JP2004344138A - Microorganism in activated sludge of water treatment plant for removing nitrogen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new microorganisms presenting in the activated sludge of a water treatment plant for reducing nitrogen and the new base sequences of genes of nitrous acid-reducing enzyme NiR K and NiR S. <P>SOLUTION: It is possible to specify the microorganisms associated with the removal of nitrogen by detecting the nitrous acid-reducing enzymes NiR K and NiR S and also based on base sequences obtained by elucidating the base sequences of parts of the above genes. It is also possible to establish a new method for removing nitrogen by using the activated sludge containing these specified microorganisms. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００４３】
使用するｐｒｉｍｅｒは以下の表２より選択する。
【００４４】
【表２】

【００４５】
４．サーマルサイクラーＴ３ Ｔｈｅｒｍｏｃｙｃｌｅｒ（Ｂｉｏｍｅｔｒａ）またはＧｅｎｅＡｍｐ９６００（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ）で、目的に応じたプログラム条件でＰＣＲを行う。
【００４６】
５．アガロースゲル（アガロースを１×ＴＡＥに溶かし１質量％から２質量％としたもの）を用いて１００Ｖで１５分間電気泳動を行い、その後１５分間のエチジウムブロマイド染色を行い、ＵＶトランスイルミネーター（東洋紡）でＰＣＲ産物が得られたかどうか確認をする。
【００４７】
クローニング
複合微生物系で、ある遺伝子を標的としたＰＣＲを行いそのＰＣＲ産物をテンプレートとしてクローニングを行うと、系の中に存在する様々な微生物がある確率で単離した状態で得られ、さらにクローンライブラリーを作成することで、存在微生物の全体像を知ることができる。硝化脱窒型の窒素除去を行う水処理プロセスの活性汚泥中の微生物について、ｎｉｒＫ遺伝子ののｐｒｉｍｅｒで得たＰＣＲ産物からクローンライブラリーを作成した。
【００４８】
クローニングは、大きく分けて次の２段階の反応からなる。
ライゲーション
ライゲーションとは、プラスミドベクターにインサート（特定の遺伝子産物（ＰＣＲ産物））を組み込む操作である。クローニングに用いられるプラスミドベクターは、インサートを組み込む必要があるため、制限酵素で切断されるクローニングサイトをもつ必要がある。また、最終的にプラスミドを大腸菌に感染させて、大腸菌を増やすことでプラスミドに組み込まれたＰＣＲ産物を増やすことをするので、プラスミドベクターは大腸菌に認識される複製開始点を持ち、さらにプラスミドを持つ大腸菌を選択的に増殖させるために薬剤耐性遺伝子を持つ必要がある。プラスミドベクターの調製は自分でも行うことはできるが、上記のように調製済みのベクターが市販されている。
【００４９】
ライゲーションの際には、ＰＣＲ産物の末端の修飾方法やベクター／インサート比に注意が必要である。末端の修飾方法にはいくつかあるが、比較的よく行われているのは、ＰＣＲでのＤＮＡ合成の際に付加されるデオキシアデノシン（ｄＡ）を利用したＴＡクローニングという方法である。また、ベクター／インサート比については、効率よくライゲーションが行われるように通常は１／１から１／１０程度にする必要がある。
【００５０】
トランスフォーメーション（形質転換）
トランスフォーメーションとは、ライゲーションを行ったプラスミドを、ＤＮＡの取り込む能力のある大腸菌のコンピテント細胞に感染させる操作である。トランスフォーメーションした細胞を薬剤の含んだプレート上に播くことにより、薬剤耐性を持つ大腸菌、すなわちプラスミドを含む大腸菌が選択的に増殖してコロニーを形成する。一つのコロニーは一種類のみのインサートを含む大腸菌から成るので、複合微生物系において微生物ごとに遺伝子を分けることができる。
【００５１】
１）クローニングの方法の検討
インサートの末端修飾には幾つかの方法があり、それに伴いライゲーションの方法もいくつかあるが、本実験では、操作が比較的簡単で、形質転換体が７０％程度の確率で得られるといわれるＴＡクローニング法の変法を採用した。ＴＡクローニング法は、ＰＣＲでのＤＮＡ合成の際に付加されるデオキシアデノシン（ｄＡ）を利用した方法であり、鋳型ＤＮＡの配列によってはＰＣＲ産物にｄＡが付加しない平滑状のものが混ざった状態なので、ＰＣＲの鋳型ＤＮＡ等の条件によりクローニング効率が変わることに注意を要する。
【００５２】
ＴＡクローニング法として市販されているものの中で、具体的にはＱＩＡＧＥＮ ＰＣＲ Ｃｌｏｎｉｎｇ Ｓｙｓｔｅｍ（ＵＡクローニング）のＫｉｔを使用した。これは、ＴＡクローニング法の変法で、Ｔの代わりにＵの付いたベクターを用いたものである。Ｔは非相補的な塩基（Ｇ，Ｃ，Ｔ）とハイブリダイズしやすく、Ｔが付加されたベクターはベクター同士のアニーリング等の結果をもたらす可能性が有るが、Ｕは非特異的なベースペアを形成しにくい為、クローニング効率が高いと言われている（ＱＩＡＧＥＮプロダクトガイド）。本実験においても、ＴＡクローニングとＵＡクローニングの比較を行った。ＴＡクローニングはインサート率（出現コロニー当たりのインサートが入ったコロニーの割合）が半分程度であったが、ＵＡクローニングはインサート率が９割程度と高く、ＵＡクローニング法の方がクローニング効率がよいことが明らかとなった。
【００５３】
２）適切なベクター／インサート比の検討
ベクター／インサート比については、通常１／１から１／１０程度にする。これは、インサートが少ないとインサート率が低くなり、逆にインサートが多すぎると複数のインサートが一つのベクターに入ってしまうという可能性も出てくるためである。本実験では、ＱＩＡＧＥＮ ＰＣＲ Ｃｌｏｎｉｎｇ ＳｙｓｔｅｍのＫｉｔで推奨されている１／５と１／１０の両方について実験を行った。その結果、１／５と１／１０の両者で１／１０の方がコロニー数が１、２割程度多かったものの、顕著な差ではなかったため、１／５や１／１０程度の比であればどちらでもよいという結論に達した。
【００５４】
３）インサートの確認方法の検討
インサートの確認には、コロニーを液体培養で増やし、ミニプレップでプラスミドの抽出・精製を行った後にＰＣＲまたは制限酵素処理を行い、アガロースゲル電気泳動をする方法を使用した。
【００５５】
以下、手順を説明する。
【００５６】
１）ＰＣＲ産物（インサート）の精製
１．クローニングしたい目的のＰＣＲ断片を含むＰＣＲ産物を１サンプルにつき数本用意する。
２．それらを集め、Ｍｉｃｒｏｃｏｎ（Ｍｉｌｌｉｐｏｒｅ）を用いて過剰なプライマーやｄＮＴＰを除去して精製を行う。
３．精製したサンプルのＤＮＡ濃度を測定する。
【００５７】
２）クローニング操作
クローニングの基本的操作は、ＱＩＡＧＥＮ ＰＣＲ Ｃｌｏｎｉｎｇ Ｓｙｓｔｅｍのプロトコルに準じた。
１．Ｌｉｇａｔｉｏｎ ｍｉｘｔｕｒｅを作成する。（ライゲーション反応液内のベクター／インサート比については、各サンプルにつき１／５と１／１０の両方を調製する。）
２．ＧｅｎｅＡｍｐ９６００（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ）で、１６℃で３０分間インキュベーションし、ライゲーション反応を行う。
３．トランスフォーメーションの操作をプロトコル通りに行う。
４．トランスフォーメーション液を寒天プレートに播く。
５．でき上がったプレートを３７℃で１７時間程度（一晩）インキュベートする。
【００５８】
３）プラスミド抽出
１．コロニーの生えたプレートを１時間程度４℃でコールドインキュベーションする。
２．プラスミドが入っているホワイトコロニーのみを楊枝でピックアップし、液体培地に入った１５ｍｌチューブに楊枝ごと入れる。
３．２を振とうしながら８〜１０時間培養する。
【００５９】
４．ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ Ｋｉｔ（ＱＩＡＧＥＮ）を用いて付属のプロトコルに従いプラスミド抽出を行う。（このＫｉｔでは、大腸菌からプラスミドを溶出させ、精製、濃縮を行っていることになり、フェノール／クロロホルム抽出やエタノール沈殿等の操作を改めて行う必要はない。）
【００６０】
４）Ｍ１３ ＰＣＲ
得られたプラスミド抽出液をテンプレートとし、インサートよりも外側に位置している以下の表３に示すＭ１３プライマーを用いてＰＣＲを行う。ＰＣＲ条件はインサートの長さにより変える必要がある。
【００６１】
【表３】

