A water purifying agent for aquaculture based on composite microorganisms and a preparation method thereof

Abstract

The present application relates to the technical field of microbial application, and particularly relates to a water purification agent for aquaculture based on composite microorganisms and a preparation method thereof, the present application obtains microsphere A by combining modified composite microorganisms, carboxylated hollow silica microspheres and composite microorganisms, wherein the modified composite microorganisms are prepared by taking modified chitosan as a shell and composite microorganisms as a core, microsphere B is obtained by fixing nano titanium dioxide particles on silica microspheres, and microsphere C is obtained by mixing activated biochar and bentonite, the water purification agent for aquaculture based on composite microorganisms is obtained by compounding microsphere A, microsphere B and microsphere C, which can not only purify water quality, effectively remove organic pollutants and heavy metals in water, and provide good water quality conditions for aquaculture, but also can improve the stability and durability of the composite microorganisms, reduce the feeding frequency of the water purification agent, and has a wide application prospect.

Description

Technical Field

[0001] The present 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 aquaculture, water purification is crucial for ensuring the healthy growth of aquaculture organisms. Under natural conditions, indigenous bacteria in water bodies maintain normal material circulation through self-purification. However, with the development of high-density aquaculture, the organic load in aquaculture waters has far exceeded the self-purification capacity of the original bacterial community. Unutilized organic matter has become the largest source of water pollution, consuming large amounts of dissolved oxygen and creating an oxygen debt. This in turn inhibits the complete oxidation of organic matter, resulting in a large number of incomplete oxidation intermediates. 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 in farmed animals, and in severe cases, large-scale fish mortality, resulting in heavy economic losses.

[0003] Currently, the main water purification methods widely used in the market include physical filtration, chemical treatment, and biological purification. Among them, biological purification has attracted widespread attention due to its environmentally friendly and sustainable characteristics. In recent years, microbial preparations have begun to be widely used in the aquaculture sector for purposes such as purifying water, increasing feed intake of farmed animals, and reducing the incidence of diseases. Beneficial microorganisms released into water bodies grow and reproduce in large numbers, secreting abundant extracellular enzymes that effectively decompose organic matter. During their growth and metabolism, microorganisms can utilize ammonia nitrogen, metabolize nitrite, and oxidize hydrogen sulfide, thereby achieving the goals of degrading toxic substances, purifying water quality, and improving aquaculture efficiency.

[0004] In the prior art, patent document CN106629890A discloses a fish pond water purifier comprising 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; this invention can effectively remove harmful substances from water bodies by compounding the components, but the microbial components used are easily affected by external environmental factors in the water body, resulting in decreased activity, and the ability to exert a sustained and stable water purification effect needs to be further improved.

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

[0006] Therefore, the present application aims to provide a water body purifying agent for aquaculture based on composite microorganisms and a preparation method thereof to solve the problem of poor stability and durability of microorganisms in the prior art.

[0007] Based on the above purpose, the present application provides a water body purifying agent for aquaculture based on composite microorganisms and a preparation method thereof.

[0008] A water body purifying agent for aquaculture based on composite microorganisms is prepared from the following mass parts of raw materials:

[0009] Microspheres A 47-55 parts, microspheres B 2.8-5.3 parts; microspheres C 5-8 parts;

[0010] The microspheres A are prepared from modified composite microorganisms, carboxylated hollow silica microspheres and composite microorganisms;

[0011] The microspheres B are prepared from titanium dioxide and silica microspheres;

[0012] The microspheres C are prepared from activated biochar and bentonite;

[0013] The modified composite microorganisms are prepared from modified chitosan as a shell and composite microorganisms as a core;

[0014] The modified chitosan is prepared from chitosan and bentonite;

[0015] The composite microorganisms are obtained by mixing Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria and Brevibacillus laterosporus.

[0016] Preferably, the preparation method of the modified chitosan is as follows:

[0017] Step A1. Dissolve chitosan in a 2% mass fraction acetic acid aqueous solution, stir uniformly, then add bentonite and mix uniformly to obtain a mixture A;

[0018] Step A2. Dissolve 2-amino terephthalic acid in a sodium hydroxide solution, then add the mixture A and mix uniformly, centrifuge and dry to obtain modified chitosan.

[0019] Preferably, the amount 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;

[0020] The mass ratio of 2-amino terephthalic acid, sodium hydroxide and mixture A in step A2 is 8-13: 10-15: 6-10;

[0021] The concentration of the sodium hydroxide solution is 0.9-1.2 mol / L;

[0022] The drying temperature is 50-60° C. and the drying time is 12-14 hours.

[0023] Preferably, the preparation method of the composite microorganism is as follows:

[0024] Step B1. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis, cellulose-degrading bacteria, and Brevibacillus laterosporus were inoculated into LB liquid medium, and cultured at 37°C until the logarithmic growth phase to obtain a suspension of Pseudomonas stutzeri, a suspension of Rhodopseudomonas palustris, a suspension of Bacillus subtilis, a suspension of cellulose-degrading bacteria, and a suspension of Brevibacillus laterosporus, respectively;

[0025] 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 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;

[0026] The LB liquid culture medium in step B1 is formulated as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and a pH of 7.0-7.2.

[0027] The mass ratio of the Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and Brevibacillus laterosporus suspension in step B2 is 1:1-2:3-5:2.5-3.8:1-2.

[0028] Preferably, the preparation method of the microspheres A is as follows:

[0029] 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 rotation speed of 130-160 r / min for 20-24 hours. After removal, let it stand for 2-3 hours, filter, rinse with sterile water, and filter to obtain the modified composite microorganism;

[0030] Step C2. γ-aminopropyltriethoxysilane and succinic anhydride were added to a flask containing N,N-dimethylformamide and heated with stirring at 55-65°C for 2-3 hours to obtain a mixture B. The hollow silica microspheres were added to N,N-dimethylformamide and ultrasonically dispersed. The mixture was heated with stirring at 55-65°C for 4-5 hours and washed by centrifugation to obtain carboxylated hollow silica microspheres.

