Bacteriophage and bactericidal composition and application

Abstract

The present invention relates to the field of bacteriophages, and in particular to bacteriophages and bactericidal compositions and applications. The present invention provides a bacteriophage, and the phage has a preservation number of CGMCC45383. The best infection multiplicity MOI of the bacteriophage provided by the present invention is 0.001, and the one-step growth curve shows that the incubation period of SRB7757 is 4 hours, the lysis period is 32 hours, and the maximum burst volume is about 100 PFU / cell; it is active in the pH range of 2 to 12, and is still active after being treated at 70°C for 60 minutes; it can significantly reduce the inhibition of metal corrosion caused by SRB; and it is insensitive to chloroform.

Description

Technical Field

[0001] The present invention relates to the field of bacteriophages, and in particular to bacteriophages and bactericidal compositions and applications. Background Art

[0002] Microbiologically induced corrosion (MIC) refers to the corrosion of metals caused directly or indirectly by the life of various microorganisms and their metabolites. Many microorganisms, including bacteria, archaea and fungi, have been found to cause corrosion. The bacteria related to microbiological corrosion are mainly iron bacteria (FB), sulfate-reducing bacteria (SRB), and saprophytic bacteria (TGB). Among them, SRB is the most serious.

[0003] SRB is a type of anaerobic microorganism that uses organic carbon sources as electron donors to reduce sulfur oxides such as sulfate, sulfite, and thiosulfate as electron acceptors of organic matter to hydrogen sulfide under anaerobic or locally anoxic conditions. SRB has a corrosive effect on almost all common metal materials such as steel, aluminum, zinc, copper, and alloy metals. de Queiroz et al. found that microorganisms easily form biofilms on metal surfaces, causing local corrosion. In oilfield reinjection water, high concentrations of sulfate ions can cause a large number of SRB to multiply, produce H2S and FeS, and seriously corrode water injection pipelines and drilling equipment. This process is also called microbial souring in oilfields. Hydrogen sulfide accelerates the corrosion rate, contaminates crude oil, reduces crude oil quality, and increases subsequent processing costs. In addition, it is highly toxic and poses a serious threat to the lives of workers. The resulting ferrous sulfate precipitation will cause the oil layer to be blocked, thereby reducing the oil layer leakage rate, making it much more difficult to mine. Reservoir acidification causes the normal production of the oil field to be compromised, resulting in huge economic losses. Metal corrosion causes economic losses of 2.5 trillion US dollars worldwide each year, accounting for about 3.4% of the world's GDP, and microbially induced corrosion (MIC) accounts for 30% of this. MIC often occurs in pipeline systems in crude oil production, tap water industry, fermentation workshops, etc., as well as in environments such as docks, ports and steel piles on docks. Studies have shown that the economic losses caused by SRB corrosion in my country's oil industry each year are as high as hundreds of millions of dollars. In addition, the production pipeline system, especially the pipelines of the power water system, including the heat exchange system of the equipment, is severely corroded, resulting in great production losses.

[0004] Chlorine, formaldehyde, glutaraldehyde and quaternary ammonium salts are commonly used in oil fields to inhibit SRB. Although chemical fungicides have high sterilization efficiency against bacteria and are easy to operate, they can cause environmental pollution and harm human health. Long-term use of fungicides can make SRB resistant. In recent years, people have found that competition, antagonism and parasitism between microorganisms can kill or inhibit SRB. Biological competitive inhibition technology has the characteristics of being environmentally friendly, low-cost, wide treatment range and simple operation. However, SRB will form a biofilm when growing in the pipeline. For SRB fixed bacteria growing in the biofilm, fungicides and biological competitive inhibition technology have limited effect. Summary of the invention

[0005] In order to achieve the above-mentioned purpose, the present invention provides a bacteriophage and a bactericidal composition and application. The bacteriophage provided by the present invention can effectively kill and / or inhibit SRB, thereby significantly reducing or inhibiting various effects caused by SRB.

[0006] Therefore, the first aspect of the present invention provides a bacteriophage, the deposit number of the bacteriophage is CGMCC45383.

[0007] The second aspect of the present invention provides a bactericidal composition, which contains the bacteriophage as described above.

[0008] The third aspect of the present invention provides use of the bacteriophage as described above or the bactericidal composition as described above in killing sulfate-reducing bacteria and / or inhibiting the growth of sulfate-reducing bacteria.

