Lysinibacillus sp. And application thereof in inhibiting sulfate reducing bacteria

Abstract

The invention relates to lysinibacillus sp.GWN-3 and application thereof in inhibiting sulfate reducing bacteria in the technical field of biological treatment of oilfield produced water, the lysinibacillus sp.GWN-3 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO 13904, the preservation date is March 20, 2017, and the preservation number is CGMCC NO 13904. The lysinibacillus fusiformis is classified and named as lysinibacillus fusiformis, and is named as Lysinibacillus fusiformis in Latin literature. The 16S rDNA (ribosomal deoxyribonucleic acid) gene sequence of the lysinibacillus sp. GWN-3 is as shown in SEQ ID NO: 1. The lysinibacillus sp. Is separated from oilfield produced water, is used for inhibiting proliferation of high-concentration SRB in an oilfield produced water ground treatment system, is long in inhibition time, and has a relatively good biological inhibition effect on SRB at the rear end of the oilfield ground produced water treatment system compared with a chemical sterilization technology; the content of SRB at the rear end of the system is effectively controlled, and the treatment cost of produced water is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of biological treatment of oilfield produced water, and in particular to an application of lysine bacillus separated and screened from oilfield produced water in inhibiting sulfate-reducing bacteria in an oilfield produced water treatment system. Background Art

[0002] Oilfield produced water refers to the oily wastewater that is extracted along with crude oil during the oilfield exploitation process. In order to increase the crude oil recovery rate, it is often necessary to use various physical, chemical and other technologies to treat the oily wastewater so that it meets the reservoir wastewater quality standards and is reinjected into the formation to increase the formation pressure. Oilfield produced water changes with environmental conditions. Sulfate-reducing bacteria (SRB) often breed in large numbers in surface sewage treatment systems. Their growth and metabolism process can cause corrosion and perforation of metal pipelines. At the same time, H2S produced during the metabolism of SRB will also reduce the quality and economic value of crude oil and natural gas, and increase the cost of subsequent treatment. The generation, spillage and diffusion of H2S will also harm the health and even life of front-line operators. S produced during the growth and metabolism of SRB 2- It can form sulfide precipitation (such as FeS) with other ions, thereby reducing the permeability of the reservoir and even blocking the reservoir formation, thereby increasing the difficulty and development cost of crude oil extraction. Studies have shown that the economic losses caused by SRB corrosion in my country's oil industry alone are as high as hundreds of millions of dollars each year. Therefore, SRB content has always been an important control indicator in water quality control standards. Generally, the SRB content in sewage before treatment is 2500-11000 / mL, and the SRB content in sewage after treatment is less than 25 / mL in the recommended control indicator.

[0003] At present, in the field of oilfield produced water treatment technology, the control methods of microorganisms generally include physical, chemical and microbiological methods. The physical method mainly uses ionizing rays in the ultraviolet (260nm) wavelength band to kill bacteria such as SRB in oilfield injection water. The chemical method mainly adds chemical fungicides to the produced water. The chemical method is currently the most common method used by oilfield enterprises to control the growth and reproduction of SRB. The mechanism of chemical sterilization technology is to use quaternary ammonium salts or diquaternary ammonium salts and other bactericidal chemicals to destroy the cell structure, that is, the bactericide acts on the bacterial cell wall, cell membrane, cell nucleus, etc., so that the functions of these structures are destroyed, resulting in bacterial poisoning and death; or interfere with its metabolic process, that is, interfere with the biological oxidation process and biosynthesis process, causing the bacteria to die. However, there are three disadvantages of chemical sterilization technology in actual applications. First, the drug resistance characteristics of SRB make it difficult to ensure the sustained and effective sterilization effect of chemical agents. Since the added fungicide is unevenly distributed in the various complex water environments on site, the SRB in local locations are in a low-concentration antibiotic environment for a long time and cannot be killed. A few of them eventually develop resistance to the fungicide, which gradually increases the required concentration of the fungicide, increasing the treatment cost while failing to guarantee the bactericidal effect. Second, SRB are wrapped in biofilm substances such as polysaccharide glue, which makes it difficult for fungicides to penetrate and achieve a killing effect. Third, in on-site applications, due to the drug resistance of bacteria, it is often necessary to frequently replace different types of chemical fungicides, which increases the workload of personnel and the operating cost of water treatment, and at the same time cannot guarantee continuous and effective sterilization.

