Alkali-resistant biomineralization bacterium composition and application thereof

Abstract

The invention provides an alkali-resistant biologically mineralized bacterium combination and an application of the alkali-resistant biologically mineralized bacterium combination. The alkali-resistant biologically mineralized bacterium combination is composed of Sporosarcina pasteurii DC419 and Bacillus pseudofirus DCG02, and the alkali-resistant biologically mineralized bacterium combination is composed of Sporosarcina pasteurii DC419 and Bacillus pseudofirus DCG02. The invention firstly provides a mixed culture process and application of two alkali-resistant biomineralized bacteria. The method comprises the following steps: activating strains, adding the strains into a fermentation medium step by step to enrich the strains, and finally fermenting to obtain the biomineralization mixed bacterial liquid with high urease activity. Then, the cementing liquid containing calcium ions and the mixed bacterium liquid are mixed in different environments for mineralization, and the result shows that the mixed bacterium system has high mineralization yield and can be biologically mineralized in strong alkaline and high-temperature environments. And finally, adding the cementing liquid containing the calcium ions and the mixed bacteria liquid into the soft soil for curing to generate calcium carbonate, so that the strength of the soft soil is improved. The invention further provides a method for generating magnesium calcite through combined induction of the alkali-resistant biomineralization bacteria, and a new thought is provided for microorganism induction of carbonate precipitation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an alkali-resistant biomineralizing bacteria combination and its application. Background Technology

[0002] Microbial-induced carbonate precipitation (MICP) is an emerging method for soil solidification. This process promotes the sequestration of atmospheric carbon dioxide by forming calcium carbonate crystals, which then fill soil pores and enhance particle bonding. After MIP treatment, key geotechnical properties such as strength, permeability, and compressibility are significantly improved. MICP is characterized by its rapid reaction rate, low environmental requirements, wide application range, and significant greenhouse gas emission reduction effect, and is widely used in various fields including geology, civil engineering, water conservancy, and environment. The calcium carbonate minerals induced by MICP are mostly calcite. *Sporosarcina pasteurii* DC419 is a widely used strain in MIP technology, known for its high urease activity and ability to promote efficient carbonate precipitation. However, its application is subject to strict cultivation conditions and it has poor survival ability under high temperature or strong alkaline conditions. Bacillus pseudofirmus DCG02 is less commonly used in MIPs due to its relatively low mineralization yield, but it exhibits stronger environmental tolerance.

[0003] Soft clayey soils are characterized by high water content, low strength, high compressibility, significant sensitivity, and susceptibility to disturbance, and are widely distributed in coastal areas and river valleys. Rapid urbanization has generated large quantities of this type of soil. If not properly managed, these soils may pose geological engineering risks, including foundation settlement and slope instability, potentially damaging structural integrity and transportation infrastructure. Therefore, improving soft soil textures is an urgent problem to be solved. Magnesium calcite, a variant of calcite (CaCO3), has a crystal structure in which some calcium ions are replaced by magnesium ions, and its general chemical formula is Ca. 1-x Mg x Compared to calcite, magnesia-calcite has higher hardness, stronger dissolution resistance, greater biocompatibility and functional scalability, and has a wide range of applications. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an alkali-resistant biomineralizing bacteria combination and its application, which addresses the shortcomings of the prior art.

[0005] To address the aforementioned technical problems, this invention discloses an alkali-resistant biomineralizing bacteria combination and its application. Specifically, it includes the following technical solutions:

[0006] The present invention first provides a strain of Bacillus pseudofirmus, which is classified and named Bacillus pseudofirmus, strain number DCG02, accession number CCTCC NO:M 20222037, and accession date December 23, 2022.

[0007] Secondly, the present invention provides an alkali-resistant biomineralizing bacteria combination, comprising *Sporosarcina pasteurii* and the *Pseudomonas stolonifer* described in the first aspect. Preferably, the *Sporosarcina pasteurii* comprises *Sporosarcina pasteurii* DC419, with accession number CCTCC NO:M 20222038.

[0008] Thirdly, the present invention provides the application of the alkali-resistant biomineralizing bacteria combination described in the second aspect in the solidification of soft soil and / or the preparation of magnesium calcite.

