JP4095682B2 - Rare earth element accumulation microorganism - Google Patents

Description

【００１４】
また、培養上清中に残存するイッテルビウム（Ｙｂ）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、培養した後の培養液サンプル約１．４ｍｌをエッペンドルフ遠心管に入れ、遠心分離（４℃、１２，０００ｒｐｍ×１０分間）することによって得られた培養上清１ｍｌを新しい試験管に入れる。さらに、この試験管に、予め調製しておいた０．１％アルセナゾIII (Arsenazo III)溶液及び塩酸・塩化カリウム緩衝液（２５ｍｌ ０．２Ｍ ＫＣｌ、５９ｍｌ ０．２Ｍ ＨＣｌ及び１６ｍｌ 脱塩水を混合して１００ｍｌに定容した溶液）を各々０．５ｍｌずつ加えた後、最後に純水１ｍｌを加え、ボルテクスにより良く撹拌した。この混合溶液を暗所で３０〜６０分間放置した後、サンプル溶液の６５５ｎｍの吸光度を測定し、以下のようにして作成された標準直線に基づいて各サンプル中に含まれるイッテルビウム（Ｙｂ）の濃度を求めた。標準直線は、試験管に各々０、１、２、３、４、５μｌずつＹｂ（ＮＯ₃ ）₃ 溶液を加え、そこに純水１ｍｌを加えたものをサンプルとしかつ遠心分離を行わない以外は上記と同様の比色操作方法を行うことによって、作成した。なお、この際、Ｙｂ（ＮＯ₃ ）₃ 溶液としては、原子吸光用の硝酸イッテルビウム溶液［１モル／リットルの硝酸溶液におけるＹｂ（ＮＯ₃ ）₃ 濃度＝１．０ｍｇＹｂ／ｍｌ］（和光純薬製）を使用した。
【００１５】
上記スクリーニングによって、Ｙｂ含有培地中のイッテルビウム濃度を他の菌株と比べて著しく減少させる１菌株が得られた。以下に、この菌株の菌学的性質を示す。
【００１６】
（ａ）形態
１） 細胞の形および大きさ： 長桿菌で長さ１．０〜２．０μｍ、直径３．０〜４．０μｍ
２） 細胞の多形性の有無： 無し
３） 運動性の有無： 有り
４） 胞子の有無： 無し
（ｂ）培養的性質
１） 肉汁寒天平板培養（３０℃）： コロニーは円形で不透明なピンク色でかつ凸状であり、表面は滑らかで光沢がある。コロニーの大きさは、３０℃で５日間培養した際、直径約２ｍｍであり、希釈肉汁寒天培地では、３０℃で５日間培養した際、直径約１ｍｍである。
２） ベネット寒天培地（３７℃）： ＋
ベネット寒天培地（４１℃）： 弱い＋
ベネット寒天培地（４５℃）： −
なお、ベネット寒天培地(Bennett′s agar) の組成は、酵母エキス０．１％、牛肉エキス０．１％、ＮＺアミンＡ０．２％、ブドウ糖１．０％、寒天２．０％（ｐＨ７．３）である。
【００１７】
（ｃ）生理学的性質（３０℃、１４日間）
１） グラム染色性： −
２） カタラーゼ： ＋
３） オキシダーゼ： −
４） Ｏ−Ｆテスト： −
５） 酸素に対する態度： 好気性
（ｄ） その他の生理学的性質
１） メタノールの利用： ＋
２） グルコースの利用： −
３） フラクトースの利用： ＋
４） 酢酸塩の利用： ＋
５） ベタインの利用： −
６） メチルアミンの利用： −
７） Ｎ，Ｎ−ジメチルホルムアミドの利用： −
８） 水酸化テトラメチルアンモニウムの利用： −
９） トリメチルアミンの利用： −
１０） アスパラギン酸塩の利用： 僅かに＋
１１） グルタミン酸塩の利用： ＋
１２） フコースの利用： ＋
１３） アラビノースの利用： ＋
１４） クエン酸の利用： ＋
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 ）だけでは分類不可能であったため、本菌の同定をザ ナショナル コレクションズ 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 National Collections of Industrial and Marine Bacteria Limited)に依頼（参照番号：ＩＤ ２６６０／ＮＣＩＤ ４２９５、日付：１９９５年３月２３日の報告書類における菌株３−１）したところ、非定型の陰性のグルコース反応を有するメチロバクテリウム フジサワエンス(Methylobacterium fujisawaense) に近似することが分かった［参考文献：グラム−ネガティブ メチロトロフィック ロッズ アンド コッキー(Gram-negative Methylotrophic Rods and Cocci) 、ピーエヌ グリーン(P.N. Green)、アイデンティフィケーション メソッズ イン アプライド アンド エンヴァーロメンタル マイクロバイオロジー(Identification Methods in Applied and Environmental Microbiology)１９９２年、アールジー ボード(R.G. Board)、ディ ジョーンズ(D. Jones)及びエフエー スキナー(F.A. Skinner)、ソサイエティ フォー アプライド バクテリオロジー テクニカル シリーズ(Society for Applied Bacteriology Technical Series) ２９、ブラックウェル サイエンティフィック(Blackwell Scientific)における］。
【００１８】
従来まで、メチロバクテリウム フジサワエンス(Methylobacterium fujisawaense) 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、本菌株をメチロバクテリウム フジサワエンス(Methylobacterium fujisawaense) ３−１株 （以下、単に３−１株と称する）と命名した。また、この３−１株は、平成７年９月２９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−１５２１１号である。
【００１９】
本発明では３−１株を使用したが、この菌株に限られることなく、希土類元素を集積することが可能なメチロバクテリウム属(Methylobacterium)に属する微生物であれば使用できる。
【００２０】
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、使用する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。
【００２１】
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、使用する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、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、グリセロール、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。
