A method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface

Abstract

This invention relates to the field of microbial mineralization technology, providing a method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface. The method includes the following steps: S1, immersing at least two steel substrates in a test bacterial solution containing the bacteria to be tested, and incubating for 3 days; S2, detecting the calcium signal intensity on the substrate surface: taking at least one steel substrate and sequentially performing TPEN immersion treatment, calcium ion fluorescent probe immersion treatment, and room temperature incubation in the dark, then detecting the fluorescence signal intensity on the steel substrate surface; S3, detecting bacterial adhesion: taking at least one steel substrate and performing cleaning, crystal violet staining, cleaning, and immersion in anhydrous ethanol, then detecting the absorbance value of the anhydrous ethanol; S4, determining the ability of the test bacteria to form a calcium carbonate protective layer on the steel surface based on the fluorescence signal intensity and absorbance value. Compared to existing technologies, this invention, through the combined detection of "calcium signal at the steel substrate interface + bacterial adhesion," shortens the detection cycle by more than 57%.

Description

Technical Field

[0001] This invention relates to the field of microbial mineralization, and in particular to a method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface. Background Technology

[0002] In marine environments, most metal materials, such as carbon steel, face varying degrees of corrosion risks in seawater. While steel structures, used in ships and offshore platforms, offer lower economic costs, their corrosion resistance is poor. Existing anti-corrosion technologies primarily involve antifouling and anti-corrosion paints, but these are prone to causing chemical pollution in the ocean. Microorganisms readily adhere to material surfaces, forming biofilms. Some biofilms accelerate metal surface corrosion, particularly localized corrosion. However, some biofilms can maintain pH, ion activity, and organic matter concentration, inducing inorganic mineral nucleation to form a biomineralized layer covering the surface. This alters interfacial mass transfer and electrochemical reaction processes, effectively inhibiting corrosion.

[0003] Bacterial-mediated calcium carbonate mineralization in marine environments is the core mechanism for green protection of carbon steel, but the mineralization capacity of different bacteria varies significantly. Currently, the main steps for assessing bacterial mineralization capacity include: surface treatment of the steel surface such as grinding and cleaning, followed by immersion in a mineralization solution containing candidate bacteria for 7–14 days to allow the bacteria to grow and metabolize sufficiently on the steel surface to form a biofilm. Then, the surface morphology, thickness, and corrosion resistance of the biofilm are detected by scanning electron microscopy, electrochemical impedance spectroscopy, and dynamic potential polarization to evaluate the mineralization performance of the candidate bacteria.

[0004] However, this method has the following significant drawbacks: the detection cycle is long, requiring more than 7 days to observe obvious mineralization signals; the detection method is complex; and the throughput of a single screening is low, which cannot meet the needs of rapid screening of strains with high mineralization capacity. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to overcome the defects or deficiencies of the prior art and provide a rapid preparation process for marine microbial mineralization layers.

[0006] This invention is achieved through the following technical solution:

[0007] A method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface includes the following steps: S1. Inoculate the bacteria to be tested into a liquid culture medium to obtain the bacterial solution to be tested; immerse at least two steel substrates in the bacterial solution to be tested and incubate them statically for 3 days; S2. Detection of calcium signal intensity on the substrate surface: Take at least one steel substrate and sequentially clean it, soak it in N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) solution, clean it, immerse it in a solution containing a calcium ion fluorescent probe, incubate it at room temperature in the dark, and then detect the fluorescence signal intensity D on the surface of the steel substrate. S3. The bacterial adhesion OD of the test bacteria was determined using the crystal violet staining method. 595 ; S4. Determine the ability of the bacteria to form a calcium carbonate protective layer on the steel surface based on the fluorescence signal intensity D in step S2 and the absorbance value in step S3. Steps S2 and S3 are not in any particular order.

[0008] Compared to existing technologies, this invention, through a combination of indicators—"calcium signal at the steel substrate interface + bacterial adhesion"—shortens the detection cycle from 7 days to 3 days, a reduction of over 57%. Simultaneously, this three-indicator combination effectively avoids the problem of misinterpretation in existing technologies.

[0009] In one embodiment, in step S4, when the bacteria to be tested simultaneously meet the following conditions (a1) and (a2), it is determined that the bacteria to be tested can form a calcium carbonate protective layer on the steel surface: (a1) Calcium signal intensity D on the matrix surface > 140 au / mm 2 ; (a2) Bacterial adhesion OD 595 ≤0.5.

[0010] In one embodiment, the calcium ion fluorescent probe is Fluo-4 with a concentration of 3~7 µg / mL.

[0011] In one embodiment, the concentration of the TPEN solution is 5~15 µM.

