JP4112082B2 - Method for promoting growth of Burkholderia cepacia 1A and bioreactor using the microorganism - Google Patents

Description

【００１１】
前記自然環境中のリンと窒素の濃度の測定値に基づき、本発明者らは目的とする有機炭素の濃度が高く、その他のリンと窒素の栄養塩濃度のみが低い条件で生育し、汚染物質を分解することのできる微生物を得るための培地は、リン濃度が約５ｐｐｍ以下で、かつ窒素濃度が約５０ｐｐｍ以下のものであることを見出し、この知見に基づき、低栄養条件下で芳香族化合物を分解する微生物としてＢｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａ（ＦＥＲＭ Ｐ−１５５６６）を先に提案している。
【００１２】
さらに本発明者らは、栄養塩類を添加することなしに、低栄養状態の排水を効率よく処理するためには、低栄養性微生物は、菌体生育量が少ないため、菌体量を高めたバイオリアクターを構築する必要があると考え、低栄養条件である製油所や化学工場などの低栄養排水について低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａを用いたバイオリアクターについて菌体濃度を高めるため実験を多く行った。
【００１３】
すなわち、低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａを培養する際に、培養液にポリスチレン、テフロン、ポリプロピレン、デルリン、アルミナ、エポキシ樹脂、ナイロン、ポリエチレンの各種素材で作成したビーズを添加し、該微生物の生育量とフェノール分解量について検討した。その結果、培養液中にポリスチレンを添加することによって、低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育とこれに伴うフェノールの分解活性が著しく向上することを見出し、本発明に到達することができた。
従って、本発明の特徴の第１は、Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ１Ａの生育を低栄養条件でポリエスチレンの共存下で行うことを特徴とするＢｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育促進方法に関する。
【００１４】
培地にプラスチック製品を添加して微生物を生育させる研究は、本発明前に服部らの報告がある〔森崎久雄ら、界面と微生物、学会出版センター（１９８８）〕。服部らは、培養液にプラスチック製の陰イオン交換樹脂を添加して、微生物の生育至適ｐＨがイオン交換樹脂を添加しない場合と比べてアルカリ側に移動することを見い出した。そして、この現象は陰イオン交換樹脂の表面近傍における培養液のｐＨが陰イオン交換樹脂から遠くに存在する培養液のｐＨと異なることに起因するものと考察している。
しかしポリスチレンには、イオン交換能と言ったような特別な機能はなく、陰イオン交換樹脂の添加効果と本発明で使用するポリスチレンの作用とは、まったく異なるものである。
【００１５】
一方、菌体の固定化法については、従来より多く研究がなされており、アルギン酸、ポリビニールアルコール、κ−カラギーナン、ゲランガム、アガロース、セルロース、デキストラン等のゲル状物質に包括固定する方法や、ガラス、活性炭、ポリスチレン、ポリエチレン、ポリプロピレン、木材、シリカゲル等の表面に吸着固定する方法を挙げることができる［扇元 敬司、「バイオテクノロジーテキストシリーズ 微生物学」、講談社、Ｐ１６８（１９９４）、相田 浩ら、「新版 応用微生物学ＩＩ」、朝倉書店、Ｐ１１６（１９９５）］。
【００１６】
また、フェノールの排水処理に於いてもＲｈｃｄｏｃｏｃｃｕｓ属細菌［Ｓ−Ｌ．Ｐａｉ外ら、Ｂｉｏｒｅｓｏｕｒｃｅ Ｔｅｃｈ．，５１，Ｐ３７（１９９５）］やＰｓｅｎｄｏｍｏｎａｓ ｐｕｔｉｄａ［渡辺一哉ら、水処理技術、３６，Ｐ７７（１９９４）、Ａ．Ｍｏｒｓｅｎら、Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈ．，３３，Ｐ２０６（１９９０）］等の細菌、Ｃａｎｄiｄａｔｒｏｐｉｃａｌｉｓ［Ｈ．Ｗｅｎｈｕａ外ら、Ｗａｔｅｒ Ｔｒｅｎｔ，８，Ｐ１１９（１９９３）］やＣｒｙｐｔｏｃｏｃｃｕｓ ｅｌｉｎｏｖｉｒ［Ａ．Ｍｏｒｓｅｎら、Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈ．，３３，Ｐ２０６（１９９０）］等の酵母及び活性汚泥［Ｆ．Ｓ．Ｌａｋｈｗａｌａ ら、Ｂｉｏｐｒｏｃｅｓｓ Ｅｎｇｉ．，８，Ｐ９（１９９２）］をアルギン酸やポリビニルアルコール等による包括法及び活性炭や粘土鉱物による吸着法にて固定化して用いる例が報告されている。
【００１７】
しかしながら、本発明における前記ポリスチレンの効果は、その効果からみて、担体としての菌体固定化のための効果のみではなく、Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａ培養液中の浮遊菌体の量をも高める他の素材には見られない増殖触媒作用であると考えられる。
前記ポリスチレンの増殖触媒作用は、ポリスチレンが水に不溶なプラスチックであること、低栄養性微生物Ｂｕｒｋｈｏｄｅｒｉａ ｃｅｐａｃｉａ １Ａがポリスチレン製のビーズを炭素源として利用できないこと、培養液中に加えるポリスチレン製のビーズの数に比例することより、ポリスチレンの表面積に依存するものと考えられる（実施例７，９）。
【００１８】
培養液中に加えるポリスチレンの形状は、ビーズ状のものに限られるものではなく、シート状、ハニカム状、パイプ状等の種々の形状のものを用いることができるが、前記のようにその表面積の大きいものが好ましい。さらにポリスチレンは、他のプラスチック、ガラス、シリカゲル、アルミナ、金属等の表面にポリスチレンをコーティングした形でも用いることができる。
【００１９】
さらに、本発明者らは培養液中に加えるポリスチレンの使用態様について検討した結果、ポリスチレンに物理的な力を加え、該ポリスチレン表面に保持されて増殖した菌体の少なくとも一部を積極的に剥離させ、浮遊菌体とすることにより、ポリスチレンの増殖作用が向上することを見出した。この増殖作用の向上の原因としては、図４に示すように、ポリスチレン表面から菌体剥離され、該剥離された浮遊菌体もフェノール分解活性を有する菌体であり、さらに菌体が剥離されたポリスチレン表面には、また菌体が形成されるので、リアクター全体としてはフェノール分解活性が向上する。
【００２０】
前記培養工程中にポリスチレン表面から増殖した菌体を剥離するためには、菌体剥離型リアクターを用いるのが好ましい。該菌体剥離型リアクターとしては、例えば菌体剥離型ーエアーリフト型リアクター、小型ジャー型リアクターおよびビーズインボール型リアクター等が挙げられる。
【００２１】
本発明のバイオリアクターに用いるポリスチレンは耐水性に優れた半永久的な材質であり、長期間使用することが可能である。長期間使用すれば、ポリスチレンそのものは廃棄物とならないので、総合的な観点からは環境負荷が少ない。
また、本発明で言うポリスチレンとは、スチレンの単独重合体のみを指すのではなく、本発明の意図する増殖触媒作用を奏する限り、スチレン以外に他のモノマー成分を含有するものであっても良い。
本発明のバイオリアクターに用いるポリスチレンは耐水性に優れた半永久的な材質であり、長期間使用することが可能である。長期間使用すれば、ポリスチレンそのものは廃棄物とならないので、総合的な観点からは環境負荷が少ない。
【００２２】
本発明で使用するＢｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａは、リン濃度５ｐｐｍ以下でかつ窒素濃度５０ｐｐｍ以下の培地を用いて、フェノール１０００ｐｐｍを炭素源とした条件で検索することによって得られた。
【００２３】
リン源は実質的に微生物がリン源として利用可能な物質であればなんでも良く、リン酸一アンモニウム、リン酸一ナトリウム、リン酸一カリウム、リン酸水素アンモニウムカリウム、リン酸水素二ナトリウム、リン酸水素二カリウム、リン酸二水素アンモニウム、リン酸二水素ナトリウム、リン酸二水素カリウム等の無機のリン酸塩およびこれらの塩の水和物、酵母エキス、肉汁エキス等のリンを含む天然物やリン酸エステル等の有機リン化合物等をあげることができる。
【００２４】
同様に窒素源も実質的に微生物が窒素源として利用可能な物質であればなんでも良く、硫酸アンモニウム、リン酸一アンモニウム、硝酸アンモニウム、リン酸水素アンモニウムカリウム、塩化アンモニウム等のアンモニウム塩およびこれらの塩の水和物、硝酸アンモニウム、硝酸ナトリウム、硝酸カリウム等の硝酸塩、亜硝酸ナトリウム等の亜硝酸塩、尿素、アミノ酸、酵母エキス、肉汁エキス、ペプトン等の窒素を含む天然物やアミド化合物等の有機含窒素化合物等をあげることができる。
【００２５】
前記Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａは、リン５ｐｐｍ以下でかつ窒素５０ｐｐｍ以下と言う低栄養条件下で生育することができるので、栄養塩類を添加しない環境調和型の環境浄化や排水処理に応用することが可能である。ただし、該微生物がリン５ｐｐｍ以下でかつ窒素５０ｐｐｍ以下と言う低栄養条件下で生育することができると言う性質を有することは、該微生物がリン５ｐｐｍ以上あるいは窒素５０ｐｐｍ以上の条件で生育できないことを意味するのではなく、前記Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａは、低栄養条件下のみならず、高栄養条件下でも生育できる通性低栄養性微生物である。
【００２６】
【実施例】
実施例１
低リン濃度、低窒素濃度条件（自然環境）で生育するフェノール資化性菌の検索
（検索用培地の作成）
検索用培地として、下表２に示した培地（以下、Ｅ−培地）を１／１０に希釈したもの（１／１０Ｅ−培地：リン約５ｐｐｍ、窒素約５０ｐｐｍ）を用いた。これに、炭素源としてフェノール１，０００ｐｐｍを加えた。また、各培地のｐＨは７．０とした。
【００２７】
【表２】

