JP3591886B2 - Rare earth element accumulating microorganism - Google Patents

Description

【００１４】
また、培養上清中に残存するイットリウム（Ｙ）濃度の測定方法を以下に記載する。
スクリーニングする菌株を接種、培養した後のＹ含有培地１．５ｍｌをエッペンドルフ遠心管に入れ、遠心分離（１２，０００ｒｐｍ、４℃、１０分間）することによって得られた培養上清１ｍｌに、０．５ｍｌ ０．１％アルセナゾＩＩＩ （Ａｒｓｅｎａｚｏ ＩＩＩ）溶液、０．５ｍｌ 塩酸・塩化カリウム緩衝溶液（３０ｍｌ ０．２Ｍ ＫＣｌ、２０ｍｌ ０．２Ｍ ＨＣｌ及び５０ｍｌ 脱塩水を混合して１００ｍｌに定容し、ｐＨが約１．５となった溶液）および１ｍｌ 脱塩水を加え、この混合溶液の６５５ｎｍの吸光度を測定することによって、標準直線に基づいてイットリウム（Ｙ）の濃度を求めた。なお、この際、イットリウムの標準直線は、原子吸光用の硝酸イットリウム溶液［１モル／リットルの硝酸溶液におけるＹ（ＮＯ_３ ）_３ 濃度＝１．０ｍｇＹ／ｍｌ］（和光純薬製）を使用する以外同様の操作を用いることによって作成した。
【００１５】
上記スクリーニングによって得られた菌株の菌学的性質を以下に示す。
【００１６】
（ａ）形態
１） 細胞の形および大きさ： かん菌で（０．６〜０．９）μｍ×（３．０〜４．０）μｍ
２） 細胞の多形性の有無： 無し
３） 運動性の有無： 無し
４） 胞子の有無： 無し

（ｃ）生理学的性質（３０℃、７２時間）
１） グラム染色： −
２） カタラーゼ： ＋
３） オキシダーゼ： ＋
４） Ｏ−Ｆテスト： ＋（若干酸化発酵）
５） 硝酸塩の還元： ＋
６） インドールの生成： −
７） グルコースからの酸の生成： −
８） アルギニンデヒドロラーゼ： −
９） ウレアーゼ： −
１０） エスクリン加水分解： −
１１） ゼラチン加水分解： −
１２） β−ガラクトシダーゼ： −
１３） グルコース同化： −
１４） アラビノース同化： −
１５） マンノース同化： −
１６） マンニトール同化： −
１７） Ｎ−アセチルグリコサミン同化： −
１８） マルトース同化： −
１９） グルコネート同化： ＋
２０） カプレート同化： ＋
２１） アジペート同化： ＋
２２） マレート同化： ＋
２３） シトレート同化： −
２４） フェニルアセテート同化： −
２５） チトクロムオキシターゼ： ＋
（ｄ）生理的性質の補足（３０℃、７日）
１） Ｔｗｅｅｎ８０加水分解： ＋
２） グルコースＯＦからの酸の生成： ＋（僅かに）
３） フルクトースＯＦからの酸の生成： ＋
４） キシロースＯＦからの酸の生成： −
５） ３．５％ＮａＣｌ中での生育： −
６） ＮＯ_２ −Ｎ_２ ： −
７） アセトアミドのアルカリ化： ＋
８） アラントインのアルカリ化： ＋
９） 酒石酸のアルカリ化： ＋
１０） ウレアーゼ： ＋（僅かに）
１１） ゼラチン分解： −
１２） デオキシリボヌクレアーゼ： −
１３） リシンデカルボキシラーゼ： −
１４） シトレートの利用： ＋
１５） グルコースの利用： ＋（僅かに）
本菌は、バージーズ マニュアル オブ システマティック バクテリオロジー（Ｂｅｒｇｅｙ´ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ ８ｔｈ ）による分類が不可能であったため、本菌の同定をザ インターナショナル コレクション オブ インダストリアル アンド マリーン バクテリア リミテッド（ＮＣＩＭＢ）（Ｔｈｅ Ｎｔｉｏｎａｌ Ｃｏｌｌｅｃｔｉｏｎ ｏｆ Ｉｎｄｕｓｔｒｉａｌ ａｎｄ Ｍａｒｉｎｅ Ｂａｃｔｅｒｉａ Ｌｉｍｉｔｅｄ）に依頼 （参照番号：ＩＤ ２５９１／ＮＣＩＤ ３４１５、日付：１９９４年７月１８日の報告書類における菌株Ｆ）したところ、バリオボラックス パラドクス（Ｖａｒｉｏｖｏｒａｘ ｐａｒａｄｏｘｕｓ）に近似していることが分かった（参考文献：インターナショナル ジャーナル オブ システマティック バクテリオロジー（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ）１９９１年４１（３）、４４５〜４５０頁、インターナショナル ジャーナル オブ システマティック バイオテクノロジー（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ）１９９１年４１（３）、４２７〜４４４頁、ジャーナル オブ クリニカル マイクロバイオロジー（Ｊｏｕｒｎａｌ ｏｆ Ｃｌｉｎｉｃａｌ ＭｉｃｒｏＢａｉｏｌｏｇｙ ）１９８６年２３巻、９２０〜９２３頁）。
