A strong alkali-resistant urease-producing terrigenous bacillus and application thereof in microbial mineralization

Abstract

This invention discloses a highly alkali-resistant, urease-producing soil-derived Bacillus and its application in microbial mineralization, relating to the field of microbial technology. This soil-derived Bacillus was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37020. This strain can stably survive under extremely alkaline conditions, maintaining effective enzyme production activity and exhibiting significant biomineralization efficiency. This strain and its derived biomineralization agents and compositions have irreplaceable application value in the fields of alkaline soil reinforcement, heavy metal pollution remediation, and self-healing of building materials in highly alkaline environments, providing an efficient and green microbial solution for environmental remediation in extremely alkaline environments, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strongly alkali-resistant soil-derived urease-producing Bacillus and its application in microbial mineralization. Background Technology

[0002] Microbially Induced Carbonate Precipitation (MICP) is a novel environmentally friendly technology that utilizes microbial metabolic activity to promote mineral formation. With its core advantages of being green, efficient, and environmentally friendly, it has become a research hotspot and technological frontier in fields such as soil improvement, environmental pollution control, and building material repair. This technology regulates the mineral crystallization process through microbial metabolic activity, inducing the formation of highly stable mineral precipitates such as calcium carbonate. This enables functions such as soil reinforcement, heavy metal ion fixation, and crack self-repair. Compared to traditional chemical treatment technologies, it has significant advantages such as low energy consumption, no secondary pollution, and wide applicability, demonstrating enormous development potential in ecological environmental protection and engineering applications.

[0003] In the MIP (Microbial Infusion Propagation) technology system, urease-producing microorganisms are the core functional carriers. They catalyze the hydrolysis of urea to generate ammonia and carbonate ions, raising the pH of the reaction system. This ammonia then combines with calcium ions in the environment to form calcium carbonate precipitate. This reaction pathway has been proven to be one of the most efficient and widely used mineralization mechanisms. Some Bacillus strains, due to their high urease production capacity, can catalyze the hydrolysis of urea to generate carbonate ions, inducing calcium carbonate precipitation in the presence of calcium ions. They have become key functional strains in MIP technology.

[0004] However, the growth and enzyme activity of conventional Bacillus strains are often inhibited in alkaline environments (pH>10.0), limiting their application in alkaline soils or highly alkaline industrial environments. Therefore, screening novel strains with strong alkali tolerance and high urease activity is of great significance for expanding the applicable environments of microbial mineralization technology. Summary of the Invention

[0005] The purpose of this invention is to provide a highly alkali-resistant, urease-producing soil-derived Bacillus and its application in microbial mineralization, thereby addressing the problems existing in the prior art. This strain can survive stably under extremely alkaline conditions, maintain effective enzyme production activity, and exhibits significant biomineralization efficiency.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a soil-derived Bacillus strain that is strongly resistant to alkali and produces urease ( SolibacillusThe soil-derived Bacillus sp. was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37020.

[0008] The present invention also provides the application of the above-mentioned soil-derived Bacillus in microbial mineralization.

[0009] The present invention also provides the application of the above-mentioned soil-derived Bacillus in the preparation of biomineralizing agents.

[0010] Furthermore, the biomineralizing agent is suitable for microbial mineralization treatment in an alkaline environment with a pH of not less than 10.

[0011] The present invention also provides a biomineralizing agent, the active ingredient of which includes the above-mentioned soil-derived Bacillus.

[0012] Furthermore, the biomineralizing agent also includes agent excipients.

[0013] Furthermore, the excipients for the microbial agent include carriers, activity protectants, nutritional supplements, or dosage form optimization excipients.

[0014] Furthermore, the dosage form optimization excipients include dispersants, binders, wetting agents, or fillers.

[0015] The present invention also provides a composition for use in microbial mineralization, comprising the substances described in (1) and (2) below:

[0016] (1) Urea;

[0017] (2) The above-mentioned soil-derived Bacillus or biomineralizing agents.

[0018] The present invention also provides the application of the above-mentioned biomineralizing agents or compositions in microbial mineralization.

[0019] The present invention discloses the following technical effects:

[0020] This invention provides a soil-derived Bacillus strain ( Solibacillus MIP07 (sp.) breaks through the technical bottleneck of conventional Bacillus growth and limited enzyme activity in a strongly alkaline environment, demonstrating three core technological advantages:

[0021] (1) Outstanding alkali resistance: This strain can survive stably under extreme alkaline conditions of pH 12.0-12.5, which solves the problem that traditional strains are difficult to function in high alkaline environments and greatly expands the applicable scenarios of microbial mineralization technology.

[0022] (2) Excellent urease activity: It can still maintain efficient urea hydrolysis capacity at pH 12.0, providing sufficient reaction substrate for calcium carbonate precipitation induction, and still maintain effective enzyme production activity at pH 12.5.

