Coal slime mineralization microorganism double-urease-producing-bacterium synergistic dust suppressant, screening method and application

Abstract

The invention belongs to the technical field of microbial dust suppressants, and discloses a coal slime mineralization microbial double-urease-producing-bacterium synergistic dust suppressant, a screening method and application. The dust suppressant is prepared from bacillus pasteurii and indigenous urease producing bacteria through inoculation, and the inoculation ratio of the bacillus pasteurii to the indigenous urease producing bacteria is 1: 1. The two strains are subjected to compound culture, the influence of different inoculation time on the growth of compound microorganisms is researched, and the result shows that the BM microbial agent system is the optimal compound microbial agent system. The particle size range of precipitates generated by mixed strain culture is larger, the wetting effect is better than that of a single strain, and when the wind speed is 10m. S <-1 >, the wind erosion resistant efficiency is 93.1%. The BM double-production urease complex microbial inoculant system shows an excellent dust suppression effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial dust suppressant, and particularly relates to a coal slime mineralization microbial dual urease bacteria synergistic dust suppressant, a screening method and application. BACKGROUND

[0002] In the process of large-scale exploitation and utilization of coal resources, how to realize the safe and green environmental protection exploitation and transportation of coal mines is the key research direction. The existing technology has studied some dust suppression methods for coal dust, but the dust suppressant for suppressing coal dust still has certain research prospects and depth in environmental protection, economy, degradation and high efficiency, etc. Therefore, it is a current research hotspot to study a high-efficiency, environmentally-friendly and low-cost coal dust suppressant.

[0003] The microbial induced calcium carbonate precipitation technology is a process of using the metabolism of microorganisms to produce urease to decompose urea, and the carbonate ions produced after the decomposition of urea combine with free metal cations to generate gelatinous crystals. The technology has low energy consumption, low production cost, sustainable development and high environmental protection, and in the development process of the strategic goal of strengthening occupational health, the microbial induced calcium carbonate precipitation technology has been gradually researched and applied to the coal dust suppression.

[0004] Some related researches have been carried out on non-indigenous urease bacteria for coal dust suppression: Shi et al. used Bacillus pasteurii and cementing liquid to carry out coal dust suppression research, studied the influence of bacterial solution on dust capture and cementation, and the influence of urea concentration on the urease of Bacillus pasteurii, and the optimal cementing liquid concentration was 0.6 mol·L -1The weight loss rate of single spraying dust suppression is 3.44%. Shi et al. studied the influence of different calcium sources and different concentrations of cementing solution on the dust suppression effect of bacillus pasteurii bacterial agent. The experimental results show that the best calcium source is calcium chloride, and the yield reaches 81.11%, and the content of calcite in the mineralized product is high. Shi et al. studied the dust suppression effect of bacillus pasteurii dust suppressant on different particle size coal particles and different types of coal dust. When the particle size of coal dust is greater than 200 μm, the dust suppression effect is better, and the agglomeration performance of the mineralized product on lignite is better than that on other coal. The research on dust suppression by screening bacteria from the environment is relatively less. At present, Song et al. isolated a strain of staphylococcus succinum J3 from the soil of a mine area. Through research, it is found that the bacteria have high urease activity and tolerance to coal dust. The influence of initial bacterial biomass, calcium concentration, urea concentration and spraying frequency on the staphylococcus MICP process is studied by single factor experiment method. Song et al. screened and isolated two urease-producing bacteria SZS1-3 and SZS1-5 in the coal mine soil in order to popularize the application of microbial induced carbonate precipitation (MICP) technology in coal mine soil dust suppression. The mineralized product of the microorganism is vanadium stone type and calcite type calcium carbonate, but the whole microbial induced calcium carbonate precipitation needs a long time. The research on dust suppression by composite culture microorganism is less. Zhu et al. co-cultured two non-indigenous urease-producing bacteria bacillus pasteurii and bacillus cereus to study the dust suppression effect. The experimental results show that the continuous inoculation with an interval of 14 h is the best growth inoculation condition, and the urease activity is the highest. The addition of cementing liquid in the bacterial solution can successfully solidify the coal powder, and the mineralized product of the composite bacteria is vanadium stone type and calcite type calcium carbonate. Hu et al. co-cultured urease-producing bacteria bacillus sphaericus and carbonic anhydrase-producing bacteria bacillus mucilaginosus for dust suppression experiment. The double bacteria system can effectively improve the mineralization yield of single bacteria, and has no effect on the mineralized product.

[0005] Microbial dust suppressant has gradually begun to be applied in the field of coal mine dust suppression due to its advantages of no pollution, economy, high efficiency and the like. However, microbial dust suppression is a new research direction, and there are still problems worthy of further research and improvement in the use of MICP technology to suppress coal dust. First, only a few strains are suitable for coal dust suppression, and the types of indigenous urease-producing bacteria need to be increased. Second, most of the current microbial dust suppression research uses single strain, but in fact, due to the hydrophobicity of coal dust and the complexity of the coal mine environment, single strain dust suppression has some shortcomings, and the advantages of mixed strains in growth and dust suppression need to be further studied. SUMMARY

[0006] To overcome the problems in the related art, the present application discloses a coal slime mineralization microbial double urease-producing bacteria synergistic dust suppressant, a screening method and an application, and specifically relates to a coal slime mineralization microbial screening-separation and double urease-producing bacteria synergistic dust suppression performance and mechanism research.

[0007] The technical solution is as follows: a slime mineralization microbial dual urease-producing bacteria synergistic dust suppressant, which is inoculated with Bacillus pasteurii and indigenous urease-producing bacteria to achieve dust suppression; the inoculation ratio of Bacillus pasteurii and indigenous urease-producing bacteria is 0.25%, the concentration of Bacillus pasteurii is 4.8*10 9 CFU / mL, and the concentration of indigenous urease-producing bacteria is 7.1*10 9 CFU / mL.

[0008] Further, the inoculation time of Bacillus pasteurii and indigenous urease-producing bacteria includes:

[0009] Bacillus pasteurii and indigenous urease-producing bacteria MN-5 are inoculated at the same time, which is recorded as BM; indigenous urease-producing bacteria is inoculated 5 hours and 24 hours after Bacillus pasteurii is inoculated, which is recorded as B5M and B 24 M; the indigenous urease-producing bacteria is MN-5 mineralization bacteria, and Bacillus pasteurii is referred to as B mineralization bacteria; then, culture is carried out to the stable period, and the time length is 24h, at which time OD 600 basically remains stable.

[0010] Further, the inoculation time of Bacillus pasteurii and indigenous urease-producing bacteria further includes:

[0011] Bacillus pasteurii is inoculated 5 hours and 16 hours after indigenous urease-producing bacteria is inoculated, which is recorded as M5B and M 16 B.

[0012] Another object of the present application is to provide a screening method of a slime mineralization microbial dual urease-producing bacteria synergistic dust suppressant, which is used for screening the slime mineralization microbial dual urease-producing bacteria synergistic dust suppressant, and the method comprises the following steps:

[0013] S1, culturing Bacillus pasteurii in culture medium material;

[0014] S2, screening indigenous urease-producing bacteria from a slime sample, and screening indigenous urease-producing bacteria belonging to lysine bacillus, named MN-5;

[0015] S3, different inoculation times are set for the cultured Bacillus pasteurii and the screened urease-producing indigenous bacteria named MN-5 to obtain different compound microbial community samples, and growth test analysis is performed, and the inoculation ratio of Bacillus pasteurii and the screened urease-producing indigenous bacteria named MN-5 is 1:1.

[0016] In step S2, the urease-producing indigenous bacteria belonging to Bacillus licheniformis and named MN-5 are screened, including:

[0017] S201, selection and pretreatment of coal samples, and preparation of coal slime samples;

[0018] S202, treatment and separation of the prepared coal slime samples, and preliminary screening of urease-producing indigenous bacteria strains with urease-producing capacity;

[0019] S203, Gram staining and DNA sequencing of the urease-producing indigenous bacteria strains with urease-producing capacity, screening of the urease-producing indigenous bacteria belonging to Bacillus licheniformis and named MN-5, preservation in the China Center for Type Culture Collection, Wuhan, Wuhan University, China, preservation number CCTCC NO: M 20251465, preservation date July 1, 2025, detection result survival, classification and naming Lysinibacillus sp. MN-5.

