Sporosarcina lutea j4 and its application

Abstract

The application discloses a kind of light yellow sporosarcina J4 and its application, belong to microbial technical field.The strain preservation number is GDMCC No.67388, its core feature is in: first, the strain has high urease activity, but growth does not depend on urea, can be cultured in conventional medium with high efficiency and low cost;Second, the strain can quickly promote the formation of stable calcite crystal form during the induction of calcium carbonate precipitation, and the relative content of calcite in the product mineralized for 4 hours is significantly higher than that of common comparative strains, thereby greatly improving the long-term stability of the mineralized product.The application also provides a microbial preparation comprising the strain, a method for preventing wind and sand fixation using the strain, and a method for improving the stability of MICP products by using the strain.The strain has excellent comprehensive performance and has broad industrial application prospects in the fields of desertification prevention and control, geotechnical engineering reinforcement, etc.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a kind of light yellow spore eight ball J4 and its application, more particularly to a kind of light yellow spore eight ball J4 with high urease activity and excellent calcium carbonate precipitation capacity and its application in microbial induced calcium carbonate precipitation (MICP) technology, especially in wind prevention and sand fixation, rock-soil reinforcement, ecological restoration and improvement of mineral product stability. BACKGROUND

[0002] Land desertification is the most serious ecological and environmental problem in the world today. This process leads to a sharp decline in vegetation cover, a sharp decline in biodiversity, soil structure destruction, loss of soil fertility, and ultimately the evolution of land with production capacity into desert.

[0003] To address this challenge, a variety of sand-fixing technologies, including physical, chemical and biological, have been developed and applied. Physical sand fixation (such as setting up grass grid sand barriers, high vertical sand barriers, and covering gravel) can quickly and effectively fix the surface sand, creating conditions for plant restoration, but it has high material cost, large engineering quantity, easy aging and difficulty in improving soil quality. Chemical sand fixation is a method that forms a solidified shell on the surface of sand by spraying chemical consolidating agents (such as asphalt emulsion, synthetic polymer, etc.) to achieve the purpose of wind resistance and erosion resistance. This method has a quick effect on sand fixation, but most chemical agents are expensive, cause secondary pollution, and damage soil permeability. Compared with the above two methods, biological sand fixation (mainly through planting drought-tolerant plants) is considered the most fundamental and sustainable solution. It can effectively restore vegetation, improve soil, and rebuild ecosystems, but its success is highly dependent on harsh water conditions. In extremely dry and barren sandy lands, plants are difficult to establish, have low survival rates, and grow slowly, especially in the early stages, which are easily affected by wind erosion and severely affect their growth, greatly limiting their application effect. Therefore, using a certain sand-fixing technology to fix the sand first and then planting plants is a more effective way of biological sand fixation.

[0004] Under this background, microbial induced calcium carbonate precipitation technology (MICP) as a new and environmentally friendly biological sand-fixing technology has received widespread attention in recent years. This technology uses the metabolic activity of specific microorganisms to produce carbonate, which combines with calcium ions in the environment to form calcium carbonate crystals, thereby cementing loose sand particles together and significantly enhancing the wind erosion resistance of sand. Microbial sand fixation has unique advantages such as natural material source, eco-friendly process, adjustable cementing strength, and to some extent, improvement of soil microenvironment.

[0005] However, the large-scale application of MICP technology still faces bottlenecks:

[0006] Economic efficiency and convenience of strain cultivation: Many reported high urease-active strains (such as certain Pasteurella multocida and other related strains) are highly dependent on urea as a nitrogen source during growth. Urea is unstable at high temperatures and cannot be autoclaved with conventional culture media. It must be added aseptically after being filtered and sterilized separately, which significantly increases the process complexity, operating costs, and contamination risks of large-scale industrial cultivation.

[0007] Long-term stability of mineralized products: Microbially induced calcium carbonate is often predominantly metastable aragonite, which gradually transforms into thermodynamically stable calcite under natural environmental conditions (especially wet-dry cycles and temperature changes). This transformation process is accompanied by changes in volume and structure, which may lead to microcracks or strength reduction in the solidified body, affecting the durability of engineering results.

