JP5969309B2 - Selective control method for sulfate-reducing bacteria - Google Patents
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Description
本発明は、他の菌の増殖を抑制することなく、硫酸還元菌の増殖のみを抑制する硫酸還元菌の選択的制菌方法に関するものである。 The present invention relates to a selective sterilization method for sulfate-reducing bacteria that suppresses only the growth of sulfate-reducing bacteria without inhibiting the growth of other bacteria.
硫酸還元菌は、硫酸イオンを電子受容体として有機物を酸化し、エネルギーを獲得する細菌である。前記硫酸還元菌は種類によってその生育温度域が異なり、高温性菌、中温性菌、低温性菌および好冷性菌が存在する。また、生育ｐＨの違いによって、中性付近で良く生育する菌と、酸性あるいはアルカリ性の条件で良く生育する菌が存在する。また、生存域も海洋泥、一般土壌、熱水噴出域、パイプラインの中等と広域にわたる。 A sulfate-reducing bacterium is a bacterium that acquires energy by oxidizing an organic substance using sulfate ions as an electron acceptor. The growth temperature range of the sulfate-reducing bacteria varies depending on the type, and there are thermophilic bacteria, mesophilic bacteria, psychrophilic bacteria and psychrophilic bacteria. In addition, depending on the growth pH, there are bacteria that grow well near neutrality and bacteria that grow well under acidic or alkaline conditions. In addition, the survival area covers a wide area such as marine mud, general soil, hydrothermal eruption area, and pipeline.
硫酸還元菌のエネルギー獲得の過程においては、硫酸イオンの還元体である硫化水素が生成される。この硫化水素は毒性や腐食性が強く、悪臭を放つ物質であるため、多量に生成されると問題となる。例えば、自然水、肥料（硫酸アンモニウム等）、不法投棄された石膏製品等に含まれる硫酸イオンを電子受容体として硫酸還元菌がエネルギーを獲得する際に硫化水素が発生し、農地において作物の生育を阻害し、或いは鉄材（埋設管等）を腐食する等の問題が知られている。 In the process of obtaining energy by sulfate-reducing bacteria, hydrogen sulfide, which is a reduced form of sulfate ions, is generated. Since this hydrogen sulfide is highly toxic and corrosive and emits a bad odor, it causes a problem if it is produced in a large amount. For example, hydrogen sulfide is generated when sulfate-reducing bacteria acquire energy by using sulfate ions contained in natural water, fertilizers (such as ammonium sulfate), and illegally dumped gypsum products as electron acceptors. Problems such as obstruction or corrosion of iron materials (such as buried pipes) are known.
そこで、硫酸還元菌の増殖を抑制し、硫化水素の生成を抑制することが試みられている。例えば、本出願人の一は、石膏を主材とする土壌処理材に、アントラキノン化合物を添加することにより、硫酸還元菌の増殖を抑制し、硫化水素の生成を抑制する方法を開示している（特許文献１参照）。 Therefore, attempts have been made to suppress the growth of sulfate-reducing bacteria and suppress the production of hydrogen sulfide. For example, one of the present applicants discloses a method for suppressing the growth of sulfate-reducing bacteria and suppressing the production of hydrogen sulfide by adding an anthraquinone compound to a soil treatment material mainly composed of gypsum. (See Patent Document 1).
しかし、特許文献１に記載の技術は、非常に高価なアントラキノン化合物を用いるために、製造コストの面で問題があった。特に土壌処理材等の大量に使用される用途においては、コストが障害となって利用が拡大されないという実情があった。 However, the technique described in Patent Document 1 has a problem in terms of manufacturing cost because an extremely expensive anthraquinone compound is used. In particular, in applications that are used in large quantities, such as soil treatment materials, there has been a situation where the cost is an obstacle and the use is not expanded.
そこで、本出願人の一は、石膏組成物に、アルミニウム硫酸塩水和物［Ａｌ2（ＳＯ4）3・ｎＨ2Ｏ（ｎ＝６、１０、１６、１８、２７）］等の特定のアルミニウム化合物を添加することにより、硫酸還元菌の増殖を抑制し、硫化水素の生成を抑制する方法も開示している（特許文献２参照）。 Therefore, one of the applicants of the present application is that a specific aluminum such as aluminum sulfate hydrate [Al 2 (SO 4 ) 3 .nH 2 O (n = 6, 10, 16, 18, 27)] is used in the gypsum composition. A method of suppressing the growth of sulfate-reducing bacteria and adding hydrogen sulfide by adding a compound is also disclosed (see Patent Document 2).
特許文献２に記載の方法は、アントラキノン化合物と比較して安価で入手し易いアルミニウム化合物を用いるため、特許文献１に記載の技術が有する製造コストという問題を解決し得るものである。しかし、以下の点において未だ改良の余地を残すものであった。 Since the method described in Patent Document 2 uses an aluminum compound that is inexpensive and easily available as compared with an anthraquinone compound, the method described in Patent Document 1 can solve the problem of manufacturing cost. However, there is still room for improvement in the following points.
