Pseudomonas stutzeri yc4+5-1, microbial preparation, biomineralization microbial agent, and preparation method and application of pseudomonas stutzeri yc4+5-1

Abstract

The invention belongs to the field of microbial remediation, and particularly relates to pseudomonas stutzeri yc4 + 5-1, a microbial preparation, a biomineralization microbial agent, a preparation method and application. The preservation number of the pseudomonas stutzeri yc4 + 5-1 is CGMCC (China General Microbiological Culture Collection Center) NO.29371, and the preservation number of the pseudomonas stutzeri yc4 + 5-1 is CGMCC NO.29371. Carbonic anhydrase produced by the pseudomonas stutzeri yc4 + 5-1 can repair a CO2 dissipation channel of a reservoir during CO2 oil displacement and improve the oil displacement efficiency, so that the oil and gas recovery efficiency is improved, the adverse effect of CO2 dissipation in the oil and gas field development process is prevented, part of CO2 can be solidified and buried, and the carbon reduction effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial carriers, and more specifically to Pseudomonas stutzeri yc4+5-1, microbial preparations, biomineralization bacterial agents, and preparation methods and applications thereof. Background Art

[0002] Currently, CO 2 Oil recovery has been widely used as an efficient technology to improve oil recovery. This technology improves oil recovery efficiency by expanding the swept area, expanding the volume of crude oil, reducing crude oil viscosity and interfacial tension. 2 During the oil recovery process, some CO 2 Sequestration in the strata to achieve CO 2 Solidification and storage to obtain stored CO 2 Therefore, CO 2 Miscible flooding technology has become an important means of reservoir development. However, during the development process, due to problems such as gravity overburden and viscous fingering, CO 2 It is very easy to form gas channeling, and as the reservoir heterogeneity increases, CO 2 The more likely it is to advance along the high permeability layer, the more likely it is to advance along the high permeability layer, which significantly reduces the oil recovery efficiency. 2 The existence of escape channels will cause serious CO 2 Emission phenomenon causes air pollution and many environmental problems.

[0003] Currently suppressing CO 2 The methods of escape are mainly gas-water alternation, foam plugging and gel plugging. Gas-water alternating injection is the most commonly used technology to plug escape channels. The principle is to adjust the injection profile of the fluid by alternating injection of water and gas phases, expand the swept volume, and thus improve the recovery rate. The foam system uses nitrogen, natural gas and CO 2 The water-in-air system formed by mixing gases and foaming agents can be used for selective plugging. Gel-based sealing systems such as polyacrylamide gel and pre-crosslinked gel particles can be used to plug fractures and highly heterogeneous reservoirs. The main problems of these types of anti-emission systems are low plugging strength and poor stability of the plugging agents. Therefore, compared with traditional repair methods, microbial mineralization-based fracture repair technology with low viscosity, adjustable reaction speed, high permeability, adjustable cementation strength, simple construction, and green and efficient is the most popular choice for CO2 repair. 2 The restoration of the escape channel provides a new idea. However, there are many research reports on the restoration of concrete cracks, marble cracks and cultural relics based on microorganisms in normal atmospheric environment. However, there are no reports on the research of microorganisms in oil reservoir environment. Summary of the invention

[0004] In order to solve the above problems, the present invention provides Pseudomonas stutzeri yc4+5-1, microbial preparations, biomineralization agents, preparation methods and applications thereof, for repairing CO2 in oil reservoirs. 2 Escape channel.

[0005] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a Pseudomonas stutzeri (Stutzerimonas stutzeri) yc4+5-1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 21, 2023, with a deposit number of CGMCCNO.29371.

[0007] The second aspect of the present invention provides a microbial preparation, wherein the Pseudomonas stutzeri yc4+5-1 is inoculated into a carbonic anhydrase culture medium and cultured to prepare the microbial preparation.

[0008] The Pseudomonas stutzeri yc4+5-1 or the microbial preparation is used in producing carbonic anhydrase.

[0009] Preferably, the Pseudomonas stutzeri yc4+5-1 is inoculated into a carbonic anhydrase medium and fermented for 20 h to 25 h at a fermentation temperature of 25° C. to 35° C.

[0010] The third aspect of the present invention provides a biomineralization agent, which comprises the Pseudomonas stutzeri yc4+5-1 and a carbonic anhydrase culture medium; in the biomineralization agent, the number of live bacteria of Pseudomonas stutzeri yc4+5-1 is 10 5 CFU~10 7 CFU.