[0008] Insufficient overall performance of strains: An ideal MIP engineered strain needs to possess high urease activity, rapid growth capacity, wide environmental adaptability (such as alkali and temperature tolerance), and the ability to induce the formation of stable mineralization products. Existing strains often fail to meet all of these requirements simultaneously, or have significant shortcomings in one aspect.

[0009] Therefore, developing a high-performance MICP strain that can overcome the above-mentioned defects, namely, grow efficiently in inexpensive culture media that do not rely on urea and can dominate the rapid formation of stable calcite precipitates, is of great significance for promoting this technology from the laboratory to engineering practice. Summary of the Invention

[0010] To address the problems of existing technologies, the purpose of this invention is to provide a strain of pale yellow spore-forming *Dystrophus occulta* with high urease activity and excellent calcium carbonate precipitation ability. Sporosarcina luteola J4 and its application in microbial induced calcium carbonate precipitation (MICP) technology, especially in windbreak and sand fixation, soil and rock reinforcement, ecological restoration and improving the stability of mineral products.

[0011] This invention is achieved through the following technical solution:

[0012] In a first aspect, the present invention provides a high-performance light yellow spore-forming *Dystrophus occulta* (… Sporosarcina luteola The strain J4 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on December 1, 2025, with accession number GDMCC No. 67388. Its taxonomic name is Sporosarcina luteola.

[0013] The 16S rRNA gene sequence of the strain is shown in SEQ ID NO.1;

[0014] The strain J4 of the present application has a clear genetic identity, and is identified as Paenibacillus luteus by 16S rRNA gene sequence analysis Sporosarcina luteola ).

[0015] At 37℃, the conductivity change value of its bacterial solution reacting with urea solution for 5 minutes can reach 988 mS / cm, which has a high urea decomposition ability, indicating that it has high urea enzyme activity.

[0016] The strain can grow well in a conventional bacterial culture medium (such as NB culture medium) without urea, and the OD 600 peak value reaches 1.39, and the lag phase is short. This feature completely avoids the complex sterilization step of urea-dependent strains during the culture process, and greatly reduces the production cost.

[0017] The strain can effectively induce calcium carbonate precipitation, and the precipitation amount is about 0.3g in 24 hours; when used for sand fixation, the surface strength of sand soil can be increased to more than 1.38 kg / cm², and has high mineralization and consolidation ability.

[0018] The strain can significantly promote the rapid formation of stable calcite crystal form. After mineralization for 4 hours, the relative content of calcite in the product is higher than 26.6%, which is much higher than that of the comparative strain at the same period, so as to greatly improve the long-term durability of the MICP reinforced body.

[0019] The strain can still effectively mineralize at a wide range of pH 5.0-9.84 and a temperature as high as 50℃, which is especially suitable for arid and hot environments, indicating that it has strong environmental adaptability.

[0020] In a second aspect, the present application provides a microbial preparation comprising live bacteria, spores or culture of the Paenibacillus luteus J4.

[0021] In a third aspect, the present application provides the use of the strain J4 or the microbial preparation in microbial induction of calcium carbonate precipitation, including but not limited to wind prevention and sand fixation, soil reinforcement, crack repair, historical building protection or heavy metal fixation.

[0022] In a fourth aspect, the present application provides a method for microbial mineralization sand fixation, comprising the following steps:

[0023] (1) activating culture of the Paenibacillus luteus J4 to obtain a bacterial suspension with OD 600 ≥ 0.8;

[0024] (2) apply the bacterial suspension obtained in step (1) and a cementing solution to the sand to be consolidated or the surface; the cementing solution is a mixed solution of calcium chloride and urea, with a calcium ion concentration of 0.4-0.6 mol / L; the volume ratio of the bacterial suspension to the cementing solution is 0.5-1:1;

[0025] (3) maintain at a temperature of 20-50℃ for 2-5 days to induce the bacterial strain to generate calcium carbonate precipitate and cement the sand particles.

[0026] Preferably, the activated culture in step (1) uses a culture medium without urea. In step (2), the bacterial suspension and the cementing solution can be mixed in advance before application, or applied sequentially.