[0031] Step C3. The modified composite microorganism, carboxylated hollow silica microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were heated and stirred at 35-42°C for 3-4 hours, filtered, washed with sterile water, and then the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160 r / min and cultured for 20-24 hours. After taking out, it was allowed to stand for 2-3 hours, filtered, rinsed with sterile water, and filtered to obtain microspheres A.

[0032] Preferably, the mass ratio of the composite microorganism to the modified chitosan in step C1 is 35-42:3-4;

[0033] The molar ratio of γ-aminopropyltriethoxysilane, succinic anhydride and N,N-dimethylformamide in step C2 is 1:1:3-4;

[0034] The mass ratio of the mixture B, hollow silica microspheres and N,N-dimethylformamide is 8-12:4-7:10-14;

[0035] The mass ratio of the modified composite microorganism, carboxylated hollow silica microspheres, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and composite microorganism in step C3 is 15-25:7-12:7-12:10-14.

[0036] Preferably, the preparation method of the microspheres B is as follows:

[0037] Step D1. Sodium silicate was added to deionized water and stirred for 30-40 minutes. The mixture was passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor. Nano-titanium dioxide particles were added to deionized water and ultrasonically dispersed for 150-180 minutes. PEG-400 was then added and stirred for 25-35 minutes to obtain an aqueous dispersion of titanium dioxide nanoparticles. The aqueous dispersion of titanium dioxide nanoparticles was then added to the transparent aqueous solution of the silica precursor and mixed uniformly to obtain a mixture C.

[0038] Step D2. Abil EM-90 was added to n-tetradecane, mixed evenly, and then the mixture C was added thereto and homogenized with a homogenizer for 50-65 seconds to obtain a mixture D;

[0039] Step D3. The mixture D is dried, then washed with hexane 3-4 times, dried at room temperature for 26-30 hours, and then calcined at high temperature to obtain microspheres B.

[0040] Preferably, the volume ratio of sodium silicate to deionized water in step D1 is 1:1.8-2.3;

[0041] The mass ratio of the nano-titanium dioxide particles, deionized water and PEG-400 is 0.25-0.32:10:0.03-0.04;

[0042] The volume ratio of the titanium dioxide nanoparticle aqueous dispersion to the silicon dioxide precursor transparent aqueous solution is 4-5:3-5;

[0043] In step D2, the mass ratio of Abil EM-90, n-tetradecane, and mixture C is 0.25-0.35:10:8-12;

[0044] The drying temperature in step D3 is 80-90° C. and the drying time is 90-110 min;

[0045] The temperature during the high-temperature calcination is 455-500°C, and the calcination time is 5-6h.

[0046] Preferably, the preparation method of the microspheres C is as follows:

[0047] Step E1. Soak the biochar in a 40% by mass KOH solution, stir for 12-13 hours, let it stand for 20-24 hours, and then dry it at 110-120°C for 48-50 hours. After cooling, wash it twice with a 1 mol / L hydrochloric acid solution and deionized water, and then dry it at 60-70°C for 3-4 hours to obtain activated biochar.

[0048] Step E2. Evenly mixing the activated biochar and bentonite to obtain microspheres C;

[0049] In step E1, the mass ratio of the biochar to the 40% KOH solution is 1:4.5-5.5;

[0050] The mass ratio of the activated biochar to bentonite in step E2 is 15-20:6-9.

[0051] A method for preparing a water purifier for aquaculture based on composite microorganisms comprises the following steps:

[0052] The microspheres A, B and C are mixed evenly to obtain a water purifier for aquaculture based on composite microorganisms.

[0053] Beneficial effects of the present invention:

[0054] The present invention provides a water purifier for aquaculture based on composite microorganisms and a preparation method thereof. The present invention obtains microspheres A by combining modified composite microorganisms, carboxylated hollow silica microspheres and the composite microorganisms; obtains microspheres B by immobilizing nano-titanium dioxide particles by silica microspheres; obtains microspheres C by mixing activated biochar with bentonite; and obtains a water purifier for aquaculture based on composite microorganisms by compounding microspheres A, microspheres B and microspheres C. The water purifier can purify water and effectively remove organic pollutants and heavy metals in water, thereby providing good water quality conditions for aquaculture, and can also improve the stability and durability of the composite microorganisms and reduce the frequency of adding water purifiers.

[0055] Among them, the modified composite microorganisms in microspheres A are prepared with modified chitosan as the shell and the composite microorganisms as the core. They can effectively adsorb heavy metals and other pollutants in water, and synergize with carboxylated hollow silica microspheres to further improve the stability and durability of the composite microorganisms. Microspheres A can better achieve water purification effects through physical adsorption and decomposition of composite microorganisms.

[0056] The titanium dioxide particles in microspheres B can decompose organic pollutants into small carbon dioxide molecules, while the silicon dioxide microspheres can reduce the agglomeration of titanium dioxide particles and improve stability. On the one hand, microspheres B can purify water bodies, and on the other hand, they can also reduce the adverse effects of organic pollutants on complex microorganisms; microspheres C purify water quality through physical adsorption, which helps to delay the action time of complex microorganisms and thus exert a lasting purification effect; compared with existing technologies, it has broad application prospects. DETAILED DESCRIPTION

[0057] 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 with reference to specific embodiments.

[0058] The sources and properties of some of the raw materials used in the present invention are as follows:

[0059] 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; and n-tetradecane was purchased from Zhongshan Dixin Chemical Co., Ltd.

