JP2018093744A - Base material for culturing magnetotactic bacteria - Google Patents
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Abstract
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本発明は、磁性細菌培養用基材及びこれを用いた磁性細菌の集積方法に関する。 The present invention relates to a magnetic bacteria culture substrate and a method for accumulating magnetic bacteria using the same.
磁性細菌は、細胞内に単体鉄やフェライトの小粒を作り、磁性を持つ細菌である。この磁性細菌が有する磁性微粒子は、磁気記録材料、医療分野における薬剤キャリア等として期待されている（特許文献１〜３）。また、磁性細菌をバイオレメディエーションに応用しようとする研究もされている。 A magnetic bacterium is a bacterium that has magnetism by making small particles of simple iron or ferrite in the cell. The magnetic microparticles possessed by the magnetic bacteria are expected as magnetic recording materials, drug carriers in the medical field, etc. In addition, research has been conducted to apply magnetic bacteria to bioremediation.
このような磁性細菌培養用の培地としては、コハク酸、ガラクトース、アミノ酸類、硝酸塩、キナ酸鉄、チオグリコール酸ナトリウムを含有する培地（特許文献１、３）、ＭＳＧＭ培地（非特許文献１、２）等が用いられている。 As a medium for culturing such magnetic bacteria, a medium containing succinic acid, galactose, amino acids, nitrate, iron quinate, sodium thioglycolate (Patent Documents 1 and 3), an MSGM medium (Non-Patent Documents 1, 2) etc. are used.
しかしながら、従来の磁性細菌培養培地組成は、複雑でかつ高価な試薬を複数種使用するため、大量培養には応用できず、また磁性細菌の選択性が低く、自然界に他の多くの菌とともに存在する細菌叢の中から磁性細菌を選択的に集積させることは困難であった。
本発明の課題は、磁性細菌を選択的に集積培養することができる磁性細菌培養用基材を提供することにある。
However, the conventional magnetic bacteria culture medium composition uses multiple types of complex and expensive reagents, so it cannot be applied to mass culture, and the selectivity of magnetic bacteria is low, and it exists with many other bacteria in nature. It was difficult to selectively accumulate magnetic bacteria from the flora.
The subject of this invention is providing the base material for magnetic bacteria culture | cultivation which can carry out selective accumulation culture | cultivation of magnetic bacteria selectively.
そこで本発明者は、磁性細菌を選択的に増殖させることができる材料について種々検討したところ、全く意外にも、炭酸塩と鉄化合物と硫黄とを含有する基材を用いれば、磁性細菌が選択的に増殖し、多種の菌が存在する菌叢の中から磁性細菌を集積させることができることを見出し、本発明を完成した。 Therefore, the present inventor has conducted various studies on materials that can selectively propagate magnetic bacteria. Surprisingly, if a substrate containing carbonate, iron compound, and sulfur is used, magnetic bacteria can be selected. The present invention was completed by finding that magnetic bacteria can be accumulated from a bacterial flora that proliferates and has various types of bacteria.
すなわち、本発明は、次の〔１〕〜〔７〕を提供するものである。 That is, the present invention provides the following [1] to [7].