[0027] In a fifth aspect, the present application provides a method for improving the stability of the product of microbial-induced calcium carbonate precipitation, characterized in that the mineralization is performed using the S. luteola J4 to promote the rapid formation of stable calcite crystal form.

[0028] Compared with the prior art, the present application has the following significant advantages:

[0029] 1. The strain J4 successfully solves the common coupling problem of "high urease activity" and "urea dependence". Its excellent growth ability in urea-free medium enables the production of bacterial agents to completely use conventional and inexpensive culture medium and sterilization process, significantly simplifying the process and reducing costs, and removing a key economic obstacle for large-scale engineering application of MICP technology.

[0030] 2. The present application first realizes effective regulation of the crystal form of the MICP product by providing a specific strain (J4). J4 can quickly generate a high proportion of stable calcite, reducing the content of metastable vaterite from the source, thereby potentially fundamentally improving the long-term weathering and aging resistance of the consolidated body and ensuring the durability of the reinforcement effect.

[0031] 3. The J4 strain integrates high activity, high growth efficiency, strong environmental tolerance, and excellent immediate consolidation strength, and its comprehensive performance is superior to or equivalent to that of existing high-performance model strains (such as S. pasteurii), and it exhibits unique advantages in product stability, making it an ideal MICP engineering strain.

[0032] 4. Based on its excellent characteristics, the strain and the corresponding method are not only suitable for desertification prevention and control, but also can be widely used in slope stabilization, foundation improvement, cultural relic restoration, heavy metal contaminated site solidification / stabilization, and other fields.

[0033] The strain J4 involved in the present application has been preserved in Guangdong Microbial Culture Collection Center (GDMCC) on December 1, 2025, the address of the preservation center is: Experimental Building, No. 100, Martyrs' Road, Yuexiu District, Guangzhou, Guangdong Province, China, the preservation number is: GDMCC No. 67388, and the taxonomic name is: Sporosarcina luteola. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Comparison results of urease activities of different strains (conductivity method);

[0035] Figure 2 is a comparison result of calcium carbonate precipitation abilities of different strains;

[0036] Figure 3 is a comparison result of surface strength of sand consolidation of different strains;

[0037] Figure 4 Growth curves of different strains in NB medium (a) and NBU medium (b);

[0038] Figure 5 Microscopic morphologies of mineralization products of different strains at 4h (a) and 20h (b);

[0039] Figure 6 is an XRD pattern of mineralization products of different strains: (a) SP; (b) J4; (c) T2; (d) relative contents of calcite and vaterite;

[0040] Figure 7 A phylogenetic tree of strain J4 constructed based on 16S rRNA gene sequences;

[0041] Figure 8 Colony morphology photos, scanning electron microscope pictures and gram staining photos of strain J4 on NBU medium;

[0042] Figure 9 Results of mineralization condition optimization in Example 3: (a) biomass; (b) calcium ion concentration; (c) pH of cementation liquid; (d) time; (e) temperature. DETAILED DESCRIPTION

[0043] The present application will be further described in detail below in combination with the drawings and examples, but the present application is not limited in any way by the following description, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0044] The process, conditions, reagents, experimental methods, etc. for implementing the present application are all general knowledge and common sense in the art, and the present application does not have special limitations. The experimental methods not specified in the examples are usually performed according to the conventional conditions or the conditions recommended by the manufacturers.

[0045] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions provided in this specification and those provided in the patents, applications, and other literature incorporated by reference, the definition provided in this specification controls.

[0046] The solutions and media used in the following examples were prepared as follows:

[0047] (1) Solution Preparation

[0048] 1) Sterile Urea Solution: Urea 500 g, distilled water to 1000 mL volumetric flask, 0.22 mm sterile filter membrane filtration when used.

[0049] 2) Urea Solution: Urea 10 g, distilled water to 1000 mL volumetric flask.

[0050] 3) Glue solution: Take 55 g of calcium chloride and use distilled water to make up to 500 mL volumetric flask, 30 g of urea and use distilled water to make up to 500 mL volumetric flask, mix equal volume of calcium chloride solution and urea solution and store at 4°C. The concentration of glue solution is calculated as calcium ion.