[0060] The Pseudomonas stutzeri used in the present invention is disclosed in the invention patent with the authorization publication number “CN114806959B”, entitled “A microbial preparation and aquaculture water purification method”, and the deposit number is CGMCC1.3184;

[0061] The Rhodopseudomonas palustris used in the present invention is disclosed in the invention patent with the authorization publication number "CN106148218B" and the name "Photosynthetic Bacteria Microecology for Aquaculture and Its Application", and the deposit number is CGMCC No.9136;

[0062] The Bacillus subtilis used in the present invention is Bacillus subtilis N9-1-35, which is disclosed in the invention patent with the authorization publication number "CN105481101B" and the name "A composite water purifier for aquaculture and its preparation method", and the deposit number is CCTCC M 2011301;

[0063] 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 publication number "CN103468618B" and the name "Cellulose-degrading bacteria with phosphate-solubilizing ability and its application", and the deposit number is CGMCC No. 7923;

[0064] The Brevibacillus laterosporus used in the present invention is Brevibacillus laterosporus HY30, which is disclosed in the invention patent with authorization publication number “CN111394272B” and titled “A strain of Brevibacillus laterosporus and its application”, and the deposit number is CCTCCNo: M20191101.

[0065] Example 1: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps:

[0066] S1. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 40°C until completely dissolved. Adjust the pH to 7. Add 15 g of agar and heat at 50°C until completely dissolved. Filter and autoclave at 121°C for 15 min to obtain LB solid medium. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 40°C until completely dissolved. Adjust the pH to 7. Filter and autoclave at 121°C for 15 min to obtain LB liquid medium.

[0067] S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12, and Brevibacillus laterosporus HY30 were activated, plated, and cultured at 37°C. Single colonies were picked and streaked on LB solid medium for further purification until the colonies were uniform and free of contaminants. A single colony of the activated bacteria was picked with an inoculating loop and inoculated into LB liquid medium. The culture was 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. The suspensions were then washed three times with sterile water.

[0068] 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. The suspensions were centrifuged at 4000 rpm for 20 min and mixed with sterile water to form a uniform bacterial suspension. The viable bacteria were counted under a microscope, and the number of viable bacteria was calculated to be 2.56 × 10 9 cfu / mL, composite microorganisms were obtained;

[0069] S4. 0.45 g of chitosan was dissolved in 45 mL of a 2% aqueous acetic acid solution, stirred, and then 0.12 g of bentonite was added and mixed to obtain a mixture A;

[0070] S5. 8g of 2-aminoterephthalic acid was dissolved in 10g of 0.9mol / L sodium hydroxide solution, and then 6g of mixture A was added and mixed uniformly, centrifuged and dried at 50°C for 14h to obtain modified chitosan;

[0071] S6 was added to 35g of the composite microorganism 3g of modified chitosan, and the conical flask was placed in a constant temperature shaking incubator set to 25 ° C, the speed was set to 160r / min and cultured for 24h, removed and allowed to stand for 3h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism;

[0072] S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 3 mol of N,N-dimethylformamide and heated with stirring 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 ultrasonically dispersed. The mixture was heated with 8 g of the mixture B at 55 ° C for 4 h and washed by centrifugation to obtain carboxylated hollow silica microspheres.

[0073] 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, and washed with sterile water. Then, 10 g of the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set to 25 ° C and a speed of 130 r / min and incubated for 20 h. After removal, the mixture was allowed to stand for 2 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A;

[0074] 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 was added to 10 g of deionized water and ultrasonically dispersed for 150 min. 0.03 g of PEG-400 was then added and stirred for 25 min to obtain an aqueous dispersion of titanium dioxide nanoparticles. 4 mL of the aqueous dispersion of titanium dioxide nanoparticles was then added to 3 mL of the transparent aqueous solution of the silica precursor and mixed to obtain a mixture C.

[0075] S10. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed well, and then 8 g of mixture C was added thereto and homogenized with a homogenizer for 50 s to obtain a mixture D;

[0076] S11. The mixture D was dried at 80 ° C for 90 min, then washed three times with hexane, dried at room temperature for 26 h, and calcined at 455 ° C for 5 h to obtain microspheres B;

[0077] S12. 1 g of biochar was immersed in 4.5 g of a 40% KOH solution, stirred for 12 h, allowed to stand for 20 h, and then dried at 110°C for 48 h. After cooling, the solution was washed twice with 1 mol / L hydrochloric acid solution and then with deionized water, and then dried at 60°C for 3 h to obtain activated biochar.

[0078] S13. 15 g of activated biochar was mixed with 6 g of bentonite to obtain microspheres C;

[0079] S14. Evenly mix 47 g of microspheres A, 2.8 g of microspheres B, and 5 g of microspheres C to obtain a water purifier for aquaculture based on composite microorganisms.

[0080] Example 2: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps:

[0081] S1. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 45°C until completely dissolved. Adjust the pH to 7.1. Add 15 g of agar and heat at 55°C until completely dissolved. Filter and autoclave at 121°C for 15 min to obtain LB solid medium. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 45°C until completely dissolved. Adjust the pH to 7.1. Filter and autoclave at 121°C for 15 min to obtain LB liquid medium.

[0082] S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12, and Brevibacillus laterosporus HY30 were activated, plated, and cultured at 37°C. Single colonies were picked and streaked on LB solid medium for further purification until the colonies were uniform and free of contaminants. A single colony of the activated bacteria was picked with an inoculating loop and inoculated into LB liquid medium. The culture was 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. The suspensions were then washed three times with sterile water.

