Inhibition bacterium for inhibiting sulfate reducing bacteria and anti-corrosion application thereof

Abstract

The invention discloses an inhibiting bacterium for inhibiting sulfate reducing bacteria and anti-corrosion application thereof, and belongs to the field of microbial anti-corrosion technology and application of oilfield reinjection water, the inhibiting bacterium is bacillus licheniformis NRB-12, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 24531. The bacillus licheniformis disclosed by the invention is used for treating oilfield reinjection water with high sulfate reducing bacteria content, and has the characteristics of environment friendliness, low price, large treatment range and simplicity in operation; effective inhibition of sulfate reducing bacteria is achieved by providing a nutrient system optimized by nitrate reducing bacteria, and safe production of oil fields and oil extraction is achieved.

Description

[0001] This application is a divisional application of the invention patent application filed on April 25, 2022, with application number 202210438737.6 and entitled "An Inhibitor of Sulfate-Reducing Bacteria and Its Corrosion-Preventive Application". Technical Field This invention pertains to the microbial corrosion prevention technology and application of oilfield reinjection water, specifically involving inhibitory bacteria that inhibit sulfate-reducing bacteria. This invention also relates to the corrosion prevention application of these inhibitory bacteria. Background Technology

[0002] Microbiologically influenced corrosion (MIC) refers to corrosion of metals caused directly or indirectly by the life activities and metabolic products of various microorganisms. In nature, microorganisms form biofilms on various materials, providing an environment for microbial corrosion. The defense mechanisms of biofilms protect microorganisms from environmental damage, making it difficult for common bactericides to penetrate and achieve a bactericidal effect.

[0003] In oil and gas systems, oxidizing and non-oxidizing bactericides are widely used. While bactericides are simple to use and maintain, they cannot penetrate the polysaccharide membranes produced by microorganisms. Sulfate-reducing bacteria (SRB) sometimes coexist within these membranes, making sterilization difficult. In a long-term reducing environment with H2S, the sterilization efficiency of oxidizing bactericides decreases, failing to meet standards. Furthermore, prolonged use of large quantities of bactericides leads to antibiotic resistance in microorganisms, requiring increasingly higher concentrations to achieve the desired sterilization effect, thus increasing treatment costs. The toxicity of the bactericides themselves also causes environmental pollution. Biological competitive exclusion technology, with its environmentally friendly, low-cost, wide-ranging, and simple-to-operate characteristics, is an important method for controlling sulfate-reducing bacteria in oilfield water injection systems and reservoirs. Therefore, the use of biological competitive methods to inhibit sulfate-reducing bacteria growth is becoming increasingly common. Summary of the Invention

[0004] The purpose of this invention is to provide an inhibitory bacterium that inhibits the activity of sulfate-reducing bacteria. This bacterium can inhibit sulfate-reducing bacteria in oilfield reinjection water, thereby reducing corrosion caused by sulfate-reducing bacteria.

[0005] Another object of the present invention is to provide the application of inhibiting sulfate-reducing bacteria in oilfield reinjection water in inhibiting corrosion caused by sulfate-reducing bacteria in oilfield reinjection water.

[0006] The technical solution adopted in this invention is as follows: an inhibitory bacterium that inhibits the activity of sulfate-reducing bacteria, wherein the inhibitory bacterium is Bacillus licheniformis NRB-12, with accession number CGMCC No.24531.

[0007] A method for screening inhibitory bacteria that inhibit the activity of sulfate-reducing bacteria, comprising the following steps: S1: Enrich the inoculum and separate and purify the product; S2: Screening for NO3-containing compounds from isolated and purified cultures. - Bacteria with reducing properties; S3: By drawing a diagram with NO3 - Growth curves of bacteria with reducing properties were used to screen for bacteria that grow rapidly and have a short growth period. S4: Further screen the bacteria screened in step S3 by utilizing their antagonistic inhibitory effect with sulfate-reducing bacteria (SRB) to obtain inhibitory bacteria that inhibit the activity of sulfate-reducing bacteria. S5: Predict the inhibition rate of the inhibitory bacteria that inhibit the activity of sulfate-reducing bacteria screened in step S4 on the corrosion caused by sulfate-reducing bacteria using static corrosion plate experiments.

