Inhibiting bacteria for inhibiting sulfate-reducing bacteria and anticorrosion application thereof

Abstract

The application discloses inhibiting bacteria for inhibiting sulfate-reducing bacteria and anti-corrosion application thereof, belongs to the technical field and application of microbial corrosion prevention of oilfield reinjection water, and the inhibiting bacteria is Paenibacillus sp. NRB-7 with a preservation number of CGMCC No. 24530 or Bacillus licheniformis NRB-12 with a preservation number of CGMCC No. 24531. The application has the characteristics of environmental friendliness, low price, wide treatment range and simple operation in the treatment of oilfield reinjection water with high content of sulfate-reducing bacteria; the effective inhibition of sulfate-reducing bacteria is realized by providing a nutrient system optimized for nitrate-reducing bacteria, so that the safety production of oilfields and oil extraction is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field and application of microbial corrosion prevention of oilfield reinjection water, and particularly relates to an inhibitory bacterium for inhibiting sulfate-reducing bacteria and application thereof in corrosion prevention. BACKGROUND

[0002] Microbiological influenced corrosion (MIC) refers to corrosion caused by the life activities and metabolic products of various microorganisms directly or indirectly on metals. Microorganisms in nature form biofilms on various materials, providing an environment for MIC. The defense mechanism of the biofilm protects microorganisms from environmental damage, making it difficult for general bactericides to achieve bactericidal effect.

[0003] In oil and gas systems, oxidative bactericides and non-oxidative bactericides are widely used. Although bactericides are simple to use and easy to maintain, bactericides cannot penetrate the polysaccharide film produced by microorganisms, and sulfate-reducing bacteria (SRB) sometimes coexist in the polysaccharide film produced by other microorganisms, making sterilization difficult. In a long-term reducing environment of H2S, the bactericidal efficiency of oxidative bactericides will decrease and cannot meet the standards. In addition, a large amount of long-term use of bactericides will cause the microorganisms to develop drug resistance, and only by continuously increasing the concentration of bactericides can the bactericidal effect be achieved, increasing the treatment cost. The toxicity of bactericides themselves will also cause environmental pollution. The biological competitive exclusion technology has the characteristics of environmental friendliness, low price, large treatment range and simple operation, and is an important method for treating sulfate-reducing bacteria in oilfield injection systems and oil reservoirs. Therefore, the use of biological competition method to inhibit the growth of sulfate-reducing bacteria is becoming more and more common. SUMMARY

[0004] The present application aims at the above-mentioned problems, and provides an inhibitory bacterium for inhibiting the activity of sulfate-reducing bacteria and application thereof in inhibiting corrosion caused by sulfate-reducing bacteria in oilfield reinjection water, so as to achieve the purpose of safe production of oilfield reinjection water and oil production.

[0005] The technical solution adopted by the present application is as follows: an inhibitory bacterium for inhibiting the activity of sulfate-reducing bacteria, wherein the inhibitory bacterium is Paenibacillus sp. NRB-7 with a preservation number of CGMCC No.24530, or Bacillus licheniformis NRB-12 with a preservation number of CGMCC No.24531.

[0006] A screening method of an inhibitory bacterium for inhibiting the activity of sulfate-reducing bacteria, comprising the following steps:

[0007] S1: enrichment culture is carried out on the inoculum, and the product is separated and purified;

[0008] S2: bacteria with NO3 - reduction characteristics are screened from the separated and purified culture;

[0009] S3: bacteria with fast growth and short growth period are screened by drawing growth curves of bacteria with NO3 - reduction characteristics;

[0010] S4: the bacteria screened in step S3 are further screened by antagonistic inhibition of sulfate-reducing bacteria (SRB), i.e. to obtain inhibitory bacteria that inhibit the activity of SRB;

[0011] S5: the inhibitory bacteria screened in step S4 that inhibit the activity of SRB are used to predict the inhibition rate of corrosion caused by SRB through static corrosion coupon test.

[0012] Preferably, the inoculum in step S1 is oilfield reinjection water; the method of enrichment culture in step S1 is: inoculate 5-15% of the inoculum with a weight percentage of NRB and SRB mixed culture medium into sterile NRB and SRB mixed culture medium, and culture at 30-35℃ under normal pressure for 3-5d; then take 5-15% of the culture solution that has not turned black in the mixed culture medium, and inoculate into new NRB culture medium for enrichment culture, and repeat the above culture step 2-4 times; the separation and purification in step S1 uses the method of dilution coating separation of single bacterial colony.

