JP2023146134A - Selenium reduction treatment method using selenium-contaminated samples containing selenium-reducing bacteria - Google Patents
Selenium reduction treatment method using selenium-contaminated samples containing selenium-reducing bacteria Download PDFInfo
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Abstract
Description
本発明は、セレン還元菌を含むセレン汚染試料を用いたセレンの還元処理方法に関する。 The present invention relates to a selenium reduction treatment method using a selenium-contaminated sample containing selenium-reducing bacteria.
セレンは自然界に存在する元素であり、硫化物又は硫黄の鉱床に含有されている。当該元素は人体にとって必須元素の一つである一方、多量に摂取した場合には健康被害をもたらすことから、環境基本法の水質汚濁に係る環境基準や土壌汚染対策法の第二種特定有害物質に指定されている。そのため、基準値以上の汚染地下水やセレンが溶出する汚染土壌への対策として、原位置における汚染水の浄化処理や不溶化処理の措置が取られる場合がある。自然由来のセレンが問題となるケースの一つとして、トンネル工事において発生する掘削ずりや、涌水等により発生するトンネルの廃水処理、搬出や運搬及び保管中に掘削ずりから飛散や流出したことによる周辺土壌や地下水及び河川の汚染対策、産廃処理場や汚泥処理等のプラントで処理する際の廃水の処理が挙げられる。しかし、既存の処理材のセレンに対する効果は低く、また汚染水を浄化するためには煩雑な処理設備が必要となるため、セレン汚染試料に対する効果の高い処理技術が求められている。 Selenium is a naturally occurring element found in sulfides or sulfur deposits. While this element is one of the essential elements for the human body, it can cause health damage if ingested in large quantities, so it is classified as a Class 2 Specified Hazardous Substance under the Environmental Standards for Water Pollution under the Basic Environment Law and the Soil Contamination Countermeasures Law. specified. Therefore, as a countermeasure for contaminated groundwater that exceeds the standard value or contaminated soil from which selenium is leached, in-situ purification treatment or insolubilization treatment of contaminated water may be taken. One of the cases where naturally occurring selenium becomes a problem is the excavation slag generated during tunnel construction, tunnel wastewater treatment generated by spring water, etc., and the surrounding area due to scattering or leakage from excavation slag during removal, transportation, and storage. These include countermeasures against pollution of soil, groundwater, and rivers, and treatment of wastewater when treated at industrial waste treatment plants, sludge treatment plants, etc. However, existing treatment materials have low effects on selenium, and complicated treatment equipment is required to purify contaminated water, so there is a need for highly effective treatment techniques for selenium-contaminated samples.
自然環境中のセレンの形態は４価の亜セレン酸（ＳｅＯ３ ２-）或いは６価のセレン酸（ＳｅＯ４ ２-）であり、自然環境下では混在しているケースが多い。このうち６価セレンの還元処理が難しいことが、セレンの処理を困難にしていると考えられている。６価セレンを処理する場合、まず４価セレンに還元してから処理する方法が一般的であるが（例えば特許文献１）、二段階の処理が必要になるため、いずれの価数にも効果的な還元処理方法が望まれている。 The form of selenium in the natural environment is tetravalent selenite (SeO 3 2- ) or hexavalent selenic acid (SeO 4 2- ), and in many cases they are mixed in the natural environment. It is believed that the difficulty in reducing hexavalent selenium makes it difficult to process selenium. When treating hexavalent selenium, it is common to first reduce it to tetravalent selenium and then treat it (for example, Patent Document 1), but since it requires two stages of treatment, it is not effective for either valence. A reduction treatment method is desired.
本発明は、セレン還元菌を含むセレン汚染試料を利用したセレンの還元処理方法を提供することを目的とする。 An object of the present invention is to provide a method for reducing selenium using a selenium-contaminated sample containing selenium-reducing bacteria.
本発明者らは、セレンの還元処理について鋭意検討を行ったところ、セレン還元菌を含むセレン汚染試料を、乳酸の存在下、微好気条件下で静置又は撹拌することにより、極めて低い相対存在量（％）である当該セレン還元菌が活動することで、４及び６価のセレンを還元できるという知見を得、この知見に基づき、本発明を完成させるに至った。 The present inventors conducted intensive studies on selenium reduction treatment, and found that by leaving selenium-contaminated samples containing selenium-reducing bacteria at rest or stirring them under microaerobic conditions in the presence of lactic acid, a very low relative We have obtained the knowledge that selenium-reducing bacteria in abundance (%) can reduce tetravalent and hexavalent selenium through their activity, and based on this knowledge, we have completed the present invention.
すなわち、本発明は以下の１）～７）に係るものである。
１）セレン還元菌を含むセレン汚染試料を、乳酸の存在下、微好気的条件下で静置又は撹拌する、セレン汚染試料のセレンの還元処理方法であって、セレン還元菌が、配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ａ、配列番号：２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｂ、配列番号：３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｃ、配列番号：４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｄ、配列番号：５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｅ、配列番号：６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｆ、配列番号：７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｇ、配列番号：８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｈ、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｉ、配列番号：１０の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｊ、配列番号：１１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｋ、配列番号：１２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｌ、配列番号：１３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｍ、配列番号：１４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｎ、配列番号：１５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｏ、配列番号：１６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｐ、配列番号：１７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｑ、及び配列番号：１８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｒからなる群から選択される少なくとも１種である、方法。
２）微好気的条件の下、１０～４０℃で、１２時間以上静置又は撹拌する、１）に記載の方法。
３）セレン還元菌が、配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ａ、配列番号：６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｆ、配列番号：８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｈ、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｉ、配列番号：１０の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｊ、配列番号：１２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｌ、及び配列番号：１７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｑからなる群から選択される少なくとも１種を含む、１）又は２）に記載の方法。
４）セレン還元菌が、配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ａ、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｉ、及び配列番号：１２との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｌからなる群から選択される少なくとも１種を含む、１）～３）のいずれかに記載の方法。
５）処理されるセレン汚染試料が、セレンを含む掘削ずり、土砂、汚泥、水、焼却灰及び工場廃水から選ばれる１種以上である、１）～４）のいずれかに記載の方法。
６）処理されるセレン汚染試料にリン酸、乳酸以外の有機酸、アルコール類及び糖類から選ばれる１種以上を加える工程を含む、１）～５）のいずれかに記載の方法。
７）セレン汚染試料のセレン還元化能を評価する方法であって、試料中の配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ａ、配列番号：２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｂ、配列番号：３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｃ、配列番号：４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｄ、配列番号：５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｅ、配列番号：６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｆ、配列番号：７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｇ、配列番号：８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｈ、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｉ、配列番号：１０の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｊ、配列番号：１１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｋ、配列番号：１２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｌ、配列番号：１３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｍ、配列番号：１４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｎ、配列番号：１５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｏ、配列番号：１６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｐ、配列番号：１７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｑ、及び配列番号：１８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｒからなる群から選択される少なくとも１種の存在を確認する工程を含む、方法。
That is, the present invention relates to the following 1) to 7).
1) A selenium reduction treatment method for a selenium-contaminated sample, in which a selenium-contaminated sample containing selenium-reducing bacteria is left standing or stirred under microaerobic conditions in the presence of lactic acid, the selenium-contaminated sample containing selenium-reducing bacteria having SEQ ID NO. :Selenium-reducing bacteria A having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 1, Selenium-reducing bacteria A having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 2 B. A selenium-reducing bacterium having a 16S rRNA gene that has a 97% or more identity with the base sequence of SEQ ID NO: 3. C. A 16S rRNA gene that has a 16S rRNA gene that has a 97% or more identity with the base sequence of SEQ ID NO: 4. Selenium-reducing bacteria D having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 5.Selenium-reducing bacteria E having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 6. Selenium-reducing bacteria F having a 16S rRNA gene, with an identity of 97% or more to the base sequence of SEQ ID NO: 7. Selenium-reducing bacteria G having a 16S rRNA gene, having an identity of 97% to the base sequence of SEQ ID NO: 8. % or more, selenium-reducing bacteria H having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 9, selenium-reducing bacteria I having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 10. A selenium-reducing bacterium J having a 16S rRNA gene with an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 11, a selenium-reducing bacterium K having a 16S rRNA gene having an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12. A selenium-reducing bacterium L having a 16S rRNA gene with a nucleotide sequence of 97% or more, and a selenium-reducing bacterium M having a 16S rRNA gene having a 16S rRNA gene with a 97% or more identity with the nucleotide sequence of SEQ ID NO: 13. Selenium-reducing bacterium N having a 16S rRNA gene that has a 97% or more identity with the base sequence of number: 14, selenium-reducing bacteria N that has a 16S rRNA gene that has a 16S rRNA gene that has a 97% or more identity with the base sequence of SEQ ID NO: 15 Bacterium O, a selenium-reducing bacterium P having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 16, a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 17 and selenium-reducing bacterium R, which has a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 18.