[0020] In step S202, treatment and separation of the prepared coal slime samples, and preliminary screening of urease-producing indigenous bacteria strains with urease-producing capacity, including:

[0021] The coal slime sample is placed in 5 mol / L high-concentration urea medium for acclimation and enrichment culture for 24 hours, wherein the urea medium is composed of 200 mL sterile water and 5 mol / L urea; the rotation speed is set to 150 rpm, and the temperature is set to 35°C.

[0022] The sterile water is added to the coal slime mixed suspension for dilution, and the dilution is performed in gradients of 10 -6 , 10 -7 , 10 -8 , 10 -9 Coating screening is performed, and the coating is inoculated on the urea-agar medium, and the culture is performed at 30°C for 24-48 h, and the colored colonies around the urea-agar medium are selected and streaked, and after multiple purification culture, the single colonies are obtained.

[0023] The morphologically different colonies appearing are separated and screened, and 5 bacteria are separated and labeled as #1, #2, #3, #4 and #5; after culture under the same conditions for 48 hours, the #5 bacteria have urease-producing capacity.

[0024] Further, urea-agar medium: proteose peptone 15 g / L, NaCl 5 g / L, KH2PO3 2 g / L, glucose 0.1 g / L, phenol red 0.1 g / L, urea 20 g / L, agar 20 g / L, water 1 L, pH = 7;

[0025] Bacterial solid medium: same as liquid medium, additionally 20 g / L agar; specifically: casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH = 7, agar 20 g / L.

[0026] In step S202, after the indigenous urease-producing bacterial strain with urease-producing ability is preliminarily screened, growth test, urease activity determination and characterization experiments are further performed.

[0027] In step S3, different inoculation times are set for the cultured Bacillus pasteurii and the screened indigenous urease-producing bacteria named MN-5, to obtain different compound bacterial samples, including inoculating MN-5 after 5 hours of inoculating Bacillus pasteurii B. pasteurii, sterilizing the completed bacterial liquid medium to room temperature, inoculating 0.5 mL of Bacillus pasteurii liquid bacterial liquid into 100 mL of liquid medium in a sterile operation table by using a pipette; then, the inoculated sample is placed in a constant-temperature shaking incubator for culture, and the culture condition is set as 25 DEG C and 180 rmp, and is recorded as sample B5M.

[0028] Bacterial liquid medium: casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH = 7; used for growth culture of indigenous bacteria, to form a liquid bacterial liquid, and the liquid culture is mainly used in subsequent performance experiments.

[0029] Another purpose of the present application is to provide an application of the slime mineralization microbial double urease-producing bacteria synergistic dust suppressant in coal mine dust suppression, and the slime mineralization microbial double urease-producing bacteria synergistic dust suppressant is applied.

[0030] In combination with all the above technical solutions, the present application has the following beneficial effects:

[0031] First, for the currently researched microbial dust suppression technology, the present application innovatively proposes a double urease-producing bacterial strain compound culture dust suppression bacterial agent, one of which is Bacillus pasteurii, and the other is an indigenous urease-producing bacteria isolated and screened from coal slime in a coal mine, which is identified as lysine bacillus through 16s rDNA sequencing and gene sequence comparison, and is named MN-5. The optimal growth pH experiment of the MN-5 strain is performed, and the optimal mineralization Ca 2+Concentration experiment, the experimental results of MN-5 are as follows: the optimum growth pH is 7-8; the optimum mineralized Ca 2+ The concentration is 0.6mol·L -1 . MN-5 strain is in the lag phase within 0-5h; the logarithmic growth phase is 5-16h; after 16h, it enters the stationary phase. The strain has the ability to induce calcium carbonate precipitation, and the maximum urease activity is 5.15mmol·L -1 ·min -1 during the experiment. The precipitate produced by mineralization is mostly calcite type. The complex culture of the two strains is explored to explore the influence of different inoculation times on the growth of complex microorganisms. The results show that the BM microbial agent system is the best complex microbial agent system. The precipitate produced by mixed strain culture has a larger particle size range, and the wetting effect is better than that of single strain. When the wind speed is 10m·s -1 , the anti-erosion efficiency is 93.1%. The BM double urease complex microbial agent system shows excellent dust suppression effect.

[0032] Second, the core advantage of double urease complex microbial dust suppressant is high-efficiency and environmentally friendly dust suppression. It can be widely used in construction, mining, port logistics and other fields. It can quickly occupy the market with 93.1% anti-erosion efficiency, and the low raw material cost and long service life can significantly improve the profit space.

[0033] The core ability of the dust suppressant to induce calcium carbonate precipitation has broad development space: in the field of ecological restoration, it can be used for soil solidification, desertification control and ancient building stone repair, and can stabilize loose substrates through biomineralization; in the field of construction engineering, it is expected to replace traditional cementitious materials for light building material production or crack self-repair, and reduce carbon emissions. The multi-field reusability will greatly improve its commercial value and form a "dust suppression as the basis, multi-domain extension" profit pattern.

[0034] Third, the double urease complex microbial dust suppressant fills the technical gap of double-strain complex microbial dust suppression at home and abroad with double-strain complex culture technology, and has irreplaceable competitiveness in dust suppression scenes. At the same time, its core ability to induce calcium carbonate precipitation extends to the fields of ecological restoration (soil solidification, desertification control), construction engineering (building material production, crack repair), etc.

[0035] The dual-urease-producing compound bacteria dust suppressant has successfully overcome the long-standing technical challenges of limited effectiveness and insufficient wind erosion resistance in physical, chemical, and single-microbial dust suppression methods. While previous attempts to suppress dust through microorganisms have shown that single-strain mineralization precipitates have a narrow particle size range and poor wetting effect, making stable dust control difficult in high-wind-velocity environments. This new system, with its synergistic effect of dual strains, not only improves wind erosion resistance but also addresses the poor adaptability of microbial mineralization in complex environments. This breakthrough not only meets the urgent need for efficient dust suppression in industrial settings but also clears the way for the large-scale application of biomineralization technology across multiple fields. Attached Figure Description

[0036] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure;

[0037] Figure 1 This is a flowchart of the screening method for synergistic dust suppressants by dual-producing urease bacteria in coal slime mineralization microorganisms provided in the embodiments of the present invention;

[0038] Figure 2 This invention provides a process flow chart and bacterial strain diagram for screening indigenous urease-producing bacteria in coal slime; (A) shows the screening process, (B) shows the isolation and culture process, and (C) shows the bacterial strain and Gram staining results.

[0039] Figure 3 This is a schematic diagram of the phylogenetic tree of the strains provided by the present invention;

[0040] Figure 4 This invention provides growth curves and urease activity diagrams of MN-5 bacteria at different pH levels.

[0041] Figure 5 These are the SEM and EDS test results provided by this invention;

[0042] Figure 6 The image shows the infrared test results of the mineralization products of Bacillus pasteurii and MN-5 provided by this invention.

[0043] Figure 7 This is an XRD pattern of the microbial mineralization products provided by the present invention;

[0044] Figure 8 This is a growth curve and urease activity assay diagram of a single bacterium provided by the present invention;

[0045] Figure 9 This is the growth curve and urease activity assay diagram of the compound bacteria provided by the present invention;

[0046] Figure 10 This is a characteristic analysis diagram of the compound bacterial mineralization products provided by the present invention;

[0047] Figure 11 is a contact change and penetration test result graph provided by the present application;

[0048] Figure 12 is a consolidation effect electron microscope graph provided by the present application;

[0049] Figure 13 is an anti-erosion test result graph provided by the present application;

[0050] Figure 14 is a double urease-producing bacteria synergistic dust suppression mechanism graph provided by the present application. DETAILED DESCRIPTION

[0051] In order to make the above-mentioned purposes, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0052] Example 1, a slime mineralization microbial double urease-producing bacteria synergistic dust suppressant, the dust suppressant is inoculated by Bacillus pasteurii and indigenous urease-producing bacteria, so as to realize dust suppression; the inoculation ratio of Bacillus pasteurii and indigenous urease-producing bacteria is 0.25%, the concentration of Bacillus pasteurii is 4.8x10 9 CFU / mL, and the concentration of indigenous urease-producing bacteria is 7.1x10 9 CFU / mL.