[0004] From an environmental perspective, it is necessary to find new methods from microbiology itself to prevent and control SRB. By using the method of biological competition elimination, harmful microbial problems can be turned into favorable factors through the replacement of microbial populations. The microbial method is to add nitrates or nitrites to the injection water of oil production wells under the premise of limited nutrients and space, and use the competition and antagonism between microorganisms for nutrients and space to achieve the effect of inhibiting microbial corrosion. Some bacteria are very similar to SRB in living habits, but the bacteria do not produce H2S during the growth and metabolism process. Injecting the bacteria into the formation with the inflow of injection water allows them to live in the same ecological environment as SRB in the environment, and they can compete with native SRB for living space and nutrients, thereby inhibiting the growth and reproduction of SRB; some bacterial metabolites also contain substances similar to antibiotics, which can directly kill sulfate-reducing bacteria and reduce corrosion. Studies have shown that the decaaminocyclic peptide gramicidin S secreted by Bacillus brevis can inhibit SRB. Bacillus subtilis ATCC663, Bacillus brevis ATCC 35690 and B. brevis 18 can inhibit the formation of SRB colonies for up to 7 days. After supplementing the culture medium, except for the conical flask inoculated with B. brevis 18, the rest turned black within 24 hours, and ferrous sulfide was detected. Bacillus brevis 18 inhibited SRB for up to 28 days. Bacillus brevis 18, which produces antibiotics, can inhibit the adsorption of SRB on 304 stainless steel, thereby delaying the growth of SRB on SAE1018 mild steel to form biofilms. The experimental results show that the addition of gramicidin S before SRB forms colonies can effectively inhibit the growth of SRB. Compared with the use of high doses of fungicides, the production of gramicidin S in biofilms to inhibit the growth of SRB is a better choice. However, this type of method is currently only used in the oil-water environment system of the oil well casing annulus rich in SRB. This technology has not been applied to the produced water treatment system on the surface of the oil field, especially the use of a single specific bacterial agent for inhibition in the back-end of the oil field produced water biochemical treatment system.

[0005] As mentioned above, in the field of biological treatment of SRB in oil fields, based on considerations such as technical requirements, use costs, and research progress, most of the current biological treatment technology fields for SRB in wastewater at home and abroad are mostly in the annular space of oil wells with high water content. By adding nutrients required by microorganisms in the oil wells, trace amounts of denitrifying bacteria such as Acinetobacter, Serratia marcescens, Pseudomonas stutzeri, and Geobacillus existing in the environment are activated, or microbial bacteria and inhibitors are added at the same time to change the composition of the biological community in the environmental system of the annular space of the oil well, so that denitrifying bacteria become the dominant bacteria therein, and the antagonistic effect of microorganisms is used to achieve the purpose of inhibiting SRB in the wastewater of the annular space of the oil well, thereby realizing the SRB treatment of the oil well wellbore.

[0006] In the field of surface SRB treatment technology for oilfield produced water, after searching, it was found that this technology has not yet been discovered, and there are a large number of field applications in this field. In the Chinese patent database, the search results for related patents with "oilfield sewage and denitrifying bacteria and ground" as keywords are "none"; with "oilfield sewage and treatment and denitrifying bacteria" as keywords, two patents were detected, of which "Oilfield water biological treatment agent and its preparation method and use method" was rejected; the other patent is "Oilfield water biological treatment agent and its preparation method and use method" which has expired due to non-renewal after authorization. Although this patent uses the addition of denitrifying bacteria, from the content of its patent claims, the biological treatment agent contains diquaternary ammonium salts, potassium permanganate and potassium dichromate, and the main bactericidal effect of the agent mainly comes from this, and it cannot be judged as a technical idea of ​​denitrifying bacteria or biological inhibition. Using "denitrifying bacteria in oilfield wastewater" as the keyword, three patents were detected, among which a new patent was added, "A viscosity protector for preparing polyacrylamide solution from oilfield wastewater". The invention disclosed a polymer solution for preparing oilfield wastewater, which mainly includes a viscosity protector, which is composed of a chelating scale inhibitor, a composite bacterial culture and an activator. Its purpose is to inhibit SRB and maintain the viscosity of the aqueous solution in the preparation of polymer oil recovery solution on the ground, which is similar to the application scenario of the present invention, but not the same.

