JP4754700B2 - Novel strain having volatile aromatic hydrocarbon resolving ability and use thereof - Google Patents
Novel strain having volatile aromatic hydrocarbon resolving ability and use thereof Download PDFInfo
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- JP4754700B2 JP4754700B2 JP2001044871A JP2001044871A JP4754700B2 JP 4754700 B2 JP4754700 B2 JP 4754700B2 JP 2001044871 A JP2001044871 A JP 2001044871A JP 2001044871 A JP2001044871 A JP 2001044871A JP 4754700 B2 JP4754700 B2 JP 4754700B2
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【0001】
【発明の属する技術分野】
本発明は揮発性芳香族炭化水素分解能を有する微生物及びそれを用いた環境の浄化方法に関する。
【0002】
【従来の技術】
石油による土壌や海洋、大気等の環境汚染は、近年特に増大している。石油成分は生物に対する毒性を有するため、製油工場からの漏出、タンカー座礁等により環境中へ石油が流出すると、人間のみならず、動物や魚、鳥、海草、貝類等の生物に対して悪影響を及ぼす。特に、石油成分の中でもベンゼン、トルエン、エチルベンゼン、キシレンに代表される揮発性芳香族炭化水素類は毒性が高く、また、揮発性を有するゆえに大気から拡散するとともに、若干の水溶性を有するために、地下水からの拡散の危険性もあり、大きな問題となっている。また、塗料工場等から排出される排水には原材料として使用される揮発性芳香族炭化水素類が多量に含有されており、その処理も大きな問題となっている。
【0003】
実際の汚染環境における処理現場では、これまで、揮発性芳香族炭化水素を機械的に揮発させ、回収する方法が主として用いられてきたが、コストが高い、あるいは回収された炭化水素を何らかの方法で再度処理しなければならない等の問題点があり、有効な手段ではなかった。これに対して、近年、微生物を利用した環境修復、いわゆるバイオレメディエーションの適用が論じられるようになってきた。しかし、バイオレメディエーションの過程で増殖する菌の種類や、中間代謝物の安全性等不明な点が多く、実施にはなかなか到らないのが現状である。特に、オルト-キシレンは他の揮発性芳香族炭化水素類に比較して分解が難しいため、分解菌が単離された例は非常に少なく、また、分解能も低い。このため、バイオレメディエーションあるいは排水処理の過程で高い揮発性芳香族炭化水素分解能を有する微生物の開発が待たれていた。
【0004】
【発明が解決しようとする課題】
本発明は、揮発性芳香族炭化水素分解能を有する微生物を利用し、揮発性芳香族炭化水素により汚染された環境を迅速に浄化する手段を提供することを目的とする。
【0005】
【課題を解決するための手段】
本発明者は、上記課題を解決するため、小規模なバイオレメディエーション実験を行い、この時の細菌相の変化とオルト-キシレンの分解を解析した結果、オルト-キシレンが分解されるときに菌数の増加する菌株を見いだし、本発明を完成した。
【0006】
すなわち、本発明は、揮発性芳香族炭化水素分解能を有するロドコッカス属に属する微生物である。
【0007】
また、本発明は、上記微生物で、揮発性芳香族炭化水素により汚染された環境を浄化することを特徴とする環境の浄化方法である。
【0008】
【発明の実施の形態】
以下、本発明を詳細に説明する。
【0009】
本発明の微生物は、ロドコッカス(Rhodococcus)属に属し、揮発性芳香族炭化水素を分解する能力を有するものである。本発明の微生物には、揮発性芳香族炭化水素、例えば、オルト−キシレンを単一炭素源として分解することのできる微生物が含まれる。このような微生物としては、本発明者により単離されたKN-14株、KN-45株、KN-46株などを例示することができる。
【0010】
これらの菌株は、オルト-キシレンにより汚染された土壌からオルト-キシレン分解能を持つ微生物を単離すると同時に、土壌中で優占化していると考えられる微生物をDGGE法を用いることにより遺伝子レベルで解析し、DGGE法で得られた遺伝子配列と99%以上の相同性を持つ微生物を単離された微生物から選抜することで得られた新規な菌株である。
【0011】
