An immobilized microbial promoter for mining based on biosurfactant bacteria and its preparation method

Abstract

The present invention relates to an immobilized microbial promoter for mining based on biosurfactant bacteria, which comprises immobilized biosurfactant bacteria, immobilized mineralizing bacteria, an activation solution and a binder. First, the biosurfactant bacteria and the mineralizing bacteria are inoculated into the activation solution to obtain the fermentation broth of the biosurfactant bacteria and the mineralizing bacteria; then, the biosurfactant bacteria and the mineralizing bacteria are selectively adsorbed and enriched by an immobilization material respectively to obtain the immobilized biosurfactant bacteria and the immobilized mineralizing bacteria. The immobilized microbial promoter for mining can not only improve the hydrophobicity when the microbial promoter is applied to coal dust, but also realize the improvement of the mineralization ability and the transformation of the calcium carbonate crystal shape through the mutualistic symbiosis between the two strains of bacteria. In addition, the surface of the biosurfactant bacteria has negative charges, which can serve as the nucleation sites for carbonate precipitation and accelerate carbonate precipitation.

Description

Technical Field

[0001] The present invention relates to an immobilized mine microbial promoter based on biosurfactant bacteria and a preparation method thereof, belonging to the technical field of coal mine dust prevention and suppression. Background Art

[0002] To solve the problem of coal dust pollution, it is urgent to research and develop a dust suppressant that has no secondary pollution, good dust suppression effect and low price. Chemical dust suppressants are composed of a variety of chemical substances, which will inevitably have an adverse impact on the growth characteristics of microorganisms in the original environment, and are not easily degraded and prone to cause secondary pollution. Microbial-induced carbonate precipitation (MICP) technology is a common phenomenon in nature. It is reported that by applying the MICP technology to coal dust, the generated carbonate precipitate with gelling effect can bond coal dust particles to form a consolidated layer, ultimately achieving coal dust prevention and control. However, the surface of coal dust contains many hydrophobic groups (such as aliphatic hydrocarbons and aromatic hydrocarbons), resulting in poor wettability and difficult penetration when the dust suppressant is applied to coal dust. At present, a large number of studies have shown that surfactants need to be added in the application of dust suppressants in the field of coal dust prevention and control. Surfactants can improve the wettability of the solution, thereby increasing the contact between the dust suppression material and coal dust. When spraying microbial dust suppressants, if surfactants are not added, the dust suppressant is difficult to penetrate, and the generated carbonate precipitate can only consolidate the surface of coal dust, with poor consolidation effect and easy to cause secondary dust flying.

[0003] Biosurfactant is a natural surface-active compound secreted and metabolized by microorganisms, and is a heterogeneous secondary metabolite with the ability to improve the surface wetting performance of substances. Compared with chemical surfactants, biosurfactants have the characteristics of environmental friendliness and easy degradation. Therefore, the addition of biosurfactants can enhance the penetration, adsorption and retention of microbial dust suppressants in coal dust. How to apply the technology of combining biosurfactant bacteria with mineralization technology to the field of microbial coal dust prevention and control has become an urgent problem to promote the development of microbial dust suppressants in the field of dust prevention and control. Summary of the Invention

[0004] Aiming at the above problems, the present invention provides an immobilized mine microbial promoter based on biosurfactant bacteria and a preparation method thereof. The immobilized mine microbial promoter can produce biosurfactant during the fermentation process to improve the hydrophobicity of coal dust itself; at the same time, the symbiosis of bacterial cells promotes the mineralization bacteria to play a mineralization role and improve the dust suppression effect. In addition, the biosurfactant bacteria with negative charges on the surface can serve as the nucleation sites of carbonate precipitation during the process of microbial-induced carbonate precipitation, accelerating carbonate precipitation.

[0005] To achieve the above invention purposes, the present invention provides the following technical solutions:

[0006] An immobilized microbial promoter for mining based on biosurfactant bacteria, comprising immobilized biosurfactant bacteria, immobilized mineralizing bacteria, an activation solution, and a cementing agent; the activation solution and the cementing agent are independently packaged before use.

[0007] Preferably, the ratio of the total mass of the immobilized biosurfactant bacteria and the immobilized mineralizing bacteria to the volumes of the activation solution and the cementing agent is (1 - 3) g : (65 - 75) mL : (5 - 10) mL.