[0007] The biochemical treatment technology for oil-bearing produced water in oil fields is a new type of oil field produced water treatment technology with complete intellectual property rights owned by Jiangsu Oilfield. It mainly adopts the contact oxidation sewage treatment process. Due to the advantages of less sludge, simple production management, and low energy consumption, the contact oxidation process is currently the main one in this technical field. This technology has a good effect on the removal of sulfides and petroleum hydrocarbons in sewage. However, due to the improvement of the microbial environment of produced water after treatment by this process and the change from aerobic to anaerobic environment in the process flow, the problem of high SRB content is more common in the back end of the biochemical pool. How to use microbial technology to achieve effective, stable and continuous control of SRB in produced water through the community structure regulation of contact oxidation biological systems is an important topic and problem, and it is also a technical gap in the back end of SRB biological treatment of oilfield wastewater microbial oil removal technology. At present, in the field of oilfield biological anti-corrosion technology, the technology of effectively inhibiting SRB in the annular space of the oil casing of the oil well by adding denitrifying bacteria and inhibitors in the wellbore of the oil well has been well applied. However, in the field of oilfield produced water SRB microbial control technology, especially the SRB biological inhibition at the back end of the oilfield biochemical oil removal technology, there is still a blank.

[0008] Using "Lysinibacillus" as the keyword, a search in the Chinese patent database found 225 patents, all in the technical fields of bioremediation, plant growth promotion, pest control, etc., and no application in this technical field was found. Further search of Lysinibacillus found 41 patents in the Chinese patent database, all in the technical fields of plant growth promotion, protease production, bioremediation, tailings treatment, fertilizer production, etc., and no application in this field was found.

[0009] The water treatment technology of the surface treatment system of oilfield produced water currently mainly adopts the water treatment process of flocculation sedimentation and filtration, and partly adopts the water treatment process technology of biological oil removal and filtration. From the perspective of water quality changes along the surface water treatment process, after oil removal treatment, the suspended solid content in the produced water is effectively reduced in the purification and impurity removal process of the filtration unit, but in the unit sewage treatment process, SRB often proliferates in the water retention area of ​​the filter or under the filter material scale, so that during the water treatment process, accompanied by the growth and metabolism of SRB, the biofilm will periodically fall off. The changes in the sampling and testing water quality data show that the periodic SRB content rises sharply. Since the water injection tank unit generally adopts the process of maintaining a certain liquid level at the same time, there is often a long time interval for the replacement of a part of the water in the water injection tank, which also causes the proliferation of SRB in the water of the water injection tank. Therefore, the filter and water injection tank positions of the surface water treatment system are areas where harmful bacteria such as SRB are easy to breed and difficult to be stably managed. Since the nutrients in the water increase and harmful substances decrease after biological oil removal treatment, this feature is more prominent in the process of biological oil removal and filtration.

[0010] In summary, the current oilfield produced water treatment system, especially the high-concentration SRB proliferation problem at the filters and water injection tanks at the back end of the oil-containing produced water biochemical treatment system, cannot be effectively and cost-effectively treated in a sustained manner with existing physical and chemical sterilization technologies. Therefore, the research and application of biological inhibition technology can not only meet the actual technical needs of the site, but also reduce the cost of produced water treatment and achieve sustained and effective inhibition of harmful bacteria in produced water. Summary of the invention

[0011] The present invention aims at the problems existing in the prior art in the treatment of sulfate-reducing bacteria in oilfield produced water treatment systems, provides a lysine Bacillus obtained by culturing and separating from produced water for inhibiting sulfate-reducing bacteria, and further provides a fermentation method of lysine Bacillus and its application in the high-salt environment of oilfield produced water.

[0012] To achieve the above object, the present invention first provides a lysinibacillus, characterized in that it is lysinibacillus GWN-3, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC NO13904, a deposit date of March 20, 2017, and a classification name of: fusiform lysinibacillus, and a Latin literary name of: Lysinibacillus fusiformis .

[0013] The above-mentioned lysine Bacillus of the present invention is separated and obtained from oil field produced water, and is used to inhibit the proliferation of high-concentration SRB in the surface treatment system of oil field produced water, and the inhibition time is long. Compared with chemical sterilization technology, the lysine Bacillus of the present invention has a better biological inhibition effect on the high-concentration SRB at the rear end of the oil field surface produced water treatment system, effectively controls the content of SRB at the rear end of the system, and greatly reduces the cost of produced water treatment.

