A high-strength mineralizing strain and its cultivation method and application

Abstract

The invention discloses a high-strength mineralization strain and a cultivation method and application thereof. The high-strength mineralization strain is classified and named as Staphylococcus aureus Staphylococcus simulans ZN1#, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32123. Its optimal culture conditions are: culture temperature 30°C, culture speed 200 r / min, and culture time 24 h. This strain has a high soil mineralization intensity and can be widely used in soil microbial mineralization reinforcement. This strain has the characteristics of small bacterial size and small induced calcite crystal structure, and is particularly suitable for the mineralization and reinforcement of fine-grained soils. It has high strength and good stability in fine-grained soil solidification, which solves the bottleneck problem of the difficulty in applying microbial induced mineralization technology in fine-grained soils.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of high-strength mineralizing strains, and in particular to a high-strength mineralizing strain and a cultivation method and application thereof. Background Art

[0002] With the rapid development of the national economy, problems such as soil loosening and desertification have arisen. How to effectively solve these problems has become a hot issue in the field of geotechnical engineering. Traditional soil reinforcement methods often have high material costs and great limitations, causing damage to the original soil structure and having a great impact on the environment. Therefore, microbial induced calcium carbonate precipitation (MICP) technology is widely used in engineering problems such as soil reinforcement and soil desertification prevention. MICP technology uses high-yield urease bacteria that are widely present in nature, such as Bacillus pasteurianus: it uses its own metabolism to produce urease, promotes urea hydrolysis, and forms a large amount of CO3 2- Free Ca in the environment 2+ Formation of calcium carbonate precipitation. MICP technology uses high-yield urease bacteria to attach to the surface of soil particles or sand particles to form calcium carbonate precipitation to cement the soil particles or sand particles, thereby improving the strength of the soil. However, in actual engineering problems, the effect of improving soil strength is affected by many factors, among which the particle size of soil particles and the size of high-yield urease microbial cells are important factors affecting soil strength. Many studies at home and abroad have shown that traditional MICP technology using Bacillus pasteurianus in the reinforcement of fine-grained soil has low strength and poor solidification effect. This is because the pores between fine-grained soil particles are small, and the calcium carbonate or microbial cells induced by microorganisms are difficult to pass through the pores and form effective cementation, so the solidification effect is poor. Summary of the invention

[0003] The purpose of the present invention is to provide a high-strength mineralizing strain and a cultivation method thereof, which can achieve mineralization and reinforcement in fine-grained soil, and has high strength, improves the mechanical properties of the soil to meet engineering applications, and solves the problems raised in the above background technology. The purpose of the present invention is achieved through the following technical solutions:

[0004] A high-strength mineralization strain, the high-strength mineralization strain is classified and named as Staphylococcus aureus ( Staphylococcus simulans )ZN1 # , deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32123 and the deposit date September 30, 2024. Its 16S rRNA gene sequence is shown in SEQID NO.1. Staphylococcus simulans MK 148 (or the same genus) has a homology of 99.09%.

[0005] The high-strength mineralizing strain is isolated from saline soil. The strain belongs to Gram-positive bacteria, belongs to halotolerant bacteria and facultative alkaliphilic bacteria, and also belongs to urease-producing mineralizing bacteria. The high-strength mineralizing strain has a salt tolerance range of 0% to 30% and an alkali tolerance range of pH ≤ 13. The strain is in the form of regular liquid water droplets on the solid culture medium, with a milky white color, a spherical body, and a length of 0.5 μm to 0.8 μm. Figure 1 shown.

[0006] The cultivation method of the high-strength mineralizing strain is as follows: the logarithmic growth phase of the high-strength mineralizing strain is 24 hours, and the optimal cultivation conditions are: cultivation temperature 30 °C, cultivation speed 200 r / min, and cultivation time 24 hours.

