CN111024594B - An observation method for microbial adhesion and corrosion of metal materials - Google Patents
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Abstract
本发明提供了一种微生物附着腐蚀金属材料的观测方法，依次包括以下步骤：将金属样品打磨后进行表面抛光；将金属样品以高氯酸乙醇溶液为电解液进行电解双喷；将金属样品立即用乙醇溶液清洗后无氧干燥后真空保存；金属样品静置在海洋微生物溶液中；金属样品用PBS缓冲溶液冲洗后于质量分数2.5％～2.7％的戊二醛溶液中浸泡10min～15min；除去金属样品附着液体后用TEM观测。本发明的观测方法能够实现微生物附着腐蚀金属材料表面的微生物腐蚀界面的动态过程的观测记录，上述的观测方法将金属样品先打磨后再进行微生物腐蚀观测，与之前的先进行微生物腐蚀后再打磨样品相比，避免了微生物信息的丢失，获得了更多的微生物附着腐蚀金属材料的信息。
The invention provides an observation method for microbial adhesion and corrosion of metal materials, which sequentially includes the following steps: grinding the metal sample and then carrying out surface polishing; using perchloric acid ethanol solution as electrolyte for double-spray electrolysis of the metal sample; immediately After washing with ethanol solution, anaerobic drying, and then vacuum storage; the metal sample was placed in the marine microorganism solution; the metal sample was washed with PBS buffer solution and then soaked in glutaraldehyde solution with a mass fraction of 2.5% to 2.7% for 10min to 15min; removed The metal sample was observed by TEM after adhering to the liquid. The observation method of the present invention can realize the observation record of the dynamic process of the microorganism corrosion interface where microorganisms attach and corrode the surface of the metal material. The above observation method first grinds the metal sample and then conducts the microbial corrosion observation, which is different from the previous microbial corrosion and then polishing. Compared with the samples, the loss of microbial information is avoided, and more information on the adhesion and corrosion of metal materials by microorganisms is obtained.
Description
技术领域technical field
本发明涉及材料微观检测领域，具体涉及一种微生物附着腐蚀金属材料的观测方法。The invention relates to the field of material microscopic detection, in particular to an observation method for microbial adhesion and corrosion of metal materials.
背景技术Background technique
21世纪是海洋的世纪。海洋资源开发、海上运输、海港和海防建设等领域都需要大量的表面防护材料。据统计，与海洋微生物附着有关的材料破坏占到涉海材料总量的70％-80％，每年因微生物附着而造成的能源消耗及腐蚀的损失高达上千亿美元，因此，对于微生物附着腐蚀的海洋表面防护材料的研究格外重要，需要一种相应的方法来研究微生物附着腐蚀的海洋表面防护材料，但是现有的微生物附着腐蚀的海洋表面防护材料的检测方法在样品处理过程中会导致微生物信息的丢失。The 21st century is the century of the ocean. The development of marine resources, marine transportation, seaport and coastal defense construction and other fields all require a large amount of surface protection materials. According to statistics, the destruction of materials related to the attachment of marine microorganisms accounts for 70%-80% of the total sea-related materials, and the annual energy consumption and corrosion losses caused by the attachment of microorganisms are as high as hundreds of billions of dollars. The research of marine surface protection materials is particularly important, and a corresponding method is needed to study the marine surface protection materials of microbial attachment and corrosion, but the existing detection methods of marine surface protection materials of microbial attachment and corrosion may lead to microorganisms in the sample processing process. loss of information.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于克服现有技术存在的不足之处而提供一种微生物附着腐蚀金属材料的观测方法。The purpose of the present invention is to overcome the deficiencies of the prior art and provide an observation method for microbial adhesion and corrosion of metal materials.