2) The method according to 1), wherein the method is left standing or stirring under microaerobic conditions at 10 to 40°C for 12 hours or more.
3) The selenium-reducing bacteria has a 16S rRNA gene that has a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 1, and has a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 6. A selenium-reducing bacterium F having a certain 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 8. A selenium-reducing bacterium H having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 9. Selenium-reducing bacteria I having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 10, Selenium-reducing bacteria J having a 16S rRNA gene that is 97% or more identical to the nucleotide sequence of SEQ ID NO: 10, and the nucleotide sequence of SEQ ID NO: 12. From the group consisting of selenium-reducing bacteria L, which has a 16S rRNA gene that has an identity of 97% or more, and selenium-reducing bacteria Q, which has a 16S rRNA gene that has a 16S rRNA gene that has 97% or more identity to the base sequence of SEQ ID NO: 17. The method according to 1) or 2), comprising at least one selected type.
4) The selenium-reducing bacteria has a 16S rRNA gene that has a 16S rRNA gene that has an identity of 97% or more with the base sequence of SEQ ID NO: 1, and a selenium-reducing bacteria that has 97% or more identity with the base sequence of SEQ ID NO: 9. 1) containing at least one species selected from the group consisting of a selenium-reducing bacterium I having a certain 16S rRNA gene and a selenium-reducing bacterium L having a 16S rRNA gene having 97% or more identity with SEQ ID NO: 12; The method described in any of -3).
5) The method according to any one of 1) to 4), wherein the selenium-contaminated sample to be treated is one or more selected from selenium-containing excavation slag, earth and sand, sludge, water, incineration ash, and factory wastewater.
6) The method according to any one of 1) to 5), which includes the step of adding one or more selected from phosphoric acid, organic acids other than lactic acid, alcohols, and sugars to the selenium-contaminated sample to be treated.
7) A method for evaluating the selenium reducing ability of a selenium-contaminated sample, comprising: a selenium-reducing bacterium A having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 1 in the sample; :Selenium-reducing bacteria B having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 2, Selenium-reducing bacteria B that has a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 3 C, a selenium-reducing bacterium having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 4; D, a 16S rRNA gene that has 97% or more identity with the nucleotide sequence SEQ ID NO: 5; Selenium-reducing bacteria E having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 6.Selenium-reducing bacteria F having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 7. Selenium-reducing bacteria G having a 16S rRNA gene, having an identity of 97% or more with the base sequence of SEQ ID NO: 8 Selenium-reducing bacteria H having a 16S rRNA gene, having an identity of 97% with the base sequence of SEQ ID NO: 9 Selenium-reducing bacteria I having a 16S rRNA gene with an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 10, Selenium-reducing bacteria J having a 16S rRNA gene with an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 11. Selenium-reducing bacteria K having a 16S rRNA gene with an identity of 97% or more, selenium-reducing bacteria L having a 16S rRNA gene having an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13. A selenium-reducing bacterium M having a 16S rRNA gene with a nucleotide sequence of 97% or more, a selenium-reducing bacterium N having a 16S rRNA gene having a 97% or more identity with the nucleotide sequence of SEQ ID NO: 14, the sequence A selenium-reducing bacterium O having a 16S rRNA gene that has a 97% or more identity with the base sequence of number: 15, a selenium-reducing bacterium that has a 16S rRNA gene that has a 16S rRNA gene that has a 97% or more identity with the base sequence of SEQ ID NO: 16. Bacterium P, selenium-reducing bacterium Q having a 16S rRNA gene that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 17, and 16S rRNA that has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 18. A method comprising the step of confirming the presence of at least one species selected from the group consisting of selenium-reducing bacteria R having the gene.
本発明によれば、セレン汚染試料中のセレン還元菌に基づくセレン還元力を利用することにより、処理材を用いることなくセレンの還元処理が可能になる。 According to the present invention, by utilizing the selenium-reducing power based on selenium-reducing bacteria in a selenium-contaminated sample, selenium reduction treatment can be performed without using a treatment material.
本明細書において、範囲を示す「Ｘ～Ｙ」は「Ｘ以上Ｙ以下」を意味する。また、特記しない限り、操作及び物性等の測定は室温ｃｖ（２０～２５℃）／相対湿度４０～５０％ＲＨの条件で測定する。 In this specification, the range "X to Y" means "more than or equal to X and less than or equal to Y." Further, unless otherwise specified, operations and physical properties are measured under conditions of room temperature CV (20 to 25° C.)/relative humidity 40 to 50% RH.
後述する実施例に記載のとおり、トンネル建設現場から採掘された掘削ずりにおける微生物群集の状態と４及び６価セレンの還元活性との関係を検討したところ、微生物群集から、セレンの還元に関与する１８種の微生物（セレン還元菌）が同定された。これらのセレン還元菌は、微生物群集全体に対して、極めて低い相対存在量（例えば１．６％以下）であったが、微好気条件下において、乳酸を加えることにより、その相対存在量が増加し、当該掘削ずりのセレン還元活性が上昇することが見出された。
したがって、１８種のセレン還元菌を含むセレン汚染試料は、嫌気条件ではない微好気条件下でもセレン還元能を有していると考えられ、試料中の当該セレン還元菌のセレン還元力を利用することによりセレンの還元処理が可能となる。また、試料中のセレン還元菌の存在を指標として、当該試料のセレン還元能を評価することができる。
As described in the Examples below, we investigated the relationship between the state of the microbial community in excavated slag excavated from a tunnel construction site and the reduction activity of tetravalent and hexavalent selenium. Eighteen types of microorganisms (selenium-reducing bacteria) were identified. The relative abundance of these selenium-reducing bacteria was extremely low (for example, 1.6% or less) relative to the entire microbial community, but by adding lactic acid under microaerophilic conditions, the relative abundance could be reduced. It was found that the selenium reduction activity of the drilling slag increased.
Therefore, the selenium-contaminated sample containing 18 types of selenium-reducing bacteria is considered to have selenium-reducing ability even under microaerobic conditions rather than anaerobic conditions, and the selenium-reducing ability of the selenium-reducing bacteria in the sample is utilized. By doing so, reduction treatment of selenium becomes possible. Furthermore, the selenium reducing ability of the sample can be evaluated using the presence of selenium-reducing bacteria in the sample as an indicator.
［セレン還元菌］
本発明において、セレン還元菌とは、４及び６価セレンを電子受容体基質として還元できる菌を意味する。
本発明に係るセレン還元菌としては、配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ａ（本明細書中、単に「セレン還元菌Ａ」とも称す）、配列番号：２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｂ（本明細書中、単に「セレン還元菌Ｂ」とも称す）、配列番号：３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｃ（本明細書中、単に「セレン還元菌Ｃ」とも称す）、配列番号：４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｄ（本明細書中、単に「セレン還元菌Ｄ」とも称す）、配列番号：５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｅ（本明細書中、単に「セレン還元菌Ｅ」とも称す）、配列番号：６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｆ（本明細書中、単に「セレン還元菌Ｆ」とも称す）、配列番号：７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｇ（本明細書中、単に「セレン還元菌Ｇ」とも称す）、配列番号：８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｈ（本明細書中、単に「セレン還元菌Ｈ」とも称す）、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｉ（本明細書中、単に「セレン還元菌Ｉ」とも称す）、配列番号：１０の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｊ（本明細書中、単に「セレン還元菌Ｊ」とも称す）、配列番号：１１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｋ（本明細書中、単に「セレン還元菌Ｋ」とも称す）、配列番号：１２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｌ（本明細書中、単に「セレン還元菌Ｌ」とも称す）、配列番号：１３の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｍ（本明細書中、単に「セレン還元菌Ｍ」とも称す）、配列番号：１４の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｎ（本明細書中、単に「セレン還元菌Ｎ」とも称す）、配列番号：１５の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｏ（本明細書中、単に「セレン還元菌Ｏ」とも称す）、配列番号：１６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｐ（本明細書中、単に「セレン還元菌Ｐ」とも称す）、配列番号：１７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｑ（本明細書中、単に「セレン還元菌Ｑ」とも称す）、及び配列番号：１８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有する菌Ｒ（本明細書中、単に「セレン還元菌Ｒ」とも称す）、からなる群から選択される菌が挙げられる。
[Selenium reducing bacteria]
In the present invention, selenium-reducing bacteria refers to bacteria that can reduce tetravalent and hexavalent selenium as electron acceptor substrates.
