Preparation method and application of an algistatic bacteria immobilization preparation

Abstract

This invention discloses a method for preparing an immobilized algae-inhibiting bacterium formulation and its application. The preparation method includes the following steps: S1, preparing the bacterial solution: obtaining the algae-inhibiting bacterium solution through streak plating and liquid culture; S2, preparing the mixed solution: adding an embedding wall material and a carrier solution to the algae-inhibiting bacterium solution and mixing; S3, preparing the immobilized formulation: adding the mixed solution obtained in step S2 dropwise to a crosslinking agent solution for solidification, obtaining a spherical immobilized formulation product; Application: anticipatory prevention and emergency control of red tides caused by algae in water bodies. The preparation method of the immobilized formulation disclosed in this invention is convenient to operate and has low production cost. This immobilized formulation improves the stability of algae-inhibiting bacteria in water bodies, promoting their efficient and long-lasting algae-inhibiting ability; the immobilized algae-inhibiting bacterium formulation of this invention can be widely used in the anticipatory prevention and emergency control of red tides caused by algae in water bodies, and has high practical application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for preparing and applying an immobilized algae-inhibiting bacterium formulation. Background Technology

[0002] Immobilized microorganisms are a biotechnology that involves fixing selected microorganisms onto chosen carriers, achieving high density and maintaining biological activity, enabling rapid and large-scale proliferation under suitable conditions. Immobilized microorganisms offer advantages such as large specific surface area, thorough and uniform contact, strong shock resistance, and high volumetric loading, making them widely used in environmental engineering. This technology helps increase the concentration of microorganisms within bioreactors and reduces their resistance to adverse environmental conditions. The key to immobilized microorganism technology lies in the selection of the immobilization carrier and the choice of immobilization process flow.

[0003] Red tides are a harmful ecological phenomenon caused by the explosive and rapid proliferation of plankton in the ocean under specific environmental conditions, resulting in abnormal seawater color. Red tides cause significant damage to aquatic resources and, in severe cases, can affect port construction due to sediment formation. Currently, there are few reports on the application of immobilized microbial technology to control harmful algae in eutrophic waters. Therefore, the preparation of an immobilized algae-inhibiting bacterium formulation for the predictive prevention and emergency control of algae-induced red tides is an urgent problem to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing and applying an immobilized algae-inhibiting agent, which addresses the shortcomings of the prior art.

[0005] The technical solution adopted by this invention to solve its technical problem is: a method for preparing an immobilized algae-inhibiting bacterium preparation, comprising the following steps:

[0006] S1. Preparation of bacterial solution: Streaking the algae-inhibiting bacteria on a solid culture medium to obtain colonies, picking a single colony and shaking it in a liquid culture medium to obtain an algae-inhibiting bacterial solution with a concentration of 1×10~9×10 CFU / mL.

[0007] S2. Preparation of the mixture: Add the embedding wall material to the algae-inhibiting bacteria solution obtained in step S1, stir for the first time and let stand to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 10-20 g / L; take the immobilized carrier in water to obtain a carrier solution with a solid content of 15-30 g / L, mix it with the algae-inhibiting bacteria-packaging material mixture at a volume ratio of 1:(0.8-1.2), stir for the second time and let stand to obtain an algae-inhibiting bacteria-packaging material-carrier mixture;

[0008] S3. Preparation of immobilized formulation: Take the crosslinking agent in water to obtain a crosslinking agent solution with a solid content of 10-30 g / L. Add 2-5 mL of the algae-inhibiting bacteria-packaging material-carrier mixture to 40-50 mL of the crosslinking agent solution for curing to obtain the finished spherical immobilized formulation.

[0009] Preferably, in step S1, the solid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, 15-20g agar, and 1L water, with a pH of 7.4-7.6; the liquid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, and 1L water, with a pH of 7.4-7.6.

