JP2003079364A - New polycyclic aromatic hydrocarbon-decomposing bacterium and new polycyclic aromatic hydrocarbon- decomposing agent containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire a microorganism effective for decomposition treatment of benzo[a]pyrene by carrying out screening of a bacterium capable of efficiently decomposing benzo[a]pyrene from a sample obtained from a crude oil-gushing ground and to provide a treating agent for toxic polycyclic aromatic hydrocarbon which was untreatable by an existing microorganism formulation or microorganism material. SOLUTION: This bacterium is a bacillus obtained by culturing a bacterium which inhabits in soil collected from the crude oil-gushing ground by using benzo[a]pyrene represented by chemical formula (1) as a carbon source and having properties which is gram-positive and positive against oxidase activity and positive against catalase activit and has a polycyclic aromatic hydrocarbon- decomposing property. This polycyclic aromatic hydrocarbon-decomposing agent comprises the bacterium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism, particularly a bacillus having a polycyclic aromatic hydrocarbon decomposing property, and a polycyclic aromatic hydrocarbon decomposing agent containing the same.

[0002]

2. Description of the Related Art Currently, for petroleum that has leaked to the environment,
Prevention of diffusion by oil fences and collection by collection machines are taken as temporary measures, but it is known that effectiveness is lost depending on waves and contaminated area, and effective removal of petroleum that cannot be collected by oil fences , A decomposing means is needed. Therefore, even if a chemical method such as addition of a dispersant or a neutralizing agent is used, the chemical itself may have a bad influence on the natural world. in addition,
It is necessary to treat contaminated soil and secure a disposal site. Therefore, these existing methods have limitations as a practical environmental purification method. Therefore, an environmental restoration technique using the self-cleaning action of microorganisms such as bacteria and mold is currently drawing attention. The first method is a method of improving by degrading microorganisms living in a polluted environment by adding a nutrient source or the like, and the second method is a method of improving high resolution for a specific substrate. This is a method of improving the microorganisms by administering them to a polluted environment. The first method is a method based on the bioactivity of microorganisms, and is not effective in a low temperature environment where the bioactivity is remarkably reduced, or when it contains a large amount of hardly decomposable components such as heavy oil of distillation residual oil. In particular, high molecular weight compounds such as polycyclic aromatic hydrocarbons, resins, and asphaltene in petroleum are difficult to decompose, and at present, it is difficult to treat them when they flow into the environment.

[0003]

An object of the present invention is to screen microorganisms capable of decomposing benzo [a] pyrene efficiently by using a sample obtained from a crude oil source, and to analyze benzo [a] pyrene It is intended to obtain microorganisms effective for decomposition treatment and to provide a treatment agent for toxic polycyclic aromatic hydrocarbons which cannot be dealt with by existing microbial preparations and microbial materials.

[0004]

[Means for Solving the Problems] The first aspect of the present invention is to obtain a bacterial cell inhabiting a soil sampled from a crude oil source from a chemical formula (1).

