A method for preparing a biological filler for ammonia degradation

Abstract

The present application belongs to the technical field of biological purification of waste gas, and particularly relates to a preparation method of a biological filler for ammonia degradation. The preparation method of the biological filler for ammonia degradation comprises the following steps: placing methyl methacrylate anhydride gelatin, a photoinitiator and bacteria for ammonia degradation in water to prepare biological ink, then performing 3D printing on the biological ink, and performing solidification after the 3D printing is completed to obtain the biological filler with a porosity of 60-80%. The biological filler with a three-dimensional structure is obtained by using the 3D printing layering principle, the methyl methacrylate anhydride gelatin has the characteristics of natural and synthetic biomaterials, the biological filler has high specific surface area and porosity, which provides a good growth environment for the bacteria, and the biological filler has high water absorption performance, which can improve the ammonia adsorption capacity in the environment and greatly improve the ammonia degradation rate.

Description

Technical Field

[0001] This invention relates to the field of biological purification technology for waste gas, and specifically to a method for preparing a biological packing material for ammonia degradation. Background Technology

[0002] With industrialization and urbanization, the emission of waste gas and pollutants from human activities has increased dramatically, leading to increasingly serious air pollution problems, such as natural disasters, common smog, and severe pollution incidents. Ammonia (NH3) can react with acidic gases such as sulfuric acid and nitric acid in the atmosphere to form ammonium salts, creating secondary inorganic aerosol particles that pollute air quality. Large amounts of ammonia emissions can cause eutrophication of lakes and other near-shore waters, and soil acidification. Ammonia also irritates the mucous membranes of the upper respiratory tract, nose, and eyes; excessive ammonia can increase irritation symptoms and affect the central nervous system. Therefore, ammonia control is urgently needed.

[0003] Compared to physical and chemical technologies, biological deodorization and purification processes have advantages such as simple operation, low cost, and no secondary pollution. It works by using microorganisms to absorb and degrade pollutants, converting them into nutrients and metabolizing them out of the body, ultimately breaking them down into small, non-polluting molecules such as water and carbon dioxide.

[0004] Packing materials, as the core component of biological waste gas purification processes, serve as carriers for microbial attachment, growth, and reproduction. Based on their source and preparation, they can be categorized into three types: natural, artificial, and composite. Natural packing materials include pebbles, zeolite, and charcoal, while artificial packing materials include ceramsite, ceramics, and sponges. These materials possess large specific surface areas and porosity, providing an environment for microbial growth and biofilm formation. However, these three types of packing materials have poor adhesion to microorganisms, easily causing biofilm detachment when waste gas flow is high. Furthermore, most have poor water absorption and adsorption capacity for waste gas in the environment, requiring continuous circulation of nutrient solution to maintain moisture, resulting in poor ammonia degradation rates. Summary of the Invention

[0005] This invention provides a method for preparing a biological packing material for ammonia degradation, solving the problem of poor ammonia degradation rate in existing technologies.

[0006] To solve the above-mentioned technical problems, the technical solution of the preparation method of the biological packing material for ammonia degradation of the present invention is as follows:

[0007] A method for preparing a biofiller for ammonia degradation includes the following steps: placing methacrylic anhydride gelatin, a photoinitiator, and bacteria for ammonia degradation in water to prepare a bioink; then 3D printing the bioink; and finally curing the biofiller after 3D printing to obtain a biofiller with a porosity of 60-80%.

[0008] This invention improves upon existing technologies by providing a method for preparing a bio-filler for ammonia degradation. The method utilizes methacrylic anhydride-modified gelatin, a photoinitiator, and a bio-ink formulated with bacteria for ammonia degradation. The bio-filler is obtained using the 3D printing layer-by-layer principle. Methacrylic anhydride-modified gelatin combines the characteristics of both natural and synthetic biomaterials, forming a three-dimensional structure after photocuring. This structure possesses a suitable three-dimensional structure for bacterial growth, excellent biocompatibility, biodegradability, and bacterial reactivity. The high specific surface area and porosity of the bio-filler provide a favorable growth environment for bacteria. Simultaneously, the bio-filler exhibits high water absorption capacity, enhancing the adsorption capacity of ammonia in the environment. Bacteria embedded within the filler can also utilize the nutrient solution absorbed by the bio-filler for growth, thus more effectively treating ammonia and significantly improving the ammonia degradation rate. The preparation method provided by this invention is simple, low-cost, and utilizes 3D printing to meet the needs for different shapes of the bio-filler, making it suitable for large-scale industrial production.

