Method for inducing bacillus subtilis to produce calcium carbonate by calcium lignosulfonate and application thereof

Abstract

The present application belongs to the field of microorganisms and environmental remediation technology, and specifically relates to a method for replacing urea with calcium lignosulfonate to induce Bacillus subtilis to generate calcium carbonate and application thereof. In order to solve the technical problems of existing MICP technology, such as serious ammonia pollution, high pathogenicity risk of strains, low precipitation efficiency and narrow adaptability, the present application uniformly mixes a calcium lignosulfonate solution and a bacterial additive solution containing Bacillus subtilis, as a MICP system, for generating calcium carbonate precipitation. The MICP system constructed by the present application, through the synergistic effect of calcium lignosulfonate and Bacillus subtilis, not only solves the environmental protection and safety pain points of traditional technology, but also improves the precipitation efficiency and scene adaptability, providing a new technical solution of "environmental protection, high efficiency and low toxicity" for building engineering repair, soil and water conservation and ecological governance, and widening the industrial application scenarios of MICP technology. The composite system prepared by the present application has a mature process and controllable cost, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial and environmental remediation technology, specifically relating to a method for inducing Bacillus subtilis to produce calcium carbonate by replacing urea with calcium lignin sulfonate and its application. Background Technology

[0002] Microbially Induced Calcium Carbonate Precipitation (MICP) is a novel biomineralization method that utilizes the metabolic activity of microorganisms to generate carbonate ions, which then combine with calcium ions in the environment to form calcium carbonate precipitates. This technology can achieve material bonding and reinforcement, structural repair, and crack filling, offering significant advantages such as ease of operation, environmental friendliness, and strong sustainability. MICP has been widely applied in engineering fields such as concrete crack repair, soil reinforcement, soil aggregate structure optimization, and soil and water conservation, providing a green alternative to traditional mineral consolidation and chemical remediation methods. In recent years, it has become a research hotspot in geotechnical engineering, soil and water conservation, and ecological restoration.

[0003] Currently, the mainstream MICP technology is based on the urea hydrolysis pathway. Its mechanism involves urease-producing strains catalyzing the hydrolysis of urea to generate ammonia and carbonate ions. The carbonate ions then combine with calcium ions to form calcium carbonate precipitate. This method can rapidly achieve cementation and reinforcement, but it has several significant limitations: 1. Poor environmental compatibility: The large amount of ammonia produced by the urea hydrolysis pathway is easily released in gaseous form or forms ammonia nitrogen, polluting water bodies and leading to air pollution, eutrophication, and aquatic ecotoxicity, violating the core concept of ecological restoration and affecting regional environmental quality. 2. Biosafety risks: Commonly used functional strains (such as Bacillus cereus, Streptococcus pasteurellis, Salmonella, etc.) often pose pathogenic risks, easily causing environmental biological pollution and safety hazards, making it difficult to meet the safety requirements of large-scale engineering applications. 3. Limited reaction efficiency: The concentration of calcium ions in the system significantly affects the calcium carbonate precipitation efficiency. High concentrations of calcium ions inhibit the metabolic activity and enzyme secretion capacity of microorganisms, while low concentrations of calcium ions make effective precipitation difficult. Currently, there are no unified industry standards for calcium source selection and calcium ion concentration control, affecting the engineering adaptability and stability of the technology. 4. Unstable crystal structure: The crystal morphology and structural stability of calcium carbonate precipitates are affected by multiple factors such as substrate, strain, and environmental conditions, which can easily lead to uneven crystal distribution and insufficient cementation strength, making it difficult to meet the engineering requirements of fine concrete repair and differentiated soil reinforcement.

[0004] To mitigate the risk of ammonia emissions, some studies have attempted to use non-urea hydrolysis pathways such as nitrate reduction and sulfate reduction. However, these methods require a strict anaerobic environment, have poor adaptability to natural engineering scenarios, low microbial metabolic efficiency, long reaction cycles, and are difficult to regulate in engineering, making it difficult to achieve industrialization and large-scale application.

[0005] In summary, existing MIP (Microbiologically Injectable Catalytic Pulsation) technologies generally lack synergistic solutions combining environmentally friendly substrates with highly efficient non-pathogenic bacterial strains. Key technical bottlenecks include: poor substrate environmental compatibility, severe byproduct contamination, difficulty in balancing the safety and metabolic efficiency of functional strains, insufficient structural stability of calcium carbonate precipitates, and a lack of standardized specifications for key reaction conditions and concentration ratios. These issues hinder the synergistic optimization of the environmental benefits, reaction efficiency, and engineering adaptability of MIP technology, severely impeding its transformation from laboratory research to industrial and engineering applications.

[0006] Therefore, finding suitable environmentally friendly substrates and highly efficient non-pathogenic strains to address the aforementioned shortcomings of existing MIP technology is of significant academic importance and has broad industrial prospects for the environmentally friendly, efficient, standardized, and large-scale application of MIP technology. Summary of the Invention

[0007] To address the technical problems of severe ammonia pollution, high pathogenicity risk of strains, low precipitation efficiency, and narrow compatibility of existing MIP technology, this invention provides a method for inducing Bacillus subtilis to produce calcium carbonate by replacing urea with calcium lignin sulfonate. This method can achieve environmentally friendly, efficient, and safe implementation of concrete crack repair and cementation of various soil types.

[0008] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a method for inducing Bacillus subtilis to produce calcium carbonate, comprising the following steps: A calcium lignosulfonate solution was mixed with a bacterial additive solution containing Bacillus subtilis 168 to form a MICP system for generating calcium carbonate precipitate.

[0009] In some embodiments, the concentration of the calcium lignosulfonate solution is 8–10 g / L.

[0010] In some implementations, the bacterial additive solution is obtained by preparing activated Bacillus subtilis 168 bacterial culture, nutrient broth, and calcium chloride to a McFarland concentration of 4.0.

