Microbial corrosion control method based on biological membrane attachment area regulation and control

Abstract

The invention discloses a microbial corrosion control method based on biological membrane attachment area regulation, which comprises the following steps: under the condition of keeping the volume of a liquid culture medium and a bacterial strain unchanged, introducing solid fillers which account for a fixed total volume but have different specific surface areas into a closed anaerobic reactor so as to form a multi-stage attachable area; a pretreated metal sample is placed in the closed anaerobic reactor, and the surface of the sample is only in contact with the culture medium and the biological membrane distribution area determined by the filler; and culturing at the same temperature and time, and synchronously monitoring the growth, corrosion rate and gas production rate of the biological membrane, so as to establish a quantitative relationship among biological membrane distribution area-corrosion behavior-mechanical property, and realize directional control of the microbial corrosion process by regulating and controlling area parameters.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of material corrosion, and particularly relates to a microbial corrosion control method based on biofilm attachment area regulation. BACKGROUND

[0002] Microbiologically Influenced Corrosion (MIC) is an accelerated corrosion phenomenon of metal materials under the action of microorganisms and their metabolites, in which sulfate-reducing bacteria (SRB) is considered to be one of the most destructive bacteria groups. Desulfovibrio vulgaris (D. vulgaris) as a typical SRB widely exists in oil and gas fields, seabed sediments and anaerobic soil, and the hydrogen sulfide (H2S) and hydrogen (H2) produced by its metabolism can cause local pitting, hydrogen-induced cracking and mechanical property degradation, which seriously threatens the safe operation of high-grade pipeline steels such as X80.

[0003] Therefore, detailed corrosion analysis of microorganisms is a technical problem to be solved in the prior art. SUMMARY

[0004] The application aims to provide a microbial corrosion control method based on biofilm attachment area regulation.

[0005] The technical scheme of the application is a microbial corrosion control method based on biofilm attachment area regulation, comprising: a) under the condition that the volume of liquid medium and the strain are kept unchanged, introducing solid fillers with different specific surface areas into a closed anaerobic reactor to form a multi-stage attachable area; b) placing a pretreated metal sample in the closed anaerobic reactor, so that the sample surface is only in contact with the medium and the biofilm distribution area determined by the fillers; c) culturing under the same temperature and time and synchronously monitoring the biofilm growth, corrosion rate and gas production to establish a quantitative relationship among the biofilm distribution area, corrosion behavior and mechanical property, so as to realize directional control of the microbial corrosion process by regulating the area parameter.

[0006] Optionally, the solid fillers include epoxy resin solid blocks, single-particle-size glass beads or a combination of two or more particle sizes of glass beads.

[0007] Optionally, by adjusting the glass bead diameter 2r and the number n, the total specific surface area A=4πr²n of different closed anaerobic reactors forms a multi-stage attachable area, and the volume of liquid medium and headspace is kept constant to exclude the interference of volume effect on the area variable.

[0008] Optionally, the closed anaerobic reactor is formed with three levels of attachable area, i.e. small, medium and large.

[0009] Optionally, the three levels of attachable area are obtained by solid fillers, i.e. 100 cm², 390 cm², 640 cm² in a fixed volume of 50 cm³.

[0010] Optionally, the three levels of attachable area are realized by the following glass bead-volume combinations: a) Small area level: no glass beads added, only 50 cm³ of epoxy resin solid block, theoretical attachable area 100 cm²; b) Medium area level: add glass beads with a diameter of 12 mm, single bead volume 0.904 cm³, number 55±1, total glass bead volume 50 cm³, theoretical attachable area 390 cm²; c) Large area level: add glass beads with a diameter of 6 mm, single bead volume 0.113 cm³, number 442±2, total glass bead volume 50 cm³, theoretical attachable area 640 cm².

[0011] Optionally, the closed anaerobic reactor is formed with three levels of attachable area, i.e. small, medium and large.

[0012] Optionally, after step c), further comprising: Establishing a three-dimensional calibration curve of "unit area bacterial amount-biofilm thickness-gas production" for predicting the microbial corrosion rate and hydrogen embrittlement risk under any given area.