これらの菌株の16S rRNAをコードするDNAの塩基配列の一部を決定し、NCBIのプログラム BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)により同定を行った。その結果、これらの菌株は、ロドコッカス属に属することが判明した。しかし、ロドコッカスに属する公知の菌株とはオルト-キシレン分解能において異なる性質を示すことから、これらの菌株を新菌種と認め、ロドコッカス・エスピーKN-14株(Rhodococcus sp. KN-14株)、ロドコッカス・エスピーKN-45株(Rhodococcus sp. KN-45株)、ロドコッカス・エスピーKN-46株(Rhodococcus sp. KN-46株)と命名した。
【0012】
ロドコッカス・エスピーKN-14株(以下、「KN-14株」という)、ロドコッカス・エスピーKN-45株(以下、「KN-45株」という)、及びロドコッカス・エスピーKN-46株(以下、「KN-46株」という)は、産業技術総合研究所生命工学工業技術研究所に以下の受託番号で寄託されている。
【0013】
ロドコッカス・エスピーKN-14株:FERM P-18195(寄託日:平成13年2月2日、識別表示:Rhodococcus sp. KN-14)
ロドコッカス・エスピーKN-45株:FERM P-18197(寄託日:平成13年2月2日、識別表示:Rhodococcus sp. KN-45)
ロドコッカス・エスピーKN-46株:FERM P-18196(寄託日:平成13年2月2日、識別表示:Rhodococcus sp. KN-46)
KN-14株、KN-45株、及びKN-46株の菌学的性質を以下に示す。
A. 形態学的性質
形態的にはKN-14株、KN-45株、KN-46株ともにほぼ共通であり、細胞の形は糸状性である。また、1/10TSA培地(Tryptic Soy Broth w/o Dextrose(Difco社製) 2.75g/L、グルコース 0.25g/L)を用いて寒天プレートにより培養した場合、コロニーの色は培養初期はクリーム色であるが、培養後期にはピンク色に変化し、コロニーの透明度はない。さらに、基質特異性は表1のとおりである。
【0014】
【表1】
B. 16S rRNAに対応するDNA(以下、「16S rRNA遺伝子」という)及びジャイレースサブユニットをコードするDNA(以下、「gyrB遺伝子」という)の塩基配列 KN-14株、KN-45株、KN-46株の16S rRNA遺伝子の塩基配列を、それぞれ配列番号1、配列番号2、配列番号3に示す。また、KN-14株、KN-45株、KN-46株のgyrB遺伝子の塩基配列を、それぞれ配列番号4、配列番号5、配列番号6に示す。
【0015】
なお、塩基配列の決定は、Current Protocols in Molecular Biology (eds.)( Greene Publishing Associates and Wiley-Interscience, N.Y.(1987) ) に準じて行った。DNAのシーケンスはautomated DNA sequencer (ABI 377; Perkin Elmer, Inc., USA)を用いて行った。
【0016】
本発明の微生物には、KN-14株、KN-45株、KN-46株のほか、これらの菌株と一定の類似性を示す微生物も含まれる。一定の類似性を示す微生物とは、例えば、微生物の分類に利用されている16S rRNA遺伝子やgyrB遺伝子の塩基配列が、上記3菌株と類似している微生物をいう。具体的には、16S rRNA遺伝子の塩基配列が配列番号1、配列番号2、又は配列番号3に記載の塩基配列と90%以上、好ましくは97%以上相同である微生物やgyrB遺伝子の塩基配列が配列番号4、配列番号5、又は配列番号6に記載の塩基配列と73%以上、好ましくは85%以上相同である微生物が、上記菌株と一定の類似性を示す微生物に含まれる。
【0017】
このような上記菌株と一定の類似性を示す微生物は、揮発性芳香族炭化水素により汚染された土壌などから配列番号1〜6に記載の塩基配列を指標として単離してくることができる。
【0018】
本発明の微生物を増殖させるには、通常の培養法が挙げられる。培養は、好気的条件で行うことが好ましく、有機物、無機塩、窒素源、その他栄養源を含む1/10TSA培地等に本発明の微生物を接種し、例えば振とう培養法、通気撹拌培養法などにより培養を行う。
【0019】
上記培養における温度条件は、使用する微生物の生育温度の範囲、好ましくは最適生育温度の範囲に設定する。例えば20〜30℃、好ましくは25℃に設定することができる。なお、培地のpHは、6.5〜7.5の範囲に設定すればよい。
【0020】
無機塩として培地に添加する物質としては、リン酸塩、マグネシウム塩、カルシウム塩、鉄塩、その他必要に応じて微量金属塩が挙げられる。また、窒素源としては、本発明の微生物が資化し得るものであればよく、例えば、ペプトン、カシトン、尿素、硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、硝酸アンモニウム、各種アミノ酸などが挙げられる。これらの窒素源は1種でもよく、2種以上を適宜組み合わせても良い。さらに、本発明の微生物の増殖を促進するための栄養源として、ビタミン、酵母エキス、麦芽エキスなどを適量添加しても良い。
【0021】
