Ammonia nitrogen degrading biological agent, preparation method thereof and application thereof in sewage treatment

Abstract

The present application relates to a kind of ammonia nitrogen degradation biological inoculant and its preparation method and application in wastewater treatment, preparation method includes the following steps: the bacteria suspension formed by mixing freeze-dried powder of inner core bacteria and sodium alginate aqueous solution is dropped into calcium chloride solution, and is solidified and crosslinked into calcium alginate microspheres, and the inner core microspheres are obtained by centrifugal washing, vacuum drying;It is added to composite chitosan solution, stirring adsorption, and the primary coated microspheres are obtained by centrifugal, vacuum drying;Freeze-dried powder of middle layer strain is uniformly mixed with biomass activated carbon powder and zeolite powder by ultrasonic dispersion, then mixed with composite gelatin solution to obtain mixed suspension one, the primary coated microspheres are sprayed with mixed suspension one on the surface, and the secondary coated microspheres are obtained by drying;Freeze-dried powder of outer layer strain is mixed with composite aqueous polyacrylic acid resin solution, and the mixed suspension two is obtained by ultrasonic dispersion, the secondary coated microspheres are sprayed with mixed suspension two on the surface, and the tertiary coated microspheres are obtained by drying.The ammonia nitrogen degradation biological inoculant of the present application realizes the efficient removal of ammonia nitrogen and heavy metal.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to an ammonia nitrogen-degrading biological agent, its preparation method, and its application in wastewater treatment. Background Technology

[0002] With the acceleration of industrialization and urbanization, large amounts of wastewater containing high concentrations of ammonia nitrogen and metal ions are discharged into water bodies, leading to increasingly serious eutrophication. This not only disrupts the balance of aquatic ecosystems but also threatens the safety of human drinking water. Ammonia nitrogen is one of the main pollutants in water bodies. Its excessive presence consumes dissolved oxygen in the water, promotes the proliferation of algae, and produces toxic substances, causing serious harm to aquatic organisms. Currently, the main methods for treating ammonia nitrogen wastewater include physicochemical methods and biological methods. Among them, biological methods have become the mainstream technology for ammonia nitrogen wastewater treatment due to their advantages such as low cost, no secondary pollution, and environmental friendliness.

[0003] Biological denitrification is mainly achieved through nitrification-denitrification processes. Nitrification occurs under aerobic conditions, where ammonia-oxidizing bacteria oxidize ammonia nitrogen into nitrite, which is then further oxidized into nitrate by nitrite-oxidizing bacteria. Denitrification occurs under anoxic conditions, where denitrifying bacteria reduce nitrate into nitrogen gas, which is then removed from the water. However, traditional biological denitrification technologies have the following problems: single-species or simple mixed-species bacteria are difficult to adapt to complex and variable wastewater quality and have poor resistance to shock loads; free bacteria are easily lost, making it difficult to maintain high biomass in the reactor, resulting in unstable treatment efficiency.

[0004] In recent years, microbial immobilization technology, by embedding or adsorbing bacteria into carrier materials, has effectively improved bacterial stability, extended bacterial survival time, and enhanced resistance to environmental shocks, becoming an important means to improve biological nitrogen removal efficiency. However, existing immobilized bacterial agents mostly adopt single embedding or simple adsorption methods, which have problems such as low bacterial load, high mass transfer resistance, and single function, making it difficult to meet the needs of complex wastewater treatment. Therefore, developing a composite ammonia nitrogen degradation biological agent with a multi-layer coating structure, clear functional zoning, and strong synergistic effect is of great significance for improving ammonia nitrogen wastewater treatment efficiency, simplifying treatment processes, and reducing treatment costs. Summary of the Invention

[0005] The purpose of this invention is to provide an ammonia nitrogen-degrading biological agent, its preparation method, and its application in wastewater treatment in order to solve the above-mentioned problems.