【００２８】
（分解菌の取得方法Ａ）
分離源としては、つくば市周辺の土壌約４８０種類を用い、以下の方法で微生物のみを抽出して培地に添加した。３００ｍｌ容の三角フラスコに滅菌水３０ｍｌを入れ、ここに土壌サンプル５ｇを加えた。これを１日回転振とうして、菌の抽出を行なった。抽出後、遠心分離（２，０００ｒｐｍ，５ｍｉｎ）して土壌を沈澱させ、上清を濾紙、およびグラスフィルターにてろ過して、土壌微粒子を除いた。得られた菌液をニトロセルロースフィルター（０．２μｍ）でろ過して微生物を捕集した。このフィルターを適宜切断して培地に加え、検索を行なった。検索は以下の方法で行なった。３００ｍｌ容の三角フラスコに１／１０Ｅ−培地３０ｍｌを入れ、ここに上述のニトロセルロースフィルターを加え３０℃にて５日間振とう培養を行なった。生育の見られたものについては、１／１０Ｅ−培地８ｍｌを入れた内径２４ｍｍの大型試験管に１００μｌ植え継ぎ５日間培養した。これを２回繰り返し、菌の単離を行なった。
菌の単離には、前述の１／１０Ｅ−培地に１．２％のアガロース[ａｇａｒｏｓｅ（電気泳動用ｕｌｔｒａ−ｐｕｒｅ ｇｒａｄｅ）]を加えて作成した平板を用いた。これに、前述の培養液を適宜希釈して塗布し、３０℃にて５日間培養して、生育してきたコロニーを単離した。
【００２９】
実施例２
（実施例１で取得した分解菌の同定）
前記実施例１で得られたフェノール資化性菌１Ａ株はグラム陰性で、運動性を持ち、オキシダーゼ、カタラーゼ共に陽性であった。また、Ｏ−Ｆテストは酸化を示した。その他の各種生理試験の結果から、取得した菌株の１種類はＢｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａであると同定された。下表に、フェノール資化性菌１Ａ株の特性を示す。
【００３０】
【表３】

【００３１】
【表４】

【００３２】
実施例３
実施例１で得られた分解菌による低リン濃度、低窒素濃度条件下のフェノールの分解
（分解方法）
１Ａ株を１／１０Ｅ−平板培地（フェノール１，０００ｐｐｍ）に植菌し、３０℃にて培養した。生育した菌体を１／１０Ｅ−培地２ｍｌを入れたプラスチックチューブ（フェノールがチューブに吸着しないことは確認済み）に植菌し、振とう培養機（ＭＢＳＳ−１０、丸菱）にて培養して、培養後のフェノールの分解量を、ガスクロマトグラフィーを用いて定量した。初発フェノール濃度は１００ｐｐｍ、１０００ｐｐｍとし、それぞれ３、６日後にサンプリングを行った。ガスクロマトグラフィーの条件を以下に示す。
カラム（Ｃｏｌｕｍｎ） ：ＵｎｉｓｏｌｅＦ−２００
注入温度（Ｉｎｊ．ｔｅｍｐ） ：１８０℃
カラム温度（Ｃｏｌ．ｔｅｍｐ）：１５０℃
検出器 （Ｄｅｔｅｃｔｏｒ） ：ＦＩＤ
注入量 （ｉｎｊ．ｓｉｚｅ） ：２μｌ
内部標準物質（ＩＳ） ：ジエチレングリコール
分解試験の結果を下表５に示す。実験に供したＢｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａ株は、フェノール分解能を有していた。
【００３３】
【表５】

【００３４】
実施例４
（実施例１で得られた菌株と既存の菌株のフェノール分解能の比較実験）
供試菌株としては実施例１で得た分離株１Ａ株を用いた。また、比較のために、これまでにフェノール分解菌として報告されているＡＴＣＣ（Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ）保存菌株も用いた。このＡＴＣＣ保存のフェノール分解菌は、下記の４株である。
・Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ ＡＴＣＣ １１１７２
・Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ １４３４７
・Ｖｉｂｒｉｏ ｃｙｃｌｏｓｉｅｓ ＡＴＣＣ １４６３５
・Ｒｈｏｄｏｃｏｃｃｕｓ ｚｏｐｆｉｉ ＡＴＣＣ ５１３４９
前記供試菌株を下表６に示す培地を１／１００に希釈した１／１００Ｆ−培地（リン約１ｐｐｍ、窒素約２０ｐｐｍ）、または１／１００Ｆ−培地のリン、窒素濃度をそれぞれ約５００ｐｐｍに高めた培地２ｍｌを入れたプラスチックチューブに植菌し、振とう培養機（ＭＢＳＳ−１０、丸菱）にて３０℃、４日間培養した。培養後のフェノールの分解量を、ガスクロマトグラフィーを用いて定量した。また、生育量は６６０ｎｍにおける吸光度（Ａ６６０）として測定した。
【００３５】
【表６】

