JP5706134B2 - Purification promotion material and purification promotion method - Google Patents
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- Processing Of Solid Wastes (AREA)
- Treatment Of Biological Wastes In General (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Description
本発明は、微生物を用いて有害化学物質を浄化する有害化学物質浄化技術において、浄化対象とする廃水、汚染土壌、汚染地下水等に対して供給する浄化促進材料及び微生物による浄化促進方法に関するものである。 The present invention relates to a purification promoting material supplied to waste water, contaminated soil, contaminated groundwater, etc. to be purified in a hazardous chemical purification technology that purifies harmful chemicals using microorganisms, and a purification promotion method using microorganisms. is there.
微生物を用いる浄化技術は、排水処理における活性汚泥法や嫌気処理法などに広く利用されている。
また、近年では、有害化学物質で汚染された土壌および地下水を微生物により浄化する技術(バイオレメディエーション)も、環境負荷および浄化コストの小さい浄化方法として着目されている。
Purification technology using microorganisms is widely used for activated sludge method and anaerobic treatment method in wastewater treatment.
In recent years, a technique (bioremediation) for purifying soil and groundwater contaminated with harmful chemical substances with microorganisms has attracted attention as a purification method with low environmental burden and purification cost.
このような微生物を用いる浄化技術は、二次的汚染が生じにくく、使用エネルギー量が少ないなどの長所が有る一方で、物理・化学的な浄化方法と比較すると、浄化に適した状態を維持・管理する方法に不確定要素が多くある点に問題がある。 While purification technology using such microorganisms has the advantages of being less susceptible to secondary contamination and using less energy, it maintains a state suitable for purification compared to physical and chemical purification methods. There is a problem in that there are many uncertainties in the management method.
例えば、上記活性汚泥法によるバルキングは、曝気槽中に糸状菌が大量発生することにより活性汚泥の沈降性が阻害される現象であるが、このように処理効率の低下や阻害が生じる要因として、処理環境中の微生物群衆構造が影響していることが示されている。
すなわち、処理環境中には、有用な微生物だけでなく、浄化を阻害する微生物も同時に増殖して、影響を与えるとしている（非特許文献１）。
For example, bulking by the above activated sludge method is a phenomenon in which the sedimentation property of activated sludge is inhibited due to the occurrence of a large amount of filamentous fungi in the aeration tank. It has been shown that the microbial community structure in the processing environment is affected.
That is, in the treatment environment, not only useful microorganisms but also microorganisms that inhibit purification are simultaneously proliferated and affected (Non-Patent Document 1).
ところが、廃水処理やバイオレメディエーションの分野において存在する微生物種は、多岐にわたると同時に、その微生物群衆構造は処理を行っている場所、時間によって絶えず変化しているものである。
このため、浄化環境を適切な微生物群衆構造にリアルタイムでコントロールすることは極めて難しいと考えられている。
However, the microbial species existing in the fields of wastewater treatment and bioremediation are diverse, and the microbial community structure is constantly changing depending on the place and time of treatment.
For this reason, it is considered extremely difficult to control the purification environment to an appropriate microbial community structure in real time.
このような背景から、有用微生物を浄化対象とする場所に導入する方法が、微生物群衆構造を安定させて浄化効率を高めたり、特定の有害化学物質を浄化したりする方法として用いられている（非特許文献２．３）。 From such a background, a method of introducing useful microorganisms to a place to be purified is used as a method of stabilizing the microbial community structure to increase the purification efficiency or purifying a specific harmful chemical substance ( Non-patent document 2.3).
しかしながら、有用微生物は複雑な微生物群衆構造内で長期的に効果を発揮することが難しく、導入した微生物が十分に効果を発揮できない場合もある（非特許文献４）。また、微生物の導入は、培養等に多大なコストが必要であるため、浄化コストが大きくなることも課題となっている。 However, it is difficult for useful microorganisms to exert long-term effects within a complex microbial community structure, and the introduced microorganisms may not be sufficiently effective (Non-patent Document 4). In addition, since introduction of microorganisms requires a large amount of cost for culturing and the like, an increase in purification cost is also an issue.
本発明は、微生物を用いて有害化学物質を浄化する技術分野において、浄化に関わりの無い微生物の活動を抑制することにより浄化に関わる有用徹生物の活動を速やかに促進させる環境を形成することにより、浄化効率を高めて浄化期間を短縮することが可能な浄化促進材料及び浄化促進方法を提供することを目的とするものである。 In the technical field of purifying harmful chemicals using microorganisms, the present invention forms an environment that promptly promotes the activities of useful organisms related to purification by suppressing the activities of microorganisms that are not involved in purification. An object of the present invention is to provide a purification promoting material and a purification promoting method capable of increasing the purification efficiency and shortening the purification period.