[0010] Preferably, in step S1, the solid culture medium is incubated at 35-39℃ for 14-18 hours; the liquid culture medium is incubated at 35-39℃ for 14-18 hours at 80-120 rpm.

[0011] Preferably, in step S2, the embedding wall material is activated carbon, polyvinyl alcohol, microporous starch, or ethyl cellulose.

[0012] Preferably, the immobilization carrier is sodium alginate, and the crosslinking agent is calcium chloride.

[0013] Preferably, in step S3, after curing for 1-4 hours, a spherical immobilized formulation product with a diameter of 4-6 mm is obtained.

[0014] Preferably, the procedure further includes step S4, calculating the number of single colonies: the immobilized formulation product obtained in step S3 is stored at 4℃, 20℃, and 30℃ for 10-14 hours, respectively, and then dissolved and diluted 10... 7 ~10 9 The cells were then spread onto solid culture plates and incubated at 35-39℃ for 14-18 hours. Plates with a colony count of 30-300 CFU were then selected for counting.

[0015] Preferably, in step S4, 8-12 of the finished spherical immobilized formulation are mixed with 8-12 mL of sodium pyrophosphate solution with a concentration of 0.8-1.2% and allowed to stand for 0.8-1.2 h to dissolve.

[0016] Application of an immobilized algae-inhibiting bacterium preparation in the anticipated prevention and emergency control of algae-induced red tides in water bodies.

[0017] Preferably, the algae-inhibiting bacteria immobilization preparation is used to inhibit *Phaeocystis globosa*.

[0018] The beneficial effects of this invention are:

[0019] This invention proposes a method for preparing an immobilized algae-inhibiting bacterium formulation. After culturing and multiplying algae-inhibiting bacteria to obtain a large number of bacterial cells, an immobilization carrier and embedding wall material are combined with the target bacterial strain using an embedding method to form an immobilized formulation. The preparation method of this invention is convenient and uses inexpensive raw materials. This immobilized formulation improves the stability of algae-inhibiting bacteria in water, effectively reduces the number lost due to water flow, and promotes their efficient and long-lasting algae-inhibiting ability.

[0020] The immobilized algae-inhibiting bacteria formulation of the present invention can effectively inhibit *Phaeocystis globosa*, with an inhibition rate of over 80%. This immobilized algae-inhibiting bacteria formulation can be widely used in the prevention and emergency control of red tides caused by algae in water bodies, and has high practical application value. Attached Figure Description

[0021] Figure 1 This is a comparison of the algae inhibition rates of the immobilized algae-inhibiting bacteria formulations in Examples 1-3 of the present invention.

[0022] Figure 2 This is a comparison of the algae inhibition rates of the algae-inhibiting bacteria immobilized formulations of Example 3 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0023] To provide a clearer understanding of the technical features, objectives, and effects of this invention, the invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the invention and do not constitute a limitation on the scope of protection of this invention.

[0024] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0025] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of algae-inhibiting bacteria from the -80℃ freezer, streak the algae-inhibiting bacteria on fresh solid culture medium for purification, and incubate at 35-39℃ for 14-18h to obtain colonies. Pick a single colony and place it in 90-110mL of liquid culture medium, and incubate at 35-39℃ with constant temperature shaking at 80-120rpm for 14-18h to obtain an algae-inhibiting bacterial suspension with a concentration of 1×10~9×10CFU / mL.

[0026] The algae-inhibiting bacterium used in this invention is *Enterobacter sp.* isolated from seawater in the Hongyanhe area of ​​Liaoning Province, China. It was deposited at the China General Microbiological Culture Collection Center on June 17, 2022, with accession number CGMCC No. 25119. Understandably, other strains with algae-inhibiting activity can be used, and this is not limited to any specific strain.

[0027] The solid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, 15-20g agar, and 1L water, with a pH of 7.4-7.6. The liquid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, and 1L water, with a pH of 7.4-7.6. The preparation methods for the culture media are existing technologies and will not be described in detail hereafter.

