Microcapsule type denitrification bacterial agent and preparation method thereof

Abstract

The present application relates to a kind of microcapsule type denitrification bacterial agent and its preparation method, specifically, the present application provides a kind of microcapsule type denitrification bacterial agent, adopts asymmetric double-layer wall material structure, inner layer wall material is the composite system of low concentration sodium alginate and mass transfer enhancer, outer layer wall material is the composite system of high concentration sodium alginate and chitosan, is prepared by step crosslinking process.The double-layer wall material structure of combination of inner layer low concentration mass transfer optimization layer and outer layer high concentration mechanical protection layer is constructed, and the concentration ratio of each layer wall material, crosslinking agent concentration and crosslinking time are accurately controlled, effectively solve the technical contradiction that microcapsule wall material mass transfer resistance and mechanical strength are difficult to consider in prior art, while keeping high embedding rate (≥85%) And good mechanical strength, significantly reduce wall material mass transfer resistance, improve denitrification efficiency (ammonia nitrogen removal rate ≥90%, total nitrogen removal rate ≥85%), can be widely applied in wastewater treatment, water ecological restoration and other fields.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a microcapsule denitrifying agent for wastewater treatment. This denitrifying agent has good mass transfer performance and mechanical strength, and its preparation method is simple. Background Technology

[0002] Biological denitrification technology has become one of the core technologies in the field of wastewater treatment due to its advantages such as environmental friendliness and low cost. Microbial immobilization technology, by encapsulating functional microorganisms in specific carriers, can effectively solve problems such as easy loss and poor stability of free bacterial agents. Among them, microencapsulation method has attracted much attention because it can provide a relatively independent microenvironment for microbial growth and achieve controlled release.

[0003] Sodium alginate (SA) is one of the most widely used microcapsule wall materials. Its guluronic acid (G) units on its molecular chain can react with Ca²⁺. + Divalent cations undergo ionic cross-linking reactions to form an "egg-box" model gel with a three-dimensional network structure. Chitosan (CS), the only alkaline polysaccharide found in nature, can form a polyelectrolyte composite membrane with the negatively charged carboxyl groups of sodium alginate, effectively improving the mechanical strength and density of microcapsules. Polyvinyl alcohol (PVA) has extremely high mechanical strength and chemical stability and is often used in combination with sodium alginate to improve carrier performance.

[0004] However, existing microencapsulated denitrifying bacterial agents still face significant technical challenges in terms of wall material selection and preparation processes: On the one hand, increasing the wall material concentration or extending the cross-linking time can improve mechanical strength and encapsulation efficiency, but it can also lead to excessive densification of the gel network, significantly increasing mass transfer resistance and limiting the diffusion of substrates (nitrogen sources) and metabolites into and out of the microcapsules, thereby reducing denitrification efficiency; on the other hand, reducing the amount of wall material can improve mass transfer efficiency, but it can also lead to problems such as decreased encapsulation efficiency, severe cell leakage, fragile microcapsule structure, and insufficient mechanical strength, affecting the actual application effect of the bacterial agent.

[0005] In the prior art, CN104887647B discloses a probiotic microcapsule and its preparation method, using 2%~3% sodium alginate and 0.1%~0.3% chitosan as the wall material. However, the wall material concentration is uniformly distributed, failing to resolve the contradiction between mass transfer resistance and mechanical strength. CN107260704A discloses a method for preparing microcapsules using a sodium alginate and konjac gum composite wall material, with a sodium alginate concentration of 2.5%, which also suffers from the problem of a single wall material structure and limited mass transfer performance. In addition, existing studies have reported that the PVA-SA composite carrier typically contains 6%~12% PVA and 0.5%~2% SA. Although the performance is improved to some extent through material composite, a uniform wall material structure is still used, which cannot fundamentally solve the aforementioned technical contradictions.

[0006] Therefore, developing a novel microencapsulated denitrifying bacterial agent that achieves a synergistic improvement in mass transfer performance and mechanical strength by adjusting the wall material structure is of significant scientific and engineering value for promoting the practical application of biological denitrification technology. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a microcapsule denitrifying bacterial agent with an asymmetric double-layer wall structure and its preparation method. The core innovation of this invention lies in: employing an asymmetric double-layer wall structure design, where the inner wall material uses a low-concentration formulation and incorporates a mass transfer enhancer to optimize mass transfer channels, while the outer wall material uses a high-concentration formulation to ensure mechanical strength and encapsulation efficiency. Through a stepwise crosslinking process, the structure of the inner and outer layers is precisely controlled, thereby significantly reducing wall material mass transfer resistance and improving denitrification efficiency while maintaining high encapsulation efficiency and good mechanical strength.

