Aquaculture water purifying agent based on compound microorganisms and preparation method of aquaculture water purifying agent

Abstract

The invention relates to the technical field of microorganism application, in particular to a compound microorganism-based water purifying agent for aquaculture and a preparation method of the compound microorganism-based water purifying agent for aquaculture. Modified compound microorganisms, carboxylated hollow silicon dioxide microspheres and the compound microorganisms are combined to obtain microspheres A, the modified composite microorganism is prepared by taking modified chitosan as a shell and a composite microorganism as a core, nano titanium dioxide particles are immobilized by silicon dioxide microspheres to obtain microspheres B, activated charcoal and bentonite are mixed to obtain microspheres C, and the modified composite microorganism is prepared by compounding the microspheres A, the microspheres B and the microspheres C. The water purifying agent for aquaculture based on the compound microorganisms can purify water, effectively remove organic pollutants and heavy metals in water and provide good water quality conditions for aquaculture, can improve the stability and durability of the compound microorganisms and reduce the putting frequency of the water purifying agent, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to an aquaculture water purifier based on composite microorganisms and a preparation method thereof. Background Art

[0002] In the field of aquaculture, water purification is a key link to ensure the healthy growth of farmed organisms. Usually under natural conditions, indigenous bacteria in water bodies can maintain normal material circulation in water bodies through self-purification. However, with the development of high-density aquaculture, the organic load in aquaculture water bodies has greatly exceeded the self-purification capacity of the original bacterial community. Organic matter that is not effectively utilized has become the largest source of pollution in water bodies, consuming a large amount of dissolved oxygen, causing oxygen debt in water bodies, and then inhibiting the complete oxidation of organic matter, forming a large number of incomplete oxidation intermediates, and these incomplete oxidation products are often the most harmful toxic substances in aquaculture, such as ammonia nitrogen, nitrite and hydrogen sulfide, causing chronic and acute poisoning of farmed animals, and severely causing large-scale fish deaths, resulting in heavy economic losses.

[0003] At present, the water purification methods widely used in the market mainly include physical filtration, chemical treatment and biological purification. Among them, biological purification has attracted widespread attention due to its environmental protection and sustainability. In recent years, microbial preparations have begun to be widely used in the field of aquaculture to purify water, increase the feed intake of farmed animals, and reduce the occurrence of diseases. The beneficial microorganisms released into the water body grow and reproduce in large numbers, secrete rich extracellular enzymes, and effectively decompose organic matter. During the growth and metabolism process, microorganisms can utilize ammonia nitrogen, metabolize nitrite, and oxidize hydrogen sulfide, thereby achieving the purpose of degrading toxic substances, purifying water quality, and improving aquaculture efficiency.

[0004] In the prior art, for example, patent document CN106629890A discloses a fish pond water purifier, which contains the following components: 100-200 parts of bentonite, 50-90 parts of activated carbon, 10-30 parts of saponin extract, 3-7 parts of pectin, 50-80 parts of pebbles, 1-17 parts of microbial agents, 0.2-0.8 parts of artemisinic acid and 0.1-1.1 parts of 2-phenylchromone; the invention can effectively remove harmful substances in the water through the compounding of the components, but the microbial components used are easily affected by external environmental factors in the water, and their activity decreases, and the long-lasting and stable water purification effect needs to be further improved.

[0005] Therefore, according to the above-mentioned related technologies, it is urgent to develop a water purifier for aquaculture based on composite microorganisms and a preparation method thereof. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an aquaculture water purifier based on composite microorganisms and a preparation method thereof, so as to solve the problem of poor microbial stability and durability in the prior art.

[0007] Based on the above purpose, the present invention provides a water purifier for aquaculture based on composite microorganisms and a preparation method thereof.

[0008] A water purifier for aquaculture based on composite microorganisms is prepared from the following raw materials in parts by weight: Microsphere A 47-55 parts, Microsphere B 2.8-5.3 parts; Microsphere C 5-8 parts; The microsphere A is prepared from modified composite microorganisms, carboxylated hollow silica microspheres and composite microorganisms; The microspheres B are prepared from titanium dioxide and silicon dioxide microspheres; The microspheres C are prepared from activated biochar and bentonite; The modified composite microorganism is prepared by using modified chitosan as a shell and the composite microorganism as a core; The modified chitosan is prepared from chitosan and bentonite; The composite microorganism is obtained by mixing Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria and Brevibacillus laterosporus.

[0009] Preferably, the preparation method of the modified chitosan is as follows: Step A1. Dissolve chitosan in a 2% by mass acetic acid aqueous solution, stir evenly, then add bentonite and mix evenly to obtain a mixture A; Step A2. Dissolve 2-aminoterephthalic acid in a sodium hydroxide solution, then add the mixture A to mix evenly, centrifuge and dry to obtain modified chitosan.

[0010] Preferably, the ratio of chitosan, acetic acid aqueous solution and bentonite in step A1 is 0.45-0.6 g: 45-60 mL: 0.12-0.25 g; The mass ratio of 2-aminoterephthalic acid, sodium hydroxide and mixture A in step A2 is 8-13:10-15:6-10; The concentration of the sodium hydroxide solution is 0.9-1.2 mol / L; The drying temperature is 50-60°C and the drying time is 12-14 hours.

[0011] Preferably, the preparation method of the composite microorganism is as follows: Step B1. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria and Brevibacillus laterosporus were inoculated into LB liquid culture medium respectively, and cultured at 37°C until the logarithmic growth phase to obtain Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacteria suspension and Brevibacillus laterosporus suspension respectively; Step B2: Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were mixed evenly to prepare a suspension with a viable cell count of ≥1.0×10 9 cfu / mL of mixed bacterial solution, centrifuging the mixed bacterial solution at 4000-5000 rpm for 15-20 min, discarding the supernatant, collecting the bacteria, and preparing a bacterial suspension with sterile water to obtain a composite microorganism; The LB liquid culture medium in step B1 is formulated as follows: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, and a pH of 7.0-7.2; The mass ratio of the Pseudomonas stutzeri suspension, the Rhodopseudomonas palustris suspension, the Bacillus subtilis suspension, the cellulose-degrading bacterial suspension and the Brevibacillus laterosporus suspension in step B2 is 1:1-2:3-5:2.5-3.8:1-2.

