Sulfate reducer detection kit and sulfate reducer detection method

Abstract

To provide a sulfate reducer detection kit capable of preferentially detecting a sulfate reducer causing an electric microbial corrosion (EMIC) and a sulfate reducer detection method using the kit.SOLUTION: A sulfate reducer detection kit comprises a culture medium and a detection member containing iron. The culture medium includes water, a sulfate, which is a first component, and a second component having a positive oxidation reduction potential rather than the first component and functioning as an electron acceptor of a sulfate reducer. A concentration of an organic material having a negative oxidation reduction potential rather than the first component in the culture medium is 2 mmol / L or less.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a sulfate-reducing bacteria detection kit and a sulfate-reducing bacteria detection method. [Background technology]

[0002] Sulfate-reducing bacteria are ubiquitous in soil, seabed, seawater, river water, and other environments, and they oxidize various organic matter and hydrogen gas as electron donors and reduce sulfate to corrosive hydrogen sulfide, so they have been considered one of the main causes of corrosion of iron materials in these environments (especially anaerobic environments). Therefore, detecting sulfate-reducing bacteria in the environment has been used as a method for evaluating corrosion risk. For example, one method for detecting sulfate-reducing bacteria is to add a sample collected from the environment to a medium containing organic matter (electron donor) and sulfate (electron acceptor) that are suitable for sulfate-reducing bacteria and cultivate them.

[0003] Meanwhile, sulfate-reducing bacteria that use iron as an electron source have been isolated in recent years (Non-Patent Document 1). In the process of iron corrosion by these sulfate-reducing bacteria, a thick, conductive corrosion product is formed on the iron surface, and the sulfate-reducing bacteria progress the corrosion by directly absorbing electrons from the iron on the corrosion product. Such corrosion caused by bacteria and dependent on the extraction of electrons is called EMIC (Electrical microbially influenced corrosion). The inventors analyzed the cell membrane of sulfate-reducing bacteria in detail to analyze the electron uptake mechanism, and revealed that a specific enzyme group is involved in directly extracting electrons from iron (Patent Document 1). Furthermore, they revealed that even in the absence of this enzyme, sulfate-reducing bacteria accelerate the corrosion of iron using corrosion products (iron sulfide, etc.) (Non-Patent Document 2).

[0004] Such electrochemical microbial corrosion (EMIC) is known to have a corrosion rate several tens of times faster than non-EMIC corrosion (Non-Patent Document 3), and once it occurs, it progresses at a rapid rate of several tens of mm per year. For this reason, many sudden and serious accidents caused by EMIC have been reported in environments that cannot be visually confirmed on a daily basis, such as inside oil pipelines, facilities that handle contaminated water or treated water, and storage tanks for treated water. Enormous economic losses estimated at tens of billions of dollars per year are occurring, mainly in the energy and shipping industries of developed countries, and the detection of sulfate-reducing bacteria that cause EMIC is particularly important. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 7066224 [Non-patent literature]

[0006] [Non-Patent Document 1] Hang T. Dinh, et al. “Iron corrosion by novel anaerobic microorganisms”, Nature volume 427, p.829-832, 2004. [Non-Patent Document 2] Xiao Deng, et al. “Biogenic Iron Sulfide Nanoparticles to Enable Extracellular Electron Uptake in Sulfate-Reducing Bacteria” Angew. Chem. Int. Ed., December 25, 2019. [Non-Patent Document 3] Dennis Enning, et al. “Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust”, Environ Microbiology, 2012 July, 14(7), 1772-87. Summary of the Invention [Problem to be solved by the invention]

[0007] However, conventional methods for detecting sulfate-reducing bacteria do not provide conditions conducive to the occurrence of electrochemical corrosion (EMIC) using iron as an electron source, and therefore cannot preferentially detect sulfate-reducing bacteria that cause EMIC.

[0008] The present invention is intended to solve these problems. That is, the present invention provides a sulfate-reducing bacteria detection kit capable of preferentially detecting sulfate-reducing bacteria that cause EMIC, and a sulfate-reducing bacteria detection method using the kit. [Means for solving the problem]

[0009] As a result of intensive research into achieving the above object, the inventors have found that the above object can be achieved by the following configuration.

[0010] [1] A sulfate-reducing bacteria detection kit, comprising: A culture medium and a detection member containing iron, The medium is Water, The first component is sulfate, a second component having a more positive redox potential than the first component and functioning as an electron acceptor for sulfate-reducing bacteria; A sulfate-reducing bacteria detection kit, wherein the concentration of an organic matter having a more negative oxidation-reduction potential than the first component in the medium is 2 mmol / L or less. [2] The sulfate-reducing bacteria detection kit according to [1], wherein the concentration of organic matter having a more negative redox potential than sulfate in the culture medium is 1 mmol / L or less. [3] The sulfate-reducing bacteria detection kit according to [1], wherein the culture medium does not contain any organic matter having a more negative redox potential than the sulfate. [4] The sulfate-reducing bacteria detection kit according to any one of [1] to [3], wherein the second component is at least one selected from the group consisting of fumarate, dimethyl sulfoxide, and nitrate. [5] The sulfate-reducing bacteria detection kit according to any one of [1] to [4], wherein the second component has a standard oxidation-reduction potential (pH 7) of +20 mV to +500 mV. [6] The sulfate-reducing bacteria detection kit according to any one of [1] to [5], wherein in the medium, the ratio (M1 / M2) of the molar concentration of the first component (M1) to the molar concentration of the second component (M2) is 2 to 4. [7] The sulfate-reducing bacteria detection kit according to any one of [1] to [6], wherein the detection member is a plate-like body. [8] The sulfate-reducing bacteria detection kit according to any one of [1] to [7], wherein the detection member is carbon steel. [9] A method for detecting sulfate-reducing bacteria, comprising: Providing a detection kit according to any one of [1] to [8]; Adding a sample to the culture medium and conducting a corrosion test of the detection member in the culture medium; and evaluating a degree of corrosion of the detection member after the corrosion test.

