JP2009060795A - Method for analyzing bacterium contained in activated sludge - Google Patents

Method for analyzing bacterium contained in activated sludge Download PDF

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Publication number
JP2009060795A
JP2009060795A JP2007228704A JP2007228704A JP2009060795A JP 2009060795 A JP2009060795 A JP 2009060795A JP 2007228704 A JP2007228704 A JP 2007228704A JP 2007228704 A JP2007228704 A JP 2007228704A JP 2009060795 A JP2009060795 A JP 2009060795A
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bacteria
dna
activated sludge
sulfur
genus
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Yosuke Nakamura
洋介 中村
Hiroto Nishijima
裕人 西島
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing bacteria contained in activated sludge, by which the kinds and population of the bacteria in the activated sludge can be known quickly. <P>SOLUTION: This method for analyzing the bacteria contained in the activated sludge is characterized by extracting RNAs of the bacteria contained in the activated sludge, subjecting the extracted RNAs to a reverse transcription to obtain the reverse transcription DNAs, and then hybridizing the obtained reverse transcription DNAs with the DNA fragments of the following DNA chip. The DNA chip is such that a DNA chip is obtained, by immobilizing DNA fragments containing the DNA base sequences of Nocardia filamentous bacteria, Rhodococcus filamentous bacteria, Beggiatoa filamentous bacteria, Thiothrix filamentous bacteria, Desulfobacter sulfur metabolism bacteria, Desulfotomaculum sulfur metabolism bacteria, Desulfovibrio sulfur metabolism bacteria, Desulfomicrobium sulfur metabolism bacteria, Desulforhopalus sulfur metabolism bacteria, Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacterium, and Nitrospira nitrifying bacterium on a substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、活性汚泥に含まれる細菌の分析方法に関する。 The present invention relates to a method for analyzing bacteria contained in activated sludge.

活性汚泥を用いて、これに生息している種々の細菌により、排水に含まれる汚染物質、例
えば有機態炭素などを生物学的に分解する、いわゆる活性汚泥法は、排水を浄化する排水
処理方法は、生物学的に排水を処理する生物学的排水処理方法として広く用いられている
〔特許文献1:特開平5−237488号公報、特許文献2:特開2000−27998
2号公報など〕。
The so-called activated sludge method, which uses activated sludge to biologically decompose pollutants contained in wastewater, such as organic carbon, by various bacteria that inhabit it, is a wastewater treatment method that purifies wastewater. Is widely used as a biological wastewater treatment method for treating wastewater biologically [Patent Document 1: JP-A-5-237488, Patent Document 2: JP-A 2000-27998.
No. 2 publication etc.].

活性汚泥に含まれる細菌は、それぞれ特定の汚染物質を選択的に分解するのが通常である
。例えば硫黄代謝細菌は、無機硫黄化合物などを選択的に分解するが、有機態炭素、アン
モニア態窒素などは分解しない。しかも、活性汚泥中の細菌は、汚染物質を分解すると、
分解量に応じて生息数が増加するが、その一方で、分解すべき汚染物質が無い状態が続く
と、次第に死滅して減少する。
In general, bacteria contained in activated sludge each selectively decompose specific pollutants. For example, sulfur-metabolizing bacteria selectively decompose inorganic sulfur compounds and the like, but do not decompose organic carbon, ammonia nitrogen, and the like. Moreover, when bacteria in activated sludge break down pollutants,
The number of inhabitants increases according to the amount of decomposition, but on the other hand, if there is no pollutant to be decomposed, it will gradually die and decrease.

また、同一の排水源からの排水に含まれる汚染物質の種類やその濃度は、長期間に亘り概
ね一定の範囲内にあるのが通常である。例えば家庭から排出される生活排水は、通常、有
機態炭素を主な汚染物質とし、その濃度は概ね一定の範囲内である。
In addition, the types and concentrations of pollutants contained in the drainage from the same drainage source are usually within a certain range over a long period of time. For example, domestic wastewater discharged from households usually has organic carbon as the main pollutant, and its concentration is generally within a certain range.

このため、活性汚泥中の細菌の種類やその生息数は、長期間、同じ排水源からの排水を処
理し続けることより、その排水の処理に適切なものとなるのが通常である。
For this reason, the type of bacteria in the activated sludge and the number of inhabitants are usually more appropriate for the treatment of the wastewater than the treatment of the wastewater from the same wastewater source for a long time.

しかし、活性汚泥中の細菌が、夏場の高温、突発的な被毒物質の混入、汚染物質の急激な
増加など、様々な原因により、全て死滅する場合がある。このような場合に、他の排水源
からの排水の処理に使用している活性汚泥を投入し、排水処理を再開することがあるが、
このような活性汚泥は、処理すべき排水に含まれる汚染物質に必ずしも適したものではな
い。このため、汚染物質の分解が効率よく行われず、排水の処理が滞ると言う問題があっ
た。
However, the bacteria in the activated sludge may all be killed due to various causes such as high temperatures in summer, sudden contamination with poisonous substances, and sudden increase in pollutants. In such a case, the activated sludge used to treat wastewater from other wastewater sources may be thrown in and wastewater treatment resumed.
Such activated sludge is not necessarily suitable for pollutants contained in the wastewater to be treated. For this reason, there was a problem that the pollutants were not efficiently decomposed and the wastewater treatment was delayed.

かかる問題を解決するには、処理すべき排水に対して適切な種類の細菌を適切な生息数で
含む活性汚泥を選択して投入し、排水処理を再開すればよい。このため、活性汚泥中の細
菌の種類や、その生息数を速やかに、精度よく、知りうる方法が求められている。
In order to solve such a problem, it is only necessary to select and input activated sludge containing an appropriate type of bacteria with an appropriate number of inhabitants for the wastewater to be treated, and restart the wastewater treatment. For this reason, there is a need for a method that can quickly and accurately know the types of bacteria in activated sludge and the number of inhabitants.

活性汚泥中の細菌を分析する方法としては、従来から、顕微鏡により細菌の形状や個数を
観察する方法が知られている〔特許文献3:特開平5−146791号公報〕。
As a method for analyzing bacteria in activated sludge, a method of observing the shape and number of bacteria with a microscope has been conventionally known [Patent Document 3: Japanese Patent Laid-Open No. Hei 5-14691].

しかし、顕微鏡による従来の分析方法は、細菌を速やかに、精度よく分析しうるものでは
なかった。
However, the conventional analysis method using a microscope cannot analyze bacteria quickly and accurately.

特開平5−237488号公報JP-A-5-237488 特開2000−279982号公報JP 2000-279982 A 特開平5−146791号公報Japanese Patent Laid-Open No. 5-146791 SCAS NEWS 2005−II〔株式会社住化分析センター(2005年8月22日)発行、第3頁〜第6頁〕SCAS NEWS 2005-II (Sumitomo Analysis Center Co., Ltd. (August 22, 2005), pages 3-6)

そこで本発明者は、活性汚泥中の細菌の種類や生息数を速やかに知るべく鋭意検討した結
果、本発明に至った。
Therefore, the present inventor has intensively studied to quickly know the type and number of bacteria in the activated sludge, resulting in the present invention.

すなわち本発明は、活性汚泥に含まれる細菌のRNA〔リボ核酸〕を抽出し、抽出されたRNAの逆転写を行って逆転写DNA〔逆転写デオキシリボ核酸〕を得、得られた逆転写DNAを以下のDNAチップのDNA断片と交雑させることを特徴とする前記細菌の分析方法を提供するものである。 That is, the present invention extracts bacterial RNA [ribonucleic acid] contained in activated sludge, performs reverse transcription of the extracted RNA to obtain reverse transcription DNA [reverse transcription deoxyribonucleic acid], and obtains the reverse transcription DNA obtained The present invention provides a method for analyzing bacteria, which is characterized by crossing with a DNA fragment of the following DNA chip.