[0012] In one embodiment, in step S4, when the bacteria to be tested simultaneously meet the following conditions (a1), (a2), and (a3), it is determined that the bacteria to be tested can form a calcium carbonate protective layer on the steel surface: (a1) Fluorescence signal intensity D > 140 au / mm 2 ; (a2) Absorbance value OD 595 ≤0.5; (a3) Calcium signals are uniformly dispersed on the surface of the steel substrate.

[0013] In one embodiment, in step S4, when the bacteria to be tested meet conditions (b1) and (b2), it is determined that the bacteria to be tested cannot form a calcium carbonate protective layer on the steel surface. (b1) Fluorescence signal intensity D≤30 au / mm 2 ; (b2) Absorbance value OD 595 ≥0.8.

[0014] In one embodiment, in step S4, when the bacteria to be tested meet conditions (c1) and (c2), it is determined that the bacteria to be tested have a weak ability to form a calcium carbonate protective layer on the steel surface. (c1) Either condition (a1) or (a2) is not satisfied; (c2) Either of conditions (b1) or (b2) is not satisfied.

[0015] In one embodiment, in step S1, the liquid culture medium is 2216E liquid culture medium, and the calcium content of the liquid culture medium is 1~3 g / L, OD 600 It ranges from 0.7 to 0.9.

[0016] In one embodiment, the steel substrate sample is made of Q235 marine steel.

[0017] In one embodiment, step S1 further includes: polishing the sample surface until there are no obvious scratches, cleaning it with alcohol, drying it, and then immersing it in the bacterial solution to be tested and incubating it at 20~24 ℃.

[0018] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0019] Figure 1 The calcium signals of VN bacteria and PD bacteria on the steel surface and in the test bacterial solution after static culture for 3 days in Example 1 are shown. Among them, a1 and a2 represent the calcium signals of VN bacteria and PD bacteria on the steel surface, respectively; b1, b2, and b3 represent the calcium signals of the matrix PD bacteria (green), the bacteria themselves (red), and the fusion diagram, respectively; and c1 and c2 represent the calcium signals of VN bacteria and PD bacteria in the test bacterial solution, respectively.

[0020] Figure 2 The image shows the morphology of the steel surface after 7 days of static culture in Example 1. Here, a1~a3 and b1~b3 represent the morphology formed on the steel surface by VN bacteria after 7 days and the morphology formed on the steel surface by PD bacteria after 7 days (whether or not calcium carbonate is formed).

[0021] Figure 3 In Example 1, the product components formed on the steel surface by VN bacteria and PD bacteria within 7 days (V.sp represents VN bacteria, P.sp represents PD bacteria).

[0022] Figure 4 The Ca after removing the TPEN working fluid in Example 22+ Signal and bacterial detection status, where green represents Ca. 2+ Signal, red indicates bacteria.

[0023] Figure 5 In Example 3, the calcium ion detection time was changed to the second day. 2+ Signal detection status.

[0024] Figure 6 The results of mineralization capacity testing for different strains in Example 4 are shown, where a and b represent... P. tetraodonis and V. fortis Calcium signal in steel matrix after 3 days of culture, c and d represent P. tetraodonis and V. fortis Calcium carbonate formed on the steel surface after 3 days of cultivation.

[0025] Figure 7 In Example 5, the Ca ion detection reagent was modified. 2+ Signal detection status.

[0026] Figure 8 In the test example, the corrosion resistance of the calcium carbonate protective layer in a high-salt environment is shown. In the test example, a1 and a2 represent sterile culture solutions, b1 and b2 represent VN bacteria, c1 and c2 represent Nyquist and Bode plots of steel substrates immersed in PD bacteria solution, d represents polarization curves of PD bacteria and VN bacteria, and e and f represent pitting corrosion on the substrate surface of PD bacteria and VN bacteria, respectively.

[0027] Figure 9 The surface morphology of Q235 after immersion in VN bacteria and PD bacteria for 3 days in the test case. Detailed Implementation

[0028] To improve strain screening efficiency, existing technologies attempt to monitor Ca²⁺ in mineralized solutions. + Indirect indicators such as concentration and pH changes are used to assess the mineralization capacity of the bacterial strain. However, while this method reduces the complexity of the detection process to some extent, it cannot shorten the culture period of the steel substrate in the bacterial solution (it still requires more than 7 days), and the accuracy of the assessment is also low. In-depth analysis reveals that the reasons for its insufficient accuracy mainly include the following three aspects: (1) The indicators have poor specificity and are prone to false positives and false negatives. Ca 2+ Decreased concentration and increased pH are not specific markers of biomineralization. Similar changes can occur due to non-mineralization processes such as nutrient consumption in the culture medium and bacterial metabolic acid / alkali production, leading to false positives. Furthermore, some highly mineralizing strains may not cause significant Ca2+ changes during the detection window due to their slower mineralization rate or higher solubility of mineralization products.2+ pH changes can also lead to false negatives.