[0009] To further improve the degradation efficiency of ammonia, preferably, the mass ratio of the methacrylic anhydride gelatin, photoinitiator, and bacteria for ammonia degradation is (6-12):(0.2-0.5):(0.1-0.3), and the curing is photocuring. The colony count of the bacteria for ammonia degradation is (2-3) × 10⁻⁶. 10 CFU / g. More preferably, the mass ratio of the methacrylic anhydride gelatin, the photoinitiator, and the bacteria for ammonia degradation is 12:0.2:0.1. Methacrylic gelatin is a photosensitive biomaterial that, when used in conjunction with a photoinitiator, can rapidly crosslink and solidify under blue or ultraviolet light to form a three-dimensional structure with a certain strength.

[0010] To further improve the degradation efficiency of ammonia and the mechanical properties of the bio-filler, preferably, the bio-ink includes sodium alginate, and the mass ratio of the methacrylic anhydride gelatin, sodium alginate, photoinitiator, and bacteria for ammonia degradation is (6-12):(1-2):(0.2-0.5):(0.1-0.3), and the curing is photocuring and chemical cross-linking curing. The colony count of the bacteria for ammonia degradation is (2-3) × 10⁻⁶. 10 CFU / g. More preferably, the mass ratio of the methacrylic anhydride gelatin, sodium alginate, photoinitiator, and bacteria for ammonia degradation is 6:2:0.5:0.3. Sodium alginate will undergo a cross-linking reaction with calcium ions to further improve the compressive strength of the biofiller.

[0011] To further improve the porosity of the three-dimensional structure and the efficiency of photocuring, preferably, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and the photocuring is performed by irradiation with 405nm blue light for 15-20s.

[0012] To further improve the porosity of the three-dimensional structure and the efficiency of chemical cross-linking curing, preferably, the chemical cross-linking curing is performed by immersion in CaCl2 solution for 30-35 seconds.

[0013] To further improve the bioactivity of methacrylic anhydride gelatin, preferably, the methacrylic anhydride gelatin is obtained by reacting gelatin and methacrylic anhydride in D-PBS buffer, followed by dialysis and freeze-drying, wherein the molecular weight cutoff of the dialysis bag used during dialysis is 13,000 to 14,000.

[0014] To further enhance the adhesion to bacteria, preferably, the mass ratio of the gelatin to the volume ratio of methacrylic anhydride is (1-2) g: 3 mL.

[0015] To further improve the reaction efficiency of gelatin and methacrylic anhydride, preferably, the reaction temperature is 50–60°C and the reaction time is 2.5–3 hours. Since methacrylic anhydride-modified gelatin is a photosensitive material, more preferably, the reaction is carried out in a light-protected environment.

[0016] To further improve the degradation efficiency of ammonia, the 3D printing parameters are preferably set as follows: line spacing of 1.3–1.7 mm, first layer height of 0.25–0.3 mm, remaining layer height of 0.18–0.22 mm, and extrusion speed of 0.20–0.30 mm / s². 3 The needle movement speed is 2.00–3.00 mm / s. By controlling the printing parameters, the three-dimensional structure of the biological packing material is controlled, as well as its porosity and pore size. The more pores and the smaller the pore size of the biological packing material, the greater the amount of bacteria it can embed and the higher its water content, which can greatly improve the degradation efficiency of ammonia.

[0017] To further improve 3D printing performance, preferably, the ambient temperature during 3D printing is 21–23°C, the needle temperature is 20–23°C, and the printing platform temperature is 16–17°C. During 3D printing, temperature significantly affects the viscosity of the bio-ink, which in turn affects the performance of the filaments extruded from the needle. When the viscosity is high, the bio-ink becomes too viscous, causing the needle to clog or coil at the needle tip; when the viscosity is low, the bio-ink tends to form droplets.

[0018] To further improve the degradation rate of ammonia by bacteria, preferably, the bacteria used for ammonia degradation are Bacillus subtilis or a mixed bacterial community, wherein the mixed bacterial community consists of Bacillus subtilis and yeast in a mass ratio of (1-5):1; and the colony count of Bacillus subtilis is (2-3) × 10⁻⁶. 10 CFU / g, the colony count of the yeast is (2-3) × 10⁻⁶. 9 CFU / g. Attached Figure Description

[0019] Figure 1 A photograph of the GelMA object prepared in Example 1 of this invention;

[0020] Figure 2 Flowchart for preparing bio-ink according to Embodiment 1 of the present invention;

[0021] Figure 3 Photographs showing the state of the needle when the viscosity of the bio-ink is too low during the 3D printing process.

[0022] Figure 4 Photographs showing the state of the needle when the viscosity of the bio-ink is too high during the 3D printing process.