The selenium-reducing bacterium according to the present invention includes Bacterium A having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1 (herein also simply referred to as "Selenium-reducing bacterium A"). , Bacterium B having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 2 (herein also simply referred to as "Selenium-reducing bacterium B"), and the nucleotide sequence of SEQ ID NO: 3. Bacterium C (herein also simply referred to as "Selenium-reducing bacterium C") having a 16S rRNA gene with an identity of 97% or more to the base sequence of SEQ ID NO: 4, which has an identity of 97% or more to the nucleotide sequence of SEQ ID NO: 4. Bacterium D having a 16S rRNA gene (herein also simply referred to as "Selenium-reducing bacterium D"), Bacterium E having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 5 (in this specification). Bacteria F, which has a 16S rRNA gene having a 97% or more identity with the base sequence of SEQ ID NO: 6 (also simply referred to as "Selenium-reducing bacteria E" in the specification); F), Bacterium G having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 7 (herein also simply referred to as "Selenium-reducing bacterium G"), SEQ ID NO: Bacterium H having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 8 (herein also simply referred to as "Selenium-reducing bacterium H"); Bacterium I having a 16S rRNA gene of 97% or more (herein also simply referred to as "Selenium reducing bacterium I"), a 16S rRNA gene having a 97% or more identity with the base sequence of SEQ ID NO: 10. Bacterium J having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 11 (herein, also referred to simply as "Selenium-reducing bacterium J") Bacteria L having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 12 (also simply referred to as "Selenium reducing bacterium L" herein) ), Bacterium M having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 13 (herein also simply referred to as "Selenium-reducing bacterium M"), nucleotide sequence of SEQ ID NO: 14 Bacterium N having a 16S rRNA gene that has 97% or more identity with the nucleotide sequence of SEQ ID NO: 15 (herein also simply referred to as "Selenium-reducing bacterium N"), Bacterium O having a certain 16S rRNA gene (herein also simply referred to as "Selenium reducing bacterium O"), Bacterium P having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 16 ( In this specification, it is also simply referred to as "Selenium reducing bacterium P"), Bacterium Q having a 16S rRNA gene having 97% or more identity with the nucleotide sequence of SEQ ID NO: 17 (herein simply referred to as "Selenium reducing bacterium P"), Bacterium R (herein also simply referred to as "Selenium-reducing bacterium R"), which has a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 18. Examples include bacteria selected from the group consisting of:
本発明において、塩基配列との同一性とは、特に記載した場合を除き、いずれの塩基配列においても、２つの配列を最適の態様で整列させた場合に、２つの配列間で共有する一致したヌクレオチドの個数の百分率を意味する。すなわち、同一性（％）＝（一致した位置の数／位置の全数）×１００で算出でき、市販されているアルゴリズムを用いて計算することができる。また、このようなアルゴリズムは、Altschul et al., J. Mol. Biol. 215(1990)403-410に記載されるＮＢＬＡＳＴ及びＸＢＬＡＳＴプログラム中に組込まれている。より詳細には、塩基配列の同一性に関する検索・解析は、当業者には周知のアルゴリズム又はプログラム（例えばＢＬＡＳＴＮ、ＣｌｕｓｔａｌＷなど）により行うことができる。プログラムを用いる場合のパラメータは、当業者であれば適切に設定することができ、また各プログラムのデフォルトパラメーターを用いてもよい。これらの解析方法の具体的な手法もまた、当業者には周知である。 In the present invention, identity with a nucleotide sequence refers to the identity shared between two nucleotide sequences when the two sequences are aligned in an optimal manner, unless otherwise specified. It means the percentage of the number of nucleotides. That is, it can be calculated as identity (%) = (number of matched positions/total number of positions) x 100, and can be calculated using a commercially available algorithm. Such algorithms have also been incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) 403-410. More specifically, searches and analyzes regarding the identity of base sequences can be performed using algorithms or programs (eg, BLASTN, ClustalW, etc.) well known to those skilled in the art. Parameters when using a program can be appropriately set by those skilled in the art, and default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.
配列番号：１～１８の塩基配列を含む１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌を表１に示す。これらのセレン還元菌は、後述する「セレン還元菌の同定」で示した方法により同定された菌である。下記表１において、「同一性（％）」は、配列番号：１～１８の塩基配列について、ＮＣＢＩ（Ｔｈｅ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｍａｔｉｏｎ）の塩基配列データベースのＢＬＡＳＴプログラムを用いて決定した。 Table 1 shows selenium-reducing bacteria having a 16S rRNA gene containing the base sequence of SEQ ID NOs: 1 to 18. These selenium-reducing bacteria were identified by the method described in "Identification of selenium-reducing bacteria" below. In Table 1 below, "identity (%)" was determined for the nucleotide sequences of SEQ ID NOs: 1 to 18 using the BLAST program of the nucleotide sequence database of NCBI (The National Center for Biotechnology Information).
表１に示すとおり、１８種のセレン還元菌は、１６Ｓ ｒＲＮＡ遺伝子の塩基配列同一性が９７～１００％であり、いずれも既知の微生物種又はこれと系統学的に同一又は近縁の微生物であると考えられる。 As shown in Table 1, the 18 types of selenium-reducing bacteria have 97-100% nucleotide sequence identity in their 16S rRNA genes, and all of them are known microbial species or phylogenetically identical or closely related microorganisms. It is believed that there is.
本発明のセレン還元菌を含むセレン汚染試料は、その微生物群集中に、上記のセレン還元菌を含むものである。
本発明のセレン還元菌を含むセレン汚染試料は、本発明のセレン還元菌を含む限りその由来は限定されないが、セレンを含有し微好気状態が維持され得る地下水、地盤や岩盤等の土壌（具体的にはトンネル等の地中構造物を建設する際の掘削ずり）、及び地下構造物の建設工事で発生する土砂や汚泥、廃水が挙げられる。本発明のセレン還元菌を含むセレン汚染試料は、上記セレン還元菌を含む微生物群集を含んでいれば、細菌に加えて糸状菌、原生動物が混在していてもよい。
The selenium-contaminated sample containing selenium-reducing bacteria of the present invention contains the above-mentioned selenium-reducing bacteria in its microbial community.
The origin of the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is not limited as long as it contains the selenium-reducing bacteria of the present invention; Specifically, these include excavation slag during construction of underground structures such as tunnels), and earth, sand, sludge, and wastewater generated during construction of underground structures. The selenium-contaminated sample containing selenium-reducing bacteria of the present invention may contain filamentous fungi and protozoa in addition to bacteria, as long as it contains a microbial community containing the selenium-reducing bacteria.
本発明のセレン還元菌を含むセレン汚染試料は、セレン還元能の観点から、好ましい実施態様として、配列番号：１の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ａ、配列番号：６の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｆ、配列番号：８の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｈ、配列番号：９の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｉ、配列番号：１０の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｊ、配列番号：１２の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｌ、及び配列番号：１７の塩基配列との同一性が９７％以上である１６Ｓ ｒＲＮＡ遺伝子を有するセレン還元菌Ｑからなる群から選択される少なくとも１種を含むものが挙げられる。
これら７種類のセレン還元菌は、既往の知見において微好気環境で嫌気呼吸能を有することが知られている。
From the viewpoint of selenium-reducing ability, the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention is preferably a selenium-reducing bacterium having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 1. A, a selenium-reducing bacterium having a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 6, F, a 16S rRNA gene that has 97% or more identity with the base sequence of SEQ ID NO: 8; Selenium-reducing bacteria H having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 9.Selenium-reducing bacteria I having a 16S rRNA gene, which has an identity of 97% or more with the nucleotide sequence of SEQ ID NO: 10. Selenium-reducing bacteria J having a 16S rRNA gene, selenium-reducing bacteria L having a 16S rRNA gene having 97% or more identity with the base sequence of SEQ ID NO: 12, and selenium-reducing bacteria L having 97% or more identity with the base sequence of SEQ ID NO: 17. Examples include those containing at least one species selected from the group consisting of selenium-reducing bacteria Q having 97% or more of the 16S rRNA gene.