[10] The evaluation of the degree of corrosion of the detection member is checking the presence or absence of a corrosion product layer on the surface of the detection member; Evaluating the surface shape of the detection member; and and evaluating the change in weight of the detection member before and after the corrosion test. Effect of the Invention

[0011] The sulfate-reducing bacteria detection kit of the present invention is equipped with a culture medium having a specific composition that promotes electrochemical microbial corrosion (EMIC), and is therefore capable of preferentially detecting sulfate-reducing bacteria that cause EMIC, and also of shortening the detection time. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a schematic cross-sectional view of the sulfate-reducing bacteria detection kit of the present embodiment. [Diagram 2] 1 is a flowchart illustrating a method for detecting sulfate-reducing bacteria according to the present embodiment. [Diagram 3] 1 is a photograph of the top surface of the detection member after a corrosion test in Experiment 1-1. [Figure 4A] 13 is a top view photograph of the detection member after the corrosion test in Experiment 1-7. [Figure 4B] 13 is a side view of the detection component (with bacteria) after the corrosion test in Experiment 1-7. [Figure 4C] 1 is a side view of the detection component (with bacteria) after the corrosion test in Experiment 1-6. [Figure 5A] 13 is a photograph of the top surface of the detection member after the corrosion products were removed after a corrosion test in Experiments 1-1 to 1-7. [Figure 5B] 5B is an enlarged view of the portion surrounded by the dotted line in Fig. 5A, that is, a photograph of the top surface of the detection member after the corrosion products were removed after the corrosion test in Experiments 1-5 to 1-7. [Figure 6] FIG. 13 is a diagram showing the surface shape evaluation results (measured cross-sectional curves) of the detection member after the corrosion products were removed after the corrosion test in Experiments 1-1 and 1-5 to 1-7. [Figure 7] FIG. 13 is a diagram showing the evaluation results of the weight change of the detection member before and after the corrosion test in Experiments 1-1 to 1-7. [Figure 8] FIG. 13 is a diagram showing the evaluation results of the weight change of the detection member before and after the corrosion test in Experiments 2-1 to 2-7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described in detail below. The following description of the constituent elements may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits.

[0014] [Sulfate-reducing bacteria detection kit] As shown in Fig. 1, the sulfate-reducing bacteria detection kit (hereinafter, sometimes simply referred to as a "detection kit" or "kit") 100 of this embodiment includes a culture medium 10 and an iron-containing detection member 20. A specimen (sample) is added to the culture medium 10, and the degree (state) of corrosion of the detection member 20 by the sulfate-reducing bacteria is evaluated, thereby making it possible to detect sulfate-reducing bacteria in the sample (see Fig. 2).

[0015] Examples of sulfate-reducing bacteria detected by the sulfate-reducing bacteria detection kit of this embodiment, that is, sulfate-reducing bacteria that cause EMIC, include Desulfovibrio vulgaris Hildenborough, D. ferrophilus IS5, and Desulfobacterium corrodens IS4.

[0016] The medium 10 of this embodiment includes a first component, sulfate, and a second component having a more positive redox potential than the first component. The second component functions as an electron acceptor for sulfate-reducing bacteria together with the first component (sulfate). The inventors have surprisingly found that by including the second component in the medium together with the first component, EMIC is significantly promoted (the corrosion rate is significantly increased), and have arrived at the present invention. As shown in the examples described later, the rate of EMIC when only the first component is included as the electron acceptor is almost the same as the rate of EMIC when only the second component is included as the electron acceptor. Therefore, it is presumed that in the medium 10 of this embodiment, a synergistic effect between the first and second components occurs, promoting EMIC. The mechanism is presumed to be as follows. When an electron acceptor (second component) with a redox potential more positive than sulfate is present, the electrons taken up into the bacterial cell are branched to the second component with a positive potential (step-down electron transfer) and to NADH or sulfate with a negative potential (step-up electron transfer). The intracellular electron branching reaction promotes the reduction of negatively charged substances (sulfates). As a result, EMIC in the entire system is promoted. Note that the mechanism described above is merely speculation and does not limit the present invention in any way. Furthermore, in culture medium 10, the concentration of an organic substance (hereinafter, appropriately referred to as a "specific organic substance") that can act as an electron donor for sulfate-reducing bacteria and has a more negative oxidation-reduction potential than the first component is 2 mmol / L or less. When the concentration of the specific organic substance that can act as an electron donor in culture medium 10 is low (or culture medium 10 does not contain a specific organic substance), the sulfate-reducing bacteria use the iron in detection member 20 as an electron donor, and EMIC is further promoted. Thus, by providing culture medium 10 with a composition that promotes electrical microbial corrosion (EMIC), kit 100 of this embodiment can preferentially detect sulfate-reducing bacteria that cause EMIC and can also shorten the detection time.

[0017] The first component, sulfate, functions as an electron acceptor (first electron acceptor) for sulfate-reducing bacteria. Sulfate ions derived from sulfate are reduced by sulfate-reducing bacteria to generate hydrogen sulfide. The first component is not particularly limited as long as it is a sulfate. Examples of sulfate include sodium sulfate, magnesium sulfate, and iron sulfate. The first component may be composed of only one type of sulfate, or may be composed of two or more types of sulfate.