[0053] For example, the inoculation time of Bacillus pasteurii and indigenous urease-producing bacteria includes: simultaneous inoculation of Bacillus pasteurii and indigenous urease-producing bacteria MN-5 strains, denoted as BM; 5 hours and 24 hours after inoculation of Bacillus pasteurii, indigenous urease-producing bacteria is inoculated respectively, denoted as B5M and B 24 M; the indigenous urease-producing bacteria is MN-5 mineralization strain, and Bacillus pasteurii is denoted as B mineralization bacteria; then, culture to the stable period, the time length is 24h, at this time, OD 600 basically remains stable.

[0054] The inoculation time of Bacillus pasteurii and indigenous urease-producing bacteria further includes:

[0055] Inoculating the indigenous urease-producing bacteria 5 hours after inoculating the Bacillus pasteurii, denoted as M5B, M 16 B.

[0056] As shown in Example 2, the present application provides a screening method for a slime mineralization microbial dual urease-producing bacteria synergistic dust suppressant, which comprises: Figure 1

[0057] S1, culturing the Bacillus pasteurii in a culture medium;

[0058] S2, screening the indigenous urease-producing bacteria from the slime sample, and screening the indigenous urease-producing bacteria belonging to Bacillus licheniformis, named MN-5;

[0059] S3, setting different inoculation times for the cultured Bacillus pasteurii and the screened indigenous urease-producing bacteria named MN-5, obtaining different composite bacterial community samples, and performing growth test analysis, and the inoculation ratio of the Bacillus pasteurii and the screened indigenous urease-producing bacteria named MN-5 is 1:1.

[0060] The different inoculation times for the cultured Bacillus pasteurii and the screened indigenous urease-producing bacteria named MN-5 include inoculating the MN-5 strain 5 hours after inoculating the Bacillus pasteurii, cooling the sterilized bacterial liquid culture medium to room temperature, and inoculating 0.5 mL of the Bacillus pasteurii liquid bacterial solution into 100 mL of the liquid culture medium in a sterile operation table using a pipette gun for inoculation operation; then placing the inoculated sample in a constant temperature shaking incubator for culture, setting the culture conditions as 25℃ and 180 rmp, and denoting the sample as B5M.

[0061] Bacterial liquid culture medium: casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH = 7; used for indigenous bacteria growth culture, forming a liquid bacterial solution, and the liquid culture is the main form used for subsequent performance experiments.

[0062] For example, in step S2, screening the indigenous urease-producing bacteria named MN-5 includes:

[0063] S201, selection and pretreatment of the coal sample, and preparation of the slime sample;

[0064] S202, using the prepared slime sample for treatment and separation, and preliminarily screening the indigenous urease-producing bacteria strains with urease-producing ability;

[0065] ​The indigenous urease-producing bacterial strains with urease-producing capacity are preliminarily screened by treating and separating a coal slime sample, including the following steps:

[0066] The coal slime sample is taken and placed in a 5 mol / L high-concentration urea culture medium for acclimation enrichment culture for 24 hours, wherein the urea culture medium is composed of 200 mL sterile water and 5 mol / L urea; the rotation speed is set to 150 rpm, and the temperature is set to 35 DEG C.

[0067] The sterile water is taken and added to the coal slime mixed suspension for dilution, and gradient dilution is performed in 10 -6 , 10 -7 , 10 -8 , 10 -9 Coating screening is performed, and the coating is inoculated on the urea-agar culture medium, and the culture is performed at 30 DEG C for 24-48 h; the colored colonies around the urea-agar culture medium are selected and streaked, and after multiple purification culture, the single colonies are obtained.

[0068] The colonies with different morphologies are separated and screened, and five bacteria are separated and labeled as #1, #2, #3, #4 and #5; after culture for 48 hours under the same conditions, the #5 bacteria have urease-producing capacity.

[0069] The urea-agar culture medium: proteose peptone 15 g / L, NaCl 5 g / L, KH2PO32 g / L, glucose 0.1 g / L, phenol red 0.1 g / L, urea 20 g / L, agar 20 g / L, water 1 L, pH = 7;

[0070] The bacterial solid culture medium: same as the liquid culture medium, and additionally adding agar 20 g / L; specifically: casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH = 7, agar 20 g / L.

[0071] After the indigenous urease-producing bacterial strains with urease-producing capacity are preliminarily screened, growth test, urease activity determination and characterization experiments are further performed.

[0072] S203, the indigenous urease-producing bacterial strains with urease-producing capacity are subjected to gram staining and DNA sequencing, and the indigenous urease-producing bacteria belonging to lysinibacillus sp. named MN-5 are screened out, and are preserved in the China Center for Type Culture Collection, Wuhan, Wuhan University, China, with the preservation number CCTCC NO: M 20251465 and the preservation date July 1, 2025; the detection result is survival; and the classification and naming is Lysinibacillus sp. MN-5.

[0073] Exemplary, the application also provides a coal slime mineralization microorganism double urease-producing bacteria synergistic dust suppressant for dust suppression in coal mines, and application of the coal slime mineralization microorganism double urease-producing bacteria synergistic dust suppressant.

[0074] Application example: The application screens indigenous urease-producing bacteria from coal slime in a certain coal mine, determines the DNA sequence of the bacteria, optimizes the growth conditions of the bacteria, determines the characteristics of the mineralized products, and determines the dust suppression advantages of the bacteria. Since Bacillus pasteurii has been proven to have high urease activity, produce calcite calcium carbonate, and have strong environmental adaptability and other dust suppression advantages, the application complexly cultivates the indigenous urease-producing bacteria screened from the coal slime and Bacillus pasteurii, analyzes the optimal growth conditions for synergistic dust suppression, and proposes a synergistic dust suppression mechanism of the two bacteria. The complex cultivation of Bacillus pasteurii and the indigenous urease-producing bacteria can reduce the cost of using bacteria, retain the dust suppression advantages of Bacillus pasteurii, and synergistically suppress dust, promote dust suppression in coal mines from the aspects of wetting and consolidation, and strengthen the dust suppression effect on the basis of environmental protection.

[0075] In the above embodiments, the description of each embodiment has its own focus, and the parts not described or recorded in a certain embodiment can be referred to the related description of other embodiments.

[0076] To further illustrate the related effects of the embodiments of the application, the following experiments are performed.

[0077] 1. Materials and methods.

[0078] 1.1 Selection and pretreatment of coal samples: The coal samples used in the experiment are from a certain coal mine. The coal samples are crushed by a crusher, and coal samples with a particle size of 80-100 mesh are selected and dried for use. The component and elemental analysis of the coal samples are shown in Table 1:

[0079] Table 1 Component and elemental analysis of coal samples

[0080]

[0081] 1.2 Source of Bacillus pasteurii and culture medium materials.

[0082] The preparation of the culture medium is 3g casein peptone, 20g / L urea solution, 5g / L soybean peptone, 5g / L sodium chloride, 200mL of deionized water, and the pH is adjusted to about 7.3, and shaken thoroughly. The solid culture medium is the same as above, and additionally 3g / L agar is added. The culture dishes, culture conical flasks and inoculation instruments are placed in a high-temperature sterilization pot and sterilized at 121℃ for 20min; after sterilization, they are placed in a super-clean bench, and the ultraviolet lamp and fan are turned on for sterilization and cooling treatment. When the solid culture medium is not solidified, pour it into the culture dishes until the bottom layer is completely covered, and then invert the culture medium on the table top after the culture medium is completely solidified. Take out the activated strain, and use the streak method to purify and culture beside the alcohol lamp, and place it in a biochemical incubator at 35℃ and culture for 24-48h.

[0083] Prepare the liquid culture medium, use the sterilized forceps and toothpicks to dip the single colonies formed after purification, and inoculate. After inoculation, place it in a shaking incubator at a speed of 180rmp and a temperature of 35℃, and set the culture time according to the test needs.