[0023] Figure 5 This is a photograph of the state of the needle extruding filaments during the 3D printing process of Embodiment 1 of the present invention;

[0024] Figure 6 Photograph of the biological packing material prepared in Example 1 of this invention;

[0025] Figure 7 Fourier transform infrared spectra of the gelatin and GelMA prepared in this invention;

[0026] Figure 8 This is a structural diagram of the ammonia degradation rate testing device. Detailed Implementation

[0027] The technical concept of the preparation method of the biological packing material for ammonia degradation of the present invention is as follows:

[0028] The present invention provides a method for preparing a biofiller for ammonia degradation, comprising the following steps: placing methacrylic anhydride gelatin, a photoinitiator and bacteria for ammonia degradation in water to prepare a bioink, then 3D printing the bioink, and curing it after 3D printing to obtain a biofiller with a porosity of 60-80%.

[0029] Immobilized microbial technology, applied in the treatment of malodorous gases and VOCs, can help functional microorganisms withstand harsh environments, increase the concentration of functional microorganisms within bioreactors, and shorten treatment time. Existing methods for immobilizing microorganisms mainly include adsorption, cross-linking, and encapsulation. This invention is based on the encapsulation method, using methacrylic anhydride gelatin, sodium alginate, a photoinitiator, and a bio-ink formulated with bacteria for ammonia degradation. A three-dimensional bio-filler is obtained using the principle of 3D printing layering. Methacrylic anhydride gelatin combines the characteristics of natural and synthetic biomaterials, possessing a three-dimensional structure suitable for bacterial growth, excellent biocompatibility, biodegradability, and bacterial reactivity. The high specific surface area and porosity of the bio-filler provide a favorable growth environment for bacteria. Simultaneously, the bio-filler has high water absorption capacity, enhancing the adsorption capacity of ammonia in the environment. The bacteria encapsulated in the filler can also utilize the nutrient solution absorbed by the bio-filler for growth, thus more effectively treating ammonia and significantly improving the ammonia degradation rate. The preparation method provided by this invention is simple, low-cost, and, using 3D printing, can meet the needs of different shapes of the bio-filler, making it suitable for large-scale industrial production.

[0030] In a specific embodiment, the rotation speed of the reaction system during the reaction is 1000-1500 rpm.

[0031] In a specific embodiment, the dialysis is performed in a light-protected environment using a dialysis bag with a molecular weight cutoff of 14,000, at a temperature of 40–50°C, for a duration of 7 days; more preferably, the dialysis is also performed in a light-protected environment.

[0032] In a specific embodiment, the dialysis bag is heated at 100-120°C for 15-30 minutes before use to remove harmful substances.

[0033] In a specific embodiment, after the light-protected dialysis, the pH is adjusted to neutral using an alkaline solution, wherein the alkaline solution is a NaOH solution with a concentration of 1 to 1.5 M.

[0034] In a specific embodiment, the freeze-drying involves first freezing at -100 to -80°C for 12 to 16 hours, and then continuously freeze-drying at -55°C to -45°C for 4 to 6 days in a freeze dryer. More preferably, the freeze-drying is carried out in a light-protected environment.

[0035] In a specific embodiment, the preparation process of the bio-ink is as follows: First, prepare an aqueous solution of photoinitiator and a bacterial suspension. Then, add methacrylic anhydride gelatin, the aqueous solution of photoinitiator, and the bacterial suspension to water. After the methacrylic anhydride gelatin is dissolved at 35-45°C, sodium alginate is added. After dissolution, the bio-ink is obtained. Sodium alginate is added to the bio-ink to adjust its viscosity, which has a significant impact on 3D printing.

[0036] In a specific embodiment, the mass concentration of the photoinitiator aqueous solution is 2-5%, the mass concentration of the bacterial suspension is 1-3%, and the mass concentrations of methacrylic anhydride gelatin, sodium alginate, photoinitiator, and bacteria in the bio-ink are 6-12%, 0-2%, 0.2-0.5%, and 0.1-0.3%, respectively. Here, mass concentration refers to the ratio of solute mass to solvent volume.

[0037] In a specific implementation, a cube model is selected for 3D printing, with a length × width × height of (10~12)mm × (10~12)mm × (10~12)mm.

[0038] In a specific embodiment, the CaCl2 solution is an aqueous solution of CaCl2 with a mass fraction of 5-10%.

[0039] In a specific embodiment, the reaction temperature of gelatin and methacrylic anhydride is 50-60°C, and the reaction time is 2.5-3 hours.

[0040] It should be noted that line spacing refers to the distance of the filament extruded by the needle during printing; the model is sliced ​​during printing, the first layer height refers to the height of the slice closest to the bottom of the platform, and the layer height refers to the height of each layer other than the first layer height, which refers to the height of one layer of filament extruded by the needle during printing.

[0041] The embodiments of the present invention will be further described below with reference to specific examples. Unless otherwise specified, all chemical reagents involved in the following examples are commercially available products.