[0018] The second component functions as an electron acceptor (second electron acceptor) for sulfate-reducing bacteria. The second component is not particularly limited as long as it has a more positive redox potential than the first component in the medium and functions as an electron acceptor for sulfate-reducing bacteria, and examples of the second component include fumarate, nitrate, dimethyl sulfoxide (DMSO), iron citrate, sodium dichromate, manganese oxide, etc., and from the viewpoint of further promoting EMIC, fumarate, nitrate, and dimethyl sulfoxide (DMSO) are preferred. The fumarate is not particularly limited, and examples of the fumarate include sodium fumarate, iron fumarate, magnesium fumarate, etc. The nitrate is not particularly limited, and examples of the nitrate include sodium nitrate, potassium nitrate, magnesium nitrate, etc. In addition, nitrate promotes EMIC, but when reduced, it produces nitrite (HNO 2 ), which may be toxic to sulfate-reducing bacteria. In this case, the activity of sulfate-reducing bacteria may be suppressed. Therefore, from the viewpoint of further promoting EMIC, it is preferable to use a compound that is not toxic to sulfate-reducing bacteria, such as a fumarate salt or dimethyl sulfoxide (DMSO), as the second component. The second component may be composed of only one type of compound, or may be composed of two or more types of compounds.

[0019] The standard redox potential of a representative second component (pH 7) and the standard redox potential of sulfate ion are shown in the table below. If the second component has a standard redox potential that is more positive than the standard redox potential of sulfate ion, it can be determined that the second component has a more positive redox potential than the first component in the medium 10. From the viewpoint of further promoting EMIC, the standard redox potential of the second component is preferably, for example, 0 mV or more, or +20 mV to +500 mV.

[0020] [Table 1]

[0021] In the medium 10 of this embodiment, the concentration of the organic matter (specific organic matter) having a more negative redox potential than the first component is 2 mmol / L or less, and preferably 1 mmol / L or less. Some specific organic matters are compounds that act as electron donors for sulfate-reducing bacteria. By setting the concentration of the specific organic matter to the above upper limit or less, conditions are created that make it easier for electromicrobial corrosion (EMIC) using iron as an electron source to occur, and sulfate-reducing bacteria that cause EMIC can be preferentially detected. The medium 10 of this embodiment may not contain the specific organic matter (i.e., the concentration of the specific organic matter may be 0 (zero) mmol / L).

[0022] Examples of specific organic substances that act as electron donors for sulfate-reducing bacteria include lactate, glucose, hydrogen, propionic acid, etc. As described above, the concentration of these compounds is 2 mmol / L or less, but from the viewpoint of making EMICs more likely to occur, it is preferably 1 mmol / L or less, and more preferably zero (0 mmol / L).

[0023] The medium of this embodiment may contain a specific organic substance of 2 mmol / L or less for a purpose other than the electron donor for sulfate-reducing bacteria. Examples of such specific organic substances include yeast containing trace elements and amino acids, carbon sources (acetate, bicarbonate ion, etc.), ascorbic acid used as a reducing agent, cysteine, etc. The medium 10 of this embodiment may or may not contain these specific organic substances as necessary. Furthermore, these specific organic substances can exert a sufficient effect at a low concentration of 2 mmol / L or less, or 1 mmol / L or less.

[0024] If the substance has a standard oxidation-reduction potential that is more negative than the standard oxidation-reduction potential of sulfate ion, it can be determined that the substance has a more negative oxidation-reduction potential than the first component in the culture medium 10, that is, that the substance is a specific organic substance.

[0025] When the medium 10 of this embodiment contains a plurality of types of specific organic substances, the total concentration (total blend amount) of the plurality of types of specific organic substances is 2 mmol / L or less, and preferably 1 mmol / L or less.

[0026] The medium of this embodiment contains water. The water may be pure water, ion-exchanged water, etc. The first component and the second component are preferably dissolved or dispersed in water.

[0027] The medium of this embodiment contains water, the first component, and the second component, and the concentration of the specific organic matter is 2 mmol / L or less, so the composition is not particularly limited. The medium of this embodiment may be composed of only water, the first component, and the second component, or may contain other components other than water, the first component, and the second component. Examples of other components include carbon sources, nitrogen sources, trace nutrients (vitamins, amino acids, etc.), inorganic salts (minerals), pH adjusters, reducing agents, etc. In addition, the medium of this embodiment may be, for example, artificial seawater to which the first component and the second component have been added. The artificial seawater may contain, for example, multiple types of inorganic salts mainly composed of sodium chloride, pH adjusters, etc.

[0028] The concentration (amount) M1 of the first component (sulfate) and the concentration (amount) M2 of the second component in the medium are not particularly limited and may be appropriately adjusted depending on the type of sulfate-reducing bacteria to be detected. The concentration M1 of the first component may be, for example, 10 mmol / L to 25 mmol / L. The concentration M2 of the second component may be, for example, 3 mmol / L to 8 mmol / L.

[0029] In the medium, the ratio (M1 / M2) of the molar concentration of the first component (M1) to the molar concentration of the second component (M2) is not particularly limited, but from the viewpoint of further promoting EMIC by sulfate-reducing bacteria, it is preferably 1 to 10, more preferably 2 to 8, and even more preferably 2 to 4.

[0030] In the medium, there is no particular limitation on the concentration (mixture amount) of other components other than water, the first component, and the second component. The concentration of the other components may be, for example, 0.1% by mass to 10% by mass.

[0031] The medium 10 may be produced by uniformly mixing water, the first component (sulfate), the second component, and, if necessary, other components, by a known method.