【００１７】
従来まで、バリオボラックス パラドクス（Ｖａｒｉｏｖｏｒａｘ ｐａｒａｄｏｘｕｓ）が希土類元素を集積するという報告はなく、本菌は明らかに公知の菌種と区別されるため、本発明の微生物を新規な微生物であると判断し、本菌株をバリオボラックス パラドクス Ｒ−１（Ｖａｒｉｏｖｏｒａｘ ｐａｒａｄｏｘｕｓ Ｒ−１）（以下、単にＲ−１株と称する）と命名した。また、このＲ−１株は、平成６年８月２６日付で工業技術院生命工学工業技術研究所に寄託され、その受託番号はＦＥＲＭ Ｐ−１４４９２号である。
【００１８】
本発明の菌株の培養に使用する培地は、固体または液体培地のいずれでもよく、また、本細菌が資化しうる炭素源、適量の窒素源、無機塩及びその他の栄養素を含有する培地であれば、合成培地または天然培地のいずれでもよい。
【００１９】
本発明の菌株の培養において使用できる炭素源としては、本菌株が資化できる炭素源であれば特に制限されない。具体的には、微生物の資化性を考慮して、グルコース、フラクトース、マルトース、ガラクトース、デンプン、デンプン加水分解物、糖蜜、廃糖蜜などの糖類、肉エキス、ペプトン、麦、米などの天然物、グルセロール、メタノール、エタノール等のアルコール類、酢酸、グルコン酸、ピルピン酸、クエン酸等の脂肪酸類、グリシン、グルタミン酸、アスパラギン酸等のアミノ酸などを１種または２種以上選択して使用することができる。
【００２０】
本発明の菌株の培養において使用できる窒素源としては、肉エキス、ペプトン、ポリペプトン、酵母エキス、大豆加水分解物、大豆粉末、ミルクカゼイン、カザミノ酸、各種アミノ酸、コーンスティープリカー、その他の動物、植物、微生物の加水分解物等の有機窒素化合物、アンモニア、硝酸アンモニウム、硫酸アンモニウム、塩化アンモニウムなどのアンモニウム塩、硝酸ナトリウムなどの硝酸塩、尿素等の無機窒素化合物より使用する微生物の資化性を考慮して、１種または２種以上選択して使用する。
【００２１】
本発明において使用できる無機塩としては、マグネシウム、マンガン、カルシウム、ナトリウム、カリウム、銅、鉄及び亜鉛などのリン酸塩、塩酸塩、硫酸塩及び酢酸塩等から選ばれた１種または２種以上を使用することができる。また、培地中に、必要に応じて、植物油、界面活性剤等を添加してもよい。
【００２２】
本発明の微生物に効率よく希土類元素を集積させるために、培地中に希土類元素、より好ましくはイットリウム、ランタン、セリウム、プラセオジムおよびネオジムを、１〜２０ｐｐｍ、より好ましくは２〜１０ｐｐｍの濃度で、添加することが好ましい。この際、添加する希土類元素は２種以上の混合物またはミッシュメタル等の合金であってもよい。
【００２３】
本発明において、培養は、本発明の細菌は好気性であるため、好気的条件下で行われ、その際の培養条件は、培地の組成や培養法によって適宜選択され、本菌株が増殖できる条件であれば特に制限されない。通常は、培養温度が、２０〜７０℃、好ましくは２５〜４０℃であり、また、培養に適当な培地のｐＨは、５〜９、好ましくは６〜８である。
【００２４】
また、本発明において、希土類元素の微生物からの分離、精製方法としては、上記培養条件下で培養を行った後、菌体を瀘過あるいは遠心分離等によって集め、例えば、凍結融解処理、超音波処理、加圧処理、浸透圧差処理、磨砕処理等の物理的手段またはリゾチーム等の細胞壁溶解酵素処理若しくは界面活性剤との接触処理等の化学的処理を単独または組み合わせて行うことにより菌体を破砕し、破砕した菌体から目的とする希土類元素を、分別結晶法、分別沈殿法、イオン交換クロマトグラフィー法および溶媒抽出法等の既知の方法を単独若しくは組み合わせて用いて精製する方法が挙げられる。破砕した菌体からの希土類元素の精製法としては、手間、時間および費用の点から、溶媒抽出法、特に溶媒抽出操作を連続して行う向流多段抽出法が好ましく用いられる。
【００２５】
本発明の微生物は、希土類元素を効率よく集積する特性を有するが、従来、バリオボラックス属（Ｖａｒｉｏｖｏｒａｘ）に属し、上記特性を有する微生物に関する報告はなかった。
【００２６】
【実施例】
以下、実施例によって本発明をさらに具体的に説明する。
【００２７】