[0008] Preferably, the preparation method of the immobilized biosurfactant bacteria or the immobilized mineralizing bacteria comprises the following steps:

[0009] (1) Adjust the activation solution to neutral pH with sodium hydroxide, sterilize it, and then inoculate the activated biosurfactant bacteria or mineralizing bacteria into the sterilized activation solution, and culture it in an incubator to obtain a biosurfactant bacteria fermentation broth or a mineralizing bacteria fermentation broth;

[0010] (2) Sequentially subject the biosurfactant bacteria fermentation broth or the mineralizing bacteria fermentation broth obtained in step (1) to centrifugation, removal of the supernatant, and addition of sterile physiological saline, and repeat the operation at least three times to obtain a uniform biosurfactant bacteria suspension or mineralizing bacteria suspension;

[0011] (3) After sterilizing the immobilization material, add it to the biosurfactant bacteria suspension or mineralizing bacteria suspension, and then add the sterilized activation solution. After sealing and culturing at a constant temperature, obtain an immobilized biosurfactant bacteria solution or an immobilized mineralizing bacteria solution;

[0012] (4) Filter the immobilized biosurfactant bacteria solution or the immobilized mineralizing bacteria solution through a filter sieve and perform freeze-drying using a freeze dryer to obtain immobilized biosurfactant bacteria or immobilized mineralizing bacteria.

[0013] More preferably, the sterilization conditions in step (1) are: sterilize at 121 °C for 20 min; the culture conditions are: culture at 30 °C and a stirring speed of 150 rpm for 48 h.

[0014] More preferably, the concentrations of the biosurfactant bacteria suspension and the mineralizing bacteria suspension in step (2) are 1×10 7 ~1×10 8 CFU / mL, 1×10 8 ~1×10 10 CFU / mL.

[0015] More preferably, the volume ratio of the biosurfactant bacteria suspension or the mineralizing bacteria suspension to the activation solution in step (3) is 1 : (99 - 105).

[0016] More preferably, the culture conditions in step (3) are: culturing at 25°C with a stirring speed of 150 rpm for 24 h.

[0017] More preferably, the preparation method of the immobilized material in step (3) is as follows:

[0018] S1. The immobilized object is successively washed with distilled water, soaked in an aqueous NaOH solution, washed with deionized water, and dried to obtain an alkali-treated immobilized object;

[0019] S2. The nano-titanium dioxide particles and the superhydrophobic coating material are added to ethanol as the activation solution and dispersed evenly to obtain a milky white suspension;

[0020] S3. The alkali-treated immobilized object described in step S1 is soaked in the suspension described in step S2 and then dried to obtain the immobilized material.

[0021] Even more preferably, in step S1, the concentration of the aqueous NaOH solution is 1 - 1.2 mol / L, the soaking time is 24 h; the number of washings is 3 - 5 times; the drying method is drying to constant weight at 65°C in a vacuum freeze-drying oven.

[0022] Even more preferably, in step S1, the immobilized object is at least one of loofah sponge, straw, bagasse, and corncob; the particle size of the immobilized object is 30 - 40 mm.

[0023] Even more preferably, in step S2, the superhydrophobic coating material is at least one of paraffin, polytetrafluoroethylene, and graphene.

[0024] Even more preferably, in step S2: 2 - 5 g of nano-titanium dioxide particles and 1 g of superhydrophobic coating material are respectively taken and added to 100 mL of ethanol, and ultrasonic dispersion is carried out for 2 - 4 h to obtain a milky white suspension.

[0025] Even more preferably, in step S3, the soaking time is 12 h; the drying is carried out in an oven at 60°C for 24 h.

[0026] Preferably, the biosurfactant bacteria are selected from at least one of Brevibacillus brevis, Pseudomonas aeruginosa, Bacillus licheniformis, or Corynebacterium.

[0027] Preferably, the mineralizing bacteria are selected from at least one of Bacillus mucilaginosus, Bacillus subtilis, Sarcina pasteurii, Bacillus amyloliquefaciens, or Bacillus sphaericus.

[0028] Preferably, the activation solution includes NH 4 Cl, MnSO 4 ·H 2 O and NiCl 2 ·6H2 O, and further includes yeast extract or peptone. More preferably, the activation solution includes the following components in parts by weight: yeast extract or peptone 2000 - 3000 parts, NH 4 Cl 1000 - 2000 parts, MnSO 4 ·H 2 O 100 - 150 parts, NiCl 2 ·6H 2 O 200 - 300 parts.

[0029] Preferably, the binder contains soluble calcium salt and urea.

[0030] More preferably, the soluble calcium salt is at least one of calcium formate, calcium chloride, calcium lactate, and calcium acetate.

[0031] Even more preferably, the molar concentration ratio of the soluble calcium salt to urea is 1:1.

[0032] Even more preferably, the molar concentration of the soluble calcium salt is 0.8 - 1.0 mol / L.