[0008] Preferably, the inoculum in step S1 is oilfield reinjection water; the enrichment culture method in step S1 is as follows: 5-15% by weight of the inoculum in a mixed NRB and SRB medium is added to a sterile mixed NRB and SRB medium and cultured at 30-35℃ and normal pressure for 3-5 days; then, 5-15% of the culture solution that has not turned black in the mixed medium is taken out and inoculated into a new NRB medium for enrichment culture, and the above culture steps are repeated 2-4 times; the separation and purification in step S1 are carried out by dilution and plating to separate single colonies.

[0009] Preferably, in step S2, samples containing NO3 are screened out. - The method for determining the reducing properties of bacteria is the nitrate reagent test. The nitrate reagent test involves sequentially inoculating the bacteria obtained from the isolation, purification, and screening in step S1 into NRB medium and culturing them. Then, the NO2 in the NRB medium is oxidized using Griess' reagent. - The NO3 in NRB culture medium was detected using diphenylamine reagent. - The test was conducted to screen out bacteria that tested positive, i.e., those containing NO3. -Nitrate-reducing bacteria. The NRB and SRB mixed culture medium (g / L) consisted of: NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, KNO3 3, NaSO4 4, sodium lactate 6 ml, and yeast extract 1, adjusted to pH 7.0-7.4. The nitrate-reducing bacteria (NRB) culture medium (g / L) consisted of: NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, KNO3 3, sodium lactate 6 ml, and yeast extract 1, adjusted to pH 7.0-7.4.

[0010] Preferably, the method for plotting the growth curve in step S3 is as follows: The growth curves selected in step S2 that exhibit NO3+ are plotted... - The bacteria with reducing properties were inoculated into fresh NRB medium. During the culture process, samples were taken every 10-14 hours. The NRB culture was centrifuged and washed, and then its optical density value (OD600) was measured using a UV-Vis spectrophotometer and its growth curve was plotted.

[0011] Preferably, step S4 utilizes the antagonistic inhibitory effect of nitrate-reducing bacteria and sulfate-reducing bacteria to further screen the bacteria screened in step S3. The specific steps of this method are as follows: S4-1: Determine the nutritional evaluation system for inhibiting sulfate-reducing bacteria; S4-2: After activation culture, the bacteria screened in step S3 are inoculated into the nutritional evaluation system for sulfate-reducing bacteria determined in step S4-1 in the same amount. Parallel and blank experiments are set up and cultured in a constant temperature incubator at 30-35℃. The concentration change of S2- is measured regularly every day to screen out bacteria with good inhibitory effect on sulfate-reducing bacteria.

[0012] Preferably, the specific method for determining the nutritional evaluation system for inhibiting sulfate-reducing bacteria is as follows: S4-11: By adding oilfield reinjection water samples to the sulfate-reducing bacteria culture medium at a weight ratio of (9:1)-(1:9), multiple experimental groups and corresponding control groups are designed; S4-12: The S2- content in the experimental and control groups is measured daily, and on the 1st and 5th days of cultivation, a bio-activator is added to the experimental group at a weight ratio of (1-3):20; S4-13: The curve of S2- content change with cultivation time is plotted, and the control group is determined to have stable sulfate-reducing bacteria growth and the experimental group has a significant inhibitory effect as the nutritional evaluation system for inhibiting sulfate-reducing bacteria. Sulfate-reducing bacteria (SRB) enrichment medium (g / L): NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, NaSO4 4, sodium lactate 6ml, yeast powder 1, adjust the pH to 7.0-7.4.

[0013] Preferably, the bioactivator is composed of NO3 in a weight ratio of (0-9):(1-10):(0-15). - NO2 - and MoO4 - composition.

[0014] The above-mentioned inhibitory bacterium, Bacillus licheniformis NRB-12, is used to inhibit corrosion caused by sulfate-reducing bacteria in oilfield reinjection water.

