Bacillus megaterium 1-9-1, microbial agent and application of bacillus megaterium 1-9-1 and microbial agent

Abstract

The invention belongs to the technical field of microbial remediation, and particularly relates to bacillus megatherium 1-9-1, a microbial agent and application of the bacillus megatherium 1-9-1 and the microbial agent. The bacillus megaterium 1-9-1 is preserved in the China General Microbiological Culture Collection Center on December 21, 2023, and the preservation number of the bacillus megaterium 1-9-1 is CGMCC (China General Microbiological Culture Collection Center) No.29373. According to the application of the bacillus megatherium 1-9-1 in the aspects of repairing the overburden cracks and preventing the gas layer from escaping, the bacillus megatherium 1-9-1 is attached to the surface of the rock, carbonate precipitates are mineralized and deposited around the rock pores and the cracks for repairing, and meanwhile CO2 is absorbed and sealed. After the microbial agent provided by the invention is injected into a cover layer, the metabolite of the microbial agent can block pores and tiny cracks in the cover layer, so that the purposes of better carbon sequestration effect and oil and gas reservoir protection are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial remediation, and specifically relates to Bacillus megaterium 1-9-1, a microbial agent and its application. Background Art

[0002] As a technology with great potential for emissions reduction, carbon capture, utilization and storage (CCUS) technology, like CO2 flooding technology, is an essential emissions reduction technology. Injecting the captured CO2 into underground storage bodies for geological utilization and storage is a key link. However, the CO2 injected underground may break through the storage body or caprock and leak into the soil and the surface, which not only has a huge impact on human health and the ecological environment, but also directly affects the actual emissions reduction and oil displacement effects of storage. Therefore, it is necessary to use plugging technology to plug the CO2 injected underground.

[0003] Existing plugging technologies include chemical reaction precipitation plugging, foam system plugging, polymer resin plugging, polymer gel plugging, etc. These methods have poor permeability for low-permeability rocks and low efficiency in repairing micro-cracks. Different from other plugging technologies, microbial-induced calcium carbonate precipitation (MICP) technology has better permeability for low-permeability rocks, can repair micro-cracks, and is more conducive to filling micro-pores and reducing CO2 leakage. Therefore, it is necessary to use microbial-induced calcium carbonate precipitation (MICP) technology to develop more microorganisms for actual microbial remediation applications.

[0004] Currently, the microorganisms used in actual microbial remediation applications include Pseudomonas, Bacillus subtilis, and Sporosarcina pasteurii. These microbial strains are difficult to mineralize and deposit in extremely small pores and maintain the remediation effect for a long time. Summary of the Invention

[0005] To solve the defect that the microbial strains used in actual microbial remediation applications in the prior art are difficult to mineralize and deposit in extremely small pores and maintain the remediation effect for a long time, the present invention provides a strain of Bacillus megaterium 1-9-1, a microbial agent and its application. To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a strain of Bacillus megaterium 1-9-1, which was deposited at the China General Microbiological Culture Collection Center on December 21, 2023, with the deposit number CGMCC No. 29373 and the taxonomic name Bacillus megaterium.

[0007] The Bacillus megaterium 1-9-1 is a strain of Bacillus megaterium producing carbonic anhydrase isolated and screened from oilfield produced water, and it has been experimentally verified to have the performance of high-yield carbonic anhydrase; at the same time, this bacterium can effectively plug the large pores and micro-fractures of the caprock, and it can carry out mineralization deposition in extremely small pores and maintain the repair effect for a long time.

[0008] The present invention also provides a microbial inoculum, which includes the Bacillus megaterium 1-9-1 and a strain culture medium.

[0009] The strain culture medium is composed of the following materials at the final concentrations: beef extract 3 g·L -1 ~4 g·L -1 、tryptone 10 g·L -1 ~11 g·L -1 、sodium chloride 4 g·L -1 ~5 g·L -1 、zinc sulfate 1 μmol·L -1 ~1.2 μmol·L -1 , and the solvent is water.

[0010] The present invention also provides a preparation method of the microbial inoculum, which includes the following steps: Inoculate the bacterial liquid of the Bacillus megaterium 1-9-1 into the strain culture medium, and perform fermentation culture to obtain a fermentation broth; filter the fermentation broth and collect the filtrate to obtain the microbial inoculum.

[0011] Preferably, the conditions for the fermentation culture are: culture at 34°C to 36°C for 23 h to 25 h.

