JP4363624B2 - Pesticide degradation method using novel pesticide-degrading bacteria and complex microbial system - Google Patents

Description

また、同様にして、菌株ＣＳＢ１又はＣＤＢ２１を用いてシマジン分解能を調べた。結果を次の表２に示す。
【００１４】

【００１５】
この結果、速やかなシマジン分解を示したＣＤＢ２１株と異なり、ＣＤ７ＷＬ株及びＣＳＢ１株ではシマジン濃度が対照とほとんどかわらなかった。このことからＣＤ７ＷＬ株及びＣＳＢ１株はシマジン分解能を有しないと結論した。
しかし、ＣＤ７ＷＬ、ＣＤＢ２１、ＣＳＢ１はいずれも単独でシマジン５ｍｇ／Ｌを含む無機塩寒天培地平板に接種したところ明確なコロニーを形成しなかった。そこで、これらの微生物を組合せて同無機塩培地に接種したところ、ＣＤＢ２１とＣＳＢ１の組合せのみにコロニーが出現した。得られたＣＤＢ２１とＣＳＢ１よりなる複合微生物系はシマジン５ｍｇ／Ｌを含む無機塩寒天培地に接種することにより継代維持できることもわかった。
即ち、単離されたＣＤＢ２１は、単独でもシマジンを分解することができるが、これを継代培養する場合はＣＤＢ２１とＣＳＢ１よりなる複合微生物系が好ましいことがわかった。
【００１６】
次に単離したＣＤＢ２１の菌学的位置づけを行うために、形態的性質、培地における生育の様相および生理学的性質について検討した。培養温度は特記しない限り３０℃で実施し、次の表３に示す形態、培養、生理学的性質が認められた。
【００１７】

【００１８】
上記の結果をBergey’s Manual of Determinative Bacteriology 第９版に従って同定を行なったところ、本発明のＣＤＢ２１株はシュードモナス群(Pseudomonas group)に属する菌株と推定された。
【００１９】
同様に、単離されたＣＳＢ１の菌学的位置づけを行うために、形態的性質、培地における生育の様相および生理学的性質について検討した。培養温度は特記しない限り３０℃で実施し、次の表４に示す形態、培養、生理学的性質が認められた。
【００２０】

【００２１】
以上の結果をBergey's Manual of Determinative Bacteriology 第９版に従って同定を行なったところ、本発明のＣＳＢ１株はシュードモナス群（Pseudomonas group）に属する菌株と推定された。
【００２２】
シュードモナス群に属する細菌は遺伝子的に多様な細菌を含む細菌群であることが知られており、本発明のＣＤＢ２１及びＣＳＢ１の菌学的性状のみから分類を同定することは困難と考え、ＣＤＢ２１及びＣＳＢ１の菌学的位置づけを遺伝子工学的側面から特徴付けるために、１６Ｓ−ｒＲＮＡ遺伝子の塩基配列の決定（解析）を行った。
本発明のＣＤＢ２１及びＣＳＢ１の菌体を用いてｃＤＮＡを調製し、このｃＤＮＡを鋳型ＤＮＡとして、公知のユーバクテリア１６Ｓ−ｒＲＮＡ増幅用プライマーを用いてＰＣＲ法によりＣＤＢ２１及びＣＳＢ１のそれぞれの１６Ｓ−ｒＲＮＡ遺伝子断片を増幅し、塩基配列を決定（解析）した。
以上により得られたＣＤＢ２１の１６Ｓ−ｒＲＮＡ遺伝子断片の塩基配列より、両端のＰＣＲ用プライマー配列を除いた１４８８塩基を配列表の配列番号１に示す。更に、ＣＳＢ１について得られた１６Ｓ−ｒＲＮＡ遺伝子断片の塩基配列より、両端のＰＣＲ用プライマー配列を除いた１４６２塩基を配列表の配列番号２に示す。
【００２３】
配列番号１に示したＣＤＢ２１の１６Ｓ−ｒＲＮＡ遺伝子断片塩基配列に対してジェネティクスＰＤＢ（GENETYX PDB）を用いてＤＮＡ塩基配列データベース(GenBank) に登録された他の微生物が保有する１６Ｓ−ｒＲＮＡ遺伝子の配列とＦＡＳＴＡ法により相同性比較を行なった。
その結果、ＣＤＢ２１は、未培養のバクテリアクローンＩ１２ （uncultured bacterium clone I12, ACCESSION:AY187895）と９９．９％の相同性（1369/1370、INT.Score = 5474、OPT.Score = 5474）を示した。これ以外では多くの未培養ベータプロテオバクテリア（uncultured beta proteobacterium）、未同定ベータプロテオバクテリア、チオバシラス属(Thiobacillus)、バークホルデリア属(Burkholderia)、ラルストニア属(Ralstonia)などのベータプロテオバクテリアと９０％以上の相同性が得られた。
近隣結合法（ＮＪ法）により近縁株と系統樹を作成し、類縁関係を解析したところ、ベータプロテオバクテリアの範疇に属することが明らかとなった。以上より、本発明者らは本発明の菌株ＣＤＢ２１を、これまで単離培養されたことのない、いかなる既知の属種にも帰属しない新規なベータプロテオバクテリアに属するＣＤＢ２１株と同定し、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ ＢＰ−１０４０３として寄託した。
【００２４】
同様に、配列番号２に示したＣＤＢ２１の１６Ｓ−ｒＲＮＡ遺伝子断片塩基配列に対してジェネティクスＰＤＢ（GENETYX PDB）を用いてＤＮＡ塩基配列データベース（GenBank）に登録された他の微生物が保有する１６Ｓ−ｒＲＮＡ遺伝子の配列とＦＡＳＴＡ法により相同性比較を行なった。ＣＳＢ１はブラディリゾビウム ジャポニカム （Bradyrhizobium japonicum strain USDA 62, ACCESSION:AF208517）と９９．６％の相同性（1456/1462、INT.Score = 5812、OPT.Score = 5812）を示した。ＤＮＡ塩基配列データベースに登録された他のブラディリゾビウム ジャポニカム１６Ｓ−ｒＲＮＡ遺伝子塩基配列とも９９％以上の相同性を示した。
近隣結合法（ＮＪ法）により系統樹を作成し、比較したところ、ＣＳＢ１はブラディリゾビウム ジャポニカムの範疇に属する事が明らかとなった。以上より、本発明者らは本発明の菌株ＣＳＢ１をブラディリゾビウム ジャポニカム ＣＳＢ１株と同定し、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ ＢＰ−１０４０２として寄託した。
【００２５】
次に本発明のＣＤＢ２１株によるシマジンの分解と菌の活性を更に詳細に検討した。バクトトリプトン１ｇ／Ｌ、シマジン５ｍｇ／Ｌを添加した培地に、ＣＤＢ２１株を接種して、３０℃、１２０ｒｐｍにて振盪培養を行い、菌体の生育、シマジンの分解とそれに伴う塩素イオンの発生を測定した。結果を図２に示す。図２の白丸印（○）はシマジンの残存率（％）を示し、白四角印（□）は生成した塩素イオンの濃度（ｍｇ／Ｌ）を示す。図２のグラフの縦軸は左側がシマジンの残存率（％）を示し、右側が生成した塩素イオンの濃度（ｍｇ／Ｌ）を示す。横軸は培養時間（日）を示す。
シマジンは、培養開始後３日目に殆ど分解され、４日目にはほぼ完全に分解された。また、シマジンの分解量に見合った塩素イオンの生成が認められ、さらに菌体の増殖が認められた。このように本発明のＣＤＢ２１株は、約３日から４日でほぼ完全にシマジンを分解することができ、かつ培養条件によってはＣＤＢ２１株単独であっても菌体が増殖しながらシマジンを分解することができる。
【００２６】
次に、ＣＤＢ２１株との複合微生物系によるシマジンの分解を検討した。
Ｎａ_２ＨＰＯ_４・１２Ｈ_２Ｏを１．２ｇ／Ｌ、ＫＨ_２ＰＯ_４を０．５ｇ／Ｌ、さらにシマジン５ｍｇ／Ｌを含有する平板培地にＣＤＢ２１株菌体を接種し、次いで他の菌株を接種した後に中央で両菌株を混合し、３０℃で７日間培養した。それぞれ単独で接種した部分には生育せず、培養器の中央部で混合した部分にコロニーが生じた組み合せを複合微生物系とした。このようにして複合微生物系を形成したコロニーから得られた複合微生物系をシマジン含有無機塩液体培地に接種し、３０℃、１２０ｒｐｍにて４日間振盪培養を行い、シマジンの分解を測定した。結果を表５に示す。検討した菌株のうち５種類との組み合せで、ＣＤＢ２１複合微生物系の構築が可能であり、いずれも無機塩培地中でシマジンを分解した。
【００２７】
【表５】