[0029] (2) It cannot reflect the actual mineralization of the steel surface. The Ca in the solution phase... 2+ Consumption and pH changes can only indirectly infer the mineralization trend. They cannot distinguish whether the mineralization products are actually deposited on the steel surface, nor can they assess key quality parameters such as the crystallinity, density, coverage, and adhesion to the substrate of the mineralization layer—which directly determine the anti-corrosion effect of the mineralization layer.

[0030] (3) Inability to distinguish between biomineralization and chemical precipitation. In high-Ca environments... 2+ Under high pH conditions, calcium carbonate can spontaneously precipitate in the solution. Existing indirect indicators cannot effectively distinguish this abiotic process from bacterial-mediated biomineralization, further reducing the reliability of the assessment.

[0031] In view of the above-mentioned defects, the present invention proposes a new technical approach: by detecting the destination of calcium ions, it can be distinguished whether calcium ions are captured and enriched on the steel surface by bacteria, or whether most of them remain in the solution or precipitate at the bottom of the container, thereby predicting whether bacteria can form an effective calcium carbonate protective layer on the steel surface.

[0032] Based on this, the present invention proposes a method for detecting calcium signals in biofilms on steel surfaces: immersing the steel substrate in the bacterial solution to be tested and culturing it for a period of time, then using a fluorescent probe to label calcium ions on the biofilm on the surface of the steel substrate, and then judging or predicting the mineralization capacity of the bacteria to be tested by detecting the distribution of calcium ions.

[0033] However, during experiments, this invention revealed that although calcium signals were detected on the steel surface, the signals were dispersed, with no obvious calcium accumulation, inconsistent with the continuous mineralization layer observed by scanning electron microscopy (SEM). Analysis suggests this phenomenon may be due to the presence of other metal ions (such as Fe) in the tested bacterial solution that can bind to the fluorescent probe. 3+ These metal ions (etc.) produce non-specific fluorescence interference, leading to distorted colorimetric results.

[0034] To address this, the present invention eliminates interference from other metal ions in calcium signal detection and improves the specificity of calcium ion detection by adding other chemical solvents and changing the type of probe. After screening, treatment with TPEN followed by labeling with a Fluo-4 probe significantly improved the specificity and accuracy of the calcium signal.

[0035] After eliminating interference, this invention uses bacteria with different mineralization abilities to conduct mineralization experiments on steel for different times, and systematically detects the calcium signal intensity and distribution characteristics on the steel surface to explore the quantitative relationship between calcium signal distribution law, calcium signal intensity change and bacterial mineralization ability and biofilm corrosion protection performance.

[0036] Based on the research results, this invention classifies the mineralization ability of bacteria into "able to form calcium carbonate on the surface of steel substrate", "having a weak ability to form calcium carbonate on the surface of steel substrate" and "unable to form calcium carbonate on the surface of steel substrate".

[0037] The phrase "able to form calcium carbonate on the surface of a steel substrate" indicates that the tested bacteria can form a uniform, dense, and highly corrosion-resistant calcium carbonate protective layer on the surface of the steel substrate. Specifically, the thickness of the calcium carbonate protective layer formed after the steel substrate is immersed in the test bacterial solution for 7 days ranges from 5 to 10 μm, and its resistance to salt spray at 35 to 45 ℃ exceeds 500 h.

[0038] "Weak ability to form calcium carbonate on steel substrate surface" means that the tested bacteria can form a calcium carbonate protective layer on the steel substrate surface, but the protective layer is discontinuous, uneven, or has weak corrosion resistance. Specifically, the thickness of the calcium carbonate protective layer formed on the steel substrate after immersion in the tested bacterial solution for 7 days is less than 5 μm, and the salt spray resistance at 35~45 ℃ does not exceed 500 h.

[0039] "Cannot form calcium carbonate on steel substrate surface" means that bacteria cannot form a calcium carbonate protective layer on the surface of steel substrate.

[0040] Based on the classification of mineralization capacity, this invention attempts to establish a method and standard for assessing bacterial mineralization capacity through biofilm calcium signals. Results show that this method can significantly shorten the detection cycle from 7 days to 3 days, and its accuracy is higher than that of Ca... 2+ The pH index method has significantly improved performance, but a certain percentage of false positive and false negative results still exist.