[Chemical 2] And a bacillus having positive gramase, positive oxidase activity, and positive catalase activity obtained by culturing using benzo [a] pyrene as a carbon source. A second aspect of the present invention relates to a bacillus that is Gram-positive, oxidase-active, and catalase-positive and has the following biochemical properties. Biochemical test Gram stain + Oxidase + Oxidase + Catalase + Glycerol + Erythritol-D-arabinose-L-arabinose (L-arabinose) Ribose) -D-xylose (D-Xylose) -L-xylose (L-Xylose) -Adonitiol (Adonitiol) -β-methyl-D-xylose (β-Methyl-D-Xylose) -galactose (Galactose) -Glucose (Glucose). Glucose) -Fructose + Mannose-Sorbose- Rhamnose-Dulcitol-Mannitol + sorbitol-Sorbitol-α-methyl-D-mannose (α-Methyl-D-Mannoside) -N-acetyl-glucosamine (N-Acetyl-Glucyl-Glucyl). Amygdalin-Arubutin + Escline + Salicin- Cellobiose- Maltose- Maltose-Lactose (Melaciose) + Mellibiose (Melubose) -Inuline -Melizetose-Raffinose-Starch-Glycogen-Xylitol-Gentiobiose-D-Turanose (D-Turanolyse-D-Duranalyse) -D-Tagatose + D-Fucose (D-Fucose) + L-Fucose (L-Fucose) -D-Arabitol (D-Arabitol) -L-Arabitol (L-Arabitol) + Gluconate (Gluconate) + 2 -Keto-gluconate (2-Keto-Gluconate) + 5-keto-gluconate (5-Keto-Gluconate) In the above test, "-" means negative, "+" indicates positive. The third of the present invention is 16 shown in SEQ ID NO: 1.
The bacillus according to claim 2, which has a base sequence of SrDNA. The fourth aspect of the present invention is the patent biological depository center deposit number FERM P-18, National Institute of Advanced Industrial Science and Technology.
458 relates to the bacillus according to claim 3. A fifth aspect of the present invention relates to a polycyclic aromatic hydrocarbon decomposing agent containing the bacillus according to any one of claims 1 to 4. A sixth aspect of the present invention relates to the polycyclic aromatic hydrocarbon decomposing agent according to claim 5, wherein the polycyclic aromatic hydrocarbon according to claim 5 is benzo [a] pyrene.

As the microorganism of the present invention, benzo [a] pyrene was used as a carbon source to isolate and select a microorganism having an affinity for benzo [a] pyrene from a soil sample collected from a crude oil source. The composition of the separation / medium used for the separation and culture of the polycyclic aromatic hydrocarbon degrading microorganism is potassium dihydrogen phosphate 0.2 g dipotassium hydrogen phosphate 0.8 g ammonium nitrate 0.2 g magnesium sulfate heptahydrate. 0.02 g Yeast extract (Difco) 0.1 g Bactopeptone (Difco) 0.1 g Mineral solution 2 ml Distilled water 1.0 l Agar (only during plate preparation) 15 g pH (adjusted with dilute hydrochloric acid) 7.0 The composition of the above mineral solution is nitrilotriacetic acid 3.0 g magnesium sulfate / heptahydrate 6.0 g manganese sulfate / monohydrate 1.0 g sodium chloride 2.0 g iron (II) sulfate / heptahydrate 0.2 g cobalt chloride -Hexahydrate 0.2 g Calcium chloride 0.2 g Zinc sulfate-heptahydrate 0.02 g Copper sulfate-pentahydrate 0.02 g Potassium aluminum sulfate Um 0.02g Boric acid 0.02g Sodium molybdate 0.02g Distilled water 1.0l Further, powdered benzo is used as a carbon source for growing bacterial cells.
A solution of [a] pyrene dissolved in dimethyl sulfoxide was used. Thus, the shape of the isolated strain of the present invention was observed using a transmission electron microscope. It was found that the size of the bacillus was about 0.1 μm to 1.2 μm and the width was about 0.5 μm.

Example 1 Gram stain, oxidase test and catalase test were performed on the strain of the present invention. Gram staining was carried out by preparing a bacterial suspension, (1) smearing a small amount of the bacterial suspension on a preparation, allowing it to air dry, and then fixing the flame with a pilot fire of a gas burner. (2) In the fixed portion of the bacterial cells on the preparation,
A small amount of crystal violet solution was dropped, left for 1 minute to dye, and then washed with distilled water. (3) A small amount of Lugol's solution was dropped, left for 1 minute, treated with iodine, and then washed with distilled water. (4) The cells stained blue were decolorized with 70% ethanol, left for 30 seconds, and washed with distilled water. (5) After dyeing with a safranine solution, it was left to stand for 10 to 30 seconds, washed with distilled water, and then naturally dried. (6) After drying, the staining result was confirmed under a phase contrast optical microscope. At this time, if the bacterial cells are stained purple,
Gram-positive bacteria, and if stained red, it was considered to be Gram-negative bacteria. In the oxidase test, a filter paper impregnated with a 1% tetramethyl-p-phenylenediamine HCl aqueous solution was used as an oxidase test paper. The collected fresh cells were smeared on the test paper with a platinum loop, and if a dark blue color was exhibited within 10 seconds, the oxidase activity was determined to be positive. For the catalase test, drop a few drops of 3% hydrogen peroxide on a glass slide,
After applying the collected fresh cells with a platinum loop, if bubbles were observed, it was determined to be positive for catalase activity. As a result, the bacterial cells of the present invention were Gram-positive when Gram-stained, and both oxidase activity and catalase activity were positive.

