Microorganism immobilization material, method for preparing the same, and method for immobilizing microbial inoculants

Abstract

This invention discloses a microbial immobilization material and its preparation method, as well as a method for immobilizing microbial agents, belonging to the field of microbial technology. The specific steps involve uniformly mixing activated carbon powder, flour, and a protective agent in a mass ratio of 3-5:1:1, and then using the microbial immobilization material for microbial agent immobilization. The microbial immobilization material of this invention, by providing nutrients (flour, glucose, skim milk powder) and a stable microenvironment (the inclusion effect of β-cyclodextrin and the adsorption and immobilization by activated carbon), can significantly delay the activity decay of microbial strains during storage.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a microbial immobilization material and its preparation method and a method for immobilizing microbial agents. Background Technology

[0002] With the rapid development of industrialization and urbanization, water pollution has become an increasingly serious problem, and bioremediation technology has become a research hotspot due to its environmental friendliness and low cost. The core of microbial remediation technology lies in maintaining the high activity of microbial agents during storage, transportation, and application. However, existing microbial agents are often susceptible to contamination by other microorganisms and environmental factors under non-sterile conditions, resulting in problems such as rapid decline in activity, short shelf life, and poor dispersibility in water.

[0003] Currently used immobilization carriers (such as sodium alginate, polyvinyl alcohol gels, etc.) suffer from drawbacks such as high mass transfer resistance, complex preparation processes, or damage to bacterial cells. Simple adsorption carriers (such as activated carbon) often lack the function of protecting bacterial activity and are prone to sedimentation in water, resulting in short shelf-life of the bacterial agent. Therefore, developing an immobilization material and method that combines good water dispersibility, effective protection of bacterial activity, long shelf life, and simple preparation is of great significance for promoting the practical application of microbial remediation technology. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this invention provides a microbial immobilization material and its preparation method, as well as a bacterial agent immobilization method. The microbial immobilization material prepared by this method can effectively protect the activity of microorganisms and has good dispersibility in water. The bacterial agent immobilization method obtained can extend the shelf life of the bacterial agent under non-sterile conditions.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: a method for preparing microbial immobilization material is provided, the innovation of which is: to uniformly mix activated carbon powder, flour and protective agent in a mass ratio of 3-5:1:1.

[0006] Preferably, the protective agent is prepared by mixing glucose, skim milk powder and β-cyclodextrin.

[0007] Preferably, in the protective agent, the mass ratio of glucose, skim milk powder and β-cyclodextrin is 2-4:1:1.

[0008] This invention also provides a microbial immobilization material prepared based on the above-described preparation method. The microbial immobilization material of this invention utilizes a synergistic effect of a ternary composite system. Activated carbon provides a large specific surface area for adsorbing and immobilizing microorganisms, while flour and a protective agent provide the microorganisms with carbon and nitrogen sources and other nutrients and protection during storage, together forming a microenvironment conducive to microbial survival.

[0009] This invention also provides a method for immobilizing microbial agents based on the above-mentioned microbial immobilization materials, the innovation of which lies in the following steps: (1) Cultivate the bacterial culture to OD 600 It is 1.0; (2) Add sodium thiamycin to the bacterial culture in step (1); (3) The bacterial solution containing sodium thiamycin obtained in step (2) is mixed with the microbial fixation material to obtain an immobilized bacterial agent.

[0010] Preferably, the method for obtaining the bacterial solution in step (1) is as follows: take 1 mL of the bacterial strain and transfer it to 100 mL of culture medium, and culture it under suitable conditions to obtain the cultured bacterial solution.

[0011] Furthermore, the OD of the bacterial solution 600 The method to adjust the value to 1.0 is as follows: If the bacterial culture OD 600 If the value is greater than 1.0, dilute the bacterial culture with culture medium to OD. 600 The value is 1.0; if the bacterial culture OD 600 If the value is less than 1.0, continue incubation on a shaker until the OD value of the bacterial culture is greater than or equal to 1.0 (Note: OD values ​​are measured by sampling). 600 (Performed under aseptic conditions).

