Bacillus pasteurii and application thereof

Abstract

The bacillus pasteurii is preserved in the China Center for Type Culture Collection, and the preservation number of the bacillus pasteurii is CCTCC M 20241864; the urealyticum mutant strain with high urease activity is successfully screened by adopting a normal-pressure and room-temperature plasma technology, and the capability of inducing precipitation of calcium carbonate can be remarkably enhanced, so that the mineralization efficiency of microorganism-induced calcium carbonate deposition and the sandy soil reinforcement effect are improved; in addition, an improved bacterium-mediated microorganism-induced mineralization reaction kinetic model is established, and an improved process and method for reinforcing sandy soil through strains are provided according to the requirement for efficient control of ammonium emission. The research result of the invention has important scientific significance and wide application prospect for improving the practical application effect of the microbial soil reinforcement technology and improving the environmental problem caused by the byproduct waste ammonium solution.

Description

Technical field:

[0001] The invention relates to the technical field of microbial mineralization reinforcement, and in particular to a Bacillus pasteurianus and an application of the microbial mineralization reinforcement technology thereof. Background technology:

[0002] Soil reinforcement is one of the key issues in geotechnical engineering practice. However, the traditional solidification method is to use adhesives such as cement and chemical materials, which has limited its widespread application due to its significant carbon emissions and negative impact on the ecological environment. In recent years, microbial induced calcium carbonate precipitation (MICP) technology has improved the geotechnical properties of soil through biocementation, providing a low-carbon, environmentally friendly and sustainable comprehensive solution to solve geotechnical engineering-related problems in the environment.

[0003] The urease-mediated MICP mineralization process is based on the urease-catalyzed hydrolysis of urea in microbial metabolic activities to produce CO2 and NH3. The hydrolysis of NH3 produces OH ﹣ , causing the pH value of the solution to rise, which in turn causes CO2 to convert to CO3 2- At the same time, the negatively charged groups on the surface of bacterial cell membranes adsorb Ca in the environment. 2+ (provided by the cementing fluid), which promotes the nucleation of CaCO3 crystals at the bacterial deposition site to form a calcium carbonate biocement skeleton, thereby enhancing the bridging properties between soil particles and improving the mechanical properties of the soil.

[0004] However, the formation of biominerals depends on specific environmental conditions, and the process is highly regulated by the mineralization environment. In fact, the biomineralization efficiency of MICP technology is not ideal. Some studies have used higher concentrations of urea (≥1 mol / L) to improve the mechanical properties of MICP-treated soil. However, this high concentration of urea will produce a large amount of ammonium ions. Untreated wastewater ammonium may infiltrate the soil and pollute groundwater, threatening the drinking water safety of surrounding residents; in addition, wastewater ammonium may generate greenhouse gas N2O through ammonia oxidation, causing potential harm to the ecological environment. Summary of the invention:

[0005] In view of this, the present invention provides a strain of Bacillus pasteurianus and its application, which enhances the ability of Bacillus pasteurianus to induce the precipitation of calcium carbonate, and has important scientific significance and broad application prospects for improving the practical application of microbial soil reinforcement technology and improving the environmental problems caused by the by-product waste ammonium liquid.

[0006] A Bacillus pasteurianus, deposited in China Center for Type Culture Collection with the deposit number CCTCC M20241864.

[0007] Application of Bacillus pasteurianus with a deposit number of CCTCC M 20241864 in microbial-induced calcium carbonate precipitation.

[0008] A Bacillus pasteurianus, comprising a 16S rDNA sequence as shown in SEA ID NO. 1;

[0009] A use of Bacillus pasteurianus comprising a 16S rDNA sequence as shown in SEA ID NO.1 in microbial-induced calcium carbonate precipitation.

[0010] A Bacillus pasteurianus, comprising the sequence shown in SEA ID NO.2.

[0011] A method for using Bacillus pasteurianus comprising a sequence as shown in SEA ID NO.2 in microbial-induced calcium carbonate precipitation.

[0012] A microorganism-induced calcium carbonate precipitation component comprises Bacillus pasteurianus, urea and a calcium source. The Bacillus pasteurianus is the Bacillus pasteurianus with a preservation number of CCTCC M 20241864.

[0013] Preferably, the urea concentration is 0.2 mol / L, the calcium source is calcium acetate with a concentration of 0.27 mol / L, and the inoculation ratio of Bacillus pasteurianus is 1.1%;

[0014] Preferably, the calcium source is at least one of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, calcium acetyl, calcium silicate, calcium aluminate, and dicalcium acetyl.

[0015] A microorganism-induced calcium carbonate precipitation component comprises Bacillus pasteurianus, urea and a calcium source, wherein the Bacillus pasteurianus comprises a 16S rDNA sequence as shown in SEA ID NO.1.

[0016] Preferably, the urea concentration is 0.2 mol / L, the calcium source is calcium acetate with a concentration of 0.27 mol / L, and the inoculation ratio of Bacillus pasteurianus is 1.1%;

[0017] Preferably, the calcium source is at least one of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, calcium acetyl, calcium silicate, calcium aluminate, and dicalcium acetyl.

