Recombinant escherichia coli positioning carbonic anhydrase in periplasmic space and method for preparing microbial cement thereof

Abstract

The application discloses a recombinant escherichia coli for positioning carbonic anhydrase in periplasm space and a method for preparing microbial cement by using the same, and the carbonic anhydrase is expressed by being fused with a periplasm signal peptide, so that the enzyme is enriched in the periplasm space of the escherichia coli, and the carbonic anhydrase catalyzes the hydration of carbon dioxide to generate carbonate, and the calcium ion is induced to deposit calcium carbonate to cement sand particles to prepare microbial cement. Three groups of preferred combinations are obtained through screening, and the induction temperature, the IPTG concentration and the ratio of the bacterial liquid to the cementing liquid are optimized, so that efficient expression and solidification are realized. The gelatin enhancer is added in the cementing system to enhance the unconfined compressive strength of the microbial cement brick. The application adopts a non-urease path, has no ammonia nitrogen pollution, does not need an external source of pure enzyme, has the advantages of green environmental protection, low cost and great engineering application potential, and is suitable for sand solidification and preparation of low-carbon cementing materials.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology, microbial mineralization and biogeological engineering technology, and specifically relates to a recombinant Escherichia coli that expresses carbonic anhydrase through periplasmic spatial localization and a method for preparing microbial cement using this engineered bacterium. Background Technology

[0002] Against the backdrop of global climate change, countries have successively proposed "dual-carbon" development goals to promote the low-carbon transformation and sustainable development of their economies and societies. Traditional cement-based grouting reinforcement and soil consolidation technologies are core materials for infrastructure construction; however, cement production involves large amounts of carbon dioxide emissions, making it a significant source of carbon emissions in the industrial sector. Therefore, developing low-carbon, green, and sustainable engineering alternative materials has become a critical issue that urgently needs to be addressed in the fields of building materials and geotechnical engineering.

[0003] In recent years, the interdisciplinary integration of microbiology, geochemistry, and civil engineering has driven the rapid development of microbially induced carbonate precipitation (MICP) technology. MICP is a widely existing biomineralization process in nature. Microorganisms produce carbonate ions through metabolism, inducing calcium ions to form calcium carbonate crystals outside the cells, thus cementing loose soil and rock particles into a solidified body with mechanical strength, namely "microbial cement." Compared with traditional cement, microbial cement can fix carbon dioxide, reduce the carbon footprint of engineering projects, and align with the development trend of low-carbon building materials.

[0004] Currently, the mainstream MICP technology primarily utilizes the Bacillus pasteurization urease system, achieving mineralization through urease-catalyzed urea hydrolysis. However, this pathway has significant drawbacks: the ammonia produced during hydrolysis significantly raises the system's pH, easily causing ammonia nitrogen pollution; the reaction is accompanied by an irritating odor and additional carbon dioxide emissions, contradicting green environmental protection goals. In contrast, carbonic anhydrase catalyzes the hydration of carbon dioxide to produce bicarbonate / carbonate ions, introducing no nitrogenous pollutants and thus exhibiting greater environmental friendliness; furthermore, carbonic anhydrase has high catalytic efficiency, demonstrating promising application potential in fields such as biomineralization, low-carbon building materials, and carbon dioxide capture.

[0005] Existing research has confirmed that carbonic anhydrase can effectively mediate the deposition of calcium carbonate in microbial cement (MICP), but key bottlenecks remain: the enzyme preparations are expensive, and complex pretreatment processes such as cell disruption and protein purification are required, hindering their engineering and widespread application. To address this, some researchers have proposed a whole-cell catalytic strategy that targets heterologous carbonic anhydrase within the periplasmic space of *E. coli*, achieving calcium carbonate precipitation without enzyme purification. The periplasmic space can both enrich carbonic anhydrase and provide a suitable nucleation microenvironment, significantly improving mineralization efficiency and offering a new approach for low-cost microbial cement preparation.