即ち、特許文献２に記載の方法によれば、硫酸還元菌の増殖を抑制し、硫化水素の生成を抑制することは可能であるものの、硫酸還元菌以外の菌（以下、「他の菌」と記す。）の増殖を抑制してしまう場合があった。他の菌の増殖が抑制された場合、土壌等の微生物相が破壊され、悪臭の発生、植物の生育障害等の問題を生ずることがある。従って、他の菌の増殖を抑制することなく、硫酸還元菌の増殖を選択的に抑制することが可能な方法を確立することが切望されている。 That is, according to the method described in Patent Document 2, although it is possible to suppress the growth of sulfate-reducing bacteria and suppress the production of hydrogen sulfide, bacteria other than sulfate-reducing bacteria (hereinafter referred to as “other bacteria”). In some cases). When the growth of other bacteria is suppressed, the microflora such as soil may be destroyed, resulting in problems such as the generation of malodors and impaired plant growth. Therefore, it is anxious to establish a method capable of selectively suppressing the growth of sulfate-reducing bacteria without suppressing the growth of other bacteria.
本発明は、前記従来技術の課題を解決するためになされたものである。即ち、本発明は、他の菌の増殖を抑制することなく、硫酸還元菌の増殖を選択的に抑制することが可能な硫酸還元菌の選択的制菌方法を提供するものである。 The present invention has been made to solve the above-described problems of the prior art. That is, the present invention provides a selective sterilization method for sulfate-reducing bacteria that can selectively inhibit the growth of sulfate-reducing bacteria without inhibiting the growth of other bacteria.
本発明者らは前記課題について鋭意検討を行った結果、キレート化Ａｌに硫酸還元菌の増殖を選択的に抑制する作用があることを見出して、本発明を完成するに至った。 As a result of intensive studies on the above problems, the present inventors have found that chelated Al has an action of selectively suppressing the growth of sulfate-reducing bacteria, and have completed the present invention.
即ち、本発明によれば、硫酸還元菌の存在する環境に、キレート化Ａｌを併存させることにより、前記硫酸還元菌の増殖を選択的に抑制することを特徴とする硫酸還元菌の選択的制菌方法が提供される。 That is, according to the present invention, the selective control of sulfate-reducing bacteria is characterized by selectively inhibiting the growth of the sulfate-reducing bacteria by coexisting chelated Al in the environment where the sulfate-reducing bacteria are present. A fungal method is provided.
本発明においては、前記硫酸還元菌の存在する環境に、Ａｌ3+源及びキレート剤を添加して、前記環境中で前記キレート化Ａｌを生成させることが好ましい。また、本発明においては、前記Ａｌ3+源が、Ａｌ2Ｏ3、ＡｌＣｌ3、Ａｌ（ＯＨ）3及びＡｌ2（ＳＯ4）3からなる群より選択される少なくとも一種のＡｌ化合物であることが好ましい。更に、本発明においては、前記硫酸還元菌が、中温域（２０〜４５℃）かつ中性域（ｐＨ５〜９）で生息する菌であることが好ましい。
In the present invention, it is preferable to add an Al 3+ source and a chelating agent to the environment where the sulfate-reducing bacteria are present to produce the chelated Al in the environment. In the present invention, the Al 3+ source is at least one Al compound selected from the group consisting of Al 2 O 3 , AlCl 3 , Al (OH) 3 and Al 2 (SO 4 ) 3. Is preferred. Furthermore, in the present invention, the sulfate-reducing bacteria are preferably bacteria that inhabit in a medium temperature range (20 to 45 ° C.) and a neutral range (
本発明に係る選択的制菌方法は、硫酸還元菌の増殖を選択的に抑制することができる。より具体的には、硫酸還元菌の増殖を抑制し、硫化水素の生成を抑制することができる。一方、本発明に係る選択的制菌方法は、他の菌の増殖が抑制されないため、土壌等の微生物相が破壊されることもなく、悪臭の発生、植物の生育障害等の問題を生ずるおそれはない。 The selective sterilization method according to the present invention can selectively suppress the growth of sulfate-reducing bacteria. More specifically, the growth of sulfate-reducing bacteria can be suppressed and the production of hydrogen sulfide can be suppressed. On the other hand, the selective sterilization method according to the present invention does not inhibit the growth of other bacteria, so that the microflora such as soil is not destroyed, and problems such as generation of malodor and disturbance of plant growth occur. It is not.
以下、本発明について詳細に説明する。但し、本発明は下記の実施形態に限定されず、その発明特定事項を有する全ての対象を含むものである。 Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following embodiment, and includes all objects having the invention-specific matters.
［１］硫酸還元菌の選択的制菌方法：
本発明は、硫酸還元菌の増殖を選択的に抑制する選択的制菌方法に関するものである。
[1] Method for selective sterilization of sulfate-reducing bacteria:
The present invention relates to a selective sterilization method that selectively suppresses the growth of sulfate-reducing bacteria.