These seven types of selenium-reducing bacteria are known to have anaerobic respiration ability in a microaerobic environment based on past findings.
また、更に好ましい実施形態として、セレン汚染試料は、セレン還元能の観点から、セレン還元菌Ａを含むものが挙げられ、より好ましくはセレン還元菌Ａ、セレン還元菌Ｉ及びセレン還元菌Ｌを含むものが挙げられる。 Furthermore, in a more preferred embodiment, the selenium-contaminated sample contains selenium-reducing bacteria A, more preferably selenium-reducing bacteria A, selenium-reducing bacteria I, and selenium-reducing bacteria L from the viewpoint of selenium-reducing ability. Things can be mentioned.
本発明のセレン還元菌を含むセレン汚染試料には、約４，０００種～約５，０００種の微生物種が含まれ、これらの微生物群集における、本発明の上記セレン還元菌の相対存在量は特に制限されず、微生物種全体に対して、極めて低くてもよい。セレン還元菌の相対存在量（％）は、微生物群集全体に対して、例えば０．００１％以下～０．６７％である。相対存在量の値は、実施例に記載の方法により求めた値である。 The selenium-contaminated sample containing the selenium-reducing bacteria of the present invention contains about 4,000 to about 5,000 microbial species, and the relative abundance of the selenium-reducing bacteria of the present invention in these microbial communities is It is not particularly limited and may be extremely low relative to all microbial species. The relative abundance (%) of selenium-reducing bacteria is, for example, 0.001% or less to 0.67% with respect to the entire microbial community. The value of relative abundance is a value determined by the method described in Examples.
なお、上記セレン還元菌は、試料中の存在量が極めて少ないため、これらを単離することは、技術的理由により困難である。そのため、寄託機関に寄託を行っていない。しかし、本出願人は、本発明に係るセレン還元菌を含むセレン汚染試料について、日本国特許法施行規則第２７条の３各号に該当する場合、各法令の順守を条件に、第三者に分譲することを保証する。 In addition, since the selenium-reducing bacteria described above exist in extremely small amounts in the sample, it is difficult to isolate them for technical reasons. Therefore, it has not been deposited with a depositary institution. However, in cases where the selenium-contaminated sample containing the selenium-reducing bacteria according to the present invention falls under Article 27-3 of the Japanese Patent Law Enforcement Regulations, the applicant may request that a third party provide the We guarantee that the property will be distributed to
［セレン還元菌の同定］
複数の微生物を含む試料（例えばセレン汚染土壌）中のセレン還元菌の同定は、以下に示す工程Ａ～Ｃによって行うことができる。
工程Ａ：セレンを含有する未培養の試料から核酸を回収すること；及び、当該核酸に存在する標的遺伝子の塩基配列を次世代シークエンサーにより解析し、各微生物の相対存在量（１）を求める工程、
工程Ｂ：４及び６価セレン並びに乳酸の存在下で前記試料の培養を開始し、培養物を得ること；当該培養物から核酸を回収すること；及び、当該核酸に存在する標的遺伝子の塩基配列を次世代シークエンサーにより解析し、各微生物の相対存在量（２）を求める工程、
工程Ｃ：前記相対存在量（２）が前記相対存在量（１）より多い微生物をセレン還元菌であると判断する工程。
[Identification of selenium-reducing bacteria]
Identification of selenium-reducing bacteria in a sample containing multiple microorganisms (for example, selenium-contaminated soil) can be performed by steps A to C shown below.
Step A: Recovering nucleic acid from an uncultured sample containing selenium; and analyzing the base sequence of the target gene present in the nucleic acid using a next-generation sequencer to determine the relative abundance (1) of each microorganism. ,
Step B: Starting the culture of the sample in the presence of tetravalent and hexavalent selenium and lactic acid to obtain a culture; Recovering the nucleic acid from the culture; and base sequence of the target gene present in the nucleic acid. a step of analyzing the microorganisms using a next-generation sequencer and determining the relative abundance (2) of each microorganism;
Step C: A step of determining that microorganisms in which the relative abundance (2) is greater than the relative abundance (1) are selenium-reducing bacteria.
４及び６価セレンを還元したセレン還元菌は、還元が起こる前に比べて相対存在量が大きくなる。すなわち、未培養時点の試料と４及び６価セレン並びに乳酸により培養した試料とを用意し、それぞれの試料から得られたＤＮＡの解析・比較を行う。セレン還元と乳酸濃度の減少が観察された試料において、未培養時点の試料のＤＮＡにおける相対存在量よりも、４及び６価セレン並びに乳酸による培養後の試料における相対存在量が大きい微生物は、乳酸の存在下で４及び６価セレンを還元して生育した微生物であると解される。これにより、乳酸の存在下で４及び６価セレンを還元する微生物（セレン還元菌）を同定することができる。 The relative abundance of selenium-reducing bacteria that reduced tetravalent and hexavalent selenium becomes greater than before the reduction occurs. That is, a sample at the time of unculture and a sample cultured with tetravalent and hexavalent selenium and lactic acid are prepared, and the DNA obtained from each sample is analyzed and compared. In samples in which selenium reduction and decrease in lactic acid concentration were observed, microorganisms whose relative abundance in the sample after culture with tetravalent and hexavalent selenium and lactic acid was larger than the relative abundance in the DNA of the uncultured sample were It is understood that this microorganism grows by reducing tetravalent and hexavalent selenium in the presence of . This makes it possible to identify microorganisms (selenium-reducing bacteria) that reduce tetravalent and hexavalent selenium in the presence of lactic acid.
工程Ａ及び工程Ｂにおいて、セレン含有試料中の微生物の相対存在量を求めるには、以下の３つの工程が行われる。
（ｉ）４及び６価セレン並びに乳酸をセレン含有試料に追添加して、試料に含まれる微生物を培養すること；
（ｉｉ）未培養時点と４及び６価セレン並びに乳酸添加による培養後のそれぞれの試料から核酸（例えばＤＮＡ）を回収すること；
（ｉｉｉ）回収した核酸に存在する標的遺伝子（例えば１６Ｓ ｒＲＮＡ遺伝子）の塩基配列を次世代シークエンサーにより解析すること。
In Step A and Step B, the following three steps are performed to determine the relative abundance of microorganisms in the selenium-containing sample.
(i) additionally adding tetravalent and hexavalent selenium and lactic acid to a selenium-containing sample and culturing microorganisms contained in the sample;
(ii) recovering nucleic acids (e.g., DNA) from each sample before culture and after culture with the addition of tetravalent and hexavalent selenium and lactic acid;
(iii) Analyzing the base sequence of the target gene (eg, 16S rRNA gene) present in the recovered nucleic acid using a next-generation sequencer.
以下、上記（ｉ）～（ｉｉｉ）について具体的に説明する。
（ｉ）４及び６価セレン並びに乳酸をセレン含有試料に添加して、試料に含まれる微生物を培養すること
培養に用いるセレン含有試料は、微生物と４及び６価セレンとの反応性を良くするとの観点から、水分を添加するのが好ましい。
微生物の培養は、大気環境で行った後に、気密容器内で行われる。すなわち、添加した乳酸の有機物分解により、試料内の酸素を消費させ、微好気（一部嫌気）状態を形成し、これにより生物学的なセレン還元が誘導される。
Below, the above (i) to (iii) will be specifically explained.
(i) Adding 4- and 6-valent selenium and lactic acid to a selenium-containing sample and culturing the microorganisms contained in the sample. From this point of view, it is preferable to add water.
Cultivation of microorganisms is carried out in an air-tight container after being carried out in an atmospheric environment. That is, the added lactic acid decomposes organic matter, consuming oxygen in the sample and forming a microaerobic (partially anaerobic) state, which induces biological reduction of selenium.
