Microbial gelation enhancing fluid for low temperature cementing slurry and preparation method thereof

Abstract

The present application belongs to the field of oil and gas well cementing technology, and particularly relates to a microbial gel-increasing cementing fluid for low-temperature well cementing and a preparation method thereof. The microbial gel-increasing cementing fluid, according to 100 parts of total weight, comprises: 10-30 parts by weight of spore dry powder of a microbial strain which is domesticated to be alkali-resistant, temperature-resistant and low-oxygen-resistant; 20-50 parts by weight of nitrogen source nutrients; and 20-60 parts by weight of calcium source. The present application utilizes the in-situ generation of calcium carbonate by microbial mineralization, not only improves the macroscopic cementing strength of the interface, but also performs self-repair on the micro-cracks inside the cement sheath, significantly enhances the ability of the cement sheath to resist external disturbance and prevent interlayer fluid channeling, and has important value for guaranteeing the long-term sealing performance of deep water shallow well cementing and the wellbore integrity.

Description

Technical Field

[0001] This invention belongs to the field of oil and gas well cementing technology, specifically relating to a microbial cementitious binder for low-temperature cementing slurry and its preparation method. Background Technology

[0002] In the entire process of oil and gas resource development, cementing is a crucial step in ensuring wellbore integrity, preventing inter-layer fluid cross-flow, and protecting the casing, directly determining the service life and development safety of oil and gas wells. As oil and gas exploration and development advances into deep-water, shallow formations, the severity of the cementing environment has increased significantly. Current cementing technologies mainly rely on "chemically modified cement slurry," but this has inherent limitations.

[0003] like Figure 1 As shown, the interface between the cement stone and the casing is one interface, and the interface between the cement stone and the formation is the second interface. Traditional chemical admixtures have poor low-temperature adaptability. Although they can slightly improve low-temperature hydration efficiency, they easily lead to uncontrolled cement slurry thickening time and cannot solve the problem of insufficient bonding strength at the second interface. Under low-temperature conditions, the bonding strength at the interface is insufficient, and traditional resin-based and expansion agent-based cementing materials are difficult to achieve effective strength improvement, failing to meet the needs of cementing operations in deep water and shallow formations. As deep water and shallow oil and gas reservoirs become important areas for future oil and gas reserve and production increases, existing cementing technologies can no longer meet the comprehensive requirements of "low-temperature rapid isolation and effective bonding at the second interface." The industry usually pays more attention to the bonding strength of the cementing interface in the producing zone, which is typically at medium to high temperatures. This has led to a long-term lack of attention to low-temperature cementing materials, and the industry still lacks cementing agents specifically designed for low-temperature environments.

[0004] Preliminary research has been conducted on the application of microbial mineralization technology in well cementing. Microbial self-healing cement slurry can effectively enhance the self-healing ability of cement stone through calcium carbonate produced by microbial metabolism, and it also has a significant advantage in reducing heat of hydration. The acid-producing, halophilic, alkali-loving, and denitrifying activities of calcifying microorganisms have laid the foundation for the successful application of microbial cementing completion technology. The self-healing of biological metabolites is one of the important development directions of self-healing cement technology.

[0005] However, existing research mainly focuses on the application of microbial self-repair under normal or medium-high temperature conditions, and lacks systematic research on key issues such as the maintenance of microbial activity, strain domestication, and environmental adaptability under low temperature conditions. Summary of the Invention

[0006] In response to the technical problems of insufficient bonding strength between cement stone and casing and formation interface in existing deepwater low-temperature cementing operations, poor low-temperature adaptability of traditional chemical admixtures and inability to effectively improve the interface bonding quality, and lack of targeted low-temperature cementing enhancement materials, this invention provides a microbial cementing enhancement fluid for low-temperature cementing slurry and its preparation method.

[0007] Specifically, the present invention is achieved through the following technical solutions: A microbial binder for low-temperature cementing slurry, comprising, by weight 100 parts: Dry powder of microbial strains acclimated to alkali resistance, temperature resistance, and low oxygen resistance: 10~30 parts by weight; Nitrogen source nutrients: 20-50 parts by weight; Calcium source: 20-60 parts by weight.

[0008] The microbial gelling solution mentioned above contains *Bacillus pasteurellii* strain.