前記のように、「硫酸還元菌」とは、硫酸イオンを電子受容体として有機物を酸化し、エネルギーを獲得する細菌である。この細菌は、一般土壌や下水汚泥等の嫌気環境に広く分布している。本発明の適用対象となる硫酸還元菌の種類は特に限定されない。例えば、グラム陰性菌であってもよいし、グラム陽性菌であってもよいし、古細菌でもよい。 As described above, a “sulfate-reducing bacterium” is a bacterium that acquires energy by oxidizing an organic substance using sulfate ions as electron acceptors. This bacterium is widely distributed in anaerobic environments such as general soil and sewage sludge. The kind of sulfate-reducing bacteria to which the present invention is applied is not particularly limited. For example, it may be a Gram negative bacterium, a Gram positive bacterium, or an archaea.
具体的には、デスルホビブリオ属（Desulfovibrio；グラム陰性嫌気性桿菌、同らせん菌）、デスルフロモナス属（Desulfuromonas；グラム陰性嫌気性桿菌、同らせん菌）、デスルフィトバクテリウム属（Desulfitobacterium；グラム陰性偏性嫌気性菌）、デスルホトマキュラム属（Desulfotomaculum；グラム陽性内生胞子形成桿菌）等の硫酸還元菌を挙げることができる。 Specifically, the genus Desulfovibrio (Desulfovibrio; Gram-negative anaerobic gonococcus, Helicobacteria), Desulfuromonas (Desulfuromonas; Gram-negative anaerobic gonococcus, Helicobacteria), Desulfitobacterium (Gram-negative) Examples include sulfate-reducing bacteria such as obligately anaerobic bacteria and Desulfotomaculum (Gram-positive endospore-forming rods).
デスルホビブリオ属の細菌の具体例としては、デスルホビブリオ・ブルガリス（Desulfovibrio vulgaris）、デスルホビブリオ・アフリカヌス（Desulfovibrio africanus）、デスルホビブリオ・デスルフリカンス（Desulfovibrio desulfuricans）、デスルホビブリオ・ギガス（Desulfovibrio gigas）等を挙げることができる。デスルホトマキュラム属の細菌の具体例としては、デスルホトマキュラム・ルミニス（Desulfotomaculum ruminis）等を挙げることができる。 Specific examples of bacteria of the genus Desulfovibrio include Desulfovibrio vulgaris, Desulfovibrio africanus, Desulfovibrio desulfuricans, Desulfovibrio desulfuricans, Desulfovibrio vulgaris (Desulfovibrio vulgaris) Desulfovibrio gigas) and the like. Specific examples of bacteria belonging to the genus Desulfotomaculum include Desulfotomaculum ruminis and the like.
本発明の方法は、前記硫酸還元菌全般に対して抑制効果を奏する。中でも、中温域（２０〜４５℃）かつ中性域（ｐＨ５〜９）で生息する硫酸還元菌に対して好適に用いることができる。既述のデスルホビブリオ・ブルガリス、デスルホビブリオ・アフリカヌス、デスルホビブリオ・デスルフリカンス、デスルホビブリオ・ギガス、デスルホトマキュラム・ルミニスは、「中温域（２０〜４５℃）かつ中性域（ｐＨ５〜９）で生息する硫酸還元菌」である。 The method of the present invention has an inhibitory effect on the sulfate-reducing bacteria in general. Especially, it can use suitably with respect to the sulfate reduction bacteria which inhabit in a middle temperature range (20-45 degreeC) and a neutral range (pH 5-9). The above-mentioned desulfobibrio bulgaris, desulfobibrio africanus, desulfobibrio desulfuricans, desulfobibrio gigas, desulfotomacurum luminis are “medium temperature range (20 to 45 ° C.) and neutral range. "Sulphate-reducing bacteria that inhabit (pH 5-9)".
本発明の方法は、キレート化Ａｌが硫酸還元菌の硫酸還元機構に作用し、これにより硫酸還元菌の硫酸還元機構が停止し、結果として硫酸還元菌の増殖を選択的に抑制していると考えられる。「選択的」とは、増殖抑制効果が硫酸還元菌に対して特異的であること、即ち、硫酸還元菌に対する増殖抑制効果と比較して、硫酸還元菌以外の菌（他の菌）に対する増殖抑制効果が殆ど無いことを意味する。 In the method of the present invention, the chelated Al acts on the sulfate reduction mechanism of the sulfate-reducing bacteria, thereby stopping the sulfate-reducing mechanism of the sulfate-reducing bacteria and, as a result, selectively suppressing the growth of the sulfate-reducing bacteria. Conceivable. “Selective” means that the growth inhibitory effect is specific to sulfate-reducing bacteria, that is, the growth of bacteria other than sulfate-reducing bacteria (other bacteria) compared to the growth inhibitory effect on sulfate-reducing bacteria. It means that there is almost no suppression effect.
