Composite filler for synergistically increasing oxygen and degrading aquaculture pollutants and preparation method thereof

Abstract

The application discloses a kind of composite fillers of synergic oxygen-increasing degradation aquaculture pollutant and preparation method thereof, belong to aquaculture water treatment technical field.For the problem that the activity of bacteria is inhibited, the functions of oxygen-increasing and degradation cannot be synergized in the existing composite filler, the bacteria are directly contacted with cobalt porphyrin, the bacteria body and the first chitosan, sodium alginate are crosslinked to form bacteria agent microspheres by Bacillus subtilis, lactic acid bacteria, nitrifying bacteria mixed bacteria body;Carboxylated mesoporous silica nanoparticles are first covalently fixed in polyvinyl alcohol-sodium alginate-second chitosan gel network containing amino, and then cobalt porphyrin is loaded in the pore channel of the fixed mesoporous silica nanoparticles, to obtain cobalt porphyrin functionalized gel liquid;The bacteria agent microspheres are immersed in the gel liquid to form core-shell structure microspheres, and are loaded on the carrier to obtain the composite filler.The composite filler can be effectively used for water quality purification of industrialized aquaculture, pond engineering aquaculture, small desktop fish tank and other aquaculture systems.

Description

Technical Field

[0001] This invention relates to the field of aquaculture water treatment technology. More specifically, this invention relates to a composite filler for synergistic oxygenation and degradation of aquaculture pollutants, and its preparation method. Background Technology

[0002] In recent years, my country's aquaculture industry has developed rapidly towards high-density, intensive, and factory-style operations, resulting in significant increases in aquaculture output and economic benefits. However, under high-density farming models, uneaten feed, aquatic animal metabolic waste, dead algae, and other organic matter accumulate in the water, easily leading to a rapid increase in toxic and harmful substances such as ammonia nitrogen, nitrite, and nitrate. This causes eutrophication, insufficient dissolved oxygen, and water quality deterioration, which in turn triggers stress, decreased immunity, and frequent disease outbreaks in aquatic animals, seriously hindering the healthy and sustainable development of aquaculture.

[0003] Currently, aquaculture water purification and water quality control technologies mainly include physical purification, chemical bottom improvement, and microbial remediation. Physical purification often employs sedimentation, filtration, and aeration, which can improve water transparency to some extent, but its effectiveness in removing dissolved pollutants such as ammonia nitrogen and nitrite is limited. Furthermore, aeration equipment has high energy consumption and operating costs. While chemical amendments are fast-acting, they can easily cause secondary pollution, and long-term use can disrupt the aquatic microecological balance, affecting the quality and safety of aquatic products.

[0004] Microbial remediation technology has become the mainstream technology for water quality control in aquaculture due to its advantages such as environmental friendliness, low treatment cost, and no secondary pollution. However, the following problems are commonly encountered in practical applications: commonly used single strains or simple compound bacterial agents have limited functions and low degradation efficiency for complex pollutants, making it difficult to achieve the synergistic degradation of ammonia nitrogen, nitrite, and organic matter simultaneously; key functional bacteria such as nitrifying bacteria and nitrite-oxidizing bacteria are mostly strict aerobic bacteria, and their activity decreases significantly when the aquaculture water is hypoxic or when dissolved oxygen fluctuates greatly, resulting in unstable degradation effects; free bacteria are easily lost after being directly introduced into the water, have difficulty forming biofilms, poor colonization ability, short lifespan, and require frequent replenishment.

[0005] To overcome the aforementioned shortcomings, existing research has attempted to combine biomimetic oxygen-carrying agents with functional bacterial agents. Cobalt porphyrin, with its heme-like structure, possesses excellent biomimetic oxygen-carrying and transport capabilities, enabling continuous and stable oxygenation without external power, thus providing a suitable living environment for aerobic functional microorganisms. In recent years, researchers have attempted to load cobalt porphyrin onto mesoporous silica nanoparticles (MSN), utilizing the nanopores of MSN to encapsulate cobalt porphyrin, thereby delaying its release and reducing toxicity. However, while the particle size of MSN (50-100 nm) is larger than that of conventional gels (20-40 nm), which can to some extent prevent direct contact between cobalt porphyrin and bacterial cells, simply mixing MSN@CoP with the bacterial agent still carries the risk of MSN@CoP adhering to the surface of the bacterial agent and detaching during long-term use, making it difficult to achieve stable physical isolation and long-term synergistic effects.

[0006] Furthermore, cobalt porphyrin itself is a known photosensitive antibacterial agent that generates reactive oxygen species under light, exhibiting strong bactericidal effects, especially against Gram-positive bacteria (such as Bacillus subtilis and lactic acid bacteria). However, directly mixing cobalt porphyrin or its loadings with bacterial cells or simply co-immobilizing them can easily lead to a significant decrease in bacterial activity or even death, thus losing the pollutant degradation function. Therefore, how to utilize the oxygenation function of cobalt porphyrin while avoiding its toxicity to bacterial cells, achieving a synergistic effect of "oxygenation-degradation," is a pressing technical challenge in this field. Summary of the Invention

[0007] One objective of this invention is to address at least the aforementioned problems and provide a composite filler for synergistic oxygenation and degradation of aquaculture pollutants, along with its preparation method. This method involves first covalently fixing carboxylated mesoporous silica nanoparticles to an amino-containing polyvinyl alcohol-sodium alginate-second chitosan gel network via an amidation reaction. Then, cobalt porphyrin is physically loaded into the pores of the fixed mesoporous silica nanoparticles to form an outer shell layer. Simultaneously, a composite bacterial agent of Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria is cross-linked with a first chitosan and sodium alginate to form an inner layer of bacterial agent microspheres. A second cross-linking process constructs core-shell structured microspheres, achieving physical isolation between the cobalt porphyrin and the bacteria. This structure utilizes the biomimetic oxygen-carrying capacity of cobalt porphyrin to promote aerobic bacterial metabolism while avoiding its antibacterial toxicity from harming the bacteria. Furthermore, bacterial metabolites can diffuse outwards and synergistically enhance pollutant degradation with cobalt porphyrin, forming a spatially ordered positive feedback loop. This solves the problems of easy bacterial inactivation, low degradation efficiency, and poor functional coupling in existing technologies.