[0011] In some preferred embodiments, the bacterial additive solution contains 12.0 × 10⁻⁶. 8 CFU / mL Bacillus subtilis 168 bacterial suspension, 0.1–0.3 g / L nutrient broth, 8–12 g / L calcium chloride.

[0012] In some implementations, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2.

[0013] In some preferred embodiments, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 1:0.20 to 0.21.

[0014] Secondly, the present invention provides an application of the MICP system in mineral consolidation, concrete crack repair, soil reinforcement, soil aggregate structure optimization and / or soil and water conservation. The MICP system is composed of calcium lignosulfonate solution as substrate and bacterial additives containing Bacillus subtilis as functional strains.

[0015] In some implementations, the MICP system is applicable to any soil type or soil, such as Yunnan red soil, Henan loess, Sichuan purple soil, Jiangsu green soil, Heilongjiang black soil, composite sandy clay, saline-alkali soil, or silty soil for roadbed.

[0016] In some implementation schemes, the concrete crack repair application involves adjusting the cement to a just-set state with water, adding a bacterial additive solution and a calcium lignosulfonate solution, mixing them thoroughly, pouring the mixture into the pre-repaired crack, and curing it at 28–31°C and 40% RH for 25–32 days.

[0017] In some preferred embodiments, the concentration of the bacterial additive solution in the concrete is 200–300 μL / g.

[0018] In some implementation schemes, the soil reinforcement application involves adjusting the moisture content of the filtered soil to 10-20%, adding a bacterial additive solution and a calcium lignosulfonate solution, mixing them thoroughly, smoothing the surface, and curing at 28-31°C and 40% RH for 25-32 days.

[0019] In some preferred embodiments, the concentration of the bacterial additive solution in the soil is 50–150 μL / g.

[0020] In some preferred embodiments, to ensure that the MIP system achieves the best application effect, soil moisture content, humidity, compaction or cementation status, maintenance environment, pH value, salinity or appearance changes are tested every 7 days during the maintenance period.

[0021] In some preferred embodiments, the soil filter screen is 2–4 mm.

[0022] In some implementations, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2.

[0023] In some preferred embodiments, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 1:0.20 to 0.21.

[0024] Beneficial effects: This invention deeply integrates environmentally friendly calcium lignosulfonate substrate with the safe strain of Bacillus subtilis 168 to construct a novel MIP system that is "ammonia-free, low-toxicity, and highly efficient," effectively overcoming the limitations of traditional MIP technology. Specific advantages are as follows: 1. Substrate-Strain Synergistic Innovation: Abandoning the urea substrate and potential pathogens used in traditional MIP technology, calcium lignosulfonate that meets the relevant standards for concrete / soil additives is selected as the energy supplier for the microbial cells, and paired with the non-pathogenic probiotic Bacillus subtilis 168, ammonia pollution and biosafety hazards are eliminated from the source. The two have excellent compatibility, and calcium lignosulfonate can effectively regulate the stability of the system and further improve the precipitation efficiency of calcium carbonate, solving the core pain points of substrate pollution and insufficient strain safety in traditional systems.

[0025] 2. Innovative Metabolic Mechanism: A novel metabolic pathway for the conversion of Ca(OH)2 to CaCO3 was discovered for the first time. Bacillus subtilis 168 can achieve this through BsDyP enzyme, ssu enzyme, and CaCO3. 2+ Five metabolic pathways, including channel enzymes, efficiently decompose lignin sulfonate and synergistically induce stable calcite-type calcium carbonate crystals, filling the technological gap in the existing MIP technology regarding the metabolic mechanism of lignin substrates. 3. Integrative Structural-Functional Innovation: Constructing an integrated functional system of "substrate metabolism-ion adsorption-targeted precipitation," calcium lignosulfonate and calcium ions work synergistically to achieve a concrete crack filling rate of over 90% and increase the unconfined compressive strength of soil by 56.5%. It is suitable for five typical eroded soils, including sandy soil and saline-alkali soil, as well as different types of concrete repair scenarios, achieving a dual improvement in repair effect and structural stability.

[0026] 4. Application Adaptability Innovation: Balancing environmental protection and engineering practicality, the entire preparation process requires no special equipment and the curing conditions are mild. It is suitable for both large-scale engineering applications and field operations, effectively expanding the industrial application scenarios of MIP technology and solving the problem of traditional technologies being difficult to implement on a large scale.

[0027] The MIP system constructed in this invention, through the synergistic effect of calcium lignosulfonate and Bacillus subtilis 168, not only solves the environmental and safety pain points of traditional technologies but also improves precipitation efficiency and application adaptability. It provides a novel "environmentally friendly, efficient, and low-toxicity" technical solution for building restoration, soil and water conservation, and ecological restoration, thus broadening the industrial application scenarios of MIP technology. The composite system prepared by this invention has a mature process, controllable cost, and broad application prospects. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation and characterization process of calcium carbonate production induced by calcium lignosulfonate in Bacillus subtilis according to the present invention. Figure 2 This is a sulfonate pathway analysis diagram of Bacillus subtilis 168 lignin KEGG in this invention; Figure 3 The VITEK MS mass spectrum for the identification of Bacillus subtilis 168 in this invention; Figure 4 Comparison of the appearance of Bacillus subtilis producing calcium carbonate induced by different concentrations of calcium lignin sulfonate according to the present invention. Figure 5 This is a macroscopic characterization diagram of typical soil treated with the MIP system according to Example 2 of the present invention; Figure 6 SEM microstructure characterization of concrete samples after treatment with the MICP system; Figure 7 This is a schematic diagram showing the content of different elements in concrete samples treated with the 10 g / L calcium lignosulfonate-MICP system, as detected by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) of this invention. Figure 8 The XRD crystal characterization spectra of the concrete samples in the calcium lignosulfonate group based on Example 1 of the present invention are shown. Figure 9 This is a comparison diagram of the CaCO3 crystal structure of different groups of concrete samples obtained based on Example 1 of the present invention; Figure 10 Schematic diagram of FT-IR testing of different groups of concrete samples after treatment with the MICP system; Figure 11 This is a schematic diagram of the RAMAN results for different groups of concrete samples after treatment with the MICP system. Figure 12 This is a schematic diagram of the RAMAN microscopic results of different groups of concrete samples after treatment with the MICP system. Detailed Implementation

