An aquaculture microbial purifying agent and its preparation method

Abstract

The present invention discloses an aquaculture microbial purifying agent and a preparation method thereof, relating to the technical field of water treatment; the aquaculture microbial purifying agent is composed of a survival carrier loaded with microorganisms and an adsorption matrix material; the adsorption matrix material is composed of sodium alginate, polyvinyl alcohol, polyglutamic acid, geopolymers and calcium chloride; active bacteria are attached to a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface to form a survival carrier loaded with microorganisms, and then attached to the adsorption matrix material, which fully ensures the attachment, fixation and growth of microorganisms, reduces the adverse effects of temperature and pH on their activity, and at the same time, through the dual actions of microbial degradation and material adsorption, effectively reduces the content of antibiotics in water, achieving an efficient, effective and stable purification effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of water treatment, and specifically refers to an aquaculture microbial purifying agent and a preparation method thereof. Background Art

[0002] The aquaculture industry provides a large amount of high-quality aquatic products for humans. In order to improve the breeding efficiency, the improper use of antibiotics will lead to their accumulation in water, causing pollution to the aquatic ecosystem, including changes in biological communities, the reproduction of antibiotic-resistant bacteria, and the spread of resistance genes. It can also threaten human health through the food chain. Therefore, effectively removing antibiotics in aquaculture has become an urgent problem to be solved.

[0003] Tetracycline, as a broad-spectrum antibiotic, is commonly used to treat bacterial infections in aquaculture, and antibiotic types such as quinolones, sulfonamides, macrolides, and chloramphenicol are also often detected. The methods for removing antibiotics in water mainly include chemical precipitation, biodegradation, photocatalysis, and adsorption. Among them, microorganisms can degrade antibiotics through enzymatic degradation, and can also use antibiotics as carbon or nitrogen sources and completely decompose them through metabolic pathways. Microorganisms can improve water quality by decomposing organic waste and other pollutants. Microorganisms can help balance the intestinal flora of aquatic animals, promote digestion and nutrient absorption, prevent the growth of pathogenic bacteria, and reduce the risk of infection. They can also ferment feed, eliminate anti-nutritional factors in the feed, promote the digestion and absorption of aquatic animals, improve feed utilization rate and growth rate. Therefore, the use of microorganisms has a unique role in aquaculture purification treatment.

[0004] Currently, the existing technologies mainly have the following problems:

[0005] The activity of microorganisms is easily affected by the temperature and pH in water, reducing the effectiveness and stability of antibiotic decomposition, and the efficiency of microorganisms in degrading antibiotics such as tetracycline is low. Therefore, the effect of purifying treatment of antibiotics is limited. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the existing technology, the present invention provides an aquaculture microbial purifying agent, which comprises the following components in parts by weight: 50-60 parts of a survival carrier loaded with microorganisms, and 20-30 parts of an adsorption matrix material.

[0007] The survival carrier loaded with microorganisms comprises the following components in parts by weight: 20-30 parts of coconut shell biochar chitosan composite microspheres, 30-40 parts of metal-organic framework materials, and 30-40 parts of active bacterial cells.

[0008] The adsorptive matrix material includes the following components in parts by weight: 20-30 parts of sodium alginate, 20-25 parts of polyvinyl alcohol, 8-10 parts of polyglutamic acid, 8-10 parts of geopolymer, and 4-8 parts of calcium chloride.

[0009] The preparation method of the survival carrier loaded with microorganisms specifically includes the following steps:

[0010] (1) Wash the coconut shell with water, dry it, crush it through a 80-100 mesh sieve, weigh 2.0-3.0 g of coconut shell powder, add it to 100 mL of water, stir for 5-6 h, then add 30 mL of ammonia water, stir evenly and let it stand for 4 h, filter, wash the filter residue with anhydrous ethanol 3-5 times, transfer the washed filter residue to a hydrothermal reaction kettle, add 100 mL of water, react at 180-200 °C for 8-12 h, after the reaction is completed, cool, collect the product, and dry it. The porous structure of the coconut shell biochar provides an ideal habitat for microorganisms, which helps their colonization and growth. The organic substances on the surface of the biochar can also serve as the nutrient source for microorganisms, ensuring the activity and content of the bacterial cells, and obtaining coconut shell biochar;

[0011] (2) Dissolve chitosan in 50 mL of acetic acid solution with a mass fraction of 5%, add 3.0-4.0 g of copper nitrate trihydrate and the coconut shell biochar described in step (1) to it, stir magnetically for 3-4 h, use a disposable sterile syringe with a specification of 5 mL to extract the mixed solution, and drop it into 300 mL of sodium hydroxide solution with a mass fraction of 4%, let it stand for 4-5 h, collect the formed microspheres, and wash them with deionized water until the pH of the eluate is 7.0. The addition of coconut shell biochar increases the surface roughness of the chitosan microspheres, thereby improving the attachment amount and attachment stability of microorganisms, which is beneficial to enhancing the protection effect on the activity of microorganisms. The introduced copper ions provide conditions for the subsequent distribution of metal-organic framework materials on the microspheres, and obtain coconut shell biochar chitosan composite microspheres;

[0012] (3) Immerse the coconut shell biochar chitosan composite microspheres described in step (2) and 3.0-4.0 g of 2-aminoterephthalic acid in 30 mL of N,N-dimethylformamide, place them in a reactor and react at 160-180 °C for 1-2 h, wash the product with N,N-dimethylformamide 3-5 times, and dry it under vacuum. With Cu 2+Using in-situ synthesis method, composite microspheres with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface were generated with a metal center ion and 2-aminoterephthalic acid as the organic ligand. The addition of the metal-organic framework material further enhanced the rough structure and specific surface area of the microsphere surface, increased the adsorption sites and adsorption stability, which was not only beneficial to the attachment of microorganisms, but also could effectively adsorb antibiotics such as tetracycline and toxic substances in water, showing excellent water purification effect. Moreover, the metal-organic framework material was evenly dispersed on the microsphere, reducing the aggregation of metal-organic framework particles, and also improving the stability and loading capacity of the microsphere, resulting in a stable microsphere carrier;

[0013] (4) Inoculate 1.0 - 2.0 g of microorganisms into LB medium, and culture at 37 °C and 100 rpm until the viable bacteria content reaches 13 - 14 LogCFU / g to obtain a bacterial solution. Centrifuge, discard the supernatant, and freeze-dry to obtain active bacterial cells for use. Then soak the stable microsphere carrier described in step (3) in phosphate buffer for 6 - 8 h, take it out and mix it with the active bacterial cells, and culture it in a constant temperature shaker at 60 - 100 rpm and 36 °C for 1 - 2 d to ensure that the active bacterial cells are fully attached, grown, and fixed on the microsphere surface. The microsphere structure can resist the adverse effects of external conditions such as temperature and pH, and can also serve as a nutrient carrier to continuously provide nutrients for microorganisms, enhancing their stability in the environment and effectively improving the activity of microorganisms, thus obtaining a survival carrier loaded with microorganisms;

[0014] Preferably, in step (2), the addition amount of chitosan is 1.5 - 2.5 g. Chitosan can chelate metal ions, increase the absorption of essential trace elements by microorganisms, can serve as a carbon source and nitrogen source for microorganisms, promote their growth, and help microorganisms form biofilms, providing protection and enhancing their activity;

[0015] Preferably, in step (4), the microorganisms are composed of one or two mixtures of Bacillus subtilis and Pseudomonas. Bacillus subtilis and Pseudomonas degrade tetracycline through various ways such as enzymatic degradation, biosorption, metabolic pathways, and gene regulation.