[0008] One of the objectives of this invention is to provide a microencapsulated denitrifying agent: The microcapsule denitrifying bacterial agent has a double-layered asymmetric wall structure and is prepared through a stepwise cross-linking process. The inner wall material is first pre-cross-linked to form the inner wall, and then the outer wall material is coated on the inner wall and cross-linked again. The inner wall material consists of: an inner wall material composed of sodium alginate with a mass volume concentration of 0.8% to 1.5% and a mass transfer enhancer, which is polyethylene glycol or xanthan gum with a mass volume concentration of 0.3% to 1.0%; and an outer wall material composed of sodium alginate with a mass volume concentration of 2.0% to 3.5% and chitosan with a mass volume concentration of 0.3% to 0.8%. The mass ratio of the inner and outer wall materials of the microcapsule is 1:1.5 to 1:3. In the above scheme, the mass-volume concentration of sodium alginate in the inner wall material is 1.0%~1.2%, the mass-volume concentration of polyethylene glycol is 0.5%~0.8%, or the mass-volume concentration of xanthan gum is 0.3%~0.5%.

[0009] In the above scheme, the mass-volume concentration of sodium alginate in the outer wall material is 2.5%~3.0%, and the mass-volume concentration of chitosan is 0.4%~0.6%.

[0010] In the above scheme, the inner wall material also contains polyvinyl alcohol with a mass-volume concentration of 0.5% to 2.0% to enhance the inner skeleton structure.

[0011] In the above scheme, the particle size of the microcapsules is 1.5mm~4.0mm, the thickness of the inner wall material is 30%~45% of the total wall material thickness, and the thickness of the outer wall material is 55%~70% of the total wall material thickness.

[0012] Another objective of this invention is to provide a method for preparing a microencapsulated denitrifying bacterial agent: the specific steps are as follows, (1) Preparation of bacterial suspension: The denitrifying functional strain was inoculated into liquid culture medium and cultured for 18h~36h. The bacterial cells were collected by centrifugation and resuspended in physiological saline to prepare a suspension with a concentration of 1.0×10⁻⁶. 9 CFU / mL ~1.0×10 11 CFU / mL bacterial suspension; (2) Preparation of inner wall material solution: Dissolve sodium alginate in deionized water to prepare a sodium alginate solution with a mass-volume concentration of 0.8%~1.5%, add polyethylene glycol or xanthan gum and stir evenly, sterilize and cool to 30℃~40℃; (3) Preparation of outer wall material solution: Sodium alginate is dissolved in deionized water to prepare a sodium alginate solution with a mass-volume concentration of 2.0%~3.5% and chitosan is dissolved in acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.3%~0.8%. Both are sterilized and used for later use. (4) Preparation of inner layer microcapsules: The bacterial suspension obtained in step (1) and the inner wall material solution obtained in step (2) are mixed in proportion and stirred evenly. Then, the mixture is dripped into the CaCl2 pre-crosslinked solution by dripping or injection to form pre-crosslinked inner layer microcapsules with porous structure. (5) Outer layer coating and final crosslinking: The pre-crosslinked inner layer microcapsules obtained in step (4) are transferred into the outer wall material solution obtained in step (3) and soaked for 5 min to 10 min to allow the outer wall material to fully adhere. Then, they are dropped into the CaCl2 final crosslinking solution to form microcapsules with an asymmetric double wall material structure. (6) Post-processing: Wash the microcapsules obtained in step (5) with sterile physiological saline 2-3 times and store them at 4°C for later use.

[0013] In the above scheme, the mass-volume concentration of polyethylene glycol or xanthan gum in step (2) is 0.3%~1.0%.

[0014] In the above scheme, the mass volume concentration of the CaCl2 pre-crosslinked solution in step (4) is 1.2%~1.5%, and the crosslinking time is 8min~12min.

[0015] In the above scheme, the mass volume concentration of the CaCl2 final crosslinking solution in step (5) is 3.5%~4.5%, and the crosslinking time is 20min~25min; when the outer layer is coated, the volume ratio of the pre-crosslinked inner layer microcapsule to the outer wall material solution is 1:3 to 1:6.

[0016] In the above scheme, the denitrifying functional strain is one or a mixed strain of Pseudomonas stutzeri, Acinetobacter pittii, and Marinobacter sp.