[0018] A microorganism-induced calcium carbonate precipitation component comprises Bacillus pasteurianus, urea and a calcium source, wherein the sequence of the Bacillus pasteurianus is shown in SEA ID NO.2.

[0019] Preferably, the urea concentration is 0.2 mol / L, the calcium source is calcium acetate with a concentration of 0.27 mol / L, and the inoculation ratio of Bacillus pasteurianus is 1.1%;

[0020] Preferably, the calcium source is at least one of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, calcium acetyl, calcium silicate, calcium aluminate, and dicalcium acetyl.

[0021] A method for inducing calcium carbonate precipitation by using Bacillus pasteurianus, comprising adding any one of the three microorganism-induced calcium carbonate precipitation components to the object to be reinforced.

[0022] A method for microbial induced calcium carbonate precipitation was proposed. The three independent variables A (urea concentration), B (calcium concentration) and C (microbial inoculation ratio) were subjected to multiple regression fitting by Design Expert software, and the second-order regression model equation of the response values ​​Y1 (compressive strength) and Y2 (ammonium concentration) on the three independent variables was established.

[0023] Through the second-order regression model equation, the urea concentration, calcium concentration and microbial inoculation ratio added to the reinforced object are determined according to the required compressive strength;

[0024] Alternatively, the urea concentration, calcium concentration and microbial inoculation ratio to be added to the reinforced object can be determined according to the required ammonium concentration through a second-order regression model equation.

[0025] Preferably, the inoculated microorganism is Bacillus pasteurianus with a deposit number of CCTCC M 20241864, and the calcium concentration is a calcium acetate concentration;

[0026] Preferably, the inoculated microorganism is Bacillus pasteurianus including a 16S rDNA sequence as shown in SEA ID NO.1, and the calcium concentration is a calcium acetate concentration;

[0027] Preferably, the inoculated microorganism is Bacillus pasteurianus including a sequence as shown in SEA ID NO.2, and the calcium concentration is a calcium acetate concentration.

[0028] A method for screening Bacillus pasteurianus for microbial-induced calcium carbonate precipitation uses normal pressure and room temperature plasma to induce mutations in Bacillus pasteurianus and screen out mutants with improved urease activity compared with the original strain.

[0029] Preferably, the parameters of the atmospheric pressure and room temperature plasma mutagenesis test are power 120W, gas flow 10SLM, radiation distance 2mm, and mutagenesis treatment time 50s to 60s.

[0030] A culture medium for culturing Bacillus pasteurianus comprises 17 g / L casein peptone, 3 g / L soy peptone, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate and 20 g / L urea.

[0031] Preferably, the pH of the culture medium is 7.5.

[0032] Preferably, it is used to cultivate Bacillus pasteurianus with a deposit number of CCTCC M 20241864.

[0033] Preferably, it is used to culture Bacillus pasteurianus comprising a 16S rDNA sequence as shown in SEA ID NO.1.

[0034] Preferably, the culture medium is used for culturing Bacillus pasteurianus comprising the sequence shown in SEA ID NO.2.

[0035] The present invention aims to successfully screen out urea-lytic bacteria mutants with high urease activity by using atmospheric pressure room temperature plasma (ARTP) technology, significantly enhancing the precipitation ability of induced calcium carbonate, so as to improve the mineralization efficiency of microbial induced calcium carbonate deposition and the sand reinforcement effect. The present invention establishes an improved bacteria-mediated microbial induced mineralization reaction kinetic model, and proposes a process and method for improving sand reinforcement by strains in response to the demand for efficient control of ammonium emissions. The research results of the present invention have important scientific significance and broad application prospects for improving the practical application effect of microbial soil reinforcement technology and improving the environmental problems caused by byproduct waste ammonium liquid. Description of the drawings:

[0036] Figure 1 This is an electron microscope scanning image of the morphology of the starting Bacillus pasteurianus strain.

[0037] Figure 2 This is the lethality curve of Bacillus pasteurianus ARTP induced mutation.

[0038] Figure 3 This is the control before ARTP mutagenesis and the determination of urease activity of some strains after ARTP mutagenesis.

[0039] Figure 4 This is the control before ARTP mutagenesis and the determination of calcium carbonate precipitation induced by some strains after ARTP mutagenesis.

[0040] Figure 5 This is an electron microscope scanning image of the morphology of the mutant strain M55-1 Bacillus pasteurianus.

[0041] Figure 6 Construction of the evolutionary tree of Bacillus pasteurianus mutant strain M55-1.

[0042] Figure 7 To determine the genetic stability of the urease activity of mutant strain M55-1.

[0043] Figure 8 This is a genetic stability test of the optical density value of the mutant strain M55-1.

[0044] Fig. 9 Schematic diagram of the biopumping reinforcement device for loess samples.