[0016] The present invention also provides a preparation method of an aquaculture microbial purifying agent, which specifically includes the following steps:

[0017] S1. Load 50.0 g of silica and 300.0 g of slag into a blender for mixing. The stirring speed is 600 - 800 rpm, and keep the mass ratio of silica to alumina at 3:1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, then pour it into the blender and continue stirring for 5 - 10 min. Pour the uniform slurry into a 40 mm×40 mm×40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, and place it in a standard curing box for 1 d. Then perform crushing treatment through a 0.074 mm sieve. Geopolymer is an amorphous three-dimensional network gel containing crystalline and amorphous phases formed by aluminosilicate minerals under the action of a chemical activator. It has the advantages of high strength, good durability, wide raw material sources, low-carbon environmental protection, etc. It can be used as an adsorption material and has a good removal effect on antibiotics, heavy metals, and pollutants in aquaculture water bodies. It can also decompose sediments, organic matter, and biological metabolites at the bottom of the pond, improve the permeability and oxidation state of the bottom mud, and obtain geopolymer;

[0018] S2. Dissolve 2.0 - 2.5 g of polyvinyl alcohol in 100 mL of water, then add 2.0 - 3.0 g of sodium alginate and stir for 6 - 8 h. Then add polyglutamic acid and stir for 1 - 2 h. Use a syringe to drop it into 400 mL of a calcium chloride solution with a mass fraction of 1.0 - 2.0% drop by drop. After the dropping is completed, add the geopolymer described in step S1 and soak for 12 h. Geopolymer can adjust the pH of the water body and neutralize the acidic environment. The addition of polyglutamic acid and geopolymer significantly refines the gel structure, which has both a membrane pore structure and a small pore structure, improving the denseness and complexity of the gel, enhancing the mechanical properties and adsorption properties of the gel, and thus enhancing the adsorption and decomposition performance of pollutants such as antibiotics, to obtain an adsorption-type matrix material;

[0019] S3. Add the survival carrier loaded with microorganisms to the adsorption-type matrix material described in step S2, and then perform ultrasonic treatment for 1 - 2 h. The ultrasonic temperature is 5 - 6 °C, and the ultrasonic power is 600 - 800 W. Then perform freeze-drying. The complex and dense structure of the gel provides a large specific surface area, increasing the contact between the survival carrier loaded with microorganisms and the gel. The holes and channels in the gel can also serve as physical anchor points for the microsphere-shaped survival carrier loaded with microorganisms, improving the attachment stability of the microspheres, and thus further enhancing the protection effect on the bacteria. Through the double protection of the matrix material and the growth carrier, the inactivation of microorganisms caused by water temperature and pH is effectively reduced, the activity of the bacteria is improved, and then the purification function of microorganisms and adsorption materials on aquaculture water bodies can be more fully exerted, to obtain an aquaculture microbial purifier;

[0020] Preferably, in step S2, the addition amount of polyglutamic acid is 0.8 - 1.0 g. Polyglutamic acid can promote the formation of a membranous pore structure in the gel system. Its long-chain structure serves as a crosslinking point in the gel system, improving the stability of the gel material. Moreover, polyglutamic acid can be decomposed and utilized by microorganisms to support the growth and metabolism of bacteria.

[0021] The beneficial effects achieved by the present invention are as follows:

[0022] In the present invention, by attaching active bacteria to a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface, a survival carrier for loaded microorganisms is formed. Then, it is attached to an adsorption-type matrix material, which fully ensures the attachment, fixation, and growth of microorganisms, reduces the adverse effects of temperature and pH on their activity. At the same time, through the dual effects of microbial degradation and material adsorption, the content of antibiotics in the water body is effectively reduced, achieving an efficient, effective, and stable purification effect; in the survival carrier for loaded microorganisms, a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface is generated by in-situ synthesis method. Then, active bacteria are attached, grown, and fixed on the surface of the microsphere, which can not only resist the adverse effects of external conditions such as temperature and pH on the activity of bacteria, but also the microsphere can serve as a nutrient carrier to continuously provide nutrients for microorganisms, thus effectively enhancing the activity of microorganisms. Among them, the uniform dispersion of the metal-organic framework material further enhances the roughness and specific surface area of the microsphere surface, providing more adsorption sites and a more stable adsorption effect, which is beneficial to the attachment of microorganisms and can also adsorb antibiotics such as tetracycline and toxic substances in the water body, enhancing the purification treatment effect; in the adsorption-type matrix material, by introducing polyglutamic acid and geopolymers, the gel structure of polyvinyl alcohol and sodium alginate is significantly refined, which not only promotes the formation of a membrane pore structure but also is rich in more small pore structures, improving the denseness and complexity of the gel, enhancing the mechanical properties and adsorption properties of the gel, being able to effectively adsorb antibiotics and toxic substances, reducing water pollution. At the same time, the gel matrix with a large specific surface area increases the contact area of the survival carrier for loaded microorganisms, and its pores and channels can also serve as physical anchor points for the microsphere-shaped survival carrier for loaded microorganisms, improving the attachment amount and attachment stability. Thus, through the dual protection of the matrix material and the growth carrier, the adverse effects of temperature and pH on the activity of microorganisms are reduced, effectively enhancing the activity of bacteria, which is beneficial to better exerting the degradation effect of microorganisms on tetracycline antibiotics and achieving a more excellent purification treatment effect; the present invention uses a survival carrier for loaded microorganisms and an adsorption-type matrix material to make an aquaculture microbial purifier, which can not only maintain the activity of microorganisms but also enhance the adsorption effect on antibiotics, achieving an efficient, effective, and stable water purification effect. Description of the Drawings

[0023] Figure 1Scanning electron micrograph of the survival carrier loaded with microorganisms prepared in Example 1 of the present invention;

[0024] Figure 2 Scanning electron micrograph of the adsorptive matrix material prepared in Example 1 of the present invention;

[0025] Figure 3 Graph of viable cell counts for Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0026] Figure 4 Graph of tetracycline adsorption amounts for Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0027] Figure 5 Graph of tetracycline removal rates for Examples 1-4 and Comparative Examples 1-3 of the present invention. Detailed implementation manners

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes and cannot limit the content of this application.

[0030] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0031] Example 1

[0032] This example presents an aquaculture microbial purifying agent, which includes the following components in parts by weight: 60 parts of a survival carrier loaded with microorganisms and 30 parts of an adsorptive matrix material.

[0033] The survival carrier loaded with microorganisms includes the following components in parts by weight: 30 parts of coconut shell biochar-chitosan composite microspheres, 40 parts of metal-organic framework materials, and 40 parts of active bacterial cells.

[0034] The adsorptive matrix material includes the following components in parts by weight: 30 parts of sodium alginate, 25 parts of polyvinyl alcohol, 10 parts of polyglutamic acid, 10 parts of geopolymers, and 8 parts of calcium chloride.