【００３６】
なお、表６中の微量金属（ｔｒａｃｅ ｍｅｔａｌ）の組成は下表７の通りである。
【表７】

【００３７】
（結果と考察）
分解試験の結果を下表８〜９に示した。表８に示すように、取得した分解菌は１／１００Ｆ−培地に良好に生育し、添加した１００ｐｐｍのフェノールはほとんどが４日以内に完全に分解されていた。また、培地中のリン、窒素濃度を高めても、生育、フェノール分解共に阻害は全く見られなかった。
一方、ＡＴＣＣ保存株の方は、全ての菌株が基本培地には全く生育が見られなかった。表中でフェノールのわずかな減少が見られるのは、植菌した菌体への吸着であると思われる。なお、培地中のリン、窒素濃度をそれぞれ５００ｐｐｍに高めた培地を用いた表９の場合にも、表８に示すように、フェノールの分解が観察された。この結果から、前記実施例１のフェノール分解菌は、これまでに知られているフェノール分解菌が全く生育できないような低リン低窒素条件下で生育し、良好なフェノール分解能を有する新規な菌であると考えられる。
【００３８】
【表８】

【００３９】
【表９】

【００４０】
実施例５
低リン濃度、低窒素濃度条件で生育するフェノール資化性菌を用いた各種芳香族化合物の分解
供試菌株としては実施例１で得た分離株１Ａを用いてフェノール、ベンゼン、トルエン、キシレン（異性体混合物）の資化性を確認した。基質である各種芳香族化合物の量は１００ｐｐｍとし、培地は１／１００Ｆ−培地を用い、３０℃で４日間培養行った。
その結果を下表１０に示す。
【００４１】
【表１０】

前表の結果より、何れの基質（炭素源）も炭素源が無い場合より、微生物の生育が高いことが確認できた。
【００４２】
実施例６
低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの固定化担体の材質を検討するため、ポリスチレン、テフロン、アルミナ、ナイロン、デルリン、ポリエチレン、エポキシ樹脂、ポリプロピレンを用いてψ６．３ｍｍのビーズを作成した。
種母培養した１Ａ株は他の培養実験と同様に滅菌水で懸濁し、５８０ｎｍ吸光度（Ａ５８０）で０．０１になるように植菌した。培地１ｍｌとビーズ４個をネジ付ガラス試験管に入れ、３０℃、６００ｒｐｍの回転振盪下で１日間培養を行い、フェノールの分解能を検討した。
結果は図１に示した。ナイロンとデルリンはフェノールの吸着或いは吸収量が高かったため、以降の実験には供しなかった。
培養時にはポリスチレンビーズを添加した時に、最もフェノールは速く分解された。他の材質ビーズはビーズ無添加（浮遊菌体）に比べフェノール分解を僅かしか促進せず、その程度にはほとんど差がなかった。
【００４３】
実施例７
１Ａ株の培養時にψ６．３ｍｍのポリスチレンビーズを試験管あたり１〜４個添加してポリスチレンビーズの添加個数とフェノール分解に対する影響を検討した。
種母培養した１Ａ株は他の培養実験と同様に滅菌水で懸濁し、Ａ５８０で０．０１になるように植菌した。培地１ｍｌとビーズ４個をネジ付きガラス試験管に入れ、３０℃、６００ｐｐｍの回転振盪下で１日間培養を行い、フェノールの分解能を検討した。
その結果を図２に示した。ポリスチレンビーズの添加個数が増加するに従い、浮遊菌体量も増加し、これに伴ってフェノール分解量も増加した。
【００４４】
実施例８
ポリスチレンビーズ（ψ６．３ｍｍ）を用いて図３に示すようなバイオリアクターを２台作成した。
１台にはポリスチレンビーズを３００個添加し、もう１台にはポリスチレンビーズを全然添加せずに両者を比較検討した。
培地は各々８００ｍｌ添加し、種母培養した１Ａ株は滅菌水に懸濁してＡ５８０で０．００１になるように植菌した。
培養は回転バネを２５０ｒｐｍで回転攪拌しながら２５℃で行った。反応は回分式で行った。
その結果、図３に示すようなポリスチレンビーズを添加したものは浮遊菌体量も増加し、これに伴ってフェノール分解能もポリスチレンビーズ無添加に比べ高かった。
【００４５】
実施例９
低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育に及ぼすポリスチレンビーズの表面積の影響について検討した。
直径の異なるポリスチレンビーズ（ψ３．２ｍｍおよびψ６．３５ｍｍ）の０〜３５個を栄養系に添加して、その生育に及ぼす影響を検討した。培養は３０゜Ｃ、６００ｒｐｍで一日間行った。ポリスチレンビーズ表面に吸着している菌体はアルカリ加水分解してタンパク質量として測定した。
その結果を図５に示した。ポリスチレンビーズの大小に関わらず、浮遊菌体量と吸着菌体量は共にポリスチレンビーズの実面積に依存して増加した。また、浮遊菌体量は表面積３００ｍｍ２付近、吸着菌体量は表面積６００ｍｍ２付近で飽和に達した。前記結果より、低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育促進効果はビーズの大小ではなくビーズの表面積に依存することが解る。
【００４６】
Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａ株の場合、乾燥菌体の約４５％がタンパク質であり、１細胞の乾燥重量は８．１×１０−８μｇである。また、図５に示したデータよりビーズの表面積に吸着している菌体量はタンパク質として０．０５μｇ／ｍｍ２と算出される。前記１Ａ株の設置面積を０．５×１．５μｍと仮定すると、１ｍｍ２に１．３×１０６ｃｅｌｌが吸着し得ることになり、この値は先に求めた１．３７×１０６ｃｅｌｌ／ｍｍ２にほぼ等しい。従って、ポリスチレンの表面積には１Ａ株がほぼ飽和に近い状態で吸着しているものと考えられる。
【００４７】
ポリスチレンビース表面に吸着した菌体量は、アルカリ加水分解した後にタンパク質量として測定した。培養後のポリスチレンビーズを超純水で１回洗い、０．１ＮのＮａＯＨを加えて８０℃で一晩加熱して菌体を加水分解した。加水分解後の溶液に含まれるタンバク質はローリー法によりウシ血清アルブミンを標準として定量した（Ｒ．Ｆ．Ｓｃｈｌｅｉｆら（川上正也ら監訳）、『分子生物学マニュアル』講談社サイエンティフック、ｐ９０（１９８３））
【００４８】
実施例１０
各種ビースの表面親水性と仕と低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育に及ぽす影響
φ６．３〜６．４ｍｍのガラス、テフロン及びポリスチレン製の各種ビースを用いて表面親水性と１Ａ株の生育に及ぼす影響を検討した。培養は３０℃、６００ｒｐｍで１日間行った。各種ビーズの表面親水性はＭ−Ｏ、Ｓａｍｕｅｌｓｓｏｎらの方法に従いビース表面に滴下した水滴とビーズ表面の接触角より求め。親水性（ＷＡ値）は、
【数１】
ＷＡ＝Ｙ_LV０（１＋ｃｏｓθ）
（θは接触角、Ｙ_LV０は飽和蒸気内での水の表面張力であり、７２．８ｍＮｍ^-1である）
で与えられる（Ｍ−Ｏ、Ｓａｍｕｅｌｓｓｏｎら、Ｅｎｖｉｒｏｎ.Ｍｉｃｒｏｂｉｏｌ.、５６、ｐ３６４３(１９９０)）。
各種ビース表面に吸着した菌体量は、アルカリ加水分解した後にタンパク質量として測定した。
前記の実験結果と各種ビーズの親水性を下表１１に示した。テフロンビーズが最も疎水性であり、ガラスビースは親水性てあった。ポリスチレンビースの親水性はテフロンビースとガラスビースの中間の値を示した。
【００４９】
【表１１】