[0013] Preferably, the method of screening bacteria with NO3 - reduction characteristics in step S2 is to use nitric acid reagent test method, which is to inoculate the bacteria screened in step S1 into NRB culture medium for culture, and then detect NO2 - in the NRB culture medium by Griess reagent, and detect NO3 - in the NRB culture medium by diphenylamine reagent, and screen the bacteria with positive test results, i.e. bacteria with NO3 -The bacteria with reduction characteristics. The mixed culture medium (g / L) of NRB and SRB: NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, KNO33, NaSO44, sodium lactate 6ml, yeast powder 1, and the pH value is adjusted to 7.0-7.4; the NRB culture medium (g / L): NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, KNO33, sodium lactate 6ml, yeast powder 1, and the pH value is adjusted to 7.0-7.4.

[0014] Preferably, the method for drawing the growth curve in step S3 is as follows: the bacteria screened out in step S2 with the NO3 - The bacteria with reduction characteristics are inoculated into the new NRB culture medium, and during the cultivation process, the NRB culture solution is taken out every 10-14 hours, centrifuged and washed, and then the optical density value (OD 600 ) is measured by using the ultraviolet-visible spectrophotometer and the growth curve is drawn.

[0015] Preferably, the method for further screening the bacteria screened out in step S3 by using the antagonistic inhibition of the NRB and the SRB in step S4 is as follows: S4-1: determining the nutrition evaluation system for inhibiting the SRB; S4-2: after the bacteria screened out in step S3 are activated and cultivated, the bacteria are inoculated into the nutrition evaluation system for inhibiting the SRB determined in S4-1 in the same amount, parallel and blank experiments are set, and the cultivation is carried out in the constant temperature incubator at 30-35℃, the concentration change of S 2- is measured every day, and the bacteria with good inhibiting effect on the SRB are screened out.

[0016] Preferably, the specific method for determining the nutrition evaluation system for inhibiting the SRB is as follows: S4-11: adding the oilfield reinjection water sample in the weight ratio of (9:1)-(1:9) in the SRB culture medium to design multiple experimental groups and corresponding control groups; S4-12: the S 2- content in the experimental groups and the control groups is measured every day, and the biological activator is added in the experimental groups in the weight ratio of (1-3):20 on the first day and the fifth day of the cultivation; S4-13: the change curve of S2-content with the cultivation time is drawn, and the nutrition evaluation system for inhibiting the SRB is determined as the stable growth of the SRB in the control group and the obvious inhibiting effect in the experimental group. The SRB enrichment culture medium (g / L): NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, NaSO44, sodium lactate 6ml, yeast powder 1, and the pH value is adjusted to 7.0-7.4.

[0017] Preferably, the bioactivator is composed of NO3 in a weight ratio of (0-9):(1-10):(0-15). - NO2 - and MoO4 - composition.

[0018] The above-described inhibitory bacteria, Paenibacillus sp. NRB-7 and / or Bacillus licheniformis NRB-12, are used to inhibit corrosion caused by sulfate-reducing bacteria in oilfield reinjection water.

[0019] 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.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention provides Paenibacillus sp. NRB-7 or 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

[0022] Figure 1 When S is 90 mL of SRB culture medium + 10 mL of water sample, S 2- Changes in content;

[0023] Figure 2 When S is 80 mL of SRB culture medium + 20 mL of water sample, S 2- Changes in content;

[0024] Figure 3 When S is 70 mL of SRB culture medium + 30 mL of water sample, S 2-Content variation;

[0025] Figure 4 S when SRB medium 60 mL + water sample 40 mL 2- Content variation;

[0026] Figure 5 S when SRB medium 50 mL + water sample 50 mL 2- Content variation;

[0027] Figure 6 S when SRB medium 40 mL + water sample 60 mL 2- Content variation;

[0028] Figure 7 S when SRB medium 30 mL + water sample 70 mL 2- Content variation;

[0029] Figure 8 S when SRB medium 20 mL + water sample 80 mL 2- Content variation;

[0030] Figure 9 S when SRB medium 10 mL + water sample 90 mL 2- Content variation;

[0031] Figure 10 Isolation and purification of nitrate-reducing bacteria;