〔１〕炭酸カルシウム及び炭酸マグネシウムから選ばれる１種以上の炭酸塩と、鉄化合物と、硫黄とを含有する磁性細菌培養用基材。
〔２〕形態が粒状物又は塊状物であり、当該各粒状物又は各塊状物中に前記炭酸塩、鉄化合物及び硫黄が含まれている〔１〕記載の磁性細菌培養用基材。
〔３〕前記鉄化合物が、硫酸第一鉄、塩化第一鉄、硝酸第一鉄及びクエン酸第一鉄から選ばれる１種以上である〔１〕又は〔２〕記載の磁性細菌培養用基材。
〔４〕炭酸カルシウム及び炭酸マグネシウムから選ばれる１種以上の炭酸塩と鉄化合物と硫黄とを含有する混合物を加熱溶融後急冷固化し、次いで得られた固化物を粉砕及び／又は造粒することを特徴とする磁性細菌培養用基材の製造方法。
〔５〕得られる磁性細菌培養用基材の形態が粒状物又は塊状物であり、当該各種粒状物又は各塊状物中に前記炭酸塩、鉄化合物及び硫黄が含まれている〔４〕記載の磁性細菌培養用基材の製造方法。
〔６〕前記鉄化合物が、硫酸第一鉄、塩化第一鉄、硝酸第一鉄及びクエン酸第一鉄から選ばれる１種以上である〔４〕又は〔５〕記載の磁性細菌培養用基材の製造方法。
〔７〕〔１〕〜〔３〕のいずれか１項記載の磁性細菌培養用基材を含有する培地中で、磁性細菌を含有する微生物混合物を培養することを特徴とする磁性細菌の集積方法。
[1] A magnetic bacterial culture substrate containing at least one carbonate selected from calcium carbonate and magnesium carbonate, an iron compound, and sulfur.
[2] The substrate for cultivating magnetic bacteria according to [1], wherein the form is granular or massive, and the carbonate, iron compound and sulfur are contained in each granular or massive.
[3] The magnetic bacterial culture base according to [1] or [2], wherein the iron compound is at least one selected from ferrous sulfate, ferrous chloride, ferrous nitrate, and ferrous citrate. Wood.
[4] A mixture containing at least one carbonate selected from calcium carbonate and magnesium carbonate, an iron compound, and sulfur is heated and melted, and then rapidly solidified, and then the obtained solidified product is pulverized and / or granulated. A method for producing a substrate for culturing magnetic bacteria, characterized by comprising:
[5] The form of the obtained substrate for cultivating magnetic bacteria is a granule or a lump, and the carbonate, iron compound and sulfur are contained in the various granule or lump. A method for producing a substrate for culturing magnetic bacteria.
[6] The magnetic bacterial culture substrate according to [4] or [5], wherein the iron compound is at least one selected from ferrous sulfate, ferrous chloride, ferrous nitrate and ferrous citrate. A method of manufacturing the material.
[7] A method for accumulating magnetic bacteria, comprising culturing a microorganism mixture containing magnetic bacteria in a medium containing the substrate for culturing magnetic bacteria according to any one of [1] to [3] .
本発明の磁性細菌培養用基材を用いれば、磁性細菌を選択的に増殖させることができるため、自然界等の多種の菌を含有する菌叢の中から磁性菌を集積培養できる。 By using the substrate for culturing magnetic bacteria of the present invention, magnetic bacteria can be selectively grown, and therefore, magnetic bacteria can be cultivated from a flora containing various bacteria such as the natural world.
本発明の磁性細菌培養用基材は、（Ａ）炭酸カルシウム及び炭酸マグネシウムから選ばれる１種以上の炭酸塩と、（Ｂ）鉄化合物と、（Ｃ）硫黄とを含有する。 The substrate for culturing magnetic bacteria of the present invention contains (A) one or more carbonates selected from calcium carbonate and magnesium carbonate, (B) an iron compound, and (C) sulfur.
本発明に用いられる（Ａ）炭酸塩は、炭酸カルシウム及び炭酸マグネシウムから選ばれる１種以上である。これらの炭酸塩は、１種を用いてもよいし２種を混合して用いてもよい。
（Ａ）炭酸塩としては、これらの炭酸塩を含有する材料、例えば重質炭酸カルシウム、軽質炭酸カルシウム、石灰石粉末、苦灰石粉末、貝化石粉末、炭酸マグネシウム、炭酸マグネシウムカルシウムを用いることができる。
(A) carbonate used for this invention is 1 or more types chosen from calcium carbonate and magnesium carbonate. These carbonates may be used alone or in combination of two.