[0012] The above method can quickly and accurately measure the OD of bacterial culture in the experiment. 600 The values ​​were adjusted to a uniform value to provide bacterial solutions with consistent concentration and stable state for subsequent experiments and activity detection, ensuring the reliability of experimental results and the consistency of activity detection.

[0013] Preferably, in (2), sodium thiamycin is added to the bacterial solution at a ratio of 5-25 g / L to prolong the storage time of the immobilized bacterial agent.

[0014] Preferably, in (3), the solid-liquid ratio of the microbial fixation material and the natamycin-containing bacterial solution in the immobilized bacterial agent is 2-4 g: 2 mL.

[0015] Preferably, the immobilized bacterial agent is stored at 2-8°C.

[0016] This invention provides a microbial immobilization material and its preparation method, as well as a method for immobilizing microbial agents, which has the following beneficial effects: (1) High storage stability: This invention adds sodium natamycin to the microbial fixation material. As a polyene antifungal agent, sodium natamycin binds to ergosterol in the fungal cell membrane, destroys membrane permeability and inhibits spore germination, and has a highly selective inhibitory effect on molds. Combined with low temperature storage, it reduces the metabolic rate and enzyme reaction rate of all microorganisms. However, the target bacteria maintain basic energy balance due to the "metabolic buffer state", while the miscellaneous bacteria have difficulty establishing a dominant population under the dual pressure of low temperature and antibacterial agent, forming a selective ecological barrier. Under non-strict aseptic operation conditions, it can effectively inhibit the growth of miscellaneous bacteria (especially molds), greatly extending the shelf life and practical application window of the agent.

[0017] (2) Superior Activity Protection: The microbial immobilization material of this invention provides nutrients (flour, glucose, and skim milk powder can maintain the basal metabolism and energy balance of the bacteria under low water activity conditions) and a stable microenvironment (the inclusion effect of β-cyclodextrin can reduce the damage of lipid oxidation products and metabolic byproducts to cell membranes and proteins, and the adsorption and immobilization of activated carbon can adsorb metabolic products, reactive oxygen species, and inhibitory small molecules and regulate the local oxygen diffusion environment). These factors work synergistically to change the physiological state of the bacteria during storage, significantly delaying the activity decay of the strain during storage. The target bacteria maintain basal metabolism and energy balance in the microenvironment composed of the above materials, forming a low-activity stable state, i.e., a metabolic buffer state.

[0018] (3) Good water dispersibility: The immobilized bacterial agent prepared by the microbial immobilization material of the present invention can be rapidly and uniformly dispersed in water and can spread rapidly in the aquatic environment without the need for complex equipment and additional energy consumption. While ensuring activity, it increases the convenience of bacterial storage and reduces application costs. This is beneficial for the application of the bacterial agent in water remediation sites, ensuring full contact between microorganisms and pollutants and improving remediation efficiency. It is suitable for large-scale water pollution remediation projects and has good economic efficiency and operability.

[0019] (4) Simple preparation and application: Material preparation and microbial agent immobilization can be carried out under non-strict aseptic conditions. The process does not require expensive equipment or complex procedures. The conditions are mild and the operation is simple, which is conducive to industrial production and field promotion and application.

[0020] (5) Environmentally friendly and low cost: All raw materials of this invention are common, safe, biodegradable or environmentally friendly substances. The overall solution is low in cost and has significant economic and environmental benefits. Attached Figure Description

[0021] Figure 1 This is a microscopic illustration of activated carbon adsorbing bacteria and nutrients.

[0022] Figure 2 This is a flowchart of the preparation process of the immobilized bacterial agent of the present invention.

[0023] Figure 3 This is a test chart showing the dispersibility of immobilized materials in water.

[0024] Figure 4 This is a graph showing the test results of the effects of immobilization materials and protective agents on the activity of bacterial strains.

[0025] Figure 5 The graph shows the test results of the effect of contaminating bacteria on the immobilization effect under non-sterile conditions. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described.