为实现上述目的，本发明采取的技术方案为：一种微生物附着腐蚀金属材料的观测方法，所述方法包括以下步骤：In order to achieve the above purpose, the technical scheme adopted in the present invention is: a method for observing the corrosion of metal materials by microorganism attachment, and the method comprises the following steps:
(1)将金属样品打磨后进行机械表面抛光；(1) Mechanical surface polishing is performed after the metal sample is ground;
(2)在抛光电压为20V～25V和温度为零下20℃～零下22℃的条件下，将步骤(1)得到的金属样品以高氯酸乙醇溶液为电解液进行电解双喷；(2) under the condition that the polishing voltage is 20V～25V and the temperature is minus 20 ℃～minus 22 ℃, the metal sample obtained in step (1) is electrolyzed double-spray with perchloric acid ethanol solution as electrolyte;
(3)将步骤(2)处理后的金属样品立即用乙醇溶液清洗后无氧干燥；(3) the metal sample processed in step (2) is immediately cleaned with an ethanol solution and then dried anaerobic;
(4)将盐卤溶解在去离子水中得到盐度为2.8％～3.5％的溶液A，向灭菌冷却后的溶液A中加入海洋微生物及培养基得到海洋微生物溶液；(4) dissolving the brine in deionized water to obtain a solution A with a salinity of 2.8% to 3.5%, adding marine microorganisms and a culture medium to the sterilized and cooled solution A to obtain a marine microorganism solution;
(5)将步骤(3)处理后的金属样品浸没在步骤(4)的海洋微生物溶液中静置24～72小时；(5) immersing the metal sample treated in step (3) in the marine microorganism solution of step (4) and standing for 24-72 hours;
(6)将步骤(5)处理后的金属样品用PBS缓冲溶液冲洗后于质量分数2.5％～2.7％的戊二醛溶液中浸泡10min～15min；(6) washing the metal sample processed in step (5) with a PBS buffer solution and then soaking it in a glutaraldehyde solution with a mass fraction of 2.5% to 2.7% for 10 min to 15 min;
(7)将步骤(6)处理后的金属样品除去附着液体后用TEM观测。(7) The metal sample treated in step (6) was removed from the attached liquid and observed by TEM.
上述的观测方法能够实现微生物附着腐蚀金属材料表面的微生物腐蚀界面的动态过程的观测记录，上述的观测方法将金属样品先打磨后再进行微生物腐蚀观测，与之前的先进行微生物腐蚀后再打磨样品相比，避免了微生物信息的丢失，获得了更多的微生物附着腐蚀金属材料的信息。The above observation method can realize the observation record of the dynamic process of the microorganism corrosion interface on the surface of the metal material. In contrast, the loss of microbial information is avoided, and more information on microbial attachment and corrosion of metal materials is obtained.
优选地，所述金属样品的材料为碳钢、低合金钢、不锈钢或者含铜钢。Preferably, the material of the metal sample is carbon steel, low alloy steel, stainless steel or copper-containing steel.
发明人经过研究发现，上述的观测方法能够适用于所有可适用于海洋工程应用的金属材料，且对于金属样品的材料为碳钢、低合金钢、不锈钢或者含铜钢时效果更好。The inventor found through research that the above observation method can be applied to all metal materials applicable to marine engineering applications, and the effect is better when the material of the metal sample is carbon steel, low alloy steel, stainless steel or copper-containing steel.
优选地，所述金属样品的材料为EH32船板钢、316L不锈钢、含铜量1.0wt％的316L不锈钢。Preferably, the material of the metal sample is EH32 ship plate steel, 316L stainless steel, and 316L stainless steel with a copper content of 1.0 wt%.
优选地，所述海洋微生物为需钠弧菌或者铜绿假单胞菌。Preferably, the marine microorganism is Vibrio natriureus or Pseudomonas aeruginosa.
发明人经过研究发现，上述的观测方法能够适用于所有可以培养的海洋微生物，当适用于需钠弧菌或者铜绿假单胞菌时，观测效果更好。The inventors have found through research that the above observation method can be applied to all marine microorganisms that can be cultivated, and the observation effect is better when applied to Vibrio natriureus or Pseudomonas aeruginosa.
优选地，所述步骤(2)中，高氯酸乙醇溶液的质量分数为5％～10％。Preferably, in the step (2), the mass fraction of the perchloric acid ethanol solution is 5% to 10%.