[0012] Preferably, the volume ratio of the bacterial liquid of the Bacillus megaterium 1-9-1 to the strain culture medium is 0.9 to 1.1:50.

[0013] Preferably, the bacterial liquid of the Bacillus megaterium 1-9-1 is the supernatant after enrichment of the water sample produced from the oilfield.

[0014] The present invention also provides the application of the microbial inoculum in the repair of caprock cracks to prevent the escape of gas from the gas layer.

[0015] Preferably, the microbial inoculum is used to prepare microbial cement, so as to repair the caprock cracks and prevent the escape of gas from the gas layer.

[0016] Preferably, the method for preparing microbial cement includes the following steps: Inject the microbial inoculum into the caprock rock to induce the formation of microbial cement.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides Bacillus megaterium 1-9-1. The present invention provides a carbonic anhydrase-producing bacterium, namely Bacillus megaterium 1-9-1. This Bacillus megaterium 1-9-1 is a carbonic anhydrase-producing Bacillus megaterium strain isolated and screened from produced water in oilfields, and through experiments, it has been found to have the performance of high-yield carbonic anhydrase production; at the same time, this bacterium can effectively plug the large pores and micro-fractures in the caprock. The strain adheres to the fractures after being injected into the caprock and generates calcium carbonate precipitation at the nucleation sites, which can effectively plug the pores and fractures in the caprock, improve the recovery rate, and significantly enhance the repair of the integrity of the caprock rock, greatly reducing the permeability of the caprock. The Bacillus megaterium 1-9-1 provided by the present invention solves the defect in the prior art that it is difficult for microbial strains used in actual microbial remediation applications to carry out mineralization deposition in extremely small pores and maintain the remediation effect for a long time.

[0018] 2. The research on Bacillus megaterium in the field of biotechnology mainly focuses on aspects such as enzyme production, heavy metal removal, and genetic engineering. In recent years, with the progress of carbon capture, utilization, and storage (CCUS) technology, its potential in microbial-induced calcium carbonate precipitation (MICP) has gradually attracted attention. However, current research mostly focuses on the repair of surface or shallow fractures. In the repair of deep underground or large-scale fractures, how to ensure that the strain can effectively penetrate and carry out mineralization deposition in extremely small pores and maintain the repair effect for a long time is still a challenge. Based on this, the present invention also provides an application of a microbial agent, which includes Bacillus megaterium 1-9-1 and a strain culture medium. This microbial agent generates calcium carbonate precipitation through the metabolic activities of Bacillus megaterium 1-9-1, that is, the microbial-induced calcium carbonate precipitation (MICP) technology, which can fill the large pores, fractures, and cement soil particles in the caprock, thereby improving the properties such as permeability, strength, and stiffness of the caprock. The microbial agent provided by the present invention can prepare microbial cement through Bacillus megaterium 1-9-1 and is used to repair the caprock fractures, achieving the effect of preventing the escape of gas layers.

[0019] 3. The key to the preparation of microbial cement lies in the selection of microorganisms. There are many metabolic activities that can achieve the MICP technology. Bacillus megaterium 1-9-1 utilizes the production of carbonic anhydrase and its mechanism to induce the reaction of carbonate ions at the nucleation sites in the microenvironment to generate calcium carbonate precipitation, which can further repair the integrity of the caprock. Different from other plugging technologies, the present invention uses the MICP technology to have better extensibility for low-permeability or difficult-to-enter rocks. Its product has a low viscosity, can repair microcracks, and is more conducive to filling microvoids and reducing CO2 leakage. By screening out Bacillus megaterium 1-9-1 that can produce carbonic anhydrase, the large pores and cracks in the caprock are plugged to reduce the escape of CO2 and improve the capture effect. At the same time, as a method in the field of microbial remediation, the application of the MICP technology has less impact on the environment and relatively low cost, and is a sustainable method for repairing and strengthening the integrity of the caprock, with broad application prospects. Description of the Drawings

[0020] Figure 1 It is a morphological diagram of the strain Bacillus megaterium 1-9-1 in the present invention.

[0021] Figure 2 It is a phylogenetic tree of Bacillus megaterium 1-9-1 in the present invention.

[0022] Figure 3 It is the strain concentration of Bacillus megaterium 1-9-1 at different culture times in the present invention.

[0023] Figure 4 It is the strain concentration of Bacillus megaterium 1-9-1 at different temperatures in the present invention.

[0024] Figure 5 It is the strain concentration of Bacillus megaterium 1-9-1 at different pH values in the present invention.

