Immobilized microbial inoculum capable of degrading toluene as well as preparation method and application of immobilized microbial inoculum

Abstract

The invention relates to the technical field of toluene degradation treatment, and discloses an immobilized microbial inoculum capable of degrading toluene as well as a preparation method and application of the immobilized microbial inoculum. The preparation method comprises the following steps: adding a bacillus megatherium activated bacterium solution into a liquid culture medium, and adding nano carbon black powder to obtain carbon black immobilized bacteria; heating and stirring the carboxymethyl xylan zinc complex and the nano magnesium lithium silicate until the mixture is molten; after cooling, adding the nano carbon black immobilized bacteria to obtain a mixed solution; slowly dropwise adding the mixed solution into a calcium chloride solution to form embedded pellets; and washing to obtain the immobilized microbial inoculum. According to the immobilized microbial inoculum capable of degrading toluene and the preparation method and application thereof, a strain is embedded in CXY-Zn / nLMS gel and gradually released through gel beads, the biological activity of the strain is better maintained, meanwhile, carbon black of a porous structure provides a place for attachment and growth of the strain, the service life of the strain is prolonged, the degradation capacity of microorganisms is improved, and the immobilized microbial inoculum can be used for degrading toluene. The prepared immobilized microbial agent can be used for sewage treatment and soil improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of toluene degradation treatment, and particularly to an immobilized microbial agent capable of degrading toluene, a preparation method thereof, and an application thereof. Background Art

[0002] Toluene, also known as methylbenzene, is an organic compound widely used in industrial production and daily life and belongs to one of the benzene series (BTEX). Toluene is a colorless and transparent liquid under normal temperature and pressure, with an aromatic odor, and its chemical formula is C7H8. It is an important petrochemical product and is widely used in the production of solvents, fuels, and chemical raw materials. In nature, toluene often exists in crude oil and coal tar and is generated through the production process of the petrochemical industry. Due to its relatively stable chemical properties, if not properly managed during production, use, and transportation, toluene is likely to leak into the environment, especially into soil and groundwater, posing potential hazards to the environment and human health.

[0003] As an industrial chemical, the environmental pollution problem of toluene cannot be ignored. During production, storage, use, and transportation, the leakage of toluene may cause pollution of air, soil, and water bodies. Especially in the groundwater system, toluene is difficult to degrade naturally, may exist and accumulate for a long time, and ultimately pose a threat to human health through the drinking water system. In order to prevent the impact of toluene on the environment and human health, many countries and regions have formulated strict standards and regulations to control the emission and use of toluene. In addition, with the development of environmental protection technologies, many new pollution treatment technologies have also been applied to the treatment process of toluene pollution, such as biodegradation technology, adsorption technology, and photocatalysis technology, all of which can effectively reduce the environmental hazards of toluene.

[0004] Biological treatment is an environmental protection technology that uses microorganisms to degrade, transform, or stabilize pollutants, relying on the metabolic activities of microorganisms to convert harmful pollutants into non-toxic or low-toxic substances, such as water, carbon dioxide, and inorganic salts. Compared with chemical and physical treatment methods, the cost of biological treatment is usually lower. Bacillus megaterium is a common bacterium in soil and aquatic environments and has attracted attention in environmental pollution remediation due to its excellent biodegradation ability, especially having significant advantages in toluene degradation.

[0005] Microbial embedding materials are technical materials that immobilize microorganisms (such as bacteria, fungi, enzymes, etc.) in specific matrices through physical or chemical means. Its core function is to form a stable microenvironment by encapsulating microbial cells, protecting the microorganisms from interference by external adverse conditions (such as extreme pH, toxic substances, mechanical shear forces), while allowing the selective permeation of nutrients and metabolites, thereby maintaining the activity and function of the microorganisms. Such materials are widely used in fields such as sewage treatment, bioremediation, and bioenergy production (such as hydrogen production, methane production). For example, in wastewater treatment, the embedding materials can immobilize highly efficient degrading bacteria, extend their service life, and improve the removal efficiency of pollutants; in biofuel production, the embedding technology can protect enzyme-producing microorganisms and achieve continuous production. Traditional embedding materials mainly include natural polymer materials (such as sodium alginate, gelatin, chitosan) and synthetic polymer materials (such as polyvinyl alcohol, polyacrylamide). Although these materials have been widely used, their limitations are significant. For example, traditional sodium alginate gels are prone to rupture in dynamic fluids, resulting in microbial leakage; gelatin is easily dissolved in high-temperature or high-salt environments, with poor stability; natural materials (such as chitosan) have a fast degradation rate and are difficult to achieve long-term slow release; synthetic materials (such as polyvinyl alcohol) are difficult to degrade and may cause environmental pollution; traditional materials are difficult to simultaneously meet the multifunctional requirements of mechanical support, nutrient transfer, and antibacterial properties, and have poor adaptability to complex environments (such as high-salt, highly acidic wastewater).

[0006] Currently, the main adsorption and immobilization carriers are natural carriers and synthetic carriers. Among them, natural carriers include peanut shells, corn cobs, etc. Bacteria immobilized by natural carriers have a better degradation efficiency for pollutants than free bacteria and stronger environmental adaptability, with broad application prospects. The embedding and immobilization technology uses embedding agents to retain microorganisms in polymer gels, preventing the leakage of microorganisms with degradation ability, and at the same time can block the contact between microorganisms and harmful substances in the system, thereby achieving the effects of enhancing the purification effect and immobilizing microorganisms.