DNAチップ:基板上に、
Nocardia属糸状性細菌、Rhodococcus属糸状性細菌、
Beggiatoa属糸状性細菌硫黄代謝細菌、Thiothrix属糸状性細菌硫黄代謝細菌、
Desulfobacter属硫黄代謝細菌、Desulfotomaculum属硫黄代謝細菌、Desulfovibrio属硫黄
代謝細菌、Desulfomicrobium属硫黄代謝細菌、Desulforhopalus属硫黄代謝細菌、
Nitrosomonas属硝化細菌、Nitrosospira属硝化細菌およびNitrospira属硝化細菌
のDNA塩基配列を含むDNA断片が固定されてなるDNAチップ
DNA chip: on the substrate,
Nocardia spp., Rhodococcus spp.,
Beggiatoa filamentous bacteria sulfur metabolizing bacteria, Thiothrix filamentous bacteria sulfur metabolizing bacteria,
Desulfobacter genus sulfur-metabolizing bacteria, Desulfotomaculum genus sulfur-metabolizing bacteria, Desulfovibrio genus sulfur-metabolizing bacteria, Desulfomicrobium genus sulfur-metabolizing bacteria, Desulforhopalus genus sulfur-metabolizing bacteria,
Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacteria, and a DNA chip on which DNA fragments containing DNA base sequences of Nitrospira genus nitrifying bacteria are fixed

本発明の分析方法は、活性汚泥に含まれる細菌の中でも、硝化細菌の分析に好適である。 The analysis method of the present invention is suitable for analysis of nitrifying bacteria among bacteria contained in activated sludge.

本発明の分析方法によれば、活性汚泥中の細菌の種類を知り、また、その生息数を見積もる分析を、速やかに、精度よく、行うことができる。 According to the analysis method of the present invention, the type of bacteria in the activated sludge can be known and the analysis for estimating the number of inhabitants can be performed quickly and accurately.

〔DNAチップを構成する基板〕
本発明の分析方法に用いられる上記DNAチップを構成する基板として通常は、無機ガラス製の板が挙げられる。基板のサイズとしては、取扱いが容易である点で、通常は厚み0.5mm〜2mmで、短辺が10mm〜20mm、長辺が50mm〜100mmのものが使用される。このような基板としては、光学顕微鏡観察に広く用いられているスライドグラスが、入手が容易である点で、好ましく用いられ
[Substrate constituting the DNA chip]
The substrate constituting the DNA chip used in the analysis method of the present invention usually includes an inorganic glass plate. As the size of the substrate, one having a thickness of 0.5 mm to 2 mm, a short side of 10 mm to 20 mm, and a long side of 50 mm to 100 mm is usually used because it is easy to handle. As such a substrate, a slide glass widely used for optical microscope observation is preferably used because it is easily available.

〔細菌〕
かかる基板の上に固定されるDNA断片は、上記した糸状性細菌、硫黄代謝細菌および硝
化細菌のDNA塩基配列を含むものである。
[Bacteria]
The DNA fragment fixed on such a substrate contains the DNA base sequences of the filamentous bacteria, sulfur-metabolizing bacteria and nitrifying bacteria described above.

〔糸状性細菌〕
糸状性細菌は、活性汚泥による汚染物質の分解に対して障害となりうる細菌である。糸状
性細菌の中でも、上記したNocardia属、Rhodococcus属のものは、過剰に繁殖すると活性
汚泥が浮上する膨化と呼ばれる現象の原因となって活性汚泥による分解に対する障害とな
り易いものとして、多くの活性汚泥に含まれうるものである。
[Filamentous bacteria]
Filamentous bacteria are bacteria that can hinder the degradation of pollutants by activated sludge. Among the filamentous bacteria, those of the genus Nocardia and Rhodococcus mentioned above are considered to be prone to hindrance to decomposition by activated sludge as a cause of the phenomenon called activated sludge that rises when excessively propagated. Can be included.

上記DNAチップは、上記した糸状性細菌以外の糸状性細菌のDNA塩基配列を含むDNA断片が固定されていてもよく、中でもSphaerotilus属糸状性細菌は、比較的多くの活性汚泥に含まれうることから、これのDNA塩基配列を含むDNA断片が固定されていることが好ましい。 The DNA chip may be fixed with a DNA fragment containing a DNA base sequence of a filamentous bacterium other than the filamentous bacterium described above, and among them, the genus Sphaerotilus can be contained in a relatively large amount of activated sludge. Therefore, it is preferable that a DNA fragment containing the DNA base sequence is fixed.

〔硫黄代謝細菌〕
硫黄代謝細菌は、硫黄または無機硫黄化合物を酸化しうる細菌であり、排水が硫黄または
無機硫黄化合物を含む場合に、活性汚泥に多く含まれるものである。硫黄代謝細菌の中で
も上記したDesulfobacter属、Desulfotomaculum属、Desulfovibrio属、Desulfomicrobium
属およびDesulforhopalus属のものは、活性汚泥により、その量が大きく変化しうるもの
である。
[Sulfur metabolizing bacteria]
A sulfur-metabolizing bacterium is a bacterium that can oxidize sulfur or an inorganic sulfur compound, and is abundant in activated sludge when waste water contains sulfur or an inorganic sulfur compound. Among the sulfur-metabolizing bacteria, the genus Desulfobacter, Desulfotomaculum, Desulfovibrio, and Desulfomicrobium described above
The genus and those of the genus Desulforhopalus can vary greatly depending on the activated sludge.

上記DNAチップは、上記した硫黄代謝細菌以外の硫黄代謝細菌のDNA塩基配列を含むDNA断片が固定されていてもよく、中でもDesulfococcus属硫黄代謝細菌、Desulfomonas属硫黄代謝細菌、Desulfuromonas属硫黄代謝細菌、Desulfurella属硫黄代謝細菌、Desulfomonile属硫黄代謝細菌、Desulfosarcina属硫黄代謝細菌、Lawsonia属硫黄代謝細菌は、活性汚泥により、その量が比較的大きく変化しうることから、そのDNA塩基配列を含むDNA断片が固定されていることが好ましい。 The DNA chip may be fixed with a DNA fragment containing a DNA base sequence of a sulfur-metabolizing bacterium other than the above-described sulfur-metabolizing bacterium, among which, Desulfococcus genus sulfur-metabolizing bacteria, Desulfomonas genus sulfur-metabolizing bacteria, Desulfuromonas genus sulfur-metabolizing bacteria, Desulfurella genus sulfur-metabolizing bacteria, Desulfomonile genus sulfur-metabolizing bacteria, Desulfosarcina genus sulfur-metabolizing bacteria, and Lawsonia genus sulfur-metabolizing bacteria can vary in amount by activated sludge. It is preferably fixed.

〔糸状性細菌硫黄代謝細菌〕
糸状性細菌硫黄代謝細菌は、糸状性細菌の性質と、硫黄代謝細菌の性質を併せ持つ細菌で
あり、例えばBeggiatoa属糸状性細菌硫黄代謝細菌、Thiothrix属糸状性細菌硫黄代謝細菌
などが挙げられる。
[Filamentous bacteria sulfur-metabolizing bacteria]
Filamentous bacteria sulfur-metabolizing bacteria are bacteria having both the properties of filamentous bacteria and the properties of sulfur-metabolizing bacteria, and examples thereof include Beggiatoa filamentous bacteria, sulfur-metabolizing bacteria, and Thiothrix genus filamentous bacteria.

〔硝化細菌〕
硝化細菌は、アンモニア態窒素または亜硝酸態窒素を酸化する菌であって、排水がアンモ
ニア態窒素を含む場合に、活性汚泥に多く含まれるものであり、アンモニア態窒素を亜硝
酸に酸化するアンモニア酸化細菌、または亜硝酸を硝酸に酸化する亜硝酸酸化細菌である
。上記したNitrosomonas属硝化細菌(アンモニア酸化細菌)、Nitrosospira属硝化細菌(
アンモニア酸化細菌)およびNitrospira属硝化細菌(亜硝酸酸化細菌)は、活性汚泥によ
り、その量が大きく変化するものである。
[Nitrifying bacteria]
Nitrifying bacteria are bacteria that oxidize ammonia nitrogen or nitrite nitrogen, and are abundant in activated sludge when wastewater contains ammonia nitrogen, and ammonia that oxidizes ammonia nitrogen to nitrite Oxidizing bacteria or nitrite oxidizing bacteria that oxidize nitrite to nitric acid. Nitrosomonas genus nitrifying bacteria (ammonia-oxidizing bacteria), Nitrosospira genus nitrifying bacteria (
Ammonia-oxidizing bacteria) and Nitrospira genus nitrifying bacteria (nitrite-oxidizing bacteria) vary greatly depending on the activated sludge.

