Method for reinforcing high fill body by nanogel and microbial mineralization in cooperation

Abstract

The application belongs to the technical field of geotechnical engineering, and discloses a method for reinforcing high fill body by nanogel and microbial mineralization, which comprises the following steps: proliferative culture and activation of bacillus, preparation of responsive gel, mixing NaHCO3 buffer, activated bacillus, nanoclay-chitosan composite sol, responsive gel and cementing liquid into the soil in sequence, and layer compaction to realize dense reinforcement of the soil body. The application solves the technical problem of the existing chemical grouting method, i.e. poor controllability of mineral deposition space, easy plugging of pore throat, influence on drainage, and single migration to pollute the environment.

Description

Technical Field

[0001] This invention belongs to the field of geotechnical engineering technology and relates to a method for reinforcing high fill soil by nanogel synergistic microbial mineralization. Background Technology

[0002] During road reconstruction and expansion, the excavation and filling of the original roadbed inevitably damages the original soil structure. High fill, as an artificial accumulation, initially exhibits a disordered and loose structure, requiring mechanical compaction to increase its density and meet engineering bearing capacity requirements. Insufficient compaction will lead to a significant reduction in the soil's shear strength, subsequently inducing a series of safety issues such as localized collapses or differential settlement. The essence of this problem lies in the fact that after the soil structure is disturbed, the original mechanical equilibrium between particles is broken, and rebuilding a stable structure requires overcoming its inherent spatiotemporal variability.

[0003] In existing construction techniques, methods such as chemical grouting, layered compaction, and biological reinforcement are commonly used. Grouting involves injecting chemical grout under pressure to fill soil pores, thus reinforcing the soil. Layered compaction relies on mechanical energy to compact the soil layer by layer, increasing its density. Microbial-induced calcium carbonate precipitation technology is a novel biological reinforcement technique that utilizes the metabolic activities of specific microorganisms to reinforce soil. By spraying or injecting a mixed grout consisting of a microbial solution and a reaction solution into the soil to be reinforced, the urease released by the microorganisms hydrolyzes the urea in the reaction solution to generate ammonium carbonate. The ammonium carbonate then reacts with calcium chloride in the cementing solution to form calcium carbonate cementing material, thereby binding the originally loose soil particles together. This biomineralization process effectively improves soil strength, reduces soil deformation, and lowers soil permeability. Compared to traditional reinforcement methods, microbial-induced calcium carbonate precipitation technology has significant green and environmentally friendly characteristics. Its core lies in utilizing the widely existing biomineralization process in nature to generate the required reinforcing material in situ.

[0004] However, existing chemical grouting methods lack spatial control over the mineral deposition process, easily leading to pore blockage in the soil throat, weakening the foundation's drainage function, and causing environmental problems due to the migration of organic monomers. Conventional microbial reinforcement technologies are limited by the migration and loss of microorganisms in fine-grained soils and the inability to precisely control the reaction rate, resulting in uneven spatial distribution of cementation. These shortcomings restrict the universality of the technology in engineering scenarios. Summary of the Invention

[0005] This invention provides a method for reinforcing high-fill soil by nanogel synergistic microbial mineralization, which solves the technical problems of existing chemical grouting methods, such as poor controllability of mineral deposition space, easy blockage of pore throats, affecting drainage, and individual particle migration polluting the environment.

[0006] To address the aforementioned technical problems, the present invention employs a method for reinforcing high-fill soil using a nanogel-assisted microbial mineralization process, the specific steps of which are as follows:

[0007] S1. Cultivate and activate Bacillus;

[0008] S2. Preparation of a responsive gel; the method for preparing the responsive gel includes:

[0009] S2.1: Modify gelatin with hydrazide and graft polyvinylimide;

[0010] S2.2: Mix modified gelatin, modified polyethyleneimine, and polyethylene glycol dialdehyde in a mass ratio of 1:(1.2~2.5):(0.1~0.25), and add pre-loaded Ca. 2+ Nano-zeolite was stirred to obtain a hydrogel precursor solution;

[0011] S2.3: Preparation of polylactic acid microspheres encapsulated with calcium citrate; the method for preparing the polylactic acid microspheres encapsulated with calcium citrate includes:

[0012] S2.3.1: Using 5 wt% calcium citrate aqueous solution as the inner aqueous phase and 5 wt% polylactic acid-dichloromethane solution as the oil phase, mix them at a volume ratio of 1:5 and shear emulsify at 10000~25000 rpm for 2~5 minutes to form a W / O primary emulsion;

[0013] S2.3.2: Add the primary emulsion to an external aqueous phase containing 1-3 wt% polyvinyl alcohol, and homogenize at 3000-8000 rpm for 1-3 minutes to form a W / O / W double emulsion;

[0014] S2.3.3: The dichloromethane in the W / O / W double emulsion is volatilized, and the polylactic acid is solidified to obtain the polylactic acid microspheres encapsulated with calcium citrate;

[0015] S2.4: Crosslink polylactic acid microspheres encapsulated with calcium citrate with a hydrogel precursor solution to obtain a responsive gel;

[0016] S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve dense reinforcement of the high fill soil.

[0017] Further, S1.1: Prepare a microbial proliferation culture medium, wherein the microbial proliferation culture medium is composed of 8-12 g / L tryptone, 4-6 g / L yeast extract, 4-6 g / L ammonium chloride, and the remainder is distilled water;

[0018] S1.2: Thaw the frozen bacterial strain, inoculate it into the microbial proliferation medium, and culture it with shaking at 28-32℃ and 170-190rpm for 22-26h;

[0019] S1.3: The culture medium was subjected to an ice bath to terminate metabolism, and after standing, the OD was measured using a blank culture medium as a control. 600 Value, pending OD 600 When the value reaches 0.5-0.7, it is transferred to a urea-containing differential medium containing phenol red to verify urease activity;

[0020] S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 7 -10 9 The inoculum of CFU / g was resuspended in the activation solution and activated at room temperature for 1.5-2.5 h.

[0021] Furthermore, the specific steps for modifying gelatin with hydrazide in S2.1 are as follows:

[0022] S2.1.1: Dissolve gelatin in deionized water with a pH of 4.5–5.0 to prepare a solution;

[0023] S2.1.2: Place the solution in an ice bath at 4°C, and add the reactants sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent = 1:(1.2~2.0):(0.5~1.0):(5~20). React for 2~6 hours to obtain the reaction product.