微生物の培養は、セレン含有試料と４及び６価セレン並びに乳酸とを混合して、４及び６価セレン並びに乳酸の存在下で行われる。
微生物の培養は、微生物群集の状態を維持できるとの観点から、セレン含有試料に対して、４及び６価セレンと共に、乳酸を添加して培養することが挙げられるが、必要に応じて、リン酸等その他の成分を適宜添加してもよい。特に、リン酸（リン酸水素カリウム等）、乳酸以外の有機酸（酢酸等）、アルコール類（エタノール等）、糖類（グルコース等）を添加することがセレン還元菌の増殖に好ましい。
Cultivation of microorganisms is performed in the presence of tetravalent and hexavalent selenium and lactic acid by mixing the selenium-containing sample with tetravalent and hexavalent selenium and lactic acid.
From the perspective of maintaining the state of the microbial community, microorganisms can be cultured by adding lactic acid to selenium-containing samples along with tetravalent and hexavalent selenium. Other components such as acids may be added as appropriate. In particular, it is preferable for the growth of selenium-reducing bacteria to add phosphoric acid (potassium hydrogen phosphate, etc.), organic acids other than lactic acid (acetic acid, etc.), alcohols (ethanol, etc.), and sugars (glucose, etc.).
４及び６価セレンは、実環境での濃度変動（０．００００１５～０．０４ｍＭ）を踏まえて、適宜調整することができる。本発明の一実施形態では、０．０００１２７～０．０２ｍＭの４及び６価セレンの存在下で前記複数の微生物の培養を開始することが挙げられる。未培養の時点において、前記４及び６価セレンの濃度が０．０００１２７ｍＭ以上であることにより、セレン還元菌が下記の好ましい培養時間においても、４及び６価セレンを還元できる。前記４及び６価セレンの濃度が０．０２ｍＭ以下であることにより、微生物群集の状態変化を抑制できる。よって、本発明のセレン還元菌を同定する方法において、０．０００１２７～０．０２ｍＭの前記４及び６価セレンの存在下で前記の微生物の培養を開始することが好ましい。
また、化学量論として１Ｍの６価セレンを０価までに還元するために必要とされる乳酸は１．５Ｍであるが、汚染試料への乳酸の添加は、化学量論の必要濃度に対して１倍～５００倍とするのが好ましい。
Tetravalent and hexavalent selenium can be adjusted as appropriate based on concentration fluctuations in the actual environment (0.000015 to 0.04mM). One embodiment of the present invention includes starting the culture of the plurality of microorganisms in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium. By setting the concentration of 4- and 6-valent selenium to 0.000127 mM or more before culturing, the selenium-reducing bacteria can reduce 4- and 6-valent selenium even during the preferred culture time described below. When the concentration of tetravalent and hexavalent selenium is 0.02 mM or less, changes in the state of the microbial community can be suppressed. Therefore, in the method for identifying selenium-reducing bacteria of the present invention, it is preferable to start culturing the microorganism in the presence of 0.000127 to 0.02 mM of tetravalent and hexavalent selenium.
In addition, the lactic acid required to reduce 1M of hexavalent selenium to zero valence is 1.5M in terms of stoichiometry, but the addition of lactic acid to the contaminated sample is less than the required stoichiometric concentration. It is preferable to increase the amount by 1 to 500 times.
微生物の培養温度は、実環境での年間温度変化を踏まえて、設定すればよい。微生物の培養温度は、実環境で活動するセレン還元菌を検出できるとの観点から、好ましくは１０～４０℃の範囲、より好ましくは２０～３０℃である。培養温度が１０℃以上であることにより、４及び６価セレンの還元を確認できる。培養温度が４０℃以下であることにより、微生物群集の変化を抑制できる。 The culture temperature of microorganisms may be set based on annual temperature changes in the actual environment. The culture temperature of the microorganism is preferably in the range of 10 to 40°C, more preferably 20 to 30°C, from the viewpoint of detecting selenium-reducing bacteria active in a real environment. By setting the culture temperature to 10° C. or higher, reduction of tetravalent and hexavalent selenium can be confirmed. By setting the culture temperature to 40° C. or lower, changes in the microbial community can be suppressed.
微生物を培養する間、培養液を静置又は撹拌してもよい。微生物の培養には、従来公知の培養器具を用いることができる。
微生物の培養時間は、例えば１２時間以上であり、好ましくは２４時間以上である。
While culturing the microorganisms, the culture solution may be left standing or stirred. Conventionally known culture equipment can be used for culturing microorganisms.
The culture time of the microorganism is, for example, 12 hours or more, preferably 24 hours or more.
（ｉｉ）培養物から核酸を回収すること
（ｉ）で得られた培養物から核酸を回収する方法は、特に制限されず、従来公知の方法を用いることができる。回収する核酸としては、ＤＮＡ及びＲＮＡのいずれであってもよい。
ＤＮＡを回収する方法としては、例えば、ビーズ（ガラスビーズ、ジルコニアとシリカの混合ビーズなど）による物理的破砕方法、超音波処理による破砕方法、フレンチプレスによる破砕方法、液体窒素で凍結した試料を乳鉢と乳棒とで破砕する方法などを用いて、微生物細胞を破壊し、その後、従来公知の方法（フェノール・クロロホルム抽出など）を用いて、タンパク質を除去し、ＲＮａｓｅを用いてＲＮＡを消化し、ＤＮＡを回収することが挙げられる。
(ii) Recovering nucleic acids from the culture The method for recovering nucleic acids from the culture obtained in (i) is not particularly limited, and conventionally known methods can be used. The nucleic acid to be recovered may be either DNA or RNA.
Methods for recovering DNA include, for example, physical crushing using beads (glass beads, mixed beads of zirconia and silica, etc.), crushing using ultrasonication, crushing using a French press, and crushing a sample frozen with liquid nitrogen in a mortar. Microbial cells are destroyed using a method such as crushing with a pestle and a pestle, and then proteins are removed using conventionally known methods (phenol/chloroform extraction, etc.), RNA is digested using RNase, and DNA is extracted. One example is to collect the
（ｉｉｉ）回収した核酸に存在する標的遺伝子の塩基配列を次世代シークエンサーにより解析すること
セレン還元菌を同定するために用いる標的遺伝子としては、大規模な系統解析に適するとの観点から、好ましくは１６Ｓ ｒＲＮＡ遺伝子Ｖ４領域である。
次世代シークエンサーとは、サンガー法を利用した蛍光キャピラリーシークエンサーである「第１世代シークエンサー」と対比させて用いられている用語であり、ＤＮＡポリメラーゼ又はＤＮＡリガーゼによる逐次的ＤＮＡ合成法を用いて、数千万から数億のＤＮＡ断片に対して数十～数千ｂｐのリード長の断片を網羅的に解析することによって超並列的に塩基配列を決定するための装置を意味する。次世代シークエンサーでは、ジデオキシヌクレオチドを用いてＤＮＡポリメラーゼの伸長を止めるサンガー法を用いた第１世代シークエンサーと異なるシーケンシング原理が用いられている。このような原理として、合成シーケンシング法、パイロシーケンシング法、リガーゼ反応シーケンシング法などが挙げられる。これまでに、多くの企業や研究機関などから、多様な次世代シークエンサーが提供されており、例えば、ＨｉＳｅｑ２５００（イルミナ株式会社）、ＭｉＳｅｑ（イルミナ株式会社）、５５００ｘｌ ＳＯＬｉＤ（登録商標）（サーモフィッシャーサイエンティフィック）、Ｉｏｎ Ｐｒｏｔｏｎ（登録商標）（サーモフィッシャーサイエンティフィック）、Ｉｏｎ ＰＧＭ（登録商標）（サーモフィッシャーサイエンティフィック）、ＧＳ ＦＬＸ＋（ロシュ・ダイアグノスティックス社）などが挙げられる。
次世代シークエンサーによる解析方法としては、付属説明書に従って実施することができる。これにより、数万から数十万の微生物種（ＯＴＵ）を同定、及び各微生物種の相対存在量（％）を解析できる。
(iii) Analyzing the base sequence of the target gene present in the recovered nucleic acid using a next-generation sequencer From the viewpoint of being suitable for large-scale phylogenetic analysis, the target gene used to identify selenium-reducing bacteria is preferably 16S rRNA gene V4 region.
