetobacter genus microorganism according to (3), wherein the aquatic plant is a Lemnaceae plant.

(5) The microorganism belonging to the genus Acinetobacter according to (4), wherein the duckweed plant is Lemna perpusilla or Spirodela polyrrhiza.

(6) Acinetobacter genus microorganisms deposited under the deposit number NITE P-523.

(7) A monocyclic aromatic compound having a hydroxyl group, including the Acinetobacter microorganism according to any one of (1) to (6), or a polycyclic group in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction Biologics for breaking down aromatic compounds.

(8) A monocyclic aromatic compound having a hydroxyl group comprising an Acinetobacter genus microorganism according to any one of (1) to (6) and a Lemaceae plant, or an aromatic ring by an aromatic ring cleavage reaction A biopharmaceutical for decomposing a polycyclic aromatic compound on which a hydroxyl group is formed.

(9) The biologic for degrading phenol and / or naphthalene according to (8), wherein the duckweed plant is duckweed (Lemna perpusilla) or duckweed (Spirodella polyrrhiza).

(10) Acinetobacter microorganism according to any one of (1) to (6) and a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic group in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction On the aromatic ring by the cleavage reaction of the monocyclic aromatic compound or aromatic ring having a hydroxyl group contained in the contaminated water or soil, comprising the step a) of contacting the contaminated water or soil with the compound A method of decomposing and removing a polycyclic aromatic compound in which a hydroxyl group is generated.

(11) Step a) is a monocyclic aromatic compound having a hydroxyl group of Acinetobacter and a hydroxyl group by administering the biologic according to any one of (7) to (9) to the contaminated water or soil. Alternatively, the method according to (10), which is a step of contacting water or soil contaminated with a polycyclic aromatic compound in which a hydroxyl group is generated on the aromatic ring by an aromatic ring cleavage reaction.

(12) Acynetobacter microorganism according to any one of (1) to (6) and a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic group in which a hydroxyl group is generated on the aromatic ring by a cleavage reaction of the aromatic ring A) contacting a contaminated water or soil with a compound, and a monocyclic aromatic compound having a hydroxyl group contained in the contaminated water or soil or a hydroxyl group on an aromatic ring by a cleavage reaction of the aromatic ring. A method for purifying the contaminated water or soil, comprising the step b) of decomposing and removing the polycyclic aromatic compound from which water is generated.

(13) Step a) comprises administering the biologic according to any one of claims 7 to 9 to the contaminated water or soil, whereby a monocyclic aromatic compound or fragrance having a hydroxyl group of Acinetobacter and a hydroxyl group The purification method according to (12), which is a step of contacting water or soil contaminated with a polycyclic aromatic compound in which a hydroxyl group is generated on the aromatic ring by an aromatic ring cleavage reaction.

  The present invention can purify the environment by decomposing and removing environmental pollutants such as phenol using microorganisms that are completely harmless to nature. In particular, the biologics of the present invention can purify the environment by decomposing and treating environmental pollutants such as phenol leaked in the environment, particularly in lakes and rivers, in the natural environment efficiently and extremely inexpensively.

  The present invention provides a microorganism belonging to the genus Acinetobacter having a resolution with respect to a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is generated on an aromatic ring by a cleavage reaction of the aromatic ring. To do.

  The Acinetobacter microorganism of the present invention has the microbiological properties shown in Table 1 below. A preferred example thereof is Acinetobacter sp. P23 strain deposited at the National Institute of Advanced Industrial Science and Technology as a deposit number NITE P-523.

  Acinetobacter microorganisms having the above microbiological characteristics, wherein the monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction Acinetobacter microorganisms having a resolution, in particular, Acinetobacter microorganisms that are rhizosphere microorganisms of duckweed family plants, any Acinetobacter microorganisms are also included in the present invention, and are deposited under the accession number NITE P-523. Although not limited to the Acinetobacter genus microorganism strain, the preferred Acinetobacter genus microorganism in the present invention is Acinetobacter sp. P23 strain deposited under accession number NITE P-523. Hereinafter, Acinetobacter sp. P23 strain is referred to as A-P23 strain.

  As a nutrient source of the medium used for culturing the Acinetobacter genus microorganism of the present invention, any carbon source, nitrogen source, and so on as long as it is necessary for normal microorganism growth and can be assimilated by the bacterium Inorganic salts may be used.