[0084] The Bacillus pasteurii used in the present application is purchased from the Guangdong Microbial Culture Collection Center, and the strain number is CGMCC 1.803. The casein peptone (BR), soybean peptone (BR), agar, urea (AR) are purchased from Beijing Shuangxuan Microbial Culture Product Factory; calcium chloride (AR), sodium chloride (AR), sodium hydroxide (AR), phenol red (AR), glucose (AR), KH2PO3 (AR) are purchased from Tianjin Yongda Chemical Reagent Co., Ltd.

[0085] 1.3 Screening of indigenous urease-producing bacteria

[0086] 1.3.1 Screening experiment: The coal slime sample used in the experiment is sampled from a certain coal mine. 1g of coal slime sample is placed in 100mL of 5mol / L high-concentration urea culture medium, and domesticated and enriched for 24h at a speed of 150rmp and a temperature of 35℃. 9mL of sterile water is added to 1mL of coal slime mixed suspension for dilution, and gradient dilution is performed in 10 -6 , 10 -7 , 10 -8 , 10 -9 Coating screening, coating inoculation on urea-agar medium, 30℃ culture for 24-48h. Select the red colonies around the culture medium and streak culture, and obtain single colonies after multiple purification culture. The screening process is shown in Figure 2 The process of screening indigenous urease-producing bacteria from coal slime and the strain diagram (A) in the figure shows the screening process.

[0087] In order to determine which strain has urease-producing ability, the morphologically different colonies appearing on the plate are separated and screened, and the colonies with red halos are selected and streaked on the urea-agar medium.Figure 2 As can be seen in the (B) diagram in the process of screening slime indigenous urease-producing bacteria and bacterial strains, about 5 kinds of bacteria are separated, and are respectively marked as #1, #2, #3, #4 and #5. After culturing for 48 hours under the same conditions, it can be seen that the #1 and #5 strains can make the phenol red culture medium red, and the color change of the phenol red of the #5 strain is the deepest. Therefore, the #5 strain is subjected to primary screening, and the urease-producing ability of the #5 strain is the best. Therefore, the present application mainly aims at purifying and culturing the #5 strain and dust suppression experiment.

[0088] Urea-agar culture medium: 15 g / L of proteose peptone, 5 g / L of NaCl, 2 g / L of KH2PO3, 0.1 g / L of glucose, 0.1 g / L of phenol red, 20 g / L of urea, 20 g / L of agar, 1 L of water, and pH=7;

[0089] Bacterial liquid culture medium: 15 g / L of casein peptone, 20 g / L of urea, 5 g / L of soybean peptone, 5 g / L of sodium chloride, 1 L of water, and pH=7;

[0090] Bacterial solid culture medium: same as the liquid culture medium, and additionally 20 g / L of agar.

[0091] 1.3.2 Gram staining results of indigenous urease-producing bacteria: Figure 2 As can be seen in the (C) diagram in the process of screening slime indigenous urease-producing bacteria and bacterial strains, the (a) diagram of the bacterial strain is a bacterial culture dish after purification of the #5 strain. According to the isoelectric point of the bacteria and the composition of the bacterial cell wall, the Gram staining of the bacteria is determined. The negative charge of the Gram-positive bacteria is more than that of the Gram-negative bacteria, and the combination with ammonium oxalate crystal violet is more compact, and the resistance to decolorization of the ethanol solution is strong. The Gram-positive bacteria have high content of peptidoglycan in the cell wall, and the ethanol decolorization and dehydration make the pore size of the cell wall smaller, which prevents the ethanol decolorization, and further shows purple. According to this principle, the Gram staining of the bacteria is observed, and the test results are as follows Figure 2 As can be seen in the (C) diagram in the process of screening slime indigenous urease-producing bacteria and bacterial strains, the Gram staining condition (b) diagram shows that the bacterial strain belongs to Gram-positive bacteria.

[0092] 1.3.3 DNA sequencing of urease-producing indigenous bacteria: The sequencing of the strain was completed in Shanghai Meiji Testing Company. The sample form of the strain was a solid culture medium after purification. The 16S rDNA sequence was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplification primer 27F can be found in the literature Song, W., Yang, Y., Qi, R., Li, J., Pan, X., 2019. Suppression of coal dust by microbially induced carbonate precipitation using Staphylococcus succinus. Environmental Science and Pollution Research 26, 35968-35977.

[0093] The Blast program was used to search for matching sequences (>99% similarity) in the NCGIBank database. The phylogenetic tree was constructed by the neighborhood-joining model and the Bootstrap method (repeated comparison of matching sequences 1000 times) using MEGA7.0 software, and the test results are as follows Figure 3 The strain phylogenetic tree is shown in the schematic diagram. After comparison, it was found that the strain belongs to Bacillus, belongs to Lysinibacillus, and is named MN-5 (urease-producing indigenous bacteria).

[0094] 1.4 Growth test and urease activity determination.

[0095] (1) Growth curve determination: The absorbance value of the bacterial solution at 600 nm was measured to represent the growth of the microorganism, and the specific test process is as follows: the inoculated microbial liquid medium was placed in a 180rmp, 30℃ shaking incubator, and the OD 600 value of the bacterial solution was measured every 2 hours with a UV spectrophotometer (752N type). When the OD 600 value was less than 0.8, the concentration of the microorganism could be calculated by formula (1):

[0096] Y = 8.59 x 10 7 x Z 1.326 (1)

[0097] Where Y is the number of bacteria, cells / mL; Z is the OD 600 value. The growth of the microorganism in the present application is represented by the OD 600 value, and the growth curve is drawn using the OD 600 value.

[0098] (2) Urease activity determination: Urease activity refers to the ability of urease to hydrolyze urea per unit of time. The conductivity test method is used in the present application, and the specific test procedure is as follows: the urea hydrolysis rate is directly proportional to the conductivity of the solution, and 1 ms / min of conductivity change corresponds to 11.11 ms / min of urea hydrolysis amount. 10 mL of bacterial solution is placed in 90 mL of standard urea solution, and the change in conductivity within 5 min is measured. The value is converted to the urea hydrolysis amount per minute, and then multiplied by the dilution factor 10 to obtain the urea hydrolysis amount per minute of the bacterial solution. The value represents the urease activity, and the calculation formula is shown as (2):

[0099] U = C x 11.11 x 10 (2)

[0100] Wherein, U is the urease activity (mmol / L / min); C is the average conductivity change per minute (mS / min); 11.11 is the urease activity coefficient at room temperature.

[0101] 1.5 Characterization experiment: Scanning electron microscopy (JSM-IT800, Thermo Fisher Scientific, Beijing) is used to observe the morphology of the bacterial mineralization product; EDS energy dispersive spectroscopy (JSM-IT800, Thermo Fisher Scientific, Beijing) is used to determine the content and proportion of C, O, Ca elements in the mineralization product, to judge the mineralization product; Fourier infrared spectroscopy (NICOLETiS50, Zhongke Ruijie Technology Co., Ltd., Tianjin) is used to determine the functional groups contained in the microbial mineralization product and the vibration contraction of each absorption peak; X-ray diffraction analyzer (D-MAX 2500 / PC, Japan) is used to analyze the diffraction peak and crystal form of the mineralization product.

[0102] 1.6 Compound bacteria growth experiment: Bacillus pasteurii is referred to as B mineralization bacteria. Indigenous urease-producing bacteria are MN-5 mineralization bacteria screened from a certain coal mine slurry. According to the growth characteristics of the two mineralization bacteria over time, different inoculation time compound bacteria samples are set for growth test analysis. Specifically, Bacillus pasteurii is inoculated for 5 hours, and then MN-5 bacteria (indigenous urease-producing bacteria) are inoculated, which is recorded as B5M. Different samples are set according to the inoculation time, which are BM, B5M, B 24 M, M5B, M 16 B. The inoculation ratio is 1:1. The growth conditions and urease activity of the compound bacteria and single bacteria are analyzed to determine the interaction between the two bacteria and the influence of the compound bacteria on the urease production capacity.

[0103] For example, 5 hours after inoculation of Bacillus pasteurii, MN-5 was inoculated, including: the sterilized bacterial liquid medium was cooled to room temperature, and inoculation was performed in a sterile operation table using a pipette, 0.5 mL of Bacillus pasteurii liquid bacterial liquid was inoculated into 100 mL of bacterial liquid medium. Then, the inoculated sample was placed in a constant temperature shaking incubator for culture, and the culture conditions were set to 25°C, 180 rpm, and the sample was recorded as sample 1.