[0035] Preparation method of a survival carrier loaded with microorganisms, specifically including the following steps:

[0036] (1) Wash the coconut shell with water, dry it, crush it through a 100-mesh sieve, weigh 3.0 g of coconut shell powder, add it to 100 mL of water, stir for 6 h, then add 30 mL of ammonia water, stir evenly and let it stand for 4 h, filter, wash the filter residue with anhydrous ethanol 5 times, transfer the washed filter residue to a hydrothermal reaction kettle, add 100 mL of water, react at 200 °C for 12 h, after the reaction is over, cool, collect the product and dry it. The porous structure of the coconut shell biochar provides an ideal habitat for microorganisms, helps their colonization and growth, and the organic substances on the surface of the biochar can also serve as a nutrient source for microorganisms, ensuring the activity and content of the bacterial cells, and obtaining coconut shell biochar;

[0037] (2) Dissolve chitosan in 50 mL of acetic acid solution with a mass fraction of 5%. The addition amount of chitosan is 2.5 g. Chitosan can chelate metal ions, increase the absorption of essential trace elements by microorganisms, and can serve as a carbon source and nitrogen source for microorganisms to promote their growth. Chitosan helps microorganisms form a biofilm, provides protection and enhances their activity. Add 4.0 g of copper nitrate trihydrate and the coconut shell biochar described in step (1) to it, stir magnetically for 4 h, use a disposable sterile syringe with a specification of 5 mL to extract the mixed solution, and drop it into 300 mL of sodium hydroxide solution with a mass fraction of 4%, let it stand for 5 h, collect the formed microspheres, and wash them with deionized water until the pH of the eluate is 7.0. The addition of coconut shell biochar increases the surface roughness of the chitosan microspheres, thereby improving the attachment amount and attachment stability of microorganisms, which is beneficial to enhancing the protective effect on the activity of microorganisms. The introduced copper ions provide conditions for the distribution of the subsequent metal-organic framework material on the microspheres, and obtain coconut shell biochar chitosan composite microspheres;

[0038] (3) Immerse the coconut shell biochar chitosan composite microspheres described in step (2) and 4.0 g of 2-aminoterephthalic acid in 30 mL of N,N-dimethylformamide, place them in a reactor and react at 180 °C for 2 h. Wash the product with N,N-dimethylformamide 5 times and dry it in vacuum. Using Cu 2+ as the metal center ion and 2-aminoterephthalic acid as the organic ligand, a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface is generated by the in-situ synthesis method. The addition of the metal-organic framework material further enhances the rough structure and specific surface area of the microsphere surface, increases the adsorption sites and adsorption stability, which is not only beneficial to the attachment of microorganisms, but also can effectively adsorb antibiotics such as tetracycline and toxic substances in water, and has excellent water purification effect. And the metal-organic framework material is evenly dispersed on the microsphere, reducing the aggregation of metal-organic framework particles, and also improving the stability and loading capacity of the microsphere, and obtaining a stable microsphere carrier;

[0039] (4) Inoculate 2.0 g of microorganisms into LB medium. The microorganisms are composed of a mixture of Bacillus subtilis and Pseudomonas. Bacillus subtilis and Pseudomonas degrade tetracycline through various ways such as enzymatic degradation, biosorption, metabolic pathways, and gene regulation. Cultivate at 37 °C and 100 rpm until the viable cell content reaches 14 LogCFU / g to obtain a bacterial solution. Centrifuge and discard the supernatant, then freeze-dry to obtain active bacterial cells for later use. Then soak the stable microsphere carrier described in step (3) in phosphate buffer for 8 h. After taking it out, mix it with the active bacterial cells, and incubate it in a constant temperature shaker at 100 rpm and 36 °C for 2 d to ensure that the active bacterial cells fully adhere to, grow on, and are fixed on the surface of the microspheres. The microsphere structure can resist the adverse effects of external conditions such as temperature and pH, and can also serve as a nutrient carrier to continuously provide nutrients for the microorganisms, enhance their stability in the environment, effectively improve the activity of the microorganisms, and obtain a survival carrier loaded with microorganisms.

[0040] This example provides a preparation method of an aquaculture microbial purifying agent, which specifically includes the following steps:

[0041] S1. Put 50.0 g of silica and 300.0 g of slag into a blender for mixing, with a stirring speed of 800 rpm, and keep the mass ratio of silica to alumina at 3:1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, then pour it into the blender and continue stirring for 10 min. Pour the uniform slurry into a 40 mm × 40 mm × 40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h and then remove the mold, put it into a standard curing box for curing for 1 d, and perform crushing treatment through a 0.074 mm sieve. Geopolymer is an amorphous three-dimensional network gel containing crystalline and non-crystalline phases formed by aluminosilicate minerals under the action of a chemical activator. It has the advantages of high strength, good durability, wide raw material sources, low carbon and environmental protection, etc. It can be used as an adsorption material and has a good removal effect on antibiotics, heavy metals, and pollutants in aquaculture water bodies. It can also decompose the sediments, organic matters, and biological metabolites at the bottom of the pond, improve the permeability and oxidation state of the bottom mud, and obtain geopolymer.

[0042] S2. Dissolve 2.5 g of polyvinyl alcohol in 100 mL of water, then add 3.0 g of sodium alginate and stir for 8 h. Then add polyglutamic acid and stir for 2 h. The addition amount of polyglutamic acid is 1.0 g. Polyglutamic acid can promote the formation of a membranous pore structure in the gel system. Its long-chain structure serves as a cross-linking point in the gel system, improving the stability of the gel material. Moreover, polyglutamic acid can be decomposed and utilized by microorganisms to support the growth and metabolism of bacteria. Dropwise add it into 400 mL of calcium chloride solution with a mass fraction of 2.0% using a syringe. After the dropping is completed, add the geopolymer described in step S1 and soak for 12 h. The geopolymer can adjust the pH of the water body and neutralize the acidic environment. The addition of polyglutamic acid and the geopolymer significantly refines the gel structure, having both a membranous pore structure and a small pore structure, improving the compactness and complexity of the gel, enhancing the mechanical properties and adsorption properties of the gel, and thus enhancing the adsorption and decomposition properties of pollutants such as antibiotics, obtaining an adsorption-type matrix material;

[0043] S3. Add the survival carrier loaded with microorganisms into the adsorption-type matrix material described in step S2, and then perform ultrasonic treatment for 2 h at an ultrasonic temperature of 6 °C and an ultrasonic power of 800 W. Then freeze-dry. The complex and compact structure of the gel provides a large specific surface area, increasing the contact between the survival carrier loaded with microorganisms and the gel. The holes and channels in the gel can also serve as physical anchor points for the microsphere-shaped survival carrier loaded with microorganisms, improving the attachment stability of the microspheres, and thus further enhancing the protective effect on the bacteria. Through the dual protection of the matrix material and the growth carrier, the inactivation of microorganisms by water temperature and pH is effectively reduced, the activity of the bacteria is improved, and further the purification function of microorganisms and adsorption materials on aquaculture water bodies can be more fully exerted, obtaining an aquaculture microbial purifier.

[0044] In this example, the survival carrier loaded with microorganisms and the adsorption-type matrix material prepared are subjected to scanning electron microscopy to observe their microtopographies. Figure 1 Figure 8 is a SEM image of the survival carrier loaded with microorganisms prepared in Example 1 magnified 1000 times. Figure 2 Figure 10 is a SEM image of the adsorption-type matrix material prepared in Example 1 magnified 10000 times. As Figure 1 , the spherical structure surface of the survival carrier loaded with microorganisms prepared in this example is relatively rough. As Figure 2 , the gel structure of the adsorption-type matrix material prepared in this example shows compactness and complexity.

[0045] Example 2

[0046] This example provides an aquaculture microbial purifier, which includes the following components in parts by weight: 50 parts of the survival carrier loaded with microorganisms and 20 parts of the adsorption-type matrix material.

[0047] A survival carrier loaded with microorganisms, comprising the following components in parts by weight: 20 parts of coconut shell biochar-chitosan composite microspheres, 30 parts of metal-organic framework materials, and 30 parts of active bacterial cells.

[0048] An adsorption matrix material, comprising the following components in parts by weight: 20 parts of sodium alginate, 20 parts of polyvinyl alcohol, 8 parts of polyglutamic acid, 8 parts of geopolymers, and 4 parts of calcium chloride.