[0023] (3) Significant mineralization efficiency: When working synergistically with urea and calcium ions in an alkaline system, the conversion rate of insoluble calcium is as high as 67.8% under pH 12.0 conditions, and can reach 35.6% even in the harsh environment of pH 12.5, which can efficiently induce the formation of calcium carbonate precipitation.

[0024] This strain and its derived biomineralizing agents and compositions have irreplaceable application value in the fields of microbial mineralization, such as alkaline soil reinforcement, heavy metal pollution remediation, and self-healing of building materials in highly alkaline environments. They provide efficient and green microbial solutions for environmental governance in extremely alkaline environments and have broad application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0026] Figure 1 This is a colony morphology diagram of strain MICP07.

[0027] Figure 2 This is a plate diagram of urease production by strain MICP07.

[0028] Figure 3 A phylogenetic tree diagram;

[0029] Figure 4 The graph shows the results of the urease activity and alkali resistance tests. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Terminology Explanation:

[0036] Microbial mineralization refers to the technology that uses the metabolic activities of microorganisms to regulate the mineral crystallization process, inducing inorganic ions (such as calcium ions) in the environment to combine with metabolic products (such as carbonate ions) to form stable mineral precipitates (such as calcium carbonate). It has the characteristics of being green and environmentally friendly, having a mild reaction, and being widely adaptable, and is widely used in soil reinforcement, pollution remediation, building self-healing and other fields.

[0037] Urease: A nickel-containing metalloenzyme that catalyzes the hydrolysis of urea. It can decompose urea into ammonia and carbonate ions, raising the pH of the reaction system and providing the necessary conditions for calcium carbonate precipitation. It is the core functional enzyme in MICP technology, and its activity directly determines the mineralization efficiency.

[0038] Enrichment culture: A culture method that utilizes the selectivity of culture medium components and culture conditions (such as pH, temperature, and substrate concentration) to promote the rapid proliferation of target microorganisms and increase their proportion in mixed bacterial communities. In this invention, highly alkali-resistant urease-producing strains are enriched under high concentrations of urea and strongly alkaline conditions.

[0039] Single colony purification: The process of isolating a single colony with a uniform genetic background from a mixed microbial community by methods such as streak plating or dilution plating. It is a key step in the isolation and preservation of microbial strains and can ensure the purity and stability of the strain.

[0040] TSA (Tryptic Soy Agar): A medium rich in tryptone soy agar, containing tryptone, soy peptone, agar, etc. It is nutritionally complete and suitable for the culture, preservation, and purification of a variety of bacteria. It is a commonly used general-purpose culture medium in microbiology laboratories.

[0041] 16S rRNA gene sequencing: Based on the high conservation and species specificity of bacterial 16S rRNA genes, it is a molecular biological method for identifying bacterial species through PCR amplification, sequencing and sequence alignment analysis, and is one of the gold standards for microbial classification and identification.

[0042] Phylogenetic tree: An evolutionary relationship diagram constructed based on the homology differences of biological macromolecules (such as 16S rRNA gene sequences). It can intuitively reflect the kinship between the target strain and known species, and provide an important basis for the classification and attribution of strains.

[0043] Biomineralizing agents: Microbial preparations made with functional microorganisms as the core active ingredients and appropriate excipients. They can be directly applied to microbial mineralization scenarios and have the characteristics of convenient use, stable activity, and controllable mineralization efficiency.

[0044] Microbial excipients: A general term for auxiliary components in biomineralized microbial agents other than the core functional strains. They are used to optimize the physicochemical properties of microbial agents, protect the activity of strains, and improve application effects. According to their functions, they can be divided into categories such as carriers, activity protectants, nutritional supplements, and dosage form optimization excipients.

[0045] Carrier: An inert or functional matrix in bacterial agents that carries microbial cells. It can provide attachment sites for the cells and maintain the morphological stability of the bacterial agent. Common types include natural or modified materials such as diatomaceous earth, vermiculite, corn cob powder, and attapulgite.

[0046] Active protectants: These are components used to protect microorganisms and maintain their metabolic activity during storage, transportation, and application. They can alleviate damage to the microbial cells caused by environmental stresses (such as high temperature, high alkali, and dryness). Common types include glycerol, sucrose, trehalose, and skim milk powder.

[0047] Nutritional supplements: These are ingredients that provide nutritional support for the rapid proliferation and metabolic function of microorganisms in the application environment. They typically contain carbon sources, nitrogen sources, and minerals, such as glucose, peptone, and potassium dihydrogen phosphate, which can improve the mineralization efficiency and duration of action of microbial agents.

[0048] Formulation optimization excipients: These are components used to improve the formulation characteristics (such as dispersibility, binding and wetting) of microbial agents to adapt to different application scenarios. They include dispersants (such as sodium lignosulfonate), binders (such as sodium carboxymethyl cellulose), wetting agents (such as Tween-80), fillers (such as talc), etc., which can expand the application adaptability of microbial agents in different carriers such as soil and building materials.