「他の菌」の代表例としては、エセルシア・コリ（Escherichia coli；通性嫌気性グラム陰性桿菌、和名：大腸菌）、クロストリジウム・ブチリウム（Clostridium butyricum；グラム陽性内生胞子形成桿菌、和名：酪酸菌)等を挙げることができる。 Representative examples of “other bacteria” include Escherichia coli (facultative anaerobic gram-negative bacillus, Japanese name: E. coli), Clostridium butyricum (gram-positive endospore-forming bacillus, Japanese name: Butyric acid bacteria).
なお、本発明に言う「制菌」とは、細菌の増殖を抑制することを意味する。即ち、殺菌や滅菌のように直接的に細菌数を減少させることまでは必要としない。 The “antibacterial” referred to in the present invention means to suppress bacterial growth. That is, it is not necessary to reduce the number of bacteria directly like sterilization or sterilization.
［１−１］キレート化Ａｌ：
本発明に係る方法は、硫酸還元菌の存在する環境に、キレート化Ａｌを併存させる点に特徴がある。「硫酸還元菌の存在する環境」は、硫酸還元菌の存在が確認可能な限り、特に制限はない。例えば一般土壌や下水汚泥等の嫌気環境を挙げることができる。「キレート化Ａｌ」とは、中心原子となるＡｌ3+にキレート剤が配位した錯体を指す。キレート化Ａｌは、後述するＡｌ3+源とキレート剤を混合することで速やかに形成される。
[1-1] Chelated Al:
The method according to the present invention is characterized in that chelated Al coexists in an environment where sulfate-reducing bacteria are present. The “environment where sulfate-reducing bacteria are present” is not particularly limited as long as the presence of sulfate-reducing bacteria can be confirmed. For example, anaerobic environments such as general soil and sewage sludge can be mentioned. “Chelating Al” refers to a complex in which a chelating agent is coordinated to Al 3+ serving as a central atom. Chelated Al is rapidly formed by mixing an Al 3+ source and a chelating agent described later.
「キレート剤」とは、Ａｌ3+に配位してキレート化Ａｌを形成する多座配位子である。本発明において、キレート剤の種類は特に限定されない。例えば鎖状配位子であってもよいし、環状配位子であってもよい。 A “chelating agent” is a multidentate ligand that coordinates to Al 3+ to form chelated Al. In the present invention, the type of chelating agent is not particularly limited. For example, it may be a chain ligand or a cyclic ligand.
鎖状配位子としては、例えばシュウ酸、マロン酸、酒石酸、グルタル酸、リンゴ酸、クエン酸、マレイン酸（いずれも２座配位子）等の多価カルボン酸類；エチレンジアミン（ＥＤＡ、２座配位子）等の多価アミン類；エチレンジアミン四酢酸（ＥＤＴＡ、６座配位子）等のアミノポリカルボン酸類；２，２’−ビピリジン、１，１０−フェナントロリン（いずれも２座配位子）等のビピリジン類；等を挙げることができる。 Examples of chain ligands include polyvalent carboxylic acids such as oxalic acid, malonic acid, tartaric acid, glutaric acid, malic acid, citric acid, and maleic acid (both are bidentate ligands); ethylenediamine (EDA, bidentate) Ligands), etc .; aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA, hexadentate ligand); 2,2′-bipyridine, 1,10-phenanthroline (both bidentate ligands) ) And the like; and the like.
環状配位子としては、例えばポルフィリン類（４座配位子）；クラウンエーテル類（化合物により配座数が異なる。例えば１８−クラウン−６は６座配位子）；等を挙げることができる。 Examples of the cyclic ligand include porphyrins (tetradentate ligand); crown ethers (the number of conformations varies depending on the compound. For example, 18-crown-6 is a hexadentate ligand); and the like. .
前記キレート剤はＡｌ3+に配位して容易にキレート化Ａｌを形成するキレート剤であることが好ましい。また、本発明は自然環境中で実施することが多いため、環境に悪影響を与えないキレート剤であることが更に好ましい。具体的には、クエン酸、マロン酸、酒石酸、グルタル酸、リンゴ酸、マレイン酸等を挙げることができる。 The chelating agent is preferably a chelating agent that coordinates to Al 3+ and easily forms chelated Al. Further, since the present invention is often carried out in a natural environment, a chelating agent that does not adversely affect the environment is more preferable. Specific examples include citric acid, malonic acid, tartaric acid, glutaric acid, malic acid, maleic acid and the like.
Ａｌ3+とキレート剤の量比については特に限定されない。キレート剤の種類により、Ａｌ3+と安定な錯体を形成するモル比は異なる。但し、添加したＡｌ3+源の全量がキレート化し、Ａｌ3+が溶存するように、キレート剤の添加量（モル比）を調節することが望ましい。例えば、Ａｌ3+源とシュウ酸が安定な錯体を形成するモル比は１：３、Ａｌ3+源とクエン酸が安定な錯体を形成するモル比は１：２、Ａｌ3+源とＥＤＴＡが安定な錯体を形成するモル比は１：１である。 The amount ratio of Al 3+ to the chelating agent is not particularly limited. The molar ratio for forming a stable complex with Al 3+ varies depending on the type of chelating agent. However, it is desirable to adjust the addition amount (molar ratio) of the chelating agent so that the total amount of the added Al 3+ source is chelated and Al 3+ is dissolved. For example, the molar ratio in which an Al 3+ source and oxalic acid form a stable complex is 1: 3, the molar ratio in which an Al 3+ source and citric acid form a stable complex is 1: 2, and the Al 3+ source and EDTA The molar ratio for forming a stable complex is 1: 1.
