A pseudomonas alternaria culture medium for corrosion experiment of steel material

Abstract

The application belongs to the field of microbial corrosion, and particularly relates to a Pseudoalteromonas culture medium for steel material corrosion experiment, which is composed of the following components: glucose 0.5-1 g / L, yeast extract powder 0.5-0.8 g / L, ammonium salt 0.002-0.005 g / L, magnesium chloride 3-5 g / L, sodium chloride 20-24 g / L, calcium chloride 2-3 g / L, dipotassium hydrogen phosphate 0.2-0.6 g / L, ferric ammonium citrate 0.01-0.08 g / L, growth factor 0.035-0.045 g / L, and the rest is deionized water. The culture medium can accelerate the corrosion process of steel material under the action of Pseudoalteromonas, and effectively improve the corrosion test efficiency of steel material under the action of Pseudoalteromonas.

Description

Technical Field

[0001] This invention belongs to the field of microbial corrosion, and in particular relates to a pseudoalternating monoclonal bacteria culture medium for corrosion experiments on steel materials. Background Technology

[0002] Microbiologically induced corrosion (MIC) is a corrosion process involving the life activities of microorganisms. This process is widespread in the petroleum industry, marine equipment, fluid transportation, and water treatment, and the resulting corrosion failures are increasingly attracting attention from engineering and research fields. Steel is the most widely used material in the national economy, and its microbiological corrosion has become a research hotspot. Because the factors influencing microbial corrosion processes in real-world environments are complex and variable, and difficult to control and quantify, studying the microbial corrosion behavior of steel materials under laboratory conditions is of great significance for developing novel microbiologically resistant steel materials and protective technologies.

[0003] *Pseudomonas aeruginosa* is widely distributed in aquatic environments such as oceans and lakes, and is one of the more common microorganisms in the corrosion products of steel materials in marine service environments. The adsorption and growth of bacteria on material surfaces involves complex biological, physical, and chemical processes, which are influenced by many factors. Among these, nutrients play a crucial role in bacterial reproduction and biofilm attachment and turnover. Different corrosive bacteria have different nutrient requirements, and the growth activity of the same bacteria varies greatly in different culture media, with some even failing to grow. Furthermore, the concentration of nutrients in the culture medium also significantly affects bacterial life activities. Too low a nutrient concentration can lead to a reduction in bacteria or hinder cellular synthesis, while too high a concentration may inhibit bacterial growth. Therefore, the culture medium has a significant impact on bacterial life activities, cell density, and the generation of metabolic products, thus significantly influencing the behavior of microbial corrosion.

[0004] Currently, in studies on the corrosion behavior of steel materials under the action of *Pseudomonas*, 2216E medium is often used. Its main components include peptone, yeast extract, ferric citrate, sodium chloride, magnesium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium carbonate, potassium bromide, strontium chloride, boric acid, sodium silicate, sodium fluoride, ammonium nitrate, and disodium hydrogen phosphate. This medium is a broad-spectrum medium for marine bacteria. Studies on the corrosion behavior of steel materials under the action of *Pseudomonas* using 2216E medium are often time-consuming. The study "The Influence of *Pseudomonas* on the Galvanic Corrosion of EH40 / B10" (Equipment Environmental Engineering, 2018, 15(10): 77-83.) shows that, under the conditions of 2216E medium, the experiment to investigate the galvanic corrosion behavior between EH40 steel and B10 copper alloy lasted 21 days. The study in the literature "Study on the mechanism of melanin secretion by marine pseudoalteromonas lipolytica accelerating the corrosion of 316L stainless steel" (Chinese Journal of Corrosion and Protection, 2022, 42(5): 743-751.) shows that obvious pitting corrosion only appears on the surface of 316L stainless steel after soaking in 2216E medium for 14 days. Patent publication number: CN115975872A discloses a culture medium for pseudoalteromonas that is different from 2216E medium. This culture medium contains: sodium chloride, magnesium chloride, sodium sulfate, calcium chloride, potassium chloride, hydroxyethylpiperazine ethionine, peptone and yeast extract. However, the purpose of this invention is to mix the target gene donor and the recipient pseudoalteromonas in the culture medium for growth, so as to effectively transfer the shuttle plasmid containing the target gene into the recipient strain through the donor strain, thereby better realizing the transfer of genetic material. It is not suitable for corrosion tests of steel materials. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the purpose of this invention is to provide a Pseudomonas alterniflora culture medium for corrosion experiments on steel materials, thereby solving the technical problems of slow corrosion process initiation, long experimental cycle, and low efficiency when using the general-purpose 2216E culture medium for corrosion experiments on steel materials under the action of Pseudomonas alterniflora.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials, the culture medium comprising the following components: Glucose 0.5~1g / L, yeast extract 0.5~0.8g / L, ammonium salt 0.002~0.005g / L, magnesium chloride 3~5g / L, sodium chloride 20~24g / L, calcium chloride 2~3g / L, dipotassium hydrogen phosphate 0.2~0.6g / L, ferric ammonium citrate 0.01~0.08g / L, growth factor 0.035~0.045g / L, balance deionized water.

