Method for evaluating microbial corrosion risk and evaluation kit for evaluating microbial corrosion risk

Abstract

To provide an evaluation method and an evaluation kit that enable microbial corrosion risk to be evaluated more easily and more accurately.SOLUTION: The present invention provides a method for evaluating microbial corrosion risk in an environment, the method comprising: preparing an evaluation sample that includes a material collected from the environment; and detecting outer membrane vesicles having redox characteristics in the evaluation sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for evaluating a risk of microbial corrosion and an evaluation kit for evaluating the risk of microbial corrosion. [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, a method for detecting sulfate-reducing bacteria is to add a sample (collected matter) taken 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] In addition, 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 corrosion by directly ingesting electrons from the iron on the corrosion product (EEU: Extracellular Electron Uptake). Corrosion caused by such bacteria and dependent on the abstraction 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 group of enzymes is involved in directly abstracting electrons from iron (Patent Document 1, Non-Patent Document 2).

[0004] This type of electrochemical microbial corrosion (EMIC) is known to have a corrosion rate several tens of times faster than non-EMIC corrosion, and once it occurs, it progresses at a rapid rate of several tens of millimeters per year. For this reason, many sudden and serious accidents caused by EMIC have been reported in environments that cannot be visually checked on a daily basis, such as inside oil pipelines, facilities that handle contaminated water or treated water, and storage tanks for treated water. Huge 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.

[0005] On the other hand, various microorganisms are known to secrete outer membrane vesicles (OMVs). OMVs were discovered in pathogenic bacteria about 40 years ago, and they have been found to be deeply involved in bacterial pathogenicity and interactions with the host, attracting much attention in the medical field. However, discussions on the role of OMVs in non-pathogenic bacteria are very limited, and in particular, there have been no studies on OMVs secreted by bacteria that cause EMIC (electromicrobial corrosion). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 7066224 [Non-patent literature]

[0007] [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. “Multi-heme cytochromes provide a pathway for survival in energy-limited environments” Science Advances. 4 [2] (2018). Summary of the Invention [Problem to be solved by the invention]

[0008] Conventional methods for evaluating corrosion risk based on bacterial detection have had the following problems, for example. Because the type of culture medium limits the types of bacteria that grow, there is a risk that only specific bacteria can be detected, and it takes a long time, at least one week, for the bacteria to grow. In addition, EMIC progresses at an accelerated rate in a biofilm formed on a metal, but once a biofilm is formed, bacteria are not necessarily released outside the biofilm. Therefore, bacterial detection cannot grasp the presence of bacteria in an amount sufficient to form a biofilm, and therefore cannot accurately evaluate corrosion risk.

[0009] The present invention is intended to solve these problems. That is, the present invention provides an evaluation method capable of evaluating the risk of microbial corrosion more simply and accurately, and an evaluation kit utilizing said evaluation method. [Means for solving the problem]

[0010] 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.

[0011] [1] A method for assessing microbial corrosion risk in an environment, comprising: preparing an evaluation sample comprising a sample taken from the environment; An evaluation method comprising detecting outer membrane vesicles having redox properties in the evaluation sample. [2] The evaluation method described in [1], wherein detecting the outer membrane vesicles is determining whether or not the outer membrane vesicles are present and / or quantifying the outer membrane vesicles. [3] The evaluation method described in [1] or [2], wherein the detection of the outer membrane vesicles is carried out by a staining method. [4] The evaluation method according to [3], wherein a redox color-developing reagent is used in the staining method. [5] The evaluation method described in [1] or [2], wherein the detection of the outer membrane vesicles is carried out by measuring an oxidation-reduction potential. [6] The evaluation method described in [1] or [2], wherein the detection of the outer membrane vesicles is carried out by absorbance measurement. [7] The evaluation method according to any one of [1] to [6], wherein the redox potential of the outer membrane vesicle is more positive than the redox potential of the iron material. [8] The evaluation method according to any one of [1] to [7], wherein the redox potential (25°C, pH 7) of the outer membrane vesicle is more positive than -0.6. [9] The evaluation method according to any one of [1] to [8], wherein the standard redox potential (25°C, pH 7) of the outer membrane vesicle is more positive than the redox potential of the iron material and more negative than +0.77 V (SHE).

