JP2008215852A - Evaluation method of corrosion resistance of iron or iron containing alloy - Google Patents

Evaluation method of corrosion resistance of iron or iron containing alloy Download PDF

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JP2008215852A
JP2008215852A JP2007049837A JP2007049837A JP2008215852A JP 2008215852 A JP2008215852 A JP 2008215852A JP 2007049837 A JP2007049837 A JP 2007049837A JP 2007049837 A JP2007049837 A JP 2007049837A JP 2008215852 A JP2008215852 A JP 2008215852A
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iron
corrosion
sulfate
corrosion resistance
aqueous solution
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JP4741532B2 (en
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Kimio Ito
公夫 伊藤
Osamu Miki
理 三木
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Nippon Steel Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluation method of the corrosion resistance of iron or an ion-containing alloy capable of evaluating the microbial corrosion high in the advance speed of corrosion observed in an actual environment. <P>SOLUTION: Iron is allowed to exist in an aqueous solution containing a carbonate substance, sulfuric acid ions and chlorine ions, and held to an anaerobic condition as an electron doner and methane producing bacteria cultured using the carbonate substance as a carbon source, and sulfate reducing bacteria are also allowed to exist in the aqueous solution to bring the aqueous solution containing the methane producing bacteria and the sulfate reducing bacteria into contact with iron or the iron-containing alloy or iron, or the iron-containing alloy is immersed in the aqueous solution containing both bacteria to be corroded under the anaerobic condition. Alternatively, after the above process, air or oxygen is supplied to the aqueous solution to increase the concentration of dissolved oxygen in the aqueous solution to set the aqueous solution to an aerobic condition and, and iron or the iron-containing alloy is corroded, and then the corrosion quantity thereof is measured to evaluate the corrosion resistance of iron or the iron-containing alloy. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、鉄または鉄を含む合金に対して腐食能を有する微生物による鉄または鉄を含む合金の耐食性評価方法に関する。   The present invention relates to a method for evaluating the corrosion resistance of iron or an iron-containing alloy by a microorganism having corrosion ability with respect to iron or an iron-containing alloy.

2001年米国のＦＨＷＡ（The US Federal Highway Administration）により金属の腐食に関わるコストの調査結果が報告された（非特許文献１）。本報告によると、米国では金属腐食による損失は年間2760億ドルに達し、国内総生産（GDP）の3.1%に相当すると報告されている。また、米国のガス産業において、パイプラインなどの腐食に掛かるコストが年間134億ドルに達し、このうちの約20億ドル（約15％）は微生物腐食によるものと報告されている（非特許文献２）。わが国においても腐食防食協会と日本防錆技術協会を中心とする腐食コスト調査委員会の調査により、1997年にわが国の腐食対策に講じた費用は3兆9千億円で、わが国の国内総生産（GDP）の0.8％に相当すると報告されている（非特許文献３）。以上のように、腐食による被害額は甚大であり、これを防ぐことは資源の乏しい我が国にとって重要な課題である。   In 2001, the US FHWA (The US Federal Highway Administration) reported the results of a survey of costs related to metal corrosion (Non-patent Document 1). According to the report, metal corrosion losses amount to $ 276 billion annually in the United States, accounting for 3.1% of gross domestic product (GDP). Also, in the US gas industry, the cost of corrosion of pipelines reaches $ 13.4 billion annually, of which about $ 2 billion (about 15%) is reported to be due to microbial corrosion (Non-patent literature) 2). In Japan, the cost of our anti-corrosion measures in 1997 was 3.9 trillion yen, based on a survey by the Corrosion Cost Research Committee led by the Corrosion and Corrosion Protection Association and the Japan Anticorrosion Technology Association. It is reported that it corresponds to 0.8% of (GDP) (Non-patent Document 3). As described above, the amount of damage caused by corrosion is enormous, and preventing this is an important issue for Japan, which has few resources.

微生物腐食はこれまで鉄鋼材料を中心に多く報告されている。酸素が利用できる好気条件と、酸素が利用できない嫌気条件でそれぞれ異なる種類の微生物が鉄鋼材料の腐食作用を示すことが知られている。嫌気条件の微生物腐食の原因微生物として硫酸塩還元菌に関する多くの報告がある。硫酸塩還元菌は、海水などに含まれる硫酸塩を硫化物に還元する活性を有する。その結果発生する硫化水素は、鉄をはじめとしてさまざまな金属と硫化物をつくるため、強い腐食性が知られている。また、硫酸塩還元菌には、水素原子あるいは水素分子をプロトンに酸化できる酵素、ヒドロゲナーゼを有するものがある。嫌気条件下、すなわち酸化還元電位の低い還元的な環境条件では、中性条件においても水の分解により発生するプロトンを用いて、鉄表面でカソード反応が起こり、水素原子さらに水素分子が形成される（この反応にカップルして、アノードでは、鉄の酸化がおこり、Fe(II)が生成する）。この際、ヒドロゲナーゼ活性を有する硫酸塩還元菌は、カソード反応で生成する水素原子あるいは水素分子を、カソード反応で電子受容体となるプロトンに酸化し、鉄表面を復極させて、カソード反応を促進する。この結果、電子の授受が円滑にすすむため、嫌気条件における鉄の酸化、鉄のアノード溶解が促進される。このようなヒドロゲナーゼを有する硫酸塩還元菌による腐食促進メカニズムは、カソード復極説として知られている（非特許文献４）。   So far, microbial corrosion has been reported mainly in steel materials. It is known that different types of microorganisms exhibit the corrosive action of steel materials under aerobic conditions where oxygen can be used and anaerobic conditions where oxygen cannot be used. There are many reports on sulfate-reducing bacteria as the cause of microbial corrosion under anaerobic conditions. Sulfate-reducing bacteria have an activity of reducing sulfate contained in seawater or the like to sulfide. The resulting hydrogen sulfide is known to be highly corrosive because it forms sulfides with various metals including iron. Some sulfate-reducing bacteria have an enzyme or hydrogenase that can oxidize hydrogen atoms or hydrogen molecules to protons. Under anaerobic conditions, that is, reductive environmental conditions with a low redox potential, cathodic reactions occur on the iron surface using protons generated by water decomposition even in neutral conditions, forming hydrogen atoms and hydrogen molecules. (Coupled with this reaction, iron is oxidized at the anode and Fe (II) is produced). At this time, sulfate-reducing bacteria with hydrogenase activity promote the cathode reaction by oxidizing the hydrogen atoms or hydrogen molecules produced in the cathode reaction to protons that become electron acceptors in the cathode reaction and depolarizing the iron surface. To do. As a result, since the transfer of electrons proceeds smoothly, the oxidation of iron and the anodic dissolution of iron under anaerobic conditions are promoted. Such a mechanism for promoting corrosion by sulfate-reducing bacteria having hydrogenase is known as a cathode reversal theory (Non-patent Document 4).

例えば油井など、石油環境では、硫酸塩還元菌による腐食影響は、大きな課題になっている（非特許文献５）。段階的な希釈により硫酸塩還元菌を検出、存在量をモニタリングするための簡易なキットなどが、石油生産に関わる産業分野では使用されている（非特許文献６）。   For example, in oil environments such as oil wells, the corrosive effect of sulfate-reducing bacteria is a major issue (Non-Patent Document 5). Simple kits for detecting sulfate-reducing bacteria by stepwise dilution and monitoring their abundance are used in the industrial field related to oil production (Non-patent Document 6).

以上のように、硫酸塩還元菌による鉄鋼材料の微生物腐食は、広く知られており、その検出や存在量測定のための技術が報告されている。また、硫酸塩還元菌の増殖を抑制する方法なども考案されている。例えば、非特許文献７では、抗生物質を生産する微生物を共存させることで、硫酸塩還元菌の増殖を抑制する方法などが報告されている。   As described above, microbial corrosion of iron and steel materials by sulfate-reducing bacteria is widely known, and techniques for its detection and abundance measurement have been reported. A method for suppressing the growth of sulfate-reducing bacteria has also been devised. For example, Non-Patent Document 7 reports a method for suppressing the growth of sulfate-reducing bacteria by allowing microorganisms that produce antibiotics to coexist.

また、硫酸塩還元菌のほかにも、嫌気環境に棲息する微生物生態系を構成する主要な微生物として、メタン生成菌がある。メタン生成菌を嫌気環境における腐食原因菌としては、一般的にこれまで認知されてはいない。例えば、非特許文献８には、微生物腐食の原因菌が紹介されているが、この中でメタン生成菌は、腐食原因菌として説明されていない。これは、硫酸塩還元菌の生成する硫化水素が極めて強力な腐食原因物質であるのに対して、メタン生成菌が生成するメタンは、腐食原因物質ではないことが、メタン生成菌が腐食原因菌としてみなされてこなかったことの、大きな要因として考えられる。   In addition to sulfate-reducing bacteria, methanogenic bacteria are the main microorganisms that make up the microbial ecosystem that lives in anaerobic environments. Methanogens are not generally recognized as corrosive bacteria in anaerobic environments. For example, Non-Patent Document 8 introduces causative bacteria of microbial corrosion, but methanogens are not described as corrosive causative bacteria. This is because hydrogen sulfide produced by sulfate-reducing bacteria is an extremely strong causative substance, whereas methane produced by methanogens is not a causative substance. It is considered as a big factor of not being regarded as.

Report FHWA-RD-01-156, September 2001.Report FHWA-RD-01-156, September 2001. National Energy Technology Laboratory, DE-FC26-01NT41158National Energy Technology Laboratory, DE-FC26-01NT41158 わが国における腐食コスト（腐食防食協会、日本防錆技術協会）(1997)Corrosion costs in Japan (Corrosion and Corrosion Protection Association, Japan Rust Prevention Technology Association) (1997) Von Wolzogen Kuehr and van der Vlugt, Water 16, 147 (1934)Von Wolzogen Kuehr and van der Vlugt, Water 16, 147 (1934) Petroleum Microbiology, edited by Atlas,R.M., Macmillan Publishing Company (1984)Petroleum Microbiology, edited by Atlas, R.M., Macmillan Publishing Company (1984) Microbiologically Influenced Corrosion, NACE InternationalMicrobiologically Influenced Corrosion, NACE International

p.43(1997) p.43 (1997)

Zuo R, Wood TK. Appl Microbiol Biotechnol. 65: 747(2004)Zuo R, Wood TK. Appl Microbiol Biotechnol. 65: 747 (2004) 腐食反応とその制御（第３版）ユーリック、レヴィー共著（産業図書）（１９８９）Corrosion reaction and its control (Third Edition) Co-authored by Euryk and Levy (Industry Books) (1989) Aspects of Microbially induced corrosion, papers from EUROCORR’96 and The EFC Working party on Microbial Corrosion, edited by Thierry,D., The Institute of Materials P.4(1997)Aspects of Microbially induced corrosion, papers from EUROCORR’96 and The EFC Working party on Microbial Corrosion, edited by Thierry, D., The Institute of Materials P.4 (1997) Pankhania,I.P., Moosavi,A.N. and Hamilton,W.A., J.Gen.Microbiol 132, 3357-3365 (1986)Pankhania, I.P., Moosavi, A.N. and Hamilton, W.A., J. Gen. Microbiol 132, 3357-3365 (1986) Aspects of Microbially induced corrosion, papers from EUROCORR’96 and The EFC Working party on Microbial Corrosion, edited by Thierry,D., The Institute of Materials P.11-P.37(1997)Aspects of Microbially induced corrosion, papers from EUROCORR’96 and The EFC Working party on Microbial Corrosion, edited by Thierry, D., The Institute of Materials P.11-P.37 (1997) Daniels,L., Belay,N., Rajagopal,B.S. and Weimer,P.J., Science 237, 509-511(1987)Daniels, L., Belay, N., Rajagopal, B.S. And Weimer, P.J., Science 237, 509-511 (1987)

以上のように、嫌気条件で微生物腐食の原因となる微生物として、硫酸塩還元菌については広く知られており、硫酸塩還元菌に対する検出方法や、腐食対策方法も報告されている。水やスラッジ等の環境試料から硫酸塩還元菌を検出する方法は、例えば米国のＡＳＴＭによって ASTM D4412-84(2002) Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits が定められている。   As described above, sulfate-reducing bacteria are widely known as microorganisms that cause microbial corrosion under anaerobic conditions, and detection methods for sulfate-reducing bacteria and corrosion countermeasure methods have also been reported. For example, ASTM D4412-84 (2002) Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits is established by the US ASTM as a method for detecting sulfate-reducing bacteria from environmental samples such as water and sludge. .