[0015] Paenibacillus sp. NRB-7, accession number CGMCC No. 24530, accession date: March 15, 2022, depositary institution: China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus licheniformis NRB-12, accession number CGMCC No. 24531, accession date: March 15, 2022, depositary institution: China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides Bacillus licheniformis NRB-12 to treat oilfield reinjection water with high sulfate-reducing bacteria content. It is environmentally friendly, inexpensive, has a wide treatment range, and is easy to operate. By providing an optimized nutrient system for nitrate-reducing bacteria, it effectively inhibits sulfate-reducing bacteria, thereby achieving the goal of safe production in oilfields and oil extraction. Attached Figure Description

[0017] Figure 1 When S is 90 mL of SRB culture medium + 10 mL of water sample, S 2- Changes in content; Figure 2 When S is 80 mL of SRB culture medium + 20 mL of water sample, S 2- Changes in content; Figure 3 When S is 70 mL of SRB culture medium + 30 mL of water sample, S 2- Changes in content; Figure 4 When S is 60 mL of SRB culture medium + 40 mL of water sample, S 2- Changes in content; Figure 5 When S is 50 mL of SRB culture medium + 50 mL of water sample, S 2-Changes in content; Figure 6 When S is 40 mL of SRB culture medium + 60 mL of water sample, S 2- Changes in content; Figure 7 When S is 30 mL of SRB culture medium + 70 mL of water sample, S 2- Changes in content; Figure 8 When S is 20 mL of SRB culture medium + 80 mL of water sample, S 2- Changes in content; Figure 9 When S10mL of SRB culture medium + 90mL of water sample is used, S 2- Changes in content; Figure 10 For the isolation and purification of nitrate-reducing bacteria; Figure 11 The growth curves of nitrate-reducing bacteria under anaerobic conditions are shown. Figure 12 To add nitrate-reducing bacteria to inhibit S 2- The impact; Figure 13 The results are from scanning electron microscopy of NRB-7 bacteria. Figure 14 The results are from scanning electron microscopy of NRB-12 bacteria. Figure 15 To add different proportions of NO3 - NO2 - , for S 2- The impact; Figure 16 To add different concentrations of molybdate to S 2- The effect of concentration; Figure 17 This is a comparison of the static corrosion rates of the original water sample and the sterilized water sample. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of the invention, and not all of them.

[0019] Example 1: Determination of the nutritional evaluation system for inhibiting sulfate-reducing bacteria To better observe the corrosive and inhibitory effects of sulfate-reducing bacteria (SRB), sulfate-reducing bacteria culture medium was added to oilfield reinjection water samples (pH = 7.055, salinity 131-137 mg / L, conductivity 153.8 mS / cm). These growth conditions are favorable for SRB, thus amplifying their effects. The influence of different concentrations of SRB on the number of SRB and their sulfate-reducing activity was investigated by adding different concentrations of SRB to the reinjection water samples. To determine the optimal system for evaluating the growth and inhibition of sulfate-reducing bacteria (SRB), experiments were conducted on the ratio of SRB culture medium to water sample: 90 mL SRB culture medium + 10 mL water sample, 80 mL SRB culture medium + 20 mL water sample, 70 mL SRB culture medium + 30 mL water sample, 60 mL SRB culture medium + 40 mL water sample, 50 mL SRB culture medium + 50 mL water sample, 40 mL SRB culture medium + 60 mL water sample, 30 mL SRB culture medium + 20 mL water sample, and 10 mL SRB culture medium + 90 mL water sample. During the experiments, on days 1 and 5, a prepared bioactivator with a weight ratio of NO3 was added. - NO2 - Add 10mL; the optimal ratio is chosen when the sulfate-reducing bacteria are in a stable growth phase. After adding the nutrient, the sulfate-reducing bacteria show a significant inhibitory effect.

[0020] from Figure 1-9 It can be seen that when 20 mL of SRB medium is added to 80 mL of oilfield reinjection water sample, SRB is in a stable growth period and the inhibitory effect is obvious. Therefore, 20 mL of SRB medium plus 80 mL of oilfield reinjection water sample is selected as the nutritional evaluation system for inhibiting sulfate-reducing bacteria.

[0021] The medium for sulfate-reducing bacteria (SRB) (g / L) consisted of: NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, NaSO4 4, sodium lactate 6 ml, and yeast extract 1 ml. The pH was adjusted to 7.0-7.4.