[0014] Furthermore, the 16S rDNA gene sequence of the lysinic Bacillus GWN-3 is shown in SEQ ID NO: 1.

[0015] Furthermore, the isolation method of the lysinic Bacillus GWN-3 is divided into the following steps: Step 1: Pipette 1 mL of oilfield produced water into a conical flask containing 50 mL of sterile GWN-3 culture medium, add 10 mL of mineral oil and seal it, place the conical flask in a 35°C incubator, and culture it for 1 month; Pipette the bacterial suspension in the conical flask, dilute it in a gradient, and evenly spread 100 μL of the suspension on a sterile solid LB plate for each gradient, and then culture it in a 35°C incubator for 24 h; When the colonies grow, randomly select bacteria on the plates cultured in each gradient for purification; In the second step, all the single colonies purified in the first step were suspended and aspirated into graduated transparent penicillin bottles containing 9 mL SRB culture medium. After saturation with nitrogen, they were sealed and stored. They were cultured at 35°C for 3 days. The strains that can grow stably in the test bottles were selected for further testing and screening: the screened single colony suspension was inoculated into the SRB test bottle. After culturing at 35°C for 3 days, 1 mL of oil well produced water was inoculated for co-culture. No strain was inoculated as a control. After culturing at 35°C for 7 days, the blackening degree of the test bottle was observed. The strain with less blackening in the test bottle was selected as the target bacteria: Bacillus lysinicola GWN-3.

[0016] Furthermore, the components of the GWN-3 culture medium are: 1 g / L glucose, 2 g / L sucrose, 1 g / L corn steep liquor, 0.2 g / L calcium carbonate, 1 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.5 g / L yeast extract.

[0017] Furthermore, the fermentation method of the above-mentioned lysinic Bacillus GWN-3 of the present invention is: Lysinibacillus GWN-3 was inoculated aseptically on a sterile solid culture medium, and cultured at 30-35°C for 2-3 days to obtain a single colony of activated lysinibacillus GWN-3; The solid culture medium is composed of the following components by mass percentage: 0.4-0.6% yeast extract, 0.8-1.2% peptone, 1.5-2.0% agar powder, 3-5% NaCl, and the balance is water, with a pH of 7.0-7.2.

[0018] Take several sterile square bottles with a capacity of 250 mL and solid culture medium inside, inoculate activated single colonies on the inclined surface of the solid culture medium in each bottle, and culture them at 30-35°C for 46 days to form bacterial spores; wash the spores on the surface of the solid culture medium in 3-4 square bottles with sterile water to obtain bacterial cell or spore suspension as fermentation seed liquid. The solid culture medium is the same as the solid culture medium in ①.

[0019] The seed liquid prepared in ② was inoculated into the sterile liquid culture medium of a fermentation tank with a capacity of 1000L. The liquid culture medium filling volume was 500L. The fermentation temperature conditions were as follows: inoculation temperature 35℃; inoculation spore germination period of 1-8h, control temperature 34℃; inoculation 8-15h logarithmic growth period, control temperature 30℃, inoculation 16-35h hysteresis period, control temperature 28-30℃; ventilation: fermentation ventilation: inoculation 1-8h early fermentation 0.1m 3 / min, the fermentation tank pressure is maintained at 0.2MPa, in the middle and late stages of 9-35h fermentation, the ventilation volume is 3 m 3 / min, the tank pressure was maintained at 0.3Mpa; during the fermentation process, constant stirring was adopted at 180rpm. After 35 hours of fermentation, oval spores with high density and uniform morphology were obtained through microscopic observation. The spore rate was more than 90%. The pH of the culture medium was 7.5-8. The fermentation culture medium was collected to obtain a high concentration of lysine Bacillus liquid. The blood cell counting plate count reached 1×10 9 cfu / mL or above.

[0020] Furthermore, the liquid culture medium of the fermentation tank is composed of the following components in mass percentage: NaNO3 2-5%, starch 0.5-1.0%, corn steep liquor 0.4-0.6%, magnesium sulfate 0.05-0.1%, sodium chloride 0.1-0.2%, dipotassium hydrogen phosphate 0.05-0.1%, zinc sulfate 0.01-0.015%, and the balance is water, with a pH value of 7.0-7.2.

[0021] Furthermore, the sterilization conditions of the solid culture medium in ① are: 120°C, 0.11Mpa for 25 to 35 minutes.