[0007] The separation and screening method of the high-strength mineralizing strain comprises the following steps:

[0008] Step 1: Take n grams of saline soil from the root system of surface plants in Inner Mongolia saline soil, put it into 10n grams of sterile distilled water, shake it for 1 hour, let it stand for 12 hours, and take the supernatant for later use;

[0009] Step 2: inoculate the supernatant into liquid culture medium and perform constant temperature shaking culture;

[0010] Step 3: Prepare the liquid from the constant temperature shaking culture in step 2 into 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The diluted solutions of different concentrations are coated on the plates of solid culture medium respectively, and then cultured at a constant temperature for 1 to 2 days at a temperature of 30 to 37°C;

[0011] Step 4: Pick a clear single colony on the solid culture medium and continue to inoculate it into the liquid culture medium for shaking culture. The shaking temperature is 37-40°C, the shaking frequency is 150-200 r / min, the pH is the natural pH of the culture medium, and the culture time is 24-48 hours;

[0012] Step 5: Take the bacterial solution to verify the mineralization performance;

[0013] Step 6: Select the single colony with the best mineralization effect and continue to inoculate it into the solid culture medium. After repeated streaking and purification, the obtained single colony is the highly mineralized strain imitating Staphylococcus aureus;

[0014] The solid culture medium comprises: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of agar per 1000 mL of deionized water;

[0015] The liquid culture medium comprises: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution per 1000 mL of deionized water.

[0016] Further, the mineralization performance verification: after different single colonies are placed in liquid culture medium and cultured for 24-48 hours, 5-10 ml of bacterial solution is inoculated into the calcium source solution, the mixed solution with white precipitation is allowed to stand for 24 hours, the supernatant is poured out, and the residual bacteria in the precipitation are washed with sterile distilled water, and the sediment sample is obtained at 40-50°C for 12-24 hours; the sediment is detected with 1 mol / L dilute hydrochloric acid. If a large number of bubbles are generated immediately after adding dilute hydrochloric acid to the sediment, the sediment is judged to be calcium carbonate precipitation, and the single colony is a colony with mineralization performance; the calcium source solution includes 0.5-1 mol / L urea and 0.5-1 mol / L dihydrate calcium chloride solution. The microscopic morphology of the sediment is observed and judged by scanning electron microscopy (SEM); the crystal structure of the sediment is analyzed by X-ray diffractometer (XRD); and the chemical composition of the sediment is analyzed by energy dispersive spectrometer (EDS).

[0017] The high-strength mineralizing strain is used in soil biological induced mineralization reinforcement. The microbial mineralization particle size of the high-strength mineralizing strain is widely applicable, suitable for coarse-grained soil and fine-grained soil, and the adapted soil particle size range is ≤10mm. In particular, it performs better in fine-grained soil, the maximum particle size of the fine-grained soil is not more than 4.75mm, and the nominal maximum particle size is not more than 2.36mm. The microbial mineralization particle size of the high-strength mineralizing strain is suitable for clay, silt and sand. The high-strength mineralizing strain has a wide range of environmental tolerance, not only suitable for normal soil environment, but also suitable for degraded soil environment such as high salt and high alkali. The salt environment tolerance range of the high-strength mineralizing strain is 0-32% (in mass percentage), and the alkaline environment tolerance range is: pH≤13.

[0018] Furthermore, the application of the high-strength mineralizing strain in the biologically induced mineralization reinforcement of fine-grained soil includes the following steps:

[0019] Step 1: Prepare a high-strength mineralizing bacterial solution and a cementing solution for fine-grained soil biological induced mineralization: The high-strength mineralizing bacterial solution is prepared by expanding the high-strength mineralizing strain, and the OD of the bacterial solution is 600The value is 0.5~2.5, and the urease activity is 2~4 mMurea / min; the composition of the cementing liquid is: urea (Urea) 30~60 g / L, CaCl2·2H2O 73.5~147 g / L; the solvent of the cementing liquid is water;

[0020] Step 2: Perform in-situ microbial mixing to form a microbial mineralization layer: including the following steps:

[0021] 1) mixing the high-strength mineralizing bacterial solution and the cementing solution at a volume ratio of 1:1 to 1:3 and stirring evenly to obtain a bacterial solution-cementing solution mixture;

[0022] 2) Mixing the bacterial liquid-cementing liquid mixture with fine-grained soil in a ratio of 1:2 to 1:4 and stirring evenly;

[0023] 3) Compact the fine-grained soil obtained by mixing and allow it to dry naturally for 7 to 14 days;

[0024] The induced mineralization reinforced fine-grained soil improves the strength of the fine-grained soil and improves the mechanical properties of the fine-grained soil by cementing and solidifying the fine-grained soil. The high-strength mineralizing strain has the characteristics of small bacterial size and small induced calcite crystal structure, which can effectively improve the strength of the microbial mineralized solidified soil. The unconfined compressive strength of fine-grained saline soil solidified by the high-strength mineralizing strain can reach 4.39MPa, which is 192.7% higher than the average strength of traditional microbial solidification; the unconfined compressive strength of fine standard sand solidified by the high-strength mineralizing strain is 3.97MPa, which is 98.5% higher than the average strength of traditional microbial solidification.