[0033] The application of the immobilized mine - used microbial promoter based on biosurfactant - producing bacteria is used for coal dust consolidation, and the steps are as follows:

[0034] Respectively take the immobilized biosurfactant - producing bacteria and the immobilized mineralizing bacteria and add them into the sterilized activation solution. After constant - temperature culture, a composite microbial fermentation broth is obtained. Then spray the composite microbial fermentation broth and the sterilized binder onto the coal dust in sequence.

[0035] Preferably, the way of adding the immobilized biosurfactant - producing bacteria and the immobilized mineralizing bacteria into the sterilized activation solution is: first add 1 part of the immobilized biosurfactant - producing bacteria into the sterilized activator, culture for 14 h, then add 1 part of the immobilized mineralizing bacteria, and continue to culture for 10 h.

[0036] Preferably, the volume ratio of the composite microbial fermentation broth to the binder is (13 - 15):(1 - 2).

[0037] The present invention also provides a production device for the above - mentioned immobilized biosurfactant - producing bacteria or immobilized mineralizing bacteria, which sequentially includes a constant - temperature shaking incubator, a connecting pipe, an immobilized bacterial solution storage tank, a filtering vibrating screen, a conveyor belt, and a freeze - dryer according to the direction of the production process flow; the outlet of the constant - temperature shaking incubator is connected to the inlet of the connecting pipe, and the outlet of the connecting pipe is vertically arranged above the opening of the immobilized bacterial solution storage tank; the filtering vibrating screen is arranged below the outlet of the immobilized bacterial solution storage tank, one end of the conveyor belt is arranged below the filtering vibrating screen, and the other end is arranged above the inlet of the freeze - dryer.

[0038] In the present invention, the process of preparing immobilized biosurfactant bacteria or immobilized mineralized bacteria using the above production device is as follows:

[0039] (1) Take 1 part of the immobilized material, add 13 - 15 parts of the activation solution and 1 - 2 parts of the biosurfactant bacteria suspension or mineralized bacteria suspension, and culture for 48 h under the culture conditions of 30 °C and 150 rpm using a constant temperature shaker to obtain the immobilized biosurfactant bacteria solution or immobilized mineralized bacteria solution;

[0040] (2) Transfer the immobilized biosurfactant bacteria solution or immobilized mineralized bacteria solution to the immobilized bacteria solution storage tank through a connecting pipe, and separate the immobilized biosurfactant bacteria or immobilized mineralized bacteria from the fermentation broth by falling into a filtering vibrating screen;

[0041] (3) The immobilized biosurfactant bacteria or immobilized mineralized bacteria are conveyed to a freeze dryer through a conveyor belt to achieve freeze - drying of the immobilized biosurfactant bacteria or immobilized mineralized bacteria.

[0042] The immobilized mine - used microbial promoter based on biosurfactant bacteria in the present invention mainly utilizes the fact that biosurfactant bacteria can produce biosurfactants during the growth of bacteria. The biosurfactant can improve the hydrophobicity of coal dust itself, enhance the wetting and penetration ability of the microbial promoter. The biosurfactant can increase the adhesion effect, enabling a large number of microorganisms to adhere to each other to form aggregates. And the surface of the biosurfactant bacteria is negatively charged, which can increase the nucleation sites and accelerate carbonate precipitation.

[0043] By adding a urea reagent, that is, adding the decomposition substrate of urease in the mineralized bacteria in the liquid phase environment, ammonium ions and carbonate ions can be decomposed. Soluble calcium salts can provide calcium ions for the precipitation of carbonates, generating carbonate precipitates with gelling effects. That is, the immobilized mine - used microbial promoter provided by the present invention can effectively provide a basic guarantee for microbially induced calcium carbonate precipitation, solve the problems such as hydrophobicity faced by the microbial promoter when applied to coal dust, and at the same time achieve the purposes of environmental protection, pollution - free, and low cost.

[0044] The present invention has the following advantages compared with the prior art:

[0045] (1) The immobilized mine - used microbial promoter of the present invention contains a biosurfactant that is non - toxic to the application environment and microorganisms, increasing the penetration of the bacteria solution and mineralized substrate during use and increasing the thickness of the coal dust consolidation layer.

[0046] (2) The immobilized material prepared by the present invention has hydrophobicity, is easy to clean and recyclable, and has a large specific surface area, which is conducive to the uniform distribution of bacteria.

[0047] (3) The mineralizing bacteria will produce urease during the cultivation process. The urease will decompose urea to produce carbonate ions, and the produced carbonate ions will combine with calcium ions in the mineralizing solution to form calcium carbonate during use, which is green, environmentally friendly, and pollution-free.