[0041] Analysis revealed a limitation of using a single calcium signal indicator: although some bacteria accumulate Ca on the steel surface... 2+ However, due to weak adhesion leading to easy biofilm detachment, or insufficient enzyme activity resulting in weak mineralization driving force, effective continuous mineralization layer cannot be formed in the end. Such bacteria may have a strong calcium signal, but their actual mineralization ability is weak (false positive). Conversely, some bacteria may have high enzyme activity and strong mineralization driving force, but due to weak adhesion and difficulty in colonizing the steel surface, their calcium signal may be low, but their actual mineralization potential is not bad (false negative).

[0042] To this end, this invention systematically screened and analyzed the correlation of multiple potential indicators, such as bacterial motility, bacterial adhesion, urease activity, carbonic anhydrase activity, EPS production, biofilm surface charge, and solution turbidity. Ultimately, it was determined that bacterial adhesion is the indicator with the strongest complementarity to calcium signals and the simplest operation. It can be combined with calcium signals to construct a dual-indicator judgment model, which can eliminate false positives and false negatives to the greatest extent while shortening the detection cycle and simplifying the detection method.

[0043] Based on the above research, this invention finally proposes a method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface, comprising the following steps: S1. Pretreatment: Inoculate the bacteria to be tested into a solution containing calcium ions (Ca). 2+ In the seawater liquid culture medium, the bacterial concentration was adjusted to OD0.05 600 The concentration is approximately 0.5~1.0, which yields the bacterial solution to be tested.

[0044] Meanwhile, after grinding, cleaning, and drying, the steel substrate sample was immersed in the bacterial solution to be tested and incubated at 25-30 ℃ for 3 days.

[0045] S2. Detection of calcium signal intensity on the substrate surface: After cultivation, the steel substrate sample was removed, and the surface was gently rinsed with deionized water to remove loosely attached, non-deposited calcium salts. The calcium salts on the biofilm were then labeled with Fluo-4 Pentapotassium Salt (Fluo-4 Bio-enhanced). 2+ The distribution of bacteria was determined, and the total bacteria on the surface were visualized using PI working solution. After rinsing, the sample was treated with TPEN working solution for 5 minutes, which showed its effect on Fe... 3+ Heavy metal ions have a strong chelating ability, especially for Ca. 2+ The affinity is relatively weak. After rinsing three times with PBS, Fluo-4 working solution was added, and the mixture was incubated at room temperature in the dark for 15 min. The surface stain was then washed away with PBS buffer before microscopic examination. The concentrations of the storage solution and working solution, as well as the excitation and emission wavelengths (Ex / Em) of TPEN and Fluo-4, are shown in Table 1.

[0046] Table 1. Concentration of storage fluid and working fluid and excitation / emission wavelength

[0047] S3. The bacterial adhesion OD of the test bacteria was determined using the crystal violet staining method. 595 : A modified 96-well plate crystal violet titration method was used to determine bacterial adhesion ability. 200 µL of OD200 was added... 595 Bacterial culture medium at a concentration of approximately 0.5–1.0 μL was inoculated into 96-well plates. After incubation at 22 °C, the bacterial culture was removed, and the plates were washed three times with PBS buffer. The plates were then stained with 1.0 wt.% crystal violet solution for 15 min. After washing away any excess stain, the adhering crystal violet stain was eluted with anhydrous ethanol, and the OD was measured. 595 (Subtract the blank control).

[0048] S4, Combination Determination: Based on the interfacial calcium signal intensity obtained in step S2 and the bacterial adhesion obtained in step S3, the following rules are used to determine whether bacteria can form a calcium carbonate protective layer on the steel surface: The bacteria to be tested are deemed to "be able to form calcium carbonate on the surface of a steel substrate" when they simultaneously meet the following conditions: (a1) Calcium signal intensity D on the matrix surface > 140 au / mm 2 ; (a2) Bacterial adhesion OD 595 ≤0.5.

[0049] When the bacteria to be tested meet conditions (b1) and (b2), it is determined that the bacteria to be tested "cannot form calcium carbonate on the surface of the steel substrate": (b1) Fluorescence signal intensity D≤30 au / mm 2 ; (b2) Absorbance value OD 595 ≥0.8.

[0050] When the test bacteria meet conditions (c1) and (c2), the test bacteria are judged to have "a weak ability to form calcium carbonate on the steel substrate surface": (c1) Either condition (a1) or (a2) is not satisfied; (c2) Either of conditions (b1) or (b2) is not satisfied.

[0051] Steps S2 and S3 are not in any particular order.

[0052] The calcium signal intensity is expressed as the average fluorescence intensity per unit area (au / mm²). 2 ).