[0104] After 5 hours of culture, sample 1 was taken out again and placed in a sterile operation table, and MN-5 (urease-producing bacteria) was inoculated again, 0.5 mL of MN-5 liquid bacterial liquid was inoculated into sample 1. The inoculated sample was recorded as B5M, and B5M was placed in a constant temperature shaking incubator for culture, and the culture conditions were set to 25°C, 180 rpm. The inoculation ratio of the two bacteria was 1:1.

[0105] 1.7 Dust suppression effect experiment.

[0106] The dust suppression experiment was carried out using the composite bacterial liquid, and the dust suppression effects of water solution, cementation liquid and single bacterial liquid were compared. The wind erosion resistance of the test solution after acting on the coal dust was tested, the consolidation effect was observed, and the penetration effect and wetting effect of different solutions were tested.

[0107] 1.7.1 Solution penetration capacity and contact angle test: In order to test the wetting of the composite bacteria, the forward penetration and contact angle experiment were used to test the wetting ability of the solution to the coal dust.

[0108] (1) 10 g of dry coal powder was weighed and placed in multiple 25 mL test tubes, in order to ensure uniform dispersion of the dust and facilitate observation, the test tubes containing the coal powder were shaken horizontally to the 15 mL mark. At the same time, 1.5 mL of bacterial liquid was added, and the penetration of the solution was observed after a period of time. The penetration rate within 30 min was calculated. The penetration time was set to 30 min, and the penetration volume was V 30 The penetration speed is represented by P, and the calculation formula is shown in equation (3):

[0109]

[0110] Where P is the penetration speed (mL·min -1 ); V 30 is the penetration volume (mL).

[0111] (2) 0.5 g of dry coal powder was weighed and pressed into a coal cake using a tablet press with a pressure of 20 MPa and an action time of 1 min. The contact angle change of water, cementation liquid, single bacterial liquid and composite bacterial liquid acting on the coal cake was tested using a DSA100 contact angle measuring instrument.

[0112] 1.7.2 Observation of microscopic consolidation characteristics: Scanning electron microscopy was used to test coal dust samples after consolidation in different solutions to observe the microscopic characteristics of the consolidation effect of single and compound microbial dust suppressants.

[0113] 1.7.3 Wind Erosion Resistance Experiment: A wind erosion resistance experiment of the dust suppressant was conducted using a simulated wind turbine. 50g of coal powder sample was placed in petri dishes of the same size, and aqueous solution, cementing liquid, single-strain dust suppressant, and compound-strain dust suppressant were sprayed on each sample. Different wind speeds were simulated according to the distance from the wind turbine. The dust suppression rate was measured after 20 minutes of wind blowing. A coal sample without any spraying was set as a blank group. The experimental results are expressed as dust suppression rate γ, and the calculation formula is shown in (4):

[0114]

[0115] Wherein, γ represents dust suppression efficiency; M1 is the mass of coal dust before air blowing (g); M2 is the mass of coal dust after air blowing (g); and M0 is the mass of blank dust (g).

[0116] 2. Analysis of experimental results.

[0117] 2.1 Growth characteristics of MN-5 bacteria.

[0118] 2.1.1 Analysis of Growth at Different pH Levels: To investigate the optimal pH for the growth of the screened indigenous urease-producing bacteria, liquid culture media with different pH values ​​were prepared and strain MN-5 was cultured. Growth curves and urease activity test results are shown below. Figure 4 The growth curves and urease activity graphs of MN-5 bacteria at different pH levels are shown in Figure (a) and Figure (b) respectively. Figure 4 As can be seen, from 0 to 5 hours, the MN-5 strain is in the lag phase of growth, during which the bacteria, newly inoculated into the culture medium, need a period of adaptation to the new environment; from 5 to 16 hours, the bacteria enter the logarithmic growth phase, at which time the bacteria begin to grow rapidly and have vigorous metabolism; from 16 to 35 hours, this is the stationary phase of bacterial growth, with the growth rate at its maximum and the bacterial population trending towards a stable level; from 35 to 48 hours, the bacteria gradually begin to enter the death phase, with OD... 600The value gradually decreased. It is worth noting that when the culture medium pH = 5, the time it takes for bacteria to enter the logarithmic growth phase is significantly slower than that of bacteria growing at other pH values. This is because the environment is acidic, and bacteria cannot quickly adapt to the acidic environment, thus their growth is slower at the beginning. When the pH is set to 9 and 10, the rate of bacterial death decreases slightly faster when entering the decline phase compared to other pH values. During growth, bacteria also provide an alkaline environment through their own metabolic activities via autotrophic processes, which leads to pH superposition. Excessively high pH values ​​negatively impact bacterial growth activity. Therefore, pH 7-8 is the optimal culture condition for strain MN-5, allowing for rapid bacterial adaptation without affecting the later growth rate. Urease activity was measured on bacteria in the stationary phase after 24 hours of culture. The results show that urease activity is significantly higher at pH 7 and 8 than under other pH conditions, and the test results are generally consistent with the growth curve.

[0119] 2.2 Characteristics of mineralization products.

[0120] 2.1.1 Analysis of mineralized products: such as Figure 5 The SEM and EDS images shown are the scanning electron microscope (SEM) images and EDS results of mineralization products from MN-5 bacteria and Bacillus pasteurii bacteria screened from coal slime. Figure 5 Figure (a) shows scanning electron microscope (SEM) images of mineralization products of Bacillus pasteurellium at different magnifications. From Figure 5 As shown in Figure (a), the mineralization products of *Bacillus pasteurellii* are mostly spherical precipitate crystals of varying sizes with relatively smooth surfaces, ranging from approximately 2 to 20 μm in size. Figure 5 As can be seen in Figure (b), most of the mineralization products of MN-5 bacteria are spherical, such as... Figure 5 Figure (b) shows larger particles, with a general particle size range of 1-30 μm. A small portion consists of ellipsoidal particles, shown in the lower figure, with a particle length range of 3.7 μm-5.5 μm. The surface of the crystals reveals that the precipitates produced by strain MN-5 have a small portion with rough, blocky crystals, which are polycrystalline aragonite composed of nano-sized microcrystalline spheroids. Aragonite is mostly hollow or porous, possessing advantages such as high hydrophilicity and a large specific surface area. The smooth crystals are calcite-type calcium carbonate, which are single crystals. Strain MN-5 provides a wider particle size range for the mineralized consolidation layer. This allows the mineralized deposits to penetrate deeper into the coal dust and adhere to suppress dust.

[0121] Figure 5 (c) diagram and Figure 5Figure (d) shows the EDS test results of the mineralization products of Bacillus pasteurii and MN-5 bacteria, respectively. The precipitate produced by Bacillus pasteurii contained 15.33% C, 64.5% O, and 20.17% Ca, respectively. The precipitate produced by MN-5 bacteria contained 21.21% C, 61.07% O, and 16.72% Ca, with a Ca:C:O ratio of approximately 1:1:3, confirming that the products of microbial mineralization are both calcium carbonate crystals.