[0009] The aforementioned microbial gelling solution, wherein the spore powder of the microbial strain acclimated to alkali, temperature, and low oxygen conditions is prepared by the following method: (1) Microbial strains were cultured in an alkaline environment with a pH gradient of 10 to 13 to screen for mutant strain A with alkali resistance; (2) Based on the mutant strain A, the strain was cultured in an environment where the temperature gradient decreased from 37°C to 2°C, and mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was placed in an environment with an oxygen concentration gradient decreasing from 21% to 0.1% for low oxygen adaptation culture, and the mutant strain C that is alkali-resistant, temperature-resistant and low oxygen-resistant was screened to obtain the mutant strain C. (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

[0010] The aforementioned microbial gelling solution, wherein the spore powder of the microbial strain acclimated to alkali, temperature, and low oxygen conditions is prepared by the following method: (1) Pasteurella multocida was inoculated into liquid culture medium and cultured in an alkaline environment with pH ranging from 10 to 13 at 30°C and in air. Each batch was subcultured 5 times and cultured for 24 hours each time to screen out alkali-resistant mutant strain A. (2) The mutant strain A was inoculated into liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out in an air atmosphere with the temperature decreasing from 37°C to 2°C. Each batch was continuously passaged 5 times, and each culture lasted 24 hours. The mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was inoculated into a liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out at 30°C in a gradient environment with oxygen concentration decreasing from 21% to 0.1% to screen for alkali-resistant, temperature-resistant and hypoxia-resistant mutant strain C. (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

[0011] The aforementioned microbial gelling solution contains spore powder with a viable count of not less than 1 × 10⁻⁶. 8 CFU / g.

[0012] The nitrogen source nutrients in the aforementioned microbial gelling solution include one or more of urea, peptone, yeast extract, and sodium acetate.

[0013] The calcium source in the aforementioned microbial gelling solution includes one or more of calcium chloride, calcium lactate, and calcium nitrate.

[0014] A method for preparing a microbial cementitious binder for low-temperature cementing slurry includes the following steps: (1) Prepare dry powder of microbial strain spores that have been acclimatized to be alkali-resistant, temperature-resistant, and low-oxygen-resistant; (2) The microbial strain spore powder is mixed with the nitrogen source nutrient and calcium source according to the ratio to obtain microbial gelling solution.

[0015] A method for using a microbial binder for low-temperature cementing slurry includes: mixing the microbial binder into the cementing slurry system, mixing it evenly, and then pumping it into the wellbore for setting.

[0016] In the above method, the amount of the microbial cementitious agent added to the cement slurry is 30~40 kg / cubic meter of cement slurry.

[0017] The technical solution of the present invention has the following beneficial effects: (1) The microbial strains obtained by the present invention through a specific composite domestication process can maintain high urease activity and metabolic rate in deep water environments of 2-37℃, pH 10-13, and 0-30MPa, continuously inducing calcium carbonate precipitation, effectively solving the problems of insufficient activity and poor cementing effect of traditional additives at low temperatures. Experimental data show that, compared with the blank example without the addition of cementing agent, the test example with the addition of the microbial cementing agent of the present invention can increase the cementing strength of the two interfaces by more than 40% after 24 hours, reaching a maximum of 56%.

[0018] (2) This invention utilizes microbial mineralization to generate calcium carbonate in situ, which not only improves the macroscopic bonding strength of the interface, but also enables the self-repair of microcracks inside the cement sheath, significantly enhancing the cement sheath's ability to resist external disturbances and prevent interlayer fluid crossflow. It is of great value in ensuring the long-term sealing and wellbore integrity of deep-water shallow-layer cementing.

[0019] (3) The main components of the microbial cementitious liquid of the present invention are microorganisms and their metabolites, which are green and environmentally friendly. It has good compatibility with various oil well cement admixtures and will not have an adverse effect on the conventional engineering properties of cement slurry such as thickening time, rheology, and compressive strength. Attached Figure Description

[0020] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0021] Figure 1 A schematic diagram of the cementitious interface bonding structure in cementing wells. Figure 2 A schematic diagram of the method for measuring the bonding strength of the cement stone-casing interface; Figure 3 This is a schematic diagram of a method for measuring the bonding strength of the cement stone-formation interface. Detailed Implementation

[0022] To fully understand the purpose, features, and effects of this invention, the following detailed embodiments are provided. Except as described below, the process methods of this invention employ conventional methods or apparatus in the art. Unless otherwise specified, the terms and expressions used below have the meanings commonly understood by those skilled in the art.