請求項１に係る発明は、微生物を用いて有害化学物質を浄化するに際し、廃水、汚染土壌、汚染地下水等の浄化対象に対して供給する浄化促進材料において、該材料が、浄化に関わりの無い微生物の活動を抑制し、浄化に必要な有用微生物を優占化して活動を促進させるものから構成されている浄化促進材料とした。 The invention according to claim 1 is a purification promoting material supplied to a purification target such as waste water, contaminated soil, and contaminated ground water when purifying harmful chemical substances using microorganisms, and the material is not involved in purification. It was made into the purification promotion material comprised from what suppresses the activity of microorganisms, dominates useful microorganisms required for purification, and promotes activity.
請求項２に係る発明は、微生物を用いて有害化学物質を浄化するに際し、廃水、汚染土壌、汚染地下水等の浄化対象に対して供給する浄化促進材料おいて、該材料が、ホップ、ホップ抽出成分、又はホップを含む材料である浄化促進材料とした。 The invention according to claim 2 is a purification promoting material that is supplied to a purification target such as waste water, contaminated soil, and contaminated ground water when purifying harmful chemical substances using microorganisms. It was set as the purification | cleaning promotion material which is a material containing a component or a hop.
請求項３に係る発明は、有害化学物質で汚染された廃水、汚染土壌、汚染地下水等の浄化対象を微生物を用いて浄化するに際し、前記浄化対象に対して浄化促進材料を添加して微生物による浄化を促進する浄化促進方法において、前記浄化促進材料として、浄化に関わりの無い微生物の活動を抑制し、浄化に必要な有用微生物を優占化して活動を促進させるもの、あるいはホップ、ホップ抽出成分、又はホップを含む材料を添加する浄化促進方法とした。 In the invention according to claim 3, when purifying a purification target such as waste water, contaminated soil, and contaminated groundwater contaminated with a harmful chemical substance using a microorganism, a purification promoting material is added to the purification target and the microorganism is used. In the purification promotion method for promoting purification, the purification promoting material suppresses the activity of microorganisms not related to purification, predominates useful microorganisms necessary for purification, or promotes the activity, or hops, hop extraction components Or a purification promoting method of adding a material containing hops.
請求項４に係る発明は、請求項３に記載された浄化促進方法において利用する微生物が、デハロコッコイデス属細菌であることを特徴としている。 The invention according to claim 4 is characterized in that the microorganism used in the purification promotion method according to claim 3 is a bacterium belonging to the genus Dehalococcides.
請求項５に係る発明は、請求項３に記載された浄化促進方法において利用する微生物が、vcrA遺伝子を有する細菌であることを特徴としている。 The invention according to claim 5 is characterized in that the microorganism used in the purification promotion method according to claim 3 is a bacterium having the vcrA gene.
請求項６に係る発明は、請求項３に記載された浄化促進方法において浄化する有害化学物質が、揮発性有機塩素化合物であることを特徴としている。 The invention according to claim 6 is characterized in that the harmful chemical substance to be purified in the purification promoting method according to claim 3 is a volatile organic chlorine compound.
請求項１に係る発明によれば、微生物を用いて有害化学物質を浄化するに際し、廃水、汚染土壌、汚染地下水等の浄化対象に対して供給する浄化促進材料において、該材料を浄化に関わりの無い微生物の活動を抑制し、浄化に必要な有用微生物を優占化して活動を促進させるものとしたので、浄化を阻害する微生物の増殖を抑制してその影響を極力低減し、有害化学物質を短時間に浄化して浄化効率を高めて、浄化期間を短縮して浄化コストを下げることができる。
また、浄化に必要な有機資材等の微生物活性資材の投入量を低減できることから、この面でも浄化コストを低減可能な浄化促進材料を提供することができる。
According to the first aspect of the present invention, in the purification promoting material to be supplied to the purification target such as waste water, contaminated soil, and contaminated ground water when purifying harmful chemical substances using microorganisms, the material is related to purification. Since the activity of non-existing microorganisms is suppressed and the useful microorganisms necessary for purification are predominated to promote the activity, the growth of microorganisms that inhibit the purification is suppressed, and the effects are reduced as much as possible. Purification can be performed in a short time to increase the purification efficiency, and the purification period can be shortened to reduce the purification cost.
Moreover, since the input amount of microbially active materials such as organic materials necessary for purification can be reduced, it is possible to provide a purification promoting material capable of reducing the purification cost also in this aspect.