【００２８】
次に、ＣＤＢ２１株若しくはＣＳＢ１株単独、又はＣＤＢ２１及びＣＳＢ１株との複合微生物系によるシマジンの分解を検討した。
シマジン含有無機塩液体培地にＣＤＢ２１株単独、若しくはＣＳＢ１株単独、又はＣＤＢ２１株及びＣＳＢ１株より構成される複合微生物系を接種した。３０℃、１２０ｒｐｍにて振盪培養を行い、菌体の生育、シマジンの分解とそれに伴う塩素イオンの発生を測定した。結果を図３に示す。図３の（Ａ）はシマジンの残存率（％）を示し、図３の（Ｂ）は生成した塩素イオンの濃度（ｍｇ／Ｌ）を示す。図３の（Ａ）のグラフの縦軸はシマジンの残存率（％）を示し、図３の（Ｂ）は生成した塩素イオンの濃度（ｍｇ／Ｌ）を示す。横軸はいずれも培養時間（日）を示す。図３中の黒丸印（●）はＣＤＢ２１株を単独で接種した場合を示し、黒三角印（▲）はＣＳＢ１株を単独で接種した場合を示し、黒四角印（■）はＣＤＢ２１株及びＣＳＢ１株より構成される複合微生物系を接種した場合を示す。
この結果、ＣＤＢ２１株ではシマジンは培養開始後７日目に殆ど分解され、シマジンの分解量に見合った塩素イオンの生成が認められたが、この場合には菌体の増殖は殆ど認められなかった。ＣＳＢ１株では培養期間中、シマジンの分解、塩素イオンの生成、菌体の増殖のいずれも認められなかった。ＣＤＢ２１株とＣＳＢ１株より成る複合微生物系では、シマジンは培養開始後、１日間で速やかにシマジンが分解され、２日目には完全に分解された。また、シマジンの分解量に見合った塩素イオンの生成が認められ、複合微生物系の菌体の速やかな増殖が認められた。
【００２９】
次に、本発明のＣＤＢ２１複合微生物系によるシマジンの分解によりトリアジン環が開裂し無機化されるか否かについて検討した。まずシマジン含有無機塩培地にトリアジン環の炭素原子を^１４Ｃで標識したシマジン（simazine-ring-UL-^１４Ｃ）を添加した。本培地中には^１４Ｃ標識シマジン０．７５ｍｇ／L及び非標識シマジン３．４ｍｇ／Ｌが含まれる。これにＣＤＢ２１株とＣＳＢ１株を含む複合微生物系を接種し、水酸化ナトリウムトラップを通過させた空気を２ｍｌ／分で連続的に通気させながら２５℃暗所にて８日間静置培養した。培養期間中、培養液中の^１４Ｃ標識シマジン及びその代謝物濃度をラジオアナライザー付高速液体クロマトグラフで、捕集した^１４Ｃ標識二酸化炭素を液体シンチレーションカウンターで定量した。結果を図４に示す。図４の黒丸印（●）は^１４Ｃ標識シマジン濃度（ｍｇ／Ｌ）を、黒四角印（■）は^１４Ｃ標識シアヌール酸濃度（ｍｇ／Ｌ）を、黒三角（▲）は発生した^１４Ｃ標識二酸化炭素の理論量に対する割合（％）を示す。図４の縦軸は左側が^１４Ｃ標識シマジン及び^１４Ｃ標識シアヌール酸の濃度（ｍｇ／Ｌ）を、右側が発生した^１４Ｃ標識二酸化炭素量の理論量に対する割合（％）を示す。横軸は培養期間（日）を示す。
この結果、シマジンは培養６日目に大部分が分解し、８日目には完全に分解していた。シマジンの分解に伴い分解中間代謝産物であるシアヌール酸濃度が上昇したが、大量に蓄積する様子はなく、完全分解物である^１４Ｃ標識二酸化炭素の発生が認められた。このことは本発明のＣＤＢ２１株や、ＣＤＢ２１株とＣＳＢ１株を含む複合微生物系によるシマジン分解は、トリアジン環も完全に分解し、シマジンを無害化することができることを示している。
【００３０】
次に、ＣＤＢ２１複合微生物系による各種のトリアジン系除草剤の分解について検討した。シマジン含有無機塩培地にシマジンの代りに各種のトリアジン系農薬５ｍｇ／Ｌを添加し、ＣＤＢ２１株とＣＳＢ１株より構成される複合微生物系を接種した。３０℃、１２０ｒｐｍにて振盪培養を行い、これらの除草剤の分解を測定した。結果を次の表６に示す。
【００３１】
【表６】