[0025] Figure 6 It is the strain concentration of Bacillus megaterium 1-9-1 with different carbon sources in the culture medium in the present invention.

[0026] Figure 7 It is the strain concentration of Bacillus megaterium 1-9-1 with different nitrogen sources in the culture medium in the present invention.

[0027] Figure 8 It is the standard curve graph for calculating the carbonic anhydrase activity in the present invention.

[0028] Figure 9 It is the indoor evaluation result of the carbonic anhydrase production by Bacillus megaterium 1-9-1 in the present invention. Detailed Embodiments

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0030] Example 1 I. Enrichment screening and isolation of strains 1. Collection of environmental samples The water sample produced from the Yanchang Oilfield is the obtained oilfield water sample.

[0031] 2. Primary screening of strains First, prepare a single-strain isolation medium. Fill 50 mL of the single-strain isolation medium into each vial and number them, with three vials in each group. Then, place them in an autoclave and sterilize at 121 °C. Weigh 5 mL of the collected oilfield water sample, put it into 45 mL of sterile water, shake and let it stand. Use a syringe to take 1 mL of the supernatant and inject it into the corresponding vial, and then place it in a constant-temperature incubator for shaking culture at a temperature of 35 °C. After 24 hours of sample culture, take 1 mL of the supernatant and inject it into the newly prepared single-strain isolation medium. The above is called one enrichment cycle. The entire enrichment process is carried out for 5 cycles to obtain an enriched sample.

[0032] Among them, the formula of the single-strain isolation medium is: beef extract 3 g·L -1 、tryptone 11 g·L -1 、sodium chloride 5 g·L -1 、calcium carbonate 50 g·L -1 、zinc sulfate 1 μmol·L -1 , and the remaining component is water.

[0033] Use a pipette to aspirate 100 μL from the enriched sample into a centrifuge tube, add 900 μL of distilled water, dilute the culture solution by 10 times, mix well, and then use a pipette to aspirate 100 μL from the diluted solution into 900 μL of distilled water to dilute the culture solution again. Repeat the above operation until the concentration of the culture solution is diluted to 10 -6 . Use the three dilution concentrations of 10 -4 、10 -5 and 10 -6 as the target liquids to coat plates in an anaerobic chamber and place them in a constant-temperature incubator for culturing and observing records at 35 °C.

[0034] Pick out some colonies on the plate, put them into a basic medium for culture, and then dilute and coat the plate and streak the colonies again until single colonies appear on the plate. Place the isolated single strains in glycerol tubes and store them at -4 °C.

[0035] Among them, the formula of the basal medium is: beef extract 3 g·L -1 , tryptone 11 g·L -1 , sodium chloride 5 g·L -1 , agar 20 g·L -1 , and the pH is adjusted to 7.5.

[0036] 3. Re-screening of strains After primary screening and multiple rounds of enrichment, a strain capable of producing carbonic anhydrase was discovered. This strain was named 1-9-1 and is referred to as strain 1-9-1.

[0037] II. Strain identification The single strain obtained above was cultured at 35 °C using a solid medium in an incubator at a temperature of 35 °C. After culturing for one to three days, colonies with a diameter of 3 ± 1 mm appeared on the surface of the solid medium. The colonies were circular with neat edges, white as a whole, with a wet and wrinkled surface, as Figure 1 shown.

[0038] Among them, the formula of the solid medium is: beef extract 3 g·L -1 , tryptone 11 g·L -1 , sodium chloride 5 g·L -1 , agar 20 g·L -1 , zinc sulfate 1 μmol·L -1 , with water as the solvent, and the pH is adjusted to 7.5.

[0039] By sequencing the 16S rDNA of this strain 1-9-1 and comparing the 16S rDNA gene sequence of this strain 1-9-1 with the 16S rDNA sequence of Bacillus megaterium ( Bacillus megaterium ), it was found that the homology of the 16S rDNA of this strain 1-9-1 with the Bacillus megaterium sequence was greater than 99%. The experimental results are as Figure 2 shown. Therefore, this strain 1-9-1 was identified as Bacillus megaterium 1-9-1.