[0022] Example 2: Screening method for nitrate reduction inhibitory bacteria of the present invention 1.1 Enrichment culture of nitrate-reducing bacteria Three culture media were prepared according to the formula of the nitrate-reducing bacteria screening medium. The three culture media are: Nitrate-reducing bacteria (NRB) medium (g / L): NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, KNO3 3, sodium lactate 6 ml, yeast extract 1, adjusted pH to 7.0-7.4; Sulfate-reducing bacteria (SRB) medium (g / L): NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, NaSO4 4, sodium lactate 6 ml, yeast extract 1, adjusted pH to 7.0-7.4; NRB / SRB mixed medium (g / L): NaCl 5, MgCl2 1.8, CaCl2 0.02, NH4Cl 0.3, K2HPO4 0.2, KCl 0.5, KNO3 3, sodium lactate 6 ml, yeast extract 1, adjusted pH to 7.0-7.4; Add 0.5 ml of KNO3, 3 ml of NaSO4, 4 ml of sodium lactate, and 1 ml of yeast powder to adjust the pH to 7.0-7.4.

[0023] The specific experimental steps are as follows: ① The three culture media were placed into 100mL anaerobic culture bottles, and a small iron nail was placed in each bottle. After removing the oxygen in the bottles by blowing nitrogen gas from the bottles, the anaerobic bottles were sealed. ② Place the sealed anaerobic culture bottle in an autoclave for sterilization at 121°C for 20 minutes; ③ After sterilization, remove the anaerobic culture bottles and place them on a laminar flow hood to cool. Turn on the ultraviolet lamp to sterilize the laminar flow hood. After cooling, inoculate the culture bottles with oilfield reinjection water, adding 10% of the oilfield reinjection water sample. Perform three replicates for each group. After nitrogen deoxygenation, seal the bottles. ④ Place the anaerobic culture bottle in a 35℃ constant temperature incubator for four days, and observe and record the changes in the culture medium every day; ⑤ After four days of culture, select the culture medium that has not turned black from the mixed culture medium, take 10% of the culture medium and inoculate it into fresh NRB medium for transfer enrichment culture, and repeat the process three times.

[0024] In the experiment, NRB was enriched in oilfield reinjection water samples. After adding water samples to three different culture media, the media showed different changes after 4 days of enrichment: the NRB medium became turbid after 4 days, with a high bacterial concentration, indicating a large amount of NRB growth; the SRB medium became turbid and showed signs of iron nail corrosion and blackening after 4 days, indicating a large amount of SRB growth; in the mixed medium, the water sample was turbid but did not turn black, and the iron nail did not corrode, indicating that NRB in the water sample grew and multiplied extensively and inhibited the growth of SRB. The comparison shows that a large number of nitrate-reducing bacteria were enriched in the mixed medium, and these nitrate-reducing bacteria have an inhibitory effect on SRB, inhibiting SRB growth. 2- The generation of this is precisely the target bacterial species in this experiment.

[0025] 1.2 Isolation and purification of nitrate-reducing bacteria After multiple rounds of enrichment, the enriched NRB medium was then used to isolate single colonies via dilution and plating, thus isolating facultative anaerobic NRB bacteria. The specific experimental steps are as follows: ① Prepare NRB solid culture medium in a 500mL Erlenmeyer flask, sterilize it in an autoclave, then place it in a laminar flow hood and wait for it to cool to about 50-60℃. Pour the NRB solid culture medium into the sterilized agar plate, about one-half to one-third the thickness of the plate. Cover the plate and place it in a laminar flow hood to cool and solidify. ② Take a 2mL centrifuge tube, pipette 0.9mL of sterile distilled water into the tube, and then pipette 100mL of water into the centrifuge tube. The NRB culture medium was placed in it and mixed well, thus diluting the culture medium 10 times. Then, the above procedure was repeated to continue diluting until the concentration reached 10:10. -6 Or 10 -7 ; ③ Dilute to a concentration of 10 -4 10 -5 and 10 -6 Spread the diluted solution onto a plate, and then draw 200 ml of the solution in a clean bench. The target liquid is evenly coated on the plate; ④ Picking single colonies: Take a petri dish with an appropriate colony growth rate, pick a few single colonies, and streak them on the petri dish. Repeat this process several times until you are sure that they are single colonies.

[0026] The enriched mixed culture medium contained a large number of nitrate-reducing bacteria that could inhibit SRB. Therefore, a portion of the culture medium was taken from the mixed medium and plated for culture. Sixteen colonies were then picked (e.g., Figure 10The bacteria were streaked multiple times until no contaminants were found, and then preserved and named NRB-1, NRB-2, NRB-3, NRB-4, NRB-5, NRB-6, NRB-7, NRB-8, NRB-9, NRB-10, NRB-11, NRB-12, NRB-13, NRB-14, NRB-15, and NRB-16, respectively.