[0011] Preferably, the formula of the carbonic anhydrase culture medium per liter is as follows: 3g / L-3.5g / L maltose, 10g / L-12g / L tryptone, 5g / L-6g / L sodium chloride, 1μmol / L-1.2μmol / L zinc sulfate, water is used to make the volume to 1L, and the pH value is 6-9.

[0012] Preferably, the method for preparing the biomineralizing agent comprises the following steps:

[0013] The bacterial liquid of Pseudomonas stutzeri yc4+5-1 is inoculated into the culture medium and fermented for 25 hours to 27 hours at a fermentation temperature of 25° C. to 35° C. to obtain a biomineralizing bacterial agent.

[0014] The Pseudomonas stutzeri yc4+5-1, the microbial preparation or the biomineralization bacterial agent is used to repair the CO 2 Application of escape channels.

[0015] The invention relates to repairing the CO2 in oil reservoirs by the carbonic anhydrase produced by the metabolism of Pseudomonas stutzeri yc4+5-1. 2 Escape channel.

[0016] In some embodiments of the present invention, the Pseudomonas stutzeri yc4+5-1 repairs the oil reservoir CO by producing carbonic anhydrase through metabolism. 2 Escape channel.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention isolates and screens a carbonic anhydrase-producing Pseudomonas stutzeri yc4+5-1 from an oil reservoir environment. By evaluating its carbonic anhydrase activity, it is found that it can efficiently and stably produce carbonic anhydrase, which can promote CO 2 Converted to CO 3 2- and HCO 3- , combined with Ca in the formation 2+ , forming microbial cement mineralization and precipitation, the chemical formula is CaCO 3 , repair CO 2 CO in the reservoir during oil displacement 2 The escape channel improves the oil displacement efficiency, thereby increasing the oil and gas recovery rate and preventing CO 2 The adverse effects of CO escaping can also be solidified and buried 2 , which plays a role in reducing carbon emissions. 2 The repair experiment of the escape channel shows that the repair efficiency is higher from the 1st to the 7th day; the repair efficiency from the 8th to the 15th day is slower than that of the previous 5 days, but the repair is still ongoing, and on the 15th day the escape repair rate can reach more than 98%.

[0019] Biomaterial Deposit

[0020] Pseudomonas stutzeri (Stutzerimonas stutzeri) yc4+5-1 was deposited in the General Microbiology Center (GMCC) of the China Culture Collection Administration on December 21, 2023, with the deposit number CGMCC NO.29371, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 is a developmental tree diagram of the strain yc4+5-1 of the present invention;

[0023] Figure 2 is the temperature resistance curve of Pseudomonas stutzeri yc4+5-1 in the present invention;

[0024] Figure 3 The acid and alkali resistance of Pseudomonas stutzeri yc4+5-1 in the present invention;

[0025] Figure 4 is the growth curve of Pseudomonas stutzeri yc4+5-1 under the optimal culture conditions in the present invention;

[0026] Figure 5 is the carbonic anhydrase activity of Pseudomonas stutzeri yc4+5-1 in the present invention;

[0027] Figure 6 is an X-ray diffractometer analysis spectrum of the microbial cement of the present invention;

[0028] Figure 7 The scanning electron microscope analysis spectrum of the microbial cement of the present invention;

[0029] Figure 8 This is a graph showing the repair rate of the escape channel by Pseudomonas stutzeri yc4+5-1 over time. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] In order to better illustrate the present invention, the present invention is further described below in conjunction with the embodiments. The following are specific embodiments.

[0033] Example 1: Enrichment, screening, separation and purification of Pseudomonas stutzeri yc4+5-1

[0034] (1) Reservoir rock sample collection: Some rock cuttings samples from the Chang 4+5 layer of the Yanchang Wuqi Oilfield were collected and stored in a -4°C refrigerator.

[0035] (2) Strain enrichment: Prepare the enrichment medium and put it into vials for numbering, and sterilize it in a high-pressure steam autoclave at 121°C. Take 2.0g of the Chang 4+5 rock cuttings sample from the Yanchang Wuqi Oilfield, place it in 20.0mL of sterile water and shake it for 24 hours. After standing for a period of time, use a syringe to take 1.0mL of the supernatant and inject it into the vial containing the enrichment medium. After the sample is cultured for 48 hours, extract 1.0mL of the supernatant and inject it into the prepared enrichment medium, and place it in an incubator to continue shaking. After the sample is cultured for another 48 hours, extract 1.0mL of the supernatant and inject it into the prepared enrichment medium, and place it in an incubator to continue shaking. Repeat the culture operation and perform 7 rounds of enrichment.