上記DNAチップは、上記した以外の硝化細菌のDNA塩基配列を含むDNA断片が固定されていてもよく、中でもNitrosolobus属硝化細菌(アンモニア酸化細菌)、Nitrobacter属硝化細菌(亜硝酸酸化細菌)は、活性汚泥により、その量が比較的大きく変化することから、そのDNA塩基配列を含むDNA断片が固定されていることが好ましい。 The DNA chip may have a DNA fragment containing a DNA base sequence of a nitrifying bacterium other than those described above, among which Nitrosolobus genus nitrifying bacteria (ammonia oxidizing bacteria), Nitrobacter genus nitrifying bacteria (nitrite oxidizing bacteria), Since the amount of activated sludge changes relatively greatly, it is preferable that the DNA fragment containing the DNA base sequence is fixed.

〔その他の細菌〕
上記DNAチップは、上記した糸状性細菌、硫黄代謝細菌、硝化細菌以外の細菌のDNA塩基配列を含むDNA断片が固定されていてもよい。かかる細菌は、好気性細菌であってもよいし、嫌気性細菌であってもよく、例えばAcinetobacter属ポリリン酸蓄積細菌、Arthrobacter属ポリリン酸蓄積細菌、Microlunatas属ポリリン酸蓄積細菌などが挙げられる。また、上記した以外の糸状性細菌、硫黄代謝細菌、糸状性細菌硫黄代謝細菌、硝化細も挙げられる。
[Other bacteria]
The DNA chip may have a DNA fragment containing a DNA base sequence of bacteria other than the filamentous bacteria, sulfur-metabolizing bacteria, and nitrifying bacteria described above. Such a bacterium may be an aerobic bacterium or an anaerobic bacterium, and examples thereof include Acinetobacter genus polyphosphate accumulating bacteria, Arthrobacter genus polyphosphate accumulating bacteria, Microlunatas genus polyphosphate accumulating bacteria and the like. Other examples include filamentous bacteria, sulfur-metabolizing bacteria, filamentous bacteria, sulfur-metabolizing bacteria, and nitrification cells.

〔DNAチップの調製〕
かかるDNAチップは、例えば
(1)上記各細菌のDNA塩基配列を含むDNA断片を調製するDNA断片調製工程および
(2)DNA断片調製工程で得られたDNA断片を基板上に固定するDNA断片固定工程
を経て調製することができる。
[Preparation of DNA chip]
Such a DNA chip is, for example,
(1) a DNA fragment preparation step for preparing a DNA fragment containing the DNA base sequence of each of the above bacteria;
(2) It can be prepared through a DNA fragment fixing step in which the DNA fragment obtained in the DNA fragment preparing step is fixed on a substrate.

〔DNA断片調製工程〕
DNA断片は、通常の方法、例えば目的とする細菌を溶菌し、DNAを抽出し、得られた
DNAを増幅する方法、例えばポリメラーゼ連鎖反応(PCR反応)により調製すること
ができる。DNA断片は両端がDNAプライマーで固定されていてもよい。
[DNA fragment preparation step]
The DNA fragment can be prepared by an ordinary method, for example, a method for lysing a target bacterium, extracting DNA, and amplifying the obtained DNA, for example, a polymerase chain reaction (PCR reaction). Both ends of the DNA fragment may be fixed with a DNA primer.

〔DNA断片固定工程〕
上記DNA断片調製工程で得られたDNA断片は、通常の方法、例えば上記で得られたD
NA断片を溶媒で希釈し、基板上にスポット状に付着させ、次いで乾燥すればよい。
[DNA fragment fixing step]
The DNA fragment obtained in the DNA fragment preparation step can be obtained by a conventional method, for example, D obtained above.
The NA fragment may be diluted with a solvent, attached to the substrate in a spot shape, and then dried.

得られたDNAチップは、通常0℃以下、好ましくは−15℃以下で保存される。 The obtained DNA chip is usually stored at 0 ° C. or lower, preferably −15 ° C. or lower.

〔細菌の分析〕
本発明の方法は、かかるDNAチップを用いて活性汚泥に含まれる細菌の種類および量を分析するものである。
[Bacteria analysis]
The method of the present invention analyzes the type and amount of bacteria contained in activated sludge using such a DNA chip.

具体的には、
(1)活性汚泥から細菌を抽出する細菌抽出工程、
(2)細菌抽出工程で得た細菌を溶菌したのちRNAを抽出する溶菌・RNA抽出工程、
(3)溶菌・RNA抽出工程で抽出されたRNAの逆転写を行い逆転写DNAを得る逆転写工程
(4)逆転写工程で得た逆転写DNAを蛍光色素と結合させて標識化する標識化工程、および
(5)標識化工程で標識化した逆転写DNAをDNAチップ上のDNA断片と交雑させる交雑工程
を経て活性汚泥に含まれる細菌を分析することができる。
In particular,
(1) Bacterial extraction process for extracting bacteria from activated sludge,
(2) Lysis / RNA extraction process for extracting RNA after lysis of bacteria obtained in the bacterial extraction process,
(3) Reverse transcription step of reverse transcription of RNA extracted in the lysis / RNA extraction step to obtain reverse transcription DNA
(4) a labeling step in which the reverse transcription DNA obtained in the reverse transcription step is labeled with a fluorescent dye, and
(5) Bacteria contained in the activated sludge can be analyzed through a hybridization step in which the reverse-transcribed DNA labeled in the labeling step is crossed with a DNA fragment on the DNA chip.

〔細菌抽出工程〕
活性汚泥から細菌を抽出するには、例えば活性汚泥を生理食塩水で希釈したのち、遠心分
離処理すればよい。遠心分離により、細菌は沈降して沈殿物となるので、遠心処理後の沈
殿物として、細菌を得ることができる。
[Bacteria extraction process]
In order to extract bacteria from the activated sludge, for example, the activated sludge may be diluted with physiological saline and then centrifuged. By centrifugation, the bacteria settle to become a precipitate, so that the bacteria can be obtained as a precipitate after centrifugation.

〔溶菌・RNA抽出工程〕
得られた細菌を溶菌するには、例えば細菌を溶媒中、粉砕媒体と共に震盪すればよい。粉
砕媒体としては。例えばガラスビーズなどが用いられる。震盪することにより、細菌が粉
砕媒体に衝突するなどして破壊され、細菌中のRNAが溶媒中に分散する。その後、粉砕
媒体を分離し、遠心分離により固形分を除去し、塩化ナトリウムおよびエタノールを加え
てRNAを沈降させ、遠心分離により沈降物としてRNAを得ることができる。
[Bacteria / RNA extraction process]
In order to lyse the obtained bacteria, for example, the bacteria may be shaken with a grinding medium in a solvent. As a grinding medium. For example, glass beads are used. By shaking, the bacteria are destroyed, for example, by colliding with the grinding medium, and the RNA in the bacteria is dispersed in the solvent. Thereafter, the grinding medium is separated, the solid content is removed by centrifugation, sodium chloride and ethanol are added to precipitate RNA, and RNA can be obtained as a precipitate by centrifugation.

なお、上記で得られる沈降物には細菌中のDNAも含まれるので、通常は、この沈降物をDNA分解酵素〔Dnase〕と接触させることにより、沈降物中のDNAを分解する。 In addition, since the DNA in bacteria is contained in the sediment obtained above, normally, the DNA in the sediment is degraded by contacting the sediment with a DNA degrading enzyme [Dnase].

〔逆転写工程〕
溶菌・RNA抽出工程で得たRNAの逆転写は、例えば得られたRNAを鋳型として逆転写DNAを精製する通常の方法により行うことができる。
[Reverse transcription process]
Reverse transcription of RNA obtained in the lysis / RNA extraction step can be performed by, for example, an ordinary method for purifying reverse transcription DNA using the obtained RNA as a template.

〔標識化工程〕
逆転写DNAを標識化するには、例えばインドカルボシアニン(Cy)色素などの蛍光色素で標識したプライマーをPCR法により結合させればよい。
[Labeling step]
In order to label the reverse transcription DNA, for example, a primer labeled with a fluorescent dye such as indocarbocyanine (Cy) dye may be bound by the PCR method.