Example 2 An API50 identification kit (manufactured by BIOMERIEUX) was used to identify the microbial species of the strain of the present invention. After washing the collected fresh cells, API50CHB Mediu
resuspended in m. This bacterial suspension was inoculated into a specific 50-item cup on the plate of the identification kit. 2 after inoculation
After culturing at 5 ° C for 2 days and observing the color change of the plate of the kit, it was judged negative or positive, and the microbial species was identified. The identification results are Gram stain + Oxidase + Oxidase + Catalase + Glycerol + Erythritol-D-arabinose D-Arabinose-L-arabinose-L-arabine-L-arabine-L-arabine-L-arabine-L-arabine-L-arabine-L-arabine-L-arabine-L-arabine- (Ribose) -D-xylose (D-Xylose) -L-xylose (L-Xyrose) -Adonitiol (Adonitiol) -β-methyl-D-xylose (β-Methyl-D-Xyrose) -galactose (Galactose) -Glucose (Glucose) -Fructose (Fructose) + Mannose (Mannose) -Sorbose (Sorbose) Rhamnose-Dulcitol-Mannitol + sorbitol-Sorbitol-α-methyl-D-mannose (α-Methyl-D-Mannoside) -N-acetyl-glucosamine (N-Acetyl-Glucyl-Glucyl). Amygdalin-Arubutin + Escline + Salicin- Cellobiose- Maltose- Maltose-Lactose (Melaciose) + Mellibiose (Melubose) -Inuline -Melizetose-Raffinose-Starch-Glycogen-Xylitol-Gentiobiose-D-Turanose (D-Turanolyse-D-Duranalyse) -D-Tagatose + D-Fucose (D-Fucose) + L-Fucose (L-Fucose) -D-Arabitol (D-Arabitol) -L-Arabitol (L-Arabitol) + Gluconate (Gluconate) + 2 -Keto-gluconate (2-Keto-Gluconate) + 5-keto-gluconate (5-Keto-Gluconate) -In the above test, "-" indicates negative and "+" indicates positive.

Example 3 The strain of the present invention was cultured at 25 ° C. for 3 days in the following growth medium. Potassium dihydrogen phosphate 0.2 g Dipotassium hydrogen phosphate 0.8 g Ammonium nitrate 0.2 g Magnesium sulfate heptahydrate 0.02 g Yeast extract (Difco) 1.2 g Bactopeptone (Difco) 1.2 g Mineral solution 2 ml distilled water 1.0 l pH (adjusted with dilute hydrochloric acid) 7.0, and the composition of the above mineral solution is nitrilotriacetic acid 3.0 g magnesium sulfate / heptahydrate 6.0 g manganese sulfate / monohydrate 1.0 g Sodium chloride 2.0g Iron (II) sulfate / heptahydrate 0.2g Cobalt chloride / hexahydrate 0.2g Calcium chloride 0.2g Zinc sulfate / heptahydrate 0.02g Copper sulfate / pentahydrate 0.02g Sulfuric acid It is potassium aluminum 0.02 g boric acid 0.02 g sodium molybdate 0.02 g distilled water 1.0 l.

