JP4931451B2 - New microorganism - Google Patents

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JP4931451B2
JP4931451B2 JP2006081919A JP2006081919A JP4931451B2 JP 4931451 B2 JP4931451 B2 JP 4931451B2 JP 2006081919 A JP2006081919 A JP 2006081919A JP 2006081919 A JP2006081919 A JP 2006081919A JP 4931451 B2 JP4931451 B2 JP 4931451B2
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toluene
benzene
xylene
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hydrocarbons
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JP2007252285A (en
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浩二 辻
伸也 宮地
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Cosmo Oil Co Ltd
Japan Petroleum Energy Center JPEC
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Japan Petroleum Energy Center JPEC
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Description

本発明は、石油製品由来の炭化水素類、特に、ベンゼン、トルエン、キシレン等の芳香族炭化水素類を効率よく分解する微生物に関する。   The present invention relates to a microorganism that efficiently decomposes hydrocarbons derived from petroleum products, in particular, aromatic hydrocarbons such as benzene, toluene, and xylene.

石油製品由来の炭化水素類を含む土壌中の地下水を浄化する技術として、微生物による浄化技術が検討されている。
しかしながら、一般的に微生物による石油製品由来の炭化水素類の分解速度は比較的遅く、石油製品由来の炭化水素類を含む土壌中の地下水が浄化されるには数ヶ月から数年の期間が必要となっており、短期間で分解できる微生物が求められている。
As a technique for purifying groundwater in soil containing hydrocarbons derived from petroleum products, purification techniques using microorganisms are being studied.
However, the degradation rate of hydrocarbons derived from petroleum products by microorganisms is generally relatively slow, and it takes several months to several years to purify the groundwater in the soil containing hydrocarbons derived from petroleum products. Therefore, there is a demand for microorganisms that can be degraded in a short period of time.

土壌や地下水中には石油製品由来の炭化水素類を分解する微生物が含まれていることはよく知られている(非特許文献1参照)。
これまでにいくつかの石油製品由来の炭化水素類を分解する微生物種について報告がなされており、ベンゼン、トルエン、あるいはキシレン等の芳香族炭化水素類を分解する微生物も得られている(特許文献1、非特許文献2参照)。しかし、前記微生物のベンゼン、トルエン、キシレン等の成分ごとに対しての分解性は確認されているが、石油製品由来の芳香族炭化水素類であれば、通常複数の芳香族炭化水素類が混在している場合が多く、ベンゼン、トルエン及びキシレンの3成分が共存している状況下で、それぞれがどの程度分解されるかを確認するまでには至っていない。
また、別の微生物に関して、ベンゼン、トルエン及びキシレンが共存している状況下では、トルエンとキシレンだけが分解され、ベンゼンについては全く分解されないことが報告されている(非特許文献3参照)。
特開平11−266858号公報 バイオレメディエ−ションエンジニア 〜設計と応用〜、第104−114頁,エヌ・ティー・エス 1997年1月23日発行 Lee, S.K., Appl Microbiol Biotechnol, 56, 270−275 (2001) Prenafeta-Boldu., Applied and Environmental Microbiology., 2660−2665 (2002)
It is well known that soil and groundwater contain microorganisms that decompose hydrocarbons derived from petroleum products (see Non-Patent Document 1).
So far, several species of microorganisms that decompose hydrocarbons derived from petroleum products have been reported, and microorganisms that decompose aromatic hydrocarbons such as benzene, toluene, or xylene have been obtained (patent documents). 1, refer to Non-Patent Document 2). However, the degradability of the microorganisms for each component such as benzene, toluene, xylene, etc. has been confirmed. However, if the aromatic hydrocarbons are derived from petroleum products, usually a plurality of aromatic hydrocarbons are mixed. In many cases, it has not yet been confirmed to what extent each of the three components of benzene, toluene, and xylene coexists.
In addition, regarding another microorganism, it has been reported that, in a situation where benzene, toluene and xylene coexist, only toluene and xylene are decomposed, and benzene is not decomposed at all (see Non-Patent Document 3).
JP-A-11-266858 Bioremediation Engineer -Design and Application-, pp. 104-114, published on January 23, 1997 Lee, SK, Appl Microbiol Biotechnol, 56, 270-275 (2001) Prenafeta-Boldu., Applied and Environmental Microbiology., 2660-2665 (2002)