[0048] (4) The present invention utilizes the mineralization and emulsification effects of bacteria for coal powder solidification, which has no secondary pollution, low cost, and good effects, and has a good application prospect in open-pit coal mines and is worthy of wide promotion.

[0049] (5) The immobilized mine microbial promoter of the present invention has the characteristics of convenient construction, green and pollution-free compared with chemical dust suppressants and ordinary biological dust suppressants. Description of the Drawings

[0050] Figure 1 is the growth curve of bacteria in the composite microbial fermentation broth under different inoculation sequences;

[0051] Figure 2 is the calcium carbonate production amount of the immobilized mine microbial promoter under different inoculation sequences;

[0052] Figure 3 is the electron microscopy image of calcium carbonate produced by the immobilized mine microbial promoter under different inoculation sequences;

[0053] Figure 4 is the contact angle of the immobilized mine microbial promoter on coal under different inoculation sequences;

[0054] Figure 5 is the wind erosion resistance of coal powder treated with the immobilized mine microbial promoter under different inoculation sequences;

[0055] Figure 6 is the material production device described in Embodiment 1 of the present invention, wherein: 1. Constant temperature shaking incubator; 2. Connecting pipe; 3. Immobilized bacteria solution storage tank; 4. Filter vibrating screen; 5. Conveyor belt; 6. Freeze dryer. Detailed Embodiments

[0056] The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer as the description progresses. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0057] Embodiment 1

[0058] An immobilized mine microbial promoter based on biosurfactant bacteria and its preparation method.

[0059] The preparation method of the immobilized mining microbial promoter based on biosurfactant bacteria comprises the following steps:

[0060] Ⅰ. Preparation method of immobilized material, the steps are as follows:

[0061] S1. Wash the loofah sponge with distilled water, place it in 1 mol / L NaOH aqueous solution, and soak for 24 h. Then wash the loofah sponge with deionized water 5 times to remove the residual NaOH. Then place the alkali-treated loofah sponge in a vacuum freeze dryer at 65 °C and dry it to constant weight.

[0062] S2. Respectively take 2 g of nano-titanium dioxide particles and 1 g of paraffin wax and add them to 100 mL of ethanol, and ultrasonically disperse for 2 - 4 h to obtain a milky white suspension.

[0063] S3. Immerse the alkali-treated loofah sponge in the suspension for 12 h, and then place it in an oven at 60 °C and dry for 24 h to obtain the immobilized material.

[0064] Ⅱ. Preparation of immobilized biosurfactant bacteria and immobilized mineralizing bacteria, the steps are as follows:

[0065] (1) Adjust 2000 parts of yeast extract, 1000 parts of NH 4 Cl, 100 parts of MnSO 4 ·H 2 O, 200 parts of NiCl 2 ·6H 2 O to pH = 7 with 1 mol / L sodium hydroxide, place it in a vertical autoclave at 121 °C, and sterilize for 20 min. Use a pipette to inoculate the activated Pseudomonas aeruginosa and Sarcina ventriculi respectively into the sterilized activation solution, and place it in a constant temperature shaker 1 for culturing for 48 h. The culturing conditions are 150 rpm and 30 °C to obtain the fermentation broth of biosurfactant bacteria and mineralizing bacteria respectively.

[0066] (2) Respectively take 30 mL of the fermentation broth of biosurfactant bacteria and mineralizing bacteria, centrifuge at 8000 rpm for 5 min to remove the supernatant; add 5 mL of sterile normal saline and centrifuge at 8000 rpm for 5 min. Wash the bacterial precipitate twice, then remove the supernatant and add 5 mL of sterile normal saline to obtain a uniform bacterial suspension of biosurfactant bacteria and mineralizing bacteria.

[0067] (3) Weigh two portions of 1.0 g, 3.0 g, 5.0 g, 7.0 g, and 10.0 g of the washed and dried immobilized materials respectively and place them in two 100 mL conical flasks. Sterilize them in an autoclave for 20 min. Then, use a pipette to add 1 mL of the bacterial suspensions of biosurfactant-producing bacteria and mineralizing bacteria into the conical flasks respectively. Add 99 mL of the activation solution to each conical flask, seal the bottle mouths with sealing films, and culture them in a constant temperature shaker at 25 °C and 150 rpm for 24 h to obtain immobilized biosurfactant-producing bacteria solution and immobilized mineralizing bacteria solution.