[0028] S2. Preparation of the mixture: Add the embedding wall material to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 5-15 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 10-20 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 15-30 g / L. Mix the carrier solution with the algae-inhibiting bacteria-packaging material mixture at a volume ratio of 1:(0.8-1.2). After the second stirring, let it stand for 15-25 minutes to obtain the algae-inhibiting bacteria-packaging material-carrier mixture.

[0029] The embedding wall material is activated carbon, polyvinyl alcohol, microporous starch, or ethyl cellulose. Activated carbon is a specially treated carbon. Organic raw materials (fruit shells, coal, wood, etc.) are heated in the absence of air to reduce non-carbon components (this process is called carbonization). Then, it reacts with gases, causing surface erosion and creating a highly porous structure (this process is called activation). Activation is a process of point-like erosion of the surface of numerous molecular carbides, resulting in countless tiny pores on the activated carbon surface. Activated carbon has a high specific surface area, and the diameter of its surface micropores is mostly between 2 and 50 nm, which is conducive to the adsorption and immobilization of microorganisms on its surface. The immobilized preparations prepared can maintain high activity.

[0030] Encapsulation is a commonly used immobilization method in microbial immobilization technology. Its principle involves trapping biological cells within a network of pores in a water-insoluble gel polymer. Cells are retained through polymerization, ion networking, precipitation, or by altering the temperature and pH of the solvent, while simultaneously allowing the matrix to infiltrate and products to diffuse out, thus releasing the effective substances. This invention employs the encapsulation method to immobilize a target strain of algae-inhibiting bacteria using an immobilization carrier and encapsulation wall material, forming an immobilized formulation.

[0031] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10-30 g / L. Add 2-5 mL of the algae-packaging material-carrier mixture to 40-50 mL of the crosslinking agent solution. After curing for 1-4 h, a spherical immobilized formulation product with a diameter of 4-6 mm is obtained.

[0032] For the selection of immobilization carrier materials, based on the characteristics of the immobilization carrier and considering its impact on the activity of algae-inhibiting bacteria, sodium alginate was chosen as the carrier and calcium chloride as the cross-linking agent. Compared with other immobilization carriers, sodium alginate has low toxicity to the bacterial strains, and sodium alginate and the cross-linking agent calcium chloride can work together to form the optimal immobilization method. In aqueous solution, sodium alginate electrolytically generates anions, and calcium chloride generates divalent calcium ions. The calcium ions attract each other through electrostatic interactions with the anions, thus agglomerating and precipitating, i.e., physical cross-linking occurs, forming a gel.

[0033] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 4℃, 20℃, and 30℃ for 10-14 hours respectively. Take 8-12 spherical immobilized formulation samples and mix them with 8-12 mL of 0.8-1.2% sodium pyrophosphate solution, allowing them to stand for 0.8-1.2 hours to dissolve. Dilute the solution 10... 7 ~10 9 The bacteria were multiplied and spread onto solid culture plates, then incubated at 35-39℃ for 14-18 hours. Plates with colony counts of 30-300 CFU were selected for counting. Sodium pyrophosphate solution was used as the solvent for dissolving the immobilized algae-inhibiting bacteria and had no inhibitory effect on them.

[0034] Specifically, the steps for diluting the coating plate are as follows:

[0035] a. Take nine sterile 1.5 mL EP tubes, each containing 900 μL of sterile water, and dispense them according to the following steps: 1 Up to 10 9 Number them in sequence;

[0036] b. Take 100 μL of the immobilized preparation solution and dissolve it in 900 μL of sterile water. Vortex for 10–20 seconds to mix thoroughly, and prepare 10 μL of the solution. 1 Diluted solution;

[0037] c. From 10 1 Pipette 100 μL of the diluent into a test tube that has been diluted 10 times, and inject 10 μL of the solution. 2 In test tubes diluted 10 times, and so on, to prepare 10... 2 10 3 10 4 10 5 10 6 10 7 10 8 and 10 9 Diluted solution;

[0038] d. Take 100 μL of 10 7 10 8 and 10 9Add the diluted solution dropwise to the surface of the beef extract peptone solid medium, and spread the bacterial solution evenly with a spreader until it is dry. Spread at least 3 plates for each dilution.