土壌や地下水には、通常複数の石油系炭化水素類が共存している場合が多いが、上述したとおり、ベンゼン、トルエン及びキシレンの3成分が共存している状況下でこれら全ての成分を効率よく分解する能力を有する微生物は知られていない。
従って、本発明は、複数の石油系炭化水素類が共存する状況下でも、芳香族炭化水素類、特にベンゼン、トルエン及びキシレンを効率的に分解できる新規微生物を提供することを目的とする。
Soil and groundwater usually have multiple petroleum hydrocarbons in common, but as mentioned above, all these components are efficiently used in the situation where the three components of benzene, toluene and xylene coexist. No microorganism is known that has the ability to degrade well.
Accordingly, an object of the present invention is to provide a novel microorganism capable of efficiently decomposing aromatic hydrocarbons, particularly benzene, toluene and xylene, even in the situation where a plurality of petroleum hydrocarbons coexist.

本発明者らは、このような状況に鑑み、既知の微生物及び未知の微生物を日本国内各地の土壌からスクリーニングした結果、カプリアビダス(Cupriavidus)属に属する微生物が石油系炭化水素類、特にベンゼン、トルエン、キシレン等の芳香族炭化水素類に対する分解能力が高く、複数の石油系炭化水素類が共存する土壌や地下水においても、ベンゼン、トルエン及びキシレン全てを効率良く分解できることを見出し、本発明を完成されるに至った。   In view of such a situation, the present inventors screened known microorganisms and unknown microorganisms from soils in various places in Japan. As a result, microorganisms belonging to the genus Capriavidus are petroleum hydrocarbons, particularly benzene, toluene. The present invention was completed by finding that benzene, toluene, and xylene can be efficiently decomposed even in soil and groundwater in which a plurality of petroleum hydrocarbons coexist, having a high ability to decompose aromatic hydrocarbons such as xylene. It came to be.

すなわち、本発明は、石油製品由来の炭化水素類を分解する能力を有するカプリアビダス(Cupriavidus)属に属するC−102株(FERM P−20819)又はC−112株(FERM P−20820)を提供するものである。 That is, the present invention provides a C-102 strain (FERM P-20819) or a C-112 strain (FERM P-20820) belonging to the genus Capriavidus, which has the ability to decompose hydrocarbons derived from petroleum products. Is.

本発明の微生物は、ガソリン、灯油、軽油、重油、潤滑油などの石油製品由来の炭化水素類、特にベンゼン、トルエン、キシレン等の芳香族炭化水素類を効率よく分解できる。また、本発明の微生物は、ベンゼン、トルエン及びキシレンの3成分が共存している状況下でも、これらの成分全てを分解できるので、当該微生物を用いることにより、石油製品由来の炭化水素類を含有する土壌や地下水を効率よく浄化することができる。   The microorganism of the present invention can efficiently decompose hydrocarbons derived from petroleum products such as gasoline, kerosene, light oil, heavy oil and lubricating oil, particularly aromatic hydrocarbons such as benzene, toluene and xylene. In addition, since the microorganism of the present invention can decompose all of these components even in the presence of three components of benzene, toluene and xylene, it contains hydrocarbons derived from petroleum products by using the microorganism. Soil and groundwater can be efficiently purified.

本発明において石油製品とは、ガソリン、灯油、軽油、重油、潤滑油等をいい、石油製品由来の炭化水素類としては、例えばアルカン、アルケン等の脂肪族炭化水素類;ベンゼン、トルエン、キシレン等の単環芳香族炭化水素類、又は、ナフタレン、アンスラセン等の多環芳香族炭化水素類が挙げられる。   In the present invention, petroleum products refer to gasoline, kerosene, light oil, heavy oil, lubricating oil, etc., and hydrocarbons derived from petroleum products include aliphatic hydrocarbons such as alkanes and alkenes; benzene, toluene, xylene, etc. Monocyclic aromatic hydrocarbons, or polycyclic aromatic hydrocarbons such as naphthalene and anthracene.