(A) As a carbonate, materials containing these carbonates, for example, heavy calcium carbonate, light calcium carbonate, limestone powder, dolomite powder, shell fossil powder, magnesium carbonate, magnesium carbonate calcium can be used. .
本発明に用いられる（Ｂ）鉄化合物としては、無機酸第一鉄が好ましく、硫酸第一鉄、塩化第一鉄、硝酸第一鉄及びクエン酸第一鉄から選ばれる１種以上がより好ましい。これらの鉄化合物は、１種を用いてもよいし、２種以上を混合して用いてもよい。 As the iron compound (B) used in the present invention, ferrous inorganic acid is preferable, and at least one selected from ferrous sulfate, ferrous chloride, ferrous nitrate and ferrous citrate is more preferable. . These iron compounds may be used alone or in combination of two or more.
本発明に用いられる（Ｃ）硫黄は、磁性細菌の基質になると考えられる。 (C) Sulfur used in the present invention is considered to be a substrate for magnetic bacteria.
本発明の磁性細菌培養用基材は、前記（Ａ）炭酸塩と（Ｂ）鉄化合物と（Ｃ）硫黄とを含有するが、その含有比は磁性細菌の選択的増殖促進効果の点から、重量比（Ａ：Ｂ：Ｃ）で５０〜１５０：０．１〜１０：１５０〜５０が好ましく、８０〜１２０：０．１〜１０：１２０〜８０がより好ましく、９０〜１１０：０．１〜１０：１１０〜９０がさらに好ましい。 The substrate for culturing magnetic bacteria of the present invention contains the above-mentioned (A) carbonate, (B) iron compound, and (C) sulfur, but the content ratio is from the point of selective growth promoting effect of magnetic bacteria, The weight ratio (A: B: C) is preferably 50 to 150: 0.1 to 10: 150 to 50, more preferably 80 to 120: 0.1 to 10: 120 to 80, and 90 to 110: 0.1. 10 to 110: 90 is more preferable.
本発明の磁性細菌培養用基材中には、磁性細菌の選択的増殖促進効果の点から、前記（Ａ）炭酸塩、（Ｂ）鉄化合物及び（Ｃ）硫黄が均一に分散しているのが好ましく、一粒子中にこれらの３成分を含有しているのがより好ましい。かかる観点から、本発明磁性細菌培養用基材は、粒状物又は塊状物であって、各粒状物又は各塊状物中に前記３成分が含まれている形態が好ましい。 In the base for culturing magnetic bacteria of the present invention, the (A) carbonate, (B) iron compound and (C) sulfur are uniformly dispersed from the viewpoint of the selective growth promoting effect of magnetic bacteria. It is more preferable that one particle contains these three components. From this viewpoint, the substrate for culturing magnetic bacteria of the present invention is preferably a granular material or a massive material, and each granular material or each massive material contains the three components.
本発明の磁性細菌培養用基材の粒子径は、磁性細菌の選択的増殖促進効果の点から、１０μｍ〜３０ｍｍが好ましく、１００μｍから３０ｍｍがより好ましく、１００μｍから２０ｍｍがさらに好ましい。 The particle diameter of the magnetic bacteria culture substrate of the present invention is preferably 10 μm to 30 mm, more preferably 100 μm to 30 mm, and even more preferably 100 μm to 20 mm, from the viewpoint of the selective growth promoting effect of magnetic bacteria.
本発明の磁性細菌培養用基材は、例えば、（Ａ）炭酸カルシウム及び炭酸マグネシウムから選ばれる１種以上の炭酸塩と、（Ｂ）鉄化合物と、（Ｃ）硫黄とを含有する混合物を加熱溶融後急冷固化し、次いで得られた固化物を粉砕及び／又は造粒することにより製造できる。 The base material for culture of magnetic bacteria of the present invention is, for example, a mixture containing at least one carbonate selected from (A) calcium carbonate and magnesium carbonate, (B) an iron compound, and (C) sulfur. It can be manufactured by quenching and solidification after melting, and then pulverizing and / or granulating the obtained solidified product.
