A method for synergistically inducing calcium carbonate deposition solidification by bacillus subtilis and paenibacillus peoriae

Abstract

The application discloses a method for synergistically inducing calcium carbonate deposition and solidification by bacillus subtilis and sphaerotilus paratus. The method comprises the following steps: 1) adding a composite bacterial solution containing bacillus subtilis and sphaerotilus paratus to treat loose particles; and 2) adding a calcification solution to post-treat the treated loose particles. The application also provides bacillus subtilis with a preservation number of CCTCC NO: M 20231896. The composite bacterial solution containing bacillus subtilis and sphaerotilus paratus synergistically promotes the induced calcification efficiency of microorganisms, and improves the cementation performance, toughness and hardness of the calcification material.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microbial and material reinforcement engineering, specifically relating to a method for the synergistic induction of calcium carbonate deposition and solidification by Bacillus subtilis and Bacillus pasteurellus. Background Technology

[0002] Microbial-induced carbonate precipitation (MICP) is a common microbial mineralization phenomenon in nature. Currently, MICP is widely used in research fields such as building foundation reinforcement, concrete crack repair, and water conservancy project seepage prevention due to its good ecological compatibility.

[0003] The mechanisms of microbial-induced calcium carbonate deposition include photosynthetic organism-induced deposition, sulfate-reducing bacteria-induced deposition, nitrogen cycle-induced deposition, and other biochemical processes. Photo-nitrogen cycle-induced deposition further includes urea degradation and denitrification mechanisms. Representative microorganisms in the urea degradation mechanism are *Sporosarcina pasteurii* and *Bacillus sphaericus*. These microorganisms produce urease, a key protease with mineralization-inducing capabilities, through metabolism. When environmental urea levels are high, urea molecules are hydrolyzed into ammonia and carbamate molecules by urease within the bacteria. As an intermediate product, one molecule of carbamate spontaneously hydrolyzes into one molecule of carbonic acid and one molecule of ammonia within a short time. In this reaction, ammonia reacts with water, increasing the alkalinity of the aquatic environment. This increased alkalinity promotes the conversion of dissolved inorganic carbon into carbonate ions, ultimately leading to an increase in the concentration of carbonate ions in the environment, which, in the presence of a calcium source, results in the formation of calcium carbonate. Meanwhile, microbial cell surfaces typically carry a large number of negatively charged functional groups, thereby adsorbing positively charged Ca2+ from the solution. 2+ Ca 2+ When exposed to high concentrations of carbonate ions, calcium carbonate crystals are formed with organic matter such as bacteria as the core. After the crystals are deposited together, they play a role in strengthening and repairing the material. The model of inducing carbonate deposition by hydrolyzing urea with urease is the most direct and easiest to control in MICP. It is highly efficient and can induce the generation of a large amount of carbonate in a short time.

[0004] Two key enzymes in the microbial-induced calcification consolidation process are urease, which decomposes urea to produce NH3 and CO2, and carbonic anhydrase, which increases CO2 solubility and promotes CO2 dissolution. Previous research has mainly focused on the optimization and application of consolidation conditions using *Bacillus pasteurii*, *Bacillus spheroidae*, and *Bacillus pasteurii*. For example, CN101644047 discloses the use of a bacterial solution formed by *Bacillus pasteurii*, which, when mixed with urea and calcium chloride, is injected into sand particles, either separately or together, to cement and solidify the sand particles. The compressive strength of the solidified sand can reach 2 MPa. CN103725288A discloses a method for solidifying soil using *Bacillus mucilaginous* and MgO, with the compressive strength of the solidified soil sample reaching 4.8 MPa.

[0005] Two key enzymes in the microbial-induced calcification consolidation process are urease, which decomposes urea to produce NH3 and CO2, and carbonic anhydrase, which increases CO2 solubility and promotes CO2 dissolution. Previous studies have mainly focused on the optimization and application of consolidation conditions in *Bacillus pasteurellii*. However, it is worth noting that *Bacillus pasteurellii* has high urease activity but slightly low carbonic anhydrase activity. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for synergistic induction of calcium carbonate deposition and solidification by Bacillus subtilis and Bacillus pasteurellus, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention specifically adopts the following technical solution.

[0008] The first aspect of this invention protects a method for microbially induced calcium carbonate deposition and solidification, comprising the following steps:

[0009] 1) Add a compound bacterial solution containing Bacillus subtilis and Bacillus pasteurellium to treat loose particles;

[0010] 2) Add calcification solution to the loose particles after treatment for post-treatment.