发明人经过研究发现，上述方法的步骤(2)中电解双喷的电解液为质量分数为5％～10％的高氯酸乙醇溶液时，观测方法获得的信息量更大，且信息更清楚。The inventor found through research that when the electrolyte of the double-spray electrolysis in step (2) of the above method is a perchloric acid ethanol solution with a mass fraction of 5% to 10%, the amount of information obtained by the observation method is larger and the information is clearer. .
优选地，所述步骤(2)中，电解液的流量为9ml/cm2～11ml/cm2。Preferably, in the step (2), the flow rate of the electrolyte is 9ml/cm 2 to 11ml/cm 2 .
优选地，所述步骤(1)中，金属样品打磨的方法为逐步使用目数依次增大至2000目的砂纸打磨，机械表面抛光至经金相显微镜观察无划痕，机械表面抛光后进行清洗及无氧干燥，机械表面抛光后清洗的清洗剂为乙醇溶液，机械表面抛光后无氧干燥的方法为氮气吹干。Preferably, in the step (1), the method of grinding the metal sample is to use sandpaper with a mesh number gradually increased to 2000 mesh for grinding, the mechanical surface is polished until no scratches are observed by a metallographic microscope, and the mechanical surface is polished after cleaning and polishing. Oxygen-free drying, the cleaning agent for cleaning after mechanical surface polishing is ethanol solution, and the method for anaerobic drying after mechanical surface polishing is nitrogen drying.
上述的观测方法通过将金属样品机械表面抛光至经金相显微镜观察无划痕，避免了金属样品表面状况对观测结果的影响，使得观测方法获得的信息量更大，且信息更清楚。The above observation method avoids the influence of the surface condition of the metal sample on the observation result by polishing the mechanical surface of the metal sample until there is no scratches observed by a metallographic microscope, so that the observation method obtains a larger amount of information and the information is clearer.
优选地，所述步骤(3)中，无氧干燥后将金属样品真空保存，无氧干燥的方法为氮气吹干。Preferably, in the step (3), the metal sample is vacuum-stored after anaerobic drying, and the anaerobic drying method is nitrogen drying.
上述的观测方法将步骤(3)处理后的样品真空保存后避免了金属样品表面被氧化。The above observation method prevents the surface of the metal sample from being oxidized after the sample processed in step (3) is stored in vacuum.
优选地，所述步骤(4)中，海洋微生物溶液的OD值为2.0～4.8，所述培养基为2216E液体培养基。Preferably, in the step (4), the OD value of the marine microorganism solution is 2.0-4.8, and the medium is 2216E liquid medium.
优选地，所述步骤(4)中，灭菌的温度为120～121℃，灭菌的时间为15～20min，灭菌冷却后的温度为20～25℃。Preferably, in the step (4), the temperature of sterilization is 120-121°C, the time of sterilization is 15-20min, and the temperature after sterilization and cooling is 20-25°C.
优选地，所述步骤(1)中，处理前金属样品的厚度为0.5mm～0.7mm。Preferably, in the step (1), the thickness of the metal sample before treatment is 0.5 mm to 0.7 mm.
上述的观测方法使用厚度为0.5mm～0.7mm的金属样品能够获得更多且更清楚的信息。The above observation method can obtain more and clearer information by using metal samples with a thickness of 0.5 mm to 0.7 mm.
优选地，所述步骤(7)中，步骤(6)处理后的金属样品除去附着液体的方法为：将骤(6)处理后的金属样品依次置于体积分数从50％逐渐增大至100％的乙醇溶液中。Preferably, in the step (7), the method for removing the adhering liquid from the metal sample processed in the step (6) is: placing the metal sample after the step (6) treatment in sequence, and the volume fraction gradually increases from 50% to 100%. % in ethanol solution.
优选地，所述步骤(7)中，步骤(6)处理后的金属样品除去附着液体的方法为：将骤(6)处理后的金属样品依次置于体积分数为50％、60％、70％、80％、90％和100％的乙醇溶液中脱水。Preferably, in the step (7), the method for removing the attached liquid from the metal sample processed in the step (6) is: placing the metal sample processed in the step (6) in a volume fraction of 50%, 60%, 70% in turn. %, 80%, 90% and 100% ethanol solutions.