[0008] To achieve these objectives and other advantages according to the present invention, a method for preparing a composite packing material for synergistic oxygenation and degradation of aquaculture pollutants is provided, comprising the following steps: Step 1: Mix the activated Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria to obtain an activated mixed bacterial agent; mix the activated mixed bacterial agent with the first chitosan and the first sodium alginate at a mass ratio of 100:(0.1-0.3):(1.5-2.5), and add the first calcium chloride solution dropwise to form bacterial agent microspheres; Step 2: Carboxylation surface modification of mesoporous silica nanoparticles to obtain carboxylated mesoporous silica nanoparticles; Step 3: Add polyvinyl alcohol, sodium alginate 2, and chitosan 2 to water at a mass ratio of (3-6):(1.5-2.5):(0.1-0.3), where the mass-to-volume ratio of polyvinyl alcohol to water is (3-6):100 g / mL. After dissolution, a gel precursor solution is obtained. Add carboxylated mesoporous silica nanoparticles, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide to the gel precursor solution and stir until homogeneous to obtain an MSN-functionalized gel solution. Dissolve cobalt porphyrin in anhydrous ethanol and add it to the MSN-functionalized gel solution. Stir for 12-24 h to obtain a cobalt porphyrin-functionalized gel solution. The concentration of cobalt porphyrin in the cobalt porphyrin-functionalized gel solution is 0.5%-2.0% w / v, and the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.05%-0.1%. The concentration of N-hydroxysuccinimide is 0.03%-0.06% w / v; Step 4: Immerse the microspheres of the bacterial agent in cobalt porphyrin functionalized gel solution, remove them after 1-5 min, add the second calcium chloride solution to form core-shell microspheres; load the core-shell microspheres onto a synthetic fiber porous carrier, and dry and solidify to obtain a composite filler.

[0009] Preferably, in step one, the molecular weight of the first chitosan is 10-100 kDa and the degree of deacetylation is ≥90%; the first calcium chloride solution is added dropwise, and the dropping rate is controlled so that the system completes cross-linking within 5-10 minutes.

[0010] Preferably, in step two, the carboxylation surface modification is performed as follows: mesoporous silica nanoparticles are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added, refluxed at 60°C for 6 h, centrifuged and washed, and then reacted with succinic anhydride to introduce carboxyl groups, thereby obtaining carboxylated mesoporous silica nanoparticles. The particle size of the carboxylated mesoporous silica nanoparticles is 50-100 nm, and the pore size is 2-5 nm.

[0011] Preferably, in step three, polyvinyl alcohol, sodium alginate, and chitosan are added to water, heated to 45-60°C and stirred to dissolve, and then cooled to 25-30°C to obtain the gel precursor solution.

[0012] Preferably, the synthetic fiber porous carrier is subjected to plasma surface activation treatment before loading; the power of the plasma surface activation treatment is 100-300 W, and the treatment time is 2-5 min; the drying and curing is carried out under vacuum conditions at 25-35℃ for 4-8 h.

[0013] Preferably, in step one, the preparation of the activated compound microbial agent is as follows: Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria are mixed in a mass ratio of 3:2:4, inoculated into a nutrient solution, and cultured at 28-32℃ for 1.5-3 days to obtain the activated compound microbial agent; The method for preparing the nutrient solution is as follows: 1-2 parts by weight of yeast extract, 2-4 parts by weight of sucrose, 1-2 parts by weight of ammonium sulfate, 0.5-1.5 parts by weight of potassium dihydrogen phosphate, 0.3-0.8 parts by weight of magnesium sulfate, 0.1-0.3 parts by weight of manganese sulfate, 0.1-0.2 parts by weight of calcium chloride, and 0.5-1 parts by weight of B complex vitamins are added to 12-16 parts by weight of water and mixed thoroughly to obtain the nutrient solution.

[0014] Preferably, in step three, the cobalt porphyrin is polyethylene glycol cobalt porphyrin, which is prepared by reacting cobalt porphyrin with methoxy polyethylene glycol succinimide ester in a molar ratio of 1:3-1:5, and has a molecular weight of 2000-5000 Da; after the mesoporous silica nanoparticles are covalently fixed to the gel network, the polyethylene glycol cobalt porphyrin is physically adsorbed and loaded into the pores of the mesoporous silica nanoparticles.

[0015] Preferably, in step three, when loading polyethylene glycol cobalt porphyrin into the pores of mesoporous silica nanoparticles, the solution pH is first adjusted to 7.0-7.5 and stirred for 10-30 min, then the pH is adjusted to 6.0-6.5 and stirred for another 20-60 min, so that the polyethylene glycol segments form a dynamic network structure with pH responsiveness.

[0016] Preferably, in step four, the core-shell microspheres are prepared using a four-layer structure: The microspheres of the bacterial agent prepared in step one are used as the inner layer of the bacterial agent core; Catalase was mixed with a blank gel solution to form the catalase layer in the middle and inner layers, wherein the amount of catalase added was 0.5%-2.0% of the mass of polyvinyl alcohol; Glucose oxidase was mixed with a blank gel solution to form the glucose oxidase layer, which was added at an amount of 0.5%-2.0% of the mass of polyvinyl alcohol. The functionalized gel liquid obtained in step three is used as the outer catalytic layer; First, the inner bacterial agent core is immersed in the catalase layer, then removed and cross-linked once with a second calcium chloride solution to form microspheres coated with the catalase layer. Next, the microspheres are immersed in the glucose oxidase layer, then removed and cross-linked twice with a second calcium chloride solution to form microspheres with the middle layer consisting of the catalase layer and the glucose oxidase layer from the inside out. Finally, the microspheres are immersed in the outer catalytic layer, then removed and cross-linked three times with a second calcium chloride solution to form a four-layer core-shell microsphere with the inner layer being the bacterial agent, the middle layer consisting of the catalase layer and the glucose oxidase layer from the inside out, and the outer layer being a cobalt porphyrin functionalized gel.

[0017] The present invention also provides a method for preparing a composite packing material that synergistically oxygenates and degrades pollutants in aquaculture.

[0018] The present invention has at least the following beneficial effects: First, this invention loads cobalt porphyrin into mesoporous silica nanoparticles. The carboxylated mesoporous silica nanoparticles are first covalently fixed to a polyvinyl alcohol-sodium alginate-second chitosan gel network via an amidation reaction. Then, cobalt porphyrin is physically loaded into the pores of the fixed mesoporous silica nanoparticles to form an outer shell layer. Simultaneously, a composite bacterial agent of Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria is cross-linked with the first chitosan and sodium alginate to form an inner layer of bacterial agent microspheres. Through secondary cross-linking, a core-shell structure microsphere is constructed, achieving physical isolation between cobalt porphyrin and the bacteria. This structure effectively avoids the inhibition of bacterial activity by the antibacterial toxicity of cobalt porphyrin, solving the technical problem in existing technologies where direct contact between cobalt porphyrin and bacteria leads to bacterial inactivation and loss of degradation function. This allows the bacteria to maintain high activity for a long time, significantly extending the service life of the filler.