[0039] e. Incubate the plate at 35-39℃ for 14-18 hours until the colonies grow to a complete morphology. Select plates with colony counts of 30-300 CFU for counting. Effective viable count CFU / g = (A1+A2+A3)÷3×dilution factor×10, where A1, A2, and A3 represent the number of single colonies on the three plates.

[0040] The operating steps for the dilution coating plate method are the same below.

[0041] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0042] Determination of algae inhibition rate: 0.4-1.2g of immobilized preparation was added to 15-25mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, and the light intensity was 2200-2600 Lux. The culture was carried out at 18-22℃ for 1-3 days. The chlorophyll a content in the immobilized preparation-*Phaeocystis globosa* culture medium was measured, and the algae inhibition rate was calculated. The experimental group used an immobilized algae-inhibiting bacteria preparation, while the blank control group used an immobilized preparation without encapsulated algae-inhibiting bacteria. Each experiment was conducted in triplicate.

[0043] Chlorophyll a was extracted using an ethanol extraction method, which is an existing technology and will not be described in detail here. The formula for calculating the chlorophyll a content and algae-inhibiting activity is as follows:

[0044] Chlorophyll a content (Ca, mg / L) = 11.084 (A) 645nm -A 663nm )-3.914(A 750nm -A 645nm )

[0045] In the formula: A 645nm A is the absorbance at a wavelength of 645 nm. 663nm A is the absorbance at a wavelength of 663 nm. 750nm The absorbance value is at a wavelength of 750 nm.

[0046] Algae inhibition rate % = (C0 - C) e )×100% / C0

[0047] In the formula: C0 is the chlorophyll a content of the blank control group, C e The chlorophyll a content of the experimental group.

[0048] To optimize the preservation activity of immobilized algae-inhibiting bacteria formulations, considering that excessively long immobilization times may lead to bacterial cell death, while excessively short times may result in poor immobilization; if the solid content of the cross-linking agent is too low, immobilization will be difficult to form, while if the solid content is too high, the immobilized formulation will be brittle; and storage temperature mainly affects the activity of the strains, an orthogonal experiment was conducted targeting three factors: immobilization time (1-4 h), cross-linking agent solid content (10-30 g / L), and storage temperature of the finished cross-linking agent (4℃, 20℃, 30℃). The effective viable bacteria count in the diluted immobilized formulation was calculated using the dilution plating method. The experimental results are shown in Table 1.

[0049] Table 1. Orthogonal Experiment of Immobilized Algae-Inhibiting Bacteria Formulation

[0050]

[0051]

[0052] Table 1 shows that when the immobilization time is 1 hour, the target strain is not firmly immobilized, and the number of effective viable bacteria is low. When the immobilization time is 2 hours or 4 hours, the activity of the target strain decreases with increasing cross-linking agent concentration. Higher storage temperatures of the immobilized preparation are more conducive to the survival of the target strain. Range R-squared results indicate that the order of influence of the three factors on the storage activity of the immobilized algae-inhibiting bacteria is: immobilization time > cross-linking agent concentration > storage temperature. Therefore, the optimal conditions for each level of the three factors are: immobilization time of 2 hours, cross-linking agent concentration of 10 g / L, and storage temperature of 30℃. Immobilized algae-inhibiting bacteria prepared under these conditions exhibit high storage activity.

[0053] The following is an illustration through specific examples:

[0054] Example 1

[0055] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0056] S1. Preparation of bacterial culture: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 35℃ for 18h to obtain colonies. Pick a single colony and put it into 90mL of liquid culture medium, and incubate at 35℃ with constant temperature shaking at 80rpm for 18h to obtain an algae-inhibiting bacterial culture with a concentration of 1×10CFU / mL.