[0017] The beneficial effects of the technical solution provided by this invention include at least the following: (1) This invention utilizes an asymmetric double-layer wall material structure design. The inner layer, with a low-concentration wall material (0.8%~1.5% sodium alginate) and a mass transfer enhancer, effectively reduces mass transfer resistance, allowing ammonia nitrogen and nitrate nitrogen to diffuse smoothly into the microcapsule. The outer layer, with a high-concentration wall material (2.0%~3.5% sodium alginate + 0.3%~0.8% chitosan), provides sufficient mechanical protection, ensuring an encapsulation rate ≥85% and a microcapsule breakage rate <5%. Furthermore, the reduced mass transfer resistance allows the denitrifying bacteria within the microcapsule to obtain a sufficient substrate supply, and metabolic products can be promptly excreted, maintaining high metabolic activity. The ammonia nitrogen removal rate of the microcapsule denitrifying bacteria agent of this invention is ≥90%, and the total nitrogen removal rate is ≥85%, which is 15%~25% higher than that of traditional uniform wall material microcapsules.

[0018] (2) The dense outer wall material effectively prevents the leakage of bacteria. After 10 batches of continuous use, the denitrification efficiency of the microcapsules still remains above 80% of the initial efficiency, which has good operational stability and enhances the stability and reusability of the bacterial agent.

[0019] (3) The preparation process is simple and the cost is controllable: the raw materials used in this invention are all commercially available conventional products, and the preparation process does not require special equipment, making it easy to scale up production. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0021] Example 1: Preparation of microencapsulated denitrifying bacterial agent (1) Preparation of bacterial suspension: Pseudomonas stutzeri was inoculated into beef extract peptone liquid medium and cultured at 30℃ and 150 rpm for 24 h. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 5.0 × 10⁻⁶. 9 CFU / mL.

[0022] (2) Preparation of inner wall material solution: Weigh 1.0g sodium alginate and dissolve it in 100mL deionized water to prepare a sodium alginate solution with a mass volume concentration of 1.0%. Add 0.5g polyethylene glycol (PEG-4000) to make its mass volume concentration 0.5%. Stir evenly, sterilize at 121℃ for 15min, and cool to 35℃.

[0023] (3) Preparation of outer wall material solution: Weigh 2.5g of sodium alginate and dissolve it in 100mL of deionized water to prepare a sodium alginate solution with a mass-volume concentration of 2.5%; separately weigh 0.5g of chitosan (degree of deacetylation ≥90%) and dissolve it in 100mL of 1.0% acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%. Sterilize at 115℃ for 20min and cool for later use.

[0024] (4) Preparation of inner layer microcapsules: Mix 20 mL of bacterial suspension with 60 mL of inner layer wall material solution, stir evenly, and then drop 10 mL of sterile syringe into 200 mL of CaCl2 pre-crosslinking solution with a mass volume concentration of 1.5%. Stir magnetically and crosslink for 10 min to form pre-crosslinked inner layer microcapsules.

[0025] (5) Outer layer coating and final cross-linking: The pre-cross-linked inner layer microcapsules were transferred into 150 mL of outer wall material solution (sodium alginate and chitosan solution were mixed at a volume ratio of 1:1), soaked for 8 min, and then dripped into 300 mL of CaCl2 final cross-linking solution with a mass volume concentration of 4.0% using a sterile syringe, and stirred for cross-linking for 20 min.

[0026] (6) Post-processing: The obtained microcapsules were washed three times with sterile physiological saline and stored at 4°C.

[0027] The obtained microcapsules were regularly spherical with a particle size of 2.5 mm ± 0.3 mm, an encapsulation rate of 88.5%, and a mechanical strength (compressive strength) of 0.85 MPa. In a simulated wastewater denitrification experiment, with an initial ammonia nitrogen concentration of 50 mg / L, the ammonia nitrogen removal rate was 92.3% and the total nitrogen removal rate was 87.6% after 24 hours. After 10 consecutive batches, the ammonia nitrogen removal rate remained at 85.2%.

[0028] Example 2: The difference between this embodiment and Embodiment 1 is that the mass transfer enhancer in the inner wall material is xanthan gum.

[0029] (2) Preparation of inner wall material solution: Weigh 1.2g sodium alginate and dissolve it in 100mL deionized water, add 0.4g xanthan gum to make its mass-volume concentration 0.4%, stir evenly, sterilize at 121℃ for 15min, and cool to 35℃.