[0022] The present invention also provides the above-mentioned lysinic Bacillus for inhibiting the proliferation of sulfate-reducing bacteria SRB.

[0023] The present invention further provides the use of the above-mentioned lysine Bacillus for inhibiting the proliferation of sulfate-reducing bacteria SRB in an oilfield produced water treatment system. Long-term antibacterial treatment is performed according to the proliferation of SRB in the oilfield produced water surface treatment system, which can effectively prevent the proliferation of SRB in the water at the filter and water injection tank.

[0024] Biological Deposit The desulfurization bacteria agent involved in the present invention has been submitted for biological deposit, and the deposit information is as follows: . BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the colony morphology of the front side of the lysinic Bacillus GWN-3 plate.

[0026] Figure 2 This is a picture of Bacillus lysinicus observed under a GWN-3 microscope (1000X).

[0027] Figure 3 This is a gel electrophoresis plate diagram of the 16S rDNA gene sequence of Bacillus lysinicus GWN-3.

[0028] Figure 4 This is the molecular evolutionary tree of Bacillus lysinicus GWN-3 strain based on the 16S rDNA gene sequence.

[0029] Figure 5This is a diagram showing the inhibitory effect of Bacillus GWN-3 on SRB. DETAILED DESCRIPTION

[0030] The culture medium involved in the following examples is as follows: GWN-3 medium: glucose 1 g / L, sucrose 2 g / L, corn syrup 1 g / L, calcium carbonate 0.2 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, yeast extract 0.5 g / L.

[0031] SRB culture medium: dipotassium hydrogen phosphate: 0.10%, ammonium chloride: 0.03%, magnesium sulfate heptahydrate: 0.20%, anhydrous sodium sulfate: 0.085%, glucose 0.1%, yeast extract powder: 0.15%, sodium chloride: 1%~5%, L-cysteine ​​hydrochloride: 0.003%, iron 1%~5%, and the balance is deionized water.

[0032] LB medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.

[0033] Example 1: Isolation of Lysinibacillus GWN-3 Share it in the following ways: In the area with high SRB concentration at the oilfield produced water treatment site, 1 mL of produced water was drawn from the oilfield and dropped into a conical flask containing 50 mL of sterile GWN-3 culture medium, and 10 mL of mineral oil was added to seal the flask. The conical flask was placed in a 35°C incubator for incubation for 1 month. The bacterial suspension in the conical flask was drawn and diluted in gradients. 100 μL of the suspension was drawn for each gradient and evenly spread on a sterile solid LB plate, and incubated in a 35°C incubator for 24 h. After the colonies grew out, the colonies on each LB plate were randomly selected for purification to obtain purified single colonies.

[0034] The purified single colony was suspended and aspirated into a graduated transparent vial containing 9 mL of SRB culture medium. After being saturated with nitrogen, it was sealed and stored, and cultured at 35°C for 3 days. The strains that could grow stably in the test bottle were selected for further testing. The screened single colony suspension was inoculated into the SRB test bottle. After culturing at 35°C for 3 days, 1 mL of oil well produced water was inoculated for co-culture. No strain was inoculated in the control. After culturing at 35°C for 7 days, the blackening degree of the test bottle was observed, and the strain with less blackening in the test bottle was selected as the target bacteria, that is, Bacillus lysinicus GWN-3 was obtained.

[0035] Example 2: Identification of Lysinibacillus GWN-3 that inhibits the proliferation of SRB 1. Strain morphology observation GWN-3 was inoculated in three zones on an LB plate and cultured at 35°C. Observe the colony morphology, such as Figure 1The colonies are smooth milky white with bacterial film on the surface, wrinkled and raised in the center in a ring-like shape, neat and without notches on the edges, and the colonies grow quickly. After 24 hours, the colonies are 3-4 mm in diameter. Use an inoculation loop to pick up an appropriate amount of bacterial moss, spread it on a clean glass slide with a drop of distilled water, air-dry and fix it, then stain it with crystal violet mixture, iodine solution, 95% ethanol and safranin solution in turn, wash with water and air-dry, and examine under a microscope. Figure 2 The strain has a short and thick vegetative body and meso-spores.