[0027] Example 1 (In this example, except for the culture and collection of bacteria, which are performed under aseptic conditions, the remaining steps are prepared under non-strict aseptic conditions) In this embodiment, 0.3g of glucose, 0.1g of skim milk powder, and 0.1g of β-cyclodextrin were weighed and mixed evenly in a mass ratio of 3:1:1 to prepare a protective agent. 2g of activated carbon powder, 0.5g of flour, and 0.5g of the protective agent were weighed and mixed evenly in an Erlenmeyer flask in a mass ratio of 4:1:1 to obtain the microbial fixation material.

[0028] In this embodiment, the activated carbon powder is analytical grade 200 mesh activated carbon powder from Zhonglian Reagent; the flour is ordinary wheat flour; the glucose is anhydrous glucose (analytical grade); the β-cyclodextrin is Aladdin drug, used for cell culture, with a purity ≥98%; and the skim milk powder is skim milk powder from Duyou Biotechnology with a purity of 99.0%.

[0029] The above-mentioned microbial fixation materials are immobilized with bacterial agents, such as... Figure 2 As shown, it includes the following steps: (1) Provide bacterial culture and measure its optical density OD 600 Adjust the value to 1.0; 1 mL of WZ bacteria (short bacillus) was transferred to 100 mL of LB medium and cultured at 30°C and 180 rpm for 10 h to obtain a cultured WZ bacterial suspension. The LB medium consisted of the following components: 5.0 g of yeast extract, 10.0 g of trypsinized peptone, and 10.0 g of sodium chloride; 1 L of ultrapure water was added and the pH was adjusted to 7.0. The medium was then sterilized at 121°C for 20–25 min. The cultured bacterial solution was preserved using the glycerol preservation method. The steps of the glycerol preservation method are as follows: the bacterial solution is mixed with 50% glycerol at a volume ratio of 1:1 and stored in a refrigerator at -80℃ for later use. The OD of the cultured or stored WZ bacterial solution will be taken.600 Adjust the value to 1.0; if the OD of WZ bacteria... 600 If the value is greater than 1.0, dilute the bacterial culture with LB medium to obtain the OD value of WZ bacteria. 600 The value is 1.0; if the bacterial OD... 600 If the OD value is less than 1.0, continue incubation on a shaker until the OD value of the bacterial culture equals 1.0 (Note: OD values ​​are measured by sampling). 600 (Performed under aseptic conditions).

[0030] (2) Add 0.03g of sodium thiamycin to 2mL of the adjusted bacterial culture; (3) Mix 2 mL of the bacterial solution containing sodium thiamycin obtained in step (2) with 3 g of microbial fixation material in an Erlenmeyer flask to obtain the immobilized bacterial agent.

[0031] (4) The immobilized bacterial agent was stored at a low temperature of 4°C. The resulting immobilized bacterial agent was named WZ-3.

[0032] The order of preparation of the natamycin-containing bacterial solution and the microbial fixation material in this invention can be different; the microbial fixation material can be prepared first, or the natamycin-containing bacterial solution can be prepared first.

[0033] The immobilized bacterial agent prepared in Example 1 was subjected to a water dispersibility test to verify the water solubility of the immobilized material. The process is as follows: The experimental setup consisted of a 200 mL beaker. 100 mL of water was added to the beaker, and 1 g of immobilized bacterial agent was weighed and dissolved in the water. After stirring thoroughly, the liquid was poured into a disposable agar plate for observation. The microscopic display of activated carbon adsorbing bacterial cells and nutrients is shown in the image below. Figure 1 As shown.

[0034] The dispersibility test results of the immobilized material in water are as follows: Figure 3 As shown, by Figure 3 It is known that the immobilized bacterial agent is evenly dispersed in water and has good water dispersibility, enabling it to spread rapidly in the aquatic environment without the need for complex equipment or additional energy consumption. While ensuring activity, it increases the convenience of bacterial storage and reduces application costs, making it suitable for large-scale water pollution remediation projects. It has good economic efficiency and operability.