[0023] When a range of values ​​is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0024] The technical concept of this invention is as follows: alkali-resistant, pressure-resistant, and low-temperature-resistant microbial strains are obtained through a composite domestication process, and these strains are compounded with nitrogen and calcium sources to form a cementing agent. This agent is then directly mixed into the cementing slurry system. By utilizing the metabolism of microorganisms to induce calcium carbonate precipitation, in-situ filling and cementing enhancement of the micropores and microcracks in the cement stone body and interface are achieved, thereby significantly improving the cementing strength of the first and second interfaces of cementing under deep-water low-temperature conditions.

[0025] Specifically, the low-temperature cementing slurry microbial binder provided by this invention, based on a total weight of 100 parts, comprises: Dry powder of microbial strains acclimated to alkali resistance, temperature resistance, and low oxygen resistance: 10~30 parts by weight; Nitrogen source nutrients: 20-50 parts by weight; Calcium source: 20-60 parts by weight.

[0026] In practice, when the mass of the spore powder of the domesticated microbial strains is less than 10 parts by weight, the microbial concentration is too low to effectively improve the bonding strength of the cementing interface. When the mass of the spore powder of the domesticated microbial strains is greater than 30 parts by weight, the microbial concentration is too high, and they compete for growth space, thus failing to effectively improve the bonding strength of the cementing interface. When the mass of nitrogen source nutrients is less than 20 parts by weight, it cannot provide the nutrients required for microbial growth, thus failing to effectively improve the bonding strength of the cementing interface. When the mass of nitrogen source nutrients is greater than 50 parts by weight, excessive nitrogen source nutrients will significantly reduce the mechanical properties of the cement stone. When the mass of calcium source is less than 20 parts by weight, it cannot provide sufficient calcium ions required for microbial-induced calcium carbonate deposition, thus failing to effectively improve the bonding strength of the cementing interface. When the mass of calcium source is greater than 60 parts by weight, excessive calcium source will significantly reduce the mechanical properties of the cement stone.

[0027] In some preferred embodiments, the starting microbial strain used in this invention is *Bacillus pasteurellus* (…). Sporosarcina pasteurii This strain was purchased from the American Type Culture Collection (ATCC, accession number ATCC11859).

[0028] In some preferred embodiments, the composite acclimatization of the starting microbial strain includes the following steps: (1) Microbial strains were cultured in an alkaline environment with a pH gradient of 10 to 13 to screen for mutant strain A with alkali resistance; (2) Based on the mutant strain A, the strain was cultured in an environment where the temperature gradient decreased from 37°C to 2°C, and mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was placed in an environment with an oxygen concentration gradient decreasing from 21% to 0.1% for low oxygen adaptation culture to obtain alkali-resistant, temperature-resistant and low oxygen-resistant mutant strain C; (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

[0029] More preferably, the composite domestication includes the following steps: (1) Pasteurella multocida was inoculated into liquid culture medium and cultured in an alkaline environment with pH ranging from 10 to 13 at 30°C and in air. Each batch was subcultured 5 times and cultured for 24 hours each time to screen out alkali-resistant mutant strain A. (2) The mutant strain A was inoculated into liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out in an air atmosphere with the temperature decreasing from 37°C to 2°C. Each batch was continuously passaged 5 times, and each culture lasted 24 hours. The mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was inoculated into a liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out at 30°C in a gradient environment with the oxygen concentration decreasing from 21% to 0.1% to obtain the alkali-resistant, temperature-resistant and hypoxia-resistant mutant strain C. (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

[0030] In practice, the microbial strains domesticated according to the method of this invention can be cultured for 48 hours in a simulated downhole environment, namely: pH 10-13, temperature 2-37℃, and pressure 0-30MPa, and their growth index OD... 600 When the concentration reaches 0.15 or higher, the urease activity increases to 0.48 mM / min or higher, and the calcium ion utilization rate reaches 40% or higher.

[0031] The mutant strain C domesticated according to the method of the present invention is *Bacillus pasteurellus* (…). Sporosarcina pasteurii The sample was deposited on July 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39900.

[0032] In some preferred embodiments, the drying is freeze-drying, and the prepared domesticated spore powder is sealed and stored under low-temperature conditions.

[0033] In some preferred embodiments, the liquid culture medium consists of: 20 g / L yeast extract, 10 g / L ammonium sulfate, and Tris buffer (0.13 mol / L, pH 9.0).

[0034] In some preferred embodiments, the viable count of the domesticated spore powder of the present invention is not less than 1×10⁻⁶. 8 CFU / g.

[0035] In some preferred embodiments, the nitrogen source nutrients used in this invention include one or more of urea, peptone, yeast extract, and sodium acetate; the calcium source used in this invention includes one or more of calcium chloride, calcium lactate, and calcium nitrate.