[0053] Furthermore, in order to improve the accuracy of the method, the conditions for determining that the bacteria to be tested "can form calcium carbonate on the surface of the steel substrate" also include: (a3) ​​the calcium signal is uniformly dispersed on the surface of the steel substrate.

[0054] Furthermore, the present invention also provides a method for verifying the determination result, comprising the following steps: The culture was extended to day 7 under the same conditions. Scanning electron microscopy was used to observe whether a continuous calcium carbonate protective layer formed on the steel surface to verify the accuracy of the judgment rule in step S4. The steel sample, after soaking for 7 days, was rinsed once with deionized water and fixed in 2.5% glutaraldehyde solution for 20–40 min. Then, it was dehydrated sequentially in 30%, 50%, 70%, 80%, 90%, and anhydrous ethanol for 10–20 min. After the sample was dried, gold was sputtered onto the sample surface for 60–90 s using a vacuum sputtering instrument. The corrosion morphology / biomineralization layer structure on the steel sample surface was observed using scanning electron microscopy.

[0055] Compared to the closest existing technology (the traditional MIP method requires more than 7 days of cultivation and SEM morphology observation), this invention shortens the detection cycle from 7 days to 3 days, a reduction of more than 57%, through a three-indicator combination detection of "calcium signal intensity at the steel substrate interface + calcium signal distribution + bacterial adhesion." Its working principle is as follows: the calcium signal on the steel surface directly reflects the ability of bacteria to enrich calcium ions and induce nucleation at the steel-solution interface (mineralization ability); adhesion reflects the ability of bacteria to reach and colonize the steel surface (colonization ability). Their synergistic effect determines whether a mineralized layer can form, and both can be detected within 3 days, without waiting for the mineralized layer to grow to an observable thickness. The experiment in Example 1 shows that the judgment results on the 3rd day are completely consistent with the SEM verification results on the 7th day. At the same time, the three-indicator combination effectively avoids the problem of misjudgment in existing technologies.

[0056] Unless otherwise specified, all experimental materials used in the embodiments of this invention can be obtained from commercially available sources. P. distincta , V. neocaledonicus, P. tetraodonis, V. fortis It can be obtained through commercial channels or from the Third Institute of Oceanography (MCCC) of the Ministry of Natural Resources.

[0057] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0058] Example 1 This example uses the following strain: P. distincta (hereinafter referred to as PD bacteria) V. neocaledonicus (hereinafter referred to as VN bacteria) is the bacteria to be tested, and the operation steps and judgment rules of the method of the present invention are fully demonstrated.

[0059] Among them, the bacteria to be tested, PD bacteria and VN bacteria, were both isolated from the waters off Guishan Island in the South China Sea.

[0060] The steel substrate sample was made of Q235 carbon steel, processed into a 10 mm × 10 mm × 5 mm sheet, and polished with 120# and 1000# sandpaper in sequence. It was then ultrasonically cleaned with anhydrous ethanol for 5 min, rinsed with deionized water, and dried for later use.

[0061] The culture medium was 2216E liquid medium with a calcium content of 1.8 g / L and a pH of 7.0-7.2. It was sterilized at 121 °C for 15 min.

[0062] S1. Pretreatment: Co-culture of bacterial culture and sample.

[0063] Each strain was inoculated into the above-mentioned culture medium and cultured at 22 ℃ with shaking at 120 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to OD. 600=0.8. Take 1 mL of bacterial culture and place it in 200 mL of culture medium. Immerse 3 steel substrate samples in each bottle and incubate at 22 °C for 3 days.

[0064] S2. Detection of calcium signal at the steel matrix interface: On day 3 of cultivation, the steel sheet was removed and gently rinsed three times with deionized water. It was then treated with TPEN working solution (10 µM) for 5 min to determine its effect on Fe. 3+ Heavy metal ions have a strong chelating ability, especially for Ca. 2+ The affinity was relatively weak. After rinsing three times with PBS, Fluo-4 working solution (concentration 5 µg / mL) was added, and the mixture was incubated at room temperature in the dark for 15 min. After washing away the excess color with PBS, the calcium signal on the steel surface was detected using CLSM. The calcium intensity was expressed as the fluorescence signal intensity. Analysis using CLSM Ceiss software showed that the D of the PD bacteria on the substrate was approximately 162.7 au / mm². 2 The D of VN bacteria is approximately 18.2 au / mm. 2 Ca 2+ Distribution details are shown below. Figure 1 .