[0045] Fig.10 3D response surface diagram of the interaction between calcium acetate concentration (B) and inoculation ratio (C) on the effluent ammonium concentration of sand mediated by MICP treatment by mutant strain M55-1.

[0046] Fig.11 3D response surface diagram of the interaction between urea concentration (A) and calcium acetate concentration (B) on the effluent ammonium concentration of sand mediated by MICP treatment by mutant strain M55-1.

[0047] Fig.12 3D response surface diagram of the interaction between urea concentration (A) and inoculation ratio (C) on the effluent ammonium concentration of sand mediated by MICP treatment by mutant strain M55-1.

[0048] Fig.13 3D response surface diagram of the interaction effect of calcium acetate concentration (B) and inoculation ratio (C) on the compressive strength of sand treated with MICP mediated by mutant strain M55-1.

[0049] Fig.14 3D response surface diagram of the interaction effect of urea concentration (A) and inoculation ratio (C) on the compressive strength of sand treated with MICP mediated by mutant strain M55-1.

[0050] Fig.15 3D response surface diagram of the interactive effect of urea concentration (A) and calcium acetate concentration (B) on the compressive strength of sand mediated by MICP treatment of mutant strain M55-1.

[0051] Fig.16 This is a graphic showing the result after the sand column solidifies during the grouting process.

[0052] in Fig. 9 Middle: mutant strain M55-1 bacterial solution 1, cementing liquid 2, water stop clamp 3, peristaltic pump 4, rubber stopper 5, sand column 6, acrylic split mold 7, effluent 8. Specific implementation method:

[0053] Atmospheric Room Temperature Plasma (ARTP) technology induces mutations in microbial cells through high-energy ion beams. This technology uses a specific discharge device to generate a plasma beam in a gas channel and exposes the microorganisms to this plasma. The plasma beam can penetrate the bacterial culture, cause DNA damage, and significantly change its gene sequence and metabolic pathways. After subsequent treatment and screening, mutant strains with ideal traits can be obtained. Compared with traditional mutagenesis methods, ARTP has the advantages of being environmentally friendly, easy to operate, and having a high mutation rate.

[0054] The present invention is described in detail below in conjunction with the accompanying drawings and examples. It should be noted that the following examples are only for further explanation of the present invention, and those skilled in the art can make non-essential improvements and adjustments based on the content of the invention. The materials, reagents and instruments used in the following examples, unless otherwise specified, are obtained through commercial channels, and the experimental methods used are conventional methods unless otherwise specified.

[0055] (I) Screening of Bacillus pasteurianus

[0056] Prepare experimental materials:

[0057] Bacteria: Sporosarcina pasteurii (11859) was purchased from the American Tissue Culture Collection (ATCC).

[0058] CASO medium preparation: casein peptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 2.5g / L, urea 20g / L, pH 7.5, distilled water to 1L. If solid medium is prepared, add 2% agar powder. Sterilize at 121℃ for 30min.

[0059] Binder: Binder is a liquid medium mixed with urea and calcium-based solution. In order to provide a calcium source, the calcium-based solution is mainly used to provide sufficient urea and calcium ions for the microbial induced calcium carbonate precipitation (MICP) process, and at the same time provide nutrients for the growth, reproduction and metabolism of microorganisms. The present invention selects calcium acetate to provide a calcium source for the microbial induced calcium carbonate precipitation (MICP) technology, and calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, acetyl calcium, calcium silicate, calcium aluminate, and dicalcium acetyl can all be used as calcium sources. Urea and calcium acetate are both analytically pure reagents.

[0060] CASO medium is formulated to be a medium suitable for the growth of Bacillus pasteurianus (Sporosarcina pasteurii, 11859).

[0061] Specific experimental process:

[0062] (1) ARTP mutagenesis of Bacillus pasteurianus

[0063] Bacillus pasteurii is a Gram-positive facultative anaerobic bacterium (non-pathogenic), with a negatively charged cell surface, good urease synthesis behavior, and strong environmental adaptability. It is rod-shaped, about 2-3 μm in length and about 0.7 μm in diameter. The SEM image of the starting strain Bacillus pasteurii is shown in Figure 1. Figure 1 shown.

[0064] The starting strain Bacillus pasteurianus was cultured in a shake flask at a temperature of 30°C and a frequency of 180 r / min for 18 hours. The original bacterial solution in the logarithmic phase was taken and diluted to 10 -3 times, take out the autoclaved mutagenesis slide, place the slide in the flame of an alcohol lamp to burn, after the slide cools down, draw 10 μL of the diluted bacterial solution and apply it on the mutagenesis slide, place the slide in the groove of the mutagenesis chamber for atmospheric pressure room temperature plasma (ARTP) mutagenesis, and the above process is carried out in a sterile environment.

[0065] The parameters of the atmospheric pressure room temperature plasma (ARTP) mutagenesis test were as follows: power 120 W, gas flow 10 SLM, radiation distance 2 mm, mutagenesis treatment time 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s, and the unmutated treatment was used as the control group.