[0027] 1.3 Reduction performance test of nitrate-reducing bacteria To confirm that the selected single colonies are nitrate-reducing bacteria with nitrate-reducing capabilities, nitrate-reducing performance tests need to be performed on the selected single colonies. If the selected strains have the ability to reduce nitrates, they can reduce NO in the culture medium. 3- Restore to NO 2- NH3 or N2, etc., so NO can be detected by measuring NO. 2- The presence of nitrates indicates the nitrate-reducing capacity of the selected strains, which can be tested using nitrate reagent.

[0028] (1) Reagents used in the experiment ①Griess' reagent: Solution A: Dissolve 0.5g of p-aminobenzenesulfonic acid in 150mL of 10% acetic acid solution, mix well, and then store in the dark and refrigerated.

[0029] Solution B: Dissolve 0.1g of methylnaphthylamine in 150mL of 10% acetic acid solution, and dilute with 20mL of distilled water. Mix well and store in the dark.

[0030] ② Diphenylamine reagent: Dissolve 0.5g of diphenylamine in 100mL of concentrated sulfuric acid and add 20mL of distilled water.

[0031] (2) Cultivation and Testing Observation Methods: Several selected strains were inoculated into NRB medium and cultured in a 35℃ incubator. Samples were taken periodically for testing. Several small test tubes were used, and a few drops of different culture media were added to each tube (not too much, just enough to cover the bottom of the tube). The first test tube contained blank medium as a control. 1-2 drops of Griess' reagent A were added to the test tube, followed by 1-2 drops of reagent B. If the medium turned red, orange, or brown in a short time, it indicated NO. 3- It has been restored to NO 2- If the test result is positive, the culture medium will not change color. Then, add about two drops of diphenylamine reagent. If the culture medium turns blue, it indicates that NO is still present. 3- This indicates that there is no nitrate reduction, and the test result is negative; if no blue color is shown, it indicates NO. 3- and the generated NO 2-All of them have been reduced to substances such as N2 and NH3, thus indicating that the test result is positive.

[0032] Table 1. Results of nitrate reduction performance test of 16 bacterial strains after 4 days of culture ( / indicates no data).

[0033] All 16 bacterial strains turned red and orange after the addition of Griess' reagent, indicating that the NO in the culture medium was high. 3- All were restored to NO 2- The test result was positive, indicating that all 16 strains of bacteria possessed nitrate-reducing properties; the lighter the color, the lower the nitrite content in the culture medium. After adding diphenylamine reagent, the culture media of NRB-1, NRB-3, NRB-4, NRB-5, NRB-6, NRB-8, NRB-9, NRB-10, NRB-11, NRB-13, and NRB-14 turned blue, indicating that nitrate in the culture medium was not completely reduced and the nitrate-reducing ability was poor; the culture media of NRB-2, NRB-7, NRB-12, NRB-15, and NRB-16 remained the original color or colorless, without the formation of blue, indicating the absence of NO. 3- NO in the culture medium 3- NO 2- All five strains of bacteria have been consumed and tested positive for nitrate reduction.

[0034] 1.4 Determination of growth curves of nitrate-reducing bacteria Since nitrate-reducing bacteria are facultative anaerobic bacteria, their growth rate is relatively slower than that of aerobic bacteria. By plotting their growth curves, we can understand the growth time of the selected strains.

[0035] The anaerobic bottles containing NRB culture medium were sterilized in an autoclave. A 2% bacterial suspension, selected and preserved, was then inoculated and placed in an incubator at 35°C. During the incubation period, samples were taken every 12 hours. Two mL of the collected NRB culture medium was centrifuged and washed three times, and then its OD value was measured using a UV-Vis spectrophotometer. 600 And its growth curve was plotted.