[0025] The advantages and beneficial effects of the present invention are:

[0026] 1. The high-strength mineralization strain provided by the present invention is a strain that imitates Staphylococcus aureus ( Staphylococcus simulans )ZN1# strain is a high-performance mineralization strain that can induce mineralization and reinforce soil and has a high mineralization strength. This strain is particularly suitable for biomineralization reinforcement of fine-grained soil and can significantly improve the mechanical properties of soil. It is a breakthrough in the application of microbial induced mineralization in fine-grained soil and has great application potential and value.

[0027] 2. Imitate Staphylococcus aureus ( Staphylococcus simulans ) ZN1# strain is a soil self-mineralizing strain, suitable for soil mineralization and improvement, and can avoid the impact of exogenous bacteria on the soil environment.

[0028] 3. The use of high-strength mineralizing strains combined with microbial induced mineralization technology to achieve consolidation of fine-grained soil and then repair and improve fine-grained soil is a new, effective, sustainable and environmentally friendly fine-grained soil management and improvement measure. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Figure 1 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )ZN1# colony morphology and SEM scanning electron microscopy images;

[0031] Figure 2 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )Phylogenetic tree of ZN1#;

[0032] Figure 3 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )SEM, EDS and XRD spectra of ZN1# induced mineralization products; Figure 3 (a) is SEM, Figure 3 (b) for EDS, Figure 3 (c) is the XRD pattern;

[0033] Figure 4 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )Growth curve of ZN1#;

[0034] Figure 5 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )ZN1# environmental adaptability-chloride salt resistance characteristic curve;

[0035] Figure 6 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )ZN1# environmental adaptability-sulfate resistance characteristic curve;

[0036] Figure 7 The present invention provides a simulation of Staphylococcus aureus ( Staphylococcus simulans )ZN1# environmental adaptability-alkali resistance characteristic curve;

[0037] Figure 8 Strength characteristic (UCS) curve of fine-grained saline soil solidified by high-strength mineralizing strains provided in the examples of this application;

[0038] Figure 9 This is the strength characteristic (UCS) curve of fine-grained standard sand solidified by the high-strength mineralizing strain provided in the examples of the present application. DETAILED DESCRIPTION

[0039] Example 1

[0040] A high-strength mineralization strain, the high-strength mineralization strain is classified and named as Staphylococcus aureus ( Staphylococcus simulans ) ZN1#, deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No.32123 and the deposit date September 30, 2024. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. Staphylococcus simulant MK 148 (or the same genus) has a homology of 99.09%.

[0041] Mimicking Staphylococcus aureus ( Staphylococcus simulans )ZN1# bacteria 16S rRNA gene sequence:

[0042]

[0043] The high-strength mineralizing strain is isolated from saline soil. The strain belongs to Gram-positive bacteria, halotolerant bacteria and facultative alkaliphilic bacteria, and also belongs to urease-producing mineralizing bacteria. The high-strength mineralizing strain has a chloride concentration tolerance range of 0% to 30%; a sulfate concentration tolerance range of 0 to 3.0 mol / L; and an alkali tolerance range of pH 5.5 to 12. The strain appears in a regular liquid water droplet shape on a solid culture medium, is milky white in color, and has a spherical body with a length of 0.5 μm to 0.8 μm. Figure 1 shown.

[0044] In this embodiment, the separation and screening of the high-strength mineralizing strain includes the following steps:

[0045] Step 1: Isolation and screening of microorganisms from saline soil, specifically including the following steps:

[0046] The samples used in this experiment mainly come from saline soil. The saline soil was taken from a saline wasteland in the Hetao Irrigation Area of ​​Inner Mongolia.

[0047] Step 1: Take 10 grams of saline soil from the root system of surface plants in Inner Mongolia saline soil, put it into 100 grams of sterile distilled water, shake it for 1 hour, let it stand for 12 hours, and take the supernatant for later use;

[0048] Step 2: inoculate the supernatant into liquid culture medium and perform constant temperature shaking culture;

[0049] The liquid culture medium is LB liquid culture medium: composed of 1000 mL of deionized water, including 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution;

[0050] Step 3: Prepare the liquid from the constant temperature shaking culture in step 2 into 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The diluted solutions of different concentrations are coated on the plates of solid culture medium respectively, and then cultured at a constant temperature for 1 to 2 days at a temperature of 30 to 37°C;