[0066] After mutagenesis, the slides were placed in 990 μL of CASO liquid culture medium and vortexed for 45-60 seconds to obtain ARTP mutagenesis bacterial solution; then 100 μL of mutagenesis bacterial solution was immediately applied to CASO solid culture medium (three parallel tests) and placed in a constant temperature incubator for 48 hours (temperature was 30°C); finally, the colonies were counted, the lethality was calculated, and the ARTP mutagenesis treatment lethality curve was drawn, as shown in Fig. Figure 2 shown.

[0067] The lethality rate was calculated as follows: lethality rate % = (the number of colonies on the plate without mutagenesis treatment - the number of colonies on the plate with mutagenesis treatment) / the number of colonies on the plate without mutagenesis treatment × 100%

[0068] from Figure 2 It can be seen that with the increase of ARTP mutagenesis treatment time, the lethality rate continued to increase and finally stabilized. Compared with the control group, the lethality rates of ARTP mutagenesis treatment for 50s, 55s and 60s were 96.54%, 97.01% and 96.66% respectively. Finally, it was determined that the solid culture medium with a lethality rate of more than 90% was selected for the next screening work.

[0069] (2) Screening of mutants by urease activity

[0070] The strains with larger growth area than other strains in the solid culture medium after mutagenesis treatment for 50s, 55s and 60s were selected, and the single strains were inoculated into 5mL CASO liquid culture medium, cultured in a constant temperature incubator for 24h, and subcultured for 6 generations to ensure the stability of the genetic performance of the strains. Subsequently, the strains cultured for 6 generations were inoculated into CASO liquid culture medium at a 1% inoculation ratio and cultured at a constant temperature and shaking for 24h; then the urease activity of the selected strains was tested respectively.

[0071] Urease activity detection method: 6 mL of bacterial solution and 54 mL of 1.1 mol / L urea solution were mixed at 30°C to obtain a mixed solution. The conductivity change of the mixed solution within 5 minutes was measured using a conductivity meter. The average conductivity change of the mixed solution per minute was calculated. The conversion formula was used to characterize the urease activity. The calculation formula is as follows:

[0072] Urease activity mM·(L·min) -1 = Conductivity change per minute / 5min×11.11×10

[0073] Under the same mutagenesis time, the calculation formula for the degree of improvement of the urease activity of the strain relative to the urease activity of the control group is as follows:

[0074] Relative increase rate of urease activity = (average urease activity of the mutagenesis group - average urease activity of the control group) / average urease activity of the control group

[0075] Three strains with the highest urease activity were selected from each plate, a total of 9 strains. The urease activity test results are as follows: Figure 3 Among them, M0-1, M0-2, and M0-3 are controls before ARTP mutagenesis, M50-1, M50-2, and M50-3 are the three strains with the highest relative increase in urease activity after ARTP mutagenesis for 50S, M55-1, M55-2, and M55-3 are the three strains with the highest relative increase in urease activity after ARTP mutagenesis for 55S, and M60-1, M60-2, and M60-3 are the three strains with the highest relative increase in urease activity after ARTP mutagenesis for 60S.

[0076] The results showed that the average urease activity of strain M0 without mutation treatment was 1.64 mM·(L·min) -1 The average urease activity of the strains treated with mutation for 50s, 55s, and 60s was 2.49 mM·(L·min) -1 、3.75mM·(L·min) -1 、2.47mM·(L·min) -1 Overall, the relative increase rates of urease activity were 51.8%, 128.6% and 50.6%, respectively. The urease activity of the M55-1 mutant was the highest, reaching 4.31 mM·(L·min) -1 Compared with the starting strain M0-1, the average urease activity increased by 1.63 times.

[0077] (3) Screening of mutant strains by inducing calcium carbonate precipitation

[0078] In order to clarify the mineralization efficiency of calcium carbonate precipitation induced by mutants, the amount of calcium carbonate deposition mediated by the 9 selected mutant strains was detected. The detection method was as follows: take a 10mL centrifuge tube, dry it at 105℃ for 3h, and weigh the mass of the centrifuge tube, recorded as m1; take 5mL of the bacterial solution of the 9 mutants and add it to a 1mol / L cementing solution (mixed with urea and calcium acetate at a molar concentration of 1:1), and induce calcium carbonate mineralization at room temperature. After standing for 24h, discard the supernatant in the centrifuge tube, place the centrifuge tube containing the precipitate in a 105℃ drying oven, and dry it to a fixed weight. At this time, the mass of the centrifuge tube containing the precipitate is recorded as m2, and the mass of calcium carbonate m=m2-m1.