[0012] Preferably, the preparation method of the microsphere A is as follows: Step C1. Add modified chitosan to the composite microorganism, and place the conical flask in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160r / min for 20-24h, take it out and let it stand for 2-3h, filter it, rinse it with sterile water, and filter it to obtain a modified composite microorganism; Step C2. γ-aminopropyltriethoxysilane and succinic anhydride are added to a flask containing N,N-dimethylformamide, heated and stirred at 55-65°C for 2-3h to obtain a mixture B, the hollow silica microspheres are added to N,N-dimethylformamide, ultrasonically dispersed, heated and stirred with the mixture B at 55-65°C for 4-5h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; Step C3. The modified composite microorganism, carboxylated hollow silica microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are heated and stirred at 35-42°C for 3-4 hours. After filtering and washing with sterile water, the composite microorganism is added and the conical flask is placed in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160r / min and cultured for 20-24 hours. After taking out, let it stand for 2-3 hours, filter, rinse with sterile water, and filter to obtain microspheres A.

[0013] Preferably, the mass ratio of the composite microorganism to the modified chitosan in step C1 is 35-42:3-4; The molar ratio of γ-aminopropyltriethoxysilane, succinic anhydride and N,N-dimethylformamide in step C2 is 1:1:3-4; The mass ratio of the mixture B, hollow silica microspheres and N,N-dimethylformamide is 8-12:4-7:10-14; The mass ratio of the modified composite microorganism, carboxylated hollow silica microspheres, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the composite microorganism in step C3 is 15-25:7-12:7-12:10-14.

[0014] Preferably, the preparation method of the microsphere B is as follows: Step D1. Sodium silicate is added to deionized water, stirred for 30-40 minutes, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor, nano-titanium dioxide particles are added to deionized water, and ultrasonically dispersed for 150-180 minutes, and then PEG-400 is added and stirred for 25-35 minutes to obtain a titanium dioxide nanoparticle aqueous dispersion, and then the titanium dioxide nanoparticle aqueous dispersion is added to the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C; Step D2. Add Abil EM-90 to n-tetradecane, mix well, add mixture C thereto, and homogenize with a homogenizer for 50-65 seconds to obtain mixture D; Step D3. The mixture D is dried, then washed with hexane for 3-4 times, dried at room temperature for 26-30 hours, and then calcined at high temperature to obtain microspheres B.

[0015] Preferably, the volume ratio of sodium silicate to deionized water in step D1 is 1:1.8-2.3; The mass ratio of the nano titanium dioxide particles, deionized water and PEG-400 is 0.25-0.32:10:0.03-0.04; The volume ratio of the titanium dioxide nanoparticle aqueous dispersion to the silicon dioxide precursor transparent aqueous solution is 4-5:3-5; The mass ratio of Abil EM-90, n-tetradecane and mixture C in step D2 is 0.25-0.35:10:8-12; The drying temperature in step D3 is 80-90° C. and the drying time is 90-110 min. The temperature during the high-temperature calcination is 455-500°C, and the calcination time is 5-6h.

[0016] Preferably, the preparation method of the microsphere C is as follows: Step E1. Soak the biochar in a 40% KOH solution, stir for 12-13 hours, let stand for 20-24 hours, dry at 110-120°C for 48-50 hours, cool, wash twice with 1 mol / L hydrochloric acid solution and deionized water, and dry at 60-70°C for 3-4 hours to obtain activated biochar; Step E2. Evenly mix the activated biochar and bentonite to obtain microspheres C; In step E1, the mass ratio of the biochar to the 40% KOH solution is 1:4.5-5.5; The mass ratio of the activated biochar to bentonite in step E2 is 15-20:6-9.

[0017] A method for preparing a water purifier for aquaculture based on composite microorganisms comprises the following steps: The microspheres A, B and C are uniformly mixed to obtain a water purifier for aquaculture based on composite microorganisms.

[0018] Beneficial effects of the present invention: The invention provides a water purifier for aquaculture based on composite microorganisms and a preparation method thereof. The invention obtains microsphere A by combining modified composite microorganisms, carboxylated hollow silica microspheres and composite microorganisms, obtains microsphere B by immobilizing nano titanium dioxide particles by silica microspheres, obtains microsphere C by mixing activated biochar with bentonite, and obtains a water purifier for aquaculture based on composite microorganisms by compounding microsphere A, microsphere B and microsphere C. The water purifier can purify water, effectively remove organic pollutants and heavy metals in water, provide good water quality conditions for aquaculture, and improve the stability and durability of the composite microorganisms, thereby reducing the frequency of adding water purifiers.

[0019] Among them, the modified composite microorganism in microsphere A is prepared with modified chitosan as the shell and composite microorganism as the core, which can effectively adsorb pollutants such as heavy metals in water, and synergize with carboxylated hollow silica microspheres to further improve the stability and durability of composite microorganisms. Microsphere A can achieve better water purification effect through physical adsorption and decomposition of composite microorganisms. The titanium dioxide particles in microsphere B can decompose organic pollutants into small molecules of carbon dioxide, while the silicon dioxide microspheres can reduce the agglomeration of titanium dioxide particles and improve stability. On the one hand, microsphere B can purify water bodies, and on the other hand, it can also reduce the adverse effects of organic pollutants on complex microorganisms; microsphere C purifies water quality through physical adsorption, which is beneficial to delay the action time of complex microorganisms and thus exert a lasting purification effect; compared with the existing technology, it has broad application prospects. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0021] The sources and properties of some of the raw materials used in the present invention are as follows: Chitosan was purchased from Hebei Kelongduo Biotechnology Co., Ltd.; bentonite was purchased from Zhejiang Fenghong New Materials Co., Ltd.; 2-aminoterephthalic acid was purchased from Beijing Biolab Technology Co., Ltd.; sodium silicate was purchased from Shandong Xuchen Chemical Technology Co., Ltd.; nano-titanium dioxide was purchased from Zhejiang Yamei Nanotechnology Co., Ltd.; Abil EM-90 was purchased from Evonik Degussa; n-tetradecane was purchased from Zhongshan Dixin Chemical Co., Ltd.; The Pseudomonas stutzeri used in the present invention is disclosed in the invention patent with the authorization announcement number of "CN114806959B" and the name of "a microbial preparation and aquaculture water purification method", and the deposit number is CGMCC1.3184; The Rhodopseudomonas palustris used in the present invention is disclosed in the invention patent with the authorization announcement number of "CN106148218B" and the name of "Photosynthetic Bacteria Microecology for Aquatic Products and Its Application", and the deposit number is CGMCC No.9136; The Bacillus subtilis used in the present invention is Bacillus subtilis N9-1-35, which is disclosed in the invention patent with the authorization announcement number of "CN105481101B" and the name of "A composite water purifier for aquaculture and its preparation method", and the deposit number is CCTCC M 2011301; The cellulose degrading bacteria used in the present invention are cellulose degrading bacteria JSD-12, which is disclosed in the invention patent with the authorization announcement number of "CN103468618B" and the name of "Cellulose degrading bacteria with phosphate solubilization ability and its application", and the deposit number is CGMCC No.7923; The Brevibacillus laterosporus used in the present invention is Brevibacillus laterosporus HY30, which is disclosed in an invention patent with authorization announcement number “CN111394272B” and name “A strain of Brevibacillus laterosporus and its application”, and the deposit number is CCTCCNo:M20191101.