[0057] The solid culture medium, with a total volume of 1L, comprises the following components: 2g beef extract, 8g peptone, 4g sodium chloride, 15g agar, and 1L water, with a pH of 7.4. The liquid culture medium, with a total volume of 1L, comprises the following components: 2g beef extract, 8g peptone, 4g sodium chloride, and 1L water, with a pH of 7.4.

[0058] S2. Preparation of the mixture: Add activated carbon, the embedded wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 5 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 10 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 15 g / L. Take 10 mL of the carrier solution and mix it with 8 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 15 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0059] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 2 mL of the algae-packaging material-carrier mixture to 40 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 4 mm is obtained.

[0060] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 10 hours. Mix 8 spherical immobilized formulations with 8 mL of 0.8% sodium pyrophosphate solution and let stand for 0.8 hours to dissolve. Dilute the solution 10... 7 The culture medium was spread on solid culture plates and incubated at 35°C for 18 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0061] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0062] Determination of algae inhibition rate: 0.4g of immobilized agent was added to 15mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2200Lux, and the culture was carried out at 19℃ for 3 days. The chlorophyll a content in the immobilized agent-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0063] Example 2

[0064] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0065] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 39℃ for 14h to obtain colonies. Pick a single colony and place it in 110mL of liquid culture medium, and incubate at 39℃ with constant temperature shaking at 100rpm for 14h to obtain an algae-inhibiting bacterial suspension with a concentration of 9×10 CFU / mL.

[0066] The solid culture medium, with a total volume of 1L, comprises the following components: 4g beef extract, 12g peptone, 6g sodium chloride, 20g agar, and 1L water, with a pH of 7.6. The liquid culture medium, with a total volume of 1L, comprises the following components: 4g beef extract, 12g peptone, 6g sodium chloride, and 1L water, with a pH of 7.6.

[0067] S2. Preparation of the mixture: Add activated carbon, the embedded wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 15 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 20 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 30 g / L. Take 10 mL of the carrier solution and mix it with 12 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 25 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0068] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 5 mL of the algae-packaging material-carrier mixture to 50 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 6 mm is obtained.

[0069] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 14 hours. Mix 12 spherical immobilized formulations with 12 mL of 1.2% sodium pyrophosphate solution and let stand for 1.2 hours to dissolve. Dilute the solution 10... 9 The culture medium was spread on solid culture plates and incubated at 39°C for 14 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0070] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0071] Determination of algae inhibition rate: 0.8g of immobilized agent was added to 25mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2600Lux, and the culture was carried out at 21℃ for 1 day. The chlorophyll a content in the immobilized agent-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0072] Example 3

[0073] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0074] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 37℃ for 16h to obtain colonies. Pick a single colony and place it in 100mL of liquid culture medium, and incubate at 37℃ with constant temperature shaking at 100rpm for 16h to obtain an algae-inhibiting bacterial suspension with a concentration of 5×10 CFU / mL.

[0075] The solid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, 17g agar, and 1L water, with a pH of 7.5. The liquid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, and 1L water, with a pH of 7.5.

[0076] S2. Preparation of the mixture: Add activated carbon, the embedded wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 10 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 15 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 20 g / L. Mix 10 mL of the carrier solution with 10 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 20 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0077] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 3 mL of the algae-packaging material-carrier mixture to 45 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 5 mm is obtained.