請求項２に係る発明によれば、微生物を用いて有害化学物質を浄化するに際し、廃水、汚染土壌、汚染地下水等の浄化対象に対して供給する浄化促進材料おいて、該材料をホップ、ホップ抽出成分、又はホップを含む材料としたので、請求項１に記載された発明の効果に加えて、原料が入手しやすく浄化促進材料の取り扱いが容易である。 According to the invention of claim 2, when purifying harmful chemical substances using microorganisms, in the purification promoting material supplied to the purification target such as waste water, contaminated soil, contaminated groundwater, the material is hop, hop Since it is a material containing an extraction component or hops, in addition to the effect of the invention described in claim 1, raw materials are easily available and the handling of the purification promoting material is easy.
請求項３に係る発明によれば、汚染土壌や汚染地下水中に浄化に関わりの無い微生物の活動を抑制し、浄化に必要な有用微生物を優占化して活動を促進させるもの、あるいはホップ、ホップ抽出成分、又はホップを含む材料を添加するのみで、請求項１、２に記載された発明の効果に加えて、浄化環境を長期間に亘って適切な微生物群衆構造に制御することができ、また、有用微生物を導入することなく浄化に必要な有用微生物を優占化して活動を促進させることができるから、培養等に多大なコストを要することもない。 According to the invention according to claim 3, the activity of microorganisms not related to purification is suppressed in contaminated soil or contaminated groundwater, and the activity is promoted by dominating useful microorganisms necessary for purification, or hops, hops In addition to the effects of the inventions described in claims 1 and 2, the purification environment can be controlled to an appropriate microbial community structure over a long period of time simply by adding an extract component or a material containing hops. Moreover, since useful microorganisms necessary for purification can be predominated and activities can be promoted without introducing useful microorganisms, culturing and the like do not require significant costs.
請求項４乃至請求項６に係る発明よれば、デハロコッコイデス属細菌あるいはvcrＡ遺伝子を有する細菌は、ホップ、ホップ抽出成分、又はホップを含む材料の存在下においても増殖が抑制されることなく、その活動が速やかに促進されて有用な脱塩素化作用を呈し、有害な揮発性有機塩素化合物を塩素が存在しない無害な化合物まで変換することができる。 According to the inventions according to claims 4 to 6, the growth of the genus Dehalococcides or the bacteria having the vcrA gene is not suppressed even in the presence of hops, hop extract components, or hop-containing materials. The activity is promptly promoted to exhibit a useful dechlorination action, and harmful volatile organic chlorine compounds can be converted to harmless compounds that do not contain chlorine.
本発明は、抗菌材料であるホップ、又はホップの抽出成分を浄化環境に導入することにより、浄化に関わりの無い微生物の活動を抑制して環境中の有用微生物が従来方法に比較して短時間で優占化され、その有用微生物の活動を促進させて、浄化期間を短縮するものであり、この点に特徴がある。 The present invention introduces hops that are antibacterial materials, or hop extract components into the purification environment, thereby suppressing the activity of microorganisms that are not involved in purification, so that useful microorganisms in the environment can be produced in a shorter time than conventional methods. This is characterized by the fact that it is dominated by, promoting the activity of its useful microorganisms and shortening the purification period.
本発明では、微生物を用いて浄化する場合に、ホップ、ホップ抽出液、または、ホップ成分を含む材料からなる浄化促進材料を浄化対象とする水、土壌、地下水に導入するものである。 In the present invention, when purification is performed using microorganisms, a purification promoting material composed of hops, hop extract, or a material containing hop components is introduced into water, soil, or groundwater to be purified.
以下、塩素化エチレン汚染地下水を嫌気微生物により浄化（脱塩素化）する方法について、上記した浄化促進材料を用いた場合と用いない場合の浄化促進効果を比較した実施例について説明する。
一般的に、テトラクロロエチレンやトリクロロエチレンは還元的環境下で、微生物の働きによりエチレン、エタンにまで脱塩素化される。
この浄化方法では、汚染地下水中の有用な脱塩素化菌が、トリクロロエチレンをシス-1,2-ジクロロエチレンに、シス-1,2-ジクロロエチレンを塩化ビニルモノマーに、塩化ビニルモノマーをエチレンに順次脱塩素していく。脱塩素化菌の活動により、最終的には全ての塩素化エチレンが無害なエチレンまで変換される。
還元的環境下でテトラクロロエチレンやトリクロロエチレンを脱塩素化する微生物種のうち、エチレン、エタンまでの還元脱塩素化の反応に関与する微生物種として、いくつかのデハロコッコイデス（Dehalococcoides）属細菌が特定されている（参考文献１〜２）。
Hereinafter, with respect to a method of purifying (dechlorinating) chlorinated ethylene-contaminated groundwater with anaerobic microorganisms, an example in which the purification promotion effect is compared between the case where the above-described purification promotion material is used and the case where it is not used will be described.
