An immobilized bacterial agent capable of degrading toluene, and its preparation method and application

Abstract

The present invention relates to the technical field of toluene degradation treatment, and discloses an immobilized bacterial agent capable of degrading toluene, and a preparation method and application thereof. The preparation method comprises the following steps: adding Bacillus megaterium activated bacterial solution to a liquid culture medium, adding nano-carbon black powder to obtain carbon black immobilized bacteria; heating and stirring a carboxymethyl xylan zinc complex and nano-lithium magnesium silicate until they are melted; adding nano-carbon black immobilized bacteria after cooling to obtain a mixed solution; slowly dripping the mixed solution into a calcium chloride solution to form embedded pellets; and rinsing to obtain an immobilized bacterial agent. The present invention adopts an immobilized bacterial agent capable of degrading toluene, and a preparation method and application thereof, wherein the strain is embedded in a CXY‑Zn / nLMS gel, and is gradually released through the gel pellets to better maintain its biological activity. At the same time, the porous carbon black provides a place for the strain to attach and grow, thereby extending the life 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.

Description

Technical Field

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

[0002] Toluene, also known as methylbenzene, is an organic compound widely used in industrial production and daily life, belonging to the benzene series (BTEX). At room temperature and pressure, toluene is a colorless, transparent liquid with an aromatic odor. Its chemical formula is C7H8. It is an important petrochemical product, widely used in the production of solvents, fuels, and chemical raw materials. In nature, toluene is commonly found in crude oil and coal tar and is produced through the production processes of the petrochemical industry. Due to its relatively stable chemical properties, toluene can easily leak into the environment, particularly into soil and groundwater, if not properly managed during production, use, and transportation, posing potential hazards to the environment and human health.

[0003] As an industrial chemical, toluene poses significant environmental risks. During production, storage, use, and transportation, toluene leaks can lead to air, soil, and water pollution. Particularly in groundwater systems, toluene is difficult to degrade naturally and can persist and accumulate for extended periods, ultimately posing a threat to human health through drinking water systems. To prevent the impact of toluene on the environment and human health, many countries and regions have established strict standards and regulations to control its emissions and use. Furthermore, with the advancement of environmental protection technologies, many new pollution control technologies are being applied to the treatment of toluene pollution, such as biodegradation, adsorption, and photocatalysis, all of which can effectively reduce the environmental hazards of toluene.

[0004] Biological treatment is an environmentally friendly technology that uses microorganisms to degrade, transform, or stabilize pollutants. Relying on the metabolic activity of microorganisms, harmful pollutants are converted into non-toxic or low-toxic substances, such as water, carbon dioxide, and inorganic salts. Compared with chemical and physical treatment methods, biological treatment is generally less expensive. Bacillus megaterium, a common bacterium found in soil and aquatic environments, has attracted attention in environmental remediation due to its excellent biodegradation capabilities, particularly its significant advantages in toluene degradation.

[0005] Microbial encapsulation materials are materials that physically or chemically immobilize microorganisms (such as bacteria, fungi, and enzymes) within a specific matrix. Their core function is to encapsulate microbial cells, creating a stable microenvironment that protects them from adverse external conditions (such as extreme pH, toxic substances, and mechanical shear), while allowing the selective permeation of nutrients and metabolites, thereby maintaining microbial activity and function. These materials are widely used in wastewater treatment, bioremediation, and bioenergy production (such as hydrogen and methane production). For example, in wastewater treatment, encapsulation materials can immobilize highly efficient biodegrading bacteria, extending their lifespan and improving pollutant removal efficiency. In biofuel production, encapsulation technology can protect enzyme-producing microorganisms and enable continuous production. Traditional encapsulation materials primarily include natural polymers (such as sodium alginate, gelatin, and chitosan) and synthetic polymers (such as polyvinyl alcohol and polyacrylamide). Although these materials have been widely used, they have significant limitations. For example, traditional sodium alginate gels are prone to rupture in dynamic fluids, leading to microbial leakage; gelatin is easily soluble in high temperature or high salt environments and has poor stability; natural materials (such as chitosan) degrade quickly, making it difficult to achieve long-term sustained release; synthetic materials (such as polyvinyl alcohol) are difficult to degrade and may cause environmental pollution; traditional materials are difficult to simultaneously meet multifunctional requirements such as mechanical support, nutrient delivery, and antibacterial properties, and have poor adaptability to complex environments (such as high-salt, highly acidic wastewater).

[0006] Currently, the main adsorption immobilization carriers are natural carriers and synthetic carriers. Natural carriers include peanut shells and corn cobs. Bacteria immobilized on natural carriers are more efficient at degrading pollutants than free bacteria and have stronger adaptability to the environment, thus having broad application prospects. Encapsulation immobilization technology uses an encapsulating agent to trap microorganisms in a gel-like polymer, preventing the release of degrading microorganisms and also blocking their contact with harmful substances in the system, thereby enhancing purification and immobilizing the microorganisms.