【００６２】
ＤＧＧＥ
ＤＧＧＥはｄｅｎａｔｕｒｉｎｇ ｇｒａｄｉｅｎｔ ｇｅｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（変性剤濃度勾配ゲル電気泳動法）の略であり、元来染色体ＤＮＡの点変異検出に用いられた技術である。ＤＧＧＥ法は、ＰＣＲと組み合わせることで、微量のサンプルから環境中に存在する微生物の遺伝子を培養過程を経ずに検出することができるということで、微生物生態分野において急速に普及した。ＤＧＧＥ法は、培養できない微生物の遺伝子でも検出できるという点ではクローニングと似ているが、バンドとして可視化出来るため、環境中にどれくらいの種類の微生物が存在するのかを容易に判断することができるという利点をもっている。
【００６３】
ＤＧＧＥは、ＤＮＡ変性剤（尿素とホルムアルデヒド）の濃度勾配をつけたポリアクリルアミドゲル中でＤＮＡ電気泳動を行うことにより、長さのそろった複数種の２本鎖ＤＮＡ（例えば、ＰＣＲ産物）を、塩基配列の違いにより分離できる方法である。
【００６４】
ＧＣクランプ（配列：ＣＧＣＣＣＧＣＣＧＣＧＣＣＣＣＧＣＧＣＣＣＧＴＣＣＣＧＣＣＧＣＣＣＣＣＧＣＣＣＧ）付きのプライマーセットを用いて、ＰＣＲで増幅した２本鎖ＤＮＡを変性剤の濃度勾配をつけたポリアクリルアミドゲルで電気泳動すると、ＤＮＡ変性剤の濃度上昇とともに２本鎖ＤＮＡ間の水素結合が切断され、二重らせん構造から１本鎖ＤＮＡに変性する。しかし、ＧＣクランプ部分は結合力が強いため、ＤＮＡは３方向に伸びた形になる。このように変性したＤＮＡは、ゲルを移動する速度が著しく小さくなるため、ある場所に集まり、バンドを形成する。配列の異なる複数の２本鎖（異なる微生物）のＤＮＡはＡ、Ｔ、Ｇ、Ｃ間の水素結合の数及び配列の違いにより、異なるＤＮＡ変性剤濃度で解離するため、異なる位置にバンドが形成され、その結果、微生物を種類ごとに分離することができる。このようにして、サンプルごとにバンドプロファイルが得られ、そのパターンにより群集を評価することができるということになる。
【００６５】
以下、手順を説明する。
１）ポリアクリルアミドゲルの作成
１．変性剤について低濃度ゲル溶液、高濃度ゲル溶液、さらに０質量％のゲル溶液を調製する。ポリアクリルアミド濃度は８質量％とする。ゲルの組成を以下の表４に記す。
【００６６】
【表４】

【００６７】
２．１をそれぞれ脱気したものについて、低濃度・高濃度ゲル溶液を１４ｍｌずつ用意する。
３．用意した高濃度ゲルにＤｙｅを加え、低濃度高濃度のそれぞれに架橋剤である０．５質量％ＡＰＳと０．０５質量％ＴＥＭＥＤを加える。
４．変性剤の濃度勾配をつけられる装置を用いてゲルキャスティングを行う。
５．１２時間放置することで重合を促進させる。
【００６８】
２）電気泳動
ＤＧＧＥの泳動装置には、Ｄ ｃｏｄｅ ｓｙｓｔｅｍ（Ｂｉｏ Ｒａｄ）を使用した。
１．サンプルを用意する。
２．サンプル２０μｌと６×Ｄｙｅ４μｌを混ぜ、調製する。
３．１×ＴＡＥ７Ｌを６０℃まで加温したチャンバーにゲルをセットし、サンプルをアプライする。
４．１３０Ｖで５〜６時間、電気泳動を行う。
【００６９】
３）画像取得と解析
画像取得とその解析はＦｌｕｏｒｏＩｍａｇｅｒ５９５（ＭｏｌｅｃｕｌａｒＤｙｎａｍｉｃｓ）Ｓｃａｎｎｅｒ ＣｏｎｔｒｏｌとＩｍａｇｅ Ｑ ｗａｎｔ、ＦｒａｇｍｅＮＴを使用し行った。
【００７０】
１．泳動し終わったゲルを１万倍に希釈したＶｉｓｔｒａ Ｇｒｅｅｎで１５分間染色する。
２．余分な染色液を取り除き、蛍光イメージアナライザーＦｌｕｏｒｏＩｍａｇｅｒ５９５（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ）のＳｃａｎｎｅｒ Ｃｏｎｔｒｏｌを用いて、画像解析ソフトＩｍａｇｅ Ｑ ｗａｎｔで画像を取り込む。
【００７１】
３．塩基配列決定の為、バンドを切り出す必要のある場合はバンドを切り出す。
４．バンド強度をもとめる等の解析をする場合は、画像解析ソフトＦｒａｇｍｅＮＴを利用して付属のマニュアルに従い解析を行う。
【００７２】
シーケンシング
シーケンシングの一例として、ＳＱ−５５００（日立製作所）を用いた場合を例に、シーケンシングの方法を説明する。
【００７３】
１）オートシーケンサーＳＱ−５５００（日立製作所）によるシーケンシング
１．プロトコルに従いポリアクリルアミドゲルを作成する。
２．Ｖｉｓｔｒａ ｓｅｑｕｅｎｃｉｎｇ ｋｉｔ（Ａｍａｓｈａｍ Ｐｈａｒｍａｓｈｉａ）を用い、そのプロトコルに従ってシーケンシング反応のｍｉｘｔｕｒｅを調製する。
３．Ｔ３ Ｔｈｅｒｍｏｃｙｃｌｅｒ（Ｂｉｏｍｅｔｒａ）またはＧｅｎｅＡｍｐ９６００（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、シーケンシング反応を行う。シーケンシング反応のプログラムは、増幅部位の長さに関わらず、＜９５℃５ｍｉｎ、〔９５℃：３０ｓｅｃ、６０℃：３０ｓｅｃ〕×２５回、４℃＞を使用する。
【００７４】
４．ＨＩＴＡＣＨＩ ＳＱ−５５００で１時間予備泳動したゲルに、３のサンプルをアプライする。
５．適当な時間、電気泳動を行う。
６．ｆｏｒｗａｒｄ鎖とｒｅｖｅｒｓｅ鎖のａｓｓｅｍｂｌｅを行い、必要に応じて修正し塩基配列を決定する。
【００７５】
【実施例】
実施例１：パイロットプラントの運転
高濃度のアンモニア態窒素とＣＯＤ成分として、フェノールと、チオ硫酸、チオシアンといった硫黄化合物を含む原水を調製し、この原水から硝化脱窒による窒素除去を行うパイロットプラントを運転した。水質データのモニタリングを行い、また定期的に複合微生物解析のため、活性汚泥の採取を行った。図１に、パイロットプラントの概要を、そして以下の表５に運転条件を示す：
【００７６】
【表５】

【００７７】
パイロットプラントは、安水のＣＯＤ削減とアンモニア除去を行うため硝化液循環型の硝化脱窒活性汚泥法を採用した。原水の構成成分を以下の表６に示す：
【００７８】
【表６】

【００７９】
原水は前段で、嫌気槽である脱窒槽を、後段で、好気槽である硝化槽を通り、硝化液は脱窒槽へ戻って循環処理される。処理水は、沈殿槽を経て放流される。硝化液の循環率は２００％、汚泥返送率は１００％である。汚泥の引き抜きを行い、ＳＲＴが約３０日間となるように運転した。各水質の測定項目を以下の表７に示す：
【００８０】
【表７】

【００８１】
微生物群集解析用の活性汚泥は硝化槽の２槽めから採取した。
【００８２】
以下にパイロットプラントの運転状況を示す。
まず、窒素除去性能であるが、図２に原水と処理水の全窒素濃度及び窒素除去性能の目安となる原水と処理水の全窒素濃度の差の推移を示す。
【００８３】
図２に示すように経時的に良好な窒素除去性能が得られるようになった。
また、窒素除去が少ない２００２年８月２１日の時点と、窒素除去が良好な２００２年１１月１３日の時点における、脱窒槽でのフェノール、チオ硫酸、チオシアンの消費速度を計算した結果を、以下の表８に示す：
【００８４】
【表８】