[0036] 2. 16S rDNA sequence analysis The DNA of GWN-3 was extracted using a kit, and PCR amplification was performed using primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-TACGGTACCTTGTTACGACTT-3′. The PCR reaction system consisted of: 2×Taq PCR Master Mix 12.5μL, 1μL each of primers 27F and 1492R, 1μL of template DNA, and finally supplemented to 25μL with sterile double distilled water. The reaction procedure was: 93℃5min; 93℃30s, 58℃30s, 72℃5min, a total of 32 cycles; finally 72℃10min, and 4℃ to end the reaction. The PCR amplification products were subjected to 1% (w / v) agarose gel electrophoresis. Figure 3 The target product was sent to General Biotech (Anhui) Co., Ltd. for sequencing analysis after gel excision. The 16S rDNA gene sequence of SRB inhibitory bacterium Lysinic Bacillus GWN-3 is shown in SEQ No. 1. The sequence was compared by Blast on Genbank, and strains with high similarity to the compared sequence were selected. The phylogenetic tree was constructed using MEGA 7 software, as shown in the figure. Figure 4 Morphological observation and 16S rDNA gene sequence comparison showed that the 16S rDNA sequence of strain GWN-3 was 99.93% homologous to the 16S rDNA sequence of Lysinibacillus fusiformis strain. This strain was highly homologous to Lysinibacillus boronitolerans and was identified and named Lysinibacillus GWN-3.

[0037] Example 3: Fermentation and preparation of Bacillus lysinicus GWN-3 Lysinibacillus is inoculated on a sterile solid culture medium by aseptic streaking, and is statically cultured at 30-35°C for 2-3 days to obtain a single colony of activated lysinibacillus; the solid culture medium used is composed of the following components by mass percentage: 0.4-0.6% yeast extract, 0.8-1.2% peptone, 1.5-2.0% agar powder, 3-5% NaCl, and the balance is water, pH 7.0-7.2, and the sterilization conditions of the solid culture medium are: 120°C, 0.11Mpa for 25-35 minutes; Take several sterile square bottles with a capacity of 250 mL, put solid culture medium inside, inoculate activated single colonies on the slope of solid culture medium, and culture at 30-35°C for 46 days to form bacterial spores; wash the spores on the surface of solid culture medium in 3-4 square bottles with sterile water to obtain bacterial cell or spore suspension as fermentation seed liquid. The solid culture medium in this step is the same as the solid culture medium in ①; ③Inoculate the seed solution prepared in ② into the sterile liquid culture medium of a 1000L fermenter, and the culture medium filling volume is 500L. The fermentation conditions are as follows: temperature: inoculation temperature 35℃; spore germination period (inoculation 1-8h) control temperature 34℃; logarithmic growth period (inoculation 8-15h) control temperature 30℃, hysteresis period (16-35h) control temperature 28-30℃; ventilation: fermentation ventilation in the early stage (inoculation 1-8h) is 0.1m 3 / min, the fermentation tank pressure was maintained at 0.2MPa, and the ventilation volume in the middle and late stages of fermentation (9-35h) was 3 m 3 / min, the tank pressure was maintained at 0.3MPa. Constant stirring was used during the fermentation process, with a rotation speed of 180rpm.

[0038] In this step, the liquid culture medium of the fermentation tank is composed of the following components by mass percentage: NaNO3 2-5%, starch 0.5-1.0%, corn steep liquor 0.4-0.6%, magnesium sulfate 0.05-0.1%, sodium chloride 0.1-0.2%, dipotassium hydrogen phosphate 0.05-0.1%, zinc sulfate 0.01-0.015%, and the balance is water, with a pH value of 7.0-7.2.

[0039] After culturing the seed liquid culture medium in step ③ for 35 hours, the microscopic staining showed that the spores were oval in shape with high density and uniform morphology, and the spore rate was more than 90%. The pH of the culture medium was 7.5-8. The fermentation culture medium was collected to obtain a high concentration of Lysinibacillus GWN-3 liquid, and the blood cell count reached 1×10 9 cfu / mL or above.

[0040] Example 4: Inhibitory effect of Bacillus lysinicola GWN-3 on SRB According to the extinction dilution method in SY / T5329, the SRB is tested by injecting the water sample to be tested into the test bottle step by step with a sterile syringe for inoculation and dilution. According to the actual temperature of the water sample, it is cultured in the laboratory bacterial incubator for 7 days. According to the positive reaction of the bacterial bottle and the dilution ratio, the bacterial content is calculated.