実施例１
０．２μｍのポリサルホン製滅菌済フィルターで濾過滅菌したＹ含有培地（Ｙ濃度：５ｐｐｍ）５ｍｌに斜面培地よりＲ−１株を一白金耳接種して、３０℃で２４時間振盪培養することによって前培養を行った。このようにして得られた培養液５ｍｌを、同様にして瀘過滅菌したＹ含有培地１００ｍｌの入った５００ｍｌの枝付き坂口フラスコに入れ、３０℃で１５日間本培養を行った。この培養期間中、２４あるいは４８時間おきに培養液を５ｍｌずつ採取し、採取した培養液について、作用において記載した測定方法にしたがってＹ濃度を測定すると同時に、６６０ｎｍの波長での濁度（ＯＤ_６６０ ）および培養液のｐＨを測定した。なお、濁度については、フラスコの側面にある試験管状部分に培養液を流し込み、ここに測定器を差し込んで計測を行った。
【００２８】
このようして得られた培養時間に対するＲ−１株の培養液のＹ濃度の変化および濁度及びｐＨの変化を図１に示す。図１に示されるように、Ｒ−１株の培養によるＹ濃度は、培養開始後、短時間（６日間）で濃度が０付近に到達し、その後はほぼ一定を保つことから、Ｙ濃度の変化が指数曲線に近い形を描いていることが分かる。また、細菌は指数関数状に増殖し、Ｒ−１株によるＹ濃度の減少曲線が上記増殖曲線と対称的であることから、Ｙ濃度の減少はＲ−１株の生物学的活動によるものであると考えられる。
【００２９】
実施例２
０．２μｍのポリサルホン製滅菌済フィルターで濾過滅菌したＹ含有培地（Ｙ濃度：５ｐｐｍ）５ｍｌにＲ−１株を斜面培地より一白金耳接種し、３０℃で７日間振盪培養する。７日間培養した後のＹ含有培養液１．５ｍｌをエッペンドルフ遠心管に入れ、遠心分離（１２，０００ｒｐｍ、４℃、１０分間）することによって培養上清液を得た。続いて、上記遠心分離によって得られた沈殿（菌体）に１５０ｍＭのＮａＣｌを１ｍｌを加えて懸濁させ、これを再度遠心分離にかけ、上清液を得た。この操作を計３回繰り返し、上清液を合わせて洗浄液とした。
【００３０】
このようにして洗浄された沈殿（菌体）に１５０ｍＭのＮａＣｌを１ｍｌを加えて懸濁させた。この懸濁液を超音波破砕機（ブランソン製（ＢＲＡＮＳＯＮ）、デューティ（Ｄｕｔｙ）：３０、出力：３、３０秒×５回）を用いて破砕処理を行なうことによって菌体を破砕し、０．５ｍｌの１５０ｍＭ ＮａＣｌをさらに加えて遠心分離し、細胞抽出液を得た。そして遠心後の菌体の残渣に０．５ｍｌの濃硫酸、０．５ｍｌの濃硝酸を加え加熱し酸分解させ、これに１５０ｍＭのＮａＣｌを１ｍｌ加え、菌体の残渣液を得た。
【００３１】
なお、比較として、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＩＦＯ １０４１）を用いて同様の評価を行なった。
【００３２】
得られた各画分に含まれるＹ濃度を誘導結合プラズマＩＣＰを用い測定した。その結果を図２に示す。
【００３３】
この結果より明らかなように、大腸菌では全く集積されていなかったＹが、Ｒ−１株では初期濃度の約７０％近く菌体に集積されていることがわかる。
【００３４】
実施例３
図３に記載の各希土類元素［ランタノイド（Ｌａ〜Ｌｕ）及びイットリウム （Ｙ）］の硝酸塩溶液を硝酸イットリウム溶液の代わりに用いる以外は表１と同様の培地組成を有する希土類元素含有液体培地を孔径０．２μｍのポリサルホン製滅菌済フィルターで瀘過滅菌した。この希土類元素含有液体培地（各希土類元素濃度：５ｐｐｍ）５ｍｌに、斜面培地よりＲ−１株を一白金耳ずつ接種して、３０℃で７日間振盪培養し、培養液中に含まれる各希土類元素濃度を測定し、初濃度に対する減少率を算出した。なお、各希土類元素の濃度の測定方法は、使用する希土類元素が異なる以外は作用において記載されたイットリウム濃度の測定方法と同様であるが、６５５ｎｍの波長における感度が元素ごとに異なるため、標準曲線を各元素ごとに作成した。
【００３５】
結果を図３に示す。図３に示されるように、原子量の小さい方（図３の左側）に減少率の高い物質が偏っており、プラセオジム（Ｐｒ）がやや低い（２２％）が、イットリウムからネオジムまでが４０％前後の比較的高い減少率を示している。また、高い減少率を示すランタンからネオジムの元素はすべて原子価が３以上に限定されることから、イットリウムおよび軽希土のうち原子価が３以上に限定される元素を用いた場合に減少率が高くなっていると考えられる。
【００３６】
【発明の効果】