[0013] Optionally, in step c), "synchronous monitoring" includes: a) Measuring open circuit potential OCP and linear polarization resistance LPR every 24 h; b) Collecting electrochemical impedance spectroscopy EIS on the 1st, 3rd and 7th day; c) Measuring sample weight loss and pitting depth on the 3rd and 7th day; d) Detecting headspace H2S and H2 concentration and converting to dissolved [H2S] on the 1st, 3rd and 7th day; e) Performing uniaxial tensile test on the 7th day to obtain cross-section shrinkage and hydrogen embrittlement index.

[0014] Optionally, before the sample is installed, the metal surface is polished to 600# and ultraviolet sterilized for 20 min to ensure that the initial surface state and the bacteria source are consistent, and only the area variable is a single factor difference.

[0015] In summary, the method of the application, under the premise of constant liquid medium volume and strain conditions, realizes gradient control of biofilm attachable area by introducing solid fillers with different specific surface areas, avoiding experimental errors caused by variable cross interference in traditional methods; the metal sample only contacts the culture medium and the biofilm distribution area determined by the filler, ensuring the consistency and repeatability of the corrosion environment, providing a stable and controllable experimental platform for studying the mechanism of microbial corrosion; by simultaneously monitoring biofilm growth, corrosion rate and gas production, the quantitative relationship between "biofilm distribution area-corrosion behavior-mechanical properties" is systematically constructed, providing data support for microbial corrosion prediction and evaluation; this scheme can change the biofilm distribution characteristics by simply adjusting the specific surface area of the filler, and then regulate the corrosion process, providing a new idea for the optimization of material microbial corrosion resistance and the development of protection technology; this method is suitable for various metal materials and microbial systems, and the experimental device is simple and easy to operate, with good universality and promotional value. The experimental results show that: the larger the biofilm distribution area, the less the amount of D. vulgaris attached per unit area, and the lower the corrosion rate, but the gas production (H2S, H2) increases significantly; the corrosion mechanism changes with time: in the early stage (first 3 days), electron transfer mediated microbial corrosion (EET-MIC) is dominant, and in the later stage (last 4 days), H2S corrosion is enhanced; the degradation of mechanical properties is negatively correlated with the biofilm area: large-area biofilm leads to more hydrogen atoms penetrating into the steel matrix, significantly reducing the ductility of X80 steel and increasing the brittleness; by regulating the biofilm distribution area, the corrosion rate and material brittleness can be controlled, providing an effective means for microbial corrosion prevention and control. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A step flow chart of the experimental method for studying microbial corrosion provided by the application; Figure 2 A schematic diagram of the construction of three different biofilm coverage areas in the anaerobic culture system structure; Figure 3 A comparison chart of the number of D. vulgaris cells attached to the surface of X80 steel under different biofilm coverage areas (3 days and 7 days); Figure 4 A curve graph of the concentration of dissolved H2S in the culture solution changing with time; Figure 5 A curve graph of the concentration of H2 in the headspace changing with time; Figure 6 A mechanism diagram of the redox reaction area of the sample in the culture solution with different diffusion areas; Figure 7 A unit area weight loss chart of X80 steel under different biofilm coverage areas for 3 days and 7 days; Figure 8 SEM images of biofilm thickness and density on X80 steel surface under different coverage areas; Figure 9 CLSM images of biofilms on X80 steel surfaces with different coverage areas; green indicates live cells. Figure 10 SEM images of pitting morphology on X80 steel surface after removal of corrosion products; Figure 11 CLSM images and pitting depths of X80 square samples after 7 days of cultivation in D. vulgar bacterial culture with different diffusion areas; Figure 12 The pitting depth curves of X80 tensile specimens after 7 days of cultivation in D. vulgar bacterial culture with different diffusion areas; Figure 13 The curves showing the change of open circuit potential of X80 steel over time under different coverage areas; Figure 14 EIS spectra (including Nyquist and Bode plots) of X80 steel on days 1, 3, and 7 under different coverage areas. Figure 15 For fitting Figure 14 Equivalent circuit model of EIS data; Figure 16 The trend of linear polarization resistance of X80 steel over time under different coverage areas; Figure 17 Potential dynamic polarization curves of X80 steel on day 7 under different coverage areas; Figure 18 A comparison of the stress-strain curves of the original X80 steel with different corrosion coverage areas; Figure 19 Comparison of the reduction of area after tensile fracture of X80 steel with different coverage areas; Figure 20 A summary table of mechanical property losses of X80 steel under different coverage areas. Detailed Implementation