[0022] Example 1: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps: S1. Weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, add 15 g of agar, heat at 50°C until completely dissolved, filter, and sterilize at 121°C for 15 min to obtain LB solid culture medium; weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, filter, and sterilize at 121°C for 15 min to obtain LB liquid culture medium; S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 were activated, spread on plates, and cultured at 37°C. Single colonies were picked and streaked on LB solid culture medium for further purification until the colonies were uniform and free of foreign bacteria. Single colonies were picked with an inoculation loop from the activated bacteria, inoculated into LB liquid culture medium, and cultured at 37°C until the logarithmic growth phase to obtain bacterial suspensions of Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30, respectively, and washed 3 times with sterile water; S3. Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose-degrading bacteria JSD-12 suspension and Brevibacillus laterosporus HY30 suspension were poured into a centrifuge tube at a mass ratio of 1:1:3:2.5:1, centrifuged at 4000 rpm for 20 min, mixed with sterile water to prepare a uniform bacterial suspension, and counted the live bacteria under a microscope. The number of live bacteria was calculated to be 2.56×10 9 cfu / mL, composite microorganisms were obtained; S4. 0.45 g of chitosan was dissolved in 45 mL of a 2% aqueous acetic acid solution, stirred evenly, and then 0.12 g of bentonite was added and mixed evenly to obtain a mixture A; S5. 8 g of 2-aminoterephthalic acid was dissolved in 10 g of 0.9 mol / L sodium hydroxide solution, and then 6 g of mixture A was added and mixed evenly, centrifuged and dried at 50 ° C for 14 h to obtain modified chitosan; S6. 3g of modified chitosan was added to 35g of the composite microorganism, and the conical flask was placed in a constant temperature shaking incubator set at 25 ° C and the speed was set at 160r / min for 24h, and then taken out and allowed to stand for 3h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism; S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 3 mol of N,N-dimethylformamide, heated and stirred at 55 ° C for 2 h to obtain a mixture B, 4 g of hollow silica microspheres were added to 10 g of N,N-dimethylformamide, and after ultrasonic dispersion, 8 g of the mixture B was heated and stirred at 55 ° C for 4 h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; S8. 15 g of the modified composite microorganism, 7 g of carboxylated hollow silica microspheres and 7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were heated and stirred at 35 ° C for 3 h, filtered, washed with sterile water, and then 10 g of the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set at a temperature of 25 ° C and a speed of 130 r / min and cultured for 20 h. After taking it out, it was allowed to stand for 2 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A; S9. 1 mL of sodium silicate was added to 1.8 mL of deionized water, stirred for 30 min, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor. 0.25 g of nano-titanium dioxide particles were added to 10 g of deionized water, and ultrasonically dispersed for 150 min, and then 0.03 g of PEG-400 was added and stirred for 25 min to obtain a titanium dioxide nanoparticle aqueous dispersion. Then 4 mL of the titanium dioxide nanoparticle aqueous dispersion was added to 3 mL of the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C. S10. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed evenly, and then 8 g of mixture C was added thereto and homogenized with a homogenizer for 50 s to obtain a mixture D; S11. The mixture D was dried at 80 ° C for 90 min, then washed with hexane three times, dried at room temperature for 26 h, and calcined at 455 ° C for 5 h to obtain microspheres B; S12. Take 1g of biochar and soak it in 4.5g of 40% KOH solution, stir it for 12h, let it stand for 20h, dry it at 110℃ for 48h, cool it, wash it twice with 1mol / L hydrochloric acid solution and deionized water, and then dry it at 60℃ for 3h to obtain activated biochar; S13. 15 g of activated biochar and 6 g of bentonite were mixed evenly to obtain microspheres C; S14. 47 g of microsphere A, 2.8 g of microsphere B and 5 g of microsphere C are mixed evenly to obtain a water purifier for aquaculture based on composite microorganisms.

[0023] Embodiment 2: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps: S1. Weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 45°C until completely dissolved, adjust the pH to 7.1, add 15 g of agar, heat at 55°C until completely dissolved, filter, and sterilize at 121°C for 15 min to obtain LB solid culture medium; weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 45°C until completely dissolved, adjust the pH to 7.1, filter, and sterilize at 121°C for 15 min to obtain LB liquid culture medium; S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 were activated, spread on plates, and cultured at 37°C. Single colonies were picked and streaked on LB solid culture medium for further purification until the colonies were uniform and free of foreign bacteria. Single colonies were picked with an inoculation loop from the activated bacteria, inoculated into LB liquid culture medium, and cultured at 37°C until the logarithmic growth phase to obtain bacterial suspensions of Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30, respectively, and washed 3 times with sterile water; S3. Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were poured into a centrifuge tube at a mass ratio of 1:1.5:4:3:1.5, centrifuged at 4500 rpm for 18 min, mixed with sterile water to prepare a uniform bacterial suspension, and counted the live bacteria under a microscope. The number of live bacteria was calculated to be 1.77×10 9 cfu / mL, composite microorganisms were obtained; S4. 0.53 g of chitosan was dissolved in 50 mL of a 2% aqueous acetic acid solution, stirred evenly, and then 0.18 g of bentonite was added and mixed evenly to obtain a mixture A; S5. 8 g of 2-aminoterephthalic acid was dissolved in 10 g of 1 mol / L sodium hydroxide solution, and then 6 g of mixture A was added and mixed evenly, centrifuged and dried at 55 ° C for 13 h to obtain modified chitosan; S6. 3.5 g of modified chitosan was added to 38 g of the composite microorganism, and the conical flask was placed in a constant temperature shaking incubator set at 28 ° C and a speed set at 150 r / min for 22 h, and then allowed to stand for 2.5 h, filtered, rinsed with sterile water, and filtered to obtain a modified composite microorganism; S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 3.5 mol of N,N-dimethylformamide, heated and stirred at 60 ° C for 2.5 h to obtain a mixture B, 5 g of hollow silica microspheres were added to 12 g of N,N-dimethylformamide, and after ultrasonic dispersion, heated and stirred with 10 g of mixture B at 60 ° C for 4.5 h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; S8. 20 g of the modified composite microorganism, 9 g of carboxylated hollow silica microspheres and 9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were heated and stirred at 38 ° C for 3.5 h, filtered, washed with sterile water, and then 12 g of the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set at a temperature of 28 ° C and a speed of 150 r / min and cultured for 22 h. After taking it out, it was allowed to stand for 2.5 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A; S9. 1 mL of sodium silicate was added to 2 mL of deionized water, stirred for 35 min, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor, 0.28 g of nano-titanium dioxide particles were added to 10 g of deionized water, and ultrasonically dispersed for 160 min, and then 0.035 g of PEG-400 was added and stirred for 30 min to obtain a titanium dioxide nanoparticle aqueous dispersion, and then 4.5 mL of the titanium dioxide nanoparticle aqueous dispersion was added to 4 mL of the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C; S10. 0.3 g Abil EM-90 was added to 10 g n-tetradecane, mixed evenly, and then 10 g of mixture C was added thereto, and homogenized with a homogenizer for 60 s to obtain a mixture D; S11. The mixture D was dried at 85 ° C for 100 min, then washed with hexane 4 times, dried at room temperature for 28 h, and calcined at 480 ° C for 5.5 h to obtain microspheres B; S12. Take 1g of biochar and soak it in 5g of 40% KOH solution, stir it for 12h, let it stand for 22h, dry it at 115℃ for 49h, cool it, wash it twice with 1mol / L hydrochloric acid solution and deionized water, and then dry it at 65℃ for 3.5h to obtain activated biochar; S13. 18 g of activated biochar and 8 g of bentonite were mixed evenly to obtain microspheres C; S14. 51 g of microsphere A, 3.7 g of microsphere B and 6.6 g of microsphere C are mixed evenly to obtain a water purifier for aquaculture based on composite microorganisms.

