Microorganism 3D printing embedding immobilization method based on double-network hydrogel
By using a 3D printing embedding and immobilization method with a dual-network hydrogel, the limitations of mass transfer performance and insufficient stability in traditional gel embedding technology have been solved. This method achieves microbial immobilization with high stability and mechanical strength, and optimizes the hydrodynamics and mass transfer efficiency of wastewater treatment reactors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-03
AI Technical Summary
The dense three-dimensional network structure of existing gel embedding technology limits the mass transfer performance of the carrier, making it prone to swelling and rupture with long-term use. Furthermore, bio-inks are difficult to balance printability, biocompatibility, and long-term stability, affecting the stability and mechanical strength of immobilized microorganisms.
A microbial 3D printing embedding and immobilization method based on dual-network hydrogel was adopted. Microorganisms were embedded in a biocompatible material in the first network premixed liquid. Combining the toughness of physical cross-linking and the strength of chemical cross-linking, a stable dual-network hydrogel structure was formed. 3D printing technology was used to manufacture a macroscopic structure with regular channels and large specific surface area.
It improves the stability and mechanical strength of the encapsulated product, optimizes the fluid dynamics and mass transfer efficiency within the reactor, maintains microbial activity, and extends service life.
Smart Images
Figure BDA0005755688550000072 
Figure BDA0005755688550000081 
Figure HDA0005755688570000011
Abstract
Description
Technical Field
[0001] This application relates to the field of bioengineering technology, and in particular to a method for 3D printing and embedding microorganisms based on dual-network hydrogels. Background Technology
[0002] Microbial immobilization is a biotechnology that uses physical or chemical methods to confine selected free-living microorganisms within a limited spatial area to maintain high density and good biological activity, thereby improving the stability and reusability of bioreactors. It is widely used in wastewater treatment. Existing technologies mainly include physical adsorption, covalent bonding, cross-linking, and gel embedding, with gel embedding being the mainstream method due to its simplicity and minimal impact on cell activity. However, the three-dimensional network structure formed by cross-linking in traditional gel embedding technology is relatively dense, limiting the mass transfer performance of the carrier. After long-term use, it is prone to swelling and rupture, making it difficult to apply in wastewater treatment. It is typically only possible to produce small spheres, which can easily cause blockage and uneven mass transfer when filled into reactors, requiring further improvement. 3D printing technology provides a new approach for customized and structured microbial immobilization. However, existing bio-inks struggle to simultaneously meet the requirements of printability, biocompatibility, and long-term stability, thus affecting the stability and mechanical strength of the resulting embedded products. Summary of the Invention
[0003] To improve the stability and mechanical strength of embedded products, this application provides a method for microbial 3D printing embedding and immobilization based on dual-network hydrogel.
[0004] This application provides a method for embedding and immobilizing microorganisms in 3D printing based on a dual-network hydrogel, which adopts the following technical solution: A method for microbial 3D printing embedding and immobilization based on dual-network hydrogel includes the following steps: Preparation of the first network premix: Polyvinyl alcohol and sodium alginate were heated and dissolved in water, then nanomaterials were added, stirred and mixed evenly, cooled, and then acrylamide, crosslinking agent and microbial inoculant were added in sequence and mixed evenly to obtain the first network premix. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; 3D printing and first network forming: The first network premix liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing to obtain a gel structure; Second network solidification: The gel structure is removed from the coagulation liquid and immersed in a solution containing an initiator to initiate polymerization and form a stable double network hydrogel; Post-processing: The double-network hydrogel was removed and cleaned to obtain the final embedded microbial product.
[0005] By adopting the above technical solutions, the raw materials in the first network premix are all biocompatible materials, the encapsulation process is mild, the damage to microorganisms is small, and their activity can be effectively maintained; the dual network structure of the first network and the second network combines the toughness of physical cross-linking and the strength of chemical cross-linking, resulting in a product that is not easy to break, swell or disintegrate, and has a long service life, thus improving the stability and mechanical strength of the encapsulated product; in addition, 3D printing technology can be used to manufacture macroscopic structures with regular channels and large specific surface area, optimizing the fluid dynamics and mass transfer efficiency in the reactor.