[0035] Example 2 The difference between this embodiment and embodiment 1 is that the amount of sodium thiamycin added in step (2) is 0.01g.

[0036] The rest is the same as in Example 1, and the resulting immobilized bacterial agent is named WZ-4.

[0037] Example 3 The difference between this embodiment and embodiment 1 is that the amount of sodium thiamycin added in step (2) is 0.05g.

[0038] The rest is the same as in Example 1, and the obtained immobilized bacterial agent is named WZ-5.

[0039] Comparative Example 1 The difference between this comparative example and Example 1 is that: no microbial fixation material was prepared; instead, pure bacterial solution was directly prepared and sodium thiamycin was added. The prepared result was named WZ-1. The preparation process was the same as in Example 1.

[0040] Comparative Example 2 The difference between this comparative example and Example 1 is that no protective agent is added when preparing the microbial fixation material. 2 g of activated carbon powder (200 mesh) and 0.5 g of flour are weighed into an Erlenmeyer flask in a 4:1 ratio, mixed evenly, and the microbial fixation material is prepared.

[0041] The remaining steps are the same as in Example 1. The immobilized bacterial agent prepared in this example is named WZ-2.

[0042] Activity assays for WZ-1, WZ-2, and WZ-3 immobilized bacteria: The activity assay was performed using the dilution plating method. 1 g of each of the immobilized bacterial agents WZ-1, WZ-2, and WZ-3 were dissolved in 10 mL of physiological saline. The bacterial strains were eluted from the activated carbon by repeated pipetting with a syringe. Then, 1 mL of the suspension was added to 9 mL of physiological saline, and the process was repeated with pipetting. This dilution was repeated until a final dilution of 10 mL was achieved. -6 Take 0.1 mL of the diluted solution and spread it on an LB solid plate medium until the plate is completely dry. Place it in a constant temperature incubator at 30℃ for incubation and observation.

[0043] result Figure 4 As shown, Figure 4 CK was the blank control group (i.e., the group with blank culture medium), WZ-1 was the pure bacterial culture group (i.e., the group without immobilization material), WZ-2 was the experimental group (the group without protective agent), and WZ-3 was the experimental group (the group with immobilization material with protective agent).

[0044] In each treatment group, no bacteria grew in the blank control group, and after two weeks of storage, activity testing showed that the number of colonies in the WZ-1 group was approximately 1×10⁻⁶. 8 CFU / g, the number of colonies in group WZ-2 was approximately 3 × 10⁻⁶. 9 CFU / g, at four weeks, WZ-1 and WZ-2 groups were diluted to 10. -6 The drawing board showed almost no bacterial colonies, indicating that the activity of the bacterial strain decreased over time, with the activity of the pure bacterial solution decreasing more rapidly. Immobilizing the bacteria with immobilization materials can maintain the activity of the bacterial strain to a certain extent and slow down the rate of decline in bacterial activity.

[0045] The colony count in group WZ-3 was approximately 3 × 10⁻⁶ during the two-week activity assay.10 The CFU / g count was higher than that of group WZ-2. In the fourth week of activity testing, group WZ-2 was diluted to 10... -6 There were almost no colonies in the first group, while the number of colonies in the WZ-3 group was approximately 2 × 10⁻⁶. 10 Although the number of colonies detected in the activity test in the second week was reduced, it was still much higher than that in the WZ-2 group, indicating that the addition of the protectant played a good protective role for the immobilized strain during storage. The strain in this example has a long shelf life.

[0046] Comparative Example 3 The difference between this example and Example 1 is that the immobilized bacterial agent prepared in step (3) is stored at room temperature (25°C). The rest is the same as in Example 1. The immobilized bacterial agent prepared in this example is named WZ-x.