[0006] The present invention achieves the above objectives through the following technical solutions: This invention provides a method for preparing an ammonia nitrogen-degrading biological agent, comprising the following steps: (1) Mix the freeze-dried core bacterial cells with sodium alginate aqueous solution in a certain proportion, disperse them by ultrasonication to form a bacterial suspension, drop the bacterial suspension into calcium chloride solution, solidify and crosslink to form calcium alginate microspheres, and obtain core microspheres after centrifugation, washing and vacuum drying; (2) The core microspheres are added to the composite chitosan solution, stirred and adsorbed, and then centrifuged and vacuum dried to obtain the initially coated microspheres; (3) The freeze-dried powder of the middle layer strain is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed on the surface of the microspheres initially coated for secondary coating. After fluidized drying, the microspheres are obtained with secondary coating. (4) The outer layer strain freeze-dried powder and the composite aqueous polyacrylic acid resin solution are mixed in proportion and ultrasonically dispersed to obtain a mixed suspension II. The mixed suspension II is sprayed onto the surface of the secondary coated microspheres for a third coating. After fluidized drying, the three-coated microspheres are obtained. (5) The microspheres coated three times are vacuum dried and sealed in packaging to obtain the ammonia nitrogen degradation biological agent.

[0007] As a further optimization of the present invention, in step (1), the mass concentration of the sodium alginate aqueous solution is 2-8%, and the mass concentration of the calcium chloride solution is 1-3%. The mass ratio of the freeze-dried kernel bacterial cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:(5-10):(10-20); the freeze-dried kernel bacterial cell powder is *Pseudomonas schistosomiasis*. The curing and crosslinking temperature is 15-30℃, and the time is 30-50 min.

[0008] As a further optimization of the present invention, in step (2), the mass ratio of the composite chitosan solution to the core microspheres is 5-10:1; The raw materials for preparing the composite chitosan solution, by weight percentage, include 80-90% of a chitosan aqueous solution with a mass concentration of 2-8%, 1-3% mannitol, 0.5-2% sodium L-glutamate, 2-5% glycerol, and 5-10% starch; The stirring and adsorption temperature is 25-35℃, and the time is 30-60 min.

[0009] As a further optimization of the present invention, in step (3), the method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder with a mass ratio of (1-2):(1-2) are crushed and sieved, and then subjected to heat treatment at 450-650℃ for 2-4 hours, grinding and sieving in a nitrogen atmosphere, and then microwave activated treatment at 600-750℃ for 1-3 hours using K2CO3 solution with a mass concentration of 8-12%; the resulting product is cleaned, dried, ground and sieved to obtain biomass activated carbon powder.

[0010] As a further optimization of the present invention, in step (3), the mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:(0.5-1):(0.5-1.2):(4-8). The freeze-dried powder of the middle layer bacterial cells is denitrifying Roseobacterium; The composite gelatin solution, by weight percentage, comprises 80-90% gelatin aqueous solution with a mass concentration of 3-8%, 1-4% D-sorbitol, 2-8% glycerol, and 5-10% starch.

[0011] As a further optimization of the present invention, in step (4), the mass ratio of the outer layer strain freeze-dried powder to the composite aqueous polyacrylic acid resin solution is 1:(2-5). The freeze-dried powder of the outer layer strain is *Pseudomonas schistosomiasis*. The composite aqueous polyacrylic acid resin solution comprises 80-90% aqueous polyacrylic acid resin emulsion (5-15% by mass), 1-3% D-sorbitol, 2-7% glycerol, 2-8% starch, 2-7% sucrose, and 2-5% polyethylene glycol.

[0012] As a further optimization of the present invention, in steps (3) and (4), the secondary coating and the tertiary coating are both carried out by fluidized bed coating, the secondary coating increases the weight by 20-30%, and the tertiary coating increases the weight by 10-20%; the fluidized bed drying inlet air temperature is 25-40℃, and the fluidized bed drying time is 20-60min.

[0013] The present invention also provides an ammonia nitrogen degrading biological agent, which is prepared by the preparation method described above.

[0014] The present invention also provides an application of the ammonia nitrogen degrading biological agent as described above in wastewater treatment.

[0015] As a further optimization of the present invention, the dosage of the ammonia nitrogen degradation biological agent is ≥20g / L of domestic sewage.