[0006] In one specific implementation, the first network premix contains: polyvinyl alcohol at a mass-volume concentration of 8-10%, sodium alginate at a mass-volume concentration of 0.5-1%, nanomaterials at a mass-volume concentration of 1-3%, acrylamide at a mass-volume concentration of 10-15%, crosslinking agent at a mass-volume concentration of 0.5-2%, and microbial inoculant at a volume concentration of 10-40%.
[0007] By adopting the above technical solution, the proportion of each raw material in the first network premix is further defined, thereby improving the performance of the obtained first network premix.
[0008] In one specific implementation, the nanomaterial comprises nano-silica.
[0009] In one specific implementation, the crosslinking agent comprises N,N'-methylenebisacrylamide.
[0010] By adopting the above technical solution, under the action of the initiator, acrylamide and N,N'-methylenebisacrylamide undergo a copolymerization reaction to form a stable double-network hydrogel.
[0011] In one specific implementation, the method for preparing the microbial inoculant includes the following steps: The acclimatized nitrified sludge was centrifuged at 4000 r / min for 10 min, then the supernatant was removed, and the precipitate was washed with phosphate buffer at pH 7.0. Finally, phosphate buffer was added to obtain a concentrated solution with a dry weight of 2-5%, which is the microbial inoculant.
[0012] By adopting the above technical solution, the acclimatized nitrified sludge is first centrifuged and separated into layers, then the lower sediment is washed, and finally phosphate buffer is added to adjust the dry weight to obtain microbial inoculants.
[0013] In one specific implementation, the mass-volume concentration of calcium chloride in the coagulation solution is 1.5-2.5%.
[0014] In one specific implementation, in the 3D printing and first network forming step, the printing temperature is 20-25℃, the extrusion pressure is 0.1-0.5MPa, the printing speed is 5-20mm / s, and the diameter of the printing needle is 0.2-1.0mm.
[0015] In one specific implementation, in the second network curing step, the initiator in the initiator solution includes ammonium persulfate, and the mass-volume concentration is 0.1-0.5%.
[0016] By adopting the above technical solution, the parameters in the 3D printing and first network forming steps, as well as the concentration of the initiator in the initiator solution, are further defined, thereby improving the final effect of obtaining the embedded microbial product.
[0017] In one specific implementation, the loss of the encapsulated microbial product after 14 days of shaking is 18-20 mg / g.
[0018] In one specific feasible implementation, the fragmentation rate of the encapsulated microbial product after 14 days of shaking is 5.5-7%.
[0019] By adopting the above technical solution, the microbial 3D printing embedding and immobilization method in this application has a good embedding effect on microorganisms.
[0020] In summary, this application includes at least one of the following beneficial technical effects: 1. The raw materials in the first network premix in this application are all biocompatible materials. The encapsulation process is mild, causing little damage to microorganisms and effectively maintaining their activity. The dual-network structure of the first and second networks combines the toughness of physical cross-linking with the strength of chemical cross-linking, resulting in a product that is not easily broken, swollen, or disintegrated, and has a long service life, thus improving the stability and mechanical strength of the encapsulated product. In addition, 3D printing technology can be used to manufacture macroscopic structures with regular channels and large specific surface area, optimizing the fluid dynamics and mass transfer efficiency in the reactor. 2. In this application, under the action of an initiator, acrylamide and N,N'-methylenebisacrylamide undergo a copolymerization reaction to form a stable double-network hydrogel; 3. In this application, the acclimated nitrified sludge is first centrifuged and separated into layers, then the lower sediment is washed, and finally phosphate buffer is added to adjust the dry weight to obtain the microbial inoculant. Attached Figure Description
[0021] Figure 1 Line graphs showing product loss in the experimental and control groups.
[0022] Figure 2 Line graphs showing the product breakage rate in the experimental and control groups.
[0023] Figure 3 This is a line graph showing the ammonia nitrogen concentration in the experimental and control groups. Detailed Implementation
[0024] The present application will be further described in detail below with reference to the embodiments.
[0025] All raw materials used in the examples are commercially available.