[0049] A preparation method of a survival carrier loaded with microorganisms, specifically comprising the following steps:

[0050] (1) Wash the coconut shell with water, dry it, crush it through an 80-mesh sieve, weigh 2.0 g of coconut shell powder, add it to 100 mL of water, stir for 5 h, then add 30 mL of ammonia water, stir evenly and let it stand for 4 h, filter, wash the filter residue with absolute ethanol 3 times, transfer the washed filter residue to a hydrothermal reaction kettle, add 100 mL of water, react at 180 °C for 8 h, after the reaction is completed, cool, collect the product, and dry it. The porous structure of the coconut shell biochar provides an ideal habitat environment for microorganisms, helps their colonization and growth, and the organic substances on the surface of the biochar can also serve as a nutrient source for microorganisms to ensure the activity and content of the bacterial cells, obtaining coconut shell biochar;

[0051] (2) Dissolve chitosan in 50 mL of acetic acid solution with a mass fraction of 5%, the addition amount of chitosan is 1.5 g. Chitosan can chelate metal ions, increase the absorption of essential trace elements by microorganisms, can serve as a carbon source and nitrogen source for microorganisms to promote their growth, and chitosan helps microorganisms form a biofilm to provide protection and enhance their activity. Add 3.0 g of copper nitrate trihydrate and the coconut shell biochar described in step (1) thereto, stir magnetically for 3 h, use a disposable sterile syringe with a specification of 5 mL to extract the mixed solution, and drop it into 300 mL of sodium hydroxide solution with a mass fraction of 4%, let it stand for 4 h, collect the formed microspheres, and wash them with deionized water until the pH of the eluate is 7.0. The addition of coconut shell biochar increases the roughness of the surface of the chitosan microspheres, thereby improving the attachment amount and attachment stability of microorganisms, which is beneficial to enhancing the protective effect on the activity of microorganisms. The introduced copper ions provide conditions for the subsequent distribution of metal-organic framework materials on the microspheres, obtaining coconut shell biochar-chitosan composite microspheres;

[0052] (3) Immerse the coconut shell biochar-chitosan composite microspheres described in step (2) and 3.0 g of 2-aminoterephthalic acid in 30 mL of N,N-dimethylformamide, place them in a reactor and react at 160 °C for 1 h, wash the product 3 times with N,N-dimethylformamide, and dry it under vacuum. With Cu 2+Using 2-aminoterephthalic acid as the metal central ion and the organic ligand, a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface was generated by the in-situ synthesis method. The addition of the metal-organic framework material further enhanced the rough structure and specific surface area of the microsphere surface, increased the adsorption sites and adsorption stability, which was not only beneficial to the attachment of microorganisms, but also could effectively adsorb antibiotics such as tetracycline and toxic substances in water, showing excellent water purification effect. Moreover, the metal-organic framework material was evenly dispersed on the microsphere, reducing the aggregation of metal-organic framework particles, and also improving the stability and loading capacity of the microsphere, thus obtaining a stable microsphere carrier;

[0053] (4)Inoculate 1.0 g of microorganisms into the LB medium. The microorganisms are composed of Bacillus subtilis. Bacillus subtilis degrades tetracycline through multiple ways such as enzymatic degradation, biosorption, metabolic pathways, and gene regulation. Cultivate at 37 °C and 100 rpm until the viable bacteria content reaches 13 LogCFU / g to obtain the bacterial liquid. Centrifuge, discard the supernatant, and freeze-dry to obtain the active bacteria for use. Then soak the stable microsphere carrier obtained in step (3) in phosphate buffer for 6 h, take it out and mix it with the active bacteria, and culture it in a constant temperature shaker at 60 rpm and 36 °C for 1 d to ensure that the active bacteria are fully attached, grown, and fixed on the microsphere surface. The microsphere structure can resist the adverse effects of external conditions such as temperature and pH, and can also serve as a nutrient carrier to continuously provide nutrients for microorganisms, enhancing their stability in the environment and effectively improving the activity of microorganisms, thus obtaining a survival carrier loaded with microorganisms.

[0054] This example provides a preparation method of an aquaculture microbial purifier, which specifically includes the following steps:

[0055] S1. Put 50.0 g of silica and 300.0 g of slag into a blender for mixing, with a stirring speed of 600 rpm, and keep the mass ratio of silica to alumina at 3:1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, pour it into the blender and continue stirring for 5 min. Pour the uniform slurry into a 40 mm × 40 mm × 40 mm triple mold, vibrate on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, and put it into a standard curing box for curing for 1 d. Crush it through a 0.074 mm sieve. Geopolymer is an amorphous three-dimensional network gel containing crystalline and non-crystalline phases generated by aluminosilicate minerals under the action of a chemical activator. It has the advantages of high strength, good durability, wide raw material sources, low carbon and environmental protection, etc. It can be used as an adsorption material, having a good removal effect on antibiotics, heavy metals, and pollutants in aquaculture water bodies, and can also decompose the sediments, organic matters, and biological metabolites at the bottom of the pond, improving the permeability and oxidation state of the bottom mud, thus obtaining the geopolymer;

[0056] S2. Dissolve 2.0 g of polyvinyl alcohol in 100 mL of water, then add 2.0 g of sodium alginate and stir for 6 h. Then add polyglutamic acid and stir for 1 h. The addition amount of polyglutamic acid is 0.8 g. Polyglutamic acid can promote the formation of a membranous pore structure in the gel system. Its long-chain structure serves as a cross-linking point in the gel system, improving the stability of the gel material. Moreover, polyglutamic acid can be decomposed and utilized by microorganisms, supporting the growth and metabolism of bacteria. Dropwise add it into 400 mL of a calcium chloride solution with a mass fraction of 1.0% using a syringe. After the dropping is completed, add the geopolymer described in step S1 and soak for 12 h. The geopolymer can adjust the pH of the water body and neutralize the acidic environment. The addition of polyglutamic acid and the geopolymer significantly refine the gel structure, having both a membranous pore structure and a small pore structure, enhancing the compactness and complexity of the gel, improving the mechanical properties and adsorption properties of the gel, and thus enhancing the adsorption and decomposition properties of pollutants such as antibiotics, obtaining an adsorption-type matrix material;

[0057] S3. Add the survival carrier loaded with microorganisms to the adsorption-type matrix material described in step S2, then perform ultrasonic treatment for 1 h at an ultrasonic temperature of 5°C and an ultrasonic power of 600 W, and then freeze-dry. The complex and compact structure of the gel provides a large specific surface area, increasing the contact between the survival carrier loaded with microorganisms and the gel. The pores and channels in the gel can also serve as physical anchor points for the microsphere-shaped survival carrier loaded with microorganisms, improving the attachment stability of the microspheres, and thus further enhancing the protection effect on the bacteria. Through the dual protection of the matrix material and the growth carrier, the inactivation of microorganisms caused by water temperature and pH is effectively reduced, the activity of the bacteria is improved, and further, the purification function of microorganisms and adsorption materials on aquaculture water bodies can be more fully exerted, obtaining an aquaculture microbial purifier.

[0058] Example 3

[0059] This example proposes an aquaculture microbial purifier, which includes the following components in parts by weight: 55 parts of the survival carrier loaded with microorganisms and 25 parts of the adsorption-type matrix material.

[0060] The survival carrier loaded with microorganisms includes the following components in parts by weight: 25 parts of coconut shell biochar-chitosan composite microspheres, 35 parts of metal-organic framework materials, and 35 parts of active bacteria.

[0061] The adsorption-type matrix material includes the following components in parts by weight: 25 parts of sodium alginate, 22.5 parts of polyvinyl alcohol, 9 parts of polyglutamic acid, 9 parts of geopolymer, and 6 parts of calcium chloride.