[0083] 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. Centrifuge at 4500 rpm for 18 minutes. Mix with sterile water to form a uniform bacterial suspension. Count the viable bacteria under a microscope, and the number of viable bacteria was calculated to be 1.77 × 10 9 cfu / mL, composite microorganisms were obtained;

[0084] S4. 0.53 g of chitosan was dissolved in 50 mL of a 2% aqueous acetic acid solution, stirred, and then 0.18 g of bentonite was added and mixed to obtain a mixture A;

[0085] S5. 8g of 2-aminoterephthalic acid was dissolved in 10g of 1mol / L sodium hydroxide solution, and then 6g of mixture A was added and mixed uniformly, centrifuged and dried at 55°C for 13h to obtain modified chitosan;

[0086] S6 was added to 38g of the composite microorganism 3.5g of modified chitosan, and the conical flask was placed in a constant temperature shaking incubator set to 28 ° C, the speed was set to 150r / min and cultured for 22h, removed and allowed to stand for 2.5h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism;

[0087] 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 and heated with stirring 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 ultrasonically dispersed. The mixture was heated with 10 g of the mixture B at 60 ° C for 4.5 h and washed by centrifugation to obtain carboxylated hollow silica microspheres.

[0088] 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 to 28 ° C and the speed was set to 150 r / min for 22 h. After removal, it was allowed to stand for 2.5 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A;

[0089] 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 was added to 10 g of deionized water and ultrasonically dispersed for 160 min. 0.035 g of PEG-400 was then added and stirred for 30 min to obtain an aqueous dispersion of titanium dioxide nanoparticles. 4.5 mL of the aqueous dispersion of titanium dioxide nanoparticles was then added to 4 mL of the transparent aqueous solution of the silica precursor and mixed to obtain a mixture C.

[0090] S10. 0.3 g Abil EM-90 was added to 10 g of 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;

[0091] S11. The mixture D was dried at 85 ° C for 100 min, then washed with hexane four times, dried at room temperature for 28 h, and calcined at 480 ° C for 5.5 h to obtain microspheres B;

[0092] S12. 1 g of biochar was immersed in 5 g of a 40% KOH solution, stirred for 12 h, allowed to stand for 22 h, and then dried at 115°C for 49 h. After cooling, the solution was washed twice with 1 mol / L hydrochloric acid solution and then with deionized water, and then dried at 65°C for 3.5 h to obtain activated biochar.

[0093] S13. 18 g of activated biochar was mixed with 8 g of bentonite to obtain microspheres C;

[0094] S14. Evenly mix 51 g of microspheres A, 3.7 g of microspheres B, and 6.6 g of microspheres C to obtain a water purifier for aquaculture based on composite microorganisms.

[0095] Example 3: A method for preparing a water purifier for aquaculture based on composite microorganisms, comprising the following steps:

[0096] S1. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 50°C until completely dissolved. Adjust the pH to 7.2. Add 15 g of agar and heat at 60°C until completely dissolved. Filter and autoclave at 121°C for 15 min to obtain LB solid medium. Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add 1000 mL of distilled water, and heat at 50°C until completely dissolved. Adjust the pH to 7.2. Filter and autoclave at 121°C for 15 min to obtain LB liquid medium.

[0097] S2. Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12, and Brevibacillus laterosporus HY30 were activated, plated, and cultured at 37°C. Single colonies were picked and streaked on LB solid medium for further purification until the colonies were uniform and free of contaminants. A single colony of the activated bacteria was picked with an inoculating loop and inoculated into LB liquid medium. The culture was 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. The suspensions were then washed three times with sterile water.

[0098] 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. The suspensions were centrifuged at 5000 rpm for 15 min. The suspensions were mixed with sterile water to form a uniform bacterial suspension. The viable bacteria were counted under a microscope, and the number of viable bacteria was calculated to be 3.55 × 10 9 cfu / mL, composite microorganisms were obtained;

[0099] S4. 0.6 g of chitosan was dissolved in 60 mL of a 2% aqueous acetic acid solution, stirred, and then 0.25 g of bentonite was added and mixed to obtain a mixture A;

[0100] S5. 13g of 2-aminoterephthalic acid was dissolved in 15g of 1.2mol / L sodium hydroxide solution, and then 10g of the mixture A was added and mixed, centrifuged and dried at 60°C for 12h to obtain modified chitosan;

[0101] S6 was added to 42g of the composite microorganism 4g of modified chitosan, and the conical flask was placed in a constant temperature shaking incubator set to 30 ° C, the speed was set to 160r / min and cultured for 20h, removed and allowed to stand for 3h, filtered and rinsed with sterile water, filtered to obtain a modified composite microorganism;

[0102] S7. 1 mol of γ-aminopropyltriethoxysilane and 1 mol of succinic anhydride were added to a flask containing 4 mol of N, N-dimethylformamide and heated with stirring 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 ultrasonically dispersed. The mixture was heated with 12 g of B at 65 ° C for 5 h and washed by centrifugation to obtain carboxylated hollow silica microspheres.

[0103] 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 to 30 ° C and the speed was set to 160 r / min for 24 h. After removal, it was allowed to stand for 3 h, filtered, rinsed with sterile water, and filtered to obtain microspheres A;

[0104] 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 was added to 10 g of deionized water and ultrasonically dispersed for 180 min. 0.04 g of PEG-400 was then added and stirred for 35 min to obtain an aqueous dispersion of titanium dioxide nanoparticles. 5 mL of the aqueous dispersion of titanium dioxide nanoparticles was then added to 5 mL of the transparent aqueous solution of the silica precursor and mixed to obtain a mixture C.

[0105] S10. 0.35 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed well, and then 12 g of mixture C was added thereto and homogenized with a homogenizer for 65 s to obtain a mixture D;

[0106] S11. The mixture D was dried at 90 ° C for 110 min, then washed with hexane four times, dried at room temperature for 30 h, and calcined at 500 ° C for 6 h to obtain microspheres B;

[0107] S12. 1 g of the biochar was soaked in 5.5 g of a KOH solution with a mass fraction of 40%, stirred for 13 h, and then left to stand for 24 h. The mixture was dried at 120°C for 50 h, and then cooled. The cooled mixture was washed twice with a 1 mol / L hydrochloric acid solution and deionized water, and then dried at 70°C for 4 h to obtain the activated biochar;

[0108] S13. 20 g of the activated biochar was mixed with 9 g of the bentonite to obtain the microspheres C;

[0109] S14. 55 g of the microspheres A, 5.3 g of the microspheres B, and 8 g of the microspheres C were mixed to obtain the water purifier for aquaculture based on the composite microorganisms.