  As the carbon source, glucose, starch and other saccharides, natural products such as yeast extract, and the like can be used. However, a monocyclic aromatic compound having a hydroxyl group or a polyhydric group in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction. Cyclic aromatic compounds can also be used as a carbon source.

  As the nitrogen source, ammonium salts such as ammonium sulfate, nitrates such as sodium nitrate, various amino acids, yeast extract, meat extract, malt extract, peptone and other natural products can be used. Moreover, potassium salt, calcium salt, sodium salt, magnesium salt, iron salt, manganese salt, phosphate, etc. can be used as an inorganic component.

  Examples of a medium suitable for culturing the Acinetobacter microorganism of the present invention containing the above nitrogen source and carbon source include L medium, Hoagland medium (Hoagland et al., Soil Sci., 1940, 50, 463-463). 485), a BM (Basal salt Medium) medium, and the like, but are not limited thereto.

  The Acinetobacter microorganism of the present invention is cultured aerobically in a temperature range of 20 to 40 ° C, preferably a temperature range of 25 to 35 ° C, a pH range of 5 to 9, and preferably a pH range of 6 to 8. Can do.

  In the present invention, the monocyclic aromatic compound having a hydroxyl group is a compound in which a hydroxyl group is directly bonded to a ring of an aromatic compound and means a compound represented as a phenol. Representative examples include phenol, m-cresol, bisphenol F, salicylic acid, p-hydroxybenzoic acid and the like. Further, in the present invention, a polycyclic aromatic compound in which a hydroxyl group is generated on an aromatic ring by an aromatic ring cleavage reaction is the sharing of atoms in an aromatic ring constituting a part of the polycyclic aromatic compound. It means a polycyclic aromatic compound in which a hydroxyl group is newly formed on the aromatic ring by cutting the bond. For example, naphthalene, biphenyl, anthracene, pyrene. The microorganism of the present invention can remove such a compound from the medium or the environment by decomposing such a compound. Hereinafter, in the present invention, a covalent bond between atoms in an aromatic ring constituting a part of a monocyclic aromatic compound having a hydroxyl group and a polycyclic aromatic compound is broken to newly form an aromatic ring. The polycyclic aromatic compounds in which hydroxyl groups are formed are collectively represented as “contaminating compounds”, respectively.

  The Acinetobacter genus microorganism of the present invention is wastewater from factories, sales offices or households, polluted rivers and lakes, seawater and other waters, and it is expected or confirmed to contain “polluting compounds”. Contact with water (hereinafter referred to as “contaminated water”) or soil that is predicted or confirmed to contain “polluting compounds” (hereinafter referred to as “contaminated soil”) The “compound” can be decomposed, ie decomposed, to purify “contaminated water” and / or “contaminated soil”. The decomposition treatment of “contaminating compound” and the purification of “contaminated water” and / or “contaminated soil” are preferably performed by culturing the Acinetobacter microorganism of the present invention in “contaminated water” or “contaminated soil”, It is done by growing.

  The microorganism of the genus Acinetobacter of the present invention can be subjected to decomposition treatment of “contaminating compounds” by culturing it as it is in “contaminated water”. In addition, an immobilized microorganism is prepared by immobilizing the Acinetobacter genus microorganism of the present invention on an appropriate carrier by a known method, and using this, batch-wise or continuously “contaminated water” becomes an immobilized microorganism. The “contaminating compound” may be decomposed by contact. When the “contaminated water” is poor in nutrient sources suitable for culturing and growing the Acinetobacter microorganisms of the present invention, a suitable nutrient source may be appropriately added to the “contaminated water”.

  Further, the Acinetobacter genus microorganism of the present invention can be subjected to decomposition treatment of “contaminating compound” by spraying it on “contaminated soil” as it is. Alternatively, an immobilized microorganism obtained by immobilizing the Acinetobacter genus microorganism of the present invention on an appropriate carrier by a known method may be prepared, and this may be sprayed on “contaminated soil” to decompose the “contaminating compound”. . When the “contaminated soil” is poor in nutrient sources suitable for culturing and growing the Acinetobacter microorganisms of the present invention, a suitable nutrient source is sprayed on the “contaminated soil” or dispersed together with the Acinetobacter microorganisms of the present invention. May be.