[0078] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 12 hours. Mix 10 spherical immobilized formulations with 10 mL of 1% sodium pyrophosphate solution and let stand for 1 hour to dissolve. Dilute the solution 10... 8 The culture medium was spread on solid culture plates and incubated at 37°C for 16 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0079] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0080] Determination of algae inhibition rate: 1.2g of immobilized agent was added to 20mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2400Lux, and the culture was carried out at 20℃ for 2 days. The chlorophyll a content in the immobilized agent-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0081] The experimental results showed that the algae inhibition rates of Examples 1-3 were 56.2%, 72.7%, and 81%, respectively. The algae inhibition rates were comparable to those of... Figure 1 As shown, the immobilized algae-inhibiting bacteria preparation prepared in Example 3 showed the best algae-inhibiting effect on Phaeocystis globosa, with an inhibition rate of over 80%, effectively inhibiting the activity of Phaeocystis globosa.

[0082] Comparative test

[0083] To verify that the embedding wall material in the immobilized algae-inhibiting bacteria formulation of the present invention can effectively improve the survival of the embedded strains and significantly act on *Phaeocystis globosa*, comparative examples 1-3 were set up for the selection of embedding wall materials. The activated carbon of the embedding wall material was replaced with polyvinyl alcohol, microporous starch and ethyl cellulose respectively, while the rest were the same as in Example 3. The effects of different embedding wall materials on the algae inhibition rate of the immobilized formulation were studied.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 3 is that polyvinyl alcohol is used as the embedding wall material.

[0086] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0087] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 37℃ for 16h to obtain colonies. Pick a single colony and place it in 100mL of liquid culture medium, and incubate at 37℃ with constant temperature shaking at 100rpm for 16h to obtain an algae-inhibiting bacterial suspension with a concentration of 5×10 CFU / mL.

[0088] The solid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, 17g agar, and 1L water, with a pH of 7.5. The liquid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, and 1L water, with a pH of 7.5.

[0089] S2. Preparation of the mixture: Add polyvinyl alcohol, the embedding wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 10 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 15 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 20 g / L. Mix 10 mL of the carrier solution with 10 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 20 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0090] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 3 mL of the algae-packaging material-carrier mixture to 45 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 5 mm is obtained.

[0091] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 12 hours. Mix 10 spherical immobilized formulations with 10 mL of 1% sodium pyrophosphate solution and let stand for 1 hour to dissolve. Dilute the solution 10... 8 The culture medium was spread on solid culture plates and incubated at 37°C for 16 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0092] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0093] Determination of algae inhibition rate: 1.2g of immobilized agent was added to 20mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2400Lux, and the culture was carried out at 20℃ for 2 days. The chlorophyll a content in the immobilized agent-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0094] Comparative Example 2

[0095] The difference between this comparative example and Example 3 is that microporous starch is used as the embedding wall material.

[0096] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0097] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 37℃ for 16h to obtain colonies. Pick a single colony and place it in 100mL of liquid culture medium, and incubate at 37℃ with constant temperature shaking at 100rpm for 16h to obtain an algae-inhibiting bacterial suspension with a concentration of 5×10 CFU / mL.

[0098] The solid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, 17g agar, and 1L water, with a pH of 7.5. The liquid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, and 1L water, with a pH of 7.5.

[0099] S2. Preparation of the mixture: Add microporous starch, the embedded wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 10 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 15 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 20 g / L. Mix 10 mL of the carrier solution with 10 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 20 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0100] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 3 mL of the algae-packaging material-carrier mixture to 45 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 5 mm is obtained.

[0101] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 12 hours. Mix 10 spherical immobilized formulations with 10 mL of 1% sodium pyrophosphate solution and let stand for 1 hour to dissolve. Dilute the solution 10... 8 The culture medium was spread on solid culture plates and incubated at 37°C for 16 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0102] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0103] Determination of algae inhibition rate: 1.2g of immobilized agent was added to 20mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2400Lux, and the culture was carried out at 20℃ for 2 days. The chlorophyll a content in the immobilized agent-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0104] Comparative Example 3

[0105] The difference between this comparative example and Example 3 is that ethyl cellulose is used as the embedding wall material.