[0024] Example 3: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps: S1. Weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 50°C until completely dissolved, adjust the pH to 7.2, add 15 g of agar, heat at 60°C until completely dissolved, filter, and sterilize at 121°C for 15 min to obtain LB solid culture medium; weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 50°C until completely dissolved, adjust the pH to 7.2, filter, and sterilize at 121°C for 15 min to obtain LB liquid culture medium; S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 were activated, spread on plates, and cultured at 37°C. Single colonies were picked and streaked on LB solid culture medium for further purification until the colonies were uniform and free of foreign bacteria. Single colonies were picked with an inoculation loop from the activated bacteria, inoculated into LB liquid culture medium, and cultured at 37°C until the logarithmic growth phase to obtain bacterial suspensions of Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30, respectively, and washed 3 times with sterile water; S3. Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were poured into a centrifuge tube at a mass ratio of 1:2:5:3.8:2, centrifuged at 5000 rpm for 15 min, mixed with sterile water to prepare a uniform bacterial suspension, and counted the live bacteria under a microscope. The number of live bacteria was calculated to be 3.55×10 9 cfu / mL, composite microorganisms were obtained; S4. 0.6 g of chitosan was dissolved in 60 mL of a 2% aqueous acetic acid solution, stirred evenly, and then 0.25 g of bentonite was added and mixed evenly to obtain a mixture A; S5. 13 g of 2-aminoterephthalic acid was dissolved in 15 g of 1.2 mol / L sodium hydroxide solution, and then 10 g of mixture A was added and mixed, centrifuged and dried at 60 ° C for 12 h to obtain modified chitosan; S6. 4g of modified chitosan was added to 42g of the composite microorganism, and the conical flask was placed in a constant temperature shaking incubator set at 30 ° C and the speed was set at 160r / min for 20h, and then taken out and allowed to stand for 3h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism; S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 4 mol of N,N-dimethylformamide, heated and stirred at 65 ° C for 3 h to obtain a mixture B, 7 g of hollow silica microspheres were added to 14 g of N,N-dimethylformamide, and after ultrasonic dispersion, heated and stirred with 12 g of mixture B at 65 ° C for 5 h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; S8. 25 g of the modified composite microorganism, 12 g of carboxylated hollow silica microspheres and 12 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were heated and stirred at 42 ° C for 3 h, filtered, washed with sterile water, and then 14 g of the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set at a temperature of 30 ° C and a speed of 160 r / min and cultured for 24 h. After taking out, let it stand for 3 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A; S9. 1 mL of sodium silicate was added to 2.3 mL of deionized water, stirred for 40 min, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor, 0.32 g of nano-titanium dioxide particles were added to 10 g of deionized water, and ultrasonically dispersed for 180 min, and then 0.04 g of PEG-400 was added and stirred for 35 min to obtain a titanium dioxide nanoparticle aqueous dispersion, and then 5 mL of the titanium dioxide nanoparticle aqueous dispersion was added to 5 mL of the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C; S10. 0.35 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed evenly, and then 12 g of mixture C was added thereto, and homogenized with a homogenizer for 65 s to obtain a mixture D; S11. The mixture D was dried at 90 ° C for 110 min, then washed with hexane 4 times, dried at room temperature for 30 h, and calcined at 500 ° C for 6 h to obtain microspheres B; S12. Take 1g of biochar and soak it in 5.5g of 40% KOH solution, stir it for 13h, let it stand for 24h, dry it at 120℃ for 50h, cool it, wash it twice with 1mol / L hydrochloric acid solution and deionized water, and then dry it at 70℃ for 4h to obtain activated biochar; S13. 20 g of activated biochar and 9 g of bentonite were mixed evenly to obtain microspheres C; S14. Evenly mix 55 g of microsphere A, 5.3 g of microsphere B and 8 g of microsphere C to obtain a water purifier for aquaculture based on composite microorganisms.

[0025] Comparative Example 1: Compared with Example 1, the modified composite microorganism was not added during the preparation of microsphere A in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, an aquaculture water purifier based on composite microorganisms was obtained.

[0026] Comparative Example 2: Compared with Example 1, the microspheres B were not added in the preparation process of the water purifier for aquaculture based on composite microorganisms in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, the water purifier for aquaculture based on composite microorganisms was obtained.

[0027] Comparative Example 3: Compared with Example 1, the microsphere C was not added in the preparation process of the water purifier for aquaculture based on composite microorganisms in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, the water purifier for aquaculture based on composite microorganisms was obtained.

[0028] Comparative Example 4: Compared with Example 1, this comparative example only replaces "modified chitosan" with "chitosan", and the remaining steps and parameters are the same, which will not be repeated in this comparative example. Finally, an aquaculture water purifier based on composite microorganisms is obtained.