[0026] Preparation Example Preparation Example 1 Preparation Example 1 provides a method for preparing a microbial inoculant, comprising the following steps: Ordinary activated sludge was continuously enriched and cultured with a retention time of 24 hours. Ammonium chloride and sodium bicarbonate were used to provide ammonia nitrogen and alkalinity to the influent, with an ammonia nitrogen:alkalinity concentration ratio of 1:7.5. The enrichment culture was conducted in three stages: Stage 1: Ammonia nitrogen concentration was adjusted to 75 mg / L, and the effluent ammonia nitrogen was reduced to below 10 mg / L; Stage 2: Ammonia nitrogen concentration was adjusted to 175 mg / L, and the effluent ammonia nitrogen was reduced to below 10 mg / L; Stage 3: Ammonia nitrogen concentration was adjusted to 275 mg / L. During the enrichment culture, the pH was maintained at 7.5–8, the temperature at 25–30℃, and the dissolved oxygen concentration at 3–5 mg / L. When the sludge concentration was 3 g / L and the temperature was 25℃, the ammonia oxidation rate was ≥25 mg NH₄⁺-N / (L·h), indicating that the sludge had been acclimated and well-acclimated nitrified sludge was obtained. The acclimatized nitrified sludge was centrifuged at 4000 r / min for 10 min, and then the supernatant was removed. The precipitate was washed with phosphate buffer solution at pH 7.0, and the washing was repeated three times. Finally, phosphate buffer solution was added to obtain a concentrated solution with a dry weight of 2%, which is the microbial inoculant.
[0027] Preparation Example 2 The difference between Preparation Example 2 and Preparation Example 1 is that the acclimated nitrified sludge was centrifuged at 4000 r / min for 10 min, then the supernatant was removed, and the precipitate was washed with phosphate buffer at pH 7.0 three times. Finally, phosphate buffer was added to obtain a concentrated solution with a dry weight of 3.5%, which is the microbial inoculant. The remaining steps are the same as those in Preparation Example 1.
[0028] Preparation Example 3 The difference between Preparation Example 3 and Preparation Example 1 is that the acclimated nitrified sludge was centrifuged at 4000 r / min for 10 min, the supernatant was removed, and the precipitate was washed with phosphate buffer at pH 7.0 three times. Finally, phosphate buffer was added to obtain a concentrated solution with a dry weight of 5%, which is the microbial inoculant. The remaining steps are the same as those in Preparation Example 1. Example
[0029] Example 1 Example 1 provides a method for embedding and immobilizing microorganisms in 3D printing based on a dual-network hydrogel, comprising the following steps: Preparation of the first network premix: Polyvinyl alcohol and sodium alginate were heated to 90°C and dissolved in water. Then, nanomaterials were added, stirred and mixed evenly, and cooled to room temperature. Acrylamide, crosslinking agent, and microbial inoculant from Preparation Example 1 were added sequentially and mixed evenly to obtain the first network premix. In the first network premix: the mass-volume concentration of polyvinyl alcohol was 8%, the mass-volume concentration of sodium alginate was 0.5%, the mass-volume concentration of nanomaterials was 1%, the mass-volume concentration of acrylamide was 10%, the mass-volume concentration of crosslinking agent was 0.5%, and the volume concentration of microbial inoculant was 20%. The nanomaterials were nano-silica, and the crosslinking agent was N,N'-methylenebisacrylamide. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; wherein the mass-volume concentration of calcium chloride in the coagulation solution is 2%; 3D printing and first network formation: The first network premix liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing; the printed lines achieve preliminary cross-linking and shaping in the coagulation liquid through the instantaneous complexation of PVA and borate ions, and the immobilization and cross-linking is carried out for 2 hours to obtain a gel structure; the printing temperature is 25℃, the extrusion pressure is 0.3MPa, the printing speed is 15mm / s, and the diameter of the printing needle is 1.0mm; Second network curing: The gel structure is removed from the coagulation liquid and immersed in a solution containing an initiator to initiate polymerization. The polymerization is carried out at room temperature for 6 hours to form a stable double-network hydrogel. The initiator solution is a mixture of ammonium persulfate and water, and the mass-volume concentration of ammonium persulfate in the initiator solution is 0.1%. Post-processing: The double-network hydrogel is removed and washed with deionized water or phosphate buffer to obtain the final embedded microbial product.