[0007] Activated carbon, a commonly used physical adsorbent, derives its adsorption capacity from its highly developed pore structure and extremely large specific surface area. Zero-dimensional carbon-based nanomaterials refer to carbon materials with three spatial dimensions in the nanometer range. These materials are typically spherical or quasi-spherical, and primarily include fullerenes (C60), carbon black (CB), and carbon quasi-dots (CQDs). Summary of the Invention

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

[0009] Carbon black, as a zero-dimensional carbon material, possesses an extremely high specific surface area (typically reaching 300-1000 m² / g) and a rich porous structure. This high specific surface area provides a large number of surface adsorption sites, which is crucial for the adsorption and entrapment of microorganisms. Microorganisms can attach to the carbon black surface through physical or chemical adsorption, forming a stable entrapment. Carbon black's surface adsorption capacity also improves the contact efficiency between microorganisms and pollutants, enhancing their ability to degrade or transform pollutants. Zero-dimensional carbon-based nanomaterials typically exhibit very high thermal and chemical stability, meaning that carbon black maintains its structure and functionality under a variety of environmental conditions, such as pH fluctuations, temperature fluctuations, and oxidation. This stability not only ensures that carbon black is resistant to environmental disturbances during the microbial entrapment process, but also maintains microbial activity and entrapment effectiveness during subsequent applications. Compared to other materials, carbon black's zero-dimensional structure effectively prevents strain loss or entrapment structure disruption due to environmental changes. Because carbon black's zero-dimensional nanostructure is conductive, microorganisms conducting metabolic activities on its surface can utilize the electron transfer pathways provided by carbon black, thereby improving metabolic efficiency. For example, some electrochemically active bacteria (such as electrobioremediation bacteria and nitrogen-reducing bacteria) can more efficiently exchange electrons with the help of carbon black, accelerating metabolic processes and enhancing the microbial ability to degrade pollutants. This type of metabolic activity promoted by electron transfer has important applications in environmental remediation and biocatalysis.

[0010] After carboxymethylation, the main chain of carboxymethyl xylan is rich in carboxylic acid groups (-COOH), which can react with zinc ions ( ) form strong coordination bonds and build a three-dimensional network structure. The strong coordination cross-linking significantly improves the compressive strength of the material and is suitable for high shear environments (such as industrial wastewater treatment). The concentration can precisely control the cross-linking density to achieve a degradation cycle from several days to several months to meet the needs of different scenarios.

[0011] Zinc ions have natural antibacterial properties and can reduce bacterial contamination in the embedding system; xylan is a natural polysaccharide that is non-toxic, can be degraded by microorganisms, and is environmentally friendly.

[0012] Nanolithium magnesium silicate (nLMS) is a layered silicate mineral with a high specific surface area and ion exchange capacity. The surface of its lamellar structure is rich in hydroxyl groups (-OH) and negative charges. nLMS is embedded in the CXY-Zn network through hydrogen bonds and electrostatic interactions to form an "organic-inorganic hybrid structure" to further resist swelling and rupture. The nano-layered structure of nLMS can form directional microchannels, promote the diffusion of nutrients and metabolites, and block toxic substances (such as heavy metal ions). nLMS can still maintain structural stability under high salt, acidic or high temperature conditions, expanding the application range of encapsulation materials (such as saline wastewater, acidic mine drainage). The combination of CXY-Zn and nLMS realizes a double cross-linked network—— The coordination bond works synergistically with nLMS physical adsorption to give the material both high elasticity and rigidity.

[0013] The preparation of the carboxymethyl xylan zinc complex (CXY-Zn) of the present invention comprises the following steps:

[0014] (1) Preparation of carboxymethyl xylan:

[0015] Carboxymethylated xylan was prepared using a sodium hydroxide-chloroacetic acid method. 3.0 g of xylan was weighed, added to 50 mL of isopropanol and 25 mL of a 20% aqueous NaOH solution, and incubated in an ice-water bath for 3 hours. Then, 15.0 g of chloroacetic acid was weighed, added to 50 mL of isopropanol and 25 mL of a 20% aqueous NaOH solution, mixed thoroughly, and slowly added dropwise to the xylan reaction system. The reaction was continued at 60°C for 4 hours. After cooling, the pH was adjusted to neutral with 1 M hydrochloric acid, dialyzed against running water for 48 hours, and freeze-dried to obtain carboxymethyl xylan (CXY).