[0068] (4) Transport the immobilized biosurfactant-producing bacteria solution and immobilized mineralizing bacteria solution to the immobilized bacteria solution storage tank 3 through the connecting pipe 2, filter them using the filtering vibrating screen 4, and convey them through the conveyor belt 5 to the freeze dryer 6 for freeze-drying to obtain immobilized biosurfactant-producing bacteria and immobilized mineralizing bacteria.

[0069] Ⅲ. Take 1 g of immobilized biosurfactant-producing bacteria, 1 g of immobilized mineralizing bacteria, 75 mL of the activation solution, and 5 mL of the binder to obtain the immobilized microbial promoter for mine use, where the activation solution and the binder are independently packaged.

[0070] Experiment 1

[0071] Let the immobilized biosurfactant-producing bacteria solution and immobilized mineralizing bacteria solution obtained in step II(3) stand still, collect the supernatant, centrifuge it at 2000 rpm for 10 min, and collect the final sample 1 cm below the surface of the supernatant. By measuring the OD value of the sample, calculate the number of microbial cells adsorbed on the surface of the immobilized material. The results are shown in Table 1.

[0072] Table 1 Microbial adsorption amounts of immobilized materials with different masses

[0073]

[0074] Table 1 shows that: as the mass of the immobilized material increases, the number of microbial cells adsorbed by the immobilized material shows a trend of increasing first and then decreasing. As the mass of the coal dust increases, the adsorption capacity first increases and then decreases. This may be because the number of microbial cells is insufficient to adapt to the increasing immobilized material, resulting in a decrease in the overall adsorption amount. Among them, when the mass of the immobilized material is 3.0 - 5.0 g, the number of microbial cells adsorbed by the immobilized material reaches the highest values of 39.18 - 40.29 mg / g and 26.73 - 28.52 mg / g respectively. Therefore, when preparing immobilized biosurfactant-producing bacteria and immobilized biomineralizing bacteria, the mass of the immobilized material selected in 100 mL of the bacterial solution (1×10 8 CFU / mL) is 3.0 - 5.0 g.

[0075] Experiment 2

[0076] The immobilized biosurfactant-producing bacteria and the immobilized mineralizing bacteria are the same as those in Example 1;

[0077] Weigh 1.0 g, 2.0 g, 3.0 g, 4.0 g, 5.0 g, 6.0 g, 7.0 g, 8.0 g of the immobilized biosurfactant-producing bacteria and the immobilized mineralizing bacteria according to a mass ratio of 1:1, and place them in 75 mL of the activation solution after sterilization. Seal the bottle mouth with a sealing film, and culture them in a constant temperature shaker at 25 °C and 150 rpm for 48 h. Measure the OD value of the sample, and according to the OD value, reflect the growth characteristics of the immobilized bacteria under different masses. The results are shown in Table 2.

[0078] Table 2 Growth of immobilized bacteria under different masses

[0079]

[0080] Table 2 shows that: with the increase in the mass of the immobilized bacteria, the growth of the immobilized bacteria under different masses shows a trend of first increasing and then decreasing. This may be because with the increase in the mass of the immobilized bacteria, the growth of the bacteria in the activation solution increases. However, the nutrients in the activation solution are limited, which leads to a competitive relationship between the bacteria. When the initial addition amount of the immobilized bacteria is too high, it will instead lead to a decrease in the growth of the bacteria. Therefore, when selecting the immobilized bacteria for culture, the total mass of the immobilized biosurfactant-producing bacteria and the immobilized mineralizing bacteria is selected to be 1.0 - 3.0 g.

[0081] Experiment 3

[0082] Take 1.0 g of the immobilized biosurfactant-producing bacteria and / or 1.0 g of the immobilized mineralizing bacteria and inoculate them into 75 mL of the activation solution after sterilization, and set up a group of microbial fermentation broths according to the inoculation time of the bacteria: X, X 14 P, X 24 P, P, P 14 X, P 24 X, PX, where P represents inoculating the immobilized biosurfactant-producing bacteria, X represents inoculating the immobilized mineralizing bacteria, 14 means inoculating one strain of bacteria and then inoculating another strain of bacteria 14 h later, and 24 means inoculating one strain of bacteria and then inoculating another strain of bacteria 24 h later. Place the above microbial fermentation broths in a constant temperature shaking incubator and culture them for 48 h. The culture conditions are 150 rpm and 30 °C. At regular intervals within 48 h, take 200 μL of the microbial fermentation broth in the conical flask in a laminar flow hood, and measure the absorbance at a wavelength of 600 nm with an enzyme-labeled instrument to reflect the growth curve of the bacteria. The measured results are as Figure 1 shown, Figure 1 which are the growth curves of the bacteria in the microbial fermentation broth under different inoculation orders.