After culturing for 3 days, genome extraction was performed and a program incubator (Gene Amp P) was used.
CR System 9600, PERKINE LMER
Universal primer (1F New, 5R)
The nucleotide sequence encoding 16S rDNA was PCR (Polymerase ChainRe) using
amplification). The obtained PCR product is subjected to ultrafiltration (Centricon 100, MILLIPORE
It was used as a template DNA by being purified by the company. The template DNA was amplified again by the PCR method using the cycle sequence method. The reaction solution composition of the cycle sequence method is

[Table 1] And the reaction conditions are

[Table 2] Is. The sample obtained by the cycle sequence method was purified by ethanol precipitation. The base sequence of the purified product was analyzed by a capillary electrophoresis type DNA sequencer (ABI, manufactured by Applied Biosystems).
PRISM ^™ 310 Genetic Analyze
It was analyzed by r) and the base sequence was determined. As a result of analysis by a DNA sequencer, 16S of the strain of the present invention
The gene encoding rDNA is 16 shown in SEQ ID NO: 1.
It was confirmed to consist of 87 base pairs.

From these experiments, it was confirmed that the strain of the present invention is a gram-positive bacillus by the identification test for knowing the morphological properties and biochemical characteristics. In addition, a microbial species identification test using API50 showed that it was closely related to the genus Bacillus, and analysis of the gene sequence encoded by 16S rDNA revealed that Paenibacillusl
It was confirmed that the homology with the entimor was the highest (92.953%). However, in the phylogenetic tree, the strain of the present invention has the highest homology, Paenibacillu.
It was confirmed to be a new type of bacterial cell because the genetic distance from s lentimor was large.

The strain of the present invention is deposited under the deposit number F at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology.
Deposited as ERM P-18458.

Example 4 Regarding the decomposition of polycyclic aromatic hydrocarbons by the strain of the present invention, experiments were conducted with (1) benzo [a] pyrene, (2) pyrene and (3) anthracene. (1) Regarding benzo [a] pyrene In this experiment, a standard product having a known concentration was used as benzo [a] pyrene in order to measure an accurate decomposition amount. As a standard product, 1 mg of benzo [a] pyrene was obtained, diluted with dimethyl sulfoxide or acetonitrile to 10 ml, and further diluted with a medium or the like to be used as a carbon source having a known concentration and a standard solution. For culturing the strain of the present invention, 10 ml of a growth medium was dispensed into a 50 ml Erlenmeyer flask, and the strain of the present invention was inoculated at 1.0 × 10 ⁶ cells / ml. The concentration of benzo [a] pyrene in the medium was accurately set to 0.2 ppm by adding 20 μl of a solution prepared by dissolving a standard product in dimethyl sulfoxide as a carbon source.
Shaking culture was carried out at 5 ° C. for 7 days at 100 rpm. In addition, as a control example, the same medium that had not been inoculated was prepared, and shake culture was performed under the same conditions. To investigate the ability of the strain of the present invention to resolve benzo [a] pyrene, high performance liquid chromatography equipped with a fluorescence detector (SIL-
10A, manufactured by SHIMADZU).

The amount of degradation was measured by comparing the area values of benzo [a] pyrene of the control example in which the inoculation was not performed and the experimental example in which the inoculation was performed, which were calculated by high performance liquid chromatography. A graph of the residual ratio is shown in FIG. 1, where the area value of benzo [a] pyrene of the control example is 100%. In addition, the strain of the present invention was added to a growth medium containing benzo [a] pyrene at a concentration of 0.2 ppm at a specified temperature (4 ° C, 10 ° C,
The cells were cultured at 15 ° C., 20 ° C. and 35 ° C.) for 7 days, and the residual ratio of benzo [a] pyrene was obtained by the same method as at 25 ° C. The residual rate of benzo [a] pyrene at the specified temperature is shown in FIG. Furthermore, at 35 ° C., where the bacterial cell growth amount of the strain of the present invention was highest, the initial bacterial cell concentration to be inoculated (1.0 × 10 ⁶
cells / ml, 1.0 × 10 ⁷ cells / ml,
1.0 × 10 ⁸ cells / ml) and changed to 0.2p
Decomposition amount of benzo [a] pyrene in pm for 1 day, 3 days, 5 days,
I surveyed every 7 days. At this time, the area value in the control example in which the inoculation was not performed on day 0 was set as the residual rate of 100% and used as a reference. The result is shown in FIG.