[0079] Under the same mutation time, the calculation formula of mineralization increase rate is as follows:

[0080] Mineralization increase rate = (average amount of calcium carbonate precipitation in the induced treatment group - average amount of calcium carbonate precipitation in the control group) / average amount of calcium carbonate precipitation in the control group

[0081] The results of the mineralization of calcium carbonate precipitation are as follows Figure 4 As shown, the mineralization effect of the strain M55 group was the most significant, showing the highest mineralization increase rate. Specifically, the average calcium carbonate precipitation of the M55 group was 858.7 mg, which was a significant increase of 78.1% compared with the average value of 482.1 mg of the control group M0. This may indicate that the M55 mutant has stronger metabolic or biochemical activity in the mineralization process, resulting in a significant increase in mineralization. In contrast, the mineralization increase rates of the M50 and M60 groups were 41.9% and 25.9%, respectively, indicating that these two groups of strains also had a certain improvement in mineralization efficiency, but far less than the M55 group. Among them, the M55-1 mutant induced the largest amount of calcium carbonate precipitation, reaching 873.5 mg, which was 0.78 times higher than the average value of the calcium carbonate precipitation induced by the M0-1 starting strain.

[0082] (4) Genetic stability determination

[0083] The mutant strain M55-1 was subcultured for 5 times, inoculated into CASO liquid medium at a 1% inoculation ratio and cultured in a shaking incubator for 24 h (temperature 30°C, frequency 180 r / min). The electron microscope scanning image of the strain morphology of the Bacillus pasteurianus mutant strain M55-1 is as follows: Figure 5 shown.

[0084] Total DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the genomic DNA was used as a template to identify the strain molecularly based on 16S rRNA primers (27F-AGAGTTTGATCCTGGCTCAG and 1492R-TACGGYTACCTTGTTACGACTT). Reaction system (50 μL): Mix solution 25 μL, upstream and downstream primers 2 μL each, DNA template DNA 2 μL, ddH2O11 μL. Reaction procedure: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 40 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, a total of 30 cycles, 72℃ extension for 8 min, and 4℃ storage. The qualified PCR amplification products were electrophoresed with 1% agarose gel at 120 V for 32 min, and the gel imager was used for detection and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing of the qualified PCR products.

[0085] The sequence results of the gene fragment obtained based on 16S rRNA primers (27F-AGAGTTTGATCCTGGCTCAG and 1492R-TACGGYTACCTTGTTACGACTT) are shown in SEA ID NO.2. After NCBIBlast comparison, it can be seen that the sequence of the 16S region of strain ΔM55-1 is shown in SEA ID NO.1.

[0086] Then construct a phylogenetic tree, such as Figure 6 As shown, strain ΔM55-1 was clustered with multiple strains including Sporosarcinaterrae and Sporosarcina pasteurii, with a homology of 99%. Therefore, mutant strain M55-1 was identified as Sporosarcina (Bacillus pasteurianus).

[0087] The mutant strain M55-1 of Bacillus pasteurianus was deposited in the China Type Culture Collection (CCTCC) located at Wuhan University on August 28, 2024, and the deposit number of the mutant strain M55-1 of Bacillus pasteurianus (Sporosarcina pasteurii) is CCTCC M 20241864.

[0088] The optical density and urease activity of Bacillus pasteurianus, a mutant strain of the genus Sporosarcina, were tested. The results of the genetic stability test of urease activity were as follows: Figure 7 The results of genetic stability determination of optical density values ​​are shown in Figure 8 The results showed that the urease activity of mutant strain M55-1 was maintained at 4.22-4.51 mM·(L·min) -1, the optical density value remained between 2.31 and 2.45, indicating that the genetic performance of the mutant strain M55-1 was relatively stable and could be applied to induce calcium carbonate precipitation and mineralization in cemented soil.

[0089] (II) Application of Bacillus pasteurianus mutant M55-1 to induce calcium carbonate deposition in sandy soil cementation

[0090] The use of high-concentration microbial induced calcium carbonate precipitation (MICP) treatment process may lead to the outflow of excessive ammonia in the byproduct, posing a potential threat to coastal sand and its adjacent sea areas and local underground ecosystems. To solve this problem, the present invention proposes an improved microbial induced calcium carbonate precipitation (MICP) treatment method.

[0091] Based on the response surface methodology (RSM), the low molar concentrations of urea and calcium acetate were optimized to reduce the ammonia concentration in the MICP effluent while ensuring the mechanical properties of the treated sand.

[0092] The specific steps are as follows:

[0093] Based on the previous experiments, this example uses the three key factors that significantly affect the mechanical strength of sand and the ammonium concentration of the effluent during the process of calcium carbonate deposition induced by the mutant strain M55-1, namely A (urea concentration), B (calcium acetate concentration) and C (inoculation ratio) as independent variables, and Y1 (compressive strength) and Y2 (ammonium concentration) as response values. The three-factor three-level Box-Behnken response surface optimization test is designed using the Design Expert 13.0 software to comprehensively evaluate the influence of each factor on the mechanical properties of the cemented sand induced by calcium carbonate deposition by the M55-1 mutant strain and the ammonium concentration of the effluent. The coding and level of each factor in the Box-Behnken design are shown in Table 1.