[0009] The alkali-resistant biomineralizing bacteria combination is used in the form of mixed bacterial fermentation broth to solidify soft soil and / or prepare magnesium calcite.

[0010] The mixed-culture fermentation broth is prepared according to the following steps:

[0011] (1) Inoculate the Bacillus pasteurellus seed liquid into the fermentation medium and ferment at 28-35℃ for 16-24h to obtain Bacillus pasteurellus fermentation broth; wherein the fermentation medium contains 1-10g / L urea.

[0012] (2) Inoculate the Bacillus pseudostrongylus seed liquid into the Bacillus pasteuri fermentation broth at an inoculation rate of 0.5% to 2% v / v, and ferment at 28 to 35°C and 100 to 200 rpm for 10 to 14 hours to obtain a mixed fermentation broth.

[0013] Preferably, the *Bacillus pasteurellium* seed culture is obtained by inoculating *Bacillus pasteurellium* into LB medium at pH 9 and culturing it at 28–35°C for 10–20 h. The *Bacillus pseudostrongylus* seed culture is obtained by inoculating *Bacillus pseudostrongylus* into LB medium at pH 10 and culturing it at 28–35°C and 100–200 rpm for 10–20 h.

[0014] The solidification of soft soil is carried out by the following method: the mixed bacterial fermentation liquid and the first cementing liquid are mixed to obtain a mixed liquid, the mixed liquid is mixed evenly with soft soil, and left to stand to achieve solidification of soft soil.

[0015] The first cementing solution comprises 0.3–1.2 mol / L urea and 0.3–1.2 mol / L calcium ions; the pH of the first cementing solution is 8–12; preferably, the calcium ions are prepared in the form of calcium formate or calcium chloride; more preferably, the first cementing solution further comprises 0.3–1.2 mol / L magnesium ions, which are prepared in the form of magnesium chloride.

[0016] More preferably, the first cementing solution is a mixture of 0.9 mol / L urea and 0.9 mol / L calcium formate, with the remainder being distilled water, and the pH is 9. Alternatively, the first cementing solution is a mixture of 1 mol / L urea, 0.5 mol / L calcium chloride, and 0.5 mol / L magnesium chloride, with the remainder being distilled water, and the pH is 9. In this case, the formulation of the first cementing solution is the same as that of the second cementing solution described below.

[0017] The mixing volume ratio of the first cementing liquid and the mixed fermentation liquid is 1-2:1-2; preferably 2:1.

[0018] The added mass of the mixture is 15% to 20% of the mass of the soft soil; preferably 16.5%.

[0019] The aforementioned standing period refers to standing at room temperature for 5 to 7 days.

[0020] The preparation of magnesium calcite is carried out by the following method: the mixed bacterial fermentation broth and the second cementing broth are mixed and reacted to obtain magnesium calcite.

[0021] The second cementing solution comprises 0.3–1.2 mol / L urea, 0.3–1.2 mol / L calcium ions, and 0.3–1.2 mol / L magnesium ions, and has a pH of 8–12. Preferably, the calcium ions are calcium chloride, and the magnesium ions are magnesium chloride. More preferably, the second cementing solution is a mixture of 1 mol / L urea, 0.5 mol / L calcium chloride, and 0.5 mol / L magnesium chloride, with the remainder being distilled water, and has a pH of 9.

[0022] The volume ratio of the second cementing solution to the mixed fermentation broth is 8-12:1; preferably 10:1.

[0023] The reaction is carried out at a temperature of 28–32°C for a time of 36–72 h. Preferably, the reaction is carried out at 30°C for 48 h.

[0024] More preferably, the reactants are dried after the reaction to obtain magnesium calcite.

[0025] Beneficial effects:

[0026] This invention provides an alkali-tolerant biomineralizing bacteria combination and its application. The alkali-tolerant biomineralizing bacteria combination is a combination of Sporosarcina pasteurii DC419 and Bacillus pseudofirmus DCG02. This invention provides a mixed culture process for the two alkali-tolerant biomineralizing bacteria and its application. On the one hand, the combination of the two bacteria exhibits stronger alkali tolerance and high-temperature resistance. On the other hand, the combination of the two bacteria provides a soft soil solidification scheme based on a mixed bacterial system. The mixed bacterial system has high urease activity and the ability to produce calcium carbonate and magnesium calcite precipitates. This invention provides a method for generating magnesium calcite, providing a new product for biomineralization. Attached Figure Description

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0028] Figure 1 The soft soil molding mold used in this invention has a diameter of 50 mm and a height of 100 mm.