In general, tetrachlorethylene and trichlorethylene are dechlorinated to ethylene and ethane by the action of microorganisms in a reducing environment.
In this purification method, useful dechlorinated bacteria in contaminated groundwater are dechlorinated in turn by trichlorethylene to cis-1,2-dichloroethylene, cis-1,2-dichloroethylene to vinyl chloride monomer, and vinyl chloride monomer to ethylene. I will do it. The activity of dechlorinated bacteria eventually converts all chlorinated ethylene to harmless ethylene.
Among the microbial species that dechlorinate tetrachlorethylene and trichlorethylene in a reductive environment, several Dehalococcoides bacteria have been identified as microbial species involved in the reductive dechlorination reaction up to ethylene and ethane (References 1-2).
本試験では、トリクロロエチレンおよびシス-1,2-ジクロロエチレンで汚染された人工地下水を作成し、ホップ抽出成分を添加して、ホップ抽出成分が微生物浄化に与える影響を把握することを目的とした室内培養試験を実施した。人工地下水の組成を表１に示す。 In this experiment, indoor culture was conducted to create artificial groundwater contaminated with trichlorethylene and cis-1,2-dichloroethylene, and to add the hop extract component to understand the effect of the hop extract component on microbial purification. The test was conducted. Table 1 shows the composition of artificial groundwater.
120mLのガラスバイアル瓶に上記人工地下水を90mL、トリクロロエチレンを10μmol/L、シス-1,2-ジクロロエチレンを30μmol/L、乳酸ナトリウムを400mg/L(有機物濃度で150mg/L)、塩素化エチレン汚染地下水から採取した土壌1gと蒸留水10gを混合後に得られた土壌上澄み液を1mL、をそれぞれ添加してテフロンコーティング処理済みのブチルゴム栓とアルミシール栓を用いて密栓した。 90 mL of the above artificial ground water in a 120 mL glass vial, 10 μmol / L of trichlorethylene, 30 μmol / L of cis-1,2-dichloroethylene, 400 mg / L of sodium lactate (150 mg / L in organic matter concentration), chlorinated ethylene contaminated ground water 1 g of soil supernatant obtained after mixing 1 g of soil collected from 10 g of distilled water and 10 g of distilled water was added, and each was sealed using a Teflon-coated butyl rubber plug and an aluminum seal plug.
次に、乾燥ホップ(粉末)を1%(w/v)で蒸留水に混合して作成したホップ成分抽出水を、個々のガラスバイアル瓶に、O%(v/v)(コントロール)、3％(v/v)、1％(v/v)、0.1％(v/v)添加して、20℃の恒温室内で82日間振とう培養した。 Next, hop component extraction water prepared by mixing dry hop (powder) with distilled water at 1% (w / v) is added to each glass vial, O% (v / v) (control), 3 % (V / v), 1% (v / v), and 0.1% (v / v) were added, and cultured with shaking in a thermostatic chamber at 20 ° C. for 82 days.
人工地下水中のトリクロロエチレン、シス-1,2-ジクロロエチレン、および塩化ビニルモノマーの培養期間中の経時変化を図１に、43日目におけるガラスバイアル瓶中の気相中のエチレン濃度を図２に、それぞれ示す。
この結果、全ての培養条件で浄化（脱塩素化）の傾向が確認されたが、培養開始から約1ヶ月後（36日目、43日目）では、浄化傾向に大きな差があることが確認できた。
培養開始から43日目の時点では、ホップ成分抽出水を添加していない条件と0.1％添加した条件ではシス-1,2-ジクロロエチレンの脱塩素化は確認されず、最終生成物であるエチレンの気相への生成も確認されなかった。
一方、ホップ成分抽出水を3％、および1％添加した条件では、脱塩素化傾向と最終生成物であるエチレンの気相への生成が確認され、ホップ成分抽出水の添加量の多い条件で脱塩素化が速く進行することが示された。
Figure 1 shows the time course of trichlorethylene, cis-1,2-dichloroethylene, and vinyl chloride monomer in the artificial ground water during the culture period, and Figure 2 shows the ethylene concentration in the gas phase in the glass vial on the 43rd day. Each is shown.