[0036] The enrichment medium comprises: each L of the nutrient medium contains 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 60 g of calcium carbonate, 1 μmol of zinc sulfate and 4 mL of phenol red, and the balance is water.

[0037] (3) Primary screening of strains: The samples after 7 rounds of enrichment were diluted with sterile water until the sample was diluted to 10 4 times. Take 10 4 The solid culture medium includes: each liter of the nutrient medium contains 11g of tryptone, 5.5g of yeast extract, 11g of sodium chloride, 16g of agar, and the balance is water, and the pH value of the culture medium is adjusted to 8.0. The coated plate is placed in a 35°C constant temperature rotary shaker for incubation. After 48 hours of incubation, the microbial colonies on the plate are picked out and placed in the nutrient culture medium for incubation. After 24 hours of incubation, the sample is diluted again to 10 6 times. Take 10 6 The plate was coated with a dilution of 100 times, and the coated plate was placed in a 35°C constant temperature rotary shaker for incubation. The above dilution, plate coating and incubation operations were repeated until a single colony appeared on the plate.

[0038] (4) Preservation of strains: The strains screened above were cultured in a 35°C constant temperature rotary shaker for 24 h, and the strains were preserved in a clean bench. First, 1.0 mL of glycerol was transferred into the glycerol tube with a pipette, and then 1.0 mL of the strain cultured for 24 h was transferred with a pipette. The tube was covered with a stopper and shaken to mix well. The isolated single bacteria were stored at -4°C.

[0039] (5) Strain rescreening: The single strain obtained by the above operation was inoculated into a liquid culture medium containing phenol red. The liquid culture medium containing phenol red comprises: 11 g of tryptone, 5.5 g of yeast extract, 11 g of sodium chloride and 4 mL of phenol red per liter of the nutrient medium, and the balance is water. The pH value of the culture medium is adjusted to 8.0. The culture is placed in a constant temperature rotary shaker at 35°C for 48 hours, and the bacterial liquid is observed to see whether it turns yellow.

[0040] After the above re-screening operation, the bacterial liquid turned yellow and passed the re-screening.

[0041] Therefore, it can be determined that a strain capable of producing carbonic anhydrase was found in the rock fragments of the Chang 4+5 reservoir of the Yanchang Wuqi Oilfield and that the strain can also turn the liquid culture medium containing phenol red yellow. The strain can be named yc4+5-1.

[0042] It should be noted that the screening operation can be carried out when the nutrient medium contains 11g to 12g of tryptone, 5.5g to 6.5g of yeast extract, 11g to 13g of sodium chloride, 16g to 18g of agar, and the pH value of the culture medium is adjusted to a numerical range of 7.5 to 8.5. The above Example 1 is a specific implementation method.

[0043] Example 2, strain identification

[0044] The Pseudomonas stutzeri yc4+5-1 obtained in the above Example 1 was tested and its Gram staining was positive; it could be cultured in LB medium at 35°C, and after the culture, pale yellow colonies with dots on the surface could be obtained.

[0045] The 16S rRNA gene is a gene sequence corresponding to the coding rRNA in microorganisms and exists in all microbial genomes. The 16S rRNA gene sequencing uses the 16S rRNA gene sequence as a marker. By sequencing the 16S rDNA of the strain of the present invention, the 16S rRNA thereof is compared with the 16S rDNA sequence of Pseudomonas stutzeri. It is found that the homology between the 16S rRNA sequence of the strain and the 16S rRNA sequence of Pseudomonas stutzeri is greater than 99%. The full sequence of the 16S rRNA of the yc-4+5-1 strain is shown in SEQ ID NO.1:

[0046] SEQ ID NO.1:

[0047]

[0048] The phylogenetic tree was constructed based on the 16S rDNA gene sequence using MEGA software. Figure 1 As shown, from the phylogenetic tree, it can be seen that the strain yc-4+5-1 is very similar to OR452310.1Stutzerimonas stutzeri, confirming that yc-4+5-1 is Pseudomonas stutzeri in the genus Pseudomonas.