なお、後述するように、キレート化Ａｌは、安価で入手し易いアルミニウム化合物から形成することが可能であり、アントラキノン化合物を用いる場合に比して製造コストの低廉化を図ることができる。特に、キレート剤として、安価で入手し易い２〜４価の有機酸を用いた場合、製造コストの低廉化の効果が大きい。 As will be described later, the chelated Al can be formed from an inexpensive and readily available aluminum compound, and the production cost can be reduced as compared with the case of using an anthraquinone compound. In particular, when a divalent to tetravalent organic acid that is inexpensive and easily available is used as the chelating agent, the effect of reducing the manufacturing cost is great.
［１−２］Ａｌ3+源：
キレート化Ａｌは、それ自体（即ち、既にキレート化されているＡｌ3+）を硫酸還元菌の存在する環境に添加してもよいし、前記環境に、Ａｌ3+源及びキレート剤を添加して、前記環境中で前記キレート化Ａｌを生成させてもよい。Ａｌ3+源とキレート剤を個別に添加しても前記環境中で速やかにキレート化Ａｌが形成される。
[1-2] Al 3+ source:
The chelated Al may add itself (that is, Al 3+ already chelated) to an environment where sulfate reducing bacteria are present, or an Al 3+ source and a chelating agent may be added to the environment. The chelated Al may be generated in the environment. Even if the Al 3+ source and the chelating agent are added separately, chelated Al is rapidly formed in the environment.
「Ａｌ3+源」とは、水の存在下においてキレートの中心原子となるＡｌ3+を生成し得る物質である。具体的な物質の種類は特に限定されない。但し、Ａｌ2Ｏ3、ＡｌＣｌ3、Ａｌ（ＯＨ）3及びＡｌ2（ＳＯ4）3からなる群より選択される少なくとも一種のＡｌ化合物であることが好ましい。なお、「少なくとも一種」であるから、前記化合物の一種を単独で用いてもよいし、前記化合物を二種以上併用してもよい。 The “Al 3+ source” is a substance that can generate Al 3+ that becomes a central atom of a chelate in the presence of water. Specific types of substances are not particularly limited. However, it is preferably at least one Al compound selected from the group consisting of Al 2 O 3 , AlCl 3 , Al (OH) 3 and Al 2 (SO 4 ) 3 . In addition, since it is “at least one”, one of the above compounds may be used alone, or two or more of the above compounds may be used in combination.
「Ａｌ2Ｏ3、ＡｌＣｌ3、Ａｌ（ＯＨ）3及びＡｌ2（ＳＯ4）3」には、これらの無水物は勿論のこと、水和物も含まれる。また、これらの化合物の形態は、結晶であってもよいし、アモルファス（例えば非晶質アルミナ等）であってもよい。更に、これらの化合物として、純物質を用いる必要はなく、混合物を用いてもよい。例えば、これらの化合物を含有する鉱物をＡｌ3+源として用いることもできる。 “Al 2 O 3 , AlCl 3 , Al (OH) 3 and Al 2 (SO 4 ) 3 ” includes these anhydrides as well as hydrates. Further, the form of these compounds may be a crystal or amorphous (for example, amorphous alumina or the like). Furthermore, it is not necessary to use pure substances as these compounds, and mixtures may be used. For example, minerals containing these compounds can also be used as the Al 3+ source.
以下、実施例及び比較例により、本発明を更に具体的に説明する。但し、本発明は、下記の実施例の構成のみに限定されるものではない。なお、以下の記載における「部」、「％」は特に断らない限り質量基準である。 Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples. However, the present invention is not limited to the configurations of the following examples. In the following description, “parts” and “%” are based on mass unless otherwise specified.
［前培養］
実施例及び比較例においては、代表的な基準株である以下の菌株を用いた。Desulfovibrio vulgariss DSM 644Tについては、ドイツ国の標準菌株保存機関であるＤＳＭＺ（Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH）から、その他の菌については製品評価技術基盤機構バイオテクノロジーセンターから入手した。これらの菌株については分譲機関指定の培地を用いて前培養を行った。前培養期間は約１週間とした。
（１）硫酸還元菌：Desulfovibrio vulgariss DSM 644T
（２）大腸菌：Escherichia coli NBRC 102203T
（３）酪酸菌：Clostridium butyricum NBRC 13949T
[Pre-culture]
In the examples and comparative examples, the following strains, which are representative reference strains, were used. Desulfovibrio vulgariss DSM 644 T was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), a standard strain preservation organization in Germany, and other bacteria were obtained from the Biotechnology Center, National Institute for Product Evaluation Technology. These strains were pre-cultured using a medium designated by the distribution agency. The preculture period was about 1 week.