【００２２】
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。
【００２３】
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。
【００２４】
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は、培地中に希土類元素を添加することが好ましい。添加される希土類元素の種類及び添加量は、使用する微生物が集積しやすい希土類源の種類によるが、例えば、３−１株を使用する場合には、イッテルビウム、ランタン、ネオジム、テルビウム及びツリウムを、１〜２０ｐｐｍ、より好ましくは２〜１０ｐｐｍの濃度で、添加することが望ましい。この際、添加する希土類元素は２種以上の混合物または合金であってもよい。
【００２５】
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。
【００２６】
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。
【００２７】
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、メチロバクテリウム属(Methylobacterium)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。
【００２８】
【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。
【００２９】
実施例１
以下の実験を行うことにより、本発明の３−１株によるＹｂ濃度の経時的な変化を観察した。
【００３０】
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Ｙｂ含有培地 （Ｙｂ濃度：５ｐｐｍ）５ｍｌに斜面培地より３−１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前培養液５ｍｌを、同様にして瀘過滅菌したＹｂ含有培地９５ｍｌの入った５００ｍｌの枝付き坂口フラスコに入れ、３０℃で１０日間本培養を行った。この培養期間中、２４時間おきに培養液を３ｍｌ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従ってＹｂ濃度を測定すると同時に、菌の生育を６６０ｎｍの波長における濁度（ＯＤ₆₆₀ ）として求め、さらに、培養液のｐＨの変化をｐＨメーターにて測定した。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。
【００３１】
このようして得られた培養時間に対する３−１株の培養液のＹｂ濃度の変化および濁度及びｐＨの変化を図１に示す。図１より、３−１株は、３日目から急激に濁度が上がる（即ち、急速に増殖する）とともに、ｐＨの上昇及び急激なＹｂ濃度の減少が示され、その後は、５日目から生育が定常状態となり、ｐＨは６日目から、Ｙｂ濃度は９日目から一定になることが示された。Ｙｂ濃度の減少が逆Ｓ字曲線を描いており、菌株の成長と共にＹｂ濃度が減少していることから、菌株の増殖とＹｂの集積とは関連が有る、即ち、培養中に菌体内にイッテルビウムが蓄積されたのではないかと考えられる。
【００３２】
実施例２
以下の実験を行うことにより、本発明の３−１株の各種希土類元素に対する選択性を調べた。
【００３３】
図２に記載の各種希土類元素［プロメチウム（Ｐｍ）を除くランタノイド（Ｌａ〜Ｌｕ）及びイットリウム （Ｙ）］の硝酸溶液を硝酸イッテルビ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、希土類元素含有液体培地を作製した。
【００３４】
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希土類元素を含まない希釈肉汁培地５ｍｌに斜面培地より３−１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前培養液１００μｌを、上記したように予め別に調製しておいた各希土類元素含有液体培地５ｍｌに入れ、３０℃で１０日間振盪培養することによって本培養を行い、所定時間培養した後の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テルビ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に、標準曲線を各元素ごとに作成した。
【００３５】
また、比較対照として、大腸菌(E. coli) ＪＭ１０９株を各種希土類元素含有培地で培養して、各希土類元素濃度の減少率を求めた。
【００３６】
本発明の３−１株を用いた結果を図２に示し、比較として大腸菌(E. coli) ＪＭ１０９株を用いた結果を図３に示す。その結果、図２に示されるように、３−１株は極めて高い選択性を示した。すなわち、イッテルビウムのみが唯一５０％以上の減少率を示したのみである他は、ランタン及びネオジムが３０％程度の減少率でそれ以外の元素は１０〜２０％の低い減少率であるあるいは全く減少していないものさえ確認された。また、大腸菌(E. coli) ＪＭ１０９株を用いた図３の結果との比較から、イットリウム、セリウム、サマリウム、ユウロピウム、ガドリウム、ホルミウム及びルテチウムの濃度の減少率は誤差の範囲である可能性が高いことが示唆される。
【００３７】
【発明の効果】
以上述べたように、本発明によるメチロバクテリウム属(Methylobacterium)に属する微生物は、希土類元素、特に重希土類であるイッテルビウムを効率よく集積できる新規な微生物である。
【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テルビウム（Ｙｂ）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２は、本発明の微生物の各種希土類元素に対する選択性を示す図である。
【図３】図３は、大腸菌 ＪＭ１０９株の各種希土類元素に対する選択性を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel rare earth element-accumulating microorganism. More specifically, the present invention relates to a rare earth element-integrating microorganism having the ability to efficiently accumulate a specific rare earth element.
[0002]
[Prior art]