しかしながら、硫酸塩還元菌に対する耐食性を評価する方法は確立されていない。硫酸塩還元菌を検出するために培養する際には、電子供与体として、水素ガスか、有機酸などの有機物を添加する必要がある。非特許文献９には、硫酸塩還元菌の電子供与体として用いられる物質が報告されている。水素の他に、有機酸（蟻酸、酢酸、プロピオン酸、乳酸、ブチル酸、オキザル酸、コハク酸、マレイン酸、ピルビン酸など）、脂肪酸（炭素数１８まで）、アルコール（メタノール、エタノール、プロパノール、ペンタノール、など）、アミノ酸（アラニン、グルタミン酸、p-アミノベンゼン酸など）、糖類（フルクトース、グルコース、グリセロールなど）、炭化水素類（ノルマルアルカン、トルエン、キシレンなど）などが記載されている。硫酸塩還元菌を用いて、鉄や鉄を含む合金の腐食を調べる場合には、上記のように電子供与体を供給して硫酸塩還元菌を培養しながら、例えば腐食生成物をＸ線等を用いて解析して、硫酸塩還元菌による腐食生成物の特徴である硫化鉄を検出したり、電気化学的に腐食電流等を測定して、腐食速度を測定したりすることが報告されている（非特許文献１０、非特許文献１１）。電子供与体として主に用いられるのは有機酸や水素である。   However, a method for evaluating the corrosion resistance against sulfate-reducing bacteria has not been established. When culturing to detect sulfate-reducing bacteria, it is necessary to add hydrogen gas or an organic substance such as an organic acid as an electron donor. Non-Patent Document 9 reports a substance used as an electron donor for sulfate-reducing bacteria. In addition to hydrogen, organic acids (formic acid, acetic acid, propionic acid, lactic acid, butyric acid, oxalic acid, succinic acid, maleic acid, pyruvic acid, etc.), fatty acids (up to 18 carbon atoms), alcohols (methanol, ethanol, propanol, Pentanol, etc.), amino acids (alanine, glutamic acid, p-aminobenzene acid, etc.), saccharides (fructose, glucose, glycerol, etc.), hydrocarbons (normal alkane, toluene, xylene, etc.) and the like are described. When investigating the corrosion of iron or an iron-containing alloy using a sulfate-reducing bacterium, while supplying the electron donor as described above and culturing the sulfate-reducing bacterium, for example, the corrosion product is X-rayed or the like. It is reported that iron sulfide, which is a feature of corrosion products by sulfate-reducing bacteria, is detected, and the corrosion rate is measured electrochemically by measuring the corrosion current etc. (Non-patent document 10, Non-patent document 11). Organic acids and hydrogen are mainly used as electron donors.

しかしながら、培養した硫酸塩還元菌を用いて、鉄又は鉄を含む合金を腐食させても、その腐食は実環境で観察される腐食と比較すると一般にきわめて軽微である。したがって、培養した硫酸塩還元菌のみを用いた腐食では、微生物腐食を過小評価してしまうため、鉄又は鉄を含む合金の耐食性を評価する方法として適当でない。また、硫酸塩還元菌以外の嫌気性微生物であるメタン生成菌については、腐食の原因微生物として必ずしも一般的には認識されていないのが現状である。   However, even if iron or an alloy containing iron is corroded using cultured sulfate-reducing bacteria, the corrosion is generally very slight compared to the corrosion observed in the real environment. Therefore, corrosion using only cultured sulfate-reducing bacteria underestimates microbial corrosion and is not suitable as a method for evaluating the corrosion resistance of iron or an alloy containing iron. In addition, the present situation is that methanogens that are anaerobic microorganisms other than sulfate-reducing bacteria are not generally recognized as causative microorganisms.

しかしながら、メタン生成菌が腐食原因菌であることを示す学術的な報告はある。ダニエルス（Ｄａｎｉｅｌｓ）らは、メタン生成菌が鉄を腐食する性質を有することを報告している（非特許文献１２）。ただし、非特許文献７のようにメタン生成菌の腐食原因性に関しては、学術的には一部報告があるが、一般的には認知されていない状況である。したがって、メタン生成菌を用いて、鉄又は鉄を含む合金の耐食性を評価している報告はない。実際の嫌気環境には、メタン生成菌と硫酸塩還元菌が共存していることが考えられる。メタン生成菌と硫酸塩還元菌の両方による微生物腐食が生じていることが考えられる。   However, there are academic reports showing that methanogens are causative agents. Daniels et al. Report that methanogens have the property of corroding iron (Non-patent Document 12). However, as is the case with Non-Patent Document 7, there are some reports on the causative properties of methanogenic bacteria, but they are not generally recognized. Therefore, there is no report evaluating the corrosion resistance of iron or iron-containing alloys using methanogens. In an actual anaerobic environment, methanogens and sulfate-reducing bacteria may coexist. It is thought that microbial corrosion by both methanogen and sulfate-reducing bacteria occurs.

そこで、本発明では、メタン生成菌と硫酸塩還元菌の両方を用いることで、実環境で観察されるような腐食の進行が速い微生物作用に起因する腐食に対しても評価可能な、鉄又は鉄を含む合金の耐食性の評価方法を提供する。   Therefore, in the present invention, by using both a methanogen and a sulfate-reducing bacterium, iron or iron that can be evaluated against corrosion caused by microbial action that is rapidly progressed in corrosion as observed in an actual environment. A method for evaluating the corrosion resistance of an alloy containing iron is provided.

上記課題を解決するため鋭意検討を行なった結果、メタン生成菌と硫酸塩還元菌を共存させることにより、鉄又は鉄を含む合金が実環境で生じるような激しい腐食を生じることを見い出した。これにより、メタン生成菌と硫酸塩還元菌の両方を一緒に用いて、鉄又は鉄を含む合金の耐食性を評価する方法を確立し、本発明を完成するに至った。本発明の要旨とするところは次の（１）〜（５）である。
（１） 炭酸物質、硫酸イオン、及び塩素イオンを含む嫌気条件の水溶液中に、鉄を電子供与体として、かつ、前記炭酸物質を炭素源として培養可能なメタン生成菌及び硫酸塩還元菌を存在させ、当該メタン生成菌及び硫酸塩還元菌を含む水溶液と鉄又は鉄を含む合金とを接触させ、又は、前記微生物を含む水溶液中に鉄又は鉄を含む合金を浸漬して、前記鉄又は鉄を含む合金を嫌気条件で腐食させた後、又は、更にその後、空気又は酸素を供給して、前記水溶液中の溶存酸素濃度を高めることにより好気条件として、前記鉄又は鉄を含む合金を腐食させた後、当該腐食量を測定して、前記鉄又は鉄を含む合金の耐食性を評価することを特徴とする鉄又は鉄を含む合金の耐食性の評価方法。
（２） 前記腐食量の測定を、２種類以上の鉄又は鉄を含む合金について行い、当該鉄又は鉄を含む合金それぞれの腐食量の測定結果を比較して、前記鉄又は鉄を含む合金それぞれの前記メタン生成菌及び硫酸塩還元菌に対する耐食性を相対的に評価することを特徴とする（１）の鉄又は鉄を含む合金の耐食性の評価方法。
（３） 前記水溶液中の全炭酸濃度が１００ｍｇ/Ｌ以上、硫酸イオン濃度が１００ｍｇ/Ｌ以上７０００ｍｇ/Ｌ以下、ｐＨが５以上９以下、塩素イオン濃度が１０００ｍｇ/Ｌ以上３００００ｍｇ/Ｌ以下、嫌気条件における溶存酸素濃度が０．２ｍｇ/Ｌ未満であり、空気あるいは酸素を供給した後の好気条件における溶存酸素濃度が２ｍｇ/Ｌ以上であることを特徴とする（１）又は（２）の鉄又は鉄を含む合金の耐食性の評価方法。
（４） 前記メタン生成菌がメタノコッカレス（Ｍｅｔｈａｎｏｃｏｃｃａｌｅｓ）目メタノコッカシアエ（Ｍｅｔｈａｎｏｃｏｃｃａｃｅａｅ）科に属する微生物であり、前記硫酸塩還元菌がデスルホビブリオナレス（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ）目デスルホビブリオナシアエ（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｃｅａｅ）科に属する微生物であることを特徴とする（１）〜（３）のいずれかの鉄又は鉄を含む合金の耐食性の評価方法。
（５） 前記メタン生成菌が受託番号ＮＩＴＥ ＢＰ−２５２で特定される微生物であることを特徴とする（４）の鉄又は鉄を含む合金の耐食性の評価方法。 As a result of intensive studies to solve the above problems, it has been found that iron or an alloy containing iron causes severe corrosion that occurs in a real environment by coexisting methane-producing bacteria and sulfate-reducing bacteria. As a result, a method for evaluating the corrosion resistance of iron or an alloy containing iron using both a methanogen and a sulfate-reducing bacterium together is established, and the present invention has been completed. The gist of the present invention is the following (1) to (5).
(1) An anaerobic aqueous solution containing a carbonate substance, sulfate ion, and chloride ion contains methanogens and sulfate-reducing bacteria that can be cultured using iron as an electron donor and the carbonate substance as a carbon source. Contacting the aqueous solution containing the methanogen and sulfate-reducing bacteria with an iron or iron-containing alloy, or immersing the iron or iron-containing alloy in the aqueous solution containing the microorganism, Corrosion of the iron or iron-containing alloy as an aerobic condition by supplying air or oxygen and then increasing the dissolved oxygen concentration in the aqueous solution Then, the corrosion amount of the alloy containing iron or iron is evaluated by measuring the corrosion amount and evaluating the corrosion resistance of the iron or iron-containing alloy.
(2) The measurement of the corrosion amount is performed on two or more kinds of iron or alloys containing iron, and the measurement results of the respective corrosion amounts of the iron or iron-containing alloys are compared. (1) The method for evaluating the corrosion resistance of iron or an iron-containing alloy according to (1), wherein the corrosion resistance to the methanogenic bacteria and sulfate-reducing bacteria is relatively evaluated.
(3) Total carbonic acid concentration in the aqueous solution is 100 mg / L or more, sulfate ion concentration is 100 mg / L or more and 7000 mg / L or less, pH is 5 or more and 9 or less, chloride ion concentration is 1000 mg / L or more and 30000 mg / L or less, anaerobic (1) or (2), wherein the dissolved oxygen concentration in the conditions is less than 0.2 mg / L, and the dissolved oxygen concentration in the aerobic conditions after supplying air or oxygen is 2 mg / L or more A method for evaluating the corrosion resistance of iron or an alloy containing iron.
(4) The methanogen is a microorganism belonging to the family Methanococcalae, Methanococceae, and the sulfate-reducing bacterium is Desulfobibrionales Desulfivobrionae. The method for evaluating the corrosion resistance of iron or an alloy containing iron according to any one of (1) to (3), wherein the microorganism is a microorganism belonging to the family.
(5) The method for evaluating corrosion resistance of iron or an iron-containing alloy according to (4), wherein the methanogen is a microorganism specified by the accession number NITE BP-252.

尚、本発明で言うところの水溶液中の全炭酸濃度とは、溶存した二酸化炭素と炭酸物質の濃度の総和のことである。   The total carbonic acid concentration in the aqueous solution referred to in the present invention is the total concentration of dissolved carbon dioxide and carbonic acid substances.

本発明により、メタン生成菌と硫酸塩還元菌を共に存在させることにより、鉄又は鉄を含む合金の腐食を促進させることができるようになり、実環境の微生物腐食で観察されるような激しい腐食に対する耐食性を評価することが可能となる。   According to the present invention, the presence of both a methanogen and a sulfate-reducing bacterium makes it possible to promote the corrosion of iron or an alloy containing iron, and the severe corrosion observed in microbial corrosion in a real environment. It becomes possible to evaluate the corrosion resistance against.

これにより、耐食性の鉄鋼材料開発や防食技術の開発に有効活用することができる。   Thereby, it can utilize effectively for development of a corrosion-resistant steel material and anti-corrosion technology.

まず、本発明で使用するメタン生成菌と硫酸塩還元菌について説明する。本発明で使用するメタン生成菌と硫酸塩還元菌は、鉄を電子供与体として、かつ、二酸化炭素若しくは炭酸物質を炭素源として培養可能なメタン生成菌及び硫酸塩還元菌を使用する。ここで炭酸物質とは、二酸化炭素が水に溶解した形態の炭酸水素イオン、炭酸イオンおよびこれらの塩を意味する。たとえば、表１の鉄炭酸培地をもちいて、気相には窒素と二酸化炭素の混合ガス（Ｎ2（８０％）＋ＣＯ2（２０％））を充填した嫌気条件で培養可能なメタン生成菌及び硫酸塩還元菌であれば、本発明の耐食性評価に使用することができる。 First, the methanogen and sulfate-reducing bacterium used in the present invention will be described. The methanogen and sulfate-reducing bacterium used in the present invention use methanogen and sulfate-reducing bacterium that can be cultured using iron as an electron donor and carbon dioxide or a carbon dioxide as a carbon source. Here, the carbonic acid substance means hydrogen carbonate ions, carbonate ions and their salts in a form in which carbon dioxide is dissolved in water. For example, using the iron carbonate medium shown in Table 1, a methanogen that can be cultured under anaerobic conditions in which the gas phase is filled with a mixed gas of nitrogen and carbon dioxide (N 2 (80%) + CO 2 (20%)) and Any sulfate-reducing bacteria can be used for the corrosion resistance evaluation of the present invention.