[0036] The changes in the NRB growth curve are as follows: Figure 11 As shown. The growth curves of nitrate-reducing bacteria were determined by inoculating five strains of nitrate-reducing bacteria into NRB medium and measuring them under anaerobic conditions at 35°C. Figure 11It can be seen that within 10-20 hours, the bacterial concentration increases rapidly, with a high growth rate, indicating the logarithmic growth phase. After 20 hours, the growth curve flattens out, and NRB enters the stationary phase, which lasts for a relatively long time without a decline phase. This shows that NRB grows relatively slowly. While aerobic bacteria typically complete their entire growth cycle within 48 hours, NRB, being a facultative anaerobe, grows more slowly and has a longer growth period. The growth curves show that NRB-15 and NRB-16 bacteria have very low bacterial concentrations and poor growth, thus they were eliminated.

[0037] 1.5 Simulation Experiment of Nitrate-Reducing Bacteria Corrosion Following the sulfate-reducing bacteria culture medium and water sample ratio in Implementation Case 1, 20 mL of freshly prepared SRB culture medium and 80 mL of water sample were dispensed into 120 mL anaerobic bottles, nitrogen gas was introduced, and then sterilization was performed. After activation culture of the above 7 strains, 5% activated bacterial solution was added to the anaerobic bottles. The bacterial solution was added on days 1, 5, and 7, with three replicates. Finally, all anaerobic bottles were placed in a 35℃ constant temperature incubator for dark and static incubation. Water samples were taken regularly each day to measure S... 2- The concentration changes were investigated. The antagonistic inhibitory effects of nitrate-reducing bacteria and enriched sulfate-reducing bacteria were studied.

[0038] Depend on Figure 12 It can be seen that when activated nitrate-reducing bacteria are added alone, S 2- The concentration of nitrate-reducing bacteria was reduced to some extent compared with the blank experiment, indicating that nitrate-reducing bacteria can play a certain inhibitory role in the antagonistic effect between nitrate-reducing bacteria and sulfate-reducing bacteria. Among them, NRB-7 and NRB-12 bacteria showed the best inhibitory effect on sulfate-reducing bacteria.

[0039] 1.6 Static Corrosion Coating Test Corrosion tests were conducted on NRB-7 and NRB-12 bacteria in 20 mL of sulfate-reducing bacteria culture medium and 80 mL of water sample, as described above in the optimal system. The results were compared with a blank reagent bottle without added bacteria (NRB-7 and NRB-12 bacteria) to calculate the inhibition rate of nitrate-reducing bacteria on sulfate-reducing bacteria corrosion.

[0040] (1) Experimental materials Experimental materials and instruments: A3 steel sheet (Type II, 72.4×11.5×2mm), acetone, anhydrous ethanol, hydrochloric acid, sodium hydroxide, hexamethylenetetramine, 100mL anaerobic flask, constant temperature incubator, electronic balance.

[0041] Hydrochloric acid solution: 1+4; Sodium hydroxide solution: 60g / L; Pickling solution: Weigh 8g of hexamethylenetetramine and dissolve it in 1000mL of hydrochloric acid solution.

[0042] (2) Experimental methods The laboratory static corrosion test was conducted using the national standard GB / T18175-2000. The test temperature was 35℃, and the test period was 14 days. The specific steps are as follows: ① Wipe the grease off the A3 steel sheet with filter paper, place it in a beaker containing acetone and wipe the steel sheet with degreased cotton, then wipe it with ethanol to remove the grease. After that, dry the steel sheet with filter paper and then put it in a desiccator to dry it. After constant weight, weigh it and store the steel sheet in the desiccator for later use.

[0043] ② Perform primary filtration on the water sample to remove suspended solids, measure 100mL and inject it into the anaerobic bottle. Place three experimental steel plates in each bottle, blow air into the anaerobic bottle with a nitrogen bottle to remove oxygen, and then seal the bottle.

[0044] ③ Place the anaerobic bottle in a constant temperature incubator at 35℃ for incubation. Use the trace dilution method to determine the amount of SRB in the water sample. After incubation, observe the appearance of the corroded steel sheet and record its corrosion status.

[0045] ④ Treatment of corroded steel sheets: Wash the steel sheets with a brush, then soak them in pickling solution for about four minutes. After removing the steel sheets, rinse them quickly with water, then immediately immerse them in NaOH solution for about thirty seconds. After removing them, rinse them with distilled water, wipe them clean with filter paper, place them in anhydrous ethanol for about three minutes, place them on filter paper, and put them in a desiccator for more than four hours. After the steel sheets have reached a constant weight, weigh them and calculate the weight loss of the steel sheets. Calculate the corrosion rate using the following formula.