(2) With respect to pyrene, a solution prepared by diluting a standard product of pyrene to 0.2 ppm with acetonitrile was analyzed by high performance liquid chromatography and measured in the same manner as in the case of benzo [a] pyrene. The initial pyrene concentration of the control example without inoculation and the experimental example with inoculation was 0.2 ppm.
Culturing was carried out at 25 ° C. for 7 days in the medium set as above. For the measurement of the amount of decomposition, the area value of pyrene without inoculation calculated by high performance liquid chromatography analysis was set as a residual rate of 100% and used as a standard. The result is shown in FIG.

(3) Regarding anthracene, a solution of an anthracene standard product diluted to 0.2 ppm with acetonitrile was analyzed by high performance liquid chromatography and measured in the same manner as in the case of benzo [a] pyrene. In the medium in which the initial concentration of anthracene in the control example without inoculation and the experimental example with inoculation was set to 0.2 ppm,
Culture was carried out for 7 days. For the measurement of the amount of decomposition, the area value of anthracene without inoculation calculated by high performance liquid chromatography analysis was used as a standard with the residual rate of 100%. The result is shown in FIG.

[0016]

[Sequence list] <110> Eichii Tamiya             GATE Corporation Limited <120> New bacteria for biodegradati on of polycyclic aromatic hydrocarbo n and biogradation agent of polycy CLICK aromatic aromatic carbon using the same. <130> HP003029-5 <160> 1 <210> 1 <211> 1687 <212> DNA <213> Bacillus bacteria <400> 1     1 tagagtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag    61 cgagggtttt cggaccctag cggcggacgg gtgagtaaca cgtaggcaac ctgcctctca   121 gaccgggata acatagggaa acttatgcta ataccggata ggtttttgga ttgcatgatc   181 cgaaaagaaa agatggcttc ggctatcact gggagatggg cctgcggcgc attagctagt   241 tggtggggta acggcctacc aaggcgacga tgcgtagccg acctgagagg gtgaccggcc   301 acactgggac tgagacacgg cccacactcc tacgggaggc agcagtaggg aattttccac   361 aatggacgaa agtctgatgg agcaacgccg cgtgaacgat gaaggtcttc ggattgtaaa   421 gttctgttgt cagggacgaa cacgtgccgt tcgaataggg cggtaccttg acggtacctg   481 acgagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt   541 tgtccggatt tattgggcgt aaagcgcgcg caggcggcta tgtaagtctg gtgttaaagc   601 ccggagctca actccggttc gcatcggaaa ctgtgtagct tgagtgcaga agaggaaagc   661 ggtattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc   721 ggctttctgg tctgtaactg acgctgaggc gcggcccgga gctcaactcc ggttcgcatc   781 ggaaactgtg tagcttgagt gcagaagagg aaagcggtat tccacgtgta gcggtgaaat   841 gcgtagagat gtggaggaac accagtggcg aaggcggctt tctggtctgt aactgacgct   901 gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac   961 gatgagtgct aggtgttggg ggtttcaata ccctcagtgc cgcagctaac gcaataagca  1021 ctccgcctgg ggagtactgc tcgcaagtag tgaaactcaa aggaattgac gggggcccgc  1081 acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga  1141 catcccgctg accgctctgg agacagagct tcccttcggg gcagcggtga caggtggtgc  1201 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc  1261 ttatctttag ttgccagcat tcagttgggc actctagaga gactgccgtc gacaagacgg  1321 aggaaggcgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct  1381 acaatggttg gtacaacggg atgctacctc gcgaggggac gccaatctct gaaaaccaat  1441 ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg  1501 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg  1561 ggagtttgca acacccgaag tcggtgaggt aaccgcaagg agccagccgc cgaaggtggg  1621 gtagatgact ggggtgaagt cgtaacaagg tatccgtacc ggaaggtgcg gttggatcac  1681 ctcctta

[0017]

Industrial Applicability According to the present invention, a novel microorganism decomposing a polycyclic aromatic hydrocarbon decomposing agent, and a decomposing agent containing the same can be provided.