[0062] The preparation method of the survival carrier loaded with microorganisms specifically includes the following steps:

[0063] (1)The coconut shell was washed with water, dried, crushed through a 90-mesh sieve. 2.5 g of coconut shell powder was weighed and added to 100 mL of water, stirred for 5.5 h, then 30 mL of ammonia water was added, stirred evenly and left to stand for 4 h, filtered, and the filter residue was washed 4 times with absolute ethanol. The washed filter residue was transferred to a hydrothermal reactor, 100 mL of water was added, and the reaction was carried out at 190 °C for 10 h. After the reaction ended, it was cooled, the product was collected and dried. The porous structure of the coconut shell biochar provided an ideal habitat for microorganisms, facilitating their colonization and growth. The organic substances on the surface of the biochar could also serve as a nutrient source for microorganisms, ensuring the activity and content of the bacterial cells, and coconut shell biochar was obtained;

[0064] (2)Chitosan was dissolved in 50 mL of acetic acid solution with a mass fraction of 5%. The addition amount of chitosan was 2.0 g. Chitosan could chelate metal ions, increase the absorption of essential trace elements by microorganisms, and could serve as a carbon source and nitrogen source for microorganisms to promote their growth. Chitosan helped microorganisms form biofilms, providing protection and enhancing their activity. 3.5 g of copper nitrate trihydrate and the coconut shell biochar described in step (1) were added to it, magnetically stirred for 3.5 h, the mixed solution was drawn with a disposable sterile syringe with a specification of 5 mL, and dropped into 300 mL of sodium hydroxide solution with a mass fraction of 4%. It was left to stand for 4.5 h, the formed microspheres were collected, and washed with deionized water until the pH of the eluate was 7.0. The addition of coconut shell biochar increased the roughness of the surface of the chitosan microspheres, thereby enhancing the attachment amount and attachment stability of microorganisms, being beneficial to enhancing the protective effect on the activity of microorganisms. The introduced copper ions provided conditions for the subsequent distribution of metal-organic framework materials on the microspheres, and coconut shell biochar chitosan composite microspheres were obtained;

[0065] (3)The coconut shell biochar chitosan composite microspheres described in step (2) and 3.5 g of 2-aminoterephthalic acid were soaked in 30 mL of N,N-dimethylformamide, placed in a reactor and reacted at 170 °C for 1.5 h. The product was washed 4 times with N,N-dimethylformamide and dried in vacuo. Using Cu 2+ as the metal center ion and 2-aminoterephthalic acid as the organic ligand, a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface was generated by the in-situ synthesis method. The addition of the metal-organic framework material further enhanced the rough structure and specific surface area of the microsphere surface, increased the adsorption sites and adsorption stability, not only being beneficial to the attachment of microorganisms, but also being able to effectively adsorb antibiotics such as tetracycline and toxic substances in water, having excellent water purification effects. And the metal-organic framework material was evenly dispersed on the microspheres, reducing the aggregation of metal-organic framework particles, and also improving the stability and loading capacity of the microspheres, and stable microsphere carriers were obtained;

[0066] (4) Inoculate 1.5 g of microorganisms, which are composed of Pseudomonas, into LB medium. The Pseudomonas degrades tetracycline through various ways such as enzymatic degradation, biosorption, metabolic pathways, and gene regulation. Cultivate at 37 °C and 100 rpm until the viable bacteria content reaches 13 LogCFU / g to obtain a bacterial solution. Centrifuge and discard the supernatant, then freeze-dry to obtain active bacteria for use. Then, soak the stable microsphere carrier described in step (3) in phosphate buffer for 7 h. After taking it out, mix it with the active bacteria, and cultivate it in a constant-temperature shaker at 80 rpm and 36 °C for 1.5 d to ensure that the active bacteria fully attach, grow, and are fixed on the surface of the microspheres. The microsphere structure can resist the adverse effects of external conditions such as temperature and pH, and can also serve as a nutrient carrier to continuously provide nutrients for the microorganisms, improve their stability in the environment, effectively enhance the activity of the microorganisms, and obtain a survival carrier loaded with microorganisms.

[0067] This example provides a preparation method of an aquaculture microbial purifying agent, which specifically includes the following steps:

[0068] S1. Load 50.0 g of silica and 300.0 g of slag into a blender for mixing, with a stirring speed of 700 rpm, and keep the mass ratio of silica to alumina at 3:1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, then pour it into the blender and continue stirring for 7.5 min. Pour the uniform slurry into a 40 mm × 40 mm × 40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, put it into a standard curing box for curing for 1 d, and perform crushing treatment through a 0.074 mm sieve. Geopolymer is an amorphous three-dimensional network gel containing crystalline and non-crystalline phases formed by aluminosilicate minerals under the action of a chemical activator. It has the advantages of high strength, good durability, wide raw material sources, low carbon and environmental protection, etc. It can be used as an adsorption material and has a good removal effect on antibiotics, heavy metals, and pollutants in aquaculture water bodies. It can also decompose the sediments, organic matter, and biological metabolites at the bottom of the pond, improve the permeability and oxidation state of the bottom mud, and obtain geopolymer;

[0069] S2. Dissolve 2.25 g of polyvinyl alcohol in 100 mL of water, then add 2.5 g of sodium alginate and stir for 7 h. Then add polyglutamic acid and stir for 1.5 h. The addition amount of polyglutamic acid is 0.9 g. Polyglutamic acid can promote the formation of a membranous pore structure in the gel system. Its long-chain structure serves as a cross-linking point in the gel system, improving the stability of the gel material. Moreover, polyglutamic acid can be decomposed and utilized by microorganisms, supporting the growth and metabolism of bacteria. Dropwise add it into 400 mL of a calcium chloride solution with a mass fraction of 1.5% using a syringe. After the dropping is completed, add the geopolymers described in step S1 and soak for 12 h. Geopolymers can adjust the pH of the water body and neutralize the acidic environment. The addition of polyglutamic acid and geopolymers significantly refines the gel structure, which has both a membranous pore structure and a small pore structure, enhancing the denseness and complexity of the gel, improving the mechanical properties and adsorption properties of the gel, and thus enhancing the adsorption and decomposition properties of pollutants such as antibiotics, to obtain an adsorption-type matrix material;

[0070] S3. Add the survival carrier loaded with microorganisms to the adsorption-type matrix material described in step S2, and then perform ultrasonic treatment for 1.5 h. The ultrasonic temperature is 5.5 °C and the ultrasonic power is 700 W. Then freeze-dry. The complex and dense structure of the gel provides a large specific surface area, increasing the contact between the survival carrier loaded with microorganisms and the gel. The pores and channels in the gel can also serve as physical anchor points for the survival carrier of microsphere-loaded microorganisms, improving the attachment stability of the microspheres, and thus further enhancing the protection effect on bacteria. Through the dual protection of the matrix material and the growth carrier, the inactivation of microorganisms by water temperature and pH is effectively reduced, the activity of bacteria is improved, and further the purification function of microorganisms and adsorption materials on aquaculture water bodies can be more fully exerted, to obtain an aquaculture microbial purifier.

[0071] Example 4

[0072] This example proposes an aquaculture microbial purifier, which includes the following components in parts by weight: 60 parts of the survival carrier loaded with microorganisms and 20 parts of the adsorption-type matrix material.

[0073] The survival carrier loaded with microorganisms includes the following components in parts by weight: 30 parts of coconut shell biochar chitosan composite microspheres, 30 parts of metal-organic framework materials, and 30 parts of active bacteria.

[0074] The adsorption-type matrix material includes the following components in parts by weight: 30 parts of sodium alginate, 25 parts of polyvinyl alcohol, 8 parts of polyglutamic acid, 8 parts of geopolymers, and 8 parts of calcium chloride.