[0011] This invention utilizes *Bacillus pasteurellii* to express urease, which hydrolyzes urea in the calcification solution to produce CO2 and NH3. Simultaneously, *Bacillus subtilis* expresses carbonic anhydrase to promote the reaction between CO2 and water, thereby rapidly increasing the pH and carbonate concentration of the bacterial cell microenvironment. This creates the alkaline environment required to induce calcium carbonate precipitation, leading to the deposition and solidification of calcium ions in the calcification solution into calcium carbonate. Furthermore, microbial cell surfaces typically possess numerous negatively charged functional groups, which adsorb positively charged calcium ions from the solution. 2+ Ca2+ When exposed to high concentrations of carbonate ions, calcium carbonate crystals are formed with organic matter such as bacteria as the core. After the crystals are deposited together, they can strengthen and repair the material.

[0012] In a first aspect of the invention, the loose particles are selected from soil particles or sand and gravel. The soil particles are selected from the soil from the pits containing cultural relics. The soil from the pits containing cultural relics refers to the soil on the surface of unearthed archaeological relics and the soil surrounding them, which conforms to the requirements of the "WW / T0081-2017 Standard for Testing Chemical Indicators of Soil at Archaeological Sites".

[0013] In a first aspect of the present invention, the Bacillus subtilis comprises a strain with accession number CCTCC NO: M 20231896. The carbonic anhydrase activity of the Bacillus subtilis is not less than 3.65 U / min.

[0014] In a first aspect of the present invention, the *Pasteurella multocida* comprises a strain with accession number ATCC11859.

[0015] In the first aspect of the present invention, the ratio of viable Bacillus subtilis to Bacillus pasteurellium in the composite bacterial solution can be (0.5–3):1, (0.5–1.5):1, (1.1–2.5):1, or (2.2–3):1. In some specific embodiments, the ratios are 0.5:1, 1:1, 2:1, and 3:1.

[0016] In a first aspect of the present invention, the number of viable Bacillus subtilis bacteria in the compound bacterial solution is ≥1×10⁻⁶. 8 cfu / mL.

[0017] In a first aspect of the present invention, the viable count of *Bacillus pasteurellii* in the composite bacterial solution is ≥1×10⁻⁶. 8 cfu / mL.

[0018] In a first aspect of the present invention, the total number of viable bacteria in the compound bacterial solution is ≥2×10⁻⁶. 8 cfu / mL.

[0019] In the first aspect of the present invention, the mass-to-volume ratio of the loose particles and the composite bacterial solution can be 1g:(5-20)mL, 1g:(5-12)mL, 1g:(10-15)mL, or 1g:(13-20)mL. In one specific embodiment, it is 1g:15mL.

[0020] In the first aspect of the present invention, the method for preparing the composite bacterial solution is as follows: 1) Bacillus subtilis is cultured in a first liquid culture medium to obtain Bacillus subtilis bacterial solution; Bacillus pasteurellii is cultured in a second liquid culture medium to obtain Bacillus pasteurellii bacterial solution;

[0021] 2) Centrifuge the Bacillus subtilis culture to obtain a bacterial precipitate; mix the bacterial precipitate with the Bacillus pasteurellium culture to obtain the composite culture.

[0022] This invention selects Bacillus subtilis with a carbonic anhydrase activity of 3.65 U / min and Bacillus pasteurellis with a urease activity of 4.16 mM / min. The two are compounded at a live bacteria ratio of (0.5-3):1. The resulting compound bacterial solution maintains high carbonic anhydrase and urease activity, with carbonic anhydrase activity of 2.45-3.35 U / min and urease activity of 1.84-4.12 mM / min. This is beneficial for the deposition and solidification of calcium carbonate in loose particles, while also improving the hardness of the loose particles.

[0023] Preferably, the first liquid culture medium has the following composition: yeast extract (2-8) g / L, peptone (6-14) g / L, and NaCl (6-14) g / L. In one specific embodiment, it is: yeast extract 5 g / L, peptone 10 g / L, and NaCl 10 g / L.

[0024] Preferably, the culture temperature of Bacillus subtilis can be (25-35)℃, (25-31)℃, (27-33)℃, or (30-35)℃. In one specific embodiment, it is 30℃.

[0025] Preferably, the culture time of Bacillus subtilis is (16-24) h, or (16-21) h, or (18-22) h, or (20-24) h. In one specific embodiment, it is 20 h.