[0106] A method for preparing an immobilized algae-inhibiting bacterium formulation includes the following steps:

[0107] S1. Preparation of bacterial suspension: Take out the cryopreservation tube of Enterobacter (CGMCC No. 25119) from the -80℃ freezer, purify the algae-inhibiting bacteria by streaking on fresh solid culture medium, and incubate at 37℃ for 16h to obtain colonies. Pick a single colony and place it in 100mL of liquid culture medium, and incubate at 37℃ with constant temperature shaking at 100rpm for 16h to obtain an algae-inhibiting bacterial suspension with a concentration of 5×10 CFU / mL.

[0108] The solid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, 17g agar, and 1L water, with a pH of 7.5. The liquid culture medium, with a total volume of 1L, comprises the following components: 3g beef extract, 10g peptone, 5g sodium chloride, and 1L water, with a pH of 7.5.

[0109] S2. Preparation of the mixture: Add ethyl cellulose, the embedding wall material, to the algae-inhibiting bacteria solution obtained in step S1. After the first stirring, let it stand for 10 minutes to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 15 g / L. Dissolve the immobilized carrier sodium alginate in purified water to obtain a carrier solution with a solid content of 20 g / L. Mix 10 mL of the carrier solution with 10 mL of the algae-inhibiting bacteria-packaging material mixture. After the second stirring, let it stand for 20 minutes to obtain an algae-inhibiting bacteria-packaging material-carrier mixture.

[0110] S3. Preparation of immobilized formulation: Dissolve calcium chloride, a crosslinking agent, in purified water to obtain a crosslinking agent solution with a solid content of 10 g / L. Add 3 mL of the algae-packaging material-carrier mixture to 45 mL of the crosslinking agent solution. After curing for 2 h, a spherical immobilized formulation product with a diameter of 5 mm is obtained.

[0111] S4. Calculate the number of single colonies: Store the immobilized formulation obtained in step S3 at 30℃ for 12 hours. Mix 10 spherical immobilized formulations with 10 mL of 1% sodium pyrophosphate solution and let stand for 1 hour to dissolve. Dilute the solution 10... 8 The culture medium was spread on solid culture plates and incubated at 37°C for 16 hours. Plates with colony counts of 30-300 CFU were selected for counting.

[0112] The present invention also proposes the application of the above-mentioned immobilized algae-inhibiting bacteria preparation in the expected prevention and emergency control of red tides caused by algae in water bodies. The immobilized algae-inhibiting bacteria preparation can be used to inhibit Phaeocystis globosa.

[0113] Determination of algae inhibition rate: 1.2g of algae-inhibiting bacteria immobilization preparation was added to 20mL of *Phaeocystis globosa* culture medium and cultured under constant temperature and light conditions. The light-dark ratio was 12h:12h, the light intensity was 2400Lux, and the culture was carried out at 20℃ for 2 days. The chlorophyll a content in the immobilization preparation-*Phaeocystis globosa* culture medium was detected, and the algae inhibition rate was calculated.

[0114] The experimental results showed that the algae inhibition rates of Comparative Examples 1-3 were 20%, 56.5%, and 51.7%, respectively. The algae inhibition rates of Comparative Examples 1-3 compared to Example 3 were... Figure 2As shown, in Comparative Example 2, the algae-inhibiting bacteria immobilization preparation made from microporous starch showed a better algae-inhibiting effect on Phaeocystis globosa, reaching 56.5%. However, compared with the algae-inhibiting rate of 81% in Example 3, the algae-inhibiting bacteria immobilization preparation made from activated carbon as the embedding wall material in Example 3 showed a better algae-inhibiting effect. Therefore, activated carbon is the preferred embedding wall material in this invention.

[0115] In summary, the preparation method of the immobilized formulation of the present invention is convenient to operate and has low production cost. The immobilized formulation improves the stability of algae-inhibiting bacteria in water, effectively reduces the number of bacteria lost with water flow, and promotes their efficient and long-lasting algae-inhibiting ability.