Next-generation sequencer is a term used in contrast to "first-generation sequencer," which is a fluorescent capillary sequencer that uses the Sanger method. It refers to a device that determines base sequences in massively parallel manner by comprehensively analyzing fragments with read lengths of several tens to several thousand bp for tens of millions to hundreds of millions of DNA fragments. Next-generation sequencers use a different sequencing principle from first-generation sequencers, which use the Sanger method, which uses dideoxynucleotides to stop DNA polymerase elongation. Such principles include synthetic sequencing methods, pyrosequencing methods, ligase reaction sequencing methods, and the like. To date, many companies and research institutes have provided a variety of next-generation sequencers, such as HiSeq2500 (Illumina Corporation), MiSeq (Illumina Corporation), 5500xl SOLiD (registered trademark) (Thermo Fisher Science Ion Proton (registered trademark) (Thermo Fisher Scientific), Ion PGM (registered trademark) (thermo Fisher Scientific), GS FLX+ (Roche Diagnostics), and the like.
The analysis method using a next-generation sequencer can be carried out according to the attached instructions. This makes it possible to identify tens of thousands to hundreds of thousands of microbial species (OTU) and to analyze the relative abundance (%) of each microbial species.
工程Ｃのセレン還元菌であるとの判断は、未培養の時点における微生物の相対存在量（１）と４及び６価セレン並びに乳酸添加による培養後における微生物の相対存在量（２）とを対比することで行うことができる。
例えば、ＯＴＵのうち、セレン還元が観察された培養物において、培養後で培養開始時点よりも２倍以上に上昇している微生物を特定する。
４及び６価セレン還元が観察された培養物において、未培養の時点よりも２倍以上に上昇している微生物は、乳酸の存在下で４及び６価セレンを還元して生育したため、そのような微生物をセレン還元菌であると判断できる。
The judgment that it is a selenium-reducing bacterium in Step C is made by comparing the relative abundance of microorganisms at the time of unculture (1) and the relative abundance of microorganisms after culturing with the addition of tetravalent and hexavalent selenium and lactic acid (2). This can be done by doing this.
For example, among OTUs, in a culture in which selenium reduction has been observed, microorganisms in which the increase in selenium reduction after culture is twice or more than at the time of culture initiation are identified.
In cultures in which 4- and 6-valent selenium reduction was observed, the microorganisms whose increase was more than twice that of the uncultured state were due to growth by reducing 4- and hexavalent selenium in the presence of lactic acid. microorganisms can be determined to be selenium-reducing bacteria.
［セレン汚染試料のセレンの還元処理]
本発明によれば、上記本発明のセレン還元菌を含むセレン汚染試料を、乳酸の存在下、微好気的条件下で静置又は撹拌する工程を含む、セレン汚染試料のセレンの還元処理方法が提供される。
ここで、処理されるセレン汚染試料としては、前述したセレン還元菌を含むセレン汚染試料自体であってもよく、それとは別のセレン汚染試料（例えば、セレン還元菌を含まない別のセレン汚染試料、本発明のセレン還元菌とは異なるセレン還元菌を含む別のセレン汚染試料）が含まれていてもよい。また、単離・抽出し培養した上記セレン還元菌を、セレン汚染試料（例えば、セレン還元菌を含まない別のセレン汚染試料、本発明のセレン還元菌とは異なるセレン還元菌を含む別のセレン汚染試料）に添加したものでもよい。ここで、セレン汚染試料としては、セレンが含まれるものであれば特に限定されず、掘削ずり、土砂、汚泥、水、焼却灰等の廃棄物、工場廃水等いずれでもよい。
[Selenium reduction treatment of selenium-contaminated samples]
According to the present invention, a selenium reduction treatment method for a selenium-contaminated sample, which includes the step of leaving the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention still or stirring under microaerobic conditions in the presence of lactic acid. is provided.
Here, the selenium-contaminated sample to be processed may be the selenium-contaminated sample itself containing the selenium-reducing bacteria described above, or it may be another selenium-contaminated sample (for example, another selenium-contaminated sample that does not contain selenium-reducing bacteria). , another selenium-contaminated sample containing selenium-reducing bacteria different from the selenium-reducing bacteria of the present invention). In addition, the isolated, extracted and cultured selenium-reducing bacteria may be used in a selenium-contaminated sample (for example, another selenium-contaminated sample that does not contain selenium-reducing bacteria, or another selenium-contaminated sample that contains selenium-reducing bacteria different from the selenium-reducing bacteria of the present invention). It may also be added to a contaminated sample. Here, the selenium-contaminated sample is not particularly limited as long as it contains selenium, and may be any waste such as excavation slag, earth and sand, sludge, water, incineration ash, factory wastewater, etc.
斯かるセレン汚染試料のセレンの還元処理は、乳酸の存在下に行われる。乳酸は汚染試料中の本発明のセレン還元菌が十分に増殖し還元力を発揮できる量であれば、その使用量は限定されないが、汚染試料中（乾燥状態）の乳酸の含有量が０.０１４質量％以上となるように行われるのが好ましい。
例えば、乳酸の汚染試料中の含有量を、好ましくは０.０１４～０.７０質量％、より好ましくは０.１４～０．７０質量％とすることが挙げられる。
The selenium reduction treatment of such a selenium-contaminated sample is performed in the presence of lactic acid. The amount of lactic acid used is not limited as long as the selenium-reducing bacteria of the present invention in the contaminated sample can sufficiently proliferate and exert its reducing power, but if the lactic acid content in the contaminated sample (dry state) is 0. It is preferable that the amount is 0.014% by mass or more.
For example, the content of lactic acid in the contaminated sample is preferably 0.014 to 0.70% by mass, more preferably 0.14 to 0.70% by mass.
乳酸を汚染試料に添加する方法は、特に制限はなく、乳酸をそのまま散布機等で汚染試料に散布する方法や、水等の溶媒に溶解させた溶液状のものを、散水ノズル、灌注機等で汚染試料に散布したりする方法が採用できる。
乳酸を水等の溶媒に溶解させる場合には、例えば０．９～９０．０８ｇ／Ｌ、好ましくは０．９～４５．０ｇ／Ｌ、より好ましくは９．０～４５．０ｇ／Ｌ濃度の溶液とするのが好ましい。
There are no particular restrictions on the method of adding lactic acid to the contaminated sample, and there are methods such as spraying lactic acid directly onto the contaminated sample using a sprayer, or adding a solution of lactic acid dissolved in a solvent such as water using a water spray nozzle, irrigation machine, etc. A method of spraying contaminated samples with water can be adopted.
When lactic acid is dissolved in a solvent such as water, the concentration is, for example, 0.9 to 90.08 g/L, preferably 0.9 to 45.0 g/L, more preferably 9.0 to 45.0 g/L. Preferably, it is a solution.
処理条件は、本発明のセレン還元菌を含むセレン汚染試料中のセレン還元菌が増殖し還元力を発揮できる条件であればよく、微好気的条件下で静置又は撹拌すればよいが、好ましくは１０～４０℃、より好ましくは２０～３０℃で、１２時間以上、好ましくは１８時間以上、より好ましくは２４時間以上静置又は撹拌することが挙げられ、また、好ましくは、４５００時間以下、より好ましくは１５００時間以下である。
また、上記セレン汚染試料には、セレン還元菌の還元力が発揮されやすいように、鉄塩や亜硫酸塩、シュウ酸、その他の還元剤を添加して、還元状態に変化させてもよい。
The treatment conditions may be any conditions that allow the selenium-reducing bacteria in the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention to proliferate and exhibit reducing power, and may be left standing or stirring under microaerobic conditions. Preferably at 10 to 40°C, more preferably at 20 to 30°C, for 12 hours or more, preferably for 18 hours or more, more preferably for 24 hours or more, and preferably for 4,500 hours or less. , more preferably 1500 hours or less.
In addition, iron salts, sulfites, oxalic acid, or other reducing agents may be added to the selenium-contaminated sample to change it to a reduced state so that the reducing power of selenium-reducing bacteria can be easily exerted.
例えば、本発明のセレン還元菌を含むセレン汚染試料へ乳酸を添加した後、１０～４０℃で、１２時間以上静置又は撹拌することにより、当該試料中のセレンを還元処理できる。また、本発明のセレン還元菌を含むセレン汚染試料及び乳酸を添加して収容した槽に、それとは別のセレン汚染試料を加えることで、当該別のセレン汚染試料についてもセレンの還元処理が可能である。 For example, by adding lactic acid to a selenium-contaminated sample containing the selenium-reducing bacteria of the present invention and then allowing it to stand or stir at 10 to 40°C for 12 hours or more, the selenium in the sample can be reduced. Furthermore, by adding another selenium-contaminated sample to the tank containing the selenium-contaminated sample containing the selenium-reducing bacteria of the present invention and lactic acid, it is possible to perform selenium reduction treatment on the other selenium-contaminated sample. It is.