【００３２】
この結果、トリアジン環の２位が塩素化されたシマジンとアトラジンは３日以内に速やかに分解されたが、塩素を持たない２位がチオメチル化されたシメトリン、プロメトリン、ジメタメトリンはこの培養条件では分解されなかった。
【００３３】
以上のように、本発明は、シマジンやアトラジンなどのハロゲン含有トリアジン系除草剤を効率良く分解し、無害化できる微生物、並びにそれを用いたハロゲン含有トリアジン系除草剤の分解方法、及びそのための前記微生物含有組成物を提供するものである。特に本発明は、シマジンを生物学的手法で効率良く分解できる単一の菌株が存在することを初めて報告するものであり、シマジンを分解、好ましくはほぼ完全に無害化できる能力を有する微生物又は複合微生物系を提供するものである。
本発明のシマジン分解菌ＣＤＢ２１株は、土壌からシマジンに対する集積培養後、単離したものであるが、これに限定されるものではなく、ベータプロテオバクテリアに属する細菌であってシマジンの分解能を有する細菌、好ましくはさらに前記したベータプロテオバクテリアＣＤＢ２１株の菌学的特徴の一部又は全部を有する細菌が含まれる。
また、本発明のシマジン分解菌は、１６Ｓ−ｒＲＮＡ遺伝子の塩基配列によっても特徴付けられるものである。即ち、本発明のシマジン分解菌は、１６Ｓ−ｒＲＮＡ遺伝子の塩基配列が、配列表の配列番号１に記載された塩基配列、又は当該塩基配列と相同性、好ましくは５０％以上、７０％以上、より好ましくは８０％以上、又は９０％以上の相同性を有する塩基配列を有する細菌であって、シマジンの分解能を有する、好ましくはさらに前記したベータプロテオバクテリアＣＤＢ２１株の菌学的特徴の一部又は全部を有するものが含まれる。
【００３４】
本発明のシマジン分解菌の具体的な一例としては、独立行政法人産業技術総合研究所特許生物寄託センターに微生物の表示（寄託者が付した識別のための表示）「ＣＤＢ２１」、受託番号「ＦＥＲＭ ＢＰ−１０４０３」として寄託されている新規ベータプロテオバクテリアを挙げることができるがこれに限定されるものではない。
【００３５】
更に本発明は、本発明の前記したシマジン分解菌と当該分解菌のシマジン分解及び／又は生育を補助する能力を持った微生物との組合せにより構成される複合微生物系を提供するものである。本発明の複合微生物系としては、ベータプロテオバクテリアに属する細菌からなるシマジン分解菌の１種以上と、シマジンの分解及び／又は生育を補助する能力を有する１種以上の共生菌を含有してなる複合微生物系が挙げられる。本発明の複合微生物系は、トリアジン系農薬好ましくはハロゲン含有トリアジン系農薬の分解能を有する。
本発明の好ましい複合微生物系としては、シマジン分解菌が、ベータプロテオバクテリアＣＤＢ２１株である複合微生物系が挙げられる。
本発明のシマジン分解菌とこのような複合微生物系を構成することができる共生菌としては、ブラディリゾビウム・ジャポニカム、ロドコッカス・ロドクラウス、ステノトロフォモナス・マルトフィリア、ノカルディオイデス・ジェンセニ、ノカルディオイデス・フルヴス、ノカルディオイデス・シンプレックス、シュードモナス・エルギノーサなどが挙げられるが、本発明のシマジン分解菌と当該分解菌のシマジン分解及び／又は生育を補助する能力を持った微生物であれば、前記で例示した微生物に限定されるものではない。
このような複合微生物系を構成できるか否かは、シマジンを含有する寒天培地において、本発明のシマジン分解菌と他の菌を混合して接種して、培養後、コロニーを形成できるか否かにより判定することができる。コロニーを形成することができる菌の組み合わせは本発明の複合微生物系を構成できるものである。
【００３６】
本発明のシマジン分解菌と共に複合微生物系を構築することができる微生物の好ましい例としては、独立行政法人産業技術総合研究所特許生物寄託センターに微生物の表示（寄託者が付した識別のための表示）「ＣＳＢ１」、受託番号「ＦＥＲＭ ＢＰ−１０４０２」として寄託されている新規な細菌を挙げることができる。
また、ブラディリゾビウム ジャポニカムに属する菌であって、本発明のシマジン分解菌のシマジンの分解及び／又は生育を補助する能力を有する細菌、好ましくはさらに前記した菌学的特徴の一部又は全部を有する細菌が含まれる。さらに、その１６Ｓ−ｒＲＮＡ遺伝子の塩基配列が、配列表の配列番号２に記載する塩基配列、又は当該塩基配列と相同性、好ましくは５０％以上、７０％以上、より好ましくは８０％以上、又は９０％以上の相同性を有する塩基配列を有する細菌であって、本発明のシマジン分解菌のシマジンの分解及び／又は生育を補助する能力を有する、好ましくはさらに前記した菌学的特徴の一部又は全部を有するものが含まれる。
【００３７】
また、本発明は、ブラディリゾビウム ジャポニカムに属する細菌であって、ベータプロテオバクテリアに属する細菌からなるシマジン分解菌のシマジンの分解及び／又は生育を補助する能力を有する細菌、好ましくはさらに前記したブラディリゾビウム ジャポニカムＣＳＢ１株の菌学的特徴の一部又は全部を有する細菌を提供する。本発明のブラディリゾビウム ジャポニカムに属する細菌としては、例えば前記してきたブラディリゾビウム ジャポニカムＣＳＢ１株などが挙げられる。
さらに、本発明は、ベータプロテオバクテリアに属する細菌であってシマジン分解能を有する細菌の１種以上を単独で、またはこれと１種以上の共生菌と組み合わせてなる複合微生物系を用いることを特徴とするトリアジン系農薬、好ましくはハロゲン含有トリアジン系農薬の分解方法を提供する。ベータプロテオバクテリアに属する細菌であってシマジン分解能を有する細菌としては、前記したシマジン分解菌およびその変異株から選ばれる細菌が挙げられる。このようなシマジン分解菌と組み合わされる共生菌としては、ブラディリゾビウム ジャポニカムＣＳＢ１株、ロドコッカス ロドクラウス、ステノトロフォモナス マルトフィリア、ノカルディオイデス ジェンセニ、ノカルディオイデス フルヴス、ノカルディオイデス シンプレックス、及びシュードモナス エルギノーサなどから選ばれる細菌が好ましいが、本発明のシマジン分解菌と当該分解菌のシマジン分解及び／又は生育を補助する能力を持った微生物であれば、前記で例示した微生物に限定されるものではない。好ましい共生菌としては、例えばブラディリゾビウムジャポニカムＣＳＢ１株が挙げられる。また、好ましい複合微生物系としては、ベータプロテオバクテリアＣＤＢ２１株とブラディリゾビウム ジャポニカムＣＳＢ１株からなる複合微生物系が挙げられる。
分解培養の際に使用する培地としてはシマジンを始めとするトリアジン系農薬を含有している無機塩培地を使用することが出来る。更に本発明分解菌単独で用いる場合は窒素源を添加できる。窒素源としてはトリプトン、ペプトン、酵母エキス、肉エキス、アンモニウム塩、硝酸塩を使用することが出来る。炭素源の添加は必要ないが、シマジンを始めとするトリアジン系農薬が消費された後に菌体を維持するためにはＤＬ−リンゴ酸等の本分解菌が資化可能な炭素源を添加することが出来る。
本発明のトリアジン系農薬の分解方法におけるトリアジン系農薬としては、トリアジン環と共にハロゲン原子を含有するハロゲン含有トリアジン系農薬が挙げられる。当該ハロゲン含有トリアジン系農薬におけるハロゲン原子は、分子中のいずれに存在していてもよいが、好ましくはトリアジン環に直接結合するものが挙げられる。本発明のハロゲン含有トリアジン系農薬におけるハロゲンとしては、塩素原子、臭素原子、フッ素原子などが挙げられるが、塩素原子が好ましい。また、トリアジン環としては３個の窒素原子を有する６員環からなるものであれば単環式、多環式、又は縮合環式のいずれでもよいが、好ましくはトリアジン環を１個含有するトリアジン系農薬が挙げられる。好ましいハロゲン含有トリアジン系農薬としては、ｓ−トリアジン骨格を有するシマジンまたはアトラジンなどが挙げられる。
【００３８】
また、本発明は、前記してきた本発明のシマジン分解菌又は本発明の複合微生物系の中から選ばれる少なくとも１種を微生物保持体に保持させた浄化用資材を提供する。本発明の微生物保持体としては、本発明のシマジン分解菌又は本発明の複合微生物系を安定に保持することができるものであれば特に制限はないが、多孔質素材、更に具体的には当該細菌又は複合微生物系を集積させた多孔質の木質炭化材料、竹炭材料、綿布材料などが挙げられる。より具体的には特開平１０−２２５２８８号（特許第３０３０３７０号公報）又は特開平１１−３１８４３５号（特許第２９０４４３２号公報）に記載の多孔質材料が挙げられ、これらの公報に記載の内容を本明細書に全て取り込む。
本発明の浄化用資材の製法としては、まず培地、例えば1／10LB液体培地に多孔質資材、例えば炭、煉石あるいはバーミキュライトを入れて滅菌し、これに本発明で見出したシマジン分解菌を植菌して振とう培養し、培養終了後に培養液を除去することでシマジン分解菌を定着させた浄化用資材を得ることができる。
本発明の浄化用資材の大きさには特に制限はないが、１〜５０ｍｍ、好ましくは５〜２０ｍｍ、５〜１０ｍｍ程度の大きさが好ましい。本発明の浄化用資材は、このままトリアジン系農薬、好ましくはハロゲン含有トリアジン系農薬が残留している可能性のある土壌や用水などに混合して使用することができる。また、使用前に無機塩溶液で処理するか、無機塩溶液と共に使用することもできる。
本発明の浄化用資材は、図１に示される土壌層タンク１に単独で又は土壌と共に収納して汚染水などを浄化するための浄化装置とすることもできる。即ち、本発明は、前記した本発明の浄化用資材を含有してなるハロゲン含有トリアジン系農薬の浄化装置を提供する。
【００３９】
【実施例】
以下、本発明のシマジン分解菌及び本発明複合微生物系を構成する共存細菌の単離培養、分類同定、更に本発明分解菌及び本発明複合微生物系を用いたシマジンを始めとするトリアジン系農薬分解の実験例を示して、本発明を更に詳細かつ具体的に説明するが、本発明は以下の実施例に限定されるものではない。
【００４０】
実施例１ （単離培養）
図１に示すように、土壌層タンク１に集積用土壌層２を形成した。この集積用土壌層２は、５〜１０mm程度に砕片化した木質炭化材を人工マイクロハビタットとして用い、これを分解菌が生息する土壌に５重量％混入させて形成した。そしてシマジンのみを炭素源及び窒素源とする無機塩培地３を貯液タンク４から集積用土壌層２に還流させた。還流液中のシマジンの残留濃度やシマジンの分解による塩化物イオンの濃度を測定し、所定の集積レベルに達した集積開始３週間後に集積用土壌層２から人工マイクロハビタットを取り出した。そしてこの分解菌集積済の人工マイクロハビタットを別に用意した人工マイクロハビタットに接種、つまり混入して人工マイクロハビタットだけによる集積用層を形成し、この集積用層に上記と同様な還流を行なった。そしてこの操作を３回繰り返した後、人工マイクロハビタットを取り出し、生理食塩リン酸緩衝液（ＰＢＳ）で十分に洗浄後粉砕し、ＰＢＳで希釈後、シマジン５ｍｇ／Ｌを含む無機塩寒天培地平板に接種し、２５℃で７日間培養を行なった。生じたコロニーをシマジン５ｍｇ／Ｌを含む無機塩液体培地に接種し、２５℃で５日間培養を行なった後、シマジン分解の認められた培養液を再度シマジンを５ｍｇ／Ｌ含む無機塩寒天培地平板に接種し、シマジン分解コロニーＣＤ７Ｗを単離した。ＣＤ７Ｗをシマジン５ｍｇ／Ｌを含む０．１％バクトトリプトン寒天培地平板に接種し、同様に単コロニー分離を行なったところ形態の異なる３種のコロニーが得られた。それぞれを、ＣＤ７ＷＬ、ＣＤＢ２１、ＣＳＢ１と名付けた。
【００４１】
実施例２ （ＣＤ７ＷＬ、ＣＤＢ２１、及びＣＳＢ１のシマジン分解能の検定）シマジン約５ｍｇ／Ｌを含む１／１０Ｔ培地１０ｍｌを１００ｍｌの三角フラスコに入れ、これにシマジン分解コロニーから単離した各株を植菌して３０℃、１２０ｒｐｍで、４日間又は５日間振とう培養した後の培養液中のシマジン濃度を測定した。結果を前記表１及び表２に示す。
この結果、ＣＤＢ２１株のみが速やかなシマジン分解能を有することがわかった。
【００４２】
実施例３ （ＣＤＢ２１の菌学的性質）
実施例１にて単離したＣＤＢ２１の菌学的位置づけを行うために、形態的性質、培地における生育の様相および生理学的性質についてエヌシーアイエムビー・ジャパン社にて分析を行った。培養温度は特記しない限り３０℃で実施し、前記表３に示したとおりの形態、培養、生理学的性質が認められた。
上記の結果、本発明のＣＤＢ２１株はシュードモナス群(Pseudomonas group)に属する菌株と推定された。
【００４３】
さらに、実施例１で得られたＣＤＢ２１の菌学的性状から分類同定することは困難と考え、ＣＤＢ２１の菌学的位置づけを遺伝子工学的側面から特徴付けるために、１６Ｓ−ｒＲＮＡ遺伝子の塩基配列の決定（解析）を行った。
（１）実施例１で得られたＣＤＢ２１の菌体より、DNeasy Tissue Kit (株式会社キアゲン製)を用いてＤＮＡを調製した。
（２）上記、ＤＮＡを鋳型ＤＮＡとして、公知のユーバクテリア１６Ｓ−ｒＲＮＡ増幅用プライマーを用いてＰＣＲによりＣＤＢ２１の１６Ｓ−ｒＲＮＡ遺伝子断片を増幅し、QIAquick PCR Purification Kit(株式会社キアゲン製)を用いてＤＮＡを精製した。
（３）上記精製ＤＮＡを鋳型ＤＮＡとして蛍光ラベルした公知のユーバクテリア１６Ｓ−ｒＲＮＡ塩基配列解析用プライマーを用いてサイクルシーケンス反応を行い、日立ＤＮＡシーケンサＳＱ５５００Ｅにより塩基配列の決定（解析）を行った。
【００４４】
以上により得られたＣＤＢ２１の１６Ｓ−ｒＲＮＡ遺伝子断片の塩基配列より、両端のＰＣＲ用プライマー配列を除いた１４８８塩基を配列番号１に示す。
近隣結合法（NJ法）により近縁株と系統樹を作成し、類縁関係を解析したところ、ベータプロテオバクテリアの範疇に属することが明らかとなった。以上より、本発明者らは本発明のＣＤＢ２１株をこれまで単離培養されたことのない、いかなる既知の属種にも帰属しない新規なベータプロテオバクテリアに属するＣＤＢ２１株と同定し、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ ＢＰ−１０４０３として寄託した。
【００４５】
実施例４ （ＣＳＢ１の菌学的性質）
実施例１にて単離したＣＳＢ１の菌学的位置づけを行うために、形態的性質、培地における生育の様相および生理学的性質についてエヌシーアイエムビー・ジャパン社にて分析を行った。培養温度は特記しない限り３０℃で実施し、前記表４に示したとおりの形態、培養、生理学的性質が認められた。
以上の結果から、本発明のＣＳＢ１株はシュードモナス群(Pseudomonas group)に属する菌株と推定された。
【００４６】
実施例３と同様にしてＣＳＢ１について得られた１６Ｓ−ｒＲＮＡ遺伝子断片の塩基配列より、両端のＰＣＲ用プライマー配列を除いた１４６２塩基を配列番号２に示す。
さらに、実施例３と同様に、近隣結合法（NJ法）により系統樹を作成し、比較したところ、ＣＳＢ１はブラディリゾビウム ジャポニカムの範疇に属する事が明らかとなった。以上より、発明者らは本菌株をブラディリゾビウム ジャポニカム ＣＳＢ１株と同定し、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ ＢＰ−１０４０２として寄託した。
【００４７】
実施例５ （ＣＤＢ２１株によるシマジンの分解培養）
精製水１リットルあたりバクトトリプトン１ｇを溶解し、メタノール１ｍｌに懸濁したシマジン５ｍｇを添加した後、１２１℃、２０分間蒸気滅菌した。本培地５０ｍｌを滅菌した５００ｍｌの三角フラスコに分注し、１白金耳のＣＤＢ２１株を接種した。３０℃、１２０ｒｐｍにて振盪培養を行い、菌体の生育、シマジンの分解とそれに伴う塩素イオンの発生を測定した。結果を図２に示す。
【００４８】
実施例６ （ＣＤＢ２１株との複合微生物系の作製）
精製水１リットルあたりＮａ_２ＨＰＯ_４・１２Ｈ_２Ｏ １．２ｇ、ＫＨ_２ＰＯ_４ ０．５ｇを溶解し、メタノール１ｍｌに懸濁したシマジン５ｍｇを添加し、寒天(AGAR NOBLE , DIFCO)１．５ｇを加えた後、１２１℃、２０分間蒸気滅菌した。滅菌後、微量要素液１０ｍｌを添加してシマジン含有無機塩寒天培地を作製し平板とした。微量要素液は精製水１リットルあたりＥＤＴＡ ０．５ｇ、ＭｇＳＯ_４・７Ｈ_２Ｏ ２ｇ、ＦｅＳＯ_４・７Ｈ_２Ｏ ０．２ｇ、ＺｎＳＯ_４・７Ｈ_２Ｏ １０ｍｇ、ＭｎＳＯ_４・Ｈ_２Ｏ ５ｍｇ、Ｈ_３ＢＯ_３ ３０ｍｇ、ＣｏＳＯ_４・７Ｈ_２Ｏ ２４ｍｇ、ＣｕＳＯ_４・７Ｈ_２Ｏ ５ｍｇ、Ｎａ_２ＭｏＯ_４ ５ｍｇ、Ｃａ（ＯＨ）_２ ５０ｍｇを溶解した後、１２１℃、２０分間蒸気滅菌して調製した。
１白金耳のＣＤＢ２１株菌体を平板の半分に塗抹し、残りの半分に他の菌株を塗抹した後に中央で両菌株を混合し、３０℃で７日間培養した。それぞれ単独で塗抹した部分には生育せず、中央で混合した部分にコロニーが生じた組み合せを複合微生物系とし、１白金耳をシマジン含有無機塩寒天培地から寒天を除いたシマジン含有無機塩液体培地１０ｍｌに接種し、１００ｍｌの三角フラスコ中で３０℃、１２０ｒｐｍにて４日間振盪培養を行い、シマジンの分解を測定した。結果を前記した表５に示す。検討した菌株のうち５種類との組み合せで、ＣＤＢ２１複合微生物系の構築が可能であり、いずれも無機塩培地中でシマジンを分解した。
【００４９】
実施例７ （ＣＤＢ２１複合微生物系によるシマジン分解培養）
実施例６記載のシマジン含有無機塩液体培地５０ｍｌを滅菌した５００ｍｌ三角フラスコに分注し、１白金耳のＣＤＢ２１株あるいはＣＳＢ１株あるいはＣＤＢ２１株とＣＳＢ１株より構成される複合微生物系を接種した。３０℃、１２０ｒｐｍにて振盪培養を行い、菌体の生育、シマジンの分解とそれに伴う塩素イオンの発生を測定した。結果を図３に示す。
【００５０】
実施例８（ＣＤＢ２１複合微生物系によるトリアジン環の分解）
実施例６記載のシマジン含有無機塩培地にトリアジン環の炭素原子を^１４Ｃで標識したシマジン（simazine-ring-UL-^１４Ｃ）を添加した。本培地中には^１４Ｃ標識シマジン０．７５ｍｇ／Ｌ、非標識シマジン３．４ｍｇ／Ｌが含まれる。これにＣＤＢ２１株とＣＳＢ１株を含む複合微生物系を接種し、水酸化ナトリウムトラップを通過させた空気を２ｍｌ／分で連続的に通気させながら２５℃暗所にて８日間静置培養した。培養期間中、培養液中の^１４Ｃ標識シマジン及びその代謝物濃度をラジオアナライザー付高速液体クロマトグラフで、捕集した^１４Ｃ標識二酸化炭素を液体シンチレーションカウンターで定量した。結果を図４に示す。図４の黒丸印（●）は^１４Ｃ標識シマジン濃度（ｍｇ／Ｌ）を、黒四角印（■）は^１４Ｃ標識シアヌール酸濃度（ｍｇ／Ｌ）を、黒三角（▲）は発生した^１４Ｃ標識二酸化炭素の理論量に対する割合（％）を示す。図４の縦軸は左側が^１４Ｃ標識シマジン及び^１４Ｃ標識シアヌール酸の濃度（ｍｇ／Ｌ）を、右側が発生した^１４Ｃ標識二酸化炭素量の理論量に対する割合（％）を示す。横軸は培養期間（日）を示す。
この結果、シマジンは培養６日目に大部分が分解し、８日目には完全に分解していた。シマジンの分解に伴い分解中間代謝産物であるシアヌール酸濃度が上昇したが、大量に蓄積する様子はなく、完全分解物である^１４Ｃ標識二酸化炭素の発生が認められた。このことは本発明のＣＤＢ２１株や、ＣＤＢ２１株とＣＳＢ１株を含む複合微生物系によるシマジン分解は、トリアジン環も完全に分解し、シマジンを無害化することができることを示している。
【００５１】
実施例９（ＣＤＢ２１複合微生物系によるシマジン分解の安定性）
実施例６記載のシマジン含有無機塩液体培地２００ｍｌと木炭をAudusの還流装置（1950, Nature, 166, 356）の所定の位置に入れ、ＣＤＢ２１株とＣＳＢ１株を含む複合微生物系を接種した後、本液体培地を還流させた。７日目に還流液を新規なものと交換した後に、木炭上に水田土壌あるいはバーク堆肥を重層し、さらに還流を計５８日間継続した。５、９、１２、１６，１９、２７、３３、４０、４７、５４日目には５０００ｍｇ／Lに調製したシマジンのアセトン溶液を適量添加した。また７、１４、２２、３０、３７、４４及び５１日目に還流液そのものを新規なものと交換した。還流期間中、還流液を適宜採取してそのシマジン濃度を高速液体クロマトグラフ法にて測定した。結果を図５に示す。図５の黒四角印（■）は７日目に水田土壌を重層した場合の還流液中シマジン濃度（ｍｇ／L）を、黒丸印（●）７日目にバーク堆肥を重層した場合の還流液中シマジン濃度（ｍｇ／L）を示す。図５の縦軸はシマジン濃度（ｍｇ／L）を、横軸は培養期間（日）を示す。
この結果、ＣＤＢ２１株とＣＳＢ１株を含む複合微生物系は、木炭の存在下で速やかにシマジンを分解した。この複合微生物系のシマジン分解能は、木炭の上部に多様な微生物を含む水田土壌やバーク堆肥を重層した後であってもその影響を全く受けなかった。以上の結果より、本発明のＣＤＢ２１株や、ＣＤＢ２１株とＣＳＢ１株を含む複合微生物系によるシマジン分解は、一旦それが形成されれば他の微生物の影響を受けにくく、非常に安定であることを示している。
【００５２】
実施例１０ （ＣＤＢ２１複合微生物系によるトリアジン系除草剤の分解）
実施例６記載のシマジン含有無機塩培地にシマジンの代りに各種トリアジン系農薬５ｍｇ／Ｌを添加し、１０ｍｌの培地に１白金耳のＣＤＢ２１株とＣＳＢ１株より構成される複合微生物系を接種した。１００ｍｌ三角フラスコ中で３０℃、１２０ｒｐｍにて振盪培養を行い、シマジンの分解を測定した。結果を前記した表６に示す。２位が塩素化されたシマジンとアトラジンは３日以内に速やかに分解されたが、２位がチオメチル化されたシメトリン、プロメトリン、ジメタメトリンはこの培養条件では分解されなかった。
【００５３】
【発明の効果】
本発明の分解菌によりトリアジン系農薬、特にシマジンを分解する事ができる。更に本発明の複合微生物系を用いる事により広範囲な培養条件下でより速やかに完全に当該農薬を分解することが可能であり、環境に残留することにより、環境汚染の原因となる農薬の分解法が提供される。
【００５４】
【配列表】