[0024] S2.1.3: The reaction product was purified by dialysis and lyophilized to obtain hydrazide-modified gelatin.

[0025] Furthermore, the grafting modification of polyethyleneimine in S2.1 specifically involves:

[0026] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added in a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : (1.2~1.5) : (0.5~0.8). The mixture was activated in an ice bath. Subsequently, polyethyleneimine was added so that the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid was (0.8~1.2):1. The mixture was reacted at room temperature for 12~24 hours to obtain modified polyethyleneimine.

[0027] Furthermore, the specific steps for S3 are as follows:

[0028] S3.1: Mix 0.5~1.0wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1, and stir for 1.5~2.5 hours to form nano clay-chitosan composite sol;

[0029] S3.2: 0.8~1.2 mol·L -1 CaCl2 solution and 0.8~1.2 mol·L -1 The urea solution is mixed to obtain a cementitious solution;

[0030] S3.3: Based on the dry soil mass, add the following in sequence: 10%~20% of 0.5 M NaHCO3 buffer solution, 20%~30% of activated Bacillus bacterial solution, 1%~3% of nano-clay-chitosan composite sol, 2%~5% of responsive gel, and 10%~20% of cementing liquid, and mix evenly. Then, compact the mixture in layers to achieve soil compaction and reinforcement.

[0031] Furthermore, the activation solution in S1.4 consists of 20 mM CaCl2, 0.5 M urea and 5% trehalose.

[0032] Furthermore, in S2.2, the preload Ca 2+ The nano-zeolite has a particle size of 50-200 nm and a calcium loading of 50-80 mg / g.

[0033] The beneficial effects of this invention are as follows: This invention utilizes the synergistic effect of nano-clay and chitosan to form a composite sol system, effectively solving key technical challenges in traditional chemical grouting and microbial reinforcement. On one hand, nano-clay agglomerates fine soil particles into stable large aggregates through flocculation, optimizing pore distribution and significantly enhancing the overall strength and stability of the soil. On the other hand, chitosan, with its abundant functional groups, electrostatically coordinates with calcium ions, precisely guiding the directional nucleation and deposition of calcium carbonate at the contact points with soil particles, effectively inhibiting pore blockage caused by disordered mineral deposition and ensuring the drainage function of the foundation. Simultaneously, the viscous properties of chitosan enhance the physical retention capacity of microorganisms in fine-grained soil, improve the enrichment stability of microorganisms at the solid-liquid interface, and significantly reduce cell migration loss and adhesion failure.

[0034] Furthermore, by introducing benzimidazole-aldehyde dynamic covalent crosslinking points and polylactic acid-calcium citrate responsive gel, this invention achieves precise control over the release and nucleation process of calcium ions, avoiding uneven nucleation and differences in the spatial distribution of cementation caused by excessively rapid reactions, and ultimately obtaining a uniformly distributed and stable microbial mineralized cemented soil. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 These are the unconfined compressive strength test results of the soil at 7 days and 28 days in this invention;

[0037] Figure 2 It is the 28-day permeability coefficient of the soil in this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] This invention provides a method for reinforcing high-fill soil with nanogel synergistic microbial mineralization, comprising the following steps:

[0040] S1. Bacillus proliferation culture and activation;

[0041] The specific steps for S1 are as follows:

[0042] S1.1: Prepare a microbial proliferation culture medium, wherein the microbial proliferation culture medium is composed of 8-12 g / L tryptone, 4-6 g / L yeast extract, 4-6 g / L ammonium chloride, and the remainder is distilled water;

[0043] Specifically, in the specific embodiments of the present invention, tryptone is selected as the source of organic nitrogen and essential amino acids in the culture medium, yeast extract is selected to provide growth factors and trace elements required for microbial growth, and ammonium chloride is selected as the inorganic nitrogen source and to act as a pH buffer. All components are dissolved in distilled water, and after thorough stirring and dissolution, they are subjected to high-pressure sterilization at a temperature of 121℃±0.5℃, a pressure of 0.11±0.01 MPa, and a time of 30 min±5 min. After sterilization, the culture medium is placed in a sterile operating table to cool and prepare for use.

[0044] S1.2: Thaw the frozen bacterial strain in an ice bath at 4℃, inoculate it into a microbial proliferation medium, and culture it with shaking at 28-32℃ and 170-190rpm for 22-26h;

[0045] S1.3: Take 1.5 mL of culture medium and stop metabolism by incubating in an ice bath at 4℃. Let it stand for 30 s to eliminate the influence of air bubbles. After standing, use blank culture medium as a control to measure the OD of the culture medium. 600 Value, pending OD 600 When the value reaches 0.5-0.7, it is transferred to a urea identification medium containing 0.1% (w / v) phenol red to verify urease activity;

[0046] S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 7 -10 9 The inoculum of CFU / g was resuspended in an activation solution consisting of 20 mM CaCl2, 0.5 M urea and 5% trehalose, and activated at room temperature for 1.5-2.5 h.

[0047] S2. Preparation of responsive gel;

[0048] The specific steps for S2 are as follows:

[0049] S2.1: Modify gelatin with hydrazide and graft polyvinylimide;

[0050] The specific steps for modifying gelatin with hydrazide in S2.1 are as follows:

[0051] S2.1.1: Dissolve gelatin in deionized water with a pH of 4.5–5.0 to prepare a solution;

[0052] S2.1.2: Place the solution in an ice bath at 4°C, and add the reactants sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent = 1:(1.2~2.0):(0.5~1.0):(5~20). React for 2~6 hours to obtain the reaction product.

[0053] In a specific embodiment of the present invention, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) play a catalytic role.

[0054] S2.1.3: The reaction product is purified by dialysis and then lyophilized to obtain the hydrazide-modified gelatin.

[0055] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0056] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) were added in a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC): 1-hydroxybenzotriazole (HOBt) = 1: (1.2~1.5): (0.5~0.8). The mixture was activated in an ice bath at 4°C for 15~30 minutes. Subsequently, polyethyleneimine was added, bringing the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid to (0.8~1.2):1. The mixture was reacted at room temperature for 12~24 hours to obtain modified polyethyleneimine.