[0041] The GWN-3 bacterial agent was inoculated into the test bottle SRB-HX-7 and cultured at 35℃ for 3 days. Then, 1 mL of oil well produced water was inoculated for co-culture. No strain was inoculated into the control. After 7 days of culture at 35℃, the blackening degree of the test bottle was observed. Figure 5 The left two rows are inoculated with GWN-3 bacteria, cultured at 35℃ for 3 days, and then inoculated with 1 mL of oil well produced water for co-culture. The right two rows are controls without any strains. The test bottles inoculated with GWN-3 bacteria only turned black at level 1, while the control test bottles turned black at levels 5-6. The inhibition efficiency of GWN-3 bacteria on SRB reached 99.8%.

[0042] Example 5: Application of GWN-3 bacterial agent in oil field oil well produced water biological treatment system Treatment method: At the front end of the SRB-proliferation areas such as the filter and water injection tank of the produced water surface water treatment system, the bactericide is added in an impact manner to make its concentration reach 2 times the normal bactericidal concentration, and the dosing pump is used simultaneously to continuously pump in the bactericide that reaches the killing concentration of harmful bacteria such as SRB. After 48 hours, the sampling test is carried out, and the SRB content in the water samples at the filter outlet and the water injection tank outlet is lower than 1000 / mL, and the flow channel pretreatment along the water flow channel is ended. The use of GWN inhibitory bacteria to inhibit the growth of sewage SRB is actually the antagonism between GWN and SRB bacteria. In actual field applications, SRB is easy to gather in local locations under the scale of the filter water injection tank, and the local concentration is high. At the same time, there are other types of microorganisms in the system, and the interaction relationship between various bacteria is relatively complex. Before adding GWN bacteria, the use of bactericides can effectively reduce the number of miscellaneous bacteria in the implementation environment, which is conducive to the growth and reproduction of GWN bacteria, and quickly establishes a dominant position in the water treatment unit environment. At the same time, it can effectively improve the effect of GWN bacteria in inhibiting SRB and reduce the dosage and dosage cycle of GWN bacteria.

[0043] Method for starting and establishing the lysinophilic Bacillus inhibition system: The inhibitory liquid system is added in an impact manner at the front end of the SRB-proliferation area such as the filter and water injection tank of the surface water treatment system of the oil field produced water, which mainly includes lysinophilic Bacillus fermentation liquid or dry powder at a rate of 100-200 mL (g) / m 3 , Glucose 500g / m 3 , Inorganic phosphate 100g / m 3 , Inorganic ammonium salt 200g / m 3; Continue to drip 100 mg / L of lysine Bacillus fermentation liquid or dry powder for 3 days. Monitor the changes in SRB content for 7 consecutive days during the dosing period, and read the changes in the number of SRB after culture every day. If a downward trend appears after 48 hours, continue to add the drug, otherwise continue to drip and double the concentration; when the SRB content drops to 25 / mL, stop dripping the inhibitory bacteria system and complete the establishment of the SRB biological inhibition system. From the implementation effect, the effective inhibition period of one treatment is more than 60 days.

[0044] ③ Periodic supplementation method of the inhibition system: As the water quality of produced water entering the surface water treatment system changes, track the changes in the SRB community and content in the produced water inhibition system and the water injection tank filter once a week. When the SRB content is significantly higher than 600 / mL, add lysine Bacillus biological inhibition to the front-end system again according to step ②. After the reinjection water is treated, the number of SRB in the filter and water injection tank outlet water drops to less than 25 / mL.

[0045] Four days after applying Bacillus lysinicioides, it was found that the content of SRB before the pump decreased significantly, and compared with the previous highest value, it decreased by 77.3% to 99.7%.

[0046] After on-site application, it can effectively prevent the occurrence of high SRB concentrations in the water at the filter and water injection tank, such as greater than 11,000 / mL.

Claims

1. A lysinibacillus, characterized in that It is Lysinibacillus GWN-3, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO 13904 and a deposit date of March 20, 2017. It is classified and named as: Spindle-shaped Lysinibacillus, and its Latin literary name is: Lysinibacillus fusiformis .

2. The lysinibacillus according to claim 1, characterized in that The 16S rDNA gene sequence of the lysinic Bacillus GWN-3 is shown in SEQ ID NO:

1.