[0040] Among them, the nucleotide sequence of the 16S rDNA of this strain 1-9-1 is as shown in SEQ ID NO.1: ATGCAGTCGAGCGGATGAAGAGAGCTTGCTCTCAGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGCTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGG CAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAGCTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGGACG。

[0041] III. Performance Evaluation of Bacillus megaterium 1-9-1 1. Evaluation of Strain Growth Ability Take 1 mL of the Bacillus megaterium 1-9-1 bacterial solution and add it to the sterilized enrichment medium. Place it in a constant temperature incubator and culture it at 35°C. Measure the absorbance OD of the cultured bacterial solution at 600 nm. 600, used to characterize the growth of bacterial strains. It is measured every 3 hours for a total of 72 hours, and after obtaining the results, a microbial growth curve is plotted. The results are as Figure 3 shown. When it is 24 hours, the growth of the strain is optimal.

[0042] Among them, the formula of the enrichment medium is: beef extract 3 g·L -1 、tryptone 11 g·L -1 、sodium chloride 5 g·L -1 、calcium carbonate 50 g·L -1 、zinc sulfate 1 μmol·L -1 、the solvent is water, and the pH is adjusted to 7.5.

[0043] (1)Effect of temperature on the growth of the strain Take 1 mL of Bacillus megaterium 1-9-1 bacterial liquid and add it to an anaerobic flask containing 50 mL of nutrient liquid medium, and place it in a constant temperature gyratory shaker at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C respectively, and shake and culture at 120 r / min for 24 h.

[0044] The formula of the nutrient liquid medium is: beef extract 4 g·L -1 、yeast extract powder 10 g·L -1 、sodium chloride 5 g·L -1 、zinc sulfate 1 μmol·L -1 ,the solvent is water, and the pH value is 7.5.

[0045] Use an ultraviolet spectrophotometer to measure the OD 600 value of the bacterial liquid after culturing for 48 h, and plot the growth curve of the bacterial liquid.

[0046] The results are as Figure 4 shown. Under the condition of 35 °C, the growth ability of Bacillus megaterium 1-9-1 reaches the highest.

[0047] (2)Effect of pH on the growth of the strain Take 1 mL of Bacillus megaterium 1-9-1 bacterial liquid and add it to an anaerobic flask containing 50 mL of nutrient liquid medium. The environmental temperature is 35 °C, and the pH of the medium is adjusted to 5, 6, 7, 8, 9, and 10 in sequence, and then placed in a constant temperature gyratory shaker and shaken and cultured at 120 r / min for 24 h.

[0048] The formula of the nutrient liquid medium is: beef extract 4 g·L -1 、yeast extract powder 10 g·L -1 、sodium chloride 5 g·L -1 、zinc sulfate 1 μmol·L -1 ,the solvent is water.

[0049] Use an ultraviolet spectrophotometer to measure the OD 600Value, and plot the growth curve of the bacterial solution.

[0050] The results are as Figure 5 shown. Under the condition of pH 8, the growth ability of Bacillus megaterium 1-9-1 reaches the highest.

[0051] (3)Effect of carbon source on the growth of the strain Prepare the medium. Add 1 mL of the Bacillus megaterium 1-9-1 bacterial solution into 5 anaerobic bottles containing 50 mL of nutrient liquid medium respectively. The environmental temperature is 35 °C. Adjust the carbon source of the medium, which are beef extract, glucose, sucrose and maltose, and soluble starch in turn. Place them in a constant temperature gyratory shaker and shake culture at 120 r / min for 24 h.

[0052] Among them, the method of adjusting the carbon source of the medium with beef extract, glucose, sucrose and maltose, and soluble starch in turn is to add different carbon sources for culture each time, and the dosage of each carbon source is 3 g·L -1 .

[0053] The formula of the nutrient liquid medium except for the carbon source is: yeast extract powder 10 g·L -1 , sodium chloride 5 g·L -1 , zinc sulfate 1 μmol·L -1 , the solvent is water, and the pH value is 8.

[0054] Use an ultraviolet spectrophotometer to measure the OD of the bacterial solution after culture 600 value, and plot the growth curve of the bacterial solution.

[0055] The results are as Figure 6 shown. Under the condition of choosing beef extract as the carbon source, the growth ability of strain 1-9-1 reaches the highest.

[0056] (4)Effect of nitrogen source on the growth of the strain Prepare the medium. Add 1 mL of the Bacillus megaterium 1-9-1 bacterial solution into 5 anaerobic bottles containing 50 mL of nutrient liquid medium respectively. The environmental temperature is 35 °C. Adjust the nitrogen source of the medium, which are peptone, yeast extract powder, tryptone, ammonium chloride, and ammonium sulfate in turn. Place them in a constant temperature gyratory shaker and shake culture at 120 r / min for 24 h.