〔交雑工程〕
標識化した後の逆転写DNAを、DNAチップ上のDNA断片と交雑させるには、この逆転写DNAをDNAチップ上に塗布すればよい。塗布は、例えば逆転写DNAを溶媒中に分散させ、滴下したのち、乾燥することにより行われる。塗布により、逆転写DNAは、これに対応するDNA断片と結合する。
[Mating process]
In order to cross-link the reverse transcribed DNA after labeling with the DNA fragment on the DNA chip, the reverse transcribed DNA may be applied on the DNA chip. The application is performed, for example, by dispersing reverse-transcribed DNA in a solvent, dripping and then drying. By application, reverse transcribed DNA binds to the corresponding DNA fragment.

交雑工程の後、水洗するなどして、結合しなかった余分の逆転写DNAを除去し、紫外線照射下に蛍光を観察することにより、細菌を分析することができ、蛍光の有無から、活性汚泥に含まれる細菌の有無を分析することができ、蛍光強度から、その細菌の量を分析することができる。 Bacteria can be analyzed by removing excess reverse-transcribed DNA that was not bound by washing with water after the hybridization step, and observing fluorescence under ultraviolet irradiation. From the presence or absence of fluorescence, activated sludge Can be analyzed for the presence or absence of bacteria, and the amount of the bacteria can be analyzed from the fluorescence intensity.

本発明の分析方法によれば、活性汚泥に含まれる細菌、好ましくは硝化細菌の種類、量を速やかに、精度よく、分析することができるので、例えば活性汚泥中の細菌が死滅した場合に、他の活性汚泥を用いて排水処理、好ましくはアンモニア態窒素または亜硝酸態窒素を含む排水の処理を再開するにあたり、最適な活性汚泥を速やかに選択することができる。 According to the analysis method of the present invention, since the type and amount of bacteria contained in the activated sludge, preferably nitrifying bacteria, can be analyzed quickly and accurately, for example, when the bacteria in the activated sludge are killed, In resuming wastewater treatment using other activated sludge, preferably wastewater containing ammonia nitrogen or nitrite nitrogen, the optimum activated sludge can be quickly selected.

以下、実施例によって本発明をより詳細に説明するが、本発明は、かかる実施例により限
定されるものではない。
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this Example.

〔実施例1〕
〔DNAチップの調製〕
以下の糸状性細菌、糸状性細菌硫黄代謝細菌、硫黄代謝細菌、硝化細菌を用いてDNAチ
ップを調製した。
[Example 1]
[Preparation of DNA chip]
A DNA chip was prepared using the following filamentous bacteria, filamentous bacteria, sulfur-metabolizing bacteria, sulfur-metabolizing bacteria, and nitrifying bacteria.

なお、用いた細菌の中には属が明らかであるが、種が明らかでないものがあるので、この
ような細菌には、それぞれ(*)を付した。また、2種類と記載している細菌は、異なる
種であることが判明している2種類の細菌のDNAをそれぞれ用いたことを示す。
In addition, although the genus is clear in the used bacterium, but the species is not clear, each such bacterium was marked with (*). In addition, the bacteria described as two types indicate that DNAs of two types of bacteria that have been found to be different species were used, respectively.

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
糸状性細菌
────────────────────────────────────────
Nocardia属 :Nocardia 1(*)、Nocardia 2(*)、Nocardia 3(*)、Nocardia 4(*)
Rhodococcus属 :Rhodococcus 1(*)、Rhodococcus 2(*)
Thiothrix属 :eikelbloomii 1、eikelbloomii 2、fructosivorans、
Thiothrix sp.1,EJ1M-B(*)、unzii、Eikelbloom Type 1701
Sphaerotilus属 :Sphaerotilus(*)
上記以外の属 :Microthrix、Haliscomenobacter、Nostocoida 1、
Nostocoida 2(*)、Nostocoida 3(*)、Achromatium 1(*)、
Achromatium 2(*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Filamentous bacteria ────────────────────────────────────────
Nocardia genus: Nocardia 1 (*), Nocardia 2 (*), Nocardia 3 (*), Nocardia 4 (*)
Rhodococcus genus: Rhodococcus 1 (*), Rhodococcus 2 (*)
Thiothrix genus: eikelbloomii 1, eikelbloomii 2, fructosivorans,
Thiothrix sp.1, EJ1M-B (*), unzii, Eikelbloom Type 1701
Genus Sphaerotilus: Sphaerotilus (*)
Other genera: Microthrix, Haliscomenobacter, Nostocoida 1,
Nostocoida 2 (*), Nostocoida 3 (*), Achromatium 1 (*),
Achromatium 2 (*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
糸状性細菌硫黄代謝細菌
────────────────────────────────────────
Beggiatoa属 :Beggiatoa1、Beggiatoa2、Beggiatoa3、Beggiatoa4、Beggiatoa5、
Beggiatoa6
Thiothrix属 :Thiothrix1、Thiothrix2、Thiothrix3、Thiothrix4、Thiothrix5、
Thiothrix6
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━






━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Filamentous bacteria Sulfur metabolizing bacteria ─────────────────────────────────────────
Beggiatoa genus: Beggiatoa1, Beggiatoa2, Beggiatoa3, Beggiatoa4, Beggiatoa5,
Beggiatoa6
Thiothrix genus: Thiothrix1, Thiothrix2, Thiothrix3, Thiothrix4, Thiothrix5,
Thiothrix6
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━






━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
硫黄代謝細菌
────────────────────────────────────────
Desulfobacter属 :Desulfobacter1(*)、Desulfobacter2(*)、Desulfobacter3(*)、
Desulfobacter4(*)
Desulfotomaculum属:D. acetoxidance、D. aeronauticum、D. alkaliphilum、
D. auripigmentum、D. guttoideum、D. halophilum、
D. kuznetsovii、D. nigrificans 1、D. nigrificans 2、
D. reducens、D. ruminis、D. thermobenzoicum
Desulfovibrio属 :Desulfovibrio 1(2種類)(*)、Desulfovibrio 2(2種類)(*)
、Desulfovibrio 3(2種類)(*)、Desulfovibrio 4(*)、
Desulfovibrio 5(*)、Desulfovibrio 6(*)、Desulfovibrio 7(*)
Desulfomicrobium属:Desulfomicrobium、D. apsheronum、D. norvegicum
Desulforhopalus属 :Desulforhopalus 1(*)、Desulforhopalus 2(*)、
Desulforhopalus 3(*)
Desulfococcus属 :Desulfococcus 1(biacutus)(*)、Desulforhopalus 2(*)
Desulfomonas属 :Desulfomonas
Desulfuromonas属 :Desulfuromonas acetoxidance、D. chloroethanica、
Desulfuromonas 1(*)、Desulfuromonas 2(*)、
Desulfuromonas 3(*)、Desulfuromonas 4(*)、
Desulfuromonas 5(*)、Desulfuromonas 6(*)
Desulfurella属 :Desulfurella
Desulfomonile属 :Desulfomonile limimaris、Desulfomonile sp.(*)、
Desulfomonile sp.1(*)、Desulfomonile sp.2(*)、
Desulfomonile sp.3(*)
Desulfosarcina属 :Desulfosarcina
Lawsonia属 :Lawsonia intracellularis
上記以外の属 :Bilophila wadsworthia、Desulfobotulus、
Desulfobulbus 1(*)、Desulfobulbus 2(*)、
Desulfobulbus 3(*)、Desulfobulbus 4(*)、
Desulfocapsa 1(*)、Desulfocapsa 2(*)、Desulfocapsa 3(*)、
Desulfokalobium、Desulfonatronum、
Desulfonosporus thiosulfogenes、Desulfotalea 1(*)、
Desulfotalea 2(*)、Desulfuromusa 1(*)、
Desulfuromusa 2(*)、Desulfuromusa 3(*)、
Thermosulfovibrio、Thermoterrabacterium ferrireducens
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━