[0042] I. Specific Embodiments of the Preparation Method of the Biological Packing Material for Ammonia Degradation of the Present Invention

[0043] The raw materials and instruments used in the specific embodiments provided by this invention are all commercially available products. Specific product information is as follows:

[0044] D-PBS buffer: 500ml, calcium and magnesium ion-free, filtered sterile, enzyme-free, for cell culture, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Gelatin: 100g, type A 300g Bloom, brand VETEC; Methacrylic anhydride: 100ml, 94%, containing 0.2% topanol stabilizer, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Sodium hydroxide standard solution: 1L, concentration 1M, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP photoinitiator): 1g, purchased from Shanghai Yinchang New Materials Co., Ltd.; Sodium alginate: 25g, analytical grade (AR), brand, purchased from Aladdin; Calcium chloride: 500g, analytical grade (AR), purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; Bacillus subtilis: 5000g, colony count 2×10⁻⁶. 10 CFU / g, cooperating company: Henan Xinyangshao Biotechnology Co., Ltd.; Yeast: 500g, colony count 2×10 9 CFU / g, partner company: Henan Xinyangshao Biotechnology Co., Ltd.; 3D printer: Shanghai Boyuan's bio-3D printer.

[0045] Example 1

[0046] The method for preparing the biological packing material for ammonia degradation provided in this embodiment includes the following steps:

[0047] 1) Preparation of methacrylic anhydride gelatin: Prepare aluminum foil to completely wrap the beaker except for the bottle mouth. At the same time, prepare a piece of aluminum foil that can completely cover the bottle mouth. Add 40 ml of D-PBS buffer to the beaker, then add 4 g of gelatin. Place the beaker on a constant temperature magnetic stirrer and heat it in a water bath to 50°C. Stir at low speed to reduce bubbles in the solution. After the gelatin is completely dissolved, increase the stirring speed to 1000 rpm. Then add 1 ml of methacrylic anhydride (MA) along the beaker wall every minute for a total of 12 times. Cover the beaker with aluminum foil and react completely in the dark at 50°C and 1000 rpm for 2.5 h.

[0048] Prepare dialysis bags MD44 (14000) (flattened width 44mm, molecular weight cutoff 14000), cut into 6 segments of 15cm each, heat at 100℃ for 15 minutes to remove harmful substances from the surface, then place two 2000ml light-proof beakers in a constant temperature water bath, wrap the beakers with tin foil to completely block light, then fill each dialysis bag with 7ml of the reacted methacryloyl gelatin (GelMA) solution, clamp them with dialysis clamps, add ultrapure water to the two beakers to immerse the dialysis bags in the beakers for dialysis, change the ultrapure water 3 times a day (morning, noon and evening), and dialyze at 40℃ for 7 days;

[0049] Pour the dialyzed solution from the dialysis bag into a light-proof beaker, wrap the beaker with aluminum foil, and adjust the pH to neutral with 1M NaOH solution. Prepare three petri dishes (10cm in diameter and 2cm in height) wrapped with aluminum foil. Pour the prepared solution into the petri dishes, ensuring it does not exceed the height of the dishes, and cover them completely with aluminum foil to protect them from light. Freeze at -80℃ for 12 hours. After freezing, poke some small holes in the aluminum foil covers with a needle, and freeze-dry at -50℃ for 4 days to produce methacrylic anhydride gelatin (GelMA). A photo of the finished GelMA is shown below. Figure 1 As shown.

[0050] 2) Preparation of bio-ink: The preparation process of bio-ink is as follows... Figure 2 As shown, add 0.2g of LAP powder to 4ml of ultrapure water and stir well to prepare a solution containing 5% photoinitiator LAP. Then add 0.09g of a mixed bacterial culture of Bacillus subtilis and yeast in a 1:1 mass ratio to 3ml of ultrapure water and stir well to prepare a bacterial suspension containing 3% bacterial culture. Weigh 0.18g of GelMA obtained in step 1) and put it into a 4ml centrifuge tube. Add 2.4ml of ultrapure water, then add 0.3ml of bacterial suspension and 0.3ml of photoinitiator solution. GelMA will dissolve at high temperature and become solid at low temperature. The process is reversible. GelMA is dissolved at 40°C. After dissolution, a small amount of foam will appear on the surface of the liquid in the centrifuge tube. To remove the foam, the centrifuge tube is placed in a 4°C freezer for 45 minutes. After that, the foam on the top is scraped off with a spatula. The solution is then liquefied again at 40°C. 0.06g of sodium alginate is added, and the solution is stirred evenly in the centrifuge tube with a spatula until the sodium alginate is completely dissolved and the solution is mixed evenly. Thus, 3ml of bio-ink is prepared in a 4ml centrifuge tube with the following mass concentrations: GelMA, sodium alginate, LAP photoinitiator, and mixed bacterial community: 6%, 2%, 0.5%, and 0.3%, respectively.