培養時間は、栄養源の量や種類により異なるが、通常1日以上、好ましくは2〜3日間である。
【0022】
本発明の微生物は、揮発性芳香族炭化水素に汚染された環境の浄化に利用することができる。ここでいう「環境」には、土壌、海洋、大気のほか、排水なども含まれる。環境の浄化は、上記条件で培養した微生物の培養液、あるいは、微生物を凍結乾燥処理した乾燥粉末を汚染環境に散布することにより行われる。この際、乾燥粉末と増殖を補助する無機塩類を混合・造粒し、粉末状及び顆粒状等に製剤化したものを汚染環境に散布しても良い。処理に用いる微生物の量は、土壌及び海水の汚染状況等に応じ、任意に定めることができるが、通常、汚染土壌1m3あるいは汚染海域100m2に培養液であれば1L、乾燥菌体であれば5g程度である。また、汚染された大気の浄化は、上記培養液あるいは乾燥菌体を固定化担体を設置した気体の浄化装置に添加し、これに汚染大気を通気することで行う。さらに、汚染された排水の浄化は、同じく上記培養液あるいは乾燥菌体を汚染排水と混合し、好気条件で1〜7日程度培養することで行う。
【0023】
【実施例】
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらの実施例にその技術的範囲が限定されるものではない。
100mlの茶褐色ガラスバイアルに土壌(岩手県釜石市の畑地より採取)7g、表2に示した無機塩培地2mlを添加し、テフロンコートされたブチルゴム栓により密封した。さらに、オルト-キシレン濃度が水中で200μLとなるようにオルト-キシレンを添加し、20℃で1日間振とうした後、KN-14株を植菌し、20℃にて10日間培養を行った。培養期間中、適宜、バイアル中の気相部から気体を少量回収し、これを直接GCにより解析を行い、バイアル内の水相におけるオルト-キシレン濃度を測定することで、初期添加濃度に対する残存率を計算した。その結果を図1に示す。図1に示すように、土壌のみでは分解に約10日間必要であるが、KN-14株を植菌した系では植菌から約2日で100%分解することができ、本発明の微生物を用いることにより、迅速なオルト-キシレン分解を行うことができることが分かる。
【0024】
【表2】
【本発明の効果】
本発明は、揮発性芳香族炭化水素分解能を有する新規な微生物を提供する。この微生物によりオルト-キシレン等の揮発性芳香族炭化水素で汚染された環境を効率的に浄化処理することができる。
【0025】
【配列表】
【図面の簡単な説明】
【図1】本発明の微生物を添加した場合と添加しなかった場合のオルト-キシレン残存率の経時的変化を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microorganism having a resolution of volatile aromatic hydrocarbons and an environmental purification method using the microorganism.
[0002]
[Prior art]
In recent years, environmental pollution such as soil, ocean, and air due to oil has been increasing. Petroleum components are toxic to living organisms, so if oil spills into the environment due to leaks from refineries, tanker stranding, etc., it will adversely affect not only human beings, but also animals such as animals, fish, birds, seaweeds and shellfish. Effect. In particular, volatile aromatic hydrocarbons represented by benzene, toluene, ethylbenzene, and xylene are highly toxic among petroleum components, and because they are volatile, they diffuse from the atmosphere and have some water solubility. There is also a danger of diffusion from groundwater, which is a big problem. In addition, wastewater discharged from paint factories and the like contains a large amount of volatile aromatic hydrocarbons used as raw materials, and the treatment thereof is also a big problem.