[0029] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0030] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0031] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0032] Methods for inducing Bacillus subtilis to produce calcium carbonate This invention provides a method for inducing Bacillus subtilis to produce calcium carbonate, which involves replacing urea with a calcium lignin sulfonate solution and mixing it with a bacterial additive solution containing Bacillus subtilis to form a MICP system for generating calcium carbonate precipitate.

[0033] As used in this article, "calcium lignosulfonate" is a byproduct of the wood pulping industry, which, after sulfonation modification, becomes a water-soluble natural polymer derivative. Its molecular structure contains phenolic hydroxyl groups and sulfonic acid groups, exhibiting excellent calcium ion complexing ability, good water solubility, and environmental compatibility. It meets relevant Chinese national standards for concrete additives and soil amendments. The raw materials are readily available and inexpensive, and it has already been successfully applied in fields such as concrete water-reducing agents and soil structure amendments. Calcium lignosulfonate not only serves as a carbon source and energy supplier for microorganisms, supporting their metabolism, but also guides the morphology of calcium carbonate precipitation through calcium ion complexation, enhancing the stability and density of the precipitated crystals and providing an efficient substrate basis for microbial mineralization processes.

[0034] As used in this article, "Bacillus subtilis" or "Bacillus subtilis 168" is an internationally recognized non-pathogenic probiotic that has passed relevant safety certifications. It contains no pathogenic genes or toxin coding sequences and can tolerate the highly alkaline environment of concrete and various complex soil matrices. This strain possesses the ability to efficiently secrete lignin-degrading enzymes and carbonic anhydrases, exhibiting excellent metabolic adaptability to lignin sulfonates and effectively promoting calcium carbonate precipitation. Simultaneously, Bacillus subtilis 168 can produce antimicrobial lipopeptides, inhibiting harmful microorganisms through spatial competition mechanisms, achieving a dual function of bio-binding and bio-inhibition, thus providing a safe and efficient functional strain selection for MIP (Microbial Inhibition-Proofing) technology.

[0035] This invention discloses a method for inducing Bacillus subtilis to produce calcium carbonate. This method utilizes the surface adsorption and metabolism of Bacillus subtilis to enrich calcium ions, while simultaneously using carbon dioxide produced during metabolism or from the environment to form carbonate ions, thereby inducing nucleation on the cell surface and generating calcium carbonate precipitate. Furthermore, calcium lignin sulfonate can act as a substrate to participate in metabolism, further promoting the release and accumulation of calcium ions, thus synergistically accelerating the biomineralization process of calcium carbonate. This novel biomineralization method enables the bonding and reinforcement of materials, structural repair, and crack filling, and has significant advantages such as ease of operation, environmental friendliness, and strong sustainability.

[0036] As mentioned in the background section, MICP has been widely used in engineering fields such as concrete crack repair, soil reinforcement, soil aggregate structure optimization, and soil and water conservation. It provides a green alternative to traditional mineral consolidation and chemical remediation methods and has become a research hotspot in geotechnical engineering, soil and water conservation, and ecological restoration in recent years.

[0037] Therefore, in some embodiments of the present invention, a MICP complex system is provided, comprising a bacterial additive solution containing Bacillus subtilis 168 and a calcium lignosulfonate solution, the ratio of which is optimized based on the metabolic characteristics of Bacillus subtilis 168 and the complexation mechanism of calcium lignosulfonate. In some examples, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2.

[0038] In some preferred examples, the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 1:0.20 to 0.21.

[0039] In some examples, the preparation method of the bacterial additive solution includes: taking Bacillus subtilis 168 lyophilized powder (an internationally recognized type strain, certified as a GRAS-level safe microorganism, free of pathogenic genes and toxin coding sequences), inoculating it into a nutrient broth medium (containing an appropriate ratio of carbon and nitrogen sources and inorganic salts to meet the metabolic needs of the bacteria), and incubating it at a suitable temperature and appropriate rotation speed under constant temperature shaking until the logarithmic growth phase, preferably incubating it at 31°C and 180 rpm for 24 h to complete the activation of bacterial metabolism; adding the activated bacterial solution to a solution prepared with nutrient broth, anhydrous calcium chloride and ultrapure water, shaking to mix evenly so that calcium ions are uniformly dispersed to avoid the inhibition of bacterial activity by high concentrations of calcium ions, calibrating it to a suitable McFarland concentration by spectrophotometry, and adjusting the pH of the resulting bacterial additive solution to a suitable range, which has both high bacterial activity and stable calcium source supply capacity.

[0040] In some examples, the bacterial additive solution was adjusted to a McFarland concentration of 4.0 (12.0 × 10⁻⁶). 8 The advantages of the CFU / mL method are: moderate cell density, which can ensure that the bacteria maintain high activity in the high alkaline environment of concrete, so that the precipitation efficiency of MICP calcite is the highest and the crystal form is stable. At the same time, Ca(OH)2 can be completely converted into CaCO3, which is convenient for standardized engineering applications.