[0007] Activated carbon, as a commonly used physical adsorbent, has adsorption ability due to its highly developed pore structure and large specific surface area. Zero-dimensional carbon-based nanomaterials refer to carbon materials with all three spatial dimensions in the nanometer range, usually with a spherical or spherical-like structure, mainly including fullerenes (C60), carbon black (CB), and CQDs, etc. Summary of the Invention

[0008] The object of the present invention is to provide an immobilized bacterium agent capable of degrading toluene, its preparation method and application. First, the zero-dimensional carbon-based nanomaterial carbon black is used to adsorb strains, and then the carbon black-strain is embedded in carboxymethylxylan zinc complex (CXY-Zn) / nanometer lithium magnesium silicate (nLMS), and gradually released through gel beads to better maintain its biological activity. At the same time, the carbon black with porous structure provides a place for the strains to attach and grow, prolongs the lifespan of the strains, improves the degradation ability of microorganisms, and the prepared immobilized bacterium agent can be used for sewage treatment and soil improvement.

[0009] As a zero-dimensional carbon material, carbon black has an extremely high specific surface area (usually reaching 300-1000 m 2 / g), and its surface usually has a rich pore structure. The high specific surface area enables carbon black to provide a large number of surface adsorption sites, which is crucial for the adsorption and embedding of microorganisms. Microorganisms can attach to the surface of carbon black through physical adsorption or chemical adsorption, thus forming a stable embedding body. The surface adsorption ability of carbon black can also improve the contact efficiency between microorganisms and pollutants, increasing the ability of microorganisms to degrade or transform pollutants. Zero-dimensional carbon-based nanomaterials usually exhibit very high thermal stability and chemical stability, which means that carbon black can maintain its structure and function under various environmental conditions (such as changes in acidity and alkalinity, temperature fluctuations, oxidation, etc.). This stability can not only ensure that carbon black is not disturbed by the environment during the microorganism embedding process, but also maintain the activity of microorganisms and the embedding effect in subsequent applications. Compared with other materials, the zero-dimensional structure of carbon black can effectively avoid the loss of strains or the destruction of the embedding structure caused by environmental changes. Due to the conductivity of the zero-dimensional nanostructure of carbon black, when microorganisms carry out metabolic activities on the surface of carbon black, they can utilize the electron transfer channels provided by carbon black, thereby improving the metabolic efficiency. For example, some electrochemically active bacteria (such as electrobioremediation bacteria, nitrogen-reducing bacteria, etc.) can carry out more efficient electron exchange with the help of carbon black, accelerate the metabolic process, and improve the degradation ability of microorganisms to pollutants. This metabolic activity promoted by electron transfer has important applications in the fields of environmental remediation and biocatalysis.

[0010] After carboxymethylxylan is carboxymethylated and modified, the main chain is rich in carboxylic acid groups (-COOH), which can form strong coordination bonds with zinc ions (Zn 2+ ), constructing a three-dimensional network structure. The strong coordination crosslinking of Zn 2+ significantly improves the compressive strength of the material and is suitable for high shear force environments (such as industrial wastewater treatment). By adjusting the concentration of Zn 2+ , the crosslinking density can be precisely regulated to achieve a degradation cycle from several days to several months, meeting the requirements of different scenarios. Zinc ions have natural antibacterial properties, which can reduce the contamination of miscellaneous bacteria in the embedding system; xylan is a natural polysaccharide, non-toxic and can be degraded by microorganisms, being environmentally friendly.

[0011] Nanosized lithium magnesium silicate (nLMS) is a layered silicate mineral with a high specific surface area and ion exchange capacity. Its lamellar structure surface is rich in hydroxyl groups (-OH) and negative charges. nLMS is embedded in the CXY-Zn network through hydrogen bonding and electrostatic interactions to form an "organic-inorganic hybrid structure", further resisting swelling and rupture. The nanolayered structure of nLMS can form oriented microchannels to promote the diffusion of nutrients and metabolites, while blocking toxic substances (such as heavy metal ions). nLMS can still maintain structural stability under high-salt, acidic or high-temperature conditions, expanding the application scope of embedding materials (such as saline wastewater, acid mine drainage). The combination of CXY-Zn and nLMS realizes a double cross-linked network - Zn 2+ The coordination bond and the physical adsorption of nLMS act synergistically, endowing the material with both high elasticity and rigidity.

[0012] The preparation of the carboxymethyl xylan-zinc complex (CXY-Zn) of the present invention includes the following steps:

[0013] (1) Preparation of carboxymethyl xylan:

[0014] Carboxymethylated xylan was prepared by the sodium hydroxide-chloroacetic acid chemical method. Weigh 3.0 g of xylan, add 50 mL of isopropanol and 25 mL of 20% NaOH aqueous solution, and place it in an ice-water bath for 3 h. Then weigh 15.0 g of chloroacetic acid, add 50 mL of isopropanol and 25 mL of 20% NaOH aqueous solution, mix well and slowly drop it into the xylan reaction system, and react at 60 °C for 4 h. After cooling, adjust the pH to neutral with 1 M hydrochloric acid, dialyze against running water for 48 h, and freeze-dry to obtain carboxymethyl xylan (Carboxymethyl xylan, CXY).