[0009] A fourth aspect of the present invention provides use of the bacteriophage as described above or the fungicide composition as described above in reducing the sulfide content in the environment.

[0010] A fifth aspect of the present invention provides use of the bacteriophage or the bactericidal composition as described above in alleviating, repairing or inhibiting oil reservoir acidification and / or metal pipeline corrosion.

[0011] A sixth aspect of the present invention provides a method for alleviating, repairing or inhibiting sulfate-reducing bacteria corrosion, characterized in that the bacteriophage as described above or the bactericidal composition as described above is added to an environment where sulfate-reducing bacteria corrosion is to be alleviated, repaired or inhibited.

[0012] A seventh aspect of the present invention provides a method for alleviating, repairing or inhibiting oil reservoir acidification, the method comprising: adding the bacteriophage as described above or the bactericidal composition as described above to the oil reservoir.

[0013] Through the technical solution of the present invention, the following beneficial effects can be obtained:

[0014] 1. The optimal infection multiplicity MOI of the phage provided by the present invention is 0.001. The one-step growth curve shows that the incubation period of SRB7757 is 4 hours, the lysis period is 32 hours, and the maximum burst volume is about 100 PFU / cell.

[0015] 2. The bacteriophage provided by the present invention has good acid-base tolerance and temperature tolerance, is active in the pH range of 2 to 12, and is still active after being treated at 70°C for 60 minutes, indicating that it has a wide acid-base application range and good temperature tolerance. This property is conducive to its application in practical environments.

[0016] 3. The results of the bacteriophage simulation corrosion experiment provided by the present invention show that the addition of bacteriophage can significantly reduce and inhibit the metal corrosion caused by SRB.

[0017] 4. The bacteriophage provided by the present invention is insensitive to chloroform. Therefore, during the enrichment and purification of phage particles, chloroform can be added to dissolve PEG8000 and bacterial debris in the solution and remove impurities.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0019] Biological Deposit

[0020] The bacteriophage (Caudovirales myoviridae) provided by the present invention was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on January 4, 2023, with a deposit number of CGMCC45383, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), referred to as SRB7757. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0022] Figure 1A This is the single plaque morphology of the bacteriophage SRB7757 of the present invention on the culture medium.

[0023] Figure 1B The morphology of bacteriophage SRB7757 under transmission electron microscopy.

[0024] Figure 2 This is the one-step growth curve of the bacteriophage SRB7757 of the present invention.

[0025] Figure 3A This is the stability curve of the bacteriophage SRB7757 of the present invention at different pH values.

[0026] Figure 3B This is a thermal stability trend diagram of the bacteriophage SRB7757 of the present invention.

[0027] Figure 3C The bacteriophage SRB7757 of the present invention is shown to be sensitive to chloroform.

[0028] Figure 3D Effect of metal ion concentration on phage activity

[0029] Figure 4 The results of nucleic acid type identification of the bacteriophage SRB7757 of the present invention are shown.

[0030] Figure 5 A functional model diagram of the bacteriophage SRB7757 of the present invention is shown.

[0031] Fig. 6A This is the phylogenetic tree of the large subunit of the terminase of bacteriophage SRB7757 of the present invention.

[0032] Figure 6B This is the phylogenetic tree of the major capsid proteins of bacteriophage SRB7757 of the present invention.

[0033] Figure 7 Scanning electron microscope images of the microscopic corrosion morphology under different treatments in the simulated corrosion test.

[0034] Figure 8 The effect of the bacteriophage SRB7757 of the present invention on the sulfide production after treating SRB is shown. DETAILED DESCRIPTION

[0035] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0036] In a first aspect, the present invention provides a bacteriophage, the deposit number of the bacteriophage is CGMCC No.45383.

[0037] The bacteriophage provided by the present invention was isolated from a water sample from a sewage treatment plant and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on January 4, 2023. Its deposit number is CGMCC No. 45383, and its deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), abbreviated as SRB7757.

[0038] The bacteriophage SRB7757 provided by the present invention can be obtained in large quantities by culturing, for example, by adding it to an SRB bacterial suspension for culturing, a large amount of bacteriophage SRB7757 can be obtained. Specifically, the logarithmic host bacterial solution and the bacteriophage are mixed according to the optimal infection multiplicity, cultured at 37°C, and samples are taken at regular intervals and immediately filtered with a 0.22 um sterile filter to obtain the bacteriophage SRB7757.