[0017] Microbiologically Influenced Corrosion (MIC) is a process in which microorganisms form biofilms on the surface of metals, and then significantly accelerate the dissolution of metal anodes through extracellular electron transfer (EET), deposition of metabolic products (H2S, H2, organic acids, etc.), or local hypoxia / acidification mechanisms. In the oil and gas transportation environment, sulfate-reducing bacteria (SRB), especially Desulfovibrio vulgaris (D. vulgaris), are considered to be the primary culprits of pitting corrosion, stress corrosion cracking (SCC), and hydrogen-induced cracking (HIC) of X80 pipeline steel, due to their facultative anaerobic, high hydrogen sulfide production rate, and ability to form conductive biofilms. Field failure cases show that only 1 µm deep microbial pitting can become a "trap" for hydrogen accumulation, inducing micro-cracks under subsequent service stress, and reducing the critical stress intensity factor by more than 20 %. However, most current laboratory studies still remain at the level of binary comparison of "with / without bacteria" or "with / without inhibitors", and lack quantitative description of the key parameter of biofilm spatial distribution, making it difficult to correspond the research results with the complex morphology in the field.

[0018] Existing MIC experimental devices usually use anaerobic bottles or reaction kettles with fixed volume, and the attachable area between the metal sample and the container wall is determined by the geometric size once and for all, and cannot form an area gradient within the same batch. If the number of test pieces is increased or decreased to change the area, new variables such as "anode / cathode area ratio" and "solution resistance distribution" are introduced, making the data interpretation complicated. Some scholars try to use porous ceramic or sponge carriers to increase the surface area, but the pore size distribution is uneven, the dead volume cannot be ignored, and the carrier itself may adsorb H2S or release ions, interfering with the corrosion electrochemical signal. More importantly, the biofilm thickness and area are inversely coupled: the smaller the area, the more bacteria are forced to accumulate, the thicker the biofilm, and the stronger the EET-MIC effect; the larger the area, the thinner the film, but the total H2S gas production increases, and the hydrogen penetration path increases. The "area-thickness-gas production" three-way coupling effect has not been systematically quantified, and an "area control" experimental method that can accurately set, repeat, and interfere with the area is needed to independently study the influence of the area single factor on microbial corrosion and subsequent mechanical degradation under the premise of constant bacteria amount, constant nutrition, and constant volume.

[0019] The present scheme provides a microbial corrosion control method based on biofilm attachment area regulation, comprising: a) under the condition of keeping the volume of liquid medium and the strain unchanged, introducing solid fillers with different specific surface areas into a closed anaerobic reactor to form multiple attachable areas; Optionally, the solid fillers include epoxy resin solid blocks, single-particle-size glass beads, or a combination of two or more particle sizes of glass beads.

[0020] Optionally, by adjusting the glass bead diameter 2r and the number n, the total specific surface area A = 4πr²n of different closed anaerobic reactors forms a multi-stage attachable area, and the liquid medium and headspace volume remain constant to exclude the interference of volume effect on the area variable.

[0021] Optionally, the closed anaerobic reactor forms a "small, medium, large" three-stage attachable area. Of course, other multi-stage attachable area schemes are also within the protection scope of the present application.

[0022] Specifically, by solid fillers, 100 cm², 390 cm², 640 cm² three-stage gradient attachable areas are obtained in a fixed volume of 50 cm³.

[0023] The three-stage gradient attachable area is precisely realized by the following combination: The small area stage does not add glass beads, only a 50 cm³ epoxy resin solid block, with a theoretical attachable area of 100 cm². The medium area stage adds glass beads with a diameter of 12 mm, a single bead volume of 0.904 cm³, a number of 55±1, a total glass bead volume of 50 cm³, and a theoretical attachable area of 390 cm². The large area stage adds glass beads with a diameter of 6 mm, a single bead volume of 0.113 cm³, a number of 442±2, a total glass bead volume of 50 cm³, and a theoretical attachable area of 640 cm².