[0016] (2) Preparation of carboxymethylated xylan zinc complex:

[0017] 1.0 g of carboxymethylated xylan was dissolved in 450 mL of distilled water and stirred at a certain temperature for 10 min. 0.88 g of ZnSO4·7H2O (dissolved in 20 ml of 0.1 mol / L HCl) was added, and the pH was adjusted to 5.5 with 1 mol / L NaOH (adjusting the pH to 5-7.5 using HCl / NaOH is more conducive to the complexation of polysaccharide and zinc). The reaction was carried out at 60°C for 2 h, and the mixture was dialyzed against running water for 48 h. The mixture was concentrated and freeze-dried to obtain carboxymethyl xylan-zinc complex (CXY-Zn).

[0018] The present invention provides a novel immobilized material capable of degrading toluene and a method for preparing the material, comprising the following steps:

[0019] Step 1: inoculating a frozen solution of Bacillus megaterium into a sterilized liquid culture medium, and then culturing the culture medium in a 35° C. constant temperature incubator for 24 hours to obtain an activated bacterial solution;

[0020] Step 2: Add the activated bacterial solution prepared in step 1 to the sterilized liquid culture medium, add carbon black powder, culture on a shaker for 24 hours, and then centrifuge to obtain a solid, which is then washed with sterile water to obtain carbon black-immobilized bacteria;

[0021] Step 3: Add 4 g of CXY-Zn to a beaker containing 120 mL of deionized water and vibrate and stir repeatedly at 90°C until white particles are removed. Heat the transparent solution for 10 minutes, then add 2.8 g of LMS. Once the solution becomes a milky white, uniform, viscous liquid, continue stirring for 2 minutes, then ultrasonically disperse for 10 minutes. Continue stirring for 20 minutes, maintaining the temperature at 90°C, to obtain a CXY-Zn / nLMS mixture.

[0022] Step 4: When the CXY-Zn / nLMS mixture is cooled to 30°C, the carbon black immobilized bacteria from step 2 are quickly added and stirred to uniformly distribute the immobilized bacteria in the gel containing CXY-Zn / nLMS to obtain a mixture;

[0023] Step 5: Prepare a calcium chloride solution using ultrapure water and stir until the calcium chloride is completely dissolved to obtain a calcium chloride solution;

[0024] Step 6: Stir the calcium chloride solution in step 5 on a magnetic stirrer, draw the mixture in step 4 with a syringe, and slowly drip the mixture in the syringe into the calcium chloride solution to form embedded beads;

[0025] Step 7: Filter out the embedded beads and rinse them with ultrapure water 2-3 times to remove surface impurity ions to obtain an immobilized bacterial agent.

[0026] Furthermore, in step 1, the Bacillus megaterium is Bacillus megaterium ATCC14581, and the density of the activated bacterial solution is OD 600 =0.6-0.8.

[0027] Furthermore, in step 2, the inoculation amount of the activated bacterial solution is 5-10 v / v%, and the concentration of the carbon black powder is 5-10 w / v%.

[0028] Furthermore, in step 2, the ratio of the activated bacterial solution to the carbon black powder is 1-2:1 v / w%.

[0029] Furthermore, in step 2, the parameters of the shaker are 35° C., 130 r / min, and 24 h; the parameters of the centrifuge are 35° C., 3000 r / min, and 10 min.

[0030] Furthermore, the concentration of the calcium chloride solution in step 5 is 3-5 w / v%.

[0031] Furthermore, in step 6, the rotation speed of the magnetic stirrer is 800-1100 rpm, and the specification of the syringe is a 1 ml syringe.

[0032] The present invention also provides an immobilized bacterial agent capable of degrading toluene, which is prepared by the above preparation method.

[0033] The present invention also provides the use of the above-mentioned immobilized bacterial agent capable of degrading toluene in sewage treatment, improving soil pollution and soil improvement.

[0034] The advantages and positive effects of the novel immobilized bacterial agent capable of degrading toluene and its preparation method and application described in the present invention are:

[0035] 1. The microporous structure of the zero-dimensional nanocarbon black in the present invention (pore size 1-3 nm) can efficiently adsorb bacterial surface proteins and polysaccharides, and the adsorption capacity is significantly improved compared with traditional activated carbon. After acid washing and purification, there is no residual activator (such as ), the survival rate of bacteria will be greatly increased, avoiding the toxic inhibition of traditional activated carbon.

[0036] 2. The zinc ion coordination bonds of CXY-Zn and the layered silicate of nLMS in this invention synergistically crosslink through hydrogen bonds to form an "organic-inorganic hybrid gel" with significantly improved compressive resistance compared to traditional embedding agents. In wastewater treatment, the dual-encapsulated microbial agent can remain intact even at high flow rates, with low fragmentation, significantly outperforming traditional carriers.