[0016] The beneficial effects of this invention are as follows: 1) The ammonia nitrogen degradation biological agent of the present invention, through the design of a three-layer coating structure, fixes Pseudomonas schlegelii and Roseobacter denitrifying in functional zones. Combined with the adsorption of biomass activated carbon and zeolite, it achieves efficient and simultaneous removal of ammonia nitrogen, total nitrogen, total phosphorus and heavy metals, and has broad application prospects in the field of wastewater treatment. 2) The ammonia nitrogen degradation biological agent of the present invention has excellent purification effect on domestic sewage. The three-layer coating structure can effectively exert the synergistic denitrification effect of each layer of functional strains. The continuous maintenance of the core bacteria, the enhanced degradation of the middle bacteria and the rapid start-up function of the outer bacteria are fully utilized. 3) The biomass activated carbon prepared by combining tobacco stems and rice husks in the ammonia nitrogen degradation biological agent of the present invention is used to load the middle layer of bacteria, which can provide a better habitat for the middle layer strains to achieve the best enhanced degradation effect. Detailed Implementation

[0017] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0018] 1. Core bacterial cell lyophilized powder and outer bacterial strain lyophilized powder: Pseudomonas stutzeri, purchased from Wuhan Ruichen Standard Materials Technology Co., Ltd., product number BNCC139708, effective viable count ≥5 billion / g; 2. Mid-layer lyophilized bacterial powder: Roseobacter denitrificans, purchased from Henan Daizheng Trading Co., Ltd., product number BNCC373339, with an effective viable count ≥ 5 billion CFU / g. 3. Composite chitosan solution: The raw materials, by weight percentage, include 80-90% chitosan aqueous solution with a mass concentration of 2-8%, 1-3% mannitol, 0.5-2% sodium L-glutamate, 2-5% glycerol, and 5-10% starch (preferably, 87% chitosan aqueous solution with a mass concentration of 5%, 2% mannitol, 1.2% sodium L-glutamate, 2.8% glycerol, and 7% starch). 4. Composite gelatin solution: The raw materials for preparation include, by weight percentage, 80-90% of a gelatin aqueous solution with a mass concentration of 3-8%, 1-4% of D-sorbitol, 2-8% of glycerol, and 5-10% of starch (preferably, 84% of a gelatin aqueous solution with a mass concentration of 6%, 3% of D-sorbitol, 5% of glycerol, and 8% of starch). 5. Composite aqueous polyacrylic acid resin solution: The raw materials for preparation, by weight percentage, include 80-90% aqueous polyacrylic acid resin emulsion with a mass concentration of 5-15%, 1-3% D-sorbitol, 2-7% glycerol, 2-8% starch, 2-7% sucrose, and 2-5% polyethylene glycol (preferably, 81.2% aqueous polyacrylic acid resin emulsion with a mass concentration of 8%, 1.8% D-sorbitol, 4.5% glycerol, 5.5% starch, 4% sucrose, and 3% polyethylene glycol). Note: Unless otherwise specified, the preferred schemes were used in the following verification tests; Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products. Example 1

[0019] In this embodiment, a method for preparing an ammonia nitrogen-degrading biological agent is provided, comprising the following steps: Step 1: Mix the freeze-dried core bacteria powder (Pseudomonas schrenckii) with a 5% sodium alginate aqueous solution in a certain proportion, disperse it by ultrasonication to form a bacterial suspension, drop the bacterial suspension into a calcium chloride solution, and solidify and crosslink at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 10:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: The freeze-dried powder of the middle layer strain (Roseobacterium denitrification) is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with the composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed on the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating is 20%. After fluidized drying (inlet air temperature of 32℃, time of 50min), the secondary coated microspheres are obtained. The mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:0.5:0.5:8. The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of 2:1 are crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, the powder is activated by microwave treatment at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Step 4: Mix the freeze-dried outer strain powder (Pseudomonas schrenckii) with the composite aqueous polyacrylic acid resin solution in a certain proportion, and ultrasonically disperse to obtain mixed suspension II. Spray mixed suspension II onto the surface of the secondary coated microspheres for a third coating. The weight gain of the third coating is 20%. After fluidized drying (inlet air temperature is 28℃, time is 35min), the three-coated microspheres are obtained. The mass ratio of the outer layer strain freeze-dried powder to the composite aqueous polyacrylic acid resin solution is 1:2. Step 5: Vacuum dry the triple-coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent. Example 2