[0024] Specifically, the small area stage only includes the reactor inner wall + liquid surface exposed area: A_S = πr² + 2πrh = π(2.5 cm)² + 2π(2.5 cm)(5.01 cm) ≈ 98.3 cm² ≈ 100 cm².

[0025] Specifically, the medium area stage adds the outer surface area of 12 mm glass beads based on the small area stage: Single bead radius r_M = 0.6 cm, single bead volume V_1M = (4 / 3)πr_M³ ≈ 0.904 cm³; Number of beads n_M = 50 cm³ / 0.904 cm³ ≈ 55.3 ≈ 55; Total bead surface area A_beads,M = 55 × 4πr_M² = 55 × 4π(0.6 cm)² ≈ 248.7 cm²; A_M = A_S + A_beads,M ≈ 98.3 + 248.7 ≈ 347 cm² (including the additional lateral area of ​​the sidewall increased to 7.64 cm, approximately 42 cm², totaling approximately 390 cm²).

[0026] Specifically, for large-area applications, 6 mm glass beads are used, with a single bead radius r_L = 0.3 cm and a single bead volume V_1L = (4 / 3)πr_L³ ≈ 0.113 cm³. The number of beads, n_L = 50 cm³ / 0.113 cm³ ≈ 442; The total surface area of ​​the beads, A_beads,L = 442 × 4πr_L² = 442 × 4π(0.3 cm)² ≈ 499.6 cm²; A_L = A_S + A_beads,L + Additional lateral surface area ≈ 98.3 + 499.6 + 42 ≈ 640 cm².

[0027] Of course, the above are just examples; other schemes that can form gradient attachment areas are also within the scope of protection of this application.

[0028] Optionally, the headspace to liquid volume ratio of the closed anaerobic reactor is fixed at 3:1, and the total pressure is recorded in real time to correct H2S and H2 concentrations and standardize hydrogen permeation flux.

[0029] b) Place the pretreated metal sample in the closed anaerobic reactor so that the sample surface is in contact only with the culture medium and the biofilm distribution area determined by the packing material. Optionally, before sample installation, the metal surface is uniformly polished to 600# and sterilized with ultraviolet light for 20 min to ensure that the initial surface state is consistent with the bacterial source, with only the area variable being a single-factor difference.

[0030] c) Cultivate and monitor biofilm growth, corrosion rate and gas production at the same temperature and time to establish a quantitative relationship between "biofilm distribution area – corrosion behavior – mechanical properties" and realize the directional control of the microbial corrosion process by adjusting the area parameter.

[0031] In this step, "synchronous monitoring" includes: a) Measure the open circuit potential (OCP) and linear polarization resistance (LPR) every 24 hours; b) Electrochemical impedance spectroscopy (EIS) was collected on days 1, 3, and 7; c) Measure the weight loss and pitting depth of the sample on days 3 and 7; d) Headspace H2S and H2 concentrations were measured on days 1, 3, and 7 and converted to dissolved [H2S]; e) On day 7, a uniaxial tensile test was performed to obtain the reduction of area and hydrogen embrittlement index.

[0032] Optionally, after step c), the method further includes: A three-dimensional calibration curve of "bacterial count per unit area – biofilm thickness – gas production" was established to predict the microbial corrosion rate and hydrogen embrittlement risk under any set area.

[0033] The method described in this application, under the premise of constant liquid culture medium volume and strain conditions, achieves gradient control of the biofilm adhesion area by introducing solid fillers with different specific surface areas, thus avoiding experimental errors caused by cross-interference of dependent variables in traditional methods. The metal sample only contacts the culture medium and the biofilm distribution area determined by the filler, ensuring the consistency and reproducibility of the corrosion environment and providing a stable and controllable experimental platform for studying microbial corrosion mechanisms. By simultaneously monitoring biofilm growth, corrosion rate, and gas production, a quantitative relationship between "biofilm distribution area – corrosion behavior – mechanical properties" is systematically constructed, providing data support for the prediction and assessment of microbial corrosion. This scheme can directionally change the biofilm distribution characteristics by simply adjusting the specific surface area of ​​the filler, thereby regulating the corrosion process and providing new ideas for optimizing the microbial corrosion resistance of materials and developing protective technologies. This method is applicable to a variety of metal materials and microbial systems, with simple experimental equipment, convenient operation, and good versatility and promotion value.