[0037] 3. The CXY-Zn in this invention degrades slowly in a pH range of 6-8, maintaining its biological activity and ensuring a long-lasting degradation effect. The ion exchange capacity of nLMS allows for the adsorption of heavy metals, protecting the strain from toxic interference. The inoculant can be recycled, maintaining a high degradation rate for benzene series compounds.

[0038] 4. The present invention adjusts the ratio of CXY-Zn, nLMS and carbon black to regulate the density, mechanical strength and mass transfer rate of the gel beads, so that the immobilized bacterial agent can adapt to different environmental conditions and has a wide adaptability. It can not only complete the initial degradation of pollutants in a relatively short time, but also maintain long-term degradation ability through a sustained release effect.

[0039] 5. The raw materials of the immobilized bacterial agent in the present invention are widely available and inexpensive, and can be produced on a large scale with high economic benefits.

[0040] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a morphology diagram of the immobilized bacterial agent in Example 1 of the present invention;

[0042] Figure 2 This is the effect curve diagram in Example 2 of the present invention;

[0043] Figure 3 This is a design diagram for the simulation experiment in Example 3 of the present invention;

[0044] Figure 4 is the toluene degradation efficiency along the groundwater flow direction in Example 3 of the present invention;

[0045] Figure 5 This is a graph showing the degradation of toluene in the first 48 hours of the well group in Example 4 of the present invention;

[0046] Figure 6 This is a graph showing the degradation rate of toluene as an organic matter in each well of the well group in Example 4 of the present invention;

[0047] Figure 7 The degradation of toluene in soil by each treatment group in Example 5 of the present invention is shown;

[0048] Figure 8 The following are the changes in sucrase activity in the soil of each treatment group in Example 6 of the present invention. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0050] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0051] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not indicated are all commercially available raw materials.

[0052] The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then the steps are carried out in accordance with the general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0053] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0054] Bacillus megaterium.sp was purchased from Mingzhou Biotechnology, with the strain number ATCC 14581. The liquid culture medium was LB liquid medium.

[0055] Example 1

[0056] The preparation method of the new carbon black-Bacillus megaterium immobilized bacterial agent with dual CXY-Zn / nLM embedding is as follows:

[0057] Step 1: Inoculate 60 μL of the stored frozen solution of Bacillus megaterium into the sterilized liquid culture medium, and place the inoculated culture medium in a constant temperature incubator at 35°C for 24 hours to obtain an activated bacterial solution (OD 600 =0.8);

[0058] Step 2: Add 5 v / v% activated bacterial solution to 50 mL of sterilized culture medium, then add 10 w / v% carbon black powder, incubate on a shaker for 24 h, and then centrifuge (shaker parameters are 35°C, 130 r / min, 24 h; centrifuge parameters are 35°C, 3000 r / min, 10 min), and wash the solid with sterile water to obtain carbon black-immobilized bacteria;

[0059] Step 3: Add 4 g of CXY-Zn to a beaker containing 120 mL of deionized water and stir repeatedly with vibration at 90°C until white particles are formed. Heat the transparent solution for 10 minutes, then add 2.8 g of nLMS. Once the solution becomes a milky white, uniform, viscous liquid, stir continuously for 2 minutes, then ultrasonically disperse for 10 minutes. Continue stirring for 20 minutes, maintaining the temperature at 90°C.

[0060] Step 4: When the CXY-Zn / nLMS mixed solution is cooled to 30° C., the carbon black immobilized bacteria in step 2 are quickly added and stirred evenly to ensure that the immobilized bacteria are evenly distributed in the gel containing CXY-Zn / nLMS to obtain a mixture;

[0061] Step 5: Prepare a 4 w / v% calcium chloride solution using ultrapure water and stir until the calcium chloride is completely dissolved;

[0062] Step 6: Place the calcium chloride solution prepared in step 5 on a magnetic stirrer and add a magnet. Start the magnet to rotate at 800 rpm. Use a 1 mL syringe to draw the mixture prepared in step 4 and then slowly drip it into the calcium chloride solution to form embedded spheres.

[0063] Step 7: Filter out the fixed beads and rinse them with ultrapure water 2-3 times to remove surface impurities and obtain the immobilized bacterial agent. Figure 1 shown.

[0064] Example 2

[0065] (1) Proportion optimization:

[0066] Sixteen ratios of carboxymethylated xylan zinc complex (CXY-Zn), nanolithium magnesium silicate (nLMS), and carbon black (CB) were selected to prepare immobilized blank encapsulated beads (without encapsulating Bacillus megaterium). The density, mechanical strength, and mass transfer performance of each bead were determined using the following methods:

[0067] 1) Density: Weigh a certain volume of water using a graduated cylinder, record the initial mass, place 50 small balls of similar size and shape into the cylinder, record the difference in volume before and after, and calculate the density of the balls using the density calculation formula.