[0041] In some examples, the preparation method of calcium lignosulfonate solution includes: dissolving industrial-grade calcium lignosulfonate (solid content meeting relevant requirements, ≥90%) in water, stirring to promote the full hydration and dissolution of phenolic hydroxyl groups and sulfonic acid groups, and cooling to room temperature to form a homogeneous and transparent solution. In some preferred examples, a calcium lignosulfonate solution with a concentration of 8–10 g / L is prepared. This concentration has been optimized and verified through multiple rounds of experiments, effectively complexing calcium ions and providing a continuous carbon source for cell metabolism, while avoiding abnormal system viscosity caused by excessively high concentrations.

[0042] In practical applications, such as concrete repair, silicate cement is mixed with water to a plastic state before initial setting. Then, bacterial additive solution and calcium lignosulfonate solution are immediately added and stirred quickly to ensure that the bacteria and substrate are evenly distributed at the cement paste interface, and at the same time, the directional deposition process of calcium carbonate at the interface is initiated.

[0043] In soil reinforcement scenarios, soil samples (covering various soil types) are taken and mixed with bacterial additive solution and calcium lignosulfonate solution at a volume ratio of 0.5–2:0.1–2, with a preferred volume ratio of 1:0.20–0.21, to achieve uniform coating of the soil sample at the microscale. After mixing, the synergistic complexation of phenolic hydroxyl groups and sulfonic acid groups in calcium lignosulfonate guides the nucleation and growth of calcite crystals at the contact points with soil particles. Optimization and verification have shown that a volume ratio of bacterial additive solution to calcium lignosulfonate solution of 1:0.20–0.21 can significantly increase the amount of calcium carbonate precipitation and crystal density while ensuring a high survival rate of Bacillus subtilis 168. This provides quantifiable and replicable engineering implementation parameters for the self-repair of microcracks in concrete and the reinforcement of various soil structures.

[0044] Both the concrete and soil substrates were cured under standard conditions with suitable temperature and humidity. Bacillus subtilis 168 continuously secreted lignin-degrading enzymes and carbonic anhydrase in the alkaline environment of the substrate. Molecular docking revealed that Bacillus subtilis contains proteases BsDyP, ssu, and Ca, which participate in the metabolism of lignin precursors. 2+Channel enzymes exhibit a high-strength binding mode. This explains the high calcium aggregation and rapid metabolism of Bacillus subtilis. Bacillus subtilis enriches calcium ions through surface adsorption and metabolism, while simultaneously utilizing carbon dioxide produced during metabolism or from the environment to form carbonate ions, thereby inducing nucleation on the cell surface and generating calcium carbonate precipitates. Furthermore, calcium lignin sulfonate can act as a substrate in metabolism, further promoting the release and accumulation of calcium ions, thus synergistically accelerating the biomineralization process of calcium carbonate. Directional induction of calcite-type calcium carbonate nucleation and growth at concrete crack interfaces or soil particle contact points forms a dense micron-sized crystal network. All parameters of this process were optimized through multiple rounds of orthogonal experiments. During curing, the cell survival rate remained consistently high, and the amount of calcium carbonate deposited was significantly improved compared to the traditional urea-based MIP system. Crystallization and matrix interface bonding strength were both significantly enhanced, providing a quantifiable, replicable, and engineering-practice-compliant technical implementation plan for the self-repair of concrete microcracks and the strengthening of various soil structures.

[0045] The novel MIP system, which utilizes the synergistic effect of calcium lignosulfonate and Bacillus subtilis 168 as disclosed in this paper, deeply integrates an environmentally friendly substrate with a safe and efficient bacterial strain. This addresses the dual risks of ammonia emissions and the application of pathogenic bacteria, optimizes calcium carbonate precipitation efficiency and crystal structure stability, and significantly enhances the cementing effect, meeting the diverse engineering needs of typical soil solidification and concrete crack repair in China. A systematic search of domestic and international literature and patent databases revealed no publicly available reports on the synergistic application of calcium lignosulfonate and Bacillus subtilis 168 in MIP technology. This system overcomes the environmental and safety bottlenecks of traditional MIP technology, providing a new path for the environmentally friendly, efficient, standardized, and large-scale application of MIP technology, and possesses significant academic value and broad industrial prospects.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. The advantages and features of this invention will become clearer with this description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0047] The nutritional broth composition and ratios in the following examples include: 10.0 g / L peptone, 5.0 g / L sodium chloride, 3.0 g / L beef extract, 4.0 g / L glucose, and pH 7.2±0.4.

[0048] The Bacillus subtilis strain 168 used in the following examples was commercially available.

[0049] Example 1: Calcium lignosulfonate (10 g / L) for the repair of cracks in PO 42.5 concrete. (1) Preparation of bacterial additive solution: Commercially available Bacillus subtilis 168 activated lyophilized powder was inoculated into 100 mL of nutrient broth medium and cultured in a constant temperature shaker at 31℃ and 180 rpm for 24 h to complete activation; after activation, it was inoculated onto blood agar plates in a biosafety cabinet, with a final pH of 7.4±0.2, and grown at 31℃ for 24 h. Bacillus subtilis 168 was scraped off, and distilled water was added to prepare a McFarland concentration of 4.0 (12.0×10⁻¹⁰) using a DEN1 McFarland turbidimeter. 8 A Bacillus subtilis 168 bacterial culture (CFU / mL) was prepared, followed by the addition of 0.022 g of nutrient broth, 1.0543 g of calcium chloride, and 100 mL of ultrapure water. The mixture was then stirred at 500 rpm for 12 min on a magnetic stirrer. The McFarland concentration was adjusted to 4.0 and the pH to 8.0, ultimately yielding a culture containing 12.0 × 10⁻⁶ CFU / mL. 8 Prepare a bacterial additive solution of Bacillus subtilis 168 (CFU / mL), 0.22 g / L nutrient broth, and 10.543 g / L calcium chloride, and seal for later use.