[0039] In a second aspect, the present invention provides a bactericidal composition comprising the bacteriophage as described above.

[0040] In the present invention, there is no particular requirement for the concentration of the bacteriophage in the bactericidal composition, as long as it can effectively infect and lyse the SRB host bacteria. For example, in the bactericidal composition, the concentration of the bacteriophage SRB7757 can be 10 4 CFU / mL.

[0041] The bactericidal composition provided by the present invention may also contain other components that can kill target bacteria but will not have an adverse effect on bacteriophage SRB7757. For example, it may also contain other types of bacteriophages that can kill other targets in the sterilization environment, or another example, it may also contain agents that promote bacteriophage SRB7757 to infect all targets; another example, it may also contain nutritional components that help bacteriophage SRB7757 maintain its viability during storage of the bactericidal composition, and so on.

[0042] In the present invention, the dosage form of the bactericidal composition is not particularly limited, as long as it can ensure that the bacteriophage SRB7757 has a predetermined survival rate. For example, when used, the survival rate of bacteriophage SRB7757 is not less than 50%, not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%. In some embodiments, the dosage form of the bactericidal composition is a liquid dosage form. In some embodiments, the bactericidal composition is a powder. In some embodiments, the bactericidal composition is a tablet. It can be understood that no matter what dosage form it is in, the bactericidal composition can include the excipients required for preparing the corresponding dosage form, but it is necessary to minimize the impact on bacteriophage SRB7757.

[0043] In a third aspect, the present invention provides use of the bacteriophage as described above or the bactericidal composition as described above in killing sulfate-reducing bacteria and / or inhibiting the growth of sulfate-reducing bacteria.

[0044] As described above, the bacteriophage SRB7757 provided by the present invention can infect SRB to complete the lysis of SRB, so as to achieve the purpose of killing it. On the other hand, after the bacteriophage SRB7757 infects SRB, it will release the contents such as the phage lytic enzyme system into the environment, thereby lysing the bacterial cells and achieving the effect of inhibiting the growth of SRB. In some embodiments, after the bacteriophage SRB7757 infects SRB, it will not cause the lysis of SRB, but will coexist with SRB. However, since the bacteriophage SRB7757 needs to reproduce, it will also compete for nutrients, and therefore, it will also inhibit the growth of SRB to a certain extent.

[0045] In a fourth aspect, the present invention provides use of the bacteriophage as described above or the fungicide composition as described above in reducing the sulfide content in the environment.

[0046] As shown in Example 10 of the present invention, no H2S was found in the SRB group treated with bacteriophage SRB7757, while a considerable amount of H2S was produced in the control group without bacteriophage SRB7757. Therefore, the bacteriophage SRB7757 provided by the present invention can reduce the sulfide content in the environment.

[0047] In a preferred embodiment of the present invention, the sulfide in the environment is caused by SRB. Therefore, the environment is an environment containing SRB.

[0048] In some embodiments of the present invention, the environment containing SRB is solid, and the bacteriophage of the present invention as described above or the bactericidal composition as described above can be directly applied to the surface of the environment containing SRB. Examples of the solid environment may include, but are not limited to, underground pipeline systems of metal equipment for sewage treatment plants, metal equipment for oil field development, metal equipment for water industry, metal equipment for docks and ports, and metal equipment for fermentation workshops. In some embodiments, the environment containing SRB is liquid, and the bacteriophage of the present invention as described above or the bactericidal composition as described above can be directly added to the liquid phase containing SRB. Examples of liquid environments containing SRB include, but are not limited to, mine wastewater, oil and gas reservoirs, coal reservoirs, and industrial pipeline systems.

[0049] In a fifth aspect, the present invention provides use of the bacteriophage as described above or the bactericidal composition as described above in alleviating, repairing or inhibiting oil reservoir acidification and / or metal corrosion.

[0050] Preferably, the reservoir acidification and / or metal corrosion is caused by sulfate-reducing bacteria.

[0051] In a sixth aspect, the present invention provides a method for alleviating, repairing or inhibiting sulfate-reducing bacteria corrosion, wherein the bacteriophage as described above or the bactericidal composition as described above is added to an environment where sulfate-reducing bacteria corrosion is to be alleviated, repaired or inhibited.

[0052] In the present invention, the environment to be alleviated, repaired or inhibited by sulfate-reducing bacteria corrosion is generally a metal surface where SRB grows, for example, various metal piping systems, etc., the surface of metal equipment, etc.