[0030] Example 2 Example 2 provides a method for microbial 3D printing embedding and immobilization based on dual-network hydrogel, including the following steps: Preparation of the first network premix: Polyvinyl alcohol and sodium alginate were heated to 90°C and dissolved in water. Then, nanomaterials were added, stirred and mixed evenly, and cooled to room temperature. Acrylamide, crosslinking agent, and microbial inoculant from Preparation Example 1 were added sequentially and mixed evenly to obtain the first network premix. In the first network premix: the mass-volume concentration of polyvinyl alcohol was 9%, the mass-volume concentration of sodium alginate was 0.75%, the mass-volume concentration of nanomaterials was 2%, the mass-volume concentration of acrylamide was 12.5%, the mass-volume concentration of crosslinking agent was 1.3%, and the volume concentration of microbial inoculant was 10%. The nanomaterials were nano-silica, and the crosslinking agent was N,N'-methylenebisacrylamide. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; wherein the mass-volume concentration of calcium chloride in the coagulation solution is 2%; 3D printing and first network formation: The first network premix liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing; the printed lines achieve preliminary cross-linking and shaping in the coagulation liquid through the instantaneous complexation of PVA and borate ions, and the immobilization and cross-linking is carried out for 2 hours to obtain a gel structure; the printing temperature is 25℃, the extrusion pressure is 0.3MPa, the printing speed is 15mm / s, and the diameter of the printing needle is 1.0mm; Second network curing: The gel structure is removed from the coagulation liquid and immersed in a solution containing an initiator to initiate polymerization. The polymerization is carried out at room temperature for 6 hours to form a stable double-network hydrogel. The initiator solution is a mixture of ammonium persulfate and water, and the mass-volume concentration of ammonium persulfate in the initiator solution is 0.3%. Post-processing: The double-network hydrogel is removed and washed with deionized water or phosphate buffer to obtain the final embedded microbial product.
[0031] Example 3 Example 3 provides a method for microbial 3D printing embedding and immobilization based on dual-network hydrogel, including the following steps: Preparation of the first network premix: Polyvinyl alcohol and sodium alginate were heated to 90°C and dissolved in water. Then, nanomaterials were added, stirred and mixed evenly, and cooled to room temperature. Acrylamide, crosslinking agent, and microbial inoculant from Preparation Example 1 were added sequentially and mixed evenly to obtain the first network premix. In the first network premix: the mass-volume concentration of polyvinyl alcohol was 10%, the mass-volume concentration of sodium alginate was 1%, the mass-volume concentration of nanomaterials was 3%, the mass-volume concentration of acrylamide was 15%, the mass-volume concentration of crosslinking agent was 2%, and the volume concentration of microbial inoculant was 40%. The nanomaterials were nano-silica, and the crosslinking agent was N,N'-methylenebisacrylamide. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; wherein the mass-volume concentration of calcium chloride in the coagulation solution is 2%; 3D printing and first network formation: The first network premix liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing; the printed lines achieve preliminary cross-linking and shaping in the coagulation liquid through the instantaneous complexation of PVA and borate ions, and the immobilization and cross-linking is carried out for 2 hours to obtain a gel structure; the printing temperature is 25℃, the extrusion pressure is 0.3MPa, the printing speed is 15mm / s, and the diameter of the printing needle is 1.0mm; Second network curing: The gel structure is removed from the coagulation liquid and immersed in a solution containing an initiator to initiate polymerization. The polymerization is carried out at room temperature for 6 hours to form a stable double-network hydrogel. The initiator solution is a mixture of ammonium persulfate and water, and the mass-volume concentration of ammonium persulfate in the initiator solution is 0.5%. Post-processing: The double-network hydrogel is removed and washed with deionized water or phosphate buffer to obtain the final embedded microbial product.
[0032] Example 4 The difference between Example 4 and Example 1 lies in the preparation of the first network premix: polyvinyl alcohol and sodium alginate are heated to 90°C and dissolved in water. Then, nanomaterials are added, stirred and mixed evenly, cooled to room temperature, and acrylamide, crosslinking agent, and microbial inoculant from Example 2 are added sequentially and mixed evenly to obtain the first network premix. In the first network premix: the mass-volume concentration of polyvinyl alcohol is 8%, the mass-volume concentration of sodium alginate is 0.5%, the mass-volume concentration of nanomaterials is 1%, the mass-volume concentration of acrylamide is 10%, the mass-volume concentration of crosslinking agent is 0.5%, and the volume concentration of microbial inoculant is 20%. The nanomaterials are nano-silica, and the crosslinking agent is N,N'-methylenebisacrylamide. The remaining steps are consistent with those in Example 1.