[10] An evaluation kit for evaluating microbial corrosion risk in an environment, comprising: An evaluation sample introduction section configured to contact an evaluation sample including a sample collected from the environment; A detection unit configured to detect outer membrane vesicles having redox properties in the evaluation sample; an evaluation kit comprising: a connection section configured to connect the evaluation sample introduction section and the detection section and to enable the evaluation sample to move from the evaluation sample introduction section to the detection section.

[11] The evaluation kit described in

[10] , wherein the detection unit contains a redox color-developing reagent and is configured to detect the outer membrane vesicles by a staining method. Effect of the Invention

[0012] The present invention provides an evaluation method capable of evaluating the risk of microbial corrosion more simply and accurately, and an evaluation kit utilizing the evaluation method. [Brief description of the drawings]

[0013] [Figure 1] 3 is a flowchart illustrating a method for evaluating a microbial corrosion risk according to the first embodiment. [Diagram 2] FIG. 13 is a schematic diagram of an evaluation kit according to a second embodiment. [Diagram 3] Figure 3(a) is a transmission electron microscope (TEM) image of OMV isolated from a bacterial (IS5) culture medium in the examples, and Figure 3(b) is a TEM image at a higher magnification. [Figure 4] FIG. 2 shows the particle size distribution of OMVs isolated from a bacterial (IS5) culture medium in an example. [Diagram 5] In the examples, TEM images of OMVs stained with cytochrome-reactive DAB (3,3'-diaminobenzidine)-H2O2 are shown. Figure 5(a) shows the result of negative DAB staining in the absence of H2O2, and Figure 5(b) shows the result of positive DAB staining in the presence of H2O2. [Figure 6] FIG. 1 shows the results (OMV protein profile) of CBB (Coomassie Brilliant Blue) staining and heme-reactive TMBZ (3,3',5,5'-tetramethylbenzidine)-H2O2 staining (heme staining) of a gel after electrophoresis of OMV in an example. [Figure 7] Fig. 7(a) is a schematic diagram of a poly-L-lysine (PLL)-coated ITO electrode used in the examples, and Fig. 7(b) is a diagram showing the results of measuring the redox potential of OMV in the examples. [Figure 8] FIG. 1 shows the results of electrochemical measurements using OMV in an example. [Figure 9] 1 shows confocal laser microscope images of a biofilm formed on an electrode after electrochemical measurement in an example. [Figure 10] In the example, this is an SEM image of a biofilm formed on an electrode after electrochemical measurement. [Figure 11]FIG. 1 shows a schematic model of a biofilm formed on an ITO electrode. [Figure 12] 1 is a graph showing the weight loss of a test piece after a corrosion test in an example. [Figure 13] FIG. 13(a) is an SEM image of the test piece surface after the corrosion test in the embodiment (with biofilm and corrosion products), and FIG. 13(b) is an SEM image of the test piece surface after the biofilm and corrosion products have been peeled off. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 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.

[0015] [First embodiment] In the method for evaluating the risk of microbial corrosion of this embodiment, outer membrane vesicles secreted by bacteria causing EMIC are used as a novel corrosion biomarker, and the risk of microbial corrosion is evaluated by detecting the outer membrane vesicles. Outer membrane vesicles (OMVs) are microvesicles produced by constricting a part of the cell membrane of a bacterial cell to the outside of the bacterial body.

[0016] The present inventors have been studying bacteria that cause EMIC (electrical microbial corrosion), including sulfate-reducing bacteria, and have discovered that these bacteria secrete outer membrane vesicles (OMVs) with diameters of 40 to 120 nm. Furthermore, they have clarified that the surface of the secreted OMVs contains redox-active proteins that can directly mediate electron uptake, that is, OMVs have redox properties. They have also found that these outer membrane vesicles with redox properties (hereinafter, appropriately referred to as "specific OMVs") promote the formation of conductive biofilms and the extracellular electron uptake (EEU) of bacteria, thereby accelerating EMIC (see the Examples below and Figures 9 to 13). Although it is said that microorganisms universally secrete OMVs, there have been no reports on the secretion of OMVs in iron-corroding bacteria that cause EMIC, or the role of OMVs in the iron corrosion process. The present invention was made based on these findings in the research of the present inventors. That is, in the present invention, outer membrane vesicles (specific OMVs) with redox properties secreted by bacteria that cause EMIC are used as novel corrosion biomarkers to detect corrosion risk.