[0122] 2.1.2 Infrared Analysis of Mineralized Products: Fourier transform infrared (FTIR) analysis was performed on the mineralized products of Bacillus pasteurii and MN-5. The test results are as follows: Figure 6 Infrared spectroscopy results of mineralization products from Bacillus pasteurii and MN-5 are shown. Figure 6 The results show that the mineralization products of the two bacteria are observed at a wavelength of 3446.39 cm⁻¹. -1 and 3436.21cm -1 The peak value is due to the stretching vibrations of free -NH- groups and hydroxyl groups. This occurs at a wavelength of 1425 cm⁻¹. -1 Strong characteristic absorption peaks appeared on both sides, which is related to the CO antisymmetric stretching vibration (v3) in calcite. MN-5 bacteria showed a peak at 1080.72 cm⁻¹. -1 A peak intensity greater than that of the Bacillus pasteurii bacterium mineralization product was observed, which is due to the presence of some aragonite in the MN-5 bacterium mineralization product. The Bacillus pasteurii bacterium exhibits a peak intensity greater than that of the characteristic peak of the Bacillus pasteurii bacterium mineralization product at 1083.25 cm⁻¹. -1 There is a relatively weak characteristic peak at 875.64 cm⁻¹, which belongs to the CO symmetric stretching vibration (v1). Bacillus pasteurii and MN-5 bacteria showed peaks at 875.64 cm⁻¹. -1 and 871.28cm -1 The presence of characteristic peaks indicates that it belongs to CO3. 2- Out-of-plane bending vibration (v2). MN-5 bacteria at 744.29 cm -1 and 709.88cm -1 The characteristic peaks observed belong to the in-plane bending vibration (v4) of CO in aragonite and calcite, respectively. *Bacillus pasteurii* at 711.94 cm⁻¹ -1 The characteristic peak is the in-plane bending vibration (v4) of calcite calcium carbonate. Comparative analysis of the infrared signatures of the mineralization products of the two bacteria revealed that the MN-5 bacteria mineralization products contain characteristic peaks of both calcite and aragonite, with the calcite peak being stronger and the aragonite peak being weaker.

[0123] 2.1.3 Mineralization product XRD analysis: The X-ray diffraction analysis of the mineralization product of MN-5 bacteria was carried out and compared with the mineralization product of Bacillus pasteurii bacteria, and the test results are shown in Table 2. Figure 7 XRD test of the microbial mineralization product. The XRD pattern of the mineralization product was analyzed for phase using analysis software MDI Jade 6.0. From the analysis of the crystal face of the mineralization product, it can be seen that Bacillus pasteurii bacteria has diffraction peaks at 23.09°, 29.37°, 31.46°, 35.96°, 39.46°, 43.22°, 47.48°, 48.47°, 57.40°, and the crystal face corresponding to calcite type calcium carbonate is (012), (104), (006), (110), (113), (202), (018), (116) and (122) by comparing with PDF #47-1743 card. The mineralization product of Bacillus pasteurii bacteria is calcite type calcium carbonate, and no vaterite diffraction peak appears. According to PDF #83-1762, it can be seen that MN-5 bacteria has diffraction peaks of calcite calcium carbonate at 23.05°, 29.39°, 31.435°, 35.96°, 39.40°, 43.16°, 47.11°, 48.503°, and the corresponding crystal face is (012), (104), (006), (110), (113), (202), (024), (018), (116) and (122). According to PDF #33-0268, it can be seen that the diffraction peaks of vaterite appear at 2θ of 24.90°, 27.05°, 31.78°, 43.84°, 50.07°, and the corresponding crystal face is (110), (112), (114), (300), (118). The molar content of calcite calcium carbonate in the mineralization product of MN-5 bacteria can be calculated by crystal type analysis formulas (5) and (6) as 99.18%, and the content of vaterite is 0.82%.

[0124]

[0125] X C =1-X V (6)

[0126] wherein X V and X C represent the molar content of vaterite and calcite in the sample, respectively, and represent the XRD pattern of vaterite (110) crystal face and calcite (104) crystal face, respectively.

[0127] 2.3 Growth and Urease Activity Tests of Compound Bacteria: Growth curves and urease activity tests of compound bacteria with different inoculation sequences are shown below. Figure 8 Growth curves and urease activity assays for single bacteria. Figure 9 The growth curve and urease activity assay of the compound bacteria are shown in the figure. Figure 8 , Figure 9 The data shows that Bacillus pasteurii exhibits a delayed growth phase from 0-5 hours; a logarithmic growth phase from 5-24 hours; a stationary phase from 24-35 hours, where bacterial growth stabilizes; and a gradual decline in OD600 values ​​from 35-48 hours. The growth curve of the composite bacteria falls between that of MN-5 and Bacillus pasteurii. Compared to single Bacillus pasteurii strains, the composite culture allows the bacteria to enter the logarithmic growth phase earlier. Furthermore, the composite bacteria show a slow increase in growth from 35-48 hours, while single strains begin to decline during this period. The composite culture has a longer stationary phase than single strains, thus exhibiting a greater dust suppression advantage. Notably, the composite bacterium BM enters a stationary phase around 20 hours. Its growth is greater than other composite bacteria because both MN-5 and Bacillus pasteurii were inoculated from the beginning, allowing them to adapt to composite growth in the early stages. 24 M, M5B, M 16 B is another type of bacteria inoculated at different times during the growth process. The bacteria need a certain amount of time to adapt. After a short period of adaptation, the compound strains can all resume normal growth and ultimately have a longer stable growth period than the single strains.

[0128] Growth curves and urease activity tests of complex bacteria with different inoculation sequences are as follows: Figure 7 As shown in the figure, Bacillus pasteurii bacteria are in the lag phase from 0-5 hours; in the logarithmic growth phase from 5-24 hours; in the stationary phase from 24-35 hours, the bacterial population stabilizes; and from 35-48 hours, the bacteria begin to decline. 600The values began to gradually decrease. The growth curve of the composite bacteria was between MN-5 bacteria and Bacillus pasteurii bacteria. Compared with the single Bacillus pasteurii strain, the culture of the composite strain made the bacterial reproduction enter the logarithmic growth phase in advance. Moreover, the growth amount of the composite strain was slowly rising during 35-48 h, and the single strain began to show a downward trend during this period. The composite culture strain had a longer stable growth period than the single strain, and thus had a better dust suppression advantage. It is worth noting that the composite bacteria BM entered the stable period around 20 h. The growth amount of this composite bacteria was greater than that of other composite bacteria. This was because the two bacteria were inoculated at the beginning and had the adaptability of the composite strain in the early stage. The B5M, B 24 M, M5B, and M 16 B were inoculated at different times during the growth process. The bacteria needed a certain amount of time to adapt. After a short period of adaptation, the composite strain could restore normal growth, and ultimately had a longer stable growth period than the single strain.

[0129] A time during the stable period after inoculation was selected to measure the urease activity of the bacterial solution. It can be seen that the Bacillus pasteurii strain and the MN-5 strain measured at 32 hours were 11.22 mmol·L -1 ·min -1 and 5.03 mmol·L -1 ·min -1 , respectively. The urease activities of BM, B5M, B 24 M, M5B, and M 16 B were 15.7 mmol·L -1 ·min -1 , 11.25 mmol·L -1 ·min -1 , 11.65 mmol·L -1 ·min -1 , 11.83 mmol·L -1 ·min -1 , and 13.42 mmol·L -1 ·min -1 , respectively. The urease activity of BM was higher than that of other samples during the entire process of 5 h, 16 h, and 32 h, which once again proved the synergistic growth relationship of the composite bacteria.

[0130] 2.4 Mineralization characteristics of composite bacteria: The mineralization products of the composite bacteria were tested by scanning electron microscopy, and the test results are shown in Figure 10 Figures (a)-(e) of the composite bacteria mineralization product characteristic analysis. Figure 10 Figures (a)-(e) of the composite bacteria mineralization product characteristic analysis correspond to BM, B5M, B24 M, M5B, M 16 SEM images of mineralization products of B. Figure 10 As can be seen from the figure (a), the mineralization precipitate produced by the composite bacteria BM has the advantages of large particle size span and good binding effect. The non-uniformity of particle size is conducive to more dense filling of the gaps between coal dust particles, thereby improving the effectiveness of the microbial dust suppressant. Figure 10 As can be seen from the figures (b), (c), (d) and (e), the mineralization products form block-like binding bodies autonomously with bacteria as nucleation sites, and the CaCO3 particles are closely adsorbed and adhered.