[0047] Comparative Example 4 The difference between this example and Example 1 is that: no sodium thiamycin was added to the bacterial culture, i.e., step (2) was omitted, and the OD was... 600 The bacterial solution with a value adjusted to 1.0 was directly mixed with the microbial immobilization material to allow the bacterial solution to adsorb onto the material; the rest was the same as in Example 1. The immobilized bacterial agent prepared in this example was named WZ-6.

[0048] The immobilized bacterial activity of WZ-3, WZ-x, WZ-4, WZ-5, and WZ-6 was tested using the methods and procedures described above. The experiments showed that no mold grew on WZ-3, WZ-4, WZ-5, and WZ-6. The test results for WZ-x are as follows: Figure 5 As shown (the experiment lasted for 4 weeks). Figure 5 The immobilized bacteria in group WZ-x showed mold growth, indicating that the material contained fungi or spores from the environment. Under suitable conditions (room temperature), these microorganisms rapidly multiplied, affecting the original immobilized species. Group WZ-6 did not show mold growth, allowing the experiment to continue for four weeks. The colony count at week four was approximately 2 × 10⁻⁶. 9 This indicates that low temperature can inhibit the growth of miscellaneous bacteria and prolong the retention time of fixed bacterial strains.

[0049] Figure 4 The WZ-3 (low temperature + natamycin) in the sample had a colony count of approximately 3 × 10⁻⁶ at two weeks of activity testing. 10 In the fourth week of activity testing, the CFU / g count in group WZ-3 was approximately 2 × 10⁻⁶. 10 CFU / g; while the viable bacterial count in the WZ-6 (low temperature only) group was approximately 2 × 10⁻⁶ at two weeks of activity testing. 9 CFU / g, the colony count at week 4 was approximately 1×10⁻⁶. 9The CFU / g is much smaller than the number of viable bacteria in WZ-3. Therefore, it can be concluded that storing sodium natamycin at 4°C in the dark can extend the storage time of the prepared immobilized bacterial agent to four weeks. Natamycin is easily photodegraded and can only inhibit fungal growth but cannot kill fungi. Low temperature slows down the growth rate of miscellaneous bacteria. The combination of the two can extend the storage time of the immobilized bacterial agent and keep the immobilized bacterial strains in good activity.

[0050] Comparative Example 5 The difference between this comparative example and Example 1 is as follows: 0.3g of glucose, 0.1g of skim milk powder, and 0.1g of β-cyclodextrin were weighed and mixed evenly in a mass ratio of 3:1:1 to prepare a protective agent. 2g of activated carbon powder, 0.5g of flour, and 0.5g of the protective agent were weighed and placed in an Erlenmeyer flask in a ratio of 4:1:1, mixed evenly, sealed with sealing film, and sterilized at 121°C under high temperature and pressure for 20 min to prepare the microbial fixation material. Furthermore, the preparation of the fixation agent was also carried out under aseptic conditions; that is, both the preparation of the microbial fixation material and the fixation agent steps were performed under aseptic conditions.

[0051] The rest is the same as in Example 1. The immobilized bacterial agent prepared in this example is named WZ-7.

[0052] The immobilized bacterial activity of WZ-7 was tested using the methods and procedures described above. The colony count was approximately 2.6 × 10⁻⁶ after two weeks of activity testing. 10 CFU / g, the colony count in the fourth week was approximately 1.8 × 10⁻⁶. 10 CFU / g.

[0053] Comparing Comparative Example 5 and Example 1, it can be seen that the bacterial agents obtained from WZ-7 in Comparative Example 5 and WZ-3 in Example 1 have similar activity. This shows that the material preparation and bacterial agent immobilization of the present invention can be carried out under non-strict aseptic conditions, without the need for expensive equipment or complex processes, and the conditions are mild and the operation is simple.

[0054] Example 4 In this embodiment, 0.2g of glucose, 0.1g of skim milk powder, and 0.1g of β-cyclodextrin were weighed and mixed evenly in a mass ratio of 2:1:1 to prepare a protective agent. 1.5g of activated carbon powder, 0.5g of flour, and 0.5g of the protective agent were weighed and mixed evenly in an Erlenmeyer flask in a ratio of 3:1:1 to obtain the microbial fixation material.