[0051] 3) Preparation of bio-filler: The bio-ink prepared in step 2) was loaded into a 5ml syringe with a 25G needle and fixed on a Shanghai Boyuan entry-level desktop bio-3D printer. A 60mm × 15mm bacterial culture dish was placed on the printing platform as the printing carrier. BiomakerV2 software was opened on the computer, the cube model was selected for printing, and the printing parameters were set as follows: length, width, and height: 10mm, 10mm, 10mm; line spacing: 1.5mm; first layer height: 0.25mm; layer height: 0.20mm; extrusion speed: 0.20mm. 3 / s, needle movement speed: 2.00mm / s;

[0052] Adjust the room temperature to 21℃ using air conditioning. Set the initial temperature of the printing needle to 21℃ and the printing platform to 16℃. Begin pre-extrusion at 21℃, observing whether the solution in the syringe forms threads or droplets. Figure 3 As shown, this indicates that if the viscosity is too low, the temperature needs to be lowered; if the solution is too viscous, it will clog the needle or cause it to swirl at the needle tip. Figure 4 As shown; if the viscosity is too low, the temperature should be lowered; if the viscosity is too high, the temperature should be increased; adjust by 1°C and wait 10 minutes to allow the solution viscosity to change, then adjust the temperature appropriately until the printing needle temperature is set to 20°C and the printing platform temperature is set to 16°C. When extruded, it should form filaments, as shown. Figure 5 As shown, the needle is moved 0.2 mm onto the printing carrier, and printing begins. After the filler is printed, it is irradiated with 405 nm blue light for 15 seconds for photocuring, and then immersed in a 10% CaCl2 solution for 30 seconds to cross-link and cure the filler. The moisture is then wiped dry to obtain the biological filler. The structural diagram of the biological filler is shown below. Figure 6 As shown.

[0053] It should be noted that when not in use, this biological packing material should be stored at 4°C in a refrigerator. When in use, after absorbing the nutrient solution, it should be placed in an environment contaminated with ammonia, where it can automatically adsorb and degrade ammonia in the air; alternatively, after absorbing the nutrient solution, it can be applied to the packing layer of a bioreactor. The nutrient solution composition is: C6H 12 O6 5~6g / L, KH2PO4 1.5~3g / L, K2HPO4 3~4g / L, CaCl20.1~0.2g / L, MgSO4 0.25~0.3g / L, MnSO4 0.01~0.02g / L, FeCl3 0.1~0.2g / L.

[0054] Example 2

[0055] The method for preparing the biological packing material for ammonia degradation provided in this embodiment includes the following steps:

[0056] 1) The methacrylic anhydride gelatin in this embodiment is prepared according to the preparation method of Example 1.

[0057] 2) Preparation of bio-ink: Add 0.08g of LAP powder to 4ml of ultrapure water and stir well to prepare a solution containing 2% photoinitiator LAP. Then add 0.06g of a mixed bacterial culture of Bacillus subtilis and yeast in a mass ratio of 3:1 to 3ml of ultrapure water and stir well to prepare a bacterial suspension containing 2% bacterial culture. Weigh 0.27g of GelMA and put it into a 4ml centrifuge tube. Add 2.4ml of ultrapure water, then add 0.3ml of bacterial suspension and 0.3ml of photoinitiator solution. Dissolve GelMA at 35℃. After dissolving, a small amount of foam will appear on the surface of the liquid in the centrifuge tube. To remove the foam, place the centrifuge tube in a 4°C freezer for 45 minutes. Then, remove it and scrape off the top layer of foam with a spatula. Let it liquefy again at 35°C, add 0.03g of sodium alginate, and stir it evenly in the centrifuge tube with a spatula until the sodium alginate is completely dissolved and the solution is mixed evenly. Thus, 3ml of bio-ink containing GelMA, sodium alginate, LAP photoinitiator, and mixed bacterial flora with mass concentrations of 9%, 1%, 0.2%, and 0.2% respectively is prepared in a 4ml centrifuge tube.

[0058] 3) Preparation of bio-filler: The bio-ink prepared in step 2) was loaded into a 5ml syringe with a 25G needle and fixed on a Shanghai Boyuan entry-level desktop bio-3D printer. A 60mm × 15mm bacterial culture dish was placed on the printing platform as the printing carrier. BiomakerV2 software was opened on the computer, the cube model was selected for printing, and the printing parameters were set as follows: length, width, and height: 10mm, 10mm, 10mm; line spacing: 1.3mm; first layer height: 0.25mm; layer height: 0.18mm; extrusion speed: 0.20mm. 3 / s, needle movement speed: 2.00mm / s;

[0059] Set the room temperature to 22°C using an air conditioner. Initially set the printing needle temperature to 22°C and the printing platform temperature to 16°C. Begin pre-extrusion at 22°C, observing whether the solution in the syringe forms filaments. If the solution is more like droplets, the viscosity is too low, requiring a temperature reduction. If the solution is too viscous, it will clog the needle or cause it to swirl at the needle. If the viscosity is too low, the temperature should be lowered; if the viscosity is too high, the temperature should be increased. Adjust the temperature by 1°C and wait 10 minutes to allow the solution to change viscosity. Adjust the temperature appropriately until the printing needle temperature is 20°C and the printing platform temperature is 16°C. Once the extrusion forms filaments, move the needle 0.2 mm onto the printing carrier and begin printing. After the filler is printed, irradiate with 405nm blue light for 15 seconds for photocuring, then soak in a 5% CaCl2 solution for 30 seconds to cross-link and cure the filler. Wipe away the moisture to obtain the biological filler.