[0029] Figure 2 XRD analysis of the magnesium calcite prepared in Example 6;

[0030] Figure 3 The magnesium calcite prepared in Example 6 is characterized by scanning electron microscopy. Detailed Implementation

[0031] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0032] In the following examples, the Sporosarcina pasteurii DC419 was obtained from the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20222038, and its detailed information has been disclosed in Chinese Patent CN116445343A.

[0033] The *Bacillus pseudofirmus* DCG02, with accession number CCTCC NO: M20222037, was deposited on December 23, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The 16S rDNA sequencing sequence of *Bacillus pseudofirmus* DCG02 is shown in SEQ ID NO.1.

[0034] Example 1: Preparation of bacterial suspension in mixed bacterial system

[0035] (1) Preparation of culture medium:

[0036] The LB medium used for *S. pasteurii* DC419 consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with the remainder being distilled water. The pH was adjusted to 9 with 4M sodium hydroxide solution, with a total volume of 5 mL, and then sterilized.

[0037] The LB medium 2 used for B. pseudofirmus DCG02 consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with the remainder being distilled water. The pH was adjusted to 10 with 4M sodium hydroxide solution, with a total volume of 5 mL, and then sterilized.

[0038] Fermentation medium: 12 g / L peptone, 24 g / L yeast extract, 16.43 g / L dipotassium hydrogen phosphate, 0.49 g / L anhydrous magnesium sulfate, 10 g / L glycerol, with the remainder being distilled water. Adjust the pH to 9 with 4M sodium hydroxide solution, bringing the total volume to 100 mL, and then sterilize.

[0039] (2) Preparation of seed solution:

[0040] Remove the bacterial culture tubes from the -80℃ freezer and inoculate 50 μL of Bacillus pasteurii DC419 and 100 μL of urea (urea concentration of 200 g / L) into LB medium 1 in a clean bench; inoculate 50 μL of Bacillus pseudofirmus DCG02 into LB medium 2. After inoculation, both are placed in a constant temperature shaker at 30℃ and 180 r / min for 18 h to activate the culture, and the seed cultures of the two strains are obtained and stored in a 4℃ freezer.

[0041] (3) Fermentation enrichment:

[0042] Take out the S. pasteurii DC419 seed liquid from step (2), add 1 mL of seed liquid and 2 mL of urea (urea concentration of 200 g / L) to 100 mL of fermentation medium in a clean bench for enrichment, and then place it in a constant temperature shaker at 30℃ and 180 r / min for 24 h to obtain Bacillus pasteurii fermentation broth.

[0043] (4) Mixed culture:

[0044] Take the B. pseudofirmus DCG02 seed culture from step (2) and the Bacillus pasteurellium fermentation broth from step (3). In a clean bench, add 1 mL of the B. pseudofirmus DCG02 seed culture to the Bacillus pasteurellium fermentation broth for enrichment. Then, incubate at 30℃ and 180 r / min in a constant temperature shaker for 12 h to obtain a mixed culture fermentation broth. After enrichment, measure the concentration of the mixed culture using a spectrophotometer to OD. 600 =2.5.

[0045] In addition, the present invention also prepared a single-strain bacterial suspension, the specific method of which is as follows:

[0046] Single-strain culture of *Bacillus pasteurii*: The inoculum was removed from the -80℃ freezer and inoculated with 50 μL of *Pasteurii* DC419 strain and 100 μL of urea (200 g / L) into 5 mL of LB medium 1. The culture was then activated at 30℃ and 180 rpm for 12 h using a constant temperature shaker to obtain the seed culture. 1 mL of the seed culture and 2 mL of urea (200 g / L) were added to 100 mL of fermentation medium for enrichment. The culture was then incubated at 30℃ and 180 rpm for 20 h using a constant temperature shaker. The bacterial concentration was measured using a spectrophotometer at OD500. 600 =2.5, to obtain a single bacterial culture of Bacillus pasteurellii.