また、当該還元処理には、必要に応じて、リン酸等その他の成分を適宜添加してもよい。特に、リン酸（リン酸水素カリウム等）、乳酸以外の有機酸（酢酸等）、アルコール類（エタノール等）、糖類（グルコース等）の１種以上を添加する工程を含むのがセレン還元菌の増殖を促進する点から好ましい。 In addition, other components such as phosphoric acid may be appropriately added to the reduction treatment, if necessary. In particular, the process of adding one or more of phosphoric acid (potassium hydrogen phosphate, etc.), organic acids other than lactic acid (acetic acid, etc.), alcohols (ethanol, etc.), and sugars (glucose, etc.) It is preferable from the viewpoint of promoting proliferation.
[セレン汚染試料のセレン還元化能の評価]
本発明の一実施形態によれば、セレン汚染試料のセレン還元化能を評価する方法であって、試料中の、セレン還元菌Ａ、セレン還元菌Ｂ、セレン還元菌Ｃ、セレン還元菌Ｄ、セレン還元菌Ｅ、セレン還元菌Ｆ、セレン還元菌Ｇ、セレン還元菌Ｈ、セレン還元菌Ｉ、セレン還元菌Ｊ、セレン還元菌Ｋ、セレン還元菌Ｌ、セレン還元菌Ｍ、セレン還元菌Ｎ、セレン還元菌Ｏ、セレン還元菌Ｐ、セレン還元菌Ｑ、及びセレン還元菌Ｒからなる群から選択される少なくとも１種の存在を確認する工程を含む、方法が提供される。
[Evaluation of selenium reduction ability of selenium-contaminated samples]
According to one embodiment of the present invention, there is provided a method for evaluating the selenium reducing ability of a selenium-contaminated sample, which comprises selenium-reducing bacteria A, selenium-reducing bacteria B, selenium-reducing bacteria C, selenium-reducing bacteria D, Selenium reducing bacteria E, selenium reducing bacteria F, selenium reducing bacteria G, selenium reducing bacteria H, selenium reducing bacteria I, selenium reducing bacteria J, selenium reducing bacteria K, selenium reducing bacteria L, selenium reducing bacteria M, selenium reducing bacteria N, A method is provided that includes a step of confirming the presence of at least one species selected from the group consisting of selenium-reducing bacteria O, selenium-reducing bacteria P, selenium-reducing bacteria Q, and selenium-reducing bacteria R.
当該セレン還元菌の確認手段は、特に限定されないが、好適には、前述した菌叢ゲノムＤＮＡに含まれる１６Ｓ ｒＲＮＡ遺伝子の塩基配列に基づいて、試料中に存在する菌種を解析する方法が挙げられる。
すなわち、上述したとおり、試料から核酸を回収し、当該核酸に存在する標的遺伝子の塩基配列を次世代シークエンサーにより解析することにより、相対存在量（％）を確認することが挙げられる。
The means for confirming the selenium-reducing bacteria is not particularly limited, but preferably includes a method of analyzing the bacterial species present in the sample based on the base sequence of the 16S rRNA gene contained in the bacterial flora genomic DNA described above. It will be done.
That is, as described above, the relative abundance (%) can be confirmed by collecting a nucleic acid from a sample and analyzing the base sequence of a target gene present in the nucleic acid using a next-generation sequencer.
これにより、セレン汚染試料のセレン還元化能を評価することができる。ここで、試料のセレン還元化能とは、当該試料が有するセレン還元菌に基づくセレン還元能を意味する。 Thereby, the selenium reduction ability of the selenium-contaminated sample can be evaluated. Here, the selenium-reducing ability of a sample means the selenium-reducing ability based on the selenium-reducing bacteria that the sample has.
以下に、実施例によって本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.
実施例１ セレン還元菌の同定
（１）使用した掘削ずり
サンプルとして、建設工事で発生したトンネル掘削ずりを用いた。使用したずりの物性を表２に示す。
Example 1 Identification of selenium-reducing bacteria (1) Excavation slag used Tunnel excavation slag generated during construction work was used as a sample. Table 2 shows the physical properties of the shear used.
セレン、ひ素、鉛、ふっ素の溶出特性は、環境庁告示第１８号「土壌溶出量調査に係る測定方法を定める件」に記載されている方法に準拠して検液作成を行い、「ＪＩＳ Ｋ ０１０２：２０１３（工場排水試験方法）」に準拠して測定した。 The elution characteristics of selenium, arsenic, lead, and fluorine were determined by preparing test solutions in accordance with the method described in the Environment Agency Notification No. 18, "Measurement methods related to soil elution survey." 0102:2013 (factory wastewater test method)".
（２）培養条件
培養条件を表３に示す。
試料作製は、２０℃試験室内で行い、汚染試料５gを滅菌水又は乳酸（１００ｍＭ、５００ｍＭ）を添加した滅菌水で２．７６ｍｌ容量にして、５６ｍｌ容ガラスバイアル瓶に収容した。気相部分は大気（Ｏ２:２０％）とした。汚染試料に対して、６価セレン溶液（亜セレン酸ナトリウム；和光純薬工業株式会社製）を６価セレンの終濃度０．０１２７ｍＭとなるように添加した。その後、２０℃、暗所にて２８日まで静置培養を行った。
(2) Culture conditions The culture conditions are shown in Table 3.
Sample preparation was carried out in a 20° C. test chamber, and 5 g of the contaminated sample was diluted to 2.76 ml with sterile water or sterile water added with lactic acid (100 mM, 500 mM), and placed in a 56 ml glass vial. The gas phase portion was set to the atmosphere (O 2 :20%). A hexavalent selenium solution (sodium selenite; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the contaminated sample so that the final concentration of hexavalent selenium was 0.0127 mM. Thereafter, static culture was performed at 20° C. in the dark until 28 days.
（３）試験方法
セレン溶出量は、上記のとおり、環境庁告示第18号「土壌溶出量調査に係る測定方法を定める件」に記載されている方法に準拠して測定した。結果を図１に示す。
微好気条件にて、乳酸添加によってセレン溶出量が大幅に低下した。掘削ずり中に存在する微生物によりセレン還元が起こり、乳酸の添加によりその還元反応が加速されることが確認された。
(3) Test method As mentioned above, the amount of selenium eluted was measured in accordance with the method described in Environment Agency Notification No. 18, ``Measurement method related to soil eluted amount investigation''. The results are shown in Figure 1.
Under microaerobic conditions, the amount of selenium eluted was significantly reduced by the addition of lactic acid. It was confirmed that selenium reduction occurs due to microorganisms present in the excavation slag, and that the reduction reaction is accelerated by the addition of lactic acid.
（３）微生物学解析
未培養時点（培養０日）及び６価セレンによる培養後（培養７日）の試料について、試料を遠心分離し、遠心沈殿物を得た。前記遠心沈殿物に対して、ジルコニアとシリカの混合ビーズ（Ｚｉｒｃｏｎｉａ／Ｓｉｌｉｃａ Ｂｅａｄｓ；平均粒子径０．１ｍｍ）を加え、シェイクマスターオート（株式会社バイオメディカルサイエンス製）を用いて固相中の微生物細胞を破砕した後、フェノールとクロロホルムとを用いた精製ステップを経ることで共存するタンパク質を除去した。その後、ＲＮａｓｅ Ａ（ベックマン社製）の処理を行うことでＲＮＡを消化し、ＤＮＡを精製した。
この精製ＤＮＡを鋳型に用い、１６Ｓ ｒＲＮＡ遺伝子Ｖ４領域（約３００塩基対）を標的として、Ｑ５ Ｈｉｇｈ－Ｆｉｄｅｌｉｔｙ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ・Ｑ５
ＤＮＡポリメラーゼ（NEB社）による遺伝子増幅反応（Ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ［ＰＣＲ］）を行った。得られたＰＣＲ産物をＡＭＰｕｒｅ ＸＰ キット（ベックマン・コールター社製）及びＷｉｚａｒｄ（登録商標）ＳＶ ｇｅｌ
ａｎｄ ＰＣＲ ｃｌｅａｎ－ｕｐキット（プロメガ社製）を用いて精製し、その濃度をＱｕａｎｔ－ｉＴ ＰｉｃｏＧｒｅｅｎ ｄｓＤＮＡ 試薬・キット（サーモフィッシャーサイエンティフィック株式会社製）と蛍光定量装置Ｎａｎｏｄｒｏｐ ３３００（サーモフィッシャーサイエンティフィック株式会社製）とにより測定した。
(3) Microbiological analysis The samples were centrifuged to obtain centrifugal precipitates at the time of uncultivation (day 0 of culture) and after culture with hexavalent selenium (day 7 of culture). Mixed beads of zirconia and silica (Zirconia/Silica Beads; average particle diameter 0.1 mm) were added to the centrifugal precipitate, and microbial cells in the solid phase were isolated using Shake Master Auto (manufactured by Biomedical Science Co., Ltd.). After crushing, coexisting proteins were removed by a purification step using phenol and chloroform. Thereafter, RNA was digested by treatment with RNase A (manufactured by Beckman), and DNA was purified.