[0046] The formula for calculating the corrosion rate of the experimental steel sheet is as follows:

[0047] Where: X—corrosion rate, mm / a; M—loss mass of steel sheet, g; M0—Mass loss value of blank steel sheet experiment, g; P—Density of the steel sheet, g / cm³ 2 ; T—Experimental time, h; 8760 — hours equivalent to one year, h / a; 10 — the number of millimeters equivalent to 1 cm, mm / cm.

[0048] Table 2 Corrosion Rate of Water Samples in Static Anaerobic Simulated Steel Corrosion Test

[0049] The corrosion rate calculation results of the static anaerobic simulated steel sheet corrosion test are shown in Table 2. With an inoculum size of 5%, the corrosion rate of the three blank samples was 0.02569 mm / a, while the corrosion rate of the sample with NRB-7 bacteria was 0.01156 mm / a, a decrease of 55.00%. The corrosion rate of the sample with NRB-12 bacteria was 0.01146 mm / a. The corrosion rate decreased by 55.39% after the addition of denitrifying bacteria. This is because the relatively high amount of NRB in the water sample inhibited the corrosion of SRB bacteria, thus mitigating the corrosion effect.

[0050] 1.7 Molecular biological species identification of nitrate-reducing bacteria The nitrate-reducing bacteria NRB-7 and NRB-12 were identified by 16S rDNA sequencing. Activated NRB-7 and NRB-12 bacterial cultures were sent to Shanghai Sangon Biotech for 16S rDNA sequencing. The sequences were then compared for homology in the NCBI database to analyze the species composition. NRB-7 was identified as belonging to the genus *Bacillus*, and the sequencing results were as follows: NRB-12 is a licheniform Bacillus, and the sequencing results are as follows: (8) Observation of the bacterial morphology of nitrate-reducing bacteria The bacteria NRB-7 and NRB-12, which possess nitrate-reducing capabilities, were observed using scanning electron microscopy. The experimental steps are as follows: ① Collect and fix the bacterial cells: After culturing in NRB medium for about 4 days, when the bacterial cells are in the logarithmic growth phase, take the culture medium and place it in a centrifuge tube. Centrifuge at 8000 rpm for 10 minutes. After discarding the supernatant, wash the cells three times with phosphate buffer for 15-20 minutes each time. Then add 2.5% glutaraldehyde solution and fix the cells overnight at 4°C for 12 hours. ② Dehydration: After fixation, wash three times with PBS buffer, and then perform gradient dehydration with ethanol, using 30%, 50%, 60%, 70%, 85%, 95%, and 100% (volume ratio) ethanol solutions respectively, for 15-20 minutes each time.

[0051] ③ Replacement: Replace the ethanol with tert-butanol twice, 15 minutes each time.

[0052] ④ Vacuum freeze drying: Place the sample in a vacuum freeze dryer for about 24 hours until the tert-butanol has completely evaporated.

[0053] ⑤ Sputtering: Sputtering gold onto the surface of a dry sample in a vacuum.

[0054] ⑥ Place it under a scanning electron microscope for observation and photography.

[0055] After treating NRB-7 and NRB-12 bacterial cultures, images were taken and observed under SEM. The scanning electron microscope results are as follows: Figure 13-14 As shown.

[0056] Depend on Figure 13 and 14 It can be seen that both strains are bacilli, not spiral-shaped, with slender cells of varying lengths. NRB-7 bacteria typically range in length from 800-1400 nm, while NRB-12 bacteria typically range in length from 2500-6000 nm. They possess spores but lack flagella and capsules. This is consistent with the general characteristics of Bacillus species.

[0057] Example 3: Inhibitory effect of bioactivator formulation on SRB 1.1 Adding different proportions of NO 3- NO 2- SRB inhibition effect The combined use of nitrates and nitrites showed better inhibitory effects on SRB. This example investigated different NO... 3- / NO 2- For S 2-The effect of generation, that is, the effect on the suppression of SRB.