[0047] Single-strain culture of *Bacillus pseudofirmus*: The inoculum was removed from the -80℃ freezer and inoculated with 50 μL of *B. pseudofirmus* DCG02 strain into 5 mL of LB medium 2. The culture was then activated at 30℃ and 180 rpm for 12 h using a constant-temperature shaker to obtain the seed culture. 1 mL of this seed culture was added to 100 mL of fermentation medium for enrichment, and the culture was incubated at 30℃ and 180 rpm for 20 h using a constant-temperature shaker. The bacterial concentration was measured using a spectrophotometer at OD500 after enrichment. 600 =2.5, to obtain a single bacterial culture of Bacillus pseudosturcium.

[0048] Example 2 Urease Activity Assay

[0049] Prepare a 0.2 mol / L phosphate buffer solution with pH=8, and then add 1.1 mol / L urea to obtain a urea buffer solution.

[0050] Mix 1 mL of the mixed-culture fermentation broth prepared in Example 1 (or the single-culture system broth prepared in Example 1) with 9 mL of phosphate buffer, and react in a 30°C constant temperature water bath for three minutes. Observe the change in conductivity using a conductivity meter and calculate the urease activity. Urease activity is expressed as U = change in conductivity / 3 * 11.11 * 10.

[0051] The results showed that the urease activity of the mixed bacterial system was approximately 39.38 U / mL. The urease activity of a single Bacillus pasteurellium culture was 34.07 U / mL, and the urease activity of a single Bacillus pseudosternum culture was 28.39 U / mL.

[0052] Example 3: Determination of calcium carbonate yield

[0053] (1) First cementing solution formula: 0.9M urea and 0.9M calcium formate, with the remainder being distilled water, and the pH is adjusted to 9 with 4M / L sodium hydroxide solution.

[0054] (2) Wash the 50mL centrifuge tube, dry it, and weigh its original weight m0. Mix 20mL of the first cementing solution and 2mL of the mixed fermentation broth prepared in Example 1 (or the single-strain system broth prepared in Example 1), and let it stand in a constant temperature incubator at 30℃ for 48h to obtain the mixed solution.

[0055] (3) After standing, the mixture was centrifuged at 6000 rpm for 5 minutes in a high-speed rotary centrifuge. The waste liquid after centrifugation was discarded, and the obtained calcium carbonate sample was dried in an oven and weighed m1.

[0056] (4) Calculate the calcium carbonate content produced: m2 = m1 - m0.

[0057] The results showed that the calcium carbonate yield of the mixed bacterial system was about 2.93 g, the calcium carbonate yield of the Bacillus pasteurellis single bacterial system was 2.45 g, and the calcium carbonate yield of the Bacillus pseudosturcium single bacterial system was only 0.24 g.

[0058] Example 4: High Temperature and Alkali Resistance Test

[0059] (1) Prepare the cementing solution: 0.9M / L urea and 0.9M / L calcium formate, with the remainder being distilled water. Adjust the pH to 9, 11, or 12 using 4M / L sodium hydroxide solution.

[0060] (2) Wash the 50mL centrifuge tubes, dry them, and weigh them to their original weight m0. Mix 20mL of the cementing solution with 2mL of the mixed fermentation broth prepared in Example 1 (or the single-strain system broth prepared in Example 1). Place the experimental group with cementing solution pH=9 in a constant temperature incubator at 50℃ and let it stand for 48h. Place the experimental groups with cementing solution pH=11 and 12 in a constant temperature incubator at 30℃ and let them stand for 48h to obtain the mixed solution.

[0061] (3) After standing, the mixture was centrifuged at 6000 rpm for 5 minutes in a high-speed rotary centrifuge. The waste liquid after centrifugation was discarded, and the obtained calcium carbonate sample was dried in an oven and weighed m1.

[0062] (4) Calculate the calcium carbonate content produced: m2 = m1 - m0.

[0063] The results showed that for the mixed bacterial system, the calcium carbonate yield was approximately 2.48 g at 50℃ and pH=9, approximately 2.89 g at 30℃ and pH=11, and approximately 2.47 g at 30℃ and pH=12.