[0033] Example 5 The difference between Example 5 and Example 1 lies in the preparation of the first network premix: polyvinyl alcohol and sodium alginate are heated to 90°C and dissolved in water. Then, nanomaterials are added, stirred and mixed evenly, cooled to room temperature, and acrylamide, crosslinking agent, and the microbial inoculant from Example 3 are added sequentially and mixed evenly to obtain the first network premix. In the first network premix: the mass-volume concentration of polyvinyl alcohol is 8%, the mass-volume concentration of sodium alginate is 0.5%, the mass-volume concentration of nanomaterials is 1%, the mass-volume concentration of acrylamide is 10%, the mass-volume concentration of crosslinking agent is 0.5%, and the volume concentration of microbial inoculant is 20%. The nanomaterials are nano-silica, and the crosslinking agent is N,N'-methylenebisacrylamide. The remaining steps are consistent with those in Example 1.
[0034] Comparative Example Comparative Example 1 Comparative Example 1 provides a method for embedding and immobilizing microorganisms in 3D printing, comprising the following steps: Preparation of the premix: Polyvinyl alcohol and sodium alginate were heated to 90°C and dissolved in water. Then, nanomaterials were added, stirred and mixed evenly, cooled to room temperature, and the microbial inoculant from Preparation Example 1 was added and mixed evenly to obtain the premix. In the premix, the mass-volume concentration of polyvinyl alcohol was 8%, the mass-volume concentration of sodium alginate was 0.5%, the mass-volume concentration of nanomaterials was 1%, and the volume concentration of microbial inoculant was 20%. The nanomaterials were nano-silica. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; wherein the mass-volume concentration of calcium chloride in the coagulation solution is 2%; 3D printing and molding: The premixed liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing; the printed lines achieve preliminary cross-linking and shaping in the coagulation liquid through the instantaneous complexation of PVA and borate ions, and the immobilization and cross-linking is carried out for 2 hours to obtain a gel structure; the printing temperature is 25℃, the extrusion pressure is 0.3MPa, the printing speed is 15mm / s, and the diameter of the printing needle is 1.0mm; Post-processing: The gel structure is removed and washed with deionized water or phosphate buffer to obtain the final embedded microbial product.
[0035] Performance testing stability: The embedded microbial product was placed in an Erlenmeyer flask containing distilled water and shaken in a shaker at 30℃ and 200 rpm. Appropriate water samples were taken periodically to measure its chemical oxygen demand (COD). COD is a chemical method used to measure the amount of reducing substances in a water sample that need to be oxidized, and then the loss (L) is calculated. oss , Where L oss The carrier loss per unit mass of product is expressed in mg / g; COD represents the carrier loss per unit volume in mg / L; V represents the volume of distilled water in L; and m represents the mass of the product in g. Lower loss indicates better chemical stability. Results are as follows: Figure 1 As shown, the experimental group is Example 1, and the control group is Comparative Example 1.
[0036] Mechanical strength: Under shaking conditions of 30℃ and 100r / min, the embedded microbial product was placed in 200ml of distilled water. The number of broken gel beads was observed and recorded, and the breakage rate was calculated. The lower the breakage rate, the higher the mechanical strength. Results are as follows: Figure 2 As shown, the experimental group is Example 1, represented by the gray line in the figure; the control group is Comparative Example 1, represented by the red line in the figure.
[0037] Nitrification performance: The encapsulated microbial product was placed in a 500 mL Erlenmeyer flask, and 150 mL of simulated wastewater containing NH4+-N (NH4+-N concentration of 50 mg / L) was added. The flask was then placed in a constant-temperature shaker at 200 r / min and 25 °C. Samples were taken every 0.5 h and measured using a spectrophotometer. Results are as follows: Figure 3 As shown, the experimental group was Example 1, and the control group was 2 g / L nitrified sludge.