[0032] The amount of culture medium 10 in the sulfate-reducing bacteria detection kit 100 of this embodiment is not particularly limited. For example, the amount is preferably such that detection member 20 can be completely immersed in culture medium 10 during a detection test for sulfate-reducing bacteria (corrosion test of detection member 20). From a practical standpoint, the amount of culture medium 10 may be appropriately adjusted to, for example, 5 mL or more, or in the range of 5 mL to 1000 mL.

[0033] The detection member 20 is not particularly limited as long as it contains iron, and may be pure iron (purity of about 99.90 to 99.95%) or an iron alloy. The iron alloy preferably contains iron as the main component (50 mass% or more). Examples of the iron alloy include carbon steel (Fe-C), stainless steel (Fe-Ni-Cr), chromium molybdenum steel (Fe-Cr-Mo), manganese molybdenum steel (Fe-Mn-Mo), etc. From the viewpoint of low cost and easy availability, the detection member 20 is preferably carbon steel.

[0034] The size, shape, etc. of the detection member 20 are not particularly limited as long as they are suitable for use in a sulfate-reducing bacteria detection test (corrosion test). For example, the detection member 20 may be a plate-like body, which allows easy evaluation of the corroded surface (surface roughness, etc.). When the detection member 20 is a plate-like body, its size may be, for example, L: 5 to 20 mm, W: 5 to 20 mm, and H: 0.5 to 5 mm. The detection member 20 may be in powder form. When the detection member 20 is in powder form, the degree of corrosion can be evaluated by visually observing a color change (blackening).

[0035] The sulfate-reducing bacteria detection kit 100 may be composed of only the culture medium 10 and the detection member 20, or may further include other components. For example, the sulfate-reducing bacteria detection kit 100 may have a container 30 capable of holding the culture medium 10 and the detection member 20 (see FIG. 1). The material of the container 30 is not particularly limited as long as it achieves the effects of this embodiment, but a material that is transparent (has high transmittance) to visible light, such as glass or acrylic, is preferred so that the inside can be visually observed.

[0036] 1 shows a state in which a detection test for sulfate-reducing bacteria (corrosion test of the detection member 20) is performed using the sulfate-reducing bacteria detection kit 100. For this reason, the detection member 20 is immersed in the culture medium 10, but the form of the detection kit 100 of this embodiment is not limited to this. For example, the detection member 20 and the culture medium 10 of the detection kit 100 may not be in contact with each other when shipped from the factory, sold, stored, etc., and may be brought into contact with each other only during the sulfate-reducing bacteria detection test. By not bringing the culture medium 10 and the detection member 20 into contact with each other before the detection test, deterioration of the culture medium 10 and the detection member 20 can be prevented, and the detection sensitivity of sulfate-reducing bacteria can be increased.

[0037] [Detection method for sulfate-reducing bacteria] An example of a method for detecting sulfate-reducing bacteria according to this embodiment will be described with reference to Figures 1 and 2. The detection method includes, for example, the following steps S1 to S3, and detects sulfate-reducing bacteria in a specimen (sample). Step S1: preparing a sulfate-reducing bacteria detection kit 100 Step S2: Adding a sample to the culture medium 10 and conducting a corrosion test of the detection member 20 in the culture medium 10; and Step S3: A step of evaluating the degree of corrosion of the detection member 20 after the corrosion test.

[0038] Process S1: First, prepare a sulfate-reducing bacteria detection kit 100. Since the configuration of the sulfate-reducing bacteria detection kit 100 has already been described, a description thereof will be omitted here.

[0039] Process S2: Next, a sample is added to the culture medium 10, and a corrosion test of the detection member 20 is performed in the culture medium 10. The sample is not particularly limited, but may be, for example, a sample collected from the environment, specifically, seawater, marine soil, sludge, groundwater, river water, or soil. The amount of the sample added to the culture medium 10 is also not particularly limited, but the substantial amount may be, for example, 0.5 to 10% (v / v) of the culture medium 10.

[0040] In the sulfate-reducing bacteria detection kit 100 shown in FIG. 1, the detection member 20 is immersed in the culture medium 10, but as described above, the form of the detection kit 100 of this embodiment is not limited to this. Therefore, at the stage of step S1, the detection member 20 may not be in contact with the culture medium 10. In this case, in this step S2 (corrosion test), the detection member 20 is immersed in (contacted with) the culture medium 10. The order of adding the sample and the detection member 20 to the culture medium 10 is not particularly important. The detection member 20 and the sample may be added to the culture medium 10 in this order, or conversely, the sample and the detection member 20 may be added in this order, or the sample and the detection member 20 may be added to the culture medium 10 simultaneously.

[0041] In the corrosion test, for example, the detection element 20 is immersed in the culture medium 10 to which the sample has been added and is maintained for a predetermined time (corrosion test time). If sulfate-reducing bacteria are present in the sample, electrochemical microbial corrosion (EMIC), which will be described below, occurs during the corrosion test. First, due to the metabolism of the sulfate-reducing bacteria, electrons are extracted (oxidized) from the iron in the detection element 20, and iron ions (Fe 2+ ) is produced, while sulfate ions (SO 4 2- ) is reduced to sulfide ions (S 2- ) are produced. These then combine to produce iron sulfide (FeS), and a conductive corrosion product layer mainly composed of this iron sulfide is formed on the surface of the detection member 20. Furthermore, the sulfate-reducing bacteria continue to extract electrons from the iron in the detection member 20 through this corrosion product layer (continues to corrode). The culture medium 10 of this embodiment contains the first component and the second component as electron acceptors, and further has a composition in which the concentration of the specific organic matter is 2 mmol / L or less, thereby promoting this electrical microbial corrosion (EMIC).

