JP2019025379A - Method for producing microbe-produced manganese oxide, heavy metal adsorption method, and heavy metal adsorbent - Google Patents
Method for producing microbe-produced manganese oxide, heavy metal adsorption method, and heavy metal adsorbent Download PDFInfo
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- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 title claims abstract description 99
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 23
- 229910001385 heavy metal Inorganic materials 0.000 title claims description 46
- 239000003463 adsorbent Substances 0.000 title claims description 25
- 238000001179 sorption measurement Methods 0.000 title claims description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 63
- 229910001437 manganese ion Inorganic materials 0.000 claims abstract description 44
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 27
- 239000004575 stone Substances 0.000 claims description 23
- 241000862991 Leptothrix <Bacteria> Species 0.000 claims description 3
- 239000011572 manganese Substances 0.000 description 88
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 68
- 229910052748 manganese Inorganic materials 0.000 description 68
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- -1 cadmium and zinc Chemical class 0.000 description 2
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- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- 235000002867 manganese chloride Nutrition 0.000 description 1
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- 239000011702 manganese sulphate Substances 0.000 description 1
- PPNAOCWZXJOHFK-UHFFFAOYSA-N manganese(2+);oxygen(2-) Chemical class [O-2].[Mn+2] PPNAOCWZXJOHFK-UHFFFAOYSA-N 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Processing Of Solid Wastes (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Water Treatment By Sorption (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
Description
本発明は、マンガン酸化細菌が産生する微結晶性のマンガン酸化物（以下、微生物産生マンガン酸化物という）の製造方法と、微生物産生マンガン酸化物を利用した重金属吸着方法と重金属吸着剤に関する。 The present invention relates to a method for producing microcrystalline manganese oxide (hereinafter referred to as microorganism-produced manganese oxide) produced by manganese-oxidizing bacteria, a heavy metal adsorption method using a microorganism-produced manganese oxide, and a heavy metal adsorbent.
廃棄物や自然土壌中には、鉛や水銀、カドミウムといった有害な重金属などが含まれている場合がある。これらの有害な重金属は、雨水の浸透等による洗い出しに伴って浸出水中に溶出して環境を汚染する。このような環境汚染対策、すなわち重金属対策には、キレート剤、カルシウム化合物、硫化物、鉄粉等の薬剤を用いた不溶化、セメント固化による土壌からの溶出抑制など様々な方法が知られている。 Waste and natural soil may contain harmful heavy metals such as lead, mercury, and cadmium. These harmful heavy metals are eluted in the leachate and contaminate the environment as they are washed out by rainwater infiltration. For such environmental pollution countermeasures, that is, heavy metal countermeasures, various methods are known such as insolubilization using chemicals such as chelating agents, calcium compounds, sulfides, iron powder, and suppression of elution from soil by cement solidification.
重金属対策のなかでも、重金属吸着剤を利用する方法が知られている。ここで、微生物が産生する微生物産生マンガン酸化物は、カドミウム、亜鉛等の金属陽イオンの吸着能力が大きく、重金属吸着剤として機能する。一方で、微生物産生マンガン酸化物は、ヒ酸や亜ヒ酸のように陰イオンとして存在する金属に対する吸着能力は一般的に高くないが、酸化剤として機能するため、毒性の高い亜ヒ酸を毒性が低いヒ酸に速やかに酸化することができる。 Among the measures for heavy metals, a method using a heavy metal adsorbent is known. Here, the microorganism-produced manganese oxide produced by the microorganism has a large adsorption capacity for metal cations such as cadmium and zinc, and functions as a heavy metal adsorbent. On the other hand, microbially produced manganese oxides generally do not have a high adsorption capacity for metals that exist as anions, such as arsenic acid and arsenous acid. It can be rapidly oxidized to less toxic arsenic acid.
本発明者らは、非特許文献１において、Ｕ９−１ｉ株が、α−プロテオバクテリア綱に属する新規のマンガン酸化細菌であり、液体培地や寒天培地を用いた単独での培養時には増殖は遅く難培養性であるが、種々の真菌や細菌類等を共存させると容易に増殖できることを報告している。また、本発明者らは、特許文献１、２において、Ｕ９−１ｉ株等の微生物産生酸化マンガンを利用した重金属吸着剤を提案している。 In the non-patent document 1, the present inventors show that the U9-1i strain is a novel manganese-oxidizing bacterium belonging to the α-proteobacteria class, and its growth is difficult and slow when cultured alone using a liquid medium or an agar medium. Although it is cultivatable, it has been reported that it can be easily proliferated when various fungi and bacteria coexist. In addition, in the Patent Documents 1 and 2, the present inventors have proposed a heavy metal adsorbent using a microorganism-produced manganese oxide such as U9-1i strain.
マンガン酸化細菌が産生する微生物産生マンガン酸化物を重金属吸着剤として利用するためには、マンガン酸化細菌を大量に培養し、安定的に大量の微生物産生マンガン酸化物を得る必要がある。しかし、マンガン酸化細菌は、通常マンガンが５５ｍｇＭｎ／Ｌ（１ｍＭ）程度以上となるとマンガン酸化を行わなくなるか、マンガン酸化速度が非常に遅くなるため、微生物産生マンガン酸化物を大量に作成することは困難であった。 In order to use the microorganism-produced manganese oxide produced by manganese-oxidizing bacteria as a heavy metal adsorbent, it is necessary to culture a large amount of manganese-oxidizing bacteria and stably obtain a large amount of microorganism-produced manganese oxide. However, manganese-oxidizing bacteria usually do not oxidize manganese when manganese reaches about 55 mg Mn / L (1 mM) or higher, or the rate of manganese oxidation becomes very slow, making it difficult to produce a large amount of microorganism-produced manganese oxide. Met.
本発明は、微生物産生マンガン酸化物の高速な製造方法を提供することを課題とする。 An object of the present invention is to provide a high-speed method for producing a microorganism-produced manganese oxide.
１．マンガン酸化細菌を担持した担体に、二価マンガンイオンを供給することを特徴とする微生物産生マンガン酸化物の製造方法。
２．前記担体が、砕石、または砂利であることを特徴とする１．に記載の製造方法。
３．二価マンガンイオン濃度が１０ｍｇ／Ｌ以上であることを特徴とする１．または２．に記載の製造方法。
４．前記マンガン酸化細菌が、Ｕ９−１ｉクラスターに属する菌、または、レプトスリックス（Ｌｅｐｔｏｔｈｒｉｘ）属であることを特徴とする１．〜３．のいずれかに記載の製造方法。
５．前記マンガン酸化細菌が、Ｕ９−１ｉ株、または、ＳＰ−６株であることを特徴とする１．〜４．のいずれかに記載の製造方法。
６．マンガン酸化細菌を担持した担体を、重金属吸着剤とすることを特徴とする重金属吸着方法。
７．１．〜５．のいずれかに記載の製造方法で生成した微生物産生マンガン酸化物を、担体表面に付着したままの状態で、重金属吸着剤とすることを特徴とする重金属吸着方法。
８．前記担体が、砕石または砂利であることを特徴とする６．または７．に記載の重金属吸着方法。
９．担体と、該担体表面上に蓄積した微生物産生マンガン酸化物とを有することを特徴とする重金属吸着剤。
１０．前記担体が、砕石または砂利であることを特徴とする９．に記載の重金属吸着剤。
1. A method for producing a microorganism-produced manganese oxide, comprising supplying divalent manganese ions to a carrier supporting manganese-oxidizing bacteria.
2. The carrier is crushed stone or gravel. The manufacturing method as described in.
3. 1. The divalent manganese ion concentration is 10 mg / L or more. Or 2. The manufacturing method as described in.
4). The manganese-oxidizing bacterium is a bacterium belonging to the U9-1i cluster or a genus of Leptothrix. ~ 3. The manufacturing method in any one of.
5). 1. The manganese-oxidizing bacterium is U9-1i strain or SP-6 strain. ~ 4. The manufacturing method in any one of.