━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Sulfur metabolizing bacteria────────────────────────────────────────
Desulfobacter genus: Desulfobacter1 (*), Desulfobacter2 (*), Desulfobacter3 (*),
Desulfobacter4 (*)
Genus Desulfotomaculum: D. acetoxidance, D. aeronauticum, D. alkaliphilum,
D. auripigmentum, D. guttoideum, D. halophilum,
D. kuznetsovii, D. nigrificans 1, D. nigrificans 2,
D. reducens, D. ruminis, D. thermobenzoicum
Desulfovibrio genus: Desulfovibrio 1 (2 types) (*), Desulfovibrio 2 (2 types) (*)
, Desulfovibrio 3 (2 types) (*), Desulfovibrio 4 (*),
Desulfovibrio 5 (*), Desulfovibrio 6 (*), Desulfovibrio 7 (*)
Desulfomicrobium genus: Desulfomicrobium, D. apsheronum, D. norvegicum
Desulforhopalus genus: Desulforhopalus 1 (*), Desulforhopalus 2 (*),
Desulforhopalus 3 (*)
Desulfococcus genus: Desulfococcus 1 (biacutus) (*), Desulforhopalus 2 (*)
Genus Desulfomonas: Desulfomonas
Desulfuromonas genus: Desulfuromonas acetoxidance, D. chloroethanica,
Desulfuromonas 1 (*), Desulfuromonas 2 (*),
Desulfuromonas 3 (*), Desulfuromonas 4 (*),
Desulfuromonas 5 (*), Desulfuromonas 6 (*)
Desulfurella genus: Desulfurella
Desulfomonile genus: Desulfomonile limimaris, Desulfomonile sp. (*),
Desulfomonile sp.1 (*), Desulfomonile sp.2 (*),
Desulfomonile sp.3 (*)
Desulfosarcina genus: Desulfosarcina
Lawsonia genus: Lawsonia intracellularis
Other genera: Bilophila wadsworthia, Desulfobotulus,
Desulfobulbus 1 (*), Desulfobulbus 2 (*),
Desulfobulbus 3 (*), Desulfobulbus 4 (*),
Desulfocapsa 1 (*), Desulfocapsa 2 (*), Desulfocapsa 3 (*),
Desulfokalobium, Desulfonatronum,
Desulfonosporus thiosulfogenes, Desulfotalea 1 (*),
Desulfotalea 2 (*), Desulfuromusa 1 (*),
Desulfuromusa 2 (*), Desulfuromusa 3 (*),
Thermosulfovibrio, Thermoterrabacterium ferrireducens
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━











━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
硝化細菌(アンモニア酸化細菌)
────────────────────────────────────────
Nitrosomonas属 :Nitrosomonas、Nitrosomonas sp.(2種類)(*)、
N. aestuarii、N. communis、N. cryotolerans、N. europaea、
N. halophila、N. nitrosa、N. oligotropha、N. ureae
Nitrosospira属 :Nitrosospira、N. briensis、N. multiformis
Nitorosolobus属 :Nitrosolobus
上記以外の属 :Nitrosococcus、Nitrosococcus 1(*)、Nitrosococcus 2(*)、
Nitrosococcus 3(*)、Anammoxoglobus 1(*)、
Anammoxoglobus 2(*)、Brocadia、Kuenenia、Scalindua 1(*)、
Scalindua 2(*)、Scalindua 3(*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Nitrifying bacteria (ammonia oxidizing bacteria)
────────────────────────────────────────
Nitrosomonas genus: Nitrosomonas, Nitrosomonas sp. (2 types) (*),
N. aestuarii, N. communis, N. cryotolerans, N. europaea,
N. halophila, N. nitrosa, N. oligotropha, N. ureae
Nitrosospira genus: Nitrosospira, N. briensis, N. multiformis
Nitorosolobus genus: Nitrosolobus
Other genera: Nitrosococcus, Nitrosococcus 1 (*), Nitrosococcus 2 (*),
Nitrosococcus 3 (*), Anammoxoglobus 1 (*),
Anammoxoglobus 2 (*), Brocadia, Kuenenia, Scalindua 1 (*),
Scalindua 2 (*), Scalindua 3 (*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
硝化細菌(亜硝酸酸化細菌)
────────────────────────────────────────
Nitrospira属 :Nitrospira、N. marina、N. moscoviensis
Nitrobacter属 :Nitrobacter、Nitrobacter 1(*)、Nitrobacter 2(*)、
Nitrobacter 3(*)、Nitrobacter 4(*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Nitrifying bacteria (nitrite oxidizing bacteria)
────────────────────────────────────────
Nitrospira genus: Nitrospira, N. marina, N. moscoviensis
Nitrobacter genus: Nitrobacter, Nitrobacter 1 (*), Nitrobacter 2 (*),
Nitrobacter 3 (*), Nitrobacter 4 (*)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

〔DNA断片の調製〕
各細菌はのDNA断片は、各種の遺伝子データベースに掲載されている16SrRNA(
リボゾームRNA)塩基配列情報から固有の遺伝子領域を選出し、そのクローンを合成す
ることにより得た。
[Preparation of DNA fragment]
The DNA fragments of each bacterium are 16S rRNA (
It was obtained by selecting a unique gene region from ribosomal RNA) base sequence information and synthesizing its clone.

〔DNA断片の固定〕
上記で得たDNA断片をそれぞれスライドガラス上の所定位置にスポット状に付着させ、
乾燥することにより、固定してDNAチップを得た。得られたDNAチップは、以下の交
雑に使用するまで−20℃で保管した。
[Immobilization of DNA fragments]
Each of the DNA fragments obtained above is attached to a predetermined position on a slide glass in a spot shape,
The DNA chip was obtained by fixing by drying. The obtained DNA chip was stored at −20 ° C. until used for the following hybridization.

〔細菌の分析〕
上記で得たDNAチップを用いて、化学工場の生物学的排水処理施設から採取した活性汚泥4検体を用いて、それぞれの活性汚泥に含まれる細菌の種類数および量を以下の手順で分析した。活性汚泥は、生物学的排水処理施設に流入する排水の性状が変化したと見込まれる場合について(第1回採取検体〜第3回採取検体)、また生物学的排水処理施設の水温が数℃上昇したと見込まれる場合(第4回採取検体)にそれぞれ採取した。
[Bacteria analysis]
Using the DNA chip obtained above, the number and type of bacteria contained in each activated sludge were analyzed by the following procedure using 4 activated sludge samples collected from the biological wastewater treatment facility of the chemical factory. . For activated sludge, the temperature of the wastewater flowing into the biological wastewater treatment facility is expected to change (1st to 3rd collected samples), and the temperature of the biological wastewater treatment facility is several degrees Celsius. Each sample was collected when expected to have risen (fourth sample collected).

〔細菌の抽出〕
活性汚泥4mLに生理食塩水〔大塚製薬社製〕36mLを加えたのち振盪した。その後、
2mLを分取し、15000回転/分の遠心分離を行ったのち、上澄み液を分離して沈降
物を得、緩衝水溶液〔エーエムアール社(岐阜市)製「ライシス・バッファー」〕150
μLを加えて希釈した。なお、使用した器具は全て滅菌処理したものである。
[Bacteria extraction]
After adding 36 mL of physiological saline [Otsuka Pharmaceutical Co., Ltd.] to 4 mL of activated sludge, it was shaken. afterwards,
After separating 2 mL and centrifuging at 15,000 rpm, the supernatant was separated to obtain a precipitate, and a buffered aqueous solution [“Lysis Buffer” manufactured by AMR Co., Ltd. (Gifu City)] 150
μL was added to dilute. All instruments used were sterilized.