【図面の簡単な説明】
【図１】図１は、実施例１の集積方法で用いる装置の模式図である。
【図２】図２は、実施例５におけるＣＤＢ２１株によるシマジンの分解を示すグラフ図である。
【図３】図３は、実施例７におけるＣＤＢ２１株とＣＳＢ１株より構成される複合微生物系によるシマジンの分解及びＣＤＢ２１株、ＣＳＢ１株との比較を示すグラフ図である。
【図４】図４は、実験例８における炭素１４標識シマジンの分解を示すグラフ図である。
【図５】図５は、実験例９におけるシマジンの分解を示すグラフ図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a triazine-based pesticide, more specifically a method for degrading a halogen-containing triazine-based pesticide and a triazine-based pesticide resolution, more specifically a halogen-containing triazine-based pesticide resolution, and more particularly a novel microorganism having a simazine-degrading ability The present invention relates to a complex microbial system containing microorganisms, a purification material holding them, and a purification device containing the same.
[0002]
[Prior art]
Agricultural chemicals are indispensable for maintaining today's agricultural production, and agricultural chemicals are often used to maintain the flora in the field at golf courses. On the other hand, pesticides are becoming a pollutant and have an unfavorable impact on the environment, and in particular, they are a concern for groundwater contamination. This is not a major problem in Japan, where the use of groundwater as drinking water is decreasing, but it is a major problem in Europe and the United States, where about 70% of drinking water is dependent on groundwater. In addition, there is a problem of soil contamination due to residual pesticides. In Japan, organochlorine insecticides with high soil persistence (half-life of 1 year or more) are prohibited, but even in pesticides with relatively short half-lives. There is a concern about the problem of secondary contamination by degradation substances (metabolites, etc.) in the soil. Furthermore, in recent years, it has been reported that even if these pollutants are in very small amounts (about several tens of ppt), they have a great influence on the reproductive organs of organisms, and it is desired to completely detoxify the remaining agricultural chemicals. ing.
For this reason, it is desirable to develop a technology that effectively prevents pesticides that have completed their functions from becoming contaminants and remaining in the environment or spreading.
[0003]
Triazines such as simazine (2-chloro-4,6-bis (ethylamino) -1,3,5-triazine) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) Agricultural chemicals have been used extensively all over the world for many years and have become a problem as environmental pollutants. Simazine has been widely used for weed management in golf courses, etc., but its usage has decreased since it was designated as a water-polluting pesticide in 1994. However, simazine is one of the most frequently detected pesticides in groundwater and wastewater due to its long half-life (70-110 days) and low soil adsorption coefficient. In Europe and the United States, contamination of groundwater by atrazine has become an environmental problem. All of these pesticides contain chlorine and nitrogen atoms in the molecule and are hardly decomposable.
As a method for removing these pesticides, a method of supplementing triazine pesticides such as simazine and atrazine using a receptor polymer synthesized by a template polymerization method such as bulk polymerization or suspension polymerization (see Patent Document 1), proton free radical There has been reported a method of decomposing a triazine compound using a polymer synthesized by a template polymerization method using a triazine compound as a template in the presence of a prepolymer (see Patent Document 2). However, contamination by residual agricultural chemicals is extremely difficult to remove completely by these physical and chemical methods because the contamination range is wide and the contamination concentration is extremely low, and the chemical substances used Therefore, there is a possibility of secondary contamination, and a new method is required.
[0004]
Biological techniques have been attracting attention in place of physical and chemical techniques against a wide range of low-concentration chemical substances.
A wide variety of microorganisms inhabit the soil, and in these microorganisms, organic compounds that are the functional skeleton in many pesticides and the like are decomposed and made harmless, or lost from the environment. There are degrading bacteria that have the ability to It is possible to remove pollutants such as agricultural chemicals from the environment by utilizing the ability of such degrading bacteria. However, in its natural state, the density of degrading bacteria capable of degrading specific organic compounds is low, and it does not effectively prevent pollutants from remaining or spreading in the environment. It is normal.
[0005]
For example, atrazine, a triazine pesticide, is known to degrade many microorganisms, but many of them are unable to completely degrade atrazine alone, and secondary by incomplete degradation products. Concern about environmental pollution. For this reason, a method for completely degrading atrazine by combining a plurality of microorganisms has been attempted. On the other hand, simazine, which is also a triazine pesticide, is more persistent and is a concern as a more serious environmental pollutant. Many atrazine-degrading bacteria degrade simazine, but there is no report that completely decomposes the triazine ring of simazine, and secondary contamination by incomplete degradation products may occur. Thus, it has been desired to find a degrading bacterium or a combination of degrading bacteria capable of completely degrading triazine pesticides, particularly chlorinated triazine pesticides, specifically simazine and atrazine. In particular, regarding simazine, it has been desired to find a decomposing bacterium or a combination of degrading bacteria that leads to complete decomposition without worrying about secondary environmental pollution.
Therefore, it is considered effective to selectively accumulate and isolate such degrading bacteria from the soil and then to decompose organic pollutants such as agricultural chemicals alone or in combination. The present inventors have developed a method for accumulating degrading bacteria that degrade organic pollutants in soil using a porous wood carbonized material (see Patent Documents 3 and 4).
[0006]
[Patent Document 1]
JP-A-10-239293
[Patent Document 2]
JP 2001-79418 A
[Patent Document 3]
JP-A-10-225288 (Patent No. 3030370)
[Patent Document 4]
JP-A-11-318435 (Patent No. 2904432)
[0007]
[Problems to be solved by the invention]
The present invention provides a novel bacterium capable of degrading simazine and a complex microbial system combining the bacterium with other microorganisms, and a method for degrading triazine pesticides using these bacterium or complex microbial system, in particular simazine. It is an object of the present invention to provide a complete decomposition method.
[0008]
[Means for Solving the Problems]
The present inventors refluxed an inorganic salt medium containing a pesticide such as simazine as a sole carbon source and nitrogen source using a porous wood carbonized material, and the rate of disappearance of the pesticide increased. Group CD7W was isolated. And it succeeded in isolating a novel simazine degrading bacterium from this degrading bacterium group CD7W.
[0009]
  As a means for solving the above problems, the present invention provides a simazine-degrading bacterium that can be isolated after culturing simazine from soil, a complex microbial system containing the same, and a halogen-containing triazine-based pesticide using the same. A method for decomposing, an apparatus therefor, and the like are provided. The simazine-degrading bacterium of the present invention is a novel bacterium belonging to beta-proteobacteria, and specifically has a 16S ribosomal RNA (16S-rRNA) gene containing the nucleotide sequence set forth in SEQ ID NO: 1. Strain CDB21 belonging to(FERM BP-10403)Etc.
[0010]
That is, the present invention relates to a method for degrading halogen-containing triazine pesticides, characterized in that bacteria belonging to beta-proteobacteria are used alone or in combination with one or more symbiotic bacteria.
The present invention also relates to a bacterium belonging to betaproteobacteria having simazine-degrading ability, that is, a simazine-degrading bacterium.
Furthermore, the present invention relates to a complex microbial system comprising at least one simazine-degrading bacterium composed of bacteria belonging to beta-proteobacteria and at least one symbiotic bacterium having an ability to assist the decomposition and / or growth of simazine. About.
The present invention also provides a purification material in which at least one selected from among the microorganisms comprising the simazine-degrading bacterium of the present invention or a complex microorganism system is retained in a microorganism carrier, and a halogen-containing composition comprising the same. The present invention relates to a purification apparatus for triazine-based pesticides. The present invention also relates to a bacterium belonging to Bradyrizobium japonica, which has the ability to assist the decomposition and / or growth of simazine, a simazine-degrading bacterium composed of bacteria belonging to betaproteobacteria.
[0011]
In order to search for simazine-degrading bacteria, the present inventors refluxed an inorganic salt medium containing a pesticide such as simazine as a sole carbon source and nitrogen source using a porous wood carbonized material (see Patent Documents 3 and 4). Thus, the simazine-degrading bacteria group CD7W was separated from the increase in the disappearance rate of the pesticide. Specifically, the integrated device shown in FIG. 1 was used. A soil layer 2 for accumulation is formed in the soil layer tank 1 of the accumulation device, and 5% by weight of artificial microhabitat which is a wood carbonized material fragmented to about 5 to 10 mm is inhabited in this soil layer 2 for accumulation. Then, it was filled with the soil mixed in the soil that seems to be. Then, an inorganic salt medium 3 containing only simazine as a carbon source and a nitrogen source was refluxed from the storage tank 4 to the accumulation soil layer 2. The residual concentration of simazine in the reflux liquid and the concentration of chloride ions due to decomposition of simazine were measured, the collecting device that reached a predetermined accumulation level was recovered, and the artificial microhabitat was taken out from the accumulation soil layer 2.
In this accumulator, there is a high possibility that bacteria using simazine as the only carbon source and nitrogen source are accumulated, and the artificial microhabitat of the soil layer 2 for accumulation is an environment in which bacteria are liable to live. Therefore, the possibility that the target simazine-degrading bacteria are accumulated in the artificial microhabitat should be high. Therefore, an attempt was made to form a colony of simazine-degrading bacteria from this artificial microhabitat. As a result, we succeeded in isolating simazine-degrading colony CD7W from an inorganic salt agar plate containing 5 mg / L simazine.
[0012]
In order to isolate a simazine-degrading bacterium from this simazine-degrading colony CD7W, this was inoculated on a 0.1% bactotryptone agar plate containing simazine 5 mg / L, and single colonies were similarly separated. As a result, three types of colonies with different forms were obtained. Each was named CD7WL, CDB21, and CSB1.
These strains CD7WL or CDB21 were inoculated into a medium containing about 5 mg / L of simazine, shaken at 30 ° C. and 120 rpm for 4 days, and cultured, the concentration of simazine in the culture solution was as shown in Table 1 below. It was. In addition, the thing which does not inoculate a strain was used as a control.
[0013]