【００８５】
脱窒による窒素除去の高まりと対応して、フェノール、チオ硫酸、チオシアンが多く消費されることが明らかとなった。フェノール、チオ硫酸、チオシアンをＣＯＤ成分として消費する硝酸態窒素からの脱窒反応、及び、亜硝酸態窒素からの脱窒反応は、それぞれ以下のように表すことができると考えられる。
【００８６】
まず、硝酸態窒素からの脱窒反応については、以下のように表せる。
フェノールを用いる硝酸態窒素からの脱窒反応は、 ：
５Ｃ_６Ｈ_５ＯＨ ＋ ２８ ＮＯ_３− → １４Ｎ_２ ＋ ３０ＣＯ_２ ＋ Ｈ_２Ｏ ＋ ２８ＯＨ−
のように表すことができ、フェノール１ｇあたり硝酸態窒素を０．８３ｇ脱窒できることが計算される。
【００８７】
チオ硫酸を用いる硝酸態窒素からの脱窒反応は、
５Ｓ_２Ｏ_３２− ＋ ８ＮＯ_３− ＋ Ｈ_２Ｏ → ４Ｎ_２ ＋ １０ＳＯ_４２− ＋ ２Ｈ＋
のように表すことができ、チオ硫酸１ｇあたり硝酸態窒素を０．２ｇ脱窒できることが計算される。
【００８８】
チオシアンを用いる硝酸態窒素からの脱窒反応は、
５ＳＣＮ− ＋ ３ＮＯ_３− ＋ Ｈ_２Ｏ → ４Ｎ_２ ＋ ５ＳＯ_４２− ＋ Ｈ_２Ｏ ＋ ２Ｈ＋
のように表すことができ、チオシアン１ｇあたり硝酸態窒素を０．１４ｇ脱窒できることが計算される。
【００８９】
次に、亜硝酸態窒素からの脱窒反応については、以下のように表せる。
フェノールを用いる亜硝酸態窒素からの脱窒反応は、
３ Ｃ_６Ｈ_５ＯＨ ＋ ２８ ＮＯ_２− ＋ ２８ Ｈ＋ → １４ Ｎ_２ ＋ １８ ＣＯ_２ ＋ ２３ Ｈ_２Ｏ
のように表すことができ、フェノール１ｇあたり亜硝酸態窒素を１．４ｇ脱窒できることが計算される。
【００９０】
チオ硫酸を用いる亜硝酸態窒素からの脱窒反応は、
３ Ｓ_２Ｏ_３２− ＋ ８ ＮＯ_２− ＋ ２ Ｈ＋ → ４ Ｎ_２ ＋ ６ ＳＯ_４２− ＋ Ｈ_２Ｏ
のように表すことができ、チオ硫酸１ｇあたり亜硝酸態窒素を０．３３ｇ脱窒できることが計算される。
【００９１】
チオシアンを用いる亜硝酸態窒素からの脱窒反応は、
３ ＳＣＮ− ＋ １１ ＮＯ_２− ＋ ８ Ｈ＋ → ７ Ｎ_２ ＋ ３ ＳＯ_４２− ＋ ３ ＣＯ_２ ＋ ４ Ｈ_２Ｏ
のように表すことができ、チオシアン１ｇあたり亜硝酸態窒素を０．８８ｇ脱窒できることが計算される。
【００９２】
したがって、本パイロットプラントの結果から、硝酸態窒素又は亜硝酸態窒素からの脱窒が、原水由来のフェノール、チオ硫酸、チオシアンを用いて可能であることが実証できた。上記の計算結果から、化学量論的に、亜硝酸態窒素とフェノール又はチオシアンを基質とする脱窒、あるいは硝酸態窒素とフェノールを基質とする脱窒反応が効率的に進んだことが考えられる。
【００９３】
実施例２：クローニングによる亜硝酸還元酵素遺伝子ｎｉｒＫの塩基配列決定と、当該遺伝子を有する微生物の特定
クローニングでは、ＰＣＲで増幅することができさえすれば、サンプル中に存在する微生物全てが、ある確率でクローンとして得られることになる。そこで、窒素除去性能が良好な時点のパイロットプラントから活性汚泥を採取して、その中に存在する脱窒菌を亜硝酸還元酵素遺伝子ｎｉｒＫを有することを手掛かりに、特定することを試みた。まず、活性汚泥に存在する微生物に対して、亜硝酸還元酵素遺伝子ｎｉｒＫの約５１５ｂｐの領域のクローニングを行い、その塩基配列を決定した。
【００９４】
方法
クローニングの操作は、先に説明したプロトコルに従って行った。窒素除去性能が良好な時点のパイロットプラントから採取した活性汚泥について、ＤＮＡ抽出液から亜硝酸還元酵素遺伝子ｎｉｒＫの約５１５ｂｐの領域をＰＣＲ増幅した（ＰＣＲ条件＜９５℃：１０ｍｉｎ、〔９４℃：３０ｓｅｃ、５０℃：３０ｓｅｃ、７２℃：２ｍｉｎ〕×２５回、７２℃：１０ｍｉｎ、４℃＞）。ＰＣＲ ｐｒｏｄｕｃｔｓをＴＡ Ｃｌｏｎｉｎｇ ｋｉｔ （ｐＴ７ｂｌｕｅ Ｔ−Ｖｅｃｔｏｒ （Ｔａｋａｒａ， Ｔｏｋｙｏ， Ｊａｐａｎ）， ＤＨ５α ｃｏｍｐｅｔｅｎｔ ｈｉｇｈ ｃｏｍｐｅｔｅｎｔ ｃｅｌｌ ｋｉｔ （ＴＯＹＯＢＯ， Ｔｏｋｙｏ， Ｊａｐａｎ））を使用してクローニングした。寒天プレート上に得られたコロニーのうち、白いコロニーのみ、滅菌した爪楊枝で採取し、予め用意したＰＣＲ ｍｉｘｔｕｒｅ（１００μｌ）に懸濁した。ＰＣＲ ｍｉｘｔｕｒｅの組成、ＰＣＲ条件は、春日らの報告（春日ほか （２００１）水環境学会誌， ２４， ８５６−８６４）に従ったが、ＰＣＲ ｐｒｉｍｅｒにはベクターをターゲットとしたＭ１３ ｐｒｉｍｅｒを用い、同プライマーによりインサートを増幅した。なお得られたＰＣＲ ｐｒｏｄｕｃｔｓに対しアガロース電気泳動を行い、増幅を確認した。ＰＣＲ ｐｒｏｄｕｃｔｓをＭｉｃｒｏｃｏｎ（Ｍｉｌｌｉｐｏｒｅ Ｔｏｋｙｏ Ｊａｐａｎ）により精製し、ｐｒｉｍｅｒ ｄＮＴＰを除去した。
【００９５】
次に精製したＰＣＲ ｐｒｏｄｕｃｔｓ（２０ｎｇ）を表３に記載のＭ１３ ｐｒｉｍｅｒ、ＡＢＩ Ｂｉｇ ｄｙｅ ｔｅｒｍｉｎａｔｏｒ ｋｉｔ ｖｅｒｓｉｏｎ ３．１ （Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使って、シーケンシング反応を行った。反応条件はＡＢＩ３１０ ＤＮＡシーケンサー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）のマニュアルに従った。シーケンシング反応で得られた産物中に含まれるｄｙｅ ｔｅｒｍｉｎａｔｏｒを Ｃｅｎｔｒｉ−ｓｅｐ ｓｐｉｎ ｃｏｌｕｍｎｓ （Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）で除去した後、ＡＢＩ３１０ＤＮＡシーケンサーでシーケンシングを行った。
【００９６】
決定した塩基配列を用いて、相同性検索プログラムＢＬＡＳＴ（ｈｔｔｐ：／／ｓｐｉｒａｌ． ｇｅｎｅｓ．ｎｉｇ．ａｃ．ｊｐ／ｈｏｍｏｌｏｇｙ／ｂｌａｓｔ．ｓｈｔｍｌ）を用いて既知種の中から相同性の高い塩基配列を検索し、相同性を調べ、微生物の特定を行った。
【００９７】
結果・考察
亜硝酸還元酵素遺伝子ｎｉｒＫの塩基配列として、配列表の配列番号１〜１７に記載の塩基配列を決定した。相同性解析の結果を表９に記す。
【００９８】
【表９】

【００９９】
全てデータベース上に１００％一致のものがなく、新規なｎｉｒＫ遺伝子の塩基配列を有する微生物であることを実証した。
【０１００】
実施例３：ＰＣＲ−ＤＧＧＥによる亜硝酸還元酵素遺伝子ｎｉｒＳの塩基配列決定と、当該遺伝子を有する微生物の特定
ＰＣＲ−ＤＧＧＥは、環境サンプル等の培養できない微生物の遺伝子でも検出し、バンドとして可視化でき、さらにはバンドを切り出すことでそのバンドの塩基配列情報を得ることができるため、近年、複合微生物系である環境サンプルの解析などにおいて急速に普及してきた技術である。また、微生物の変化を系時的にバンドの変化として追うことができるという利点を有している。そこで、窒素除去性能が良好な時機のパイロットプラントから活性汚泥を採取して、その中に存在する脱窒菌を亜硝酸還元酵素遺伝子ｎｉｒＳを有することを手掛かりに、特定することを試みた。まず、活性汚泥に存在する微生物に対して、亜硝酸還元酵素遺伝子ｎｉｒＳの一部塩基配列を決定した。
【０１０１】
以下、手順を説明する。
窒素除去性能が良好な時機のパイロットプラントから採取した活性汚泥からＤＮＡを抽出し、以下の表１０に記載したＧＣクランプ付きプライマーセットを用いて亜硝酸還元酵素遺伝子ｎｉｒＳの一部領域を増幅して解析対象とした。
【０１０２】
【表１０】

【０１０３】
ＰＣＲ条件は、＜９５℃：１０ｍｉｎ、〔９４℃：３０ｓｅｃ、５３℃：３０ｓｅｃ、７２℃：３０ｓｅｃ〕×３５回、７２℃：１０ｍｉｎ、４℃＞であった。その後、ＤＧＧＥのプロトコルに従い４０質量％から６０質量％の変性剤濃度勾配をつけたゲルを用いて、１３０Ｖで６時間電気泳動を行った。
【０１０４】
塩基配列解読によるＤＧＧＥバンドの近縁種決定
得られたバンドについて、それぞれどのような微生物なのかを確認し、パイロットプラント内に存在する微生物の全体像を把握するため、各バンドの近縁種の決定を試みた。
【０１０５】
方法
ＤＧＧＥのゲルから主要なバンドを切り出し、滅菌ｍｉｌｌｉＱとともに凍結融解を３回繰り返すことで、ゲル片からＤＮＡを回収し、それをテンプレートとしてＰＣＲ−ＤＧＧＥを行うという精製の操作を繰り返し、ＤＧＧＥのゲル上で一本のバンドにした。最増幅のＰＣＲ条件は、＜９５℃：１０ｍｉｎ、〔９４℃：３０ｓｅｃ、５３℃：３０ｓｅｃ、７２℃：３０ｓｅｃ〕×２５回又は２０回、７２℃：１０ｍｉｎ、４℃＞である。その後、精製したＤＮＡをテンプレートとしてＧＣクランプ無しのプライマーで亜硝酸還元酵素遺伝子ｎｉｒＳの当該領域を再増幅（ＰＣＲ条件：＜９５℃：１０ｍｉｎ、〔９４℃：３０ｓｅｃ、５３℃：３０ｓｅｃ、７２℃：３０ｓｅｃ〕×２５回、７２℃：１０ｍｉｎ、４℃＞）し、シーケンシング反応のテンプレートとした。シーケンシング反応は先に説明したプロトコルに従い、またシーケンサーはＨＩＴＡＣＨＩ（ＳＱ−５５００）を用いた。塩基配列を決定した後、相同性検索プログラムＢＬＡＳＴ（ｈｔｔｐ：／／ｓｐｉｒａｌ．ｇｅｎｅｓ．ｎｉｇ．ａｃ．ｊｐ／ｈｏｍｏｌｏｇｙ／ｂｌａｓｔ．ｓｈｔｍｌ）を用いて既知種の中から相同性の高い塩基配列を検索し、ＤＧＧＥ各バンドの近縁種を決定した。
【０１０６】
結果・考察
亜硝酸還元酵素遺伝子 ｎｉｒＳの一部塩基配列として、配列表の配列番号１８から３９に記載の塩基配列を決定した。相同性解析の結果を以下の表１１に示す：
【０１０７】
【表１１】