[0015] (2) Preparation of carboxymethyl xylan-zinc complex:

[0016] Dissolve 1.0 g of carboxymethyl xylan in 450 mL of distilled water, stir and dissolve it at a certain temperature for 10 min, add 0.88 g of ZnSO4·7H2O (dissolved in 20 ml of 0.1 mol / L HCl), adjust the pH to 5.5 with 1 mol / L NaOH (adjusting the pH to 5 - 7.5 with HCl / NaOH is more conducive to the complexation of polysaccharide and zinc), react at 60 °C for 2 h, dialyze against running water for 48 h, concentrate, and freeze-dry to obtain carboxymethyl xylan-zinc complex (Carboxymethyl xylan-zinc complex, CXY-Zn).

[0017] The present invention provides a novel immobilized material capable of degrading toluene and a preparation method of the material, including the following steps:

[0018] Step 1: Inoculate the Bacillus megaterium freezing solution into the sterilized liquid medium, and then place it in an incubator at a constant temperature of 35°C for 24 hours to obtain an activated bacterial solution.

[0019] Step 2: Add the activated bacterial solution from Step 1 to the sterilized liquid medium, add carbon black powder, place it in a shaker for 24 hours, then centrifuge and separate to obtain a solid, and wash it with sterile water to obtain carbon black immobilized bacteria.

[0020] Step 3: Take 4 g of CXY-Zn in small amounts and add it to a beaker containing 120 mL of deionized water for vibrating and stirring. Set the temperature to 90°C to obtain a solution without white particles. Continue to heat the transparent solution for 10 minutes, then add 2.8 g of nLMS. When the whole solution becomes a milky white, uniform and viscous liquid, continue to stir for 2 minutes, then perform ultrasonic dispersion for 10 minutes, and continue to stir evenly for 20 minutes, controlling the temperature at 90°C all the time to obtain a CXY-Zn / nLMS mixed solution.

[0021] Step 4: When the CXY-Zn / nLMS mixed solution cools to 30°C, quickly add the carbon black immobilized bacteria from Step 2 and stir evenly to make the immobilized bactericide evenly distributed in the gel containing CXY-Zn / nLMS to obtain a mixed solution.

[0022] Step 5: Configure a calcium chloride solution using ultrapure water and stir until the calcium chloride is completely dissolved to obtain a calcium chloride solution.

[0023] Step 6: Place the calcium chloride solution from Step 5 on a magnetic stirrer and stir. Use a syringe to suck the mixed solution from Step 4, and slowly drip the mixed solution in the syringe into the calcium chloride solution to form embedded beads.

[0024] Step 7: Filter out the embedded beads, rinse them 2 - 3 times with ultrapure water to remove surface impurity ions to obtain the immobilized bactericide.

[0025] Further, the Bacillus megaterium in Step 1 is Bacillus megaterium ATCC14581, and the density of the bacterial solution in the activated bacterial solution is OD 600 = 0.6 - 0.8.

[0026] Further, the inoculation amount of the activated bacterial solution in Step 2 is 5 - 10 v / v%, and the carrier concentration of the carbon black powder is 5 - 10 w / v%.

[0027] Further, the ratio of the activated bacterial solution to the carbon black powder in Step 2 is 1 - 2:1 v / w%.

[0028] Further, the parameters of the shaker in Step 2 are 35°C, 130 r / min, 24 h; the parameters of the centrifuge are 35°C, 3000 r / min, 10 min.

[0029] Furthermore, the concentration of the calcium chloride solution in step 5 is 3-5 w / v%.

[0030] Furthermore, in step 6, the rotational speed of the magnetic stirrer is 800-1100 rpm, and the syringe specification is a 1 ml syringe.

[0031] The present invention also provides an immobilized bacterium agent capable of degrading toluene, which is prepared by the above preparation method.

[0032] The present invention also provides the application of the above-mentioned immobilized bacterium agent capable of degrading toluene in sewage treatment, improving soil pollution and soil improvement.

[0033] The advantages and positive effects of the novel immobilized bacterium agent capable of degrading toluene, its preparation method and application of the present invention are as follows:

[0034] 1. The microporous structure (pore size 1-3 nm) of zero-dimensional nanocarbon black in the present invention can efficiently adsorb the surface proteins and polysaccharides of bacteria, and the adsorption capacity is significantly improved compared with traditional activated carbon. After the nanocarbon black is pickled and purified, there is no residual activator (such as ZnCl2), and the survival rate of bacteria will be greatly increased, avoiding the toxicity inhibition of traditional activated carbon.

[0035] 2. The zinc ion coordination bond of CXY-Zn and the layered silicate of nLMS in the present invention are cross-linked synergistically through hydrogen bonds to form an "organic-inorganic hybrid gel", and the compressive strength is significantly improved compared with traditional embedding agents. In wastewater treatment, the double-embedded bacterium agent can remain intact under higher flow rate conditions, with a low fragmentation rate, which is significantly better than traditional carriers.