[0068] 2) Mechanical strength: Take 5 blank embedded immobilized beads of similar shape and size from each group and place them on a balance. Place a glass slide horizontally on the beads. After zeroing the balance, slowly press the slide until the beads are deformed and cannot return to their original shape. Record the maximum mass M that the beads can withstand. i , the mechanical strength of a single embedded ball is F i =10M i / 10, measure three groups and take the average value, that is, the average mechanical strength of the ball is:

[0069] .

[0070] 3) Mass Transfer Performance: The mass transfer performance of the particles was tested using the color infiltration method. The immobilized beads were immersed in white ink (10 ml of white ink added to 10 ml of water). The beads were removed and sliced ​​every 1 minute, and the penetration of the beads was observed. The slices were removed six times in total, and the number 1-6 was used to qualitatively describe the minute in which the beads were completely soaked. Lower numbers indicate better mass transfer performance. The results are shown in Table 1.

[0071] Table 1 Effects of different embedding agent concentrations on the performance of gel beads

[0072]

[0073] As shown in Table 1, when the concentration of the xylan-zinc complex is too low and the difference between it and the concentration of nano-magnesium lithium silicate is too large, it is difficult to form gel spheres and the spheres that form tend to tail. When the concentration of the xylan-zinc complex is too high, the mass transfer performance of the spheres is poor. When the xylan-zinc complex concentration is controlled at a low concentration, the higher the mass fraction of nano-magnesium lithium silicate and carbon black, the higher the overall mechanical strength of the spheres. When the mass fractions of carbon black and nano-magnesium lithium silicate are high, the mass transfer performance of the spheres is poor. Taking into account the density, mechanical strength, and mass transfer performance of each group of spheres, the optimized proportions of the components were selected as xylan-zinc complex: 3%, 3.5%, and 4%; nano-magnesium lithium silicate: 2%, 2.5%, and 3%; and carbon black: 0.5%, 1%, and 1.5%.

[0074] Combined with the above-selected optimized ratio, three influencing factors were established: xylan zinc complex concentration A (%), nano magnesium lithium silicate concentration B (%), and carbon black concentration C (%). Each factor was set at three levels. The orthogonal experimental factor levels are shown in Table 2.

[0075] Table 2 Factor level table

[0076]

[0077] (2) Orthogonal experimental results:

[0078] Contaminated groundwater was prepared to test the degradation of toluene in the contaminated groundwater at different levels of factors. The main components of the simulated contaminated groundwater were: KCl (0.02w / v%), NaCl (0.02w / v%), MgSO4·7H2O (0.02w / v%), CaCl2 (0.01w / v%), MnSO4·H2O, and FeCl2 in trace amounts. The water was diluted to volume with distilled water and the pH was adjusted to 7.0. At the same time, a certain amount of toluene was added, the addition amount was 8w / v%, and the water was placed in a dark place for later use. Immobilized embedded beads containing Bacillus megaterium were prepared, and the inoculation amount of the strain was controlled to 5v / v%. 150ml of simulated contaminated groundwater was taken into a conical flask, and 30 small beads of uniform size and shape were added to each conical flask. The flask was kept at room temperature for 5 days. Using the toluene degradation rate as the evaluation indicator, 2×9 parallel tests were conducted. The test results are as follows:

[0079] Table 3 Orthogonal experimental design scheme and results

[0080]

[0081] The range R can reflect the size of the difference in test results when the factor level changes. According to the range values ​​in Table 3, it can be seen that the order of priority of each factor is: A (carbon black) > B (xylan zinc complex) > C (nano-lithium magnesium silicate), indicating that the concentration of carbon black has the most significant effect on the degradation ability of the embedded beads, and nano-lithium magnesium silicate and xylan zinc complex have a lower effect on the degradation ability of the embedded beads. From the table, it can be judged that the A level is best at 1, the B level is best at 1, and the C level is best at 2, that is, the optimal conditions are xylan zinc complex 3%, nano-lithium magnesium silicate 2%, and carbon black 1%. The effect curves of each factor are shown in Figure 2 .

[0082] Figure 2 The following is the effect curve of the three factors as their concentration levels change. Figure 2 As shown, the concentration-effect curve of xylan-zinc complex has a downward trend with increasing concentration, the concentration-effect curve of nano-magnesium lithium silicate shows a trend of first decreasing and then increasing, and the carbon black mass fraction curve reaches its maximum value at a mass fraction of 1%. The concentration-effect curve of nano-magnesium lithium silicate is generally stable.