【０１０８】
全てデータベース上に１００％一致のものがなく、新規なｎｉｒＫ遺伝子の塩基配列を有する微生物であることを実証した。
【０１０９】
【発明の効果】
本発明は、窒素含有排水から窒素除去する作用を有する微生物を明らかにすると共に、ＤＮＡの塩基配列を用いて当該微生物を検出する方法を確立した。これにより、窒素除去方法及びその評価方法を提供することを可能にした。
【配列表】

【図面の簡単な説明】
【図１】図１は、硝化脱窒による窒素除去パイロットプラントの概略図である。
【図２】図２に原水と処理水の全窒素濃度及び窒素除去の性能の目安となる原水と処理水の全窒素濃度の差の推移を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a microorganism present in activated sludge of a water treatment plant for reducing nitrogen, a base sequence of a gene for nitrite reductases NiRK and NiRS, and nitrogen using activated sludge containing the microorganism. It relates to a water treatment method for reduction.
[0002]
[Prior art]
The nitrification denitrification method is widely used for the treatment of water containing nitrogen such as ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen such as urban sewage, agriculture, and livestock wastewater. However, nitrification denitrification has not been applied to the treatment of water containing a high concentration of nitrogen, such as wastewater (ammonified water) generated from a coke oven. At present, nitrification denitrification has recently begun to be attempted on wastewater containing high concentration of nitrogen such as low-temperature water (Patent Document 1: JP-A-2003-53383). Reference).
In addition, when removing nitrogen from wastewater containing high concentrations of nitrogen, it is not clear what kind of microorganisms are involved in nitrogen removal in the activated sludge used for nitrification and denitrification. There are no studies analyzing this.
[0003]
Nitrogen denitrification is widely used in nitrogen removal using microorganisms. Nitrification and denitrification is the action of microorganisms that oxidizes ammonia nitrogen in wastewater through nitrite nitrogen to nitrate nitrogen, then reduces it from nitrate nitrogen to nitrite nitrogen to nitrogen molecules, This is a method for removing nitrogen that is released into the atmosphere as a gas. The reaction of oxidizing ammonia nitrogen to nitrate nitrogen is called nitrification, and the reaction of reducing nitrate nitrogen to nitrogen molecules is called denitrification. That is, nitrification and denitrification are combined to form a nitrification denitrification method. In general, nitrification is a reaction that proceeds in an aerobic tank that supplies oxygen, and denitrification is a reaction that proceeds in an anaerobic tank that does not supply oxygen.
[0004]
Various attempts have been made to make nitrification denitrification efficient. The Barnard method is a nitrification denitrification method in which an anaerobic tank for denitrification is installed in the first stage, and an aerobic tank for nitrification is installed in the second stage, and the nitrification liquid generated in the second stage is circulated so as to return to the first stage. It is. In the denitrification reaction, it is necessary to simultaneously consume a COD component serving as a substrate. Since the COD component derived from raw water can be used directly for the denitrification reaction, the Barnard method is an extremely efficient nitrification denitrification method. If the nitrification reaction tank is installed at the preceding stage, the COD component derived from the raw water is easily oxidized and decomposed by the oxygen in the aerobic tank. For this reason, the COD component required for the denitrification reaction becomes insufficient.
[0005]
Nitrification denitrification is not a reaction that can be performed by a single microorganism, but is carried out by a complex microorganism system present in activated sludge. First, regarding nitrification, it is known that there are ammonia oxidizing bacteria that oxidize ammonia nitrogen to nitrite nitrogen and nitrite oxidizing bacteria that oxidize nitrite nitrogen to nitrate nitrogen. Nitrosomonas and the like are known as ammonia-oxidizing bacteria. As the nitrite-oxidizing bacteria, Nitrobacter and the like are known.
[0006]
Regarding the denitrification reaction, various types of denitrifying bacteria that reduce nitrate nitrogen or nitrite nitrogen to nitrogen molecules are known. In the denitrification reaction, which is a nitrogen removal reaction, participation of various enzymes possessed by living organisms is known. Nitrate (salt) reductase NaR (nitrate reduce) to reduce nitrate nitrogen to nitrite nitrogen, nitrite (salt) reduced oxygen NiR (nitrite reducease) to reduce nitrite nitrogen to nitric oxide, nitric oxide NOR (Nitric Oxide Reduced) that reduces nitrogen to nitrous oxide, and oxygen N that reduces nitrous oxide to nitrogen molecules₂OR (Nitrous Oxide Reduced). Under the catalytic action of these enzymes, nitrate nitrogen or nitrite nitrogen in the wastewater can be finally converted into harmless nitrogen gas and released into the atmosphere.
Of these, the reaction controlled by the nitrite reductase NiR may be rate-limiting in the denitrification reaction, and is a very important reaction in the denitrification reaction.
[0007]
Generally, in analyzing microbial communities, the culturability of microorganisms in activated sludge is low. Therefore, the microbial community structure in the activated sludge that controls nitrogen removal cannot be grasped by microbial analysis by culture.
[0008]
In particular, a microorganism having nitrite reductase involved in nitrogen removal has not yet been identified.
[Patent Document 1]
JP 2003-53383 A
[0009]
[Problems to be solved by the invention]
From the above background, from the wastewater containing high concentration of nitrogen, grasp the microbial community of activated sludge that can be used for water treatment to remove nitrogen, and discover useful microorganisms involved in nitrogen removal, There is a need to provide a new water treatment method for efficiently removing nitrogen from wastewater containing high concentrations of nitrogen using microorganisms.
[0010]
[Means for Solving the Problems]
In analyzing microbial communities, the cultureability of microorganisms in activated sludge is generally low. Therefore, it is difficult to understand the microbial community structure in activated sludge that controls nitrogen removal by microbial analysis by culture. Therefore, the present inventor, by removing nitrogen by nitrification denitrification using activated sludge from wastewater containing a high concentration of nitrogen, the microorganisms present in the sludge and performing nitrogen removal, the functional enzymes involved in nitrogen removal, We focused on genes and tried to clarify them. That is, we focused on nitrite reductase (NiR), which controls the reaction of converting nitrite nitrogen into nitric oxide, which is an important reaction in the nitrogen removal reaction. It is known that there are two types of nitrite reductases, called NiRK and NiRS. The present inventors identified microorganisms involved in nitrogen removal by detecting the genes for nitrite reductases NiRK and NiRS. In addition, by elucidating the partial nucleotide sequence of this gene, we succeeded in specifying microorganisms involved in nitrogen removal based on the nucleotide sequence. The inventors succeeded in establishing a nitrogen removal method using activated sludge containing these microorganisms, and completed the present invention.
[0011]
That is,
According to the present invention, the following [1] to [48] are provided:
[1] Nitrite reductase (NiRK) of bacteria related to Uncultured bacterium clone U13 present in activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 1 or 3 DNA fragment of the gene.
[2] The nitrite reductase (NiRK) gene of Unrelated Bacterium clone M17 related bacteria present in the activated sludge of the water treatment plant by the activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 2 DNA fragment.
[3] Mesorhizobium having the sequence shown in SEQ ID NOs: 4, 6, 7, 9, 10, 12, 13, 14, or 16, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, which is used for nitrogen reduction. sp. DNA fragment of the nitrite reductase (NiRK) gene of 4FB11 related bacteria.
[0012]
[4] Alcaligenes sp. Having the sequence shown in SEQ ID NO: 5, which is present in the activated sludge of a water treatment plant by the activated sludge method used for reducing nitrogen. DNA fragment of the nitrite reductase (NiRK) gene of a bacterium closely related to STC1.
[5] a DNA fragment of the nitrite reductase (NiRK) gene of a bacterium related to Alcaligenes xylosoxidans present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 8 .
[6] The nitrite reductase (NiRK) gene of Unrelated Bacterium clone M27-related bacteria present in the activated sludge of the water treatment plant by the activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 11 DNA fragment.
[7] a DNA fragment of the nitrite reductase (NiRK) gene of a Pseudomonas mendocina-related bacterium having the sequence shown in SEQ ID NO: 15 and present in activated sludge of a water treatment plant by an activated sludge method used for reducing nitrogen .
[0013]
[8] The nitrite reductase (NiRK) gene of Unrelated Bacterium clone M58-related bacteria present in the activated sludge of the water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 17 DNA fragment.
[9] Nitrite reductase (NiR S) of bacteria closely related to Paracoccus denitrificans Pd1222, which has the sequence shown in SEQ ID NO: 18, 33 or 39 and is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. A) DNA fragment of the gene.
[10] A nitrite reductase (NiRS) gene of a bacterium closely related to Thauera aromatica strain AR1 which has the sequence shown in SEQ ID NO: 19 and is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment.
[11] Nitrite reductase (NiRS) of a bacterium closely related to Unculturized bacterium which has the sequence shown in SEQ ID NO: 20, 22, or 24 and is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment of the gene.
[0014]
[12] A.1 present in activated sludge of a water treatment plant having the sequence shown in SEQ ID NO. A DNA fragment of the nitrite reductase (NiRS) gene of eutrophus-related bacteria.
[13] A DNA fragment of the nitrite reductase (NiRS) gene of a bacterium related to Azoarcus toluvorans present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 23 .
[14] The nitrite reductase (NiRS) gene of bacteria related to Unculturized bacterium clone nir SAK4, which is present in the activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 25 DNA fragment.
[15] The nitrite reductase (NiRS) gene of a bacterium closely related to Unculturized bacterium clone neirSAK2, which has the sequence shown in SEQ ID NO: 26 and is present in the activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction. DNA fragment.
[16] Nitrite reductase (NiRS) of bacteria related to Unculturized bacterium clone ANIS-76 present in activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction having the sequence shown in SEQ ID NO: 27 DNA fragment of the gene.
[0015]
[17] A bacterium related to Unculturized bacterium clone HNIS-6 having the sequence shown in SEQ ID NO: 28, 29, 30, 36 or 38 and present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment of nitrite reductase (NiRS) gene.
[18] a DNA fragment of the nitrite reductase (NiRS) gene of a paracoccus pantotrophus-related bacterium having the sequence shown in SEQ ID NO: 31 and present in activated sludge of a water treatment plant by an activated sludge method used for reducing nitrogen .