[0019] Secondly, this invention utilizes the spatial order of the core-shell structure to establish a positive feedback loop of "inner layer bacterial agent metabolism - outer layer cobalt porphyrin catalysis": oxygen generated by the outer layer cobalt porphyrin catalysis can freely diffuse into the inner layer, promoting the metabolism of aerobic bacteria (Bacillus subtilis, nitrifying bacteria); small molecules such as glucose produced by bacterial metabolism diffuse outward to the outer shell, synergistically enhancing the degradation of organic pollutants with cobalt porphyrin. This synergistic mechanism overcomes the limitations of the simple superposition of oxygenation and degradation functions in existing technologies, achieving a synergistic effect of 1+1>2, and significantly improving the removal efficiency of aquaculture pollutants such as ammonia nitrogen, nitrite, COD, and BOD.

[0020] Third, this invention encapsulates cobalt porphyrin in mesoporous silica nanoparticles (50-100 nm in particle size, 2-5 nm in pore size) and covalently fixes the carboxylated mesoporous silica nanoparticles into an amino-containing gel network (provided by a second chitosan) using an amidation reaction, achieving a firm anchoring of the mesoporous silica nanoparticles. Subsequently, cobalt porphyrin is loaded into its pores through physical adsorption, achieving stable loading and controlled release of cobalt porphyrin. This design prevents the migration and loss of cobalt porphyrin during use, ensures the continuous and stable performance of its catalytic oxygenation function, and avoids the potential risks to aquaculture organisms and other beneficial microorganisms in the water from direct exposure of cobalt porphyrin, demonstrating good biocompatibility and environmental safety.

[0021] Fourth, this invention employs a plasma-surface-activated porous synthetic fiber carrier to support core-shell microspheres. The plasma treatment introduces polar groups onto the fiber surface, significantly improving hydrophilicity and the bonding strength with the gel layer. This effectively prevents the filler from detaching due to water erosion during use, enhancing the mechanical stability and service life of the filler. Simultaneously, the microspheres are formed by cross-linking chitosan, sodium alginate, and calcium chloride. The natural antibacterial properties of chitosan inhibit the growth of other bacteria, while the calcium alginate gel network provides a favorable microenvironment for the bacteria, further ensuring their long-term activity.

[0022] Fifth, the preparation method of this invention is feasible, uses readily available raw materials, and requires no complex equipment. The prepared composite filler can be directly placed in the filter box, filter tank, or bottom filter area of ​​the aquaculture system, making it convenient to use without additional energy consumption. It is suitable for various aquaculture scenarios such as factory farming, pond-based aquaculture, and small desktop aquariums, and has broad application prospects. Other advantages, objectives, and features of this invention will be partly apparent from the following description and partly understood by those skilled in the art through research and practice of this invention.

[0023] Sixth, by controlling the gel crosslinking density of the microspheres, this invention forms a dense network that traps mesoporous silica nanoparticles while maintaining high permeability to small molecules. This achieves both physical isolation between cobalt porphyrin and the bacteria and ensures the normal exchange of substances required for bacterial metabolism, providing a structural basis for the realization of the "oxygenation-degradation" synergistic function. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0025] <Example 1> This embodiment provides a method for preparing a composite packing material for synergistic oxygenation and degradation of pollutants in aquaculture. The specific steps are as follows.

[0026] (1) Preparation of activated compound microbial agent Prepare the nutrient solution as follows: Take 1.5 g of yeast extract, 3 g of sucrose, 1.5 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.2 g of manganese sulfate, 0.15 g of calcium chloride, and 0.8 g of B vitamins, add them to 14 g of water, and stir until completely dissolved to obtain the nutrient solution.

[0027] Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria were mixed at a mass ratio of 3:2:4 to obtain a mixed bacterial agent. This mixed bacterial agent was inoculated into the above nutrient solution at a mass ratio of 1:9 and cultured at 30℃ for 2 days to obtain an activated compound bacterial agent. The bacterial cells were collected by centrifugation (8000 r / min, 10 min), washed twice with sterile physiological saline, and set aside for later use.

[0028] (2) Preparation of the core of the microbial agent Take 100 g of the activated composite bacterial cells obtained in step (1), add 0.2 g of chitosan (molecular weight 50 kDa, degree of deacetylation 95%) and 2.0 g of sodium alginate, stir for 5 min until uniform to obtain a mixed slurry. Prepare 2000 mL of a 2% (w / w) calcium chloride solution. Use a peristaltic pump to drop the mixed slurry into the calcium chloride solution at a rate of 5 mL / min. During the droplet addition, gently stir at 100 r / min to ensure uniform dispersion of the microspheres. Control the droplet addition rate so that the entire droplet addition process is completed within 8 min. After the droplet addition is completed, let it stand for 30 min to solidify, discard the supernatant, and wash 3 times with sterile physiological saline (add 1000 mL each time, let stand for 10 min and then discard), to obtain bacterial microspheres with a diameter of about 1 mm, and store at 4℃ for later use.

[0029] (3) Preparation of carboxylated mesoporous silica nanoparticles Mesoporous silica nanoparticles were prepared using the sol-gel method: 1 g of hexadecyltrimethylammonium bromide was dissolved in 480 mL of deionized water, and 3.5 mL of sodium hydroxide solution (2 mol / L) was added. The mixture was heated to 80 °C, and 5 mL of tetraethyl orthosilicate was added while stirring at 300 r / min. Stirring was continued at 300 r / min for 2 h. After the reaction was completed, the mixture was centrifuged (10000 r / min, 20 min), washed three times with anhydrous ethanol (200 mL each time), and calcined at 550 °C for 6 h to remove the template, yielding mesoporous silica nanoparticles. The particle size was determined to be 70 nm, and the pore size was 3.5 nm.

[0030] 10 g of the above-mentioned mesoporous silica nanoparticles were dispersed in 300 mL of anhydrous ethanol, and 5 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 60 °C for 6 h, centrifuged, and washed twice with anhydrous ethanol to obtain aminated mesoporous silica nanoparticles. The aminated mesoporous silica nanoparticles were dispersed in 200 mL of dimethylformamide, and 5 g of succinic anhydride and 3 mL of triethylamine were added. The mixture was stirred at room temperature for 12 h, centrifuged, washed three times with anhydrous ethanol, and dried under vacuum at 35 °C for 6 h to obtain carboxylated mesoporous silica nanoparticles.