[0051] (2) Medium Components

[0052] 1) NB Medium: Tryptone 5 g / L, Yeast extract 1.5 g / L, Sodium chloride 5 g / L, Beef extract 1.5 g / L. Solid medium is added with 20 g / L agar.

[0053] 2) NBU Medium: Tryptone 5 g / L, Yeast extract 1.5 g / L, Sodium chloride 5 g / L, Beef extract 1.5 g / L, after sterilization and cooling, add filtered urea solution to make the final concentration of urea 2% (w / v). Solid medium is added with 20 g / L agar.

[0054] 3) NBU solid screening medium: Add 8 mL of 0.2% phenol red solution as indicator to NBU solid medium.

[0055] Example 1 Strain Selection

[0056] 1. Acclimation and Screening

[0057] Collect the soil where plants are planted all year round and urea is applied, and use urea solution to continuously acclimate and enrich strains with high urease activity. Take 1 g of urea acclimated soil and dissolve it in 9 mL of sterile water, and use gradient dilution method to dilute the stock solution to 10 -2 ~10 -7, coated on NBU solid screening medium, 3 parallel for each gradient, and the plates were placed in a 30°C incubator for 48h, the growth of the colonies was observed, and the strains that could make the medium red and grow faster were selected. Single colonies were selected by plate streaking method, and the purified single bacteria could still make NBU medium turn red. Eight strains that grew faster and made NBU solid screening medium quickly change color were screened, and further screening was carried out.

[0058] Urea is decomposed into NH3 and CO2 under the action of urease, and further hydrolyzed in aqueous solution to generate NH4 + and CO3 2- , which increases the ion and charge concentration in the solution and enhances the conductivity. Therefore, the change of the conductivity is proportional to the decomposition amount of urea at a certain environmental temperature, so the activity of urease can be roughly inferred by the change of the conductivity. The strains obtained above and the purchased model strain SP were cultured in NBU liquid medium at 30°C and 180rpm for 24h, 4mL of bacterial solution was mixed with 36mL of urea solution in a 50mL centrifuge tube, and the initial conductivity value of the mixed solution was measured by a conductivity meter. The conductivity value was measured after 5min in a 37°C water bath, and the change of the conductivity of the mixed solution was calculated. Figure 1 It can be seen that the conductivity change values of strains SP, J4 and T2 within 5min are 1196mS / cm, 988mS / cm and 1474mS / cm respectively, which are significantly higher than those of other strains, so it is judged that the urease activity of these three strains is higher.

[0059] 2. Calcium carbonate precipitation capacity

[0060] Strains SP, J4 and T2 were cultured in NBU liquid medium at 30°C and 180rpm, 2mL of bacterial solution was added to 30mL of cementation liquid in a 50mL centrifuge tube and mixed, the mass of the centrifuge tube was accurately weighed and recorded as m1, the reaction system was placed in a 30°C incubator for mineralization, and after reaching the mineralization time, it was placed in a centrifuge at 8000rpm for 8min, and after removing the supernatant, it was resuspended with distilled water. NH4Cl was removed three times, the centrifuge tube was placed in an oven at 100°C and dried to constant weight, and the mass was weighed and recorded as m2. The mass of the precipitate calcium carbonate was calculated as m2-m1.

[0061] The calcium carbonate precipitation capacity of model strain SP and J4 and T2 screened in 1 above within 72h is as follows: Figure 2As shown, the mineralized calcium carbonate precipitates gradually increased over time, and the 24-hour mineralized precipitates were 0.3 g, 0.3 g, and 0.47 g, respectively. In combination with the conductivity changes, it was shown that T2 had higher urease activity and stronger urea decomposition ability, and produced more calcium carbonate precipitates in the same time. The experimental results of SP and J4 showed that although the urease activity of SP was higher, the amounts of calcium carbonate precipitates produced by SP and J4 were equal at 24 h, indicating that urease activity was not the only factor that could determine the amount of calcium carbonate precipitates. In addition to relying on urease activity to promote urea decomposition to further catalyze the conversion of calcium ions into calcium carbonate precipitates, other strain-specific factors such as extracellular proteins and strain zeta potential may also affect the conversion of precipitates. At 72 h, the amounts of calcium carbonate precipitates in the three systems were close to the maximum theoretical precipitate, i.e., the calcium ions in the cementing liquid were completely converted into calcium carbonate precipitates.