(1) Sulfate-reducing bacteria: Desulfovibrio vulgariss DSM 644 T
(2) Escherichia coli NBRC 102203 T
(3) Butyric acid bacteria: Clostridium butyricum NBRC 13949 T
［継代培養］
前培養を行った後、菌体のみを回収し、各菌について表１〜表３に示す組成の培地にて継代培養を行った。前記継代培養後、集菌・洗浄した菌体を、実施例及び比較例にて使用した。なお、表中の「脱イオン水／Ａｌ水溶液」は、調製したＡｌ水溶液の濃度が０ｍＭの系では脱イオン水を用い、調製したＡｌ水溶液の濃度が２ｍＭ、２０ｍＭの系では後述するＡｌ溶液を用いたことを示す。
[Subculture]
After pre-culture, only the cells were collected, and subculture was performed for each bacterium in a medium having the composition shown in Tables 1 to 3. After subculture, the collected and washed cells were used in Examples and Comparative Examples. In the table, “deionized water / Al aqueous solution” means deionized water in a system in which the prepared Al aqueous solution has a concentration of 0 mM, and an Al solution described later in a system in which the prepared Al aqueous solution has a concentration of 2 mM and 20 mM. Indicates that it was used.
［Ａｌ溶液（ＡｌＣｌ3水溶液）の調整］
所定量のＡｌＣｌ3・６Ｈ2Ｏを蒸留水に添加し、オートクレーブにて加熱溶解した後、ＫＯＨ水溶液を適量添加して、ｐＨを７．０付近に調整した。これにより、ＡｌＣｌ3の濃度が２ｍＭ、２０ｍＭの培地添加用ＡｌＣｌ3水溶液を調製した。このＡｌＣｌ3水溶液を比較例１の方法に供した。
[Preparation of Al solution (AlCl 3 aqueous solution)]
A predetermined amount of AlCl 3 .6H 2 O was added to distilled water, heated and dissolved in an autoclave, and then an appropriate amount of KOH aqueous solution was added to adjust the pH to around 7.0. Thus, the concentration of the AlCl 3 was prepared 2 mM, the AlCl 3 solution for 20mM media added. This AlCl 3 aqueous solution was subjected to the method of Comparative Example 1.
［Ａｌ溶液（キレート化Ａｌ水溶液）の調整］
所定量のＡｌＣｌ3・６Ｈ2Ｏを蒸留水に添加し、オートクレーブにて加熱溶解した後、加熱溶解したＡｌＣｌ3・６Ｈ2Ｏ水溶液に、前記水溶液中のＡｌＣｌ3と等モル量のクエン酸、及び水を添加し、１時間撹拌した。その後、ＫＯＨ水溶液を適量添加して、ｐＨを７．０付近に調整した。これにより、クエン酸・Ａｌキレートの濃度が２ｍＭ、２０ｍＭの培地添加用キレート化Ａｌ水溶液を調製した。このキレート化Ａｌ水溶液を実施例１の方法に供した。
[Preparation of Al solution (chelated Al aqueous solution)]
It was added AlCl 3 · 6H 2 O in a predetermined amount of distilled water, dissolved by heating in an autoclave, the AlCl 3 · 6H 2 O aqueous solution were heated and dissolved, an equimolar amount of citric acid and AlCl 3 in the aqueous solution, And water were added and stirred for 1 hour. Thereafter, an appropriate amount of an aqueous KOH solution was added to adjust the pH to around 7.0. Thus, a chelating Al aqueous solution for adding a medium having a citric acid / Al chelate concentration of 2 mM and 20 mM was prepared. This chelated Al aqueous solution was subjected to the method of Example 1.
加熱溶解したＡｌＣｌ3・６Ｈ2Ｏ水溶液に、前記水溶液中のＡｌＣｌ3と等モル量のシュウ酸、及び水を添加することを除いては、実施例１と同様にして、シュウ酸・Ａｌキレートの濃度が２ｍＭ、２０ｍＭの培地添加用キレート化Ａｌ水溶液を調製した。このキレート化Ａｌ水溶液を実施例２の方法に供した。 In the same manner as in Example 1, except that oxalic acid and water in an equimolar amount with AlCl 3 in the aqueous solution were added to the AlCl 3 · 6H 2 O aqueous solution dissolved by heating, the oxalic acid · Al chelate A concentration of 2 mM and 20 mM of chelating Al aqueous solution for medium addition was prepared. This chelated Al aqueous solution was subjected to the method of Example 2.
加熱溶解したＡｌＣｌ3・６Ｈ2Ｏ水溶液に、前記水溶液中のＡｌＣｌ3と等モル量のＥＤＴＡ、及び水を添加することを除いては、実施例１と同様にして、ＥＤＴＡ・Ａｌキレートの濃度が２ｍＭ、２０ｍＭの培地添加用キレート化Ａｌ水溶液を調製した。このキレート化Ａｌ水溶液を実施例３の方法に供した。 The concentration of EDTA · Al chelate was the same as in Example 1 except that EDTA in an equimolar amount with AlCl 3 in the aqueous solution and water were added to the AlCl 3 · 6H 2 O aqueous solution dissolved by heating. Prepared 2 mM and 20 mM chelating Al aqueous solutions for medium addition. This chelated Al aqueous solution was subjected to the method of Example 3.