[0006] However, current research on periplasmic expression systems is mostly at the proof-of-concept stage, lacking systematic screening of signal peptide-carbonic anhydrase combinations, overall optimization of induction expression conditions and curing formulations. This makes it difficult to support the controllable preparation of high-strength, highly homogeneous microbial cement, and its engineering application potential has not been fully explored. Therefore, there is an urgent need to develop a microbial cement preparation technology that enables efficient screening, precise control, and stable mass production of periplasmic spatially localized expression of carbonic anhydrase. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing microbial cement that is free from ammonia nitrogen pollution, requires no exogenous pure enzymes, has uniform mineralization, and high strength. This method involves constructing recombinant Escherichia coli that expresses carbonic anhydrase in the periplasmic space with high activity, and optimizing the induction and solidification system to achieve low-cost, green, and high-strength microbial cement preparation.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one embodiment, the present invention provides a recombinant Escherichia coli expressing carbonic anhydrase via periplasmic spatial localization, wherein the recombinant Escherichia coli expresses a protein fused from a periplasmic spatial localization signal peptide and carbonic anhydrase, wherein the signal peptide is selected from TorA and PhoA, and the carbonic anhydrase is selected from ngCA, hmCA, and tdCA.

[0010] In another embodiment, the signal peptide and carbonic anhydrase of the present invention are any combination of the following: TorA-ngCA, TorA-hmCA, and PhoA-tdCA.

[0011] In another embodiment, the recombinant Escherichia coli of the present invention is an engineered strain JXb6 carrying the TorA-ngCA plasmid, an engineered strain JXb12 carrying the TorA-hmCA plasmid, or an engineered strain JXb17 carrying the PhoA-tdCA plasmid.

[0012] In one embodiment, the present invention provides a method for preparing microbial cement using the recombinant Escherichia coli, comprising: inducing and culturing the recombinant Escherichia coli to obtain a bacterial solution; mixing the bacterial solution with a cementing liquid and then injecting it into a sand matrix; and solidifying it in a CO2-containing environment to form a calcium carbonate precipitate and cement the sand particles to obtain a solidified microbial cement body.

[0013] In another embodiment, the induction conditions of the present invention are as follows: JXb6 uses 16-18℃ and 0.5-1 mM IPTG; JXb12 uses 18-20℃ and 0.25-0.5 mM IPTG; and JXb17 uses 16-18℃ and 0.5-1 mM IPTG.

[0014] In another embodiment, the cementing solution of the present invention comprises a CaCl2 and Tris-HCl buffer solution with a pH of 7.5.

[0015] In another embodiment, the volume ratio of the bacterial solution to the cementing solution of the present invention is 0.5-3:1, preferably 1:1.

[0016] In another embodiment, the curing environment of the present invention is room temperature, relative humidity of 70-80%, and CO2 volume fraction of 5-8%.

[0017] In another embodiment, gelatin is added as a reinforcing agent to the bonding system of the present invention, and the amount of gelatin added is 5%-15% of the total mass of the bonding system.

[0018] In one embodiment, the present invention provides a microbial cement prepared by the method, wherein the microbial cement is a sand column or cement brick with an unconfined compressive strength ≥10 MPa.

[0019] Compared with the prior art, the present invention has the following significant advantages:

[0020] (1) Green and environmentally friendly, with no secondary pollution: The present invention adopts a non-urease mineralization pathway mediated by carbonic anhydrase. The catalytic process does not produce ammonia nitrogen or release irritating gases, thus avoiding the environmental pollution problems of the traditional urease system from the source, which meets the requirements of low-carbon and environmentally friendly engineering materials development.

[0021] (2) No need for pure enzymes, significantly reducing costs: The whole-cell catalytic system with periplasmic spatial localization expression eliminates the need for cell disruption, protein separation and enzyme purification steps, simplifying the production process, greatly reducing the cost of enzyme preparations, and making it more suitable for engineering promotion.

[0022] (3) Slow-release mineralization, uniform and dense cementation: The spatial positioning of the permamaterial makes the carbonic anhydrase exhibit a mild and continuous catalytic release characteristic, which can reduce the risk of pore blockage caused by local rapid supersaturation precipitation and improve the uniformity of calcium carbonate distribution and the density of cementation between sand grains.

[0023] (4) Higher enzyme activity and better curing performance: By optimizing the combination of signal peptide and carbonic anhydrase, the enzyme activity of the whole cell is significantly improved; compared with the intracellular expression system, periplasmic localization can significantly improve the calcium carbonate deposition efficiency and the mechanical strength of microbial cement.