As a result, the tendency of purification (dechlorination) was confirmed under all culture conditions, but it was confirmed that there was a large difference in the purification tendency after about one month (36th and 43rd days) from the start of culture. did it.
At the time of the 43rd day from the start of culture, dechlorination of cis-1,2-dichloroethylene was not confirmed under the conditions where hop component extraction water was not added and when 0.1% was added, and the final product of ethylene was not confirmed. Formation in the gas phase was not confirmed.
On the other hand, under the conditions where 3% and 1% of hop component extraction water was added, dechlorination tendency and the formation of ethylene as the final product in the gas phase were confirmed. It was shown that dechlorination proceeds rapidly.
浄化に関わる細菌の増加傾向を把握するため、デハロコッコイデス属（Dehalococcoides属、参考文献１〜２）の16S rRNA遺伝子コピー数、およびシス-1,2-ジクロロエチレン以降の脱塩素化に関わる公知の機能遺伝子（参考文献３〜６）であるvcrA遺伝子コピー数をリアルタイムPCR法により求めた。
培養試料からのゲノムDNAの取得方法、およびデハロコッコイデス属の16S rRNA遺伝子数のリアルタイムPCRによる決定方法については、次に示すとおりである。
16S rRNA gene copy number of Dehalococcoides genus (References 1-2) and known chlorination after cis-1,2-dichloroethylene to grasp the increasing tendency of bacteria related to purification The number of copies of the vcrA gene, which is a functional gene (Reference Documents 3 to 6), was determined by real-time PCR.
A method for obtaining genomic DNA from a culture sample and a method for determining the number of 16S rRNA genes of the genus Dehalococcides by real-time PCR are as follows.
ゲノムDNAを取得するため、培養試料5mlを濾過したポリカーボネート製メンブレンフィルター（孔径0.2 mm）を20mg/ml-プロテナーゼK（3μL）、10％-Sodium dodecyl sulfate（30μL）、TEバッファー（567μL）を含む溶液内において50℃で1時間静置して、続いて5M-NaCl(100μL)、CATB/NaCl溶液（80μL）を加えて、65℃で10分間静置培養した。 In order to obtain genomic DNA, a polycarbonate membrane filter (pore size 0.2 mm) filtered with 5 ml of culture sample contains 20 mg / ml-proteinase K (3 μL), 10% -Sodium dodecyl sulfate (30 μL), and TE buffer (567 μL) The solution was allowed to stand at 50 ° C. for 1 hour, followed by addition of 5M-NaCl (100 μL) and CATB / NaCl solution (80 μL), followed by stationary culture at 65 ° C. for 10 minutes.
次に、クロロホルム/イソアミルアルコール（800μL）を加えて混合後、15000rpm、5分間の遠心分離後に上清液600μLを回収し、フェノール（300μL）、クロロホルム/イソアミルアルコール（24：1，300μL）を加えて、15000rpm、5分間の遠心分離後、上清550μLを回収した。そこに、3M-酢酸ナトリウム（55μL）、イソプロピルアルコール（550μL）を加え、15000rpm、10分間の遠心分離後に上精液を捨て、70％エタノール（1mL）を加え、15000rpm、10分間の遠心分離後に上精液を捨て、DNAを回収した。 Next, after adding chloroform / isoamyl alcohol (800 μL) and mixing, after centrifugation at 15000 rpm for 5 minutes, collect 600 μL of the supernatant and add phenol (300 μL) and chloroform / isoamyl alcohol (24: 1, 300 μL). After centrifugation at 15000 rpm for 5 minutes, 550 μL of the supernatant was recovered. 3M-sodium acetate (55 μL) and isopropyl alcohol (550 μL) were added, and after centrifuging at 15000 rpm for 10 minutes, the upper semen was discarded, 70% ethanol (1 mL) was added, and after centrifugation at 15000 rpm for 10 minutes, Semen was discarded and DNA was collected.
続いて、リアルタイムPCR法により、デハロコッコイデス属16S rRNA遺伝子コピー数を以下の方法で定量した。リアルタイムPCR法により増幅させる遺伝子領域は、デハロコッコイデス属細菌に共通する配列となる16S rDNA上の特異的領域を増幅させるプライマーとして「5’-CAGCAGGAGAAAACGGAATT-3’」と「5’-GACAGCTTTGGGGATTAGC-3’」を用い、リアルタイムPCRでの遺伝子断片検出法はSYBR Green Iを使用したインターカレーター方式とした。 Subsequently, the number of dehalococcides 16S rRNA gene copy was quantified by the following method by real-time PCR. The gene region amplified by the real-time PCR method is `` 5'-CAGCAGGAGAAAACGGAATT-3 '' 'and `` 5'-GACAGCTTTGGGGATTAGC- 3 '"and the gene fragment detection method by real-time PCR was an intercalator method using SYBR Green I.