[0049] Example 3, Cultivation of Pseudomonas stutzeri yc4+5-1

[0050] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 25℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the absorbance of the bacterial solution at a wavelength of 600nm, that is, OD 600 The absorbance value can characterize the growth of the strain.

[0051] Example 4, Cultivation of Pseudomonas stutzeri yc4+5-1

[0052] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 30℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the bacterial solution OD 600 The absorbance value can characterize the growth of the strain.

[0053] Example 5: Cultivation of Pseudomonas stutzeri yc4+5-1 at different temperatures

[0054] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 35℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the bacterial solution OD 600 The absorbance value can characterize the growth of the strain.

[0055] Example 6, Cultivation of Pseudomonas stutzeri yc4+5-1

[0056] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 40℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the bacterial solution OD 600 Value, namely OD 600 The absorbance value can characterize the growth of the strain.

[0057] Example 7, Cultivation of Pseudomonas stutzeri yc4+5-1

[0058] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 45℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the bacterial solution OD 600 The absorbance value can characterize the growth of the strain.

[0059] Example 8, Cultivation of Pseudomonas stutzeri yc4+5-1

[0060] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase liquid culture medium, place it in a constant temperature incubator at 55℃ for culture, after culturing for 26h, shake the bacterial solution and pipette 2mL into a quartz cuvette, use sterile carbonic anhydrase liquid culture medium as a blank sample, and measure the bacterial solution OD 600 Value, namely OD 600 The absorbance value can characterize the growth of the strain.

[0061] The test results of Examples 3 to 8 were used to draw a microbial temperature resistance growth curve. Figure 2 As shown in Figure 2, when the temperature rises from 25℃ to 35℃, the OD 600 The value showed an increasing trend, indicating that the strain grew well; it reached the maximum value at 35°C, indicating the best growth state of the strain; then, as the temperature increased, the OD 600The value keeps decreasing, and the growth performance of the strain decreases. It can be seen that 35℃ is the optimal growth temperature for Pseudomonas stutzeri yc4+5-1.

[0062] Example 9, Cultivation of Pseudomonas stutzeri yc4+5-1

[0063] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 4 and sterilize it in a high-pressure steam sterilizer at 121°C. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35°C for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0064] Example 10: Cultivation of Pseudomonas stutzeri yc4+5-1

[0065] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 5 and sterilize it in a high-pressure steam sterilizer at 121°C. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35°C for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0066] Example 11, Cultivation of Pseudomonas stutzeri yc4+5-1

[0067] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 6 and sterilize it in a high-pressure steam sterilizer at 121℃. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35℃ for cultivation. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0068] Example 12: Cultivation of Pseudomonas stutzeri yc4+5-1

[0069] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 7 and sterilize it in a high-pressure steam sterilizer at 121℃. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35℃ for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0070] Example 13, Cultivation of Pseudomonas stutzeri yc4+5-1

[0071] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 8 and sterilize it in a high-pressure steam sterilizer at 121°C. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35°C for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0072] Example 14: Cultivation of Pseudomonas stutzeri yc4+5-1

[0073] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 9 and sterilize it in a high-pressure steam sterilizer at 121℃. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35℃ for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0074] Example 15: Cultivation of Pseudomonas stutzeri yc4+5-1

[0075] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. Adjust the pH value of the culture medium to 10 and sterilize it in a high-pressure steam sterilizer at 121℃. Transfer 2.0mL of bacterial solution to the sterilized carbonic anhydrase liquid culture medium and place it in a constant temperature incubator at 35℃ for culture. Use a UV spectrophotometer to measure the OD of the bacterial solution 600 After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette, and a sterile carbonic anhydrase liquid culture medium is used as a blank sample. After culturing for 24 hours, the OD of the bacterial solution is measured. 600 value.

[0076] The test results of Examples 9 to 15 were used to draw an acid and alkali resistance performance curve. Figure 3 As shown in the figure, Pseudomonas stutzeri yc4+5-1 basically does not grow when pH is less than 5.0; Pseudomonas stutzeri yc4+5-1 grows rapidly when pH is 5-6; Pseudomonas stutzeri yc4+5-1 grows steadily when pH is 6-9; Pseudomonas stutzeri yc4+5-1 grows slowly when pH is greater than 9. 600 The value dropped and the strain began to die. It can be seen that the pH value with higher carbonic anhydrase activity is 6-9, and the strain growth performance is highest at pH=8.