前記１Ａ株はポリスチレンビースの表面に最も良く吸着し、テフロンビース表面に吸着した菌体量はポリスチレンビーズの約１／４であつた．またガラスビーズ表面には全く吸着を示さなかった。
【００５０】
実施例１１
小型ジャー型リアクターによるフェノール分解
本実施例は、図６に示すような菌体剥離型リアクターとして小型ジャー型リアクターを用いてフェノールを分解する実施例である。該小型ジャー型リアクターは、カルスター社製の動物細胞培養用フラスコ（５００ｍｌ、ダブルアーム付）を改造して作製した。主な改造点は、フラスコ内部にステンレス製カゴを設置し、ポリスチレンビーズ（住友ベークライト製ＥＬＩＳＡ(ＲＩＡ)用ボールＨ５００個）を添加出来るように、またリアクター内の水面が水平になるようにテフロン製のバッフルを設置し、さらに該攪拌羽根の軸にステンレス製の棒を２本取付、カゴの中のポリスチレンビーズに物理的な衝撃を与えられるようにした。このリアクターを用いて、浮遊菌体量とフェノール分解量を検討した。
培地はリン０．５ｐｐｍ、窒素５ｐｐｍおよびフェノール１００ｐｐｍを含むものを用い、また、培地の流加速度は３０ｍｌ／ｈｒ（Ｔ_1/2＝１３．３ｈｒ）である。その結果を図７に示した。ステンレス製の棒を取付けることにより浮遊菌体量は著しく増大した。また、これに伴いフェノールもすべてが分解された。
【００５１】
実施例１２
エアーリフト型リアクターによるフェノール分解
本実施例では、エアーリフト型リアクターは、（株）丸菱バイオエンジ社製のＭＤＰ型５Ｌステンレス製の棒を装着したカゴを設置し、落下するポリスチレンビーズがステンレス製の棒に衝突するように改造したものを使用した。
前記菌体剥離型ーエアーリフト型リアクターの実容量は、４，０００ｍｌであり、該リアクターに２ＳＬ／ｍｉｎの通気量で通気し、コンプレッサーエアーをフィルターで除菌してこれを用いた。
【００５２】
本実施例ではポリスチレンビーズを添加しないものをコントロールとして、ポリスチレンビーズの添加しないものをコントロールとして、ポリスチレンビーズの添加効果とカゴの設置効果を比較検討した。培養は３０゜Ｃで４８時間の回分培養を行った。フェノールの初発濃度を１００ｐｐｍとし、培養２４時間後に更に１００ｐｐｍ相当量を添加した。
【００５３】
いずれのリアクターにおいても培養２４時間後には、初めに与えた１００ｐｐｍのフェノールは完全に分解されていた。１００ｐｐｍ相当のフェノールを添加した後（培養３０時間後）の結果を図８に示した。ポリスチレンビーズを添加することにより、浮遊菌体量が増加し、これに伴ってフェノール分解能も向上した。更に、ステンレス製の棒を装着したカゴを設置することにより、浮遊菌体量の増加とフェノール分解能の向上が観察された。
【００５４】
実施例１３
ビーズインボール型リアクターによるフェノール分解
本実施例は、菌体剥離型リアクターとしてビーズインボール型リアクター（出願と同時に提出した物件提出書）を用いてフェノールを分解する実施例である。本実施例では、プラスチック製の球形のカゴの中に約８０個のポリスチレンビーズを入れ、培地の攪拌によりカゴが回転することで中に収納されたポリスチレンビーズが互いにぶっかり合うようにした。培養は３０゜Ｃで２４時間の回分培養と２０ｍｌ／ｈ（Ｔ_1/2＝２０ｈｒ）と連続培養を行った。
【００５５】
その結果は図９に示した。ジャー型リアクター菌体剥離型リアクターリアクターと同様にコントロール（ポリスチレンビーズ無添加）のリアクターに比べて浮遊菌体量の増加が確認され、フェノールの分解活性も向上した。
ビーズインボール型とエアーリフト型リアクターでも菌体剥離型リアクターとなったので、菌体を剥離させる力はさほど大きくなくてもよいことが理解される。
【００５６】
【効果】
栄養塩類を添加することなしに、低栄養条件で生育し、芳香族化合物を分解することのできる低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａの生育促進方法、該微生物を用いた芳香族化合物、例えばフェノール含有排水処理用バイオリアクター、および該バイオリアクターを用いた排水処理技術、例えば製油所や化学工場などのフェノール含有排水処理技術を提供することができた。
【図面の簡単な説明】
【図１】実施例６の結果を示す図である。
【図２】実施例７の結果を示す図である。
【図３】本発明で使用するバイオリアクターの概念図の１例である。
【図４】本発明の菌体剥離型リアクターの概念を説明した図である。
【図５】低栄養性微生物Ｂｕｒｋｈｏｌｄｅｒｉａ ｃｅｐａｃｉａ １Ａ株の生育に及ぼすポリスチレンピースの表面積の影響を説明した図である。（Ａ）浮遊菌体濃度（Ｂ）ビーズ表面への吸着タンパク室濃度
【図６】小型ジャー型リアクターの模式的断面図である。
【図７】小型ジャー型リアクターによるフェノール分解結果を示した図である。
【図８】菌体剥離型ーエアーリフト型リアクターによるによるフェノール分解結果を示した図である。
【図９】ピーズインーボル型リアクターによるフエノール分解結果を示した図である。
【符号の説明】
１ ポリスチレンビーズ保持用のカゴ
２ カゴ中に保持されたポリスチレンビーズ
３ バッフル（じゃま板）
４ 攪拌羽根
５ 回転軸[0001]
【Technical field】
INDUSTRIAL APPLICABILITY The present invention is a method for promoting the growth of a low-nutrient microorganism Burkholderia cepacia 1A that grows under low nutrient conditions such as phosphorus and nitrogen and that can decompose wastewater containing aromatic compounds, and the microorganism. The present invention relates to a bioreactor for treating wastewater containing aromatic compounds such as phenol, and a wastewater treatment technique using the bioreactor.
[0002]
[Prior art]
The natural environment is generally considered to be under nutrient conditions, and wastewater from refineries and chemical factories is low nutrient water that is poor in phosphorus and nitrogen, so normally known microorganisms cannot grow. In the conventional environmental purification technology, (NH _Four ) ₂ SO _Four , NH _Four Cl, Na _Three PO _Four , Na ₂ HPO _Four And the like, and a method for increasing the activity of microorganisms has been carried out [(DA Graves et al., Applied Biochemistry and Biotechnology, vol. 28, P813 (1991): Shigeaki Harayama, “Small Earth Creatures ", Gihodo Publishing, P109 (1995): Minoru Nishimura, Soil and Microorganisms, No. 46, P19 (1995): CH Nelson et al.," Hydrocarbon Bioremediation ", CRC Press, P125 (1994)].
[0003]
Also in wastewater treatment, it is considered necessary to control the ratio of carbon: phosphorus: nitrogen in order to maintain the growth and activity of microorganisms. In order to control the ratio of carbon: phosphorus: nitrogen, it is common to add nutrient salts to wastewater [“Wastewater Treatment Technical Guide”, Petroleum Federation, P129 (1975): “Technology for Pollution Prevention Laws and Regulations, Industrial Pollution Prevention Association, P215 (1992)].
Further, Japanese Patent Laid-Open No. 6-500049 describes a method for efficiently and continuously providing a nutrient salt to a polluted environment, and Japanese Patent Laid-Open No. 7-507208 simultaneously with the supply of a nutrient substance by adding a phosphate ester. A method for emulsifying pollutants is described.
[0004]
[Problems to be solved by the invention]
As described above, the natural environment is generally considered to have low nutrient conditions, and wastewater from refineries and chemical factories is low nutrient wastewater that is poor in phosphorus and nitrogen. Under such conditions, normally known microorganisms cannot grow, so it is necessary to add nutrient salts to enhance the activity of the microorganisms for environmental purification and treatment of low nutrient wastewater.
However, nutrient salts added to the polluted environment and wastewater are one of the costs for environmental purification and wastewater treatment. In particular, when the contaminated place to be purified is in a wide range or the amount of wastewater to be treated is large, the cost of the added nutrient salt becomes enormous.
Further, excessively added nutrient salts cause secondary pollution such as nitric acid contamination of groundwater and eutrophication of the environment. Therefore, it is important to control the addition method and addition amount.
[0005]
That is, the nitrogen source is oxidized by the action of soil microorganisms to become a nitric acid body. Therefore, if an excessive nitrogen source is given, the concentration of nitrate ions in the soil and groundwater will increase. Nitrate ions have been pointed out to be carcinogenic, and mixing with drinking water has become a problem [Michiji Tsurumaki, “Present state of groundwater contamination and soil contamination and purification measures”, Industrial Technology Association, P98 (1993) ].
Moreover, when nutrients added excessively, especially phosphorus and nitrogen sources, flow into rivers and lakes by rainwater and groundwater, they cause eutrophication of the environment. Since the eutrophication of the environment causes abnormal occurrence of blue sea bream and musty odor of drinking water, many methods for removing phosphorus and nitrogen in domestic wastewater have been proposed [Kazunori Nakamura, “Microbiology 17”, Academic Publishing Center, P31 (1991): Tetsuro Fukase, “Small creatures that protect the earth”, Gihodo Publishing, P133 (1995)].
P. Morgan et al. Point out that the addition of nutrient salts to the soil environment inhibits the degradation activity of organic substances of indigenous microorganisms [Wat. Sci. Tech. , Vol. 6, P63 (1990)].
[0006]
In order to avoid secondary contamination by the added nutrients, it is necessary to add the nutrients while controlling them. However, installing such a control device in a wastewater treatment facility makes the facility more expensive and increases the wastewater treatment cost.
Furthermore, since contaminated environments such as contaminated soil and groundwater are open systems, it is necessary to close the contaminated environment in some way in order to efficiently add nutrients while controlling nutrients. However, closing the polluted environment by a civil engineering method requires a large amount of funds, which is technically difficult especially when the polluted environment is wide. Therefore, it must be said that it is virtually impossible to prevent secondary contamination by added nutrients in a contaminated environment.
[0007]
The existence of undernourished microorganisms has been known for a long time, and many studies have been made. So far, low-nutrient microorganisms have been discussed only in terms of the amount of organic carbon in the growth conditions, and microorganisms that can grow at organic carbon concentrations of 1 to 15 ppm or less have been studied as low-nutrient microorganisms [( Tsuyoshi Enomoto, “Skilled Lifestyle of Bacteria Living in Special Environments”, Kyoritsu Publishing, P7 (1993): Mitsuru Eguchi, “Marine Microorganisms and Biotechnology”, Gihodo Publishing, P68 (1991): Yuichi Suwa et al., “Microorganisms Separation method ", R & D Planning, P545 (1990)].
However, the natural polluted environment or the wastewater from refineries and chemical factories has a high concentration of organic carbon as a pollutant and a low concentration of other nutrients such as phosphorus and nitrogen. Microorganisms that grow under the conditions of high organic carbon concentration and other low nutrient concentrations such as phosphorus and nitrogen and decompose pollutants are not applicable to the conventional concept of undernourished microorganisms. There is no report so far.
[0008]
An object of the present invention is to grow a low-nutrient microorganism Burkholderia cepacia 1A capable of growing under low nutrient conditions and decomposing aromatic compounds without adding nutrient salts, and an aromatic using the microorganism It is to provide a bioreactor for treating a compound, for example, a phenol-containing wastewater, and a wastewater treatment technique using the bioreactor, for example, a phenol-containing wastewater treatment technique for an oil refinery or a chemical factory.
The low nutrient condition referred to in the present invention means that the concentration of organic carbon is high as described above, for example, 15 ppm or more, and the concentration of nutrient salts such as phosphorus and nitrogen is low, for example, phosphorus is 5 ppm or less and nitrogen is 50 ppm. It refers to the following conditions.
[0009]
[Means for Solving the Problems]
Although the normal natural environment is considered to be undernutrition, the present inventors paid attention to phosphorus and nitrogen that are particularly important for the growth of microorganisms, and measured the concentrations of phosphorus and nitrogen in the natural environment. As a result, in soil and groundwater, as shown in Table 1 below, the nitrogen concentration is about 60 ppm or less and the phosphorus concentration is about 3 ppm or less. In the ocean, the concentration of nutrient components is lower, the nitrogen concentration is about 1.0 ppm or less, and the phosphorus concentration is about 0.1 ppm or less.
[0010]
[Table 1]