[0042] The composition of the culture medium 10 of this embodiment promotes EMIC, so that the corrosion test time can be shortened. The corrosion test time is not particularly limited, but may be appropriately adjusted within a range of, for example, 3 days or more, or 3 days to 3 weeks. The temperature of the culture medium 10 during the corrosion test is not particularly limited, and may be set according to the environment in which the sample is obtained, the type of sulfate-reducing bacteria to be detected, etc., and may be appropriately selected, for example, at 1° C. or more, or within a range of 4° C. to 100° C. In addition, it is preferable to place the sulfate-reducing bacteria detection kit 100 under anaerobic conditions during the corrosion test.

[0043] Process S3: After the corrosion test, the degree of corrosion of the detection member 20 is evaluated. The method of evaluating the degree of corrosion is not particularly limited. For example, the presence or absence of a corrosion product layer on the surface of the detection member 20 may be confirmed after the corrosion test. Since corrosion products formed from iron sulfide and the like are formed by electrochemical microbial corrosion (EMIC), if a corrosion product layer is formed, it can be determined that sulfate-reducing bacteria are present in the sample. In addition, the thickness of the corrosion product layer may be measured as a method of evaluating the degree of corrosion. The thicker the corrosion product, the more advanced the corrosion is, and it can be determined that the sulfate-reducing bacteria in the sample have a high ability to cause EMIC.

[0044] In addition, the surface shape of the detection member 20 after the corrosion test may be evaluated (for example, the surface roughness is measured) to evaluate the degree of corrosion. The surface shape may be evaluated by visual inspection, optical evaluation methods, etc., and for example, the presence or absence of corrosion pits is judged. If corrosion pits are formed, it can be determined that sulfate-reducing bacteria are present in the sample. Furthermore, the larger the size of the corrosion pits (width, diameter, area, etc. on the surface of the detection member 20) and / or the deeper the corrosion depth, the more advanced the corrosion is, and it can be determined that the sulfate-reducing bacteria in the sample have a higher ability to cause EMIC. The surface shape may be evaluated after removing the corrosion product layer.

[0045] In the conventional method for detecting sulfate-reducing bacteria using a culture medium, even if sulfate-reducing bacteria are present in the sample, it takes, for example, several months of corrosion test time until a corrosion product layer having a certain thickness is formed. In addition, in the conventional method for detecting sulfate-reducing bacteria using a culture medium, it is almost impossible for deep and / or large corrosion holes that can be observed visually to be formed in the detection member 20 in about one week. In contrast, if the kit 100 of this embodiment having a culture medium that promotes EMIC is used, as shown in the examples described later, for example, a thick corrosion product layer that can be observed visually is formed in a corrosion test time of about one week, and large and deep corrosion holes that can be observed visually are formed. In this way, by using the sulfate-reducing bacteria detection kit 100 of this embodiment, it is possible to significantly shorten the sulfate-reducing bacteria detection time.

[0046] Furthermore, the degree of corrosion may be evaluated by evaluating the weight loss (weight change) WL of the detection member 20 before and after the corrosion test. For example, first, the weight of the detection member 20 before the corrosion test (WB) is subtracted from the weight of the detection member 20 after the corrosion test (WA) to obtain the difference WL1 (=WB-WA). Next, a similar corrosion test is performed using a sample that does not contain sulfate-reducing bacteria as a reference to obtain the weight loss WL2 of the detection member 20 before and after the corrosion test. Then, the difference Δ (=WL1-WL2) is obtained by subtracting the weight loss WL2 of the reference from the weight loss WL1. The difference Δ means the weight loss of the detection member 20 due to electrochemical microbial corrosion (EMIC). Therefore, it can be determined that the greater the difference Δ, the more advanced the corrosion is, and the higher the ability of the sulfate-reducing bacteria in the sample to cause EMIC.

[0047] The degree of corrosion may also be evaluated by evaluating the change in color of the detection member (powder) 20 before and after the corrosion test, using a powder containing iron as the detection member 20. When the sample contains sulfate-reducing bacteria, the iron in the detection member (powder) 20 becomes iron sulfide, causing the detection member to turn black.

[0048] Although an example of the method for evaluating the degree of corrosion of the detection member 20 has been described above, in this embodiment, only one type of evaluation method may be adopted as the method for evaluating the degree of corrosion, or multiple types of evaluation methods may be adopted. An appropriate evaluation method may be selected as appropriate based on the composition of the culture medium (particularly the type of the second component), the type of sulfate-reducing bacteria to be detected, etc. EXAMPLES

[0049] The present invention will be described in more detail below based on examples. The materials, amounts, ratios, processing contents, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the following examples.

[0050] [Experiments 1-1~1-7] Seven types of sulfate-reducing bacteria detection kits for use in Experiments 1-1 to 1-7 were prepared in six kits each by the method described below. Therefore, a total of 42 kits (7 types x 6 kits) were prepared.

[0051] (1) Preparation of medium First, each compound was uniformly mixed and dissolved in water so as to have the composition shown in Table 2, to prepare solution (I). Next, we prepared aqueous solutions of four compounds that function as electron acceptors for sulfate-reducing bacteria. The aqueous solutions prepared were as follows: 2 SO 4 , concentration: 300mmol / L), sodium fumarate aqueous solution (Na-fumarate, concentration: 100mmol / L), dimethyl sulfoxide (DMSO, concentration: 100mmol / L), sodium nitrate (NaNO 3 , concentration: 100mmol / L). Solution (I), water, and at least one of the four aqueous solutions were mixed uniformly so as to obtain the compositions shown in Table 3, thereby obtaining seven types of media used in Experiments 1-1 to 1-7.