The rare earth element is the largest element group existing in nature in the periodic table, and scandium (Sc) and yttrium are included in 15 elements (lanthanoids) from lanthanum (La) having atomic number 57 to lutetium (Lu) having 71 atomic number. (Y) is a generic name for 17 element groups, and varies depending on the magnetic properties based on the behavior of 4f electrons in an incompletely packed state of ions, optical properties such as color, and chemical properties unique to rare earths. It is used in a wide range of fields. Specifically, the 4f electron properties include the red phosphor (europium) of color tube televisions and magnets (samarium and neodymium) in small electronic devices such as quartz watches and walkmans. Examples of utilizing chemical properties include catalysts (lanthanum and cerium), high-temperature oxide superconductors, hydrogen storage alloys, ceramics, and reactor control materials used in the production of gasoline.
[0003]
These rare earth elements are contained in minerals such as monazite, bastnaesite, zenotime, euxenite, and gadolinite. Of these, monazite and bastonesite are mainly decomposed as resources of light rare earth (lanthanum to europium), and xenotime is decomposed on an industrial scale as resources of heavy rare earth (gadolinium to lutetium plus yttrium). By refining, each rare earth element can be obtained as a resource, but rare earth elements are very similar in physical and chemical properties and exist in the form of coexistence in minerals. There is a problem that it is extremely difficult to separate each other. For this reason, the fractional crystallization method and the fractional precipitation method, which are conventional rare earth element separation methods, have a small difference in solubility between the elements, so the operation must be repeated many times and the mutual separation is a very difficult task. It was. Therefore, in order to eliminate this drawback, an ion exchange method has been developed in which rare earth is adsorbed on a column packed with an ion exchange resin, and a complexing agent is used as an eluent. This method is not suitable as a mass processing method because it cannot be continuously processed and takes a long time for one operation, and the cost for regeneration and replacement of the resin is high. Therefore, a solvent extraction method that uses the difference in the method of dissolving ions in solvents that do not mix with each other has emerged instead of this method. However, this method has a problem that it is difficult to obtain a high-purity product by a single operation, and the operation must be repeated many times in order to increase the purity. For this reason, industrially, a countercurrent multistage extraction apparatus that can be operated continuously several tens of times has been developed and used, which has a larger processing amount, shorter operation time, and operation than the ion exchange method. It has advantages such as ease, and has become the mainstream of current rare earth element mass separation methods.