[0110] Comparative Example 1

[0111] The comparative example was the same as Example 1 except that the modified composite microorganisms were not added in the preparation of the microspheres A. The remaining steps and parameters were the same, and will not be repeated. Finally, the water purifier for aquaculture based on the composite microorganisms was obtained.

[0112] Comparative Example 2

[0113] The comparative example was the same as Example 1 except that the microspheres B were not added in the preparation of the water purifier for aquaculture based on the composite microorganisms. The remaining steps and parameters were the same, and will not be repeated. Finally, the water purifier for aquaculture based on the composite microorganisms was obtained.

[0114] Comparative Example 3

[0115] The comparative example was the same as Example 1 except that the microspheres C were not added in the preparation of the water purifier for aquaculture based on the composite microorganisms. The remaining steps and parameters were the same, and will not be repeated. Finally, the water purifier for aquaculture based on the composite microorganisms was obtained.

[0116] Comparative Example 4

[0117] The comparative example was the same as Example 1 except that the "modified chitosan" was replaced by "chitosan". The remaining steps and parameters were the same, and will not be repeated. Finally, the water purifier for aquaculture based on the composite microorganisms was obtained.

[0118] Comparative Example 5

[0119] S1. Take 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat to complete dissolution at 40°C, adjust the pH to 7, add 15 g of agar and heat to complete dissolution at 50°C, filter and high-temperature sterilize at 121°C for 15 min to obtain LB solid culture medium; take 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, add 1000 mL of distilled water, heat to complete dissolution at 40°C, adjust the pH to 7, filter and high-temperature sterilize at 121°C for 15 min to obtain LB liquid culture medium;

[0120] S2. Activate Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 respectively, plate spread, and place in a constant temperature incubator at 37°C. Pick single colonies on LB solid medium and streak for further purification until the colonies are uniform and free of contaminants. Take single colonies of the activated bacteria with a loop and inoculate them into LB liquid medium. Incubate at 37°C until the logarithmic growth phase. Obtain Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose-degrading bacteria JSD-12 suspension and Brevibacillus laterosporus HY30 suspension, and wash with sterile water 3 times;

[0121] S3. Mix the Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacteria suspension and Brevibacillus laterosporus suspension in a centrifuge tube at a mass ratio of 1:1:3:2.5:1. Centrifuge at 4000 rpm for 20 min. Mix with sterile water to obtain a uniformly distributed bacterial suspension. Count the viable bacteria under a microscope. The calculated viable bacteria count is 2.56 x 10 9 cfu / mL, to obtain a composite microorganism;

[0122] S4. Dissolve 0.45 g of chitosan in 45 mL of 2% acetic acid aqueous solution, stir until uniform, then add 0.12 g of bentonite and mix until uniform to obtain mixture A;

[0123] S5. Dissolve 8 g of 2-amino terephthalic acid in 10 g of 0.9 mol / L NaOH solution, then add 6 g of mixture A and mix until uniform. Centrifuge and dry at 50°C for 14 h to obtain modified chitosan;

[0124] S6. Add 3 g of modified chitosan to 35 g of composite microorganism, and place the conical flask in a constant temperature shaking incubator with a temperature setting of 25°C and a rotation speed setting of 160 r / min for 24 h. After removal, stand for 3 h, filter and rinse with sterile water, and filter to obtain modified composite microorganism;

[0125] 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 mixture B, 4 g of hollow silica microspheres were added to 10 g of N,N-dimethylformamide, and after ultrasonic dispersion, 8 g of mixture B was heated and stirred at 55°C for 4 h, and centrifuged and washed to obtain carboxylated hollow silica microspheres;

[0126] S8. 15 g of modified composite microorganisms, 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 and washed with sterile water to obtain microspheres A;

[0127] 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 was added to 10 g of deionized water and ultrasonically dispersed for 150 min, 0.03 g of PEG-400 was added, and stirred for 25 min to obtain a water dispersion of titanium dioxide nanoparticles, and then 4 mL of the water dispersion of titanium dioxide nanoparticles was added to 3 mL of the transparent aqueous solution of the silica precursor, and mixed uniformly to obtain mixture C;

[0128] S10. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane and mixed uniformly, and then 8 g of mixture C was added and homogenized for 50 s with a homogenizer to obtain mixture D;

[0129] S11. Mixture D was dried at 80°C for 90 min, then washed with hexane 3 times, and dried at room temperature for 26 h, and then calcined at 455°C for 5 h to obtain microspheres B;

[0130] S12. 1 g of biochar was soaked in 4.5 g of a 40% by mass KOH solution, stirred for 12 h, and allowed to stand for 20 h, and then dried at 110°C for 48 h, and then washed twice with a 1 mol / L hydrochloric acid solution and deionized water, and then dried at 60°C for 3 h to obtain activated biochar;

[0131] S13. 15 g of activated biochar was mixed uniformly with 6 g of bentonite to obtain microspheres C;

[0132] S14. 47 g of microspheres A, 2.8 g of microspheres B, and 5 g of microspheres C were mixed uniformly to obtain a water body purifying agent for aquaculture based on composite microorganisms.