[0026] Preferably, the second liquid culture medium comprises: beef extract (1-6) g / L, peptone (1-10) g / L, and urea (15-25) g / L. In one specific embodiment, it comprises: beef extract 3 g / L, peptone 5 g / L, and urea 20 g / L.

[0027] Preferably, the viable count of the *Pasteurella multocida* bacterial suspension is (0.63–2.21) × 10⁻⁶. 8 cfu / mL.

[0028] Preferably, the culture temperature for *Bacillus pasteurellus* can be (25–35)°C, (25–31)°C, (27–33)°C, or (30–35)°C. In one specific embodiment, it is 30°C.

[0029] Preferably, the culture time for *Bacillus pasteurellii* can be (24–48) h, (24–32) h, (30–38) h, or (36–48) h. In one specific embodiment, it is 40 h.

[0030] In a first aspect of the invention, the calcification solution is an aqueous solution of urea and calcium salt.

[0031] Preferably, the molar ratio of the calcium salt to urea is (1-3):1, or it can be (1-2.2):1, (1.8-2.6):1, or (2.2-3):1. In one specific embodiment, it is 1:1.

[0032] Preferably, the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium lactate, calcium formate, and calcium acetate.

[0033] More preferably, calcium chloride is used. In one specific embodiment, the concentration of urea is 0.5M and the concentration of calcium chloride is 0.5M, based on the total volume of the calcification solution.

[0034] In a first aspect of the present invention, the mass-to-volume ratio of the loose particles to the calcification solution can be 1g:(5-20)mL, 1g:(5-12)mL, 1g:(10-15)mL, or 1g:(13-20)mL. In one specific embodiment, it is 1g:15mL.

[0035] In the first aspect of the invention, steps 2) and 3) are repeated at least twice. Preferably, 12 to 18 times. In one specific embodiment, 15 times. This application achieves a layer-by-layer distribution of bacteria and calcium carbonate crystals on the surface of loose particles through multiple cycles, which is more conducive to promoting the cementation properties of loose particles and improving hardness.

[0036] In the first aspect of the present invention, before step 1) and before adding the composite bacterial solution, the loose particles are pretreated with a calcium salt aqueous solution. Pretreatment of the loose particles with a calcium salt aqueous solution before treatment facilitates the adsorption and fixation of bacteria in the subsequent composite bacterial solution onto the surface of the loose particles, preventing the composite bacterial solution from depositing at the bottom of the loose particles and causing uneven solidification of calcium carbonate deposition.

[0037] Preferably, the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium lactate, calcium formate, and calcium acetate.

[0038] More preferably, calcium chloride is used. The concentration of the calcium salt aqueous solution can be (0.01–0.2) mol / L, (0.01–0.06) mol / L, (0.04–0.12) mol / L, (0.09–0.18) mol / L, or (0.11–0.2) mol / L. In one specific embodiment, it is 0.05 M, i.e., 0.05 mol / L.

[0039] Preferably, the mass-to-volume ratio of the loose particles to the calcium salt aqueous solution can be 15g:(10-40)mL, 15g:(10-22)mL, 15g:(20-31)mL, or 15g:(30-40)mL. In one specific embodiment, it is 15g:25mL.

[0040] Preferably, the pretreatment time can be (3-7) hours, (3-5) hours, (4-6) hours, or (5-7) hours. In one specific embodiment, it is 5 hours.

[0041] The second aspect of this invention protects the Bacillus subtilis strain Sarib006, whose preservation number is CCTCC NO:M 20231896. The carbonic anhydrase activity of the Bacillus subtilis is not less than 3.65 U / min.

[0042] The method for determining the activity of the carbonic anhydrase is as follows:

[0043] 1) After mixing p-nitrophenol and water, perform serial dilutions and measure the absorbance at 400 nm to plot a standard curve;

[0044] 2) The bacterial culture, Tris-H2SO4 and p-nitrobenzene acetate were reacted, and the absorbance at 400 nm was measured. The enzyme activity of the acid anhydrase was calculated.

[0045] The enzyme activity of carbonic anhydrase is defined as the amount of enzyme required to hydrolyze 1 μmol of p-nitrophenyl acetic acid solution per minute at room temperature, which is one enzyme activity unit.

[0046] A third aspect of this invention protects a composite microorganism for inducing calcium carbonate deposition and solidification, comprising Bacillus subtilis and Bacillus pasteurellus as described above.