[0016]
[19] A DNA fragment of the nitrite reductase (NiRS) gene of a bacterium related to Thauera linalolentis present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 32 .
[20] The nitrite reductase (NiRS) gene of a bacterium closely related to Thauera aromatica strain AR1 which has the sequence shown in SEQ ID NO: 34 and is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment.
[21] The nitrite reductase (NiRS) gene of a related bacterium of Thauera terpenica strain 58Eu present in the activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 35 DNA fragment.
[22] The nitrite reductase (NiRS) gene of Pseudomonas aeruginosa strain Mi11 related bacteria present in the activated sludge of the water treatment plant by the activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 37 DNA fragment.
[0017]
[23] Water treatment by the activated sludge method used in the nitrite reductase (NiRK) gene, which contains the base sequence of the DNA fragment according to any one of [1] to [8] above and is used for nitrogen reduction Microorganisms present in activated sludge of the plant.
[0018]
[24] Water treatment by the activated sludge method used for reducing nitrogen, wherein the nitrite reductase (NiRS) gene contains the nucleotide sequence of the DNA fragment according to any of the above [9] to [23]. Microorganisms present in activated sludge of the plant.
[0019]
[25] Activated sludge of a water treatment plant for reducing nitrogen by a nitrifying liquid circulating type nitrifying denitrification activated sludge method, wherein the nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 1 or 3 Unculturized bacterium clone U13 closely related bacteria present in it.
[26] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 2 and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Existing Unculturated bacterium clone M17 related bacteria.
[27] The nitrite reductase (NiRK) gene contains the nucleotide sequence shown in SEQ ID NO: 4, 6, 7, 9, 10, 12, 13, 14 or 16 and has a nitrifying solution-circulating nitrification denitrification activity Mesorhizobium sp. Present in activated sludge of a water treatment plant for nitrogen reduction by a sludge method. 4FB11 related bacteria.
[0020]
[28] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 5, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Existing Alcaligenes sp. STC1 related bacteria.
[0021]
[29] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 8 and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Alcaligenes xylosoxidans related bacteria present.
[30] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 11 and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Uncultured bacterium clone M27 related bacteria present.
[31] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 15 and is contained in activated sludge of a water treatment plant for nitrogen reduction by the nitrification liquid circulation type nitrification denitrification activated sludge method. Pseudomonas mendocina related bacteria present.
[32] The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 17 and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Unculturated bacterium clone M58 related bacteria present.
[0022]
[33] A water treatment plant for reducing nitrogen by using the nitrifying liquid circulating type nitrification denitrification activated sludge method, wherein the nitrite reductase (NiRS) gene contains the nucleotide sequence of SEQ ID NO: 18, 33 or 39. Paracoccus denitrificans present in activated sludge
Closely related bacteria of Pd1222.
[34] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 19, and is contained in activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. Thauera aromatica strain AR1 related bacteria present.
[0023]
[35] A water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method, wherein the nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 20, 22, or 24. Uncultured bacterium-related bacteria present in activated sludge.
[36] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 21 and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Existing A. eutrophus-related bacteria.
[37] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 23, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Azoarcus toluvorans closely related bacteria present.
[0024]
[38] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 25, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Unculturated bacterium clone neirSAK4 related bacteria present.
[39] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 26, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Existing Unculturated bacterium clone neir SAK2 related bacteria.
[40] In the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulating type nitrification denitrification activated sludge method, which contains the base sequence shown in SEQ ID NO: 27 in its nitrite reductase (NiRS) gene. Unculturated bacterium clone ANIs-76 related bacteria present.
[41] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 28, 29, 30, 36 or 38, and is used for reducing nitrogen by the nitrification liquid circulation type nitrification denitrification activated sludge method. Unculturated bacterium clone HNIS-6 related bacteria present in the activated sludge of the water treatment plant.
[0025]
[42] In the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method, which contains the base sequence shown in SEQ ID NO: 31 in its nitrite reductase (NiRS) gene. Paracoccus pantotrophus related bacteria present.
[43] In the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulating-type nitrification denitrification activated sludge method, which contains the base sequence shown in SEQ ID NO: 32 in its nitrite reductase (NiRS) gene. A related bacterium of Thauera linalolentis present.
[44] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 34, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Thauera aromatica strain AR1 related bacteria present.
[45] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 35, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Thauera terpenica strain 58Eu related bacteria present.
[46] The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 37, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Pseudomonas aeruginosa strain Mi11 related bacteria present.
[0026]
[47] An activated sludge for use in water treatment for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method, comprising the microorganism or bacterium according to any of [23] to [46].
[0027]
[48] A water treatment method for reducing nitrogen using the activated sludge according to [47].
[0028]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, as will be described in detail in Examples, a pilot plant for performing nitrogen removal by nitrification and denitrification from raw water containing a high concentration of ammonia nitrogen and COD components is operated, and activated sludge collected from this pilot plant is operated. The microorganisms were analyzed.
[0029]
FIG. 1 shows an outline of a nitrogen removal pilot plant by nitrification and denitrification. Denitrifying bacteria in activated sludge responsible for nitrogen removal were identified based on the nucleotide sequence of the DNA by detecting the genes of nitrite reductases NiRK and NiRS. In the analysis of the nitrite reductase gene nirK (hereinafter, the gene for nitrite reductase NiRK is referred to as nirK), the cloning of a clone library by cloning is used to analyze the removal of nitrogen present in activated sludge. The nucleotide sequence of the nirK gene spanning about 550 bases carried by the nitrifying bacteria was determined. Analysis of the nitrite reductase gene nirS (hereinafter, the nitrite reductase NiRS gene is referred to as nirS) is performed by using PCR-DGGE to denitrify bacteria involved in removing nitrogen present in activated sludge. The partial sequence of the base sequence of the nirS gene, which is owned by NirS, was determined.
[0030]
Hereinafter, the experimental principle procedure used in the examples will be described.
[0031]
Collection and storage of activated sludge samples
The sludge collected from the plant was mixed with sludge: ethanol = 9: 1 and stored at 4 ° C. for a while. Sampling for DNA extraction was performed as follows.
Prepare several 1 ml sludges in a 1.2 ml tube and centrifuge to remove the supernatant.
2. The pellet is suspended in a TH (pH 8.0) buffer, and the supernatant is removed by centrifugation.
3. Store at -20 ° C.
[0032]
DNA extraction
Fast DNA SPIN Kit for soil (BIO101) was used.
Fast DNA SPIN Kit for soil (BIO101) is a kit that can efficiently extract DNA from soil or other environmental samples of up to 500 mg within 30 minutes. Although a homogenizing device is required, there are advantages such as a short extraction work time, no need for an organic solvent such as phenol and chloroform, and the ability to extract gram-positive bacteria which have been relatively difficult to extract.
[0033]
The operation of the Fast DNA SPIN Kit for soil is roughly divided into two steps.
1) Cell disruption, DNA solubilization, protein solubilization
The sample is dissolved in a buffer capable of homogenizing and solubilizing the protein, and mixed with beads made of ceramic and silica to disrupt cells and solubilize DNA. At this stage, DNA with almost no RNA contamination can be obtained.
[0034]
2) Purification and concentration of DNA
The mixed inhibitor is removed by adsorbing only the solubilized DNA to a silica binding matrix. Thereafter, the DNA adsorbed on the binding matrix is eluted to obtain a purified DNA.
[0035]
Hereinafter, the procedure will be described.
[0036]
1. The sample is homogenized to the sampled and stored pellet, and Sodium Phosphate Buffer and MT Buffer that can solubilize the protein are added, and the pellet is suspended.
2. Transfer 1 to Lysing Matrix E tube with beads. 3.2 is placed on Fast Prep, and treated twice at Speed 5.0, 30 sec, to crush and lyse the cells.
[0037]
4. Transfer the supernatant to a new tube, add PPS and stir by hand.
5. Protein is removed by centrifugation to precipitate the protein and transferring the supernatant to a new tube.
An equivalent amount of binding matrix to the supernatant transferred in 6.5 is added, and the mixture is reverse-infiltrated by hand and allowed to stand still for several minutes to adsorb and precipitate DNA on the binding matrix.
[0038]
7. After removing the supernatant, the Binding Matrix DNA is suspended in the remaining liquid, and the suspension is transferred to a Spin Filter.
8.7 is centrifuged, after which SEWS-M is added to wash and concentrate the Binding Matrix-DNA on the Filter.
9. DES is added to the binding matrix DNA on the filter, and the DNA is eluted by centrifugation to collect PCR-grade DNA. DNA can be stored at -20 ° C.
[0039]
PCR
In the following implementation, NiRK and NiRS which are nitrite reductases responsible for nitrite reduction important in the denitrification reaction were traced in order to trace those involved in nitrogen removal among the microorganisms present in the sample to be analyzed. PCR was performed targeting the gene of
[0040]
An experimental flow of the PCR method targeting the nirK gene and the nirS gene will be described.
[0041]
1. After DNA extraction, the DNA concentration is measured using an absorptiometer.
2. Prepare DNA as template at 50 ng / μl.
3. Prepare a PCR mixture as shown in Table 1 below.
[0042]
[Table 1]