[0017] Examples of bacteria that secrete specific OMVs, i.e., bacteria that cause EMIC, include D. ferrophilus IS5, Desulfovibrio vulgaris Hildenborough, Desulfobacterium corrodens IS4, etc. Detection of specific OMVs from the environment means that these bacteria are present in the environment and that the presence of the specific OMVs makes it easier for microbial corrosion to be accelerated.

[0018] The evaluation method of this embodiment will be described in detail below. The evaluation method of this embodiment is as shown in FIG. Step S1: Preparing an evaluation sample including a sample collected from the environment; Step S2: Detecting outer membrane vesicles (specific OMVs) having redox properties in the evaluation sample; Includes.

[0019] <Step S1: Preparation of evaluation sample> First, an evaluation sample including an object collected from the environment is prepared (step S1 in FIG. 1). The "environment" is not particularly limited, but examples include the inside of an oil pipeline, facilities (including piping) that handle contaminated water or treated water, and storage tanks for treated water. These are facilities that are partially or entirely made of metal (typically, metals including iron materials), and electrochemical microbial corrosion (EMIC) of metals is a major problem. The "objects collected from the environment" are fluids (liquids), solids (sludge), etc. that exist in these facilities. These "objects collected" may be in contact with metals in the environment, or may come into contact with metals. The "objects collected from the environment" may be, for example, seawater, marine soil, sludge, groundwater, river water, and soil.

[0020] The evaluation sample may be, for example, the material collected from the environment itself, but it is preferable to subject the collected material to some treatment and extract, separate, purify, etc., the OMV from the collected material to be used as the evaluation sample. This can improve the accuracy of detecting a specific OMV. There is no particular limitation on the method for extracting, separating, purifying, etc., the OMV, and conventionally known methods can be used. For example, the material collected from the environment may be dissolved or dispersed in a solvent (e.g., a physiological buffer solution), and then centrifuged, filtered, etc. may be performed. In addition, processing may be performed using a commercially available OMV isolation kit.

[0021] <Step S2: Detection of outer membrane vesicles with redox properties> Next, outer membrane vesicles (specific OMV) having redox properties are detected in the evaluation sample. Here, "detection of specific OMV" means at least the determination of the presence or absence of specific OMV, and may further include quantification of specific OMV. Then, based on the detection result of specific OMV, the microbial corrosion risk in the environment (the site where the sample was collected) can be evaluated. For example, if specific OMV is present in the evaluation sample, the microbial corrosion risk in the environment can be evaluated as being higher than when specific OMV is not present. When the specific OMV is quantified, the higher the detected amount of specific OMV, the higher the microbial corrosion risk in the environment can be evaluated. In addition, a standard value may be set for the amount of specific OMV, and if the detected amount exceeds the standard value, it may be determined that the microbial corrosion risk in the environment is high. A plurality of standard values ​​may be set to evaluate the microbial corrosion risk in multiple stages. Furthermore, samples (collected material) may be periodically collected from the environment to monitor the change over time in the amount of specific OMV present in the environment, and the microbial corrosion risk in the environment may be evaluated based on this.

[0022] In addition, there is a possibility that OMVs that are not involved in EMIC, i.e., OMVs that do not have redox properties, may be present in the evaluation sample (i.e., in the environment from which the sample was collected). In the evaluation method of this embodiment, only OMVs that have redox properties (specific OMVs) are detected as biomarkers.

[0023] The method for detecting the specific OMV is not particularly limited. For example, the specific OMV may be detected using a staining method. As described above, the present inventors have discovered that redox-active proteins (e.g., oxidoreductases such as cytochrome enzymes) are present on the surface of the specific OMV. The oxidoreductases (proteins) can be detected, for example, by a staining method using an oxidoreductase-based color-developing reagent (oxidoreductase staining method). Detection by the staining method makes it possible to determine the presence or absence of the specific OMV, and further to quantify it.

[0024] As a staining method, for example, the cytochrome-reactive DAB-H staining method described in the Examples below was used. 2 O 2 Figure 5(a) and (b) show the cytochrome-reactive DAB-H staining. 2 O2 Figure 5(a) shows a transmission electron microscope (TEM) image of OMV stained with H. 2 O 2 The results are shown in Fig. 5(b) as negative staining in the absence of H 2 O 2 As shown in Fig. 5(b), the OMV was positively stained with DAB in the presence of riboflavin, and was clearly visible. Since the OMV was positively stained in Fig. 5(b), it can be determined that the OMV has redox properties (i.e., it is a specific OMV).