[0007] The ammonium salt is one or both of ammonium chloride and ammonium sulfate.

[0008] The growth factor is a mixture of riboflavin and sodium 1,2-naphthoquinone-4-sulfonate, with a molar ratio of riboflavin to sodium 1,2-naphthoquinone-4-sulfonate of 1:1 to 1:3.

[0009] The pH value of the culture medium is 7.5 to 8.2.

[0010] The pH of the culture medium is adjusted using a 1 mol / L NaOH aqueous solution or a 1 mol / L HCl aqueous solution.

[0011] The culture medium was used in corrosion experiments on steel materials under the action of Pseudomonas alterniflora.

[0012] The functions of each component in the culture medium are designed as follows: Glucose serves as a carbon source, providing the necessary energy and carbon for bacterial growth. When the culture medium lacks a carbon source, bacteria will use iron as an electron donor, causing the iron to oxidize and leading to the corrosion of steel materials.

[0013] Yeast extract and ammonium salts are the main nitrogen sources required for bacterial growth, enabling bacteria to synthesize nitrogenous substances within their cells. Ferrous ammonium citrate provides both nitrogen and iron for bacterial growth.

[0014] Inorganic salts such as magnesium chloride, sodium chloride, calcium chloride, and dipotassium hydrogen phosphate maintain the stability of bacterial structure and regulate and maintain the osmotic pressure balance of cells. Among them, dipotassium hydrogen phosphate also acts as a buffer for the pH of the culture medium.

[0015] The growth factor is a mixture of riboflavin and sodium 1,2-naphthoquinone-4-sulfonate in a molar ratio of 1:1 to 1:3. When riboflavin and sodium 1,2-naphthoquinone-4-sulfonate are combined in this ratio to form the growth factor, the corrosion process of *Pseudomonas aeruginosa* using iron as an electron donor is significantly accelerated. During the microbial corrosion of metallic materials, microorganisms acquire electrons from the metal surface through extracellular electron transport. Riboflavin and sodium 1,2-naphthoquinone-4-sulfonate can act as electron carriers, playing a role in electron transport between microbial cells and the metal, thereby accelerating the microbial corrosion process. Specifically, riboflavin can promote electron transport across the inner and outer cell membranes. Quinones, such as sodium 1,2-naphthoquinone-4-sulfonate, can accept electrons and be reduced to hydroquinones. These hydroquinones are then oxidized by extracellular electron acceptors to the corresponding semiquinones, and finally oxidized back to quinones. Quinones participate in electron transport in this cyclical manner. The two substances differ in their mechanisms of functioning as electron carriers, thus creating a synergistic effect that jointly accelerates the corrosion process of metallic materials by microorganisms.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The culture medium of this invention achieves a rapid and significant acceleration of the corrosion of steel materials by *Pseudomonas aeruginosa* under laboratory conditions. It speeds up the corrosion process of steel materials under the action of *Pseudomonas aeruginosa*, effectively improving the efficiency of corrosion tests on steel materials under *Pseudomonas aeruginosa*. This provides strong technical support for the research and evaluation of steel materials resistant to microbial corrosion.

[0017] 2. This invention, through targeted optimization of the culture medium composition, successfully induced *Pseudomonas aeruginosa* to use steel materials as electron donors, thereby significantly accelerating the corrosion process and exacerbating pitting corrosion. This invention effectively shortens the experimental cycle, which traditionally requires 14–21 days to observe significant corrosion, to within 7 days, greatly improving the experimental efficiency of microbial corrosion research.

[0018] 3. This invention accelerates the corrosion process of Pseudorabialis by controlling the carbon source concentration and combining it with growth factors (riboflavin and sodium 1,2-naphthoquinone-4-sulfonate in a molar ratio of 1:1 to 1:3). Detailed Implementation

[0019] The present invention will now be described in detail, but it should be noted that the implementation of the present invention is not limited to the following embodiments.

[0020] Table 1 shows examples and comparative examples of *Pseudomonas alterniflora* culture medium suitable for microbial corrosion experiments on steel materials. Table 2 shows the specific effects of the examples and comparative examples. The experimental material was marine-grade AH32 steel.

[0021] Table 1. Embodiments and comparative examples of the present invention

[0022] Note: m v :m n It is the molar ratio of riboflavin to sodium 1,2-naphthoquinone-4-sulfonate.

[0023] Table 2. Effects of Examples and Comparative Examples

[0024] Note: 1. Corrosion tests were conducted in accordance with T / CSCP 0035.7-2017 Laboratory Corrosion Tests for Low Alloy Structural Steel. Part 7: General Procedures for Laboratory Microbial Corrosion Testing of Low Alloy Structural Steel; 2. The maximum depth of corrosion pits was characterized using laser confocal microscopy.

[0025] The experimental results of Examples 1-7 show that: The corrosion rate was significantly improved: After 7 days of experimentation, the corrosion rate of AH32 steel using the culture medium of this invention reached 0.021–0.030 mm / a, while the corrosion rate using the conventional 2216E culture medium (Comparative Example 8) was only 0.013 mm / a, showing a significant improvement. After 14 days of experimentation, the corrosion rate of the embodiments of this invention reached 0.043–0.075 mm / a, a significant improvement compared to the 2216E culture medium (0.022 mm / a).