以下に1検体当たりのPCR反応組成を示す。上記方法で抽出したゲノムDNAは50μlのTEバッファーに溶解して使用した。なお、本試験ではブランクを設け、PCRの反応液にはDNA抽出物の代わりに滅菌蒸留水を混合した。 The PCR reaction composition per sample is shown below. The genomic DNA extracted by the above method was used after dissolving in 50 μl of TE buffer. In this test, a blank was provided, and sterilized distilled water was mixed in the PCR reaction solution instead of the DNA extract.
2×SYBR Premix（タカラバイオ社製） 12.5μl
100μM 624-Fw 0.1μl
100μM 1232-Rv 0.1μl
50×ROX Dye（タカラバイオ社製） 0.5μl
DNA 抽出物 1.0μl
滅菌超純水 10.8μl
計 25.0μl
2 x SYBR Premix (Takara Bio) 12.5μl
100μM 624-Fw 0.1μl
100μM 1232-Rv 0.1μl
50 × ROX Dye (Takara Bio Inc.) 0.5μl
DNA extract 1.0 μl
Sterile ultrapure water 10.8μl
25.0μl total
リアルタイムPCR反応は、初期変性（95℃、30秒）の後、変性（95℃、5秒）・アニーリング（54℃、30秒）・伸長反応（72℃、45秒）を40回繰り返し、最後に60℃から95℃まで段階的に温度を上昇させる条件とした。地下水中のデハロコッコイデス属細菌のコピー数を定量するために、既知のデハロコッコイデス属細菌の16S rDNA領域をpGEM T-Easyベクター（Promega社製）にクローニングしたプラスミドDNAをスタンダードとして使用し、102〜108copiesの検出値について検量線を作成した。 For real-time PCR, after initial denaturation (95 ° C, 30 seconds), denaturation (95 ° C, 5 seconds), annealing (54 ° C, 30 seconds), and extension reaction (72 ° C, 45 seconds) are repeated 40 times. The temperature was raised stepwise from 60 ° C to 95 ° C. In order to quantify the copy number of Dehalococcides genus in groundwater, plasmid DNA obtained by cloning the 16S rDNA region of known Dehalococcides bacterium into pGEM T-Easy vector (Promega) is used as a standard. A calibration curve was prepared for the detected values of 10 2 to 10 8 copies.
＜参考文献＞
［参考文献１］X.Maymo−Gatell,Y.-T. Chien,J.M.Gossett,and S.H.Zinder,Sience,276:1568-1571,1997.
［参考文献２］D.E.Fennell,I.Nijenhuis,S.F.Wilson,S.H.Zinder,and M.M.Haggblom,Environ.Sci.Technol.,38:2075-2081,2004.
［参考文献３］J.He,K.M.Ritalahti,M.R.Aiello,and F.E.Loffler,Appl.Environ.Microbiol.,69:996-1003,2003.
［参考文献４］M.Duhamel,K.Mo,and E.A.Edwards,Appl.Environ.Microbiol.,70:5538-5545,2004.
［参考文献５］J.A.Muller,B.M.Rosner,G.von Abendroth,G.Meshulam-Simon,P.L.McCarty,and A.M.Spormann.,Appl.Environ.Microbiol.,70:4880-4888,2004.
［参考文献６］K.M.Ritalahti,B.K.Amos,Y.Sung,Q.Wu,S.S.Koenigsberg,and F.E.Loffler,Appl.Environ.Microbiol.72:2765-2774,2006.
<References>
[Reference 1] X. Maymo-Gatell, Y.-T. Chien, JMGossett, and SHZinder, Sience, 276: 1568-1571, 1997.
[Reference 2] DEFennell, I. Nijenhuis, SFWilson, SHZinder, and MM Haggblom, Environ. Sci. Technol., 38: 2075-2081, 2004.
[Reference 3] J. He, KMRitalahti, MRAiello, and FELoffler, Appl. Environ. Microbiol., 69: 996-1003, 2003.
[Reference 4] M. Duhamel, K. Mo, and EAEdwards, Appl. Environ. Microbiol., 70: 5538-5545, 2004.
[Reference 5] JAMuller, BM Rosner, G. von Abendroth, G. Meshulam-Simon, PLMcCarty, and AM Spormann., Appl. Environ. Microbiol., 70: 4880-4888, 2004.