6). A heavy metal adsorption method, wherein a carrier carrying manganese-oxidizing bacteria is used as a heavy metal adsorbent.
7.1. ~ 5. A heavy metal adsorption method, wherein the microorganism-produced manganese oxide produced by the production method according to any one of the above is used as a heavy metal adsorbent while remaining attached to the support surface.
8). 5. The carrier is crushed stone or gravel. Or 7. The heavy metal adsorption method according to 1.
9. A heavy metal adsorbent comprising a carrier and a microbially produced manganese oxide accumulated on the surface of the carrier.
10. 8. The carrier is crushed stone or gravel. The heavy metal adsorbent according to 1.
本発明の微生物産生マンガン酸化物の製造方法により、高速でマンガン酸化を行うことができる。また、本発明の製造方法は、二価マンガンイオン濃度が１０ｍｇ／Ｌ以上の高濃度条件下であっても、マンガン酸化を行うことができる。本発明の製造方法は、原料であるマンガンイオン濃度を高濃度とすることができるため、担体に担持していない状態でのマンガン酸化と比較して、微生物産生マンガン酸化物を短期間で大量に製造することができる。
微生物産生マンガン酸化物は、重金属吸着剤として用いることができる。本発明の製造方法により、重金属吸着剤（微生物産生マンガン酸化物）を短い時間で大量に製造することができるため、土壌汚染現場等で要求される大量の重金属吸着剤を迅速に供給することができる。
Manganese oxidation can be performed at high speed by the method for producing a microorganism-produced manganese oxide of the present invention. Moreover, the production method of the present invention can perform manganese oxidation even under high concentration conditions where the divalent manganese ion concentration is 10 mg / L or more. Since the production method of the present invention can increase the concentration of manganese ions as a raw material, a large amount of microorganism-produced manganese oxide can be produced in a short period of time compared to manganese oxidation in a state where it is not supported on a carrier. Can be manufactured.
The microbially produced manganese oxide can be used as a heavy metal adsorbent. Since the heavy metal adsorbent (microorganism-produced manganese oxide) can be produced in a large amount in a short time by the production method of the present invention, a large amount of heavy metal adsorbent required at a soil contamination site can be rapidly supplied. it can.
本発明の微生物産生マンガン酸化物の製造方法は、マンガン酸化細菌を担体に担持した状態で、二価マンガンイオンを供給することを特徴とする。 The method for producing a microorganism-produced manganese oxide of the present invention is characterized in that divalent manganese ions are supplied in a state where manganese-oxidizing bacteria are supported on a carrier.
「マンガン酸化細菌」
マンガン酸化細菌としては、特に制限されず、例えば、下記のような従来公知の細菌を使用することができる。
環境中（河川、湖沼、土壌、海洋など）に普遍的に棲息する細菌（Ｂ．Ｍ．Ｔｅｂｏ，Ｈ．Ａ．Ｊｏｈｎｓｏｎ，Ｊ．Ｋ．ＭｃＣａｒｔｈｙ，Ａ．Ｓ．Ｔｅｍｐｌｅｔｏｎ（２００５）．Ｇｅｏｍｉｃｒｏｂｉｏｌｏｇｙ ｏｆ ｍａｎｇａｎｅｓｅ（ＩＩ） ｏｘｉｄａｔｉｏｎ． Ｔｒｅｎｄｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ，１３，４２１−４２８；Ｈ．Ｌ．Ｅｈｒｌｉｃｈ，Ｄ．Ｋ．Ｎｅｗｍａｎ（２００９）．Ｇｅｏｍｉｃｒｏｂｉｏｌｏｇｙ，５ｔｈ ｅｄ．ＣＲＣ Ｐｒｅｓｓ．）。
"Manganese-oxidizing bacteria"
The manganese-oxidizing bacteria are not particularly limited, and conventionally known bacteria such as the following can be used.
Bacteria (BM Tebo, HA Johnson, JK McCarthy, AS Templeton (2005). Geomicrobiology of manganese inhabiting the environment (rivers, lakes, soils, oceans, etc.) (II) oxidation. Trends in Microbiology, 13, 421-428; HL Ehrlich, D.K. Newman (2009). Geobiology, 5th ed. CRC Press.).
湿地から分離されたＬｅｐｔｏｔｈｒｉｘ ｄｉｓｃｏｐｈｏｒａ ＳＰ−６（Ｌ．Ｆ．Ａｄａｍｓ，Ｗ．Ｃ．Ｇｈｉｏｒｓｅ（１９８７）．Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｅｘｔｒａｃｅｌｌｕｌａｒ Ｍｎ２＋−ｏｘｉｄｉｚｉｎｇ ａｃｔｉｖｉｔｙ ａｎｄ ｉｓｏｌａｔｉｏｎ ｏｆ ａｎ Ｍｎ２＋−ｏｘｉｄｉｚｉｎｇ ｐｒｏｔｅｉｎ ｆｒｏｍ Ｌｅｐｔｏｔｈｒｉｘ ｄｉｓｃｏｐｈｏｒａ ＳＳ−１．Ｊｏｕｒｎａｌ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ，１６９，１２７９−１２８５．）。 Leptothrix discophora SP-6 (L.F.Adams which is separated from the wetlands, W.C.Ghiorse (1987) .Characterization of extracellular Mn 2+ -oxidizing activity and isolation of an Mn 2+ -oxidizing protein from Leptothrix discophora SS-1. Journal of Bacteriology, 169, 1279-1285.).
淡水から分離されたＰｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ（Ｍ．Ｏｋａｚａｋｉ，Ｔ．Ｓｕｇｉｔａ，Ｍ．Ｓｈｉｍｉｚｕ，Ｙ．Ｏｈｏｄｅ，Ｋ．Ｉｗａｍｏｔｏ，Ｅ．Ｗ．ｄｅ Ｖｒｉｎｄ−ｄｅ Ｊｏｎｇ，Ｊ．Ｐ．Ｍ．ｄｅ Ｖｒｉｎｄ，Ｐ．Ｌ．Ａ．Ｍ． Ｃｏｒｓｔｊｅｎｓ（１９９７）．Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ，６３，４７９３−４７９９．）。 Pseudomonas putida isolated from fresh water (M. Okazuki, T. Sugita, M. Shimizu, Y. Oode, K. Iwamoto, EW de Vrind-de Jong, JPM de Vrind, P. Lind. AM Corstjens (1997) .Applied and Environmental Microbiology, 63, 4793-4799.).
淡水及び土壌から分離されたＰｅｄｏｍｉｃｒｏｂｉｕｍ ｓｐ．（Ｅ．Ｉ．Ｌａｒｓｅｎ，Ｌ．Ｉ．Ｓｌｙ，Ａ．Ｇ．ＭｃＥｗａｎ（１９９９）．Ｍａｎｇａｎｅｓｅ（ＩＩ） ａｄｓｏｒｐｔｉｏｎ ａｎｄ ｏｘｉｄａｔｉｏｎ ｂｙ ｗｈｏｌｅ ｃｅｌｌｓ ａｎｄ ａ ｍｅｍｂｒａｎｅ ｆｒａｃｔｉｏｎ ｏｆ Ｐｅｄｏｍｉｃｒｏｂｉｕｍ ｓｐ．ＡＣＭ ３０６７．Ａｒｃｈｉｖｅｓ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｙ，１７１，２５７−２６４．）。 Pedomicrobium sp. Isolated from fresh water and soil. (E.I.Larsen, L.I.Sly, AG McEwan (1999). Manganese (II) adsorption and oxidation by whole cells and a membrane froof. -264.).
土壌から分離されたＡｒｔｈｒｏｂａｃｔｅｒ ｓｐ．（Ｈ．Ｌ．Ｅｈｒｌｉｃｈ，Ｄ．Ｋ．Ｎｅｗｍａｎ（２００９）．Ｇｅｏｍｉｃｒｏｂｉｏｌｏｇｙ，５ｔｈ ｅｄ．ＣＲＣ Ｐｒｅｓｓ．）。 Arthrobacter sp. Isolated from soil. (HL Ehrlich, DK Newman (2009). Geobiology, 5th ed. CRC Press.).