  The Acinetobacter genus microorganism of the present invention, and particularly the A-P23 strain which is a preferred Acinetobacter genus microorganism, has the property of forming a rhizosphere by adhering to duckweed roots when cultured with a duckweed plant such as Duckweed (Lemna perpusilla). ing. In particular, the ability of the A-P23 strain to decompose “polluting compounds” increases due to the formation of the rhizosphere, and the rate of increase in the number of duckweed strains in an environment containing “polluting compounds” is also -It was confirmed that the rhizosphere by the P23 strain was increased. Therefore, the Acinetobacter microorganism of the present invention is a kind of PGPR (Plant Growth Promoting rhizobacteria) having a property of promoting growth by forming a rhizosphere and establishing a symbiotic relationship with a duckweed family plant. it is conceivable that.

  Thus, the Acinetobacter microorganism of the present invention can be used as a biologic for the purpose of decomposing “contaminating compounds” as it is or in combination with a duckweed plant. That is, the present invention is for decomposing or treating “contaminated water” and / or “contaminated water” by decomposing or treating a “contaminating compound” including the Acinetobacter spp. Microorganism or the Acinetobacter spp. It provides a biopharmaceutical for purifying "contaminated soil".

  The biologic of the present invention contains, in addition to the Acinetobacter spp., Acinetobacter spp. And duckweed plant of the present invention, a suitable excipient and / or a medium suitable for culturing Acinetobacter spp. May be. Further, the Acinetobacter genus microorganism and the duckweed plant may be separated and included in the preparation, or the Acinetobacter genus microorganism may form a rhizosphere on the root surface of the duckweed plant. When the duckweed family plant in which rhizosphere of Acinetobacter genus forms the rhizosphere is used as a biologic, the "polluting compounds" in the lakes and rivers are decomposed. Microorganisms that are no longer needed from the river can be easily recovered.

  The present invention includes “contaminated water” or “contaminated soil” comprising the step a) of bringing the Acinetobacter microorganism according to the present invention into contact with “contaminated water” or “contaminated soil”. A method of decomposing and removing the “compound” is provided. In addition, the step a) comprises administering the biologic according to the present invention to the “contaminated water” or “contaminated soil”, so that the Acinetobacter genus microorganism and the “contaminated water” or “contaminated soil” Provided is a method for decomposing and removing “contaminating compounds” contained in the “contaminated water” or “contaminated soil”, which is the step of contacting.

  Furthermore, the present invention relates to the step a) of bringing the Acinetobacter genus microorganism according to the present invention into contact with “contaminated water” or “contaminated soil”, and “contaminated water” contained in the “contaminated water” or “contaminated soil”. Provided is a method for purifying the “contaminated water” or “contaminated soil”, which comprises the step b) of decomposing and removing the “compound”. In addition, the step a) comprises administering the biologic according to the present invention to the “contaminated water” or “contaminated soil”, so that the Acinetobacter genus microorganism and the “contaminated water” or “contaminated soil” Provided is a purification method of “contaminated water” or “contaminated soil”, which is a step of contacting.

  Decomposition treatment of "polluted compounds" in "contaminated water" or purification of "contaminated water" using the Acinetobacter genus microorganism of the present invention, for example, by activated sludge method, bioreactor method, and "contaminated soil" May be carried out by spraying or injecting the microorganism of the genus Acinetobacter of the present invention onto the soil, and a method suitable for the purpose can be appropriately selected.

In the activated sludge method, the “contaminated compound” can be decomposed by bringing the Acinetobacter microorganism or the biologic of the present invention into contact with “contaminated water”. For example, in the case of decomposition treatment by the activated sludge method, the cells of the genus Acinetobacter of the present invention are added to an aeration tank at a concentration of 10 ³ cfu / mL or more, preferably 10 ⁶ cfu / mL or more. The decomposition treatment may be performed by culturing under various culture conditions. In the sprinkling filter bed method, the submerged filter bed method, and the fungus body fixing method, the “contaminated compound” is decomposed by bringing the carrier in which the Acinetobacter genus microorganism of the present invention is immobilized into contact with “contaminated water”. can do.

  According to the decomposition treatment method of the present invention, a contaminating compound (phenol) having a high concentration of 20 mg / L can be decomposed to 0.01 mg / L or less by culturing at 25 ° C. for 4 hours.

  In soil purification, “contaminated compound” in “contaminated soil” is obtained by bringing “contaminated soil” and the Acinetobacter microorganism of the present invention into contact with each other preferably under conditions that allow the Acinetobacter microorganism of the present invention to grow. ”Can be decomposed and“ contaminated soil ”can be purified.

In a preferred embodiment of the method according to the present invention, the Acinetobacter microorganism according to the present invention is cultured in "contaminated water" and grown to decompose and remove "contaminating compounds" contained in "contaminated water". Or it is the method of purifying "contaminated water". The culture conditions of the Acinetobacter microorganism according to the present invention are as described above.
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in more detail, this invention is not limited to the Example.