[0057] In a specific embodiment of the present invention, the modified polyethyleneimine is benzimidazole-modified polyethyleneimine.

[0058] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde are mixed in a mass ratio of 1:(1.2~2.5):(0.1~0.25) and reacted at 40℃ for 45~60 minutes. Then, pre-loaded Ca is added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 50-200 nm and a calcium loading of 50-80 mg / g. The amount added is 5% to 20% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40°C for 60 minutes to obtain a hydrogel precursor solution.

[0059] S2.3: Preparation of polylactic acid microspheres encapsulating calcium citrate;

[0060] The specific steps for S2.3 are as follows:

[0061] S2.3.1: Mix 5 wt% calcium citrate aqueous solution as the inner aqueous phase and 5 wt% polylactic acid-dichloromethane solution as the oil phase at a volume ratio of 1:5, and shear at 10000~25000 rpm for 2~5 minutes to form a W / O (water-in-oil emulsion) primary emulsion;

[0062] S2.3.2: Add the primary emulsion to the external aqueous phase of 1-3 wt% polyvinyl alcohol and homogenize at 3000-8000 rpm for 1-3 minutes to form a W / O / W (multiple emulsion) complex emulsion;

[0063] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0064] S2.4: The polylactic acid microspheres encapsulated with calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 4-6 hours to obtain a responsive gel.

[0065] S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve soil compaction and reinforcement.

[0066] The specific steps for S3 are as follows:

[0067] S3.1: Mix 0.5~1.0wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1, and stir for 1.5~2.5 hours to form nano clay-chitosan composite sol;

[0068] S3.2: 0.8~1.2 mol·L -1 CaCl2 solution and 0.8~1.2 mol·L -1 The urea solution is mixed to obtain a cementitious solution;

[0069] S3.3: Based on the dry soil mass, add the following in sequence: 10%~20% of 0.5 M NaHCO3 buffer solution, 20%~30% of activated Bacillus bacterial solution, 1%~3% of nano-clay-chitosan composite sol, 2%~5% of responsive gel, and 10%~20% of cementing liquid, and mix evenly. Then, compact the mixture in layers to achieve soil compaction and reinforcement.

[0070] In a specific embodiment of the present invention, the above proportion is based on the dry soil mass, and the percentage of each component is defined as the external admixture amount relative to the dry soil mass, rather than the component ratio within the mixed system.

[0071] The specific layered compaction operation is as follows: Pour one sample into the mold and use a scraper to initially level its top surface. Then, install the guide tube and use a compaction hammer to drop freely from a fixed height, performing 27 standard compactions on the first layer, ensuring the impact points are evenly distributed across the surface. After compacting the first layer, roughen its surface with a scraper to create a rough interface, preventing smooth, weak surfaces between layers. Next, insert the second sample and repeat the compaction and roughening process. The third layer is treated in the same way until all layers are compacted, achieving soil density and reinforcement.

[0072] Example 1 ( Figure 1 (referred to as e.1)

[0073] S1. Bacillus proliferation culture and activation;

[0074] The specific steps for S1 are as follows:

[0075] S1.1: Prepare a microbial proliferation culture medium, wherein the microbial proliferation culture medium is composed of 10 g / L tryptone, 5 g / L yeast extract, 5 g / L ammonium chloride, and the remainder is distilled water;

[0076] Specifically, in this embodiment, tryptone was selected as the source of organic nitrogen and essential amino acids in the culture medium, yeast extract was selected to provide growth factors and trace elements required for microbial growth, and ammonium chloride was selected as the inorganic nitrogen source and to act as a pH buffer. All components were dissolved in distilled water, and after thorough stirring and dissolution, they were subjected to high-pressure sterilization at a temperature of 121℃±0.5℃, a pressure of 0.11±0.01 MPa, and a time of 30 min±5 min. After sterilization, the culture medium was placed in a sterile operating table to cool and prepare for use.

[0077] S1.2: Thaw the frozen bacterial strain in an ice bath at 4°C, inoculate it into a microbial proliferation medium, and culture it with shaking at 30°C and 180 rpm for 24 h;

[0078] S1.3: Take 1.5 mL of culture medium and stop metabolism by incubating in an ice bath at 4℃. Let it stand for 30 s to eliminate the influence of air bubbles. After standing, use blank culture medium as a control to measure the OD of the culture medium. 600 Value, pending OD 600 When the value reaches 0.6, it is transferred to a urea identification medium containing 0.1% phenol red to verify urease activity;

[0079] S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 8 The inoculum of CFU / g was resuspended in an activation solution consisting of 20 mM CaCl2, 0.5 M urea and 5% trehalose and activated at room temperature for 2 h.

[0080] S2. Preparation of responsive gel;

[0081] The specific steps for S2 are as follows:

[0082] S2.1: Modify gelatin with hydrazide and graft polyvinylimide;

[0083] The specific steps for modifying gelatin with hydrazide in S2.1 are as follows:

[0084] S2.1.1: Dissolve gelatin in deionized water with a pH of 4.5 to prepare a solution;

[0085] S2.1.2: The solution was placed in an ice bath at 4°C, and the reactants were added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:1.2:0.5:10. The reaction was carried out for 6 hours to obtain the reaction product.

[0086] S2.1.3: The reaction product is purified by dialysis and then lyophilized to obtain the hydrazide-modified gelatin.

[0087] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0088] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole (HOBt) were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1:1.2:0.5. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0089] In this embodiment, the modified polyethyleneimine is benzimidazole-modified polyethyleneimine.

[0090] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.2:0.1 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 50 nm and a calcium loading of 50 mg / g. The amount added is 5% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0091] S2.3: Preparation of polylactic acid microspheres encapsulating calcium citrate;

[0092] The specific steps for S2.3 are as follows:

[0093] S2.3.1: A 5 wt% calcium citrate aqueous solution was used as the inner aqueous phase, and a 5 wt% polylactic acid-dichloromethane solution was used as the oil phase. They were mixed at a volume ratio of 1:5 and sheared at 10,000 rpm for 5 minutes to form a W / O (water-in-oil emulsion) primary emulsion.