[0062] 3. Surface strength of sand consolidation

[0063] In the sand consolidation process, the surface strength of sand consolidation is positively correlated with the wind erosion resistance, and the greater the surface strength of sand consolidation, the stronger the wind resistance after sand consolidation. Therefore, the surface strength of sand after sand consolidation by different strains was determined. 1180 g ± 20 g of sand was weighed and placed in a sand consolidation mold, 30 mL of cementing liquid and 30 mL of bacterial liquid were sprayed onto the surface, and the sprayed sand was placed in a 30°C incubator for 5 days. The surface hardness of the solidified soil was measured using a soil hardness meter ZP-50.

[0064] The surface strength of sand after sand consolidation by different strains is shown in Table 3. Figure 3 As shown in Table 3, the surface strengths of sand after treatment with SP, J4, and T2 were 1.39 kg / cm 2 , 1.38 kg / cm 2 , and 1.19 kg / cm 2 , respectively. The surface strengths of sand after treatment with SP and J4 were significantly higher than that after treatment with T2, and were significantly higher than that of the blank group treated with distilled water (0.254 kg / cm 2 ). Although the urease activity of J4 was slightly lower than that of SP, J4 had comparable ability to SP in catalyzing calcium carbonate precipitation and consolidating sand to improve surface strength.

[0065] 4. Growth curve

[0066] After 24 h of culture of each strain (SP, J4, and T2) in NBU liquid medium at 30°C and 180 rpm, 10 After L to 100-hole plate, 190 uL of NBU liquid medium and NB liquid medium were added, and the un-inoculated liquid medium was used as a blank control. To ensure the reliability of the experimental data, three strains were prepared in 10 parallel, and the growth curve instrument was used to measure the absorbance value at 600 nm wavelength, the temperature was set to 30°C, and the measurement was performed every 2 h. The measured data was plotted as a growth curve of the strain with time as the horizontal coordinate and OD 600 as the vertical coordinate.

[0067] The growth curve determination results are shown in Figure 4 The lag phase of J4 and T2 in NBU medium is relatively short, and the lag phase cannot be reflected in the figure. They quickly adapt to the new environment and enter the logarithmic growth phase, and the growth rate difference is not large. At 35 h, they enter the stationary phase and then the biomass decreases. The highest OD 600 value of T2 appears at 32 h (1.39), the highest value of SP appears at 12 h (1.20), and the highest value of J4 appears at 32 h (1.25). In NB medium, the lag phase of SP and J4 is short, and they quickly enter the logarithmic growth phase, which is consistent with that in NBU. The highest OD 600 value of SP appears at 38 h (1.30), and the highest OD 600 value of J4 appears at 32 h (1.39). The lag phase of T2 is longer, and the OD 600 value of T2 is only 0.487 at the end of the experiment. This experiment confirms that J4 does not depend on urea for growth, and in the urea-free medium, it grows more vigorously than the model bacteria SP, while T2 is a urea-dependent strain.

[0068] 5. Crystal type and morphology of strain mineralization precipitate

[0069] Microbial mineralization will undergo a crystal type conversion process, from amorphous calcium carbonate to metastable vaterite, and finally to stable calcite structure. The conversion rate of calcium carbonate crystal type will directly affect the stability of the calcium carbonate precipitate formed in the process. To further explore the types of mineralization products of different strains, scanning electron microscopy-energy dispersive spectrometer (SEM-EDS) and X-ray crystal diffraction (XRD) were used to characterize the mineral crystal type produced by strain mineralization. The SEM results are as follows Figure 5As shown in Figure 2, the main crystal form of the mineralization products of the three strains is all vaterite. As a metastable phase of calcium carbonate, vaterite has a typical spherical or spheroidal morphology. These spheres are mostly composed of 10-50 nanometer microcrystals that are randomly stacked, and the surface of the spheres is rough and porous. The thermodynamic stability of vaterite is low, and it is easy to gradually transform into other crystal forms in a water environment. Calcite has a rhombohedron as the core morphology, and the crystal surface is smooth and shiny with clear corners. Calcite is the most stable crystal form, and only part of the calcite morphology was observed in the 20h mineralization product of the J4 strain. No calcite was found in the mineralization products of the other two strains.