本発明の微生物は、カプリアビダス(Cupriavidus)属に属し、石油製品由来の炭化水素類を効率よく分解する微生物である。斯かるカプリアビダス属に属する微生物としては、石油製品由来の炭化水素類を分解する能力を有する微生物であれば特に制限されないが、芳香族炭化水素類を分解する能力を有する微生物が好ましく、特にベンゼン、トルエン及びキシレンのうちの2種又は3種の混合物を分解する能力を有する微生物が好ましく、さらに、ベンゼン、トルエン及びキシレンの共存下における3者に対する合計の分解率が50〜100%、特に90〜100%であるものが好ましい。具体的には、本発明者らが埼玉県内の土壌から単離したC−102株、C−112株であり、このうちC−112株が好ましい。
C−102株、C−112株は次の菌学的性質を有する。
(1)C−112株の菌学的性質
The microorganism of the present invention belongs to the genus Cupriavidus and efficiently decomposes hydrocarbons derived from petroleum products. Such a microorganism belonging to the genus Capriavidas is not particularly limited as long as it is a microorganism having the ability to decompose hydrocarbons derived from petroleum products, but a microorganism having the ability to decompose aromatic hydrocarbons is preferred, and in particular, benzene, Microorganisms having the ability to decompose two or three mixtures of toluene and xylene are preferred, and the total decomposition rate for the three in the presence of benzene, toluene and xylene is 50 to 100%, particularly 90 to What is 100% is preferable. Specifically, the C-102 strain and the C-112 strain isolated by the present inventors from soil in Saitama Prefecture, among which the C-112 strain is preferred.
The C-102 and C-112 strains have the following mycological properties.
(1) Mycological properties of C-112 strain

(2)C−102株の菌学的性質 (2) Mycological properties of C-102 strain

これらの菌学的性質、リボソームの16S rDNAの塩基配列及びその相同性から、(BRENNER, (D.J), KRIEG, (N.R), STALEY, (J.T)and GARRITY, (G.M), Bergey's manual of Systematic Bacteriology. Vol. Two, The Proteobacteria, Part C The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. 2005, Springer)を参考にして分類・同定した結果、C−112株はカプリアビダス(Cupriavidus)属に属すると同定された。しかしながら、種レベルでは硝酸塩を還元し、インドールを産生せず、ウレアーゼ及びプロテアーゼ等の活性を示さず、トルエンを資化し、グルコースを資化しない、41℃で生育性を示す、でんぷんを加水分解しない点がカプリアビダス属に含まれる既知の種と異なるため、新規な微生物と同定しカプリアビダス属 C−112株として、独立行政法人産業技術総合研究所特許生物寄託センターに寄託した(FERM P-20820)。   From these mycological properties, the base sequence of ribosomal 16S rDNA and its homology, (BRENNER, (DJ), KRIEG, (NR), STALEY, (JT) and GARRITY, (GM), Bergey's manual of Systematic Bacteriology Vol. Two, The Proteobacteria, Part C The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. 2005, Springer). As a result of classification and identification, the C-112 strain belongs to the genus Cupriavidus. Identified. However, at the seed level, nitrate is reduced, no indole is produced, no activity such as urease and protease is shown, toluene is assimilated, glucose is not assimilated, growth at 41 ° C., starch is not hydrolyzed Since the point is different from the known species contained in the genus Capriavidas, it was identified as a novel microorganism and deposited as the Capriavidas genus C-112 strain at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (FERM P-20820).

また、C−102株についても、その菌学的性質、リボソームの16S rDNAの塩基配列及びその相同性から、カプリアビダス(Cupriavidus)属に属すると同定された。しかしながら、種レベルでは、硝酸塩を還元し、インドールを産生せず、ウレアーゼ及びプロテアーゼ等の活性を示さず、トルエンを資化し、グルコースを資化しない、でんぷんを加水分解しない点がカプリアビダス属に含まれる既知の種と異なり、更に細胞の大きさ、41℃で生育性を示さない点がC−112株とも異なるため、新規な微生物と同定しカプリアビダス属 C−102株として、独立行政法人産業技術総合研究所特許生物寄託センターに寄託した(FERM P-20819)。   The C-102 strain was also identified as belonging to the genus Cupriavidus from its bacteriological properties, the base sequence of 16S rDNA of ribosome and its homology. However, at the seed level, Capriavidas genus includes the point that it does not reduce nitrate, produce indole, does not show activity such as urease and protease, assimilates toluene, does not assimilate glucose, and does not hydrolyze starch. Unlike the known species, the cell size and the point that it does not show growth at 41 ° C are also different from the C-112 strain. Therefore, it was identified as a novel microorganism and the Capriavidas genus C-102 strain was established as an independent administrative agency. Deposited at the Institute for Biological Deposits (FERM P-20819).