[0083] Experiment 4

[0084] Add 75 mL of the microbial fermentation broth prepared in Experiment 3 to 5 mL of a mixed solution of calcium chloride (36 g / L) and urea (20 g / L) sterilized by a filter head, and place it in a constant temperature shaking incubator for mineralization for 7 days. The culture conditions are 150 rpm and 30 °C. Filter the mineralized composite microbial fermentation broth through filter paper, and dry it in an oven at 100 °C. After drying, weigh the total mass of the filter paper and the precipitate, denoted as M1; wash the filter paper and the precipitate with 0.7 mol / L hydrochloric acid to remove the calcium carbonate precipitate, dry it, and weigh it, denoted as M2; the weight of calcium carbonate is M1 - M2 (Table 3).

[0085] Table 3 Calcium carbonate production of immobilized mine microbial promoters under different inoculation orders

[0086]

[0087] As Figure 2 Calcium carbonate production of immobilized mine microbial promoters under different inoculation orders; the results show that after 7 days of mineralization of single immobilized bacteria and composite immobilized bacteria, the immobilized mineralizing bacteria X and the composite immobilized bacteria P 14 X, PX have higher calcium carbonate yields. The calcium carbonate yield of immobilized mineralizing bacteria X after 7 days of mineralization is 4.19 ± 0.93 g / L, which is similar to that of the composite immobilized bacteria PX (4.31 ± 0.19 g / L). The composite immobilized bacteria P 14 X can reach a calcium carbonate yield of 10.4 ± 0.70 g / L, which is 148.21% and 141.30% higher than that of immobilized mineralizing bacteria X and composite immobilized bacteria PX, respectively. P 24 X has a calcium carbonate yield of 2.20 ± 0.22 g / L, which is lower than that of P 14 X. This indicates that inoculating the mineralizing bacteria 14 h after inoculating the biosurfactant bacteria will promote the precipitation amount of calcium carbonate.

[0088] As Figure 3 Electron micrographs of calcium carbonate produced by immobilized mine microbial promoters under different inoculation orders, where: A is X, B is X 14 P, C is X 24 P, D is P, E is P 14 X, F is P 14 Local enlarged view of X, G is P 24 X, H is PX. It can be seen from the figure that the mineralization products of single immobilized bacteria are spherical and belong to vaterite-type calcium carbonate. The mineralization products produced by composite immobilized bacteria are vaterite-type and calcite-type calcium carbonate. Among them, the composite immobilized bacteria P 14 X clearly shows the coexistence of vaterite-type calcium carbonate and calcite-type calcium carbonate, and the vaterite-type calcium carbonate gradually transforms into stable calcite-type calcium carbonate, indicating that inoculating the mineralizing bacteria 14 h after inoculating the biosurfactant bacteria will improve the stability of calcium carbonate crystals.

[0089] Experiment 5

[0090] Pulverized coal (1 g, 200 mesh) was pressed into a coal powder sample through a tablet press at a pressure of 15 MPa to form a coal cake. The contact angle (sessile drop method) test was carried out using an optical contact angle measuring instrument to compare the wettability of coal by biosurfactants produced by bacterial solutions cultured in different inoculation methods (the same as in Experiment 3). Figure 4 For the wettability of coal by the immobilized mine microbial promoter or water (W) under different inoculation sequences, the results showed that: the contact angle of water (W) on the coal surface was 78.2 - 77.59°, and the contact angle of the immobilized mineralized bacterial solution (X) on the coal surface was 78.16 - 71.33°. The contact angles of the immobilized mineralized bacterial solution (X) and water (W) on the coal surface were initially about 78°, but within 1 min, the contact angle of the immobilized mineralized bacterial solution (X) decreased. In addition, the composite bacterial solution P 14 X had the best wetting performance on coal. After 1 min, the surface tension of the coal decreased by 34.27%.

[0091] Experiment 6

[0092] Weighed a certain mass of pulverized coal and put it into a graduated cylinder with Φ×h of 10×100 mm. The pulverized coal was tamped with a glass rod to make the height of the pulverized coal in the graduated cylinder 10 cm, and 5 mL of microbial fermentation broth under different inoculation methods (the same as in Experiment 3) was added for the penetration experiment. The penetration depth within 20 min was measured with a ruler, and the results are shown in Table 4.