以上述べたように、本発明によるバリオボラックス属（Ｖａｒｉｏｖｏｒａｘ）に属する微生物は新規な微生物であり、希土類元素、特にイットリウム、ランタン、セリウム、プラセオジムおよびネオジムを効率よく集積できるものである。
【図面の簡単な説明】
【図１】図１は、本発明の微生物による希土類元素集積能を説明するための培養時間に対する培養液中のイットリウム（Ｙ）濃度の変化、および培養液の濁度及びｐＨの変化を示すものである。
【図２】図４は、Ｙの分布状況を示す図である。
【図３】図３は、本発明の微生物によるランタノイドに対する選択性を示す図である。[0001]
[Industrial applications]
The present invention relates to a novel rare earth element accumulating microorganism. More specifically, the present invention relates to a rare earth element accumulating microorganism having an ability to accumulate rare earth elements efficiently.
[0002]
[Prior art]
Rare earth elements are the largest element group existing in nature in the periodic table. Scandium (Sc) and yttrium are included in 15 elements (lanthanoids) from lanthanum (La) having an atomic number of 57 to lutetium (Lu) having an atomic number of 71. (Y) is a collective term for 17 element groups, and is contained in minerals such as monazite, bastnaesite, xenotime, yuxenite and gadolinite. I have. Of these, monazite and bastnaesite are mainly used as light rare earth (lanthanum to europium) resources, and xenotime is used as heavy rare earth (gadolium to ruthenium) resources on an industrial scale. Are obtained as resources.
[0003]
Further, rare earth elements are used in various wide fields due to their magnetic properties and optical properties such as color based on the behavior of 4f electrons in an incompletely filled state of ions, and furthermore, chemical properties unique to rare earth elements. Specifically, as a device using the property of the 4f electron, a red phosphor (europium) of a cathode ray tube of a color television receiver, a magnet (samarium or neodymium) in a small electronic device such as a quartz wristwatch or a walkman, Examples of those utilizing chemical properties include catalysts (lanthanum and cerium) used in gasoline production, oxide high-temperature superconductors, hydrogen storage alloys, ceramics, and control materials for nuclear reactors.