[0029] Comparative Example 5: S1. Weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, add 15 g of agar, heat at 50°C until completely dissolved, filter, and sterilize at 121°C for 15 min to obtain LB solid culture medium; weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, filter, and sterilize at 121°C for 15 min to obtain LB liquid culture medium; S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 were activated, spread on plates, and cultured at 37°C. Single colonies were picked and streaked on LB solid culture medium for further purification until the colonies were uniform and free of foreign bacteria. Single colonies were picked with an inoculation loop from the activated bacteria, inoculated into LB liquid culture medium, and cultured at 37°C until the logarithmic growth phase to obtain bacterial suspensions of Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30, respectively, and washed 3 times with sterile water; S3. Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were poured into a centrifuge tube at a mass ratio of 1:1:3:2.5:1, centrifuged at 4000 rpm for 20 min, mixed with sterile water to prepare a uniform bacterial suspension, and counted the live bacteria under a microscope. The number of live bacteria was calculated to be 2.56×10 9 cfu / mL, composite microorganisms were obtained; S4. 0.45 g of chitosan was dissolved in 45 mL of a 2% aqueous acetic acid solution, stirred evenly, and then 0.12 g of bentonite was added and mixed evenly to obtain a mixture A; S5. 8 g of 2-aminoterephthalic acid was dissolved in 10 g of 0.9 mol / L sodium hydroxide solution, and then 6 g of mixture A was added and mixed evenly, centrifuged and dried at 50 ° C for 14 h to obtain modified chitosan; S6. 3g of modified chitosan was added to 35g of the composite microorganism, and the conical flask was placed in a constant temperature shaking incubator set at 25 ° C and the speed was set at 160r / min for 24h, and then taken out and allowed to stand for 3h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism; S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 3 mol of N,N-dimethylformamide, heated and stirred at 55 ° C for 2 h to obtain a mixture B, 4 g of hollow silica microspheres were added to 10 g of N,N-dimethylformamide, and after ultrasonic dispersion, 8 g of the mixture B was heated and stirred at 55 ° C for 4 h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; S8. 15 g of the modified composite microorganism, 7 g of carboxylated hollow silica microspheres and 7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were heated and stirred at 35 ° C for 3 h, filtered, washed with sterile water, and microspheres A were obtained; S9. 1 mL of sodium silicate was added to 1.8 mL of deionized water, stirred for 30 min, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor. 0.25 g of nano-titanium dioxide particles were added to 10 g of deionized water, and ultrasonically dispersed for 150 min, and then 0.03 g of PEG-400 was added and stirred for 25 min to obtain a titanium dioxide nanoparticle aqueous dispersion. Then 4 mL of the titanium dioxide nanoparticle aqueous dispersion was added to 3 mL of the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C. S10. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed evenly, and then 8 g of mixture C was added thereto and homogenized with a homogenizer for 50 s to obtain a mixture D; S11. The mixture D was dried at 80 ° C for 90 min, then washed with hexane three times, dried at room temperature for 26 h, and calcined at 455 ° C for 5 h to obtain microspheres B; S12. Take 1g of biochar and soak it in 4.5g of 40% KOH solution, stir it for 12h, let it stand for 20h, dry it at 110℃ for 48h, cool it, wash it twice with 1mol / L hydrochloric acid solution and deionized water, and then dry it at 60℃ for 3h to obtain activated biochar; S13. 15 g of activated biochar and 6 g of bentonite were mixed evenly to obtain microspheres C; S14. 47 g of microsphere A, 2.8 g of microsphere B and 5 g of microsphere C are mixed evenly to obtain a water purifier for aquaculture based on composite microorganisms.

[0030] Comparative Example 6: Compared with Example 1, this comparative example only replaces "47g microsphere A, 2.8g microsphere B and 5g microsphere C" with "10g microsphere A, 10g microsphere B and 10g microsphere C", and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally a water purifier for aquaculture based on composite microorganisms is obtained.

[0031] Comparative Example 7: Compared with Example 1, this comparative example only replaces “the mass ratio of Pseudomonas stutzeri bacterial suspension, Rhodopseudomonas palustris bacterial suspension, Bacillus subtilis N9-1-35 bacterial suspension, cellulose degrading bacteria JSD-12 bacterial suspension and Brevibacillus laterosporus HY30 bacterial suspension is 1:1:3:2.5:1” with “the mass ratio of Pseudomonas stutzeri bacterial suspension, Rhodopseudomonas palustris bacterial suspension, Bacillus subtilis N9-1-35 bacterial suspension, cellulose degrading bacteria JSD-12 bacterial suspension and Brevibacillus laterosporus HY30 bacterial suspension is 1:1:1:1:1”, and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally an aquaculture water purifier based on composite microorganisms is obtained.

[0032] Comparative Example 8: S1. Weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, add 15 g of agar, heat at 50°C until completely dissolved, filter, and sterilize at 121°C for 15 min to obtain LB solid culture medium; weigh 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat at 40°C until completely dissolved, adjust the pH to 7, filter, and sterilize at 121°C for 15 min to obtain LB liquid culture medium; S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 were activated, spread on plates, and cultured at 37°C. Single colonies were picked and streaked on LB solid culture medium for further purification until the colonies were uniform and free of foreign bacteria. Single colonies were picked with an inoculation loop from the activated bacteria, inoculated into LB liquid culture medium, and cultured at 37°C until the logarithmic growth phase to obtain bacterial suspensions of Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30, respectively, and washed 3 times with sterile water; S3. Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were poured into a centrifuge tube at a mass ratio of 1:1:3:2.5:1, centrifuged at 4000 rpm for 20 min, mixed with sterile water to prepare a uniform bacterial suspension, and counted the live bacteria under a microscope. The number of live bacteria was calculated to be 2.56×10 9 cfu / mL, composite microorganisms were obtained; S4. 0.45 g of chitosan was dissolved in 45 mL of a 2% aqueous acetic acid solution, stirred evenly, and then 0.12 g of bentonite was added and mixed evenly to obtain a mixture A; S5. 8 g of 2-aminoterephthalic acid was dissolved in 10 g of 0.9 mol / L sodium hydroxide solution, and then 6 g of mixture A was added and mixed evenly, centrifuged and dried at 50 ° C for 14 h to obtain modified chitosan; S6. Add 3g of modified chitosan to 35g of the composite microorganism, and place the conical flask in a constant temperature shaking incubator set at 25 ° C and a speed set at 160r / min for 24h, take it out and let it stand for 3h, filter it, rinse it with sterile water, and filter it to obtain microspheres A; S7. 1 mL of sodium silicate was added to 1.8 mL of deionized water, stirred for 30 min, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor, 0.25 g of nano-titanium dioxide particles were added to 10 g of deionized water, and ultrasonically dispersed for 150 min, and then 0.03 g of PEG-400 was added and stirred for 25 min to obtain a titanium dioxide nanoparticle aqueous dispersion, and then 4 mL of the titanium dioxide nanoparticle aqueous dispersion was added to 3 mL of the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C; S8. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed evenly, and then 8 g of mixture C was added thereto and homogenized with a homogenizer for 50 s to obtain a mixture D; S9. The mixture D was dried at 80 ° C for 90 min, then washed with hexane three times, dried at room temperature for 26 h, and calcined at 455 ° C for 5 h to obtain microspheres B; S10. Take 1g of biochar and soak it in 4.5g of 40% KOH solution, stir it for 12h, let it stand for 20h, dry it at 110℃ for 48h, cool it, wash it twice with 1mol / L hydrochloric acid solution and deionized water, and then dry it at 60℃ for 3h to obtain activated biochar; S11. 15 g of activated biochar and 6 g of bentonite were mixed evenly to obtain microspheres C; S12. Evenly mix 47 g of microsphere A, 2.8 g of microsphere B and 5 g of microsphere C to obtain a water purifier for aquaculture based on composite microorganisms.