[0049] Example 1

[0050] 1. Isolation and purification of bacterial strains

[0051] Alkaline soil samples (pH ≈ 12) were initially screened using urea medium (urea 2 g / L, peptone 1 g / L, glucose 2 g / L, potassium dihydrogen phosphate 2 g / L, NaCl 5 g / L, agar 20 g / L, and phenol red indicator, pH 12.5). Enrichment culture was then performed using high-concentration urea medium (urea 20 g / L, peptone 1 g / L, glucose 2 g / L, potassium dihydrogen phosphate 2 g / L, NaCl 5 g / L, and phenol red indicator, pH 12.5) and incubated at 30℃ and 220 rpm for 48 hours with shaking. The enriched solution was plated onto urea plates and incubated at 30℃ for 48 hours. Single colonies with a clear color change zone (from yellow to red) were picked and repeatedly streaked for purification, yielding a purified strain named MICP07. Strain MICP07 was streaked onto TSA plates for preservation. Figure 1 As shown. The morphological characteristics of the strain are: forming milky white, opaque colonies with neat edges on nutrient agar plates; producing urease and turning red in urea-containing and phenol red-containing media. Figure 2 ).

[0052] 2. Strain identification

[0053] MIP07 was sent to a biotechnology company for 16S rRNA gene amplification and sequencing. The sequencing results are shown in SEQ ID NO.1.

[0054]

[0055] A phylogenetic tree was constructed based on the 16S rRNA gene sequence. The results are shown below. Figure 3 The results showed that the strain MIP07 of this invention is similar to soil-derived Bacillus (…). Solibacillus The similarity to sp.) GG 162 was 100%, ultimately confirming that MIP07 belongs to soil-derived Bacillus (sp.) Solibacillus sp.).

[0056] 3. Strain preservation

[0057] Soil-derived Bacillus ( Solibacillus sp.)MICP07 was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37020.

[0058] Example 2

[0059] MIP07 was inoculated into urea liquid medium at pH 12.0-12.5 and cultured at 30°C for 24 hours. The final pH of the culture medium and the urease activity (expressed as urea decomposition rate) were then measured.

[0060] The results showed that MIP07 maintained high urease activity at pH 12.0, indicating its strong alkali resistance and urea hydrolysis ability. It could also produce enzyme at pH 12.5, but the enzyme activity was lower than at pH 12.0. The test results are as follows: Figure 4 As shown, from left to right, the pH values ​​are 12, 12.5, and 9 (without urea).

[0061] Example 3

[0062] After culturing, MICP07 cells were isolated and resuspended in sterile water to 10⁻⁶. 8 CFU / mL was used to obtain bacterial suspensions. At an inoculum size of 1%, the bacterial suspensions were inoculated into liquid culture media containing urea and calcium chloride (5 g / L urea and 10 g / L CaCl2) at pH 12 and 12.5, respectively, and incubated at 30°C for 9 h. A blank control without inoculation was also provided. After incubation, the precipitate was collected by centrifugation. The conversion rate of insoluble calcium was then determined using an acid dissolution-titration method. This involved adding excess dilute hydrochloric acid solution to completely dissolve the insoluble calcium precipitate into calcium ions, centrifuging to remove the bacterial cells, and then using the supernatant. The amount of calcium ions was determined by EDTA complexometric titration, converted to the content of insoluble calcium precipitate, and then calculated.

[0063] The test results are shown in Table 1. The results show that MIP07 has the ability to induce calcium carbonate precipitation. Under pH 12 conditions, the conversion rate of insoluble calcium reaches 67.8%, and under pH 12.5 conditions, the conversion rate of insoluble calcium can also reach 35.6%.

[0064] Table 1 Results of the test on the insoluble calcium mineralization capacity of the strain

[0065]

[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A soil-derived Bacillus species with strong alkali resistance and urease production ( Solibacillus sp.), characterized in that, The soil-derived Bacillus was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37020.

2. The application of soil-derived Bacillus as described in claim 1 in microbial mineralization.

3. The application of the soil-derived Bacillus as described in claim 1 in the preparation of biomineralizing agents.

4. Use according to claim 3, characterized in that, The biomineralizing agent is suitable for microbial mineralization treatment in alkaline environments with a pH of not less than 10.

5. A biomineralization agent, characterized in that, The active ingredient includes the soil-derived Bacillus as described in claim 1.

6. The bio-mineralization agent of claim 5, wherein, The biomineralizing agent also includes agent excipients.

7. The bio-mineralization agent of claim 6, wherein the bio-mineralization agent is characterized by, The excipients for the microbial agent include carriers, activity protectants, nutritional supplements, or dosage form optimization excipients.

8. The bio-mineralization agent of claim 7, wherein, The dosage form optimization excipients include dispersants, binders, wetting agents, or fillers.

9. A composition for use in microbial mineralization, characterized in that, Including the following substances (1) and (2): (1) Urea; (2) The soil-derived Bacillus as described in claim 1 or the biomineralizing agent as described in any one of claims 5-7.

10. The use of a biomineralizing agent as described in any one of claims 5-7 or the composition as described in claim 9 in microbial mineralization.

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