<Example 1>
1-1) Isolation of microorganisms To Hoagland medium (Table 2) 100 mL / 300 mL Erlenmeyer flask containing 3 types of hydrocarbons (phenol, naphthalene, Malaysian crude oil) added to a final concentration of 20 or 200 mg / L, Hokkaido Duckweed (Lemna perpusilla) collected from Yueniwa Lake attached to the university and washed with distilled water was added, and it was cultured in a temperature-controlled room at 25 ° C., illuminance: 4500-6000 lux, light / dark conditions: 16 hours, light and dark for 8 hours. .

Three weeks later, the duckweed was taken out and washed twice with 10 mL of distilled water, and then the leaf state and roots for 10 strains were taken out into an Eppendorf tube. To this, 1 mL of 5 mg / L sodium tripolyphosphate aqueous solution was added, and ultrasonic oscillation operation (operation 1: 5 seconds × 6 times, operation 2: 20 seconds × 5 times) was performed, and serial dilution was performed to 1 to 1 × 10 ⁵ . A solution was prepared. This was applied to 50 μL of BM (Table 3, Basal salt Medium) medium or LB solid medium, and plated at 25 ° C. At this time, 100 μL of hydrocarbon added at the time of enrichment culture as a single carbon source was added to the BM medium. The colonies appearing on the plate were each inoculated into L medium or BM medium to which each hydrocarbon was added.

  As bacteria having different colony forms on the solid medium, 109 strains were obtained from the phenol-containing medium, 79 strains were obtained from the naphthalene-containing medium, and 41 strains were obtained from the Malaysian crude oil-containing medium. Colonies identified as single strains are inoculated into 5 mL of liquid L medium containing 10 mg / L of the hydrocarbon used in each, and shake cultured at 25 ° C. and 150 rpm until it is confirmed that the turbidity has sufficiently increased. did. The cells were collected by centrifugation (1900 × g, 5 minutes, 20 ° C.) and stored in a deep freezer at −80 ° C. as a 15% glycerol solution. After preparation of the glycerol solution, it was applied to the L solid medium, and it was confirmed that a single species was stored.

1-2) Evaluation of hydrocarbon-degrading activity of isolated bacteria

1-2) -1. Evaluation of phenol degradation activity
After inoculating each microorganism in 5 mL of L liquid medium / test tube to which phenol with a final concentration of 2 mg / L was added, shaking culture (25 ° C., 150 rpm) was performed until it was confirmed that the turbidity was sufficiently increased, and centrifugation was performed. The cells were collected by (1900 × g, 5 minutes, 20 ° C.), washed with 1 mL of BM medium, and suspended in the same amount of BM medium. Add 1 mL of bacterial suspension to 4 mL of BM medium supplemented with phenol at a final concentration of 10 mg / L, perform shaking culture (25 ° C., 150 rpm), and centrifuge 1 mL of culture solution after 7 days in a polypropylene tube. (13000 × g, 15 minutes, 4 ° C.) and the supernatant was recovered, and the amount of phenol remaining in the supernatant was measured by HPLC (HP1100 Series, HEWLETT PACKARD). Table 4 shows the HPLC measurement conditions.

From the above measurements, bacteria that decomposed 80% or more of the phenol added to the medium were selected. 1 mL of the preculture solution (OD ₆₀₀ = 1.0) of the selected bacteria was added to 50 mL of Hoagland medium added with phenol at a final concentration of 20 mg / L in a 100 mL flask and shake culture (25 ° C., 150 rpm). 500 μL of the medium was collected after 1.5, 3, 4.5, 6, 9, 12, and 24 hours from the start of the culture, and the cells were removed by centrifugation (13000 rpm, 15 minutes, 4 ° C.). The supernatant was subjected to HPLC under the same conditions as described above, and the change over time in the amount of phenol remaining in the medium was measured.

  Through this experiment, 5 strains were obtained as bacteria capable of degrading 80% or more of the phenol added to the medium. All of them had such a high degrading activity as to degrade 20 mg / L of phenol in a few hours.