[0011]
Based on the measured values of the concentration of phosphorus and nitrogen in the natural environment, the present inventors grow under the condition that the target organic carbon concentration is high and the other nutrient concentrations of phosphorus and nitrogen are low, and pollutants. A medium for obtaining a microorganism capable of decomposing lysine was found to have a phosphorus concentration of about 5 ppm or less and a nitrogen concentration of about 50 ppm or less, and based on this finding, an aromatic compound under low nutrient conditions Burkholderia cepacia 1A (FERM P-15565) has been previously proposed as a microorganism that degrades.
[0012]
Furthermore, the present inventors have increased the amount of microbial cells because low-nutrient microorganisms have a small amount of microbial growth in order to efficiently treat wastewater in a low nutrient state without adding nutrient salts. Considering that it is necessary to construct a bioreactor, many experiments were conducted to increase the cell concentration of the bioreactor using the low-nutrient microorganism Burkholderia cepacia 1A for low-nutrient wastewater such as refineries and chemical factories under low nutrient conditions. It was.
[0013]
That is, when cultivating the undernutritive microorganism Burkholderia cepacia 1A, beads made of various materials such as polystyrene, Teflon, polypropylene, delrin, alumina, epoxy resin, nylon, and polyethylene are added to the culture solution, and the growth amount of the microorganism And the amount of phenol degradation was examined. As a result, it was found that by adding polystyrene to the culture solution, the growth of the undernutritive microorganism Burkholderia cepacia 1A and the accompanying phenol decomposing activity were significantly improved, and the present invention was achieved.
Accordingly, the first feature of the present invention relates to a method for promoting the growth of Burkholderia cepacia 1A, characterized in that the growth of Burkholderia cepacia 1A is carried out in the presence of polyethylene under low nutrient conditions.
[0014]
There is a report by Hattori et al. (Hisashi Morisaki et al., Interfaces and Microorganisms, Society Publishing Center (1988)) on the growth of microorganisms by adding plastic products to the medium. Hattori et al. Found that a plastic anion exchange resin was added to the culture solution, and that the optimal pH for growth of microorganisms shifted to the alkali side compared to the case where no ion exchange resin was added. This phenomenon is considered to be caused by the fact that the pH of the culture solution in the vicinity of the surface of the anion exchange resin is different from the pH of the culture solution existing far from the anion exchange resin.
However, polystyrene does not have a special function such as ion exchange capacity, and the addition effect of an anion exchange resin and the action of polystyrene used in the present invention are completely different.
[0015]
On the other hand, the method for immobilizing bacterial cells has been studied much more than before, including a method of comprehensively fixing to a gel-like substance such as alginic acid, polyvinyl alcohol, κ-carrageenan, gellan gum, agarose, cellulose, dextran, and glass. , Activated carbon, polystyrene, polyethylene, polypropylene, wood, silica gel, and the like can be cited as a method of adsorption and fixation [Keiji Ogimoto, “Biotechnology Text Series Microbiology”, Kodansha, P168 (1994), Hiroshi Aida et al., “New edition Applied Microbiology II”, Asakura Shoten, P116 (1995)].
[0016]
In addition, in the drainage treatment of phenol, bacteria belonging to the genus Rhdocococcus [SL. Pai et al., Bioresource Tech. 51, P37 (1995)] and Psendomonas putida [Kazuya Watanabe et al., Water Treatment Technology, 36, P77 (1994), Morsen et al., Appl. Microbiol. Biotech. , 33, P206 (1990)], etc., Candida tropicalis [H. Wenhua et al., Water Trent, 8, P119 (1993)] and Cryptococcus elinovir [A. Morsen et al., Appl. Microbiol. Biotech. , 33, P206 (1990)] and activated sludge [F. S. Lakhwala et al., Bioprocess Engi. , 8, P9 (1992)] have been reported to be used by immobilization by an entrapment method using alginic acid, polyvinyl alcohol or the like, and an adsorption method using activated carbon or clay mineral.
[0017]
However, in view of the effect, the effect of the polystyrene in the present invention is not only an effect for immobilizing cells as a carrier, but also other materials that increase the amount of suspended cells in the Burkholderia cepacia 1A culture solution. Is considered to be a growth catalysis not seen.
The polystyrene growth catalysis is that polystyrene is a water-insoluble plastic, the low-nutrient microorganism Burkhoderia cepacia 1A cannot use polystyrene beads as a carbon source, and the number of polystyrene beads added to the culture solution. Therefore, it is considered that it depends on the surface area of polystyrene (Examples 7 and 9).
[0018]
The shape of polystyrene added to the culture solution is not limited to a bead shape, and various shapes such as a sheet shape, a honeycomb shape, and a pipe shape can be used. Larger ones are preferred. Further, polystyrene can be used in the form of polystyrene coated on the surface of other plastics, glass, silica gel, alumina, metal and the like.
[0019]
Furthermore, as a result of examining the usage mode of polystyrene added to the culture solution, the present inventors applied physical force to the polystyrene, and positively peeled off at least a part of the microbial cells held on the polystyrene surface. It was found that the proliferation effect of polystyrene was improved by making floating cells. As shown in FIG. 4, the cause of the improvement of the proliferation action was detachment of the cells from the polystyrene surface, and the detached suspended cells were also cells having phenol degrading activity, and the cells were further detached. Bacteria are also formed on the polystyrene surface, so that the phenol degradation activity is improved as a whole reactor.
[0020]
In order to peel off the cells grown from the polystyrene surface during the culturing step, it is preferable to use a cell peeling reactor. Examples of the cell peeling reactor include a cell peeling type-airlift type reactor, a small jar type reactor, and a bead-in-ball type reactor.
[0021]
Polystyrene used in the bioreactor of the present invention is a semi-permanent material with excellent water resistance, and can be used for a long time. When used for a long time, polystyrene itself does not become a waste, so that the environmental load is small from a comprehensive point of view.
The polystyrene referred to in the present invention does not only refer to a homopolymer of styrene, but may contain other monomer components in addition to styrene as long as the growth catalytic action intended by the present invention is exhibited. .
Polystyrene used in the bioreactor of the present invention is a semi-permanent material with excellent water resistance, and can be used for a long time. When used for a long time, polystyrene itself does not become a waste, so that the environmental load is small from a comprehensive point of view.
[0022]
Burkholderia cepacia 1A used in the present invention was obtained by searching using a medium having a phosphorus concentration of 5 ppm or less and a nitrogen concentration of 50 ppm or less under a condition using 1000 ppm of phenol as a carbon source.
[0023]
The phosphorus source may be any substance that can be used by a microorganism as a phosphorus source. Monoammonium phosphate, monosodium phosphate, monopotassium phosphate, potassium potassium hydrogenphosphate, disodium hydrogenphosphate, phosphoric acid Inorganic phosphates such as dipotassium hydrogen, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and the like, natural products containing phosphorus such as hydrates of these salts, yeast extracts and gravy extracts Examples thereof include organic phosphorus compounds such as phosphate esters.
[0024]
Similarly, the nitrogen source may be any substance that can be used by microorganisms as a nitrogen source. Ammonium salts such as ammonium sulfate, monoammonium phosphate, ammonium nitrate, ammonium hydrogen phosphate potassium, ammonium chloride, and water of these salts Japanese products such as nitrates such as ammonium nitrate, sodium nitrate and potassium nitrate, nitrites such as sodium nitrite, natural nitrogen-containing compounds such as urea, amino acids, yeast extract, gravy extract, peptone and organic nitrogen-containing compounds such as amide compounds I can give you.
[0025]
Since the Burkholderia cepacia 1A can grow under low nutrient conditions of 5 ppm or less of phosphorus and 50 ppm or less of nitrogen, it can be applied to environmentally friendly environmental purification and wastewater treatment without adding nutrient salts. . However, having the property that the microorganism can grow under low nutrient conditions of 5 ppm or less of phosphorus and 50 ppm or less of nitrogen means that the microorganism cannot grow under conditions of 5 ppm or more of phosphorus or 50 ppm or more of nitrogen. However, the Burkholderia cepacia 1A is a facultatively low-nutrient microorganism that can grow not only under low nutrient conditions but also under high nutrient conditions.
[0026]
【Example】
Example 1
Search for phenol-utilizing bacteria that grow under low phosphorus and nitrogen conditions (natural environment)
(Creation of search medium)
As a medium for search, a medium (hereinafter referred to as E-medium) shown in Table 2 diluted to 1/10 (1/10 E-medium: phosphorus about 5 ppm, nitrogen about 50 ppm) was used. To this, 1,000 ppm of phenol was added as a carbon source. The pH of each medium was 7.0.
[0027]
[Table 2]