[0093] Table 4 Penetration depth of pulverized coal

[0094]

[0095] Table 4 showed that: compared with the penetration depth of the single immobilized bacterial solution and the composite immobilized bacterial solution, the penetration depth of the composite immobilized bacterial solution increased significantly. The penetration depth of the composite immobilized bacterial solution also showed different penetration performances with different inoculation sequences. Among them, the composite immobilized bacterial solution P 14 X was significantly higher than other composite immobilized bacterial solutions, indicating that inoculating the immobilized mineralized bacteria 14 h after inoculating the immobilized biosurfactant bacteria would promote the production of biosurfactants.

[0096] Experiment 7

[0097] Take 18 mL of the microbial fermentation broth prepared under different inoculation methods (the same inoculation methods as in Experiment 3) and place it in a spray bottle. Weigh 30 g of 120-mesh coal powder with a petri dish. After spraying 15 mL of the microbial fermentation broth prepared under different inoculation methods onto the coal powder in the petri dish, spray 2 mL of the binder again, that is, 2 mL of a mixed solution of calcium chloride (36 g / L) and urea (20 g / L). Finally, place the coal powder treated with the immobilized mine microbial promoter at room temperature and let it air dry naturally. Repeat the above operations on the 3rd day, 7th day, and 15th day respectively. Conduct a wind erosion resistance experiment on the treated coal powder at a wind speed of 10 m / s on the 20th day, and the results are as Figure 5 shown. Figure 5 The wind erosion resistance performance of coal powder treated with the immobilized mine microbial promoter under different inoculation methods. It can be seen from the figure that compared with the samples treated with the microbial promoter, the samples treated with water (W), activator (C), and binder (J) have greater wind erosion mass loss. In addition, the wind erosion resistance of the composite immobilized bacterial solution is better than that of the single immobilized bacterial solution. This is due to the dual effects of microbial mineralization and biosurfactant. The mass loss of the samples treated with the composite immobilized bacterial solution is significantly lower than that of the samples treated with the single immobilized bacterial solution. In particular, the microbial promoter prepared by P 14 X has good wind erosion resistance (1.55 ± 0.91%), which is 25.01 ± 4.44% lower than that of the microbial promoter prepared by the single immobilized mineralizing bacteria. Due to the different abilities of microorganisms to utilize substrates, inoculating and culturing microorganisms in a certain order can not only give full play to the synergistic metabolic effect among microorganisms but also avoid the growth competition and inhibition among microorganisms, thus obtaining higher biomass and productivity.

Claims

1. An immobilized mining microbial promoter based on biosurfactant bacteria, characterized in that: It comprises immobilized biosurfactant bacteria, immobilized mineralizing bacteria, activation liquid and a binder; the activation liquid and the binder are separately packaged before use; The preparation method of the immobilized biosurfactant bacteria or the immobilized mineralizing bacteria comprises the following steps: (1) The activation solution is adjusted to a neutral pH with sodium hydroxide, sterilized, and then the activated biosurfactant bacteria or mineralizing bacteria are inoculated into the sterilized activation solution, and cultured in an incubator to obtain a biosurfactant bacteria fermentation liquid or a mineralizing bacteria fermentation liquid; (2) centrifuging the biosurfactant bacteria fermentation liquid or the mineralizing bacteria fermentation liquid described in step (1), removing the supernatant, and adding sterile physiological saline, and repeating the above operation at least three times to obtain a uniform biosurfactant bacteria suspension or mineralizing bacteria suspension; (3) After the immobilized material is sterilized, it is added to a biosurfactant bacterial suspension or a mineralized bacterial suspension, and then a sterilized activation liquid is added, and after sealing and constant temperature cultivation, an immobilized biosurfactant bacterial solution or an immobilized mineralized bacterial solution is obtained; (4) filtering the immobilized biosurfactant bacterial solution or the immobilized mineralized bacterial solution through a filter screen, and freeze-drying the solution using a freeze dryer to obtain immobilized biosurfactant bacteria or immobilized mineralized bacteria; The preparation method of the immobilized material comprises the following steps: S1. The fixed object was sequentially washed with distilled water, soaked in a NaOH aqueous solution, washed with deionized water, and dried to obtain an alkali-treated fixed object; S2. The nano-titanium dioxide particles and the super-hydrophobic coating material are added to the activation liquid ethanol and dispersed evenly to obtain a milky white suspension; S3. The alkali-treated immobilized material in step S1 is immersed in the suspension in step S2, and then dried to obtain an immobilized material.

2. The immobilized microbial promoter for mining according to claim 1, characterized in that: The ratio of the total mass of the immobilized biosurfactant bacteria and the immobilized mineralizing bacteria to the volume of the activation liquid and the binder is (1-3) g: (65-75) mL: (5-10) mL.