Similarly, the resolution of simazine was examined using strain CSB1 or CDB21. The results are shown in Table 2 below.
[0014]

[0015]
As a result, unlike the CDB21 strain, which showed rapid simazine degradation, the simazine concentration in the CD7WL strain and CSB1 strain was hardly different from the control. From this, it was concluded that the CD7WL strain and CSB1 strain do not have simazine resolution.
However, when CD7WL, CDB21, and CSB1 were all inoculated alone on an inorganic salt agar plate containing 5 mg / L of simazine, no clear colonies were formed. Therefore, when these microorganisms were combined and inoculated into the same inorganic salt medium, colonies appeared only in the combination of CDB21 and CSB1. It was also found that the obtained composite microbial system composed of CDB21 and CSB1 can be maintained for subculture by inoculating on an inorganic salt agar medium containing simazine 5 mg / L.
That is, isolated CDB21 can degrade simazine alone, but it was found that a complex microbial system consisting of CDB21 and CSB1 is preferable when subcultured.
[0016]
Next, in order to perform mycological positioning of the isolated CDB21, morphological properties, aspects of growth in the medium, and physiological properties were examined. The culture temperature was 30 ° C. unless otherwise specified, and the morphology, culture, and physiological properties shown in Table 3 below were observed.
[0017]

[0018]
When the above results were identified according to the 9th edition of the Bergey's Manual of Determinative Bacteriology, the CDB21 strain of the present invention was estimated to be a strain belonging to the Pseudomonas group.
[0019]
Similarly, in order to perform mycological positioning of isolated CSB1, morphological properties, growth aspects in culture medium and physiological properties were examined. The culture temperature was 30 ° C. unless otherwise specified, and the form, culture and physiological properties shown in Table 4 below were observed.
[0020]

[0021]
When the above results were identified according to the 9th edition of Bergey's Manual of Determinative Bacteriology, the CSB1 strain of the present invention was presumed to belong to the Pseudomonas group.
[0022]
Bacteria belonging to the Pseudomonas group are known to be a group of bacteria including genetically diverse bacteria, and it is considered difficult to identify the classification only from the mycological properties of CDB21 and CSB1 of the present invention. In order to characterize the mycological positioning of CSB1 from the viewpoint of genetic engineering, the base sequence of 16S-rRNA gene was determined (analyzed).
A cDNA is prepared using the cells of CDB21 and CSB1 of the present invention, and the 16S-rRNA gene of each of CDB21 and CSB1 is obtained by PCR using this cDNA as a template DNA and a known eubacterium 16S-rRNA amplification primer. The fragment was amplified and the nucleotide sequence was determined (analyzed).
1488 bases obtained by removing the PCR primer sequences at both ends from the base sequence of the 16S-rRNA gene fragment of CDB21 obtained as described above are shown in SEQ ID NO: 1 in the sequence listing. Furthermore, 1462 bases obtained by removing the PCR primer sequences at both ends from the base sequence of the 16S-rRNA gene fragment obtained for CSB1 are shown in SEQ ID NO: 2 in the sequence listing.
[0023]
  The 16S-rRNA gene possessed by another microorganism registered in the DNA base sequence database (GenBank) using Genetics PDB (GENETYX PDB) for the base sequence of the 16S-rRNA gene of CDB21 shown in SEQ ID NO: 1 Homology comparison was performed by sequence and FASTA method.
  As a result, CDB21 showed 99.9% homology (1369/1370, INT.Score = 5474, OPT.Score = 5474) with uncultured bacterial clone I12 (ACCESSION: AY187895). . Other than this, many uncultured beta proteobacterium, unidentified beta proteobacterium, Thiobacillus, Burkholderia, Ralstonia and more than 90% The homology of was obtained.
  A close tree and a phylogenetic tree were created by the neighbor-joining method (NJ method), and the relationship was analyzed, and it was revealed that it belongs to the category of betaproteobacteria. From the above, the present inventors have identified the strain CDB21 of the present invention as a CDB21 strain belonging to a novel betaproteobacterium that has never been isolated and cultured and does not belong to any known genus species. Accession number to Patent Organism Depositary, National Institute of Advanced Industrial Science and TechnologyFERM BP-10403As deposited.
[0024]
  Similarly, 16S- possessed by other microorganisms registered in the DNA base sequence database (GenBank) using Genetics PDB (GENETYX PDB) for the base sequence of the 16S-rRNA gene fragment of CDB21 shown in SEQ ID NO: 2 Homology comparison was performed by rSTA gene sequence and FASTA method. CSB1 showed 99.6% homology (1456/1462, INT.Score = 5812, OPT.Score = 5812) with Bradyrhizobium japonicum (Bradyrhizobium japonicum strain USDA 62, ACCESSION: AF208517). It showed 99% or more homology with other Bradyrizobium japonicam 16S-rRNA gene base sequences registered in the DNA base sequence database.
  A phylogenetic tree was created and compared by the neighbor join method (NJ method), and it was found that CSB1 belongs to the category of Bradyrizobium japonicam. Based on the above, the present inventors have identified the strain CSB1 of the present invention as Bradyrizobium japonicum CSB1 strain, and the accession number to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.FERM BP-10402As deposited.
[0025]
Next, the degradation of simazine and the fungal activity by the CDB21 strain of the present invention were examined in more detail. CDB21 strain is inoculated into a medium supplemented with 1 g / L of bactotryptone and 5 mg / L of simazine, followed by shaking culture at 30 ° C. and 120 rpm to grow the cells, decompose simazine and generate chloride ions associated therewith. Was measured. The results are shown in FIG. The white circle mark (◯) in FIG. 2 indicates the residual ratio (%) of simazine, and the white square mark (□) indicates the concentration (mg / L) of the generated chloride ion. The vertical axis of the graph of FIG. 2 shows the residual rate (%) of simazine on the left side, and the concentration (mg / L) of generated chlorine ions on the right side. The horizontal axis indicates the culture time (days).
Simazine was almost completely degraded on the third day after the start of the culture and almost completely degraded on the fourth day. Moreover, the production | generation of the chloride ion commensurate with the amount of simazine degradation was observed, and the growth of the cells was observed. As described above, the CDB21 strain of the present invention can almost completely degrade simazine in about 3 to 4 days, and depending on the culture conditions, even when the CDB21 strain alone is used, the microbial cells grow and degrade simazine. be able to.
[0026]
Next, degradation of simazine by a complex microbial system with CDB21 strain was examined.
Na₂HPO₄・ 12H₂O 1.2g / L, KH₂PO₄The CDB21 strain was inoculated on a plate medium containing 0.5 g / L of simazine and 5 mg / L of simazine, and then the other strains were inoculated, and then both strains were mixed at the center and cultured at 30 ° C. for 7 days. A combination that did not grow on the part inoculated alone but had colonies in the part mixed in the central part of the incubator was defined as a complex microorganism system. Thus, the complex microbial system obtained from the colony which formed the complex microbial system was inoculated into the simazine-containing inorganic salt liquid medium, and cultured with shaking at 30 ° C. and 120 rpm for 4 days, and the degradation of simazine was measured. The results are shown in Table 5. In combination with five of the examined strains, it was possible to construct a CDB21 complex microbial system, and all decomposed simazine in an inorganic salt medium.
[0027]
[Table 5]