[0047] In a third aspect of the present invention, the ratio of viable Bacillus subtilis to Bacillus pasteurellium is (0.5-3):1.

[0048] The fourth aspect of this invention protects the use of Bacillus subtilis or the composite bacteria described above as reinforcement and repair materials in the induction of calcium carbonate deposition and solidification.

[0049] This invention screened and obtained a Bacillus subtilis strain, Sarib006, with strong carbonic anhydrase production ability. The composite bacterial solution formed by Sarib006 and Bacillus pasteurellium, which has good urease production ability, exhibits high urease activity and high carbonic anhydrase activity. When using the composite bacterial solution of this invention to treat pit soil, Bacillus subtilis Sarib006 and Bacillus pasteurellium synergistically promoted the efficiency of microbial induced calcification and improved the toughness of the calcified material.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1) The new strain Bacillus subtilis Sarib006 screened in this invention is a naturally isolated strain, and its carbonic anhydrase activity is much higher than that reported in existing literature, reaching 3.65 U / min.

[0052] 2) When the compound bacterial solution formed by the combination of Bacillus subtilis Sarib006 and Bacillus pasteurellii is used for loose particles, the cementation degree of the calcified material is high, the extracted sample is the most intact and has strong hardness, which is 32% higher than that of using Bacillus pasteurellii alone. Attached Figure Description

[0053] Figure 1 This figure shows the sequence comparison results of Bacillus subtilis Sarib006 and related species in the GenBank database in Example 1 of the present invention.

[0054] Figure 2 The figure shown is a standard curve of p-nitrophenol in Example 2 of the present invention.

[0055] Figure 3 The diagram shown is a schematic representation of the calcification apparatus used in an embodiment of the present invention.

[0056] Figure 4 The graph shows the enzyme activity detection results of Bacillus subtilis Sarib006 bacterial suspension from Example 1, Bacillus pasteurellus bacterial suspension from Example 2, and the composite bacterial suspension from Example 3.

[0057] Figure 5 The image shown is a real photograph of the loose particles after being reinforced and repaired using Bacillus subtilis Sarib006 bacterial solution in Example 2 of the present invention.

[0058] Figure 6The image shown is a real photograph of the loose particles after being reinforced and repaired using Bacillus pasteurellium solution in Example 3 of the present invention.

[0059] Figure 7 The image shown is a real photograph of the results of using a composite bacterial solution formed by Bacillus subtilis Sarib006 and Bacillus pasteurellus to reinforce and repair loose particles, as described in Example 4 of the present invention. Detailed Implementation

[0060] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0061] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0062] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0063] In the following examples, the hardness test was performed using a Shore hardness tester of model HT6510A from Guangzhou Lantai Instrument Co., Ltd.

[0064] OD in this application 600 When the value is 1, the corresponding concentration of Bacillus subtilis or Bacillus pasteurellium is approximately 0.9 × 10⁻⁶. 8 cfu / mL.

[0065] Example 1: Isolation and screening of Bacillus subtilis Sarib006

[0066] 1.1 Initial screening

[0067] Highly alkaline soil from the outskirts of Shanghai was collected. After removing large stones and other impurities, the soil was sieved through a 10-mesh sieve to obtain a pretreated soil sample. 1g of the pretreated soil sample was added to 10mL of sterile water, shaken at 30℃ for 15min, and then allowed to stand for 30min. The supernatant bacterial solution was then diluted appropriately and spread onto LB agar plates. Single colonies were obtained by incubation at 30℃ for 24–48h. Purified cultures of different strains were picked and added to 100mL of LB agar, and incubated at 30℃ and 150rpm for 18–24h. A suitable amount of the bacterial solution was taken, acid-base indicator was added, and titrated to neutral with HCl. The CO3 production was roughly calculated based on the amount of HCl used. 2- Based on their ability, several plants with higher yields were selected for secondary screening.

[0068] 1.2 Secondary screening

[0069] The bacteria obtained from the initial screening were inoculated into 100 mL of LB medium and cultured in a shake flask at 30°C and 200 rpm for 18-24 h. Their carbonic anhydrase activity was measured to obtain a high-carbonic anhydrase-producing strain.

[0070] 1.3 Identification of strains

[0071] The colonies are small, flat, slightly transparent, white, with a moist surface and neat edges; they have been identified as Gram-positive bacteria. The culture temperature is 20–40℃, with an optimum temperature of 30℃. It is aerobic and does not require light.