[0131] Figure 10 The figure (f) is the mineralization process of single bacteria and composite bacteria. From the figure (f), it can be seen that after the addition of the cementing liquid, each bacteria solution will immediately produce a precipitate. Figure 10 As can be seen from the figure (f), after the addition of the cementing liquid, each bacteria solution will immediately produce a precipitate. After observing the mineralization process again after 10 minutes, it can be seen that the mineralization deposition rate of the single bacteria MN-5 is very slow, and in comparison, the mineralization rate of the composite bacteria is faster than that of the MN-5 single bacteria. The fast mineralization deposition rate indicates that the calcium carbonate induced by the microbial dust suppressant can find the bacterial site and form large binding bodies in a shorter time, and then settle down. For dust suppression, fast deposition rate can more quickly and accurately perform binding dust suppression. Figure 10 The figure (g) is a comparison of the mineralization deposition amount of single bacteria and composite bacteria at different times. From the figure (g), it can be seen that the deposition amount of the composite bacteria is more than that of the single bacteria. Figure 10 As can be seen from the figure (g), the deposition amount of the composite bacteria is more than that of the single bacteria. The mineralization deposition amount is related to the nucleation site and the urease activity of the bacteria solution.

[0132] 2.5 Dust suppression effect analysis.

[0133] 2.5.1 Penetration ability and contact angle test: The wetting ability of the composite bacteria solution was tested, and the test results are shown in the figures (a) and (b). Figure 11 As can be seen from the figures (a) and (b), the contact angle and penetration test results show that the wetting ability of the composite bacteria solution is better than that of the single bacteria solution. Figure 11 As can be seen from the figure (a), the contact angles of B, BM, B5M, B 24 M, M, M5B, M 16 Trend graph of contact angle of B, W, C at 60s. Figure 11 The figure (c) is a test experiment graph of the contact angle of different groups at 60s. From the figure (c), it can be seen that the contact angle of the composite bacteria solution is smaller than that of the single bacteria solution. Figure 11In the (a) figure, the contact angle of W and C can be observed to change little, and the wetting effect is poor. The contact angle of Bacillus pasteurii has a slight decrease from 79.2° at the beginning to 75.4°. The contact angle of single bacteria MN-5 changes from 75.1° at the beginning to 68.4°. The contact angle test effect of composite bacteria is better than that of single bacteria, especially BM bacteria. After 60s of wetting, the contact angle decreases from 70.2° to 51.1°. The wetting effect is good and the wetting speed is fast. This is because the composite bacteria produce more hydrophilic substances and the contact of hydrophilic groups in the coal sample accelerates the wetting of the coal dust.

[0134] Figure 11 In the (b) figure, the penetration volume of the bacteria solution in the coal dust and the average penetration rate test results for 30 minutes are shown. The experiment starts with the addition of 1.5mL of solution in the test tube, and the experimental results show that B, BM, B5M, B 24 M, M, M5B, M 16 The penetration volumes of B, W, C are 0.1, 1, 0.5, 0.6, 0.3, 0.5, 0.9, 0.02, 0.07mL respectively. The composite bacteria BM has the largest penetration volume in the same time, which is consistent with the contact angle test. It proves that the composite bacteria BM has better wetting effect on coal dust compared with single bacteria. The growth amount of composite bacteria is greater than that of single bacteria, and has better hydrophilicity.

[0135] 2.5.2 Analysis of consolidation characteristics: In order to observe the dust suppression and consolidation characteristics of each bacteria solution in more detail, the coal dust after mineralization and consolidation was observed by scanning electron microscope, and the test results are shown in Figure 12 The consolidation effect is shown in the electron microscope graph. Figure 12 The (a) figure is the mineralization and consolidation effect of B strain. The wetting effect of single strain Bacillus pasteurii is poor, and the mineralization product cannot be deposited in the coal seam for consolidation, and there are some gaps in the surface layer of coal dust. Figure 12 The (b) figure is the dust suppression effect of BM composite bacteria. The wetting property of BM bacteria solution is superior, and it can penetrate into the coal dust gap for mineralization and consolidation, and produce some shuttle-shaped and spherical-shaped deposits with uneven particle size to effectively fill the cemented coal dust gap and attach and wrap the whole coal dust particle. Figure 12 The (c) figure, (d) figure and (f) figure are B5M, B 24 M, M5B, M 16 B consolidation characteristics electron microscope, the mineralization and deposition of composite bacteria can cement the coal dust gap, but the overall consolidation effect is not completely tight. Figure 12 In the (e) figure, it is observed that single bacteria MN-5 produces shuttle-shaped and spherical-shaped deposits to adhere small particle size coal dust to large particle size coal dust, which can achieve certain dust suppression effect. Figure 12Figure (h) is the effect of water spraying on coal dust, and there is no adhesion effect between coal dust. Figure 12 Figure (i) is the dust suppression effect of the cementing fluid, and after the coal dust is dried, part of the solute crystals of CaCl2 and urea solution are attached to the surface, and the coal dust is still dispersed particles, which cannot resist wind erosion.

[0136] 2.5.3 Anti-erosion experiment: The coal dust after three two-phase cycle spraying consolidation was subjected to simulated anti-erosion experiment, and the wind speed was set to 2-10 m·s -1 , and the anti-wind four test results of different samples are shown in Figure 13 The anti-erosion test results are shown in Figure 13 From , it can be seen that the dust suppression effect of water and cementing fluid is the worst, as the dust suppression process does not form a consolidated layer, and as the wind speed decreases, the anti-erosion property accelerates to decrease, and when the wind speed is 10 m·s -1 , the anti-erosion rates of water and cementing fluid are 35.6% and 46.1%, respectively. The dust suppression effect of single bacteria MN-5 is better than that of water and cementing fluid, but as the mineralization and consolidation effect of single bacteria MN-5 is weaker than that of composite bacteria, there is also some deficiency in the dust suppression process. The anti-erosion effect of composite bacteria BM dust suppressant is the best, and as the wind speed increases, the mass loss changes little. When the wind speed is 10 m·s -1 , the anti-erosion rate is 93.1%. This is because the composite microorganism has better wetting effect, the urease activity is higher than that of single bacteria, and the mineralization precipitate with larger particle size range is generated, and the generation of shuttle-shaped precipitate is more conducive to the formation of dense anti-erosion consolidated layer.

[0137]

[0137] 3.6 Analysis of the synergistic dust suppression mechanism of composite bacteria: The dust suppressant prepared by culturing composite urease-producing bacteria has better dust suppression performance than single strain, and the dust suppression mechanism of composite bacteria is shown in Figure 14 The synergistic dust suppression mechanism of double urease-producing bacteria is shown in the figure. The two bacteria used in the experiment are gram-positive bacteria, and the cell membrane of gram-positive bacteria is mainly composed of phospholipid molecules, with the hydrophilic end on the outside and the hydrophobic end on the inside. The bacterial solution has wetting ability during bacterial growth. After spraying the bacterial solution, the bacterial solution penetrates into the coal dust gap, and the bacteria are dispersed between the coal dust gaps. The main component of bacterial cell wall is teichoic acid, which is negatively charged, and the bacteria are in an alkaline environment, and the charge on the surface of the bacteria is also negative, which provides greater adsorption force for attracting positively charged Ca 2+ . The composite culture of the two urease-producing bacteria can provide more nucleation sites, and the produced extracellular polymeric substance EPS can also serve as a calcium carbonate attachment point. After a period of time, spraying with a large amount of Ca 2+The cementing fluid attracts the microorganism-induced calcium carbonate to find the attachment point and cement faster due to the negative charge. In the electron microscope of the mineralization product, it is found that the particle size range of the calcium carbonate produced by the two microorganisms is different, and as the mineralization deposition proceeds, the calcium carbonate can adhere to the coal dust gap more densely to achieve the dust suppression effect. The screened indigenous lysine bacillus MN-5 increases the types of mineralization strains suitable for the coal mine dust suppression agent. Meanwhile, MN-5 makes up for the poor particle size and wetting effect of single pasteur bacillus, and the compound bacteria can be improved in wetting and consolidation, thereby achieving the synergistic dust suppression effect.

[0138] It can be understood that the present application mainly aims at the problem that there are few microbial dust suppression strains suitable for the coal mine environment at present, and the indigenous urease-producing mineralization bacteria are screened from the coal slime sample in the coal mine environment, the bacteria are genetically identified, and the growth and mineralization performance are tested. In order to optimize the dust suppression effect of the microbial dust suppression, the indigenous strain MN-5 and Bacillus pasteurii are cultured in combination, and the synergistic dust suppression effect of the compound bacteria is studied. The following conclusions are obtained:

[0139] (1) The indigenous urease-producing strain screened from the coal slime sample is genetically sequenced and compared, and the strain is lysine bacillus, which is named MN-5. The growth curve and urease activity of the MN-5 strain are tested, the strain starts to enter the logarithmic growth phase at 5h, and starts to enter the stationary phase at about 16h. The most suitable pH value for growth is 7-8. The highest urease activity is 5.15mmol·L -1 ·min -1 .