[0053] In a seventh aspect, the present invention provides a method for alleviating, repairing or inhibiting oil reservoir acidification, the method comprising: adding the bacteriophage as described above or the bactericidal composition as described above to the oil reservoir.

[0054] Example

[0055] The culture medium formula involved in the following examples is as follows:

[0056] Preparation Example 1

[0057] This preparation example is used to illustrate the preparation of host bacterial solution

[0058] The host bacteria SRB ATCC7757 was inoculated into an anaerobic tube containing modified postgate liquid medium (liquid medium formula A liquid: Na2SO41.0g / L, K2HPO40.5g / L, CaCl20.1g / L, NH4Cl1.0g / L, MgSO4·7H2O2.0g / L, sodium lactate3.65g / L, yeast extract1.0g / L, sterilized at 121℃ for standby use; B liquid: (NH4)2Fe(SO4)2.6H2O0.5g / L, ascorbic acid0.1g / L, prepared and used immediately, filtered and sterilized) according to a 5% inoculation amount, cultured at 37℃ for 2-3d, and 100uL of bacterial solution was taken and mixed with 4mL of semi-solid medium (semi-solid medium formula (upper layer): Na2SO41.0g / L, K2HPO40.5g / L, CaCl2 0.1g / L, NH4Cl 1.0g / L, MgSO4·7H2O2.0g / L, sodium lactate 3.65g / L, yeast extract 1.0g / L, agar powder 7g / L) and pour into the solidified lower culture medium. Place the culture dish in an anaerobic incubator and culture at 37℃ for 2-3 days. The small black colonies growing on the plate are SRB. Pick a single colony and inoculate it in the liquid culture medium. Culture at 37℃. When the solution produces a black precipitate, take it out for use.

[0059] Preparation Example 2

[0060] This preparation example is used to illustrate the enrichment and verification of phage

[0061] Take 50 mL of sewage sample and centrifuge it in a refrigerated centrifuge to remove impurities (8000 r / min, 15 min). Take the supernatant and filter it with a 0.22 um filter. The filtrate is set aside.

[0062] Take 10mL of the filtrate and add it to a 25mL anaerobic tube containing 10mL of liquid culture medium, inoculate 1mL of the host bacterial solution cultured to the logarithmic phase, and finally add 2mL of liquid paraffin, and culture it in a 37℃ incubator for 48h. Centrifuge the cultured mixed solution in a low-temperature centrifuge (8000r / min, 15min), take the supernatant and repeat once, and finally filter the supernatant with a 0.22um sterile filter. The filtrate is the phage enrichment solution. Store at 4℃ for later use.

[0063] The spotting method (Tang Ning, Yin Yin, Ren Huiying, et al. Isolation and purification of high-titer and wide-spectrum phages of Salmonella and determination of their biological characteristics [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2021, 48 (7): 2577-2583.) was used to verify the presence of phages in the enrichment solution. 500uL of host bacterial solution cultured to the logarithmic phase was mixed with 4mL of semi-solid culture medium and quickly poured into the solidified lower layer culture medium (solid culture medium formula (lower layer) A solution: Na2SO4 1.0g / L, K2HPO4 0.5g / L, CaCl2 0.1g / L, NH4Cl 1.0g / L, MgSO4·7H2O 2.0g / L, sodium lactate 3.65g / L, yeast extract 1.0g / L, agar powder 12g / L, sterilized at 121℃ for use; B solution: (NH4)2Fe(SO4)2.6H2O 0.5g / L, ascorbic acid 0.1g / L, prepare before use, filter and sterilize), after the upper culture medium solidifies, add 5uL of phage enrichment solution, and in another plate, add 5uL of saline and 5uL of bactericidal preservative as negative and positive controls, respectively. After standing at room temperature for 1h, place the culture dish in an anaerobic jar filled with nitrogen, and culture at 37℃ for 2-3d to observe whether there are plaques.

[0064] The observed plaques were purified and enriched, and deposited in the General Microbiology Center of China Microorganism Culture Collection on January 4, 2023. The deposit number is CGMCC No.45383, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), abbreviated as SRB7757.