[0004]
Conventionally, as microorganisms capable of collecting rare earth elements, there are microorganisms described in JP-A-59-118,825. However, Japanese Patent Application Laid-Open No. Sho 59-118,825 only discloses a method for selectively collecting yttrium using activated sludge, and has not yet specifically identified microorganisms.
[0005]
For this reason, there has been no report of a specified microorganism that efficiently accumulates rare earth elements to date.
[0006]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a microorganism that can efficiently accumulate rare earth elements.
[0007]
[Means for Solving the Problems]
The above objects are achieved by a rare earth element accumulating microorganism belonging to the genus Variovorax that can accumulate rare earth elements.
[0008]
The present invention shows rare earth element-enriching microorganisms that efficiently accumulate yttrium, lanthanum, cerium, praseodymium, and neodymium in particular among rare earth elements. The present invention further provides a rare earth element accumulating microorganism having an accession number of FERM P-14492.
[0009]
[Action]
The microorganism according to the present invention belongs to the genus Variovorax and is characterized by being capable of efficiently accumulating rare earth elements. Specific examples of this microorganism include Variovorax paradox. ) R-1 strain, and this strain was obtained by the following screening method. In the following screening method, screening was performed using yttrium (Y), which is most frequently present in nature, as a rare earth element.
[0010]
Based on the presumption that there is a high possibility that a target strain having the ability to accumulate rare earths in a microorganism capable of growing even in a low nutrient state exists, the screening was divided into two rounds, that is, as a primary screening, After the search, a strain that reduces the yttrium concentration in the culture solution was selected as a secondary screening. The screening method according to the present invention is specifically described below.
[0011]
In Tanigumi shrine as it is or Conradi stick one by 100μl diluted to 10 to 10 ³ times with sterile water samples taken from the soil (Gifu Tanigumi), primary screening Agar (pH which is prepared in the following manner : 7.2 to 7.4), and this agar medium was cultured at 30 ° C for 7 to 10 days. Next, the colonies formed on the agar medium were picked, suspended in 10 ml of sterile water, and further diluted 10 ² to 10 ⁴ times. Then, 100 μl of each was spread on the same agar medium, and this operation was further performed. Was repeated several times to purify the strain. By such a primary screening, a bacterium capable of growing even in a nutrient-diluted state was obtained.
Preparation method of agar medium for primary screening: 1/100 nutrient broth medium (polypeptone 0.01%, meat extract 0.01%, NaCl 0.005%, pH 7.2-7.4) (hereinafter 1/100) A 100-diluted broth medium) was prepared, and 1.5% (wt / v) of agar was added thereto and autoclaved, and about 25 ml was dispensed into a Petri dish to obtain a plate medium.
[0012]
Next, each of the isolated bacteria purified by the above-mentioned primary screening was inoculated in a platinum loop one platinum loop at a time into a sterile-treated liquid medium for secondary screening (5 ml) prepared by the following method. For 7 days with shaking. Further, the concentration of yttrium (Y) remaining in the culture supernatant was measured according to the following method, and bacteria having a Y concentration of less than 50% of the initial concentration (5 ppm) in the culture supernatant were selected as candidate bacteria. . At this time, the selected strain was again inoculated into a secondary screening medium (hereinafter, referred to as Y-containing medium), and the same operation was repeated to confirm the reproducibility of the decrease in Y concentration. In this way, strains that efficiently accumulate rare earth elements were screened.
Preparation method of secondary screening medium: Gravy (1% polypeptone, 1% meat extract, 0.5% NaCl, pH 7.2) was diluted 1 / 100-fold with demineralized water, and 5% (v / V) of a 5N sodium hydroxide solution (0.1% (v / v)) corresponding to the neutralization of an aqueous nitric acid solution (1 mol / l) was added in advance, and the pH was adjusted while stirring the solution. The yttrium nitrate solution was gradually dropped to about 7.2, made up to 1 liter with demineralized water, sterilized by filtration through a sterilized filter of 0.2 μm, and then subjected to a secondary screening medium (Y concentration: 5 ppm). ). This medium for secondary screening has the composition shown in Table 1.
[0013]
[Table 1]

[0014]
The method for measuring the concentration of yttrium (Y) remaining in the culture supernatant is described below.