[0075] The preparation method of the survival carrier loaded with microorganisms specifically includes the following steps:

[0076] (1)The coconut shell was washed with water, dried, crushed through a 100-mesh sieve. 3.0 g of coconut shell powder was weighed and added to 100 mL of water, stirred for 5 h, then 30 mL of ammonia water was added, stirred evenly and left to stand for 4 h, filtered, and the filter residue was washed 5 times with absolute ethanol. The washed filter residue was transferred to a hydrothermal reaction kettle, 100 mL of water was added, and the reaction was carried out at 200 °C for 8 h. After the reaction, it was cooled, the product was collected and dried. The porous structure of the coconut shell biochar provided an ideal habitat for microorganisms, facilitated their colonization and growth, and the organic substances on the surface of the biochar could also serve as a nutrient source for microorganisms, ensuring the activity and content of the bacterial cells, and coconut shell biochar was obtained;

[0077] (2)Chitosan was dissolved in 50 mL of acetic acid solution with a mass fraction of 5%. The addition amount of chitosan was 2.5 g. Chitosan could chelate metal ions, increase the absorption of essential trace elements by microorganisms, and could serve as a carbon source and nitrogen source for microorganisms to promote their growth. Chitosan helped microorganisms form biofilms, provided protection and enhanced their activity. 3.0 g of copper nitrate trihydrate and the coconut shell biochar described in step (1) were added to it, magnetically stirred for 3 h, the mixed solution was extracted using a disposable sterile syringe with a specification of 5 mL, and dropped into 300 mL of sodium hydroxide solution with a mass fraction of 4%. It was left to stand for 4 h, the formed microspheres were collected, and washed with deionized water until the pH of the eluate was 7.0. The addition of coconut shell biochar increased the roughness of the surface of the chitosan microspheres, thereby improving the attachment amount and attachment stability of microorganisms, being conducive to enhancing the protective effect on the activity of microorganisms, and the introduced copper ions provided conditions for the subsequent distribution of metal-organic framework materials on the microspheres, and coconut shell biochar chitosan composite microspheres were obtained;

[0078] (3)The coconut shell biochar chitosan composite microspheres described in step (2) and 3.0 g of 2-aminoterephthalic acid were soaked in 30 mL of N,N-dimethylformamide, placed in a reactor and reacted at 180 °C for 1 h. The product was washed 5 times with N,N-dimethylformamide and dried in vacuum. Using Cu 2+ as the metal central ion and 2-aminoterephthalic acid as the organic ligand, a composite microsphere with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface was generated by the in-situ synthesis method. The addition of the metal-organic framework material further enhanced the rough structure and specific surface area of the microsphere surface, increased the adsorption sites and adsorption stability, not only was conducive to the attachment of microorganisms, but also could effectively adsorb antibiotics such as tetracycline and toxic substances in water, having excellent water purification effects, and the metal-organic framework material was evenly dispersed on the microspheres, reducing the aggregation of metal-organic framework particles, and also improving the stability and loading capacity of the microspheres, and stable microsphere carriers were obtained;

[0079] (4) Inoculate 1.0 g of microorganisms into LB medium. The microorganisms are composed of a mixture of Bacillus subtilis and Pseudomonas sp. Bacillus subtilis and Pseudomonas sp. degrade tetracycline through various means such as enzymatic degradation, biosorption, metabolic pathways, and gene regulation. Cultivate at 37 °C and 100 rpm until the viable cell content reaches 14 LogCFU / g to obtain a bacterial solution. Centrifuge the bacterial solution, discard the supernatant, and freeze-dry to obtain active bacteria for use. Then, immerse the stable microsphere carrier described in step (3) in phosphate buffer for 6 - 8 h. After taking it out, mix it with the active bacteria and cultivate it in a constant temperature shaker at 100 rpm and 36 °C for 1 d to ensure that the active bacteria fully adhere to, grow on, and are fixed on the surface of the microspheres. The microsphere structure can resist the adverse effects of external conditions such as temperature and pH, and can also serve as a nutrient carrier to continuously provide nutrients for the microorganisms, enhance their stability in the environment, and effectively improve the activity of the microorganisms, thereby obtaining a survival carrier loaded with microorganisms.

[0080] This example provides a preparation method of an aquaculture microbial purifying agent, which specifically includes the following steps:

[0081] S1. Load 50.0 g of silica and 300.0 g of slag into a blender for mixing, with a stirring speed of 800 rpm, and keep the mass ratio of silica to alumina among them at 3:1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, pour it into the blender and continue stirring for 5 min. Pour the uniform slurry into a 40 mm × 40 mm × 40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, and place it in a standard curing box for curing for 1 d. Then, perform crushing treatment through a 0.074 mm sieve. Geopolymer is an amorphous three-dimensional network gel containing crystalline and non-crystalline phases generated by aluminosilicate minerals under the action of a chemical activator. It has the advantages of high strength, good durability, wide raw material sources, low carbon and environmental protection, etc. It can be used as an adsorption material and has a good removal effect on antibiotics, heavy metals, and pollutants in aquaculture water bodies. It can also decompose the sediments, organic matters, and biological metabolites at the bottom of the pond, improve the permeability and oxidation state of the bottom mud, and obtain geopolymer.

[0082] S2. Dissolve 2.5 g of polyvinyl alcohol in 100 mL of water, then add 3.0 g of sodium alginate and stir for 6 h. Then add polyglutamic acid and stir for 1 h. The addition amount of polyglutamic acid is 0.8 g. Polyglutamic acid can promote the formation of a membranous pore structure in the gel system. Its long-chain structure serves as a cross-linking point in the gel system, improving the stability of the gel material. Moreover, polyglutamic acid can be decomposed and utilized by microorganisms to support the growth and metabolism of the bacterial cells. Use a syringe to dropwise add it into 400 mL of a calcium chloride solution with a mass fraction of 2.0%. After the dropping is completed, add the geopolymer described in step S1 and soak for 12 h. The geopolymer can adjust the pH of the water body and neutralize the acidic environment. The addition of polyglutamic acid and the geopolymer significantly refines the gel structure, having both a membranous pore structure and a small pore structure, enhancing the compactness and complexity of the gel, improving the mechanical properties and adsorption properties of the gel, thereby enhancing the adsorption and decomposition properties of pollutants such as antibiotics, and obtaining an adsorption-type matrix material;

[0083] S3. Add the survival carrier loaded with microorganisms to the adsorption-type matrix material described in step S2, and then perform ultrasonic treatment for 1 h at an ultrasonic temperature of 6°C and an ultrasonic power of 800 W. Then perform freeze-drying. The complex and compact structure of the gel provides a large specific surface area, increasing the contact between the survival carrier loaded with microorganisms and the gel. The pores and channels in the gel can also serve as physical anchor points for the microsphere-shaped survival carrier loaded with microorganisms, improving the attachment stability of the microspheres, thereby further enhancing the protective effect on the bacterial cells. Through the dual protection of the matrix material and the growth carrier, the inactivation of microorganisms caused by water temperature and pH can be effectively reduced, the activity of the bacterial cells can be improved, and further, the purification function of microorganisms and adsorption materials for aquaculture water bodies can be more fully exerted, obtaining an aquaculture microbial purifier.

[0084] Comparative Example 1

[0085] This comparative example provides an aquaculture microbial purifier, which is different from Example 1 in that the survival carrier loaded with microorganisms does not contain coconut shell biochar chitosan composite microspheres; the preparation method of the survival carrier loaded with microorganisms does not include steps (1) and (2); the preparation method of the aquaculture microbial purifier is the same as that of Example 1.