[0050] (2) Preparation of 10 g / L calcium lignosulfonate solution: Weigh 1.0 g of calcium lignosulfonate, add 100 mL of ultrapure water, place it in a 40℃ constant temperature water bath magnetic stirrer, stir at 600 rpm for 3 h until completely dissolved, cool to 25℃, filter through a 0.22 μm filter membrane for sterilization and use.

[0051] (3) Concrete repair application: 25 g of PO 42.5 silicate cement and 8 mL of ultrapure water were placed in a mixing pot and stirred at 300 rpm for 5 min until just set; bacterial additive solution (0.3 mL of bacterial additive solution was added for every 1 g of cement to form a concentration of 300 μL / g) and 1.58 mL of 10 g / L calcium lignosulfonate solution were added, and the stirring speed was adjusted to 500 rpm and stirred for 1 min until uniformly mixed; the mixed concrete sample was poured into a pre-set crack mold (crack width 20.5 mm) and placed in a FYLYS100L multi-functional constant temperature chamber at 31℃ and 40%RH for 28 days. The surface condition and crack filling were observed every 7 days during the period.

[0052] Example 2: Calcium lignosulfonate (10 g / L) for cementation and reinforcement of Yunnan red soil (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0053] (2) Preparation of 10 g / L calcium lignosulfonate solution: The formula and preparation method are the same as in Example 1.

[0054] (3) Application of red soil cementation: Take 9 g of Yunnan red soil, pass it through a 2 mm sieve, adjust the moisture content to 15%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 10 g / L calcium lignosulfonate solution, stir evenly in a clockwise direction for 5 min with a sterile glass rod until there are no obvious lumps, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, during which the ambient humidity is kept stable, and the soil moisture content is measured every 7 days and a small amount of ultrapure water is added to maintain the initial humidity.

[0055] Example 3: Calcium lignosulfonate (10 g / L) used for cementation and reinforcement of loess in Henan Province (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0056] (2) Preparation of 10 g / L calcium lignosulfonate solution: The formula and preparation method are the same as in Example 1.

[0057] (3) Application of loess cementation: Take 9 g of Henan loess, pass it through a 2 mm sieve, adjust the moisture content to 12%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 10 g / L calcium lignosulfonate solution, stir with a sterile glass rod for 5 min until it is evenly mixed, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, observe the soil compaction status every 7 days during the period, and avoid excessive fluctuations in environmental humidity that may cause soil cracking.

[0058] Example 4: Calcium lignosulfonate (6 g / L) for cementation and reinforcement of Sichuan purple clay. (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0059] (2) Preparation of 6 g / L calcium lignosulfonate solution: Weigh 0.6 g of calcium lignosulfonate and prepare a 6 g / L calcium lignosulfonate solution according to the method in Example 1.

[0060] (3) Application of purple soil cementation: Take 9 g of Sichuan purple soil, pass it through a 2 mm sieve, adjust the moisture content to 14%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 6 g / L calcium lignosulfonate solution, stir with a sterile glass rod for 5 min until it is evenly mixed, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, record the changes in soil appearance every 7 days during the period, and ensure that the maintenance environment is free of odor and contamination by bacteria.

[0061] Example 5: Calcium lignosulfonate (10 g / L) used for cementation and reinforcement of Jiangsu green soil (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0062] (2) Preparation of 10 g / L calcium lignosulfonate solution: The formula and preparation method are the same as in Example 1.

[0063] (3) Application of soil compaction: Take 9 g of Jiangsu soil, pass it through a 2 mm sieve, adjust the moisture content to 16%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 10 g / L calcium lignosulfonate solution, stir with a sterile glass rod for 5 min until it is evenly mixed, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, observe the soil compaction every 7 days during the period until the end of the curing.

[0064] Example 6: Calcium lignosulfonate (10 g / L) for cementation and reinforcement of black soil in Heilongjiang Province (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0065] (2) Preparation of 10 g / L calcium lignosulfonate solution: The formula and preparation method are the same as in Example 1.

[0066] (3) Application of black soil cementation: Take 9 g of Heilongjiang black soil, pass it through a 2 mm sieve, adjust the moisture content to 18%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 10 g / L calcium lignosulfonate solution, stir evenly in a clockwise direction for 6 min with a sterile glass rod until there are no obvious lumps, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, during which a small amount of ultrapure water is added every 7 days to maintain the initial humidity and prevent the black soil from cracking due to moisture loss until the end of the curing.

[0067] Example 7: Calcium lignosulfonate (9 g / L) for cementation reinforcement of composite sandy clay (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0068] (2) Preparation of 9 g / L calcium lignosulfonate solution: Weigh 0.9 g of calcium lignosulfonate and prepare a 9 g / L calcium lignosulfonate solution according to the method in Example 1.

[0069] (3) Application of composite sandy clay cementation: Take 9 g of composite sandy clay, pass it through a 2 mm sieve, adjust the moisture content to 13%, place it in a 48-well plate, add bacterial additive solution (0.3 mL of bacterial additive solution is added for every 3 g of soil, forming a concentration of 100 μL / g) and 0.19 mL of 9 g / L calcium lignosulfonate solution, mix with mechanical stirring combined with ultrasonic dispersion, ultrasonic power 100 W, ultrasonic time 1 min until there are no obvious particles; place the 48-well plate in a FYLYS100L multi-functional constant temperature chamber at 31℃ and 40%RH for 28 days, observe the soil cementation status every 7 days during the period until the end of the curing.

[0070] Example 8: Calcium lignosulfonate (8 g / L) for the repair of cracks in PO 52.5 high-strength concrete. (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0071] (2) Preparation of 8 g / L calcium lignosulfonate solution: Weigh 0.8 g of calcium lignosulfonate and prepare a calcium lignosulfonate solution with a concentration of 8 g / L according to the method in Example 1.