「Ｕ９−１ｉ株」
マンガン酸化細菌として、本発明者らが、河川床生物膜から調製したマンガン酸化汚泥（集積培養系）から単離したＵ９−１ｉ株（非特許文献１）を好適に利用することができる。Ｕ９−１ｉ株は、微結晶性であるマンガン酸化物を産生し、この微生物産生マンガン酸化物は、Ｕ９−１ｉ株の細胞表層を覆うように蓄積される。微生物産生マンガン酸化物の生成と蓄積は、他のマンガン酸化細菌でも共通である。
"U9-1i strain"
As the manganese-oxidizing bacteria, the U9-1i strain (Non-patent Document 1) isolated by the present inventors from manganese oxide sludge (accumulated culture system) prepared from a riverbed biofilm can be suitably used. The U9-1i strain produces manganese oxide that is microcrystalline, and this microbially produced manganese oxide accumulates so as to cover the cell surface layer of the U9-1i strain. The production and accumulation of microbially produced manganese oxide is common to other manganese-oxidizing bacteria.
Ｕ９−１ｉ株は、受託番号ＮＩＴＥ Ｐ−０２４５９として、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）（千葉県木更津市かずさ鎌足２−５−８（郵便番号２９２−０８１８））に、２０１７年４月２１日付で寄託されている。
Ｕ９−１ｉ株の１６Ｓ ｒＲＮＡ遺伝子配列（部分配列：配列番号１）を図１に示す。１６Ｓ ｒＲＮＡ遺伝子に基づく分子系統解析の結果、Ｕ９−１ｉ株は、α−プロテオバクテリア綱に属する新規な細菌である。１６Ｓ ｒＲＮＡ遺伝子配列に基づいて近隣結合法で作成した系統樹を図２に示す。
The U9-1i strain has the accession number NITE P-02459, the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (NPMD) (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, postal code 292-0818) On April 21, 2017.
The 16S rRNA gene sequence of U9-1i strain (partial sequence: SEQ ID NO: 1) is shown in FIG. As a result of molecular phylogenetic analysis based on the 16S rRNA gene, the U9-1i strain is a novel bacterium belonging to the class of α-proteobacteria. FIG. 2 shows a phylogenetic tree prepared by the neighborhood joining method based on the 16S rRNA gene sequence.
「Ｕ９−１ｉクラスターに属する菌」
マンガン酸化細菌として、Ｕ９−１ｉ株と遺伝子的に近縁種であるＵ９−１ｉクラスターに属する菌を好適に利用することができる。ここで、本明細書において、Ｕ９−１ｉクラスターに属する菌とは、Ｕ９−１ｉ株と、１６Ｓ ｒＲＮＡ遺伝子全長１４０６ｂｐとクエリーカバー率（Ｑｕｅｒｙ ｃｏｖｅｒ）９９％以上（１４０６ｂｐのうち１３９２ｂｐ以上）において相同性９７％以上を示し、かつ近隣結合法で作成した分子系統樹においてＵ９−１ｉ株、ＯＴＳｚ Ａ ２７２株とともに１つのクレードを形成する細菌群を意味する。
"Bacteria belonging to U9-1i cluster"
As the manganese-oxidizing bacteria, bacteria belonging to the U9-1i cluster that is genetically related to the U9-1i strain can be preferably used. Here, in this specification, the bacteria belonging to the U9-1i cluster are homologous to the U9-1i strain, the 16S rRNA gene full length 1406 bp, and the query cover rate (Query cover) 99% or more (1392 bp of 1406 bp or more). It means a group of bacteria showing 97% or more and forming one clade together with the U9-1i strain and the OTSz A272 strain in a molecular phylogenetic tree prepared by the neighborhood binding method.
Ｕ９−１ｉ株は、１６Ｓ ｒＲＮＡ遺伝子に特異的な領域を有し、配列番号１に示される１６Ｓ ｒＲＮＡ遺伝子の１１８７〜１２０４位の領域（図１で下線を引いた部分の配列）である配列番号２に示す塩基配列１２７０Ｆ（５’−ＣＧＧＴＧＡＣＡＧＡＧＧＧＡＴＡＡＴ−３’）、またはその相補配列を含むプライマーにより、容易に検出、同定することができる。 The U9-1i strain has a region specific to the 16S rRNA gene, and is a region at positions 1187 to 1204 of the 16S rRNA gene shown in SEQ ID NO: 1 (sequence underlined in FIG. 1). It can be easily detected and identified by a primer containing the base sequence 1270F (5′-CGGTGACAGGGGATAAT-3 ′) shown in 2 or a complementary sequence thereof.
ここで、Ｕ９−１ｉ株、および、図２に示す系統樹に記載されている細菌の一部について、Ｕ９−１ｉ株の配列１２７０Ｆに相当する領域の塩基配列を図３に示す。
図３に示すように、Ｕ９−１ｉクラスターに属する菌は、Ｕ９−１ｉ株の配列１２７０Ｆに相当する領域において配列番号３〜５に示す塩基配列１２７０Ｆ ｆａｍｉｌｙを有する。配列１２７０Ｆ ｆａｍｉｌｙは、配列１２７０Ｆの１８ｂｐの塩基配列に対して１７ｂｐ以上が一致しており、９４．４％以上（＝１７ｂｐ／１８ｂｐ）の非常に高い相同性を有する。それに対し、Ｕ９−１ｉクラスターに属さない菌の当該領域における配列１２７０Ｆとの相同性は低く、遺伝子データベースに登録されている全真正細菌の約３５０万の配列のうち、配列１２７０Ｆと一致するものは僅か２００配列程度である。同様に、配列１２７０Ｆ ｆａｍｉｌｙと一致するものも同程度と僅かである。さらに図３に示すように、Ｕ９−１ｉクラスターに属さない比較的近縁の菌では８３．４％以下（１８ｂｐに対して１５ｂｐ以下）の一致度であることがほとんどである。したがって、配列番号３〜５に示される配列である１２７０Ｆ ｆａｍｉｌｙ、またはその相補配列からなるプライマーにより、Ｕ９−１ｉクラスターに属する菌の１６Ｓ ｒＲＮＡ遺伝子を、特異的に増幅することができる。
Here, the base sequence of the region corresponding to the sequence 1270F of the U9-1i strain is shown in FIG. 3 for the U9-1i strain and some of the bacteria described in the phylogenetic tree shown in FIG.
As shown in FIG. 3, the bacterium belonging to the U9-1i cluster has the base sequence 1270F family shown in SEQ ID NOs: 3 to 5 in a region corresponding to the sequence 1270F of the U9-1i strain. The sequence 1270F family matches 17 bp or more to the 18 bp base sequence of the sequence 1270F, and has a very high homology of 94.4% or more (= 17 bp / 18 bp). On the other hand, the homology with the sequence 1270F in the region of the bacterium not belonging to the U9-1i cluster is low, and among about 3.5 million sequences of all eubacteria registered in the gene database, the one that matches the sequence 1270F is There are only about 200 arrays. Similarly, the number corresponding to the array 1270F family is similar and slight. Furthermore, as shown in FIG. 3, in relatively closely related bacteria that do not belong to the U9-1i cluster, the degree of coincidence is 83.4% or less (15 bp or less compared to 18 bp) in most cases. Therefore, the 16S rRNA gene of the bacterium belonging to the U9-1i cluster can be specifically amplified with a primer comprising 1270F family, which is the sequence shown in SEQ ID NOs: 3 to 5, or its complementary sequence.