[0028]
Next, degradation of simazine by the CDB21 strain or CSB1 strain alone, or a complex microbial system with CDB21 and CSB1 strain was examined.
Simazine-containing inorganic salt liquid medium was inoculated with CDB21 strain alone, CSB1 strain alone, or a complex microbial system composed of CDB21 strain and CSB1 strain. Shaking culture was performed at 30 ° C. and 120 rpm, and the growth of bacterial cells, the decomposition of simazine, and the generation of chloride ions associated therewith were measured. The results are shown in FIG. 3A shows the residual ratio (%) of simazine, and FIG. 3B shows the concentration (mg / L) of the generated chlorine ions. The vertical axis of the graph in FIG. 3A shows the residual rate (%) of simazine, and FIG. 3B shows the concentration (mg / L) of the generated chlorine ions. The horizontal axis shows the culture time (days). In FIG. 3, black circles (●) indicate the case of inoculation with CDB21 strain alone, black triangles (▲) indicate the case of inoculation with CSB1 strain alone, and black squares (■) indicate the CDB21 strain and CSB1. The case where a complex microbial system composed of strains is inoculated is shown.
As a result, in the CDB21 strain, simazine was almost decomposed on the 7th day after the start of the culture, and generation of chloride ions corresponding to the amount of simazine was observed, but in this case, the growth of the cells was hardly observed. . In the CSB1 strain, none of simazine degradation, chloride ion production, or cell growth was observed during the culture period. In the complex microbial system consisting of the CDB21 strain and the CSB1 strain, simazine was rapidly degraded within one day after the start of culture, and completely degraded on the second day. In addition, generation of chloride ions commensurate with the amount of simazine degradation was observed, and rapid growth of complex microbial cells was observed.
[0029]
Next, it was examined whether or not the triazine ring was cleaved and mineralized by decomposition of simazine by the CDB21 complex microorganism system of the present invention. First, the carbon atom of the triazine ring was added to the simazine-containing inorganic salt medium.¹⁴Simazine-ring-UL- labeled with C¹⁴C) was added. In this medium¹⁴C-labeled simazine 0.75 mg / L and unlabeled simazine 3.4 mg / L are included. This was inoculated with a complex microbial system containing CDB21 strain and CSB1 strain, and statically cultured in a dark place at 25 ° C. for 8 days with continuous air aeration through a sodium hydroxide trap at 2 ml / min. During the culture period, in the culture solution¹⁴C-labeled simazine and its metabolite concentrations were collected using a high-performance liquid chromatograph with a radio analyzer.¹⁴C-labeled carbon dioxide was quantified with a liquid scintillation counter. The results are shown in FIG. The black circle (●) in Fig. 4¹⁴C-labeled simazine concentration (mg / L), black square mark (■)¹⁴C-labeled cyanuric acid concentration (mg / L), black triangle (▲) generated¹⁴The ratio (%) to the theoretical amount of C-labeled carbon dioxide is shown. The left side of the vertical axis in FIG.¹⁴C-labeled simazine and¹⁴Concentration of C-labeled cyanuric acid (mg / L) occurred on the right side¹⁴The ratio (%) of the amount of C-labeled carbon dioxide to the theoretical amount is shown. The horizontal axis indicates the culture period (days).
As a result, most of simazine was degraded on the 6th day of culture and completely degraded on the 8th day. The concentration of cyanuric acid, a degradation intermediate metabolite, increased with the degradation of simazine, but it does not accumulate in large quantities and is a complete degradation product¹⁴Generation of C-labeled carbon dioxide was observed. This indicates that simazine degradation by the microbial strain including the CDB21 strain of the present invention or the CDB21 strain and the CSB1 strain can also completely degrade the triazine ring and detoxify simazine.
[0030]
Next, degradation of various triazine herbicides by the CDB21 complex microbial system was examined. Instead of simazine, 5 mg / L of various triazine pesticides were added to the simazine-containing inorganic salt medium, and a complex microbial system composed of CDB21 strain and CSB1 strain was inoculated. Shaking culture was performed at 30 ° C. and 120 rpm, and the degradation of these herbicides was measured. The results are shown in Table 6 below.
[0031]
[Table 6]