[0034] The following examples illustrate the specific experimental steps.

[0035] The bacteria used in this study were *Desulfovibrio vulgaris* (common desulfovibrio vibrio), the culture medium was ATCC1249 medium, and the experimental material was X80 steel. The bacterial culture apparatus was as follows: Figure 2 As shown: Bottle 1: Filled with epoxy resin, total volume 50 cm³, used after drying; surface area available for biofilm growth is approximately 100 cm². Bottle 2: Filled with glass beads with a diameter of 12 mm, with a total volume of 50 cm³; the surface area available for biofilm growth is approximately 390 cm². Bottle 3: Filled with glass beads with a diameter of 6 mm, with a total volume of 50 cm³; the surface area available for biofilm growth is approximately 640 cm².

[0036] Each culture medium bottle contains 100 mL of culture medium and has a headspace of 300 mL (see [link]). Figure 2 ).

[0037] X80 coupons: for weight loss, cell counting and morphology observation, with an exposed area of 10x10 mm, the rest was sealed with epoxy resin; X80 tensile coupons: prepared according to ASTM E8 / E8M standard, with an exposed area of 4 cm2, the rest was sealed with epoxy resin, and the surface was polished with 600# sandpaper.

[0038] All coupons were placed in the corresponding anaerobic bottles under sterile conditions, inoculated with D. vulgaris bacterial solution of the same initial concentration, sealed, and placed in a 37°C constant temperature incubator for static culture for 7 days. Part of the plate coupons and tensile coupons were taken out at the 3rd and 7th days for subsequent analysis. Before sampling, 10 mL of gas was extracted from the headspace of the anaerobic bottle by a gas sampler for H2S and H2 concentration determination.

[0039] The removed coupons were first sterilized by ultraviolet light for 30 minutes, then the surface was gently washed with PBS (pH 7.4) to remove planktonic cells and residual culture medium. The coupons for cell counting were further brushed with a sterile brush to remove the surface biofilm, and the suspension was vortexed uniformly for microscopic counting; the coupons for weight loss analysis were immersed in Clarke solution for 1 minute to completely remove the corrosion products and biofilm, and then dried and precisely weighed; the coupons for morphology observation were fixed and dehydrated, and then characterized by SEM and CLSM, respectively; the coupons for electrochemical testing were directly transferred into the electrolytic cell to maintain the surface state unchanged; the coupons for tensile testing were immediately tested for mechanical properties after washing to avoid the influence of drying on the results.

[0040] During the entire culture period, the open circuit potential (OCP) and linear polarization resistance (LPR) were monitored daily, the electrochemical impedance spectroscopy (EIS) test was performed on the 1st, 3rd and 7th days, and the potentiodynamic polarization curve measurement was completed on the 7th day. The tensile test was performed immediately after the end of the culture, and the stress-strain curve and fracture parameters were recorded. All experiments were set up in triplicate, and the results were expressed as mean ± standard deviation to ensure the statistical significance of the data.

[0041] The following will be described by specific experimental results.

[0042] 2.2 Cell counting experiment To evaluate the adhesion of *Desulfovibrio vulgaris* to X80 steel surfaces under different biofilm coverage areas, plate samples were collected on days 3 and 7 of culture. After UV sterilization, the samples were gently rinsed with phosphate-buffered saline (PBS, pH 7.4) to remove surface-dwelling cells and residual culture medium. Subsequently, the biofilm cells attached to the sample surface were brushed into centrifuge tubes containing 10 mL PBS using a sterile brush, and vortexed for 30 seconds to fully disperse the cells. The resulting cell suspension was counted using a hemocytometer under an optical microscope (400×). Three parallel samples were used for each experiment, and results are expressed as mean ± standard deviation to ensure data reliability and repeatability.

[0043] like Figure 3 The figure shows the statistical results of the number of cells attached to the surface of X80 steel samples on day 3 and day 7 of culture under different biofilm coverage areas (small, medium, and large). Figure 3 It can be seen that, under the same culture conditions, the smaller the biofilm coverage area, the more D. vulgaris cells are attached per unit area, indicating that spatial constraints promote the dense growth of bacteria in a smaller area.