「担体」
本発明の製造方法は、マンガン酸化細菌を担体に担持した状態で二価マンガンイオンを供給し、微生物産生マンガン酸化物を産生することを特徴とする。マンガン酸化細菌は、微結晶性であるマンガン酸化物を産生し、この微生物産生マンガン酸化物は、マンガン酸化細菌の細胞表層を覆うように蓄積される。すなわち、本発明の製造方法により、担体に担持されたマンガン酸化細菌の細胞表層に微生物産生マンガン酸化物が蓄積され、マンガン酸化細菌等のバイオフィルムと、このマンガン酸化細菌の細胞表層に蓄積した微生物産生マンガン酸化物を備えたマンガン酸化物担持担体が得られる。得られるマンガン酸化物担持担体は、表面に蓄積した微生物産生マンガン酸化物により褐色または黒褐色となる。
"Carrier"
The production method of the present invention is characterized in that a divalent manganese ion is supplied in a state where a manganese-oxidizing bacterium is supported on a carrier to produce a microorganism-produced manganese oxide. Manganese-oxidizing bacteria produce manganese oxide that is microcrystalline, and this microorganism-produced manganese oxide accumulates so as to cover the cell surface of the manganese-oxidizing bacteria. That is, by the production method of the present invention, microorganism-produced manganese oxide is accumulated on the cell surface of manganese-oxidizing bacteria supported on the carrier, and biofilms such as manganese-oxidizing bacteria and microorganisms accumulated on the cell surface of the manganese-oxidizing bacteria A manganese oxide-supported carrier provided with the produced manganese oxide is obtained. The resulting manganese oxide-supported carrier becomes brown or blackish brown due to the microbially produced manganese oxide accumulated on the surface.
担体にマンガン酸化細菌を担持させる方法は特に制限されず、担体を充填したろ床へのマンガン酸化細菌の植種、マンガン酸化細菌含有培地への担体の浸漬等により行うことができる。マンガン酸化細菌は、菌体のみ、または、担体に担持された状態で、植種することができる。マンガン酸化細菌を担持する担体としては特に制限されず、砕石、砂利、ゼオライト、セラミック、木炭、ポリビニルアルコール、ポリオレフィン、ポリスチレン、等を用いることができる。これらの中で、砕石または砂利が、土壌汚染現場等で要求される大量の微生物産生マンガン酸化物を利用した重金属吸着剤を低コストで供給することができるため好ましい。 The method for supporting the manganese-oxidizing bacteria on the carrier is not particularly limited, and can be carried out by seeding the manganese-oxidizing bacteria in a filter bed filled with the carrier, immersing the carrier in a medium containing manganese-oxidizing bacteria, or the like. Manganese-oxidizing bacteria can be inoculated alone or in a state of being supported on a carrier. The carrier for supporting manganese-oxidizing bacteria is not particularly limited, and crushed stone, gravel, zeolite, ceramic, charcoal, polyvinyl alcohol, polyolefin, polystyrene, and the like can be used. Among these, crushed stone or gravel is preferable because a heavy metal adsorbent using a large amount of microbially produced manganese oxide required at a soil contamination site or the like can be supplied at low cost.
担体存在下でマンガン酸化細菌を培養する条件は、有機物、酸素、微量元素等を含み、温度等が適切な条件であれば特に制限されないが、マンガン酸化細菌が利用可能な形態の二価マンガンイオンを含むことが好ましい。マンガンイオンの存在下では、マンガン酸化細菌を優先的に増殖させることができる。好ましい条件としては、例えば、二価マンガンイオン濃度が０．５ｍｇ／Ｌ以上５００ｍｇ／Ｌ以下、有機物がＴＯＣ濃度で０．５ｍｇ／Ｌ以上１，０００ｍｇ／Ｌ以下である。酸素は、好気条件が維持されるよう供給する。より好ましくは、二価マンガンイオン濃度が５ｍｇ／Ｌ以上５００ｍｇ／Ｌ以下、ＴＯＣ濃度が０．５ｍｇ／Ｌ以上２５０ｍｇ／Ｌ以下である。 Conditions for culturing manganese-oxidizing bacteria in the presence of a carrier are not particularly limited as long as they contain organic matter, oxygen, trace elements, etc., and conditions such as temperature are appropriate, but divalent manganese ions in a form in which manganese-oxidizing bacteria can be used. It is preferable to contain. In the presence of manganese ions, manganese-oxidizing bacteria can be preferentially grown. As preferable conditions, for example, the divalent manganese ion concentration is 0.5 mg / L or more and 500 mg / L or less, and the organic substance has a TOC concentration of 0.5 mg / L or more and 1,000 mg / L or less. Oxygen is supplied so that aerobic conditions are maintained. More preferably, the divalent manganese ion concentration is 5 mg / L or more and 500 mg / L or less, and the TOC concentration is 0.5 mg / L or more and 250 mg / L or less.
原因は不明であるが、マンガン酸化細菌は、担体で担持した状態では、担体で担持されていない状態と比較して、二価マンガンイオンから微生物産生マンガン酸化物を高速で産生することができる。また、二価マンガンイオンが高濃度であっても微生物産生マンガン酸化物を産生することができる。すなわち、微生物産生マンガン酸化物の産生を、マンガン酸化細菌を担体に担持させた状態で行うことにより、同一時間でより大量の微生物産生マンガン酸化物を製造することができ、さらに、原料であるマンガンイオンを高濃度とした場合には、より大量の微生物産生マンガン酸化物を短期間で製造することができる。微生物産生マンガン酸化物の製造中における二価マンガンイオン濃度は特に制限されないが、１０ｍｇ／Ｌ以上５００ｍｇ／Ｌ以下が好ましい。 Although the cause is unknown, manganese-oxidizing bacteria can produce microbially produced manganese oxide from divalent manganese ions at a higher speed when supported by a carrier than when not supported by a carrier. Moreover, even if the divalent manganese ion is at a high concentration, the microorganism-produced manganese oxide can be produced. That is, by producing microbially produced manganese oxide in a state where manganese-oxidizing bacteria are supported on a carrier, a larger amount of microbially produced manganese oxide can be produced in the same time. When the ion concentration is high, a larger amount of microbially produced manganese oxide can be produced in a short period of time. The concentration of divalent manganese ions during production of the microbially produced manganese oxide is not particularly limited, but is preferably 10 mg / L or more and 500 mg / L or less.
「重金属吸着方法および重金属吸着剤」
マンガン酸化細菌により産生した微生物産生マンガン酸化物は、重金属吸着剤として使用することができる。微生物産生マンガン酸化物を重金属吸着剤として使用する形態は特に制限されず、例えば、微生物産生マンガン酸化物を備えるマンガン酸化物担持担体を、焼成、消毒等せず、マンガン酸化細菌が生息している状態で、重金属吸着剤として使用することができる。
"Heavy metal adsorption method and heavy metal adsorbent"
Microbial-produced manganese oxide produced by manganese-oxidizing bacteria can be used as a heavy metal adsorbent. The form in which the microorganism-produced manganese oxide is used as the heavy metal adsorbent is not particularly limited. For example, the manganese oxide-supporting carrier having the microorganism-produced manganese oxide is not baked, disinfected, and the manganese-oxidizing bacteria are inhabited. In the state, it can be used as a heavy metal adsorbent.
本発明の重金属吸着剤により、重金属で汚染された環境から重金属を除去することができる。重金属で汚染された環境とは、地下水、土壌（岩石含む）、廃棄物、排水、河川、湖沼、海あるいは汚染された環境を通過した水等を挙げることができる。
本発明の重金属吸着剤は、微生物産生マンガン酸化物が吸着可能な重金属の１種または２種以上を対象とする吸着処理に特に制限することなく使用することができる。また、本発明の重金属吸着剤は、マンガン酸化細菌によりマンガンイオンを固定化することができるため、マンガンの処理に用いることもできる。
The heavy metal adsorbent of the present invention can remove heavy metals from an environment contaminated with heavy metals. Examples of the environment contaminated with heavy metals include groundwater, soil (including rocks), waste, drainage, rivers, lakes, the sea, or water that has passed through the contaminated environment.
The heavy metal adsorbent of the present invention can be used without particular limitation to the adsorption treatment for one or more heavy metals capable of adsorbing the microorganism-produced manganese oxide. Moreover, since the heavy metal adsorption agent of this invention can fix manganese ion by manganese oxidation bacteria, it can also be used for the process of manganese.