[0033] Comparative Example 9: Compared with Example 1, this comparative example only replaces "0.45g chitosan, 45mL acetic acid aqueous solution and 0.12g bentonite" with "0.12g chitosan, 12mL acetic acid aqueous solution and 0.12g bentonite", and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally a water purifier for aquaculture based on composite microorganisms is obtained.

[0034] Comparative Example 10: Compared with Example 1, this comparative example only replaces "0.45g chitosan, 45mL acetic acid aqueous solution and 0.12g bentonite" with "0.9g chitosan, 90mL acetic acid aqueous solution and 0.12g bentonite", and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally a water purifier for aquaculture based on composite microorganisms is obtained.

[0035] Performance Test: Water quality testing: Take the wastewater from the fish and shrimp breeding pond with a high organic matter content, mix it and divide it into 8L plastic barrels, 4L per barrel, and add the composite microbial aquaculture water purifier prepared in Example 1-Example 3 and Comparative Example 1-Comparative Example 10 respectively, and measure the nitrite and ammonia nitrogen content of each group of water samples at 0d, 1d, 30d, and 90d, respectively. Each group of tests is repeated 3 times, and the average value of the three repeated tests is taken; Application effect detection: The same batch of crayfish seedlings were divided into 13 equal parts, and each part of crayfish seedlings was placed in a culture pond of the same size, and then the composite microbial aquaculture water purifier prepared in Example 1 to Example 3 and Comparative Example 1 to Comparative Example 10 was added, and 1 kg was added per mu of culture pond, and other culture conditions were all the same. Finally, the survival rate, yield and average weight of each group of crayfish were measured after one month of culture; The same batch of sea bass fry was divided into 13 equal parts, and each portion of sea bass fry was placed in a culture pond of the same size, and then the composite microbial aquaculture water purifier prepared in Example 1 to Example 3 and Comparative Example 1 to Comparative Example 10 was added, and 1 kg was added per mu of the culture pond. Other culture conditions were all the same. Finally, the survival rate, yield and weight of each group of sea bass were measured after 5 months of culture; Heavy metal detection in water quality: Take the wastewater from the fish and shrimp breeding pond with a high heavy metal content, mix it and divide it into 8L plastic barrels, 4L per barrel, record the initial heavy metal content in the water, and then add the composite microbial aquaculture water purifier prepared in Example 1-Example 3 and Comparative Example 1-Comparative Example 10 respectively, and detect the content of heavy metal copper and chromium in each group of water after 1d, 30d, and 90d, respectively, to obtain the heavy metal removal rate.