C−102株及びC−112株は、カプリアビダス属に属し、硝酸塩を還元し、インドールを産生せず、ウレアーゼ及びプロテアーゼ等の活性を示さず、トルエンを資化し、グルコースを資化しない、でんぷんを加水分解しないと云う性質を有する新種であると考えられる。C−102株及びC−112株の類縁菌としては、これらの性質を有するものが含まれる。   C-102 strain and C-112 strain belong to the genus Capriavidas, reduce nitrate, do not produce indole, do not show activity of urease and protease, etc., assimilate toluene, do not assimilate glucose, It is considered to be a new species having the property of not hydrolyzing. Examples of the related strains of the C-102 strain and the C-112 strain include those having these properties.

本発明の新規微生物を培養するための培地は、特に制限されず、微生物を培養するための培地で、かつ石油製品由来の炭化水素類の分解を妨げない培地であればいかなる組成の培地も用いることができる。炭素源としては、例えばベンゼン、トルエン、キシレン等の芳香族炭化水素類;これらを含むガソリン等の石油製品;酢酸、ピルビン酸等の有機酸;等が挙げられる。窒素源としては、例えば硫酸アンモニウム、硝酸アンモニウム、尿素等の無機窒素源;ペプトン、肉エキス、酵母エキス、カゼイン等の有機窒素源等が挙げられる。更に、無機塩類等を適宜添加することもできる。   The medium for cultivating the novel microorganism of the present invention is not particularly limited, and a medium having any composition may be used as long as it is a medium for culturing microorganisms and does not prevent the decomposition of hydrocarbons derived from petroleum products. be able to. Examples of the carbon source include aromatic hydrocarbons such as benzene, toluene and xylene; petroleum products such as gasoline containing these; organic acids such as acetic acid and pyruvic acid; and the like. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, and urea; organic nitrogen sources such as peptone, meat extract, yeast extract, and casein. Furthermore, inorganic salts and the like can be added as appropriate.

培養条件としては、特に制限されず、一般に微生物が死滅しない培養条件であればよく、例えばpH6〜8、温度20〜35℃の条件下で、1〜60日間好気的に培養するのが好ましい。   The culture conditions are not particularly limited and may be any culture conditions that generally do not kill microorganisms. For example, it is preferable to aerobically culture for 1 to 60 days under conditions of pH 6 to 8 and temperature of 20 to 35 ° C. .

本発明の新規微生物を用いて、石油製品由来の炭化水素類を含有する土壌又は土壌中の地下水を浄化する方法としては、微生物を石油製品由来の炭化水素類を含有する土壌や地下水に添加すればよい。添加する微生物は、培養により得られた培養液をそのまま用いてもよく、遠心分離等により集菌したものを用いてもよい。また、凍結乾燥処理したものや、公知の担体に担時させたものを用いてもよい。   As a method for purifying soil containing petroleum products-derived hydrocarbons or groundwater in the soil using the novel microorganism of the present invention, microorganisms may be added to soil or groundwater containing petroleum products-derived hydrocarbons. That's fine. As the microorganism to be added, a culture solution obtained by culturing may be used as it is, or a microorganism collected by centrifugation or the like may be used. Moreover, you may use what was freeze-dried, and what was carried on the well-known support | carrier.

以下、本発明について実施例をあげて具体的に説明するが、本発明はこれらによって何等限定されるものではない。   Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