ここで嫌気条件の目安としては、溶存酸素濃度が０．２ｍｇ/Ｌ未満となっていることを確認すればよい。メタン生成菌や硫酸塩還元菌は単離株でなくとも、これらの集積培養も使用可能である。もちろん、表１の培地は一例であって、このほかの培養方法によって取得した前記の条件を満足するメタン生成菌及び硫酸塩還元菌も本発明の耐食性の評価に使用することが可能である。前記の条件を満足するメタン生成菌としては、例えばメタノコッカレス（Ｍｅｔｈａｎｏｃｏｃｃａｌｅｓ）目メタノコッカシアエ（Ｍｅｔｈａｎｏｃｏｃｃａｃｅａｅ）科に属するメタン生成菌がある。さらに、受託番号ＮＩＴＥ ＢＰ−２５２で特定されるメタン生成菌メタノコッカス マルパリディスＫＡ１（Ｍｅｔｈａｎｏｃｏｃｃｕｓ ｍａｒｉｐａｌｕｄｉｓ ＫＡ１）がある。また、前記の条件を満足する硫酸塩還元菌としては、例えばデスルホビブリオナレス（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ）目デスルホビブリオナシアエ（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｃｅａｅ）科に属する硫酸塩還元菌がある。   Here, as a standard of the anaerobic condition, it may be confirmed that the dissolved oxygen concentration is less than 0.2 mg / L. Methanogens and sulfate-reducing bacteria are not isolates, and their enrichment cultures can also be used. Of course, the culture medium of Table 1 is an example, and methanogens and sulfate-reducing bacteria that satisfy the above-mentioned conditions obtained by other culture methods can also be used for the evaluation of the corrosion resistance of the present invention. Examples of the methanogen that satisfies the above-mentioned conditions include methanogens belonging to the family Methanococcales> Methanococceae. In addition, there is a methanogenic methanococcus maripaldis KA1 identified by accession number NITE BP-252. In addition, examples of sulfate-reducing bacteria that satisfy the above-described conditions include sulfate-reducing bacteria belonging to the family Desulfobibrionales, Desulfobibrionaceae.

次に、本発明の鉄または鉄を含む合金の微生物腐食に対する耐食性を評価する方法について説明する。ここでいう鉄を含む合金とは、鉄をその成分として含む合金を意味し、鉄の含有割合が低いものも含む。該当する鉄または鉄を含む合金としては、純鉄、炭素鋼、マンガン鋼、ニッケル鋼、ステンレス鋼などがある。まず、鉄若しくは鉄を含有する合金の耐食性を評価するために用いる水溶液について説明する。   Next, a method for evaluating the corrosion resistance against microbial corrosion of the iron or iron-containing alloy of the present invention will be described. As used herein, an alloy containing iron means an alloy containing iron as a component thereof, and includes an alloy having a low iron content. Examples of the iron or the alloy containing iron include pure iron, carbon steel, manganese steel, nickel steel, and stainless steel. First, an aqueous solution used for evaluating the corrosion resistance of iron or an iron-containing alloy will be described.

耐食性を評価するために用いる水溶液はメタン生成菌と硫酸塩還元菌の炭素源として用いるため、二酸化炭素若しくは炭酸物質を含む。特にメタン生成細菌ではこれら二酸化炭素およびその溶解した形態の炭酸水素イオンや炭酸イオンを還元してメタンを生成するので、二酸化炭素は重要な基質である。本水溶液の全炭酸濃度（溶存した二酸化炭素と炭酸物質の濃度の総和）は、１００ｍｇ/Ｌ以上であることが好ましい。ここで、全炭酸濃度とは水溶液に溶存した二酸化炭素と炭酸物質の濃度の総和のことである。   Since the aqueous solution used for evaluating the corrosion resistance is used as a carbon source for methanogens and sulfate-reducing bacteria, it contains carbon dioxide or a carbonate substance. In particular, carbon dioxide is an important substrate because methanogenic bacteria reduce methane by reducing these carbon dioxide and its dissolved bicarbonate ions and carbonate ions. The total carbonic acid concentration of this aqueous solution (the total concentration of dissolved carbon dioxide and carbonic acid substance) is preferably 100 mg / L or more. Here, the total carbonic acid concentration is the sum of the concentrations of carbon dioxide and carbonic acid dissolved in the aqueous solution.

炭酸物質は二酸化炭素が水に溶解した形態の炭酸水素イオン、炭酸イオンおよびこれらの塩であり、メタン生成菌と硫酸塩還元菌の炭素源として必要である。ｐＨ５以上ｐＨ９以下では、主要な存在形態は炭酸水素イオンである。また、ｐＨ緩衝作用があるため、耐食性の評価中にｐＨを変動させない効果もある。全炭酸濃度が１００ｍｇ/Ｌ未満の場合は、メタン生成菌と硫酸塩還元菌の炭素源が不足するため、腐食作用が弱くなる。また、ｐＨ緩衝作用が弱くなるため、ｐＨが低下して、メタン生成菌と硫酸塩還元菌の増殖を抑制するｐＨになる可能性がある。このため、本水溶液の全炭酸濃度は、１００ｍｇ/Ｌ以上とする。緩衝能によりｐＨを安定化させることと、メタン生成菌による腐食を促進するため、可能であれば全炭酸濃度を１０００ｍｇ/Ｌ以上とすることがより好ましい。なお、全炭酸濃度の上限については、飽和濃度まで用いることができる。   Carbonic acid substances are hydrogen carbonate ions, carbonate ions and their salts in a form in which carbon dioxide is dissolved in water, and are necessary as a carbon source for methanogens and sulfate-reducing bacteria. At pH 5 or more and pH 9 or less, the main existence form is bicarbonate ion. Further, since there is a pH buffering action, there is also an effect that the pH is not changed during the evaluation of the corrosion resistance. When the total carbonic acid concentration is less than 100 mg / L, the carbon source of methanogenic bacteria and sulfate-reducing bacteria is insufficient, so that the corrosive action is weakened. Moreover, since pH buffer action becomes weak, pH may fall and it may become pH which suppresses the growth of a methanogen and a sulfate reduction bacterium. For this reason, the total carbonic acid concentration of this aqueous solution shall be 100 mg / L or more. In order to stabilize the pH by the buffer capacity and promote corrosion by the methanogen, it is more preferable that the total carbonic acid concentration is 1000 mg / L or more if possible. The upper limit of the total carbonic acid concentration can be used up to the saturation concentration.

なお、全炭酸濃度の測定については、例えば、ＪＩＳ Ｋ ０１０１に記載の塩化ストロンチウム-塩酸滴定法や、赤外線分光法などの方法により測定することができる。   The total carbonic acid concentration can be measured, for example, by a method such as strontium chloride-hydrochloric acid titration method or infrared spectroscopy described in JIS K 0101.

また、本水溶液の硫酸イオン濃度は、１００ｍｇ/Ｌ以上であることが好ましい。硫酸イオンは硫酸塩還元菌の基質であるため、本水溶液の硫酸イオン濃度が１００ｍｇ/Ｌ未満であると、硫酸塩還元菌による腐食活性が落ちるからである。硫酸塩還元菌による腐食を促進するため、可能であれば硫酸イオン濃度を１０００ｍｇ/Ｌ以上とすることがより好ましい。なお、硫酸イオン濃度の上限については、７０００ｍｇ/Ｌ程度までが好ましい。海水の硫酸イオン濃度は２５００ｍｇ/Ｌ程度であるが、化石海水などで硫酸イオン濃度が７０００ｍｇ/Ｌ程度に達するものが報告されているからである。これ以上の硫酸イオン濃度でも耐食性評価は可能ではあるが、実際の腐食環境では考えにくいため、７０００ｍｇ/Ｌ程度までが好ましい。   Moreover, it is preferable that the sulfate ion concentration of this aqueous solution is 100 mg / L or more. This is because sulfate ions are a substrate for sulfate-reducing bacteria, and therefore, when the concentration of sulfate ions in this aqueous solution is less than 100 mg / L, the corrosive activity of the sulfate-reducing bacteria decreases. In order to promote corrosion by sulfate-reducing bacteria, the sulfate ion concentration is more preferably 1000 mg / L or more if possible. The upper limit of the sulfate ion concentration is preferably up to about 7000 mg / L. This is because the sulfate ion concentration of seawater is about 2500 mg / L, but it has been reported that fossil seawater and the like reach a sulfate ion concentration of about 7000 mg / L. Although corrosion resistance evaluation is possible even at a sulfate ion concentration higher than this, it is difficult to consider in an actual corrosive environment, and therefore it is preferably up to about 7000 mg / L.

また、本水溶液のｐＨは、ｐＨ５以上ｐＨ９以下であることが好ましい。ｐＨ５未満あるいはｐＨ９を超えるｐＨの範囲では、微生物腐食の原因となるメタン生成菌と硫酸塩還元菌が共に増殖しにくい状態となるため、これらの微生物を用いて耐食性の評価を行なうことが困難になる。このため、本水溶液は、ｐＨが５以上９以下であることが好ましい。   Moreover, it is preferable that pH of this aqueous solution is pH5 or more and pH9 or less. In the pH range below pH 5 or above pH 9, both the methanogenic bacteria and sulfate-reducing bacteria that cause microbial corrosion are difficult to grow, making it difficult to evaluate corrosion resistance using these microorganisms. Become. For this reason, it is preferable that pH of this aqueous solution is 5-9.

また、耐食性を評価するために用いる水溶液は塩素イオンを含むことが好ましい。塩素イオンは、二酸化炭素や硫酸イオンとは異なり、メタン生成菌や硫酸塩還元菌の基質ではない。しかし、塩素イオンは石油環境や、船舶のバラストタンクなど、実環境で微生物に起因する腐食が起こる環境において、かん水や海水に起因すると考えられるものが環境水中に高濃度に検出される。海水には１９０００ｍｇ/Ｌ程度の塩素イオンが含まれる。塩素イオンは腐食促進物質でもある。したがって、耐食性を評価するために用いる水溶液は塩素イオンを含むことが好ましい。塩素イオン濃度が１０００ｍｇ/Ｌ未満の場合には、微生物腐食で問題になるような激しい腐食が起きにくい。ただし、淡水の河川水環境など、１０００ｍｇ/Ｌ未満の塩素イオン濃度においても耐食性を評価することはもちろん可能である。また、かん水や海水が蒸発により濃縮した条件であっても塩素イオン濃度が３００００ｍｇ/Ｌを超えるような条件は実腐食環境ではあまり考えられない。塩素イオン濃度が３００００ｍｇ/Ｌを超えるような高塩環境では、微生物の腐食影響よりも、塩素イオンによる腐食影響が強く出ること可能性がある。また、塩素イオン濃度が３００００ｍｇ/Ｌを超える場合には、微生物腐食の原因となる硫酸塩還元菌およびメタン生成菌の生育にも阻害を及ぼす可能性がある。以上の理由により、本水溶液は塩素イオンを１０００ｍｇ/Ｌ以上３００００ｍｇ/Ｌ以下含むことが好ましい。   Moreover, it is preferable that the aqueous solution used in order to evaluate corrosion resistance contains a chlorine ion. Unlike carbon dioxide and sulfate ions, chloride ions are not substrates for methanogens and sulfate-reducing bacteria. However, in the environment where corrosion caused by microorganisms occurs in the real environment, such as a petroleum environment or a ballast tank of a ship, chloride ions are detected in environmental water at a high concentration that is considered to be caused by brine or seawater. Seawater contains about 19000 mg / L of chlorine ions. Chlorine ions are also corrosion promoters. Therefore, it is preferable that the aqueous solution used for evaluating the corrosion resistance contains chlorine ions. When the chlorine ion concentration is less than 1000 mg / L, severe corrosion that causes problems with microbial corrosion is unlikely to occur. However, it is of course possible to evaluate the corrosion resistance even at a chlorine ion concentration of less than 1000 mg / L, such as a freshwater river water environment. In addition, even under conditions where brine or seawater is concentrated by evaporation, conditions where the chlorine ion concentration exceeds 30000 mg / L are not very conceivable in an actual corrosive environment. In a high salt environment where the chlorine ion concentration exceeds 30000 mg / L, there is a possibility that the corrosive effect by the chlorine ion is stronger than the corrosive effect of microorganisms. In addition, when the chlorine ion concentration exceeds 30000 mg / L, there is a possibility of inhibiting the growth of sulfate-reducing bacteria and methanogenic bacteria that cause microbial corrosion. For the above reasons, the aqueous solution preferably contains 1000 mg / L or more and 30000 mg / L or less of chloride ions.