1-2) -2. Evaluation of naphthalene degradation activity After inoculating each microorganism in a BM liquid medium 10 mL / centrifuge tube to which naphthalene having a final concentration of 30 mg / L was added, shaking culture (25 ° C., 150 rpm, 7 days) was performed, and then 10 mL of the medium was added to the medium. An extraction solvent (hexane: acetone = 1: 1) was added and stirred, followed by centrifugation (2810 × g, 30 minutes, 4 ° C.). 1 mL of the hexane layer was transferred to a glass vial tube for GC, and the residual amount of naphthalene was measured using gas chromatography (hereinafter, GC-FID) under the conditions shown in Table 5. The residual rate and the decomposition rate were calculated from the chromatogram peak area values of naphthalene and the internal standard substance biphenyl.

  Bacteria judged to have high naphthalene resolution in the above experiment were cultured again under the same conditions as in the above experiment, and the change over time in the amount of naphthalene remaining in the medium was measured.

  Through this experiment, 6 strains were obtained as bacteria judged to have high naphthalene resolution.

1-2) -3. Standard gas oil decomposition activity evaluation After inoculating each microorganism in a 10 mL BM medium / 50 mL centrifuge tube to which standard gas oil 5000 mg / L was added, after shaking culture (25 ° C., 150 rpm, 7 days), the above In the same manner as in 2), the residual crude oil or residual standard gas oil was extracted from the culture medium and measured using GC-FID, and the changes over time such as the residual crude oil quantity in the culture medium were measured.

  Through this experiment, 5 strains were obtained as bacteria judged to have high standard gas oil resolution.

1-3) Identification of isolates About the 5 strains having phenol resolution, 6 strains having naphthalene resolution, and 5 strains having standard gas oil resolution obtained in 1-2), about 600 bases of the latter half of 16S rRNA are analyzed. The genus identification of each strain was performed.

  The colonies of each strain grown on the L solid medium were transferred to a polypropylene tube to which 10 μL of distilled water was added and suspended, and then heated at 99 ° C. for 15 minutes for lysis. Using this solution as a template, in the reaction solution composition shown in Table 5, after 1 minute at 94 ° C., a PCR reaction of [94 ° C., 30 seconds-52 ° C., 2 minutes-74 ° C., 30 seconds] × 30 cycles was performed. It was.

  The amplified DNA recovered by agarose gel electrophoresis was purified using Qiaquick Gel Extraction Kit (QIAGEN). Using the generated DNA as a template and using a BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) at a reaction station composition as shown in Table 6, after 10 minutes at 96 ° C., [50 C., 5 minutes to 60.degree. C., 4 minutes]. A sequence PCR reaction of 25 cycles was performed.

  The sequence PCR reaction product collected and purified by the ethanol precipitation method was dissolved in 12 μL of Hi-Di-Formamide (Applied Biosystems), and ABI3100 (ABI PRISM (registered trademark) 3100-Genetic Analyzer, Applied Biosystems) was used. The genes having high homology were searched using Blast Search (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Gene analysis was performed using GENETYX-WIN Ver. 5.1 (Software Development Co., Ltd.) and MEGA (Molecular Evolutionary Genetics Analysis) were used. The results of this experiment are shown in Table 7.

<Example 2> Measurement of biofilm-forming ability of isolated bacteria Each strain shown in Table 7 was inoculated into 5 mL of liquid L medium, and precultured until the turbidity OD ₆₀₀ = 0.3. Phenol-degrading five strains are L medium, Hoagland medium, and BM medium containing phenol at a final concentration of 10 mg / L. Naphthalene-degrading six strains are L medium, Hoagland medium, and BM medium each containing naphthalene at a final concentration of 30 mg / L. 300 μL was prepared in a centrifuge tube, the preculture solution was inoculated, and statically cultured in a greenhouse at 25 ° C. After 24 hours and 48 hours from the start of the culture, the culture solution in the centrifuge tube was removed and washed once with 350 μL distilled water, and then 400 μL of 0.1% crystal violet solution was added to the tube and stained for 20 minutes. The staining solution was removed, washed once with 450 μL of distilled water, and then naturally dried. Further, 500 μL of 95% ethanol was added, and extraction was performed for 60 minutes. The extracted solution was appropriately diluted with a 95% ethanol solution, and the measured absorbance at 590 nm was defined as biofilm activity.

  As a result, as shown in FIG. 1, Rhodococcus sp. P22 strain (hereinafter referred to as R-P22 strain) and Acinetobacter sp. P23 strain (referred to as A-P23 strain) showed a high biofilm formation amount when cultured for 24 hours in L medium. In addition, the amount of biofilm formed after 48 hours decreased (FIG. 2).