[0032]
As a result, simazine and atrazine, which were chlorinated at the 2-position of the triazine ring, were rapidly degraded within 3 days, but methimetriline, promethrin, and dimetamethrin that were thiomethylated at the 2-position without chlorine were degraded under these culture conditions. Was not.
[0033]
As described above, the present invention is a microorganism capable of efficiently degrading and detoxifying halogen-containing triazine herbicides such as simazine and atrazine, a method for degrading halogen-containing triazine herbicides using the same, and the method for the same A microorganism-containing composition is provided. In particular, the present invention reports for the first time that there is a single strain capable of efficiently degrading simazine by biological techniques, and is capable of degrading, preferably almost completely detoxifying simazine. A microbial system is provided.
The simazine-degrading bacterium CDB21 strain of the present invention is isolated after accumulation culture for simazine from soil, but is not limited thereto, and is a bacterium belonging to beta-proteobacteria having simazine resolution. In addition, a bacterium having some or all of the mycological characteristics of the aforementioned betaproteobacterium strain CDB21 is preferably included.
The simazine-degrading bacterium of the present invention is also characterized by the base sequence of the 16S-rRNA gene. That is, in the simazine-degrading bacterium of the present invention, the base sequence of the 16S-rRNA gene is the base sequence described in SEQ ID NO: 1 in the sequence listing, or homology with the base sequence, preferably 50% or more, 70% or more, More preferably, it is a bacterium having a base sequence having a homology of 80% or more, or 90% or more, and having a resolution of simazine, preferably further part of the mycological characteristics of the above-mentioned beta-proteobacterium strain CDB21 or Includes everything.
[0034]
  As a specific example of the simazine-degrading bacterium of the present invention, the indication of microorganisms (indication for identification given by the depositor) “CDB21”, the accession number “FERM BP-10403The novel betaproteobacteria deposited as "" can be mentioned, but is not limited thereto.
[0035]
Furthermore, the present invention provides a complex microbial system constituted by a combination of the above-described simazine-degrading bacterium of the present invention and a microorganism having an ability to assist simazine decomposition and / or growth of the lytic bacterium. The complex microorganism system of the present invention contains one or more simazine-degrading bacteria comprising bacteria belonging to beta-proteobacteria and one or more symbiotic bacteria having the ability to assist the decomposition and / or growth of simazine. A complex microbial system may be mentioned. The complex microbial system of the present invention has the resolution of a triazine pesticide, preferably a halogen-containing triazine pesticide.
A preferred complex microbial system of the present invention includes a complex microbial system in which the simazine-degrading bacterium is the beta proteobacteria strain CDB21.
Examples of the symbiotic fungi that can constitute such a complex microbial system with the simazine-degrading bacterium of the present invention include Bradyrizobium japonicam, Rhodococcus rhodochrous, Stenotrophomonas maltophilia, Nocardioides jenseni, Nocardioides fulvus, Nocardioides simplex, Pseudomonas aeruginosa and the like, but if it is a microorganism having the ability to assist simazine degradation and / or growth of the simazine-degrading bacterium of the present invention, It is not limited to the microorganisms exemplified above.
Whether or not such a complex microbial system can be configured is whether or not it can form colonies after culturing by inoculating the simazine-degrading bacterium of the present invention with another bacterium in an agar medium containing simazine. Can be determined. The combination of bacteria capable of forming a colony can constitute the complex microorganism system of the present invention.
[0036]
  As a preferable example of a microorganism capable of constructing a complex microorganism system together with the simazine-degrading bacterium of the present invention, the microorganism display (indication for identification given by the depositor) at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. ) "CSB1", Trust number"FERM BP-10402”Can be mentioned as new bacteria.
  Bradyrizobium Bacteria belonging to Japonicum and having the ability to assist the degradation and / or growth of simazine of the simazine-degrading bacterium of the present invention, preferably further including bacteria having part or all of the above-mentioned mycological characteristics It is. Furthermore, the base sequence of the 16S-rRNA gene is the base sequence described in SEQ ID NO: 2 in the sequence listing, or homology with the base sequence, preferably 50% or more, 70% or more, more preferably 80% or more, or A bacterium having a base sequence having a homology of 90% or more, and having the ability to assist the degradation and / or growth of simazine of the simazine-degrading bacterium of the present invention, preferably further part of the above-mentioned mycological characteristics Or the whole thing is included.
[0037]
The present invention also relates to a bacterium belonging to Bradyrizobium japonica, which has the ability to assist the decomposition and / or growth of simazine, a simazine-degrading bacterium comprising bacteria belonging to betaproteobacteria, preferably Provided is a bacterium having some or all of the mycological characteristics of Bradyrizobium japonicam CSB1 strain. Examples of the bacterium belonging to Bradyrizobium japonicum of the present invention include the aforementioned Bradyrizobium japonicum CSB1 strain.
Furthermore, the present invention is characterized by using a complex microbial system which is a bacterium belonging to betaproteobacterium and having simazine-degrading ability alone or in combination with one or more symbiotic bacteria. A method for decomposing a triazine pesticide, preferably a halogen-containing triazine pesticide is provided. Examples of the bacterium belonging to the betaproteobacterium and having simazine-degrading ability include the simazine-degrading bacterium described above and a bacterium selected from the mutant strains thereof. Examples of symbiotic bacteria combined with such simazine-degrading bacteria include Bradyrizobium japonicam CSB1, Rhodococcus rhodochrous, Stenotrophomonas maltophilia, Nocardioides jenceni, Nocardioides fulvus, Nocardioides simplex, and Pseudomonas Bacteria selected from aeruginosa and the like are preferred, but the simazine-degrading bacteria of the present invention and microorganisms having the ability to assist simazine degradation and / or growth of the degrading bacteria are not limited to the microorganisms exemplified above. Absent. As a preferable symbiotic fungus, for example, Bradyrizobium japonicum CSB1 strain can be mentioned. Moreover, as a preferable complex microbial system, a complex microbial system composed of the beta proteobacterium CDB21 strain and the Bradyrizobium japonicam CSB1 strain can be mentioned.
As a medium used in the decomposition culture, an inorganic salt medium containing a triazine pesticide such as simazine can be used. Furthermore, a nitrogen source can be added when the present invention-decomposing bacterium is used alone. As the nitrogen source, tryptone, peptone, yeast extract, meat extract, ammonium salt, and nitrate can be used. It is not necessary to add a carbon source, but in order to maintain the cells after consumption of triazine-based pesticides such as simazine, a carbon source that can be assimilated by the present degrading bacteria such as DL-malic acid should be added. I can do it.
Examples of the triazine pesticide in the method for decomposing a triazine pesticide of the present invention include a halogen-containing triazine pesticide containing a halogen atom together with a triazine ring. The halogen atom in the halogen-containing triazine pesticide may be present anywhere in the molecule, but preferably those directly bonded to the triazine ring. Examples of the halogen in the halogen-containing triazine-based agricultural chemical of the present invention include a chlorine atom, a bromine atom, and a fluorine atom, and a chlorine atom is preferable. Further, the triazine ring may be monocyclic, polycyclic, or condensed cyclic as long as it consists of a six-membered ring having three nitrogen atoms, but preferably a triazine containing one triazine ring. System pesticides. Preferable halogen-containing triazine pesticides include simazine or atrazine having an s-triazine skeleton.
[0038]
The present invention also provides a purification material in which at least one selected from the above-described simazine-degrading bacteria of the present invention or the complex microbial system of the present invention is held in a microorganism holder. The microbial support of the present invention is not particularly limited as long as it can stably hold the simazine-degrading bacterium of the present invention or the composite microbial system of the present invention. Examples thereof include porous wood carbonized materials, bamboo charcoal materials, cotton cloth materials and the like in which bacteria or complex microorganisms are accumulated. More specifically, porous materials described in JP-A-10-225288 (Patent No. 3030370) or JP-A-11-318435 (Patent No. 2904432) can be mentioned, and the contents described in these publications are described. All incorporated herein.
The purification material of the present invention is prepared by first sterilizing a medium such as 1/10 LB liquid medium with a porous material such as charcoal, brick or vermiculite, and planting the simazine-degrading bacteria found in the present invention. By purifying and shaking culture, and removing the culture solution after completion of the culture, a purification material having the simazine-degrading bacteria fixed can be obtained.
Although there is no restriction | limiting in particular in the magnitude | size of the purification | cleaning material of this invention, The magnitude | size of about 1-50 mm, Preferably about 5-20 mm, 5-10 mm is preferable. The purification material of the present invention can be used as it is by mixing it with soil, water, or the like in which triazine-based agricultural chemicals, preferably halogen-containing triazine-based agricultural chemicals may remain. Moreover, it can also process with an inorganic salt solution before use, or can be used with an inorganic salt solution.
The purification material of the present invention can be used as a purification device for purifying contaminated water or the like by storing it alone or together with soil in the soil layer tank 1 shown in FIG. That is, the present invention provides a halogen-containing triazine-based pesticide purification device comprising the purification material of the present invention described above.
[0039]
【Example】
Hereinafter, isolation and classification of simazine-degrading bacteria of the present invention and coexisting bacteria constituting the complex microbial system of the present invention, classification and identification, triazine-based pesticide degradation including simazine using the present degradable bacteria and the complex microbial system of the present invention The present invention will be described in more detail and specifically with reference to the following experimental examples, but the present invention is not limited to the following examples.
[0040]
Example 1 (Isolated culture)
As shown in FIG. 1, an accumulation soil layer 2 was formed in a soil layer tank 1. This accumulation soil layer 2 was formed by using 5% by weight of wood charcoal crushed to about 5 to 10 mm as artificial microhabitat and mixing it with 5% by weight in the soil where decomposing bacteria live. Then, an inorganic salt medium 3 containing only simazine as a carbon source and a nitrogen source was refluxed from the storage tank 4 to the accumulation soil layer 2. The residual concentration of simazine in the reflux liquid and the concentration of chloride ion due to the decomposition of simazine were measured, and the artificial microhabitat was taken out from the accumulation soil layer 2 three weeks after the start of accumulation. Then, the artificial microhabitat prepared with the decomposed bacteria was separately inoculated into an artificial microhabitat prepared, that is, mixed to form a layer for accumulation only by the artificial microhabitat, and the same reflux was performed on the layer for accumulation. After this operation was repeated three times, the artificial microhabitat was taken out, thoroughly washed with physiological saline phosphate buffer (PBS), pulverized, diluted with PBS, and then applied to a plate of inorganic salt agar medium containing simazine 5 mg / L. Inoculated and cultured at 25 ° C. for 7 days. The resulting colony was inoculated into an inorganic salt liquid medium containing 5 mg / L of simazine and cultured at 25 ° C. for 5 days, and then the culture solution in which simazine degradation was observed was re-stained on an inorganic salt agar medium plate containing 5 mg / L of simazine. And the simazine-degrading colony CD7W was isolated. When CD7W was inoculated on a 0.1% bactotryptone agar plate containing simazine 5 mg / L and single colony separation was performed in the same manner, three types of colonies with different morphologies were obtained. Each was named CD7WL, CDB21, CSB1.
[0041]
Example 2 (Assay for Simazine Decomposition of CD7WL, CDB21, and CSB1) 10 ml of 1 / 10T medium containing about 5 mg / L of simazine was placed in a 100 ml Erlenmeyer flask, and each strain isolated from a simazine-degrading colony was inoculated therein. Then, the simazine concentration in the culture solution after 4 or 5 days of shaking culture at 30 ° C. and 120 rpm was measured. The results are shown in Tables 1 and 2 above.
As a result, it was found that only the CDB21 strain has rapid simazine resolution.
[0042]
Example 3 (Mycological Properties of CDB21)
In order to perform the mycological positioning of CDB21 isolated in Example 1, the morphological properties, the mode of growth in the medium, and the physiological properties were analyzed at NCB Japan. The culture temperature was 30 ° C. unless otherwise specified, and the form, culture and physiological properties as shown in Table 3 were observed.
As a result, the CDB21 strain of the present invention was presumed to belong to the Pseudomonas group.
[0043]
Furthermore, it is considered difficult to classify and identify from the mycological properties of CDB21 obtained in Example 1, and in order to characterize the mycological position of CDB21 from the viewpoint of genetic engineering, determination of the base sequence of 16S-rRNA gene (Analysis) was performed.
(1) DNA was prepared from the cells of CDB21 obtained in Example 1 using DNeasy Tissue Kit (manufactured by Qiagen).
(2) Using the DNA as a template DNA, a 16S-rRNA gene fragment of CDB21 is amplified by PCR using a known primer for amplifying 16S-rRNA, and using QIAquick PCR Purification Kit (manufactured by Qiagen). DNA was purified.
(3) A cycle sequence reaction was performed using a known eubacterium 16S-rRNA base sequence analysis primer fluorescently labeled with the purified DNA as a template DNA, and the base sequence was determined (analyzed) by Hitachi DNA Sequencer SQ5500E.
[0044]
  SEQ ID NO: 1 shows 1488 bases obtained by removing the PCR primer sequences at both ends from the base sequence of the 16S-rRNA gene fragment of CDB21 obtained as described above.
  A close tree and a phylogenetic tree were created by the neighbor-joining method (NJ method), and the relationship was analyzed, and it was revealed that it belongs to the category of betaproteobacteria. Based on the above, the present inventors have identified the CDB21 strain of the present invention as a CDB21 strain belonging to a novel betaproteobacterium that has never been isolated and cultured and does not belong to any known genus species. Accession number to National Institute of Advanced Industrial Science and TechnologyFERM BP-10403As deposited.
[0045]
Example 4 (Mycological Properties of CSB1)
In order to perform the mycological positioning of CSB1 isolated in Example 1, the morphological properties, the growth mode in the medium and the physiological properties were analyzed at NC Japan. The culture temperature was 30 ° C. unless otherwise specified, and the form, culture and physiological properties as shown in Table 4 were observed.
From the above results, it was estimated that the CSB1 strain of the present invention belongs to the Pseudomonas group.
[0046]
  SEQ ID NO: 2 shows 1462 bases obtained by removing the PCR primer sequences at both ends from the base sequence of the 16S-rRNA gene fragment obtained for CSB1 in the same manner as in Example 3.
  Furthermore, as in Example 3, when a phylogenetic tree was prepared by the neighborhood joining method (NJ method) and compared, it was revealed that CSB1 belongs to the category of Bradyrizobium japonicam. Based on the above, the inventors identified this strain as Bradyrizobium japonicam CSB1 strain, and received the accession number at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.FERM BP-10402As deposited.
[0047]
Example 5 (Degradation culture of simazine by CDB21 strain)
1 g of bactotryptone was dissolved per liter of purified water, and 5 mg of simazine suspended in 1 ml of methanol was added, followed by steam sterilization at 121 ° C. for 20 minutes. 50 ml of this medium was dispensed into a sterilized 500 ml Erlenmeyer flask and inoculated with one platinum loop CDB21 strain. Shaking culture was performed at 30 ° C. and 120 rpm, and the growth of bacterial cells, the decomposition of simazine, and the generation of chloride ions associated therewith were measured. The results are shown in FIG.
[0048]
Example 6 (Production of complex microbial system with CDB21 strain)
Na per liter of purified water₂HPO₄・ 12H₂O 1.2g, KH₂PO₄  After dissolving 0.5 g, 5 mg of simazine suspended in 1 ml of methanol was added, and 1.5 g of agar (AGAR NOBLE, DIFCO) was added, followed by steam sterilization at 121 ° C. for 20 minutes. After sterilization, 10 ml of a trace element solution was added to produce a simazine-containing inorganic salt agar medium and used as a flat plate. The trace element solution is 0.5g EDTA per liter of purified water, MgSO₄・ 7H₂O 2g, FeSO₄・ 7H₂O 0.2g, ZnSO₄・ 7H₂O 10mg, MnSO₄・ H₂O 5 mg, H₃BO₃  30 mg, CoSO₄・ 7H₂O 24mg, CuSO₄・ 7H₂O 5mg, Na₂MoO₄  5mg, Ca (OH)₂ After dissolving 50 mg, it was prepared by steam sterilization at 121 ° C. for 20 minutes.
One platinum loop CDB21 strain was smeared on half of the plate, and the other half was smeared with other strains, and then both strains were mixed at the center and cultured at 30 ° C. for 7 days. Simazine-containing inorganic salt liquid medium in which a combination of colonies formed in the centrally mixed part does not grow on a single smeared portion, and a platinum loop is removed from a simazine-containing inorganic salt agar medium. 10 ml was inoculated, and cultured for 4 days at 30 ° C. and 120 rpm in a 100 ml Erlenmeyer flask, and the degradation of simazine was measured. The results are shown in Table 5 above. In combination with five of the examined strains, it was possible to construct a CDB21 complex microbial system, and all decomposed simazine in an inorganic salt medium.
[0049]
Example 7 (Shimazine degradation culture by CDB21 complex microorganism system)
50 ml of the simazine-containing inorganic salt liquid medium described in Example 6 was dispensed into a sterilized 500 ml Erlenmeyer flask and inoculated with a single microbial CDB21 strain, CSB1 strain, or a complex microbial system composed of the CDB21 strain and the CSB1 strain. Shaking culture was performed at 30 ° C. and 120 rpm, and the growth of bacterial cells, the decomposition of simazine, and the generation of chloride ions associated therewith were measured. The results are shown in FIG.
[0050]
Example 8 (Degradation of triazine ring by CDB21 complex microbial system)
The carbon atom of the triazine ring was added to the simazine-containing inorganic salt medium described in Example 6.¹⁴Simazine-ring-UL- labeled with C¹⁴C) was added. In this medium¹⁴C-labeled simazine 0.75 mg / L and unlabeled simazine 3.4 mg / L are included. This was inoculated with a complex microbial system containing CDB21 strain and CSB1 strain, and statically cultured in a dark place at 25 ° C. for 8 days with continuous air aeration through a sodium hydroxide trap at 2 ml / min. During the culture period, in the culture solution¹⁴C-labeled simazine and its metabolite concentrations were collected using a high-performance liquid chromatograph with a radio analyzer.¹⁴C-labeled carbon dioxide was quantified with a liquid scintillation counter. The results are shown in FIG. The black circle (●) in Fig. 4¹⁴C-labeled simazine concentration (mg / L), black square mark (■)¹⁴C-labeled cyanuric acid concentration (mg / L), black triangle (▲) generated¹⁴The ratio (%) to the theoretical amount of C-labeled carbon dioxide is shown. The left side of the vertical axis in FIG.¹⁴C-labeled simazine and¹⁴Concentration of C-labeled cyanuric acid (mg / L) occurred on the right side¹⁴The ratio (%) of the amount of C-labeled carbon dioxide to the theoretical amount is shown. The horizontal axis indicates the culture period (days).
As a result, most of simazine was degraded on the 6th day of culture and completely degraded on the 8th day. The concentration of cyanuric acid, a degradation intermediate metabolite, increased with the degradation of simazine, but it does not accumulate in large quantities and is a complete degradation product¹⁴Generation of C-labeled carbon dioxide was observed. This indicates that simazine degradation by the microbial strain including the CDB21 strain of the present invention or the CDB21 strain and the CSB1 strain can also completely degrade the triazine ring and detoxify simazine.
[0051]
Example 9 (Stability of Simazine Degradation by CDB21 Complex Microbial System)
After placing 200 ml of the simazine-containing inorganic salt liquid medium and charcoal described in Example 6 into a predetermined position of the Audis reflux apparatus (1950, Nature, 166, 356) and inoculating the complex microbial system including the CDB21 strain and the CSB1 strain, The liquid medium was refluxed. On the seventh day, the reflux solution was replaced with a new one, and then paddy field soil or bark compost was overlaid on the charcoal, and the reflux was continued for a total of 58 days. On days 5, 9, 12, 16, 19, 27, 33, 40, 47, and 54, an appropriate amount of an aqueous solution of simazine prepared to 5000 mg / L was added. In addition, the reflux solution itself was exchanged for a new one on days 7, 14, 22, 30, 37, 44 and 51. During the reflux period, the reflux solution was appropriately collected and the simazine concentration was measured by a high performance liquid chromatograph method. The results are shown in FIG. The black square mark (■) in Fig. 5 shows the simazine concentration (mg / L) in the reflux liquid when paddy soil is overlaid on the seventh day, and the black circle mark (●) reflux when bark compost is overlaid on the seventh day. The concentration of simazine in the liquid (mg / L) is shown. The vertical axis in FIG. 5 represents the simazine concentration (mg / L), and the horizontal axis represents the culture period (days).
As a result, the complex microbial system including CDB21 strain and CSB1 strain rapidly degraded simazine in the presence of charcoal. The simazine resolution of this complex microbial system was not affected at all even after overlaying paddy soil and bark compost containing various microorganisms on top of charcoal. From the above results, it can be seen that the degradation of simazine by the CDB21 strain of the present invention or the complex microbial system including the CDB21 strain and the CSB1 strain is hardly affected by other microorganisms once formed and is very stable. Show.
[0052]
Example 10 (Degradation of triazine herbicide by CDB21 complex microbial system)
In place of simazine, 5 mg / L of various triazine pesticides were added to the simazine-containing inorganic salt medium described in Example 6, and 10 ml of the medium was inoculated with a complex microbial system composed of one platinum loop CDB21 strain and CSB1 strain. Shake culture was performed at 30 ° C. and 120 rpm in a 100 ml Erlenmeyer flask, and the degradation of simazine was measured. The results are shown in Table 6 above. Simazine and atrazine, which were chlorinated at the 2-position, were rapidly degraded within 3 days, but methimeline, promethrin and dimetamethrin, which were thiomethylated at the 2-position, were not degraded under this culture condition.
[0053]
【The invention's effect】
Triazine-based pesticides, particularly simazine, can be decomposed by the decomposing bacteria of the present invention. Furthermore, by using the complex microbial system of the present invention, it is possible to decompose the pesticide more rapidly and completely under a wide range of culture conditions. By remaining in the environment, a method for decomposing the pesticide that causes environmental pollution Is provided.
[0054]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of an apparatus used in the integration method of Example 1. FIG.
FIG. 2 is a graph showing the degradation of simazine by CDB21 strain in Example 5.
FIG. 3 is a graph showing the degradation of simazine by a complex microbial system composed of CDB21 strain and CSB1 strain in Example 7 and comparison with CDB21 strain and CSB1 strain.
FIG. 4 is a graph showing the decomposition of carbon-14-labeled simazine in Experimental Example 8.
FIG. 5 is a graph showing the decomposition of simazine in Experimental Example 9.