[0025] The staining method used was heme-reactive TMBZ-H, as described in the Examples below. 2 O 2 Examples of staining methods that can be used for this purpose include staining with heme and CBB. FIG. 6 shows the protein profile of OMVs stained with heme and CBB after electrophoresis of the evaluation sample. Since bands stained with heme as well as CCB staining are visible, it can be determined that the OMV has redox properties (i.e., it is a specific OMV). In addition, in the staining method combined with electrophoresis, it is also possible to quantify the specific OMV based on the density of the bands using a calibration curve method or the like.

[0026] In the above-mentioned DAB staining, observation was performed using a TEM (Figures 5(a)(b)), and in the heme staining, observation was performed by staining the gel after electrophoresis (Figure 6), but this embodiment is not limited to this. For example, the evaluation sample may be directly contacted with each staining reagent to observe the presence or absence of staining. In this case, a TEM or electrophoresis device is not required, and the presence or absence of a specific OMV can be determined easily and quickly.

[0027] The staining method is not limited to the above-mentioned examples, and any conventional method can be used, such as a staining method using ortho-phenylenediamine (see Okajimas Folia Anatomica Japonica, Vol. 27, No. 5, 1955, pp. 335-343) or a commercially available peroxidase staining kit (e.g., manufactured by Nacalai Tesque, Inc.).

[0028] The detection of the specific OMV may also be performed using redox potential measurement. The redox potential of the OMV in the evaluation sample may be measured, and when the redox potential is within a specific range, the OMV may be determined to have redox properties (i.e., to be a specific OMV). For example, the redox potential of the specific OMV may be more positive than the redox potential of the iron material. In this case, electrons are more likely to move from the iron material to the specific OMV, promoting the action of iron-corroding bacteria (e.g., sulfate-reducing bacteria) to directly extract electrons from iron (EEU), and as a result, promoting EMIC. In this way, the specific OMV can be a pathway that mediates the uptake of electrons from iron by bacteria. Here, the iron material refers not only to pure iron (standard redox potential (25°C, pH 7) -0.44V (SHE)), but also to metal materials containing iron, such as carbon steel and alloy steel. The iron material may have a standard redox potential that is more negative than that of pure iron (e.g., about -0.6V (SHE)). The redox potential (25° C., pH 7) of a particular OMV may be, for example, more positive than −0.6 V (SHE), or more positive than −0.44 V (SHE).

[0029] The redox potential of the specific OMV may be more negative than the redox potential of the electron acceptor of the iron-corroding bacteria. This facilitates the flow of electrons from the specific OMV to the electron acceptor, and promotes electron donation from the iron-corroding bacteria to the electron acceptor. For example, examples of the electron donor of the iron-corroding bacteria include sulfate (-0.2 V (SHE)), iron ion (Fe 3+ / Fe 2+ The redox potential (25° C., pH 7) of a specific OMV may be, for example, more negative than +0.77 V (SHE) or more negative than −0.2 V (SHE).

[0030] The method of measuring the redox of OMV is not particularly limited. For example, as shown in FIG. 7(a), first, an electrode (e.g., a PLL-coated ITO electrode) with a surface treatment that allows OMV to be adsorbed is prepared. The electrode is brought into contact with an evaluation sample to adsorb OMV onto the electrode, thereby measuring the redox potential of OMV. FIG. 7(b) shows the results of the redox measurement of OMV performed in the examples described below. The measured OMV has a positive redox potential (−0.32 V (SHE)) compared to the redox potential of the iron material, and therefore can be determined to have redox properties (i.e., to be a specific OMV). In addition, the specific OMV can also be quantified based on the intensity of the redox peak (−0.32 V (SHE)) by a calibration curve method or the like.

[0031] Furthermore, the detection of the specific OMV may be performed using absorbance measurement. As described above, a redox-active protein is present on the surface of the specific OMV. When the absorption characteristics of this protein are known, the specific OMV can be detected by absorbance measurement. For example, the present inventors have discovered that the OMV secreted from D. ferrophilus IS5, a sulfate-reducing bacterium, has a specific cytochrome enzyme that is the same as the surface protein of the bacterium (IS5). It is known that this specific cytochrome enzyme has absorption peaks at 419 nm and 512 nm (Patent Document 1, Non-Patent Document 2). By focusing on this absorption peak, when the OMV in the evaluation sample has a similar absorption peak, it can be determined that the OMV is an OMV secreted from IS5 (i.e., a specific OMV). In addition, it is also possible to quantify the specific OMV based on the absorption peak intensity using a calibration curve method or the like.