[0051] The solid culture medium is LB solid culture medium: 1000 mL of deionized water, including 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of Agar;

[0052] Step 4: Pick a clear single colony on the solid culture medium and continue to inoculate it into the liquid culture medium for shaking culture. The shaking temperature is 37-40°C, the shaking frequency is 150-200 r / min, the pH is the natural pH of the culture medium, and the culture time is 24-48 hours;

[0053] Step 5: Take the bacterial solution to verify the mineralization performance;

[0054] The verification of the mineralization performance of the strain comprises the following steps:

[0055] a. Inoculate a single colony obtained during the isolation of any saline soil self-source mineralization strain into a liquid culture medium and culture for 24 to 48 hours, then take 5 to 10 ml of the bacterial solution and mix it with a calcium source solution, and let the mixed solution with a white precipitate stand for 24 hours, wherein the calcium source solution includes 0.5 to 1 mol / L urea and 0.5 to 1 mol / L calcium chloride dihydrate solution;

[0056] b. Pour off the supernatant, wash with sterile distilled water to remove the residual bacteria in the sediment, and dry at 40-50 °C for 12-24 h to obtain a sediment sample;

[0057] c. Use 1 mol / L dilute hydrochloric acid to detect the sediment. If a large number of bubbles are generated immediately after adding dilute hydrochloric acid to the sediment, it is judged that the sediment is calcium carbonate precipitation, and the single colony is a colony with mineralization performance;

[0058] d. Use a scanning electron microscope (SEM) to observe and determine the microscopic morphology of the sediment; use an X-ray diffractometer (XRD) to analyze the crystal structure of the sediment; use an energy dispersive spectrometer (EDS) to analyze the chemical composition of the sediment. The analysis results are as follows: Figure 3 .

[0059] Step 6: Select the single colony with the best mineralization effect and continue to inoculate it into the solid culture medium. After repeated streaking and purification, the obtained single colony is the highly mineralized strain imitating Staphylococcus aureus ( Staphylococcus simulans )ZN1#;

[0060] The mimetic Staphylococcus aureus appears in a regular liquid water drop shape on the solid culture medium, and the color is milky white. The SEM scanning electron microscope shows that the bacteria are spherical, and the length is 0.5μm to 0.8μm. Figure 1 shown.

[0061] Step 2: Identification of bacterial species. The obtained strains were screened and the 16S rDNA sequence was determined. The sequences were compared online with the known sequences in the gene bank (Gen Bank) established by the National Center for Biotechnology Information (NCBI, http: / / www.Ncbi.nlm.nih.gov) of the United States using the local alignment search tool (Blast). Based on the 16S rDNA sequencing results, a phylogenetic tree was constructed using MEGA4.1 software. This strain belongs to Gram-positive bacteria, and the sequencing results of its 16S rDNA gene are shown in SEQ ID NO.1. The phylogenetic tree constructed based on the gene sequencing results is shown in Figure 2 The bacteria screened were confirmed to be Staphylococcus aureus and were named Staphylococcus aureus ( Staphylococcus simulans ) ZN1#, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 30, 2024, and the registration number of the collection center is CGMCC No.32123.

[0062] Step 3: Imitate Staphylococcus aureus ( Staphylococcus simulans )The growth characteristics of ZN1#. Figure 4 The logarithmic growth period of ZN1# bacteria is 4 to 24 hours, and it enters the stable period after 24 hours.

[0063] Step 4: Imitate Staphylococcus aureus ( Staphylococcus simulans ) Environmental adaptability of ZN1# - Chloride resistance, add chloride to the above LB medium. Figure 5 The chloride concentration range of ZN1# bacteria is 0%~30%.

[0064] Step 5: Imitate Staphylococcus aureus ( Staphylococcus simulans ) Environmental adaptability of ZN1# - sulfate resistance, sulfate was added to the above LB medium. Figure 6 The sulfate concentration range of ZN1# bacteria is 0~3 mol / L.

[0065] Step 6: Imitate Staphylococcus aureus ( Staphylococcus simulans )ZN1# environmental adaptability - alkali resistance. Figure 7 , the alkali resistance range of ZN1# bacteria is pH 5.5 ~ 12. According to the alkali resistance characteristics of microorganisms, ZN1# bacteria are facultative alkaliphiles.

[0066] Embodiment 2:

[0067] The method for applying the high-strength mineralizing strain in the biologically induced mineralization reinforcement of fine-grained soil comprises the following steps: This embodiment uses the strain isolated by the separation and screening method of the high-strength mineralizing strain in Example 1.