[0065] Example 1

[0066] This example is used to illustrate the purification and morphological observation of bacteriophage

[0067] Pick the plaques formed by the spot test and add them to 5mL of the host bacterial solution cultured to the logarithmic phase, then add 5mL of liquid culture medium, and finally add liquid paraffin, and culture in a 37℃ incubator for 2-3 days. Centrifuge the mixed culture solution in a low-temperature centrifuge (8000r / min, 15min), filter the supernatant through a 0.22um sterile filter, and you can get the phage enrichment solution.

[0068] The phage stock solution was diluted 10 times with SM buffer, 200uL of phage lysate was mixed with 500uL of host bacteria and allowed to stand at 37℃ for 15min, then added to the upper semi-solid medium cooled to 50℃, quickly mixed and poured into the solidified lower medium, and the culture dish was placed in an anaerobic tank for 2-3d, with 3 parallel groups for each dilution. After a single plaque grows, pick a single plaque and repeat the above operation 3-5 times to complete the purification of the phage.

[0069] like Figure 1A As shown in the figure, the plaques grown on the plate are of uniform shape and size. The plaques are transparent and round, with a diameter of about 1-2 mm, and neat edges without halos. Figure 1B ), it can be observed that bacteriophage SRB7757 has a typical icosahedral head and a retractable tail structure.

[0070] Example 2

[0071] This example is used to illustrate the determination of phage lysis rate

[0072] The SRB strain ATCC7757 preserved in the laboratory was activated as an indicator bacteria, and the phage lysis rate was determined by spotting method. Take 500uL of bacterial solution cultured to the logarithmic growth phase and mix it with 5mL of semi-solid culture medium cooled to 50℃, and quickly pour it into the lower solid culture medium. After the upper culture medium solidifies, divide the plate into 4 areas, add 5uL of phage lysis solution to each area, and let it stand for 30min-60min until the liquid on the surface of the culture medium is absorbed, then put the culture dish into an anaerobic tank, culture at 37℃ for 2-3d, and observe whether there are plaques.

[0073] The results show that the bacteriophage provided by the present invention has lytic activity against ATCC7757 and has a narrow host spectrum and strong specificity.

[0074] Example 3

[0075] This example is used to illustrate the determination of the optimal infection multiplicity of bacteriophage

[0076] The optimal multiplicity of infection (MOI) refers to the ratio of phages that can be adsorbed to bacterial hosts within a specific period of time, and is an important indicator for conducting lytic phage infection tests.

[0077] (P0: number of bacteriophages per milliliter, B: number of bacteria per milliliter)

[0078] (1) Prepare the logarithmic phase host bacterial solution and dilute the concentration to 10 4 CFU / mL;

[0079] (2) Dilute the prepared phage lysate to 10 according to the infection multiplicity of 0.001, 0.01, 0.1, 1, 10, and 100. 1 PFU / mL, 10 2 PFU / mL, 10 3 PFU / mL, 10 4 PFU / mL, 10 5 PFU / mL, 10 6 PFU / mL;

[0080] (3) Mix 500uL of host fluid with 200uL of phage lysate, add 10mL of liquid culture medium, then add 2mL of liquid paraffin, and culture at 37°C for 2 days;

[0081] (4) Centrifuge and filter the supernatant. Verify using the double-layer plate method with 3 parallels per group and record the data.

[0082] (5) Calculate the MOI value and determine the optimal multiplicity of infection (MOI), as shown in Table 2.

[0083] Table 2

[0084]

[0085] As shown in Table 2, at MOI = 0.001, the highest phage titer was 1.3x10 7 PFU / mL,

[0086] From this, it can be determined that the optimal infection multiplicity of bacteriophage ATCC7757 is 0.001.

[0087] Example 4

[0088] Example is used to illustrate the determination of one-step growth curve and lysis amount

[0089] The logarithmic host bacterial solution and phage were mixed according to the optimal infection multiplicity, cultured at 37°C, and samples were taken at regular intervals and immediately filtered with a 0.22um sterile filter and stored at 4°C. After sampling, gradient dilution was performed uniformly and verified using the double-layer plate method. A one-step growth curve was drawn with the infection time as the horizontal axis and the number of phages (pfu / ml) as the vertical axis, and the lysis amount was calculated. The results are shown in 2.

[0090] from Figure 2 It can be seen that the incubation period of SRB7757 is 4 hours, the lysis period is 32 hours, and the number of phages no longer increases after 36 hours, reaching a stable period. Its maximum burst capacity is calculated to be about 100 PFU / cell.