[0032] The above-mentioned methods described as the detection method of the specific OMV, i.e., the staining method, the redox potential measurement, and the absorbance measurement, may be used to detect the specific OMV by any one method, or several methods may be combined to improve the detection accuracy. Compared with the absorbance measurement, the staining method and the redox potential measurement have a higher sensitivity for detecting the specific OMV, and are also suitable for evaluating a state with a lower corrosion risk (a state with less specific OMV). The detection method of the specific OMV is not limited to the staining method, the redox potential measurement, and the absorbance measurement, and may be, for example, a redox specific staining (e.g., DAB-H 2 O 2 Detection may also be achieved by measuring the change in OMV diameter before and after staining, directly measuring the OMV surface conductivity using an AFM or the like, or by impedance measurement, etc.

[0033] The method for evaluating microbial corrosion risk of the present embodiment described above has the following advantages. The method for evaluating microbial corrosion risk of the present embodiment uses specific OMVs as a novel corrosion biomarker. Therefore, bacterial culture is not required, and compared with the conventional method (corrosion risk evaluation method based on bacterial detection), corrosion risk can be evaluated more simply and in a short time. In addition, since OMVs are nanoparticles, they are more likely to be released outside the biofilm than the bacterial cells that form the biofilm. Therefore, the method of the present embodiment for detecting specific OMVs can evaluate microbial corrosion risk more accurately than the conventional method for detecting bacteria. Furthermore, compared with iron-corroding bacteria that require a strict anaerobic environment or an appropriate temperature, specific OMVs are much more robust and are easier to detect from the environment. From this point of view, the method of the present embodiment (detection of specific OMVs) can evaluate microbial corrosion risk more simply and accurately than the conventional method (bacterial detection).

[0034] [Second embodiment] In this embodiment (second embodiment), an evaluation kit for evaluating the risk of microbial corrosion in an environment using the evaluation method of the risk of microbial corrosion in the first embodiment will be described. As shown in FIG. 2, the evaluation kit 100 of this embodiment includes an evaluation sample introduction section 10 configured to contact an evaluation sample (Sample in FIG. 2) containing a sample collected from the environment, a detection section 20 configured to detect outer membrane vesicles (specific OMV) having redox properties in the evaluation sample, and a connection section 30 configured to connect the evaluation sample introduction section 10 and the detection section 20 and to allow the evaluation sample to move from the evaluation sample introduction section 10 to the detection section 20. The evaluation kit 100 further includes a substrate 40, and the evaluation sample introduction section 10, the detection section 20, and the connection section 30 may be provided on the substrate 40.

[0035] The materials constituting the evaluation sample introduction section 10, the detection section 20, and the connection section 30 are not particularly limited, and may be, for example, liquid absorbents such as fibers, paper, and sponges that can absorb the evaluation sample. The evaluation sample introduction section 10, the detection section 20, and the connection section 30 may be made of a continuous, integrated liquid absorbent material so that the evaluation sample absorbed in the evaluation sample introduction section 10 can move smoothly to the detection section 20. The evaluation sample introduction section 10 may be a recess formed in the substrate 40, and may not have a liquid absorbent material therein. In this case, the evaluation sample is held in the evaluation sample introduction section 10, which is a recess, and the evaluation sample is absorbed from there into the connection section 30, which is a liquid absorbent material.

[0036] The method of detecting the specific OMV in the detection unit 20 is not particularly limited, and may be, for example, a staining method. In this case, the detection unit 20 includes a staining reagent (for example, a redox coloring reagent) capable of detecting (staining) the specific OMV. The staining method and the staining reagent used therein may be the same as those described in the first embodiment, and the preferred embodiments are also the same.

[0037] Next, a method of using the evaluation kit 100 will be described. The evaluation kit 100 can evaluate the risk of microbial corrosion by a very simple method of simply contacting an evaluation sample containing a sample collected from the environment with the evaluation sample introduction section 10. The evaluation sample may be the sample collected from the environment itself, but in order to improve the accuracy of the specific OMV detection, the evaluation sample may be prepared in the same manner as in the first embodiment. The evaluation sample brought into contact with the evaluation sample introduction section 10 moves through the connection section 30 by capillary action to reach the detection section 20. If the specific OMV is present in the evaluation sample, it is stained with a staining reagent in the detection section 20 to form a stained section 21. The presence or absence of the specific OMV can be determined by visually determining the presence or absence of the stained section 21, and the specific OMV can also be quantified based on the density of the stained section 21 by a calibration curve method or the like. From the detection results of these specific OMVs, the risk of microbial corrosion in the environment can be evaluated in the same manner as in the first embodiment.