[0020] In this embodiment, a method for preparing an ammonia nitrogen-degrading biological agent is provided, comprising the following steps: Step 1: Mix the freeze-dried core bacteria powder (Pseudomonas schrenckii) with a 5% sodium alginate aqueous solution in a certain proportion, disperse it by ultrasonication to form a bacterial suspension, drop the bacterial suspension into a calcium chloride solution, and solidify and crosslink at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 8:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: The freeze-dried powder of the middle layer strain (Roseobacterium denitrification) is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with the composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed on the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating is 26%. After fluidized drying (inlet air temperature of 32℃, time of 50min), the secondary coated microspheres are obtained. The mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:0.5:0.5:6. The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of 2:1 are crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, the powder is activated by microwave treatment at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Step 4: Mix the freeze-dried outer strain powder (Pseudomonas schrenckii) with the composite aqueous polyacrylic acid resin solution in a certain proportion, and disperse it by ultrasonication to obtain a mixed suspension II. Spray the mixed suspension II onto the surface of the secondary coated microspheres for a third coating. The weight gain of the third coating is 14%. After fluidized drying (inlet air temperature is 28℃, time is 35min), the three-coated microspheres are obtained. The mass ratio of the outer layer strain freeze-dried powder to the composite aqueous polyacrylic acid resin solution is 1:3.5. Step 5: Vacuum dry the triple-coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent. Example 3

[0021] In this embodiment, a method for preparing an ammonia nitrogen-degrading biological agent is provided, comprising the following steps: Step 1: Mix the freeze-dried core bacteria powder (Pseudomonas schrenckii) with a 5% sodium alginate aqueous solution in a certain proportion, disperse it by ultrasonication to form a bacterial suspension, drop the bacterial suspension into a calcium chloride solution, and solidify and crosslink at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 10:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: The freeze-dried powder of the middle layer strain (Roseobacterium denitrification) is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with the composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed onto the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating is 30%. After fluidized drying (inlet air temperature of 32℃, time of 50min), the secondary coated microspheres are obtained. The mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:0.5:0.5:4. The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of 2:1 are crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, the powder is activated by microwave treatment at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Step 4: Mix the freeze-dried powder of the outer strain (Pseudomonas schrenckii) with the composite aqueous polyacrylic acid resin solution in a certain proportion, and disperse it by ultrasonication to obtain a mixed suspension II. Spray the mixed suspension II onto the surface of the secondary coated microspheres for a third coating. The weight gain of the third coating is 10%. After fluidized drying (inlet air temperature is 28℃, time is 35min), the three-coated microspheres are obtained. The mass ratio of the outer layer strain freeze-dried powder to the composite aqueous polyacrylic acid resin solution is 1:5. Step 5: Vacuum dry the triple-coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent. Example 4

[0022] In this embodiment, a method for preparing an ammonia nitrogen-degrading bio-initiative is provided. This method is based on Example 2, but with adjustments made to the method for obtaining biomass activated carbon powder. Specifically: The method for obtaining the biomass activated carbon powder is as follows: rice husk powder (i.e., replacing tobacco stem powder with an equal mass of rice husk powder) is crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, it is microwave activated at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Everything else is consistent with Example 2. Example 5

[0023] In this embodiment, a method for preparing an ammonia nitrogen-degrading bio-initiative is provided. This method is based on Example 2, but with adjustments made to the method for obtaining biomass activated carbon powder. Specifically: The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder (i.e., replacing rice husk powder with an equal mass of tobacco stem powder) is crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, it is microwave activated at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Everything else is consistent with Example 2.