[0072] (3) Application of high-strength concrete repair: 25 g of P.O52.5 silicate cement and 7 mL of ultrapure water were placed in a mixing pot and stirred at 350 rpm for 4 min until the plastic state before initial setting was reached; bacterial additive solution (0.3 mL of bacterial additive solution was added for every 1 g of cement to form a concentration of 300 μL / g) and 1.58 mL of 8 g / L calcium lignosulfonate solution were added, and the stirring speed was adjusted to 550 rpm and stirred for 1.5 min until the mixture was uniform; the mixed concrete sample was poured into a pre-set crack mold (crack width 0.1~0.3 mm) and placed in a FYLYS100L multi-functional constant temperature chamber at 31℃ and 40%RH for 28 days. During the period, the surface condition and crack filling were observed every 7 days until the curing was completed.

[0073] Example 9: Calcium lignosulfonate (7 g / L) for cementation and reinforcement of saline-alkali soil (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0074] (2) Preparation of 7 g / L calcium lignosulfonate solution: Weigh 0.7 g of calcium lignosulfonate and prepare a 7 g / L calcium lignosulfonate solution according to the method in Example 1.

[0075] (3) Application of saline-alkali soil cementation: Take 9 g of saline-alkali soil, pass it through a 2 mm sieve, adjust the moisture content to 14%, place it in a 48-well plate, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 7 g / L calcium lignosulfonate solution, stir evenly with a sterile glass rod for 5 min until the mixture is uniform, and smooth the surface; place the 48-well plate in a FYLYS100L multi-functional constant temperature incubator at 31℃ and 40%RH for 28 days, during which the soil pH and salt content are measured every 7 days and a small amount of ultrapure water is added to maintain the initial humidity until the end of the curing.

[0076] Example 10: Calcium lignosulfonate (10 g / L) for cementation and reinforcement of silty soil in road base courses. (1) Preparation of bacterial additive solution: The formula and preparation method are the same as in Example 1.

[0077] (2) Preparation of 10 g / L calcium lignosulfonate solution: The formula and preparation method are the same as in Example 1.

[0078] (3) Application of road base silty soil cementation: Take 9 g of road base silty soil, pass it through a 2 mm sieve, adjust the moisture content to 15%, place it in a small mixing device, add bacterial additive solution (add 0.3 mL of bacterial additive solution for every 3 g of soil to form a concentration of 100 μL / g) and 0.19 mL of 10 g / L calcium lignosulfonate solution, stir at 200 rpm for 8 min until it is evenly mixed, put the mixed silty soil into a mold and press it into shape; place the molded sample in a FYLYS100L multi-functional constant temperature chamber at 31℃ and 40%RH for 28 days, observe the appearance of the sample every 7 days during the period to prevent cracking, until the curing is completed.

[0079] Based on the above embodiments, after the curing process, the CaCO3 content and crystal morphology were detected using optical microscopy, industrial stereomicroscopy, SEM, EDS, XRD, FTIR, and Raman spectroscopy. The detection methods referenced existing conventional detection parameters, and the results are as follows: Figure 1 Flowchart of the preparation and characterization process of calcium carbonate production induced by calcium lignosulfonate in Bacillus subtilis; Assay methods: Integrating strain activation culture, MIP system construction, multi-media induced precipitation, and multi-dimensional characterization techniques, the concentration of the bacterial agent was controlled and cultured using a McFarland turbidimeter and a constant temperature incubator. Standardized activation of the strain was achieved through inoculation with hemoglobin plates and preparation of nutrient broth solution. Induced precipitation experiments were carried out using various media such as concrete and different types of soil (Yunnan red soil, Henan loess, Sichuan purple soil, Jiangsu green soil, and Heilongjiang black soil). Finally, optical microscopy, industrial stereomicroscopy, SEM, EDS, XRD, FTIR, and Raman spectroscopy were used to achieve full-process characterization and detection from macroscopic morphology to microscopic crystal structure, thus constructing a standardized preparation and characterization system covering strain-media-product.

[0080] The results showed that the process fully covered the activation of Bacillus subtilis 168, construction of the MIP system, induction of calcium carbonate formation in multiple media, and product characterization. The various detection technologies were well compatible and could systematically verify the inducing effect of calcium lignin sulfonate on calcium carbonate formation by Bacillus subtilis, providing technical support for the application of microbial induced calcium carbonate precipitation (MICP) in concrete repair, soil solidification and other fields.

[0081] Figure 2 This is a sulfonate pathway analysis diagram of Bacillus subtilis 168 lignin KEGG in this invention; Assay method: Genes related to lignin sulfonate metabolism in Bacillus subtilis 168 were extracted by computer simulation, and pathway enrichment analysis was performed based on the KEGG database to label key enzymes, intermediate products and reactions in the metabolic process, and to construct a complete regulatory network for sulfonate metabolism.

[0082] The results showed that the pathway diagram clarified the key steps of alkane sulfonate transport via the ssuACB system and catalysis by the ssuD enzyme, revealed the connection mechanism between sulfonate and sulfur metabolism and glycine-serine-threonine metabolism, and elucidated the metabolic pathway basis for calcium carbonate formation induced by calcium lignin sulfonate.

[0083] Figure 3 The image shows the results of species identification for Bacillus subtilis 168. Assay method: Activated Bacillus subtilis 168 was inoculated onto blood agar plates and incubated at 31℃ for 24 h. After observation of colony morphology, single colonies were picked and spread onto target plates. VITEK MS-CHCA matrix solution was added, and mass spectra were acquired in linear positive ion mode using a VITEK® MS system at a laser frequency of 200 Hz and a mass-to-charge ratio of 1500–12000 Da. The bacterial species were identified by comparison with the VITEK MS spectral library. The results showed that the colony surface was rough, opaque, and grayish-white with wrinkles, which is consistent with the typical colony morphology of Bacillus subtilis 168. The VITEK®MS identification result had a confidence level of 99%, confirming that the strain used was Bacillus subtilis 168, providing an accurate source of strain for subsequent MICP experiments.