また、嫌気条件における本水溶液の溶存酸素濃度は０．２ｍｇ/Ｌ未満とする。溶存酸素濃度が０．２ｍｇ/Ｌ以上であると、嫌気性微生物である、メタン生成菌と硫酸塩還元菌の増殖が抑制されるからである。このため、本水溶液の溶存酸素濃度は０．２ｍｇ/Ｌ未満とする。   Moreover, the dissolved oxygen concentration of this aqueous solution in anaerobic conditions shall be less than 0.2 mg / L. This is because when the dissolved oxygen concentration is 0.2 mg / L or more, the growth of methanogens and sulfate-reducing bacteria, which are anaerobic microorganisms, is suppressed. For this reason, the dissolved oxygen concentration of this aqueous solution shall be less than 0.2 mg / L.

本水溶液の溶存酸素濃度を０．２ｍｇ/Ｌ未満に維持して、メタン生成菌と硫酸塩還元菌により、鉄若しくは鉄を含有する合金を腐食させる際、嫌気条件に維持して腐食させる時間は２４時間以上とすることが好ましい。２４時間未満の場合には、嫌気条件の時間が短いため、メタン生成菌や硫酸塩還元菌による腐食が十分に進行しない可能性がある。ただしこのような場合であっても、腐食に伴って流れる電流を例えば無抵抗電流計などを用いて測定することにより、腐食の進行状況をモニタリングすることは可能である。   When the dissolved oxygen concentration of this aqueous solution is maintained at less than 0.2 mg / L, and corroding iron or an iron-containing alloy with methanogenic bacteria and sulfate-reducing bacteria, the time to maintain and corrode under anaerobic conditions is It is preferably 24 hours or longer. In the case of less than 24 hours, since the time for anaerobic conditions is short, there is a possibility that corrosion by methanogenic bacteria and sulfate-reducing bacteria does not proceed sufficiently. However, even in such a case, it is possible to monitor the progress of corrosion by measuring the current flowing along with the corrosion using, for example, a non-resistance ammeter.

また、上記嫌気状態での腐食だけでは、腐食程度が弱い場合等においては、更に、腐食を促進させて評価することができる。そのためには、嫌気条件で腐食させた後、本水溶液の溶存酸素濃度を上げて、鉄若しくは鉄を含有する合金の耐食性を評価すればよい。溶存酸素濃度としては、２ｍｇ/Ｌ以上に上げて維持することが好ましい。   In addition, the corrosion in the anaerobic state alone can be evaluated by further promoting the corrosion when the degree of corrosion is weak. For this purpose, after corroding under anaerobic conditions, the dissolved oxygen concentration of the aqueous solution is increased to evaluate the corrosion resistance of iron or an alloy containing iron. The dissolved oxygen concentration is preferably raised to 2 mg / L or more and maintained.

嫌気条件から溶存酸素濃度を上げて、耐食試験を行なう理由は、嫌気条件で微生物腐食させた後に、溶存酸素濃度を上げると、激しい腐食が起こる現象を、本発明者らが見い出したことによる。更には、嫌気条件での腐食程度と、その後溶存酸素濃度を上げた条件での腐食程度には正の相関関係があることを見出したことにもよる。   The reason for conducting the corrosion resistance test by increasing the dissolved oxygen concentration from the anaerobic condition is that the present inventors have found a phenomenon in which severe corrosion occurs when the dissolved oxygen concentration is increased after microbial corrosion under the anaerobic condition. Further, it is also found that there is a positive correlation between the degree of corrosion under anaerobic conditions and the degree of corrosion under conditions where the dissolved oxygen concentration is increased thereafter.

実際の腐食環境においても、例えば、原油タンカーのカーゴタンクの底板のように、原油を積んでいる時は嫌気条件であるが、空荷のバラスト航海時は、酸素の存在する好気環境となり、嫌気条件と好気条件をくりかえす腐食環境で、激しい底板の腐食が起こることが知られている。   Even in an actual corrosive environment, for example, the bottom plate of a cargo tank of a crude oil tanker is an anaerobic condition when crude oil is loaded, but during an empty ballast voyage, it becomes an aerobic environment where oxygen exists, It is known that severe corrosion of the bottom plate occurs in a corrosive environment that repeats anaerobic and aerobic conditions.

嫌気条件から、溶存酸素濃度を０．２ｍｇ/Ｌ〜２ｍｇ/Ｌの間に上げた場合には、酸素による腐食促進の影響が小さい。したがって、この場合には、本水溶液の溶存酸素濃度を２ｍｇ/Ｌ以上に上げて維持する。尚、溶存酸素濃度を２ｍｇ/Ｌ以上に上げて維持する時間についてであるが、それまでに嫌気条件で腐食した鉄若しくは鉄を含有する合金は、酸素によって容易に酸化されるため、酸素にさらされると短時間で激しく腐食する。このような酸素による腐食影響はきわめて短時間にも現れるため、溶存酸素濃度を２ｍｇ/Ｌ以上に上げて維持する時間は、どのような時間であってもかまわないが、好気条件に維持する時間も２４時間以上とすることが好ましい。酸素の腐食影響をより顕著にするためには、溶存酸素濃度を２ｍｇ/Ｌ以上に上げて維持する時間を２４時間以上とすることが好ましい。尚、嫌気条件の後の、空気あるいは酸素の供給による好気条件での試験をおこなわず、嫌気条件のみの試験で鉄又は鉄を含む合金の耐食性を評価することも可能である。また、上記の嫌気条件、好気条件を１サイクルとして、このサイクルを繰り返して鉄又は鉄を含む合金の耐食性を評価することももちろん可能である。これらの条件は、腐食の実環境に合わせて選択することで、より定量的な評価が可能となる。   When the dissolved oxygen concentration is increased between 0.2 mg / L and 2 mg / L from anaerobic conditions, the effect of corrosion promotion by oxygen is small. Therefore, in this case, the dissolved oxygen concentration of the aqueous solution is increased to 2 mg / L or more and maintained. Although it is about the time to maintain the dissolved oxygen concentration at 2 mg / L or more, iron or an alloy containing iron that has been corroded under anaerobic conditions is easily oxidized by oxygen and exposed to oxygen. If this happens, it will corrode in a short time. Since the corrosion effect due to oxygen appears in a very short time, the time for maintaining the dissolved oxygen concentration at 2 mg / L or more may be any time, but it should be maintained under aerobic conditions. The time is preferably 24 hours or more. In order to make the corrosion effect of oxygen more prominent, it is preferable to set the time for maintaining the dissolved oxygen concentration to 2 mg / L or more to be 24 hours or more. In addition, it is also possible to evaluate the corrosion resistance of iron or an alloy containing iron by a test only under anaerobic conditions without performing a test under aerobic conditions by supplying air or oxygen after anaerobic conditions. Moreover, it is of course possible to evaluate the corrosion resistance of iron or an alloy containing iron by repeating the cycle with the above-mentioned anaerobic and aerobic conditions as one cycle. These conditions can be evaluated more quantitatively by selecting them according to the actual environment of corrosion.

鉄若しくは鉄を含む合金の耐食性を評価しようとする場合は、本水溶液中にメタン生成菌と硫酸塩還元菌を共に存在させて、鉄若しくは鉄を含む合金とこれらの微生物を含む本水溶液とを接触させて耐食性を評価する。若しくは、これらの微生物を含む本水溶液中に鉄若しくは鉄を含む合金を浸漬して耐食性を評価する。ここで接触させる方法としては、例えば、窒素と二酸化炭素の混合ガスによる嫌気的なガス雰囲気中に、耐食性を評価しようとする、鉄または鉄を含む合金の表面を露出させた状態で、この露出した表面上に、メタン生成菌と硫酸塩還元菌を含む水溶液を接触させて置く、あるいは、接触させて付着させることにより、本水溶液と接触した鉄又は鉄を含む合金を腐食させることで、耐食性の評価が可能である。   When it is intended to evaluate the corrosion resistance of iron or an iron-containing alloy, both the methanogenic and sulfate-reducing bacteria are present in the aqueous solution, and the iron or iron-containing alloy and the aqueous solution containing these microorganisms are combined. Corrosion resistance is evaluated by contact. Alternatively, the corrosion resistance is evaluated by immersing iron or an alloy containing iron in the aqueous solution containing these microorganisms. As a method of contact here, for example, in the anaerobic gas atmosphere of a mixed gas of nitrogen and carbon dioxide, the exposure of the surface of iron or an alloy containing iron to be evaluated for corrosion resistance is performed. Corrosion resistance by corroding the iron or iron-containing alloy in contact with the aqueous solution by placing the aqueous solution containing the methanogen and sulfate-reducing bacteria in contact with the surface, or by bringing it in contact with the surface. Can be evaluated.

また、前記の鉄若しくは鉄を含む合金と接触させるメタン生成菌と硫酸塩還元菌を含む水溶液、あるいは、その中に鉄若しくは鉄を含む合金を浸漬させるメタン生成菌と硫酸塩還元菌を含む水溶液は、メタン生成菌及び硫酸塩還元菌を添加する前に、前記のように各成分濃度を満たすように調製した水溶液をフィルターろ過やオートクレーブ等で滅菌したものを使用する。また、人工海水やフィルターろ過やオートクレーブ等で滅菌した自然海水などを用いることも可能である。これらにメタン生成菌と硫酸塩還元菌を添加することで、耐食性の評価の試験液として用いることが可能である。   Further, an aqueous solution containing methanogens and sulfate-reducing bacteria that are brought into contact with iron or an iron-containing alloy, or an aqueous solution containing methanogens and sulfate-reducing bacteria in which an iron or iron-containing alloy is immersed. Uses a solution prepared by sterilizing an aqueous solution prepared so as to satisfy the concentration of each component as described above by filter filtration or autoclave before adding methanogenic bacteria and sulfate-reducing bacteria. It is also possible to use artificial seawater, natural seawater sterilized by filter filtration, autoclave, or the like. By adding methanogenic bacteria and sulfate-reducing bacteria to these, it can be used as a test solution for evaluating corrosion resistance.

特殊な微生物の培養液を用いずとも、また、特別な栄養塩を添加しなくとも、人工海水やフィルターろ過やオートクレーブ等で滅菌した自然海水のように一般的に使用される腐食試験液を用いることが可能なことが、本発明の耐食性の評価方法の特徴である。   Without using special microorganism culture solution or adding special nutrient salt, use commonly used corrosion test solution such as artificial seawater, natural seawater sterilized by filter filtration, autoclave, etc. It is possible to characterize the corrosion resistance evaluation method of the present invention.

市販の人工海水は、滅菌しなくとも液中にもともと微生物がわずかにしか存在しないので、メタン生成菌と硫酸塩還元菌を添加することで、そのまま耐食性の評価の試験液として用いることが可能である。   Commercially available artificial seawater does not have sterilization, and since there are only a few microorganisms in the liquid, it can be used as a test solution for evaluating corrosion resistance by adding methanogens and sulfate-reducing bacteria. is there.

メタン生成菌及び硫酸塩還元菌は、いずれも嫌気性微生物であることから、耐食性を評価するために用いる水の溶存酸素濃度が０．２ｍｇ／Ｌ未満であることをあらかじめ確認することが望ましい。尚、嫌気環境で腐食試験をするため、試験液が接する気相は、窒素ガス、アルゴンガスを不活性な嫌気性のガスとして使用するとともに、炭素源となる二酸化炭素を含むことが好ましい。この二酸化炭素の濃度については、特に限定条件は無い。ただし、メタン生成菌と硫酸塩還元菌による腐食を促進するために、二酸化炭素濃度が１％以上であることがより望ましい。二酸化炭素と混合するガスとしては、上記のように窒素ガスやアルゴンガスなどを用いることができる。例えば、窒素あるいはアルゴンに対して二酸化炭素を１９：１から１：１の比率の間で用いることがより望ましい。   Since both methanogens and sulfate-reducing bacteria are anaerobic microorganisms, it is desirable to confirm in advance that the dissolved oxygen concentration of water used for evaluating corrosion resistance is less than 0.2 mg / L. In order to conduct a corrosion test in an anaerobic environment, the gas phase in contact with the test solution preferably uses nitrogen gas and argon gas as an inert anaerobic gas and contains carbon dioxide as a carbon source. There is no particular limitation on the concentration of carbon dioxide. However, in order to promote corrosion by methanogenic bacteria and sulfate reducing bacteria, it is more desirable that the carbon dioxide concentration be 1% or more. As gas mixed with carbon dioxide, nitrogen gas, argon gas or the like can be used as described above. For example, it is more desirable to use carbon dioxide in a ratio of 19: 1 to 1: 1 with respect to nitrogen or argon.