[0043]
The primer to be used is selected from Table 2 below.
[0044]
[Table 2]

[0045]
4. PCR is performed using a thermal cycler T3 Thermocycler (Biometra) or GeneAmp9600 (PE Biosystems) under program conditions according to the purpose.
[0046]
5. Electrophoresis was performed at 100 V for 15 minutes using an agarose gel (agarose was dissolved in 1 × TAE to change from 1% to 2% by mass), followed by ethidium bromide staining for 15 minutes, and a UV transilluminator (Toyobo) Confirm whether the PCR product was obtained by.
[0047]
Cloning
In a complex microbial system, when PCR is performed targeting a gene and cloning is performed using the PCR product as a template, various microorganisms present in the system can be obtained with a certain probability of being isolated. By creating the above, the whole picture of the existing microorganisms can be known. Regarding microorganisms in activated sludge of a water treatment process in which nitrification and denitrification-type nitrogen removal was performed, a clone library was prepared from PCR products obtained with a primer of the nirK gene.
[0048]
Cloning is roughly divided into the following two steps.
Ligation
Ligation is an operation of incorporating an insert (specific gene product (PCR product)) into a plasmid vector. Since a plasmid vector used for cloning needs to incorporate an insert, it must have a cloning site that can be cleaved with a restriction enzyme. In addition, since the plasmid is finally transmitted to Escherichia coli and the number of PCR products incorporated in the plasmid is increased by increasing Escherichia coli, the plasmid vector has a replication origin recognized by Escherichia coli and further has a plasmid. In order to selectively grow E. coli, it is necessary to have a drug resistance gene. Although plasmid vectors can be prepared by oneself, vectors prepared as described above are commercially available.
[0049]
At the time of ligation, it is necessary to pay attention to the method of modifying the end of the PCR product and the vector / insert ratio. Although there are several terminal modification methods, a relatively common one is TA cloning utilizing deoxyadenosine (dA) added during DNA synthesis by PCR. The vector / insert ratio usually needs to be about 1/1 to 1/10 so that ligation can be performed efficiently.
[0050]
Transformation
Transformation is an operation of infecting competent E. coli cells capable of taking up DNA with the ligated plasmid. By seeding the transformed cells on a plate containing a drug, Escherichia coli having drug resistance, that is, Escherichia coli containing a plasmid, selectively grows to form a colony. Since one colony is composed of Escherichia coli containing only one type of insert, genes can be separated for each microorganism in a complex microbial system.
[0051]
1) Examination of cloning method
There are several methods for terminal modification of the insert, and some ligation methods are also involved. In this experiment, however, the procedure is relatively simple, and a TA is said to be obtained with a probability of about 70% in transformants. A variant of the cloning method was employed. The TA cloning method is a method using deoxyadenosine (dA) added at the time of DNA synthesis by PCR, and depending on the sequence of the template DNA, a PCR product is mixed with a blunt-form DNA that does not add dA. It should be noted that the cloning efficiency varies depending on conditions such as PCR template DNA.
[0052]
Among the commercially available TA cloning methods, specifically, Kit of QIAGEN PCR Cloning System (UA cloning) was used. This is a modification of the TA cloning method using a vector with U instead of T. T is easy to hybridize with non-complementary bases (G, C, T), and a vector to which T is added may give a result such as annealing between vectors, whereas U is a non-specific base pair. It is said that the cloning efficiency is high, since it is difficult to form (QIAGEN product guide). In this experiment, TA cloning and UA cloning were also compared. In TA cloning, the insert ratio (the ratio of colonies containing the insert per appearing colony) was about half, but in UA cloning, the insert ratio was as high as about 90%, and the UA cloning method had better cloning efficiency. It became clear.
[0053]
2) Examination of appropriate vector / insert ratio
The vector / insert ratio is usually about 1/1 to 1/10. This is because if the number of inserts is small, the insert rate is low, and if the number of inserts is too large, there is a possibility that a plurality of inserts may be included in one vector. In this experiment, the experiment was performed for both 1/5 and 1/10 recommended in the kit of QIAGEN PCR Cloning System. As a result, although the number of colonies was larger by about 10% in both 1/5 and 1/10, it was not a remarkable difference, so that the ratio was about 1/5 or 1/10. We came to the conclusion that either would be fine.
[0054]
3) Examination of insert confirmation method
To confirm the insert, a method was used in which colonies were expanded by liquid culture, plasmids were extracted and purified by miniprep, followed by PCR or restriction enzyme treatment, followed by agarose gel electrophoresis.
[0055]
Hereinafter, the procedure will be described.
[0056]
1) Purification of PCR product (insert)
1. Prepare several PCR products containing the target PCR fragment to be cloned per sample.
2. They are collected and purified using Microcon (Millipore) to remove excess primers and dNTPs.
3. Measure the DNA concentration of the purified sample.
[0057]
2) Cloning operation
The basic operation of cloning was in accordance with the protocol of QIAGEN PCR Cloning System.
1. Create a Ligation mixture. (For the vector / insert ratio in the ligation reaction solution, prepare both 1/5 and 1/10 for each sample.)
2. A ligation reaction is performed by incubating at 16 ° C. for 30 minutes with GeneAmp9600 (PE Biosystems).
3. Perform the transformation operation according to the protocol.
4. Seed the transformation solution on an agar plate.
5. Incubate the resulting plate at 37 ° C. for about 17 hours (overnight).
[0058]
3) Plasmid extraction
1. The plate with the colonies is cold-incubated at 4 ° C. for about 1 hour.
2. Only the white colony containing the plasmid is picked up with a toothpick and put into a 15 ml tube containing a liquid medium together with the toothpick.
3. Incubate for 8-10 hours with shaking.
[0059]
4. Plasmid extraction is carried out using QIAprep Spin Miniprep Kit (QIAGEN) according to the attached protocol. (In this Kit, the plasmid is eluted from Escherichia coli, purification and concentration are performed, and there is no need to perform another operation such as phenol / chloroform extraction or ethanol precipitation.)
[0060]
4) M13 PCR
Using the obtained plasmid extract as a template, PCR is performed using M13 primers shown in Table 3 below located outside the insert. PCR conditions need to be changed depending on the length of the insert.
[0061]
[Table 3]

[0062]
DGGE
DGGE is an abbreviation of denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis), and is a technique originally used for detecting a point mutation of chromosomal DNA. The DGGE method has rapidly spread in the field of microbial ecology because it can detect a gene of a microorganism existing in the environment from a very small amount of sample without a culturing process by being combined with PCR. The DGGE method is similar to cloning in that it can detect the genes of microorganisms that cannot be cultured, but since it can be visualized as a band, the advantage is that it is easy to determine how many types of microorganisms are present in the environment. Have.
[0063]
DGGE performs a plurality of double-stranded DNAs (e.g., PCR products) of a uniform length by performing DNA electrophoresis in a polyacrylamide gel with a concentration gradient of a DNA denaturant (urea and formaldehyde). This is a method that can be separated by the difference in base sequence.
[0064]
Using a primer set with a GC clamp (sequence: CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG), when the double-stranded DNA amplified by PCR is electrophoresed on a polyacrylamide gel with a gradient of denaturing agent, the double-stranded DNA is increased with the concentration of DNA denaturing agent. Hydrogen bonds between DNAs are broken, and the double helical structure is denatured to single-stranded DNA. However, since the GC clamp portion has a strong binding force, the DNA extends in three directions. The DNA denatured in this way is remarkably slow in moving through the gel, and thus gathers in a certain place to form a band. DNAs of a plurality of double-stranded DNAs having different sequences (different microorganisms) are dissociated at different DNA denaturing agent concentrations due to differences in the number of hydrogen bonds between A, T, G, and C and the sequence, so that bands are formed at different positions. As a result, microorganisms can be separated for each type. In this way, a band profile is obtained for each sample, and the pattern can be used to evaluate the crowd.
[0065]
Hereinafter, the procedure will be described.
1) Preparation of polyacrylamide gel
1. For the denaturing agent, a low concentration gel solution, a high concentration gel solution, and a 0% by mass gel solution are prepared. The polyacrylamide concentration is 8% by mass. The composition of the gel is shown in Table 4 below.
[0066]
[Table 4]