[0036] Table 1 Table 2 Table 3 Data Analysis: As can be seen from Tables 1 to 3, the water purifier for aquaculture based on composite microorganisms prepared by the present invention has a better purification effect and a long working time, so that the content of nitrite, ammonia nitrogen and heavy metals in the water is significantly reduced, and the yield of aquatic organisms is higher. This may be because the modified composite microorganism in microsphere A is prepared with modified chitosan as the shell and the composite microorganism as the core. The modified chitosan contains a large number of amino active groups, which can effectively adsorb pollutants such as heavy metals in the water and fix the composite microorganisms through metal chelation and electrostatic attraction, thereby improving the stability of the composite microorganisms; and the composite microorganisms are compounded by five different microorganisms in a specific ratio, which can not only effectively reduce the content of nitrite and ammonia nitrogen in the water, but also decompose the indigestible components in the feed, improve the feed utilization rate, and thus increase the yield of aquatic organisms; the modified composite microorganisms and carboxylated hollow silica microspheres synergistically enhance the stability and durability of the composite microorganisms, and the microspheres A can better achieve the water purification effect through physical adsorption and decomposition of the composite microorganisms; The titanium dioxide particles in microsphere B can decompose organic pollutants into small molecules of carbon dioxide, remove some pesticide residues in the water, and reduce pests and diseases of aquatic organisms. The silicon dioxide microspheres can reduce the agglomeration of titanium dioxide particles and improve stability. Microsphere B can purify water on the one hand, and reduce the adverse effects of organic pollutants on composite microorganisms on the other hand, thereby improving the stability of composite microorganisms. Microsphere C purifies water by physical adsorption, which is beneficial to delay the action time of composite microorganisms, thereby facilitating the composite microorganisms to play a long-term purification role. The present invention obtains a water purifier for aquaculture based on composite microorganisms by compounding microspheres A, microspheres B and microspheres C in a specific ratio. The water purifier can purify water, effectively remove organic pollutants and heavy metals in water, provide good water quality conditions for aquaculture, and improve the stability and durability of composite microorganisms, thereby reducing the frequency of water purifiers. As for Comparative Example 1, since no modified composite microorganisms were added, it can be seen from Tables 1 to 3 that its water purification effect is worse than that of Example 1, and the duration of action is shorter, so that the contents of nitrite, ammonia nitrogen and heavy metals in the water decrease at the initial stage of the water purifier, but the contents still increase in the later stage of the water purifier, and the aquatic biological yield is poor. This may be because the modified composite microorganisms are prepared with modified chitosan as the shell and composite microorganisms as the core, and the stability of the composite microorganisms is improved by immobilizing the microorganisms. The microspheres A in Comparative Example 1 are prepared only by composite microorganisms and carboxylated hollow silica microspheres, and the adsorption of pollutants such as heavy metals in water is greatly weakened, and the composite microorganisms are The stability of the substance is also poor, the effective purification time is shorter, and the yield is also low; since the microspheres B are not added in Comparative Example 2, it can be seen from Tables 1 to 3 that its water purification effect is worse than that of Example 1, and the effect time is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease in the early stage of the water purifier, but the content still increases in the later stage of the water purifier, and the aquatic biological yield is poor. This may be because the titanium dioxide particles can decompose organic pollutants into small molecules of carbon dioxide, such as aquaculture disinfectant methylene blue, reduce pesticide residues, and while purifying water quality, it can also reduce the adverse effects of organic pollutants on composite microorganisms, thereby improving the stability of the composite microorganisms. The water purification effect of the comparative example 3 is poorer than that of the embodiment 1, and the working time is shorter, so that the content of nitrite, ammonia nitrogen and heavy metals in the water decreases at the beginning of the water purifier, but the content still increases in the later period, and the aquatic biological yield is poor. This may be because the activated biochar and bentonite have a strong adsorption effect, which can play a role in a short time after the water purifier is added, thereby delaying the action time of the composite microorganisms and further removing pollutants that are not conducive to the survival of microorganisms; the comparative example 4 replaces the "modified chitosan" with " Chitosan", as can be seen from Tables 1 to 3, the water purification effect is worse than that of Example 1, and the effect time is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease in the early stage of the water purifier, but the contents still increase in the later stage of the water purifier, and the aquatic organism yield is poor. This may be because after the bentonite and chitosan are cross-linked, the chitosan can have a strong adsorption capacity, thereby improving the stability of the loaded composite microorganisms and the adsorption of pollutants such as heavy metals, and 2-aminoterephthalic acid as a cross-linking agent can also introduce amino groups to chitosan, and then better combine with the carboxylated hollow silica microspheres, so that the composite microorganisms are stable and play a long-term role;Comparative Example 5 Since the composite microorganisms were not added during the preparation of microspheres A, it can be seen from Tables 1 to 3 that the water purification effect is worse than that of Example 1, and the time of action is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease at the initial stage of the water purifier, but the content still increases in the later stage of the water purifier, and the aquatic biological yield is poor. This may be because when the composite microorganisms are mixed with the carboxylated hollow silica microspheres and the modified composite microorganisms, they may only be adsorbed in the hollow silica microspheres. These microorganisms may react faster when the water purifier is initially added and take effect first, or they may be adsorbed together with the modified composite microorganisms in the hollow silica microspheres to form The two synergize to form a more stable structure, and the two promote the stable and lasting effectiveness of microorganisms; in Comparative Example 6, since "47g microsphere A, 2.8g microsphere B and 5g microsphere C" are replaced by "10g microsphere A, 10g microsphere B and 10g microsphere C", it can be seen from Tables 1 to 3 that the water purification effect is worse than that of Example 1, and the effect time is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease in the early stage of the water purifier, but the content still increases in the later stage of the water purifier, and the aquatic biological yield is poor. This may be because in the water purifier of the present invention, microorganisms play an important role in removing nitrite and ammonia nitrogen in water and improving water quality. Microspheres B and microspheres The decomposition and adsorption effects of ball C on pollutants are limited. When the treatment limit is reached, the pollutants in the water will increase again, so the effect time is short and the yield is low; Comparative Example 7 replaces "the mass ratio of Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose degrading bacteria JSD-12 suspension and Bacillus laterosporus HY30 suspension is 1:1:3:2.5:1" with "Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose degrading bacteria JSD-12 suspension and Bacillus laterosporus HY30 suspension is 1:1:3:2.5:1". The mass ratio of Bacillus HY30 suspension is 1:1:1:1:1. As can be seen from Tables 1 to 3, the yield of aquatic organisms is poor. This may be due to the strong reproduction and pollutant decomposition capabilities of Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension and Bacillus subtilis N9-1-35, while the cellulose-degrading bacteria JSD-12 can efficiently decompose the cellulose components in the feed and convert them into simple sugars that are easily absorbed by the aquatic organisms, ensuring that the nutrients in the feed are maximized for the aquatic organisms. Brevibacillus laterosporus HY30 has good antibacterial properties. These five microorganisms are compounded in a specific ratio to play a better role in water purification.In Comparative Example 8, since carboxylated hollow silica microspheres were not added during the preparation of microspheres A, it can be seen from Tables 1-3 that the water purification effect is worse than that in Example 1, and the effect time is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease in the initial stage of the water purifier, but the content still increases in the later stage of the water purifier, and the aquatic biological yield is poor. This may be because the carboxylated hollow silica microspheres can cross-link with the modified chitosan in the modified composite microorganism, thereby improving the overall stability of microsphere A and further enhancing the compatibility with microspheres B and C; in Comparative Example 9, since "0.45g chitosan, 45mL acetic acid aqueous solution and 0.12g bentonite" is replaced by "0.12g chitosan, 12mL acetic acid aqueous solution and 0.12g bentonite", it can be seen from Tables 1-3 that the water purification effect is worse than that in Example 1, and the effect time is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water are in the water purifier. The content of the water purifier decreased in the early stage of the release, but the content still increased in the later stage of the release, and the aquatic organism yield was poor. This may be because the excess bentonite will cover the surface of chitosan, thereby covering its active functional groups, and the cross-linking degree with the carboxylated hollow silica will decrease, and the overall stability of microsphere A will decrease; Comparative Example 10 replaces "0.45g chitosan, 45mL acetic acid aqueous solution and 0.12g bentonite" with "0.9g chitosan, 90mL acetic acid aqueous solution and 0.12g bentonite". It can be seen from Tables 1 to 3 that the water purification effect is worse than that of Example 1, and the time of action is shorter, so that the nitrite, ammonia nitrogen and heavy metal content in the water decreases in the early stage of the water purifier release, but the content still increases in the later stage of the release, and the aquatic organism yield is poor. This may be because when the amount of bentonite is too small, the polymer chain of chitosan will self-crosslink to produce dense substances, resulting in fewer adsorption sites, and the degree of immobilization of the composite microorganisms will also decrease and the stability is poor. ;

[0037] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0038] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A water purifier for aquaculture based on composite microorganisms, characterized in that: Prepared from the following raw materials by weight: Microsphere A 47-55 parts, Microsphere B 2.8-5.3 parts; Microsphere C 5-8 parts; The microsphere A is prepared from modified composite microorganisms, carboxylated hollow silica microspheres and composite microorganisms; The microspheres B are prepared from titanium dioxide and silicon dioxide microspheres; The microspheres C are prepared from activated biochar and bentonite; The modified composite microorganism is prepared by using modified chitosan as a shell and the composite microorganism as a core; The modified chitosan is prepared from chitosan and bentonite; The composite microorganism is obtained by mixing Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria and Brevibacillus laterosporus.