[0064] For the Bacillus pasteurellosis single-strain system, the calcium carbonate yield was 2.19 g at 50℃ and pH=9, 2.66 g at 30℃ and pH=11, and 2.30 g at 30℃ and pH=12.

[0065] Example 5: Soft Soil Consolidation Strength Test

[0066] (1) Take a soft soil sample from a construction site at the Jiangpu Campus of Nanjing University of Technology, crush it with a crusher and weigh it.

[0067] (2) The first cementing liquid prepared in Example 3 and the mixed-culture fermentation broth prepared in Example 1 (or the single-culture system broth prepared in Example 1) were mixed at a volume ratio of 2:1. 16.5% of the mass of soft soil was then poured in and stirred evenly. The mixture was compacted into a mold and cured at room temperature in the laboratory. The mold is as follows: Figure 1 As shown.

[0068] (3) After 7 days, test the solidified soft soil on a universal tensile testing machine to break it, and record the required pressure P.

[0069] (4) Calculate its compressive strength p = P / A, where A is the cross-sectional area under force.

[0070] The results showed that the compressive strength of the solidified soil was 2.11 MPa for the mixed bacterial system, while the compressive strength of the solidified soil for the Bacillus pasteurellii single-strain system was only 0.630 MPa. Furthermore, since the experimental data in Example 3 showed that the calcium carbonate yield of the Bacillus pseudosturcium single-strain system was only 0.24 g, its effect on solidifying soft soil alone was predictable and its application value was low; therefore, relevant application data were not presented here.

[0071] In addition, this embodiment also tested the calcium carbonate content of the solidified soft soil using the acid-base neutralization method, as follows: The solidified soil was crushed in a pulverizer. 30g of the crushed soil sample was added to 100mL of 0.5mol / L hydrochloric acid (HCl) solution, and stirred until no bubbles were generated. Then, 20mL of the reaction solution was transferred to a 100mL volumetric flask and diluted to the mark with deionized water. 50mL of the diluted solution was transferred to a 250mL Erlenmeyer flask, 2 drops of phenolphthalein indicator were added, and the remaining hydrochloric acid was titrated with 0.25mol / L sodium hydroxide (NaOH) standard solution until the solution turned a pale pink color and remained so for 1 minute. The volume of NaOH consumed was recorded for calculation.

[0072] The mass content (W) of CaCO3 is calculated using the following formula: W=(C1V1-C2V5×V3 / V4×V1 / V2) / M / 2×MrCaCO3

[0073] In the formula: W is the mass content of CaCO3 (%); M is the mass of the soil sample (30g); C1 is the concentration of the HCl standard solution (0.5mol / L); C2 is the concentration of the NaOH standard solution (0.25mol / L); V1 is the volume of HCl added (100mL); V2 is the volume of solution transferred to the volumetric flask (20mL); V3 is the volume after dilution (100mL); V4 is the volume of sample used for titration (50mL); V5 is the volume of NaOH consumed in titration (mL); MrCaCO3 is the relative molecular mass of calcium carbonate (100g / mol).

[0074] The results showed that the calcium carbonate content was 30.07% for the mixed bacterial system and 20.07% for the Bacillus pasteurellis single bacterial system.

[0075] Based on the above experimental results, this mixed bacterial system exhibits high urease activity and mineralization yield, and can perform biomineralization in strongly alkaline and high-temperature environments, efficiently converting calcium ions into calcium carbonate, making it applicable to soft soil stabilization. Example 6: Microbial-induced precipitation of magnesium calcite.

[0076] (1) Prepare the second cementing solution: 1M urea, 0.5M calcium chloride and 0.5M magnesium chloride, with the remainder being distilled water. Adjust the pH to 9 with 4M sodium hydroxide solution.

[0077] (2) Wash the 50mL centrifuge tube, dry it, and weigh its original weight m0. Mix 20mL of the second cementing solution and 2mL of the mixed fermentation broth prepared in Example 1, and let it stand in a constant temperature incubator at 30℃ for 48h.

[0078] (3) After standing, the mixture was centrifuged at 6000 rpm for 5 minutes in a high-speed rotary centrifuge. The waste liquid after centrifugation was discarded, and the obtained magnesium calcite sample was dried in an oven and weighed m1.