[0044] 2.3 Determination of H2S and H2 gas concentrations To analyze the metabolic gas production of *D. vulgaris* under different biofilm distribution conditions, headspace gas was collected from the anaerobic flasks on days 1, 3, and 7 of culture, and the concentrations of hydrogen sulfide (H2S) and hydrogen (H2) were measured. Portable gas detectors (BW Technologies GAXT-H-DL for H2S and Forensics Detectors BH-90A for H2) were used for concentration readings. If the gas concentration exceeded the sensor range (H2S > 100 ppm, H2 > 1000 ppm), a dilution method was used for pretreatment: 10 mL of headspace gas was injected into a 250 mL sealed bottle containing 1 atm of air, and then 40 mL of gas was taken for detection. The total headspace pressure at each time point was recorded for subsequent calculation of dissolved gas concentrations.

[0045] Figure 4 The trend of dissolved H2S concentration in the culture medium of D. vulgaris over time is shown. Figure 5The change of H2 concentration in the headspace is shown. As can be seen from the figure, with the increase of biofilm coverage area, both the dissolved H2S and the headspace H2 concentration show an upward trend, indicating that a larger reaction interface promotes the generation and release of metabolic gases. Figure 6 is a mechanism diagram of the redox reaction area of the X80 square sample in the D. vulgaris culture solution with different diffusion areas (small; medium; large). As shown in the figure, a larger biofilm diffusion area provides a larger reaction surface area for H2S gas production, which helps to improve the production efficiency of H2S and H2. At the same time, as the biofilm diffuses, it becomes thinner. With the increase of diffusion area, more D. vulgaris effectively releases H2S. With the decrease of diffusion area, D. vulgaris tends to gather in a smaller space, which limits the activity of D. vulgaris at the bottom (purple), thereby reducing the production efficiency of H2S and H2. Figure 6

[0046] 2.4 Corrosion weight loss test To quantitatively evaluate the extent of microbial corrosion damage to X80 steel, samples were taken on the 3rd and 7th days for weight loss analysis. After ultraviolet sterilization, the samples were cleaned with Clarke solution (hydrochloric acid solution containing stannic chloride and antimony chloride) to completely remove surface corrosion products and biofilms. The cleaned samples were rinsed with deionized water, dried, and their mass change was measured using a precision electronic balance (precision 0.01 mg). Three parallel samples were set for each experiment, and the results were expressed in terms of unit area weight loss (mg / cm²), and the average value and standard deviation were calculated to evaluate the difference in corrosion rate.

[0047] Figure 7 The unit area weight loss results of X80 steel samples under different biofilm coverage areas on the 3rd and 7th days of culture are shown in the figure. As shown in the figure, at the early stage of culture (first 3 days), the weight loss under small area conditions is the largest, indicating that the EET-MIC mechanism dominates the corrosion process; while in the later stage (4-7 days), the weight loss under large area conditions increases rapidly, indicating that the H2S corrosion effect is enhanced.

[0048] 2.5 Biofilm and pitting morphology characterization To observe the structural characteristics and corrosion morphology of the biofilm on the surface of X80 steel under different biofilm coverage areas, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used for characterization. The samples were taken out on the 7th day of culture, pretreated by fixation and dehydration, and then observed by SEM and CLSM. SEM was used to analyze the microstructure of the biofilm surface and the morphology of the corrosion pits, and CLSM was used in combination with fluorescence staining technology to distinguish the distribution of live / dead cells, evaluate the thickness and coverage density of the biofilm. The related operation steps refer to the existing literature method. ​

[0049] Figure 8 The images are SEM images showing the differences in biofilm thickness and density under different coverage areas. The smaller the area, the thicker the biofilm and the denser the cell aggregation.

[0050] Figure 9 The image is a CLSM image, with green fluorescence representing live cells. The illustration shows that the number of live cells increases as the coverage area decreases, further validating the cell counting results.

[0051] Figure 10 The SEM image shows the pitting morphology after removing corrosion products, indicating that the pitting diameter is largest under small area conditions.