［Ａｌ添加培地の調製］
前記のように調製したＡｌＣｌ3水溶液またはキレート化Ａｌ水溶液に、予めオートクレーブにて滅菌処理した表１〜表３に記載の培地の成分を、表１〜表３に記載の組成比となるように添加し、Ａｌ添加培地を調製した。このＡｌ添加培地を水酸化カリウムと塩酸を適量添加して、表１〜表３に記載のｐＨに調整して用いた。
[Preparation of Al-added medium]
The components of the medium shown in Tables 1 to 3 previously sterilized in an autoclave in the AlCl 3 aqueous solution or the chelated Al aqueous solution prepared as described above so that the composition ratios shown in Tables 1 to 3 are obtained. After addition, an Al-added medium was prepared. An appropriate amount of potassium hydroxide and hydrochloric acid was added to this Al-added medium, and the pH was adjusted to the values shown in Tables 1 to 3 before use.
［培養］
滅菌済み１００ｍｌ（公称）バイアル瓶に、前記Ａｌ添加培地１００ｍｌを注入し、前記培地に脱酸素した窒素ガスを一定時間吹き込むことによって、前記培地内を嫌気状態とした。この培地に、菌体濃度が１０6個となるように、継代培養した各菌体（硫酸還元菌、大腸菌、酪酸菌）を植菌した。その後、ブチルゴム栓およびアルミシールによりバイアル瓶を密封し、３７℃のインキュベータ内で振盪培養を行った。振盪培養は、水酸化物が沈殿しない程度の振盪回数（１１０回／分）で行った。培養液からサンプルを抜き取り、評価に使用した。
[culture]
The Al-added medium (100 ml) was poured into a sterilized 100 ml (nominal) vial, and deoxygenated nitrogen gas was blown into the medium for a certain period of time to make the medium anaerobic. The subcultured cells (sulfuric acid-reducing bacteria, Escherichia coli, butyric acid bacteria) were inoculated into this medium so that the cell concentration was 10 6 cells. Thereafter, the vial was sealed with a butyl rubber stopper and an aluminum seal, and shaking culture was performed in a 37 ° C. incubator. The shaking culture was performed at the number of shakings (110 times / min) that does not precipitate the hydroxide. A sample was extracted from the culture and used for evaluation.
［評価方法（タンパク量測定）］
バイアル瓶から培地１ｍｌを採取し、培地成分を取り除き、滅菌蒸留水１ｍｌに懸濁させた。その懸濁液から超音波破砕機によってタンパクを抽出し、ＢＣＡ法（ビシンコニン酸法）によりタンパク量を測定した。このタンパク量により各菌体に対する増殖抑制効果（制菌効果）を評価した。
[Evaluation method (protein amount measurement)]
1 ml of the medium was collected from the vial, the medium components were removed, and the medium was suspended in 1 ml of sterile distilled water. Protein was extracted from the suspension by an ultrasonic crusher, and the amount of protein was measured by the BCA method (bicinchoninic acid method). The growth inhibitory effect (antibacterial effect) with respect to each microbial cell was evaluated by this protein amount.
［結果］
実施例１の方法（クエン酸・Ａｌキレートの添加）について、硫酸還元菌に対する制菌効果を表したグラフを図１に、大腸菌に対する制菌効果を表したグラフを図２に、酪酸菌に対する制菌効果を表したグラフを図３に示す。また、比較例１の方法（ＡｌＣｌ3の添加）について、硫酸還元菌に対する制菌効果を表したグラフを図４に、大腸菌に対する制菌効果を表したグラフを図５に、酪酸菌に対する制菌効果を表したグラフを図６に示す。これらのグラフにおいて、横軸は経過時間（単位：時間）を示し、縦軸はタンパク量（単位：ｍｇ／Ｌ）の対数値を示す。
[result]
With respect to the method of Example 1 (addition of citric acid / Al chelate), a graph showing the bactericidal effect against sulfate-reducing bacteria is shown in FIG. 1, a graph showing the bactericidal effect against E. coli is shown in FIG. A graph showing the fungal effect is shown in FIG. In addition, for the method of Comparative Example 1 (addition of AlCl 3 ), a graph showing the bactericidal effect on sulfate-reducing bacteria is shown in FIG. 4, a graph showing the bactericidal effect on E. coli is shown in FIG. The graph showing the effect is shown in FIG. In these graphs, the horizontal axis indicates the elapsed time (unit: time), and the vertical axis indicates the logarithmic value of the protein amount (unit: mg / L).