[0079] (4) Take the dried magnesium calcite sample from step (3) for XRD testing and analyze it under a scanning electron microscope, such as... Figure 2 and Figure 3 As shown.

[0080] (5) The content of the precipitate generated was calculated as m2 = m1 - m0, and the weight of magnesium calcite was measured to be approximately 1.2g.

[0081] In addition, this embodiment also tested the effect of magnesium calcite solidifying soft soil. The solidification method was the same as in Example 5, except that the first cementing liquid was replaced with the second cementing liquid described in this embodiment. The compressive strength of the solidified soft soil was tested, and the results showed that the compressive strength was 2.83 MPa, indicating that the magnesium calcite generated by the mixed bacterial fermentation broth of this invention has a better effect on solidifying soft soil. This embodiment set up a cementing liquid without magnesium ions as a control group, that is, the second cementing liquid did not contain magnesium chloride, and other components remained unchanged. The solidification test of the soft soil was conducted using this cementing liquid without magnesium ions, and the results showed that the compressive strength of the solidified soft soil was 2.48 MPa.

[0082] This invention provides a concept and method for the application of an alkali-resistant biomineralizing bacteria combination. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A strain of Bacillus pseudofirmus, which is named as Bacillus pseudofirmus, has a strain number of DCG02, a preservation number of CCTCC NO: M 20222037, and a preservation time of December 23, 2022.

2. A combination of alkali-resistant biomineralizing bacteria, characterized in that, The alkali-resistant bio-mineralization bacteria combination comprises Bacillus pasteurii and the Bacillus pseudofirmus of claim 1.

3. The alkaline-resistant biomineralization bacteria combination according to claim 2, characterized in that, The Bacillus pasteurii comprises Bacillus pasteurii DC419, which has a preservation number of CCTCC NO: M 20222038. 4.The alkali-resistant bio-mineralization bacteria combination of claim 2 or 3 is used for solidifying soft soil and / or preparing magnesium calcite.

5. Use according to claim 4, characterized in that, The alkali-resistant bio-mineralization bacteria combination is in the form of a mixed bacteria fermentation broth and is used for solidifying soft soil and / or preparing magnesium calcite.

6. Use according to claim 5, characterized in that, The mixed bacteria fermentation broth is prepared according to the following steps: (1) inoculating Bacillus pasteurii seed liquid into a fermentation medium, and performing fermentation culture at 28-35℃ for 16-24h to obtain a Bacillus pasteurii fermentation broth; wherein the fermentation medium comprises 1-10g / L urea; (2) inoculating Bacillus pseudofirmus seed liquid into the Bacillus pasteurii fermentation broth at an inoculation amount of 0.5%-2% v / v, and performing fermentation culture at 28-35℃ for 10-14h to obtain a mixed bacteria fermentation broth.

7. Use according to claim 5, characterized in that, The solidified soft soil is prepared according to the following method: mixing the mixed bacteria fermentation broth and a first cementation liquid to obtain a mixed liquid, uniformly mixing the mixed liquid with soft soil, and standing to realize solidification of the soft soil.

8. Use according to claim 7, characterized in that, The first cementation liquid comprises 0.3-1.2mol / L urea and 0.3-1.2mol / L calcium ions; and the pH of the first cementation liquid is 8-12. The mixed volume ratio of the first cementation liquid and the mixed bacteria fermentation broth is 1-2:1-2. The addition amount of the mixed liquid is 15%-20% of the mass of the soft soil. The standing is standing at room temperature for 5-7 days.

9. Use according to claim 5, characterized in that, The magnesium calcite is prepared according to the following method: mixing the mixed bacteria fermentation broth and a second cementation liquid, and reacting to obtain magnesium calcite.

10. Use according to claim 9, characterized in that, The second cementation liquid comprises 0.3-1.2mol / L urea, 0.3-1.2mol / L calcium ions, and 0.3-1.2mol / L magnesium ions; and the pH of the second cementation liquid is 8-12. The mixed volume ratio of the second cementation liquid and the mixed bacteria fermentation broth is 8-12:

1. The reaction is performed at a reaction temperature of 28-32℃ for a reaction time of 36-72h. ​

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