[0094] Table 1 Box-Behnken test design factor coding and level

[0095]

[0096] 1. Materials and Methods

[0097] (1) Test sand:

[0098] The test sand is China IOS standard sand.

[0099] (2) Preparation of mutant strain M55-1 bacterial solution:

[0100] Based on the mutant strain M55-1 obtained in the above experiment, it was inoculated into CASO liquid culture medium in a triangular flask and cultured at 30°C for 18 hours to obtain the mutant strain M55-1 bacterial solution. According to the Box-Behnken design, 500 μL, 750 μL and 1000 μL of M55-1 bacterial solution were inoculated into 50 mL of CASO liquid culture medium, cultured at 30°C for 18 hours, and set aside. CASO medium needs to be sterilized at 121°C for 30 minutes before inoculation.

[0101] (3) Preparation of cementing fluid:

[0102] According to the Box-Behnken design, a total of 17 groups of cementing fluids with different concentrations were prepared. The cementing fluid is a mixture of urea and calcium acetate solution. To avoid concentration dilution during mixing, the concentrations of urea and calcium acetate during actual preparation should be twice the design concentration. For example, for a design concentration of 0.3 mol / L urea and 0.2 mol / L calcium acetate, 0.6 mol / L urea and 0.4 mol / L calcium acetate solution should be used during preparation, and the mixture is the cementing fluid required for the Box-Behnken design combination.

[0103] (4) Grouting process:

[0104] Biopumping reinforcement loess sample device Fig. 9 As shown. Specifically, the grouting mold uses a self-made split acrylic tube device (wall thickness 10mm) to prevent disturbance damage during sampling after MICP reinforcement. The device consists of a split tube, a clamp, a latex plug and a hydraulic fabric. 285g of standard sand is loaded into the tube to form a height of 10cm, a diameter of 50cm, and an initial dry density of 1.45g / cm 3 Sand column. First, use a peristaltic pump to inject clean water at a rate of 4mL / min to remove the air in the sand sample and saturate the sample. Then inject the bacterial solution at a rate of 2mL / min. The injection volume of the bacterial solution is 50mL, which is about 1-4 times the pore volume of the sand column, and the best is about 1.5 times the pore volume of the sand column. Let it stand for 5 hours. Then inject the binder at the same rate. The volume of the binder is also 50mL, which is about 1-4 times the pore volume of the sand column, and the best is about 1.5 times the pore volume of the sand column. The above steps are one round of grouting. After 48h, the above steps are circulated and grouted again. A total of 5-8 rounds of grouting are carried out, and 8 rounds of grouting are the best. The curing method is natural air drying for 7 days. The results are as follows Fig.16 shown.

[0105] (5) Compressive strength test:

[0106] The unconfined compressive strength test was carried out using a YAW-300B micro electronic control testing machine with a loading speed of 0.5 mm / s.

[0107] (6) Detection of ammonium concentration in effluent:

[0108] The effluent after MICP treatment was diluted with deionized water to a conductivity of 0-0.5 mM. 2 mL of the dilution was mixed with 10 μL of Nessler reagent and reacted in a cuvette for 1 min. The absorbance was measured at 245 nm using a visible spectrophotometer. The absorbance value was converted to NH 4+ The calibration curve equation is Y = 0.00359X + 0.0032, where Y is the absorbance value and X is NH4 + Concentration (mg / L).

[0109] 2. Test results

[0110] The construction of the response surface model, regression analysis, and parameter optimization is to input the response value into the Design Expert software, output the response surface based on the response analysis results, and judge the significance of the influence of each factor on the response value, and finally obtain the excellent comprehensive performance of MICP cemented sand mediated by mutant strain M55-1 through parameter optimization. Table 2 shows the Box-Behnken response test combination and results.

[0111] (1) Regression model construction and variance analysis:

[0112] Table 2 Box-Behnken response test combinations and results

[0113]

[0114] Multiple regression fitting was performed on the three factors A (urea concentration), B (calcium acetate concentration) and C (inoculation ratio), and the second-order regression model equation of the response value Y1 (compressive strength) and Y2 (ammonium concentration) on the independent variable was established:

[0115] Y1=1.94375-14.975*A+3.45*B+0.0825*C-6.00*A*B+2.3*A*C-1.2*B*C+32.5*A 2 +7.5*B 2 +0.025*C 2

[0116] Y2=-89.175+1320*A+20*B-40.7*C-1300*A*B+230*A*C-50*B*C-1510*A 2 +

[0117] 890*B 2 +2.9*C 2

[0118] Table 3 Analysis of variance of compressive strength regression model

[0119] source sum of squares Degrees of Freedom Mean Square F-number P-value Significance Model 0.9566 9 0.1063 102.98 <0.0001 *** A-urea concentration 0.1426 1 0.1426 138.16 <0.0001 *** B-Calcium acetate concentration 0.0726 1 0.0726 70.34 <0.0001 *** C-Inoculation ratio 0.0435 1 0.0435 42.15 0.0003 ** AB 0.0009 1 0.0009 0.872 0.3815 AC 0.0272 1 0.0272 26.38 0.0013 * BC 0.0036 1 0.0036 3.49 0.1040 <![CDATA[A 2 ]]> 0.0199 1 0.0199 19.28 0.0032 ** <![CDATA[B 2 ]]> 0.0041 1 0.0041 3.98 0.0861 <![CDATA[C 2 ]]> 0.0002 1 0.0002 0.1594 0.7017 Residual 0.0072 7 0.001 Lack of Fit 0.0038 3 0.0013 1.5 0.3429 Pure Error 0.0034 4 0.0009 sum 0.9638 16