[0024] Comparative Example 1 This comparative example provides a method for preparing an ammonia nitrogen-degrading bio-initiative, comprising the following steps: Step 1: Mix the freeze-dried core bacteria powder (Pseudomonas schrenckii) with a 5% sodium alginate aqueous solution in a certain proportion, disperse it by ultrasonication to form a bacterial suspension, drop the bacterial suspension into a calcium chloride solution, and solidify and crosslink at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 8:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: The freeze-dried powder of the middle layer strain (Roseobacterium denitrification) is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with the composite chitosan solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed onto the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating is 26%. After fluidized drying (inlet air temperature is 32℃, time is 50min), the secondary coated microspheres are obtained. The mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite chitosan solution is 1:0.5:0.5:6. The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of 2:1 are crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, the powder is activated by microwave treatment at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Step 4: Mix the freeze-dried outer layer strain powder (Pseudomonas schrenckii) with the composite chitosan solution in a certain proportion, and disperse it by ultrasonication to obtain a mixed suspension II. Spray the mixed suspension II onto the surface of the secondary coated microspheres for a third coating. The weight gain of the third coating is 14%. After fluidized drying (inlet air temperature is 28℃, time is 35min), the three-coated microspheres are obtained. The mass ratio of the outer layer strain freeze-dried powder to the composite chitosan solution is 1:3.5. Step 5: Vacuum dry the triple-coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent.

[0025] Comparative Example 2 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided. This method is based on Example 2, with adjustments made to the three-coating solution. Specifically, the initial coating in step two, the secondary coating in step three, and the tertiary coating in step four all use a composite gelatin solution. Everything else is consistent with Example 2.

[0026] Comparative Example 3 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided. This method is based on Example 2, with adjustments made to the three-coating solution. Specifically, the initial coating in step two, the secondary coating in step three, and the tertiary coating in step four all use a composite waterborne polyacrylic acid resin solution. Everything else is consistent with Example 2.

[0027] Comparative Example 4 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided. Based on Example 2, the three coating solutions are adjusted. Specifically, the initial coating in step two uses a composite aqueous polyacrylic acid resin solution, the secondary coating in step three uses a composite gelatin solution, and the tertiary coating in step four uses a composite chitosan solution. Everything else is consistent with Example 2.

[0028] Comparative Example 5 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided. It is based on Example 2, with adjustments made to the core bacterial cell lyophilized powder, the middle layer strain lyophilized powder, and the outer layer strain lyophilized powder. Specifically, the core bacterial cell lyophilized powder in step one, the middle layer strain lyophilized powder in step three, and the outer layer strain lyophilized powder in step four are all made from Pseudomonas schrenckii. Everything else is consistent with Example 2.

[0029] Comparative Example 6 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided. It is based on Example 2, with adjustments made to the core bacterial cell lyophilized powder, the middle layer strain lyophilized powder, and the outer layer strain lyophilized powder. Specifically, the core bacterial cell lyophilized powder in step one, the middle layer strain lyophilized powder in step three, and the outer layer strain lyophilized powder in step four are all made from denitrifying Roseobacterium. Everything else is consistent with Example 2.

[0030] Comparative Example 7 In this comparative example, a method for preparing an ammonia nitrogen-degrading bio-initiator is provided, which is based on Example 2 with adjustments to the biomass activated carbon powder. Specifically, an equal mass of commercially available coconut shell activated carbon powder is used to replace the biomass activated carbon powder of this application. Everything else is consistent with Example 2.

[0031] Comparative Example 8 In this comparative example, a method for preparing an ammonia nitrogen-degrading bio-initiative is provided, which is based on Example 2 with adjustments made to step three, specifically as follows: The freeze-dried powder of the middle layer bacterial strain (Roseobacterium denitrification) was ultrasonically dispersed and mixed with zeolite powder (the zeolite powder replaced the biomass activated carbon powder by equal mass). Then, it was mixed with a composite gelatin solution in a certain proportion and ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one was sprayed onto the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating was 26%. After fluidized drying (inlet air temperature of 32℃, time of 50 min), the secondary coated microspheres were obtained. The mass ratio of the freeze-dried powder of the middle layer bacterial strain, zeolite powder and composite gelatin solution was 1:1:6. Everything else is consistent with Example 2.