[0077] Example 16

[0078] A biomineralization agent, characterized in that the biomineralization agent comprises the Pseudomonas stutzeri yc4+5-1 according to claim 1 and a carbonic anhydrase culture medium; in the biomineralization agent, the number of live bacteria of Pseudomonas stutzeri yc4+5-1 is 10 5 CFU.

[0079] The preparation method is as follows:

[0080] The bacterial liquid of Pseudomonas stutzeri yc4+5-1 with an absorbance value of 1.569 at 600 nm of ultraviolet spectrophotometry was inoculated into the culture medium and fermented for 25 hours at a fermentation temperature of 25° C. to obtain a biomineralizing bacterial agent.

[0081] Embodiment 17

[0082] A biomineralization agent, characterized in that the biomineralization agent comprises the Pseudomonas stutzeri yc4+5-1 according to claim 1 and a carbonic anhydrase culture medium; in the biomineralization agent, the number of live bacteria of Pseudomonas stutzeri yc4+5-1 is 10 7 CFU.

[0083] The preparation method is as follows:

[0084] The bacterial liquid of Pseudomonas stutzeri yc4+5-1 with an absorbance value of 1.569 at 600 nm of ultraviolet spectrophotometry was inoculated into the culture medium and fermented for 27 hours at a fermentation temperature of 35° C. to obtain a biomineralizing bacterial agent.

[0085] Experiment 1. Evaluation of the growth performance of Pseudomonas stutzeri yc4+5-1 under optimal culture conditions

[0086] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. The initial pH of the culture medium is 8.0, and the culture temperature is 35℃. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase culture medium and place it in a constant temperature incubator at 35℃ for culture. Use a UV spectrophotometer to measure the absorbance of the bacterial solution at 600nm, that is, OD 600 The absorbance value can characterize the growth of the strain. After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette. A sterile culture medium is used as a blank sample. The OD of the bacterial solution is measured every 3 hours. 600 The value is recorded, and the test is conducted for 36 hours. The microbial growth curve is drawn using the test results. Figure 4 As shown in the figure, the growth adaptation period of Pseudomonas stutzeri yc4+5-1 is 0h~9h, and the growth is relatively slow; the logarithmic growth period of Pseudomonas stutzeri yc4+5-1 is 10h~20h, and the growth and development are rapid, and the reproduction is rapid; the stable period of Pseudomonas stutzeri yc4+5-1 is 20h~30h, and the growth tends to be stable; the death period of Pseudomonas stutzeri yc4+5-1 is more than 30h, and the OD 600 The value decreased and the strain began to die gradually. It can be concluded that the optimal growth time of strain yc4+5-1 is 25h~27h. 600 The maximum value reaches 1.569.

[0087] Experiment 2: Evaluation of carbonic anhydrase production by Pseudomonas stutzeri yc4+5-1

[0088] Prepare carbonic anhydrase liquid culture medium, the culture medium formula is: maltose 3.5g / L, tryptone 10g / L, sodium chloride 5.5g / L, zinc sulfate 1μmol / L, water to 1L, pH 6. The initial pH of the culture medium is 8.0, and the culture temperature is 35℃. Pipette 2.0mL of bacterial solution into the sterilized carbonic anhydrase culture medium and place it in a constant temperature incubator at 35℃ for culture. Use a UV spectrophotometer to measure the absorbance of the bacterial solution at 400nm, that is, OD 400 , through the OD 400The value can be used to calculate the activity of carbonic anhydrase. After the bacterial solution is shaken, 2 mL is transferred to a quartz cuvette with a pipette. A sterile culture medium is used as a blank sample. The OD value of the bacterial solution is measured every 3 hours. 400 The value was recorded for 36 hours, and the carbonic anhydrase activity curve was drawn using the test results. Figure 5 As shown, Pseudomonas stutzeri yc4+5-1 grew slowly from 0h to 12h and did not produce carbonic anhydrase; Pseudomonas stutzeri yc4+5-1 grew rapidly from 12h to 18h, and the carbonic anhydrase activity increased rapidly; Pseudomonas stutzeri yc4+5-1 grew steadily from 20h to 25h, and the carbonic anhydrase activity also tended to be stable; after more than 25h, the OD 400 The value dropped, the strain began to die, and the carbonic anhydrase activity decreased sharply; it can be seen that the carbonic anhydrase activity was higher from 20h to 25h, and the activity of carbonic anhydrase was the highest at 24h.