耐食性評価に用いるための、水溶液へのメタン生成菌と硫酸塩還元菌の初期添加量については、例えば初期濃度で１０3細胞/ｍL以上１０7細胞/ｍL以下とすることが好ましい。初期の濃度が１０3細胞/ｍL未満であると、メタン生成菌と硫酸塩還元菌の増殖に時間を要するため、耐食性の評価に時間が掛かり効率的でない。また、初期の濃度が１０7細胞/ｍL以上であると、メタン生成菌と硫酸塩還元菌の濃度がすぐに飽和に達するため、これらの微生物の増殖にともなって起こる腐食影響を評価しづらくなる問題があるからである。また、本発明ではメタン生成菌と硫酸塩還元菌をともに存在させた条件で耐食性を評価するが、対照として、微生物を添加しない無菌の場合、あるいはメタン生成菌のみを添加した場合、あるいは硫酸塩還元菌のみを添加した場合についても耐食性を評価して比較することが好ましい。 The initial addition amount of the methanogen and sulfate-reducing bacteria to the aqueous solution for use in the corrosion resistance evaluation is preferably, for example, 10 3 cells / mL or more and 10 7 cells / mL or less at the initial concentration. If the initial concentration is less than 10 3 cells / mL, it takes time for the methanogenic and sulfate-reducing bacteria to grow, so it takes time to evaluate the corrosion resistance and is not efficient. In addition, when the initial concentration is 10 7 cells / mL or more, the concentrations of methanogens and sulfate-reducing bacteria quickly reach saturation, making it difficult to evaluate the corrosive effects caused by the growth of these microorganisms. Because there is a problem. Further, in the present invention, the corrosion resistance is evaluated under the condition in which both the methanogen and the sulfate-reducing bacterium are present, but as a control, aseptic, when no microorganism is added, or when only the methanogen is added, or sulfate Even when only reducing bacteria are added, it is preferable to evaluate and compare the corrosion resistance.

尚、耐食性を評価しようとする鉄若しくは鉄を含む合金の試験片の形状は、試験液と接触あるいは、試験液に浸漬できれば、どのような形状であってもかまわない。腐食をより進行させたい場合は、比表面積の大きな形状とすればよいし、入手の容易性から平板を用いることもできる。ただし、２種類以上の鉄または鉄を含む合金の耐食性を比較する場合は、同じ形状の試験片を用いることが好ましい。   The shape of the test piece of iron or an alloy containing iron to be evaluated for corrosion resistance may be any shape as long as it can be in contact with the test solution or immersed in the test solution. When it is desired to further promote the corrosion, the shape may be a large specific surface area, and a flat plate may be used for easy availability. However, when comparing the corrosion resistance of two or more types of iron or alloys containing iron, it is preferable to use test pieces of the same shape.

耐食性の評価における温度条件は、メタン生成菌及び硫酸塩還元菌の成育温度の範囲、２０℃以上４０℃以下が好ましい。温度が２０℃未満、あるいは４０℃を超える場合、メタン生成菌と硫酸塩還元菌を共に安定に成育できないため、安定に耐食性を評価するために、２０℃以上４０℃以下が好ましい。   The temperature condition in the evaluation of corrosion resistance is preferably in the range of the growth temperature of methanogens and sulfate-reducing bacteria, and 20 ° C. or more and 40 ° C. or less. When the temperature is lower than 20 ° C. or higher than 40 ° C., both the methanogen and the sulfate-reducing bacterium cannot be stably grown. Therefore, in order to stably evaluate the corrosion resistance, the temperature is preferably 20 ° C. or higher and 40 ° C. or lower.

鉄若しくは鉄を含む合金の腐食量の測定方法については、例えば、腐食試験前後の質量を測定し、その差から腐食量を求める方法がある。具体的には、試験終了後に、試験片を取り出して、腐食生成物を例えば水にといだクレンザーをつけた脱脂綿を用いて取り除いた後、水で洗浄して、例えばブロワーを用いて速やかに乾燥させた試験片の質量を測定し、試験前の試験片の質量からの変化を測定する方法である。   As a method for measuring the corrosion amount of iron or an alloy containing iron, for example, there is a method of measuring the mass before and after the corrosion test and obtaining the corrosion amount from the difference. Specifically, after the test is completed, the test piece is taken out, and the corrosion product is removed using, for example, absorbent cotton with a cleanser soaked in water, washed with water, and dried quickly using, for example, a blower. In this method, the mass of the test piece is measured, and the change from the mass of the test piece before the test is measured.

また、試験中に試験片から溶出した鉄の量をフェナントロリン法等によって測定するなどによって、腐食量を測定することが可能である。また、試験片を例えばクロスポリッシング加工等により腐食した試験片の断面を観察できるようにしておいて、電子顕微鏡等を用いて、腐食部位の断面構造や腐食部位の深さなどを観察することによっても、腐食量の測定が可能である。   Further, the amount of corrosion can be measured by measuring the amount of iron eluted from the test piece during the test by the phenanthroline method or the like. In addition, by observing the cross section of the test piece corroded by, for example, cross polishing, and observing the cross-sectional structure of the corroded portion and the depth of the corroded portion using an electron microscope or the like. In addition, the amount of corrosion can be measured.

また、鉄から溶解する鉄イオンによって電流が流れることから、電気化学的に電流量を測定することで、溶解した鉄の量すなはち腐食量の測定が可能である。   In addition, since current flows due to iron ions dissolved from iron, the amount of dissolved iron, that is, the amount of corrosion can be measured by electrochemically measuring the amount of current.

また、鉄を電子供与体とするメタン生成菌及び硫酸塩還元菌による腐食であることから、硫酸塩還元菌による腐食に関しては、腐食に伴い、硫化鉄など硫化物の量が増加する。したがって、硫化物の量を測定することによっても腐食量の測定が可能である。硫化物の量の測定方法としては、例えば、ＪＩＳ Ｋ ０１０２のよう素滴定法などがある。   In addition, since corrosion is caused by methanogens and sulfate-reducing bacteria using iron as an electron donor, the amount of sulfides such as iron sulfide increases with corrosion. Therefore, the amount of corrosion can also be measured by measuring the amount of sulfide. As a method for measuring the amount of sulfide, for example, there is an iodine titration method according to JIS K 0102.

メタン生成菌による腐食に関してはも、腐食がすすむほど、二酸化炭素の還元により発生するメタンの生成量は増加することになる。したがって、メタンの生成量を測定することによっても腐食量の測定が可能である。メタンの測定方法としては、例えば、上記、鉄または鉄を含む合金で耐食性の評価試験を行なった試験液の上部の気体を回収して、ガスクロマトグラフ分離管でメタンを分離した後、水素炎イオン化検出器に導入してメタンを測定する方法や、ガスクロマトグラフ質量分析計でメタンを測定する方法、レーザガス検知器を用いてメタンを測定する方法などがある。   Regarding the corrosion caused by the methane-producing bacteria, the amount of methane generated by the reduction of carbon dioxide increases as the corrosion progresses. Therefore, the amount of corrosion can also be measured by measuring the amount of methane produced. As a method for measuring methane, for example, the gas at the top of the test solution which has been subjected to the corrosion resistance evaluation test using iron or an alloy containing iron is recovered, and methane is separated by a gas chromatograph separation tube. There are a method of introducing methane into a detector, a method of measuring methane with a gas chromatograph mass spectrometer, a method of measuring methane with a laser gas detector, and the like.

以上のように、本発明の微生物を作用させた場合の、鉄または鉄を含む合金の腐食量を材料ごとに比較することによって、材料の耐食性を評価することが可能となる。また、鉄または鉄を含む合金と接するまたはこれらを含む水の中に、本発明の微生物の存在有無の条件で、鉄または鉄を含む合金の腐食量を比較することによって、鉄又は鉄を含む合金の微生物腐食に対する耐食性を評価することが可能である。   As described above, the corrosion resistance of a material can be evaluated by comparing the amount of corrosion of iron or an alloy containing iron when the microorganism of the present invention is used for each material. Moreover, iron or iron is contained by comparing the amount of corrosion of iron or iron-containing alloys in water in contact with or containing iron or iron-containing alloys in the presence or absence of the microorganism of the present invention. It is possible to evaluate the corrosion resistance of alloys against microbial corrosion.

尚、メタン生成菌による腐食では、炭酸鉄が形成される。したがって、腐食生成物をＸ線回折や蛍光Ｘ線分析等で解析する等により、炭酸鉄が検出されれば、メタン生成菌による腐食が生じていると判断できる。また、硫酸塩還元菌による腐食では、硫酸塩還元菌が鉄に対して腐食性の硫化水素を発生するため、硫化鉄が発生する。したがって、腐食生成物をＸ線回折や蛍光Ｘ線分析等で解析する等により、硫化鉄が検出されれば硫酸塩還元菌による腐食が生じていると判断できる。本発明のように、メタン生成菌と硫酸塩還元菌が共存して腐食する場合、炭酸鉄と硫化鉄が、共に検出されることが腐食生成物の特徴であるが、どちらかの腐食影響がより強い場合は、炭酸鉄あるいは硫化鉄のどちらかが検出されることもある。また、嫌気条件でこれらの微生物により腐食させた後、好気条件に移行して酸素による腐食影響を受ける場合は、例えば水酸化鉄が形成される等、この限りではない。   In addition, iron carbonate is formed in the corrosion by the methanogen. Therefore, if iron carbonate is detected by analyzing the corrosion product by X-ray diffraction, fluorescent X-ray analysis, or the like, it can be determined that corrosion due to methanogenic bacteria has occurred. In addition, in the corrosion by sulfate-reducing bacteria, the sulfate-reducing bacteria generate hydrogen sulfide that is corrosive to iron, and thus iron sulfide is generated. Therefore, if iron sulfide is detected by analyzing the corrosion product by X-ray diffraction, fluorescent X-ray analysis or the like, it can be determined that corrosion due to sulfate-reducing bacteria has occurred. As in the present invention, when methanogenic bacteria and sulfate-reducing bacteria coexist and corrode, it is a feature of the corrosion product that iron carbonate and iron sulfide are detected together. If stronger, either iron carbonate or iron sulfide may be detected. In addition, after being corroded by these microorganisms under anaerobic conditions, the conditions are not limited to this, for example, when iron hydroxide is formed by shifting to aerobic conditions and being affected by oxygen.

以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれら実施例にその技術的範囲が限定されるものではない。   Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

｛実施例１｝炭素鋼の耐食性評価実験
容積７５ｍＬの密栓可能なガラス容器に、表１に記載の鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源とした培地から鉄顆粒を除いた培地を耐食性評価試験液として２５ｍＬ用意した。試験開始時、試験液の全炭酸濃度は１５００ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１３０００ｍｇ/Ｌ、ｐＨ７．０であった。気相はN2:CO2 = 80%:20%として、この試験液中に、炭素鋼（鉄の質量含有率９９％）試験片（１０ｍｍ×１０ｍｍ×１ｍｍ）を浸漬した。耐食性を評価しようとする炭素鋼の試験片を試験液に浸漬することにより、試験片に含まれる鉄が微生物への電子供与体となる。表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される、石油取扱い施設の内部が腐食した鉄配管の中の腐食部位から採取した水の中に含まれていたメタン生成菌と硫酸塩還元菌を混合して含む集積培養液を本耐食性を評価する試験液に１/２０体積添加した。本耐食性評価試験液中のメタン生成菌と硫酸塩還元菌を合わせた初期濃度はＤＡＰＩ染色により計数したところ、２×１０5細胞/ｍＬであった。２５℃で１週間静置して、嫌気条件における耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 {Example 1} Experiment for evaluating corrosion resistance of carbon steel In a glass container having a volume of 75 mL, which can be sealed, iron granules are prepared from a medium containing iron as a sole electron donor and carbon dioxide and bicarbonate ions as carbon sources. 25 mL of the removed medium was prepared as a corrosion resistance evaluation test solution. At the start of the test, the total carbonate concentration of the test solution was 1500 mg / L, the sulfate ion concentration was 2700 mg / L, the chloride ion concentration was 13000 mg / L, and pH 7.0. The gas phase was N 2 : CO 2 = 80%: 20%, and a carbon steel (iron mass content 99%) test piece (10 mm × 10 mm × 1 mm) was immersed in this test solution. By immersing a test piece of carbon steel to be evaluated for corrosion resistance in a test solution, iron contained in the test piece becomes an electron donor to microorganisms. In the water collected from the corrosion site in the iron pipe where the inside of the oil handling facility is corroded by the culture medium of Table 1 where iron is the only electron donor and carbon dioxide and bicarbonate ions are used as the carbon source. 1/20 volume of the enriched culture solution containing the mixed methanogen and sulfate-reducing bacteria was added to the test solution for evaluating the corrosion resistance. The initial concentration of the methanogenic and sulfate-reducing bacteria in the corrosion resistance evaluation test solution was 2 × 10 5 cells / mL when counted by DAPI staining. It left still at 25 degreeC for 1 week, and evaluated the corrosion resistance in anaerobic conditions. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に試験片の質量を測定して、質量の減少量を試験時間（１週間）で割って腐食速度を求めた。試験後の試験片については、水にといだクレンザーをつけた脱脂綿を用いて腐食生成物を除去後、試験片を水で洗浄して空気ブロワーを用いて乾燥させた後、腐食生成物を取り除いて質量測定をおこなった。結果を表２に示す。   The test piece was measured for the corrosion rate by measuring the mass of the test piece before and after the test, and dividing the decrease in mass by the test time (one week). For test specimens after the test, remove the corrosion products using absorbent cotton with a cleanser soaked in water, wash the specimen with water and dry it with an air blower, and then remove the corrosion products. Mass measurement. The results are shown in Table 2.