[0030] The remaining steps were the same as in Example 1. The resulting microcapsules had a particle size of 2.3 mm ± 0.2 mm, an encapsulation efficiency of 86.2%, and a mechanical strength of 0.92 MPa. The ammonia nitrogen removal rate was 90.8% and the total nitrogen removal rate was 85.3% after 24 hours.

[0031] Example 3: The difference between this embodiment and Embodiment 1 lies in the concentration of the outer wall material.

[0032] (3) Preparation of outer wall material solution: Weigh 3.0g sodium alginate and dissolve it in 100mL deionized water to prepare a sodium alginate solution with a mass-volume concentration of 3.0%; Weigh 0.6g chitosan and dissolve it in 100mL 1.0% acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.6%.

[0033] The remaining steps were the same as in Example 1. The resulting microcapsules had a particle size of 2.8 mm ± 0.3 mm, an encapsulation efficiency of 90.1%, and a mechanical strength of 1.15 MPa. The ammonia nitrogen removal rate was 91.5% and the total nitrogen removal rate was 86.8% after 24 hours.

[0034] Comparative Example 1: Traditional uniform wall material microcapsules were prepared according to the existing technology (CN104887647B): the bacterial suspension was uniformly mixed with a 2.5% sodium alginate + 0.2% chitosan wall material solution, and then dropped into a 2.0% CaCl2 solution for cross-linking for 20 min.

[0035] The resulting microcapsules had an encapsulation rate of 82.3% and a mechanical strength of 0.78 MPa. The 24-hour ammonia nitrogen removal rate was 72.5%, and the total nitrogen removal rate was 68.4%. After 10 consecutive batches, the ammonia nitrogen removal rate decreased to 58.6%.

[0036] Comparative Example 2: The bacterial suspension was uniformly mixed with a 1.0% sodium alginate wall material solution and then added dropwise to a 1.5% CaCl2 solution for cross-linking for 10 min. The resulting microcapsules had an encapsulation efficiency of only 45.2% and a mechanical strength of 0.25 MPa.

[0037] Comparative Example 3: The bacterial suspension was mixed with a homogeneous wall material solution of 3.5% sodium alginate + 0.8% chitosan, and then added dropwise to a 5.0% CaCl2 solution for cross-linking for 40 min. The resulting microcapsules had an encapsulation efficiency of 91.2% and a mechanical strength of 1.45 MPa.

[0038] Mass transfer resistance measurement experiment To quantitatively evaluate the mass transfer performance of the microcapsules of this invention, the effective diffusion coefficient (De) and mass transfer enhancement factor (Ef) of ammonia nitrogen and nitrate nitrogen were determined by static diffusion method.

[0039] Assay method: Place the microcapsules (30g) to be tested in 500mL of NH4+ containing 50mg / L. + -N and 50 mg / L NO3 - In simulated wastewater containing -N, the microcapsules were shaken at 30℃ and 120 rpm, and samples were taken every 2 hours to measure the changes in nitrogen concentration inside and outside the solutions. The effective diffusion coefficient De was calculated using Fick's second law, and the mass transfer enhancement factor Ef was defined as the ratio of De of the asymmetric bilayer wall microcapsules to De of the conventional homogeneous wall microcapsules under the same conditions.

[0040] The measurement results are shown in the table below: Table 1 The results show that the NH4 in the wall material microcapsules of this invention + -N and NO3 - The effective diffusion coefficients of -N were superior to those of traditional homogeneous wall material microcapsules (Comparative Example 1), and the mass transfer enhancement factor was significantly greater than 1. Although in Comparative Example 2, NH4 + -N and NO3 - The effective diffusion coefficient and mass transfer enhancement factor of -N are superior to those of the present invention, but this is mainly due to excessive permeation caused by the low content of wall material, and the microcapsule structure is unstable and cannot be reused.

[0041] Example 4: Effect of different mass transfer enhancer concentrations on microcapsule performance Microcapsules were prepared according to the method in Example 1, with only the mass-volume concentration of PEG added being changed, and six levels were set: 0% (no PEG), 0.3%, 0.5%, 0.8%, 1.0%, and 1.5%.

[0042] The experimental results are shown in the table below: Table 2 The results showed that the microcapsules exhibited the best overall performance when the PEG concentration was between 0.5% and 0.8% (by volume): a high mass transfer coefficient, mechanical strength maintained above 0.80 MPa, encapsulation efficiency maintained above 85%, and ammonia nitrogen removal rate exceeding 90%. When the PEG concentration was below 0.3% (by volume), the mass transfer enhancement effect was not significant; when the PEG concentration exceeded 1.0% (by volume), although the mass transfer coefficient further improved, the inner gel skeleton became too porous, leading to a significant decrease in mechanical strength.