[0120] Table 4 Analysis of variance for regression model of ammonium concentration

[0121]

[0122] In this embodiment, the regression model was subjected to variance analysis and significance test. The variance analysis of the Y1 regression model and the Y2 regression model is shown in Table 3 and Table 4. The F values ​​of the Y1 and Y2 regression models were 102.98 and 37.70, respectively, and the P values ​​were both <0.0001, reaching an extremely significant level; the F values ​​of the lack of fit terms of the Y1 and Y2 regression models were 0.3429 and 0.2178, respectively, both of which were not significant, indicating that the Y1 and Y2 regression models were well fitted. The Y1 determination coefficient R 2 =0.9876, Y2 determination coefficient R 2 =0.9798, indicating that both the Y1 and Y2 regression models can fit more than 97.0% of the response value. The signal-to-noise ratios of the Y1 and Y2 regression models were 38.6055 and 20.9686, respectively, both greater than 4, indicating that the Y1 and Y2 regression models have sufficient signal strength. In addition, the coefficients of variation of the Y1 and Y2 regression models were 1.76% and 3.40%, respectively, both less than 10%, proving that the Y1 and Y2 regression models have good credibility and good fit. The adjusted correlation coefficient R of the Y1 regression model Adj 2 (0.9717) and the predicted correlation coefficient R Pred 2 (0.8536), and the adjusted correlation coefficient R of the Y2 regression model Adj 2 (0.9538) and the predicted correlation coefficient R Pred 2 The difference between them (0.7833) is less than 0.2, which proves the significance of the model.

[0123] The Y1 and Y2 regression models conform to the above test principles, indicating that the Y1 and Y2 models have good fitting and adaptability. The Y1 and Y2 regression models can be applied to the analysis, prediction and parameter optimization of the comprehensive properties of MICP-cemented sand mediated by the mutant strain M55-1.

[0124] (2) Response surface analysis and process optimization:

[0125] The response surface can reflect the main effect interaction effect of each factor on compressive strength and ammonium concentration. The results are as follows: Figures 10 to 15As shown in the figure, urea concentration (A), calcium acetate concentration (B) and inoculation ratio (C) have extremely significant effects on compressive strength (P < 0.01), and the significant order of the three factors affecting compressive strength is urea concentration (A) > calcium acetate concentration (B) > inoculation ratio (C). Urea concentration has an extremely significant effect on ammonium concentration (P < 0.01), while calcium acetate concentration (B) and inoculation ratio (C) have no significant effect on ammonium concentration (P > 0.05); the interaction term AC has a significant effect on compressive strength and ammonium concentration (P < 0.05), and the interaction terms AB and BC have no significant effect on compressive strength and ammonium concentration.

[0126] Based on the experimental results of the Y1 and Y2 regression models, it is predicted that the optimal process conditions for low ammonia and ammonium concentration in the effluent liquid under the condition of ensuring mechanical properties of MICP-cemented sand mediated by mutant strain M55-1 are: urea concentration of 0.2 mol / L, calcium acetate concentration of 0.27 mol / L, inoculation ratio of 1.1%, predicted compressive strength of 1.70 MPa, and ammonium concentration of the effluent liquid of 109 mg / L.

[0127] The test was repeated three times under the MICP process conditions to verify the accuracy of the regression model. The actual compressive strength of the MICP-bonded sand was measured to be 1.52 MPa, and the effluent liquid ammonia concentration was 116 mg / L, which were slightly different from the model prediction values ​​and had reference value.

[0128] (3) Process comparison and effect evaluation:

[0129] Compared with the traditional MICP cementation process (the strain is Bacillus pasteurianus, the cementing liquid concentration is 1 mol / L, the ratio of urea concentration to calcium acetate concentration is 1:1, and the inoculation ratio is 1%), after the optimization of the MICP cementation sand process mediated by the mutant strain M55-1 in the present invention, the ammonium concentration of the effluent is reduced by 6.24 times, while still maintaining sufficient mechanical properties.

[0130] In summary, the MICP process based on the mutant strain M55-1 in the present invention exhibits good mechanical properties and low environmental load in sandy soil cementation, and has broad application prospects.

[0131] The application of microbial induced calcium carbonate precipitation based on the mutant strain M55-1 is specifically used in the fields of embankment reinforcement, slope stabilization, wellbore leakage control, sand prevention and fixation, fractured rock reinforcement, building material repair, soil anti-seepage, mitigation of soil liquefaction, soil resistance to rainfall and wind erosion, and enhanced soil stability.