[0003]
Up to now, methods for volatilizing and recovering volatile aromatic hydrocarbons have been mainly used in the treatment site in an actual polluted environment. However, the cost is high, or the recovered hydrocarbon is recovered in some way. There were problems such as having to process again, and it was not an effective means. On the other hand, in recent years, the application of environmental remediation using microorganisms, so-called bioremediation, has come to be discussed. However, there are many unclear points such as the types of bacteria that proliferate in the process of bioremediation and the safety of intermediate metabolites, and it is difficult to implement them. In particular, ortho-xylene is difficult to decompose compared to other volatile aromatic hydrocarbons, and therefore, there are very few examples in which a degrading bacterium has been isolated and the resolution is low. For this reason, the development of microorganisms having high volatile aromatic hydrocarbon resolution has been awaited in the process of bioremediation or wastewater treatment.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide means for quickly purifying an environment contaminated with volatile aromatic hydrocarbons by using a microorganism having a volatile aromatic hydrocarbon resolution.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventor conducted a small-scale bioremediation experiment and analyzed the change in bacterial flora and the decomposition of ortho-xylene. As a result, the present inventors have completed the present invention.
[0006]
That is, the present invention is a microorganism belonging to the genus Rhodococcus having volatile aromatic hydrocarbon decomposing ability.
[0007]
The present invention is also a method for purifying an environment characterized by purifying an environment contaminated with volatile aromatic hydrocarbons by the microorganism.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0009]
The microorganism of the present invention belongs to the genus Rhodococcus and has the ability to decompose volatile aromatic hydrocarbons. The microorganisms of the present invention include microorganisms capable of decomposing volatile aromatic hydrocarbons such as ortho-xylene as a single carbon source. Examples of such microorganisms include KN-14 strain, KN-45 strain, and KN-46 strain isolated by the present inventors.
[0010]
These strains were analyzed at the gene level by using the DGGE method to isolate microorganisms with ortho-xylene-degrading ability from soil contaminated with ortho-xylene, and at the same time, microorganisms considered to be dominant in the soil. It is a novel strain obtained by selecting a microorganism having a homology of 99% or more with the gene sequence obtained by the DGGE method from the isolated microorganisms.
[0011]
A part of the base sequence of DNA encoding 16S rRNA of these strains was determined and identified by NCBI program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, these strains were found to belong to the genus Rhodococcus. However, since it has different properties in ortho-xylene resolution from known strains belonging to Rhodococcus, these strains are recognized as new strains, and Rhodococcus sp. KN-14 strain (Rhodococcus sp. KN-14 strain), Rhodococcus -Named SP KN-45 strain (Rhodococcus sp. KN-45 strain) and Rhodococcus sp KN-46 strain (Rhodococcus sp. KN-46 strain).
[0012]
Rhodococcus sp. KN-14 strain (hereinafter referred to as “KN-14 strain”), Rhodococcus sp. KN-45 strain (hereinafter referred to as “KN-45 strain”), and Rhodococcus sp. KN-46 strain (hereinafter referred to as “ KN-46 ") is deposited at the National Institute of Advanced Industrial Science and Technology (AIST) with the following accession number.
[0013]
Rhodococcus sp. KN-14 strain: FERM P-18195 (Deposit date: February 2, 2001, Identification: Rhodococcus sp. KN-14)
Rhodococcus sp. KN-45 strain: FERM P-18197 (Deposit date: February 2, 2001, Identification: Rhodococcus sp. KN-45)
Rhodococcus sp. KN-46 strain: FERM P-18196 (Deposit date: February 2, 2001, identification: Rhodococcus sp. KN-46)
The mycological properties of the KN-14, KN-45, and KN-46 strains are shown below.
A. Morphological properties Morphologically, the KN-14, KN-45 and KN-46 strains are almost common, and the cell shape is filamentous. In addition, when cultured on an agar plate using 1/10 TSA medium (Tryptic Soy Broth w / o Dextrose (Difco) 2.75 g / L, glucose 0.25 g / L), the colony color is cream at the beginning of the culture. However, it changes to pink in the later stage of culture and there is no transparency of colonies. Further, the substrate specificity is as shown in Table 1.
[0014]
[Table 1]
B. Nucleotide sequences of DNA corresponding to 16S rRNA (hereinafter referred to as “16S rRNA gene”) and DNA encoding gyrase subunit (hereinafter referred to as “gyrB gene”) KN-14 strain, KN-45 strain, KN The nucleotide sequences of the 16S rRNA gene of -46 strain are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The base sequences of the gyrB gene of KN-14, KN-45, and KN-46 are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
[0015]
The base sequence was determined according to Current Protocols in Molecular Biology (eds.) (Greene Publishing Associates and Wiley-Interscience, NY (1987)). DNA sequencing was performed using an automated DNA sequencer (ABI 377; Perkin Elmer, Inc., USA).