[0094] S2.3.2: Add the primary emulsion to the external aqueous phase of 2 wt% polyvinyl alcohol and homogenize at 5000 rpm for 3 minutes to form a W / O / W (multiple emulsion) complex emulsion;

[0095] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0096] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 4 hours to obtain a responsive gel.

[0097] S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve soil compaction and reinforcement.

[0098] The specific steps for S3 are as follows:

[0099] S3.1: Mix 0.75wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1 and stir for 2.5 hours to form nano clay-chitosan composite sol;

[0100] S3.2: Add 1 mol·L -1 CaCl2 solution and 1 mol·L -1 The urea solution is mixed to obtain a cementitious solution;

[0101] S3.3: Based on the dry soil mass, add the following in sequence: 15% of 0.5 M NaHCO3 buffer solution, 25% of activated Bacillus bacterial solution, 3% of nano-clay-chitosan composite sol, 5% of responsive gel, and 15% of cementing liquid. Mix evenly and compact in layers to achieve soil compaction and reinforcement.

[0102] Example 2 ( Figure 1 (referred to as e.2)

[0103] S1. Bacillus proliferation culture and activation;

[0104] The specific steps for S1 are as follows:

[0105] S1.1: Prepare a microbial proliferation medium. The microbial proliferation medium consists of 8 g / L tryptone, 6 g / L yeast extract, 4 g / L ammonium chloride, and the remainder is distilled water. Dissolve each component in distilled water, stir thoroughly to dissolve, and then autoclave at a temperature of 121℃±0.5℃, a pressure of 0.11±0.01 MPa, and a time of 30 min±5 min. After sterilization, place the medium in a sterile operating table to cool for later use.

[0106] S1.2: Thaw the frozen bacterial strain in an ice bath at 4°C, inoculate it into a microbial proliferation medium, and culture it with shaking at 32°C and 170 rpm for 22 h;

[0107] S1.3: Take 1.5 mL of culture medium and stop metabolism by incubating in an ice bath at 4℃. Let it stand for 30 s to eliminate the influence of air bubbles. After standing, use blank culture medium as a control to measure the OD of the culture medium. 600 Value, pending OD 600When the value reaches 0.5, it is transferred to a urea identification medium containing 0.1% phenol red to verify urease activity;

[0108] S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 7 The inoculum of CFU / g was resuspended in an activation solution consisting of 20 mM CaCl2, 0.5 M urea and 5% trehalose, and activated at room temperature for 1.5 h.

[0109] S2. Preparation of responsive gel;

[0110] The specific steps for S2 are as follows:

[0111] S2.1: Modify gelatin with hydrazide and graft polyvinylimide;

[0112] The specific steps for modifying gelatin with hydrazide in S2.1 are as follows:

[0113] S2.1.1: Dissolve gelatin in deionized water with a pH of 5 to prepare a solution;

[0114] S2.1.2: The solution was placed in an ice bath at 4°C, and the reactants were added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:1.2:0.5:15. The reaction was carried out for 6 hours to obtain the reaction product.

[0115] S2.1.3: The reaction product is purified by dialysis and then lyophilized to obtain the hydrazide-modified gelatin.

[0116] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0117] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.2 : 0.7. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 0.8:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0118] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.2:0.175 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+The nano-zeolite has a particle size of 100 nm and a calcium loading of 65 mg / g. The amount added is 5% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0119] S2.3: Preparation of polylactic acid microspheres encapsulating calcium citrate;

[0120] The specific steps for S2.3 are as follows:

[0121] S2.3.1: A 5 wt% calcium citrate aqueous solution was used as the inner aqueous phase, and a 5 wt% polylactic acid-dichloromethane solution was used as the oil phase. They were mixed at a volume ratio of 1:5 and sheared at 25,000 rpm for 2 minutes to form a W / O (water-in-oil emulsion) primary emulsion.

[0122] S2.3.2: The primary emulsion is added to the external aqueous phase of 3 wt% polyvinyl alcohol and homogenized at 3000 rpm for 3 minutes to form a W / O / W (multiple emulsion) complex emulsion;

[0123] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0124] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 4 hours to obtain a responsive gel.

[0125] S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve soil compaction and reinforcement.

[0126] The specific steps for S3 are as follows:

[0127] S3.1: Mix 0.75wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1 and stir for 2.5 hours to form nano clay-chitosan composite sol;

[0128] S3.2: Add 0.8 mol·L -1 CaCl2 solution and 0.8 mol·L -1 The urea solution is mixed to obtain a cementitious solution;

[0129] S3.3: Based on the dry soil mass, add the following in sequence: 15% of 0.5 M NaHCO3 buffer solution, 25% of activated Bacillus bacterial solution, 3% of nano-clay-chitosan composite sol, 5% of responsive gel, and 15% of cementing liquid. Mix evenly and compact in layers to achieve soil compaction and reinforcement.

[0130] Example 3 ( Figure 1(referred to as e.3)

[0131] S1. Bacillus proliferation culture and activation;

[0132] The specific steps for S1 are as follows:

[0133] S1.1: Prepare a microbial proliferation medium. The microbial proliferation medium consists of 12 g / L tryptone, 4 g / L yeast extract, 6 g / L ammonium chloride, and the remainder is distilled water. Dissolve each component in distilled water, stir thoroughly to dissolve, and then autoclave at a temperature of 121℃±0.5℃, a pressure of 0.11±0.01 MPa, and a time of 30 min±5 min. After sterilization, place the medium in a sterile operating table to cool for later use.

[0134] S1.2: Thaw the frozen bacterial strain in an ice bath at 4℃, inoculate it into a microbial proliferation medium, and culture it with shaking at 28℃ and 190 rpm for 26 h;

[0135] S1.3: Take 1.5 mL of culture medium and stop metabolism by incubating in an ice bath at 4℃. Let it stand for 30 s to eliminate the influence of air bubbles. After standing, use blank culture medium as a control to measure the OD of the culture medium. 600 Value, pending OD 600 When the value reaches 0.7, it is transferred to a urea identification medium containing 0.1% phenol red to verify urease activity;

[0136] S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 9 The inoculum of CFU / g was resuspended in an activation solution consisting of 20 mM CaCl2, 0.5 M urea and 5% trehalose and activated at room temperature for 2.5 h.