[0024] (5) Controllable strength to meet engineering applications: By optimizing the induction conditions, solidification ratio and introducing gelatin reinforcement, the unconfined compressive strength of microbial cement can reach more than 10 MPa, which meets the requirements of engineering scenarios such as sand solidification, low-carbon building materials and soil and rock reinforcement.

[0025] (6) The system is stable and has wider applicability: the engineered bacteria maintain high enzyme activity stability during the solidification period, and the reaction conditions are mild and controllable. It is suitable for various application forms such as sand columns, cement bricks, and soil grouting, which expands the engineering application scope of microbial mineralization technology. Attached Figure Description

[0026] Figure 1 : This is a schematic diagram of three macroscopic morphologies of microbial cement sand columns prepared by Escherichia coli periplasmic spatial localization carbonic anhydrase according to the present invention, wherein (a)-(c) are the three macroscopic morphologies respectively.

[0027] Figure 2 The diagram shows the functional design of the engineered periplasmic whole-cell catalytic system, the schematic diagram of the sand solidification test device, and a comparison chart of the transport efficiency and catalytic activity of different periplasmic spatially localized signal peptides for carbonic anhydrase. Specifically, (a)-(b) are schematic diagrams of the functional design of the engineered periplasmic whole-cell catalytic system and the sand solidification test device; (c)-(h) are bar charts comparing the transport efficiency and catalytic activity of different periplasmic spatially localized signal peptides for carbonic anhydrase.

[0028] Figure 3 The graph shows a comparison of carbonic anhydrase activity on days 1, 2, 3, and 7 between intracellular and periplasmic carbonic anhydrase expression strains, demonstrating the effect of the periplasmic localization strategy on improving enzyme activity stability and catalytic efficiency.

[0029] Figure 4 Figure 1 shows a comparison of the unconfined compressive strength and enzyme activity of the strains of the present invention. Specifically, (a)-(b) are comparisons of the unconfined compressive strength and enzyme activity of the preferred strains at different IPTG concentrations; (c)-(d) are comparisons of the unconfined compressive strength and enzyme activity of the preferred strains at different induction temperatures.

[0030] Figure 5 : Comparison of unconfined compressive strength of microbial cement sand columns prepared by three selected engineered bacteria under bacterial-cementing liquid volume ratios of 3:1, 1:1, 1:3 and 0:4.

[0031] Figure 6 : Comparison of unconfined compressive strength of microbial cement sand columns prepared by periplasmic localization strains carrying PhoA-tdCA and intracellular crude enzyme solution system under 2-day and 4-day curing cycles.

[0032] Figure 7 : This is a diagram illustrating the microbial cement brick of the present invention. Among them, (a) is a comparison diagram of the unconfined compressive strength of the microbial cement bricks of the base group and the gelatin-reinforced group; (b) is a macroscopic morphological diagram of the gelatin-reinforced microbial cement brick. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the experimental conditions are conventional conditions in the art.

[0034] This invention employs three carbonic anhydrase genes: ngCA from *Neisseriagonorrhoeae* (Genbank: GCA_013030075.1), hmCA from *Hydrogenovibriomarinus* (Genbank: GCA_013340845.1), and tdCA from *Thermosulfurimonas dismutans* (Genbank: GCA_001652585.1). The genes were synthesized commercially and cloned into the pET-28a expression vector. Different signal peptide sequences were fused to the N-terminus, and a 6×His tag was fused to the C-terminus for easy detection. The recombinant plasmid was first transformed into *E. coli* DH5α for amplification, and then transformed into the expression host *E. coli* BL21 (DE3) for induced expression.

[0035] The engineered bacteria in this example were cultured in LB broth, composed of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. Single colonies were inoculated into 100 mL LB broth containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD500 was reached. 600 ≈0.6, add IPTG and ZnCl2 for induction, with preferred final concentrations of 1 mM and 0.1 mM, respectively; after induction for 24 h, adjust the bacterial culture to OD. 600 ≈3.0 Reserved. Take 30 mL of bacterial culture from the intracellular expression group and the empty vector control group, sonicate on ice, centrifuge, and collect the supernatant as crude enzyme solution.