[0052] Figure 11 Confocal laser scanning microscopy (CLSM) images and pit depths of X80 square samples cultured in *D. vulgaris* for 7 days at different diffusion areas (small; medium; large); with... Figure 12 The figures show representative pit depth curves for X80 tensile specimens after 7 days of cultivation in D. vulgaris with different diffusion areas (small, medium, and large). The results show that the pitting depth is greatest (up to 10.7 μm) under small-area conditions, further confirming that the EET-MIC mechanism dominates localized corrosion.

[0053] 2.6 Electrochemical Testing To investigate the effect of biofilm coverage on the corrosion behavior of X80 steel, a three-electrode system was used for electrochemical testing. The working electrode was the X80 steel sample, the reference electrode was a saturated calomel electrode (SCE), and the auxiliary electrode was a platinum sheet. The sample exposure area was 10 mm × 10 mm, and the surface was polished with 600# sandpaper, ultrasonically cleaned, and UV sterilized before use. The test system was a 200 mL electrolytic cell containing 100 mL of culture medium and corresponding filler (epoxy resin or glass beads). The test items included: Open circuit potential (OCP): Monitored daily; Electrochemical impedance spectroscopy (EIS): Measurements were taken on days 1, 3, and 7. Linear polarization resistance (LPR): Daily measurement, scan range ±10 mV, scan rate 0.1667 mV / s; Potentiodynamic polarization curve: Measured on day 7, with a scan range of ±200 mV and a scan rate of 0.1667 mV / s.

[0054] All tests were conducted at room temperature, with the electrolytic cells sealed and stored in a 37°C incubator during the test intervals. EIS data were fitted using equivalent circuits to extract key parameters such as membrane resistance (R_f) and charge transfer resistance (R_ct) to evaluate corrosion rate and biofilm protection performance.

[0055] Figure 13 The open circuit potential (OCP) over time curve shows that the OCP is lower under the small area condition, and the corrosion tendency is stronger.

[0056] Figure 14 The EIS spectrum (Nyquist and Bode plots) shows that all samples exhibit double time constant and diffusion behavior, indicating that the biological membrane and corrosion product layer jointly affect the charge transfer process.

[0057] Figure 15 The equivalent circuit model for fitting the EIS data in FIG. 14 is shown in the figure; Figure 16 The linear polarization resistance (R_p) trend shows that the R_p value decreases significantly in the first 3 days, and the R_p is lowest under the small area condition, and the corrosion rate is fastest.

[0058] Figure 17 The potential dynamic polarization curve shows that the corrosion current density is maximum under the small area condition, further verifying that the corrosion is most serious.

[0059] 2.7 Tensile property test To evaluate the effect of microbial corrosion on the mechanical properties of X80 steel, the tensile specimen was taken out after the 7th day of culture and subjected to mechanical property test. After the specimen was cleaned with Clarke solution for 1 minute to remove the surface corrosion products and biological membrane, a uniaxial tensile test was performed using a MTS Landmark 370 universal testing machine. The test strain rate was 0.004 s -1 , until the specimen was broken, and the stress-strain curve was recorded. By comparing the tensile strength, elongation and reduction of area of the original specimen and the corroded specimen, the effect of microbial corrosion on the strength and plasticity of the material was evaluated. At least 3 parallel specimens were set for each group of experiments, the results were averaged, and the standard deviation was calculated to evaluate the data dispersion.

[0060] Figure 18 The stress-strain curve shows that the strength and elongation of all corroded specimens decrease compared with the original specimen (uncorroded), and the decrease is most significant under the small area condition.

[0061] Figure 19 The reduction of area comparison chart shows that the reduction of area gradually decreases with the increase of the biological membrane coverage area, indicating that the material brittleness increases.

[0062] Figure 20 The mechanical property loss summary table shows that the tensile strength loss under the small area condition reaches 13%, and the elongation loss reaches 13%, which is the most serious among all conditions.