本发明的有益效果在于：本发明提供了一种微生物附着腐蚀金属材料的观测方法，本发明的观测方法能够实现微生物附着腐蚀金属材料表面的微生物腐蚀界面的动态过程的观测记录，上述的观测方法将金属样品先打磨后再进行微生物腐蚀观测，与之前的先进行微生物腐蚀后再打磨样品相比，避免了微生物信息的丢失，获得了更多的微生物附着腐蚀金属材料的信息。The beneficial effects of the present invention are as follows: the present invention provides a method for observing the corrosion of metal materials by microorganisms. Compared with the previous microbial corrosion and then grinding samples, the metal samples are first polished and then subjected to microbial corrosion observation, which avoids the loss of microbial information and obtains more information on microbial adhesion and corrosion of metal materials.
附图说明Description of drawings
图1为本发明实施例的观测方法的应用效果图，其中(A)为对照图，(B)为样品图。FIG. 1 is an application effect diagram of an observation method according to an embodiment of the present invention, wherein (A) is a comparison diagram, and (B) is a sample diagram.
图2为本发明实施例的观测方法的应用效果图，其中(A)为对照图，(B)为样品图。FIG. 2 is an application effect diagram of an observation method according to an embodiment of the present invention, wherein (A) is a comparison diagram, and (B) is a sample diagram.
图3为本发明实施例的观测方法的应用效果图，其中(A)为对照图，(B)为样品图。3 is an application effect diagram of an observation method according to an embodiment of the present invention, wherein (A) is a comparison diagram, and (B) is a sample diagram.
图4为本发明实施例的观测方法的应用效果图，其中(A)为对照图，(B)为样品图。4 is an application effect diagram of an observation method according to an embodiment of the present invention, wherein (A) is a comparison diagram, and (B) is a sample diagram.
图5为本发明实施例的观测方法的应用效果图，其中(A)为对照图，(B)为样品图。FIG. 5 is an application effect diagram of the observation method according to an embodiment of the present invention, wherein (A) is a comparison diagram, and (B) is a sample diagram.
具体实施方式Detailed ways
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.
实施例1Example 1
作为本发明实施例的一种微生物附着腐蚀金属材料的观测方法，所述方法包括以下步骤：As an embodiment of the present invention, a method for observing microbial adhesion and corrosion of metal materials, the method comprises the following steps:
(1)将不锈钢金属样品切割至厚度为0.5mm～0.7mm，将厚度为0.5mm～0.7mm的不锈钢金属样品依次用150目、400目、800目、1500目和2000目的砂纸打磨后用金属试验抛光机在120rmp～150rmp的转速下进行表面抛光至经金相显微镜观察无划痕，表面抛光后用乙醇溶液进行清洗及氮气吹干；(1) Cut the stainless steel metal sample to a thickness of 0.5mm to 0.7mm, and polish the stainless steel metal sample with a thickness of 0.5mm to 0.7mm with 150-mesh, 400-mesh, 800-mesh, 1500-mesh and 2000-mesh sandpaper in turn, and then use the metal The surface of the test polishing machine is polished at a rotational speed of 120 rmp to 150 rmp until no scratches are observed by a metallographic microscope. After surface polishing, it is cleaned with an ethanol solution and dried with nitrogen;
(2)在抛光电压为20V～25V和温度为零下20℃～零下22℃的条件下，将步骤(1)得到的金属样品以质量分数为5％～10％的高氯酸乙醇溶液为电解液进行电解双喷，电解液的流量为9ml/cm2～11ml/cm2；(2) Under the conditions of a polishing voltage of 20V to 25V and a temperature of minus 20°C to minus 22°C, the metal sample obtained in step (1) is electrolyzed with a perchloric acid ethanol solution with a mass fraction of 5% to 10%. The liquid is electrolyzed by double spray, and the flow rate of the electrolyte is 9ml/cm 2 ~ 11ml/cm 2 ;