本発明の重金属吸着剤の使用方法は特に制限されないが、一般的には、吸着層として地中や掘削ずりの下方に設置する。吸着層が、重金属を吸着、除去することにより、下流への重金属の流出を防ぐことができる。この際、担体として砕石または砂利を用いることが、大量の重金属吸着剤を供給することができるため好ましい。 Although the usage method in particular of the heavy metal adsorption agent of this invention is not restrict | limited, Generally, it installs in the underground or the downward direction of excavation as an adsorption layer. The adsorption layer can adsorb and remove heavy metals, thereby preventing the heavy metals from flowing downstream. At this time, it is preferable to use crushed stone or gravel as a carrier because a large amount of heavy metal adsorbent can be supplied.
・マンガン酸化細菌
Ｕ９−１ｉ株、Ｌｅｐｔｏｔｈｒｉｘ ｄｉｓｃｏｐｈｏｒａ ＳＰ−６（ＡＴＣＣ ５１１６８。以下、ＳＰ−６株という。）は、下記表１に示すＬｅｐｔｏｔｈｒｉｘ培地を用いて室温で約１週間振とう培養し、フルグロースさせた。
「実験１：Ｕ９−１ｉ株とＰＶＡゲル担体」
「実施例１」
担体（株式会社クラレ製、製品名：クラゲール、直径約４ｍｍの球形であるポリビニルアルコールゲル）を表２に示す培地１００ｍＬに１０ｇ添加しオートクレーブ滅菌した。この培地に塩化マンガンを二価マンガンイオン濃度が６ｍｇ／Ｌとなるように添加した後、上記でフルグロースさせたＵ９−１ｉ株の培養液を体積比１０％となるように植種し、２５℃、９０ｒｐｍの条件で振とう培養を１４４時間（６日間）行った（２連）。
培養後、担体をデカンテーションにより回収して新鮮な培地に移し替え、二価マンガンイオン濃度を２０ｍｇ／Ｌに上昇させて９６時間（４日間）培養した後、さらに同様にして新鮮な培地を移し替え、二価マンガンイオン濃度を３０ｍｇ／Ｌに上昇させて９６時間（４日間）の培養を行うことにより、回分試験を行った。
“Experiment 1: U9-1i strain and PVA gel carrier”
"Example 1"
10 g of a carrier (manufactured by Kuraray Co., Ltd., product name: Kragale, spherical polyvinyl alcohol gel having a diameter of about 4 mm) was added to 100 mL of the medium shown in Table 2 and sterilized by autoclave. After adding manganese chloride to this medium so that the divalent manganese ion concentration becomes 6 mg / L, the culture solution of the U9-1i strain that has been fully grown as described above is inoculated so as to have a volume ratio of 10%. Shaking culture was performed at 144 ° C. and 90 rpm for 144 hours (6 days) (double series).
After culturing, the carrier is recovered by decantation and transferred to a fresh medium, the divalent manganese ion concentration is increased to 20 mg / L and cultured for 96 hours (4 days), and then the fresh medium is transferred in the same manner. Instead, a batch test was performed by increasing the divalent manganese ion concentration to 30 mg / L and culturing for 96 hours (4 days).
「比較例１」
担体を添加しない以外は、実施例１と同様にして、Ｕ９−１ｉ株浮遊性菌体による回分試験を行った。なお、比較例１において、１回目および２回目の回分培養終了後は、体積比１０％の培養液を新鮮な培地に植種して培養を繰り返した。
"Comparative Example 1"
A batch test was carried out using the U9-1i strain floating cells in the same manner as in Example 1 except that no carrier was added. In Comparative Example 1, after completion of the first and second batch cultures, a culture solution with a volume ratio of 10% was inoculated into a fresh medium and the culture was repeated.
「結果」
培養液上清中の二価マンガンイオン濃度をＩＣＰ質量分析法（ＩＣＰ―ＭＳ）で測定した。図４に、二価マンガンイオン濃度の経時変化を示す。
二価マンガンイオン濃度が６ｍｇ／Ｌである１回目の回分試験は、担体の有無に関わらず、試験開始直後から二価マンガンイオン濃度が減少し、４８時間までにマンガンイオン濃度はほぼ０となり、微生物産生マンガン酸化物が産生した。
"result"
The concentration of divalent manganese ions in the culture supernatant was measured by ICP mass spectrometry (ICP-MS). FIG. 4 shows the change with time of the divalent manganese ion concentration.
In the first batch test with a divalent manganese ion concentration of 6 mg / L, the divalent manganese ion concentration decreased immediately after the start of the test, regardless of the presence or absence of the carrier, and the manganese ion concentration became almost zero by 48 hours. Microbial produced manganese oxide was produced.
担体を添加した実施例１は、二価マンガンイオン濃度を２０ｍｇ／Ｌに上昇させた２回目の回分試験において、４８時間までに二価マンガンイオン濃度がほぼ０となった。また、培養前に白色であった担体は、ゲル上のバイオフィルムにマンガン酸化物が蓄積することにより、黒褐色に変化した。さらに３回目の回分試験において、３０ｍｇ／Ｌという高濃度である二価マンガンイオンの速やかな減少が観察され、微生物産生マンガン酸化物が高速で製造できた。 In Example 1 in which the carrier was added, in the second batch test in which the divalent manganese ion concentration was increased to 20 mg / L, the divalent manganese ion concentration became almost zero by 48 hours. In addition, the carrier that was white before the culture changed to blackish brown due to accumulation of manganese oxide in the biofilm on the gel. Further, in the third batch test, a rapid decrease in divalent manganese ions having a high concentration of 30 mg / L was observed, and a microbially produced manganese oxide could be produced at high speed.
それに対し、担体を添加していない比較例１では、二価マンガンイオン濃度を２０ｍｇ／Ｌに上昇させた２回目の回分試験以降は二価マンガン濃度の減少は認められず、マンガン濃度が高いと、マンガン酸化が進行しないことが確かめられた。
すなわち、マンガン酸化細菌は、担体に担持させることにより、二価マンガンイオンが高濃度であってもマンガン酸化物を産生できることが確かめられた。
On the other hand, in Comparative Example 1 in which no carrier was added, no decrease in the divalent manganese concentration was observed after the second batch test in which the divalent manganese ion concentration was increased to 20 mg / L, and the manganese concentration was high. It was confirmed that manganese oxidation did not proceed.
In other words, it was confirmed that manganese-oxidizing bacteria can produce manganese oxide even when the concentration of divalent manganese ions is high when supported on a carrier.
「実験２：ＳＰ−６株とＰＶＡゲル担体１」
「実施例２」
ＳＰ−６株を使用し、培養中の二価マンガンイオン濃度７ｍｇ／Ｌ（９６時間）、３０ｍｇ／Ｌ（８４時間）、６０ｍｇ／Ｌ（８４時間）１２０ｍｇ／Ｌ（９６時間）、１２０ｍｇ／Ｌ（９６時間）とした以外は、上記実施例１と同様にして、回分試験を行った。
“Experiment 2: SP-6 strain and PVA gel carrier 1”
"Example 2"
Using the SP-6 strain, the concentration of divalent manganese ions in the culture was 7 mg / L (96 hours), 30 mg / L (84 hours), 60 mg / L (84 hours) 120 mg / L (96 hours), 120 mg / L A batch test was conducted in the same manner as in Example 1 except that (96 hours) was set.
「比較例２」
担体を添加しない以外は、実施例２と同様にして、ＳＰ−６株浮遊性菌体による回分試験を行った。なお、培地交換時には、遠心分離（８０００ｒｐｍ、１０分）により菌体を沈殿させ、沈殿した菌体の全量を新鮮な培地に移植した。
“Comparative Example 2”
A batch test using SP-6 strain suspension cells was performed in the same manner as in Example 2 except that no carrier was added. At the time of medium exchange, the cells were precipitated by centrifugation (8000 rpm, 10 minutes), and the whole amount of the precipitated cells was transplanted to a fresh medium.