[0052] (2) Carbon steel plates (10 mm x 10 mm x 2 mm) were prepared as detection members for use in Experiments 1-1 to 1-7. Six kits were made for each type of medium by combining each of the seven types of medium (15 mL) prepared above with one sheet of carbon steel. As a result, a total of 42 kits (7 types x 6) were obtained.

[0053] [Table 2]

[0054] [Table 3]

[0055] [Corrosion test] (1) Preculture of sulfate-reducing bacteria Desulfovibrio vulgaris Hildenborough (DSM number: 644) was cultured in a glass vial with a butyl rubber stopper containing 80 mL of DSMZ medium 63 with a headspace of N 2 The culture was pre-cultured at 30°C for 5 days under anoxic conditions at 40°C. After 5 days, the culture was measured at 30°C for 1 h at a cell optical density (OD 600nm =3.

[0056] (2) Corrosion test After centrifuging the pre-cultured sulfate-reducing bacteria (D. vulgaris), the solution was resuspended in solution (I) and used as a sample containing sulfate-reducing bacteria. Of the six sulfate-reducing bacteria detection kits in Experiment 1-1, the detection member (plate-shaped carbon steel) and a sample containing sulfate-reducing bacteria (1 mL) were added to the medium of three kits, and the cell optical density (OD) in the medium was measured. 600nm The culture was adjusted to 0.5% by mass spectrometry (MSM) = 2 and cultured at 30°C for 7 days under anaerobic conditions. For comparison, a sample (Solution (I), 1 mL) not containing the detection member or sulfate-reducing bacteria was added to the culture medium of the remaining three kits in Experiment 1-1, and cultured in the same manner. For each of the sulfate-reducing bacteria detection kits in Experiments 1-2 to 1-7, as in Experiment 1-1, the detection components and a sample containing sulfate-reducing bacteria were added to the culture medium of three of the six kits and cultured, and the detection components and a sample not containing sulfate-reducing bacteria were added to the culture medium of the remaining three kits and cultured. After the corrosion test, the detection element was removed from the culture medium and subjected to the following evaluation.

[0057] [evaluation] (1) Observation of corrosion products and measurement of the thickness of the corrosion product layer Figure 3 shows a photograph of the detection members of Experiment 1-1. The detection members of Experiment 1-1 that contained the first component (sulfate) but not the second component turned black on the surface only when the sample contained sulfate-reducing bacteria (the three detection members at the bottom of Figure 3). This was due to corrosion products (iron sulfide) produced by the sulfate-reducing bacteria. However, the amount of corrosion products produced was small, and did not amount to forming a corrosion product layer (raised portion) on the detection members. Moreover, in Experiments 1-2 to 1-4, which contained the second component but not the first component (sulfate), the results were similar to those of Experiment 1-1 shown in Fig. 3. That is, in Experiments 1-2 to 1-4, when the sample contained sulfate-reducing bacteria, the surface of the detection member blackened, but a corrosion product layer (protrusion) did not form on the detection member.

[0058] Figure 4A shows a photograph of the detection elements of Experiments 1-7. The detection elements of Experiments 1-7 containing the first component (sulfate) and the second component (nitrate) were blackened on the surface only when the sample contained sulfate-reducing bacteria (the three detection elements at the bottom of Figure 4A), and a thick layer of corrosion products (ridges) was formed on the detection elements (see Figure 4B). In addition, in Experiments 1-5 and 1-6, which contained the first and second components, the results were similar to those of Experiment 1-7, and when the sample contained sulfate-reducing bacteria, a thick black corrosion product layer (protuberance) was formed on the detection element. In particular, in Experiment 1-6 (DMSO was used as the second component), the thickness of the corrosion product layer was about 1 mm, which was the thickest among Experiments 1-5 to 1-7 (see Figure 4C).

[0059] From the above observations of corrosion products, it was confirmed that EMIC is promoted when the medium contains both the first and second components (Experiments 1-5 to 1-7). On the other hand, the EMIC rate when the medium contains only the first component as an electron acceptor (Experiment 1-1) was almost the same as the EMIC rate when the medium contains only the second component as an electron acceptor (Experiments 1-2 to 1-4). Therefore, it is speculated that when the medium contains both the first and second components (Experiments 1-5 to 1-7), EMIC is promoted due to some kind of synergistic effect between the first and second components.

[0060] (2) Surface shape evaluation of the detection component The detection element was washed with 6N hydrochloric acid containing 3.5 g / L hexamethylenetetramine to wash away the corrosion products, and the surface of the detection element under the corrosion products was visually observed. As shown in Figures 5A and 5B, in Experiments 1-5 to 1-7 using media containing the first and second components, corrosion (corrosion pits) of the detection element could be visually confirmed when the sample contained sulfate-reducing bacteria. On the other hand, corrosion (corrosion pits) could not be visually confirmed for the other detection elements.

[0061] The surface roughness (measured cross-sectional curve) of the detection members in Experiments 1-5 to 1-7 (first and second components) near the corrosion pits was measured using a one-shot 3D shape measuring instrument (Keyence Corporation, controller VR-3000). The results are shown in Figure 6. In the measured cross-sectional curve in Figure 6, the part corresponding to the corrosion pit is surrounded by a dotted line. For comparison, the surface roughness of the detection member in Experiment 1-1 (first component only) was measured when the sample contained sulfate-reducing bacteria. The results are also shown in Figure 6.