[0038] By using the evaluation kit 100 of this embodiment, the risk of microbial corrosion can be evaluated by a very simple method as described above. In addition, since the specific OMV is a nanoparticle, it can be easily moved from the evaluation sample introduction section 10 to the detection section 20, and an accurate evaluation of the risk of microbial corrosion is possible. EXAMPLES

[0039] 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.

[0040] [Cultivation 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. When culturing the bacteria in large quantities, the bacterial liquid cultured for 5 days was inoculated at 10% into DSMZ medium 195c and cultured for another 5 days.

[0041] [Preparation of evaluation samples] To purify OMV from the above-mentioned culture (culture liquid) (3000 mL), bacteria were removed, and the resulting mixture was centrifuged (7,000 G for 15 minutes). The supernatant was then filtered through a filter (pore size: 0.22 μm), and the filtrate was then subjected to ultrafiltration (100 kDa) to concentrate the OMV. + The OMVs were dispersed in HEPES buffer (10 mL) by adding 100 mL of HEPES buffer (25 mM, pH 7.2) and concentrating the mixture to 10 mL by ultrafiltration three times. This dispersion was used in the experiments.

[0042] [Characterization of outer membrane vesicles (OMVs)] (1) TEM observation Transmission electron microscopy (TEM) was performed on OMVs isolated from bacterial (IS5) culture medium. TEM grids were stained with an EM stainer (manufactured by Nissin EM Co., Ltd.). Figures 3(a) and (b) show TEM images at different magnifications (Figure 3(b) is at a higher magnification). As shown in Figure 3(b), outer membrane (OM) and inner membrane (IM) layers were observed in some OMVs.

[0043] (2) Particle size distribution measurement The particle size distribution of the purified OMV (OMV in the evaluation sample) was measured using a flow nanoanalyzer. The results are shown in Figure 4. The average particle size was 62.8 nm.

[0044] (3) Redox dyeing 1 The purified OMVs (OMVs in the evaluation sample) were treated with cytochrome-reactive DAB (3,3'-diaminobenzidine)-H 2 O 2 The specimen was stained and observed by TEM. The results are shown in Figure 5(a) and (b). Figure 5(a) shows the H 2 O 2 The results are shown in Fig. 5(b) as negative staining in the absence of H 2 O2 This shows the result of positive DAB staining in the presence of cytochrome P450. Only the positive staining in Figure 5(b) was observed, and the surface of the OMV was particularly strongly stained. This confirmed that cytochrome enzyme (oxidoreductase) is present on the surface of the OMV, and that this OMV has redox properties (i.e., it is a specific OMV).

[0045] (4) Redox dyeing 2 After electrophoresis of the evaluation samples, the gel was stained with CBB (Coomassie Brilliant Blue) and heme-reactive TMBZ (3,3',5,5'-tetramethylbenzidine)-H. 2 O 2 Staining (heme staining) was performed. The results (protein profile of the OMV) are shown in Figure 6. As shown in Figure 6, bands were visible by heme staining as well as CCB staining. This confirmed that the OMV has redox properties (i.e., it is a specific OMV).

[0046] (5) Oxidation-reduction potential measurement First, as shown in Figure 7(a), an indium tin oxide (ITO) electrode was prepared that was surface-treated (coated) with poly-L-lysine (PLL) to allow OMV adsorption. An OMV sample solution was added to this ITO electrode and left to stand for 15 minutes to allow OMV to be adsorbed onto the electrode, and electrochemical measurements were performed using differential pulse voltammetry (DPV) to determine the redox potential of OMV. The results are shown in Figure 7(b) and labeled "OMV". For comparison, a similar electrochemical measurement was performed on a solution with the same composition as the evaluation sample except that it did not contain OMV. The results are also shown in Figure 7(b) and labeled "Sterile".

[0047] As shown in Figure 7(b), the OMV had a redox potential of -0.32 V (SHE), which is more positive than the redox potential of iron (-0.44 V (SHE)) and more negative than the redox potential of sulfate (-0.2 V (SHE)). From this result, it is inferred that the OMV promotes EMIC of sulfate-reducing bacteria (IS5).