培養液上清中の二価マンガンイオン濃度をＩＣＰ質量分析法（ＩＣＰ―ＭＳ）で測定した。図５に、二価マンガンイオン濃度の経時変化を示す。
ＳＰ−６株においても、二価マンガンイオンが低濃度（７ｍｇ／Ｌ）の場合は、担体の有無による差異はほとんど無くマンガン酸化が行われれた。
The concentration of divalent manganese ions in the culture supernatant was measured by ICP mass spectrometry (ICP-MS). FIG. 5 shows the change with time of the divalent manganese ion concentration.
Also in the SP-6 strain, when the divalent manganese ion was at a low concentration (7 mg / L), manganese oxidation was carried out with almost no difference depending on the presence or absence of the carrier.
担体に担持した実施例２は、３０ｍｇ／Ｌ、６０ｍｇ／Ｌ、１２０ｍｇ／Ｌの二価マンガンイオンのほぼ全量を、それぞれ２４時間、４８時間、９６時間で酸化できた。
それに対し、担体無しの比較例２は、９６時間程度（４日）をかければ３０および６０ｍｇ／Ｌの二価マンガンイオン濃度を全量酸化できたが、さらに高濃度（１２０ｍｇ／Ｌ）では、９６時間経過しても全量を酸化することはできなかった。
このことから、担体に担持した実施例２が、担体に担持されていない比較例２と比較して、マンガンイオンの消費が速くなり、担体添加により微生物産生マンガン酸化物を高速で製造できることが確かめられた。
In Example 2 supported on the carrier, almost all of the 30 mg / L, 60 mg / L and 120 mg / L divalent manganese ions could be oxidized in 24 hours, 48 hours and 96 hours, respectively.
On the other hand, Comparative Example 2 without a carrier could oxidize all the divalent manganese ion concentrations of 30 and 60 mg / L over about 96 hours (4 days), but at a higher concentration (120 mg / L), 96 The total amount could not be oxidized over time.
From this, it is confirmed that Example 2 supported on the carrier consumes manganese ions faster than Comparative Example 2 not supported on the carrier, and that the microorganism-produced manganese oxide can be produced at high speed by adding the carrier. It was.
「実験３：ＳＰ−６株とＰＶＡゲル担体２」
「実施例３」
培養中の二価マンガンイオン濃度を約６０ｍｇ／Ｌ（１０８時間）とした以外は、上記実施例２と同様にして、培養を行った。
“Experiment 3: SP-6 strain and PVA gel carrier 2”
"Example 3"
Culturing was carried out in the same manner as in Example 2 except that the divalent manganese ion concentration during the cultivation was about 60 mg / L (108 hours).
「比較例３」
担体を添加しない以外は、実施例３と同様にして、ＳＰ−６株浮遊性菌体による培養実験を行った。
“Comparative Example 3”
A culture experiment using the SP-6 strain floating cells was conducted in the same manner as in Example 3 except that no carrier was added.
培養液上清中の二価マンガンイオン濃度をＩＣＰ質量分析法（ＩＣＰ―ＭＳ）で測定した。図６に、二価マンガンイオン濃度の経時変化を示す。
ＳＰ−６株は、培養初期における二価マンガンイオンが６０ｍｇ／Ｌと高濃度であっても、マンガン酸化を行うことができた。しかし、担体に担持させた実施例３は、担体に担持させていない比較例３と比較して、より高速でマンガンが消費されており、担体に担持させることにより、微生物産生マンガン酸化物を高速で製造できることが確かめられた。
The concentration of divalent manganese ions in the culture supernatant was measured by ICP mass spectrometry (ICP-MS). FIG. 6 shows the change with time of the divalent manganese ion concentration.
The SP-6 strain was able to oxidize manganese even when the concentration of divalent manganese ions at the initial stage of culture was as high as 60 mg / L. However, in Example 3 supported on a carrier, manganese is consumed at a higher speed than in Comparative Example 3 not supported on a carrier. It was confirmed that it can be manufactured with.
「実施例４：Ｕ９−１ｉ株と砕石担体１」
プラスチック製コンテナに、容積が計６０Ｌとなるように砕石（７号砕石）を投入し、循環液２０Ｌ、循環速度０．２５Ｌ／分にて、培地をコンテナ上部から散水し、下部から回収して循環させた。培地は、下記表３で示した組成の培地を適宜濃縮、希釈し、さらに硫酸マンガン（５〜４５０ｍｇ−Ｍｎ／Ｌ）を添加したものを用いた。培地は、２４時間循環させる毎に新たな培地に交換した。これに、上記で培養しておいたＵ９−１ｉ株の培養液を、培養０日目に２５０ｍＬ、培養２１日目に２００ｍＬ植菌した。なお、コンテナ、及び、砕石は滅菌処理を行っていない。
Put crushed stone (No. 7 crushed stone) into a plastic container so that the total volume is 60L, sprinkle the culture medium from the top of the container at a circulating liquid of 20L and a circulation speed of 0.25L / min, and collect from the bottom. It was circulated. As the medium, a medium having the composition shown in Table 3 below was appropriately concentrated and diluted, and further added with manganese sulfate (5-450 mg-Mn / L). The medium was replaced with a new medium every 24 hours. Into this, 250 mL of the culture solution of the U9-1i strain that had been cultured above was inoculated on the 0th day of culture and 200 mL on the 21st day of culture. In addition, the container and the crushed stone are not sterilized.
実験初期より徐々にマンガン濃度（ｍｇ−Ｍｎ／Ｌ）およびマンガン負荷量（ｍｇ−Ｍｎ／Ｌ／日）（＝培養液中のＭｎ濃度（ｍｇ−Ｍｎ／Ｌ）×１日あたりの培養液量（Ｌ／日）／培養槽容積（Ｌ））を上昇させた。新たに循環させる培地のマンガン濃度（流入）と、この培地によるマンガン負荷量を図７に、２４時間循環後の培地のマンガン濃度（流出）を図８に示す。 Gradually manganese concentration (mg-Mn / L) and manganese loading (mg-Mn / L / day) (= Mn concentration in culture solution (mg-Mn / L) x amount of culture solution per day) (L / day) / culture tank volume (L)) was increased. FIG. 7 shows the manganese concentration (inflow) of the newly circulated medium and the manganese load by this medium, and FIG. 8 shows the manganese concentration (outflow) of the medium after 24 hours of circulation.
実験開始から５０日目までマンガン濃度及びマンガン負荷量を上昇させたところ、マンガン濃度（流入）７５ｍｇ−Ｍｎ／Ｌ、マンガン負荷２５ｍｇ−Ｍｎ／Ｌ／日まではマンガン濃度（流出）はほぼ０であった。しかし、マンガン濃度（流入）１３５ｍｇ−Ｍｎ／Ｌ、マンガン負荷量４５ｍｇ−Ｍｎ／Ｌ／日では、マンガン濃度（流出）が増加し、マンガンの酸化が十分に行われなかった。 When the manganese concentration and the manganese load were increased from the start of the experiment to the 50th day, the manganese concentration (outflow) was almost 0 until the manganese concentration (inflow) 75 mg-Mn / L and the manganese load 25 mg-Mn / L / day. there were. However, at a manganese concentration (inflow) of 135 mg-Mn / L and a manganese loading of 45 mg-Mn / L / day, the manganese concentration (outflow) increased and manganese was not sufficiently oxidized.
マンガン濃度（流入）７５ｍｇ−Ｍｎ／Ｌ、マンガン負荷量２５ｍｇ−Ｍｎ／Ｌ／日に戻して培養を続けたところ、培養１０２日目までは、時折、マンガン濃度（流出）の増加が見受けられたが、その後、マンガン濃度（流出）はほぼ０となり、マンガン酸化は安定した。
培養２１４日目以降、マンガン濃度（流入）およびマンガン負荷量を上昇させたところ、最大でマンガン濃度（流入）３００ｍｇ−Ｍｎ／Ｌ、マンガン負荷量１００ｍｇ−Ｍｎ／Ｌ／日まで２４時間後のマンガン濃度（流出）が０．５ｍｇ／Ｌ以下を示し、マンガン濃度（流入）４５０ｍｇ−Ｍｎ／Ｌ、マンガン負荷量１５０ｍｇ−Ｍｎ／Ｌ／日まで２４時間後のマンガン酸化率９９％以上を示した。
以上の結果から、担体として砕石を用いた場合でも、非常に高いマンガン濃度でのマンガン酸化が可能であり、高速での酸化マンガン生成が可能であることが確かめられた。
Manganese concentration (inflow) 75 mg-Mn / L, manganese load 25 mg-Mn / L / day, and continued culturing. Until day 102, an increase in manganese concentration (outflow) was observed occasionally. However, after that, the manganese concentration (outflow) became almost zero, and manganese oxidation was stabilized.