[0070] The XRD spectrum analysis results of the three strains are shown in Figure 3. Figure 6 As shown in Figure 3, the phase identification results show that the sediments of the three strains all exist in two forms of calcium carbonate precipitates. In terms of the relative proportion of calcite and vaterite, the relative content of calcite in the mineralization products of SP, J4 and T2 at 4h is 12.2%, 26.6% and 13.8%, respectively. The content of calcite in the mineralization products of the three strains at 20h is 32.1%, 31.2% and 26.1%, respectively. The content of calcite in the mineralization products of SP and J4 is comparable, and both are higher than that of T2. This phenomenon indicates that the crystal transformation rate of J4 is the fastest. Although vaterite will gradually transform into calcite, this transformation process usually needs to be carried out in a solution. When using this technology for wind prevention and sand fixation, because the desert environment is dry and hot, water will evaporate in a short time. The vaterite formed by SP and T2 may not have enough time to transform into calcite, and thus the vaterite exists in the form of a metastable state, thereby reducing the solidification strength and stability of the technology. Therefore, by comprehensively considering the urease activity, biomass, surface strength of the solidified sand soil and the transformation speed of calcite of the strains, J4 is selected as the sand-fixing strain.

[0071] Example 2 Strain J4 Identification

[0072] The bacterial strain was cultured in liquid NBU medium at 30°C and 180 rpm for 24 h. Bacterial DNA was extracted using a TIANGEN biological kit and used as a PCR template for chain amplification. Universal primers were used for amplification, and the fragment size of the PCR amplification products was detected by agarose gel electrophoresis (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2); 1492R: 5'-GGCTACCTTGTTACG ACTT-3' (SEQ ID NO. 3) were universal primers). The PCR reaction volume was 25 μL, and the amplification parameters were: pre-denaturation at 95°C for 5 min; followed by denaturation at 95°C for 45 s; annealing at 55°C for 45 s; extension at 72°C for 50 s; and final extension at 72°C for 10 min, for a total of 36 cycles. The successfully amplified samples were sent to Qingke Biotechnology Co., Ltd. (Beijing) for 16S rRNA sequencing. The obtained sequence results were submitted to the NCBI database for BLAST comparison analysis. The strain sequences with high similarity to the target strain sequencing results were downloaded. The target sequence and its similar sequences were aligned and cut using MEGA 11 software to find the optimal model and then a phylogenetic tree was constructed.

[0073] The 16S rRNA gene sequence of J4 is shown in SEQ ID NO.1.

[0074] BLAST sequence alignment results showed that the 16S rRNA gene sequence of strain J4 was consistent with... Sporosarcina luteola strain G2-54 showed the highest similarity (99.86%), and the gene phylogenetic tree is as follows: Figure 7 As shown, it was therefore identified as *Sclerotinia spp.* Sporosarcina luteola This strain was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on December 1, 2025, with accession number GDMCC No. 67388.

[0075] Colonies formed by strain J4 on NBU solid medium are shown below. Figure 8 As shown, the colonies have regular, small, round edges, a smooth, creamy-colored, dull, opaque surface, and are flat without any protrusions. They form spores within the cells, and the spore walls are thick. SEM images show that they are rod-shaped, with a cell length of approximately 1.5 μm. After Gram staining, the cells turn purple, indicating that they are Gram-positive bacteria.

[0076] Referring to the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification", the physiological and biochemical characteristics of strain J4 were tested, and the experimental results are shown in Table 1.