[0133] Comparative Example 6:

[0134] The comparative example is compared with example 1 only by replacing "47 g of microspheres A, 2.8 g of microspheres B and 5 g of microspheres C" with "10 g of microspheres A, 10 g of microspheres B and 10 g of microspheres C", and the rest of the steps and parameters are the same. The comparative example will not be repeated here. The final product is a water body purifier for aquaculture based on composite microorganisms.

[0135] Comparative example 7:

[0136] The comparative example is compared with example 1 only by replacing "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 rest of the steps and parameters are the same. The comparative example will not be repeated here. The final product is a water body purifier for aquaculture based on composite microorganisms.

[0137] Comparative example 8:

[0138] S1. Take 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, add 1000 mL of distilled water, heat to complete dissolution at 40℃, adjust pH to 7, add 15 g of agar, heat to complete dissolution at 50℃, filter and sterilize at 121℃ for 15 min to obtain LB solid medium; take 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, add 1000 mL of distilled water, heat to complete dissolution at 40℃, adjust pH to 7, filter and sterilize at 121℃ for 15 min to obtain LB liquid medium;

[0139] S2. Activate Pseudomonas stutzeri, Rhodopseudomonas palustris, Bacillus subtilis N9-1-35, cellulose-degrading bacteria JSD-12 and Brevibacillus laterosporus HY30 respectively, plate and incubate at 37℃, pick single colonies on LB solid medium for further purification, until the colonies are uniform and there are no impurities. Take a single colony of the activated bacteria with a loop, inoculate in LB liquid medium and incubate at 37℃ until the logarithmic growth phase. 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 are obtained, and washed with sterile water for 3 times;

[0140] S3. Pseudomonas stutzeri bacterial suspension, Rhodopseudomonas palustris bacterial suspension, Bacillus subtilis bacterial suspension, cellulose-degrading bacterial suspension, and Brevibacillus laterosporus bacterial suspension were poured into centrifuge tubes at a mass ratio of 1:1:3:2.5:1, centrifuged at 4000 rpm for 20 min, mixed with sterile water, and configured into a uniformly distributed bacterial suspension. Microscopic viable bacterial count was performed, and the calculated viable bacterial count was 2.56 x 10 9 cfu / mL, to obtain a composite microorganism;

[0141] S4. 0.45 g of chitosan was dissolved in 45 mL of a 2% by mass acetic acid aqueous solution, stirred until uniform, 0.12 g of bentonite was added and mixed until uniform, to obtain mixture A;

[0142] S5. 8 g of 2-amino terephthalic acid was dissolved in 10 g of a 0.9 mol / L sodium hydroxide solution, then added to 6 g of mixture A and mixed until uniform, centrifuged, and dried at 50°C for 14 h, to obtain modified chitosan;

[0143] S6. 3 g of modified chitosan was added to 35 g of the composite microorganism, and the conical flask was placed in a constant-temperature shaking incubator with a temperature setting of 25°C and a rotation speed setting of 160 r / min for incubation for 24 h. After removal, it was allowed to stand for 3 h, filtered, rinsed with sterile water, and filtered, to obtain microspheres A;

[0144] S7. 1 mL of sodium silicate was added to 1.8 mL of deionized water, stirred for 30 min, passed through a hydrogen-type cation exchange resin, to obtain a transparent aqueous solution of a silicon dioxide precursor. 0.25 g of nano-titanium dioxide particles was added to 10 g of deionized water, and ultrasonically dispersed for 150 min. 0.03 g of PEG-400 was added, stirred for 25 min, to obtain a water dispersion of titanium dioxide nanoparticles. 4 mL of the water dispersion of titanium dioxide nanoparticles was added to 3 mL of the transparent aqueous solution of the silicon dioxide precursor, and mixed until uniform, to obtain mixture C;

[0145] S8. 0.25 g of Abil EM-90 was added to 10 g of n-tetradecane, mixed until uniform, 8 g of mixture C was added, and homogenized for 50 s using a homogenizer, to obtain mixture D;

[0146] S9. Mixture D was dried at 80°C for 90 min, then washed with hexane 3 times, and placed at room temperature for drying for 26 h. High-temperature calcination was performed at 455°C for 5 h, to obtain microspheres B;

[0147] S10. 1 g of biochar was soaked in 4.5 g of a 40% by mass KOH solution, stirred for 12 h, allowed to stand for 20 h, dried at 110°C for 48 h, and then cooled. The biochar was washed twice with a 1 mol / L hydrochloric acid solution and deionized water, and then dried at 60°C for 3 h, to obtain activated biochar.

[0148] S11. 15 g of activated biochar was mixed with 6 g of bentonite to obtain microspheres C;

[0149] S12. 47 g of microspheres A, 2.8 g of microspheres B and 5 g of microspheres C were mixed to obtain the composite microorganism-based water body purifier for aquaculture.

[0150] Comparative Example 9:

[0151] This comparative example is compared with Example 1 only by replacing "0.45 g of chitosan, 45 mL of acetic acid aqueous solution and 0.12 g of bentonite" with "0.12 g of chitosan, 12 mL of acetic acid aqueous solution and 0.12 g of bentonite", and the rest of the steps and parameters are the same. This comparative example will not be repeated here. Finally, the composite microorganism-based water body purifier for aquaculture is obtained.

[0152] Comparative Example 10:

[0153] This comparative example is compared with Example 1 only by replacing "0.45 g of chitosan, 45 mL of acetic acid aqueous solution and 0.12 g of bentonite" with "0.9 g of chitosan, 90 mL of acetic acid aqueous solution and 0.12 g of bentonite", and the rest of the steps and parameters are the same. This comparative example will not be repeated here. Finally, the composite microorganism-based water body purifier for aquaculture is obtained.