[0036] 3. CXY-Zn in the present invention degrades slowly in the pH 6-8 environment, better maintaining its biological activity and the long-term effect of the degradation effect; the ion exchange ability of nLMS can adsorb heavy metals and protect the strain from toxicity interference. The bacterium agent can be recycled, and the degradation rate of benzene series compounds can be maintained at a high level.

[0037] 4. By adjusting the proportions of CXY-Zn, nLMS and carbon black in the present invention, the density, mechanical strength and mass transfer rate of the gel beads are adjusted, so that the immobilized bacterium agent can adapt to different environmental conditions, with a wide adaptability. It can not only complete the preliminary degradation of pollutants in a short time, but also maintain the long-term degradation ability through the slow release effect.

[0038] 5. The raw materials of the immobilized bacterium agent in the present invention are widely sourced and inexpensive, can be mass-produced, and have high economic benefits.

[0039] The technical solutions of the present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0040] Figure 1Morphology diagram of the immobilized microbial agent in Example 1 of the present invention;

[0041] Figure 2 Effect curve diagram in Example 2 of the present invention;

[0042] Figure 3 Simulated experimental design diagram in Example 3 of the present invention;

[0043] Figure 4 Toluene degradation efficiency along the direction of groundwater flow in Example 3 of the present invention;

[0044] Figure 5 Effect diagram of organic matter toluene degradation in the first 48 hours before the well group in Example 4 of the present invention;

[0045] Figure 6 Diagram of the degradation rate of organic matter toluene in each well of the well group in Example 4 of the present invention;

[0046] Figure 7 Toluene degradation situation in the soil by each treatment group in Example 5 of the present invention;

[0047] Figure 8 Change of sucrase activity in the soil of each treatment group in Example 6 of the present invention. Detailed implementation manners

[0048] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0049] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs.

[0050] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually determined according to national standards. The experimental instruments, equipment, and reagents not indicated in the following examples are all commercially available raw materials.

[0051] The equipment used in each step of the following examples is all conventional equipment. If there is no corresponding national standard, it shall be carried out according to the general international standard, conventional conditions, or the conditions recommended by the manufacturer.

[0052] Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0053] Bacillus megaterium.sp, purchased from Mingzhou Bio, with the strain number ATCC14581. The liquid medium is LB liquid medium.

[0054] Example 1

[0055] The preparation method of the novel immobilized bacterium agent of carbon black - Bacillus megaterium with CXY-Zn / nLM double embedding is as follows:

[0056] Step 1: Inoculate 60 μL of the stored frozen liquid of Bacillus megaterium into the sterilized liquid medium, and place the inoculated medium in a constant temperature incubator at 35 °C for 24 h to obtain an activated bacterial liquid (OD 600 = 0.8);

[0057] Step 2: Add 5 v / v% of the activated bacterial liquid to 50 mL of the sterilized medium, then add 10 w / v% of carbon black powder, place it in a shaker for 24 h and then centrifuge (the parameters of the shaker are 35 °C, 130 r / min, 24 h; the parameters of the centrifuge are 35 °C, 3000 r / min, 10 min), wash the solid with sterile water to obtain carbon black-immobilized bacteria;

[0058] Step 3: Take 4 g of CXY-Zn in small amounts and add it to a beaker containing 120 mL of deionized water for vibrating and stirring, set the temperature to 90 °C to obtain a solution without white particles. Continue to heat the transparent solution for 10 min, then add 2.8 g of nLMS. When the whole solution becomes a milky white, uniform and viscous liquid, continue to stir for 2 min, then perform ultrasonic dispersion for 10 min, and continue to stir evenly for 20 min, controlling the temperature at 90 °C all the time;

[0059] Step 4: When the CXY-Zn / nLMS mixture cools to 30 °C, quickly add the carbon black-immobilized bacteria in Step 2 and stir evenly to make the immobilized bacterium agent evenly distributed in the gel containing CXY-Zn / nLMS to obtain a mixed solution;

[0060] Step 5: Prepare a 4 w / v% calcium chloride solution with ultrapure water and stir until the calcium chloride is completely dissolved;

[0061] Step 6: Place the prepared calcium chloride solution in Step 5 on a magnetic stirrer and add a magnetic stir bar to make the magnetic stir bar start to rotate at a speed of 800 rpm. Use a 1 mL syringe to suck the mixed solution prepared in Step 4, and then slowly drip it into the calcium chloride solution to form embedded small balls;

[0062] Step 7: Filter out the fixed small balls, rinse them 2 - 3 times with ultrapure water to remove surface impurity ions to obtain the immobilized bacterium agent. The morphology is as Figure 1 shown.

[0063] Example 2

[0064] (1) Proportion optimization:

[0065] Select 16 groups of proportions of carboxymethylated xylan zinc complex (CXY-Zn), nanometer magnesium lithium silicate (nLMS) and carbon black (CB), and prepare immobilized blank embedding beads (without embedding Bacillus megaterium) respectively. Measure the density, mechanical strength and mass transfer performance of each group of beads. The specific measurement methods are as follows:

[0066] 1) Density: Weigh a certain volume of water with a measuring cylinder and record the initial mass. Take 50 beads with similar size and shape and put them into the measuring cylinder, record the volume difference before and after, and obtain the density of the beads through the density calculation formula.