Claims (9)

Bacteria selected from the beta proteobacteria strain CDB21 (FERM BP-10403) and mutants thereof alone or Bradyrizobium japonicam CSB1 strain (FERM BP-10402), Nocardioides Genseni, Nocardioides fulvus, A method for degrading simazine or atrazine , which is used in combination with at least one symbiotic bacterium selected from the group consisting of Cardioides simplex and Pseudomonas aeruginosa . The degradation method according to claim 1, wherein a complex microbial system of beta-proteobacterial CDB21 strain (FERM BP- 10403 ) and Bradyrizobium japonicum CSB1 strain (FERM BP-10402) is used. A beta-proteobacterium strain CDB21 (FERM BP-10403) , which has a resolution of simazine or atrazine . Beta proteobacteria CDB21 strain (FERM BP-10403) and degrading bacterium of simazine or atrazine consisting degradation of simazine or atrazine and / or Bradyrhizobium japonicum CSB1 strain having the ability to assist the growth (FERM BP-10402) A complex microbial system comprising one or more symbiotic fungi selected from Nocardioides Genseni, Nocardioides fulvus, Nocardioides simplex, and Pseudomonas aeruginosa . The complex microbial system according to claim 4, comprising a beta proteobacteria strain CDB21 (FERM BP-10403) and a Bradyrizobium japonicum CSB1 strain (FERM BP-10402) . The ability to assist the degradation and / or growth of simazine or atrazine of simazine or atrazine- degrading bacterium of Bradyrizobium japonicam CSB1 strain (FERM BP-10402) , which consists of beta-proteobacterium CDB21 strain (FERM BP-10403) Having bacteria. A purification material in which at least one selected from the bacteria or complex microorganism system according to any one of claims 3 to 5 is held in a microorganism holder. The purification material according to claim 7, wherein the microorganism holder is made of a porous wood carbonized material. A purification apparatus for a halogen-containing triazine-based agricultural chemical selected from the group consisting of simazine and atrazine containing the purification material according to claim 7 or 8 .

Images