[0084] Figure 4 Comparison of the appearance of Bacillus subtilis producing calcium carbonate induced by different concentrations of calcium lignin sulfonate according to the present invention. Assay method: Control groups (A, B, without added calcium lignosulfonate) and experimental groups (C: 2, 4, 6, 8, 10 g / L calcium lignosulfonate) were set up. Under the same culture conditions (37℃ constant temperature shaking culture for 48 h), Bacillus subtilis 168 mineralization precipitation was induced. After the precipitation was induced, the tubes were allowed to stand for 30 min. The precipitation formation and distribution in each test tube were photographed with a digital camera under a fixed light source and angle.

[0085] The results showed that no obvious precipitation was observed in the control groups (A and B), while different concentrations of calcium lignosulfonate showed varying degrees of white calcium carbonate precipitation. As the concentration of calcium lignosulfonate increased from 2 g / L to 10 g / L, the amount of precipitation initially increased and then tended towards saturation. This apparent result directly confirms the promoting effect of calcium lignosulfonate on Bacillus subtilis-induced calcium carbonate formation, showing a concentration-dependent effect, and provides a visual basis for subsequent optimization of the inducer dosage.

[0086] Figure 5 This is a macroscopic characterization diagram of typical soil treated with the MIP system according to Example 2 of the present invention; Measurement method: Referring to the method in Example 2, the calcium lignosulfonate-Bacillus subtilis 168 MICP system was applied to Yunnan red soil. Blank control groups A and B were set up, as well as calcium lignosulfonate concentration gradients of 2 g / L, 4 g / L, 6 g / L, 8 g / L, and 10 g / L. After curing for 28 days, the macroscopic appearance was photographed, and the curing state, surface integrity, and morphological characteristics were recorded.

[0087] The results showed that the concrete blocks treated with the MIP system had effective crack repair, and the Yunnan red soil achieved efficient solidification. The samples with a concentration of 8–10 g / L of calcium lignosulfonate showed the best macroscopic performance, with a dense surface and no disintegration, thus clarifying the optimal application concentration range of the system.

[0088] Figure 6 SEM microstructure characterization of concrete samples after treatment with the MICP system; Measurement method: Referring to the method in Example 1, concrete samples treated with 10 g / L calcium lignosulfonate-MICP system were taken. After sample preparation, the microstructure was observed using a ZEISS Sigma 500 scanning electron microscope at magnifications of 500X, 1000X, 5000X, 10000X, 20000X, and 50000X, and the crystal morphology, pore distribution, and interface bonding state were recorded.

[0089] The results showed that at different magnifications, the pores inside the concrete were filled with flocculent and needle-like calcium carbonate crystals. The crystals were tightly bonded to the matrix interface, and the density of the microstructure was significantly improved, confirming that the MICP system can effectively improve the microstructure of concrete.

[0090] Figure 7 This is a schematic diagram showing the content of different elements in concrete samples treated with the 10 g / L calcium lignosulfonate-MICP system, as detected by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) of this invention. Measurement method: The activated bacterial additive solution with added calcium chloride prepared in Example 1 of the logarithmic phase was fixed, dehydrated and sputter-coated with gold. The morphology of the bacteria was observed using a ZEISS Sigma 500 scanning electron microscope and the elemental composition and distribution were detected by an EDS spectrometer with a magnification of 1000×. The detection was performed in parallel 3 times.

[0091] The results showed that the rod-shaped morphology of Bacillus subtilis 168 could be clearly observed by SEM, and characteristic elements such as C, Mg, O, Si, S, K, Ca, and P were detected by EDS. Calcium was significantly enriched around the bacterial cells, providing direct elemental evidence for calcium carbonate formation.

[0092] Figure 8 The XRD crystal characterization spectra of the concrete samples in the calcium lignosulfonate group based on Example 1 of the present invention are shown. Measurement method: Referring to the method in Example 1, concrete samples treated with different concentrations of calcium lignosulfonate (2 g / L, 4 g / L, 6 g / L, 8 g / L, and 10 g / L) MICP system were ground into powder and then detected using a Japanese MiniFlex600 desktop X-ray powder diffractometer with a scanning angle 2θ of 10° to 70°. The position and intensity of the diffraction peaks were recorded.

[0093] The results showed that characteristic diffraction peaks of Ca3SiO5, Ca(OH)2, SiO2, and CaCO3 were detected in the spectrum. As the concentration of calcium lignosulfonate increased, the intensity of the characteristic peak of CaCO3 gradually increased, while the intensity of the characteristic peak of Ca(OH)2 decreased, confirming that calcium lignosulfonate can induce the formation of calcium carbonate crystals in concrete.

[0094] Figure 9This is a comparison diagram of the CaCO3 crystal structure of different groups of concrete samples obtained based on Example 1 of the present invention; Measurement method: Referring to the method in Example 1, XRD data of concrete samples from the blank group and the groups treated with different concentrations of calcium lignosulfonate (MICP) system were extracted. The types of CaCO3 crystal forms (calcite, aragonite, and aragonite) and their relative contents were analyzed, and a comparison diagram of crystal structures was drawn.

[0095] The results showed that almost no CaCO3 crystals were generated in the blank group, while the experimental groups were mainly composed of calcite-type CaCO3. The 10 g / L group had the highest crystal purity and the best crystallinity, which confirmed that the MICP system can control the crystal structure of CaCO3 and generate stable calcite crystals.