[0068] Preparation of fine-grained soil for biologically induced mineralization reinforcement of fine-grained soil: the fine-grained soil is saline soil with a particle size less than 2 mm taken from a saline-alkali wasteland in the Hetao Irrigation District of Inner Mongolia, and standard sand with a particle size less than 2 mm that meets national standards. The fine-grained saline soil and fine-grained standard sand are fully dried for later use.

[0069] Step 1: Prepare high-strength mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The high-strength mineralized bacterial solution is prepared by the high-strength mineralized strain imitating Staphylococcus aureus through expansion, and the OD of the bacterial solution is 600 The value is 0.5, and the urease activity is 2-4 mM urea / min; the composition of the cementing liquid is: Urea 30.03 g / L, CaCl2·2H2O 73.51 g / L; the solvent of the cementing liquid is water;

[0070] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0071] First, the high-strength mineralizing bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0072] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0073] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0074] Microbial induced mineralization reinforcement of fine-grained soil increases the soil strength of fine-grained soil and improves the mechanical properties of fine-grained soil by mineralizing and cementing fine-grained soil. Figure 8 This is the strength characteristic (UCS) curve of the biomineralized fine-grained saline soil by the high-strength mineralizing strain in the embodiment of the present application. The solidification strength can reach 4.39MPa, which is 192.7% higher than the average strength of the soil solidified by the traditional mineralizing strain (the traditional strain is shown in Comparative Example 1, and the parameters of this comparative example are the optimal data of the effect of Bacillus pasteurianus (ATCC 11859) within the test range); Figure 9 This is the strength characteristic (UCS) curve of the fine-grained standard sand biomineralized by the high-strength mineralizing strain in the embodiment of the present application. The solidification strength can reach 3.97MPa, which is 98.5% higher than the average strength of the soil solidified by the traditional mineralizing strain (traditional strain comparison example 2, the comparison example parameters are the optimal data of Bacillus pasteurianus (ATCC 11859) within the test range).

[0075] Example 3

[0076] The method for applying the high-strength mineralizing strain in the biologically induced mineralization reinforcement of fine-grained soil comprises the following steps: This embodiment uses the strain isolated by the separation and screening method of the high-strength mineralizing strain in Example 1.

[0077] Preparation of fine-grained soil for fine-grained soil biologically induced mineralization reinforcement: the fine-grained soil is saline soil with a particle size of less than 2 mm taken from a saline-alkali wasteland in the Hetao Irrigation Area of ​​Inner Mongolia, and the fine-grained saline soil is fully dried for later use.

[0078] Step 1: Prepare high-strength mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The high-strength mineralized bacterial solution is prepared by the high-strength mineralized strain imitating Staphylococcus aureus through expansion, and the OD of the bacterial solution is 600 The value is 1.0, and the urease activity is 2-4 mM urea / min; the composition of the cementing solution is: Urea 30.03 g / L, CaCl2·2H2O 73.51 g / L; the solvent of the cementing solution is water;

[0079] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0080] First, the high-strength mineralizing bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0081] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0082] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0083] Microbial induced mineralization and reinforcement of fine-grained soil improves the soil strength of fine-grained soil and improves the mechanical properties of fine-grained soil by mineralizing and cementing fine-grained soil. The solidification strength of this embodiment is slightly lower than that of embodiment 2, but the difference is not significant.

[0084] Example 4

[0085] The method for applying the high-strength mineralizing strain in the biologically induced mineralization reinforcement of fine-grained soil comprises the following steps: This embodiment uses the strain isolated by the separation and screening method of the high-strength mineralizing strain in Example 1.

[0086] Preparation of fine-grained soil for fine-grained soil biologically induced mineralization reinforcement: the fine-grained soil is saline soil with a particle size of less than 2 mm taken from a saline-alkali wasteland in the Hetao Irrigation Area of ​​Inner Mongolia, and the fine-grained saline soil is fully dried for later use.

[0087] Step 1: Prepare high-strength mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The high-strength mineralized bacterial solution is prepared by the high-strength mineralized strain imitating Staphylococcus aureus through expansion, and the OD of the bacterial solution is 600The value is 2.0, and the urease activity is 2-4 mM urea / min; the composition of the cementing liquid is: Urea 30.03 g / L, CaCl2·2H2O 73.51 g / L; the solvent of the cementing liquid is water;

[0088] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0089] First, the high-strength mineralizing bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0090] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0091] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0092] Microorganism-induced mineralization and reinforcement of fine-grained soil improves the soil strength of fine-grained soil and improves the mechanical properties of fine-grained soil by mineralizing and cementing fine-grained soil. The solidification strength of this embodiment is slightly lower than that of embodiment 2.