[0036] This invention utilizes the synergistic effect of specific nitrogen source nutrients and calcium source from domesticated alkali-resistant, pressure-resistant, and low-temperature-resistant microbial spores to enable the microbial cementing fluid to effectively enhance the cement stone-casing interface and cement stone-formation interface bonding strength of the cementing slurry under low-temperature conditions of 2℃~37℃ in deep water, thus meeting the high reliability isolation requirements of deep water shallow formations.

[0037] On the other hand, the present invention also provides a method for preparing the above-mentioned low-temperature cementing slurry microbial cementitious binder, comprising the following steps: (1) Microbial strains were subjected to complex domestication to obtain microbial strain spore powder; (2) The microbial strain spore powder is mixed with the nitrogen source nutrient and calcium source according to the ratio to obtain microbial gelling solution.

[0038] The preparation method of the gelling solution of the present invention is simple. It can be achieved by directly mixing the dry powder of microbial strain spores obtained by composite domestication with nitrogen and calcium sources.

[0039] In some preferred embodiments, the gelling agent of the present invention is stored in the form of dry powder or concentrated liquid after preparation.

[0040] The method of using microbial cementitious binder for low-temperature cementing slurry includes: mixing the microbial cementitious binder into the cementing slurry system, mixing it evenly, and then pumping it into the wellbore for setting.

[0041] The amount of the microbial cementitious agent added to the cement slurry is 30-40 kg / cubic meter of cement slurry, and more preferably 36 kg / cubic meter of cement slurry.

[0042] After the cement slurry is pumped into the wellbore, it sets under deep-water, low-temperature conditions. As the cement slurry hydration process proceeds, the microbial spores in the cementitious binder are activated by water and begin to metabolize. The urease produced by the microbial metabolism decomposes the urea in the system, generating carbonate ions; simultaneously, the calcium source in the system provides calcium ions, and the two undergo a mineralization reaction on or near the surface of the microbial cells, generating calcium carbonate crystals with cementing properties in situ. These crystals actively fill and cement the cement stone matrix, the first interface, and the micropores, microcracks, and weak cemented areas at the second interface, thereby improving the interface density, mechanical interlocking force, and overall cementing strength.

[0043] Example The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments, unless otherwise specified, were performed according to conventional methods and conditions. The raw materials used in the following embodiments were all commercially available.

[0044] Example 1: Microbial composite domestication The starting microbial strain used in this embodiment is Bacillus pasteurellus (Pasteurella multocida). Sporosarcina pasteurii This strain was purchased from the American Type Culture Collection (ATCC, accession number ATCC 11859).

[0045] (1) Tolerance to strong alkali acclimatization The *Bacillus pasteurellus* strain was inoculated into liquid culture medium consisting of 20 g / L yeast extract, 10 g / L ammonium sulfate, and Tris buffer (0.13 mol / L, pH 9.0). The culture was incubated at 30°C, 150 rpm on a shaker, under normal pressure and air atmosphere for 24 hours to obtain the seed culture.

[0046] The seed culture was transferred to a new, identical culture medium at a 5% (v / v) inoculation rate. The pH of the medium was adjusted to 10.0, 11.0, 12.0, and 13.0 using HCl or NaOH, respectively. Gradient culture was carried out in an alkaline environment with a pH gradient of 10 to 13, and the culture was subcultured stepwise under atmospheric conditions at 30℃, 150 rpm, normal pressure, and air atmosphere. Each pH level was subcultured five times consecutively, with each subculture lasting 24 hours. After each generation, the bacterial culture was plated onto solid agar plates with an agar concentration of 15 g / L at the corresponding pH. Single colonies with good growth were selected as the starting strain for the next generation of domestication. Finally, a mutant strain A that could stably survive at pH 13 was screened out.

[0047] (2) Temperature acclimatization Based on the mutant strain A, it was inoculated into a liquid culture medium with the same composition and pH of 10.0 as in step (1). It was cultured under shaking conditions of 37°C, 150 rpm, normal pressure, and air atmosphere for 24 hours to obtain the seed culture.

[0048] The seed culture was transferred to a new, identical culture medium at a 5% (v / v) inoculation rate. The culture was then subcultured at 37℃, 20℃, 10℃, and 2℃, with five subcultures at each temperature level, each lasting 24 hours. After each generation, the bacterial culture was plated onto solid agar plates with an agar concentration of 15 g / L at the corresponding temperature. Well-grown single colonies were selected as the starting strain for the next generation of domestication. Finally, a mutant strain B exhibiting both alkali and temperature tolerance was screened out.