[0031] (4) Preparation of cobalt porphyrin functionalized gel Weigh out 40 g of polyvinyl alcohol (degree of hydrolysis 98%, degree of polymerization 1700), 20 g of sodium alginate, and 2 g of chitosan (molecular weight 50 kDa, degree of deacetylation 95%), add them to 1000 mL of deionized water, heat to 55 °C, stir at 300 r / min to dissolve, and cool to 28 °C after complete dissolution to obtain an amino-containing gel precursor solution.

[0032] Take 12 g of carboxylated mesoporous silica nanoparticles, 0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and 0.5 g of N-hydroxysuccinimide (NHS) obtained in step (3), and add them sequentially to the above gel precursor solution. Stir at 500 r / min for 1 h and keep the reaction temperature at 28℃ to allow the carboxyl groups on the surface of the carboxylated mesoporous silica nanoparticles to undergo an amidation reaction with the amino groups in the gel network, thereby covalently fixing the mesoporous silica nanoparticles in the gel network to obtain MSN functionalized gel solution.

[0033] 1.2 g of cobalt porphyrin (tetraphenylcobalt porphyrin, purity ≥98%) was dissolved in 30 mL of anhydrous ethanol and added to the above MSN functionalized gel solution. The mixture was stirred at 200 r / min for 18 h to load the cobalt porphyrin into the pores of the mesoporous silica nanoparticles, thus obtaining the cobalt porphyrin functionalized gel solution.

[0034] (5) Construction of core-shell microspheres Take 100 g of the bacterial microspheres prepared in step (2) and immerse them in 300 g of the cobalt porphyrin functionalized gel prepared in step (4). Stir gently at 50 r / min to disperse the microspheres evenly. After immersion for 3 min, remove the microspheres using a stainless steel filter (0.5 mm pore size) and drain off the excess gel. Prepare 1000 mL of a 2% (w / w) second calcium chloride solution. Add the microspheres coated with the gel evenly to the second calcium chloride solution and stir gently at 100 r / min for 5 min. Let it stand for 30 min to solidify. Pour off the supernatant and wash twice with sterile physiological saline (add 500 mL each time, stir gently for 2 min, let stand for 10 min, and then pour off). This yields core-shell microspheres with a bacterial agent inner layer and a cobalt porphyrin functionalized gel outer layer.

[0035] It should be noted that the microspheres of the bacterial agent in this invention are formed by cross-linking calcium alginate and chitosan. By controlling the cross-linking conditions of the first sodium alginate and the first calcium chloride, a gel network with a suitable cross-linking density can be obtained. This gel network has good permeability to small molecules (such as oxygen, glucose, and hydrogen peroxide) to meet the metabolic needs of the bacteria. At the same time, since the particle size (50-100 nm) of the mesoporous silica nanoparticles is larger than the pore size of the gel network, the mesoporous silica nanoparticles cannot enter the interior of the bacterial agent microspheres through the gel channels, thereby confining the cobalt porphyrin in the outer shell layer and achieving physical isolation between the cobalt porphyrin and the bacteria. This design effectively avoids the inhibition of bacterial activity by the antibacterial toxicity of cobalt porphyrin.

[0036] (6) Load and curing PP cotton (polypropylene fiber cotton, 2 cm thick, 80% porosity) was used as a porous synthetic fiber carrier. The carrier was cut into blocks 10 cm long, 10 cm wide, and 2 cm thick, placed in a plasma treatment instrument, air was introduced, the treatment power was set to 200 W, and the treatment time was 3 min. Polar groups such as hydroxyl and carboxyl groups were introduced into the surface of the treated carrier.

[0037] Take 200 g of the core-shell microspheres obtained in step (5) and evenly spread them on the entire upper surface of 100 g of the treated PP cotton carrier. Gently press the microspheres to embed them into the fiber pores of the carrier. Place the loaded carrier in a vacuum drying oven and dry it at 30°C and 0.07 MPa for 6 h to obtain the composite filler for synergistic oxygenation and degradation of aquaculture pollutants.

[0038] <Example 2> This embodiment is basically the same as Embodiment 1, except that: in this embodiment, polyethylene glycol-modified cobalt porphyrin is used instead of ordinary cobalt porphyrin, and a pH gradient pre-assembly process is introduced in the preparation of the shell gel liquid. The specific steps are as follows.

[0039] (1) Preparation of activated compound microbial agent The same as step (1) in Example 1.

[0040] (2) Preparation of the core of the microbial agent The same as step (2) in Example 1.

[0041] (3) Preparation of polyethylene glycolated cobalt porphyrin 1 g of cobalt porphyrin (tetraphenylcobalt porphyrin, purity ≥98%) and 4 g of methoxy polyethylene glycol succinimide ester (molecular weight 3500 Da) were dissolved in anhydrous dimethyl sulfoxide at a molar ratio of 1:4. The reaction was carried out at 28°C under light-protected conditions for 18 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 1000 Da) and dialyzed in deionized water for 48 h, with the water changed every 8 h. After dialysis, the solution was freeze-dried to obtain polyethylene glycol-modified cobalt porphyrin with a molecular weight of approximately 3500 Da.

[0042] (4) Preparation of carboxylated mesoporous silica nanoparticles The same as step (3) in Example 1.

[0043] (5) Preparation of cobalt porphyrin functionalized gel Weigh 40 g of polyvinyl alcohol, 20 g of sodium alginate, and 2 g of chitosan, add them to 1000 mL of deionized water, heat to 55°C and stir to dissolve, then cool to 28°C to obtain an amino-containing gel precursor solution. Take 12 g of carboxylated mesoporous silica nanoparticles, 0.8 g of EDC, and 0.5 g of NHS obtained in step (4), add them sequentially to the gel precursor solution, and stir at 500 r / min for 1 h to allow the carboxyl groups on the surface of the carboxylated mesoporous silica nanoparticles to undergo an amidation reaction with the amino groups in the gel network, thereby covalently fixing the mesoporous silica nanoparticles in the gel network to obtain the MSN functionalized gel solution.

[0044] Dissolve 1.2 g of polyethylene glycol-modified cobalt porphyrin obtained in step (3) in 30 mL of anhydrous ethanol, add it to the above MSN functionalized gel solution, first adjust the pH of the solution to 7.2, stir at 200 r / min for 20 min, then adjust the pH to 6.3, and continue stirring for 40 min, so that the polyethylene glycol-modified cobalt porphyrin is loaded in the pores of mesoporous silica nanoparticles and forms a dynamic network structure with pH response, thus obtaining the cobalt porphyrin functionalized gel solution.