[0072] Genomic DNA was extracted from the bacterial culture of the strain to be identified after shaking flask culture using the Ezup column-based bacterial DNA extraction kit.

[0073] Amplification was performed using universal primers, and the resulting 16S rDNA sequences were compared with sequences of related species in the GenBank database. The comparison results are shown below. Figure 1 .

[0074] from Figure 1 It can be seen that Sarib-006 is similar to Bacillus subtilis, with a similarity of 98.67% to strain Bacillus subtilis T18. Therefore, this strain was finally named Bacillus subtilis Sarib006.

[0075]

[0076] 27F primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2)

[0077] 1492R primer: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3)

[0078] The Bacillus subtilis strain of this invention is classified and named as Bacillus subtilis strain Sarib006. The strain was deposited on October 16, 2023, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO:M 20231896. It was obtained by screening and isolation from soil.

[0079] Example 2: Bacillus subtilis Sarib006 induces calcium carbonate deposition and solidification

[0080] In this Example 2, an experiment was conducted to induce calcium carbonate deposition and solidification using Bacillus subtilis Sarib006 bacterial suspension. This included the following:

[0081] 2.1 Obtaining Bacillus subtilis bacterial culture

[0082] Cultivation method of Bacillus subtilis: The Bacillus subtilis strain obtained in Example 1 and preserved in the culture medium was transferred to a solid plate medium and evenly spread. It was then incubated at 30°C for 16–24 h. After single colonies grew, it was transferred to a liquid medium for subculturing once. The conditions for liquid culture and subculturing were the same as those for solid plate culture. The resulting bacterial suspension was stored at 4°C. The OD of the Bacillus subtilis bacterial suspension was... 600 =1.6.

[0083] Solid culture medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 20 g / L, autoclaved at 115℃ for 30 min.

[0084] Liquid culture medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, autoclaved at 115℃ for 30 min.

[0085] 2.2 Detection of urease and carbonic anhydrase activities in bacterial culture

[0086] 2.2.1 Detection of urease activity

[0087] When a certain concentration of urea solution is added to the bacterial culture of urease-producing microorganisms, the concentration of conductive ions in the solution increases per unit time due to the hydrolysis of urea by the urease-producing microorganisms, thereby increasing the conductivity of the solution. Furthermore, the amount of urea hydrolyzed is directly proportional to the increase in solution conductivity. Therefore, the change in solution conductivity can be used to characterize the ability of the bacterial culture to hydrolyze urea per unit time (bacterial culture urease activity).

[0088] The relationship between the amount of urea hydrolyzed and the change in conductivity is as follows:

[0089] Urea hydrolysis capacity (mM / min) = Change in conductivity (mS / cm / min) × 11.11

[0090] That is, using the method reported in the literature (Whiffin V S. Microbial CaCO3 precipitation for the production of biocemenet. Pertj, Austria: Murdoch University, 2004), the R of the standard curve is... 2 =0.9988.

[0091] The specific measurement method is as follows: Mix 1 volume of bacterial culture with 9 volumes of 1.1 mol / L urea solution, and measure the change in conductivity of the solution over 5 minutes using a conductivity meter. Multiply the average conductivity change over 5 minutes (unit: mS / cm / min) by the dilution factor (10-fold) to obtain the initial enzyme activity of the bacterial culture. This value reflects the ability of the bacterial culture to hydrolyze urea. Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 mmol of urea solution per minute at room temperature; one enzyme activity unit (U) is defined as this.

[0092] 2.2.2 Detection of carbonic anhydrase activity

[0093] Carbonic anhydrase not only catalyzes the hydration reaction of CO2, but also has esterase activity, which can be used to catalyze the hydrolysis of sulfonates, carboxylic esters, phosphate esters, etc. Therefore, the enzyme activity of carbonic anhydrase can be indirectly represented by esterase activity.

[0094] The specific method is as follows:

[0095] 1) Prepare a 3 mmol / L p-nitrobenzene acetate solution and a 15 mmol / L Tris-H2SO4 buffer solution (pH 7.6);

[0096] 2) Constructing a standard curve for p-nitrophenol: Weigh 0.0417 g of p-nitrophenol, dissolve it, and dilute to 100 mL to obtain a 3 mmol / L p-nitrophenol stock solution. Take 10 test tubes and add the solution according to the proportions in Table 1, mixing well. Simultaneously, use an equal volume of distilled water as a blank control. Measure the absorbance of the analyte at 400 nm at room temperature. Plot the standard curve with the p-nitrophenol concentration as the x-axis and the y-axis as the vertical axis. (See graph below.) Figure 2 R of the standard curve 2 =0.9906.