[0083] Example 3

[0084] Soil boxes were used to simulate the hydrogeology and pollution conditions of various contaminated sites, and cyclic degradation simulation tests were conducted to provide a theoretical basis for further practical field application. The required samples were collected in layers using soil sampling holes and injection wells in the survey area, mainly including water-bearing sand layers, top and bottom silt clay, and groundwater samples. Based on the site survey data, the average hydraulic gradient in the survey area was 8‰, and the permeability coefficient of the aquifer sand was 5.0×10 -4 m / s, according to the linear seepage law (Darcy's law) formula:

[0085] .

[0086] V: groundwater velocity; K: permeability coefficient; : hydraulic gradient;

[0087] : Head difference; L: horizontal distance

[0088] The groundwater flow rate in the survey area is about 4×10-6m / s.

[0089] (1) Preparation of simulation box:

[0090] Design the glass box according to the experimental needs and preliminarily design the specifications for The material is organic glass, 10mm thick, bonded with glass glue, with inlet and outlet water reservoirs of 10cm length designed on both sides. The water level height of the inlet and outlet simulates the hydraulic gradient of groundwater. The front view and top view are shown in the figure. Three simulated wells are designed in the box using PVC pipes, 20cm apart. The bottom is inserted into the aquifer floor, the middle opening is wrapped with gauze for water permeability, the upper opening is connected to the atmosphere for the release of bacterial agents, and peristaltic pumps are installed in the water inlet and outlet troughs to simulate the groundwater flow rate V. The design diagram is as follows Figure 3 shown.

[0091] (2) Simulation box filling:

[0092] Contaminated groundwater and soil collected from the field were placed in a soil box according to the field hydrogeological conditions. The bottom was composed of silty clay (0.1m), the middle was composed of aquifer fine sand (0.2m), and the upper part was composed of aeration zone silty clay (or silt) (0.3m). The lithologic medium was weighed and evenly spread in the simulation tank to prevent voids and faults. The contaminated groundwater was then injected into the model aquifer, saturating it from bottom to top. As the water level rose, the changes in the water level were observed until the fine sand layer was fully saturated, maintaining a hydraulic gradient of approximately 8‰.

[0093] (3) Simulation box experiment:

[0094] 200g of immobilized bacteria agent was added to the injection well, and the degradation effect of toluene in different simulation wells was compared and analyzed along the direction of water flow. The results are as follows: Figure 4 shown.

[0095] Depend on Figure 4 The addition of the immobilized bacterial agent to the injection wells significantly removed toluene, with the highest removal efficiency at the injection well and at monitoring well 1, located 20 cm from the injection well. The toluene concentration gradually decreased with the direction of water flow. The toluene degradation rate in the injection well was 80.86%, while that in monitoring well 1 was 68.56%, and that in monitoring well 2 was 56.98%.

[0096] Example 4

[0097] By analyzing the data from the laboratory small-scale simulation box, we screened out microbial agents with obvious degradation effects on the organic pollutant toluene. The microbial agents were added to the groundwater wells in the aquifer using equipment such as beler tubes to conduct on-site pilot experiments. By comparing the test data before and after remediation, the in-situ degradation efficiency of microorganisms was verified, thus forming a set of groundwater organic pollution remediation technology.

[0098] Eight wells were deployed within the toluene contaminated area. A0 and A00 were injection wells, A1 was a control well, A2, A3, and B2 were lateral diffusion monitoring wells, and A4 and A5 were downstream monitoring wells. The degradation target was a 50% degradation rate.

[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 aquifer by the bacterial agent. The results are shown in Table 4 and Figure 5 shown.

[0100] Table 4 Toluene detection data of well group

[0101]

[0102] Through the analysis of the detection data of toluene in the well group, it can be seen that (Table 4 and Figure 5 ), toluene concentrations showed an overall downward trend from 0 to 48 hours. Degradation effects were first evident at dosing wells A0 and A00, with toluene concentrations beginning to decline within the first 24 hours. Downstream monitoring wells A4 and A5 showed significant degradation effects starting at 36 hours. In terms of lateral diffusion, degradation occurred earlier at A2 than at A3, with A3 also showing a significant degradation trend after 36 hours. While degradation was detected at upstream monitoring well A1, a rebound in concentrations followed. Analysis suggests this was primarily due to infiltration and diffusion of contaminated water upstream.