[0137] S2. Preparation of responsive gel;

[0138] The specific steps for S2 are as follows:

[0139] S2.1: Modify gelatin with hydrazide and graft polyvinylimide;

[0140] The specific steps for modifying gelatin with hydrazide in S2.1 are as follows:

[0141] S2.1.1: Dissolve gelatin in deionized water with a pH of 4.8 to prepare a solution;

[0142] S2.1.2: The solution was placed in an ice bath at 4°C, and the reactants were added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:1.2:0.5:20. The reaction was carried out for 6 hours to obtain the reaction product.

[0143] S2.1.3: The reaction product is purified by dialysis and then lyophilized to obtain the hydrazide-modified gelatin.

[0144] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0145] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.2 : 0.8. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1.2:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0146] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.2:0.25 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 200 nm and a calcium loading of 80 mg / g. The amount added is 5% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40°C for 60 minutes to obtain a hydrogel precursor solution.

[0147] S2.3: Preparation of polylactic acid microspheres encapsulating calcium citrate;

[0148] The specific steps for S2.3 are as follows:

[0149] S2.3.1: A 5 wt% calcium citrate aqueous solution was used as the inner aqueous phase, and a 5 wt% polylactic acid-dichloromethane solution was used as the oil phase. They were mixed at a volume ratio of 1:5 and sheared at 15000 rpm for 3 minutes to form a W / O (water-in-oil emulsion) primary emulsion.

[0150] S2.3.2: The primary emulsion is added to the external aqueous phase of 1 wt% polyvinyl alcohol and homogenized at 8000 rpm for 1 minute to form a W / O / W (multiple emulsion) complex emulsion;

[0151] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0152] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 4 hours to obtain a responsive gel.

[0153] S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve soil compaction and reinforcement.

[0154] The specific steps for S3 are as follows:

[0155] S3.1: Mix 0.75wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1 and stir for 2.5 hours to form nano clay-chitosan composite sol;

[0156] S3.2: Add 1.2 mol·L -1 CaCl2 solution and 1.2 mol·L -1 The urea solution is mixed to obtain a cementitious solution;

[0157] S3.3: Based on the dry soil mass, add the following in sequence: 15% of 0.5 M NaHCO3 buffer solution, 25% of activated Bacillus bacterial solution, 3% of nano-clay-chitosan composite sol, 5% of responsive gel, and 15% of cementing liquid. Mix evenly and compact in layers to achieve soil compaction and reinforcement.

[0158] Example 4 ( Figure 1 (referred to as e.4)

[0159] The difference from Example 1 is as follows: S2.1.2: The solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:1.5:0.75:5. The reaction is carried out for 6 hours to obtain the reaction product.

[0160] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0161] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1:1.4:0.5. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to achieve a molar ratio of primary amino groups to 2-chlorobenzimidazole-5-carboxylic acid of 1:1. The mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0162] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.85:0.1 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca2+ The nano-zeolite has a particle size of 175 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0163] S2.4: The polylactic acid microspheres encapsulating calcium citrate were co-crosslinked with the hydrogel precursor solution at 40°C for 5 hours to obtain the responsive gel.

[0164] Everything else is the same as in Example 1.

[0165] Example 5 ( Figure 1 (referred to as e.5)

[0166] The difference from Example 1 is as follows: S2.1.2: The solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent 1:1.5:0.75:15. The reaction is carried out for 4 hours to obtain the reaction product.

[0167] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0168] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.4 : 0.7. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0169] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.85:0.175 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 175 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0170] S2.4: The polylactic acid microspheres encapsulating calcium citrate were co-crosslinked with the hydrogel precursor solution at 40°C for 5 hours to obtain the responsive gel.

[0171] Everything else is the same as in Example 1.

[0172] Example 6 ( Figure 1 (referred to as e.6)

[0173] The difference from Example 1 is as follows: S2.1.2: The solution was placed in an ice bath at 4°C, and the reactants were added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:1.5:0.75:20. The reaction was carried out for 4 hours to obtain the reaction product.

[0174] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0175] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.4 : 0.8. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0176] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:1.85:0.25 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 50 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0177] S2.4: The polylactic acid microspheres encapsulating calcium citrate were co-crosslinked with the hydrogel precursor solution at 40°C for 5 hours to obtain the responsive gel.

[0178] Everything else is the same as in Example 1.

[0179] Example 7 ( Figure 1 (referred to as e.7)

[0180] The difference from Example 1 is as follows: S2.1.2: The solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: hydrazide reagent 1:2:1:10. The reaction is carried out for 4 hours to obtain the reaction product.

[0181] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0182] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1:1.5:0.5. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to achieve a molar ratio of primary amino groups to 2-chlorobenzimidazole-5-carboxylic acid of 1:1. The mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0183] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:2.5:0.1 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 125 nm and a calcium loading of 80 mg / g. The amount added is 20% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0184] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 6 hours to obtain a responsive gel.

[0185] Everything else is the same as in Example 1.

[0186] Example 8 ( Figure 1 (referred to as e.8)

[0187] The difference from Example 1 is as follows: S2.1.2: The solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent 1:2:1:15. The reaction is carried out for 2 hours to obtain the reaction product.

[0188] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0189] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.5 : 0.7. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0190] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:2.5:0.175 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 135 nm and a calcium loading of 80 mg / g. The amount added is 20% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0191] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 6 hours to obtain a responsive gel.

[0192] Everything else is the same as in Example 1.

[0193] Example 9 ( Figure 1 (referred to as e.9)

[0194] The difference from Example 1 is as follows: S2.1.2: The solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecules: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent 1:2:1:20. The reaction is carried out for 2 hours to obtain the reaction product.

[0195] The grafting modification of polyethyleneimine in S2.1 specifically involves:

[0196] 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added at a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : 1.5 : 0.8. The mixture was activated in an ice bath at 4°C for 30 minutes. Subsequently, polyethyleneimine was added to make the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid 1:1, and the mixture was reacted at room temperature for 24 hours to obtain modified polyethyleneimine.