[0077] Table 1. Physiological and biochemical properties of the strains

[0078]

[0079] Example 3 Single-factor optimization experiment of strain J4 mineralization conditions

[0080] To determine the optimal application parameters of strain J4 in the process of microbial induced carbonate precipitation (MICP), a systematic single-factor optimization experiment was carried out, taking the mass (g) and yield of calcium carbonate precipitation as the main evaluation indexes. Unless otherwise specified, the basic reaction conditions were fixed as follows: bacterial suspension (J4, cultured in NB medium for 24 h) and cementing liquid (0.5 M CaCl2 mixed with 0.5 M urea in equal volume) were mixed at a volume ratio of 1:1, the total volume was 40 mL, and the precipitation amount was determined after standing mineralization at 30°C for 24 h. The specific method is as follows:

[0081] 1. The influence of bacterial liquid concentration (biomass, OD 600 )

[0082] The calcium ion concentration of the cementing liquid was fixed at 0.5 mol / L, and the pH was natural. J4 strain was cultured to different growth stages, and OD 600 values of 0.2, 0.4, 0.6, 0.8, and 1.0 were prepared by centrifugation and resuspension. 20 mL of bacterial suspension with different OD 600 values were taken and mixed with 20 mL of cementing liquid (0.5 M Ca² + ) at 30°C for 24 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 8 min, the supernatant was discarded, the precipitate was washed with sterile distilled water three times to remove soluble salt, and was placed in a 100°C oven to dry to constant weight. The mass of calcium carbonate precipitation was weighed, and the yield of calcium carbonate was calculated.

[0083] The results are shown in Figure 9 a. When the OD 600 <0.8 of the bacterial liquid, the conversion rate increased significantly with the increase of biomass; when OD 600 ≥0.8, the conversion rate tended to be stable and reached a peak (93%). Therefore, the OD 600 of the bacterial liquid should not be less than 0.8 when applied.

[0084] 2. The influence of calcium ion concentration in cementing liquid

[0085] The OD 600=1.0. Different concentrations of calcium chloride solution (0.4, 0.8, 1.2, 1.6, 2.0 mol / L) were mixed with equal volume and molar concentration of urea solution to obtain the cementation solution with calcium ion concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mol / L, respectively. 20 mL of bacterial suspension was mixed with 20 mL of cementation solution with different calcium ion concentrations, and reacted at 30°C for 24 h. The precipitate was treated, dried and weighed by the same method.

[0086] The results are shown in Table 1. Figure 9 As shown in Table 1, in the range of 0.2-0.4 mol / L, calcium ions were almost completely converted, but due to the low calcium content itself, the production of calcium carbonate was also low. With the increase of calcium ion concentration, there was an inhibition phenomenon on urease activity. When the calcium ion concentration increased to 0.8 mol / L and 1 mol / L, the urease activity was inhibited, although the production of calcium carbonate was high, but the conversion rate of calcium ion was significantly reduced, and there was a waste of calcium ion. Therefore, the calcium ion concentration should be close to 0.5 mol / L in the actual application process.

[0087] 3. Effect of initial pH of cementation solution

[0088] The OD 600 =1.0, and the calcium ion concentration of the cementation solution was 0.5 mol / L. A small amount of HCl (1 M) or NaOH (1 M) solution was used to adjust the initial pH of the cementation solution (CaCl2 mixed with urea solution) to 5.0, 6.0, 7.0, 8.0, 9.0, 9.84 (natural pH of urea solution). 20 mL of bacterial suspension was mixed with 20 mL of cementation solution with different pH, and reacted at 30°C for 24 h. The precipitate was treated, dried and weighed by the same method.

[0089] The results are shown in Table 3. Figure 9 As shown in Table 3, in the wide range of pH 5.0 to 9.84, the precipitation amount of calcium carbonate and the conversion rate of calcium ion did not fluctuate significantly, indicating that the urease activity and mineralization ability of strain J4 had strong tolerance to pH change, and was suitable for various environments.

[0090] 4. Effect of mineralization time

[0091] The OD 600 =1.0, and the calcium ion concentration of the cementation solution was 0.5 mol / L. 20 mL of bacterial suspension was mixed with 20 mL of cementation solution, and reacted at 30°C. The mineralization time gradient was set to 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 84 h. At each time point, the corresponding reaction system was centrifuged, washed, dried and weighed.