[0097] Table 1

[0098] Mother liquor / mL 0.15 0.3 0.45 0.6 0.75 0.9 1.05 1.2 1.35 1.5 Distilled water / mL 9.85 9.7 9.55 9.4 9.25 9.1 8.95 8.8 8.65 8.5 OD400 0.026 0.064 0.099 0.128 0.167 0.18 0.224 0.242 0.279 0.287

[0099] 3) Determination of carbonic anhydrase activity in bacterial culture:

[0100] 1.9 mL of Tris-H2SO4 buffer (pH 7.6), 0.1 mL of the test bacterial solution, and 1.0 mL of p-nitrophenyl acetate solution were added sequentially to the cuvette. After reacting for 5 min at room temperature, the absorbance was measured at 400 nm using a UV spectrophotometer. The cuvette without the test bacterial solution was used as a blank control.

[0101] Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of p-nitrophenyl acetate solution per minute at room temperature, which is one enzyme activity unit.

[0102] The results of the detection of urease and carbonic anhydrase activities in the bacterial culture are shown in [the table below]. Figure 4 As shown.

[0103] from Figure 4 It is known that Bacillus subtilis Sarib006 does not have urease activity, but its carbonic anhydrase activity is relatively high, at 3.65 U / min. Prior art reports Bacillus subtilis carbonic anhydrase activity <1.0 U / min, meaning the Bacillus subtilis Sarib006 of this application significantly exceeds the levels reported in the prior art.

[0104] 2.3 Bacillus subtilis-induced calcium carbonate deposition and solidification

[0105] Use such as Figure 3 The calcification device shown consists of a 50mL centrifuge tube with an opening at one end, used to hold soil from the pit. The lower end is plugged with cotton to control the outflow rate of waste liquid. Above the centrifuge tube is a syringe device that injects the solution into the soil through a needle.

[0106] The specific procedure for calcification is as follows:

[0107] 1) Take a 50mL centrifuge tube and add 15g of soil from the pit containing cultural relics (pit soil) up to the 25mL mark.

[0108] 2) Add 25 mL of fixative (0.05 M CaCl2) to the centrifuge tube, let it stand for 5 h, and then drain the waste liquid.

[0109] 3) Add 10 mL of the Bacillus subtilis culture (OD2.1) obtained in step 2.1 to the centrifuge tube. 600 =0.8~2) After standing for 3 hours, drain the waste liquid.

[0110] 4) Add 10 mL of calcification solution (0.5 M CaCl2 and 0.5 M urea) to the centrifuge tube, let it stand for 16 h, and then drain the waste liquid.

[0111] 5) Repeat steps 3) and 4) more than 15 times.

[0112] 6) Place the fixed soil sample into a 60℃ oven to dry.

[0113] Soil samples after carbonate deposition and solidification induced by Bacillus subtilis Sarib006, such as Figure 5 As shown.

[0114] from Figure 4 and 5 It is known that because Bacillus subtilis does not have urease activity, the carbonate ions required for calcification are entirely provided by dissolved CO2 in the environment. Therefore, the calcification rate is the slowest, and the material is almost completely broken after extraction, making it impossible to detect its hardness.

[0115] Example 3: Pasteurella multocida induces calcium carbonate deposition and solidification

[0116] In this Example 3, only *Pasteurella multocida* bacterial suspension (ATCC 11859) was used for the induced calcium carbonate deposition and solidification experiment. This included the following:

[0117] 3.1 Obtaining Pasteurella multocida bacterial suspension

[0118] The dissolved bacterial suspension of *Bacillus pasteurellii* strain was transferred to a solid culture medium and evenly spread. It was then incubated at 30°C for 24–48 hours. After individual colonies emerged, the suspension was transferred to a liquid culture medium for subculturing. The resulting bacterial suspension was stored at 4°C. The OD value of the *Bacillus pasteurellii* bacterial suspension was determined. 600 =1.2.

[0119] Solid culture medium: 3 g / L beef extract, 5 g / L peptone, 20 g / L urea, 20 g / L agar, autoclaved at 115℃ for 20 min.

[0120] Liquid culture medium: 3 g / L beef extract, 5 g / L peptone, 20 g / L urea, autoclaved at 115°C for 20 min.