[0032] Comparative Example 9 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided, which omits step four based on Example 2. Specifically: Step 1: The core bacterial lyophilized powder (Pseudomonas schrenckii; dosage is the same as in Example 2), the outer bacterial lyophilized powder (Pseudomonas schrenckii; dosage is the same as in Example 2, but it is combined in Step 1 to maintain the same dosage) are mixed with a 5% sodium alginate aqueous solution in a certain proportion, and ultrasonically dispersed to form a bacterial suspension. The bacterial suspension is then dropped into a calcium chloride solution and cured and crosslinked at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 8:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: The freeze-dried powder of the middle layer strain (Roseobacterium denitrification) is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with the composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed on the surface of the microspheres initially coated for secondary coating. The weight gain of the secondary coating is 26%. After fluidized drying (inlet air temperature of 32℃, time of 50min), the secondary coated microspheres are obtained. The mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:0.5:0.5:6. The method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of 2:1 are crushed and sieved, and then subjected to heat treatment at 560℃ for 2.5h, grinding, and sieving in a nitrogen atmosphere. Then, the powder is activated by microwave treatment at 680℃ for 2.5h using a 10% K2CO3 solution. The resulting product is then washed, dried, ground, and sieved to obtain the biomass activated carbon powder. Step 4: Vacuum dry the secondary coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent.

[0033] Comparative Example 10 In this comparative example, a method for preparing an ammonia nitrogen-degrading biological agent is provided, which omits steps three and four based on Example 2. Specifically: Step 1: The core bacterial lyophilized powder (Pseudomonas schrenckii; dosage is the same as in Example 2), the middle layer bacterial lyophilized powder (Roseobacter denitrifying; dosage is the same as in Example 2, but it is combined in Step 1 to maintain the same dosage), and the outer layer bacterial lyophilized powder (Pseudomonas schrenckii; dosage is the same as in Example 2, but it is combined in Step 1 to maintain the same dosage) are mixed with a 5% sodium alginate aqueous solution in a certain proportion. The mixture is ultrasonically dispersed to form a bacterial suspension. The bacterial suspension is then dropped into a calcium chloride solution and cured and crosslinked at 25°C for 40 min to form calcium alginate microspheres. After centrifugation, washing with deionized water, and vacuum drying, the core microspheres are obtained. The mass ratio of the lyophilized kernel cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:7:15. Step 2: Add the core microspheres to the composite chitosan solution (the mass ratio of the composite chitosan solution to the core microspheres is 8:1), stir and adsorb at 30°C for 45 min, and obtain the initially coated microspheres after centrifugation and vacuum drying. Step 3: Vacuum dry the initially coated microspheres, seal and package them to obtain the ammonia nitrogen degradation biological agent.

[0034] III. Experimental Verification (1) Experimental verification of the wastewater treatment effect of ammonia nitrogen degradation biological agent Experimental water: The experimental water used in this invention is domestic sewage. The influent concentration of domestic sewage is 450 mg / L COD (chemical oxygen demand), 50 mg / L ammonia nitrogen, 80 mg / L TN, 5.5 mg / L TP, and pH is around 7.0. The water quality indicators are mainly tested in accordance with "Methods for Testing and Analysis of Water and Wastewater (Fourth Edition)" and "GB / T5750.1-12-2006 Standard Test Methods for Drinking Water". Total nitrogen (TN): Potassium peroxide oxidation-ultraviolet spectrophotometry (HJ 636-2012); Total phosphorus (TP): Ammonium molybdate spectrophotometric method 《gB 11893-89》; Ammonia nitrogen (NH4) + -N): Nessler's reagent spectrophotometry (HJ 535-2009); COD (Chemical Oxygen Demand): Potassium Dichromate Method (HJ 828-2017) 20g of the ammonia nitrogen degradation biological agents prepared in Examples 1-5 and Comparative Examples 1-10 of this invention were respectively added to 1L of domestic sewage. The mixture was continuously stirred at 23°C for 24 hours, with aeration every 2 hours. The concentrations of COD, organic nitrogen, ammonia nitrogen, total nitrogen, nitrate nitrogen, nitrite nitrogen, and total phosphorus in the effluent were measured, and the removal rates were calculated. The ammonia nitrogen removal rate was calculated as: [(influent ammonia nitrogen concentration - effluent ammonia nitrogen concentration) / influent ammonia nitrogen concentration] × 100%; the total nitrogen (TN) removal rate was calculated as: [(influent total nitrogen concentration - effluent total nitrogen concentration) / influent total nitrogen concentration] × 100%; the total phosphorus (TP) removal rate was calculated as: [(influent total phosphorus concentration - effluent total phosphorus concentration) / influent total phosphorus concentration] × 100%. The experimental data are shown in Table 1. Table 1. Wastewater Treatment Effect Experimental Data Recording Table Experimental conclusion: The ammonia nitrogen degrading biological agent prepared by this invention has excellent purification effect on domestic sewage. The ammonia nitrogen removal rate of Examples 1-5 all reached more than 90%, the total nitrogen (TN) removal rate reached more than 90%, the total phosphorus (TP) removal rate reached more than 90%, and the COD concentration of the effluent was significantly reduced, indicating that the three-layer coating structure of this invention can effectively exert the synergistic denitrification effect of the functional strains in each layer. Among them, Example 2 performed best, with an ammonia nitrogen removal rate of 96.8%, a TN removal rate of 98.2%, a TP removal rate of 98.3%, and an effluent COD of only 1.4 mg / L. This is attributed to the optimized coating ratio and reasonable strain compatibility, which fully utilizes the continuous maintenance of the core bacteria, the enhanced degradation of the middle bacteria, and the rapid start-up function of the outer bacteria. In addition, the biomass activated carbon prepared by combining tobacco stems and rice husks can be used to load the middle layer of bacteria, which can provide a better habitat for the middle layer strains and achieve the best enhanced degradation effect.