実施例1 菌の取得:
表7に示す培地1を内径21mmの試験管に10mL入れ、121℃で15分滅菌処理し液体培地を作成した。これに埼玉県をはじめとして国内で採取した150試料の土壌をそれぞれ1g添加し、試験管振とう機により、30℃、250rpmの条件で好気的に培養を1年間行った。炭素源として、市販ガソリン10μLを3日間〜1週間ごとに添加した。1〜2ヶ月毎に培養液1mLを培地1へ新たに植え継いだ。生育の認められた培養液を栄養平板培地へ100μL塗布し、30℃で2日間培養を行った。出現したコロニーをそれぞれ白金耳に拾い、上記の培地1に植菌し、市販ガソリンを添加した条件下で2週間培養を行った。生育の認められた試料については、再び栄養平板培地へ塗布し、単一なコロニーを形成させた。この様にして、石油系炭化水素に対する分解能力が高い微生物としてC−102株、C−112株を取得した。
Example 1 Acquisition of Bacteria:
10 mL of the medium 1 shown in Table 7 was put in a test tube having an inner diameter of 21 mm, and sterilized at 121 ° C. for 15 minutes to prepare a liquid medium. 1 g each of 150 samples of soil collected in Japan including Saitama Prefecture was added thereto, and aerobic culture was performed for 1 year under conditions of 30 ° C. and 250 rpm with a test tube shaker. As a carbon source, 10 μL of commercial gasoline was added every 3 days to 1 week. Every 1 to 2 months, 1 mL of the culture solution was newly planted into the medium 1. 100 μL of the culture solution in which growth was observed was applied to a nutrient plate medium and cultured at 30 ° C. for 2 days. The appearing colonies were respectively picked up by platinum ears, inoculated into the above-mentioned medium 1, and cultured for 2 weeks under the condition where commercial gasoline was added. About the sample by which growth was recognized, it apply | coated to the nutrient plate culture medium again, and the single colony was formed. In this way, C-102 and C-112 strains were obtained as microorganisms having a high ability to decompose petroleum hydrocarbons.

実施例2 ベンゼン、トルエン、キシレンの分解試験:
全容50mlのガラス製遠心管に、培地1を10mL、C−112株、C−102株の培養液1mLを遠心分離し、上澄み液を除いた後、滅菌水で再度1mLに調製した微生物懸濁液を接種した後、ベンゼン、トルエン及びキシレンを0.04mgずつ添加して、30℃、100rpmにて15時間培養を行った。その後、培養液中に含まれる残存したベンゼン、トルエン、キシレンを測定することにより分解能力を測定した。
Example 2 Decomposition test of benzene, toluene and xylene:
In a 50 ml glass centrifuge tube, 10 mL of the medium 1, 1 mL of the culture solution of the C-112 strain and the C-102 strain was centrifuged, the supernatant was removed, and the microorganism suspension prepared to 1 mL again with sterilized water. After inoculating the solution, 0.04 mg of benzene, toluene, and xylene was added and cultured at 30 ° C. and 100 rpm for 15 hours. Thereafter, the decomposition ability was measured by measuring the remaining benzene, toluene, and xylene contained in the culture solution.

基質の残存量は、培養後の培養液10mLを塩化ナトリウム3g入りの20mLのバイアル瓶に導入、密閉し、バイアル瓶を80℃で30分加熱し、加熱後の気相部をガスクロマトグラフィーにより定量して求めた。基質の残存量と添加量を比較して、その減少率から分解率を求めた。また、対照試験として、実施例1における方法で取得する過程の初期で得られた土壌に含まれる微生物を用い、ベンゼン、トルエン、キシレンの分解能力を確認した。   The remaining amount of the substrate was determined by introducing 10 mL of the culture solution after culturing into a 20 mL vial containing 3 g of sodium chloride, sealing it, heating the vial at 80 ° C. for 30 minutes, and gas phase after heating by gas chromatography. Quantitatively determined. The remaining amount of the substrate was compared with the added amount, and the decomposition rate was determined from the decrease rate. Further, as a control test, microorganisms contained in soil obtained at the initial stage of the process obtained by the method in Example 1 were used to confirm the ability to decompose benzene, toluene and xylene.

15時間後のベンゼン、トルエン及びキシレンの残存量を測定した結果、表8に示すとおり、C−112株、C−102株を添加した系では、特にベンゼンに対して高い分解能力を有することが示された。また、ベンゼン、トルエン及びキシレンが共存する環境下においても、これら3成分同時に分解可能であることが示された。表中の数値は添加した基質に対する減少率を百分率で示している。   As a result of measuring the residual amounts of benzene, toluene and xylene after 15 hours, as shown in Table 8, the system to which C-112 strain and C-102 strain were added had particularly high decomposition ability for benzene. Indicated. It was also shown that these three components can be decomposed simultaneously in an environment where benzene, toluene and xylene coexist. The numerical values in the table indicate the percentage reduction with respect to the added substrate.

Claims (1)

石油製品由来の炭化水素類を分解する能力を有するカプリアビダス(Cupriavidus)属に属するC−102株(FERM P−20819)又はC−112株(FERM P−20820) C-102 strain (FERM P-20819) or C-112 strain (FERM P-20820) belonging to the genus Cupriavidus having the ability to decompose hydrocarbons derived from petroleum products.
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