[0057] Among them, the method of adjusting the nitrogen source of the medium with peptone, yeast extract powder, tryptone, ammonium chloride, and ammonium sulfate in turn is to add different nitrogen sources for culture each time, and the dosage of each nitrogen source is 10 g·L -1 .

[0058] The formula of the nutrient liquid medium except for the nitrogen source is: beef extract 4 g·L -1 , sodium chloride 5 g·L -1 , zinc sulfate 1 μmol·L-1 The solvent is water and the pH value is 8.

[0059] Use an ultraviolet spectrophotometer to measure the OD of the bacterial solution after cultivation. 600 value and plot the growth curve of the bacterial solution.

[0060] The results are as Figure 7 shown. Under the condition of choosing beef extract as the carbon source, the growth ability of strain 1-9-1 reaches the highest.

[0061] The optimal growth medium formula for the strain is obtained as follows: beef extract 4 g·L -1 , tryptone 11 g·L -1 , sodium chloride 5 g·L -1 , zinc sulfate 1 μmol·L -1 , pH value is 8, and the environmental temperature is 35 °C.

[0062] 2. Evaluation of carbonic anhydrase activity First, define the carbonic anhydrase activity as the amount of 1 μmol of p-nitrophenol generated per milliliter per minute, with the unit of U. The carbonic anhydrase activity is measured according to the method of esterase activity. This method uses p-nitrophenyl acetate as the substrate and utilizes the esterase activity of carbonic anhydrase to catalyze p-nitrophenyl acetate into p-nitrophenol. Since p-nitrophenol has a characteristic absorption peak at 400 nm. Therefore, measure its absorbance at 400 nm to obtain the standard curve, and the activity of carbonic anhydrase can be calculated through the linear regression equation obtained by curve fitting. The standard curve is as Figure 8 shown.

[0063] In a centrifuge tube, first add diethylmalonic acid, and then add 9.5 mL of phosphate buffer solution to prepare a solution with a concentration of 0.01 mol·L -1 . Subsequently, take 0.5 mL of absolute ethanol and prepare a p-nitrophenyl acetate solution with a concentration of 0.02 mol·L -1 . Mix these two liquids to form the required working solution.

[0064] First, dilute the Bacillus megaterium 1-9-1 bacterial solution. Use a pipette to aspirate 1 mL of the Bacillus megaterium 1-9-1 bacterial solution and mix it evenly with 1 mL of the working solution. React at room temperature for 5 min, measure the absorbance of the mixed solution at a wavelength of 400 nm under a spectrophotometer. The control group uses a sterile medium and is cultured for 36 h, and the absorbance is measured every 3 h. Calculate the enzyme activity by referring to the standard curve. The results are as Figure 9 shown.

[0065] Among them, the Bacillus megaterium 1-9-1 bacterial solution is taken from the supernatant of the strain after primary screening and multiple rounds of enrichment cultured in a single-bacterium isolation medium.

[0066] The linear regression equation is: y = 3.426x + 0.1114, and its activity can be calculated from the above definition of carbonic anhydrase activity.

[0067] IV. Physical experiment to simulate fracture repair (1)Preparation of microbial inoculum Inoculate the Bacillus megaterium 1-9-1 bacterial liquid into the strain culture medium, and culture it in an incubator at a temperature of 35°C for 24 hours. Then, extract the supernatant to obtain the microbial inoculum.

[0068] Among them, the volume ratio of the Bacillus megaterium 1-9-1 bacterial liquid to the strain culture medium is 1:50.

[0069] The strain culture medium consists of the following materials at the final concentrations: beef extract 3 g·L -1 , peptone 10 g·L -1 , sodium chloride 5 g·L -1 , zinc sulfate 1 μmol·L -1 , and the solvent is water.

[0070] (2)Gas permeability measurement method: After drying and cooling the experimental core, place it in a core holder, inject nitrogen into the core, and record data such as the time and pressure required to exclude the same volume. The permeability can be calculated according to the following formula:

[0071] ; Among them, K is the permeability, with the unit of 10 -3 μm 2 ; Q0 is the flow rate, with the unit of cm 3 / s; P is the core displacement pressure, with the unit of MPa; μ is the dynamic viscosity, with the unit of mPa·s; A is the cross-sectional area, with the unit of cm 2 ; L is the core length, with the unit of cm.

[0072] The experimental core is a fractured core with a diameter of 2.502 cm and a length of 5.894 cm.