[0055] In this embodiment, the activated carbon powder is analytical grade 200 mesh activated carbon powder from Zhonglian Reagent; the flour is ordinary wheat flour; the glucose is anhydrous glucose (analytical grade); the β-cyclodextrin is Aladdin drug, used for cell culture, with a purity ≥98%; and the skim milk powder is skim milk powder from Duyou Biotechnology with a purity of 99.0%.

[0056] The above-mentioned microbial fixation materials are immobilized with bacterial agents (under aseptic conditions), including the following steps: (1) Provide bacterial culture and measure its optical density OD 600 Adjust the value to 1.0; 1 mL of WZ bacteria was transferred to 100 mL of LB medium and cultured at 30℃ with shaking at 180 rpm for 10 h to obtain a cultured WZ bacterial suspension. The LB medium consisted of the following components: 5.0 g of yeast extract, 10.0 g of trypsinized peptone, and 10.0 g of sodium chloride; 1 L of ultrapure water was added and the pH was adjusted to 7.0. The medium was then sterilized at 121℃ for 20–25 min. The cultured bacterial solution was preserved using the glycerol preservation method. The steps of the glycerol preservation method are as follows: the bacterial solution is mixed with 50% glycerol at a volume ratio of 1:1 and stored in a refrigerator at -80℃ for later use. The OD of the cultured or stored WZ bacteria solution was measured. 600 The value was adjusted to 1.0, if the OD of WZ bacteria... 600 If the value is greater than 1.0, dilute the bacterial culture with LB medium to obtain the OD value of WZ bacteria. 600 The value is 1.0; if the bacterial OD... 600 If the OD value is less than 1.0, continue incubation on a shaker until the OD value of the bacterial culture equals 1.0 (Note: OD values ​​are measured by sampling). 600 (Performed under aseptic conditions).

[0057] (2) Add 0.01g of sodium thiamycin to 2mL of the adjusted bacterial culture; (3) Mix 2 mL of the bacterial solution containing sodium thiamycin obtained in step (2) with 2 g of microbial fixation material in an Erlenmeyer flask to obtain the immobilized bacterial agent.

[0058] (4) Store the immobilized bacterial agent at a low temperature of 2°C.

[0059] Example 5 In this embodiment, 0.4g of glucose, 0.1g of skim milk powder, and 0.1g of β-cyclodextrin were weighed and mixed evenly in a mass ratio of 4:1:1 to prepare a protective agent. 2.5g of activated carbon powder, 0.5g of flour, and 0.5g of the protective agent were weighed and mixed evenly in an Erlenmeyer flask in a ratio of 5:1:1 to obtain the microbial fixation material.

[0060] In this embodiment, the activated carbon powder is analytical grade 200 mesh activated carbon powder from Zhonglian Reagent; the flour is ordinary wheat flour; the glucose is anhydrous glucose (analytical grade); the β-cyclodextrin is Aladdin drug, used for cell culture, with a purity ≥98%; and the skim milk powder is skim milk powder from Duyou Biotechnology with a purity of 99.0%.

[0061] The above-mentioned microbial fixation materials are immobilized with bacterial agents (under aseptic conditions), including the following steps: (1) Provide bacterial culture and measure its optical density OD 600 Adjust the value to 1.0; 1 mL of WZ bacteria was transferred to 100 mL of LB medium and cultured at 30℃ with shaking at 180 rpm for 10 h to obtain a cultured WZ bacterial suspension. The LB medium consisted of the following components: 5.0 g of yeast extract, 10.0 g of trypsinized peptone, and 10.0 g of sodium chloride; 1 L of ultrapure water was added and the pH was adjusted to 7.0. The medium was then sterilized at 121℃ for 20–25 min. The cultured bacterial solution was preserved using the glycerol preservation method. The steps of the glycerol preservation method are as follows: the bacterial solution is mixed with 50% glycerol at a volume ratio of 1:1 and stored in a refrigerator at -80℃ for later use. The OD of the cultured or stored WZ bacteria solution was measured. 600 The value was adjusted to 1.0, if the OD of WZ bacteria... 600 If the value is greater than 1.0, dilute the bacterial culture with LB medium to obtain the OD value of WZ bacteria. 600 The value is 1.0; if the bacterial OD... 600 If the OD value is less than 1.0, continue incubation on a shaker until the OD value of the bacterial culture equals 1.0 (Note: OD values ​​are measured by sampling). 600 (Performed under aseptic conditions).