[0032] Figure 11 Growth curve of nitrate-reducing bacteria under anaerobic conditions;

[0033] Figure 12 Effect of adding nitrate-reducing bacteria on inhibition of S 2- ;

[0034] Figure 13 Scanning electron microscope results of NRB-7 bacteria;

[0035] Figure 14 Scanning electron microscope results of NRB-12 bacteria;

[0036] Figure 15 Effect of adding different proportions of NO3 - , NO2 - on S 2- ;

[0037] Figure 16 Effect of adding different concentrations of molybdate on S 2- concentration;

[0038] Figure 17 Comparison of static corrosion rates of water samples as is and sterilized water samples. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1: Determination of the nutritional evaluation system for inhibiting sulfate-reducing bacteria

[0041] 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.

[0042] from Figures 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.

[0043] 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.

[0044] Example 2, screening method of nitrate-reducing inhibiting bacteria of the present application

[0045] 1.1 Enrichment culture of nitrate-reducing bacteria

[0046] Three kinds of culture media were prepared according to the formula of the nitrate-reducing bacteria screening medium, which were: nitrate-reducing bacteria (NRB) medium (g / L): NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, KNO33, sodium lactate 6 ml, yeast powder 1, adjust pH to 7.0-7.4; sulfate-reducing bacteria (SRB) medium (g / L): NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, NaSO44, sodium lactate 6 ml, yeast powder 1, adjust pH to 7.0-7.4; NRB and SRB mixed medium (g / L): NaCl 5, MgCl21.8, CaCl20.02, NH4Cl 0.3, K2HPO40.2, KCl 0.5, KNO33, NaSO44, sodium lactate 6 ml, yeast powder 1, adjust pH to 7.0-7.4.

[0047] The specific experimental steps are as follows:

[0048] ① Three kinds of culture media were respectively filled into 100 mL anaerobic culture bottles, and a small iron nail was put in each bottle. After blowing nitrogen to remove oxygen in the bottle, the anaerobic bottle was sealed;

[0049] ② The sealed anaerobic culture bottles were sterilized in a sterilized pot at 121℃ for 20 minutes;

[0050] ③ After sterilization, the anaerobic culture bottles were taken out and cooled on the ultraclean bench, and the ultraclean bench was sterilized by turning on the ultraviolet lamp. After cooling, 10% of the oilfield reinjection water sample was inoculated into the culture bottle, and each group was done in triplicate. After blowing nitrogen to remove oxygen, the bottle was sealed;

[0051] ④ The anaerobic culture bottles were placed in a 35℃ constant temperature incubator for culture for four days, and the change of the culture medium was observed and recorded every day;

[0052] ⑤ After four days of culture, the culture medium in the mixed culture medium which did not turn black was selected, and 10% of the culture medium was inoculated into fresh NRB culture medium for transfer enrichment culture, which was done for three rounds.

[0053] In the experiment, the oilfield reinjection water sample was used to enrich the NRB in the water sample. After adding the water sample to three kinds of culture media respectively, after 4 days of enrichment culture, the culture media showed different changes: after 4 days of NRB culture medium culture, the culture medium became turbid, with a large bacterial concentration, indicating that a large number of NRB grew; after 4 days of SRB culture medium culture, the culture medium became turbid and the iron nail corroded, and the culture medium turned black, indicating that a large number of SRB grew; in the mixed culture medium, the water sample was turbid, and did not turn black, and the iron nail did not corrode, indicating that the NRB in the water sample grew in large numbers and inhibited the growth of SRB. By comparison, it can be concluded that a large number of nitrate reducing bacteria are enriched in the mixed culture medium, and these nitrate reducing bacteria have the effect of inhibiting SRB, which can inhibit the generation of S 2- , which is the target strain of this experiment.