表２に示したように、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌と硫酸塩還元菌の集積培養液を添加した系では、炭素鋼の腐食が促進された。無菌の系に比べて、腐食速度は約１０倍になった。   As shown in Table 2, in a system in which an enriched culture solution of methanogen and sulfate-reducing bacteria cultured using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source, Corrosion was promoted. Compared to the sterile system, the corrosion rate was about 10 times.

以上のように、本発明の微生物を用いて、鉄を含む合金の耐食性を評価できる。   As described above, the corrosion resistance of an alloy containing iron can be evaluated using the microorganism of the present invention.

｛実施例２｝純鉄の耐食性評価試験（嫌気条件における耐食性評価試験）
容積７５ｍＬの密栓可能なガラス容器に、表１に記載の鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源とした培地から鉄のみを除いた耐食性評価試験液を２０ｍＬ用意した。試験開始時、試験液の全炭酸濃度は１５００ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１３０００ｍｇ/Ｌ、ｐＨ７．０であった。 {Example 2} Pure iron corrosion resistance test (corrosion resistance test under anaerobic conditions)
In a glass container with a capacity of 75 mL, 20 mL of a corrosion resistance evaluation test solution was prepared by removing only iron from a medium containing iron as the only electron donor and carbon dioxide and hydrogen carbonate ions as listed in Table 1. At the start of the test, the total carbonate concentration of the test solution was 1500 mg / L, the sulfate ion concentration was 2700 mg / L, the chloride ion concentration was 13000 mg / L, and pH 7.0.

気相はN2:CO2 = 80%:20%として、この試験液中に、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される、デスルホビブリオナレス（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ）目デスルホビブリオナシアエ（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｃｅａｅ）科に属する硫酸塩還元菌とメタノコッカレス（Ｍｅｔｈａｎｏｃｏｃｃａｌｅｓ）目メタノコッカシアエ（Ｍｅｔｈａｎｏｃｏｃｃａｃｅａｅ）科に属するメタン生成菌（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、本耐食性を評価する試験液に１/２０体積それぞれ添加することにより、硫酸塩還元菌単独添加、メタン生成菌単独添加、メタン生成菌と硫酸塩還元菌を共に添加したもの、および両方の菌株を添加しない無菌系を対照として用意した。本耐食性評価試験液に添加したメタン生成菌と硫酸塩還元菌の初期濃度をＤＡＰＩ染色により計数したところ、それぞれ２×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食試験を行なった。尚、対照として、微生物を添加しない無菌の系でも腐食試験を行なった。腐食量は、試験液中の懸濁物を含む鉄濃度を測定することにより、算出した。結果を表３に示す。 Gas phase is N 2 : CO 2 = 80%: 20%, and pure iron (iron mass content of 99.9% or more) test piece (10 mm × 10 mm × 0.1 mm, mass 80 mg) in this test solution Soaked. Sulfate reduction belonging to the family of Desulfobibrionales (Desulfovibrioaceae), cultured with the medium of Table 1 using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source 1/20 volume each of a culture solution of a methanogen belonging to the family Methanococcalae (Methanococcasae) (Accession No. NITE BP-252) is added to the test solution for evaluating the corrosion resistance. Thus, a sulfate-reducing bacterium alone added, a methanogenic bacterium alone added, a methane-producing bacterium and a sulfate-reducing bacterium added together, and a sterile system to which both strains were not added were prepared as controls. When the initial concentrations of the methanogenic and sulfate-reducing bacteria added to the corrosion resistance evaluation test solution were counted by DAPI staining, they were 2 × 10 5 cells / mL, respectively. The corrosion test was conducted under anaerobic conditions by standing at 25 ° C. for 1 week. As a control, the corrosion test was also performed in a sterile system to which no microorganism was added. The amount of corrosion was calculated by measuring the iron concentration including the suspension in the test solution. The results are shown in Table 3.

表３に示したように、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌単離株と硫酸塩還元菌単離株を両方添加した系では、純鉄の腐食が促進された。無菌の系に比べて、腐食速度は約１５倍になった。   As shown in Table 3, in a system to which both a methanogenic isolate and a sulfate-reducing bacterium isolate, which are cultured using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source, Corrosion of pure iron was promoted. Compared to sterile systems, the corrosion rate was approximately 15 times.

｛実施例３｝純鉄の耐食性評価試験（嫌気条件に引き続き好気条件とする耐食性評価試験）
容積７５ｍＬの密栓可能なガラス容器に、表１に記載の鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源とした培地から鉄のみを除いた耐食性評価試験液を２０ｍＬ用意した。試験開始時、試験液の全炭酸濃度は１５００ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１３０００ｍｇ/Ｌ、ｐＨ７．０であった。 {Example 3} Pure iron corrosion resistance evaluation test (corrosion resistance evaluation test following anaerobic conditions followed by aerobic conditions)
In a glass container with a capacity of 75 mL, 20 mL of a corrosion resistance evaluation test solution was prepared by removing only iron from a medium containing iron as the only electron donor and carbon dioxide and hydrogen carbonate ions as listed in Table 1. At the start of the test, the total carbonate concentration of the test solution was 1500 mg / L, the sulfate ion concentration was 2700 mg / L, the chloride ion concentration was 13000 mg / L, and pH 7.0.

気相はN2:CO2 = 80%:20%として、この試験液中に、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される、デスルホビブリオナレス（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ）目デスルホビブリオナシアエ（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｃｅａｅ）科に属する硫酸塩還元菌とメタノコッカレス（Ｍｅｔｈａｎｏｃｏｃｃａｌｅｓ）目メタノコッカシアエ（Ｍｅｔｈａｎｏｃｏｃｃａｃｅａｅ）科に属するメタン生成菌（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、本耐食性を評価する試験液に１/２０体積それぞれ添加することにより、硫酸塩還元菌単独添加、メタン生成菌単独添加、メタン生成菌と硫酸塩還元菌を共に添加したもの、および両方の菌株を添加しない無菌系を対照として用意した。本耐食性評価試験液に添加したメタン生成菌と硫酸塩還元菌の初期濃度をＤＡＰＩ染色により計数したところ、それぞれ２×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食試験を行なった後、容器のフタを開け空気開放した。試験液のＤＯが２ｍｇ/Ｌ以上となっていることを確認し、さらに１週間２５℃で静置した。尚、対照として、微生物を添加しない無菌の系でも腐食試験を行なった。腐食量は、試験液中の懸濁物を含む鉄濃度を測定することにより、算出した。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。 Gas phase is N 2 : CO 2 = 80%: 20%, and pure iron (iron mass content of 99.9% or more) test piece (10 mm × 10 mm × 0.1 mm, mass 80 mg) in this test solution Soaked. Sulfate reduction belonging to the family of Desulfobibrionales (Desulfovibrioaceae), cultured with the medium of Table 1 using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source 1/20 volume each of a culture solution of a methanogen belonging to the family Methanococcalae (Methanococcasae) (Accession No. NITE BP-252) is added to the test solution for evaluating the corrosion resistance. Thus, a sulfate-reducing bacterium alone added, a methanogenic bacterium alone added, a methane-producing bacterium and a sulfate-reducing bacterium added together, and a sterile system to which both strains were not added were prepared as controls. When the initial concentrations of the methanogenic and sulfate-reducing bacteria added to the corrosion resistance evaluation test solution were counted by DAPI staining, they were 2 × 10 5 cells / mL, respectively. After standing at 25 ° C. for 1 week and conducting a corrosion test under anaerobic conditions, the container lid was opened and the air was released. After confirming that the DO of the test solution was 2 mg / L or more, the test solution was allowed to stand at 25 ° C. for 1 week. As a control, the corrosion test was also performed in a sterile system to which no microorganism was added. The amount of corrosion was calculated by measuring the iron concentration including the suspension in the test solution. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

表４に示したように、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌単離株と硫酸塩還元菌単離株を両方添加した系では、純鉄の腐食が促進された。無菌の系に比べて、腐食速度は約４０倍になった。   As shown in Table 4, in a system to which both a methanogenic isolate and a sulfate-reducing bacterium isolate, which are cultured using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source, Corrosion of pure iron was promoted. Compared to sterile systems, the corrosion rate was about 40 times.

｛実施例４｝人工海水を用いた炭素鋼の耐食性評価実験
容積７５ｍＬの密栓可能なガラス容器に、人工海水を２５ｍＬ用意した。試験開始時、試験液の全炭酸濃度は１５０ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１９０００ｍｇ/Ｌ、ｐＨ８．２であった。 {Example 4} Corrosion resistance evaluation experiment of carbon steel using artificial seawater 25 mL of artificial seawater was prepared in a glass container having a volume of 75 mL that can be sealed. At the start of the test, the total carbonic acid concentration of the test solution was 150 mg / L, the sulfate ion concentration was 2700 mg / L, the chloride ion concentration was 19000 mg / L, and pH 8.2.

気相はN2:CO2 = 80%:20%として、この試験液中に、炭素鋼（鉄の質量含有率９９％）試験片（１０ｍｍ×１０ｍｍ×１ｍｍ）を浸漬した。表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌として、デスホビブリオナシエと、同じく鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌として、メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエの初期濃度は共に、１×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させた後、さらに１週間２５℃で静置させて、耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 The gas phase was N 2 : CO 2 = 80%: 20%, and a carbon steel (iron mass content 99%) test piece (10 mm × 10 mm × 1 mm) was immersed in this test solution. With the medium shown in Table 1, as a sulfate-reducing bacterium that is cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, Desphobibrionaceae and iron as the only electron donor, carbon dioxide As a methanogen that is cultivated using carbon dioxide and hydrogen carbonate as a carbon source, a culture solution of methanococciae (accession number NITE BP-252) was added to the test solution for evaluating the corrosion resistance by 1/40 each. . When added to the corrosion resistance test solution, the initial concentrations of the sulfate-reducing bacterium Desphobibrionace and the methanogenic methanococcasia were 1 × 10 5 cells / mL. After standing at 25 ° C. for 1 week and corroding under anaerobic conditions, the container lid is once removed, the gas phase is replaced with air, and the test solution is also stirred and sufficiently brought into contact with air. It was allowed to stand at 25 ° C. for 1 week to evaluate the corrosion resistance. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。   The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を表５に示す。表５に示したように、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエを添加した系では、炭素鋼の腐食が促進された。無菌の系に比べて、腐食速度は約６０倍になった。以上のように、人工海水を用いても、メタン生成菌と硫酸塩還元菌による微生物腐食に対する耐食性を評価できることが明らかになった。   The results are shown in Table 5. As shown in Table 5, in the system to which the sulfate-reducing bacterium Desphobibrionaceae and the methanogenic methanococcasia were cultured using iron as the sole electron donor and carbon dioxide and bicarbonate ions as the carbon source, , Carbon steel corrosion was promoted. Compared to sterile systems, the corrosion rate was approximately 60 times. As described above, it was revealed that the corrosion resistance against microbial corrosion caused by methanogens and sulfate-reducing bacteria can be evaluated using artificial seawater.