[0067]
For each degassed of 2.1, prepare 14 ml of a low-concentration / high-concentration gel solution.
3. Dye is added to the prepared high-concentration gel, and 0.5% by mass APS and 0.05% by mass TEMED, which are crosslinking agents, are added to each of the low-concentration and high-concentration gels.
4. Gel casting is performed using a device capable of providing a concentration gradient of a denaturant.
5. Accelerate polymerization by leaving for 12 hours.
[0068]
2) Electrophoresis
D code system (Bio Rad) was used for the DGGE electrophoresis apparatus.
1. Prepare a sample.
2. Mix and prepare 20 μl of sample and 4 μl of 6 × Dye.
3. Set the gel in a chamber in which 7 L of 1 × TAE has been heated to 60 ° C., and apply the sample.
4. Perform electrophoresis at 130V for 5-6 hours.
[0069]
3) Image acquisition and analysis
Image acquisition and its analysis were performed using FluoroImager 595 (Molecular Dynamics) Scanner Control, Image Q Want, and FragmeNT.
[0070]
1. The gel after electrophoresis is stained for 15 minutes with Vistra Green diluted 10,000 times.
2. Excess staining solution is removed, and an image is captured using image analysis software Image Q wan using Scanner Control of a fluorescence image analyzer FluoroImager 595 (Molecular Dynamics).
[0071]
3. If it is necessary to cut out a band for base sequence determination, cut out the band.
4. When performing analysis such as obtaining band intensity, analysis is performed according to the attached manual using the image analysis software FragmeNT.
[0072]
Sequencing
As an example of the sequencing, a sequencing method will be described by using a case where SQ-5500 (Hitachi, Ltd.) is used as an example.
[0073]
1) Sequencing by Auto Sequencer SQ-5500 (Hitachi, Ltd.)
1. Prepare a polyacrylamide gel according to the protocol.
2. A mixture of sequencing reactions is prepared using the Vistra sequencing kit (Amasham Pharmacia) according to the protocol.
3. A sequencing reaction is performed using T3 Thermocycler (Biometer) or GeneAmp9600 (PE Biosystems). The sequencing reaction program uses <95 ° C for 5 min, [95 ° C: 30 sec, 60 ° C: 30 sec] x 25 times, 4 ° C> regardless of the length of the amplification site.
[0074]
4. The sample of No. 3 is applied to a gel preliminarily electrophoresed with HITACHI SQ-5500 for 1 hour.
5. Perform electrophoresis for an appropriate time.
6. Assemble the forward chain and the reverse chain, correct as necessary, and determine the base sequence.
[0075]
【Example】
Example 1: Operation of pilot plant
Raw water containing high-concentration ammonia nitrogen and sulfur compounds such as thiosulfuric acid and thiocyanate as COD components was prepared, and a pilot plant for removing nitrogen from the raw water by nitrification and denitrification was operated. Water quality data was monitored, and activated sludge was collected periodically for complex microbial analysis. FIG. 1 shows an overview of the pilot plant and Table 5 below shows the operating conditions:
[0076]
[Table 5]

[0077]
The pilot plant adopted a nitrification liquid circulation-type nitrification denitrification activated sludge method to reduce COD and remove ammonia in low-temperature water. The components of the raw water are shown in Table 6 below:
[0078]
[Table 6]

[0079]
Raw water passes through a denitrification tank, which is an anaerobic tank, in the first stage, and passes through a nitrification tank, which is an aerobic tank, in the latter stage, and the nitrification liquid is returned to the denitrification tank and circulated. Treated water is discharged through a sedimentation tank. The circulation rate of the nitrification liquid is 200%, and the sludge return rate is 100%. Sludge was extracted and the operation was performed so that the SRT was about 30 days. The measurement items for each water quality are shown in Table 7 below:
[0080]
[Table 7]

[0081]
Activated sludge for microbial community analysis was collected from the second nitrification tank.
[0082]
The operation status of the pilot plant is shown below.
First, regarding the nitrogen removal performance, FIG. 2 shows the transition of the total nitrogen concentration of the raw water and the treated water and the transition of the total nitrogen concentration of the raw water and the treated water, which is a measure of the nitrogen removal performance.
[0083]
As shown in FIG. 2, good nitrogen removal performance was obtained over time.
Also, the results of calculating the consumption rates of phenol, thiosulfuric acid, and thiocyan in the denitrification tank at the time of August 21, 2002 where nitrogen removal is small and at the time of November 13, 2002 where nitrogen removal is good, Shown in Table 8 below:
[0084]
[Table 8]

[0085]
It became clear that phenol, thiosulfuric acid, and thiocyanine were consumed in large quantities in response to the increase in nitrogen removal by denitrification. It is considered that the denitrification reaction from nitrate nitrogen, which consumes phenol, thiosulfuric acid, and thiocyanate as COD components, and the denitrification reaction from nitrite nitrogen, respectively, can be expressed as follows.
[0086]
First, the denitrification reaction from nitrate nitrogen can be expressed as follows.
The denitrification reaction from nitrate nitrogen using phenol is:
5C₆H₅OH + 28 NO₃− → 14N₂  + 30CO₂  + H₂O + 28OH-
It is calculated that 0.83 g of nitrate nitrogen can be denitrified per 1 g of phenol.
[0087]
The denitrification reaction from nitrate nitrogen using thiosulfuric acid is
5S₂O₃2- + 8NO₃− + H₂O → 4N₂  + 10SO₄2- + 2H +
It is calculated that 0.2 g of nitrate nitrogen can be denitrified per 1 g of thiosulfuric acid.
[0088]
The denitrification reaction from nitrate nitrogen using thiocyan
5SCN- + 3NO₃− + H₂O → 4N₂  + 5SO₄2- + H₂O + 2H +
It is calculated that 0.14 g of nitrate nitrogen can be denitrified per 1 g of thiocyan.
[0089]
Next, the denitrification reaction from nitrite nitrogen can be expressed as follows.
The denitrification reaction from nitrite nitrogen using phenol is
3 C₆H₅OH + 28 NO₂− + 28H + → 14N₂  +18 CO₂  + 23H₂O
It is calculated that 1.4 g of nitrite nitrogen can be denitrified per 1 g of phenol.
[0090]
The denitrification reaction from nitrite nitrogen using thiosulfuric acid
3 S₂O₃2- + 8 NO₂− + 2H + → 4N₂  +6 SO₄2- + H₂O
It is calculated that 0.33 g of nitrite nitrogen can be denitrified per 1 g of thiosulfuric acid.
[0091]
The denitrification reaction from nitrite nitrogen using thiocyan
3 SCN- + 11 NO₂− + 8H + → 7N₂  +3 SO₄2- + 3 CO₂  + 4H₂O
It is calculated that 0.88 g of nitrite nitrogen can be denitrified per 1 g of thiocyan.
[0092]
Therefore, the results of this pilot plant demonstrated that denitrification from nitrate nitrogen or nitrite nitrogen was possible using phenol, thiosulfate, and thiocyan from raw water. From the above calculation results, it is considered that stoichiometrically, denitrification using nitrite nitrogen and phenol or thiocyanate as a substrate or denitrification reaction using nitrate nitrogen and phenol as a substrate proceeded efficiently. .
[0093]
Example 2: Determination of base sequence of nitrite reductase gene nirK by cloning and identification of microorganisms having the gene
In cloning, all microorganisms present in a sample can be obtained as clones with a certain probability, as long as they can be amplified by PCR. Therefore, an attempt was made to collect activated sludge from a pilot plant at a time when the nitrogen removal performance was good, and to identify the denitrifying bacteria present therein based on having the nitrite reductase gene nirK. First, a 515 bp region of the nitrite reductase gene nirK was cloned from a microorganism present in activated sludge, and its nucleotide sequence was determined.
[0094]
Method
The cloning operation was performed according to the protocol described above. About the activated sludge collected from the pilot plant at the time when the nitrogen removal performance was good, a region of about 515 bp of the nitrite reductase gene nirK was PCR-amplified from the DNA extract (PCR conditions <95 ° C .: 10 min, [94 ° C .: 30 sec. 50 ° C .: 30 sec, 72 ° C .: 2 min] × 25 times, 72 ° C .: 10 min, 4 ° C.>). PCR products were cloned from TA Cloning kit (pT7blue T-Vector (Takara, Tokyo, Japan), DH5α competent high competent cell kit (TOYOBO, Tokyo). Of the colonies obtained on the agar plate, only white colonies were collected with a sterilized toothpick and suspended in a PCR mixture (100 μl) prepared in advance. The composition of the PCR mixture and the PCR conditions were in accordance with the report of Kasuga et al. (Kasuga et al. (2001) Journal of Japan Society on Water Environment, 24, 856-864), but the PCR primer used was an M13 primer targeting a vector. The insert was amplified by the primer. Agarose electrophoresis was performed on the obtained PCR products to confirm the amplification. PCR products were purified by Microcon (Millipore Tokyo Japan) to remove primer dNTPs.
[0095]
Next, the purified PCR products (20 ng) were subjected to a sequencing reaction using M13 primer and ABI Big dye terminator kit version 3.1 (Applied Biosystems) shown in Table 3. The reaction conditions followed the manual of ABI310 DNA sequencer (Applied Biosystems). The dye terminator contained in the product obtained by the sequencing reaction was removed with Centri-spin spin columns (Applied Biosystems), and then sequenced with an ABI310 DNA sequencer.
[0096]
Using the determined base sequence, a homologous search program BLAST (http://spiral.genes.nig.ac.jp/homology/blast.shtml) is used to search for a base sequence having high homology from known species. Then, the homology was examined and the microorganism was identified.
[0097]
Results and discussion
As the base sequence of the nitrite reductase gene nirK, the base sequences described in SEQ ID NOs: 1 to 17 in the sequence listing were determined. Table 9 shows the results of the homology analysis.
[0098]
[Table 9]

[0099]
There was no 100% match on all databases, demonstrating that the microorganism had a novel nirK gene base sequence.
[0100]
Example 3: Determination of the nucleotide sequence of the nitrite reductase gene nirS by PCR-DGGE and identification of the microorganism having the gene
In recent years, PCR-DGGE is a complex microbial system because it can detect even a gene of a microorganism that cannot be cultured such as an environmental sample and visualize it as a band, and further, by cutting out the band, the base sequence information of the band can be obtained. This is a technology that has rapidly spread in the analysis of environmental samples. It also has the advantage that changes in microorganisms can be followed as changes in bands over time. Accordingly, an attempt was made to extract activated sludge from a pilot plant with good nitrogen removal performance and to identify the denitrifying bacteria present therein based on the fact that it has the nitrite reductase gene nirS. First, a partial nucleotide sequence of the nitrite reductase gene nirS was determined for microorganisms present in activated sludge.
[0101]
Hereinafter, the procedure will be described.
DNA was extracted from activated sludge collected from a pilot plant with good nitrogen removal performance, and a partial region of the nitrite reductase gene nirS was amplified using a primer set with a GC clamp described in Table 10 below. Analyzed.
[0102]
[Table 10]