From the 214th day onward, when the manganese concentration (inflow) and manganese load were increased, the maximum manganese concentration (inflow) was 300 mg-Mn / L, manganese load was 100 mg-Mn / L / day, and the manganese was 24 hours later. Concentration (outflow) was 0.5 mg / L or less, manganese concentration (inflow) was 450 mg-Mn / L, manganese load was 150 mg-Mn / L / day, and manganese oxidation rate after 99 hours was 99% or more.
From the above results, it was confirmed that even when crushed stone was used as the carrier, manganese oxidation at a very high manganese concentration was possible and manganese oxide could be produced at high speed.
「リアルタイムＰＣＲ」
Ｕ９−１ｉ株について、配列番号２に示す配列１２７０Ｆからなるオリゴヌクレオチドプローブと、真正細菌特異的なユニバーサルプライマー（１４９２Ｒ）とのプライマーセットを用いて、下記表４に示す条件でＳＹＢＲ Ｇｒｅｅｎ法によるリアルタイムＰＣＲを行った。また、全細菌について、真正細菌特異的なユニバーサルプライマー（１０４８Ｆ、１１９４Ｒ）を用いて、下記表４に示す条件でＳＹＢＲ Ｇｒｅｅｎ法によるリアルタイムＰＣＲを行った。
For the U9-1i strain, real-time by the SYBR Green method under the conditions shown in Table 4 below using a primer set of an oligonucleotide probe consisting of the sequence 1270F shown in SEQ ID NO: 2 and a eubacterial-specific universal primer (1492R) PCR was performed. For all bacteria, real-time PCR was carried out by the SYBR Green method using the eubacterial-specific universal primers (1048F, 1194R) under the conditions shown in Table 4 below.
・Ｕ９−１ｉ株の定量
上記実施例４における砕石表面からバイオフィルムを約０．５ｇ測り採った後、１００ｍＭアスコルビン酸溶液を２００μＬ添加し、ボルテックスで５分間撹拌してＭｎ酸化物を溶解した。Ｍｎ酸化物溶解後の試料全量を土壌ＤＮＡ抽出キット（株式会社ニッポン・ジーン製、商品名：ＩＳＯＩＬ ｆｏｒ Ｂｅａｄｓ Ｂｅａｔｉｎｇ）を用いてＤＮＡを抽出した。なお、ＤＮＡ抽出は付属のマニュアルに従った。
抽出したＤＮＡを分光光度計（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、装置名：ＮａｎｏＤｒｏｐ １０００）により定量後、１０〜１００倍に希釈して定量ＰＣＲに用いた。
-Quantification of U9-1i strain After measuring about 0.5 g of a biofilm from the crushed stone surface in Example 4, 200 μL of a 100 mM ascorbic acid solution was added and stirred for 5 minutes with vortex to dissolve Mn oxide. DNA was extracted from the total amount of the Mn oxide dissolved sample using a soil DNA extraction kit (trade name: ISOIL for Beads Beating, manufactured by Nippon Gene Co., Ltd.). In addition, DNA extraction followed the attached manual.
The extracted DNA was quantified with a spectrophotometer (Thermo Fisher Scientific, apparatus name: NanoDrop 1000), diluted 10 to 100 times, and used for quantitative PCR.
定量ＰＣＲは、上記表４に示す「１２７０Ｆ」を用いたプライマーセットを用いてＳＹＢＲ Ｇｒｅｅｎ法で行った。反応にはＦａｓｔＳｔａｒｔ Ｅｓｓｅｎｔｉａｌ ＤＮＡ Ｇｒｅｅｎ Ｍａｓｔｅｒ（Ｒｏｃｈｅ社製）を用い、マニュアルで推奨された反応液組成およびプライマー濃度で反応させた。なお、ＤＮＡの定量はリアルタイムＰＣＲシステム（Ｒｏｃｈｅ社製、装置名：ＬｉｇｈｔＣｙｃｌｅｒ Ｎａｎｏシステム）により、上記表４のサーマルサイクル反応で解析した。また、Ｕ９−１ｉ株の１６Ｓ ｒＲＮＡ遺伝子を用いてスタンダードを作成し、絶対検量線法で定量した。
鋳型ＤＮＡを段階希釈して実験を行い、最も増幅効率の高かった結果をＤＮＡのコピー数とした。その結果を図９に示す。
Quantitative PCR was performed by the SYBR Green method using a primer set using “1270F” shown in Table 4 above. For the reaction, FastStart Essential DNA Green Master (manufactured by Roche) was used, and the reaction was performed with the reaction solution composition and primer concentration recommended in the manual. The quantification of the DNA was analyzed by a real-time PCR system (Roche, device name: LightCycler Nano system) by the thermal cycle reaction shown in Table 4 above. A standard was prepared using the 16S rRNA gene of the U9-1i strain and quantified by the absolute calibration curve method.
Experiments were carried out with serial dilutions of template DNA, and the result with the highest amplification efficiency was taken as the DNA copy number. The result is shown in FIG.
４０日までは配列１２７０Ｆは検出されたがコピー数の増加は確認できなかった。しかし、全細菌の１６Ｓ ｒＲＮＡ遺伝子コピー数がほぼ一定（１０８／ｇ担体）に達した５０日以後に配列１２７０Ｆのコピー数が増加し、その後培養１００日目からは１０６〜１０７コピー／ｇ担体の範囲で、安定的に維持されていることが確認できた。Ｕ９−１ｉ株のゲノム中には、１６Ｓ ｒＲＮＡ遺伝子が１コピー存在するため、１００日目以後は、担体１ｇ当たり１０６〜１０７細胞のＵ９−１ｉ株が保持されていることになる。
一方、それぞれプライマーが異なるため、単純な割り算で存在比を求めることは難しいが、１６Ｓ ｒＲＮＡ遺伝子数を目安として全細菌に占めるＵ９−１ｉ株の存在比（Ｕ９−１ｉ １６Ｓ ｒＲＮＡ遺伝子コピー数／全細菌１６Ｓ ｒＲＮＡ遺伝子コピー数×１００）を見積もった。その結果、培養開始直後から４０日後までの存在比は１％から０．１％程度と低かったが、その後増加し、配列１２７０Ｆのコピー数がほぼ一定に維持された１００日以後は数％〜１０％程度で推移していた。
Up to 40 days, the sequence 1270F was detected, but no increase in copy number could be confirmed. However, the copy number of the sequence 1270F increased after 50 days when the 16S rRNA gene copy number of all bacteria reached a substantially constant (10 8 / g carrier), and from 10 6 to 10 7 copies / It was confirmed that it was stably maintained in the range of g carrier. Since there is one copy of the 16S rRNA gene in the genome of the U9-1i strain, 10 6 to 10 7 cells of the U9-1i strain are retained per gram of the carrier after the 100th day.
On the other hand, since each primer is different, it is difficult to determine the abundance ratio by simple division, but the abundance ratio of U9-1i strain in all bacteria (U9-1i 16S rRNA gene copy number / total Bacterial 16S rRNA gene copy number × 100) was estimated. As a result, the abundance ratio from immediately after the start of culture to 40 days later was as low as about 1% to 0.1%, but then increased, and after 100 days when the copy number of the sequence 1270F was maintained almost constant, several% to It was around 10%.