[0045] (6) Construction of core-shell microspheres The same as step (5) in Example 1.

[0046] (7) Load and curing The same as step (6) in Example 1.

[0047] <Example 3> This embodiment is basically the same as Embodiment 2, except that: this embodiment adopts a four-layer core-shell structure, and introduces a double enzyme layer (inner catalase layer and outer glucose oxidase layer) between the inner bacterial agent core and the outer catalytic layer. The specific steps are as follows.

[0048] (1) Preparation of activated compound microbial agent The same as step (1) in Example 2.

[0049] (2) Preparation of the inoculum core (inner inoculum core) Same as step (2) in Example 2, microspheres of bacterial agent were prepared as the inner layer of bacterial agent core.

[0050] (3) Preparation of polyethylene glycolated cobalt porphyrin Polyethylene glycol cobalt porphyrin was prepared according to step (3) of Example 2.

[0051] (4) Preparation of carboxylated mesoporous silica nanoparticles The same as step (4) in Example 2.

[0052] (5) Preparation of cobalt porphyrin functionalized gel Cobalt porphyrin functionalized gel was prepared according to step (5) of Example 2.

[0053] Then, prepare a blank gel solution: weigh 40 g of polyvinyl alcohol, 20 g of sodium alginate, and 2 g of chitosan (molecular weight 50 kDa, degree of deacetylation 95%), add them to 1000 mL of deionized water, heat to 55°C and stir to dissolve, cool to 28°C to obtain a blank gel solution (without cobalt porphyrin and EDC / NHS). (6) Prepare the inner enzyme layer gel solution. Take 100 g of the blank gel solution obtained in step (5), add 0.2 g of catalase, stir evenly, and obtain the inner enzyme layer gel solution.

[0054] (7) Preparation of outer enzyme layer gel solution Take 100 g of the blank gel solution obtained in step (5), add 0.8 g of glucose oxidase, stir evenly, and obtain the outer enzyme layer gel solution.

[0055] (8) Construction of four-layer core-shell microspheres Take 100 g of the bacterial microspheres prepared in step (2) and immerse them in 200 g of the inner enzyme layer gel prepared in step (6). Stir gently at 50 r / min to disperse the microspheres evenly. After immersion for 3 min, remove the microspheres and add a 2% (w / w) second calcium chloride solution for cross-linking once. Let stand and solidify for 20 min to form microspheres coated with the inner enzyme layer. Immerse the microspheres in 200 g of the outer enzyme layer gel prepared in step (7). After immersion for 3 min, remove the microspheres and add a 2% (w / w) second calcium chloride solution for cross-linking twice. Let stand and solidify for 20 min to form microspheres with a middle layer consisting of a catalase layer and a glucose oxidase layer from the inside out. Immerse the microspheres in 200 g of the remaining cobalt porphyrin functionalized gel prepared in step (5). After immersion for 3 min, remove the microspheres and add a 2% (w / w) second calcium chloride solution for cross-linking three times. Let stand and solidify for 30 min. The microspheres were washed twice with sterile saline to obtain a four-layered core-shell microsphere with an inner layer of bacterial agent, a middle layer consisting of catalase and glucose oxidase layers from the inside out, and an outer layer of cobalt porphyrin functionalized gel.

[0056] (9) Load and curing The same as step (7) in Example 2.

[0057] <Comparative Example 1> The difference between this comparative example and Example 1 is that: in step (1), the preparation of the activated compound bacterial agent is not carried out, and in step (2), the first chitosan, the first sodium alginate, and an equal amount of sterile physiological saline are directly mixed to prepare blank microspheres. The remaining steps are the same as in Example 1.

[0058] <Comparative Example 2> The difference between this comparative example and Example 1 is that in step (4), only the carboxylated mesoporous silica nanoparticles are covalently fixed in the gel network to obtain the MSN functionalized gel solution. However, the subsequent cobalt porphyrin loading operation of "dissolving cobalt porphyrin in anhydrous ethanol, adding it to the MSN functionalized gel solution, and stirring to load the cobalt porphyrin into the pores of the mesoporous silica nanoparticles" is not performed, and the functionalized gel solution without cobalt porphyrin is directly obtained. The remaining steps are the same as in Example 1.

[0059] <Comparative Example 3> The difference between this comparative example and Example 1 is that: in step (4), polyvinyl alcohol, second sodium alginate, and second chitosan are not added; instead, carboxylated mesoporous silica nanoparticles are mixed with EDC and NHS and directly sprayed onto the carrier. The remaining steps are the same as in Example 1.

[0060] <Comparative Example 4> The difference between this comparative example and Example 1 is that the cobalt porphyrin functionalized gel in step (4) does not contain the second chitosan and polyvinyl alcohol; only the second sodium alginate and calcium chloride are used. That is, only the second sodium alginate is added in step (4), and polyvinyl alcohol and the second chitosan are not added. The remaining steps are the same as in Example 1.

[0061] <Comparative Example 5> The difference between this comparative example and Example 1 is that Bacillus subtilis was not added to the compound bacterial agent. That is, in step (1), only lactic acid bacteria and nitrifying bacteria were mixed and activated at a mass ratio of 2:4. The remaining steps are the same as in Example 1.

[0062] <Comparative Example 6> The difference between this comparative example and Example 1 is that no lactic acid bacteria were added to the compound microbial agent. That is, in step (1), only Bacillus subtilis and nitrifying bacteria were mixed and activated at a mass ratio of 3:4. The remaining steps are the same as in Example 1.

[0063] <Comparative Example 7> The difference between this comparative example and Example 1 is that nitrifying bacteria were not added to the compound microbial agent. That is, in step (1), only Bacillus subtilis and lactic acid bacteria were mixed and activated at a mass ratio of 3:2. The remaining steps are the same as in Example 1.

[0064] <Comparative Example 8> The difference between this comparative example and Example 1 is that cobalt porphyrin was not loaded into mesoporous silica nanoparticles. In step (4), a blank gel solution (polyvinyl alcohol, second sodium alginate, second chitosan, without EDC / NHS and MSN) was prepared. Cobalt porphyrin was directly dissolved in the blank gel solution and stirred evenly to obtain a cobalt porphyrin gel solution without MSN. The remaining steps are the same as in Example 1.