[0028]
(Degrading bacteria acquisition method A)
As a separation source, about 480 kinds of soil around Tsukuba City were used, and only microorganisms were extracted and added to the medium by the following method. 30 ml of sterilized water was placed in a 300 ml Erlenmeyer flask, and 5 g of a soil sample was added thereto. This was shaken for 1 day to extract bacteria. After extraction, the soil was precipitated by centrifugation (2,000 rpm, 5 min), and the supernatant was filtered through a filter paper and a glass filter to remove soil fine particles. The obtained bacterial solution was filtered through a nitrocellulose filter (0.2 μm) to collect microorganisms. This filter was appropriately cut and added to the culture medium, and a search was performed. The search was performed by the following method. 30 ml of 1 / 10E-medium was placed in a 300 ml Erlenmeyer flask, and the above-mentioned nitrocellulose filter was added thereto and cultured with shaking at 30 ° C. for 5 days. About the thing with which growth was seen, it transplanted 100 microliters to a large test tube with an internal diameter of 24 mm containing 8 ml of 1 / 10E-medium, and was cultured for 5 days. This was repeated twice to isolate the bacteria.
For isolation of the fungus, a plate prepared by adding 1.2% agarose (agarose (ultra-pure grade for electrophoresis)) to the 1 / 10E-medium described above was used. To this, the above-mentioned culture solution was appropriately diluted and applied, and cultured at 30 ° C. for 5 days to isolate colonies that had grown.
[0029]
Example 2
(Identification of degrading bacteria obtained in Example 1)
The phenol-assimilating bacterium strain 1A obtained in Example 1 was gram-negative, motile, and positive for both oxidase and catalase. The OF test showed oxidation. From the results of other various physiological tests, one kind of strain obtained was identified as Burkholderia cepacia. The following table shows the characteristics of the phenol-utilizing bacterium 1A strain.
[0030]
[Table 3]

[0031]
[Table 4]