[0036] Preparation of microbial cement sand columns: 50 g of river sand was used to prepare sand columns. On day 1, 10 mL of cementing solution and 10 mL of bacterial / crude enzyme solution were injected into the sand matrix; the cementing solution contained 100 mM CaCl2, 1 M Tris-HCl buffer, and pH 7.5. The columns were then placed in an incubator at 37℃, 80% relative humidity, and 5% CO2 for curing. On day 3, an equal volume of cementing solution and bacterial / crude enzyme solution was added again; the columns were allowed to stand for another 2 days, then dried in a 60℃ oven for 72 h before mechanical testing. Unconfined compressive strength was determined according to ASTM D1633 (2017), with loading at a rate of 1.5 mm / min until failure. The maximum bearing capacity was recorded and the strength was calculated.

[0037] Example 1: Construction of engineered bacteria and screening of optimal signal peptide-carbonic anhydrase combinations

[0038] To screen for the optimal signal peptide-CA combination that combines high catalytic activity and microbial cement solidification performance, three carbonic anhydrases, ngCA, hmCA, and tdCA, were fused with periplasmic spatial localization signal peptides DsbA, PelB, PhoA, and TorA, as well as an exocrine signal peptide OmpA, to construct a total of 12 periplasmic spatial localization-expressing CA engineered strains, 3 secretory-expressing CA engineered strains, and 3 intracellular CA-expressing control strains.

[0039] like Figure 1 As shown ((a)-(c) represent three macroscopic forms), the recombinantly expressed carbonic anhydrase can efficiently catalyze the hydration of CO2 to HCO3 in the periplasmic space of E. coli. - HCO3 - Transported to the extracellular space via outer membrane porins or transport proteins, where they interact with Ca in the gelling fluid. 2+ The combined components form CaCO3 precipitate; at the same time, engineered whole cells of Escherichia coli can act as a mineral nucleation carrier, promoting the uniform attachment of the generated calcium carbonate to the surface of sand particles and achieving effective bonding between sand particles, thereby forming a microbial cement solidified body with a certain mechanical strength.

[0040] The above strains were cultured in LB liquid medium until OD200. 600 ≈0.6. After inducing culture with IPTG for 24 h, the whole-cell CA enzyme activity of each strain was measured. Microbial cement sand columns were prepared using the same process, and the unconfined compressive strength was tested. The catalytic performance and solidification effect of different signal peptide-CA combinations were systematically evaluated.

[0041] Figure 2 Images (a)-(b) show schematic diagrams of the functional design of the engineered periplasmic whole-cell catalytic system and the experimental setup for sand particle solidification. The results show that, as Figure 2As shown in Figure (ch), different periplasmic spatial localization signal peptides significantly affect the transport efficiency and whole-cell catalytic activity of carbonic anhydrase. Specifically, the TorA signal peptide increased the whole-cell enzyme activity of recombinant strains overexpressing ngCA and hmCA by approximately 116% and 49%, respectively; the PhoA signal peptide increased the whole-cell enzyme activity of recombinant strains overexpressing tdCA by approximately 20%. Mechanical property testing of microbial cement sand columns showed that the whole-cell enzyme activity level of engineered bacteria was generally positively correlated with the unconfined compressive strength.

[0042] Based on the comprehensive evaluation results of whole-cell enzyme activity and unconfined compressive strength, three optimal fusion configurations, TorA-ngCA, TorA-hmCA, and PhoA-tdCA, were selected and obtained, corresponding to the engineered strain numbers JXb6, JXb12, and JXb17, respectively, for further research in subsequent embodiments.

[0043] Example 2: Evaluation of enzyme activity stability of carbonic anhydrase expressed by periplasmic spatial localization

[0044] To systematically evaluate the enzymatic stability of periplasmic spatially expressed CA under microbial cement preparation conditions, three control strains expressing CA intracellularly, JXb1, JXb7, and JXb13, and three engineered strains expressing CA periplasmic spatially, JXb6, JXb12, and JXb17, were selected as test subjects.

[0045] Under the culture conditions for microbial cement preparation, the CA enzyme activity levels of each strain were measured on days 1, 2, 3, and 7 to investigate the change in enzyme activity with curing time.