[0121] 3.2 Detection of urease and carbonic anhydrase activities in bacterial culture

[0122] The *Pasteurella multocida* bacterial suspension obtained in step 3.1 was subjected to enzyme activity detection. The method for enzyme activity detection was the same as step 2.2 in Example 2. The detection results are shown in [Figure 2]. Figure 1 As shown.

[0123] from Figure 4 It can be seen that the urease activity of the Bacillus pasteurellus bacterial solution is 4.16 mM / min and the carbonic anhydrase activity is 1.36 U / min. The urease activity is relatively high, but the carbonic anhydrase activity is relatively low.

[0124] 3.3. Pasteurella multocida-induced calcium carbonate deposition and solidification

[0125] The difference from step 2.3 in Example 2 is that in step 3) of the calcification operation, *Pasteurella multocida* bacterial solution is used instead of *Bacillus subtilis* bacterial solution. Actual photographs of the calcified samples are shown below. Figure 6 .

[0126] from Figure 4 and 6 It can be seen that, due to the low carbonic anhydrase activity of Bacillus pasteurellii, the soil in the calcified artifact pit was not completely cemented and had low hardness, resulting in some breakage after extraction. Its hardness was measured to be 4.6 kgf / cm². 2 .

[0127] Example 4: Bacillus subtilis and Bacillus pasteurellium synergistically induce calcium carbonate deposition and solidification

[0128] In Example 4, a composite bacterial solution formed by Bacillus subtilis and Bacillus pasteurellium was used to induce calcium carbonate deposition and solidification experiments. This included the following:

[0129] 4.1 Obtaining the compound bacterial solution

[0130] After centrifuging the Bacillus subtilis culture from Example 2 and removing the supernatant, the bacterial precipitate was added to the Pasteurella multocida culture obtained in Example 3 at a ratio of bacterial volume (live bacteria count) of 0.5:1, 1:1, 2:1, and 3:1 to obtain a compound bacterial culture.

[0131] 4.2 Detection of urease and carbonic anhydrase activities in bacterial culture

[0132] The enzyme activity of the composite bacterial solution obtained in step 4.1 was detected using the same method as step 2.2 in Example 2. The detection results are shown in [the table below]. Figure 4 As shown.

[0133] from Figure 4It was found that when the ratio of Bacillus subtilis to Bacillus pasteurellium was 1:1, the urease and carbonic anhydrase activities of the compound bacterial solution were the highest, at 4.04 mM / min and 3.15 U / min, respectively. Compared with the Bacillus pasteurellium bacterial solution, the urease activity decreased slightly, but the carbonic anhydrase activity increased significantly.

[0134] 4.3 Synergistic Induction of Calcium Carbonate Deposition and Solidification by Compound Bacterial Solution

[0135] The difference from step 2.3 in Example 2 is that in step 3) of the calcification operation, the compound bacterial solution obtained in 4.1 is used instead of the Bacillus subtilis bacterial solution, and the OD of the compound bacterial solution is... 600 =2.4. Actual photographs of the calcified samples are shown below. Figure 7 .

[0136] from Figure 4 and Figure 7 It can be seen that, due to the high urease and carbonic anhydrase activities in the compound bacterial solution (carbonic anhydrase activity of 2.45–3.35 U / min and urease activity of 1.84–4.12 mM / min), it exhibits the fastest calcification rate for loose particles. The calcified soil samples obtained by the synergistic induction of the two bacteria have the highest degree of cementation and the most intact samples after extraction. Furthermore, its 1:1 solution exhibits the highest hardness, with a test result of 6.1 kgf / cm². 2 The improvement was 32% compared to the calcified sample obtained by using Bacillus pasteurellus alone in Example 3.

[0137] Comparative Example 1

[0138] The difference between Comparative Example 1 and Example 4 is that commercially available Bacillus subtilis ATCC6633 was used instead of Bacillus subtilis Sarib006; otherwise, they are the same as in Example 4.

[0139] The carbonic anhydrase activity of Bacillus subtilis ATCC6633 is 0.9 U / min. It is mixed with Bacillus pasteurellii at a bacterial volume ratio of 1:1 to obtain a compound bacterial solution, which is used to reinforce and repair the pit soil.

[0140] The results were similar to those of Example 3 using only Bacillus pasteurellii, with incomplete bonding and low hardness, only 4.4 kgf / cm². 2 .