[0048] [Electrochemical measurements of OMVs] Using a three-electrode anaerobic reactor equipped with an ITO electrode maintained at a potential of -0.4 V (SHE), the cathodic current generated in the following three types of samples (solutions) 1 to 3 was measured. The results are shown in Figure 8. In this test, the ITO electrode was the only electron donor for the bacteria (IS5). Sample 1 (OMV only): OMV solution (evaluation sample described above) (2.6 × 10 9 Sample 1 was prepared by adding 0.5 mL of OMVs (particles / mL) to 4.5 mL of electrolyte in a reaction vessel. The OMV density in sample 1 was 2.6 × 10 8 particles / mL. Sample 2 (IS5 only): First, bacteria (IS5) were centrifuged from a liquid culture solution that had been cultured for 5 days, and the separated bacteria were redispersed in 0.5 mL of HEPES buffer (25 mM, pH 7.2). The redispersed solution was added to 4.5 mL of electrolyte in a reaction vessel (final OD 600nm =1) Sample 2 was prepared. Sample 3 (IS5+OMV): Bacteria (IS5) were centrifuged from a liquid culture solution that had been cultured for 5 days, and the separated bacteria were redispersed in 0.5 mL of OMV solution (the evaluation sample described above). The redispersed solution was added to 4.5 mL of electrolyte in a reaction vessel to prepare sample 3. The OMV density in sample 3 was 2.6 × 10 8 particles / mL. The electrolyte solutions used in Samples 1 to 3 were prepared in-house and had the same composition as the electrolyte solution disclosed in Non-Patent Document 2.

[0049] As shown in FIG. 8, the generation of electric current was confirmed in Sample 2 (IS5 only) and Sample 3 (IS5+OMV), which contained bacteria. In particular, the electric current value in Sample 3 (IS5+OMV) was significantly increased compared to Sample 2 (IS5 only). In Sample 3 (IS5+OMV), the OMV concentration was low (10 8Since an increase in the current value was observed despite the OMV concentration being low (cells / mL, pM order), it is speculated that the OMVs do not act as electron mediators to mediate electron transfer by diffusion, but rather promote the withdrawal of electrons (EEU) from the ITO electrode by the bacteria (IS5). Furthermore, only a small amount of current was generated in sample 1 (OMV only), confirming that the OMVs themselves do not withdraw electrons (EEU).

[0050] [Confocal scanning fluorescence microscopy and SEM observation of biofilms] In the electrochemical measurements described above (see FIG. 8), the ITO electrode (without OMV) used in sample 2 (IS5 only) and the ITO electrode (with OMV) used in sample 3 (IS5+OMV) were observed under a confocal scanning fluorescence microscope and an SEM. The confocal laser microscope images are shown in FIG. 9, and the SEM images in FIG. 10. In the confocal scanning fluorescence microscope observations, the cells were stained with a Live / Dead fluorescent dye.

[0051] As shown in Figs. 9 and 10, a thicker biofilm was formed in Sample 3 (With OMV) and many bacteria were observed compared to Sample 2 (Without OMV). In addition, as shown in Fig. 10, the formation of nanowires was observed between the bacteria and between the bacteria and the electrode surface in Sample 3 (With OMV). The inventor of the present application reported in Non-Patent Document 2 that this nanowire also has a specific cytochrome enzyme that is the same as the cell membrane of the bacteria (IS5) and promotes EEU of the bacteria (IS5). It is presumed that the presence of this nanowire caused the biofilm formed in Sample 3 (With OMV) to have high conductivity despite its thick film thickness. From the above results, it was confirmed that the presence of OMV caused the bacteria (IS5) to form a thicker and more conductive biofilm, and as a result, the extraction of electrons (EEU) from the ITO electrode by the bacteria (IS5) was accelerated (see Fig. 8). Fig. 11 shows a schematic model of the biofilm formed on the ITO electrode in Sample 2 (Without OMV) and Sample 3 (With OMV).