〔溶菌およびRNAの抽出〕
上記で希釈した後の沈降物に、ジルコニアビーズ〔エーエムアール社(岐阜市)製〕を加え、90℃で10分間保持した後、細胞破砕機〔安井機器社製「Multibeads Shocker」〕により2500rpmで60秒間振盪した。その後、ドデシル硫酸ナトリウム(SDS)水溶液〔濃度0.2質量%〕200μLを加え、1分間、ボルテックス撹拌を行い、15000回転/分で5分間、遠心分離した後、上澄み液を分取し、この上澄み液に対して0.05容量倍の塩化ナトリウム水溶液〔5モル/L〕および2容量倍のエタノール〔100%〕を加え、30秒間、ボルテックス撹拌を行った。その後、15000回転/分で5分間、遠心分離して沈殿物を分取し、55℃で5分間保持することにより乾燥して、RNAを含む沈降物を得た。この沈殿物をRNase free 滅菌蒸留水〔RNA分解酵素を含まない滅菌蒸留水〕200μLに懸濁して懸濁液とした。
[Bacterial lysis and RNA extraction]
After adding zirconia beads (manufactured by AMR Co., Ltd. (Gifu City)) to the precipitate after the above dilution and holding at 90 ° C. for 10 minutes, the cell crusher (“Multibeads Shocker” manufactured by Yasui Kikai Co., Ltd.) is used at 2500 rpm. Shake for 60 seconds. Then, 200 μL of sodium dodecyl sulfate (SDS) aqueous solution (concentration 0.2% by mass) was added, vortexed for 1 minute, centrifuged at 15000 rpm for 5 minutes, and the supernatant was collected. 0.05 volume times sodium chloride aqueous solution [5 mol / L] and 2 volume times ethanol [100%] were added to the supernatant and vortexed for 30 seconds. Thereafter, the precipitate was collected by centrifugation at 15000 rpm for 5 minutes, and dried by holding at 55 ° C. for 5 minutes to obtain a precipitate containing RNA. This precipitate was suspended in 200 μL of RNase free sterilized distilled water [sterilized distilled water not containing RNase] to prepare a suspension.

次いで、この懸濁液43μLに、緩衝水溶液〔10×DNAase buffer(インビトロジェン社製)〕5μLおよびDNA分解酵素水溶液〔DNAase I(インビトロジェン社製)〕2μLを加え、37℃にて2時間保持してDNAを分解した。その後、55℃に昇温し、同温度を5分間保持してDNA分解酵素を失活させた後、4℃に冷却した。 Next, 5 μL of a buffer aqueous solution [10 × DNAase buffer (manufactured by Invitrogen)] and 2 μL of an aqueous solution of DNA-degrading enzyme [DNAase I (manufactured by Invitrogen)] are added to 43 μL of this suspension and kept at 37 ° C. for 2 hours. DNA was degraded. Thereafter, the temperature was raised to 55 ° C., the temperature was maintained for 5 minutes to deactivate the DNA-degrading enzyme, and then cooled to 4 ° C.

〔逆転写〕
上記でDNAを分解した後の混合物5μLにRNase free 滅菌蒸留水13.5μLを加え、これに市販のPCR反応用溶液〔「TaKaRa−One Step SYBR RT−PCR Kit」(宝酒造社製)〕および「16SrRNA8UA−519Bプライマーセット」(シグマアルドリッチジャパン社)を用いて50μLの反応系で42℃で60分間保持し、98℃で5分間保持後、4℃に冷却することにより逆転写を行って、逆転写DNAを含む混合物を得た。
[Reverse transcription]
RNase free sterilized distilled water (13.5 μL) was added to 5 μL of the mixture after degrading DNA as described above, and a commercially available PCR reaction solution [“TaKaRa-One Step SYBR RT-PCR Kit” (Takara Shuzo)] and “ 16SrRNA8UA-519B primer set "(Sigma Aldrich Japan Co., Ltd.) in a 50 μL reaction system, held at 42 ° C. for 60 minutes, held at 98 ° C. for 5 minutes, and then cooled to 4 ° C. to perform reverse transcription. A mixture containing the copied DNA was obtained.

〔標識化〕
上記で得た逆転写DNAに、市販の「16SrRNA8UA−519Bプライマーセット」(シグマアルドリッチジャパン社製)を用いて100μLのPCR法により、逆転写DNAの増幅を行った。
[Labeling]
The reverse transcription DNA was amplified by the PCR method of 100 μL using the commercially available “16SrRNA8UA-519B primer set” (manufactured by Sigma Aldrich Japan).

その後、「Cy Dye 8UA−519Bプライマーセット」(シグマアルドリッチジャパン社製)(蛍光色素)を用いて100μLの反応系でPCR法により、逆転写DNAを蛍光色素と結合させて標識化した。 Thereafter, reverse transcription DNA was labeled with a fluorescent dye by PCR in a 100 μL reaction system using “Cy Dye 8UA-519B primer set” (manufactured by Sigma-Aldrich Japan) (fluorescent dye).

その後、逆転写DNAを蛍光色素と結合させた後の混合物に対して0.05容量倍の塩化ナトリウム水溶液〔5モル/L〕および2容量倍のエタノール〔100%〕を加え、30秒間、ボルテックス撹拌を行った。その後、15000回転/分で5分間、遠心分離して沈殿物を分取し、PCRグレードの純水20μLに懸濁させて懸濁液としたのち、−20℃で保管した。 Thereafter, 0.05 vol. Times aqueous sodium chloride solution [5 mol / L] and 2 vol. Times ethanol [100%] were added to the mixture after the reverse transcription DNA was bound to the fluorescent dye, and vortexed for 30 seconds. Stirring was performed. Thereafter, the precipitate was collected by centrifugation at 15000 rpm for 5 minutes, suspended in 20 μL of PCR grade pure water, and then stored at −20 ° C.

〔交雑〕
先に調製したDNAチップを、塩化ナトリウム(0.15モル/L)−クエン酸ナトリウ
ム(0.015モル/L)混合水溶液〔以下、SSC水溶液とする。〕2容量部およびS
DS水溶液(濃度0.2質量%)1容量部の混合液を用いて室温で15分間洗浄し、さら
に95℃で5分間洗浄したのち、超純水により3回洗浄した。その後、800回転/分に
て遠心乾燥した。
[Crossing]
The previously prepared DNA chip is a mixed aqueous solution of sodium chloride (0.15 mol / L) -sodium citrate (0.015 mol / L) [hereinafter referred to as SSC aqueous solution. ] 2 capacitors and S
The mixture was washed with 1 part by volume of a DS aqueous solution (concentration: 0.2% by mass) at room temperature for 15 minutes, further washed at 95 ° C. for 5 minutes, and then washed with ultrapure water three times. Thereafter, it was centrifugally dried at 800 rpm.

一方、上記〔標識化〕で得た懸濁液に純水を加えてDNA濃度が100ng/μLとなる
ように希釈し、28μLを分取し、SSC水溶液10μLおよびSDS水溶液(濃度0.
2質量%)2μLを加えた後、DNAチップ上に滴下し、次いで気泡が入らないようにカバーガラスを被せ、50℃〜65℃で12時間〜16時間保持した。その後、DNAチップを37℃にて、塩化ナトリウム(0.3モル/L)、クエン酸ナトリウム(0.03モル/L)およびSDS(濃度0.2質量%)の水溶液に1分間浸漬してカバーガラスを脱落させ、次いで、塩化ナトリウム(0.3モル/L)、クエン酸ナトリウム(0.03モル/L)およびSDS(濃度0.2質量%)の水溶液中で5分間振盪し、SSC水溶液にて洗浄後、800回転/分にて遠心乾燥した。
On the other hand, pure water was added to the suspension obtained in the above [labeling] to dilute the DNA concentration to 100 ng / μL, and 28 μL was separated, and 10 μL of SSC aqueous solution and SDS aqueous solution (concentration 0.
(2% by mass) was added dropwise to the DNA chip, and then covered with a cover glass so that bubbles did not enter, and held at 50 to 65 ° C. for 12 to 16 hours. Thereafter, the DNA chip was immersed in an aqueous solution of sodium chloride (0.3 mol / L), sodium citrate (0.03 mol / L) and SDS (concentration 0.2% by mass) at 37 ° C. for 1 minute. The cover glass was removed, and then shaken in an aqueous solution of sodium chloride (0.3 mol / L), sodium citrate (0.03 mol / L) and SDS (concentration 0.2% by mass) for 5 minutes. After washing with an aqueous solution, it was centrifugally dried at 800 rpm.

〔蛍光強度の測定〕
上記で乾燥したのちのDNAチップに、レーザースキャナー〔GSI LUMONICS
社製、「Scan Array 4000」にて紫外線を照射し、蛍光シグナルの強度を測
定し、相対強度を求めた。
[Measurement of fluorescence intensity]
After drying the DNA chip, the laser scanner [GSI LUMONICS]
Relative intensity was obtained by irradiating ultraviolet rays with “Scan Array 4000” manufactured by the company, measuring the intensity of the fluorescent signal.