[0065] <Comparative Example 9> The difference between this comparative example and Example 1 is that the carboxylated mesoporous silica nanoparticles were not covalently fixed in the gel network through an amidation reaction; that is, EDC and NHS were not added. The carboxylated mesoporous silica nanoparticles were directly physically mixed with the gel precursor liquid in step (4), and the subsequent cobalt porphyrin loading operation was the same as in Example 1. The remaining steps were the same as in Example 1.

[0066] <Comparative Example 10> The difference between this comparative example and Example 1 is that a core-shell structure was not constructed. Instead, the microbeads prepared in step (2) were simply mixed with the cobalt porphyrin functionalized gel prepared in step (4) and then directly loaded onto PP cotton. The remaining steps were the same as in Example 1.

[0067] <Comparative Example 11> The difference between this comparative example and Example 1 is that no first chitosan was added to the microbeads; only sodium alginate was used to crosslink the microbeads. The remaining steps are the same as in Example 1.

[0068] <Comparative Example 12> The difference between this comparative example and Example 1 is that the PP cotton carrier was not subjected to plasma surface activation treatment. The remaining steps are the same as in Example 1.

[0069] <Performance Index Testing Methods> The composite packing material to be tested was placed in the filter tank of the simulated aquaculture water body at a packing volume to water volume ratio of 8%. The circulating water pump was turned on to ensure full contact between the packing material and the water body. Aeration was started normally, and samples were taken to measure various indicators after 30 days of continuous operation. The initial conditions of the simulated aquaculture water body were: dissolved oxygen (DO) 3.5 mg / L, chemical oxygen demand (COD) 120 mg / L, ammonia nitrogen 5.0 mg / L, nitrite 2.0 mg / L, and water temperature 25℃. Each experiment was repeated three times, and the average value was taken.

[0070] The specific testing methods for each indicator are as follows: 1. DO after 30 days of operation: The dissolved oxygen content of the water body was determined according to the national standard "Determination of Dissolved Oxygen in Water - Iodometric Method" (GB / T 7489-1987) after 30 days of operation.

[0071] 2. Oxygen transfer efficiency: The total oxygen transfer coefficient and standard oxygen transfer efficiency were calculated with reference to the industry standard "Determination of oxygen mass transfer performance of microporous aerator in clear water" (CJ / T 475-2015), and corrected to the standard conditions of 20℃ and 101.3 kPa.

[0072] 3. COD removal rate: The initial and 30-day COD levels were determined according to the industry standard "Determination of Chemical Oxygen Demand in Water - Dichromate Method" (HJ 828-2017), and the removal rate was calculated.

[0073] 4. Ammonia nitrogen removal rate: The ammonia nitrogen content at the initial stage and after 30 days of operation was determined according to the industry standard "Determination of Ammonia Nitrogen in Water - Nessler's Reagent Spectrophotometric Method" (HJ 535-2009), and the removal rate was calculated.

[0074] 5. Nitrite removal rate: The nitrite nitrogen content was determined according to the national standard "Determination of Nitrite Nitrogen in Water - Spectrophotometric Method" (GB / T7493-1987) at the beginning and after 30 days of operation, and the removal rate was calculated.

[0075] 6. Cell survival rate: The plate colony count method was used. Microspheres of the bacterial agent in the packing material after 30 days of operation were ground, diluted, and spread on the corresponding culture medium. The number of viable cells (CFU / g) was counted, and the ratio of the number of viable cells to the initial number of viable cells was the cell survival rate.

[0076] 7. Microsphere breakage rate: After continuous operation in simulated water flow (200 L / h) for 30 days, the mass percentage of broken microspheres was determined by sieving (0.5 mm aperture).

[0077] 8. Cobalt porphyrin retention rate: The cobalt porphyrin content in the packing material after 30 days of operation was determined by ultraviolet-visible spectrophotometry (λ=420 nm) and the ratio to the initial content.

[0078] All examples and comparative examples were sampled and measured after running in the same simulated aquaculture water (initial DO 3.5 mg / L, COD 120 mg / L, ammonia nitrogen 5.0 mg / L, nitrite 2.0 mg / L, water temperature 25℃) for 30 days. Each experiment was repeated three times and the average value was taken.

[0079] <Performance Index Test Results> 1. The DO, oxygen transfer efficiency, COD removal rate, ammonia nitrogen removal rate, nitrite removal rate, and bacterial survival rate of Example 1 and Comparative Examples 1-7 were measured after 30 days of operation. The results are shown in Table 1.

[0080] Table 1 As shown in Table 1, Example 1, possessing a composite bacterial agent, cobalt porphyrin, and a complete binder system, exhibits the best performance across all indicators. Comparative Example 1, lacking the composite bacterial agent, while achieving comparable oxygenation to Example 1, shows a significant decrease in pollutant removal rate, demonstrating that the bacterial agent is the core functional unit for pollutant degradation, and its metabolic activity directly determines the conversion efficiency of ammonia nitrogen, nitrite, and organic matter. Comparative Example 2, lacking cobalt porphyrin, shows a significant reduction in dissolved oxygen and oxygen transfer efficiency. The aerobic bacteria's metabolism is hindered due to hypoxia, leading to a simultaneous decrease in pollutant removal rate, proving that cobalt porphyrin-based biomimetic oxygenation is crucial for maintaining aerobic bacterial activity. Comparative Example 3, lacking the binder, suffers from unstable core-shell structure formation and decreased performance across all aspects, demonstrating that the binder is a necessary component for constructing a stable filler structure. Comparative Example 4, lacking the second chitosan and polyvinyl alcohol, shows a decrease in bacterial survival rate and poorer long-term stability, indicating that the amino groups provided by the second chitosan are key to achieving MSN covalent fixation, while polyvinyl alcohol enhances the gel network strength. Their synergistic effect ensures the stability of the outer shell structure, thereby providing effective protection for the bacteria. Comparative examples 5-7, which lacked a single bacterial species, showed a significant decrease in the removal rate of the corresponding pollutants, demonstrating that Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria form a collaborative microbial community ecosystem during degradation: Bacillus subtilis secretes extracellular enzymes to decompose organic matter, lactic acid bacteria produce acid to regulate the microenvironment and inhibit other bacteria, and nitrifying bacteria are responsible for the conversion of ammonia nitrogen and nitrite. All three are indispensable.

[0081] 2. The removal rates of DO and ammonia nitrogen, bacterial survival rate, microsphere breakage rate, and cobalt porphyrin retention rate of Example 1 and Comparative Examples 8-12 were measured after 30 days of operation. The results are shown in Table 2.