[0049] (3) Tolerance to low oxygen acclimatization The mutant strain B was used as the starting strain and inoculated into a liquid culture medium with the same composition and pH of 10.0 as in step (1). The culture was then shaken and cultured for 24 hours at 30°C, 150 rpm on a shaker, under normal pressure and air atmosphere to obtain the seed culture.

[0050] The seed culture was transferred to a new, identical culture medium at a volume ratio of 5%, and then subcultured stepwise under oxygen concentration gradients of 21%, 10%, 5%, 1%, and 0.1%, with each oxygen concentration gradient being subcultured 5 times. The oxygen concentrations at each gradient were precisely controlled using a three-gas incubator (O2 concentrations set at 21%, 10%, 5%, 1%, and 0.1%, CO2 concentration at 5%, and the remainder N2), at a temperature of 30°C and a pressure of atmospheric pressure. Each oxygen concentration gradient was subcultured 5 times consecutively, with each incubation lasting 24 hours. After each generation, the bacterial culture was plated onto solid agar plates with an agar concentration of 15 g / L. The plates were incubated at 30°C in air, and single colonies with good growth were selected as the starting strain for the next generation of domestication. The final result was the target strain C, which could stably survive and maintain metabolic activity in a simulated downhole environment (pH 10–13, temperature 2–37°C, pressure 0–30 MPa).

[0051] (4) Preparation of spore powder of target strain C The target strain C was inoculated into liquid culture medium containing 20 g / L yeast extract, 10 g / L ammonium sulfate, and Tris buffer (0.13 mol / L, pH 9.0). The culture was carried out at 30°C, 150 rpm on a shaker, under normal pressure and air atmosphere for 48 hours to allow the strain to enter the stationary phase.

[0052] Collect the fermentation broth and centrifuge at 4°C and 6000 rpm for 15 minutes to obtain a bacterial precipitate. Wash the bacterial precipitate three times with sterile physiological saline, centrifuging under the same conditions after each wash to remove residual culture medium components.

[0053] The washed bacterial precipitate was freeze-dried into a dry powder. The freeze-drying conditions were: pre-freezing temperature -80℃ for 4 hours, followed by drying at -50℃ and a vacuum degree below 10Pa for 24 hours. The resulting spore powder was then sealed and stored at 4℃.

[0054] Example 2: C activity test of target strain 1. Growth index OD 600 test The target strain C prepared in Example 1 was inoculated into liquid culture medium and cultured with shaking for 48 hours under simulated cementing environmental conditions (pH 11-13, temperature 2-37℃). After the culture was completed, a bacterial culture sample was taken, and the absorbance was measured at 600 nm using an uninoculated blank culture medium after zeroing the culture. The OD value was recorded. 600 Value. Each sample was measured in triplicate, and the average value was taken.

[0055] Tests showed that the target strain's C growth index OD 600It is 0.23.

[0056] 2. Method for determining urease activity The indophenol blue colorimetric method was used, with the following steps: 1 mL of the bacterial culture of the target strain C prepared in Example 1, after being cultured in a simulated cementing environment for 48 hours, was added to 9 mL of phosphate buffer (pH 7.0) containing urea (final concentration 20 g / L), and reacted in a 25°C water bath for 15 minutes. After the reaction, 1 mL of phenol solution (1 g / L) and 1 mL of sodium hypochlorite solution (active chlorine content ≥5%) were added, mixed well, and the mixture was allowed to develop color at 25°C for 20 minutes. The absorbance was measured at 578 nm using a spectrophotometer, and the NH3-N content generated was calculated based on the NH3-N standard curve. Urease activity unit is defined as: the amount of enzyme that hydrolyzes urea to produce 1 μmol of NH3-N per minute per mL of bacterial culture at 25°C and pH 7.0 is defined as 1 enzyme activity unit (U / mL). Each sample was measured in triplicate, and the average value was taken.

[0057] The urease activity of the target strain C was tested to be 0.53 mM / min.

[0058] 3. Method for determining calcium ion utilization rate.

[0059] The calcium ion utilization rate was determined by EDTA titration. The specific steps were as follows: The mineralization reaction solution of the target strain C prepared in Example 1, cultured in a simulated cementing environment for 48 hours, was filtered through a 0.22 μm filter membrane, and the filtrate was collected. 10 mL of the filtrate was taken, and 2 mL of ammonia buffer solution at pH 10 and 3-4 drops of Eriochrome Black T indicator were added. The solution was titrated with standard EDTA solution (0.01 mol / L) until the solution changed from wine red to pure blue. The volume of EDTA consumed was recorded, and the remaining calcium ions in the solution were calculated. 2+ concentration.