[0060] Example 3

[0061] The method for preparing the biological packing material for ammonia degradation provided in this embodiment includes the following steps:

[0062] 1) The methacrylic anhydride gelatin in this embodiment is prepared according to the preparation method of Example 1.

[0063] 2) Preparation of bio-ink: Take 4 ml of ultrapure water and add 0.08 g of LAP powder, stir evenly to prepare a solution containing 2% photoinitiator LAP. Then take 3 ml of ultrapure water and add 0.03 g of a mixed bacterial culture of Bacillus subtilis and yeast in a mass ratio of 5:1, stir evenly to prepare a bacterial suspension containing 1% bacterial culture. Weigh 0.36 g of GelMA and put it into a 4 ml centrifuge tube, add 2.4 ml of ultrapure water, then add 0.3 ml of bacterial suspension and 0.3 ml of photoinitiator solution. Dissolve GelMA at 45℃. After dissolving, there will be a small amount of foam on the surface of the liquid in the centrifuge tube. To remove the foam, put the centrifuge tube in a 4℃ freezer for 45 minutes, then take it out and scrape off the upper layer of foam with a spatula. Then liquefy it again at 45℃. Thus, 3 ml of bio-ink with mass concentrations of 12% GelMA, 0.2% LAP photoinitiator, and 0.1% mixed bacterial culture were prepared in a 4 ml centrifuge tube.

[0064] 3) Preparation of bio-filler: The bio-ink prepared in step 2) was loaded into a 5ml syringe with a 25G needle and fixed on a Shanghai Boyuan entry-level desktop bio-3D printer. A 60mm × 15mm bacterial culture dish was placed on the printing platform as the printing carrier. BiomakerV2 software was opened on the computer, the cube model was selected for printing, and the printing parameters were set as follows: length, width, and height: 10mm, 10mm, 10mm; line spacing: 1.7mm; first layer height: 0.25mm; layer height: 0.22mm; extrusion speed: 0.20mm. 3 / s, needle movement speed: 2.00mm / s;

[0065] Adjust the room temperature to 23℃ using an air conditioner, set the printing needle temperature to 23℃, and the printing platform temperature to 16℃. Begin pre-extrusion at 23℃, observing whether the solution in the syringe forms filaments. If the solution is more like droplets, the viscosity is too low, requiring a temperature reduction. If the solution is too viscous, it will clog the needle or cause it to swirl at the needle. If the viscosity is too low, the temperature should be lowered; if the viscosity is too high, the temperature should be increased. Adjust the temperature by 1℃ and wait 10 minutes to allow the solution to change viscosity. Adjust the temperature appropriately until the printing needle temperature is 20℃ and the printing platform temperature is 16℃. Once the extrusion forms filaments, move the needle 0.2mm onto the printing carrier and begin printing. After the filler is printed, use 405nm blue light to irradiate for 15 seconds for photocuring. Wipe away the moisture to obtain the biological filler.

[0066] II. Comparative Example

[0067] Comparative Example 1

[0068] The biological packing material used in this comparative example is a commercial biological filter media purchased from Zhejiang Lvmei New Special Environmental Protection Technology Co., Ltd., model LMSW-biological filter media. The main raw materials are inorganic minerals of silicon and calcium, and the biofilm-forming bacteria are a mixed microbial community mainly composed of Bacillus subtilis and yeast.

[0069] Comparative Example 2

[0070] The biological packing material used in this comparative example is an embedded composite packing material. The preparation method of the embedded composite packing material is as follows:

[0071] Polyvinyl alcohol (32% by mass) and sodium alginate (15% by mass) as encapsulating and protective agents were added to water and heated to 100°C for 30 minutes to allow them to blend together. Then the mixture was cooled to 35°C, and a mixed bacterial suspension (0.3%) of Bacillus subtilis and yeast in a mass ratio of 1:1 was added. The mixture was then mixed in a container for 15 minutes and extruded into spherical particles with a diameter of 10±1 mm.

[0072] Comparative Example 3

[0073] The preparation method of the biological filler in this comparative example differs from that in Example 1 in that: no mixed microbial community is added during the preparation of the biological ink in step 2), while the other steps are the same.