[0197] S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde were mixed in a mass ratio of 1:2.5:0.25 and reacted at 40°C for 60 minutes. Then, pre-loaded Ca was added. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 150 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0198] S2.4: The polylactic acid microspheres encapsulating calcium citrate are co-crosslinked with the hydrogel precursor solution at 40°C for 6 hours to obtain a responsive gel.

[0199] Everything else is the same as in Example 1.

[0200] Example 10 ( Figure 1 (referred to as e.10)

[0201] The difference from Example 5 is as follows: S3.1: 0.5wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution are mixed at a volume ratio of 3:1 and stirred for 2.5 hours to form nano clay-chitosan composite sol;

[0202] Everything else is the same as in Example 5.

[0203] Example 11 ( Figure 1 (referred to as e.11)

[0204] The difference from Example 10 is as follows: S3.1: 1.0 wt% chitosan acetate solution and 2.0 wt% nano clay aqueous solution are mixed at a volume ratio of 3:1 and stirred for 2.5 hours to form nano clay-chitosan composite sol;

[0205] Everything else is the same as in Example 10.

[0206] Comparative Example 1 ( Figure 1 (Note: p.1)

[0207] The difference from Example 1 is that steps S2 and S3.1 are omitted.

[0208] Everything else is the same as in Example 1.

[0209] Comparative Example 2 ( Figure 1 (Note: p.2)

[0210] The difference from Example 1 is as follows: S2.3.1: 5 wt% calcium citrate aqueous solution as the inner aqueous phase and 5 wt% polylactic acid-dichloromethane solution as the oil phase are mixed at a volume ratio of 1:5 and sheared at 15000 rpm for 5 minutes to form a W / O primary emulsion.

[0211] S2.3.2: Add the primary emulsion to the external aqueous phase of 0.5 wt% polyvinyl alcohol and homogenize at 1000 rpm for 3 minutes to form a W / O / W complex emulsion.

[0212] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0213] Everything else is the same as in Example 1.

[0214] Comparative Example 3 ( Figure 1 (Note: p.3)

[0215] The difference from Example 1 is as follows: S2.3.1: 5 wt% calcium citrate aqueous solution as the inner aqueous phase and 5 wt% polylactic acid-dichloromethane solution as the oil phase are mixed at a volume ratio of 1:5 and sheared at 15000 rpm for 5 minutes to form a W / O primary emulsion.

[0216] S2.3.2: Add the primary emulsion to the external aqueous phase of 5 wt% polyvinyl alcohol and homogenize at 10000 rpm for 3 minutes to form a W / O / W complex emulsion.

[0217] S2.3.3: The dichloromethane in the W / O / W double emulsion slowly evaporates, and the polylactic acid solidifies to obtain polylactic acid microspheres encapsulated with calcium citrate.

[0218] Comparative Example 4 ( Figure 1 (Note: p.4)

[0219] The difference from Example 1 is as follows: S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde are mixed in a mass ratio of 1:1.0:0.05 and reacted at 40°C for 60 minutes, followed by the addition of pre-loaded Ca. 2+ Nano-zeolite, pre-loaded with Ca 2+ The nano-zeolite has a particle size of 135 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40 °C for 60 minutes to obtain a hydrogel precursor solution.

[0220] Everything else is the same as in Example 1.

[0221] Comparative Example 5 ( Figure 1 (Note: p.5)

[0222] The difference from Example 1 is as follows: S2.2: Modified gelatin, modified polyethyleneimine, and 10 wt% polyethylene glycol dialdehyde are mixed in a mass ratio of 1:2.8:0.3 and reacted at 40°C for 60 minutes, followed by the addition of pre-loaded Ca. 2+The nano-zeolite, preloaded with Ca²⁺, has a particle size of 135 nm and a calcium loading of 80 mg / g. The amount added is 10% of the total mass of the modified gelatin and modified polyethyleneimine. The mixture is stirred at 40°C for 60 minutes to obtain a hydrogel precursor solution.

[0223] Everything else is the same as in Example 1.

[0224] Comparative Example 6 ( Figure 1 (Note: p.6)

[0225] The difference from Example 5 is as follows: S3.3: Based on the dry soil mass, add 15% of 0.5 M NaHCO3 buffer solution, 25% of activated Bacillus bacterial solution, 5% of response gel, and 15% of cementing liquid in sequence, according to the dry soil mass. After mixing evenly, compact the mixture in layers to achieve soil compaction and reinforcement.

[0226] Everything else is the same as in Example 5.

[0227] The testing method after soil compaction and reinforcement is as follows:

[0228] In this embodiment, the unconfined compressive strength test was conducted using a TST-055 geotechnical testing apparatus. The mold dimensions were 50 mm in diameter and 100 mm in height. After uniform mixing according to the design dosage, the mixture was molded and compacted (compaction degree of 95%). Curing was carried out at 25℃ and relative humidity >95% for 7 days and 28 days. The loading rate was 1 mm·min. -1 Record the peak load converted to compressive strength, and the experimental results are as follows: Figure 1 As shown.

[0229] The test method for soil impermeability is as follows:

[0230] In this example, the soil permeability test was conducted using a TST-70 permeameter. The sample size was 61.8 mm in diameter and 40 mm in height. After 28 days of curing, the soil sample was cut and leveled, then placed into the sample ring of the permeameter. After degassing and saturation, a specific water head was applied. The test was conducted under constant temperature (20±1℃). The permeability coefficient of the soil sample was calculated by measuring the seepage flow rate over a fixed time period or recording the water level change in the variable head tube, based on Darcy's law. The average value of three parallel tests was taken as the final result. The experimental results are as follows: Figure 2 As shown.