比較例１の方法（ＡｌＣｌ3の添加）によれば、図４〜図６に示すように、硫酸還元菌、大腸菌、酪酸菌の全ての培養液においてタンパク量の増加が抑制された。即ち、比較例１の方法によれば、硫酸還元菌の増殖が抑制されるのみならず、大腸菌や酪酸菌の増殖も抑制された。 According to the method of Comparative Example 1 (addition of AlCl 3 ), as shown in FIGS. 4 to 6, the increase in the amount of protein was suppressed in all the culture solutions of sulfate-reducing bacteria, Escherichia coli, and butyric acid bacteria. That is, according to the method of Comparative Example 1, not only the growth of sulfate-reducing bacteria was suppressed, but also the growth of Escherichia coli and butyric acid bacteria was suppressed.
また、実施例１の方法（クエン酸・Ａｌキレートの添加）によれば、図１に示すように、硫酸還元菌の培養液においてはタンパク量の増加が抑制された。即ち、硫酸還元菌の増殖が抑制された。 Moreover, according to the method of Example 1 (addition of citric acid / Al chelate), as shown in FIG. That is, the growth of sulfate-reducing bacteria was suppressed.
一方、図２及び図３に示すように、大腸菌、酪酸菌の培養液においては、Ａｌキレートを添加しても、不添加の場合と同様にタンパク量が増加した。即ち、大腸菌および酪酸菌の増殖は抑制されておらず、実施例１の方法については、選択的な制菌効果が認められた。なお、グラフには示さなかったが、実施例２の方法（Ａｌ・シュウ酸キレートの添加）及び実施例３の方法（Ａｌ・ＥＤＴＡキレートの添加）についても、実施例１と同様の選択的制菌効果が認められた。 On the other hand, as shown in FIGS. 2 and 3, in the culture solution of Escherichia coli and butyric acid bacteria, the amount of protein increased even when Al chelate was added, as in the case of no addition. That is, the growth of Escherichia coli and butyric acid bacteria was not suppressed, and the selective bactericidal effect was observed for the method of Example 1. Although not shown in the graph, the method of Example 2 (addition of Al · oxalic acid chelate) and the method of Example 3 (addition of Al · EDTA chelate) are also selectively controlled as in Example 1. A fungal effect was observed.
本発明に係る制菌方法は、硫酸還元菌の増殖抑制、ひいては硫化水素の生成抑制に利用することができる。 The antibacterial method according to the present invention can be used for inhibiting the growth of sulfate-reducing bacteria and thus for inhibiting the production of hydrogen sulfide.
Claims (4)
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| JP2012180093A JP5969309B2 (en) | 2012-08-15 | 2012-08-15 | Selective control method for sulfate-reducing bacteria |
| PCT/JP2013/071612 WO2014027613A1 (en) | 2012-08-15 | 2013-08-09 | Method for selectively inhibiting sulfate-reducing bacterium, gypsum composition capable of inhibiting proliferation of sulfate-reducing bacterium by said method, gypsum-type solidifying material, and gypsum-type building material |
| EP13879516.6A EP2885972B1 (en) | 2012-08-15 | 2013-08-09 | Use of aluminium chelates for inhibiting sulfate-reducing bacteria |
| MX2015001956A MX2015001956A (en) | 2012-08-15 | 2013-08-09 | Method for selectively inhibiting sulfate-reducing bacterium, gypsum composition capable of inhibiting proliferation of sulfate-reducing bacterium by said method, gypsum-type solidifying material, and gypsum-type building material. |
| KR1020157005067A KR101632545B1 (en) | 2012-08-15 | 2013-08-09 | Method for selectively inhibiting sulfate-reducing bacterium, gypsum composition capable of inhibiting proliferation of sulfate-reducing bacterium by said method, gypsum-type solidifying material, and gypsum-type building material |
| US14/421,359 US9901101B2 (en) | 2012-08-15 | 2013-08-09 | Method for selectively inhibiting sulfate-reducing bacterium, gypsum composition capable of inhibiting proliferation of sulfate-reducing bacterium by said method, gypsum-type solidifying material, and gypsum-type building material |
| CN201380043319.6A CN104582491B (en) | 2012-08-15 | 2013-08-09 | The selectivity bacteriostasis method of sulfate reducting bacteria, utilize the gypsum compositions of propagation of preceding method inhibiting sulfate reduction bacterium, gypsum curing materials and gypsum construction material |
| BR112015003090A BR112015003090B8 (en) | 2012-08-15 | 2013-08-09 | method for selectively inhibiting sulfate-reducing bacteria, gypsum composition capable of inhibiting sulfate-reducing bacteria proliferation by said method, gypsum-like solification material, and gypsum-like building material |
| AU2013303570A AU2013303570B2 (en) | 2012-08-15 | 2013-08-09 | Method for selectively inhibiting sulfate-reducing bacterium, gypsum composition capable of inhibiting proliferation of sulfate-reducing bacterium by said method, gypsum-type solidifying material, and gypsum-type building material |
| TW102129261A TWI568701B (en) | 2012-08-15 | 2013-08-15 | Selective bacteriostatic method of sulfate-reducing bacteria |
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