[0032]
Example 3
Degradation of phenol under conditions of low phosphorus concentration and low nitrogen concentration by the degrading bacteria obtained in Example 1
(Disassembly method)
The 1A strain was inoculated into 1 / 10E-plate medium (phenol 1,000 ppm) and cultured at 30 ° C. The grown cells are inoculated into a plastic tube (confirmed that phenol does not adsorb to the tube) containing 1 ml of 1 / 10E-medium, and cultured in a shaking incubator (MBSS-10, Maruhishi). The degradation amount of phenol after culturing was quantified using gas chromatography. The initial phenol concentration was 100 ppm and 1000 ppm, and sampling was performed after 3 and 6 days, respectively. The conditions for gas chromatography are shown below.
Column (Column): Unisole F-200
Injection temperature (Inj.temp): 180 ° C
Column temperature (Col. temp): 150 ° C
Detector (Detector): FID
Injection volume (inj.size): 2 μl
Internal standard (IS): Diethylene glycol
The results of the decomposition test are shown in Table 5 below. The Burkholderia cepacia 1A strain subjected to the experiment had phenolic resolution.
[0033]
[Table 5]

[0034]
Example 4
(Comparison experiment of phenol resolution of strains obtained in Example 1 and existing strains)
As a test strain, the isolate 1A obtained in Example 1 was used. For comparison, ATCC (American Type Culture Collection) -preserved strain, which has been reported as a phenol-degrading bacterium, was also used. The ATCC preserved phenol-degrading bacteria are the following 4 strains.
Pseudomonas putida ATCC 11172
Rhodococcus rhodochrous ATCC 14347
Vibrio cycloses ATCC 14635
・ Rhodococcus zopfii ATCC 51349
1 / 100F-medium (about 1 ppm phosphorus, about 20 ppm nitrogen) diluted to 1/100 of the medium shown in Table 6 below, or the phosphorus and nitrogen concentrations of the 1 / 100F-medium to about 500 ppm respectively. The cells were inoculated into a plastic tube containing 2 ml of the culture medium and cultured at 30 ° C. for 4 days in a shaker (MBSS-10, Maruhishi). The amount of phenol decomposed after the culture was quantified using gas chromatography. The amount of growth was measured as the absorbance at 660 nm (A660).
[0035]
[Table 6]

[0036]
The composition of trace metals in Table 6 is as shown in Table 7 below.
[Table 7]

[0037]
(Results and discussion)
The results of the decomposition test are shown in Tables 8 to 9 below. As shown in Table 8, the obtained degrading bacteria grew well in 1/100 F-medium, and most of the added 100 ppm phenol was completely degraded within 4 days. Further, even when the phosphorus and nitrogen concentrations in the medium were increased, no inhibition was observed in both growth and phenol degradation.
On the other hand, in the case of the ATCC stock, all the strains did not grow at all on the basic medium. The slight decrease in phenol in the table appears to be due to adsorption to the inoculated cells. In the case of Table 9 using a medium in which the phosphorus and nitrogen concentrations in the medium were each increased to 500 ppm, as shown in Table 8, the decomposition of phenol was observed. From this result, the phenol-degrading bacterium of Example 1 is a novel bacterium that grows under low phosphorus and low nitrogen conditions so that no known phenol-degrading bacterium can grow at all, and has good phenol degradability. It is believed that there is.
[0038]
[Table 8]

[0039]
[Table 9]

[0040]
Example 5
Degradation of various aromatic compounds using phenol-utilizing bacteria growing under conditions of low phosphorus concentration and low nitrogen concentration
Using the isolate 1A obtained in Example 1 as a test strain, the assimilation property of phenol, benzene, toluene, xylene (isomer mixture) was confirmed. The amount of each aromatic compound as a substrate was 100 ppm, and the medium was cultured at 30 ° C. for 4 days using 1/100 F-medium.
The results are shown in Table 10 below.
[0041]
[Table 10]

From the results in the previous table, it was confirmed that the growth of the microorganisms was higher in any substrate (carbon source) than in the case where there was no carbon source.
[0042]
Example 6
In order to examine the material of the immobilization support for the undernutritive microorganism Burkholderia cepacia 1A, beads having a diameter of 6.3 mm were prepared using polystyrene, Teflon, alumina, nylon, delrin, polyethylene, epoxy resin, and polypropylene.
The 1A strain that had been seed-cultured was suspended in sterilized water in the same manner as in other culture experiments, and was inoculated to 0.01 at 580 nm absorbance (A580). 1 ml of the medium and 4 beads were placed in a glass test tube with a screw, and cultured for 1 day at 30 ° C. under rotary shaking at 600 rpm to examine the resolution of phenol.
The results are shown in FIG. Nylon and Delrin were not subjected to subsequent experiments because of their high phenol adsorption or absorption.
Phenol was most rapidly degraded when polystyrene beads were added during culture. Other material beads promoted phenol degradation only slightly compared with no beads added (floating cells), and there was almost no difference in the degree.
[0043]
Example 7
At the time of culturing the 1A strain, 1 to 4 polystyrene beads having a diameter of 6.3 mm were added per test tube, and the number of polystyrene beads added and the influence on phenol degradation were examined.
The 1A strain that had been seed-cultured was suspended in sterilized water in the same manner as in other culture experiments, and inoculated to 0.01 with A580. 1 ml of the medium and 4 beads were placed in a glass test tube with a screw, and cultured for 1 day at 30 ° C. under rotary shaking at 600 ppm to examine the resolution of phenol.
The results are shown in FIG. As the number of polystyrene beads added increased, the amount of floating cells increased, and the amount of phenol degradation increased accordingly.
[0044]
Example 8
Two bioreactors as shown in FIG. 3 were prepared using polystyrene beads (φ6.3 mm).
One unit was added with 300 polystyrene beads, and the other unit was not added with any polystyrene beads, and both were compared.
800 ml of each medium was added, and the 1A strain that had been seed-cultured was suspended in sterilized water and inoculated to 0.001 with A580.
Incubation was performed at 25 ° C. while rotating and stirring the rotary spring at 250 rpm. The reaction was carried out batchwise.
As a result, the addition of polystyrene beads as shown in FIG. 3 increased the amount of floating cells, and accordingly, the phenol resolution was higher than that without addition of polystyrene beads.
[0045]
Example 9
The influence of the surface area of polystyrene beads on the growth of the undernutritive microorganism Burkholderia cepacia 1A was examined.
0 to 35 polystyrene beads having different diameters (ψ3.2 mm and ψ6.35 mm) were added to the nutrient system, and the influence on the growth was examined. The culture was performed at 30 ° C. and 600 rpm for one day. The bacterial cells adsorbed on the polystyrene bead surface were subjected to alkaline hydrolysis and measured as the amount of protein.
The results are shown in FIG. Regardless of the size of the polystyrene beads, both the amount of floating cells and the amount of adsorbed cells increased depending on the actual area of the polystyrene beads. Further, the amount of floating cells reached saturation near the surface area of 300 mm2, and the amount of adsorbed cells reached saturation near the surface area of 600 mm2. From the above results, it can be seen that the growth promoting effect of the undernutritive microorganism Burkholderia cepacia 1A depends on the surface area of the beads, not the size of the beads.
[0046]
In the case of Burkholderia cepacia 1A strain, about 45% of the dried cells are proteins, and the dry weight of one cell is 8.1 × 10 −8 μg. From the data shown in FIG. 5, the amount of cells adsorbed on the surface area of the beads is calculated as 0.05 μg / mm 2 as protein. Assuming that the installation area of the 1A strain is 0.5 × 1.5 μm, 1.3 × 10 6 cells can be adsorbed to 1 mm 2, and this value is almost equal to the previously obtained 1.37 × 10 6 cells / mm 2. . Therefore, it is considered that the 1A strain is adsorbed on the surface area of polystyrene in a state of almost saturation.
[0047]
The amount of cells adsorbed on the surface of the polystyrene beads was measured as the amount of protein after alkaline hydrolysis. The cultured polystyrene beads were washed once with ultrapure water, 0.1N NaOH was added and heated at 80 ° C. overnight to hydrolyze the cells. Proteins contained in the hydrolyzed solution were quantified by the Raleigh method using bovine serum albumin as a standard (RF Schleif et al. (Translated by Masaya Kawakami et al.), “Molecular Biology Manual” Kodansha Scientific Hook, p90 ( 1983))
[0048]
Example 10
Surface hydrophilicity of various beads and their effects on the growth of the undernutrient microorganism Burkholderia cepacia 1A
Using various beads made of φ6.3-6.4 mm glass, Teflon and polystyrene, the surface hydrophilicity and the influence on the growth of the strain 1A were examined. The culture was performed at 30 ° C. and 600 rpm for 1 day. The surface hydrophilicity of each bead is obtained from the contact angle between the water droplet dropped on the bead surface and the bead surface according to the method of MO, Samuelsson et al. Hydrophilicity (WA value)
[Expression 1]
WA = Y _LV 0 (1 + cos θ)
(Θ is the contact angle, Y _LV 0 is the surface tension of water in saturated steam, 72.8 mNm ^-1 Is)
(MO, Samuelsson et al., Environ. Microbiol., 56, p3643 (1990)).
The amount of cells adsorbed on the surface of various beads was measured as the amount of protein after alkaline hydrolysis.
The experimental results and hydrophilic properties of various beads are shown in Table 11 below. Teflon beads were the most hydrophobic and glass beads were hydrophilic. The hydrophilicity of polystyrene beads was intermediate between Teflon beads and glass beads.
[0049]
[Table 11]