[0065] S3, Bacterial Adhesion Detection: 200 µL OD 595 Bacterial culture medium with a concentration of approximately 0.5–1.0 μL was inoculated into 96-well plates and incubated at 22 °C for 24–48 h. The bacterial culture was then removed, and the plates were washed three times with PBS buffer. The plates were stained with 1.0 wt.% crystal violet solution for 15 min, washed with deionized water to remove excess stain, and the crystal violet bound to the bacteria was dissolved in anhydrous ethanol. The OD value was then measured using a microplate reader. 595 Absorbance value. Result: OD of PD bacterial group. 595 =0.35±0.03; OD of VN bacterial group 595 =1.30±0.08.

[0066] S4, Combination Determination: PD bacteria meet the following criteria: D > 140 au / mm 2 OD 595 <0.5, therefore PD bacteria are identified as bacteria capable of forming a protective calcium carbonate layer on the substrate surface. VN bacteria meet the following condition: D < 30 au / mm 2 OD 595 >0.8, therefore VN bacteria are determined to be unable to form calcium carbonate on the substrate surface.

[0067] Then, this embodiment verified the above judgment results through an experiment: the two groups were cultured for another 7 days, the steel sheets were removed, gently rinsed with deionized water, and the surface morphology was observed using a scanning electron microscope (SEM), and the sediment phase was analyzed by X-ray diffraction (XRD).

[0068] See Figure 2 and Figure 3 The surface of PD bacteria steel is completely covered by a continuous and dense crystalline layer with typical rhombohedral morphology. XRD confirmed that it is calcite-type calcium carbonate. The surface of VN bacteria steel only has corrosion products, and most areas still expose the metal matrix. XRD showed that it did not form continuous calcium carbonate.

[0069] Example 2 Based on Example 1, while keeping the concentrations of other raw materials and the preparation process unchanged, step S2 does not use TPEN working solution.

[0070] The detection results of step S2 are shown below. Figure 4 The detection results of step S3 showed that the OD of the PD bacterial group 595 =0.56±0.10, OD of VN bacterial group 595 =1.21±0.12.

[0071] like Figure 4 As shown, the calcium signal on the substrate surface is blurred, which has a certain impact on the calculation of fluorescence intensity: no obvious calcium aggregation is observed on the substrate surface. Although there is a calcium signal, the calcium signal is in a dispersed state, which does not conform to the rules of crystal nucleation. Therefore, although the detection result can calculate the calcium ion signal, it has no reference value. It may be caused by the dye solution combining with other metal ions.

[0072] Therefore, the purpose of adding TPEN working solution is to chelate other ions and prevent them from affecting Ca. 2+ This has an impact on the removal of TPEN working fluid from Ca. 2+ The detection results of the signal have a significant impact.

[0073] Example 3 Based on Example 1, while keeping the concentrations of other raw materials and the preparation process unchanged, the cultivation time for steps S2 and S3 is the second day.

[0074] The result of step S2 is shown below. Figure 5 The results showed that the calcium signal on the substrate surface was weak at this time, and it was impossible to statistically analyze the fluorescence intensity of the calcium signal.

[0075] The results of step S3 showed that the OD of the PD bacterial group 595 =0.45±0.04, OD of VN bacterial group 595= 0.94±0.08. Compared with the results of Example 1, the bacterial adhesion was still in a continuous upward phase on the second day, so the second day is not suitable as the optimal time to detect adhesion.

[0076] Based on steps S2-S3, proceed to step S4, combination determination: PD bacteria have a weak calcium signal, i.e., D < 30 au / mm 2 OD 595 The value was <0.5, therefore PD bacteria were determined to have a weak ability to form calcium carbonate on the surface of the steel substrate.

[0077] VN bacteria have a weak calcium signal, i.e., D < 30 au / mm 2 OD 595 >0.8, therefore VN bacteria are determined to be unable to form calcium carbonate on the substrate surface.

[0078] As can be seen, on the second day, the various indicators were not yet stable (weak calcium signal, low adhesion), and judging the mineralization ability of the test bacteria at this time was prone to false positives; while on the third day, the various indicators tended to be stable (see Example 1). Therefore, the third day is the earliest reliable judgment time point of the method of the present invention.

[0079] Example 4 Based on Example 1, while keeping other raw material concentrations and preparation processes unchanged, the strain in step S2 was replaced with... P. tetraodonis (PT bacteria) and V. fortis (Abbreviated as VF bacteria).

[0080] The result of step S2 is shown below. Figure 6 Analysis using CLSM Ceiss software showed that the concentration of PT bacteria in the matrix was approximately 76.2 au / mm. 2 The D value of VF bacteria is approximately 17.5 au / mm. 2 .

[0081] The results of step S3 showed that the OD of the PT bacterial group... 595 =0.39±0.09, OD of VF bacterial group 595 =0.91±0.02.