[0035] (2) Experimental verification of the removal effect of ammonia nitrogen degradation biological agent on metal ions After adjusting the pH to 4-5 with 50 mL of 80 mg / L copper sulfate solution, lead acetate solution, zinc sulfate solution, and mercuric nitrate solution, add them to the domestic sewage used in experiment (1) at a volume ratio of 1:1. Then add 2 g of ammonia nitrogen degradation biological agent from Examples 1-5 and Comparative Examples 1-10. Place the mixture on a shaker at 25℃ and 240 r / min for 36 h. After standing for 10 min, take 5 mL of the supernatant and filter it through a 0.45 μm filter membrane. Detect the concentration of the metal solution before and after adsorption and calculate the removal rate. The removal rate is calculated as: [(concentration of metal solution before adsorption - concentration of metal solution after adsorption) / concentration of metal solution before adsorption] × 100%. The experimental data are shown in Table 2. Table 2. Experimental Data Recording Table of Metal Ion Removal Effect Experimental results: The ammonia nitrogen degradation bio-agent of the present invention not only has excellent removal effect on nitrogen pollutants, but also exhibits good adsorption and removal capacity for heavy metal ions. Examples 1-5 show good adsorption and removal capacity for Cu. 2+ Pb 2+ Zn 2+ Hg 2+ The removal rates of all samples were relatively high, with Example 2 showing the highest removal rate. The outer layer of water-based polyacrylic acid resin contains abundant carboxyl groups, which have ion exchange and chelation effects on metal ions. The biomass activated carbon (tobacco stems and rice husks in a mass ratio of 2:1) and zeolite in the middle coating layer have strong adsorption capacity and can fix heavy metal ions through physical and chemical adsorption. The amino and hydroxyl groups in chitosan molecules can form stable complexes with heavy metal ions, forming a cumulative effect and achieving a high removal rate of metal ions.

[0036] In summary, this invention, through the design of a three-layer coating structure, functionally immobilizes *Pseudomonas stearothermiae* and *Roseobacter denitrifyingus*, and combines the adsorption effects of biomass activated carbon and zeolite to achieve efficient and simultaneous removal of ammonia nitrogen, total nitrogen, total phosphorus, and heavy metals, which has broad application prospects in the field of wastewater treatment.