[0092] The results are shown in Table 4. Figure 9As shown in Figure d, the amount of calcium carbonate precipitation increases rapidly with time from 0 to 48 hours, and the growth tends to slow down after 48 hours, indicating that the main precipitation process is basically completed within 48 hours. A mineralization time of 24-72 hours can be selected based on the actual engineering requirements for strength and construction period.

[0093] 5. The Influence of Mineralization Temperature

[0094] OD of fixed bacterial solution 600 =1.0, calcium ion concentration in the cementing solution 0.5 mol / L. Mix 20 mL of bacterial suspension with 20 mL of cementing solution and react at 30℃. Set the mineralization temperature gradient to 20℃, 30℃, 40℃, 50℃, and 60℃. Place the reaction system in a constant temperature incubator at different temperatures and allow it to stand for 48 h. Determine the amount of precipitate using the same method.

[0095] The results are as follows Figure 9 As shown in e, within the temperature range of 20-50℃, the amount of calcium carbonate precipitation continuously increases with increasing temperature, reaching a maximum value (approximately 0.865g) at 50℃, demonstrating that strain J4 maintains excellent mineralization ability at high temperatures, making it particularly suitable for field applications in summer or tropical and desert regions.

[0096] Based on the above results, the optimal range of conditions for strain J4 for MIP sand fixation was determined: bacterial culture OD 600 The optimal parameters for the practical engineering application of strain J4 are: ≥ 0.8; calcium ion concentration in the cementing solution: 0.4-0.6 mol / L (preferably 0.5 mol / L); initial pH of the cementing solution: a wide range of 5.0-9.84 is feasible; mineralization time: 24-48 h; mineralization temperature: 20-50℃, with better performance at higher temperatures (50℃).

[0097] Example 4: Application Case Study - Simulated Sand Fixation

[0098] J4 was cultured in NB medium until OD. 600 =1.0 to obtain the bacterial suspension. The cementing solution was a mixture of 0.5 M CaCl2 and 0.5 M urea in equal volumes. After mixing the bacterial suspension and cementing solution at a volume ratio of 1:1, the mixture was sprayed onto the sand surface and cured at 30℃ for 3 days. The resulting consolidated layer was dense, and simulated wind erosion tests showed that its wind erosion resistance was significantly better than that of the untreated sand.

[0099] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A pale yellow spore-forming Micrococcus ( Sporosarcina luteola J4, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 1, 2025, with accession number GDMCC No. 67388.

2. A microbial preparation, characterized in that, The product comprises live cells, spores, or cultures of the pale yellow spore-forming Micrococcus J4 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The culture was obtained by culturing in a urea-free medium.

4. The use of the pale yellow spore-forming Micrococcus J4 as described in claim 1 or the microbial preparation as described in any one of claims 2-3 in microbial-induced calcium carbonate precipitation.

5. The application according to claim 4, characterized in that, The applications include, but are not limited to, windbreak and sand fixation, soil reinforcement, crack repair, protection of historical buildings, or heavy metal fixation.

6. A method for microbial mineralization and sand fixation, characterized in that, Includes the following steps: (1) Activating the S. subflavus J4 of claim 1 to obtain a bacterial suspension with OD 600 ≥ 0.8; (2) Apply the bacterial suspension and cementing solution obtained in step (1) to the sand or surface to be consolidated; (3) Cultivate under suitable conditions to induce the bacterial strain to generate calcium carbonate precipitate and cement sand particles.

7. The method according to claim 6, characterized in that, In step (1), the culture medium used for activation culture is a urea-free culture medium.

8. The method according to claim 6, characterized in that, In step (2), the cementing solution is a mixed solution of calcium chloride and urea, with a calcium ion concentration of 0.4-0.6 mol / L; the volume ratio of the bacterial suspension to the cementing solution is 0.5-1:

1. In step (2), the bacterial suspension and the cementing solution are either pre-mixed and then applied, or applied sequentially. In step (3), the conditions for maintenance are: temperature 20-50℃, maintenance time 2-5 days.

9. A method for improving the stability of microbially induced calcium carbonate precipitation products, characterized in that, Mineralization was performed using the pale yellow spore-forming Micrococcus J4 as described in claim 1 to promote the rapid formation of a stable calcite crystal form.

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