[0054] 1.2 Isolation and purification of nitrate reducing bacteria

[0055] After several rounds of enrichment, the enriched NRB culture medium was used to separate single colonies by dilution and coating. The specific experimental steps are as follows:

[0056] ① Prepare NRB solid culture medium in a 500 mL triangular flask, sterilize in a sterilization pot, then place in a clean bench, cool to about 50-60°C, pour the NRB solid culture medium into a sterilized plate culture dish, the thickness is about one-third of the plate, cover the dish cover and place it in the clean bench to cool and solidify;

[0057] ② Take 2 mL of centrifuge tube, use a pipette to suck 0.9 mL of sterile distilled water into the centrifuge tube, then suck 100 of NRB culture solution into it, mix well, so that the culture solution is diluted by 10 times, then continue to dilute according to the above operation until the dilution concentration is 10 -6 or 10 -7 ;

[0058] ③ Dilute the dilution liquid with a dilution concentration of 10 -4 , 10 -5 and 10 -6 , and suck 200 of the target liquid evenly on the plate in the clean bench;

[0059] ④ Pick single colonies: take a culture dish with appropriate number of colonies, pick several single colonies, and perform streak culture on the plate culture dish, repeat several times until it is a single colony.

[0060] A large number of nitrate reducing bacteria that can inhibit SRB exist in the enriched mixed culture solution, so the culture solution is sucked from the mixed culture medium for plate coating culture, and 16 colonies are picked out (as shown inFigure 10 ) were preserved and named as 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.

[0061] 1.3 Test of reduction performance of nitrate-reducing bacteria

[0062] In order to determine that the different single colonies screened are nitrate-reducing bacteria with the performance of reducing nitrate, the nitrate-reducing performance test is needed to be conducted on several single bacteria screened. If the screened bacteria have the ability to reduce nitrate, the NO 3- in the culture medium can be reduced to NO 2- , NH3 or N2, etc., so the nitrate-reducing performance of the screened bacteria can be inferred by detecting the presence of NO 2- , which can be tested by nitric acid reagent.

[0063] (1) Reagents used in the experiment

[0064] ① Griess reagent:

[0065] A solution: 0.5 g of p-aminobenzenesulfonic acid was dissolved in 150 mL of 10% acetic acid solution, mixed well and stored in dark and cold.

[0066] B solution: 0.1 g of methylaniline was dissolved in 150 mL of 10% acetic acid solution, and 20 mL of distilled water was added for dilution, mixed well and stored in dark and cold.

[0067] ② Diphenylamine reagent: 0.5 g of diphenylamine was dissolved in 100 mL of concentrated sulfuric acid, and 20 mL of distilled water was added.

[0068] (2) Cultivation and test observation method: several strains screened were inoculated in NRB culture medium and cultured in a 35°C incubator, and samples were taken for determination at regular intervals. Several test tubes were taken, and different culture liquids were added dropwise in the test tubes (not too much, just to cover the bottom of the test tube), and the first test tube was added with blank culture medium as a control. 1-2 drops of Griess reagent A solution were added dropwise into the test tube, followed by 1-2 drops of B solution. If the culture medium turned into red orange brown color in a short time, it indicated that NO 3- had been reduced to NO 2- ,

[0069] The determination result was positive; if the culture liquid did not change color, 2 drops of diphenylamine reagent were added dropwise, and if the culture liquid showed blue color, it indicated that NO3- , indicating that there is no nitrate reduction, the test result is negative; if no blue color is shown, it indicates that NO 3- and the generated NO 2- have been reduced to N2, NH3 and other substances, so the test result is positive.

[0070] Table 1 Test results of nitrate reduction performance of 16 strains of bacteria after 4 days of culture ( / indicates no data)

[0071]

[0072] After adding Griess reagent, the 16 strains of bacteria all turned red and orange, indicating that the NO 3- in the culture medium has been reduced to NO 2- , and the test result is positive, i.e., the 16 strains of bacteria all have nitrate reduction performance; the lighter the color, the less nitrite in the culture solution. After adding diphenylamine reagent, the culture solution of NRB-1, NRB-3, NRB-4, NRB-5, NRB-6, NRB-8, NRB-9, NRB-10, NRB-11, NRB-13, NRB-14 appeared blue, indicating that the nitrate in the culture solution was not completely reduced, and the nitrate reduction performance was poor; the culture solution of NRB-2, NRB-7, NRB-12, NRB-15 and NRB-16 remained the original color or was colorless without blue color, indicating that there was no NO 3- , the NO 3- and NO 2- in the culture solution had been consumed, and the 5 strains of bacteria all had positive nitrate reduction performance.

[0073] 1.4 Determination of growth curve of nitrate-reducing bacteria

[0074] Since nitrate-reducing bacteria are facultative anaerobic bacteria, their growth rate is slower than that of aerobic bacteria. By drawing the growth curve, the growth process of the screened strains can be understood.