After inoculating and culturing the strain to be screened, 1.5 ml of the Y-containing medium was placed in an Eppendorf centrifuge tube, and centrifuged (12,000 rpm, 4 ° C., 10 minutes). 5 ml 0.1% Arsenazo III solution, 0.5 ml hydrochloric acid / potassium chloride buffer solution (30 ml 0.2 M KCl, 20 ml 0.2 M HCl and 50 ml demineralized water) were mixed to make a constant volume of 100 ml. Then, the concentration of yttrium (Y) was determined based on a standard straight line by measuring the absorbance at 655 nm of the mixed solution. In this case, the standard straight line of yttrium uses an yttrium nitrate solution for atomic absorption [Y (NO ₃ ) ₃ concentration in a 1 mol / liter nitric acid solution = 1.0 mgY / ml] (manufactured by Wako Pure Chemical Industries, Ltd.). Except that the same operation was used.
[0015]
The bacteriological properties of the strain obtained by the above screening are shown below.
[0016]
(A) Form 1) Cell shape and size: (0.6-0.9) μm × (3.0-4.0) μm for bacillus
2) Cell polymorphism: None 3) Motility: None 4) Spore presence: None

(C) Physiological properties (30 ° C., 72 hours)
1) Gram stain:-
2) Catalase: +
3) Oxidase: +
4) OF test: + (slightly oxidative fermentation)
5) Reduction of nitrate: +
6) Indole production:-
7) Production of acid from glucose:-
8) Arginine dehydrolase:-
9) Urease:-
10) Esculin hydrolysis:-
11) Gelatin hydrolysis:-
12) β-galactosidase:-
13) Glucose assimilation:-
14) Arabinose assimilation:-
15) Mannose assimilation:-
16) Assimilation of mannitol:-
17) N-acetylglycosamine assimilation:-
18) Maltose assimilation:-
19) Assimilation of gluconate: +
20) Caprate assimilation: +
21) Adipate assimilation: +
22) Malate assimilation: +
23) Citrate assimilation:-
24) Phenyl acetate assimilation:-
25) Cytochrome oxidase: +
(D) Supplementation of physiological properties (30 ° C, 7 days)
1) Tween 80 hydrolysis: +
2) Generation of acid from glucose OF: + (slightly)
3) Generation of acid from fructose OF: +
4) Generation of acid from xylose OF:
5) Growth in 3.5% NaCl:
6) _NO 2 _-N 2: -
7) Alkalinization of acetamide: +
8) Alkalinization of allantoin: +
9) Tartaric acid alkalization: +
10) Urease: + (slightly)
11) Gelatin degradation:-
12) Deoxyribonuclease:-
13) Lysine decarboxylase:-
14) Use of citrate: +
15) Use of glucose: + (slightly)
Since this strain could not be classified by Bergey's Manual of Systematic Bacteriology (8th), the strain was identified by The International Collection of Industrial and Marine Bacterial Limited (NCIMB). of Industrial and Marine Bacteria Limited (reference number: ID 2591 / NCID 3415, date: strain F in the report on July 18, 1994), which is similar to Variovorax paradox. (Reference: International International Journal of Systematic Bacteriology, 1991, 41 (3), 445-450, International Journal of Systematic Biotechnology (International Journal of Systematic Bacteriology, pp. 41-44, 1991). Journal of Clinical MicroBiology, 1986, 23, 920-923).
[0017]
Until now, there has been no report that Variovorax paradoxus accumulates rare earth elements, and the present bacterium is clearly distinguished from known bacterial species. Therefore, the microorganism of the present invention was determined to be a novel microorganism. This strain was named Variovorax paradoxus R-1 (hereinafter simply referred to as R-1 strain). The R-1 strain was deposited on August 26, 1994 with the Institute of Biotechnology and Industrial Technology, and the deposit number is FERM P-14492.
[0018]
The medium used for culturing the strain of the present invention may be either a solid or liquid medium, and any medium containing a carbon source capable of assimilating the bacterium, an appropriate amount of a nitrogen source, inorganic salts and other nutrients. , A synthetic medium or a natural medium.