[0037] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an ammonia nitrogen-degrading biological agent, characterized in that, Includes the following steps: (1) Mix the freeze-dried core bacterial cells with sodium alginate aqueous solution in a certain proportion, disperse them by ultrasonication to form a bacterial suspension, drop the bacterial suspension into calcium chloride solution, solidify and crosslink to form calcium alginate microspheres, and obtain core microspheres after centrifugation, washing and vacuum drying; (2) The core microspheres are added to the composite chitosan solution, stirred and adsorbed, and then centrifuged and vacuum dried to obtain the initially coated microspheres; (3) The freeze-dried powder of the middle layer strain is ultrasonically dispersed and mixed with biomass activated carbon powder and zeolite powder, and then mixed with composite gelatin solution in proportion. The mixture is ultrasonically dispersed to obtain a mixed suspension one. The mixed suspension one is sprayed on the surface of the microspheres initially coated for secondary coating. After fluidized drying, the microspheres are obtained with secondary coating. (4) The outer layer strain freeze-dried powder and the composite aqueous polyacrylic acid resin solution are mixed in proportion and ultrasonically dispersed to obtain a mixed suspension II. The mixed suspension II is sprayed onto the surface of the secondary coated microspheres for a third coating. After fluidized drying, the three-coated microspheres are obtained. (5) The microspheres coated three times are vacuum dried and sealed in packaging to obtain the ammonia nitrogen degradation biological agent.

2. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 1, characterized in that, In step (1), the mass concentration of the sodium alginate aqueous solution is 2-8%, and the mass concentration of the calcium chloride solution is 1-3%. The mass ratio of the freeze-dried kernel bacterial cell powder, sodium alginate aqueous solution, and calcium chloride solution is 1:(5-10):(10-20); the freeze-dried kernel bacterial cell powder is *Pseudomonas schistosomiasis*. The curing and crosslinking temperature is 15-30℃, and the time is 30-50min.

3. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 1, characterized in that, In step (2), the mass ratio of the composite chitosan solution to the core microspheres is 5-10:1; The raw materials for preparing the composite chitosan solution, by weight percentage, include 80-90% of a chitosan aqueous solution with a mass concentration of 2-8%, 1-3% mannitol, 0.5-2% sodium L-glutamate, 2-5% glycerol, and 5-10% starch; The stirring and adsorption temperature is 25-35℃, and the time is 30-60 min.

4. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 1, characterized in that, In step (3), the method for obtaining the biomass activated carbon powder is as follows: tobacco stem powder and rice husk powder in a mass ratio of (1-2):(1-2) are crushed and sieved, and then subjected to heat treatment at 450-650℃ for 2-4 hours, grinding, and sieving in a nitrogen atmosphere. Then, they are microwave activated at 600-750℃ for 1-3 hours using a K2CO3 solution with a mass concentration of 8-12%. The resulting product is then washed, dried, ground, and sieved to obtain biomass activated carbon powder.

5. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 4, characterized in that, In step (3), the mass ratio of the freeze-dried middle layer bacterial cell powder, biomass activated carbon powder, zeolite powder, and composite gelatin solution is 1:(0.5-1):(0.5-1.2):(4-8). The freeze-dried powder of the middle layer bacterial cells is denitrifying Roseobacterium; The composite gelatin solution, by weight percentage, comprises 80-90% gelatin aqueous solution with a mass concentration of 3-8%, 1-4% D-sorbitol, 2-8% glycerol, and 5-10% starch.

6. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 1, characterized in that, In step (4), the mass ratio of the outer layer strain freeze-dried powder to the composite aqueous polyacrylic acid resin solution is 1:(2-5). The freeze-dried powder of the outer layer strain is *Pseudomonas schistosomiasis*. The composite aqueous polyacrylic acid resin solution comprises 80-90% aqueous polyacrylic acid resin emulsion (5-15% by mass), 1-3% D-sorbitol, 2-7% glycerol, 2-8% starch, 2-7% sucrose, and 2-5% polyethylene glycol.

7. The method for preparing an ammonia nitrogen-degrading biological agent according to claim 1, characterized in that, In steps (3) and (4), the secondary coating and the tertiary coating are both carried out by fluidized bed coating. The secondary coating increases the weight by 20-30%, and the tertiary coating increases the weight by 10-20%. The inlet air temperature for fluidized bed drying is 25-40℃, and the fluidized bed drying time is 20-60min.

8. A biological agent for degrading ammonia nitrogen, characterized in that, It is prepared by any of the preparation methods described in claims 1-7.

9. The application of the ammonia nitrogen degrading biological agent as described in claim 8 in wastewater treatment.

10. The application according to claim 9, characterized in that, The dosage of the ammonia nitrogen degradation biological agent is ≥20g / L of domestic sewage.

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