[0004]
Apart from the above method, a method for obtaining a microorganism capable of selectively collecting a specific rare earth element and obtaining the target rare earth element from the microorganism has been developed and proposed. Examples of such microorganisms include those described in JP-A-59-118,825. However, the above publication only discloses a method for selectively collecting yttrium using activated sludge, and has not yet specifically identified microorganisms.
[0005]
Thus, the identified microorganisms that efficiently accumulate rare earth elements are still under development, and those that are actually used industrially do not yet exist, and the development of such microorganisms is desired. Yes.
[0006]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a microorganism capable of efficiently accumulating rare earth elements.
[0007]
[Means for Solving the Problems]
The above-mentioned objects are achieved by a rare earth element-integrating microorganism belonging to the genus Methylobacterium that can accumulate rare earth elements.
[0008]
The present invention shows a rare earth element-integrating microorganism that efficiently accumulates ytterbium among rare earth elements. The present invention also shows a rare earth element-integrating microorganism in which the microorganism is Methylobacterium Fujisawaens. The present invention further shows a rare earth element-integrating microorganism having a deposit number of FERM P-15212.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The microorganism according to the present invention belongs to the genus Methylobacterium and is capable of efficiently collecting rare earth elements. Specific examples of this microorganism include Methylobacterium Fujisawaens. (Methylobacterium fujisawaense) 3-1 strain is mentioned, and this strain is obtained by the following screening method. In the following screening method, ytterbium (Yb) having divalent and trivalent valences was used as a rare earth element for screening.
[0010]
Based on the speculation that there is a high probability that the target strain with the ability to accumulate rare earths in the microbial population that can actively proliferate in a low nutrient state, the screening is divided into two stages, that is, the primary screening. As a secondary screening, a strain having the ability to accumulate ytterbium (Yb) from these bacteria, that is, reducing the Yb concentration in the culture solution, was selected. The screening method used in the present invention is specifically described below.
[0011]
In Conradi stick one by 100μl which an aqueous sample taken diluted appropriately 10 to 10 ³ times with sterile water from Hamana, following primary screening agar produced by the method (pH: 7.2.about.7.4 The agar medium was cultured at 30 ° C. for 3 to 5 days. Next, the colonies formed on the agar medium are picked, suspended in 10 ml of sterilized water and further diluted 10 ² to 10 ³ times, and then 100 μl each is applied to the same agar medium. Was repeated several times to purify the strain. By such primary screening, bacteria that can grow even in a dilute state of nutrients were obtained.
Production 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 to 7.4) (hereinafter, diluted broth (Referred to as a medium), and 1.5% (wt / v) agar added thereto and sterilized by autoclaving was dispensed into a sterilized petri dish about 30 ml at a time, and a plate medium (hereinafter referred to as diluted meat juice agar medium). Called).
[0012]
Next, the sterilized secondary screening liquid medium produced by the following method was dispensed into test tubes that were previously autoclaved with 5 ml of silicon stoppers. Each of the isolated bacterial cells purified by the primary screening was inoculated into each platinum ear with a platinum loop, and cultured with shaking at 30 ° C. for 10 days. After culturing, the ytterbium (Yb) concentration remaining in the culture supernatant is measured according to the following method, and the ytterbium (Yb) concentration remaining in the culture supernatant is selected. Bacteria whose Yb concentration in the culture supernatant was reduced by 50% or more from the initial concentration (5 ppm) were selected as candidate bacteria. At this time, the selected strain was inoculated again into the secondary screening medium, and the same operation was repeated to confirm the reproducibility regarding the decrease in the Yb concentration. In this way, strains that efficiently accumulate rare earth elements were screened.
Method for preparing secondary screening medium: Meat juice (polypeptone 1%, meat extract 1%, NaCl 0.5%, pH 7.2 to 7.4) was diluted 100 times with demineralized water, and 5% ( v / v) ytterbium nitrate [Yb (NO ₃ ) ₃ ] solution (1 mol / liter) in an amount [0.1% (v / v)] of 5N sodium hydroxide solution previously neutralized. In addition, while stirring this solution, 1,000 ppm of Yb (NO ₃ ) ₃ solution was gradually added dropwise to a pH of about 7.2, and the solution was made up to 1 liter with demineralized water, and the polysulfone having a pore size of 0.2 μm. The mixture was sterilized by filtration with a sterilized filter, and this was used as a secondary screening medium (Yb concentration: 5 ppm) (hereinafter referred to as Yb-containing medium). The composition of this Yb-containing medium is shown in Table 1.