[0060] The calcium ion utilization rate is calculated using the following formula: Calcium ion utilization rate = (C 初始 - C 剩余 ) / C 初始 × 100%.

[0061] Among them, C 初始 The Ca²⁺ content in the system at the initial stage of the reaction + Total concentration, C 剩余 The remaining Ca in the solution after the reaction is complete 2+ Concentration. Each sample was measured in triplicate, and the average value was taken.

[0062] Tests showed that the calcium ion utilization rate of the target strain C was 47%.

[0063] 4. Test for viable bacteria count in dried spore powder The method for determining the viable bacterial count was in accordance with GB / T 4789.2 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count".

[0064] The test results showed that the viable bacteria count in the spore powder prepared in Example 1 was 1.75 × 10⁻⁶. 8 .

[0065] Example 3: Preparation of the microbial gelling solution of the present invention Weigh 30 parts by weight of the spore powder of the target strain C prepared in Example 1, 50 parts by weight of the nitrogen source nutrient, and 60 parts by weight of the calcium source.

[0066] The nitrogen source consists of 40% peptone, 40% casein peptone, 12% sodium chloride, and 8% magnesium chloride. The calcium source consists of 25% calcium chloride, 70% soybean protein, 3% cellulose, 0.5% modified polyvinyl alcohol, and 1.5% polyacrylamide. All the above materials were purchased from Sinopharm Chemical Reagent Co., Ltd., are industrial grade, and have a purity of ≥96%.

[0067] The above three raw materials were mixed evenly to obtain microbial gelling solution sample 1.

[0068] Comparative Example 1: Weigh 30 parts by weight of the spore powder of the target strain C prepared in Example 1, 50 parts by weight of nitrogen source nutrient (same as in Example 3), and 0 parts by weight of calcium source.

[0069] The three raw materials were mixed evenly to obtain microbial gelling solution sample 2.

[0070] Comparative Example 2: Weigh 0 parts by weight of the spore powder of the target strain C prepared in Example 1, 0 parts by weight of the nitrogen source nutrient, and 60 parts by weight of the calcium source (same as in Example 3).

[0071] The above three raw materials were mixed evenly to obtain the cementitious liquid sample 3.

[0072] Comparative Example 3: Weigh 30 portions of the starting microbial strain *Bacillus pasteurellii* ( Sporosarcina pasteurii 50 parts by weight of nitrogen source (same as in Example 3), and 60 parts by weight of calcium source (same as in Example 3).

[0073] The three raw materials were mixed evenly to obtain microbial gelling solution sample 4.

[0074] Performance testing of microbial gelling solution To verify the effectiveness of the microbial gelling solution of the present invention, the following methods were used for testing: (1) The compressive strength of cement stone was determined with reference to the petroleum and natural gas industry standard SY / T 5546-92 "Test Method for Application Performance of Cement in Oil Wells"; (2) The bonding strength of the first interface and the second interface was determined using a self-developed bonding ring testing device. The specific testing method is as follows: Figure 2 , 3 As shown, the textual description is as follows: For the first interface test, the diameter of the indenter is slightly smaller than the casing diameter, while the outer diameter of the annular base is slightly larger than the casing diameter, and the inner diameter is slightly smaller than the casing external thread diameter. The testing principle utilizes the difference in the force-bearing area and application position of the interface test indenter and the annular base to cause the cement sample and casing to shift along the simulated cementing interface. By measuring the peak shear stress and the shear surface area, the first interface bonding strength can be calculated. The calculation of the interface bonding strength is shown in Formulas 1 and 2. For the second interface test, the diameter of the indenter is slightly smaller than the rock column diameter, while the inner diameter of the annular base is slightly larger than the rock column diameter. The mechanical testing machine applies force to the indenter, causing the cement sample and rock column to shift along the simulated cementing interface. The peak shear stress is measured, and the second interface bonding strength is calculated using the formulas.

[0075] σ=FA(1) A = πD × H (2) Where σ is the interfacial bonding strength (MPa); F is the maximum shear force (kN); A is the interfacial area (m²); D is the inner diameter of the test body (m); and H is the height of the test body (m).