  Moreover, although film formation was confirmed also about the strain | stump | stock which has naphthalene resolution | decomposability, there was little film formation amount compared with the strain | stump | stock which has phenol resolution | decomposability. FIG. 3 shows the film-forming ability (after 24-hour culture) of a strain having naphthalene resolution.

Example 3 Evaluation of Adherence of Isolated Bacteria to Duckweed 1) Measurement of Duckweed Attached Bacteria Count After gently stirring for 10 minutes in an aqueous solution containing 5% NaClO and 0.05% Triton X100, the solution was discarded, and further 1 minute stirring was performed using sterilized distilled water to sterilize duckweed. .

From the strains shown in Table 7, Pseudomonas. sp. P2 strain (hereinafter referred to as P-P2 strain), Rhodococcus sp. The P11c strain (hereinafter referred to as R-P11c strain) and the A-P23 strain are selected, colonies formed by growing on the L solid medium are inoculated into 5 mL of liquid L medium, and shaken culture (25 ° C. , 170 rpm, 24 hours) to prepare a preculture solution. Prepare a sterile 50 mL Hoagland medium, BM medium, and L medium in a 100 mL flask, add sterilized duckweed, and inoculate 1% of the preculture solution adjusted to OD ₆₀₀ = 0.3. The stationary culture was performed at ° C for 48 hours.

After culturing, duckweed was collected and placed in a tube to which 500 μL of 5 mg / L sodium tripolyphosphate solution was added, and then the solution was discarded, and 500 μL of the same solution was newly added again to wash the surface of duckweed. Furthermore, 500 μL of the same solution was added, and ultrasonic treatment and stirring treatment were performed to release bacteria adhering to duckweed into the solution. This solution was diluted with L medium, 50 μL was applied to L solid medium, and the number of colonies formed was counted. Moreover, the suspended cell turbidity (OD ₆₀₀ ) of the culture solution after the culture was measured.

  As a result, 48 hours after the start of the culture, the suspended cell turbidity of the culture solution decreased to about 1% by culturing with Duckweed in the P-P2 strain and the A-P23 strain (for the A-P23 strain, FIG. 4). ).

On the other hand, in the R-P11c strain, no change in suspended cell turbidity was confirmed. In addition, the number of bacteria attached to Duckweed was confirmed to be 1 × 10 ⁹ to 1 × 10 ¹⁰ CFUs of attached bacteria per duckweed strain in any of the three types of microorganisms. In addition, the number of bacteria adhering to duckweed increased approximately twice after 48 hours compared to 24 hours later.

  Further, after wiping off the water of duckweed collected after the cultivation, it was placed in 0.5 mL of a fluorescent dye mixture containing 250 μL of Propidium ioide (Pi, green) or Syto9 (S9, red) and fluorescently stained for 5 minutes. . By fluorescent staining using Pi and S9, the plant surface is stained orange by incorporating Pi and S9 together, viable bacteria are stained green, and dead bacteria are stained red. After 5 minutes, only Duckweed was taken out and observed (objective lens × 20) using a fluorescence microscope (BZ9000 KEYENCE). The result (photograph) is shown in FIG.

  The surface of the sterilized duckweed is stained orange (Panel E in FIG. 5), but in the P-P2 strain (Panel A and C in FIG. 5) and the A-P23 strain (Panel B and D in FIG. 5), Viable bacteria stained in green were observed on the surface of duckweed. In particular, in the P-P2 strain, it was confirmed that a large number of viable bacteria were clearly attached to Duckweed after 48 hours of culture compared to 24 hours after culture.

<Example 4> Characteristic analysis of A-P23 strain 1) Physiological test for bacterial species identification A-P23 strain was inoculated on a solid agar medium containing BactoTryptic Soy Broth (TSB) and cultured at 25 ° C for 24 hours to form. Several dozen colonies were collected and suspended in a sterile 0.85% NaCl aqueous solution. This solution was diluted to McFarland turbidity 0.5 (OD550 = 0.125), and 55 μL was injected into a kit plate for an ID 32E API system (Japan BIOMERIEUX). Further, two drops of mineral oil were dropped into ODC, ADH, LDC, URE, LARL, GAT and 5KG cups, and allowed to stand in an incubator at 37 ° C. for 24 hours. After 24 hours, James reagent was added to the IND cup. The grades were read visually according to the judgment table.