[Brief description of drawings]

FIG. 1 is a graph showing the residual rate of benzo [a] pyrene at 25 ° C. by the microorganism of the present invention.

FIG. 2 Benzo [a] according to culture temperature by the microorganism of the present invention
It is a graph which shows the residual rate of pyrene.

FIG. 3 is a graph showing the residual rate of benzo [a] pyrene according to the initial concentration of bacterial cells by the microorganism of the present invention.

FIG. 4 is a graph showing the residual rate of pyrene at 25 ° C. by the microorganism of the present invention.

FIG. 5 is a graph showing the residual rate of anthracene at 25 ° C. by the microorganism of the present invention.

Continued front page    (72) Inventor Toshifumi Sakaguchi             2-12-24, in front of the airport, Hakata-ku, Fukuoka City, Fukuoka Prefecture             Legant Onizuka II 201 (72) Inventor Kenji Yokoyama             Ishikawa Prefecture Nomi-gun Tatsuguchi-cho Asahidai 1-50 B Building No. 23 (72) Inventor Shigetaka Morita             1-50 Asahidai, Tatsuguchiguchi, Nomi-gun, Ishikawa Prefecture E Building No. 13 (72) Inventor Yuji Murakami             Ishikawa Prefecture Nomi-gun Tatsuguchi-cho Asahidai 1-50 D Bldg. 33 (72) Inventor Saturli Ramachandra Rao (Sa             thuluri Ramachandra               Rao)             5-8, 1-8 Asahidai, Tatsunokuchi-cho, Nomi-gun, Ishikawa Prefecture             issue F-term (reference) 4B065 AA01X AC12 AC20 BA23                       CA56

Claims (6)

[Claims] 1. A fungus body inhabiting soil collected from a crude oil source is represented by the chemical formula (1): A bacillus having a gram positive, an oxidase activity positive, and a catalase activity positive obtained by culturing with benzo [a] pyrene represented by the formula (1) as a carbon source. 2. A bacillus that is Gram-positive, oxidase-positive, and catalase-positive, and has the following biochemical properties. Biochemical test Gram stain + Oxidase + Oxidase + Catalase + Glycerol + Erythritol-D-arabinose-L-arabinose (L-arabinose) Ribose) -D-xylose (D-Xylose) -L-xylose (L-Xylose) -Adonitiol (Adonitiol) -β-methyl-D-xylose (β-Methyl-D-Xylose) -galactose (Galactose) -Glucose (Glucose). Glucose) -Fructose + Mannose-Sorbose- Rhamnose-Dulcitol-Mannitol + sorbitol-Sorbitol-α-methyl-D-mannose (α-Methyl-D-Mannoside) -N-acetyl-glucosamine (N-Acetyl-Glucyl-Glucyl). Amygdalin-Arubutin + Escline + Salicin- Cellobiose- Maltose- Maltose-Lactose (Melaciose) + Mellibiose (Melubose) -Inuline -Melizetose-Raffinose-Starch-Glycogen-Xylitol-Gentiobiose-D-Turanose (D-Turanolyse-D-Duranalyse) -D-Tagatose + D-Fucose (D-Fucose) + L-Fucose (L-Fucose) -D-Arabitol (D-Arabitol) -L-Arabitol (L-Arabitol) + Gluconate (Gluconate) + 2 -Keto-gluconate (2-Keto-Gluconate) + 5-keto-gluconate (5-Keto-Gluconate) In the above test, "-" means negative, "+" indicates positive. 3. The bacillus according to claim 2, which has a nucleotide sequence of 16S rDNA represented by SEQ ID NO: 1. 4. The bacillus according to claim 3, which is represented by Incorporated Administrative Agency National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Depositary FERM P-18458. 5. A polycyclic aromatic hydrocarbon decomposing agent containing the rod-shaped bacterium according to any one of claims 1 to 4. 6. The polycyclic aromatic hydrocarbon decomposing agent according to claim 5, wherein the polycyclic aromatic hydrocarbon according to claim 5 is benzo [a] pyrene.