[0103] Seven weeks of monitoring data for organic toluene in the combined well group revealed three downward trends in toluene concentrations in the aquifer. Ultimately, toluene concentrations remained stable in all monitoring wells except control well A1. In the second week, toluene concentrations rebounded. Analysis suggests this was due to a decrease in dissolved oxygen in the groundwater, which weakened microbial activity. The rebound was most pronounced in wells A0 and A00. In the third week, microorganisms adapted to the anaerobic environment and began to proliferate, leading to a subsequent decrease in toluene concentrations. However, once the dissolved oxygen and other nutrients in the aquifer were depleted, toluene concentrations rose again in the fourth week. The project team addressed this by reinvigorating the dosing of microbial agents. Subsequent monitoring revealed a further decrease in toluene concentrations. However, toluene concentrations in control well A1 increased, likely due to infiltration and diffusion of contaminated water from upstream. Toluene concentrations in the other groundwater wells remained stable through 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 the addition of 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 positive degradation rates, demonstrating that the inoculant has a good degradation effect on toluene in the aquifer. Of these, 20% of the monitoring points had degradation rates exceeding 50%, with the highest rate reaching 58.5%, indicating that the toluene contamination in the well group has been remediated.

[0105] Example 5: Improving soil pollution

[0106] Grind the collected toluene-contaminated soil sample and pass it through a 10-mesh sieve. Add 15g of the sieved toluene-contaminated soil sample to a 250mL Erlenmeyer flask and add 190mL of ultrapure water. Then, add 10mL of Bacillus culture solution and 166.8g of embedded bacterial pellets. A blank sample without culture solution was used. The sample was incubated at 18°C ​​for 40 days to obtain a contaminated soil degradation solution.

[0107] The soil degradation solution after cultivation was poured into a separatory funnel, and 10 mL of petroleum ether was added for extraction. The extract was stirred at 5000 r·min. -1 The supernatant was collected by centrifugation for 10 min under the conditions of 40 °C. The upper extract was filtered with anhydrous sodium sulfate, placed in a 25 mL volumetric flask and diluted to the mark with petroleum ether as the sample to be tested.

[0108] Toluene concentration was determined using the headspace / gas chromatography method (HJ 1067-2019, "Determination of Benzene Series in Water"). Headspace sampler heating equilibrium temperature: 60°C; heating equilibrium time: 30 min; injection valve temperature: 100°C; transfer line temperature: 100°C; injection volume: 1.0 ml (quantitative loop). Gas chromatograph inlet temperature: 200°C; detector temperature: 250°C; column temperature program: 40°C (hold for 5 min) to 80°C (hold for 5 min) at a rate of 5°C / min; carrier gas flow rate: 2.0 ml / min; combustion gas flow rate: 30 ml / min; auxiliary gas flow rate: 300 ml / min; makeup gas flow rate: 25 ml / min; split ratio: 10:1. Pre-add 3 g of sodium chloride to each of seven headspace vials. Then, accurately add 10.0 ml, 10.0 ml, 10.0 ml, 9.8 ml, 9.6 ml, 9.2 ml, and 8.8 ml of water, in sequence. Then, using a microsyringe and pipette, add 5.00 µl, 20.0 µl, 50.0 µl, 0.20 ml, 0.40 ml, 0.80 ml, and 1.2 ml of standard working solution, in sequence, to prepare a standard series with target compound concentrations of 0.050 mg / L, 0.200 mg / L, 0.500 mg / L, 2.00 mg / L, 4.00 mg / L, 8.00 mg / L, and 12.0 mg / L, respectively. Immediately seal the headspace vials and gently shake to mix. Samples were injected and analyzed sequentially from low to high concentrations according to instrument conditions. The retention times and responses of the target compounds in the standard series were recorded. A working curve was constructed with the BTEX concentration as the horizontal axis and the corresponding response value as the vertical axis. After establishing the standard curve, pre-add 3 g of sodium chloride to the headspace bottle, add 10.0 ml of the sample to be tested, immediately cover and seal, shake well, and measure. Figure 7 shown.

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

[0110] Depend on Figure 7 As can be seen, in the blank control group, without the addition of bacterial agents, the degradation rate of toluene in the soil reached 6.92%, indicating that the native microorganisms in the soil have a certain effect on toluene degradation. In the experiment, the addition of Bacillus spore liquid and immobilized bacterial agents showed significantly higher toluene degradation capabilities than the control group. The immobilized bacterial agent was more effective than the bacterial liquid alone, with a degradation rate of 65.98%, while the degradation rate of the bacterial liquid alone experimental group was 45.78%. This shows that the immobilized bacterial agent can significantly degrade toluene in the soil and improve soil pollution.

[0111] Example 6 Soil remediation

[0112] In order to study the effect of immobilized embedded beads on enzyme activity during soil remediation, four 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 solution (OD 600 =0.8)); contaminated soil + immobilized bacterial agent (5% w / w toluene + 17.42g immobilized embedded beads), and the dinitrosalicylic acid colorimetric method was used to determine the activity of protease in the soil. By measuring the activity of sucrase under different treatments, the changes in the activity of sucrase in the toluene soil by immobilized bacterial agent were clarified. The results are as follows Figure 8 shown.