[0076] The experiment was conducted under a uniform 7-day curing environment at 12℃. The reference cement slurry consisted of 100% G-grade oil well cement, 0.4% C-X60L defoamer, 8% C-G86L fluid loss reducer, 6% C-P81L weight-reducing agent, and 1.9% C-R23L retarder. All cement additives were provided by CNOOC Oilfield Services Limited. Artificial seawater with a salinity of 35 g / L was used. The slurry density was controlled at 1.55 g / cm³. 3 The six test cases differed only in the amount of cementitious agent added: Test Example 1 No microbial cementitious agent was added to each cubic meter of reference cement slurry. The samples were prepared by stirring at 200 r / min for 8 min. After curing the samples in a laboratory at a low temperature of 12℃, the compressive strength and the bonding strength at the first and second interfaces were measured. To track the early strength development, the mechanical properties were measured at 24 h and 48 h.

[0077] The results showed that after 24 hours of curing, the compressive strength of the reference cement stone was 3.8 MPa. After 48 hours of curing, its compressive strength significantly increased to 8.35 MPa. The bonding strength at the first interface was 1.27 MPa, the bonding strength at the second interface was 2.16 MPa, and the density of the cement stone was 1.8 g / cm³.3 . Serves as the basic reference data.

[0078] Test Example 2 Add 36 kg of microbial cementitious agent sample 1 to each cubic meter of standard cement slurry, with the same mixing parameters and curing conditions as test example 1.

[0079] Experimental Results: The bonding strength at the first and second interfaces showed the greatest increase. After 24 hours of curing, the compressive strength of the reference cement stone was 8.25 MPa. After 48 hours of curing, its compressive strength significantly increased to 13 MPa. The bonding strength at the first interface was 2.02 MPa, and the bonding strength at the second interface was 3.39 MPa, representing an increase of approximately 57% compared to 2.16 MPa in Test Example 1. The cement stone exhibited the best density, with a density of 2.2 g / cm³. 3 The preferred dosage for this invention is specified below.

[0080] Test Example 3 Add 36 kg of microbial cementitious binder sample 2 to each cubic meter of standard cement slurry, with the same mixing parameters and curing conditions as test example 1.

[0081] Results: After 24 hours of curing, the compressive strength of the reference cement stone was 4.4 MPa. After 48 hours of curing, its compressive strength increased to 9.06 MPa. The bond strength at the first interface was 1.34 MPa, the bond strength at the second interface was 2.2 MPa, and the density of the cement stone was 1.85 g / cm³. 3 Microbial spore powder can slightly increase the strength of cement, but without the addition of a calcium source, it cannot increase the interfacial bonding strength.

[0082] Test Example 4 Add 36 kg of microbial cementitious binder sample 3 to each cubic meter of standard cement slurry, with the same mixing parameters and curing conditions as test example 1.

[0083] Results: After 24 hours of curing, the compressive strength of the reference cement stone was 3.9 MPa. After 48 hours of curing, the compressive strength increased to 8.36 MPa. The bond strength at the first interface was 1.24 MPa, the bond strength at the second interface was 2.2 MPa, and the density of the cement stone was 1.85 g / cm³. 3 The strength of the cement itself and the interfacial bonding strength were basically the same as those in Test Example 1. Only calcium source was added, but the spore powder of the target strain C prepared in Example 1 and nitrogen source nutrients were not added, so the interfacial bonding strength could not be increased.

[0084] Test Example 5 36 kg of microbial cementitious binder sample 4 was added to each cubic meter of standard cement slurry, and the remaining test conditions were the same as those in test example 2.

[0085] Results: After 24 hours of curing, the compressive strength of the reference cement stone was 4.5 MPa. After 48 hours of curing, its compressive strength significantly increased to 8.5 MPa. The bond strength at the first interface was 1.3 MPa, the bond strength at the second interface was 2.5 MPa, and the density of the cement stone was 1.83 g / cm³. 3 At low temperatures, the activation rate of the bacterial strain is low, and the improvement in interfacial bonding is far less than that in test example 2.

[0086] Test Example 6 Add 30 kg of microbial cementitious binder sample 1 to each cubic meter of standard cement slurry, and the remaining test conditions are the same as those in test example 2.

[0087] Results: After 24 hours of curing, the compressive strength of the reference cement stone was 6.2 MPa. After 48 hours of curing, its compressive strength significantly increased to 11.4 MPa. The bond strength at the first interface was 1.93 MPa, the bond strength at the second interface was 3.06 MPa, and the density of the cement stone was 2.1 g / cm³. 3 The interfacial bonding strength was better than that of the control group, but lower than that of Test Example 2 with the optimal dosage. Insufficient dosage resulted in less mineralization products.