[0082] Table 2 Table 2 shows that in Comparative Example 8, cobalt porphyrin was not encapsulated in mesoporous silica, and the cobalt porphyrin molecules were in direct contact with the bacteria. Its photosensitive antibacterial toxicity led to a significant decrease in bacterial survival and severely impaired pollutant degradation function. Simultaneously, free cobalt porphyrin was easily lost, with extremely low retention after 30 days, resulting in a decline in oxygenation function. This demonstrates that mesoporous silica encapsulation is a key technical feature for achieving bacterial protection and stable oxygenation. Its nanopores can both load cobalt porphyrin and prevent it from directly contacting the bacteria, forming a physical barrier. In Comparative Example 9, without amidation, carboxylated mesoporous silica nanoparticles were covalently immobilized in the gel network, resulting in a significantly reduced cobalt porphyrin retention rate and a marked decrease in dissolved oxygen after 30 days. This indicates that physically embedded carboxylated mesoporous silica nanoparticles are easily detached under water erosion, while amidation allows the carboxyl groups on the MSN surface to form covalent bonds with the amino groups in the gel network, firmly anchoring the MSN in the gel framework. This is a necessary measure to ensure the durability of the filler. Comparative Example 10 did not construct a core-shell structure. The bacterial agent and cobalt porphyrin functionalized gel were randomly mixed, and the bacteria were directly exposed to the cobalt porphyrin environment. The survival rate decreased significantly, and the microsphere breakage rate increased. After long-term operation, the structure became loose and the function deteriorated. This demonstrates that the core-shell spatial isolation structure is the fundamental means to solve the core contradiction between "cobalt porphyrin antibacterial toxicity" and "maintaining bacterial activity." It achieves functional synergy of "external oxygen production and internal degradation" through physical partitioning. It should be noted that in Comparative Example 10, although cobalt porphyrin was encapsulated in mesoporous silica nanoparticles, a core-shell structure was not constructed. MSN@CoP was simply mixed and attached to the surface of the bacterial agent microspheres. It was easy to detach during long-term operation, and the MSN@CoP attached to the surface could still contact the surface bacteria through the pore interface. Therefore, the bacterial survival rate was only 45.3%. Example 1 demonstrates how an outer gel layer securely fixes MSN@CoP to the outside of microspheres, forming a stable physical isolation layer. This effectively prevents MSN@CoP from detaching and directly contacting the bacteria, significantly increasing the bacterial survival rate to 85.6%, approaching that of a cobalt-free porphyrin state (Comparative Example 2: 84.2%). This comparison fully demonstrates that the core-shell structure provides further physical isolation protection on top of MSN encapsulation, which is key to achieving a high bacterial survival rate. In Comparative Example 11, the microspheres containing the bacterial agent without the addition of chitosan showed decreased mechanical strength, weakened bacterial protection, and reduced survival rate after long-term operation. This demonstrates that the composite gel network formed by chitosan and sodium alginate significantly enhances the mechanical stability of the microspheres. Simultaneously, the antibacterial activity of chitosan itself inhibits the colonization of other bacteria, providing a more stable microbial niche for functional bacteria. Comparative Example 12, where the PP cotton carrier was not plasma activated, showed weak bonding between the microspheres and the carrier, low microsphere retention rate after 30 days, loss of effective components, and performance degradation. This demonstrates that plasma treatment introduces polar groups such as hydroxyl and carboxyl groups onto the fiber surface, significantly improving hydrophilicity and chemical affinity with the gel layer, which is a necessary step to enhance load-bearing strength.

[0083] 3. The DO of the normal pH (7.0) after 30 days of operation and the acidic pH (5.8) after 30 days of operation in Examples 1-2 were measured. The results are shown in Table 3.

[0084] Table 3 Table 3 shows that in water with normal pH, the dissolved oxygen in Example 2 was slightly better than that in Example 1 after 30 days. This is because the hydrophilic segments of polyethylene glycol-modified cobalt porphyrin can improve its dispersion uniformity in the gel network and avoid cobalt porphyrin agglomeration and deactivation. At the same time, the pH gradient pre-assembly process causes the polyethylene glycol segments to form pre-loaded stress during the assembly process, giving the dynamic network a higher structural regularity.

[0085] In acidic pH water, dissolved oxygen levels remained high in Example 2, while significantly decreased in Example 1. This is because the polyethylene glycol segments in Example 2 exhibit pH responsiveness. Under acidic conditions (simulating bacterial metabolic acid production or acidification in the later stages of aquaculture), the segments contract, causing some cobalt porphyrin to be "released" from the gel network, alleviating the inhibition of catalytic activity by local acid accumulation. When the pH rises again, the segments re-extend, and the cobalt porphyrin re-anchors. This reversible dynamic balance enables intelligent regulation of the oxygenation function, significantly improving the adaptability of the packing material in complex water quality environments.

[0086] 4. The ammonia nitrogen removal rate and bacterial survival rate after 30 days of operation in Examples 2-3 were determined. The results are shown in Table 4.

[0087] Table 4 As shown in Table 4, Example 2 has a double-layer core-shell structure (inner layer of bacterial agent + outer layer of cobalt porphyrin functionalized gel), while Example 3 introduces a double-layer middle enzyme layer (the middle layer consists of a catalase layer and a glucose oxidase layer from the inside out), constructing a four-layer gradient structure of "inner layer bacterial agent - inner layer enzyme layer - outer layer enzyme layer - outer layer cobalt porphyrin". The ammonia nitrogen removal rate and cell survival rate of Example 3 are significantly higher than those of Example 2, demonstrating that the introduction of the double-layer enzyme layer brings about a significant performance improvement.

[0088] Its mechanism of action is as follows: glucose produced by the bacterial agent's metabolism diffuses outward to the outer enzyme layer, where it is catalyzed by glucose oxidase to produce gluconic acid and hydrogen peroxide. Gluconic acid diffuses inward, triggering the contraction of polyethylene glycol-modified cobalt porphyrin chains, enhancing mass transfer efficiency; hydrogen peroxide mainly diffuses outward to form highly reactive oxide species with cobalt porphyrin, synergistically enhancing degradation. A small amount of hydrogen peroxide diffuses inward and is decomposed into water and oxygen by catalase in the inner enzyme layer before reaching the bacterial cells, effectively protecting the bacterial cells from oxidative damage.