(3)将步骤(2)处理后的金属样品在2～6s内迅速用乙醇溶液清洗后氮气吹干，将氮气吹干后的金属样品真空保存；(3) the metal sample processed in step (2) is rapidly cleaned with an ethanol solution within 2 to 6 s and then dried with nitrogen, and the metal sample dried with nitrogen is stored in a vacuum;
(4)将盐卤溶解在去离子水中得到盐度为2.8％～3.5％的溶液A，向灭菌冷却后的溶液A中加入需钠弧菌的2216E菌液得到海洋微生物溶液，海洋微生物溶液的OD值为2.0～4.8；(4) dissolving the brine in deionized water to obtain a solution A with a salinity of 2.8% to 3.5%, adding the 2216E bacterial solution of Sodium Vibrio to the solution A after the sterilization and cooling to obtain a marine microbial solution, the marine microbial solution OD value is 2.0～4.8;
(5)将步骤(3)处理后的金属样品浸没在步骤(4)的海洋微生物溶液中静置24小时；(5) immersing the processed metal sample in step (3) in the marine microorganism solution of step (4) and standing for 24 hours;
(6)将步骤(5)处理后的金属样品用PBS缓冲溶液冲洗3～4次后于质量分数2.5％～2.7％的戊二醛溶液中浸泡10min～15min；(6) washing the metal sample processed in step (5) with a PBS buffer solution for 3 to 4 times and then soaking it in a glutaraldehyde solution with a mass fraction of 2.5% to 2.7% for 10 min to 15 min;
(7)将步骤(6)处理后的金属样品依次置于体积分数为50％、60％、70％、80％、90％和100％的乙醇溶液中脱水后用TEM观测。(7) Dehydration of the metal samples treated in step (6) in 50%, 60%, 70%, 80%, 90% and 100% ethanol solutions by volume fraction and then observing by TEM.
设置对照实验，对照实验与本实施例的区别为：步骤(4)中溶液A中未加入需钠弧菌的2216E菌液且步骤(5)中将步骤(3)处理后的金属样品浸没在灭菌冷却后的溶液A中。A control experiment is set up, and the difference between the control experiment and this example is: in the step (4), the 2216E bacteria solution of Vibrio sodium need not be added to the solution A, and in the step (5), the metal sample processed in the step (3) is immersed in the solution A. Sterilize the cooled solution A.
如图1(B)所示，需钠弧菌已经牢固的附着到不锈钢样品表面，且对不锈钢样品结构(位错、晶界等)未造成破坏性影响；如图1(A)所示，对照样品腐蚀以均匀腐蚀为主，基体微观形貌无明显变化，说明通过本实施例的方法能够观察微生物对金属样品腐蚀的影响。As shown in Figure 1(B), Sodium Vibrio has been firmly attached to the surface of the stainless steel sample, and has no destructive effect on the structure of the stainless steel sample (dislocations, grain boundaries, etc.); as shown in Figure 1(A), The corrosion of the control sample is dominated by uniform corrosion, and the microscopic morphology of the substrate has no obvious change, indicating that the method of this embodiment can observe the influence of microorganisms on the corrosion of the metal sample.
实施例2Example 2
作为本发明实施例的一种微生物附着腐蚀金属材料的观测方法，本实施例与实施例1的唯一区别为：金属样品为碳钢金属样品。As a method for observing the adhesion and corrosion of metal materials by microorganisms in the embodiment of the present invention, the only difference between this embodiment and Embodiment 1 is that the metal sample is a carbon steel metal sample.
如图2(B)所示，需钠弧菌已经牢固的附着到碳钢样品表面，且对碳钢样品内部结构(位错、晶界等)已造成破坏性影响，造成了穿孔。如图2(A)所示，对照样品无明显穿孔现象，腐蚀以均匀腐蚀为主，基体微观形貌无明显变化，说明通过本实施例的方法能够观察微生物对金属样品腐蚀的影响。As shown in Figure 2(B), Sodium Vibrio has firmly attached to the surface of the carbon steel sample, and has caused destructive effects on the internal structure (dislocations, grain boundaries, etc.) of the carbon steel sample, resulting in perforation. As shown in Figure 2 (A), the control sample has no obvious perforation phenomenon, the corrosion is mainly uniform corrosion, and the microscopic morphology of the substrate has no obvious change, indicating that the method of this embodiment can observe the influence of microorganisms on the corrosion of metal samples.