[0062] (2) Add 0.03g of sodium thiamycin to 2mL of the adjusted bacterial culture; (3) Mix 2 mL of the bacterial solution containing sodium thiamycin obtained in step (2) with 4 g of microbial fixation material in an Erlenmeyer flask to obtain the immobilized bacterial agent.

[0063] (4) Store the immobilized bacterial agent at a low temperature of 2°C.

[0064] The immobilized bacterial agents obtained in Examples 4 and 5 were subjected to immobilized bacterial activity testing. The results showed that the immobilized bacterial agent obtained in Example 4 had a colony count of approximately 2.5 × 10⁻⁶ after two weeks of activity testing. 10 CFU / g, the colony count in the fourth week was approximately 1.5 × 10⁻⁶. 10 The CFU / g test result showed that the immobilized bacterial agent obtained in Example 5 had a colony count of approximately 2.8 × 10⁻⁶ after two weeks of activity testing. 10 CFU / g, the colony count at week 4 was approximately 2 × 10⁻⁶. 10 CFU / g.

[0065] The above embodiments specifically demonstrate the excellent immobilization and protection effect of the present invention on WZ bacteria. Those skilled in the art will understand that the protective materials and methods provided by the present invention are universal, and their core lies in providing a composite protection system for microorganisms consisting of a porous carrier, a nutrient source, a protectant, and an inhibitory additive. Therefore, this solution is also applicable to other microbial strains that require immobilization, preservation, and application, such as nitrifying bacteria, denitrifying bacteria, polyphosphate-accumulating bacteria, petroleum hydrocarbon-degrading bacteria, and pesticide-degrading bacteria used for wastewater treatment. Reasonable adjustments to the proportions of the activated carbon powder, flour, and protectant within the range specified in the claims, as well as the selection of the sodium thiamethoxam dosage and storage temperature within the specified range, can achieve the fundamental purpose of the present invention: extending the shelf life of the bacterial agent.

[0066] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a microbial immobilization material, characterized in that: Mix activated carbon powder, flour, and protective agent evenly in a mass ratio of 3-5:1:

1.

2. The method for preparing a microbial immobilization material according to claim 1, characterized in that: The protective agent is made by mixing glucose, skim milk powder and β-cyclodextrin.

3. The method for preparing a microbial immobilization material according to claim 2, characterized in that: In the protective agent, the mass ratio of glucose, skim milk powder and β-cyclodextrin is 2-4:1:

1.

4. A microbial immobilization material prepared by the preparation method according to any one of claims 1-3.

5. A method for immobilizing microbial agents based on the microbial immobilization material according to claim 4, characterized in that: Includes the following steps: (1) Cultivate the bacterial culture to OD 600 It is 1.0; (2) Add sodium thiamycin to the bacterial culture in step (1); (3) The bacterial solution containing sodium thiamycin obtained in step (2) is mixed with the microbial fixation material to obtain an immobilized bacterial agent.

6. The method for immobilizing microbial agents according to claim 1, characterized in that: In step (2), sodium thiamycin is added to the bacterial culture at a ratio of 5-25 g / L.

7. The method for immobilizing microbial agents according to claim 1, characterized in that: In step (3), the solid-liquid ratio of the microbial fixation material and the sodium thiamycin-containing bacterial solution in the immobilized bacterial agent is 2-4 g: 2 mL.

8. The method for immobilizing microbial agents according to claim 1, characterized in that: The immobilized bacterial agent is stored at 2-8°C.

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