[0093] Comparative Example 1

[0094] Preparation of fine-grained soil for fine-grained soil biologically induced mineralization reinforcement: the fine-grained soil is saline soil with a particle size of less than 2 mm taken from a saline-alkali wasteland in the Hetao Irrigation Area of ​​Inner Mongolia, and the fine-grained saline soil is fully dried for later use.

[0095] Step 1: Prepare mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The mineralized bacterial solution is prepared by amplification of Bacillus pasteurianus (ATCC 11859), and the average OD of the mineralized bacterial solution is 600 The value is 0.7, and the average urease activity is 6.67 mM urea / min; the composition of the cementing liquid is: Urea 30.03 g / L, CaCl2·2H2O 73.51 g / L; the solvent of the cementing liquid is water;

[0096] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0097] First, the mineralized bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0098] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0099] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0100] Bacillus pasteurianus (ATCC 11859) biomineralizes fine-grained saline soil, with an average solidification strength of up to 1.5 MPa.

[0101] Comparative Example 2

[0102] Preparation of fine-grained soil for fine-grained soil biologically induced mineralization reinforcement: the fine-grained soil is standard sand with a particle size of less than 2 mm that meets national standards, and the fine-grained standard sand is fully dried for later use.

[0103] Step 1: Prepare mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The mineralized bacterial solution is prepared by amplification of Bacillus pasteurianus (ATCC 11859), and the average OD of the mineralized bacterial solution is 600 The value is 0.7, and the average urease activity is 6.67 mM urea / min; the composition of the cementing liquid is: Urea 30.03 g / L, CaCl2·2H2O 73.51 g / L; the solvent of the cementing liquid is water;

[0104] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0105] First, the mineralized bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0106] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0107] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0108] Bacillus pasteurianus (ATCC 11859) biomineralized fine-grained standard sand, with an average solidification strength of up to 2 MPa.

[0109] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A high-strength mineralizing strain, characterized in that: The high-strength mineralization strain is classified and named Staphylococcus aureus ( Staphylococcus simulans ) ZN1#, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32123 and the deposit date September 30, 2024.

2. A method for culturing the high-strength mineralizing strain according to claim 1, characterized in that: The logarithmic growth phase of the high-strength mineralizing strain is 24 h, and the optimal culture conditions are: culture temperature 30 °C, culture speed 200 r / min, and culture time 24 h.

3. A method for culturing the high-strength mineralizing strain according to claim 1, characterized in that: The high-strength mineralization strain culture medium comprises: per 1000 mL of deionized water, 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of agar.

4. The application of the high-strength mineralizing strain described in claim 1 in soil biological induced mineralization reinforcement is suitable for soil particle size range of ≤10mm.

5. The use according to claim 4, characterized in that: The high-strength mineralizing strain is used in the biologically induced mineralization reinforcement of fine-grained soil, and the maximum particle size of the fine-grained soil is not greater than 4.75 mm.

6. The use according to claim 5, characterized in that: The following steps are involved: Step 1: Prepare a high-strength mineralizing bacterial solution and a cementing solution for fine-grained soil biological induced mineralization: The high-strength mineralizing bacterial solution is prepared by expanding the high-strength mineralizing strain, and the OD of the bacterial solution is 600 The value is 0.5~2.5, and the urease activity is 2~4 mMurea / min; the composition of the cementing liquid is: urea 30~60 g / L, CaCl2·2H2O 73.5~147 g / L; the solvent of the cementing liquid is water; Step 2: Perform in-situ microbial mixing to form a microbial mineralization layer: including the following steps: 1) mixing the high-strength mineralizing bacterial solution and the cementing solution at a volume ratio of 1:1 to 1:3 and stirring evenly to obtain a bacterial solution-cementing solution mixture; 2) Mixing the bacterial liquid-cementing liquid mixture with fine-grained soil at a mass ratio of 1:2 to 1:4 and stirring evenly; 3) Compact the fine-grained soil obtained after mixing and allow it to dry naturally for 7 to 14 days.

7. The use according to claim 4, characterized in that: The microbial mineralization particle size of the high-strength mineralizing strain is suitable for clay soil, silt soil and sandy soil.

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