[0154] Performance test:

[0155] Water quality test:

[0156] Take fish and shrimp farming pond wastewater with high organic matter content, mix well and divide into 8L plastic buckets, each bucket containing 4L, and add the composite microorganism-based water body purifier for aquaculture prepared in Example 1-Example 3 and Comparative Example 1-Comparative Example 10, respectively. Measure the nitrite and ammonia nitrogen content values of each group of water samples at 0d, 1d, 30d and 90d, respectively. Each test is repeated 3 times, and the average value of the three repeated tests is taken;

[0157] Application effect test:

[0158] The same batch of crayfish fry was divided into 13 equal parts, and each part of the crayfish fry was placed in a breeding pond of the same size. Then, the composite microorganism-based water body purifier for aquaculture prepared in Example 1-Example 3 and Comparative Example 1-Comparative Example 10 was added, and 1 kg was added per mu of breeding pond. The other breeding conditions were all the same. Finally, the survival rate, yield and average weight of each group of crayfish were measured after one month of breeding.

[0159] The same batch of sea bass fry was divided into 13 equal parts, and each part of the sea bass fry was placed in a culture pond of the same size. Then, the composite microbial aquaculture water purifier prepared in Examples 1 to 3 and Comparative Examples 1 to 10 was added, and 1 kg was added per acre of the culture pond. Other culture conditions were the same. Finally, after 5 months of culture, the survival rate, yield and weight of each group of sea bass were measured;

[0160] Water heavy metal detection:

[0161] Take fish and shrimp breeding pond wastewater 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 Examples 1 to 3 and Comparative Examples 1 to 10, respectively. After 1 day, 30 days, and 90 days, the content of heavy metal copper and chromium in each group of water was detected to obtain the heavy metal removal rate.

[0162] Table 1

[0163]

[0164] Table 2

[0165]

[0166] Table 3

[0167]

[0168] Data Analysis:

[0169] As can be seen from Tables 1 to 3, the aquaculture water purifier based on composite microorganisms prepared by the present invention has a better purification effect and a long working time, which significantly reduces the content of nitrite, ammonia nitrogen and heavy metals in the water and increases the yield of aquatic organisms. This may be because the modified composite microorganisms in microspheres A are prepared with modified chitosan as the shell and the composite microorganisms as the core. The modified chitosan contains a large number of amino active groups, which can effectively adsorb heavy metals and other pollutants in the water and fix the composite microorganisms through metal chelation and electrostatic attraction, thereby improving the stability of the composite microorganisms. The composite microorganisms are compounded by five different microorganisms in a specific proportion, which can not only effectively reduce the content of nitrite and ammonia nitrogen in the water, but also decompose the difficult-to-digest 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. Microspheres A can better achieve water purification effects through physical adsorption and decomposition of composite microorganisms.

[0170] The titanium dioxide particles in microspheres B can decompose organic pollutants into small molecules of carbon dioxide, remove some pesticide residues in the water, and reduce diseases and pests of aquatic organisms. The silicon dioxide microspheres can reduce the agglomeration of titanium dioxide particles and improve stability. On the one hand, microspheres B can purify water bodies, and on the other hand, they can also reduce the adverse effects of organic pollutants on composite microorganisms, thereby improving the stability of composite microorganisms. Microspheres C purify water quality through physical adsorption, which is beneficial to delaying 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 quality, effectively remove organic pollutants and heavy metals in water, provide good water quality conditions for aquaculture, and also improve the stability and durability of composite microorganisms, thereby reducing the frequency of water purifier administration.

[0171] As can be seen from Tables 1 to 3, since no modified composite microorganisms were added to Comparative Example 1, its water purification effect was worse than that of Example 1, and its working time was shorter, so that the contents of nitrite, ammonia nitrogen and heavy metals in the water decreased in the early stage of the water purifier, but the contents still increased in the later stage, and the aquatic production was poor. This may be because the modified composite microorganisms were prepared with modified chitosan as the shell and composite microorganisms as the core, and the stability of the composite microorganisms was improved by immobilizing the microorganisms. The microspheres A in Comparative Example 1 were prepared only by composite microorganisms and carboxylated hollow silica microspheres, and the adsorption of pollutants such as heavy metals in water was greatly weakened, and the composite microorganisms were not stable. The stability of the substance is also poor, the effective purification time is shorter, and the yield is also low; since Comparative Example 2 does not add microspheres B, it can be seen from Tables 1 to 3 that its water purification effect is worse than that of Example 1, and the time of taking effect 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 organism yield is poor. This may be because titanium dioxide particles can decompose organic pollutants into small molecules of carbon dioxide, such as aquaculture disinfectants 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 composite microorganisms. The water purification effect of Comparative Example 3 is poorer than that of Example 1 due to the lack of microspheres C. As can be seen from Tables 1 to 3, the water purification effect is shorter than that of Example 1, and the time of taking effect is shorter, so that the content of nitrite, ammonia nitrogen and heavy metals in the water decreases in the early stage of the water purifier, but the content still increases in the later stage, and the aquatic production is poor. This may be because the activated biochar and bentonite have a strong adsorption effect, which can play a role instantly within a short time after the water purifier is added, thereby delaying the action time of the composite microorganisms and further removing pollutants that are detrimental to the survival of microorganisms. In Comparative Example 4, "modified chitosan" is replaced by " Chitosan", as can be seen from Tables 1 to 3, the water purification effect is worse than that of Example 1, and the working 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 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, which then better combines with the carboxylated hollow silica microspheres, so that the composite microorganisms are stable and work for a long time;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 taking effect 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 organism 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. They may also be adsorbed in the hollow silica microspheres together with the modified composite microorganisms to form In comparative example 6, since "47g microspheres A, 2.8g microspheres B and 5g microspheres C" are replaced by "10g microspheres A, 10g microspheres B and 10g microspheres C", 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 taking effect is shorter, so that the content of nitrite, ammonia nitrogen and heavy metals in the water decreases in the early stage of the water purifier, but the content still increases in the later stage of the water purifier, and the aquatic organism 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 from water and improving water quality. Microspheres B and microspheres play an important role in removing nitrite and ammonia nitrogen from water and improving water quality. The decomposition and adsorption effects of ball C on pollutants are limited. When the treatment limit is reached, the pollutants in the water will grow again. Therefore, the working time is short and the yield is low. In comparative example 7, the "mass ratio of Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose-degrading bacteria JSD-12 suspension and Brevibacillus laterosporus HY30 suspension is 1:1:3:2.5:1" is replaced by "Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis N9-1-35 suspension, cellulose-degrading bacteria JSD-12 suspension and Brevibacillus 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 aquatic organism yield is poor. This may be due to the strong reproduction and pollutant decomposition capabilities of the Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, and Bacillus subtilis N9-1-35. 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 must be compounded in a specific ratio to play a better water purification role.Comparative Example 8 Since carboxylated hollow silica microspheres 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 taking effect 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, and the aquatic biological yield is poor. This may be because the carboxylated hollow silica microspheres can be cross-linked with the modified chitosan in the modified composite microorganism, thereby improving the overall stability of microspheres A and further enhancing the compatibility with microspheres B and microspheres C; 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 to 3 that the water purification effect is worse than that of Example 1, and the time of taking effect is shorter, so that the nitrite, ammonia nitrogen and heavy metal contents in the water decrease in the initial stage of the water purifier. The content decreased in the early stage of administration, but continued to increase in the later stage, and the aquatic organism yield was poor. This may be because the excess bentonite covers the surface of chitosan, thereby covering its active functional groups, reducing the degree of cross-linking with the carboxylated hollow silica, and reducing the overall stability of Microsphere A. In Comparative Example 10, since "0.45g chitosan, 45mL acetic acid aqueous solution, and 0.12g bentonite" was replaced with "0.9g chitosan, 90mL acetic acid aqueous solution, and 0.12g bentonite", as can be seen from Tables 1-3, the water purification effect was worse than that of Example 1, and the duration of action was shorter. As a result, the nitrite, ammonia nitrogen, and heavy metal contents in the water decreased in the early stage of water purifier administration, but continued to increase in the later stage of administration, and the aquatic organism yield was poor. This may be because when the amount of bentonite is too small, the polymer chains of chitosan will self-crosslink to form a dense substance, resulting in fewer adsorption sites, a reduced degree of immobilization of the composite microorganisms, and poor stability.