[0091] Example 5

[0092] Examples are used to illustrate pH stability

[0093] Use concentrated hydrochloric acid (1 mol / L) or sodium hydroxide (1 mol / L) to adjust the SM buffer to different pH values ​​(pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 12), and sterilize by high pressure for later use. Take 200uL of phage lysate and 500uL of host and add them to SM buffer with different pH values, mix well and place in a 37°C water bath for 1h, determine the phage titer by double-layer plate method, and draw a pH stability curve. The results are shown in Figure 3A shown.

[0094] Depend on Figure 3A The results showed that the phages were active after being treated with SM buffers of different pH values. The phage activity was high in the pH range of 2 to 9, and the titer of the phage decreased significantly in the pH range of 10 to 12. The phages were still active at pH = 2 and pH = 12, indicating that the phage SRB7757 had a wide acid-base tolerance range.

[0095] Example 6

[0096] Examples are used to illustrate thermal stability

[0097] Take 1mL of known titer phage and culture it in a water bath at 4℃, 20℃, 30℃, 50℃, 60℃, 70℃, and 80℃ for 60min, then dilute it uniformly and measure the number of plaques on a double-layer plate. Set up 3 parallels for each group, record the data and draw a thermal stability trend chart. The results are as follows: Figure 3B shown.

[0098] like Figure 3B As shown in the figure, the activity of phages varies greatly at different temperatures. The activity of phages is relatively stable in the temperature range of 4℃ to 60℃. When the temperature is 70℃, the phage titer is significantly reduced and the activity is weak. When the temperature reaches 80℃, the phage is completely inactivated. After SRB7757 was treated at 70℃ for 60min, its titer remained at 10PFU / mL, indicating that it has good tolerance to temperature.

[0099] Example 7

[0100] Example is used to illustrate the sensitivity test of bacteriophage to chloroform

[0101] Take 1 mL of phage solution, add 10% and 50% chloroform respectively, mix well and let stand at 4℃ for 24h. Set phage solution without chloroform as negative control, centrifuge at 8000r / min for 10min, take the supernatant and dilute it with SM buffer, and use double-layer plate method to determine the change of phage titer before and after chloroform treatment. The results are as follows: Figure 3C shown.

[0102] During the enrichment and purification of phage particles, chloroform is added to dissolve PEG8000 and bacterial debris in the solution and remove impurities. However, some phages contain lipids in their caps or tails, so the lipid solubility of chloroform will destroy the structure of the phage, thereby affecting the activity of the phage. Therefore, it is necessary to test the phage for chloroform sensitivity. Figure 3C , there was no significant difference in the efficacy of the phage before and after chloroform treatment, indicating that phage SRB virus 7757 is not sensitive to chloroform and the phage shell or tail does not contain lipid substances.

[0103] Example 8

[0104] The example is used to illustrate the effect of metal ion concentration on phage activity

[0105] Generally, phage proliferation requires a certain amount of Ca 2+ Mg 2+ ions, we therefore examined the phage response to Ca 2+ Mg 2+ In short, we added metal ions (Ca 2+ Mg 2+ ), with no metal ions added as control, then inoculated with gradient dilutions of phage fluid and host, and the double-layer plate method was used to observe the number of phage plaques.

[0106] The results are as follows Figure 3D As shown, SRB7757 has an effect on Ca 2+ and Mg 2+ There is an obvious need. When the ion concentration is between 5mM and 15mM, the infection efficiency of SRB7757 gradually increases with the increase of ion concentration. When the ion concentration reaches 20mM, its continuous enhancement effect is not obvious.

[0107] Example 9

[0108] This example is used to illustrate the simulated corrosion test

[0109] The material used in this test is 20 mm x 10 mm x 2 mm. # Carbon steel specimen. # , 400 # , 600 # , 1000 # Use water-abrasive sandpaper to polish the surface step by step, then soak it in acetone for 2 hours to remove grease. After drying, put it in a drying oven for 24 hours for later use.

[0110] The test was carried out in a simulated medium of oilfield produced water. The formula of the simulated medium

[16] The mixture includes 3.5g sodium bicarbonate, 1.5g sodium sulfate, 8.0g sodium chloride, 1.6g crystalline magnesium sulfate, 0.5g anhydrous calcium chloride, 0.35g potassium sulfate, 2.0g sodium nitrate, and 0.5g sodium acetate, which are fully dissolved in 1L deionized water, and the salinity is 1.6, which is consistent with the salinity of oilfield produced water. In order to allow bacteria to grow better, 10% of SRB culture medium nutrients are added to the simulated medium, the corrosion test medium is 90% v / v simulated aqueous solution + 10% v / v modified Postgate culture medium, and the pH is adjusted to 7.0±0.2 with sodium hydroxide. All chemicals used are analytically pure, dissolved in distilled water, sterilized with high-pressure steam at 121°C for 20 minutes, and cooled to room temperature for use.