[Reference 6] KMRitalahti, BKAmos, Y. Sung, Q. Wu, SS Koenigsberg, and FELoffler, Appl. Environ. Microbiol. 72: 2765-2774, 2006.
vcrA遺伝子コピー数は、ゲノムDNAの採取後にリアルタイムPCR法により以下の方法で定量した。リアルタイムPCR法により機能遺伝子vcrAの特異的領域を増幅させるプライマーとして「5’-CGGGCGGATGCACTATTTT-3’」、および「5’-GAATAGTCCGTGCCCTTCCTC-3’」を用い、5’末端を蛍光物質（FAM）で、3’末端をクエンチャー物質（TAMRA）で修飾したオリゴヌクレオチド「5’-CGCAGTAACTCAACCATTTCCTGGTAGTGG-3’」をプローブとして用いた。
リアルタイムPCR法での遺伝子断片検出方法は、蛍光プローブ法とした。
The vcrA gene copy number was quantified by the following method by real-time PCR after collecting genomic DNA. "5'-CGGGCGGATGCACTATTTT-3 '" and "5'-GAATAGTCCGTGCCCTTCCTC-3'" are used as primers to amplify a specific region of the functional gene vcrA by real-time PCR, and the 5 'end is a fluorescent substance (FAM). Oligonucleotide “5′-CGCAGTAACTCAACCATTTCCTGGTAGTGG-3 ′” whose 3 ′ end was modified with a quencher substance (TAMRA) was used as a probe.
The gene fragment detection method by the real-time PCR method was a fluorescent probe method.
以下に1検体辺りのPCR反応組成を示す。上記方法で抽出したDNAは50μlのTEバッファーに溶解して使用した。なお、本試験ではブランクを設け、PCRの反応液にはDNA抽出物の代わりに滅菌蒸留水を混合した。 The PCR reaction composition per sample is shown below. The DNA extracted by the above method was used after dissolving in 50 μl of TE buffer. In this test, a blank was provided, and sterilized distilled water was mixed in the PCR reaction solution instead of the DNA extract.
2×Premix EX Tag（タカラバイオ社製） 12.5μl
100μM 1022-Fw 0.1μl
100μM 1093-Rv 0.1μl
100μM1042-Probe 0.1μl
50×ROX Dye（２）（タカラバイオ社製） 0.5μl
DNA 抽出物 1.0μl
滅菌超純水 10.7μl
計 25.0μl
2 x Premix EX Tag (Takara Bio) 12.5μl
100μM 1022-Fw 0.1μl
100μM 1093-Rv 0.1μl
100μM1042-Probe 0.1μl
50 × ROX Dye (2) (Takara Bio Inc.) 0.5μl
DNA extract 1.0 μl
Sterile ultrapure water 10.7μl
25.0μl total
リアルタイムPCR反応は、初期変性（95℃、10秒）の後、変性（95℃、15秒）、アニーリング（58℃、1分）を40サイクル行い、特定遺伝子のコピー数を求めた。地下水中の機能遺伝子を保有する微生物種を定量するために、完全脱塩素化の培養系から既知のvcrA遺伝子領域をpGEM T-Easyベクター（Promega社製）にクローニングしたプラスミドDNAをスタンダードとして使用し、102〜108 copiesの検出値について検量線を作成した。 In the real-time PCR reaction, after initial denaturation (95 ° C., 10 seconds), denaturation (95 ° C., 15 seconds) and annealing (58 ° C., 1 minute) were performed 40 cycles to determine the copy number of a specific gene. In order to quantify microbial species possessing functional genes in groundwater, plasmid DNA obtained by cloning a known vcrA gene region into a pGEM T-Easy vector (Promega) from a complete dechlorination culture system was used as a standard. A calibration curve was prepared for the detected values of 10 2 to 10 8 copies.
本方法により、培養開始から43日目におけるデハロコッコイデス属細菌の16S rRNA遺伝子コピー数、およびvcrA遺伝子コピー数を定量した結果、両遺伝子数は図３に示されるように同様の傾向を示した。 As a result of quantifying the 16S rRNA gene copy number and the vcrA gene copy number of the dehalococcideae bacteria on the 43rd day from the start of the culture by this method, both gene numbers showed the same tendency as shown in FIG. It was.
デハロコッコイデス属細菌の16S rRNA遺伝子コピー数、およびvcrA遺伝子コピー数は、いずれも脱塩素化がみられたホップ成分抽出水を3％、および1％添加した条件で顕著に増加していることが確認できた。 Both 16S rRNA gene copy number and vcrA gene copy number of dehalococcideae bacteria are markedly increased under the condition that 3% and 1% of hop component extraction water with dechlorination was added. I was able to confirm.