・砕石バイオフィルムの真正細菌叢解析
次世代シーケンサーＭｉＳｅｑ（Ｉｌｌｕｍｉｎａ社製）用のアダプター配列を連結した真正細菌に特異的なプライマーセット（ｈｔｔｐ：／／ｗｗｗ．ｅａｒｔｈｍｉｃｒｏｂｉｏｍｅ．ｏｒｇ）（５１５Ｆ：５’−ＧＴＧＣＣＡＧＣＭＧＣＣＧＣＧＧＴＡＡ−３’、８０６Ｒ：５’−ＧＧＡＣＴＡＣＨＶＧＧＧＴＷＴＣＴＡＡＴ−３’）によりＰＣＲ増幅を行った。ＰＣＲは、ＤＮＡポリメラーゼ（タカラバイオ株式会社製、商品名：Ｅｘ Ｔａｑ）を用いてサーマルサイクラー（ＴｈｅｒｍｏＦｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、装置名：ＳｉｍｐｌｉＡｍｐ Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ）により行った。反応液の総量は４０μＬとし、組成はＥｘ Ｔａｑ付属のマニュアルに従った。
ＰＣＲ産物をＤＮＡ精製試薬（Ｂｅｃｋｍａｎ Ｃｏｕｌｔｅｒ社製、商品名：ＡＭＰｕｒｅ ＸＰ）で精製後、フルオロメーター（ＴｈｅｒｍｏＦｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、装置名：Ｑｕｂｉｔ）で定量後に各サンプルを同濃度で混合した。混合したＰＣＲ産物は、自動ＤＮＡ断片ゲル抽出装置（Ｓａｇｅ Ｓｃｉｅｎｃｅ社製、装置名：ＢｌｕｅＰｉｐｐｉｎ）で再度精製してアンプリコン解析用のライブラリーとした。
調製したライブラリーはシーケンサー（Ｉｌｌｕｍｉｎａ社製、装置名：ＭｉＳｅｑ）により配列を決定した。得られた配列は、Ｃｌａｉｄｅｎｔ ｖ０．２（ｈｔｔｐｓ：／／ｗｗｗ．ｃｌａｉｄｅｎｔ．ｏｒｇ）を用いて分子系統学的に分類した。結果を図１０に示す。
-Authentic bacterial flora analysis of crushed stone biofilm Primer set specific to eubacteria linked to adapter sequence for next-generation sequencer MiSeq (Illumina) (http://www.earthmicrobiome.org) (515F: 5'- PCR amplification was performed by GTGCCAGCMGCCGCGGTAA-3 ′, 806R: 5′-GGACTACHGGGGTTCTCAT-3 ′). PCR was performed using a DNA polymerase (manufactured by Takara Bio Inc., trade name: Ex Taq) with a thermal cycler (manufactured by ThermoFisher Scientific, apparatus name: SimpliAmp Thermal Cycler). The total amount of the reaction solution was 40 μL, and the composition was in accordance with the manual attached to Ex Taq.
After purifying the PCR product with a DNA purification reagent (Beckman Coulter, trade name: AMPure XP) and quantifying with a fluorometer (ThermoFisher Scientific, apparatus name: Qubit), each sample was mixed at the same concentration. The mixed PCR product was purified again with an automatic DNA fragment gel extraction device (manufactured by Sage Science, device name: BluePippin) to obtain a library for amplicon analysis.
The prepared library was sequenced by a sequencer (manufactured by Illumina, apparatus name: MiSeq). The resulting sequences were classified molecularly phylogenetically using Cladent v0.2 (https://www.clientent.org). The results are shown in FIG.
運転開始直後にＡｃｉｄｏｂａｃｔｅｒｉａ門、Ｆｉｒｍｉｃｕｔｅｓ門および未同定細菌群の急激な増加が確認された。運転約６０日目、Ｂａｃｔｅｒｏｉｄｅｔｅｓ門の増加とともにＦｉｒｍｉｃｕｔｅｓ門は減少し、その後細菌叢は安定した。加えて、Ｐｒｏｔｅｏｂａｃｔｅｒｉａ門は運転期間中増減を繰り返しながら常に優占した。
Ｕ９−１ｉ株は、培養直後は１％以下に減少したが、培養６０日目以降でＵ９−１ｉ株の増加が確認され、定量ＰＣＲの結果と一致した。Ｕ９−１ｉ株の増加は試験終了時まで持続しており、他の細菌の存在下でもＵ９−１ｉ株を安定的に培養できることが確かめられた。
Immediately after the start of operation, a rapid increase in the Acidobacteria gate, the Firmicutes gate and the unidentified bacterial group was confirmed. About 60 days after the operation, the Firmictes gate decreased with the increase of the Bacteroidetes gate, and then the bacterial flora became stable. In addition, the Proteobacteria gate was always dominant, repeating the increase and decrease during the operation period.
Although the U9-1i strain decreased to 1% or less immediately after the culture, an increase in the U9-1i strain was confirmed after the 60th day of culture, which was consistent with the result of quantitative PCR. The increase in the U9-1i strain continued until the end of the test, and it was confirmed that the U9-1i strain could be stably cultured even in the presence of other bacteria.
「実験５：Ｕ９−１ｉ株と砕石担体２」
ガラスカラム（φ２．２ｃｍ、長さ１５ｃｍ、容積５７ｃｍ３）に、実験４における培養１０３日目の砕石と新品の７号砕石とを等量混合したものを充填した（空隙：２５．５ｍＬ）。なお、ガラスカラムと新品の砕石とは、滅菌処理を行っていない。
実施例４と同様の培地をカラム下部からチューブポンプで送液した。
６２日目までは、上部から流出した培地は、マンガン濃度（流出）を測定後、廃棄した。
６３日目以降は、カラム上部から流出した培地は、プラスチック瓶に戻し、このプラスチック瓶からカラム下部に送液することにより循環した。培地は、１日毎に交換し、交換時にマンガン濃度（流出）を測定した。
“Experiment 5: U9-1i strain and crushed stone carrier 2”
A glass column (φ2.2 cm, length 15 cm, volume 57 cm 3 ) was filled with an equal mixture of the crushed stone on the 103rd day of culture in Experiment 4 and a new No. 7 crushed stone (void: 25.5 mL). The glass column and the new crushed stone are not sterilized.
A medium similar to that in Example 4 was fed from the bottom of the column with a tube pump.
Until the 62nd day, the medium flowing out from the upper part was discarded after measuring the manganese concentration (outflow).
From the 63rd day onward, the medium flowing out from the upper part of the column was returned to the plastic bottle and circulated by sending the liquid from this plastic bottle to the lower part of the column. The medium was changed every day, and the manganese concentration (outflow) was measured at the time of replacement.
実験初期より徐々にマンガン濃度（流入）およびマンガン負荷量を上昇させた。培地のマンガン濃度（流入）と、この培地によるマンガン負荷量を図１１に、マンガン濃度（流出）を図１２に示す。なお、６２日目までは流量、６３日目以降は循環させる培地の総量がが異なるため、マンガン濃度（流入）とマンガン負荷量とは、別々に変化している。 The manganese concentration (inflow) and manganese load were gradually increased from the beginning of the experiment. FIG. 11 shows the manganese concentration (inflow) of the medium and the manganese loading amount by this medium, and FIG. 12 shows the manganese concentration (outflow). Since the flow rate is different until the 62nd day and the total amount of the medium to be circulated is different from the 63rd day, the manganese concentration (inflow) and the manganese load are changed separately.
５０日程度でマンガン酸化は安定した。最大でマンガン濃度（流入）２００ｍｇ−Ｍｎ／Ｌ、マンガン負荷量３００ｍｇ−Ｍｎ／Ｌ／日まで安定したマンガン酸化が可能であり、砕石を用いたマンガン酸化は、高いマンガン酸化速度を持つことが確認された。 Manganese oxidation stabilized in about 50 days. Stable manganese oxidation is possible up to a manganese concentration (inflow) of 200 mg-Mn / L and a manganese load of 300 mg-Mn / L / day. Manganese oxidation using crushed stone has a high manganese oxidation rate. It was done.
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