[0016]
The microorganisms of the present invention include KN-14, KN-45, and KN-46 strains, as well as microorganisms that exhibit certain similarities to these strains. The microorganism having a certain similarity refers to, for example, a microorganism in which the base sequences of 16S rRNA gene and gyrB gene used for classification of microorganisms are similar to the above three strains. Specifically, the base sequence of a microorganism or gyrB gene whose base sequence of 16S rRNA gene is 90% or more, preferably 97% or more homologous to the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Microorganisms that are 73% or more, preferably 85% or more homologous to the base sequence described in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 are included in the microorganisms exhibiting a certain degree of similarity to the strain.
[0017]
Such a microorganism having a certain similarity to the above strain can be isolated from soil contaminated with volatile aromatic hydrocarbons using the base sequences described in SEQ ID NOs: 1 to 6 as an index.
[0018]
In order to grow the microorganism of the present invention, a usual culture method can be mentioned. Cultivation is preferably performed under aerobic conditions, and inoculated with the microorganism of the present invention in a 1/10 TSA medium containing organic matter, inorganic salt, nitrogen source, and other nutrient sources, for example, shaking culture method, aeration stirring culture method Cultivate by such as
[0019]
The temperature condition in the culture is set in the range of the growth temperature of the microorganism to be used, preferably in the range of the optimum growth temperature. For example, it can be set to 20 to 30 ° C, preferably 25 ° C. In addition, what is necessary is just to set the pH of a culture medium in the range of 6.5-7.5.
[0020]
Substances added to the medium as inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, and other trace metal salts as necessary. The nitrogen source is not particularly limited as long as it can be assimilated by the microorganism of the present invention, and examples thereof include peptone, cascitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids. These nitrogen sources may be used alone or in combination of two or more. Furthermore, as a nutrient source for promoting the growth of the microorganism of the present invention, an appropriate amount of vitamins, yeast extract, malt extract and the like may be added.
[0021]
The culture time varies depending on the amount and type of nutrient source, but is usually 1 day or longer, preferably 2 to 3 days.
[0022]
The microorganism of the present invention can be used for purification of an environment contaminated with volatile aromatic hydrocarbons. The “environment” here includes soil, ocean, air, and wastewater. Purification of the environment is performed by spraying a culture solution of microorganisms cultured under the above conditions or a dry powder obtained by freeze-drying microorganisms to a contaminated environment. At this time, dry powder and inorganic salts that assist in growth may be mixed and granulated, and the powdered and granular preparations may be dispersed in a contaminated environment. The amount of microorganisms used in the process, depending on the pollution of soil and sea water, etc., can be arbitrarily determined, there usually if culture in contaminated soil 1 m 3 or contaminated waters 100 m 2 1L, in dry cell About 5g. The contaminated air is purified by adding the culture solution or dried cells to a gas purifier equipped with an immobilizing carrier and venting the contaminated air to the gas purifier. Furthermore, the contaminated waste water is purified by mixing the culture solution or the dried cells with the contaminated waste water and culturing them for about 1 to 7 days under aerobic conditions.
[0023]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
7 g of soil (collected from the field in Kamaishi City, Iwate Prefecture) and 2 ml of the inorganic salt medium shown in Table 2 were added to a 100 ml brown glass vial and sealed with a Teflon-coated butyl rubber stopper. Furthermore, ortho-xylene was added so that the ortho-xylene concentration was 200 μL in water, and after shaking at 20 ° C for 1 day, the KN-14 strain was inoculated and cultured at 20 ° C for 10 days. . During the incubation period, a small amount of gas is recovered from the gas phase in the vial as needed, and this is analyzed directly by GC. By measuring the ortho-xylene concentration in the aqueous phase in the vial, the residual rate relative to the initial concentration added Was calculated. The result is shown in FIG. As shown in Fig. 1, the soil alone requires about 10 days for degradation, but the system inoculated with the KN-14 strain can be 100% degraded in about 2 days from the inoculation. It can be seen that rapid ortho-xylene decomposition can be carried out by using.
[0024]
[Table 2]
[Effect of the present invention]
The present invention provides a novel microorganism having volatile aromatic hydrocarbon resolution. An environment contaminated with volatile aromatic hydrocarbons such as ortho-xylene can be efficiently purified by this microorganism.
[0025]
[Sequence Listing]
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing changes over time in ortho-xylene residual rate when a microorganism of the present invention is added and when it is not added.
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