[0074] III. Experimental Examples

[0075] This experiment tested the Fourier transform infrared (FTIR) spectra of gelatin and GelMA, as well as the porosity, pore size, and ammonia degradation rate of the biological filler.

[0076] 1) Fourier transform infrared spectroscopy test

[0077] Infrared spectroscopy is generated by molecular vibrational energy level transitions. The position and intensity of each peak represent the number and nature of substituents of different functional groups; the higher the molecular symmetry, the weaker the absorption peak. To test whether there are carbon-carbon double bonds in the product, the Fourier transform infrared (FTIR) spectra of gelatin used in the synthesis of GelMA in this invention and GelMA were measured. The test parameters are as follows: sample scan 32 times, background scan 32 times, resolution 4.000, sampling gain 1.0, mirror speed 0.4747, aperture 100.0, detector DTGSKBr, spectrometer KBr. The measured Fourier transform infrared spectra of gelatin and GelMA are shown below. Figure 7 As shown. 4000~2500cm -1 The high wavenumber region is the absorption band of stretching vibrations of functional groups OH, NH, CH, and SH bonds with hydrogen atoms at one end; 1900–1350 cm⁻¹ -1 The low wavenumber region is the stretching vibration region of the double bond and the skeletal vibration region of the aromatic ring. Specifically, the stretching vibration absorption positions of the carbon-carbon double bond in alkenes are between 1680 and 1620 cm⁻¹.-1 ~3281.6cm -1 The location is mainly related to the stretching vibration of the NH bond; ~1630.2cm -1 The amide I band is caused by the stretching vibration of the C=O bond; ~1526.4 cm -1 This is due to the coupling of NH bending vibration and CN stretching vibration. A comparison of the peak shapes of gelatin and GelMA shows that the NH bond position in GelMA has higher transmittance and lower absorption intensity compared to gelatin, which may be due to the substitution of the NH bond. The absorption peak position of amide I shifts slightly to higher frequencies, indicating that the gelatin molecule has carbon-carbon double bonds.

[0078] 2) Porosity Test: The biological packing materials obtained in Examples 1-3 and Comparative Example 3 were immersed in anhydrous ethanol. No swelling occurred, and the porosity was determined using the ethanol displacement method. The freeze-dried biological packing material was weighed as M0, and then immersed in anhydrous ethanol until it sank to the bottom. The weight of the immersed biological packing material was then recorded as M1. The porosity was calculated as follows:

[0079]

[0080] Where ρ is the density of anhydrous ethanol at room temperature, and V is the volume of the biological packing material;

[0081] The porosity of Comparative Examples 1 and 2 was determined using a specific surface area analyzer. The results showed that the porosity of Comparative Example 1 was 45% and that of Comparative Example 2 was 13%.

[0082] 3) Pore size test: The biological fillers of the examples and comparative examples were freeze-dried, sliced, sputtered with gold, and the pore size was observed and measured by scanning electron microscopy.

[0083] 4) Ammonia degradation rate test

[0084] First, the prepared biological packing material was placed in a nutrient solution and soaked for 30 minutes to allow it to absorb the nutrient solution. After absorption was complete, excess moisture on the surface of the packing material was wiped dry, and an ammonia degradation rate test was performed. The nutrient solution composition was: C6H 12 O6 5g / L, KH2PO41.5g / L, K2HPO4 3g / L, CaCl2 0.1g / L, MgSO4 0.25g / L, MnSO4 0.01g / L, FeCl3 0.1g / L.

[0085] The packing material is placed in a wide-mouthed bottle of a sealed static adsorption device, keeping the biological packing material in the middle of the bottle. The structural diagram of the ammonia degradation rate testing device is shown below. Figure 8As shown. Ammonia gas was introduced into the long tube, and the ammonia concentration was measured at the outlet of the short tube to bring the ammonia concentration in the bottle to a certain value. Then, both tube openings were completely sealed, and the ammonia degradation efficiency was measured after 48 hours of reaction using a fixed continuous ammonia gas meter. The static adsorption conditions were: initial ammonia concentration of 50 ppm, room temperature, and a 10L wide-mouth bottle for the static adsorption device. The ammonia concentration was measured using a fixed ammonia gas meter, and the ammonia degradation rate was calculated as: Degradation rate (%) = [(initial ammonia concentration - ammonia concentration after degradation) / initial ammonia concentration] × 100%.

[0086] The porosity, pore size, and ammonia degradation rate of the biological packing materials used in Examples 1-3 and Comparative Examples 1-3 are shown in Table 1.

[0087] Table 1. Performance test results of the biological packing materials in the examples and comparative examples.