[0132] In specific implementation, the calcium source can be at least one of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, calcium acetyl, calcium silicate, calcium aluminate, and dicalcium acetyl.

[0133] When the binder and the bacterial solution are both in liquid form, the liquid bacterial solution and the liquid binder are mixed with the reinforced object before and after or allowed to be fully absorbed by the reinforced object. Specifically, the liquid calcium source and the liquid urea solution can be fully mixed into a binder. The binder, the bacterial solution and the reinforced object (dam, sand, rock, soil, etc.) are fully mixed and then left to stand and wait for solidification.

[0134] Urea and calcium source in the cementing fluid can be solid powders, and live bacteria are also prepared in powder form for easy carrying and mixing. Powdered calcium source, powdered urea and bacterial powder are fully mixed with the reinforced object (dam, sand, rock, soil, etc.), and then left to stand and wait for solidification.

Claims

1. A Bacillus pasteurianus, deposited in China Center for Type Culture Collection with the deposit number CCTCCM20241864.

2. Application of Bacillus pasteurianus with a deposit number of CCTCC M 20241864 in microbial-induced calcium carbonate precipitation.

3. A Bacillus pasteurianus, It is characterized in that The 16S rDNA sequence is included as shown in SEA ID NO.1; the best one includes the sequence as shown in SEA ID NO.

2.

4. Use of Bacillus pasteurianus as claimed in claim 3 in microbial induced calcium carbonate precipitation.

5. A microbial induced calcium carbonate precipitation component, It is characterized in that The invention comprises Bacillus pasteurianus, urea and a calcium source, wherein the Bacillus pasteurianus is the Bacillus pasteurianus with a deposit number of CCTCC M 20241864; preferably, the invention comprises Bacillus pasteurianus, urea and a calcium source, wherein the Bacillus pasteurianus comprises a 16S rDNA sequence as shown in SEA ID NO.1; preferably, the invention comprises Bacillus pasteurianus, urea and a calcium source, wherein the Bacillus pasteurianus comprises a sequence as shown in SEA ID NO.

2.

6. The microbial induced calcium carbonate precipitation component as claimed in claim 5, It is characterized in that The urea concentration is 0.2 mol / L, the calcium source is calcium acetate with a concentration of 0.27 mol / L, and the pasteurian Bacillus inoculation ratio is 1.1%; preferably, the calcium source is at least one of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, acetyl calcium, calcium silicate, calcium aluminate, and dicalcium acetyl.

7. A method for inducing calcium carbonate precipitation by Bacillus pasteurianus, Features: Add the microbial-induced calcium carbonate precipitation component described in any one of claims 5-6 to the object to be reinforced.

8. A method for microbial induction of calcium carbonate precipitation, Features: The Design Expert software was used to perform multiple regression fitting on the three independent variables A (urea concentration), B (calcium concentration) and C (microbial inoculation ratio), and the second-order regression model equation of the response values ​​Y1 (compressive strength) and Y2 (ammonium concentration) on the three independent variables was established; According to the urea concentration, calcium concentration and microbial inoculation ratio added to the reinforced object, the compressive strength can be predicted through the second-order regression model equation; According to the urea concentration, calcium concentration and microbial inoculum ratio added to the reinforced object, the ammonium concentration of the effluent can be predicted by the second-order regression model equation.

9. The method for microbial induced calcium carbonate precipitation as claimed in claim 8, Features: The inoculated microorganism is Bacillus pasteurianus with a deposit number of CCTCC M 20241864, and the calcium concentration is a calcium acetate concentration; preferably, the inoculated microorganism is Bacillus pasteurianus including a 16S rDNA sequence as shown in SEA ID NO.1, and the calcium concentration is a calcium acetate concentration; preferably, the inoculated microorganism is Bacillus pasteurianus including a sequence as shown in SEA ID NO.2, and the calcium concentration is a calcium acetate concentration.

10. A method for screening Bacillus pasteurianus for microbial induced calcium carbonate precipitation, It is characterized in that Bacillus pasteurianus is mutated by atmospheric pressure and room temperature plasma to screen out mutants whose urease activity is improved compared with that of the original strain; preferably, the atmospheric pressure and room temperature plasma mutagenesis test parameters are power 120W, gas flow 10SLM, radiation distance 2mm, and mutagenesis treatment time 50s-60s.

11. A culture medium for culturing Bacillus pasteurianus, Features: Including casein peptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, and urea 20g / L.

12. The culture medium according to claim 11, Features: The pH of the culture medium is 7.5; preferably, it is used to culture Bacillus pasteurianus with a deposit number of CCTCC M 20241864; preferably, it is used to culture Bacillus pasteurianus including a 16S rDNA sequence as shown in SEA ID NO.1; preferably, it is used to culture Bacillus pasteurianus including a sequence as shown in SEA ID NO.2.

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