[0141] The Bacillus subtilis strain Sarib006 of this invention is a naturally isolated strain with a carbonic anhydrase activity of 3.65 U / min, which is much higher than that of Bacillus subtilis in the prior art. When used in combination with Bacillus pasteurellii to form a compound bacterial solution, the material has a high degree of cementation after solidification by calcium carbonate deposition, and the extracted sample is the most intact and has strong hardness, which is 32% higher than that of Bacillus pasteurellii alone.

[0142] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for microbial-induced calcium carbonate deposition and solidification, characterized in that, Includes the following steps: 1) Add a compound bacterial solution containing Bacillus subtilis and Bacillus pasteurellium to treat loose particles; 2) Add calcification solution to the treated loose particles for post-treatment; The Bacillus subtilis strain mentioned is the strain with the preservation number CCTCC NO: M 20231896; The *Pasteurella multocida* strain mentioned is the strain with the preservation number ATCC11859; Steps 1) and 2) should be repeated at least twice; Step 1) Before adding the compound bacterial solution, the loose particles are pretreated with a calcium salt aqueous solution. In the compound bacterial solution, the ratio of viable Bacillus subtilis to Bacillus pasteurellium is (0.5-3):

1.

2. The method as described in claim 1, characterized in that, The calcification solution is an aqueous solution of urea and calcium salts; And / or, the loose particles are selected from soil particles or sand and gravel.

3. The method as described in claim 2, characterized in that, The calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium lactate, calcium formate, and calcium acetate; And / or, in the calcification solution, the molar ratio of the calcium salt to urea is (1-3):1; And / or, the mass-to-volume ratio of the loose particles to the compound bacterial solution is 1 g: (5-20) mL; And / or, the processing time is (1-5) h; And / or, the mass-to-volume ratio of the loose particles to the calcification solution is 1 g: (5-20) mL; And / or, the post-processing time is (11-21) h; And / or, the mass-to-volume ratio of the loose particles to the calcium salt aqueous solution is 15 g: (10-40) mL; And / or, the preprocessing time is (3-7) h.

4. The method as described in claim 2, characterized in that, The number of viable Bacillus subtilis bacteria in the compound bacterial solution is ≥1×10⁻⁶. 8 cfu / mL; And / or, the viable count of *Bacillus pasteurellus* in the compound bacterial solution is ≥1×10⁻⁶. 8 cfu / mL; And / or, the total number of viable bacteria in the compound bacterial solution is ≥2×10⁻⁶. 8 cfu / mL.

5. The method as described in claim 4, characterized in that, The method for preparing the composite bacterial solution is as follows: 1) Bacillus subtilis was cultured in the first liquid medium to obtain Bacillus subtilis bacterial suspension; Bacillus pasteurellii was cultured in the second liquid medium to obtain Bacillus pasteurellii bacterial suspension. 2) Centrifuge the Bacillus subtilis culture to obtain a bacterial precipitate; mix the bacterial precipitate with the Bacillus pasteurellium culture to obtain the composite culture.

6. The method as described in claim 5, characterized in that, The first liquid culture medium is composed of: yeast extract (2-8) g / L, peptone (6-14) g / L, and NaCl (6-14) g / L; And / or, the composition of the second liquid culture medium is: beef extract (1-6) g / L, peptone (1-10) g / L, urea (15-25) g / L; And / or, the culture temperature of the Bacillus subtilis is (25-35)℃; And / or, the culture time of the Bacillus subtilis is (16-24) h; And / or, the culture temperature of the *Pasteurella multocida* is (25–35) °C; And / or, the culture temperature of the *Pasteurella multocida* is (24–48) h.

7. Bacillus subtilis strain Sarib006, characterized in that, The Bacillus subtilis has the accession number CCTCC NO: M 20231896.

8. The strain according to claim 7, characterized in that, The carbonic anhydrase activity of the Bacillus subtilis is not less than 3.65 U / min.

9. A composite microorganism for inducing calcium carbonate deposition and solidification, characterized in that, It contains Bacillus subtilis and Bacillus pasteurellus as described in claim 7; the Bacillus pasteurellus strain is the strain with accession number ATCC11859; the live count ratio of Bacillus subtilis and Bacillus pasteurellus is (0.5-3):

1.

10. The use of Bacillus subtilis as described in claim 7 or the composite bacteria as described in claim 9 as a reinforcing and repairing material in the induction of calcium carbonate deposition and solidification.

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