[0172] 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. Within the scope 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.

[0173] 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 within the scope of protection of the present invention.

Claims

1. A water purifier for aquaculture based on composite microorganisms, characterized in that: It is prepared from the following raw materials in parts by weight: Microspheres A 47-55 parts, microspheres B 2.8-5.3 parts; microspheres C 5-8 parts; The microspheres A are 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% acetic acid aqueous solution, stir evenly, then add bentonite and mix evenly to obtain a mixture A; Step A2. Dissolve 2-aminoterephthalic acid in sodium hydroxide solution, then add it to mixture A and 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 medium, and cultured at 37°C until the logarithmic growth phase to obtain a suspension of Pseudomonas stutzeri, a suspension of Rhodopseudomonas palustris, a suspension of Bacillus subtilis, a suspension of cellulose-degrading bacteria, and a suspension of Brevibacillus laterosporus, 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 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 extract, 10 g / L NaCl, and a pH of 7.0-7.

2. The mass ratio of the Pseudomonas stutzeri suspension, Rhodopseudomonas palustris suspension, Bacillus subtilis suspension, cellulose-degrading bacterial suspension and 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 rotation speed of 130-160 r / min for 20-24 hours. After removal, let it stand for 2-3 hours, filter, rinse with sterile water, and filter to obtain the modified composite microorganism; Step C2. γ-aminopropyltriethoxysilane and succinic anhydride were added to a flask containing N,N-dimethylformamide and heated with stirring at 55-65°C for 2-3 hours to obtain a mixture B. The hollow silica microspheres were added to N,N-dimethylformamide and ultrasonically dispersed. The mixture was heated with stirring at 55-65°C for 4-5 hours 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 were heated and stirred at 35-42°C for 3-4 hours, filtered, washed with sterile water, and then the composite microorganism was added and the conical flask was placed in a constant temperature shaking incubator set at a temperature of 25-30°C and a speed of 130-160 r / min and cultured for 20-24 hours. After taking out, it was allowed to stand for 2-3 hours, filtered, rinsed with sterile water, and filtered 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 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 microspheres B is as follows: Step D1. Sodium silicate was added to deionized water and stirred for 30-40 minutes. The mixture was passed through a hydrogen-type cation exchange resin to obtain a transparent aqueous solution of a silica precursor. Nano-titanium dioxide particles were added to deionized water and ultrasonically dispersed for 150-180 minutes. PEG-400 was then added and stirred for 25-35 minutes to obtain an aqueous dispersion of titanium dioxide nanoparticles. The aqueous dispersion of titanium dioxide nanoparticles was then added to the transparent aqueous solution of the silica precursor and mixed uniformly to obtain a mixture C. Step D2. Abil EM-90 was added to n-tetradecane, mixed evenly, and then the mixture C was added thereto and homogenized with a homogenizer for 50-65 seconds to obtain a mixture D; Step D3. The mixture D is dried, then washed with hexane 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; In step D2, the mass ratio of Abil EM-90, n-tetradecane, and mixture C 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% by mass KOH solution, stir for 12-13 hours, let it stand for 20-24 hours, and then dry it at 110-120°C for 48-50 hours. After cooling, wash it twice with a 1 mol / L hydrochloric acid solution and deionized water, and then dry it at 60-70°C for 3-4 hours to obtain activated biochar. Step E2. Evenly mixing 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 mixed evenly to obtain a water purifier for aquaculture based on composite microorganisms.

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