[0088] project Porosity aperture <![CDATA[NH3 degradation rate %]]> Example 1 81.7％ 49.75~73.15μm 91.3 Example 2 74.3％ 46.41~70.61μm 87.9 Example 3 66.1％ 44.45~68.25μm 85.5 Comparative Example 1 45％ 195~205μm 61.3 Comparative Example 2 13％ 175~185μm 72.1 Comparative Example 3 82.2％ 51.15~73.55μm 45

[0089] As shown in Table 1, the porosity (66-82%) of the biological packing material prepared by the method of this invention is much higher than that of Comparative Examples 1 and 2 (45%, 13%), and the pore size (44-74 μm) is much lower than that of Comparative Examples 1 and 2 (180 μm, 200 μm). The ammonia degradation rate (85%-95%) is also much higher than that of Comparative Examples 1 and 2 (61.3%, 72.1%). This indicates that the more pores and the smaller the pore size of the biological packing material, the greater the water content of the embedded bacteria and the packing material, resulting in better adsorption and degradation performance for ammonia and effectively improving the degradation efficiency of ammonia. Compared to Comparative Example 3, Example 1 achieved a porosity and pore size comparable to Example 1, but the biological packing material of Example 1 had a higher ammonia removal rate. This is because Comparative Example 3 did not include ammonia-degrading bacteria, and the ammonia reached saturation under the adsorption of the hydrogel packing material, resulting in low degradation efficiency. This indicates that the printed biological packing material with a mixed bacterial community of Bacillus subtilis and yeast successfully degraded ammonia. In addition, compared with Examples 2-3, the bio-filler in Example 1 has higher porosity, smaller pore size, and higher ammonia degradation rate, indicating that as the mass fraction of GelMA in the bio-ink decreases, the microbial activity increases, thereby improving the ammonia degradation efficiency.

[0090] Finally, it should be noted that the above-described embodiments are only for illustrating the preparation method and are not intended to limit the technical solution of the present invention. Any equivalent substitutions or corresponding improvements made to the features of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a biological packing material for ammonia degradation, characterized in that, The process includes the following steps: preparing a bio-ink by placing methacrylic anhydride gelatin, a photoinitiator, and bacteria for ammonia degradation in water; then 3D printing the bio-ink; and finally curing it after 3D printing to obtain a bio-filler with a porosity of 60-80%.

2. The method for preparing the biological packing material for ammonia degradation as described in claim 1, characterized in that, The mass ratio of the methacrylic anhydride gelatin, the photoinitiator, and the bacteria used for ammonia degradation is (6-12):(0.2-0.5):(0.1-0.3), and the curing is photocuring.

3. The method for preparing the biological packing material for ammonia degradation as described in claim 1, characterized in that, The bio-ink includes sodium alginate, and the mass ratio of the methacrylic anhydride gelatin, sodium alginate, photoinitiator and bacteria for ammonia degradation is (6-12):(1-2):(0.2-0.5):(0.1-0.3), and the curing is photocuring and chemical cross-linking curing.

4. The method for preparing the biological packing material for ammonia degradation as described in claim 2 or 3, characterized in that, The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and the photocuring is performed by irradiation with 405nm blue light for 15-20s.

5. The method for preparing the biological packing material for ammonia degradation as described in claim 3, characterized in that, The chemical cross-linking and curing process involves immersing the sample in a CaCl2 solution for 30–35 seconds.

6. The method for preparing the biological packing material for ammonia degradation as described in claim 1, 2, or 3, characterized in that, The methacrylic anhydride gelatin is obtained by reacting gelatin and methacrylic anhydride in D-PBS buffer, followed by dialysis and freeze-drying. The dialysis bag used during dialysis has a molecular weight cutoff of 13,000 to 14,000.

7. The method for preparing the biological packing material for ammonia degradation as described in claim 6, characterized in that, The mass ratio of the gelatin to the volume ratio of the methacrylic anhydride is (1-2) g: 3 mL.

8. The method for preparing the biological packing material for ammonia degradation as described in claim 1, characterized in that, The 3D printing parameters are set as follows: line spacing of 1.3–1.7 mm, first layer height of 0.25–0.3 mm, remaining layer height of 0.18–0.22 mm, and extrusion speed of 0.20–0.30 mm / s². 3 / s, the needle movement speed is 2.00~3.00mm / s.

9. The method for preparing the biological packing material for ammonia degradation as described in claim 1, characterized in that, The ambient temperature during 3D printing is 21–23°C, the needle temperature is 20–23°C, and the printing platform temperature is 16–17°C.

10. The method for preparing the biological packing material for ammonia degradation as described in claim 1, characterized in that, The bacteria used for ammonia degradation are Bacillus subtilis or a mixed bacterial community, wherein the mixed bacterial community consists of Bacillus subtilis and yeast in a mass ratio of (1-5):1; the colony count of Bacillus subtilis is (2-3) × 10⁻⁶. 10 CFU / g, the colony count of the yeast is (2-3)×10⁻⁶. 9 CFU / g.

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