蛍光強度の測定には、画像解析ソフト〔日立ソフト社製「DNASYS ARRAY」〕
を用いた。蛍光強度は、検出された全ての蛍光シグナルの合計強度を100%とし、0.
1%未満の蛍光強度は、非検出とした。
For the measurement of fluorescence intensity, image analysis software ("DNASYS ARRAY" manufactured by Hitachi Software Co., Ltd.)
Was used. The fluorescence intensity is calculated by taking the total intensity of all the detected fluorescence signals as 100%.
A fluorescence intensity of less than 1% was not detected.

4回採取した活性汚泥の各種細菌群のRNA測定結果を図1〜図3に示す。それぞれ、硝化細菌群、糸状性細菌群及び硫黄代謝細菌群の測定結果であり、柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。糸状性細菌であり硫黄代謝細菌であるものは両方に計上した。これらの図に示されるように、当該生物学的処理装置に対して流入排水の水質が変化したり、水温が上昇したりした可能性が考えられたが、活性汚泥中のこれら細菌群の活動度とみなしうるRNAの測定結果によれば、当該生物学的処理装置の活性汚泥の機能は、菌種数、検出レベルともおおむね一定に保たれていることが判明した。 The RNA measurement results of various bacterial groups of the activated sludge collected four times are shown in FIGS. The measurement results for the nitrifying bacteria group, the filamentous bacteria group, and the sulfur-metabolizing bacteria group, respectively, the columnar graph indicates the number of detected bacterial species, and the line graph indicates the total value of the relative intensities of the fluorescent signals. Filamentous and sulfur-metabolizing bacteria were counted in both. As shown in these figures, there was a possibility that the quality of the influent wastewater changed or the water temperature increased for the biological treatment equipment, but the activity of these bacteria in the activated sludge According to the measurement result of RNA that can be regarded as a degree, it has been found that the function of the activated sludge of the biological treatment apparatus is generally kept constant both in the number of bacterial species and the detection level.

〔実施例2〕
図4に示す生物学的処理試験槽〔曝気部容積1L、沈降部容積0.3L〕(1)に化学工場の活性汚泥〔濃度5000mg−MLSS/L、MLSSは浮遊懸濁物質を意味する〕(2)を入れ、温度を任意に調節できる恒温室(インキュベータ)(図示せず)に収納した。この生物学的処理試験槽(1)は、連続通水式のものであり、エアストーン〔直径約2cm〕(3)による空気(A)の供給と、この空気による攪拌機能を有する曝気部〔容積1L、有効深さ約40cm〕(11)と、下部〔底面から約2cm高さまで〕(12)のみが曝気部と開通された沈降部(13)から構成される。この沈降部(13)は、間仕切り板(14)により曝気部(11)から仕切られている。曝気部の上部から排水(B)を連続的に流入し、沈降部(13)の上方側面に開けられた排出口(15)から処理水(C)を排出する構造である。なお、この生物学的処理試験槽の材質は全て透明塩化ビニル樹脂である。
[Example 2]
The biological treatment test tank shown in FIG. 4 [aeration part volume 1 L, sedimentation part volume 0.3 L] (1) in the activated sludge of chemical factory [concentration 5000 mg-MLSS / L, MLSS means suspended suspended solids] (2) was placed and stored in a temperature-controlled room (incubator) (not shown) where the temperature could be adjusted arbitrarily. This biological treatment test tank (1) is of a continuous water flow type, and is supplied with air (A) by an air stone (diameter of about 2 cm) (3) and an aeration section having a stirring function by this air [ Only a volume of 1 L, an effective depth of about 40 cm] (11), and a lower part (from the bottom to about 2 cm height) (12) are composed of an aeration part and a settling part (13) opened. The settling part (13) is partitioned from the aeration part (11) by a partition plate (14). In this structure, waste water (B) flows continuously from the upper part of the aeration unit, and treated water (C) is discharged from the discharge port (15) opened on the upper side surface of the settling part (13). The biological treatment test tank is made of a transparent vinyl chloride resin.

この試験槽に同工場の排水(汚濁物質として主として有機性の物質とアンモニア性の物質を含有する)を78mL/時の流速で連続的に流入して好気的処理を行った。処理成績の指標として、有機性汚濁物質に対してはTOC(総有機態炭素)濃度をTOC計(島津製作所製「TOC−5000」)によって、アンモニア性汚濁物質に対してはアンモニア態窒素濃度を紫外線吸光光度法(JIS日本工業規格K0102 45.2)によって、それぞれ経時測定した。まず試験槽を処理水温36℃の条件で運転開始し、汚濁物質処理がおおむね安定したと見られる時点で、処理水温を42℃に上昇した。温度上昇の直前、上昇後1日経過後及び42℃の条件に活性汚泥がおおむね適応(馴化)したとみなされる温度上昇後20日経過後の3時点で、試験槽から活性汚泥を採取し、実施例1に記載した手順によってRNAを抽出し、逆転写を行って、DNAチップを用いた硝化細菌、糸状性細菌及び硫黄代謝細菌の分析を行った。 The aerobic treatment was performed by continuously flowing waste water from the factory (mainly containing organic substances and ammoniacal substances as pollutants) into the test tank at a flow rate of 78 mL / hour. As an index of processing results, TOC (total organic carbon) concentration is measured for organic pollutants, and ammonia nitrogen concentration is measured for ammonia pollutants using TOC meter ("TOC-5000" manufactured by Shimadzu Corporation). Each was measured over time by an ultraviolet absorptiometry (JIS Japanese Industrial Standard K0102 45.2). First, the operation of the test tank was started at a treatment water temperature of 36 ° C., and the treatment water temperature was raised to 42 ° C. when it was considered that the treatment of contaminants was generally stable. The activated sludge was collected from the test tank immediately before the temperature rise, 1 day after the rise, and at 3 time points after 20 days after the temperature rise that the activated sludge is considered to be generally adapted (acclimated) to the condition of 42 ° C. RNA was extracted by the procedure described in 1, reverse transcription was performed, and nitrifying bacteria, filamentous bacteria, and sulfur-metabolizing bacteria were analyzed using a DNA chip.

図5に試験槽のTOC、アンモニア態窒素(NH4−N)負荷推移及び処理水温変化を示す。経過日数17日目に温度を約36℃から約42℃に上昇した。 FIG. 5 shows the TOC of the test tank, the ammonia nitrogen (NH 4 -N) load transition and the treatment water temperature change. On the 17th day, the temperature rose from about 36 ° C to about 42 ° C.

図6に試験槽のTOC除去率及びNH4−N除去率の推移を示す。経過日数17日目に汚濁物質処理がおおむね安定したと見て水温を約36℃から42℃に上昇し、水温上昇の直前(17日目)及び1日後(18日目)に活性汚泥を採取して、それぞれX−1及びX−2と命名した。特開2006−263642号公報に記載されるように、生物学的処理装置の水温が40℃を超える温度領域に到達したときには一時的に有機性排水の処理水質が悪化するが、時間の経過に伴って次第に回復することが知られている。図6において、水温を42℃に上昇の1日後に約60%であったTOC除去率が、水温上昇から約20日後の、経過日数38日目には約80%にまで回復したので活性汚泥を採取し、X−3と命名した。NH4−N除去率は、水温上昇前には50%程度であったのが水温上昇後には20%前後に低下し、TOC除去率とは異なり、時間が経過しても回復が見られなかった。 FIG. 6 shows the transition of the TOC removal rate and NH4-N removal rate of the test tank. The water temperature was increased from about 36 ° C to 42 ° C on the assumption that the treatment of pollutants was almost stable on the 17th day, and activated sludge was collected immediately before the water temperature rise (17th day) and 1 day later (18th day). They were named X-1 and X-2, respectively. As described in JP-A-2006-263642, when the water temperature of the biological treatment apparatus reaches a temperature region exceeding 40 ° C., the quality of the treated water of the organic waste water is temporarily deteriorated. It is known to recover gradually with this. In FIG. 6, the TOC removal rate, which was about 60% one day after the water temperature rose to 42 ° C., recovered to about 80% on the 38th day after about 20 days from the water temperature rise. Was collected and designated X-3. The NH4-N removal rate was about 50% before the water temperature rose but decreased to around 20% after the water temperature rose, and unlike the TOC removal rate, no recovery was seen over time. .