[0073] (3)Set the confining pressure of the fractured core to 4 MPa and the temperature in the thermostat to 35 °C. Place the fractured core in a core holder with a slit width of 0.5 mm. Then, inject water into the fractured core until saturation and the pressure reaches equilibrium. After that, inject the above microbial inoculant for 4 hours. Then, close the valves at the injection end and the outlet end of the core holder to simulate the treatment of the core by the MICP technique under shut-in conditions for 28 days. Inject the microbial inoculant into the fractures of the fractured core once a week. A total of four injections of the microbial inoculant are made from the start to the end (a total of 28 days, with the microbial inoculant injected once every 7 days, for a total of four times). After 28 days, measure the permeability of the fractured core. Calculate the rock fracture repair efficiency using the permeability of the fractured core before the experiment and the permeability of the fractured core after 28 days of treatment by the MICP technique, that is, the change in the core permeability before and after repair.

[0074] Among them, when injecting additional fluid, it is at a flow rate of 0.2 ml·min -1 three times the pore volume of the microbial inoculant.

[0075] When measuring the permeability of the fractured core, the rock fracture repair efficiency is calculated using the following formula: ; Among them, is the repair efficiency, with the unit of %; K1 and K2 are the permeabilities before and after the experiment, respectively, with the unit of μm 2 .

[0076] The results of the change in the core permeability before and after repair are shown in Table 1.

[0077] Table 1 Permeability before and after repair As shown in Table 1, the experimental results show that the permeabilities of the five groups of cores have a relatively obvious decrease, and their repair efficiencies are all above 75%, with the highest repair efficiency reaching 82.41%. The diameter of the microfractures in the formation is generally dozens of micrometers or more. Small calcium carbonate crystals continuously cement to reach a larger diameter, and these caprock fractures will be filled and blocked by the crystals and connected together, thereby preventing the passage of fluids. At the same time, it can also strengthen the mechanical properties of the caprock, enhance the formation carbon sequestration ability, and better protect the oil and gas reservoir.

[0078] In summary, the Bacillus megaterium 1-9-1 provided by the present invention has excellent ability to repair micro-pores and fractures. The obtained bacterial solution can be directly used for repair, and significantly reduces the permeability of the caprock rock, providing a very promising strain for CO2 sequestration and microbial enhanced oil recovery technology.

[0079] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0081] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A Bacillus megaterium 1-9-1, characterized in that, The Bacillus megaterium ( Bacillus megaterium ) 1-9-1 was deposited at the China General Microbiological Culture Collection Center on December 21, 2023, with the deposit number CGMCC No. 29373.

2. A microbial inoculant, characterized in that, The microbial inoculum comprises Bacillus megaterium 1-9-1 as claimed in claim 1 and a strain culture medium; The strain culture medium consists of the following materials at the following final concentrations: beef extract 3 g·L -1 ~4 g·L -1 , tryptone 10 g·L -1 ~11 g·L -1 , sodium chloride 4 g·L -1 ~5 g·L -1 , zinc sulfate 1 μmol·L -1 ~1.2 μmol·L -1 , and the solvent is water.

3. The preparation method of the microbial inoculum according to claim 2, characterized in that, comprises the following steps: Inoculate the bacterial liquid of Bacillus megaterium 1-9-1 into the strain culture medium, and perform fermentation culture to obtain a fermentation broth; filter the fermentation broth and collect the filtrate to obtain the microbial inoculum.

4. The preparation method of the microbial inoculum according to claim 3, characterized in that, The conditions for the fermentation culture are: culture at 34°C to 36°C for 23 h to 25 h.

5. The preparation method of the microbial inoculum according to claim 3, characterized in that The volume ratio of the bacterial liquid of Bacillus megaterium 1-9-1 to the strain culture medium is 0.9 to 1.1:

50.

6. The preparation method of the microbial inoculum according to claim 5, characterized in that, The bacterial liquid of Bacillus megaterium 1-9-1 is the supernatant after enrichment of the water sample produced from the oilfield.

7. Application of the microbial inoculum as claimed in claim 2 in preventing gas layer dissipation by repairing the caprock cracks.

8. The application according to claim 7, characterized in that The microbial inoculum is used for preparing microbial cement, thereby repairing the caprock cracks and preventing gas layer dissipation.

9. The application according to claim 8, wherein The method for preparing microbial cement comprises the following steps: Induce the formation of microbial cement by injecting the microbial inoculum into the caprock.

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