｛実施例５｝人工海水を用いた純鉄の耐食性評価実験（全炭酸濃度の影響）
容積７５ｍＬの密栓可能なガラス容器に、全炭酸濃度が５０ｍｇ/Ｌ、１００ｍｇ/Ｌ、５００ｍｇ/Ｌ、１０００ｍｇ/Ｌ、１５００ｍｇ/Ｌ、２０００ｍｇ/Ｌの人工海水を各２５ｍＬずつ用意した。試験開始時の硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１９０００ｍｇ/Ｌ、ｐＨ８．２であった。 {Example 5} Experiment for evaluating corrosion resistance of pure iron using artificial seawater (effect of total carbonic acid concentration)
25 mL each of artificial seawater having a total carbonic acid concentration of 50 mg / L, 100 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L and 2000 mg / L was prepared in a glass container having a capacity of 75 mL. The sulfate ion concentration at the start of the test was 2700 mg / L, the chloride ion concentration was 19000 mg / L, and pH 8.2.

気相はN2:CO2 = 80%:20%として、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。 The vapor phase was N 2 : CO 2 = 80%: 20%, and pure iron (iron mass content of 99.9% or more) test pieces (10 mm × 10 mm × 0.1 mm, mass 80 mg) were immersed therein.

表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌として、デスホビブリオナシエと、同じく鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌として、メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエの初期濃度は共に、１×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させた後、さらに１週間２５℃で静置させて、耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 With the medium shown in Table 1, as a sulfate-reducing bacterium that is cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, Desphobibrionaceae and iron as the only electron donor, carbon dioxide As a methanogen that is cultivated using carbon dioxide and hydrogen carbonate as a carbon source, a culture solution of methanococciae (accession number NITE BP-252) was added to the test solution for evaluating the corrosion resistance by 1/40 each. . When added to the corrosion resistance test solution, the initial concentrations of the sulfate-reducing bacterium Desphobibrionace and the methanogenic methanococcasia were 1 × 10 5 cells / mL. After standing at 25 ° C. for 1 week and corroding under anaerobic conditions, the container lid is once removed, the gas phase is replaced with air, and the test solution is also stirred and sufficiently brought into contact with air. It was allowed to stand at 25 ° C. for 1 week to evaluate the corrosion resistance. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。   The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を図１に示す。図１に示したように、全炭酸濃度が１００ｍｇ/Ｌ以上で、純鉄の腐食が促進された。したがって、耐食性を評価する際には、試験液の全炭酸濃度は１００ｍｇ/Ｌ以上とするべきことが判明した。
｛実施例６｝人工海水を用いた純鉄の耐食性評価実験（硫酸イオン濃度の影響）
容積７５ｍＬの密栓可能なガラス容器に、硫酸イオン濃度が５０ｍｇ/Ｌ、１００ｍｇ/Ｌ、１０００ｍｇ/Ｌ、２０００ｍｇ/Ｌ、３０００ｍｇ/Ｌ、７０００ｍｇ/Ｌの人工海水を各２５ｍＬずつ用意した。試験開始時の全炭酸濃度は１５００ｍｇ/Ｌ、塩素イオン濃度は１９０００ｍｇ/Ｌ、ｐＨ８．２であった。 The results are shown in FIG. As shown in FIG. 1, the corrosion of pure iron was promoted when the total carbonic acid concentration was 100 mg / L or more. Therefore, it was found that when evaluating corrosion resistance, the total carbonic acid concentration of the test solution should be 100 mg / L or more.
{Example 6} Corrosion resistance evaluation experiment of pure iron using artificial seawater (effect of sulfate ion concentration)
25 mL each of artificial seawater having a sulfate ion concentration of 50 mg / L, 100 mg / L, 1000 mg / L, 2000 mg / L, 3000 mg / L, and 7000 mg / L was prepared in a glass container with a capacity of 75 mL. The total carbonic acid concentration at the start of the test was 1500 mg / L, the chloride ion concentration was 19000 mg / L, and pH 8.2.

気相はN2:CO2 = 80%:20%として、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。 The gas phase N 2: CO 2 = 80% : a 20% pure iron (mass content of 99.9% or more iron) test piece (10 mm × 10 mm × 0.1 mm, mass 80 mg) was immersed.

表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌として、デスホビブリオナシエと、同じく鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌として、メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエの初期濃度は共に、１×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させた後、さらに１週間２５℃で静置させて、耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 With the medium shown in Table 1, as a sulfate-reducing bacterium that is cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, Desphobibrionaceae and iron as the only electron donor, carbon dioxide As a methanogen that is cultivated using carbon dioxide and hydrogen carbonate as a carbon source, a culture solution of methanococciae (accession number NITE BP-252) was added to the test solution for evaluating the corrosion resistance by 1/40 each. . When added to the corrosion resistance test solution, the initial concentrations of the sulfate-reducing bacterium Desphobibrionace and the methanogenic methanococcasia were 1 × 10 5 cells / mL. After standing at 25 ° C. for 1 week and corroding under anaerobic conditions, the container lid is once removed, the gas phase is replaced with air, and the test solution is also stirred and sufficiently brought into contact with air. It was allowed to stand at 25 ° C. for 1 week to evaluate the corrosion resistance. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。   The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を図２に示す。図２に示したように、硫酸イオン濃度が１００ｍｇ/Ｌ以上で、純鉄の腐食が促進されることが判明した。
｛実施例７｝人工海水を用いた純鉄の耐食性評価実験（ｐＨの影響）
容積７５ｍＬの密栓可能なガラス容器に、ｐＨが４、５、６、７、８、９、１０の人工海水を各２５ｍＬずつ用意した。試験開始時の全炭酸濃度は１５００ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１９０００ｍｇ/Ｌであった。 The results are shown in FIG. As shown in FIG. 2, it was found that the corrosion of pure iron is promoted when the sulfate ion concentration is 100 mg / L or more.
{Example 7} Experimental evaluation of corrosion resistance of pure iron using artificial seawater (effect of pH)
25 mL each of artificial seawater having a pH of 4, 5, 6, 7, 8, 9, 10 was prepared in a glass container having a capacity of 75 mL that can be sealed. The total carbonic acid concentration at the start of the test was 1500 mg / L, the sulfate ion concentration was 2700 mg / L, and the chloride ion concentration was 19000 mg / L.

気相はN2:CO2 = 80%:20%として、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。 The vapor phase was N 2 : CO 2 = 80%: 20%, and pure iron (iron mass content of 99.9% or more) test pieces (10 mm × 10 mm × 0.1 mm, mass 80 mg) were immersed therein.

表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌として、デスホビブリオナシエと、同じく鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌として、メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエの初期濃度は共に、１×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させた後、さらに１週間２５℃で静置させて、耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 With the medium shown in Table 1, as a sulfate-reducing bacterium that is cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, Desphobibrionaceae and iron as the only electron donor, carbon dioxide As a methanogen that is cultivated using carbon dioxide and hydrogen carbonate as a carbon source, a culture solution of methanococciae (accession number NITE BP-252) was added to the test solution for evaluating the corrosion resistance by 1/40 each. . When added to the corrosion resistance test solution, the initial concentrations of the sulfate-reducing bacterium Desphobibrionace and the methanogenic methanococcasia were 1 × 10 5 cells / mL. After standing at 25 ° C. for 1 week and corroding under anaerobic conditions, the container lid is once removed, the gas phase is replaced with air, and the test solution is also stirred and sufficiently brought into contact with air. It was allowed to stand at 25 ° C. for 1 week to evaluate the corrosion resistance. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。   The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を図３に示す。図３に示したように、ｐＨが５以上９以下で、純鉄の腐食が促進されることが判明した。
｛実施例８｝人工海水を用いた純鉄の耐食性評価実験（塩素イオン濃度の影響）
容積７５ｍＬの密栓可能なガラス容器に、塩素イオン濃度が１００ｍｇ/Ｌ、５００ｍｇ/Ｌ、１０００ｍｇ/Ｌ、５０００ｍｇ/Ｌ、１００００ｍｇ/Ｌ、１５０００ｍｇ/Ｌ、２００００ｍｇ/Ｌ、２５０００ｍｇ/Ｌ、３００００ｍｇ/Ｌの人工海水を各２５ｍＬずつ用意した。これらの人工海水は、塩化ナトリウムを添加せず調製した人工海水をベースに、塩化ナトリウムを塩素イオンが設定濃度になるように適当量添加することで調製した。試験開始時の全炭酸濃度は１５００ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、ｐＨは８．２であった。 The results are shown in FIG. As shown in FIG. 3, it has been found that the corrosion of pure iron is accelerated when the pH is 5 or more and 9 or less.
{Example 8} Corrosion resistance evaluation experiment of pure iron using artificial seawater (effect of chloride ion concentration)
In a glass container with a capacity of 75 mL, the chloride ion concentration is 100 mg / L, 500 mg / L, 1000 mg / L, 5000 mg / L, 10000 mg / L, 15000 mg / L, 20000 mg / L, 25000 mg / L, 30000 mg / L. 25 mL each of artificial seawater was prepared. These artificial seawaters were prepared by adding an appropriate amount of sodium chloride to a set concentration of chloride ions based on artificial seawater prepared without adding sodium chloride. The total carbonic acid concentration at the start of the test was 1500 mg / L, the sulfate ion concentration was 2700 mg / L, and the pH was 8.2.

気相はN2:CO2 = 80%:20%として、純鉄（鉄の質量含有率９９.９％以上）試験片（１０ｍｍ×１０ｍｍ×０．１ｍｍ、質量８０ｍｇ）を浸漬した。 The vapor phase was N 2 : CO 2 = 80%: 20%, and pure iron (iron mass content of 99.9% or more) test pieces (10 mm × 10 mm × 0.1 mm, mass 80 mg) were immersed therein.

表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養される硫酸塩還元菌として、デスホビブリオナシエと、同じく鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌として、メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）の培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、硫酸塩還元菌デスホビブリオナシエとメタン生成菌メタノコッカシアエの初期濃度は共に、１×１０5細胞/ｍＬであった。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させた後、さらに１週間２５℃で静置させて、耐食性を評価した。尚、対照として、微生物を添加しない無菌の系でも耐食性を評価した。 With the medium shown in Table 1, as a sulfate-reducing bacterium that is cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, Desphobibrionaceae and iron as the only electron donor, carbon dioxide As a methanogen that is cultivated using carbon dioxide and hydrogen carbonate as a carbon source, a culture solution of methanococciae (accession number NITE BP-252) was added to the test solution for evaluating the corrosion resistance by 1/40 each. . When added to the corrosion resistance test solution, the initial concentrations of the sulfate-reducing bacterium Desphobibrionace and the methanogenic methanococcasia were 1 × 10 5 cells / mL. After standing at 25 ° C. for 1 week and corroding under anaerobic conditions, the container lid is once removed, the gas phase is replaced with air, and the test solution is also stirred and sufficiently brought into contact with air. It was allowed to stand at 25 ° C. for 1 week to evaluate the corrosion resistance. As a control, the corrosion resistance was evaluated even in a sterile system to which no microorganism was added.

試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。   The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を図３に示す。図４に示したように、塩素イオン濃度が１０００ｍｇ/Ｌ以上で、純鉄試験片の腐食が促進されることが判明した。
｛実施例９｝相対的な耐食性の比較試験
容積７５ｍＬの密栓可能なガラス容器に、人工海水を２５ｍＬ用意した。試験開始時、試験液の全炭酸濃度は１５０ｍｇ/Ｌ、硫酸イオン濃度は２７００ｍｇ/Ｌ、塩素イオン濃度は１９０００ｍｇ/Ｌ、ｐＨ８．２であった。 The results are shown in FIG. As shown in FIG. 4, it was found that the corrosion of the pure iron test piece was promoted when the chlorine ion concentration was 1000 mg / L or more.
{Example 9} Comparative test of relative corrosion resistance 25 mL of artificial seawater was prepared in a glass container having a volume of 75 mL that can be sealed. At the start of the test, the total carbonic acid concentration of the test solution was 150 mg / L, the sulfate ion concentration was 2700 mg / L, the chloride ion concentration was 19000 mg / L, and pH 8.2.

気相はN2:CO2 = 80%:20%として、この試験液中に、成分組成の異なる３種類の合金鋼Ａ、Ｂ、Ｃ及び純鉄の同一形状の試験片（１０ｍｍ×１０ｍｍ×１ｍｍ）を浸漬した。 The gas phase is N 2 : CO 2 = 80%: 20%, and in this test solution, three types of alloy steels A, B, C, and pure iron having the same shape and different shapes are used (10 mm × 10 mm × 1 mm).