[0082] Based on the results of steps S2-S3, proceed to step S4, combination determination: The calcium signal of PT bacteria is weak, at 30 au / mm. 2 <D<140 au / mm 2 OD 595 <0.5, therefore, PT bacteria are judged to have a weak ability to form calcium carbonate on the surface of steel substrate.

[0083] VF bacteria have a weak calcium signal, i.e., D < 30 au / mm 2 OD 595 >0.8, therefore VF bacteria are determined to be unable to form calcium carbonate on the substrate surface.

[0084] Then, this embodiment conducted a verification experiment to verify the above judgment results: the two groups were cultured until the 7th day, the steel sheets were taken out, gently rinsed with deionized water, and the surface morphology was observed using a scanning electron microscope (SEM).

[0085] The results proved that the determination was accurate and could be applied to the screening of other strains.

[0086] Example 5 Based on Example 1, while keeping the concentrations of other raw materials and the preparation process unchanged, the calcium ion detection reagent in step S2 was replaced with 5 µM Fura-2 / AM working solution (Beyotime).

[0087] The detection results of step S2 are as follows Figure 7 As shown, the D of the matrix PD bacteria, analyzed by CLSM Ceiss software, was approximately 43.1 au / mm. 2 The D value of VN bacteria is approximately 48.6 au / mm. 2 .

[0088] The test results in step S3 showed that the OD of PD bacteria... 595 =0.40±0.07, OD of VN bacterial group 595 =1.37±0.11.

[0089] S4, Combination Determination: PD bacteria content: 30 au / mm 2 <D<140 au / mm 2 OD 595 The value was <0.5, therefore PD bacteria were determined to have a weak ability to form calcium carbonate on the surface of the steel substrate.

[0090] VN bacteria meet the following standard: 30 au / mm 2 <D<140 au / mm 2 OD 595 >0.8, therefore VN bacteria are determined to be unable to form calcium carbonate on the substrate surface.

[0091] The above results show that, compared with Example 1, the Fura-2 / AM fluorescent probe cannot effectively distinguish between PD bacteria and VN bacteria, and cannot accurately determine the mineralization ability of PD bacteria.

[0092] Test case This embodiment verifies that PD bacteria, which are determined by the present invention to be able to form a protective layer, do indeed have an antiseptic effect, while VN bacteria, which are determined not to form a protective layer, do not have an antiseptic effect, thus proving the practical value of the method of the present invention.

[0093] PD bacteria and VN bacteria were inoculated into the culture medium of Example 1, and cultured at 22 °C with shaking at 120 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to OD. 600 =0.8. Take 1 mL of bacterial culture and place it in 200 mL of culture medium. Immerse 3 steel substrate samples in each bottle of culture medium and incubate at 22 °C.

[0094] The corrosion resistance was evaluated using both electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. After copper wire bonding, the samples were embedded in epoxy resin until they were exposed to the working surface (1 cm). 2 The sample was immersed in bacterial culture medium. EIS was tested in situ using a three-electrode system, with the sample as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum sheet as the counter electrode. An electrochemical workstation (Parstat IM6e, Princeton Applied Research, USA) was used, applying a 300-s sinusoidal perturbation at open-circuit potential (OCP) with a frequency range of 10... -2 Hz to 10 5 Hz. EIS impedance was measured on days 1, 3, 5, and 7. The EIS data were fitted and analyzed using ZSimpWin. Potentiodynamic polarization curves were tested in the range of -1.5 V to +1.0 V at a scan rate of 2 mV / s.

[0095] In the sterile control, the overall changes in Nyquist and Bode responses were limited within 1–7 days, and the interfacial charge transfer resistance remained on the same order of magnitude, indicating that the system was dominated by uniform corrosion. Figure 8 a1 and Figure 8 a2). The radius of the impedance arc on the matrix surface of VN bacteria first increased and then decreased significantly over time, and |Z| decreased significantly. The fitted charge transfer resistance / polarization resistance continued to decrease (a2). Figure 8 b1 and Figure 8 (b2) This indicates that early bacterial attachment and film formation may have weakened the charge transfer rate, but the subsequent surface film accelerated the anodic and cathodic reactions. PD bacteria maintained a high impedance level, with larger and more stable fitted resistance parameters. Figure 8 c1 and Figure 8 c2). See also Figure 8 d~ Figure 8 f, the increased corrosion current density of VN bacteria indicates enhanced local corrosion sensitivity. After removing surface products, the substrate surface of PD bacteria is more uniform and smooth, while that of VN bacteria ( Figure 8Typical pitting (approximately ~31 μm deep) can be observed on the substrate surface in f), which reflects accelerated pitting induced by local film rupture.