  As a result, the P-AP23 strain was falsely positive for indole production, L-Aspartic-allylamidase, and acidification of glucose. Moreover, as a result of API system database search based on this phenotype, it was speculated that the A-P23 strain is a microorganism of a close species (99.9% homology) with Acinetobacter baumannii. However, Acinetobacter baumannii has no report on phenol degrading activity and adhesion activity to plant roots, and the A-P23 strain was judged to be a new species of Acinetobacter genus bacteria.

2) Specificity of degrading contaminants A-P23 strain colonies on solid L agar medium are inoculated into 5 mL of liquid L medium and shake cultured (25 ° C., 150 rpm, 24 hours) to prepare a preculture solution. did. Prepare 5 mL of BM liquid medium (only naphthalene, separately add 50 mg / L BM liquid medium) containing 500 mg / L of each hydrocarbon (phenol, benzene, toluene, naphthalene, ethanol, glucose) 1% inoculated and cultured with shaking (25 ° C., 150 rpm), and the presence or absence of increased turbidity was confirmed visually. Table 8 shows the presence or absence of turbidity changes on day 1 and day 5 from the start of culture.

  As shown in Table 8, the A-P23 strain showed an increase in turbidity in a medium containing phenol and naphthalene as a carbon source, but an increase in turbidity was observed in a medium containing glucose, toluene or benzene as a carbon source. Was not.

3) Phenolytic activity by co-culture with duckweed A-P23 strain colonies on solid L agar medium were inoculated into 5 mL liquid L medium, shake cultured (25 ° C., 150 rpm, 24 hours), precultured A liquid was prepared. Bacteria were collected from this precultured solution by centrifugation (1900 × g, 5 minutes, 20 ° C.), washed with 1 mL of Hoagland medium, and then a suspension / Hoagland medium with OD600 = 15 was prepared. 50 mL Hoagland medium containing 20 mg / L phenol was prepared in a 100 mL Erlenmeyer flask, and 30 strains of sterilized duckweed prepared in the same manner as in 1) of Example 3 were added. 1 mL of the suspension was added thereto, and the cells were statically cultured for 48 hours in an artificial weather apparatus (25 ° C., 8500 lux, 16 hours: light to 8 hours dark). After 48 hours, only 20 duckweed strains were transferred to a fresh 50 mL Hoagland medium supplemented with 40 mg / L phenol.

  The time when the duckweed was transferred to a new medium was set to 0 hour, 250 μL of the culture solution was collected with gentle stirring every 4 hours or 8 hours, and collected by centrifugation (13000 × g, 10 minutes, 20 ° C.). The amount of phenol remaining in the cleansing was determined according to Example 1-2) -1. In the same manner as above, measurement was performed using HPLC. When 40 hours had elapsed, an equal amount of fresh Hoagland medium was added to the collected medium, and phenol was newly added to a final concentration of 40 mg / L. This time was newly set to 0 hour, and the same sampling, measurement of the residual amount of phenol, medium and phenol supplementation were repeated twice. As controls, duckweed that was not sterilized and duckweed that were sterilized were prepared. FIG. 6 shows the change in the amount of residual phenol by this experiment.

  When only sterilized duckweed was cultured, the amount of residual phenol hardly changed. On the other hand, comparing the presence or absence of inoculation of the A-P23 strain, in the first cycle, the system inoculated with the A-P23 strain (◯ in FIG. 6) and the duckweed that has not been sterilized, that is, the duckweed containing resident bacteria ( In Fig. 6, ●, there was no difference in the removal rate, but there was a difference in the phenol removal rate in the second cycle, and in the third cycle, there was almost no decrease in phenol in duckweed that was not sterilized. I was not able to admit.

4) Effect on the growth of blue duckweed Prepare a 50 ml Hoagland medium containing 20 mg · L of phenol in a 100 ml Erlenmeyer flask, add 30 duckweed strains at a time, and add the suspension of OD600 = 15 prepared in 3) above / Hoagland medium was added, and static culture was performed in an artificial weather apparatus (25 ° C., 8500 lux, 16 hours: light to 8 hours dark). After a certain time, the number of duckweeds and the root length were measured.

  The growth of Duckweed after 160 hours in the medium inoculated with the A-P23 strain reached 1.7 times that of Duckweed in the medium that did not ingest the A-P23 strain (FIG. 7).

  Further, FIG. 8 shows the state of duckweed root elongation after 160 hours of culture. The growth of the roots of the duckweed not sterilized, the strain inoculated with the A-P23 strain, and the strain not inoculated with the A-P23 strain changed to the length of the roots after about 100 hours from the start of the culture. The average length of each of the above three strains after cultivation for 160 hours is 4.2 cm, 3.75 cm, and 6.5 cm. Was confirmed.