[0046] As shown in Figure 3, all tested strains maintained high CA activity throughout the 7-day testing period, stably catalyzing the CO2 hydration reaction and continuously providing the driving force for calcium carbonate precipitation, supporting the gradual formation and improvement of the compressive strength of the microbial cement sand column. Compared with the intracellular expression system, the periplasmic spatial localization strategy did not reduce enzyme stability but significantly enhanced the epigenetic catalytic activity of the whole cell. Among them, the engineered strain JXb17 carrying PhoA-tdCA showed approximately 32% higher CA activity than the intracellular expression control strain, indicating that periplasmic spatial localization expression can maintain long-term enzyme activity stability while enhancing the sustained catalytic ability of the mineralization reaction.

[0047] Example 3: System Optimization of Carbonic Anhydrase Expression Induction Conditions

[0048] Using IPTG induction concentration and induction temperature as key regulatory factors, the expression conditions of the preferred strains JXb6, JXb12, and JXb17 selected in Example 1 were optimized to obtain the highest whole-cell CA activity and the best microbial cement curing performance.

[0049] 1. IPTG Concentration Optimization

[0050] At an induction temperature of 25℃, four IPTG concentration gradients (0 mM, 0.25 mM, 0.5 mM, and 1 mM) were set up to investigate their effects on whole-cell CA activity and sand column compressive strength. The results are as follows: Figure 4 As shown in (ab):

[0051] JXb6 (TorA-ngCA) exhibited a whole-cell CA activity of 376.58 U / mL under 1 mM IPTG conditions, corresponding to an unconfined compressive strength of 2411.42 kPa for the microbial cement sand column.

[0052] JXb12 (TorA-hmCA) exhibited a whole-cell CA activity of 539.26 U / mL under 0.25 mM IPTG conditions, corresponding to a compressive strength of 2265.63 kPa.

[0053] JXb17 (PhoA-tdCA) exhibited a whole-cell CA activity of 665.81 U / mL under 1 mM IPTG conditions, corresponding to a compressive strength of 2627.03 kPa.

[0054] 2. Optimization of induction temperature

[0055] Expression was induced at three temperature gradients (16℃, 20℃, and 25℃) for each strain at its optimal IPTG concentration. Results are as follows: Figure 4 Shown in (cd):

[0056] The optimal induction conditions for JXb6 were 16℃ and 1 mM IPTG, with whole-cell CA activity of 519.88 U / mL and compressive strength of 2267.99 kPa.

[0057] The optimal induction conditions for JXb12 were 20℃ and 0.25 mM IPTG, with whole-cell CA activity of 538.23 U / mL and compressive strength of 2059.45 kPa.

[0058] The optimal induction conditions for JXb17 were 16℃ and 1 mM IPTG, with whole-cell CA activity of 585.40 U / mL and compressive strength of 2761.97 kPa.

[0059] Under the same induction conditions, the microbial cement solidification performance of the periplasmic spatial localization CA whole-cell catalytic system was significantly better than that of the crude enzyme solution system expressing CA intracellularly. This indicates that intact Escherichia coli cells can provide stable and continuous catalytic activity and also serve as a calcium carbonate nucleation carrier, promoting the formation of a more uniform and dense cemented structure between sand grains.

[0060] Example 4: Optimization of Microbial Cement Curing Conditions

[0061] Using the selected engineered bacterial strains JXb6, JXb12, and JXb17 as research subjects, the effect of the volume ratio of bacterial solution to cementitious liquid on the solidification effect of microbial cement was investigated. Three experimental groups with volume ratios of 3:1, 1:1, and 1:3 were set up, and a blank control was set up with only 20 mL of pure cementitious liquid added.

[0062] The results are as follows Figure 5 As shown, when the bacterial solution and cementing solution are mixed at a 1:1 volume ratio, all three engineered bacteria can prepare structurally intact and densely cemented microbial cement sand columns, with significantly better solidification effects than the 3:1 and 1:3 ratios. This ratio is beneficial for the interaction of carbonic anhydrase and Ca in the cementing solution. 2+ The buffer components are in full contact to ensure efficient and continuous calcium carbonate precipitation. The blank control group relies solely on physical consolidation, which is prone to deliquescence and structural disintegration during curing, and cannot form a stable biomineralization cementation system.