[0043] Example 5: Effect of different types of mass transfer enhancers on microcapsule performance Microcapsules were prepared using polyethylene glycol (PEG-4000), xanthan gum (XG), polyvinylpyrrolidone (PVP), and without mass transfer enhancer (blank control), respectively, according to the method in Example 1. The mass transfer enhancer concentration was uniformly 0.5%.

[0044] Table 3 The results showed that PEG and xanthan gum were the best mass transfer enhancers. PEG-4000 was slightly better than xanthan gum in terms of mass transfer coefficient and denitrification efficiency, while PVP had a relatively weak mass transfer enhancement effect.

[0045] Example 6: Effect of Inner Wall Material Concentration on Microcapsule Performance The outer wall material is fixed at 2.5% sodium alginate + 0.5% chitosan, and the mass transfer enhancer is 0.5% PEG-4000. Only the concentration of sodium alginate in the inner layer is changed, with six levels set at 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, and 2.0%.

[0046] The experimental results are shown in the table below: Table 4 The results showed that the microcapsules exhibited the best overall performance when the sodium alginate concentration in the inner layer was within the range of 1.0% to 1.2%. When the inner layer concentration was below 0.8%, the mechanical strength was insufficient and the encapsulation efficiency decreased; when the inner layer concentration was above 1.5%, the mass transfer resistance increased, leading to a significant decrease in denitrification efficiency.

[0047] Example 7: Effect of outer wall material concentration on microcapsule performance The wall material was fixed at 1.0% sodium alginate + 0.5% PEG-4000, and only the concentration of the outer wall material was changed, with six experimental groups set up.

[0048] The experimental results are shown in the table below: Table 5 The results showed that the microcapsules exhibited the best overall performance when the outer layer sodium alginate concentration was in the range of 2.5%–3.0% and the chitosan concentration was in the range of 0.4%–0.6%. When the outer layer concentration was too low (<2.0%), the mechanical strength and encapsulation efficiency were insufficient; when the outer layer concentration was too high (>3.5%), the mass transfer resistance increased and the denitrification efficiency decreased.

[0049] Comparative Example 4: The inner layer uses a high-concentration wall material (3.0% sodium alginate), and the outer layer uses a low-concentration wall material (1.0% sodium alginate + 0.5% chitosan). The remaining preparation steps are the same as in Example 1.

[0050] The resulting microcapsules had an encapsulation efficiency of 75.3% and a mechanical strength of 0.42 MPa. (NH4) + The effective diffusion coefficient of -N is 2.78 × 10⁻⁶. -13 m 2 / s, the ammonia nitrogen removal rate over 24 hours was 79.2%, and the total nitrogen removal rate was 74.6%. These results indicate that when a high-concentration wall material is placed in the inner layer and a low-concentration wall material is placed in the outer layer, although the mass transfer coefficient is similar to that of Example 1, the mechanical strength is significantly reduced (only 49.4% of that of Example 1).

[0051] The experimental data above show that the asymmetric double-layer wall material structure microcapsule denitrifying bacterial agent of the present invention is significantly superior to traditional uniform wall material microcapsules (Comparative Example 1) and low-concentration wall material microcapsules (Comparative Example 2) in terms of encapsulation efficiency, mechanical strength, mass transfer performance, and denitrification efficiency. The addition of the mass transfer enhancer can significantly improve the effective diffusion coefficient of the microcapsules (by 25% to 55%), thereby directly improving the denitrification efficiency (ammonia nitrogen removal rate increased by 6% to 10%). The effect is best when the PEG concentration is 0.5% to 0.8%. The concentration ratio of the inner and outer wall materials has a decisive influence on the performance of the microcapsules: the sodium alginate concentration of the inner layer is preferably 1.0% to 1.2%, and that of the outer layer is preferably 2.5% to 3.0%. Reversing the inner and outer layer concentrations (Comparative Example 5) will lead to a significant decrease in mechanical strength. The present invention solves the technical contradiction of difficulty in balancing wall material mass transfer resistance and mechanical strength in the prior art through the asymmetric double-layer wall material structure design and the synergistic effect of the mass transfer enhancer.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microencapsulated denitrifying agent, characterized in that: The microcapsule denitrifying agent has a double-layer asymmetric wall structure and is prepared through a stepwise cross-linking process. The inner wall material is first pre-crosslinked to form the inner wall, and then the outer wall material is coated onto the inner wall and crosslinked again. This includes: Inner wall material: composed of sodium alginate with a mass volume concentration of 0.8%~1.5% and a mass transfer enhancer, wherein the mass transfer enhancer is polyethylene glycol or xanthan gum, and the mass volume concentration of the mass transfer enhancer is 0.3%~1.0%; The outer wall material is composed of sodium alginate with a mass-volume concentration of 2.0% to 3.5% and chitosan with a mass-volume concentration of 0.3% to 0.8%; the mass ratio of the inner layer to the outer wall material of the microcapsule is 1:1.5 to 1:

3.

2. The microencapsulated denitrifying bacterial agent according to claim 1, characterized in that, The inner wall material contains sodium alginate at a mass-volume concentration of 1.0% to 1.2%, polyethylene glycol at a mass-volume concentration of 0.5% to 0.8%, or xanthan gum at a mass-volume concentration of 0.3% to 0.5%.

3. The microencapsulated denitrifying bacterial agent according to claim 1, characterized in that, The outer wall material contains sodium alginate at a mass-volume concentration of 2.5% to 3.0% and chitosan at a mass-volume concentration of 0.4% to 0.6%.

4. The microencapsulated denitrifying bacterial agent according to claim 1, characterized in that, The inner wall material also contains polyvinyl alcohol at a mass-volume concentration of 0.5% to 2.0% to enhance the inner skeleton structure.

5. The microencapsulated denitrifying bacterial agent according to claim 1, characterized in that, The microcapsules have a particle size of 1.5 mm to 4.0 mm, an inner wall material thickness of 30% to 45% of the total wall material thickness, and an outer wall material thickness of 55% to 70% of the total wall material thickness.

6. The method for preparing the microencapsulated denitrifying bacterial agent according to any one of claims 1-5, characterized in that: The specific steps are as follows: (1) Preparation of bacterial suspension: The denitrifying functional strain was inoculated into liquid culture medium and cultured for 18h~36h. The bacterial cells were collected by centrifugation and resuspended in physiological saline to prepare a suspension with a concentration of 1.0×10⁻⁶. 9 CFU / mL ~1.0×10 11 CFU / mL bacterial suspension; (2) Preparation of inner wall material solution: Dissolve sodium alginate in deionized water to prepare a sodium alginate solution with a mass-volume concentration of 0.8%~1.5%, add polyethylene glycol or xanthan gum and stir evenly, sterilize and cool to 30℃~40℃; (3) Preparation of outer wall material solution: Sodium alginate is dissolved in deionized water to prepare a sodium alginate solution with a mass-volume concentration of 2.0%~3.5% and chitosan is dissolved in acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.3%~0.8%. Both are sterilized and used for later use. (4) Preparation of inner layer microcapsules: The bacterial suspension obtained in step (1) and the inner wall material solution obtained in step (2) are mixed in proportion and stirred evenly. Then, the mixture is dripped into the CaCl2 pre-crosslinked solution by dripping or injection to form pre-crosslinked inner layer microcapsules with porous structure. (5) Outer layer coating and final crosslinking: The pre-crosslinked inner layer microcapsules obtained in step (4) are transferred into the outer wall material solution obtained in step (3) and soaked for 5 min to 10 min to allow the outer wall material to fully adhere. Then, they are dropped into the CaCl2 final crosslinking solution to form microcapsules with an asymmetric double wall material structure. (6) Post-processing: Wash the microcapsules obtained in step (5) with sterile physiological saline 2-3 times and store them at 4°C for later use.

7. The preparation method according to claim 6, characterized in that, The mass-volume concentration of polyethylene glycol or xanthan gum mentioned in step (2) is 0.3%~1.0%.

8. The preparation method according to claim 6, characterized in that, The mass-volume concentration of the CaCl2 pre-crosslinked solution in step (4) is 1.2%~1.5%, and the crosslinking time is 8min~12min.

9. The preparation method according to claim 6, characterized in that, In step (5), the mass-volume concentration of the CaCl2 final crosslinking solution is 3.5%~4.5%, and the crosslinking time is 20min~25min; when coating the outer layer, the volume ratio of the pre-crosslinked inner layer microcapsule to the outer wall material solution is 1:3 to 1:

6.

10. The preparation method according to claim 6, characterized in that, The denitrifying strain is a mixture of one or more of Pseudomonas, Acinetobacter, and Marinebacterium.

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