[0019]
The carbon source that can be used in culturing the strain of the present invention is not particularly limited as long as it can be used by the strain. Specifically, considering the assimilation of microorganisms, glucose, fructose, maltose, galactose, starch, starch hydrolysate, molasses, sugars such as molasses, meat extracts, peptone, wheat, rice and other natural products Alcohols such as glycerol, methanol, ethanol, etc., fatty acids such as acetic acid, gluconic acid, pyruvic acid, citric acid, and amino acids such as glycine, glutamic acid, aspartic acid and the like. it can.
[0020]
Nitrogen sources that can be used in culturing the strain of the present invention include meat extract, peptone, polypeptone, yeast extract, soybean hydrolyzate, soybean powder, milk casein, casamino acid, various amino acids, corn steep liquor, other animals and plants Considering the assimilation of microorganisms used from organic nitrogen compounds such as hydrolysates of microorganisms, ammonia, ammonium nitrate, ammonium sulfate, ammonium salts such as ammonium chloride, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea, One or two or more are selected and used.
[0021]
As the inorganic salt that can be used in the present invention, one or more selected from phosphates, hydrochlorides, sulfates, acetates, and the like of magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, and the like. Can be used. Further, a vegetable oil, a surfactant and the like may be added to the medium as needed.
[0022]
In order to efficiently accumulate rare earth elements in the microorganism of the present invention, rare earth elements, more preferably yttrium, lanthanum, cerium, praseodymium and neodymium, are added to the medium at a concentration of 1 to 20 ppm, more preferably 2 to 10 ppm. Is preferred. At this time, the rare earth element to be added may be a mixture of two or more kinds or an alloy such as a misch metal.
[0023]
In the present invention, the culture is performed under aerobic conditions because the bacterium of the present invention is aerobic, and the culture conditions at that time are appropriately selected depending on the composition of the medium and the culture method, and the strain can be grown. There are no particular restrictions on the conditions. Usually, the culture temperature is 20 to 70 ° C, preferably 25 to 40 ° C, and the pH of the medium suitable for culture is 5 to 9, preferably 6 to 8.
[0024]
Further, in the present invention, as a method for separating and purifying rare earth elements from microorganisms, after culturing under the above culture conditions, the cells are collected by filtration or centrifugation, for example, freeze-thaw treatment, ultrasonic wave Treatment, pressurization treatment, osmotic pressure difference treatment, physical means such as trituration treatment, or cell wall lysing enzyme treatment such as lysozyme or chemical treatment such as contact treatment with a surfactant alone or in combination to perform the bacterial cells. A method of crushing and purifying a rare earth element of interest from the crushed cells using a known method such as a fractional crystallization method, a fractional precipitation method, an ion exchange chromatography method and a solvent extraction method alone or in combination is mentioned. . As a method for purifying the rare earth element from the disrupted cells, a solvent extraction method, particularly a countercurrent multistage extraction method in which the solvent extraction operation is continuously performed, is preferably used from the viewpoint of labor, time and cost.
[0025]
Although the microorganism of the present invention has the property of accumulating rare earth elements efficiently, it has been reported that the microorganism belongs to the genus Variovorax and has no such property.
[0026]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0027]
Example 1
One loopful of the R-1 strain was inoculated from a slant medium into 5 ml of a Y-containing medium (Y concentration: 5 ppm) sterilized by filtration with a sterilized filter made of 0.2 μm polysulfone, followed by shaking culture at 30 ° C. for 24 hours. Culture was performed. 5 ml of the culture solution thus obtained was placed in a 500 ml branch-equipped Sakaguchi flask containing 100 ml of a Y-containing medium similarly filtered and sterilized, followed by main culture at 30 ° C. for 15 days. During this culture period, 5 ml of the culture solution was collected every 24 or 48 hours, and the Y concentration of the collected culture solution was measured according to the measurement method described in the action, and simultaneously, the turbidity (OD ₆₆₀₎ at a wavelength of 660 nm was measured. ) And the pH of the culture was measured. The turbidity was measured by pouring the culture solution into a test tube portion on the side of the flask and inserting a measuring instrument into the tube.
[0028]
FIG. 1 shows changes in the Y concentration, turbidity and pH of the culture solution of the R-1 strain with respect to the culture time obtained in this manner. As shown in FIG. 1, the Y concentration of the R-1 strain after cultivation reached a value close to 0 in a short time (6 days) after the start of the cultivation, and thereafter remained substantially constant. It can be seen that the change has a shape close to an exponential curve. In addition, the bacteria grow exponentially, and the decrease curve of the Y concentration by the R-1 strain is symmetrical to the above growth curve. Therefore, the decrease in the Y concentration is due to the biological activity of the R-1 strain. It is believed that there is.