[0088] Test Example 7 40 kg of microbial cementitious binder sample 1 was added to each cubic meter of standard cement slurry, and the remaining test conditions were the same as those in test example 2.

[0089] Results: After 24 hours of curing, the compressive strength of the reference cement stone was 8.5 MPa. After 48 hours of curing, its compressive strength significantly increased to 13.2 MPa. The bond strength at the first interface was 2.1 MPa, the bond strength at the second interface was 3.4 MPa, and the density of the cement stone was 2.2 g / cm³. 3 The bonding strength was not significantly improved compared to Test Example 2, and excessive addition resulted in raw material waste and increased production costs.

[0090] Experimental results show that only when the gelling agent prepared with the domesticated strain of this invention is used and the dosage is controlled at 36 kg / cubic meter of cement slurry can the microbial mineralization gelling effect be maximized. Without the domesticated strain, without adding the gelling agent, or with too high or too low dosage, efficient interface reinforcement cannot be achieved, which confirms that this formula and dosage are the key to improving the bonding strength of the first and second interfaces at low temperature.

[0091] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0092] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0093] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A microbial binder for low-temperature cementing slurry, characterized in that, Based on a total weight of 100 portions, including: Dry powder of microbial strains acclimated to alkali resistance, temperature resistance, and low oxygen resistance: 10~30 parts by weight; Nitrogen source nutrients: 20-50 parts by weight; Calcium source: 20-60 parts by weight.

2. The microbial gelling solution according to claim 1, characterized in that, The microbial strain is Pasteurella multocida.

3. The microbial gelling solution according to claim 1, characterized in that, The spore powder of the microbial strain that has been acclimatized to alkali, temperature, and low oxygen was prepared by the following method: (1) Microbial strains were cultured in an alkaline environment with a pH gradient of 10 to 13 to screen for mutant strain A with alkali resistance; (2) Based on the mutant strain A, the strain was cultured in an environment where the temperature gradient decreased from 37°C to 2°C, and mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was placed in an environment with an oxygen concentration gradient decreasing from 21% to 0.1% for low oxygen adaptation culture, and the mutant strain C that is alkali-resistant, temperature-resistant and low oxygen-resistant was screened to obtain the mutant strain C. (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

4. The microbial gelling solution according to claim 3, characterized in that, The spore powder of the microbial strain that has been acclimatized to alkali, temperature, and low oxygen was prepared by the following method: (1) Pasteurella multocida was inoculated into liquid culture medium and cultured in an alkaline environment with pH ranging from 10 to 13 at 30°C and in air. Each batch was subcultured 5 times and cultured for 24 hours each time to screen out alkali-resistant mutant strain A. (2) The mutant strain A was inoculated into liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out in an air atmosphere with the temperature decreasing from 37°C to 2°C. Each batch was continuously passaged 5 times, and each culture lasted 24 hours. The mutant strain B with both alkali resistance and temperature resistance was screened out. (3) The mutant strain B was inoculated into a liquid culture medium, the pH of the liquid culture medium was adjusted to 10, and the culture was carried out at 30°C in a gradient environment with oxygen concentration decreasing from 21% to 0.1% to screen for alkali-resistant, temperature-resistant and hypoxia-resistant mutant strain C. (4) Ferment the mutant strain C, collect the fermentation liquid, centrifuge, wash and dry it to obtain spore powder.

5. The microbial gelling solution according to claim 3 or 4, characterized in that, The spore powder contains no less than 1×10⁻⁶ live bacteria. 8 CFU / g.

6. The microbial gelling solution according to claim 1, characterized in that, The nitrogen source nutrients include one or more of urea, peptone, yeast extract, and sodium acetate.

7. The microbial gelling solution according to claim 1, characterized in that, The calcium source includes one or more of calcium chloride, calcium lactate, and calcium nitrate.

8. A method for preparing a microbial cementitious binder for low-temperature cementing slurry according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Prepare spore powder of microbial strains that have been acclimatized to alkali, temperature and low oxygen according to claim 3 or 4; (2) The microbial strain spore powder is mixed with the nitrogen source nutrient and calcium source according to the ratio to obtain microbial gelling solution.

9. The method of using the microbial cementitious binder for low-temperature cementing slurry according to any one of claims 1 to 7, characterized in that, include: The microbial cementitious agent is mixed into the cementing slurry system, and after being mixed evenly, it is pumped into the wellbore for setting.

10. The method of use according to claim 9, characterized in that, The amount of the microbial cementitious binder added to the cement slurry is 30-40 kg / cubic meter of cement slurry.

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