[0052] [Evaluation of the effect of OMV on microbial corrosion] A metal (carbon steel) corrosion test was performed using the three types of samples (OMV only, IS5 only, and IS5+OMV) prepared in the electrochemical measurement described above (see FIG. 8). 9 mL of modified DSMZ195c medium was used as a corrosion medium to disperse the above samples. The modified DSMZ195c medium is a medium obtained by removing lactic acid from DSMZ195c medium and adding 1 mM sodium acetate as a carbon source. A test piece (SPCC carbon steel coupon (10 mm×10 mm×2 mm)) was added to each of sample 1 (OMV only), sample 2 (IS5 only), and sample 3 (IS5+OMV) as the only electron donor for the bacteria (IS5). In sample 1 and sample 3, the OMV density was adjusted to 2.6×10 8 For Sample 2 and Sample 3, the starting cell concentration of IS5 in the tube was determined as OD 600nm = 0.05. Samples 1 to 3 were cultured in an anaerobic environment at 28°C for two weeks. The corroded iron plates were then observed by SEM. The results are shown in Figure 13(a). After observation, the test pieces were immersed in 6 M hydrochloric acid containing 3.5 g / L hexamethylenetetramine for five minutes to remove the corrosion products and biofilm from the test piece surfaces. The test piece surfaces after removal were observed by SEM. The results are shown in Figure 13(b).

[0053] The weight of the test piece before the culture and the weight of the test piece after the culture from which the corrosion products and biofilm were peeled off were measured, and the weight loss of the test piece due to the corrosion test was calculated from the difference between these weights. The results are shown in Figure 12. For comparison, a similar corrosion experiment was carried out using modified DSMZ195c medium (sterile) instead of samples 1 to 3. The results are shown in Figure 12 and Figures 13(a) and (b) labeled "Sterile".

[0054] As shown in Figure 12 and Figure 13(a)(b), a larger weight loss and more severe surface deterioration of the test specimen was observed in sample 3 (IS5+OMV) compared to sample 2 (IS5 only). This result confirmed that OMV promotes the pitting corrosion of iron by bacteria (IS5). Furthermore, more weight loss and corrosion pits on the surface were observed in sample 1 (OMV only) compared to Sterile, confirming that corrosion is promoted even with OMV alone. The reason for this may be that the distribution of OMV on the test specimen (carbon steel) may affect the local concentration of chemical species on the surface of the test specimen. [Industrial Applicability]

[0055] The method for evaluating the risk of microbial corrosion of the present invention can evaluate the risk of microbial corrosion more simply and accurately. This makes it possible to detect anaerobic iron corrosion present inside an oil pipeline or the like at an early stage, and to prevent accidents caused by microbial corrosion in advance. [Explanation of symbols]

[0056] 100 Evaluation Kit 10. Evaluation sample introduction section 20 Detection unit 21 Dyeing Department 30 Connecting part 40 Base material

Claims

1. A method for evaluating the risk of microbial corrosion in the environment, To prepare an evaluation sample containing materials collected from the aforementioned environment, An evaluation method comprising detecting outer membrane vesicles having redox properties in the evaluation sample.

2. The evaluation method according to claim 1, wherein detecting the outer membrane vesicles is used to determine whether or not the outer membrane vesicles are present, and / or to quantify the outer membrane vesicles.

3. The evaluation method according to claim 1 or 2, wherein the detection of the outer membrane vesicles is performed by a staining method.

4. The evaluation method according to claim 3, wherein a redox color-developing reagent is used in the staining method.

5. The evaluation method according to claim 1 or 2, wherein the detection of the outer membrane vesicles is performed by measuring the redox potential.

6. The evaluation method according to claim 1 or 2, wherein the detection of the outer membrane vesicles is performed by absorbance measurement.

7. The evaluation method according to claim 1, wherein the redox potential of the outer membrane vesicles is more positive than the redox potential of the iron material.

8. The evaluation method according to claim 1, wherein the redox potential of the outer membrane vesicles (25°C, pH 7) is greater than -0.6 V (SHE).

9. The evaluation method according to claim 1, wherein the standard redox potential (25°C, pH 7) of the outer membrane vesicles is more positive than the redox potential of the iron material and less negative than +0.77 V (SHE).

10. An evaluation kit for assessing the risk of microbial corrosion in the environment, An evaluation sample introduction unit is configured to bring an evaluation sample, including materials collected from the aforementioned environment, into contact with the evaluation sample, A detection unit configured to detect outer membrane vesicles having redox properties in the evaluation sample, An evaluation kit comprising a connecting unit that connects the evaluation sample introduction unit and the detection unit, and is configured to allow the evaluation sample to move from the evaluation sample introduction unit to the detection unit.

11. The evaluation kit according to claim 10, wherein the detection unit contains a redox colorimetric reagent and is configured to detect the outer membrane vesicles by a staining method.

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