[0075] Put the anaerobic bottle containing NRB culture medium into a sterilization pot for sterilization, inoculate 2% of the stored bacterial solution, and place it in a 35°C incubator. During the culture process, take a sample every 12 hours, centrifuge 2 mL of NRB culture solution taken out and wash it 3 times, then measure the OD 600 using a UV-visible spectrophotometer and draw the growth curve.

[0076] The change of NRB growth curve is as follows Figure 11As 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 11 It 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.

[0077] 1.5 Simulation Experiment of Nitrate-Reducing Bacteria Corrosion

[0078] 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.

[0079] 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.

[0080] 1.6 Static Corrosion Coating Test

[0081] 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.

[0082] (1) Experimental materials

[0083] Experimental materials and instruments: A3 steel sheet (type II 72.4x11.5x2mm), acetone, anhydrous ethanol, hydrochloric acid, sodium hydroxide, hexamethylenetetramine, 100mL anaerobic bottle, constant temperature box, electronic balance.

[0084] Hydrochloric acid solution: 1+4; Sodium hydroxide solution: 60g / L;

[0085] Acid pickling solution: weigh 8g of hexamethylenetetramine, dissolve it in 1000mL of hydrochloric acid solution.

[0086] (2) Experimental method

[0087] The national standard GB / T18175-2000 was used for laboratory static corrosion coupon test, the test temperature was 35℃, and the test period was 14 days. The specific steps are as follows:

[0088] ① The grease on the A3 steel sheet was wiped off with filter paper, and the steel sheet was scrubbed with degreasing cotton in a beaker containing acetone, and then scrubbed in ethanol. After removing the grease, the steel sheet was dried with filter paper, and then placed in a desiccator for drying. After constant weight, the steel sheet was weighed and stored in a desiccator for later use.

[0089] ② The water sample was subjected to primary filtration to remove suspended solids, and 100mL was taken and injected into an anaerobic bottle. Three experimental steel sheets were placed in each bottle, and the anaerobic bottle was blown with nitrogen to remove oxygen, and then sealed.

[0090] ③ The anaerobic bottle was placed in a constant temperature box at 35℃ for cultivation, and the number of SRB in the water sample was determined by absolute dilution method. After cultivation, the appearance of the corrosion steel sheet was observed, and its corrosion condition was recorded.

[0091] ④ Treatment of the corrosion steel sheet: the steel sheet was washed with a brush, then immersed in the acid pickling solution for about four minutes. After taking out the steel sheet, it was quickly rinsed with water, and then immediately placed in the NaOH solution for about thirty seconds. After taking out, it was rinsed with distilled water, wiped clean with filter paper, placed in anhydrous ethanol for about three minutes, placed on filter paper, and placed in a desiccator for four hours or more. After the steel sheet was weighed, the weight loss of the steel sheet was calculated, and the corrosion rate was calculated using the following formula.

[0092] The corrosion rate calculation formula of the experimental steel sheet is as follows:

[0093]

[0094] In the formula: X Corrosion rate, mm / a;

[0095] M Mass loss of steel sheet, g;

[0096] M0 - blank test mass loss value of steel sheet, g;

[0097] P - density of steel sheet, g / cm 2 ;

[0098] T - test time, h;

[0099] 8760 - hours corresponding to one year, h / a;

[0100] 10 - millimeters corresponding to 1 cm, mm / cm.

[0101] Table 2 Corrosion rate of static anaerobic simulation steel sheet corrosion test of water sample

[0102]

[0103] The corrosion rate calculation results of static anaerobic simulation steel sheet corrosion test are shown in Table 2. In the case of 5% inoculation amount of bacteria solution, the corrosion rate of 3 blank samples is 0.02569 mm / a, the corrosion rate of the sample added with NRB-7 bacteria is 0.01156 mm / a, and the corrosion rate decreases by 55.00%. The corrosion rate of the sample added with NRB-12 bacteria is 0.01146 mm / a. After adding denitrifying bacteria, the corrosion rate decreases, and the corrosion rate decreases by 55.39%. It is because that the number of NRB in the water sample is relatively large, which plays a certain inhibitory effect on SRB, and reduces the corrosion effect.