前記（Ａ）炭酸塩、（Ｂ）鉄化合物及び（Ｃ）硫黄の混合比は、前記含有比のとおりである。 The mixing ratio of (A) carbonate, (B) iron compound and (C) sulfur is as described above.
得られた混合物の加熱溶融条件は、例えば１２０℃〜１５０℃に加熱すればよく、より好ましくは１３０℃〜１４０℃に加熱すればよい。次に急冷固化条件は、例えば１〜１０℃／ｓｅｃの条件で急冷すればよい。具体的には、固化物を２０±５℃の水を使用して冷却すればよい。 What is necessary is just to heat the melt conditions of the obtained mixture to 120 to 150 degreeC, for example, and to heat to 130 to 140 degreeC more preferably. Next, the rapid solidification conditions may be, for example, rapid cooling under conditions of 1 to 10 ° C./sec. Specifically, the solidified product may be cooled using 20 ± 5 ° C. water.
得られた固化物の粉砕には、ジョークラッシャー、ジャイレトリークラッシャー、ハンマーミル、ボールミル等を用いることができる。また、造粒は、例えば、パンペレタイザー、ディスクペレッター、ブリケットマシン等を用いることができる。これらの粉砕及び／又は造粒後、さらに必要により篩分けにより、前記の粒子径に調整するのが好ましい。 For crushing the obtained solidified product, a jaw crusher, a gyratory crusher, a hammer mill, a ball mill or the like can be used. For granulation, for example, a pan pelletizer, a disk pelleter, a briquette machine or the like can be used. After the pulverization and / or granulation, it is preferable to adjust to the above particle diameter by sieving as necessary.
本発明の磁性細菌培養用基材を用いて、磁性細菌含有水等の微生物混合物を培養すれば、磁性細菌が選択的に増殖するため、磁性細菌を集積することができる。従って、本発明は、前記の磁性細菌培養用基材を含有する培地中で、磁性細菌を含有する微生物混合物を培養することを特徴とする磁性細菌の集積方法を提供する。 When a microorganism mixture such as magnetic bacteria-containing water is cultured using the substrate for culturing magnetic bacteria of the present invention, the magnetic bacteria are selectively grown, so that the magnetic bacteria can be accumulated. Accordingly, the present invention provides a method for accumulating magnetic bacteria, comprising culturing a microorganism mixture containing magnetic bacteria in a medium containing the above-mentioned substrate for culturing magnetic bacteria.
ここで、微生物混合物としては、磁性細菌が含まれている可能性のある水溶液であり、例えば生活排水、産業排水、畜産排水、農業排水、水産養殖排水、地下水、河川水、海水等が挙げられる。 Here, the microbial mixture is an aqueous solution that may contain magnetic bacteria, and examples include domestic wastewater, industrial wastewater, livestock wastewater, agricultural wastewater, aquaculture wastewater, groundwater, river water, seawater, and the like. .
培地には、本発明の磁性細菌培養用基材の他、コハク酸、トリプトファン、グルコース、マルトース等の炭素源、塩化アンモニウム、硝酸カリウム、リン酸カリウム等の無機塩、微量金属、水等を含有するのが好ましい。なお、ｐＨは６．５〜７．５が好ましい。培地中の本発明磁性細菌培養用基材の含有量は、１５０〜４００ｇ／Ｌが好ましく、２００〜３５０ｇ／Ｌがより好ましく、２５０〜３００ｇ／Ｌがさらに好ましい。 In addition to the magnetic bacterial culture substrate of the present invention, the medium contains carbon sources such as succinic acid, tryptophan, glucose and maltose, inorganic salts such as ammonium chloride, potassium nitrate and potassium phosphate, trace metals, water and the like. Is preferred. The pH is preferably 6.5 to 7.5. 150-400 g / L is preferable, as for content of the base material for culture | cultivation of this invention in a culture medium, 200-350 g / L is more preferable, 250-300 g / L is further more preferable.