表１の培地により、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌と硫酸塩還元菌を共に含む集積培養液を、それぞれ本耐食性評価のための試験液に１/４０体積ずつ添加した。耐食試験液に添加した際の、メタン生成菌と硫酸塩還元菌を合わせた初期濃度は１×１０3細胞/ｍＬとした。２５℃で１週間静置して嫌気条件で腐食させた後、容器のフタをいったんはずして、気相を空気と入れ換えるとともに、試験液も攪拌して、空気と十分に接触させて溶存酸素濃度を２ｍｇ/Ｌ以上とした後、さらに１週間２５℃で静置して、それぞれの試験片を腐食させて耐食性を評価した。試験片は、試験開始前と、試験後に質量を測定して、腐食速度を求めた。試験後の試験片については、腐食生成物を取り除いて質量測定をおこなった。尚、耐食性の評価では、嫌気条件で１週間、好気条件で１週間、それぞれ要し、嫌気条件と好気条件では腐食速度が異なるが、合計の試験時間２週間を用いて、平均化した腐食速度を算出した。 Using the medium shown in Table 1, an enrichment culture containing both methanogens and sulfate-reducing bacteria that are cultured using iron as the only electron donor and carbon dioxide and bicarbonate ions as the carbon source, respectively, was used for this corrosion resistance evaluation. 1/40 volume was added to the test solution. The initial concentration of methanogenic and sulfate-reducing bacteria when added to the corrosion resistance test solution was 1 × 10 3 cells / mL. After standing at 25 ° C for 1 week and corroding under anaerobic conditions, remove the lid of the container, replace the gas phase with air, stir the test solution, and bring it into sufficient contact with air to dissolve oxygen concentration Was set at 2 mg / L or more, and then allowed to stand at 25 ° C. for another week to corrode each test piece to evaluate the corrosion resistance. The test piece measured the mass before the test start and after the test, and calculated | required the corrosion rate. About the test piece after a test, the corrosion product was removed and mass measurement was performed. In addition, in the corrosion resistance evaluation, it took 1 week under anaerobic conditions and 1 week under aerobic conditions. Corrosion rates differed between anaerobic conditions and aerobic conditions, but they were averaged using a total test time of 2 weeks. Corrosion rate was calculated.

結果を表６に示す。表６に示したように、鉄を唯一の電子供与体、二酸化炭素と炭酸水素イオンを炭素源として培養されるメタン生成菌と硫酸塩還元菌を共に含む集積培養系を用いて、合金鋼Ａが最もよい耐食性を示すことがあきらかになった。以上のように、２種類以上の鉄鋼材料の耐食性を比較して評価できることが明らかになった。   The results are shown in Table 6. As shown in Table 6, using an integrated culture system containing both methanogens and sulfate-reducing bacteria that are cultured using iron as the sole electron donor and carbon dioxide and bicarbonate ions as carbon sources, alloy steel A It has become clear that shows the best corrosion resistance. As described above, it has become clear that the corrosion resistance of two or more types of steel materials can be compared and evaluated.

｛実施例１０｝人工海水を用いた塗装に欠落がある鉄板の耐食性評価実験
図５に示したように、中央部の直径２ｍｍの円形範囲を除いて、タールエポキシ塗装した１００ｍｍ×１００ｍｍ×２ｍｍの形状の炭素鋼ＳＳ４００製の鉄板の上部に、外径７０ｍｍ、内径６５ｍｍで高さ１００ｍｍのアクリル製円筒をタールエポキシ塗装した鉄板の塗装面上で接着させた鉄板の腐食試験装置を２つ用意した。円筒内に炭酸水素ナトリウムを用いて全炭酸濃度を１５００ｍｇ/Ｌとした人工海水を２００ｍＬ入れ、Ｎ２ガスでバブリングして溶存酸素濃度ＤＯ<０．２ｍｇ/Ｌとなったことを確認した。Ｎ２ガスを満たした嫌気チャンバー内で、２つある腐食試験装置のうち、一方にはメタン生成菌メタノコッカシアエ（受託番号ＮＩＴＥ ＢＰ−２５２）と硫酸塩還元菌デスホビブリオナシエを共に共に初期濃度で１×１０6細胞/ｍＬになるように添加した。円筒内の液面から上部の気相をＮ２ガスで満たした状態でゴム栓を用いて密栓した。なお、残るもう１つの腐食試験装置は、対照用に微生物の添加はおこなわず、微生物を添加した系と同様に円筒内の液面から上部の気相をＮ２ガスで満たした状態でゴム栓を用いて密栓した。両方の腐食試験装置を２５℃で２週間静置して嫌気条件で腐食させた後、ゴム栓をはずして、円筒内の人工海水にそれぞれ空気を供給して溶存酸素濃度ＤＯが約３ｍｇ/Ｌの状態で１日間おいた。直径２ｍｍの円形の塗装をしなかった部位について、腐食生成物を除去後、最大腐食深さを測定した。メタン生成菌と硫酸塩還元菌を添加した腐食試験装置と微生物を添加しなかった腐食試験装置それぞれの最大腐食深さの測定結果を表７に示す。メタン生成菌と硫酸塩還元菌を添加した場合には、これらの微生物を添加しない場合に比べて最大腐食深さは約３０倍に増加した。以上のように、塗装に欠落がある鉄板鉄板にメタン生成菌と硫酸塩還元菌を添加した腐食試験液が接触し、さらに二酸化炭素ガスを使用しなくとも試験液中に溶存した炭酸塩を使用することによって、耐食性の評価が可能であることが判明した。 {Example 10} Experiment for evaluating corrosion resistance of iron plate with lack of coating using artificial seawater As shown in FIG. 5, except for a circular range with a diameter of 2 mm at the center, 100 mm × 100 mm × 2 mm coated with tar epoxy Two steel plate corrosion test apparatuses were prepared by adhering an acrylic cylinder having an outer diameter of 70 mm, an inner diameter of 65 mm, and a height of 100 mm to the top of the steel plate made of carbon steel SS400 in the shape of tar epoxy coating. . 200 mL of artificial seawater having a total carbonic acid concentration of 1500 mg / L using sodium hydrogen carbonate was put into the cylinder, and bubbling with N 2 gas was performed to confirm that the dissolved oxygen concentration DO <0.2 mg / L. In an anaerobic chamber filled with N 2 gas, one of the two corrosion test apparatuses is a methanogen, Methaneococciae (accession number NITE BP-252), and a sulfate-reducing bacterium, Desphobibrionaceae. The initial concentration was 1 × 10 6 cells / mL. Sealed with a rubber stopper while the upper gas phase from the liquid level in the cylinder was filled with N 2 gas. In addition, the remaining corrosion test apparatus does not add microorganisms for control, and in the same manner as the system to which microorganisms are added, the rubber plug is filled with N 2 gas from the liquid level in the cylinder to the upper gas phase. The bottle was sealed with Both corrosion test apparatuses were allowed to stand at 25 ° C. for 2 weeks to be corroded under anaerobic conditions, and then the rubber stoppers were removed, and air was supplied to the artificial seawater in the cylinder, so that the dissolved oxygen concentration DO was about 3 mg / L. I stayed for one day. About the site | part which was not painted circularly 2 mm in diameter, after removing the corrosion product, the maximum corrosion depth was measured. Table 7 shows the measurement results of the maximum corrosion depths of the corrosion test apparatus to which the methanogen and sulfate-reducing bacteria were added and the corrosion test apparatus to which the microorganism was not added. When methanogenic bacteria and sulfate-reducing bacteria were added, the maximum corrosion depth increased about 30 times compared to the case where these microorganisms were not added. As described above, the corrosion test solution with added methanogen and sulfate-reducing bacteria is in contact with the iron plate with missing coating, and the carbonate dissolved in the test solution is used without using carbon dioxide gas. As a result, it was found that the corrosion resistance can be evaluated.

試験液の全炭酸濃度が鉄腐食速度に及ぼす影響。Effect of total carbonic acid concentration of test solution on iron corrosion rate. 試験液の硫酸イオン濃度が鉄腐食速度に及ぼす影響。The effect of sulfate concentration in the test solution on the iron corrosion rate. 試験液のｐＨが鉄腐食速度に及ぼす影響。Effect of pH of test solution on iron corrosion rate. 試験液の塩素イオン濃度が鉄腐食速度に及ぼす影響。Effect of chloride concentration of test solution on iron corrosion rate. 腐食試験装置の説明図。Explanatory drawing of a corrosion test apparatus.

Claims (5)

炭酸物質、硫酸イオン、及び塩素イオンを含む嫌気条件の水溶液中に、鉄を電子供与体として、かつ、前記炭酸物質を炭素源として培養可能なメタン生成菌及び硫酸塩還元菌を存在させ、当該メタン生成菌及び硫酸塩還元菌を含む水溶液と鉄又は鉄を含む合金とを接触させ、又は、前記微生物を含む水溶液中に鉄又は鉄を含む合金を浸漬して、前記鉄又は鉄を含む合金を嫌気条件で腐食させた後、又は、更にその後、空気又は酸素を供給して、前記水溶液中の溶存酸素濃度を高めることにより好気条件として、前記鉄又は鉄を含む合金を腐食させた後、当該腐食量を測定して、前記鉄又は鉄を含む合金の耐食性を評価することを特徴とする鉄又は鉄を含む合金の耐食性の評価方法。   In an anaerobic aqueous solution containing carbonate, sulfate and chloride ions, there are methanogens and sulfate-reducing bacteria that can be cultured using iron as an electron donor and the carbonate as a carbon source, An alloy containing iron or iron by contacting an aqueous solution containing methanogen and sulfate-reducing bacteria with iron or an alloy containing iron, or immersing iron or an alloy containing iron in an aqueous solution containing the microorganism After corroding under anaerobic conditions, or after further corroding the iron or iron-containing alloy as aerobic conditions by supplying air or oxygen and increasing the dissolved oxygen concentration in the aqueous solution. A method for evaluating the corrosion resistance of iron or an iron-containing alloy, characterized by measuring the corrosion amount and evaluating the corrosion resistance of the iron or iron-containing alloy. 前記腐食量の測定を、２種類以上の鉄又は鉄を含む合金について行い、当該鉄又は鉄を含む合金それぞれの腐食量の測定結果を比較して、前記鉄又は鉄を含む合金それぞれの前記メタン生成菌及び硫酸塩還元菌に対する耐食性を相対的に評価することを特徴とする請求項１に記載の鉄又は鉄を含む合金の耐食性の評価方法。   The measurement of the corrosion amount is performed on two or more kinds of iron or an alloy containing iron, and the measurement results of the corrosion amounts of the iron or the alloy containing iron are compared, and the methane of each of the iron or the alloy containing iron is compared. The method for evaluating corrosion resistance of iron or an iron-containing alloy according to claim 1, wherein the corrosion resistance against the producing bacteria and sulfate-reducing bacteria is relatively evaluated. 前記水溶液中の全炭酸濃度が１００ｍｇ/Ｌ以上、硫酸イオン濃度が１００ｍｇ/Ｌ以上７０００ｍｇ/Ｌ以下、ｐＨが５以上９以下、塩素イオン濃度が１０００ｍｇ/Ｌ以上３００００ｍｇ/Ｌ以下、嫌気条件における溶存酸素濃度が０．２ｍｇ/Ｌ未満であり、空気あるいは酸素を供給した後の好気条件における溶存酸素濃度が２ｍｇ/Ｌ以上であることを特徴とする請求項１又は２に記載の鉄又は鉄を含む合金の耐食性の評価方法。   Total carbonic acid concentration in the aqueous solution is 100 mg / L or more, sulfate ion concentration is 100 mg / L or more and 7000 mg / L or less, pH is 5 or more and 9 or less, chlorine ion concentration is 1000 mg / L or more and 30000 mg / L or less, dissolved in anaerobic conditions The iron or iron according to claim 1 or 2, wherein the oxygen concentration is less than 0.2 mg / L, and the dissolved oxygen concentration in aerobic conditions after supplying air or oxygen is 2 mg / L or more. Evaluation method of corrosion resistance of alloys containing 前記メタン生成菌がメタノコッカレス（Ｍｅｔｈａｎｏｃｏｃｃａｌｅｓ）目メタノコッカシアエ（Ｍｅｔｈａｎｏｃｏｃｃａｃｅａｅ）科に属する微生物であり、前記硫酸塩還元菌がデスルホビブリオナレス（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ）目デスルホビブリオナシアエ（Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｃｅａｅ）科に属する微生物であることを特徴とする請求項１〜３のいずれか１項に記載の鉄又は鉄を含む合金の耐食性の評価方法。   The methanogen is a microorganism belonging to the family Methanococcalae, Methanococceae, and the sulfate-reducing bacterium is a member of the Desulfobibrionales (Desulfobrionaceae) family. The method for evaluating the corrosion resistance of iron or an alloy containing iron according to any one of claims 1 to 3, wherein the microorganism belongs to the microorganism. 前記メタン生成菌が受託番号ＮＩＴＥ ＢＰ−２５２で特定される微生物であることを特徴とする請求項４に記載の鉄又は鉄を含む合金の耐食性の評価方法。   5. The method for evaluating corrosion resistance of iron or an iron-containing alloy according to claim 4, wherein the methanogen is a microorganism specified by a deposit number NITE BP-252.
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