[0013]
[Table 1]

[0014]
Moreover, the measuring method of the ytterbium (Yb) density | concentration which remains in a culture supernatant is described below.
Place approximately 1.4 ml of the culture solution sample after inoculation and culture of the strain to be screened into an Eppendorf centrifuge tube, and centrifuge (4 ° C., 12,000 rpm × 10 minutes) to obtain 1 ml of the culture supernatant as a new test. Put in a tube. In addition, a 0.1% Arsenazo III solution prepared in advance and a hydrochloric acid / potassium chloride buffer solution (25 ml 0.2 M KCl, 59 ml 0.2 M HCl and 16 ml demineralized water) were mixed in this test tube. 0.5 ml each) was added, and finally 1 ml of pure water was added and stirred well by vortexing. After this mixed solution is allowed to stand for 30 to 60 minutes in the dark, the absorbance at 655 nm of the sample solution is measured, and the concentration of ytterbium (Yb) contained in each sample based on the standard straight line prepared as follows. Asked. The standard straight line is except that 0, 1, 2, 3, 4, 5 μl of Yb (NO ₃ ) ₃ solution is added to each test tube, 1 ml of pure water is added as a sample, and no centrifugation is performed. It was created by performing a colorimetric operation method similar to that described above. At this time, as the Yb (NO ₃ ) ₃ solution, an ytterbium nitrate solution for atomic absorption [Yb (NO ₃ ) ₃ concentration in a 1 mol / liter nitric acid solution = 1.0 mgYb / ml] (manufactured by Wako Pure Chemical Industries, Ltd.) )It was used.
[0015]
The above screening yielded one strain that significantly reduced the ytterbium concentration in the Yb-containing medium as compared to other strains. The mycological properties of this strain are shown below.
[0016]
(A) Form 1) Shape and size of cells: long bacillus 1.0-2.0 μm in length, 3.0-4.0 μm in diameter
2) Presence / absence of polymorphism in cells: None 3) Presence / absence of motility: Yes 4) Presence / absence of spores: None (b) Culture characteristics 1) Meat broth agar plate culture (30 ° C): Colony is circular and opaque pink It is colored and convex, and the surface is smooth and glossy. The size of the colony is about 2 mm in diameter when cultured for 5 days at 30 ° C., and about 1 mm in diameter when cultured for 5 days at 30 ° C. in a diluted broth agar medium.
2) Bennett agar (37 ° C): +
Bennett agar (41 ℃): weak +
Bennett agar medium (45 ° C.): −
The composition of Bennett's agar is as follows: yeast extract 0.1%, beef extract 0.1%, NZ amine A 0.2%, glucose 1.0%, agar 2.0% (pH 7. 3).
[0017]
(C) Physiological properties (30 ° C., 14 days)
1) Gram stainability: −
2) Catalase: +
3) Oxidase: −
4) OF test: −
5) Attitude toward oxygen: Aerobic (d) Other physiological properties 1) Utilization of methanol: +
2) Use of glucose: −
3) Use of fructose: +
4) Use of acetate: +
5) Use of betaine: −
6) Use of methylamine: −
7) Use of N, N-dimethylformamide: −
8) Use of tetramethylammonium hydroxide: −
9) Use of trimethylamine: −
10) Use of aspartate: slightly +
11) Use of glutamate: +
12) Use of fucose: +
13) Use of arabinose: +
14) Use of citric acid: +
Since this bacterium could not be classified only by the Bergey's Manual of Systematic Bacteriology 8th, the identification of this bacterium was performed by the National Collections of Industrial and Marine Bacteria Limited (NCIMB) (The National Collections of the Industrial and Marine Bacteria Limited (reference number: ID 2660 / NCID 4295, date: strain 3-1 in the report on March 23, 1995), a methylo having an atypical negative glucose response. [Reference: Gram-negative Methylotrophic Rods and Cocci, PN Green, Identity] Identification Methods in Applied and Environmental Microbiology, 1992, RG Board, D. Jones, FA Skinner, Society for Applied In Society for Applied Bacteriology Technical Series 29, Blackwell Scientific].