[0116] The immobilized algae-inhibiting bacteria formulation of the present invention can effectively inhibit *Phaeocystis globosa*, with an inhibition rate of over 80%. This immobilized algae-inhibiting bacteria formulation can be widely used in the prevention and emergency control of red tides caused by algae in water bodies, and has high practical application value.

[0117] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing an immobilized algae-inhibiting bacterium formulation, characterized in that, Includes the following steps: S1. Preparation of bacterial suspension: The algae-inhibiting bacteria are streaked onto a solid culture medium to obtain colonies. Single colonies are picked and cultured in a liquid culture medium with shaking to obtain an algae-inhibiting bacterial suspension with a concentration of 1×10~9×10 CFU / mL; the algae-inhibiting bacteria are Enterobacteriaceae. Enterobacter sp. Its accession number is CGMCC No. 25119; S2. Preparation of the mixture: Add the embedding wall material to the algae-inhibiting bacteria solution obtained in step S1, stir for the first time to obtain an algae-inhibiting bacteria-packaging material mixture with a solid content of 10-20 g / L; take the immobilized carrier in water to obtain a carrier solution with a solid content of 15-30 g / L, mix it with the algae-inhibiting bacteria-packaging material mixture at a volume ratio of 1:(0.8-1.2), stir for the second time to obtain an algae-inhibiting bacteria-packaging material-carrier mixture; In step S2, the embedding wall material is activated carbon; the immobilization carrier is sodium alginate. S3. Preparation of immobilized formulation: Take the crosslinking agent in water to obtain a crosslinking agent solution with a solid content of 10-30 g / L. Add 2-5 mL of the algae-inhibiting bacteria-packaging material-carrier mixture to 40-50 mL of the crosslinking agent solution for solidification to obtain a spherical immobilized formulation product. The crosslinking agent is calcium chloride. The algae-inhibiting bacteria immobilization preparation is used to inhibit *Phaeocystis globosa*.

2. The method for preparing the immobilized algae-inhibiting bacteria formulation according to claim 1, characterized in that, In step S1, the solid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, 15-20g agar, and 1L water, with a pH of 7.4-7.6; the liquid culture medium, with a total volume of 1L, comprises the following components: 2-4g beef extract, 8-12g peptone, 4-6g sodium chloride, and 1L water, with a pH of 7.4-7.

6.

3. The method for preparing the immobilized algae-inhibiting bacteria formulation according to claim 1, characterized in that, In step S1, the solid culture medium is incubated at 35-39℃ for 14-18 hours; the liquid culture medium is incubated at 35-39℃ for 80-120 rpm for 14-18 hours.

4. The method for preparing the immobilized algae-inhibiting bacteria formulation according to claim 1, characterized in that, In step S3, after curing for 1-4 hours, a spherical immobilized formulation product with a diameter of 4-6 mm is obtained.

5. The method for preparing the immobilized algae-inhibiting bacteria formulation according to claim 1, characterized in that, It also includes step S4, calculating the number of single colonies: the immobilized formulation product obtained in step S3 is stored at 4℃, 20℃ and 30℃ for 10-14 hours respectively, and then dissolved and diluted 10. 7 ~10 9 The cells were then spread onto solid culture plates and incubated at 35-39℃ for 14-18 hours. Plates with 30-300 CFU of colonies were then selected for counting.

6. The method for preparing the immobilized algae-inhibiting bacteria formulation according to claim 5, characterized in that, In step S4, 8-12 of the finished spherical immobilized formulation are mixed with 8-12 mL of sodium pyrophosphate solution with a concentration of 0.8-1.2% and allowed to stand for 0.8-1.2 h to dissolve.

7. The application of an immobilized algae-inhibiting bacterium preparation in the anticipated prevention and emergency control of algae-induced red tides in water bodies, characterized in that, The algae-inhibiting bacteria immobilization preparation is prepared by the method of any one of claims 1 to 6, wherein the algae is *Phaeocystis globosum*.

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