[0231] Figure 1The unconfined compressive strength of all Examples 1 to 11 (e.1 to e.11) was significantly higher than that of the untreated soil, indicating that the method of the present invention can effectively improve the mechanical properties of the soil. As the curing period increased from 7 days to 28 days, the strength of each example generally increased further, proving that the microbial-induced calcium carbonate precipitation reaction continued, the cementitious material accumulated continuously, and the strengthening effect increased over time. Among them, Example 5 (e.5) showed the best compressive strength, indicating that the gel network formation state was optimal under this ratio, which is conducive to the microbial mineralization process. However, when the proportion of modified polyethyleneimine or crosslinking agent is too high or too low, it may lead to an unsuitable degree of crosslinking, affecting the mechanical properties of the gel and its support for the microbial environment, thereby weakening the final cementitious strength. The large permeability coefficients of Examples 1 (e.1), 2 (e.2), and 3 (e.3) can be attributed to the reduction in pH-responsive modified polyethyleneimine, the inability of the smart responsive gel to respond precisely, the inability to release calcium ions, resulting in a loose soil structure and thus a decrease in unconfined compressive strength. Comparing Examples 10 (e.10) and 11 (e.11) with Example 5 (e.5), it is evident that chitosan concentration has a significant impact on the reinforcement effect. Chitosan, through its abundant functional groups, coordinates with calcium ions, guiding the directional deposition of calcium carbonate at the contact points with soil particles, thus improving cementation efficiency. Too low a concentration weakens its guiding effect; too high a concentration may cause aggregation, affecting dispersibility and the uniformity of nucleation sites, both leading to a decrease in strength performance.

[0232] The strength of Comparative Example 1 (conventional microbial solidification method) was much lower than that of all other examples, which confirms that the measures introduced in this invention, such as nano-clay-chitosan composite sol, smart responsive gel, and polylactic acid microspheres encapsulating calcium citrate, play a key role in improving cementation uniformity, stabilizing microbial activity, and optimizing calcium carbonate deposition patterns.

[0233] The intensity of Comparative Examples 2 and 3 (where the PVA concentration and homogenization rate in the external aqueous phase were outside the range) was lower than that of Example 5. The reasons include: too low a PVA concentration and homogenization rate can lead to emulsion instability, causing microsphere adhesion, excessive size, and a significant decrease in drug encapsulation efficiency; while too high a PVA concentration and homogenization rate can easily cause excessive shearing, resulting in abnormal microsphere morphology, the formation of satellite spheres, or excessively small size. At the same time, PVA embedding may cause the microsphere surface to be rough and the structure to be porous.

[0234] Comparative Examples 4 and 5 (where the ratio of modified polyethyleneimine to crosslinking agent exceeded the range) showed that the main reason for the decrease in strength was that when the ratio was too low, the crosslinking was insufficient, the gel network strength was poor, and the content of functional groups was low; when the ratio was too high, it was easy to cause steric hindrance, which hindered effective crosslinking, or caused the gel to become brittle and the pore size to be too small, which was not conducive to microbial migration and material transport.

[0235] The strength properties of Comparative Example 6 (omitting the nano-clay-chitosan composite sol) were significantly reduced, indicating that the composite sol plays a key role in the system: nano-clay promotes the aggregation of fine particles through flocculation, optimizing the soil pore structure; chitosan guides the directional deposition of calcium carbonate at the contact points between particles through functional groups, improving the cementation efficiency and uniformity, and avoiding pore blockage caused by disordered deposition. Without this component, the fixation of microorganisms and the nucleation guidance effect decrease, resulting in a significant weakening of the cementation effect.

[0236] pass Figure 2 The permeability coefficient of medium-density soil shows that it decreases with increasing unconfined compressive strength, and the two are generally negatively correlated; that is, the higher the strength, the worse the permeability. This phenomenon is mainly caused by cementing materials blocking the pore throats.

[0237] Example 5 achieves optimal compressive strength while maintaining a permeability coefficient of 0.12 × 10⁻⁶. -3 The medium permeability level indicates that the gel-microbial system at this ratio not only promotes effective cementation of calcium carbonate but also optimizes the pore structure through the synergistic effect of the nano-clay-chitosan composite sol, achieving synergistic regulation of strength and permeability. The higher permeability coefficients in Examples 1-3 (e.1, e.2, e.3) can be attributed to the loose soil structure, leading to an increase in permeability. In Example 10 (e.10), insufficient chitosan concentration weakened the guiding effect on calcium carbonate deposition, potentially leaving local seepage channels and resulting in a significant increase in permeability. In Example 11 (e.11), excessively high concentration caused agglomeration, leading to uneven distribution of the composite sol, all of which resulted in a decrease in the permeability regulation effect.

[0238] The permeability coefficient of Comparative Example 1 (conventional microbial solidification method) is in the lower range of the figure, which confirms that its deposition process lacks spatial controllability. Calcium carbonate is prone to disordered deposition and blockage of the main pore channels, resulting in poor permeability. It is also accompanied by problems such as poor uniformity and ineffective deep reinforcement.

[0239] The permeability coefficients of Comparative Examples 2 and 3 (where the PVA concentration and homogenization rate in the external aqueous phase exceeded the scope of the claims) were higher than those of Example 5. This is because excessively low or high PVA concentrations and homogenization rates can lead to problems such as emulsion instability, abnormal microsphere morphology, and a significant decrease in calcium ion encapsulation rate, resulting in insufficient calcium ion supply in the later stages, causing problems such as decreased strength and increased permeability coefficient.

[0240] The abnormal permeability coefficients of Comparative Examples 4 and 5 (where the ratio of modified polyethyleneimine to crosslinking agent exceeds the scope of the claims) are due to insufficient crosslinking when the ratio is too low, and low content of pH-responsive groups, resulting in insufficient pH responsiveness, leading to a decrease in strength and an increase in permeability coefficient. When the ratio is too high, steric hindrance is easily caused, hindering effective crosslinking and impeding microbial migration and material transport; however, its compressive strength is lower, and its permeability coefficient is still higher than that of Example 5.

[0241] Comparative Example 6 (omitting the nano-clay-chitosan composite sol) exhibits a lower permeability coefficient. This is because the nano-clay optimizes pore distribution through flocculation, while chitosan guides the directional deposition of calcium carbonate at particle contact points, achieving effective cementation while preventing disordered blockage of pore throats. Without this component, the deposition process lacks spatial guidance, leading to disordered deposition of calcium carbonate and blockage of major pore channels, resulting in poor permeability.

[0242] This invention achieves synergistic optimization of strength and permeability by precisely controlling the location and morphology of mineralized deposits. Specifically, nano-clay optimizes the internal pore distribution structure of the soil through flocculation, providing a more rational spatial framework for mineral precipitation; chitosan, through its molecular guiding effect, promotes the preferential directional deposition of calcium carbonate at the contact points of soil particles, thereby effectively enhancing structural bonding rather than blindly filling pores; and the smart gel controls the release rate of calcium ions to achieve temporal regulation of the calcium carbonate crystallization process, avoiding premature and excessive cementation in certain areas.