[0089] Experiment 3: Pseudomonas stutzeri yc4+5-1 repairs CO 2 Applications in Escape Channels

[0090] 2.0 mL of bacterial solution was added to 40 mL of carbonic anhydrase liquid culture medium after high temperature sterilization, and cultured at 35°C with constant temperature shaking, and CO was passed through every 6 hours. 2 250 mL was introduced each time, and the growth of microbial cement in the conical flask was observed. After 7 days of cultivation, the sediment adsorbed on the side wall of the conical flask was scraped off with a medicine spoon for X-ray diffractometer, abbreviated as XRD; scanning electron microscope, abbreviated as SEM. The results of X-ray diffractometer measurement are as follows Figure 6 As shown in the figure, the corresponding analysis of the characteristic peaks of the diffraction spectrum shows that the calcium carbonate crystal produced by Pseudomonas stutzeri yc4+5-1 is calcite. Figure 7 As shown, the SEM image of the microbial cement shows that the calcium carbonate crystals induced by Pseudomonas stutzeri yc4+5-1 are calcite.

[0091] The natural core was treated with cracks, and 5 mm copper wire was added to fix the crack width. The core was wrapped with raw tape, and the core permeability was measured and recorded by liquid permeability method. The core was placed in the core holder, and a certain amount of carbonic anhydrase liquid culture medium was introduced to fully saturate the core. The outlet valve was closed and a certain amount of CO was introduced. 2 , close the inlet valve, take out the core every 24 hours, dry it, and measure the permeability. Repeat the above operation for 15 days. After 5 days of repair, the cracks are filled to a large extent and the permeability coefficient is greatly reduced. Continuous repair for 15 days can achieve excellent repair results and the repair rate can reach more than 98%. Figure 8 shown.

[0092] It should be noted that in the above-mentioned carbonic anhydrase liquid culture medium formula, maltose is 3g / L~3.5g / L, trypsin is 10g / L~12g / L, sodium chloride is 5g / L~6g / L, zinc sulfate is 1μmol / L~1.2μmol / L, water is adjusted to 1L, and the operation can be carried out in the numerical range of 6 to 9.

[0093] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0094] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0095] 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Pseudomonas stutzeri (Stutzerimonas sutzeri) yc4+5-1, characterized in that The Pseudomonas stutzeri was deposited in the General Microbiology Center of China Culture Collection Administration on December 21, 2023, with the deposit number CGMCCNO.29371.

2. A microbial preparation, characterized in that: The Pseudomonas stutzeri yc4+5-1 described in claim 1 is inoculated into a carbonic anhydrase culture medium and fermented to produce the microbial preparation.

3. Use of the Pseudomonas stutzeri yc4+5-1 according to claim 1 or the microbial preparation according to claim 2 in producing carbonic anhydrase.

4. The use according to claim 3, characterized in that The Pseudomonas stutzeri yc4+5-1 is inoculated into a carbonic anhydrase culture medium and fermented for 20 h to 25 h at a fermentation temperature of 25° C. to 35° C.

5. A biomineralizing agent, characterized in that: The biomineralization agent comprises the Pseudomonas stutzeri yc4+5-1 of claim 1 and a carbonic anhydrase culture medium; in the biomineralization agent, the number of live bacteria of Pseudomonas stutzeri yc4+5-1 is 10 5 CFU~10 7 CFU.

6. The biomineralizing agent according to claim 5, characterized in that: The formula of the carbonic anhydrase culture medium per liter is as follows: 3g / L-3.5g / L maltose, 10g / L-12g / L tryptone, 5g / L-6g / L sodium chloride, 1μmol / L-1.2μmol / L zinc sulfate, water is used to make the volume to 1L, and the pH value is 6-9.

7. The method for preparing the biomineralizing agent according to claim 6, characterized in that: The steps include: The bacterial liquid of Pseudomonas stutzeri yc4+5-1 is inoculated into the culture medium and fermented for 25 hours to 27 hours at a fermentation temperature of 25° C. to 35° C. to obtain a biomineralizing bacterial agent.

8. Use of the Pseudomonas stutzeri yc4+5-1 according to claim 1, the microbial preparation according to claim 2 or the biomineralizing bacterial agent according to claim 5 in repairing CO2 escape channels in oil reservoirs.

9. The use according to claim 8, characterized in that Repairing the CO2 escape channel of oil reservoirs through carbonic anhydrase produced by the metabolism of Pseudomonas stutzeri yc4+5-1.

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