[0026] The degree of pitting corrosion was significantly aggravated: when using the culture medium of this invention, the maximum pit depth reached 15-19 μm after 14 days of experimentation, while it was only 9 μm on the 2216E culture medium, significantly increasing the pitting depth. This means that this invention can induce more severe localized corrosion in a shorter time, facilitating rapid evaluation of the material's pitting corrosion resistance.

[0027] By controlling the carbon source concentration and combining it with growth factors (riboflavin and sodium 2-naphthoquinone-4-sulfonate in a molar ratio of 1:1 to 1:3), the corrosion process of *Pseudomonas alterniflora* was accelerated. Comparative example data further validated the key roles of each component: Carbon source: In Comparative Example 1, when the glucose concentration was increased to 2 g / L, the corrosion rate dropped to 0.017 mm / a after 7 days, indicating that excessive carbon source inhibited the corrosion process and could not accelerate corrosion.

[0028] Growth factors: In Comparative Example 3, when the total concentration of growth factors was reduced to 0.015 g / L, the corrosion rate after 7 days was only 0.015 mm / a; Comparative Example 4 (molar ratio 1:5) and Comparative Example 5 (molar ratio 1:0.5) also failed to reach the corrosion rate level of the embodiments of the present invention, proving that the type, total concentration and molar ratio range of riboflavin and sodium 2-naphthoquinone-4-sulfonate are key to achieving accelerated corrosion.

[0029] pH: Comparative Example 6 (pH 6.5) and Comparative Example 7 (pH 9.0) both deviated from the suitable growth range of Pseudomonas aeruginosa, resulting in a significant decrease in corrosion rate (only 0.015 mm / a and 0.006 mm / a, respectively, after 7 days), which verifies the necessity of strictly controlling the pH between 7.5 and 8.2 in this invention.

[0030] As can be seen from the implementation results of each embodiment, under the same experimental time conditions, the corrosion rate and maximum corrosion pit depth of the sample after corrosion test using the culture medium described in the technical solution of the present invention are significantly higher than those using 2216E culture medium. Therefore, the corrosion behavior and performance evaluation of steel materials under the action of pseudoalternating monoclonal bacteria can be carried out more efficiently. In Comparative Example 1, the carbon source content in the culture medium was too high, and *Pseudomonas aeruginosa* was not in a relatively "starved" state, so the corrosion process of the steel material was not significantly accelerated. In Comparative Example 2, the nitrogen source, ammonium salt, and inorganic salt content in the culture medium was too high, which inhibited the growth of *Pseudomonas aeruginosa* and did not accelerate the corrosion process of the steel material. In Comparative Example 3, the growth factor content in the culture medium was too low, and the corrosion process of *Pseudomonas aeruginosa* using iron as an electron donor was not significantly accelerated. In Comparative Examples 4 and 5, the molar ratio of riboflavin to sodium 1,2-naphthoquinone-4-sulfonate in the culture medium was not within the optimal range, which also resulted in the corrosion process of *Pseudomonas aeruginosa* using iron as an electron donor not being significantly accelerated. In Comparative Examples 6 and 7, the pH value of the culture medium was not within the suitable range for the growth of *Pseudomonas aeruginosa*, which inhibited the growth of *Pseudomonas aeruginosa* and did not accelerate the corrosion process of the steel material.

[0031] The present invention is only illustrated by the above embodiments. Any equivalent transformations made according to the principles of the present invention should not be excluded from the protection scope of the present invention.

Claims

1. A *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials, characterized in that, The culture medium consists of the following components: Glucose 0.5~1g / L, yeast extract 0.5~0.8g / L, ammonium salt 0.002~0.005g / L, magnesium chloride 3~5g / L, sodium chloride 20~24g / L, calcium chloride 2~3g / L, dipotassium hydrogen phosphate 0.2~0.6g / L, ferric ammonium citrate 0.01~0.08g / L, growth factor 0.035~0.045g / L, balance deionized water.

2. The *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials according to claim 1, characterized in that, The ammonium salt is one or both of ammonium chloride and ammonium sulfate.

3. The *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials according to claim 1, characterized in that, The growth factor is a mixture of riboflavin and sodium 1,2-naphthoquinone-4-sulfonate, with a molar ratio of riboflavin to sodium 1,2-naphthoquinone-4-sulfonate of 1:1 to 1:

3.

4. The *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials according to claim 1, characterized in that, The pH value of the culture medium is 7.5 to 8.

2.

5. The *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials according to claim 1, characterized in that, The pH of the culture medium is adjusted using a 1 mol / L NaOH aqueous solution or a 1 mol / L HCl aqueous solution.

6. A *Pseudomonas alterniflora* culture medium for corrosion experiments on steel materials according to any one of claims 1-5, characterized in that, The culture medium was used in corrosion experiments on steel materials under the action of Pseudomonas alterniflora.

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