[0058] Using a 120 mL anaerobic culture flask, add 20 mL of SRB medium (inoculated at 5%) and 80 mL of water sample, then add 10... Different proportions of NO 3- and NO 2- A mixture of (0:10, 1:9, 2:8, 3:7, 4:6, 5:5) makes NO 2- NO 3- The final concentration was 560.0 g / L. After deoxygenation by purging with high-purity nitrogen, the bottles were sealed. Finally, all anaerobic bottles were placed in a 35°C constant temperature incubator for static incubation in the dark. Water samples were taken daily to measure sulfur (S). 2- The concentration change.

[0059] Add different NO 3- / NO 2- For S 2- The effects of such Figure 15 shown. When NO 3- / NO 2- The inhibition effect is best when the ratio is 1:9, NO 3- / NO 2- When the ratio is 1:1, for S 2- The inhibitory effect is the worst. (By...) Figure 15 It can be seen that NO 3- / NO 2- With NO 2- The higher the proportion of NO, the better the inhibitory effect, because NO... 2- The effect of inhibiting SRB compared to NO 3- Better. Taking into account factors such as economics and the effectiveness of SRB suppression, activating NO in the system is preferable. 3- / NO 2- When the ratio is 1:9, S is suppressed. 2- It has the best effect and the best effect in inhibiting SRB.

[0060] 1.2 Synergistic effect of different concentrations of molybdate on NO 3- / NO 2- Suppressing SRB effects Molybdate has a synergistic inhibitory effect on the biological inhibition of SRB, but because molybdate contains heavy metals, the amount added should not be excessive. A 120 mL anaerobic culture flask was used, with 20 mL of SRB medium (inoculated at 5%) and 80 mL of water sample added, followed by 10 μL of the prepared activator. The experiment was conducted after adding NO... 3- / NO 2- With a ratio of 1:9, seven experimental groups were set up, adding different doses of high-concentration sodium molybdate stock solution to adjust the MoO2 content. 4-Concentrations of 0 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, and 15 g / L were used, and a blank control experiment was conducted. The concentrations of S were... 2- The concentration of molybdate was measured and the inhibition rate was calculated to study the synergistic inhibitory effect of molybdate on SRB.

[0061] Add NO 3- / NO 2- Based on a 1:9 ratio, different concentrations of molybdate were added to S 2- The impact such as Figure 16 As shown, adding different concentrations of Na₂MoO₄ resulted in longer inhibition times and better inhibition effects compared to adding only NaNO₂. 10 g / L Na₂MoO₄ showed the best inhibition effect, and 12.5 g / L Na₂MoO₄ showed the best inhibition time on day seven. 2- The concentration increase rate of [the substance] was faster than that of 10 g / L Na₂MoO₄, indicating that 10 g / L is the optimal molybdate concentration for inhibiting SRB. Experiments show that Na₂MoO₄ has a good synergistic inhibitory effect.

[0062] Example 4: Calculation of the contribution rate of microbial corrosion to oilfield corrosion Corrosion of oilfield injection water can be considered mainly composed of chemical corrosion and microbial corrosion. Corrosion caused by injection water without sterilization is considered total corrosion, while corrosion after sterilization is chemical corrosion. Microbial corrosion is calculated by subtracting chemical corrosion from total corrosion. This study uses comparative experiments to examine the contribution rate of microbial corrosion through sterilization methods. The formula for calculating the contribution rate of microbial corrosion is as follows:

[0063] in: R —Microbial corrosion rate, % V 1—Corrosion rate of the original water sample, mm / a; V 2—Corrosion rate of sterilized water sample, mm / a.

[0064] like Figure 17 Microbial corrosion rate of the two water samples R 1(IQQQ), R 2(IIIQQQ), the result is: R 1 = 34.18%, R =2 = 16.2%, which shows that adding 0.1 ml / L of the prepared activator significantly reduced the absolute corrosion rate by 65.34%. By adding the developed activator, the biological activity of sulfate-reducing bacteria was inhibited, and the biocorrosion rate was significantly reduced.

[0065] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. An inhibitory bacterium that inhibits the activity of sulfate-reducing bacteria, characterized in that, The inhibiting bacterium is Bacillus licheniformis NRB-12, with accession number CGMCC No. 24531.

2. The application of the inhibitory bacteria based on the sulfate-reducing bacteria activity described in claim 1 in inhibiting corrosion caused by sulfate-reducing bacteria in oilfield reinjection water.

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