[0018]
Until now, there has been no report that Methylobacterium fujisawaense accumulates rare earth elements, and since this bacteria is clearly distinguished from known bacterial species, the microorganism of the present invention is considered to be a novel microorganism. Judging from this, this strain was named Methylobacterium fujisawaense 3-1 strain (hereinafter simply referred to as 3-1 strain). In addition, the 3-1 shares, was deposited with the Agency of life Institute of Advanced Industrial Science and Technology dated September 29, 1995, the accession number is No. FERM P-15211.
[0019]
In the present invention, strain 3-1 was used, but is not limited to this strain, and any microorganism belonging to the genus Methylobacterium capable of accumulating rare earth elements can be used.
[0020]
The medium used for culturing the strain of the present invention may be either a solid or liquid medium, and may be a medium containing a carbon source, an appropriate amount of nitrogen source, an inorganic salt, and other nutrients that can be assimilated by the bacteria used. For example, either a synthetic medium or a natural medium may be used.
[0021]
The carbon source that can be used in the culture of the strain of the present invention is not particularly limited as long as the strain used can be assimilated. Specifically, considering the utilization of microorganisms, fructose, maltose, galactose, starch, starch hydrolysate, molasses, molasses and other sugars, meat extracts, peptone, wheat, rice and other natural products, glycerol One or more alcohols such as methanol and ethanol, fatty acids such as acetic acid, gluconic acid, pyrpinic acid and citric acid, and amino acids such as glycine, glutamic acid and aspartic acid can be selected and used.
[0022]
Nitrogen sources that can be used in the culture of the strain of the present invention include meat extract, peptone, polypeptone, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, various amino acids, corn steep liquor, other animals, plants Considering the assimilation of microorganisms used from organic nitrogen compounds such as microbial hydrolysates, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, and ammonium chloride, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea, One or more types are selected and used.
[0023]
Examples of the inorganic salt that can be used in the present invention include one or more selected from phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, hydrochlorides, sulfates and acetates. Can be used. Moreover, you may add a vegetable oil, surfactant, etc. in a culture medium as needed.
[0024]
In order to efficiently accumulate rare earth elements in the microorganism of the present invention, it is preferable to add rare earth elements to the medium. The type and amount of rare earth element added depends on the type of rare earth source in which the microorganism to be used is likely to accumulate. For example, when 3-1 strain is used, ytterbium, lanthanum, neodymium, terbium and thulium are used. It is desirable to add at a concentration of 1 to 20 ppm, more preferably 2 to 10 ppm. At this time, the rare earth element to be added may be a mixture or alloy of two or more.
[0025]
In the present invention, the culture is carried out under aerobic conditions since the bacterium of the present invention is aerobic, and the culture conditions at that time are appropriately selected according to the composition of the medium and the culture method, and the strain can be grown. If it is conditions, it will not restrict | limit in particular. Usually, the culture temperature is 20 to 70 ° C., preferably 25 to 40 ° C., and the pH of the medium suitable for the culture is 5 to 9, preferably 6 to 8.
[0026]
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 Cell bodies by performing physical treatment such as treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment or chemical treatment such as cell wall lytic enzyme treatment such as lysozyme or contact treatment with a surfactant alone or in combination Examples include a method of purifying a target rare earth element from crushed cells by 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. . As a method for purifying rare earth elements from crushed cells, a solvent extraction method, particularly a countercurrent multistage extraction method in which solvent extraction operations are continuously performed, is preferably used from the viewpoint of labor, time and cost.
[0027]
The microorganism of the present invention has the property of efficiently accumulating rare earth elements, but heretofore, there has been no report on microorganisms belonging to the genus Methylobacterium and having the above characteristics.
[0028]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0029]
Example 1
By performing the following experiment, the change with time of the Yb concentration by the strain 3-1 of the present invention was observed.