The 1A strain adsorbed best on the surface of polystyrene beads, and the amount of cells adsorbed on the surface of Teflon beads was about 1/4 of that of polystyrene beads. Also, no adsorption was shown on the glass bead surface.
[0050]
Example 11
Phenol degradation in a small jar reactor
In this example, phenol is decomposed by using a small jar type reactor as a cell exfoliation type reactor as shown in FIG. The small jar type reactor was produced by modifying an animal cell culture flask (500 ml, with double arm) manufactured by Calstar. The main remodeling points are Teflon made by placing a stainless steel basket inside the flask so that polystyrene beads (500 balls for ELISA (RIA) made by Sumitomo Bakelite) can be added and the water level in the reactor is horizontal. In addition, two stainless steel rods were attached to the shaft of the stirring blade so that the polystyrene beads in the basket could be physically impacted. Using this reactor, the amount of suspended cells and the amount of phenol degradation were examined.
A medium containing 0.5 ppm phosphorus, 5 ppm nitrogen and 100 ppm phenol is used, and the flow acceleration of the medium is 30 ml / hr (T _1/2 = 13.3 hr). The results are shown in FIG. The amount of suspended cells increased remarkably by attaching a stainless steel rod. Along with this, all phenol was also decomposed.
[0051]
Example 12
Degradation of phenol by airlift reactor
In this example, the air lift reactor is installed with a basket equipped with a MDP type 5L stainless steel rod manufactured by Maruhishi Bioengineer Co., Ltd., so that the falling polystyrene beads collide with the stainless steel rod. I used a modified one.
The actual capacity of the cell peeling type-airlift type reactor was 4,000 ml, and the reactor was ventilated at an air flow rate of 2 SL / min, and compressor air was sterilized with a filter and used.
[0052]
In this example, the addition effect of polystyrene beads and the installation effect of the cage were compared and examined with the control without the addition of polystyrene beads and the control with no addition of polystyrene beads. The culture was carried out at 30 ° C. for 48 hours. The initial concentration of phenol was 100 ppm, and an additional equivalent of 100 ppm was added after 24 hours of culture.
[0053]
In both reactors, after 24 hours of culture, 100 ppm of phenol initially given was completely decomposed. The results after adding phenol equivalent to 100 ppm (after 30 hours of culture) are shown in FIG. By adding polystyrene beads, the amount of suspended cells increased, and the phenol resolution was improved accordingly. Furthermore, an increase in the amount of suspended cells and an improvement in phenol resolution were observed by installing a basket equipped with a stainless steel rod.
[0054]
Example 13
Phenol degradation in a bead-in-ball reactor
In this example, phenol is decomposed using a bead-in-ball type reactor (a property submission file submitted at the same time as the application) as a bacterial cell peeling type reactor. In this example, about 80 polystyrene beads were placed in a plastic spherical cage, and the polystyrene beads contained therein collided with each other by rotating the cage by stirring the medium. Incubation was carried out at 30 ° C for 24 hours and 20 ml / h (T _1/2 = 20 hr) and continuous culture.
[0055]
The results are shown in FIG. Similar to the jar type reactor cell peeling type reactor reactor, an increase in the amount of suspended cells was confirmed and the phenol degradation activity was improved as compared with the control reactor (without addition of polystyrene beads).
Since the bead-in-ball type and air lift type reactors are also cell detachment type reactors, it is understood that the force for detaching the microbial cells need not be so great.
[0056]
【effect】
Method for promoting growth of low-nutrient microorganism Burkholderia cepacia 1A that can grow under low nutrient conditions and decompose aromatic compounds without adding nutrient salts, aromatic compounds using such microorganisms, for example, phenol-containing wastewater A bioreactor for treatment and wastewater treatment technology using the bioreactor, for example, phenol-containing wastewater treatment technology for refineries, chemical factories, etc., could be provided.
[Brief description of the drawings]
1 is a graph showing the results of Example 6. FIG.
2 is a graph showing the results of Example 7. FIG.
FIG. 3 is an example of a conceptual diagram of a bioreactor used in the present invention.
FIG. 4 is a diagram for explaining the concept of the cell detachment reactor according to the present invention.
FIG. 5 is a diagram illustrating the influence of the surface area of polystyrene pieces on the growth of the undertrophic microorganism Burkholderia cepacia 1A. (A) Concentration of suspended cells (B) Concentration of adsorbed protein chamber on the bead surface
FIG. 6 is a schematic cross-sectional view of a small jar reactor.
FIG. 7 is a diagram showing a result of phenol decomposition by a small jar type reactor.
FIG. 8 is a diagram showing the result of phenol decomposition by a cell peeling type-airlift type reactor.
FIG. 9 is a view showing the result of phenol decomposition by a peas-in-bolt reactor.
[Explanation of symbols]
1 Basket for holding polystyrene beads
2 Polystyrene beads held in a basket
3 Baffle
4 stirring blades
5 Rotating shaft

Claims (7)

A method for promoting the growth of Burkholderia cepacia 1A, wherein the growth of Burkholderia cepacia 1A (FERM P-15666) is carried out in the presence of polystyrene. The method for promoting growth of Burkholderia cepacia 1A according to claim 1, wherein physical force is applied to polystyrene during growth of Burkholderia cepacia 1A, and at least a part of the bacterial cells held and proliferated is actively peeled off. A bioreactor characterized in that Burkholderia cepacia 1A coexists with polystyrene. The bioreactor according to claim 3, further comprising means for positively peeling at least a part of the bacterial cells held on the polystyrene surface and proliferated. The bioreactor according to any one of claims 3 to 4, which is for wastewater treatment. The bioreactor according to claim 5, wherein the waste water contains an aromatic compound. The bioreactor according to claim 6, wherein the aromatic compound is phenol.

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