[0096] See Figure 9 The results showed that on day 3, neither of the two bacterial strains produced calcium carbonate. This, combined with the corrosion test, supports the rationality of choosing day 3 as the judgment time in this invention. On day 1, the two bacterial strains had high impedance due to vigorous biofilm growth, which could lead to false positives. After 3 days, the biofilm entered a mature and stable period. Bacteria capable of colonizing the steel surface and having the ability to complex calcium ions began to form amorphous calcium carbonate, which has a certain inhibitory effect on corrosion. However, amorphous calcium carbonate is unstable, lacks a typical structure, and is essentially undetectable. Meanwhile, the biofilms of bacteria unable to bind calcium ions to the steel surface could not form a protective layer, resulting in a significant decrease in impedance.

[0097] In summary, the present invention has the following advantages: (1) The testing cycle has been shortened from 7 days to 3 days, greatly improving screening efficiency; (2) Use a combination of two indicators, "calcium signal + adhesion", to avoid misjudgment caused by a single indicator (e.g., measuring only adhesion will misjudge highly adhesive VN bacteria as positive). (3) It does not rely on expensive large-scale equipment such as scanning electron microscopes and can be implemented in ordinary microbiology laboratories; (4) It can perform high-throughput quantitative operations and is suitable for large-scale strain screening.

[0098] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for rapidly determining whether bacteria can form a calcium carbonate protective layer on a steel surface, characterized in that, Includes the following steps: S1. Inoculate the bacteria to be tested into a liquid culture medium to obtain the bacterial solution to be tested; immerse at least two steel substrates in the bacterial solution to be tested and incubate them statically for 3 days; S2. Detection of calcium signal intensity on the substrate surface: Take at least one steel substrate and clean it in sequence, soak it in TPEN solution, clean it, immerse it in a solution containing calcium ion fluorescent probe, incubate it at room temperature in the dark, and then detect the fluorescence signal intensity D on the surface of the steel substrate. S3. The bacterial adhesion OD of the test bacteria was determined using the crystal violet staining method. 595 ; S4. Determine the ability of the bacteria to form a calcium carbonate protective layer on the steel surface based on the fluorescence signal intensity D in step S2 and the absorbance value in step S3. Steps S2 and S3 are not in any particular order.

2. The preparation process according to claim 1, characterized in that, In step S4, when the bacteria to be tested simultaneously meet the following conditions (a1) and (a2), it is determined that the bacteria to be tested can form a calcium carbonate protective layer on the steel surface: (a1) Fluorescence signal intensity D > 140 au / mm 2 ; (a2) Absorbance value OD 595 ≤0.

5.

3. The method according to claim 2, characterized in that, The calcium ion fluorescent probe is Fluo-4, with a concentration of 3~7µg / mL.

4. The method according to claim 3, characterized in that, The concentration of the TPEN solution is 5~15 µM.

5. The method according to claim 2, characterized in that, In step S4, when the bacteria to be tested simultaneously meet the following conditions (a1), (a2), and (a3), it is determined that the bacteria to be tested can form a calcium carbonate protective layer on the steel surface: (a1) Fluorescence signal intensity D > 140 au / mm 2 ; (a2) Absorbance value OD 595 ≤0.5; (a3) Calcium signals are uniformly dispersed on the surface of the steel substrate.

6. The method according to claim 2, characterized in that, In step S4, when the bacteria to be tested meet conditions (b1) and (b2), it is determined that the bacteria to be tested cannot form a calcium carbonate protective layer on the steel surface. (b1) Fluorescence signal intensity D≤30 au / mm 2 ; (b2) Absorbance value OD 595 ≥0.

8.

7. The method according to claim 6, characterized in that, In step S4, when the bacteria to be tested meet conditions (c1) and (c2), it is determined that the bacteria to be tested have a weak ability to form a calcium carbonate protective layer on the steel surface. (c1) Either condition (a1) or (a2) is not satisfied; (c2) Either of conditions (b1) or (b2) is not satisfied.

8. The method according to any one of claims 1 to 7, characterized in that, In step S1, the liquid culture medium is 2216E liquid culture medium, and the calcium content of the liquid culture medium is 1~3 g / L, OD 600 It ranges from 0.7 to 0.

9.

9. The method according to claim 7, characterized in that, The steel substrate sample was made of Q235 marine steel.

10. The method according to claim 7, characterized in that, Step S1 further includes: after polishing the sample surface until there are no obvious scratches, cleaning it with alcohol and drying it, then immersing it in the bacterial solution to be tested and incubating it at 20~24 ℃.

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