[0063] At the optimal 1:1 ratio, the microbial cement sand column prepared by the engineered strain JXb17 carrying the PhoA-tdCA plasmid exhibited the highest unconfined compressive strength, demonstrating the best mineralization efficiency and cementing performance.

[0064] Example 5: The Influence of Peripheral Space Slow-Release Effect on Curing Performance

[0065] Using the periplasmic engineered bacterium JXb17 carrying PhoA-tdCA as the experimental group and the crude enzyme solution system JXb13 expressing tdCA intracellularly as the control group, microbial cement sand columns were prepared under two solidification cycles of 2 days and 4 days, respectively, to verify the slow-release mineralization characteristics of the periplasmic spatial positioning system.

[0066] The results are as follows Figure 6As shown, both systems can achieve a certain degree of sand particle cementation within a short curing period. With prolonged curing time, the compressive strength of the periplasmic engineered bacteria system significantly increased compared to the crude enzyme solution control group. The periplasmic spatial localization expression system can release catalytic activity gently and continuously, causing carbonate to be slowly generated and uniformly deposited, effectively reducing the risk of local supersaturation and pore blockage, and improving the uniformity of calcium carbonate distribution and the density of cementation. In contrast, the intracellular crude enzyme solution system exhibits an excessively rapid early catalytic rate, easily leading to localized precipitation concentration, which is detrimental to long-term mineralization and the establishment of overall structural stability.

[0067] Example 6: Preparation and Mechanical Property Testing of Gelatin-Reinforced Microbial Cement Bricks

[0068] Based on the microbial cement sand column preparation process, using the optimal strain JXb17 as the biocatalytic component, 10% by mass of gelatin was introduced into the cementing system as a reinforcing phase to prepare reinforced microbial cement bricks, and their unconfined compressive strength was tested.

[0069] The results are as follows Figure 7 As shown in Figure (ab), the unconfined compressive strength of the basic microbial cement brick without gelatin is 3660 kPa; after adding gelatin for reinforcement, the compressive strength is significantly increased to 10910 kPa, which is approximately 10.1 MPa, meeting the strength requirements of conventional non-load-bearing structural materials in buildings.

[0070] The gelatin-reinforcing phase and the periplasmic spatial localization carbonic anhydrase-mediated MICP process exhibit a strong synergistic effect: gelatin forms a continuous, flexible reinforcing network, enhancing the material's toughness and structural integrity; the periplasmic localization system continuously generates an inorganic cementing phase dominated by calcite, achieving efficient cementation of sand particles. This synergistic effect significantly improves the mechanical properties of the microbial cementitious body. This method can be used not only for the solidification of sand to prepare sand columns but can also be extended to the preparation of reinforced microbial cement bricks and other low-carbon bio-based cementing materials.

[0071] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for preparing microbial cement using recombinant Escherichia coli, characterized in that, The process includes: obtaining a bacterial solution by inducing and culturing recombinant Escherichia coli; mixing the bacterial solution with a cementing solution and then injecting it into a sand matrix; and solidifying it in a CO2-containing environment to form a calcium carbonate precipitate that cements the sand particles, thus obtaining a microbial cement solidified body. The recombinant Escherichia coli expresses a protein fused with a periplasmic spatial localization signal peptide and carbonic anhydrase, wherein the combination of the signal peptide and carbonic anhydrase is PhoA-tdCA. Induction conditions were: 16-18℃, 0.5-1mM IPTG; The cementing solution contains a CaCl2 and Tris-HCl buffer solution with a pH of 7.5; the volume ratio of bacterial solution to cementing solution is 0.5-3:

1.

2. The method according to claim 1, characterized in that, The volume ratio of bacterial solution to cementing solution is 1:

1.

3. The method according to claim 1, characterized in that, The curing environment is room temperature, relative humidity of 70-80%, and CO2 volume fraction of 5-8%.

4. The method according to claim 1, characterized in that, Gelatin is added to the binder as a reinforcing agent, and the amount of gelatin added is 5%-15% of the total mass of the binder.

5. The microbial cement prepared by the method of claim 4, characterized in that, The microbial cement is a sand column or cement brick with an unconfined compressive strength ≥10MPa.

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