[0062] As shown in Figure 6, in experiment 1-5 (using fumarate as the second component), deep corrosion pits measuring approximately 1.5 mm in width and 14 μm in maximum depth were observed. In experiment 1-6 (using DMSO as the second component), large, deep corrosion pits measuring approximately 4 to 5 mm in width and 19 μm in maximum depth were observed. In experiment 1-7 (using nitrate as the second component), corrosion pits measuring approximately 2 to 3 mm in width and 5 μm in maximum depth were observed. On the other hand, in Experiment 1-1 (only the first component), although overall corrosion was observed, no deep corrosion (corrosion pits) was observed as assessed by visual inspection.

[0063] From the above evaluation of the surface shape of the detection material, it was confirmed that EMIC is promoted when the culture medium contains both the first and second components (Experiments 1-5 to 1-7).

[0064] (3) Change in weight of the detected parts before and after the corrosion test In Experiments 1-1 to 1-7, first, the weight difference (WB-WA) was calculated by subtracting the weight (WA) of the detection element after the corrosion test from the weight (WB) of the detection element before the corrosion test. The weight (WA) is the weight of the detection element after the corrosion products were washed away with hydrochloric acid. Figure 7 shows the difference (WB-WA) in each experiment as the weight loss. In each experiment, Figure 7 shows three weight loss values ​​and their average value WL2 when the sample did not contain sulfate-reducing bacteria (sterile), and three weight loss values ​​and their average value WL1 when the sample contained sulfate-reducing bacteria on the left side, and three weight loss values ​​and their average value WL1 when the sample contained sulfate-reducing bacteria on the right side.

[0065] This evaluation focuses on corrosion caused by sulfate-reducing bacteria (EMIC). For this purpose, the weight loss amount WL2 when the sample does not contain sulfate-reducing bacteria (sterile) was subtracted from the weight loss amount WL1 when the sample contains sulfate-reducing bacteria in each experiment, and the difference Δ (=WL1-WL2) was calculated and compared. The larger the difference Δ, the greater the degree of EMIC.

[0066] As shown in Figure 7, in Experiments 1-6 and 1-7, in which the medium contained both the first and second components, the difference Δ was larger and EMIC was more promoted than in Experiments 1-1 to 1-4, in which the medium contained only the first component or only the second component as the electron acceptor. Among them, the difference Δ (weight loss due to EMIC) was the largest in Experiment 1-6 (DMSO was used as the second component).

[0067] In addition, in experiment 1-5 (using fumarate as the second component), the difference Δ (weight loss due to EMIC) could not be confirmed. However, this does not negate the EMIC-promoting effect of the culture medium in experiment 1-5. For example, the EMIC-promoting effect of the culture medium in experiment 1-5 was confirmed in the above-mentioned (1) observation of corrosion products and (2) evaluation of the surface shape of the detection member. In the corrosion experiment in experiment 1-5, deep corrosion holes were formed in the detection member, but their width (diameter) was small (see Figure 6). For this reason, it is speculated that there were cases in which the effect of corrosion on the weight loss was small.

[0068] [Experiments 2-1~2-7] By the method described below, 6 kits each of the 7 types of sulfate-reducing bacteria detection kits used in Experiments 2-1 to 2-7 were prepared. Therefore, a total of 42 kits (7 types x 6 kits) were prepared.

[0069] (1) Preparation of medium First, each compound was uniformly mixed and dissolved in water to prepare solution (II) so as to obtain the composition shown in Table 4. Solution (II) is artificial seawater. In Table 4, the Se-W solution was prepared by dissolving each compound in water. The trace metal solution (SL-10 solution) was prepared by dissolving FeCl 2 was dissolved in hydrochloric acid, diluted with water, other salts were added and dissolved therein, and finally diluted with water to prepare a total solution of 1000.00 mL. Next, we prepared aqueous solutions of four compounds that function as electron acceptors for sulfate-reducing bacteria. The aqueous solutions prepared were as follows: 2 SO 4 , concentration: 420mmol / L), sodium fumarate aqueous solution (Na-fumarate, concentration: 100mmol / L), dimethyl sulfoxide (DMSO, concentration: 100mmol / L), sodium nitrate (NaNO 3 , concentration: 100mmol / L). Solution (II), water, and at least one of the above four aqueous solutions were mixed uniformly so as to obtain the compositions shown in Table 5, thereby obtaining seven types of media used in Experiments 2-1 to 2-7.

[0070] (2) As the detection members used in Experiments 2-1 to 2-7, carbon steel plates similar to those used in Experiments 1-1 to 1-7 were prepared. Six kits were made for each type of medium by combining each of the seven types of medium (15 mL) prepared with one sheet of carbon steel. As a result, a total of 42 kits (seven types x six) were obtained.

[0071] [Table 4]

[0072] [Table 5]

[0073] [Corrosion test] (1) Preculture of sulfate-reducing bacteria Desulfovibrio ferrophilus IS5 (DSM number: 15579) was cultured in a glass vial with a butyl rubber stopper containing 80 mL of DSMZ medium 195c, with the headspace filled with CO. 2 / N 2 (volume ratio 20 / 80) and pre-cultured at 28°C for 5 days under anoxic conditions. After 5 days, the culture was measured for cell optical density (OD 600nm =0.3.