It is a graph which shows the measurement result of the biofilm formation ability (24 hours culture | cultivation) of the microorganisms strain | stump | stock which isolate | separated from duckweed and has a phenol resolution. In the figure, P2 to P24a represent microbial strains shown in Table 7, respectively. It is a graph which shows the measurement result of the biofilm formation ability (culture 48 hours) in the culture medium containing the phenol of the bacteria isolate | separated from duckweed. In the figure, P2 to P24a represent microbial strains shown in Table 7, respectively. It is a graph which shows the measurement result of the biofilm formation ability (24 hours culture | cultivation) of the microorganisms strain which has the naphthalene resolution isolate | separated from duckweed. In the figure, N1-b to N12-b represent microbial strains shown in Table 7, respectively. It is a figure (photograph) which shows the phenomenon of floating cell turbidity in a culture solution when A-P23 strain | stump | stock is cultured with a duckweed. It is a fluorescence-microscope observation result (photograph) which shows the A-P23 stock | strain adhering to the rhizosphere of a duckweed when the A-P23 strain | stump | stock was cultured with the duckweed using fluorescent dye. It is a graph which shows a time-dependent change of a phenol degradation activity when A-P23 strain | stump | stock is co-cultured with Duckweed. In the figure, ▲ indicates a sterilized duckweed, ● indicates a duckweed having resident bacteria, and ◯ indicates a duckweed inoculated with the A-P23 strain. It is a figure (photograph) which shows the influence of A-P23 stock | strain with respect to the growth of the duckweed in the culture medium containing a phenol. In the figure, the left shows Duckweed inoculated with the A-P23 strain, and the right shows only sterilized Duckweed. It is a figure (photograph) which shows the influence of A-P23 stock | strain on the growth (root extension) of the duckweed in the culture medium containing a phenol. In the figure, a shows a duckweed having resident bacteria, b shows a duckweed inoculated with the A-P23 strain, and c shows only a duckweed sterilized.

Claims (8)

Acinetobacter sp strain P23, deposited under accession number NITE P-523. Decomposing a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is produced on an aromatic ring by a cleavage reaction of the aromatic ring, including the Acinetobacter sp strain P23 according to claim 1 Biologic for processing. A monocyclic aromatic compound having a hydroxyl group, which is composed of the Acinetobacter sp P23 strain according to claim 1 and a Lemaceae plant, or a hydroxyl group is produced on the aromatic ring by a cleavage reaction of the aromatic ring. A biologic for decomposing polycyclic aromatic compounds. 4. The biologic of claim 3 , wherein the duckweed plant is Lemna perpusilla or Spirodela polyrrhiza. The Acinetobacter sp (P23) strain according to claim 1 was contaminated by a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is formed on the aromatic ring by a cleavage reaction of the aromatic ring. A polycyclic ring having a hydroxyl group on the aromatic ring by a cleavage reaction of the monocyclic aromatic compound or aromatic ring having a hydroxyl group contained in the contaminated water or soil, comprising the step a) of contacting with water or soil A method for decomposing and removing aromatic compounds. Step a) comprises administering the biologic according to any one of claims 2 to 4 to the contaminated water or soil, so that Acinetobacter sp P23 strain and monocyclic aromatic compound having a hydroxyl group Alternatively, the method according to claim 5 , which is a step of contacting water or soil contaminated with a polycyclic aromatic compound in which a hydroxyl group is generated on the aromatic ring by an aromatic ring cleavage reaction. The Acinetobacter sp (P23) strain according to claim 1 was contaminated by a monocyclic aromatic compound having a hydroxyl group or a polycyclic aromatic compound in which a hydroxyl group is formed on the aromatic ring by a cleavage reaction of the aromatic ring. A) contacting with water or soil a), and a monocyclic aromatic compound having a hydroxyl group contained in the contaminated water or soil, or a polycyclic ring having a hydroxyl group on the aromatic ring by the cleavage reaction of the aromatic ring A method for purifying contaminated water or soil, comprising the step b) of decomposing and removing aromatic compounds. Step a) comprises administering the biologic according to any one of claims 2 to 4 to the contaminated water or soil, so that Acinetobacter sp P23 strain and monocyclic aromatic compound having a hydroxyl group Alternatively, the purification method according to claim 7 , which is a step of contacting water or soil contaminated with a polycyclic aromatic compound in which a hydroxyl group is generated on the aromatic ring by an aromatic ring cleavage reaction.