[0113] Compared to the control group, the sucrase activity in the uninoculated treatment with toluene showed a downward trend, while the sucrase activity in the treatments with free bacteria and immobilized microbes showed a long-lasting effect. The enzyme activity in the treatments with immobilized microbes was 23.52% to 25.09% higher than that in the control soil between days 14 and 21, and 33.69% to 42.48% higher than that in the soils with bacterial solution. This indicates that immobilized microbes are more effective than free microbes in restoring sucrase activity in soils affected by toluene.

[0114] Therefore, the present invention adopts the above-mentioned immobilized bacterial agent capable of degrading toluene, its preparation method and application, embeds the bacterial strain in the carboxymethylxylan zinc complex / nano-lithium magnesium silicate gel, and gradually releases the bacterial strain through the gel beads to better maintain its biological activity. At the same time, the porous structure of carbon black provides a place for the bacterial strain to attach and grow, prolongs the life of the bacterial strain, and improves 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 rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an immobilized bacterial agent capable of degrading toluene, characterized in that: The following steps are involved: Step 1: inoculating a frozen solution of Bacillus megaterium into a sterilized liquid culture medium, and then culturing the culture medium in a 35° C. constant temperature incubator for 24 hours to obtain an activated bacterial solution; Step 2: Add the activated bacterial solution in step 1 to the sterilized liquid culture medium, add nano-carbon black powder, culture on a shaker for 24 hours, and then centrifuge to obtain a solid, which is then washed with sterile water to obtain carbon black-immobilized bacteria; Step 3: Place CXY-Zn in a beaker and vibrate and stir at 90°C to obtain a transparent solution. Continue heating for 10 minutes, then add nLMS. After the entire solution becomes a milky white, uniform, viscous liquid, continue stirring for 2 minutes, then perform ultrasonic dispersion for 10 minutes, and continue uniform stirring for 20 minutes. Control the temperature at 90°C to obtain a CXY-Zn / nLMS mixed solution. Step 4: When the CXY-Zn / nLMS mixture is cooled to 30°C, the carbon black immobilized bacteria from step 2 are quickly added and stirred to uniformly distribute the immobilized bacteria in the gel containing CXY-Zn / nLMS to obtain a mixture; Step 5: Prepare a calcium chloride solution using ultrapure water and stir until the calcium chloride is completely dissolved to obtain a calcium chloride solution; Step 6: Stir the calcium chloride solution in step 5 on a magnetic stirrer, draw the mixture in step 4 with a syringe, and slowly drip the mixture in the syringe into the calcium chloride solution to form embedded beads; Step 7: Filter out the embedded beads and rinse them with ultrapure water 2-3 times to remove surface impurity ions to obtain an immobilized bacterial agent; The Bacillus megaterium in step 1 is Bacillus megaterium ATCC14581, and the density of the activated bacterial solution is OD 600 =0.6-0.8; The preparation method of the carboxymethylxylan zinc complex CXY-Zn in step 3 is as follows: Carboxymethylated xylan was prepared by a sodium hydroxide-chloroacetic acid chemical method. Carboxymethylated xylan was dissolved in distilled water and stirred to dissolve. ZnSO4·7H2O solution was added and the pH was adjusted to 5.5 with 1 mol / L NaOH. The reaction was carried out at 60°C for 2 h, dialyzed against running water for 48 h, concentrated, and freeze-dried to obtain carboxymethylated xylan zinc complex CXY-Zn.

2. The method for preparing an immobilized bacterial agent capable of degrading toluene according to claim 1, wherein: In step 2, the inoculum amount of the activated bacterial solution is 5-10 v / v%, and the concentration of the carbon black powder is 5-10 w / v%.

3. The method for preparing an immobilized bacterial agent capable of degrading toluene according to claim 1, wherein: The ratio of activated bacterial solution to carbon black powder in step 2 is 1-2:1 v / w.

4. The method for preparing an immobilized bacterial agent capable of degrading toluene according to claim 1, wherein: In step 2, the shaker parameters are 35°C, 130 r / min, and 24 h; the centrifuge parameters are 35°C, 3000 r / min, and 10 min.

5. The method for preparing an immobilized bacterial agent capable of degrading toluene according to claim 1, wherein: The concentration of the calcium chloride solution in step 5 is 3-5 w / v%.

6. The method for preparing an immobilized bacterial agent capable of degrading toluene according to claim 1, wherein: In step 6, the rotation speed of the magnetic stirrer is 800-1100 rpm, and the specification of the syringe is a 1 ml syringe.

7. An immobilized bacterial agent capable of degrading toluene, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the immobilized bacterial agent capable of degrading toluene as claimed in claim 7 in sewage treatment, improving soil pollution and soil improvement.

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