3. The lysinibacillus according to claim 1, characterized in that The isolation method of the lysinic Bacillus GWN-3 is divided into the following steps: Step 1: Pipette 1 mL of produced water from the oil field into a conical flask containing 50 mL of sterile GWN-3 culture medium, add 10 mL of mineral oil and seal it, place the conical flask in a 35°C incubator, and culture it for 1 month; Pipette the bacterial suspension in the conical flask, dilute it in a gradient, and evenly spread 100 μL of the suspension on a sterile solid LB plate for each gradient, and then culture it in a 35°C incubator for 24 hours; When the colonies grow, randomly select bacteria on the plates cultured in each gradient for purification; In the second step, all the single colonies purified in the first step were suspended and aspirated into graduated transparent penicillin bottles containing 9 mL SRB culture medium. After saturation with nitrogen, they were sealed and stored. They were cultured at 35°C for 3 days. The strains that can grow stably in the test bottles were selected for further testing and screening: the screened single colony suspension was inoculated into the SRB test bottle. After culturing at 35°C for 3 days, 1 mL of oil well produced water was inoculated for co-culture. No strain was inoculated as a control. After culturing at 35°C for 7 days, the blackening degree of the test bottle was observed. The strain whose test bottle did not turn black was screened as the target bacteria: Lysinibacillus GWN-3.

4. The lysinibacillus according to claim 3, characterized in that The components of the GWN-3 culture medium are: 1 g / L glucose, 2 g / L sucrose, 1 g / L corn steep liquor, 0.2 g / L calcium carbonate, 1 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.5 g / L yeast extract.

5. The lysinibacillus according to claim 1, characterized in that The fermentation method of the lysinic Bacillus GWN-3 is: The lysinibacillus GWN-3 was inoculated on a sterile solid culture medium by aseptic streaking, and statically cultured at 30-35°C for 2-3 days to obtain a single colony of activated lysinibacillus GWN-3; The solid culture medium is composed of the following components by mass percentage: 0.4-0.6% yeast extract, 0.8-1.2% peptone, 1.5-2.0% agar powder, 3-5% NaCl, and the balance is water, with a pH of 7.0-7.2; Take several sterile square bottles with a capacity of 250 mL and solid culture medium inside, inoculate activated single colonies on the slope of the solid culture medium in each bottle, and culture them at 30-35°C for 46 days to form bacterial spores; rinse the spores on the surface of the solid culture medium in 3-4 square bottles with sterile water to obtain bacterial cell or spore suspension as fermentation seed liquid, the solid culture medium is the same as the solid culture medium in ①; The seed liquid prepared in ② was inoculated into the sterile liquid culture medium of a fermentation tank with a capacity of 1000L. The liquid culture medium filling volume was 500L. The fermentation temperature conditions were as follows: inoculation temperature 35℃; inoculation spore germination period of 1-8h, control temperature 34℃; inoculation 8-15h logarithmic growth period, control temperature 30℃, inoculation 16-35h hysteresis period, control temperature 28-30℃; ventilation: fermentation ventilation: inoculation 1-8h early fermentation 0.1m 3 / min, the fermentation tank pressure is maintained at 0.2MPa, in the middle and late stages of 9-35h fermentation, the ventilation volume is 3 m 3 / min, the tank pressure was maintained at 0.3Mpa; during the fermentation process, a constant stirring speed of 180rpm was used. After 35h of fermentation, oval spores with high density and uniform morphology were obtained through microscopic observation. The spore rate was more than 90%, and the pH of the culture medium was 7.5-8. The fermentation culture medium was collected to obtain a high concentration of lysine Bacillus liquid, and the blood cell counting plate count reached 1×10 9 cfu / mL or more; The liquid culture medium of the fermentation tank is composed of the following components by mass percentage: NaNO3 2-5%, starch 0.5-1.0%, corn steep liquor 0.4-0.6%, magnesium sulfate 0.05-0.1%, sodium chloride 0.1-0.2%, dipotassium hydrogen phosphate 0.05-0.1%, zinc sulfate 0.01-0.015%, and the balance is water, with a pH value of 7.0-7.

2.

6. The lysinibacillus according to claim 5, characterized in that ① The sterilization conditions of solid culture medium are: 120℃, 0.11Mpa for 25 to 35 minutes.

7. A method of using the lysinibacillus according to any one of claims 1 to 6 for inhibiting the proliferation of sulfate-reducing bacteria SRB.

8. Use of the lysinibacillus described in any one of claims 1 to 6 for inhibiting the proliferation of sulfate-reducing bacteria (SRB) in an oilfield produced water treatment system.

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