2. The aquaculture water purifier based on composite microorganisms according to claim 1, characterized in that: The preparation method of the modified chitosan is as follows: Step A1. Dissolve chitosan in a 2% by mass acetic acid aqueous solution, stir evenly, then add bentonite and mix evenly to obtain a mixture A; Step A2. Dissolve 2-aminoterephthalic acid in a sodium hydroxide solution, then add the mixture A to mix evenly, centrifuge and dry to obtain modified chitosan.

3. The aquaculture water purifier based on composite microorganisms according to claim 2, characterized in that: The ratio of chitosan, acetic acid aqueous solution and bentonite used in step A1 is 0.45-0.6 g:45-60 mL:0.12-0.25 g; The mass ratio of 2-aminoterephthalic acid, sodium hydroxide and mixture A in step A2 is 8-13:10-15:6-10; The concentration of the sodium hydroxide solution is 0.9-1.2 mol / L; The drying temperature is 50-60°C and the drying time is 12-14 hours.

4. The aquaculture water purifier based on composite microorganisms according to claim 1, characterized in that: The preparation method of the composite microorganism is as follows: Step B1. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria and Brevibacillus laterosporus were inoculated into LB liquid culture medium respectively, and cultured at 37°C until the logarithmic growth phase to obtain Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacteria suspension and Brevibacillus laterosporus suspension respectively; Step B2: Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension were mixed evenly to prepare a suspension with a viable cell count of ≥1.0×10 9 cfu / mL of mixed bacterial solution, centrifuging the mixed bacterial solution at 4000-5000 rpm for 15-20 min, discarding the supernatant, collecting the bacteria, and preparing a bacterial suspension with sterile water to obtain a composite microorganism; The LB liquid culture medium in step B1 is formulated as follows: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, and a pH of 7.0-7.2; The mass ratio of the Pseudomonas stutzeri suspension, the Rhodopseudomonas palustris suspension, the Bacillus subtilis suspension, the cellulose-degrading bacterial suspension and the Brevibacillus laterosporus suspension in step B2 is 1:1-2:3-5:2.5-3.8:1-2.

5. The aquaculture water purifier based on composite microorganisms according to claim 1, characterized in that: The preparation method of the microsphere A is as follows: Step C1. Add modified chitosan to the composite microorganism, and place the conical flask in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160r / min for 20-24h, take it out and let it stand for 2-3h, filter it, rinse it with sterile water, and filter it to obtain a modified composite microorganism; Step C2. γ-aminopropyltriethoxysilane and succinic anhydride are added to a flask containing N,N-dimethylformamide, heated and stirred at 55-65°C for 2-3h to obtain a mixture B, the hollow silica microspheres are added to N,N-dimethylformamide, ultrasonically dispersed, heated and stirred with the mixture B at 55-65°C for 4-5h, and washed by centrifugation to obtain carboxylated hollow silica microspheres; Step C3. The modified composite microorganism, carboxylated hollow silica microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are heated and stirred at 35-42°C for 3-4 hours. After filtering and washing with sterile water, the composite microorganism is added and the conical flask is placed in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160r / min and cultured for 20-24 hours. After taking out, let it stand for 2-3 hours, filter, rinse with sterile water, and filter to obtain microspheres A.

6. The aquaculture water purifier based on composite microorganisms according to claim 5, characterized in that: The mass ratio of the composite microorganism to the modified chitosan in step C1 is 35-42:3-4; The molar ratio of γ-aminopropyltriethoxysilane, succinic anhydride and N,N-dimethylformamide in step C2 is 1:1:3-4; The mass ratio of the mixture B, hollow silica microspheres and N,N-dimethylformamide is 8-12:4-7:10-14; The mass ratio of the modified composite microorganism, carboxylated hollow silica microspheres, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the composite microorganism in step C3 is 15-25:7-12:7-12:10-14.

7. The aquaculture water purifier based on composite microorganisms according to claim 1, characterized in that: The preparation method of the microsphere B is as follows: Step D1. Sodium silicate is added to deionized water, stirred for 30-40 minutes, and passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor, nano-titanium dioxide particles are added to deionized water, and ultrasonically dispersed for 150-180 minutes, and then PEG-400 is added and stirred for 25-35 minutes to obtain a titanium dioxide nanoparticle aqueous dispersion, and then the titanium dioxide nanoparticle aqueous dispersion is added to the transparent aqueous solution of the silica precursor, and mixed evenly to obtain a mixture C; Step D2. Add Abil EM-90 to n-tetradecane, mix well, add mixture C thereto, and homogenize with a homogenizer for 50-65 seconds to obtain mixture D; Step D3. The mixture D is dried, then washed with hexane for 3-4 times, dried at room temperature for 26-30 hours, and then calcined at high temperature to obtain microspheres B.

8. The aquaculture water purifier based on composite microorganisms according to claim 7, characterized in that: The volume ratio of sodium silicate to deionized water in step D1 is 1:1.8-2.3; The mass ratio of the nano titanium dioxide particles, deionized water and PEG-400 is 0.25-0.32:10:0.03-0.04; The volume ratio of the titanium dioxide nanoparticle aqueous dispersion to the silicon dioxide precursor transparent aqueous solution is 4-5:3-5; The mass ratio of Abil EM-90, n-tetradecane and mixture C in step D2 is 0.25-0.35:10:8-12; The drying temperature in step D3 is 80-90° C. and the drying time is 90-110 min. The temperature during the high-temperature calcination is 455-500°C, and the calcination time is 5-6h.

9. The aquaculture water purifier based on composite microorganisms according to claim 1, characterized in that: The preparation method of the microsphere C is as follows: Step E1. Soak the biochar in a 40% KOH solution, stir for 12-13 hours, let stand for 20-24 hours, dry at 110-120°C for 48-50 hours, cool, wash twice with 1 mol / L hydrochloric acid solution and deionized water, and dry at 60-70°C for 3-4 hours to obtain activated biochar; Step E2. Evenly mix the activated biochar and bentonite to obtain microspheres C; In step E1, the mass ratio of the biochar to the 40% KOH solution is 1:4.5-5.5; The mass ratio of the activated biochar to bentonite in step E2 is 15-20:6-9.

10. A method for preparing a water purifier for aquaculture based on composite microorganisms according to any one of claims 1 to 9, characterized in that: The following steps are involved: The microspheres A, B and C are uniformly mixed to obtain a water purifier for aquaculture based on composite microorganisms.

Images