[0029]
Example 2
One loopful of the R-1 strain is inoculated from a slanted medium into 5 ml of a Y-containing medium (Y concentration: 5 ppm) sterilized by filtration with a 0.2 μm polysulfone sterilized filter, and cultured with shaking at 30 ° C. for 7 days. After culturing for 7 days, 1.5 ml of the Y-containing culture solution was placed in an Eppendorf centrifuge tube, and centrifuged (12,000 rpm, 4 ° C., 10 minutes) to obtain a culture supernatant. Subsequently, 1 ml of 150 mM NaCl was added to and suspended in the precipitate (cells) obtained by the centrifugation, and the suspension was centrifuged again to obtain a supernatant. This operation was repeated three times in total, and the supernatant was combined to obtain a washing solution.
[0030]
1 ml of 150 mM NaCl was added to the precipitate (cells) thus washed and suspended. The suspension was crushed using an ultrasonic crusher (Branson, Duty: 30, output: 3, 30 seconds × 5 times) to crush the bacterial cells. 5 ml of 150 mM NaCl was further added and centrifuged to obtain a cell extract. Then, 0.5 ml of concentrated sulfuric acid and 0.5 ml of concentrated nitric acid were added to the residue of the cells after centrifugation, and the mixture was heated and acid-decomposed, and 1 ml of 150 mM NaCl was added thereto to obtain a cell suspension.
[0031]
As a comparison, the same evaluation was performed using Escherichia coli IFO 1041.
[0032]
The Y concentration contained in each of the obtained fractions was measured using inductively coupled plasma ICP. The result is shown in FIG.
[0033]
As is clear from the results, it was found that Y, which was not accumulated at all in Escherichia coli, was accumulated in the cells of the R-1 strain at about 70% of the initial concentration.
[0034]
Example 3
A rare-earth element-containing liquid medium having the same medium composition as in Table 1 except that a nitrate solution of each of the rare-earth elements [lanthanoids (La to Lu) and yttrium (Y)] shown in FIG. 3 is used instead of the yttrium nitrate solution. The solution was sterilized by filtration with a 0.2 μm polysulfone sterilized filter. 5 ml of this rare earth element-containing liquid medium (each rare earth element concentration: 5 ppm) was inoculated with one platinum loop of the R-1 strain from the slant medium, and cultured with shaking at 30 ° C. for 7 days. The element concentration was measured, and the reduction rate with respect to the initial concentration was calculated. The method of measuring the concentration of each rare earth element is the same as the method of measuring the yttrium concentration described in the operation except that the rare earth element used is different. However, since the sensitivity at a wavelength of 655 nm differs for each element, a standard curve is used. Was prepared for each element.
[0035]
The results are shown in FIG. As shown in FIG. 3, the substance having a high reduction rate is biased toward the side with the smaller atomic weight (left side in FIG. 3), and praseodymium (Pr) is slightly lower (22%), but the range from yttrium to neodymium is about 40%. Shows a relatively high rate of decrease. In addition, since all elements from lanthanum to neodymium exhibiting a high reduction rate are limited to a valence of 3 or more, when yttrium or a light rare earth element having a valence of 3 or more is used, the reduction rate is reduced. Is thought to be higher.
[0036]
【The invention's effect】
As described above, the microorganism belonging to the genus Variovorax according to the present invention is a novel microorganism, and can efficiently accumulate rare earth elements, particularly, yttrium, lanthanum, cerium, praseodymium, and neodymium.
[Brief description of the drawings]
FIG. 1 shows a change in yttrium (Y) concentration in a culture solution and a change in turbidity and pH of a culture solution with respect to a culture time for explaining rare earth element accumulation ability by a microorganism of the present invention. It is.
FIG. 2 is a diagram illustrating a distribution state of Y;
FIG. 3 is a diagram showing selectivity for lanthanoids by the microorganism of the present invention.

Claims (1)

Variovorax paradoxus R-1 strain which is FERM P-14492 capable of accumulating rare earth elements.

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