採取した各活性汚泥から抽出したRNAのDNAチップによる測定結果を図7〜9に示す。それぞれ、硝化細菌群、糸状性細菌群及び硫黄代謝細菌群の測定結果であり、柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。糸状性細菌であり硫黄代謝細菌であるものは両方に計上した。 The measurement result by the DNA chip of RNA extracted from each collected activated sludge is shown in FIGS. The measurement results for the nitrifying bacteria group, the filamentous bacteria group, and the sulfur-metabolizing bacteria group, respectively, the columnar graph indicates the number of detected bacterial species, and the line graph indicates the total value of the relative intensities of the fluorescent signals. Filamentous and sulfur-metabolizing bacteria were counted in both.

硝化細菌(図7)は、検出菌種数、蛍光シグナルの相対強度とも、水温上昇前(試料名X−1)に比較して水温上昇1日後(X−2)には低下し、その20日後(X−3)も回復が見られず、図6に示したNH4−N除去率の推移をほぼ反映する結果が得られた。糸状性細菌(図8)及び硫黄代謝細菌(図9)は、必ずしも有機性汚濁除去を行うすべての細菌群を含むものではないが、水温が上昇した直後(X−2)に検出菌種数及び蛍光シグナルの相対強度が若干増加するものの、水温変動に伴う極端な変化はなく、42℃の高水温環境に対する活性汚泥の有機性汚濁処理機能の比較的早い適応(馴化)を裏付ける推移の傾向を示している。 In the nitrifying bacteria (FIG. 7), both the number of detected bacteria and the relative intensity of the fluorescence signal were reduced one day after the water temperature rise (X-2) compared to before the water temperature rise (sample name X-1). No recovery was observed even after (X-3), and a result almost reflecting the transition of the NH4-N removal rate shown in FIG. 6 was obtained. Filamentous bacteria (FIG. 8) and sulfur-metabolizing bacteria (FIG. 9) do not necessarily include all bacterial groups that perform organic decontamination, but immediately after the water temperature rises (X-2) Although there is a slight increase in the relative intensity of the fluorescence signal, there is no extreme change due to fluctuations in the water temperature, and the trend of supporting the relatively early adaptation (acclimation) of the organic pollution treatment function of activated sludge to a high water temperature environment of 42 ° C Is shown.

化学工場の生物学的処理装置における採取試料毎の硝化細菌群RNAの変化を示す。横軸は1〜4回目採取の活性汚泥試料をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of nitrifying bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. 化学工場の生物学的処理装置における採取試料毎の糸状性細菌群RNAの変化を示す。横軸は1〜4回目採取の活性汚泥試料をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of filamentous bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. 化学工場の生物学的処理装置における採取試料毎の硫黄代謝細菌群RNAの変化を示す。横軸は1〜4回目採取の活性汚泥試料をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of the sulfur metabolism bacteria group RNA for every collection sample in the biological treatment apparatus of a chemical factory is shown. The horizontal axis represents the activated sludge sample collected 1 to 4 times, the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total value of the relative intensities of fluorescent signals. 実施例2で用いた連続通水式の生物学的処理試験槽を模式的に示す図である。It is a figure which shows typically the continuous flow type biological treatment test tank used in Example 2. FIG. 生物学的処理試験槽のTOC、アンモニア態窒素(NH4−N)負荷推移及び処理水温変化を示す。横軸は試験開始からの経過日数、左縦軸はTOC及びNH4−Nの負荷量(1日あたりグラム数)及び右縦軸は処理水温(℃)である。The TOC of the biological treatment test tank, the ammonia nitrogen (NH4-N) load transition and the treatment water temperature change are shown. The horizontal axis is the number of days elapsed from the start of the test, the left vertical axis is the load of TOC and NH4-N (grams per day), and the right vertical axis is the treated water temperature (° C.). 生物学的処理試験槽のTOC除去率及びNH4−N除去率の推移を示す。横軸は試験開始からの経過日数、縦軸は除去率(%)である。The transition of the TOC removal rate and NH4-N removal rate of the biological treatment test tank is shown. The horizontal axis is the number of days elapsed from the start of the test, and the vertical axis is the removal rate (%). 生物学的処理試験槽における採取試料毎の硝化細菌群RNAの変化を示す。横軸は試料名(X−1、X−2及びX−3)をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of nitrifying bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals. 生物学的処理試験槽における採取試料毎の糸状性細菌群RNAの変化を示す。横軸は試料名(X−1、X−2及びX−3)をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of filamentous bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals. 生物学的処理試験槽における採取試料毎の硫黄代謝細菌群RNAの変化を示す。横軸は試料名(X−1、X−2及びX−3)をあらわし、縦軸の柱状グラフは検出菌種数を、折れ線グラフは蛍光シグナルの相対強度の合計値を示す。The change of the sulfur metabolism bacteria group RNA for every collection sample in a biological treatment test tank is shown. The horizontal axis represents sample names (X-1, X-2, and X-3), the columnar graph on the vertical axis represents the number of detected bacterial species, and the line graph represents the total relative intensity of fluorescent signals.

符号の説明Explanation of symbols

1:生物学的処理試験槽
11:曝気部 12:下部 13:沈降部 14:間仕切り板 15:排水口
2:活性汚泥
3:エアストーン
A:空気 B:排水 C:処理水
1: Biological treatment tank
11: Aeration part 12: Lower part 13: Settling part 14: Partition plate 15: Drainage port 2: Activated sludge 3: Air stone A: Air B: Drainage C: Treated water

Claims (2)

活性汚泥に含まれる細菌のRNAを抽出し、抽出されたRNAの逆転写を行って逆転写DNAを得、得られた逆転写DNAを以下のDNAチップのDNA断片と交雑させることを特徴とする前記細菌の分析方法。
DNAチップ:基板上に、
Nocardia属糸状性細菌、Rhodococcus属糸状性細菌、
Beggiatoa属糸状性細菌硫黄代謝細菌、Thiothrix属糸状性細菌硫黄代謝細菌、
Desulfobacter属硫黄代謝細菌、Desulfotomaculum属硫黄代謝細菌、Desulfovibrio属硫黄
代謝細菌、Desulfomicrobium属硫黄代謝細菌、Desulforhopalus属硫黄代謝細菌、
Nitrosomonas属硝化細菌、Nitrosospira属硝化細菌およびNitrospira属硝化細菌
のDNA塩基配列を含むDNA断片が固定されてなるDNAチップ
Extracting bacterial RNA contained in activated sludge, performing reverse transcription of the extracted RNA to obtain reverse transcription DNA, and crossing the obtained reverse transcription DNA with DNA fragments of the following DNA chip The method for analyzing bacteria.
DNA chip: on the substrate,
Nocardia spp., Rhodococcus spp.,
Beggiatoa filamentous bacteria sulfur metabolizing bacteria, Thiothrix filamentous bacteria sulfur metabolizing bacteria,
Desulfobacter genus sulfur-metabolizing bacteria, Desulfotomaculum genus sulfur-metabolizing bacteria, Desulfovibrio genus sulfur-metabolizing bacteria, Desulfomicrobium genus sulfur-metabolizing bacteria, Desulforhopalus genus sulfur-metabolizing bacteria,
Nitrosomonas nitrifying bacteria, Nitrosospira nitrifying bacteria, and a DNA chip on which DNA fragments containing DNA base sequences of Nitrospira genus nitrifying bacteria are fixed
前記活性汚泥に含まれる細菌が硝化細菌である請求項1に記載の分析方法。 The analysis method according to claim 1, wherein the bacteria contained in the activated sludge are nitrifying bacteria.
JP2007228704A 2007-09-04 2007-09-04 Method for analyzing bacterium contained in activated sludge Pending JP2009060795A (en)

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JP2008228691A (en) * 2007-03-23 2008-10-02 Sumitomo Chemical Co Ltd Dna chip

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CN108060243A (en) * 2017-12-21 2018-05-22 河海大学 A kind of high-precision detection microorganism and the method for net dirty carrier fixation intensity
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