[0038] Table 1. Performance test results of the products Combination Figure 1 , Figure 2 Example 1 and Comparative Example 1, Figure 1 The loss in the experimental group was significantly lower than that in the control group, and Figure 2 The breakage rate in the experimental group was also significantly lower than that in the control group. Thus, the embedded microbial product in Example 1 has good stability and mechanical strength. It can be seen that by using the 3D printing embedding and immobilization method in this application, the dual network structure of the first network and the second network combines the toughness of physical cross-linking and the strength of chemical cross-linking, resulting in a product that is not easy to break, swell or disintegrate, and has a long service life. Therefore, the stability and mechanical strength of the embedded product are improved.
[0039] Combination Figure 3 The nitrification rate of the control group was 21.7 mg / (L·h), while that of the experimental group was 17.7 mg / (L·h). The nitrification rate of the experimental group was slightly lower because boric acid and other substances have certain toxicity during the encapsulation process. However, the activity can be restored by long-term culture. The product has good stability and mechanical strength, as well as good nitrification effect.
[0040] Based on Examples 1-3, it can be seen that when performing the microbial 3D printing embedding and immobilization method, the embedding effect of microorganisms is better when preparing the first network premix and coagulation solution according to the raw material ratio in Examples 1-3.
[0041] Based on Examples 2, 4, and 5, it can be seen that when the dry weight of the microorganisms in the microbial inoculant is 2-5%, the performance of the encapsulated microbial product is better.
[0042] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A method for 3D printing and embedding microorganisms based on dual-network hydrogels, characterized in that: Includes the following steps: Preparation of the first network premix: Polyvinyl alcohol and sodium alginate were heated and dissolved in water, then nanomaterials were added, stirred and mixed evenly, cooled, and then acrylamide, crosslinking agent and microbial inoculant were added in sequence and mixed evenly to obtain the first network premix. Preparation of coagulation solution: Calcium chloride is dissolved in a saturated boric acid solution to obtain a coagulation solution; 3D printing and first network forming: The first network premix liquid is loaded into an extrusion 3D printer, and the print head is immersed in the coagulation liquid for printing to obtain a gel structure; Second network solidification: The gel structure is removed from the coagulation liquid and immersed in a solution containing an initiator to initiate polymerization and form a stable double network hydrogel; Post-processing: The double-network hydrogel was removed and cleaned to obtain the final embedded microbial product.
2. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 1, characterized in that: In the first network premixed solution: the mass volume concentration of polyvinyl alcohol is 8-10%, the mass volume concentration of sodium alginate is 0.5-1%, the mass volume concentration of nanomaterials is 1-3%, the mass volume concentration of acrylamide is 10-15%, the mass volume concentration of crosslinking agent is 0.5-2%, and the volume concentration of microbial inoculant is 10-40%.
3. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 2, characterized in that: The nanomaterials include nano-silica.
4. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 2, characterized in that: The crosslinking agent includes N,N'-methylenebisacrylamide.
5. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 2, characterized in that: The preparation method of the microbial inoculant includes the following steps: The acclimatized nitrified sludge was centrifuged at 4000 r / min for 10 min, then the supernatant was removed, and the precipitate was washed with phosphate buffer at pH 7.
0. Finally, phosphate buffer was added to obtain a concentrated solution with a dry weight of 2-5%, which is the microbial inoculant.
6. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 1, characterized in that: The mass-volume concentration of calcium chloride in the coagulation solution is 1.5-2.5%.
7. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 1, characterized in that: In the 3D printing and first network forming steps, the printing temperature is 20-25℃, the extrusion pressure is 0.1-0.5MPa, the printing speed is 5-20mm / s, and the diameter of the printing needle is 0.2-1.0mm.
8. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 1, characterized in that: In the second network curing step, the initiator in the initiator solution includes ammonium persulfate, and the mass-volume concentration is 0.1-0.5%.
9. The method for microbial 3D printing embedding and immobilization based on dual-network hydrogel according to claim 1, characterized in that: The loss of the encapsulated microbial product after 14 days of shaking was 18-20 mg / g.
10. The method for microbial 3D printing embedding and immobilization based on a dual-network hydrogel according to claim 1, characterized in that: The fragmentation rate of the encapsulated microbial product after 14 days of shaking was 5.5-7%.