[0067] 2) Mechanical strength: Take out 5 blank embedding immobilized beads with similar shape and size from each group and place them on a balance. Horizontally place a glass slide on the beads, zero the balance and then slowly press the glass slide until the beads are deformed and cannot return to their original shape, and record the maximum mass M that the beads can withstand i , and the mechanical strength of a single embedding bead is F i = 10M i / 10. Measure three groups and take the average value, that is, the average mechanical strength of the beads is:

[0068]

[0069] 3) Mass transfer performance: Use the color infiltration method to test the mass transfer property of the particles. Immerse the immobilized beads in white ink solution (add 10 ml of white ink solution to 10 ml of water), take out the beads every 1 minute and slice the beads, observe the penetration situation of the beads. Take out the slices 6 times in total, and qualitatively describe at which minute the beads are completely soaked with the numbers 1-6. The smaller the number, the better the mass transfer performance of the beads. The results are shown in Table 1.

[0070] Table 1 Effects of embedding agents with different concentrations on the performance of gel beads

[0071]

[0072] As can be seen from Table 1, when the concentration of xylan zinc complex is too low and the concentration difference from lithium magnesium silicate nanosheets is too large, it is difficult to form gel beads and the formed beads are prone to trailing; when the concentration of xylan zinc complex is too high, the mass transfer performance of the formed beads is poor. When controlling the xylan zinc complex at a lower concentration, the higher the mass fractions of lithium magnesium silicate nanosheets and carbon black, the higher the overall mechanical strength of the beads; when the mass fractions of carbon black and lithium magnesium silicate nanosheets are higher, the mass transfer performance of the beads is worse. Considering the three indicators of the density, mechanical strength and mass transfer performance of each group of beads comprehensively, the optimized ratios of each component selected are: xylan zinc complex: 3%, 3.5%, 4%; lithium magnesium silicate nanosheets: 2%, 2.5%, 3%; carbon black 0.5%, 1%, 1.5%.

[0073] Combined with the above selected optimized ratios, three influencing factors are set, namely the concentration of xylan zinc complex as A (%), the concentration of lithium magnesium silicate nanosheets as B (%), and the concentration of carbon black as C (%). Each factor is set at three levels, and the factor levels of the orthogonal experiment are shown in Table 2.

[0074] Table 2 Factor level table

[0075]

[0076] (2) Results of orthogonal experiment:

[0077] Prepare the groundwater in the polluted area to test the degradation of toluene in the polluted groundwater under different factor levels. The main components of the simulated polluted groundwater are: KCl (0.02 w / v%), NaCl (0.02 w / v%), MgSO4·7H2O (0.02 w / v%), CaCl2 (0.01 w / v%), both MnSO4·H2O and FeCl2 are trace amounts, make up the volume with distilled water and adjust the pH = 7.0. At the same time, add a certain amount of toluene, and the addition amount is 8 w / v%. Place it in the dark for standby. Make immobilized embedding beads containing Bacillus megaterium, control the inoculation amount of the strain at 5 v / v%, take 150 ml of the simulated polluted groundwater in a conical flask, add 30 beads with uniform size and shape to each conical flask, and incubate at room temperature for 5 d. Using the toluene degradation rate as the evaluation index, 2×9 groups of parallel experiments are carried out, and the experimental results are as follows:

[0078] Table 3 Orthogonal experiment design scheme and results

[0079]

[0080]

[0081] The range R can reflect the magnitude of the difference in test results when the factor levels change. According to the range values in Table 3, the primary and secondary order of each factor can be known as: A (carbon black) > B (xylan zinc complex) > C (nano magnesium lithium silicate), indicating that the concentration of carbon black has the most significant impact on the degradation ability of the embedded pellets, while nano magnesium lithium silicate and xylan zinc complex have a relatively lower impact on the degradation ability of the embedded pellets. It can be judged from the table that the best level for A is 1, the best level for B is 1, and the best level for C is 2, that is, the optimal conditions are 3% xylan zinc complex, 2% nano magnesium lithium silicate, and 1% carbon black. The effect curves of each factor are shown in Figure 2 .

[0082] Figure 2 The effect curve graphs showing how the three factors behave as the level concentration changes. As Figure 2 shown, the effect curve of the xylan zinc complex concentration has a tendency to decline as the concentration increases, the effect curve of the nano magnesium lithium silicate concentration shows a trend of first declining and then rising, and the carbon black mass fraction curve reaches its maximum value when the mass fraction is 1%. The effect curve of the nano magnesium lithium silicate concentration is generally relatively stable.

[0083] Example 3

[0084] A soil box was used to simulate the hydrogeological and pollution conditions of various polluted sites, and a cyclic degradation simulation test was carried out to provide a theoretical basis for further practical site applications. Samples required were collected layer by layer using the soil sampling holes and injection wells in the investigation area, mainly including water-bearing sand layers, top and bottom powder clay, and groundwater samples. According to the site investigation data, the average hydraulic gradient in the investigation area was obtained as 8‰, and the permeability coefficient of the aquifer sand was 5.0×10 -4 m / s. Based on the formula of the linear seepage law (Darcy's law):

[0085] V = K × [Δh÷L].

[0086] V: groundwater flow velocity; K: permeability coefficient; [Δh÷L]: hydraulic gradient;

[0087] Δh: head difference; L: horizontal distance

[0088] The groundwater flow velocity in the investigation area was approximately 4×10-6 m / s.