[0111] The processed 20 # The carbon steel sheet was placed in a medium containing 100 mL of 90% v / v simulated aqueous solution + 10% v / v modified Postgate culture medium. The control group, group a, was only added with simulated water medium, and group b was added with simulated medium water and a final concentration of 10 6 CFU / mL of SRB bacterial solution, group C added simulated medium water and a final concentration of 10 6 CFU / mL of SRB bacterial solution, and add phage according to MOI=0.01, and soak and culture for 7-28 days.

[0112] Corrosion morphology observation

[0113] After the sample is taken out, the surface is gently rinsed with sterile PBS buffer, fixed with 2.5% v / v glutaraldehyde (prepared with sterile PBS solution) at 4°C for 3h, and then gradient dehydration is performed with 20%, 40%, 60%, 80%, and 100% v / v ethanol prepared with sterile PBS. Each gradient dehydration takes 20 to 30 minutes. The samples of the control group do not need to be fixed with glutaraldehyde after being taken out. They can be directly observed after dehydration with different gradients of ethanol. After drying, the surface morphology of the sample is observed using a scanning electron microscope. At the same time, the surface morphology of the samples in the two systems after the corrosion products are removed is observed, and each test is randomly performed at 3 different locations of the sample.

[0114] The results of the control group and each experimental group after running in the corrosion system for 28 days are as follows: Figure 7 As shown, group a is the control group with simulated water medium, group b is the experimental group with simulated water medium and SRB bacterial solution, and group c is the experimental group with simulated water medium, SRB bacterial solution and bacteriophage. The results of scanning electron microscopy showed that no corrosion pits and cracks were observed on the surface of group a, more SRB metabolic corrosion products and obvious cracks (arrows) were observed in group b, and the surface corrosion products of group c were significantly reduced with only a very small number of tiny cracks. It can be judged that the addition of bacteriophage has a certain inhibitory effect on metal corrosion caused by SRB.

[0115] Example 10

[0116] This example is used to illustrate the detection of hydrogen sulfide concentration

[0117] First, the host bacteria were cultured to the logarithmic growth phase (106 CFU / mL), and then 1 mL of host bacteria was added to the control group and 1 mL of host bacteria (10 6 CFU / mL) and 1mL phage (104PFU / mL), and finally add 10mL liquid culture medium. Three parallel tests are set up for each group. The mixture is placed in a 37℃ incubator for culture. Samples are taken at regular intervals. The content of sulfide in the mixture is determined by the methylene blue spectrophotometry. The results are as follows Figure 8 shown.

[0118] It was determined that the initial number of SRBs was about 1.4*10 6 CFU / mL, phage titer is 1.2*10 4 From 0 to 24 hours, no H2S production was found in the phage-treated group, while the sulfide content of the control group was about 3.9 μg / mL.

[0119] Embodiment 11

[0120] Example for illustrating the taxonomic identification of bacteriophages

[0121] 9.1 Genome extraction and nucleic acid type identification

[0122] (1) Take 1 ml of phage collection solution, add 1 ul DNase and 1 ul RNase, and treat at 37°C for 30 min;

[0123] (2) Add 20% SDS, 1M Tris-Cl and EDTA. The concentrations of these three substances in the solution are 0.25% (12.5ul) SDS, 50mM (50ul) Tris-Cl and 50mM (100ul) EDTA. Treat the solution in a water bath at 80°C for 15min.

[0124] (3) Add 2 M potassium acetate to a final concentration of 0.625 M (313 μl), then mix well and place on ice for 60 min, mixing 1-2 times during the process;

[0125] (4) Centrifuge at 10,000 rpm for 15 min at 4°C to remove the SDS-K precipitate (cut the tip of the pipette slightly) and aspirate about 1.5 ml of the supernatant;

[0126] (5) Add 2 volumes of -20°C precooled anhydrous ethanol (-20°C precooled equal volume of isopropanol) and centrifuge at 10,000 rpm for 10 min at 4°C

[0127] (6) Wash the obtained DNA once with 70% pre-cooled ethanol, air dry it naturally until no visible liquid remains, then dissolve it in ddH2O (100-200ul), place it at 4℃ for at least 12h (or treat it at 65℃ for 1h) to make it completely dissolved, and detect it by electrophoresis (2ul sample, 2ul lambda phage marker, electrophoresis for 15min).