实施例3Example 3
作为本发明实施例的一种微生物附着腐蚀金属材料的观测方法，本实施例与实施例1的唯一区别为：金属样品为低合金钢金属样品。As an observation method of microorganisms attaching and corroding metal materials according to an embodiment of the present invention, the only difference between this embodiment and Embodiment 1 is that the metal sample is a low-alloy steel metal sample.
如图3(B)所示，需钠弧菌已经牢固的附着到低合金钢金属样品表面，且对低合金钢金属样品内部结构(位错、晶界等)已造成破坏性影响，造成了腐蚀缝隙。如图3(A)所示，低合金钢金属样品无明显穿孔现象，腐蚀以均匀腐蚀为主，基体微观形貌无明显变化。As shown in Fig. 3(B), Sodium Vibrio has been firmly attached to the surface of the low-alloy steel metal sample, and has caused a destructive effect on the internal structure (dislocation, grain boundary, etc.) of the low-alloy steel metal sample, resulting in Corrosion crevices. As shown in Figure 3(A), the low alloy steel metal sample has no obvious perforation phenomenon, the corrosion is mainly uniform corrosion, and the microscopic morphology of the matrix has no obvious change.
实施例4Example 4
作为本发明实施例的一种微生物附着腐蚀金属材料的观测方法，本实施例与实施例1的唯一区别为：步骤(4)中，海洋微生物为铜绿假单胞菌。As an observation method of microorganisms attaching and corroding metal materials according to an embodiment of the present invention, the only difference between this embodiment and Embodiment 1 is that in step (4), the marine microorganism is Pseudomonas aeruginosa.
如图4(B)所示，铜绿假单胞菌已经牢固的附着到不锈钢金属样品表面，且对不锈钢钢金属样品内部结构(位错、晶界等)已造成破坏性影响。如图4(A)所示，不锈钢金属样品无明显穿孔现象，腐蚀以均匀腐蚀为主，基体微观形貌无明显变化。As shown in Figure 4(B), Pseudomonas aeruginosa has firmly attached to the surface of the stainless steel metal sample, and has caused destructive effects on the internal structure (dislocations, grain boundaries, etc.) of the stainless steel metal sample. As shown in Figure 4(A), the stainless steel metal sample has no obvious perforation phenomenon, the corrosion is mainly uniform corrosion, and the microscopic morphology of the matrix has no obvious change.
实施例5Example 5
作为本发明实施例的一种微生物附着腐蚀金属材料的观测方法，本实施例与实施例1的唯一区别为：金属样品为含铜量为1.0wt％的不锈钢。As a method for observing the adhesion and corrosion of metal materials by microorganisms in the embodiment of the present invention, the only difference between this embodiment and Embodiment 1 is that the metal sample is stainless steel with a copper content of 1.0 wt%.
如图5(B)所示，铜绿假单胞菌未能附着到含铜量为1.0wt％的不锈钢表面，且对含铜量为1.0wt％的不锈钢内部结构(位错、晶界等)未造成破坏性影响。如图5(A)所示，含铜量为1.0wt％的不锈钢无明显穿孔现象，腐蚀以均匀腐蚀为主，基体微观形貌无明显变化。As shown in Figure 5(B), Pseudomonas aeruginosa failed to attach to the stainless steel surface with a copper content of 1.0 wt%, and the internal structure (dislocations, grain boundaries, etc.) of the stainless steel with a copper content of 1.0 wt% No damaging effects. As shown in Figure 5(A), the stainless steel with a copper content of 1.0 wt% has no obvious perforation phenomenon, the corrosion is mainly uniform corrosion, and the microscopic morphology of the matrix has no obvious change.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.
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