[0089] (1) Preparation of the simulation box:

[0090] Design a glass box according to the experimental requirements. The preliminary design specifications are length * width * height = 0.8m * 0.3m * 0.5m. The material is plexiglass with a thickness of 10mm, bonded with glass glue. Import and export reservoirs with a length of 10cm are designed on both sides. The hydraulic gradient of groundwater is simulated by the water level height at the import and export. The front view and top view are as shown in the figure. Three simulated wells are designed in the box using PVC pipes, spaced 20cm apart. The bottom is inserted into the aquifer floor, the middle opening is wrapped with a screen for water permeability, and the upper opening is connected to the atmosphere for adding bacteria agents. Peristaltic pumps are equipped in the inlet and outlet tanks to simulate the groundwater flow velocity V. The design drawing is as Figure 3 shown.

[0091] (2) Filling the simulation box:

[0092] Put the polluted groundwater and soil collected from the field into the soil box according to the field hydrogeological conditions. The bottom is silty clay (0.1m), the middle is aquifer fine sand (0.2m), and the upper is vadose zone silty clay (or silt) (0.3m). Weigh the lithological medium and spread it evenly in the simulation tank to prevent voids and faults. Pour the polluted groundwater into the aquifer of the model and saturate it from bottom to top. During the rising process of the water level, observe the water level change until the fine sand layer is saturated, and control the hydraulic gradient at about 8‰.

[0093] (3) Simulation box experiment:

[0094] Add 200g of immobilized bacteria agent to the injection well, and compare and analyze different simulated wells. Along the water flow direction, analyze the degradation effect of toluene. The results are as Figure 4 shown.

[0095] As Figure 4 can be seen, after adding the immobilized bacteria agent to the injection well, the removal effect of toluene is obvious. The monitoring well 1 at the injection well and 20cm away from the injection well has a good removal effect, and the concentration of toluene gradually decreases along the water flow direction. The degradation rate of toluene in the injection well is 80.86%, the degradation rate in the monitoring well 1 is 68.56%, and the degradation rate in the monitoring well 2 is 56.98%.

[0096] Example 4

[0097] Use the data analysis of the laboratory bench-scale simulation box to screen out the microbial bacteria agent with obvious degradation effect on the organic pollutant toluene. Use equipment such as a bailer to add the microbial bacteria agent to the unconfined aquifer groundwater well to carry out on-site pilot tests. By comparing the detection data before and after the remediation, verify the in-situ degradation efficiency of the microorganisms and form a set of groundwater organic pollution remediation technologies.

[0098] Eight wells are arranged in the field toluene pollution area. Among them, A0 and A00 are injection wells, A1 is a control well, A2, A3, and B2 are lateral diffusion monitoring wells, and A4 and A5 are downstream monitoring wells. The degradation rate of 50% is used as the degradation target.

[0099] The on-site pilot dosing plan was implemented based on the results of the laboratory test. The immobilized bacterial agent was added to the dosing wells A0 and A00. After the collected samples were tested, the monitoring data of the well group for seven weeks was obtained to analyze the degradation of organic pollution in the on-site aquifer by the bacterial agent. The results are shown in Table 4 and Figure 5 shown.

[0100] Table 4 Well group toluene detection data table

[0101] Time A0 (μg / l) A00 (μg / l) A1 (μg / l) A2 (μg / l) A3 (μg / l) A4 (μg / l) A5 (μg / l) 0h 63.8 68.3 62.0 54.8 58.8 44.2 43.4 12h 54.8 51.0 53.2 62.4 55.9 40.8 41.4 24h 34.7 33.7 60.1 58.8 53.3 41.7 42.4 36h 37.1 39.5 30.1 46.1 56.6 46.5 47.5 48h 29.6 31.1 42.4 41.8 31.2 23.4 33.0 Two weeks 33.2 33.8 60.8 66.8 39.5 31.4 33.4 Three weeks 39.8 39.4 46.6 48 29.1 32 32.2 Four weeks 54.9 49.8 39.4 40.1 49.4 36.8 38.6 Five weeks 34.3 38.1 46.8 32.4 28.8 38.8 38.5 Seven weeks 33.1 38.7 50.6 30.8 42.4 26.7 33.3

[0102] Through the analysis of the detection data of toluene in the well group, it can be seen (Table 4 and Figure 5 ), in the time period from 0h to 48h, the toluene concentration showed an overall downward trend. Among them, the degradation effect of the dosing wells A0 and A00 was the earliest to appear, and the toluene concentration began to decline within the first 24 hours; the downstream monitoring wells A4 and A5 showed obvious degradation effects from 36 hours. In the lateral diffusion direction, the degradation effect of A2 preceded that of A3, and the A3 monitoring well also showed a significant degradation trend after 36 hours. For the upstream monitoring well A1, although the degradation effect was detected, it rebounded immediately. Analysis shows that this is mainly caused by the infiltration and diffusion of the upstream polluted water body.