[0086] Comparative Example 2

[0087] This comparative example provides an aquaculture microbial purifier, which is different from Example 1 in that the survival carrier loaded with microorganisms does not contain metal-organic framework materials; in step (2) of the preparation method of the survival carrier loaded with microorganisms, copper(II) nitrate trihydrate is not added and step (3) is not included; the preparation method of the aquaculture microbial purifier is the same as that of Example 1.

[0088] Comparative Example 3

[0089] This comparative example provides an aquaculture microbial purifying agent, which is different from Example 1 in that the adsorption matrix material does not contain polyglutamic acid and geopolymers; the preparation method of the survival carrier for loading microorganisms is the same as that in Example 1; the preparation method of the aquaculture microbial purifying agent does not include step S1 and polyglutamic acid is not added in step S2.

[0090] Experimental Example 1

[0091] Bacterial activity experiment

[0092] Test samples: The aquaculture microbial purifying agents prepared in Examples 1-4 and Comparative Examples 1-3.

[0093] Test method: Prepare simulated aquaculture water with a water temperature of 15-20°C and a pH of 6.2-6.5. Take 1000 mL of the simulated aquaculture water as a sample, weigh 1.0 g of the test sample and add it thereto, and shake and culture at 36°C and 120 rpm for 3 h. Take 0.5 mL of the sample solution, dilute it step by step with PBS buffer solution, and measure the viable bacteria count (LogCFU / g).

[0094] Figure 3 Figure for the viable bacteria counts of Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the viable bacteria counts of Examples 1-4 are 12.8-14.0 LogCFU / g, indicating strong bacterial activity; the viable bacteria counts of Comparative Examples 1-3 are 10.1-11.6 LogCFU / g, indicating weak bacterial activity; the survival carrier for loading microorganisms in Comparative Example 1 does not contain coconut shell biochar chitosan composite microspheres, which cannot provide an attachment carrier for the active bacteria and cannot provide a nutrient carrier for them, so it cannot resist the inactivation of the bacteria by the low temperature and acidic environment in the aquaculture water body, resulting in weak bacterial activity; the survival carrier for loading microorganisms in Comparative Example 2 does not contain metal-organic framework materials, which is not conducive to enhancing the surface roughness of the microspheres, reducing the adsorption sites, and is not conducive to the attachment, fixation and growth of the active bacteria, thus reducing the resistance to the water temperature and acidic environment, resulting in weak bacterial activity; the adsorption matrix material in Comparative Example 3 does not contain polyglutamic acid and geopolymers, which cannot promote the formation of membrane pores and small pore structures, is not conducive to improving the compactness and complexity of the gel, reduces the contact area and attachment stability of the survival carrier for loading microorganisms, and thus weakens the protection effect on the active bacteria, resulting in weak bacterial activity.

[0095] Experimental Example 2

[0096] Adsorption experiment

[0097] Test samples: The aquaculture microbial purifying agents prepared in Examples 1-4 and Comparative Examples 1-3.

[0098] Test method: Prepare simulated aquaculture water with an antibiotic mass concentration of 100 mg / L. In this experiment, common tetracycline was selected as the antibiotic pollutant for the adsorption experiment. Weigh 1.0 g of the test sample and add it to 1000 mL of simulated aquaculture water. Place it in a constant temperature oscillator (303 K, 120 rpm) and react for 24 h. Then filter it through a 0.45 μm organic filter membrane. The absorbance of tetracycline in the filtrate is measured by UV at 355 nm. According to the concentration-absorbance standard curve fitting equation y = 5.5728x - 0.0186 (R 2 = 0.99834) obtained from tetracycline solutions with different mass concentrations, the mass concentration of tetracycline corresponding to the measured absorbance of the solution is obtained, and the adsorption amount (mg / g) of tetracycline is calculated according to the following formula:

[0099] Adsorption amount (mg / g) = (C0 - C e ) × V / m

[0100] Wherein, C0 is the initial tetracycline mass concentration in mg / L, C e is the tetracycline mass concentration in mg / L after the reaction, V is the solution volume in L, and m is the added amount of the test sample in g.

[0101] Figure 4 Figure for the tetracycline adsorption amount results of Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the tetracycline adsorption amount of Examples 1-4 is 78 - 88 mg / g, indicating good adsorption performance; the tetracycline adsorption amount of Comparative Examples 1-3 is 56 - 68 mg / g, indicating poor adsorption performance; in Comparative Example 1, the survival carrier of the loaded microorganism does not contain coconut shell biochar chitosan composite microspheres, and the metal-organic framework material cannot be evenly dispersed, increasing the particle aggregation, which is not conducive to the adsorption of tetracycline substances, resulting in poor adsorption performance; in Comparative Example 2, the survival carrier of the loaded microorganism does not contain the metal-organic framework material, and the adsorption performance of tetracycline cannot be exerted, relying only on the role of the adsorption-type matrix material, resulting in poor adsorption performance; in Comparative Example 3, the adsorption-type matrix material does not contain polyglutamic acid and geopolymers, which is not conducive to improving the compactness and complexity of the gel structure, thus not conducive to improving the adsorption performance of tetracycline substances, resulting in poor adsorption performance.

[0102] Experimental Example 3

[0103] Purification effect experiment

[0104] Test samples: The aquaculture microbial purifiers prepared in Examples 1-4 and Comparative Examples 1-3.

[0105] Test method: Prepare simulated aquaculture water with a water temperature of 15 - 20 °C, a pH of 6.2 - 6.5, and an antibiotic mass concentration of 100 mol / L. In this experiment, common tetracycline was selected as the antibiotic pollutant for the purification experiment. Weigh 1.0 g of the test sample and add it to 1000 mL of simulated aquaculture water. Oscillate and culture it at 36 °C and 120 rpm for 3 h, then place it in a constant temperature oscillator (303 K, 120 rpm) and react for 24 h. Then filter it through a 0.45 μm organic filter membrane. The absorbance of tetracycline in the filtrate was measured by UV at 355 nm. According to the concentration - absorbance standard curve fitting equation y = 5.5728x - 0.0186 (R 2 = 0.99834) obtained from the determination of tetracycline solutions with different mass concentrations, the mass concentration of tetracycline corresponding to the measured absorbance of the solution was obtained, and the removal rate (%) of tetracycline was calculated according to the following formula:

[0106] Removal rate (%) = (C0 - C e ) / C0 × 100%

[0107] where C0 is the initial mass concentration of tetracycline in mg / L, and C e is the mass concentration of tetracycline after the reaction in mg / L.

[0108] Figure 5 Figure 16 shows the results of the tetracycline removal rate for Examples 1 - 4 and Comparative Examples 1 - 3; as shown in the figure, the tetracycline removal rate for Examples 1 - 4 is 90 - 98%, indicating a relatively high tetracycline removal rate and better purification effect; the tetracycline removal rate for Comparative Examples 1 - 3 is 58 - 70%, indicating a relatively low tetracycline removal rate and poor purification effect; in Comparative Example 1, the survival carrier of the loaded microorganisms does not contain coconut shell biochar chitosan composite microspheres, which can neither improve the activity of the active bacteria and thus limit the degradation of tetracycline by microorganisms, nor evenly disperse the metal - organic framework material, making it difficult for it to play an adsorption and removal role, resulting in a relatively low tetracycline removal rate and poor purification effect; in Comparative Example 2, the survival carrier of the loaded microorganisms does not contain the metal - organic framework material, which is not conducive to the stable attachment of the active bacteria, increases the inactivation of the bacteria by low - temperature and acidic water bodies, limits the purification effect of the microorganisms, and at the same time cannot play the adsorption and removal role of the metal - organic framework material itself for tetracycline, resulting in a relatively low tetracycline removal rate and poor purification effect; in Comparative Example 3, the adsorption - type matrix material does not contain polyglutamic acid and geopolymers, which cannot refine the gel structure of polyvinyl alcohol and sodium alginate, is not conducive to increasing the attachment of microorganisms, and is also not conducive to improving the adsorption performance of the gel, and thus cannot better play the dual degradation role of microorganisms and materials for tetracycline, resulting in a relatively low tetracycline removal rate and poor purification effect.