[0030]
Inoculate 5 ml of Yb-containing medium (Yb concentration: 5 ppm) sterilized with a 0.2 μm polysulfone sterilized filter by inoculating one platinum ear from the slanted medium and incubate by shaking at 30 ° C. for 24 hours. Culture was performed. 5 ml of the preculture liquid thus obtained was put into a 500 ml branch Sakaguchi flask containing 95 ml of a Yb-containing medium that was similarly filter sterilized, and main culture was performed at 30 ° C. for 10 days. During this culture period, 3 ml of the culture solution was collected every 24 hours, and the Yb concentration of the collected culture solution was measured according to the measurement method described in the action, and at the same time, the growth of the bacteria was measured for turbidity (OD _{660 at} a wavelength of 660 nm). ) And the change in pH of the culture solution was measured with a pH meter. In addition, about the turbidity, the culture solution was poured into the test tubular part on the side surface of the flask, and the measurement device was inserted therein to measure.
[0031]
FIG. 1 shows changes in the Yb concentration and changes in turbidity and pH of the culture solution of the strain 3-1 with respect to the culture time thus obtained. As shown in FIG. 1, the 3-1 strain showed a sudden increase in turbidity (that is, rapid growth) from the third day, and an increase in pH and a rapid decrease in the Yb concentration. It was shown that the growth reached a steady state, and the pH became constant from the 6th day and the Yb concentration became constant from the 9th day. Since the decrease in the Yb concentration forms an inverted S-shaped curve, and the Yb concentration decreases with the growth of the strain, there is a relationship between the growth of the strain and the accumulation of Yb. May have been accumulated.
[0032]
Example 2
The selectivity for various rare earth elements of the 3-1 strain of the present invention was examined by conducting the following experiment.
[0033]
A liquid having the same medium composition as in Table 1 except that nitric acid solutions of various rare earth elements [lanthanoids (La to Lu) and yttrium (Y) excluding promethium (Pm) and yttrium (Y)] shown in FIG. 2 are used instead of the ytterbium nitrate solution. The medium was filtered and sterilized with a polysulfone sterilized filter having a pore diameter of 0.2 μm to prepare a rare earth element-containing liquid medium.
[0034]
Pre-culture by inoculating 5 ml of diluted rare broth culture medium containing no rare earth elements with a 0.2 μm sterilized filter made of polysulfone and inoculating one platinum ear of 3-1 strain from the slant medium and shaking culture at 30 ° C. for 24 hours. Went. 100 μl of the preculture solution thus obtained is placed in 5 ml of each rare earth element-containing liquid medium prepared separately as described above, and main culture is performed by shaking culture at 30 ° C. for 10 days. The concentration of each rare earth element contained in the culture solution after culturing for a time was measured, and the rate of decrease relative to the initial concentration was calculated. The method for measuring the concentration of each rare earth element is the same as the method for measuring the ytterbium concentration described in the action except that the rare earth element to be used is different, but the sensitivity at a wavelength of 655 nm differs depending on the element. Curves were created for each element.
[0035]
Further, as a comparative control, E. coli JM109 strain was cultured in various rare earth element-containing media, and the reduction rate of each rare earth element concentration was determined.
[0036]
The results using the strain 3-1 of the present invention are shown in FIG. 2, and the results using the E. coli JM109 strain are shown in FIG. 3 for comparison. As a result, as shown in FIG. 2, the strain 3-1 showed extremely high selectivity. That is, except that ytterbium only showed a reduction rate of 50% or more, lanthanum and neodymium had a reduction rate of about 30%, and other elements had a low reduction rate of 10 to 20% or not at all. Even things that were not done were confirmed. In addition, from the comparison with the results of FIG. 3 using E. coli JM109 strain, the decrease rate of the concentration of yttrium, cerium, samarium, europium, gadolinium, holmium and lutetium is likely to be in the range of error. It is suggested.
[0037]
【The invention's effect】
As described above, the microorganism belonging to the genus Methylobacterium according to the present invention is a novel microorganism that can efficiently accumulate rare earth elements, particularly ytterbium, which is a heavy rare earth.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in ytterbium (Yb) concentration in a culture solution and changes in turbidity and pH of the culture solution with respect to a culture time for explaining the ability of the microorganisms of the present invention to accumulate rare earth elements. It is.
FIG. 2 is a graph showing the selectivity of the microorganism of the present invention for various rare earth elements.
FIG. 3 is a graph showing the selectivity of Escherichia coli JM109 strain for various rare earth elements.

Claims (2)

Methylobacterium Fujisawaens , which has a deposit number of FERM P-15212 and can accumulate rare earth elements.   The methylobacterium fujisawaens according to claim 1, which accumulates particularly ytterbium among rare earth elements efficiently.

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