[0103]
The PCR conditions were <95 ° C .: 10 min, [94 ° C .: 30 sec, 53 ° C .: 30 sec, 72 ° C .: 30 sec] × 35 times, 72 ° C .: 10 min, 4 ° C.>. Thereafter, electrophoresis was performed at 130 V for 6 hours using a gel having a denaturant concentration gradient of 40% by mass to 60% by mass according to the protocol of DGGE.
[0104]
Determination of closely related species of the DGGE band by sequencing
In order to confirm the type of microorganisms in each of the obtained bands and determine the overall picture of the microorganisms present in the pilot plant, an attempt was made to determine the relative species of each band.
[0105]
Method
A major band was cut out from the DGGE gel, and the freeze-thaw was repeated three times with sterilized milliQ, thereby recovering DNA from the gel piece, and performing a PCR-DGGE using the DNA as a template to repeat a purification operation. To make one band. The maximum amplification PCR conditions are <95 ° C .: 10 min, [94 ° C .: 30 sec, 53 ° C .: 30 sec, 72 ° C .: 30 sec] × 25 or 20 times, 72 ° C .: 10 min, 4 ° C.>. Thereafter, the region of the nitrite reductase gene nirS was reamplified with primers without GC clamp using the purified DNA as a template (PCR conditions: <95 ° C: 10 min, [94 ° C: 30 sec, 53 ° C: 30 sec, 72 ° C: 30 sec] × 25 times, 72 ° C .: 10 min, 4 ° C.>) to obtain a template for the sequencing reaction. The sequencing reaction followed the protocol described above, and the sequencer used was HITACHI (SQ-5500). After determining the nucleotide sequence, a homologous search program BLAST (http://spiral.genes.nig.ac.jp/homology/blast.shtml) was used to search for a highly homologous nucleotide sequence from known species. , DGGE and related species of each band were determined.
[0106]
Results and discussion
As a partial nucleotide sequence of the nitrite reductase gene nirS, the nucleotide sequences of SEQ ID NOs: 18 to 39 in the sequence listing were determined. The results of the homology analysis are shown in Table 11 below:
[0107]
[Table 11]

[0108]
There was no 100% match on all databases, demonstrating that the microorganism had a novel nirK gene base sequence.
[0109]
【The invention's effect】
The present invention clarified a microorganism having an action of removing nitrogen from a nitrogen-containing wastewater, and established a method for detecting the microorganism using a DNA base sequence. This has made it possible to provide a method for removing nitrogen and a method for evaluating the same.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of a pilot plant for nitrogen removal by nitrification and denitrification.
FIG. 2 shows changes in the total nitrogen concentration of the raw water and the treated water and the change in the total nitrogen concentration of the raw water and the treated water, which is a measure of the nitrogen removal performance.

Claims (48)

DNA of the nitrite reductase (NiRK) gene of Unrelated Bacterium clone U13 related bacteria present in activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 1 or 3. fragment. A DNA fragment of a nitrite reductase (NiRK) gene of a bacterium related to Uncultured bacterium clone M17 present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 2. Mesorhizobium sp. Having the sequence shown in SEQ ID NO: 4, 6, 7, 9, 10, 12, 13, 14, or 16 present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment of the nitrite reductase (NiRK) gene of 4FB11 related bacteria. Alcaligenes sp. Having the sequence shown in SEQ ID NO: 5 and present in the activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction. DNA fragment of the nitrite reductase (NiRK) gene of a bacterium closely related to STC1. A DNA fragment of a nitrite reductase (NiRK) gene of a bacterium related to Alcaligenes xylosoxidans which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 8. A DNA fragment of a nitrite reductase (NiRK) gene of a bacterium related to Unculturized bacterium Clone M27 present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 11. A DNA fragment of a nitrite reductase (NiRK) gene of a Pseudomonas mendocina-related bacterium present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 15. A DNA fragment of a nitrite reductase (NiRK) gene of a bacterium related to Unculturized bacterium clone M58, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 17. The nitrite reductase (NiRS) gene of a paracoccus denitrificans Pd1222 closely related bacterium having the sequence shown in SEQ ID NO: 18, 33 or 39 and present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction. DNA fragment. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium related to Thauera aromatica strain AR1 which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 19. DNA of the nitrite reductase (NiRS) gene of bacteria related to Unculturized bacterium which is present in activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 20, 22 or 24 fragment. A. A present in activated sludge of a water treatment plant having the sequence shown in SEQ ID NO. A DNA fragment of the nitrite reductase (NiRS) gene of eutrophus-related bacteria. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium related to Azoarcus toluvorans, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 23. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium related to Unculturized bacterium clone neirSAK4, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 25. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium closely related to Unculturized bacterium clone nir SAK2, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 26. DNA of the nitrite reductase (NiRS) gene of bacteria related to Unculturized bacterium clone ANIS-76 present in the activated sludge of the water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 27 fragment. Nitrite reduction of bacteria related to Unculturized bacterium clone HNIS-6 present in activated sludge of a water treatment plant by the activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 28, 29, 30, 36 or 38. Enzyme (NiR
S) DNA fragment of the gene. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium related to Paracoccus pantotrophus, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction and having the sequence shown in SEQ ID NO: 31. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium closely related to Thauera linalolentis present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 32. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium closely related to Thauera aromatica strain AR1, which is present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 34. A DNA fragment of a nitrite reductase (NiRS) gene of a bacterium closely related to Thauera terpenica strain 58Eu present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 35. A DNA fragment of the nitrite reductase (NiRS) gene of Pseudomonas aeruginosa strain Mi11 related bacteria present in activated sludge of a water treatment plant by an activated sludge method used for nitrogen reduction, having the sequence shown in SEQ ID NO: 37. 9. Activated sludge of a water treatment plant by the activated sludge method, wherein the nitrite reductase (NiRK) gene contains the base sequence of the DNA fragment according to any one of claims 1 to 8, and is used for reducing nitrogen. Microorganisms present in. 23. Activated sludge of a water treatment plant by the activated sludge method, wherein the nitrite reductase (NiRS) gene contains the base sequence of the DNA fragment according to any one of claims 9 to 22, and is used for reducing nitrogen. Microorganisms present in. The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 1 or 3 and is present in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. Uncultured bacterium clone U13 related bacteria. The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 2 and is present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. bacterium clone M17 Closely related bacteria. The nitrite reductase (NiRK) gene contains the nucleotide sequence shown in SEQ ID NO: 4, 6, 7, 9, 10, 12, 13, 14 or 16 and is obtained by a nitrification liquid circulation type nitrification denitrification activated sludge method. Mesorhizobium sp. Present in activated sludge of a water treatment plant for nitrogen reduction. 4FB11 related bacteria. Alcaligenes containing the nucleotide sequence shown in SEQ ID NO: 5 in its nitrite reductase (NiRK) gene, and present in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. sp. STC1 related bacteria. Alcaligenes containing the nucleotide sequence shown in SEQ ID NO: 8 in its nitrite reductase (NiRK) gene, and present in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. xylosoxidans closely related bacteria. The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 11 and is present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. Bacterium clone M27 related bacteria. The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 15, and Pseudomonas present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. related bacteria. The nitrite reductase (NiRK) gene contains the base sequence shown in SEQ ID NO: 17 and is present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. bacterium clone M58 closely related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 18, 33 or 39, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. A related bacterium of Paracoccus denitrificans Pd1222 present in Japan. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 19, and is present in activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. aromatica strain AR1 related bacteria. The nitrite reductase (NiRS) gene contains the nucleotide sequence shown in SEQ ID NO: 20, 22, or 24, and is contained in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method. A related bacterium of Unculturized bacterium present in the country. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 21 and is present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. . eutrophus-related bacteria. Azoarcus, which contains the base sequence shown in SEQ ID NO: 23 in its nitrite reductase (NiRS) gene and is present in activated sludge of a water treatment plant for nitrogen reduction by a nitrification liquid circulation type nitrification denitrification activated sludge method toluvorans closely related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 25, and is present in the activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. Bacterium clone nir SAK4 related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 26, and is present in the activated sludge of a water treatment plant for reducing nitrogen by the nitrification liquid circulation type nitrification denitrification activated sludge method. Bacterium clone neirSAK2 closely related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 27, and is present in the activated sludge of a water treatment plant for reducing nitrogen by the nitrification liquid circulation type nitrification denitrification activated sludge method. Bacterium clone A closely related bacterium to ANIS-76. A water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method, wherein the nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 28, 29, 30, 36 or 38. Unculturated bacterium clone HNIS-6 closely related bacteria present in the activated sludge. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 31 and is present in the activated sludge of a water treatment plant for nitrogen reduction by the nitrification liquid circulation type nitrification denitrification activated sludge method. Pantotrophus-related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 32, and is present in activated sludge of a water treatment plant for reducing nitrogen by a nitrification liquid circulation type nitrification denitrification activated sludge method. linalolentis related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 34 and Thauera present in activated sludge of a water treatment plant for nitrogen reduction by the nitrification liquid circulation type nitrification denitrification activated sludge method. aromatica strain AR1 related bacteria. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 35 and Thauera present in activated sludge of a water treatment plant for nitrogen reduction by the nitrification liquid circulation type nitrification denitrification activated sludge method. A related bacterium of terpenica strain 58Eu. The nitrite reductase (NiRS) gene contains the base sequence shown in SEQ ID NO: 37, and Pseudomonas present in the activated sludge of a water treatment plant for reducing nitrogen by the nitrification liquid circulation type nitrification denitrification activated sludge method. aeruginosa strain Mi11 related bacteria. Activated sludge for use in water treatment for nitrogen reduction by the nitrification liquid circulation type nitrification denitrification activated sludge method, comprising the microorganism or bacterium according to any one of claims 23 to 46. A water treatment method for reducing nitrogen using the activated sludge according to claim 47.

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