[0103] The seven-week organic toluene detection data of the comprehensive well group showed that the toluene concentration in the groundwater of the aquifer showed a three-fold downward trend. In the end, except for the control well A1, the toluene concentrations of the remaining monitoring wells could be maintained at a certain level. In the second week, the toluene concentration rebounded. Analysis showed that this was due to the decrease in dissolved oxygen content in the groundwater, which led to the weakening of microbial activity. Among them, the rebound of wells A0 and A00 was the most obvious. In the third week, the microorganisms adapted to the anaerobic environment and began to proliferate, and the toluene concentration decreased accordingly. However, when the dissolved oxygen and other nutrients in the aquifer were exhausted, the toluene concentration rose again in the fourth week. In response to this situation, the project team intervened by reinforcing the addition of bacterial agents, and subsequent monitoring showed that the toluene concentration dropped again. However, the toluene concentration in the control well A1 increased. Analysis showed that the reason may be due to the infiltration and diffusion of polluted water bodies upstream, while the toluene concentration in other groundwater wells remained stable in the seventh week.

[0104] According to the test results, the toluene concentration at each time point was compared with the concentration at 0 hour (before adding the bacterial agent), and a toluene degradation rate diagram of each well in the well group was drawn ( Figure 6 ). The results showed that 80% of the monitoring points had a positive degradation rate, indicating that the bacterial agent had a good degradation effect on toluene in the aquifer. Among them, 20% of the monitoring points had a degradation rate of more than 50%, and the highest degradation rate reached 58.5%, indicating that the toluene pollution in the well group has reached the remediation target.

[0105] Example 5 Improvement of Soil Pollution

[0106] The collected toluene-contaminated soil samples were ground and passed through a 10-mesh sieve. 15 g of the sieved toluene-contaminated soil sample was added to a 250 mL conical flask, and 190 mL of ultrapure water was added. 10 mL of Bacillus bacterium liquid and 166.8 g of embedded bacteria balls were added respectively. Without adding the bacterium liquid was used as the blank treatment, and cultured at 18 °C for 40 d to obtain the contaminated soil degradation liquid.

[0107] The cultured soil degradation liquid was poured into a separating funnel, and 10 mL of petroleum ether was added for extraction. The extract was centrifuged at 5000 r·min -1 for 10 min under the condition and the supernatant was collected. The upper extract was filtered with anhydrous sodium sulfate, filled into a 25 mL volumetric flask and fixed to the scale line with petroleum ether as the sample to be measured.

[0108] HJ 1067—2019 Determination of Benzene Series in Water Headspace / Gas Chromatography was used to determine the toluene concentration. Headspace sampler heating equilibrium temperature: 60 °C; heating equilibrium time: 30 min; injection valve temperature: 100 °C; transmission line temperature: 100 °C; injection volume: 1.0 ml (quantitative loop). Gas chromatograph injection port temperature: 200 °C; detector temperature: 250 °C; chromatographic column temperature rising program: 40 °C (held for 5 min), rising at a rate of 5 °C / min to 80 °C (held for 5 min); carrier gas flow rate: 2.0 ml / min; combustion gas flow rate: 30 ml / min; combustion-supporting; gas flow rate: 300 ml / min; tail blow gas flow rate: 25 ml / min; split ratio: 10:1. 3 g of sodium chloride was pre-added to 7 headspace bottles respectively, and 10.0 ml, 10.0 ml, 10.0 ml, 9.8 ml, 9.6 ml, 9.2 ml and 8.8 ml of water were accurately added in sequence. Then, 5.00 μl, 20.0 μl, 50.0 μl, 0.20 ml, 0.40 ml, 0.80 ml and 1.2 ml of standard stock solution were added in sequence with a micro syringe and a pipette to prepare standard series with the mass concentrations of the target compounds being 0.050 mg / L, 0.200 mg / L, 0.500 mg / L, 2.00 mg / L, 4.00 mg / L, 8.00 mg / L, 12.0 mg / L respectively. Immediately seal the headspace bottle, gently shake well, and inject samples for analysis in sequence from low concentration to high concentration according to the instrument conditions, and record the retention time and response value of the standard series target substances. With the benzene series concentration as the abscissa and its corresponding response value as the ordinate, a working curve was established. After establishing the standard curve, 3 g of sodium chloride was pre-added to the headspace bottle, 10.0 ml of the sample to be measured was added, immediately capped and sealed, shaken well, and measured. The experimental results are as Figure 7 shown.

[0109] Toluene degradation rate = (blank toluene concentration - toluene concentration in the sample) / blank toluene concentration × 100%.

[0110] It can be seen from Figure 7 that without adding the bacterial agent, the toluene degradation rate in the soil of the blank control group reached 6.92%, indicating that the indigenous microorganisms in the soil had a certain effect on the degradation of toluene; in the experiment, adding Bacillus bacterial liquid and the immobilized bacterial agent had significantly higher toluene degradation ability than the control group, and the degradation effect of the immobilized bacterial agent was better than that of only adding the bacterial liquid. The degradation rate was 65.98%, while the degradation rate of the experimental group with only adding the bacterial liquid was 45.78%. It shows that the immobilized bacterial agent can significantly degrade toluene in the soil and improve soil pollution.