一方、培養開始から43日目における添加した有機物（乳酸）量に対して培養後に消費された有機物の割合を示す消費率は、図４に示されるように、ホップ成分抽出水の添加量が多いほど小さかった。 On the other hand, as shown in FIG. 4, the consumption rate indicating the ratio of the organic matter consumed after the cultivation to the amount of the added organic matter (lactic acid) on the 43rd day from the start of the cultivation is large in the amount of hop component extraction water added. It was so small.
さらに、培養開始から43日目における人工地下水中に存在する微生物全体の増殖活性を示す指標となるATPは、図５に示されるように、ホップ成分抽出水を添加すると低くなり、特に添加量が多くなるほどATP活性が低くなった。 Furthermore, as shown in FIG. 5, ATP, which is an index indicating the growth activity of all microorganisms present in the artificial ground water on the 43rd day from the start of the culture, becomes low when hop component extraction water is added, and the added amount is particularly high. The ATP activity decreased as the amount increased.
以上の試験結果から、以下の４点が明らかとなった。
（１）ホップ成分抽出水の添加量の多い条件で脱塩素化が速く進行すること。
（２）脱塩素化菌数の指標となる遺伝子コピー数は、ホップ成分抽出水を所定量以上添加した条件で顕著に増加すること。
（３）有機物の消費率は、ホップ成分抽出水の添加量が多いほど小さいこと。
（４）人工地下水中に存在する微生物全体の増殖活性を示す指標となるATPは、ホップ成分抽出水の添加量が多くなるほど活性が低くなること。
From the above test results, the following four points became clear.
(1) Dechlorination proceeds rapidly under conditions where the amount of hop component extraction water added is large.
(2) The number of gene copies serving as an index of the number of dechlorinated bacteria should be significantly increased under the condition where a predetermined amount or more of hop component extraction water is added.
(3) The consumption rate of organic matter is so small that there is much addition amount of hop component extraction water.
(4) The activity of ATP, which is an indicator of the growth activity of all microorganisms present in artificial groundwater, decreases as the amount of hop component extraction water added increases.
上記(１)と（２）から、ホップ成分抽出水の添加が、脱塩素化に関わる有用細菌数を増加させ、脱塩素化を速く進行させることが明らかである。
逆に上記（２）(３)及び（４）からは、ホップ成分抽出水の添加が、浄化に関係しない細菌の増殖は抑制されることを窺い知ることができる。
From the above (1) and (2), it is clear that the addition of hop component extraction water increases the number of useful bacteria involved in dechlorination and allows dechlorination to proceed rapidly.
Conversely, from the above (2), (3) and (4), it can be known that the addition of hop component extracted water suppresses the growth of bacteria not related to purification.
以上の結果を総合すると、ホップ抽出成分を存在させることにより、浄化に無関係な細菌の増殖は抑制され、脱塩素化に関わる有用細菌数を増加させて脱塩素化を促進させる作用効果があることが明らかとなった。 In summary, the presence of the hop extract component suppresses the growth of bacteria unrelated to purification and has the effect of promoting the dechlorination by increasing the number of useful bacteria involved in dechlorination. Became clear.
本発明は、浄化促進材料はホップ抽出成分を添加した上記実施例に限られず、ホップまたは乾燥ホップそのものを添加してもよい。
要するに、浄化対象に対しホップ中に含有される成分を添加できる材料であれば浄化促進材料とすることができる。
また、浄化に必要な有用微生物はデハロコッコイデス属細菌あるいはvcrA遺伝子をもつ細菌に限定されず、ホップ、ホップ抽出成分、又はホップを含む材料を添加することにより、活性化する微生物であればなんでもよい。
In the present invention, the purification promoting material is not limited to the above-described embodiment to which a hop extraction component is added, and hops or dried hops themselves may be added.
In short, any material that can add components contained in hops to be purified can be a purification promoting material.
In addition, useful microorganisms necessary for purification are not limited to bacteria having the genus Dehalococcides or vcrA, but any microorganism that can be activated by adding hops, hop extract components, or materials containing hops. Anything.
Claims (3)
前記浄化促進材料として、ホップ、乾燥ホップ、ホップ抽出成分、又はホップを含む材料を添加することを特徴とする浄化促進方法。 When purifying purification targets such as waste water, contaminated soil, and contaminated groundwater contaminated with volatile organic chlorinated compounds using microorganisms, a purification promotion material is added to the purification targets to promote purification by microorganisms. In the method
A purification promotion method comprising adding a hop, a dry hop, a hop extract component, or a material containing hop as the purification promotion material.
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