[0243] The synergistic effect of the above-mentioned technologies significantly improves the unconfined compressive strength of the soil while avoiding complete blockage of the pore throats, allowing the soil to maintain a certain permeability after achieving high strength, thus breaking through the constraint of the trade-off between strength and permeability in traditional solidification.

[0244] The various embodiments in this specification are described in a related manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions of the method embodiments.

[0245] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A method for reinforcing high-fill soil bodies through nanogel-assisted microbial mineralization, characterized in that, The specific steps are as follows: S1. Cultivate and activate Bacillus; S2. Preparation of a responsive gel; the method for preparing the responsive gel includes: S2.1: Modify gelatin with hydrazide and graft polyvinylimide; S2.2: Mix modified gelatin, modified polyethyleneimine, and polyethylene glycol dialdehyde in a mass ratio of 1:(1.2~2.5):(0.1~0.25), and add pre-loaded Ca. 2+ Nano-zeolite was stirred to obtain a hydrogel precursor solution; S2.3: Preparation of polylactic acid microspheres encapsulated with calcium citrate; the method for preparing the polylactic acid microspheres encapsulated with calcium citrate includes: S2.3.1: Using 5 wt% calcium citrate aqueous solution as the inner aqueous phase and 5 wt% polylactic acid-dichloromethane solution as the oil phase, mix them at a volume ratio of 1:5 and shear emulsify at 10000~25000 rpm for 2~5 minutes to form a W / O primary emulsion; S2.3.2: Add the primary emulsion to an external aqueous phase containing 1-3 wt% polyvinyl alcohol, and homogenize at 3000-8000 rpm for 1-3 minutes to form a W / O / W double emulsion; S2.3.3: The dichloromethane in the W / O / W double emulsion is volatilized, and the polylactic acid is solidified to obtain the polylactic acid microspheres encapsulated with calcium citrate; S2.4: Crosslink polylactic acid microspheres encapsulated with calcium citrate with a hydrogel precursor solution to obtain a responsive gel; S3. NaHCO3 buffer solution, activated Bacillus, nano-clay-chitosan composite sol, responsive gel, and cementing liquid are mixed into the soil in sequence, and then compacted in layers to achieve dense reinforcement of high fill soil. The specific steps for S3 are as follows: S3.1: Mix 0.5~1.0wt% chitosan acetate solution and 2.0wt% nano clay aqueous solution at a volume ratio of 3:1, and stir for 1.5~2.5 hours to form nano clay-chitosan composite sol; S3.2: 0.8~1.2 mol·L -1 CaCl2 solution and 0.8~1.2 mol·L -1 The urea solution is mixed to obtain a cementitious solution; S3.3: Based on the dry soil mass, add the following in sequence: 10%~20% of 0.5 M NaHCO3 buffer solution, 20%~30% of activated Bacillus bacterial solution, 1%~3% of nano-clay-chitosan composite sol, 2%~5% of responsive gel, and 10%~20% of cementing liquid, and mix evenly. Then, compact the mixture in layers to achieve soil compaction and reinforcement.

2. The method for reinforcing high-fill soil with nanogel synergistic microbial mineralization according to claim 1, characterized in that, The specific steps for S1 are as follows: S1.1: Prepare a microbial proliferation culture medium, wherein the microbial proliferation culture medium is composed of 8-12 g / L tryptone, 4-6 g / L yeast extract, 4-6 g / L ammonium chloride, and the remainder is distilled water; S1.2: Thaw the frozen bacterial strain, inoculate it into the microbial proliferation medium, and culture it with shaking at 28-32℃ and 170-190rpm for 22-26h; S1.3: The culture medium was subjected to an ice bath to terminate metabolism, and after standing, the OD was measured using a blank culture medium as a control. 600 Value, pending OD 600 When the value reaches 0.5-0.7, it is transferred to a urea-containing differential medium containing phenol red to verify urease activity; S1.4: Centrifuge the culture medium containing urease activity, collect the bacterial cells, and then... 7 -10 9 The inoculum of CFU / g was resuspended in the activation solution and activated at room temperature for 1.5-2.5 h.

3. The method for reinforcing high-fill soil with nanogel synergistic microbial mineralization according to claim 1, characterized in that, The specific steps for modifying gelatin with hydrazide in S2.1 are as follows: S2.1.1: Dissolve gelatin in deionized water with a pH of 4.5–5.0 to prepare a gelatin solution; S2.1.2: The gelatin solution is placed in an ice bath at 4°C, and the reactants are added sequentially according to the molar ratio of carboxyl group in gelatin molecule: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: acylhydrazide reagent = 1:(1.2~2.0):(0.5~1.0):(5~20). The reaction is carried out for 2~6 hours to obtain the reaction product. S2.1.3: The reaction product was purified by dialysis and lyophilized to obtain hydrazide-modified gelatin.

4. The method for reinforcing high-fill soil with nanogel synergistic microbial mineralization according to claim 1, characterized in that, The grafting modification of polyethyleneimine in S2.1 specifically involves: 2-Chlorobenzimidazole-5-carboxylic acid was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added in a molar ratio of 2-chlorobenzimidazole-5-carboxylic acid: EDC: HOBt = 1 : (1.2~1.5) : (0.5~0.8). The mixture was activated in an ice bath. Subsequently, polyethyleneimine was added so that the molar ratio of its primary amino group to 2-chlorobenzimidazole-5-carboxylic acid was (0.8~1.2):

1. The mixture was reacted at room temperature for 12~24 hours to obtain modified polyethyleneimine.

5. The method for reinforcing high-fill soil with nanogel synergistic microbial mineralization according to claim 2, characterized in that, The activation solution in S1.4 consists of 20 mM CaCl2, 0.5 M urea and 5% trehalose.

6. The method for reinforcing high-fill soil with nanogel synergistic microbial mineralization according to claim 1, characterized in that, Preload Ca in S2.2 2+ The nano-zeolite has a particle size of 50-200 nm and a calcium loading of 50-80 mg / g.

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