[0128] (7) The extracted nucleic acid was digested with DNaseI and RNase A for 1 h in a 37°C water bath, and the digestion products were identified by agarose gel electrophoresis. Figure 4 shown.

[0129] 9.2 Phage genome sequencing and bioinformatics analysis

[0130] Through the gene prediction and functional annotation provided by the biological company, the non-redundant protein database (Nr database) was searched using the Blastp program on NCBI, and the coding products of the genes were searched for similarity and identity with the proteins with known functions in the NR data to complete the functional annotation; the tRNA encoding genes in the genome sequence were found using the software tRNAscan-SEM. The whole genome functional pattern map of bacteriophage SRB7757 was generated using the CG View online tool (http: / / stothard.afns.ualberta.ca / cgview_server / ). The phylogenetic tree of the large subunit of the terminase and the major capsid protein was constructed using MEGA11 (http: / / www.MEGAsoftware.net), and the phylogenetic tree was beautified using iTOL online software ( https: / / itol.embl.de / ), the results are as follows Figure 5 shown.

[0131] The results showed that the genome of bacteriophage SRB7757 was 142573 bp in length, with a G+C content of 44.29%. The phage genome had 217 open reading frames (ORFs) ranging from 114 bp to 4980 bp in length, of which 129 ORFs were hypothetical proteins and 88 ORFs encoded functional proteins. The online software tRNAscan-SE 2.0 predicted that the phage contained 6 tRNAs. Figure 5As shown in the figure, according to the different functions of its open reading frame encoding, it can be divided into 5 modules: cleavage module, packaging and structure module, DNA replication and regulation module, and hypothetical protein. The phylogenetic tree of the large subunit of the terminase and its major capsid protein was constructed. The results showed that the phylogenetic tree of the major capsid protein of SSRB7757 showed that it was closely related to the phages of the order Caudovirales and was independently located in a branch. Based on the phylogenetic tree of the large subunit of the terminase, it was found that it was on the same branch as the phage MedDecM-OCT-S09-C7 of the order Caudovirales. In summary, it can be judged that SRB7757 may belong to the order Caudovirales.

[0132] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A bacteriophage, characterized in that: The deposit number of this phage is CGMCC NO.45383.

2. A bactericidal composition, characterized in that: The bactericidal composition contains the bacteriophage according to claim 1.

3. Use of the bacteriophage according to claim 1 or the bactericidal composition according to claim 2 in killing sulfate-reducing bacteria ATCC7757 and / or inhibiting the growth of sulfate-reducing bacteria ATCC7757.

4. Use of the bacteriophage according to claim 1 or the bactericidal composition according to claim 2 in reducing the sulfide content in the environment; in, The environment is an environment containing sulfate-reducing bacteria ATCC7757.

5. Use of the bacteriophage according to claim 1 or the bactericidal composition according to claim 2 in alleviating, repairing or inhibiting oil reservoir acidification and / or metal pipeline corrosion; in, The oil reservoir acidification and / or metal pipeline corrosion are caused by sulfate-reducing bacteria ATCC7757.

6. A method for alleviating, repairing or inhibiting sulfate-reducing bacteria corrosion, characterized in that: The bacteriophage described in claim 1 or the bactericidal composition described in claim 2 is added to an environment where corrosion of sulfate-reducing bacteria ATCC7757 is to be alleviated, repaired or inhibited.

7. The method according to claim 6, wherein: The environment to be alleviated, repaired or inhibited by sulfate-reducing bacteria corrosion includes at least one of the following: sewage treatment plants, mine wastewater, underground pipeline systems, metal equipment used in oil field development, metal equipment used in the water industry, docks, metal equipment used in ports, oil and gas reservoirs, coal reservoirs and fermentation workshop pipeline equipment.

8. A method for alleviating, repairing or inhibiting oil reservoir acidification, characterized in that: The method comprises: adding the bacteriophage according to claim 1 or the bactericidal composition according to claim 2 into an oil reservoir containing ATCC7757.

Images