3. The immobilized microbial promoter for mining according to claim 1, characterized in that: Step (1) Sterilization conditions: sterilize at 121°C for 20 min; culture conditions: culture at 30°C and 150 rpm for 48 h.

4. The immobilized microbial promoter for mining according to claim 1, characterized in that: The concentrations of the biosurfactant bacterial suspension and the mineralizing bacterial suspension in step (2) are 1×10 7 ~1×10 8 CFU / mL, 1×10 8 ~1×10 10 CFU / mL.

5. The immobilized microbial promoter for mining according to claim 1, characterized in that: The volume ratio of the biosurfactant bacterial suspension or the mineralized bacterial suspension to the activation solution in step (3) is 1:(99-105).

6. The immobilized microbial promoter for mining according to claim 1, characterized in that: Step (3) Cultivation conditions: Cultivate at 25°C and 150 rpm stirring speed for 24 h.

7. The immobilized microbial promoter for mining according to claim 1, characterized in that: The concentration of the NaOH aqueous solution in step S1 is 1-1.2 mol / L, and the soaking time is 24 h; the washing times are 3-5 times; and the freeze-drying method is to dry the mixture in a vacuum freeze drying oven at 65° C. to a constant weight.

8. The immobilized microbial promoter for mining according to claim 1, characterized in that: In step S1, the fixed object is at least one of loofah, straw, bagasse, and corn cob; and the particle size of the fixed object is 30-40 mm.

9. The immobilized microbial promoter for mining according to claim 1, characterized in that: The super-hydrophobic coating material in step S2 is at least one of paraffin, polytetrafluoroethylene, and graphene.

10. The immobilized microbial promoter for mining according to claim 1, characterized in that: Step S2: Take 2-5 g of nano-titanium dioxide particles and 1 g of super-hydrophobic coating material, add them into 100 mL of ethanol, and ultrasonically disperse them for 2-4 h to obtain a milky white suspension.

11. The immobilized microbial promoter for mining according to claim 1, characterized in that: The soaking time in step S3 is 12 h; the drying is carried out in an oven at 60° C. for 24 h.

12. The immobilized microbial promoter for mining according to claim 1, characterized in that: The biosurfactant bacteria are selected from at least one of Bacillus brevis, Pseudomonas aeruginosa, Bacillus licheniformis or Corynebacterium, and the bacterial concentration is 1×10 7 ~1×10 8 CFU / mL; the mineralized bacteria are selected from at least one of Bacillus jelly-like, Bacillus subtilis, Bacillus amyloliquefaciens, Sporosarcina pasteurianus, and Bacillus sphaericus, and the concentration of the bacteria is 1×10 8 ~1×10 10 CFU / mL.

13. The immobilized microbial promoter for mining according to claim 1, characterized in that: The activation solution includes NH4Cl, MnSO4∙H2O, NiCl2∙6H2O, and yeast extract or peptone.

14. The immobilized microbial promoter for mining according to claim 13, characterized in that: The activation solution comprises the following components in parts by weight: 2000-3000 parts of yeast extract or peptone, 1000-2000 parts of NH4Cl, 100-150 parts of MnSO4∙H2O, and 200-300 parts of NiCl2∙6H2O.

15. The immobilized microbial promoter for mining according to claim 1, characterized in that: The binder comprises soluble calcium salt and urea.

16. The immobilized microbial promoter for mining according to claim 15, characterized in that: The soluble calcium salt is at least one of calcium formate, calcium chloride, calcium lactate and calcium acetate.

17. The immobilized microbial promoter for mining according to claim 15, characterized in that: The molar concentration of the soluble calcium salt is 0.8-1.0 mol / L.

18. The immobilized microbial promoter for mining according to claim 15, characterized in that: The molar concentration ratio of the soluble calcium salt to urea is 1:

1.

19. Use of the immobilized mining microbial promoter according to any one of claims 1 to 18 for coal dust consolidation, characterized in that: Here are the steps: The immobilized biosurfactant bacteria and the immobilized mineralizing bacteria are respectively added to the sterilized activation solution, and the composite microbial fermentation liquid is obtained after constant temperature cultivation, and the composite microbial fermentation liquid and the sterilized binder are sprayed onto the coal dust successively; The immobilized biosurfactant bacteria and the immobilized mineralizing bacteria are added to the sterilized activation solution in the following manner: first, one portion of the immobilized biosurfactant bacteria is added to the sterilized activation agent, and after culturing for 14 hours, one portion of the immobilized mineralizing bacteria is added, and the culturing is continued for 10 hours; The volume ratio of the composite microbial fermentation liquid to the binder is (13-15):(1-2).

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