[0104] 1.7 Molecular biology species identification of nitrate-reducing bacteria

[0105] The NRB-7 bacteria and NRB-12 bacteria with nitrate-reducing ability obtained above are subjected to species identification through 16S rDNA gene sequence determination. The bacterial solution of activated NRB-7 bacteria and NRB-12 bacteria is sent to Shanghai Shengong for 16S rDNA gene sequence determination. The measured sequence is subjected to homology comparison in the database of NCBI, and the species of the strain is analyzed. The NRB-7 bacteria is Bacillothrix, and the sequencing result is as follows:

[0106]

[0107] NRB-12 is Bacillus licheniformis, and the sequencing results are as follows:

[0108]

[0109] (8) Bacterial morphological observation of nitrate-reducing bacteria

[0110] The obtained bacteria NRB-7 and NRB-12 with nitrate-reducing ability were observed by scanning electron microscope, and the experimental steps were as follows:

[0111] ① Collecting and fixing bacteria: the bacteria in the logarithmic growth phase were collected after being cultured in NRB medium for about 4 days, and the culture solution was centrifuged at 8000 rpm for 10 minutes. After the supernatant was discarded, the bacteria were washed with phosphate buffer for 3 times, 15-20 minutes each time, and then fixed with 2.5% glutaraldehyde solution at 4°C for 12 hours.

[0112] ② Dehydration: after fixation, the bacteria were washed with PBS buffer for 3 times, and then dehydrated with ethanol by gradient, using 30%, 50%, 60%, 70%, 85%, 95% and 100% (volume ratio) ethanol solution for 15-20 minutes each time.

[0113] ③ Replacement: the ethanol was replaced with tert-butyl alcohol for a total of 2 times, 15 minutes each time.

[0114] ④ Vacuum freeze-drying: the sample was placed in a vacuum freeze-drying machine for about 24 hours until the tert-butyl alcohol was completely volatilized.

[0115] ⑤ Gold spraying: gold was sprayed on the surface of the dried sample in vacuum.

[0116] ⑥ Observation and photography under scanning electron microscope.

[0117] After the treatment of NRB-7 and NRB-12, the bacteria were observed and photographed under SEM, and the scanning electron microscope results are shown in Figures 13-14 .

[0118] As can be seen from Figure 13 and 14 , both of the bacteria are bacilli, not spiral-shaped, with slender bodies of varying lengths. The length of NRB-7 bacteria is generally between 800-1400 nm, and the length of NRB-12 bacteria is generally between 2500-6000 nm. Both of them have spores, no flagella, and no capsules. This is consistent with the general characteristics of Bacillus.

[0119] Example 3, Inhibition effect of biological activator formula on SRB

[0120] 1.1 Inhibition effect of different proportions of NO 3- , NO 2- on SRB

[0121] 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.

[0122] Using a 120 mL anaerobic culture flask, add 20 mL of LSRB 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.

[0123] 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.

[0124] 1.2 Synergistic effect of different concentrations of molybdate on NO 3- / NO 2- Suppressing SRB effects

[0125] 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.

[0126] 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.

[0127] Example 4: Calculation of the contribution rate of microbial corrosion to oilfield corrosion

[0128] 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:

[0129]

[0130] in: R —Microbial corrosion rate, %

[0131] V 1—Corrosion rate of the original water sample, mm / a;

[0132] V 2—Corrosion rate of sterilized water sample, mm / a.

[0133] As Figure 17 , two aqueous microbial corrosion rate R 1 (IQQQ), R 2 (IIIQQQ), the results are: R 1 = 34.18%, R 2 = 16.2%, thus it can be seen that the absolute corrosion rate is significantly reduced by 65.34% by adding 0.1 ml / L of the prepared activator. By adding the developed activator, the biological activity of sulfate-reducing bacteria is inhibited, and the biological corrosion rate is significantly reduced.

[0134] The above examples only express the specific embodiments of the present application, which are described in detail and in detail, but cannot be understood as a limitation on the protection scope of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the technical scheme of the present application, a number of modifications and improvements can be made, which belong to the protection scope of the present application.

Claims

1. A bacteriostatic bacteria which inhibits the activity of sulfate-reducing bacteria, characterized by comprising, The inhibiting bacteria is Paenibacillus sp. NRB-7, with the preservation number of CGMCC No.24530.

2. Use of the inhibiting bacteria according to claim 1 in inhibiting corrosion caused by sulfate-reducing bacteria in oilfield reinjection water.

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