[0089] In the four-layer structure of this invention, the catalase layer is adjacent to the bacterial agent core, enabling it to decompose hydrogen peroxide as soon as it diffuses inward, providing the most effective protection for the bacteria. The glucose oxidase layer is located in the middle, converting glucose produced by bacterial metabolism into hydrogen peroxide, which mainly diffuses outward and degrades in synergy with cobalt porphyrin. The cobalt porphyrin layer is located on the outermost layer, providing both oxygenation catalysis and synergistic enhancement of degradation with hydrogen peroxide. The three functional layers in the four-layer structure are spatially ordered and have clear division of labor, achieving a unity of "synergistic hydrogen peroxide production" and "bacterial cell protection".

[0090] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing a composite packing material for synergistic oxygenation and degradation of pollutants in aquaculture, characterized in that, Includes the following steps: Step 1: Mix the activated Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria to obtain an activated mixed bacterial agent; mix the activated mixed bacterial agent with the first chitosan and the first sodium alginate at a mass ratio of 100:(0.1-0.3):(1.5-2.5), and add the first calcium chloride solution dropwise to form bacterial agent microspheres; Step 2: Carboxylation surface modification of mesoporous silica nanoparticles to obtain carboxylated mesoporous silica nanoparticles; Step 3: Add polyvinyl alcohol, sodium alginate 2, and chitosan 2 to water at a mass ratio of (3-6):(1.5-2.5):(0.1-0.3), with a polyvinyl alcohol to water mass-to-volume ratio of (3-6):100 g / mL. After dissolution, a gel precursor solution is obtained. Add carboxylated mesoporous silica nanoparticles, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide to the gel precursor solution and stir until homogeneous to obtain an MSN-functionalized gel solution. Dissolve cobalt porphyrin in anhydrous ethanol and add it to the MSN-functionalized gel solution. Stir for 12-24 h to obtain a cobalt porphyrin-functionalized gel solution. The concentration of cobalt porphyrin in the cobalt porphyrin-functionalized gel solution is 0.5%-2.0% w / v, and the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.05%-0.1%. The concentration of N-hydroxysuccinimide is 0.03%-0.06% w / v; Step 4: Immerse the microspheres of the bacterial agent in cobalt porphyrin functionalized gel solution, remove them after 1-5 min, add the second calcium chloride solution to form core-shell microspheres; load the core-shell microspheres onto a synthetic fiber porous carrier, and dry and solidify to obtain a composite filler.

2. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step one, the molecular weight of the first chitosan is 10-100 kDa and the degree of deacetylation is ≥90%; the first calcium chloride solution is added dropwise, and the dropping rate is controlled so that the system completes cross-linking within 5-10 minutes.

3. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step two, the carboxylation surface modification is as follows: mesoporous silica nanoparticles are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added, refluxed at 60°C for 6 h, centrifuged and washed, and then reacted with succinic anhydride to introduce carboxyl groups, thereby obtaining carboxylated mesoporous silica nanoparticles. The particle size of the carboxylated mesoporous silica nanoparticles is 50-100 nm, and the pore size is 2-5 nm.

4. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step three, polyvinyl alcohol, sodium alginate, and chitosan are added to water, heated to 45-60°C and stirred to dissolve, and then cooled to 25-30°C to obtain the gel precursor solution.

5. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, Before loading, the synthetic fiber porous carrier undergoes plasma surface activation treatment; the power of the plasma surface activation treatment is 100-300 W, and the treatment time is 2-5 min; the drying and curing is carried out under vacuum conditions at 25-35℃ for 4-8 h.

6. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step one, the preparation of the activated compound microbial agent is as follows: Bacillus subtilis, lactic acid bacteria, and nitrifying bacteria are mixed in a mass ratio of 3:2:4, inoculated into a nutrient solution, and cultured at 28-32℃ for 1.5-3 days to obtain the activated compound microbial agent; The method for preparing the nutrient solution is as follows: 1-2 parts by weight of yeast extract, 2-4 parts by weight of sucrose, 1-2 parts by weight of ammonium sulfate, 0.5-1.5 parts by weight of potassium dihydrogen phosphate, 0.3-0.8 parts by weight of magnesium sulfate, 0.1-0.3 parts by weight of manganese sulfate, 0.1-0.2 parts by weight of calcium chloride, and 0.5-1 parts by weight of B complex vitamins are added to 12-16 parts by weight of water and mixed thoroughly to obtain the nutrient solution.

7. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step three, the cobalt porphyrin is polyethylene glycol cobalt porphyrin, which is prepared by reacting cobalt porphyrin with methoxy polyethylene glycol succinimide ester in a molar ratio of 1:3-1:5, and has a molecular weight of 2000-5000 Da. After the mesoporous silica nanoparticles are covalently fixed to the gel network, the polyethylene glycol cobalt porphyrin is physically adsorbed and loaded into the pores of the mesoporous silica nanoparticles.

8. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 7, characterized in that, In step three, when loading polyethylene glycol cobalt porphyrin into the pores of mesoporous silica nanoparticles, first adjust the pH of the solution to 7.0-7.5 and stir for 10-30 min, then adjust the pH to 6.0-6.5 and continue stirring for 20-60 min, so that the polyethylene glycol segments form a dynamic network structure with pH responsiveness.

9. The preparation method of the composite packing material for synergistic oxygenation and degradation of aquaculture pollutants as described in claim 1, characterized in that, In step four, the core-shell microspheres are prepared using a four-layer structure: The microspheres of the bacterial agent prepared in step one are used as the inner layer of the bacterial agent core; Catalase was mixed with a blank gel solution to form the catalase layer in the middle and inner layers, wherein the amount of catalase added was 0.5%-2.0% of the mass of polyvinyl alcohol; Glucose oxidase was mixed with a blank gel solution to form the glucose oxidase layer, which was added at an amount of 0.5%-2.0% of the mass of polyvinyl alcohol. The functionalized gel liquid obtained in step three is used as the outer catalytic layer; First, the inner bacterial agent core is immersed in the catalase layer, then removed and cross-linked once with a second calcium chloride solution to form microspheres coated with the catalase layer. Next, the microspheres are immersed in the glucose oxidase layer, then removed and cross-linked twice with a second calcium chloride solution to form microspheres with the middle layer consisting of the catalase layer and the glucose oxidase layer from the inside out. Finally, the microspheres are immersed in the outer catalytic layer, then removed and cross-linked three times with a second calcium chloride solution to form a four-layer core-shell microsphere with the inner layer being the bacterial agent, the middle layer consisting of the catalase layer and the glucose oxidase layer from the inside out, and the outer layer being a cobalt porphyrin functionalized gel.

10. The composite packing prepared by the method for preparing composite packing for synergistic oxygenation and degradation of aquaculture pollutants as described in any one of claims 1-9.

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