[0096] Figure 10 This is a schematic diagram of Fourier transform infrared (FT-IR) spectroscopy of concrete samples from different groups after processing with the MICP system. Assay method: Samples of different concentrations of calcium lignosulfonate (2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L) co-cultured with the bacterial additive from Example 1 were analyzed using a THERMO-FISHER iS50R Fourier transform infrared spectrometer (USA) at a scanning wavenumber of 500 cm⁻¹. -1 ~4000cm -1 Record characteristic absorption peaks.

[0097] The results showed that the characteristic absorption peaks of carbonate and hydroxyl groups of Ca(OH)2 were clearly detected in the spectrum. As the concentration of calcium lignosulfonate increased, the intensity of the characteristic peak of carbonate gradually increased, and there were no obvious impurity peaks, which confirmed that Bacillus subtilis 168 can specifically generate carbonate substances under the induction of calcium lignosulfonate.

[0098] Figure 11 This is a schematic diagram of the RAMAN results for different groups of concrete samples after treatment with the MICP system. Determination method: Samples of different concentrations of calcium lignosulfonate (2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L) co-cultured with the bacterial additive of Example 1 were analyzed using a LabRAM HR Evolution microconfocal Raman spectrometer (France), with Raman shifts ranging from 150 to 1200 cm⁻¹. -1 Record characteristic Raman peaks.

[0099] The results show that: in the spectrum at 280cm -1 154cm -1 The characteristic Raman peaks of calcium carbonate appeared on both sides, with the 10 g / L group showing the highest intensity and sharpest peak shape, consistent with the FT-IR detection results, further confirming that calcium lignosulfonate can promote the production of calcium carbonate by Bacillus subtilis 168.

[0100] Figure 12 This is a schematic diagram of the RAMAN microscopy results of different groups of concrete samples after treatment with the MICP system. Measurement method: Using a LabRAM HR Evolution Raman microscope (France), samples with different concentrations of calcium lignosulfonate (2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L) co-cultured with the bacterial additive from Example 1 were subjected to Raman imaging and microscopic observation to simultaneously acquire the microscopic morphology of the bacterial cells and the distribution of Raman characteristic signals.

[0101] The results showed that Raman microscopy enabled the simultaneous detection of bacterial morphology and calcium carbonate characteristic signals. The calcium carbonate characteristic signals were mainly distributed around the bacterial cells, and the signal intensity gradually increased with the increase of calcium lignin sulfonate concentration, confirming that Bacillus subtilis 168 is the core carrier for calcium carbonate production, and calcium lignin sulfonate can promote the enrichment of calcium carbonate around the bacterial cells.

[0102] The above embodiments all verify the effectiveness of the technical solution of the present invention. Through the synergistic effect of calcium lignosulfonate and Bacillus subtilis 168, calcite precipitation can be efficiently induced, achieving concrete crack repair and cementation reinforcement of various types of soil. A calcium lignosulfonate concentration of 8–10 g / L is the preferred option, exhibiting strong adaptability and stable effects. During the preparation process, further control of the McFarland concentration of the bacterial additive solution, the dissolution temperature and time of calcium lignosulfonate, and the curing environment parameters (31℃, 40%RH) ensures microbial metabolic activity and calcium carbonate precipitation efficiency. All components comply with Chinese standards for concrete and soil additives. The preparation process is simple and controllable, requires no special equipment, and is easy for industrial production and engineering applications.

Claims

1. A method for inducing Bacillus subtilis to produce calcium carbonate, characterized in that, Includes the following steps: A calcium lignosulfonate solution was mixed with a bacterial additive solution containing Bacillus subtilis 168 to form a MICP system for generating calcium carbonate precipitate.

2. The method for inducing Bacillus subtilis to produce calcium carbonate according to claim 1, characterized in that, The concentration of the calcium lignosulfonate solution is 8–10 g / L.

3. The method for inducing Bacillus subtilis to produce calcium carbonate according to claim 1, characterized in that, The bacterial additive solution contains 12.0 × 10⁻⁶ 8 CFU / mL Bacillus subtilis 168 bacterial suspension, 0.1–0.3 g / L nutrient broth, 8–12 g / L calcium chloride.

4. The method for inducing Bacillus subtilis to produce calcium carbonate according to any one of claims 1 to 3, characterized in that, The volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2; preferably, the volume ratio is 1:0.20–0.

21.

5. An application of a MICP system in mineral consolidation, concrete crack repair, soil reinforcement, soil aggregate structure optimization, and / or soil erosion control, characterized in that, The MICP system consists of a substrate of calcium lignosulfonate solution and a bacterial additive containing Bacillus subtilis as the functional strain.

6. The application according to claim 5, characterized in that, The soil or soil type mentioned includes one or any combination of Yunnan red soil, Henan loess, Sichuan purple soil, Jiangsu green soil, Heilongjiang black soil, composite sandy clay, saline-alkali soil, or silty soil for road base.

7. The application according to claim 5, characterized in that, In the concrete crack repair application, the cement is adjusted to a just-set state with water, and a bacterial additive solution and calcium lignosulfonate solution are added, mixed well, poured into the pre-repaired crack, and cured at 28-31℃ and 40%RH for 25-32 days.

8. The application according to claim 7, characterized in that, The concentration of the bacterial additive solution in concrete is 200–300 μL / g; the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2; preferably, the volume ratio is 1:0.20–0.

21.

9. The application according to claim 5, characterized in that, In the aforementioned soil reinforcement application, the filtered soil is adjusted to a moisture content of 10-20%, then a bacterial additive solution and a calcium lignosulfonate solution are added, mixed thoroughly, the surface is smoothed, and cured at 28-31℃ and 40%RH for 25-32 days.

10. The application according to claim 9, characterized in that, The concentration of the bacterial additive solution in the soil is 50–150 μL / g; the volume ratio of the bacterial additive solution to the calcium lignosulfonate solution is 0.5–2:0.1–2; preferably, the volume ratio is 1:0.20–0.21.

Images