[0063] The experimental results show that the larger the biofilm distribution area, the less the amount of D. vulgaris attached per unit area, the lower the corrosion rate, but the gas production (H2S, H2) increases significantly; the corrosion mechanism changes with time: in the early stage (first 3 days), electron transfer mediated microbial corrosion (EET-MIC) is dominant, and in the later stage (last 4 days), H2S corrosion is enhanced; the degradation of mechanical properties is negatively correlated with the biofilm area: large-area biofilm leads to more hydrogen atoms penetrating into the steel matrix, significantly reducing the ductility of X80 steel and increasing the brittleness; by regulating the biofilm distribution area, the corrosion rate and the degree of material embrittlement can be controlled in a targeted manner, providing an effective means for the prevention and control of microbial corrosion.

[0064] Finally, it should be noted that: the above is only the preferred embodiment of the present application, only for the description of the technical scheme of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application is included in the protection scope of the present application.

Claims

1. A method for controlling microbial corrosion based on the regulation of biofilm attachment area, characterized in that, include: a) While keeping the volume of liquid culture medium and the strain constant, introduce solid packing materials with different specific surface areas into a closed anaerobic reactor, which occupy a fixed total volume, to form a multi-level attachable area. b) Place the pretreated metal sample in the closed anaerobic reactor so that the sample surface is in contact only with the culture medium and the biofilm distribution area determined by the packing material. c) Cultivate and monitor biofilm growth, corrosion rate and gas production at the same temperature and time to establish a quantitative relationship between "biofilm distribution area – corrosion behavior – mechanical properties" and realize the directional control of the microbial corrosion process by adjusting the area parameter.

2. The method according to claim 1, characterized in that, The solid filler includes epoxy resin solid blocks, glass beads of a single particle size, or a combination of glass beads of two or more particle sizes.

3. The method according to claim 2, characterized in that, By adjusting the glass bead diameter 2r and the number n, the total specific surface area A=4πr²n of different closed anaerobic reactors is made into a multi-level attachable area, while the liquid culture medium and headspace volume remain constant, so as to eliminate the interference of volume effect on area variables.

4. The method according to any one of claims 1-3, characterized in that, The closed anaerobic reactor has three levels of attachable area: small, medium, and large.

5. The method according to claim 4, characterized in that, By using solid fillers, three levels of adherable area of ​​100 cm², 390 cm², and 640 cm² are obtained in a fixed volume of 50 cm³.

6. The method according to claim 5, characterized in that, The three-level attachable area is precisely achieved through the following glass bead-volume combination: a) Small area scale: No glass beads added, only 50 cm³ epoxy resin solid block, theoretical adhesion area of ​​100 cm²; b) Medium area level: Add 12 mm diameter glass beads, each bead has a volume of 0.904 cm³, with a quantity of 55±1 beads, a total glass bead volume of 50 cm³, and a theoretical adhesion area of ​​390 cm². c) Large area scale: Add 6 mm diameter glass beads, each bead has a volume of 0.113 cm³, 442±2 beads in total, a total glass bead volume of 50 cm³, and a theoretical adhesion area of ​​640 cm².

7. The method according to any one of claims 1–6, characterized in that, The headspace to liquid volume ratio of the closed anaerobic reactor is fixed at 3:1, and the total pressure is recorded in real time to correct H2S and H2 concentrations and standardize hydrogen permeation flux.

8. The method according to any one of claims 1–6, characterized in that, Following step c), the method further includes: A three-dimensional calibration curve of "bacterial count per unit area – biofilm thickness – gas production" was established to predict the microbial corrosion rate and hydrogen embrittlement risk under any set area.

9. The method according to any one of claims 1–6, characterized in that, In step c), "synchronous monitoring" includes: a) Measure the open circuit potential (OCP) and linear polarization resistance (LPR) every 24 hours; b) Electrochemical impedance spectroscopy (EIS) was collected on days 1, 3, and 7; c) Measure the weight loss and pitting depth of the sample on days 3 and 7; d) Headspace H2S and H2 concentrations were measured on days 1, 3, and 7 and converted to dissolved [H2S]; e) On day 7, a uniaxial tensile test was performed to obtain the reduction of area and hydrogen embrittlement index.

10. The method according to any one of claims 1-6, characterized in that, Before sample installation, the metal surface was uniformly polished to 600# and sterilized with ultraviolet light for 20 minutes to ensure that the initial surface condition was consistent with the bacterial source, with only the area variable being a single-factor difference.

Images