培養条件は、磁性細菌が生育する条件であればよく、例えば濃度が０．１〜５ｍｇ／Ｌ、より好ましくは１〜５ｍｇ／Ｌの条件でｐＨ６．０〜８．０、１０〜４０℃の条件で行えばよい。 The culture conditions may be any conditions that allow magnetic bacteria to grow. For example, the concentration is 0.1 to 5 mg / L, more preferably 1 to 5 mg / L, and pH 6.0 to 8.0 and 10 to 40 ° C. It can be done under conditions.
なお、本発明における磁性細菌としては、Magnetovibrio blakemorei、Magnetospirillum magnetotacticum、Magnetospirillum gyphiswaldense等が挙げられる。 Examples of magnetic bacteria in the present invention include Magnetovibrio blakemorei, Magnetospirillum magnetotacticum, Magnetospirillum gyphiswaldense, and the like.
次に実施例を挙げて本発明を更に詳細に説明する。 EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.
実施例１
（１）試験方法
表１に示す配合で硫黄（粉末硫黄、Ｓ ２００メッシュ、細井化学工業社製）を１２０℃で加熱して溶融させ、炭酸カルシウム（Ｔ−２００、ニッチツ社製）、鉄化合物（硫酸第一鉄、試薬、関東化学社製）と均一に攪拌混合させた後に冷却水で急冷した。得られた固化物を破砕して篩い分けをし、粒径が５〜２０ｍｍの基材を作製した。５００ｍＬ容三角フラスコに磁性細菌Magnetospirillum magnetotacticum ＮＢＲＣ１５２７２培養液と表１の組成の基材を１００ｇ入れ、表２に示す組成の培地を４００ｍＬ添加して３０℃で２週間静置培養した（ただし比較例４は基材投入無し）。２週間後の培養液中の微生物数を計測した。培養液中の微生物数は、研究用生物顕微鏡Ｎｉ−Ｕ（Ｎｉｋｏｎ社製）を用いて顕微鏡法で計測した。
Example 1
(1) Test method Sulfur (powder sulfur, S 200 mesh, manufactured by Hosoi Chemical Co., Ltd.) was heated and melted at 120 ° C. in the formulation shown in Table 1, and calcium carbonate (T-200, manufactured by Nichetsu Co., Ltd.), iron compound (Ferrous sulfate, reagent, manufactured by Kanto Chemical Co., Inc.) was stirred and mixed uniformly, and then quenched with cooling water. The obtained solidified product was crushed and sieved to produce a substrate having a particle size of 5 to 20 mm. A 500 mL Erlenmeyer flask was charged with 100 g of the magnetic bacterium Magnetospirillum magnetotacticum NBRC15272 culture solution and a substrate having the composition shown in Table 1 and 400 mL of a medium having the composition shown in Table 2 was added, followed by standing culture at 30 ° C. for 2 weeks (Comparative Example 4). Is no base material). The number of microorganisms in the culture solution after 2 weeks was counted. The number of microorganisms in the culture solution was measured by a microscopic method using a research biological microscope Ni-U (manufactured by Nikon).
（２）試験結果
試験結果を表３に示す。実施例１〜７はいずれも比較例１及び２に比べて２週間後の細菌数が１オーダー程度高く、磁性細菌の培養基材として適していることを確認した。
(2) Test results Table 3 shows the test results. In each of Examples 1 to 7, the number of bacteria after 2 weeks was about 1 order higher than Comparative Examples 1 and 2, and it was confirmed that it was suitable as a culture substrate for magnetic bacteria.