Using this purified DNA as a template and targeting the 16S rRNA gene V4 region (approximately 300 base pairs), Q5 High-Fidelity DNA Polymerase Q5
Gene amplification reaction (Polymerase chain reaction [PCR]) using DNA polymerase (NEB) was performed. The obtained PCR product was used with AMPure XP kit (manufactured by Beckman Coulter) and Wizard (registered trademark) SV gel.
and PCR clean-up kit (manufactured by Promega), and its concentration was determined using a Quant-iT PicoGreen dsDNA reagent kit (manufactured by Thermo Fisher Scientific) and a fluorescence quantification device Nanodrop 3300 (manufactured by Thermo Fisher Scientific). Co., Ltd.).
最適量の精製産物をＭｉＳｅｑ Ｒｅａｇｅｎｔ キット３００サイクルと次世代シークエンサーＭｉＳｅｑ（イルミナ株式会社製）を用いた大規模塩基配列解析に供した。各試料から遺伝子断片を数万リード解読し、ソフトウエアｍｏｔｈuｒ（ｖｅｒ １．３１．２）を用いて、非特異的配列を除去し、ソフトウエアＱＩＩＭＥ（ｖｅｒ １．６．０）を用いて、微生物系統解析を行い、微生物種（ＯＴＵ：Ｏｐｅｒａｔｉｏｎａｌ ｔａｘｏｎｏｍｉｃ ｕｎｉｔ）の同定と相対存在量（％）の解析を行った。 The optimal amount of the purified product was subjected to large-scale base sequence analysis using MiSeq Reagent kit 300 cycles and next-generation sequencer MiSeq (manufactured by Illumina Corporation). Tens of thousands of reads of gene fragments were decoded from each sample, non-specific sequences were removed using the software mothur (ver 1.31.2), and the software QIIME (ver 1.6.0) was used to Microbial phylogenetic analysis was performed, and microbial species (OTU: Operational taxonomic unit) were identified and relative abundances (%) were analyzed.
得られたＯＴＵのうちセレン還元が観察された乳酸（１００ｍＭ、５００ｍＭ）及びセレンを添加して培養した培養物において、培養後に、培養前と比べて相対存在量が有意に２倍以上に増加した１８種の微生物を特定した（表４及び５）。ＯＴＵの近縁種と遺伝子配列同一性をＮＣＢＩ塩基配列データベースのＢＬＡＳＴプログラムにより決定した。 Among the obtained OTUs, in cultures cultured with the addition of lactic acid (100mM, 500mM) and selenium in which selenium reduction was observed, the relative abundance significantly increased by more than 2 times after culture compared to before culture. Eighteen types of microorganisms were identified (Tables 4 and 5). Gene sequence identity of OTUs to related species was determined by the BLAST program of the NCBI sequence database.
表４及び５に示すとおり、汚染試料の微生物群集において、セレン還元菌は、いずれも培養開始時点では相対存在量が１．６％以下の稀少微生物種であることが明らかになった。したがって、微好気的な雰囲気にした汚染試料中のセレン還元菌は、それぞれの試料及び乳酸の存在下で増加し、４及び６価セレンの還元に関与していることが強く示唆された。 As shown in Tables 4 and 5, it was revealed that in the microbial communities of the contaminated samples, all selenium-reducing bacteria were rare microbial species with a relative abundance of 1.6% or less at the start of culture. Therefore, the selenium-reducing bacteria in the contaminated samples exposed to a microaerobic atmosphere increased in each sample and in the presence of lactic acid, strongly suggesting that they were involved in the reduction of tetravalent and hexavalent selenium.
また、表４及び５に示すとおり、１８種のセレン還元菌は、１６Ｓ ｒＲＮＡ遺伝子の塩基配列同一性が９７～１００％であり、いずれも既知の微生物種又はこれと系統学的に同一又は近縁の微生物であると考えられる。
例えば、ＯＴＵ１７１３１（セレン還元菌にＡ属する）は、Ｈａｌｏｍｏｎａｓ ｄｅｎｉｔｒｉｆｉｃａｎｓ（ハロモナス デニトリフィカンス）と系統学的に同一である（１６Ｓ ｒＲＮＡ遺伝子配列同一性は、１００％を示した）。
また、ＯＴＵ１２５９１（セレン還元菌Ｆに属する）は、Ａｚｏａｒｃｕｓ ｔｏｌｕｌｙｔｉｃｕｓ（アゾアルカス トルリティカス）と系統学的に同一又は近縁である（１６Ｓ ｒＲＮＡ遺伝子配列同一性は、１００％を示した）。
また、ＯＴＵ８１９７（セレン還元菌Ｈに属する）、ＯＴＵ１５３７６（セレン還元菌Ｉに属する）、ＯＴＵ７０４１（セレン還元菌Ｑに属する）は、Ｍａｒｍｏｒｉｃｏｌａ ｂｉｇｅｕｍｅｎｓｉｓ（マルモリコラ ビギウメンシス）と系統学的に近縁である（１６Ｓ ｒＲＮＡ遺伝子配列同一性は、それぞれ９８．０％、９７．３％、９８．０％を示した）。
また、ＯＴＵ８２７（セレン還元菌Ｊに属する）、ＯＴＵ６３０６（セレン還元菌Ｌに属する）は、Ｐｓｅｕｄｏｎａｃａｒｄｉａ ａｎｔｉｍｉｃｒｏｂｉｃａ（シュードノカルジア アンチミクロビカ）と系統学的に近縁である（１６Ｓ ｒＲＮＡ遺伝子配列同一性は、９７．６％、９９．６％を示した）。
Furthermore, as shown in Tables 4 and 5, the nucleotide sequence identity of the 16S rRNA gene of the 18 types of selenium-reducing bacteria is 97 to 100%, and all of them are known microbial species or are phylogenetically identical or close to this. It is thought to be a marginal microorganism.
For example, OTU17131 (belonging to the selenium-reducing bacteria A) is phylogenetically identical to Halomonas denitrificans (16S rRNA gene sequence identity showed 100%).
Furthermore, OTU12591 (belonging to selenium-reducing bacteria F) is phylogenetically identical or closely related to Azoarcus tolulyticus (16S rRNA gene sequence identity showed 100%).
Additionally, OTU8197 (belongs to selenium-reducing bacteria H), OTU15376 (belongs to selenium-reducing bacteria I), and OTU7041 (belongs to selenium-reducing bacteria Q) are phylogenetically related to Marmoricola bigeumensis (16S rRNA gene sequence identity showed 98.0%, 97.3%, and 98.0%, respectively).
In addition, OTU827 (belongs to selenium-reducing bacteria J) and OTU6306 (belongs to selenium-reducing bacteria L) are phylogenetically related to Pseudonacardia antimicrobica (16S rRNA gene sequence identity is 97.6% and 99.6%).
斯かるＡ、Ｆ、Ｈ、Ｉ、Ｊ、Ｌ及びＱで示される７種のセレン還元菌については、既往の知見において微好気環境で嫌気呼吸能を有していることが報告されている。したがって、この点を加味すると、セレンの還元処理にはＡ、Ｆ、Ｈ、Ｉ、Ｊ、Ｌ及びＱから選ばれる１種以上を含むように用いるのが好ましいと云える。 The seven types of selenium-reducing bacteria represented by A, F, H, I, J, L, and Q have been reported to have anaerobic respiration ability in a microaerobic environment. . Therefore, taking this point into account, it can be said that it is preferable to use one or more selected from A, F, H, I, J, L, and Q in the reduction treatment of selenium.
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