[0111] Example 6 Soil Remediation

[0112] To study the effect of the immobilized embedding beads on the enzyme activity during soil remediation, 4 treatment groups were set up: blank group (no bacterial agent and toluene were added to the soil); contaminated soil (5% w / w toluene); contaminated soil + strain (5% w / w toluene + 1 ml Bacillus bacterial liquid (OD 600 = 0.8)); contaminated soil + immobilized bacterial agent (5% w / w toluene + 17.42 g immobilized embedding beads). The dinitrosalicylic acid colorimetric method was used to measure the protease activity in the soil. By measuring the sucrase activity under different treatments, the change in the sucrase activity during the remediation of toluene-contaminated soil by the immobilized bacterial agent was clarified, and the results are as Figure 8 shown.

[0113] Compared with the control group, the sucrase activity showed a downward trend in the treatment group without adding bacteria with toluene added, while the sucrase activity showed long-term effectiveness in the treatment groups with free bacteria and the immobilized bacterial agent added. The enzyme activity in the treatment group with the immobilized bacterial agent added was 23.52% - 25.09% higher than that of the control group soil and 33.69% - 42.48% higher than that of the soil in the group with the bacterial liquid added from 14 to 21 days. It can be seen that the immobilized bacterial agent can more effectively restore the sucrase activity in the soil affected by toluene compared with the free bacterial agent.

[0114] Therefore, the present invention adopts the above-mentioned immobilized bacterial agent capable of degrading toluene, its preparation method and application. The strain is embedded in the carboxymethyl xylan zinc complex / nanosized magnesium silicate lithium gel and gradually released through the gel beads, better maintaining its biological activity. At the same time, the carbon black with a porous structure provides a place for the strain to attach and grow, prolonging the lifespan of the strain and improving the degradation ability of the microorganism. The prepared immobilized bacterial agent can be used for sewage treatment and soil improvement.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a novel immobilized microbial agent capable of degrading toluene, characterized in that, It includes the following steps: Step 1: Inoculate the Bacillus megaterium freezing solution into the sterilized liquid medium, and then place it in an incubator at a constant temperature of 35°C for 24 h to obtain an activated bacterial solution. Step 2: Add the activated bacterial solution in Step 1 to the sterilized liquid medium, add nano-carbon black powder, place it on a shaker for 24 h, then centrifuge and separate to obtain a solid, and wash it with sterile water to obtain carbon black immobilized bacteria. Step 3: Take CXY-Zn in a beaker and stir it vibrantly, set the temperature to 90°C to obtain a transparent solution. Continue heating for 10 min, then add nLMS. When the whole solution becomes a milky white, uniform and viscous liquid, continue stirring for 2 min, then perform ultrasonic dispersion for 10 min, and continue uniform stirring for 20 min, controlling the temperature at 90°C to obtain a CXY-Zn / nLMS mixture. Step 4: When the CXY-Zn / nLMS mixture cools to 30°C, quickly add the carbon black immobilized bacteria in Step 2 and stir evenly to make the immobilized bactericide evenly distributed in the gel containing CXY-Zn / nLMS to obtain a mixture. Step 5: Prepare a calcium chloride solution using ultrapure water, stir until the calcium chloride is completely dissolved to obtain a calcium chloride solution. Step 6: Place the calcium chloride solution in Step 5 on a magnetic stirrer and stir. Use a syringe to suck the mixture in Step 4, and slowly drip the mixture in the syringe into the calcium chloride solution to form embedded beads. Step 7: Filter out the embedded beads, rinse them 2 - 3 times with ultrapure water to remove surface impurity ions to obtain the immobilized bactericide.

2. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, wherein: In step 1, the Bacillus megaterium is Bacillus megaterium ATCC14581, and the density of the bacterial liquid in the activated bacterial liquid is OD 600 = 0.6 - 0.

8.

3. The preparation method of an immobilized bacterium agent capable of degrading toluene according to claim 1, characterized in that: In Step 2, the inoculation amount of the activated bacterial solution is 5 - 10 v / v%, and the carrier concentration of the carbon black powder is 5 - 10 w / v%.

4. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, characterized in that: In Step 2, the ratio of the activated bacterial solution to the carbon black powder is 1 - 2:1 v / w%.

5. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, characterized in that: In Step 2, the parameters of the shaker are 35°C, 130 r / min, 24 h; the parameters of the centrifuge are 35°C, 3000 r / min, 10 min.

6. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, characterized in that: In Step 3, the preparation method of the carboxymethylxylan zinc complex CXY-Zn is as follows: Prepare carboxymethylated xylan by the sodium hydroxide-chloroacetic acid chemical method. Dissolve the carboxymethylated xylan in distilled water, stir to dissolve, add ZnSO4·7H2O solution, adjust the pH to 5.5 with 1 mol / L NaOH, react at 60°C for 2 h, dialyze with running water for 48 h, concentrate, and freeze-dry to obtain the carboxymethylxylan zinc complex CXY-Zn.

7. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, characterized in that: In Step 5, the concentration of the calcium chloride solution is 3 - 5 w / v%.

8. The preparation method of an immobilized microbial agent capable of degrading toluene according to claim 1, characterized in that: In Step 6, the rotation speed of the magnetic stirrer is 800 - 1100 rpm, and the syringe specification is a 1 ml syringe.

9. An immobilized microbial agent capable of degrading toluene, characterized in that: Prepared by the preparation method described in any one of Claims 1 - 8.

10. The application of an immobilized bactericide capable of degrading toluene as described in Claim 9 in sewage treatment, improving soil pollution, and soil improvement.

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