実施例２
（磁性細菌の集積培養）
（１）試験方法
水域環境より試料を採取してＤＮＡを抽出し、１６ＳｒＲＮＡ遺伝子Ｖ４領域（約２５０ｂｐ）を対象とし、次世代シークエンスを用いた微生物相の解析を実施した。
更に、採取した試料を用いて磁性細菌の集積培養を実施した。５００ｍＬ容三角フラスコに試料１０ｇと培養基材１（実施例２：硫黄：炭カル：硫酸第一鉄＝１００：１００：１）または培養基材２（比較例１：硫黄：炭カル＝１００：１００、硫酸第一鉄は添加無し）のいずれかを１００ｇ入れ、表４に示す組成の培地を４００ｍＬ添加し、３０℃で静置培養した。１週間に１回培養液の半分をデカントして新たに作製した培地を加える半回分式で実施した。集積２ヵ月後の培養基材１および２の表面に付着したバイオフィルムよりＤＮＡを抽出し、同様に次世代シークエンスを用いて微生物相を解析した。
Example 2
(Magnetic bacteria accumulation culture)
(1) Test method A sample was collected from an aquatic environment, DNA was extracted, and the 16S rRNA gene V4 region (about 250 bp) was targeted, and the microflora was analyzed using the next-generation sequence.
Furthermore, magnetic bacteria were accumulated and cultured using the collected samples. In a 500 mL Erlenmeyer flask, 10 g of sample and culture substrate 1 (Example 2: sulfur: charcoal cal: ferrous sulfate = 100: 100: 1) or culture substrate 2 (Comparative Example 1: sulfur: charcoal cal = 100: 100, no ferrous sulfate added), 400 mL of a medium having the composition shown in Table 4 was added, and static culture was performed at 30 ° C. Half a half of the culture solution was decanted once a week. DNA was extracted from the biofilm attached to the surfaces of the culture substrates 1 and 2 after 2 months of accumulation, and the microflora was similarly analyzed using the next-generation sequence.
（２）試験結果
集積培養前の試料中の分析結果を表５に、２ヶ月間の集積培養後の培養基材１の分析結果を表６、２ヶ月間の集積培養後の培養基材２の分析結果を表７に示す。集積培養前は多様な細菌種が存在し、磁性細菌であるMagnetovibrio blakemoreiの検出率は０．１８％と極めて低かった。また硫酸第一鉄を含まない培養基材２を用いて集積しても検出率は３．５３％と低くSulfurimonas denitrificansなどの硫黄脱窒菌が集積されたのに対して、硫酸第一鉄を含む培養基材１を用いた集積では８２．９４％とMagnetovibrio blakemoreiの検出率が非常に高くなり、硫酸第一鉄を含む培養基材１を用いることで磁性細菌が集積されることが確認された。
(2) Test results Table 5 shows the analysis results in the sample before enrichment culture, Table 6 shows the analysis results of the culture substrate 1 after enrichment culture for 2 months, and Table 6 shows the culture substrate 2 after enrichment culture for 2 months. Table 7 shows the analysis results. Various bacterial species existed before enrichment culture, and the detection rate of magnetic bacterium Magnetovibrio blakemorei was as low as 0.18%. Moreover, even if it accumulates using the culture base material 2 which does not contain ferrous sulfate, the detection rate is as low as 3.53%, whereas sulfur denitrifying bacteria such as Sulfurimonas denitrificans are accumulated, whereas it contains ferrous sulfate. In the accumulation using the culture substrate 1, the detection rate of Magnetovibrio blakemorei was 82.94%, and it was confirmed that magnetic bacteria were accumulated by using the culture substrate 1 containing ferrous sulfate. .
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| CN105572396A (en) * | 2016-03-17 | 2016-05-11 | 北京中科圆融生物科技发展有限公司 | Bacterial magnetic particles with recombinant protein G expression capability and application thereof |
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| BAZYLINSKI D. A. ET AL., ARCH MICROBIOL., vol. 182 (2004), JPN6020027603, pages 373 - 387, ISSN: 0004444070 * |
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