[0109] The above experimental results show that the bacterial activity, adsorption property and purification effect of Examples 1-4 of the present invention are significantly better than those of the samples of Comparative Examples 1-3. Among them, Example 1 using the survival carrier loaded with microorganisms and the adsorptive matrix material has stronger bacterial activity, better adsorption property for tetracycline and better purification effect. The active bacteria are attached to the composite microspheres with coconut shell biochar chitosan as the matrix and metal-organic framework particles distributed on the surface to form a survival carrier loaded with microorganisms, and then attached to the adsorptive matrix material, which fully ensures the attachment, fixation and growth of microorganisms, reduces the adverse effects of temperature and pH on their activity, and at the same time, through the dual effects of microbial degradation and material adsorption, effectively reduces the content of antibiotics in water, achieving a high-efficiency, effective and stable purification effect.

[0110] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

[0111] The above description of the present invention and its embodiments is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar ways and embodiments to this technical solution without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. An aquaculture microbial purifying agent, characterized in that: The aquaculture microbial purifying agent comprises the following components in parts by weight: 50-60 parts of a survival carrier loaded with microorganisms, and 20-30 parts of an adsorption matrix material; the survival carrier loaded with microorganisms comprises the following components in parts by weight: 20-30 parts of a coconut shell biochar chitosan composite microsphere, 30-40 parts of a metal-organic framework material, and 30-40 parts of active bacterial cells; the adsorption matrix material comprises the following components in parts by weight: 20-30 parts of sodium alginate, 20-25 parts of polyvinyl alcohol, 8-10 parts of polyglutamic acid, 8-10 parts of geopolymers, and 4-8 parts of calcium chloride; The preparation method of the survival carrier loaded with microorganisms specifically comprises the following steps: (1) Wash the coconut shell with water, dry it, crush it through a 80-100 mesh sieve, weigh 2.0-3.0 g of coconut shell powder, add it to 100 mL of water, stir for 5-6 h, then add 30 mL of ammonia water, stir evenly and let it stand for 4 h, filter, wash the filter residue with anhydrous ethanol 3-5 times, transfer the washed filter residue to a hydrothermal reaction kettle, add 100 mL of water, react at 180-200 °C for 8-12 h, after the reaction is completed, cool, collect the product and dry it to obtain coconut shell biochar; (2) Dissolve chitosan in 50 mL of acetic acid solution with a mass fraction of 5%, add 3.0-4.0 g of copper nitrate trihydrate and the coconut shell biochar obtained in step (1) thereto, stir magnetically for 3-4 h, use a disposable sterile syringe with a specification of 5 mL to extract the mixed solution, and drop it into 300 mL of sodium hydroxide solution with a mass fraction of 4%, let it stand for 4-5 h, collect the formed microspheres, and wash them with deionized water until the pH of the eluate is 7.0 to obtain coconut shell biochar chitosan composite microspheres; (3) Immerse the coconut shell biochar chitosan composite microspheres obtained in step (2) and 3.0-4.0 g of 2-aminoterephthalic acid in 30 mL of N,N-dimethylformamide, place them in a reactor and react at 160-180 °C for 1-2 h, wash the product with N,N-dimethylformamide 3-5 times, and dry it under vacuum to obtain a stable microsphere carrier; (4) Inoculate 1.0-2.0 g of microorganisms into an LB medium, culture at 37 °C and 100 rpm until the viable bacteria content reaches 13-14 LogCFU / g to obtain a bacterial solution, centrifuge, discard the supernatant, freeze-dry to obtain active bacterial cells for use, then immerse the stable microsphere carrier obtained in step (3) in a phosphate buffer solution for 6-8 h, take it out and mix it with the active bacterial cells, and culture it in a constant temperature shaker at 60-100 rpm and 36 °C for 1-2 d to obtain a survival carrier loaded with microorganisms; The preparation method of the aquaculture microbial purifying agent specifically comprises the following steps: S1. Load 50.0 g of silica and 300.0 g of slag into a blender for mixing. The stirring speed is 600 - 800 rpm, and maintain the mass ratio of silica to alumina at 3:

1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, then pour it into the blender and continue stirring for 5 - 10 min. Pour the uniform slurry into a 40 mm×40 mm×40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, place it in a standard curing box for curing for 1 d, and perform crushing treatment through a 0.074 mm sieve to obtain geopolymers; S2. Dissolve 2.0 - 2.5 g of polyvinyl alcohol in 100 mL of water, then add 2.0 - 3.0 g of sodium alginate, stir for 6 - 8 h, then add polyglutamic acid and stir for 1 - 2 h. Use a syringe to drop it into 400 mL of calcium chloride solution with a mass fraction of 1.0 - 2.0% drop by drop. After the dropping is completed, add the geopolymers described in step S1 and soak for 12 h to obtain an adsorption matrix material; S3. Add the survival carrier loaded with microorganisms to the adsorption matrix material described in step S2, then perform ultrasonic treatment for 1 - 2 h, the ultrasonic temperature is 5 - 6 °C, the ultrasonic power is 600 - 800 W, and then perform freeze-drying to obtain an aquaculture microbial purifier.

2. The preparation method of the aquaculture microbial purifying agent according to claim 1, characterized in that: Specifically, it includes the following steps: S1. Load 50.0 g of silica and 300.0 g of slag into a blender for mixing. The stirring speed is 600 - 800 rpm, and maintain the mass ratio of silica to alumina at 3:

1. Weigh 18 g of sodium hydroxide powder and dissolve it in 105 mL of deionized water, then pour it into the blender and continue stirring for 5 - 10 min. Pour the uniform slurry into a 40 mm×40 mm×40 mm triple mold, vibrate it on a vibrating table to discharge the internal air, let it stand for 24 h to remove the mold, place it in a standard curing box for curing for 1 d, and perform crushing treatment through a 0.074 mm sieve to obtain geopolymers; S2. Dissolve 2.0 - 2.5 g of polyvinyl alcohol in 100 mL of water, then add 2.0 - 3.0 g of sodium alginate, stir for 6 - 8 h, then add polyglutamic acid and stir for 1 - 2 h. Use a syringe to drop it into 400 mL of calcium chloride solution with a mass fraction of 1.0 - 2.0% drop by drop. After the dropping is completed, add the geopolymers described in step S1 and soak for 12 h to obtain an adsorption matrix material; S3. Add the survival carrier loaded with microorganisms to the adsorption matrix material described in step S2, then perform ultrasonic treatment for 1 - 2 h, the ultrasonic temperature is 5 - 6 °C, the ultrasonic power is 600 - 800 W, and then perform freeze-drying to obtain an aquaculture microbial purifier.

3. The preparation method of the microbial purifying agent for aquaculture according to claim 2, wherein: In step S2, the addition amount of polyglutamic acid is 0.8 - 1.0 g.

4. The preparation method of the microbial purifying agent for aquaculture according to claim 3, characterized in that: In step (2), the addition amount of chitosan is 1.5 - 2.5 g.

5. The preparation method of the aquaculture microbial purifying agent according to claim 4, characterized in that: In step (4), the microorganisms are composed of one or two mixtures of Bacillus subtilis and Pseudomonas.

Images