[0140] (2) The mineralization product of the indigenous urease-producing bacteria is analyzed, and it is found through XRD analysis that the mineralization precipitate produced by the MN-5 bacteria is mainly calcite, and contains a small amount of ball spar. The scanning electron microscope can observe that the produced precipitate is in the shape of a shuttle and a ball, and the particle size range of the mineralization product is 1-30μm.

[0141] (3) The two kinds of urease-producing bacteria are cultured in combination, and the best urease activity effect of different inoculation times is explored. The results show that the BM bacterial agent system is the best compound bacterial agent system, and the highest urease activity can reach 15.7mmol·L -1 ·min -1 , which is 3 times higher than that of the single MN-5 bacteria. The mixed culture produces precipitates with a wider particle size, and the wetting effect is better than that of the single strain. When the wind speed is 10m·s -1 , the anti-erosion efficiency is 93.1%. The MN compound bacterial agent system shows excellent dust suppression effect.

[0142] (4) The mechanism of synergistic dust suppression of the two urease-producing bacteria was analyzed. The composite microorganism has better wetting effect, higher urease activity than single bacteria, and generates mineralized precipitates with larger particle size range, and the generation of fusiform precipitates is more conducive to the formation of dense anti-erosion consolidated layer.

[0143] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement, and improvement within the technical range disclosed by the present application and within the spirit and principle of the present application should be encompassed within the protection scope of the present application.

Claims

1. A slime mineralization microbial dual urease bacteria bacteria synergistic dust suppressant, characterized in that, The dust inhibitor is inoculated by Bacillus pasteurii and indigenous urease-producing bacteria to realize dust suppression; the inoculation ratio of Bacillus pasteurii and indigenous urease-producing bacteria is 0.25%, the concentration of Bacillus pasteurii is 4.8x10 9 CFU / mL, and the concentration of indigenous urease-producing bacteria is 7.1x10 9 CFU / mL.

2. The slime mineralizing microbial dual urease bacteria bacteria synergistic dust suppressant of claim 1, characterized in that, The inoculation time of the Bacillus pasteurii and the indigenous urease-producing bacteria further comprises: Bacillus pasteurii and indigenous urease-producing bacteria MN-5 strain are inoculated at the same time, recorded as BM; Bacillus pasteurii is inoculated 5 hours and 24 hours later, and indigenous urease-producing bacteria is inoculated respectively, recorded as B5M, B 24 M; the indigenous urease-producing bacteria is MN-5 mineralization strain, and Bacillus pasteurii is recorded as B mineralization bacteria; then, culture is carried out to stable period, and the time length is 24h, at which time OD 600 is kept stable.

3. The slime mineralizing microbial dual urease bacteria bacteria synergistic dust suppressant of claim 1, characterized in that, The inoculation time of the Bacillus pasteurii and the indigenous urease-producing bacteria further comprises: Inoculation of indigenous urease producing bacteria 5, 16 hours later followed by inoculation of Bacillus pasteurii, noted M5B, M 16 B.

4. A screening method for a coal slime mineralization microbial dual urease bacteria synergistic dust suppressant, characterized in that, The screening method is used for screening the coal slime mineralization microbial dual urease-producing bacteria synergistic dust suppressant according to any one of claims 1-3, and the method comprises the following steps: S1, culturing the Bacillus pasteurii in a culture medium material; S2, screening the indigenous urease-producing bacteria from the coal slime sample, and screening the indigenous urease-producing bacteria belonging to Lysinibacillus sp. and named as MN-5; S3, setting different inoculation times for the cultured Bacillus pasteurii and the screened indigenous urease-producing bacteria named as MN-5, obtaining different composite microbial community samples, and performing growth test analysis, and the inoculation ratio of the Bacillus pasteurii and the screened indigenous urease-producing bacteria named as MN-5 is 1:

1.

5. The method for screening coal slime mineralization microbe dual urease bacteria synergistic dust suppressant according to claim 4, characterized in that, In step S2, the indigenous urease-producing bacteria belonging to Lysinibacillus sp. and named as MN-5 are screened, which comprises: S201, selection and pretreatment of the coal sample, and preparation of the coal slime sample; S202, treatment and separation of the prepared coal slime sample, and preliminary screening of the indigenous urease-producing bacteria strains with urease-producing capacity; S203, gram staining and DNA sequencing of the indigenous urease-producing bacteria strains with urease-producing capacity, screening of the indigenous urease-producing bacteria belonging to Lysinibacillus sp. and named as MN-5, preservation in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, preservation number CCTCC NO: M 20251465, and preservation date July 1, 2025; the detection result is survival; and the classification name is Lysinibacillus sp. MN-5.

6. The method for screening coal slime mineralization microbe dual urease bacteria synergistic dust suppressant according to claim 5, characterized in that, In step S202, the indigenous urease-producing bacteria strains with urease-producing capacity are preliminarily screened by treating and separating the prepared coal slime sample, which comprises: The coal slime sample is placed in a 5 mol / L high-concentration urea culture medium for acclimation and enrichment culture for 24 hours, wherein the urea culture medium is composed of 200 mL sterile water and 5 mol / L urea; the rotation speed is set to 150 rpm, and the temperature is set to 35°C; The sterile water was added to the coal slurry mixed suspension for dilution, and the dilution was performed in gradients of 10 -6 , 10 -7 , 10 -8 , 10 -9 Coating screening was performed, and the coating inoculation was performed on urea-agar medium, and the culture was performed at 30°C for 24-48h. The colored colonies around the urea-agar medium were selected and streaked, and the single colonies were obtained through multiple purification culture. The morphologically different colonies appearing are separated and screened, and five bacteria are separated and labeled as #1, #2, #3, #4 and #5; after being cultured for 48 hours under the same conditions, the #5 bacteria are preliminarily screened as the bacteria strains with urease-producing capacity.

7. The method for screening coal slime mineralization microbe dual urease bacteria synergistic dust suppressant according to claim 6, characterized in that, Urea-agar culture medium: proteose peptone 15 g / L, NaCl 5 g / L, KH2PO3 2 g / L, glucose 0.1 g / L, phenol red 0.1 g / L, urea 20 g / L, agar 20 g / L, water 1 L, pH=7; Bacterial solid culture medium: same as the liquid culture medium, and additionally containing agar 20 g / L; specifically, casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH=7, and agar 20 g / L.

8. The method for screening coal slime mineralization microbe dual urease bacteria synergistic dust suppressant of claim 6, characterized in that, Step S202, after the preliminary screening of indigenous urease-producing bacterial strains with urease-producing ability, growth test, urease activity determination and characterization experiments are further carried out.

9. The method for screening coal slime mineralization microbe dual urease bacteria synergistic dust suppressant according to claim 4, characterized in that, In step S3, different inoculation times are set for the cultured Bacillus pasteurii and the screened indigenous urease-producing bacteria named MN-5 to obtain different composite microbial community samples, including inoculating MN-5 after 5 hours of inoculating Bacillus pasteurii, cooling the sterilized bacterial liquid medium to room temperature, and inoculating 0.5 mL of Bacillus pasteurii liquid bacterial liquid into 100 mL of liquid medium in a sterile operation table using a pipette; then the inoculated sample is placed in a constant temperature shaking incubator for culture, and the culture conditions are set as 25℃ and 180 rmp, and the sample is recorded as sample B5M. Bacterial liquid medium: casein peptone 15 g / L, urea 20 g / L, soybean peptone 5 g / L, sodium chloride 5 g / L, water 1 L, pH = 7; used for growth culture of indigenous bacteria to form liquid bacterial liquid, and the liquid culture is the main form used in subsequent performance experiments.

10. The application of coal slime mineralization microbial dual urease bacteria synergistic dust suppressant in coal mine dust suppression, characterized in that, The coal slime mineralization microbial dual-urease-producing bacteria synergistic dust suppressant according to any one of claims 1-3.

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