[0074] (2) Corrosion test After centrifuging the pre-cultured sulfate-reducing bacteria (D. ferrophilus IS5), the liquid resuspended in solution (II) was used as a sample containing sulfate-reducing bacteria. Of the six sulfate-reducing bacteria detection kits in Experiment 2-1, the detection member (plate-shaped carbon steel) and a sample containing sulfate-reducing bacteria (1 mL) were added to the medium of three kits, and the cell optical density (OD) in the medium was measured. 600nm = 0.15, and the mixture was cultured at 28°C for 7 days under anaerobic conditions. For comparison, a detection member (carbon steel plate) and a sample not containing sulfate-reducing bacteria (solution (II), 1 mL of artificial seawater) were added to the culture medium of the remaining three kits in Experiment 2-1 and cultured in the same manner. For each of the sulfate-reducing bacteria detection kits in Experiments 2-2 to 2-7, as in Experiment 2-1, the detection components and a sample containing sulfate-reducing bacteria were added to the culture medium of three of the six kits and cultured, and the detection components and a sample not containing sulfate-reducing bacteria were added to the culture medium of the remaining three kits and cultured. After the corrosion test, the detection element was removed from the culture medium and subjected to the following evaluation.

[0075] [Evaluation: Change in weight of detected parts before and after corrosion test] In Experiments 2-1 to 2-7, the weight change of the detection member before and after the corrosion test was evaluated in the same manner as in Experiments 1-1 to 1-7 described above. First, the weight difference (WB-WA) was calculated by subtracting the weight of the detection part after the corrosion test (WA) from the weight of the detection part before the corrosion test (WB). Figure 8 shows the weight loss (WB-WA) of each experiment. In Figure 8, the left side of each experiment shows three weight losses when the sample did not contain sulfate-reducing bacteria (sterile), and the average value WL2, and the right side shows three weight losses when the sample contained sulfate-reducing bacteria, and the average value WL1. In this evaluation, we also focus on the difference Δ (=WL1-WL2) obtained by subtracting the weight loss WL2 when the sample did not contain sulfate-reducing bacteria (sterile) from the weight loss WL1 when the sample contained sulfate-reducing bacteria in each experiment. The larger the difference Δ, the greater the degree of EMIC.

[0076] As shown in Figure 8, in Experiments 2-5 to 2-7, in which the medium contained both the first and second components, the difference Δ was larger and EMIC was more promoted than in Experiments 2-1 to 2-4, in which the medium contained only the first component or only the second component as the electron acceptor. Among them, the difference Δ (weight loss due to EMIC) was the largest in Experiment 2-5 (using fumarate as the second component).

[0077] Thus, when D. ferrophilus IS5 was used as the sulfate-reducing bacterium, experiment 2-5 (using fumarate as the second component) had the largest difference Δ (weight loss due to EMIC) (see Figure 8). On the other hand, when D. vulgaris was used as the sulfate-reducing bacterium, experiment 1-6 (using DMSO as the second component) had the largest difference Δ (weight loss due to EMIC). Thus, the type of second component that promotes corrosion varies depending on the type of sulfate-reducing bacterium to be detected. Therefore, by selecting a second component suitable for the sulfate-reducing bacterium to be detected and preparing the medium, it is possible to more efficiently detect sulfate-reducing bacteria that cause EMIC. [Industrial Applicability]

[0078] The sulfate-reducing bacteria detection kit of the present invention can preferentially detect sulfate-reducing bacteria that cause electrochemical microbial corrosion (EMIC) and can also shorten the detection time. This makes it possible to detect anaerobic iron corrosion present inside oil pipelines, for example, at an early stage and prevent accidents caused by microbial corrosion in advance. [Explanation of symbols]

[0079] 10 Culture medium 20 Detection member 30 containers 100 Sulfate-reducing bacteria detection kit

Claims

1. A sulfate-reducing bacteria detection kit, comprising: a culture medium and a detection member containing iron, wherein the culture medium contains water, a first component, sulfate, and a second component that functions as an electron acceptor for sulfate-reducing bacteria and has a redox potential more positive than that of the first component, and the concentration of an organic substance having a redox potential more negative than that of the first component in the culture medium is 2 mmol / L or less. A sulfate-reducing bacteria detection kit.

2. The sulfate-reducing bacteria detection kit according to claim 1, wherein the concentration of an organic substance having a redox potential more negative than that of the sulfate in the culture medium is 1 mmol / L or less.

3. The sulfate-reducing bacteria detection kit according to claim 1, wherein the culture medium does not contain an organic substance having a redox potential more negative than that of the sulfate.

4. The sulfate-reducing bacteria detection kit according to claim 1, wherein the second component is at least one selected from the group consisting of fumarate, dimethyl sulfoxide, and nitrate.

5. The sulfate-reducing bacteria detection kit according to claim 1, wherein the standard redox potential (pH 7) of the second component is +20 mV to +500 mV.

6. The sulfate-reducing bacteria detection kit according to claim 1, wherein in the culture medium, the ratio (M1 / M2) of the molar concentration (M1) of the first component to the molar concentration (M2) of the second component is 2 to 4.

7. The sulfate-reducing bacteria detection kit according to claim 1, wherein the detection member is a plate-like body.

8. The sulfate-reducing bacteria detection kit according to claim 1, wherein the detection member is carbon steel.

9. A method for detecting sulfate-reducing bacteria, comprising: preparing the sulfate-reducing bacteria detection kit according to any one of claims 1 to 8; adding a sample to the culture medium and performing a corrosion test of the detection member in the culture medium; and evaluating the degree of corrosion of the detection member after the corrosion test. A method for detecting sulfate-reducing bacteria.

10. The evaluation of the degree of corrosion of the detection member includes confirming the presence or absence of a corrosion product layer on the surface of the detection member, evaluating the surface shape of the detection member, and evaluating the weight change amount of the detection member before and after the corrosion test, and is at least one selected from the group consisting of these. The method for detecting sulfate-reducing bacteria according to claim 9.

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