Preparation method and application of complex microbial inoculant for removing nitrite and sulfide in aquatic water

Abstract

The invention relates to the field of microbial products, in particular to a preparation method and application of a complex microbial inoculant for removing nitrite and sulfide in aquatic water, and the preparation method comprises the following steps: culturing and enriching a sample to obtain an enriched bacterial solution; the preparation method comprises the following steps: enriching bacteria in a water body environment, modifying the enriched bacteria liquid by adopting aluminum oxide, sodium alginate, polyvinyl alcohol and a mixed solution of the aluminum oxide, the sodium alginate and the polyvinyl alcohol to obtain different embedded bacteria liquids, and finally mixing the bacteria liquids according to a specific mass ratio to obtain the composite bacterial agent. The advantages of various materials are integrated, and the stability and activity of the strain in the complex water body environment are further enhanced; the prepared complex microbial inoculant can synchronously remove nitrite and sulfide in aquaculture water, has the advantages of being eco-friendly, not limited by dissolved oxygen, lasting in effect and the like, effectively overcomes the defects in an existing removal method, and can be widely applied to the field of aquaculture to improve the environment of the aquaculture water.

Description

Technical Field

[0001] The present invention relates to the field of microbial products, and in particular to a preparation method and application of a composite bacterial agent for removing nitrite and sulfide in aquaculture water. Background Art

[0002] The aquaculture industry is developing rapidly, but the current blind expansion of scale has led to serious negative effects. In order to increase aquaculture production, a large amount of bait is added to the aquaculture water, resulting in the accumulation of residual bait, causing serious water pollution, and the feces produced by the aquaculture organisms will also pollute the water body. Ammonia nitrogen is the most common pollutant in aquaculture wastewater, which mainly refers to nitrogen in the form of free ammonia and ammonium ions in the water. In aquaculture, maintaining good water quality is the key to ensuring the healthy growth of aquaculture organisms. Nitrite and sulfide are common harmful substances in water bodies, and their effective removal has always been a key issue of concern in the industry. At present, the common methods for removing nitrite and sulfide are mainly biological and chemical methods. The principle of biological method is based on the metabolic activities of specific microorganisms. In terms of nitrite removal, nitrifying bacteria play a key role. In an aerobic environment, bacteria can use their own enzyme system to gradually oxidize nitrite into nitrate, thereby reducing the nitrite content in the water body, while hydrogen sulfide oxidizing bacteria focus on the removal of sulfide. Under sufficient oxygen supply, they oxidize the hydrogen sulfide in the water body into sulfate to achieve the purpose of sulfur removal. This method utilizes the natural ecological cycle to a certain extent and is relatively environmentally friendly. The chemical law mainly relies on the strong oxidizing property of chemical reagents to achieve the oxidative decomposition of nitrite and sulfide. Chemical agents such as chlorine dioxide and potassium persulfate have strong oxidizing ability and can quickly react chemically with nitrite and sulfide, converting them into relatively harmless substances, thereby reducing their concentration in the water body. The advantage of the chemical method is that the reaction speed is fast and obvious results can be seen in a short time.

[0003] However, there are many shortcomings in the existing methods. For example, the biological method has the following defects: 1. Due to the limitation of dissolved oxygen, the normal metabolic activities of nitrifying bacteria and hydrogen sulfide oxidizing bacteria are highly dependent on sufficient dissolved oxygen. Once the dissolved oxygen in the water body is insufficient, the activity of these microorganisms will be greatly inhibited, resulting in a significant decrease in the removal efficiency of nitrite and sulfide. In some high-density aquaculture ponds, due to the respiration of aquaculture organisms and the decomposition of organic matter, a large amount of oxygen is consumed, and there is often a lack of dissolved oxygen, making it difficult for the biological method to achieve the desired effect; 2. Affecting the growth of farmed animals. When nitrifying bacteria and hydrogen sulfide oxidizing bacteria overgrow in the water body, they will A large amount of dissolved oxygen in the water is consumed, which will not only lead to the threat of hypoxia for farmed organisms, affecting their normal physiological activities and growth and development, but may also cause a series of diseases, increasing the risk and cost of farming; 3. It is unable to solve the nitrogen pollution in the bottom sediment. One of the sources of nitrite and sulfide is the pond bottom sediment. During the farming process, leftover bait, feces and other organic matter continue to accumulate in the bottom sediment, and nitrite and sulfide will be produced through microbial decomposition. However, current biological-based products and technologies cannot penetrate deep into the canteen bottom sediment to play a role, which makes the nitrogen pollution problem in the bottom sediment cannot be effectively solved for a long time, and becomes a hidden danger of secondary pollution of water bodies. However, the chemical method also has the following shortcomings: 1. It destroys the aquaculture ecological environment. Frequent addition of chemical agents to the water body will cause serious damage to the microbial colonies and algae in the water environment. These microorganisms and algae play an important role in the aquatic ecosystem. For example, microorganisms participate in the decomposition of organic matter and material circulation, and algae provide oxygen through photosynthesis and serve as a food source for aquaculture organisms. The use of chemical agents destroys their ecological environment, leading to ecological imbalance, which in turn affects the stability of the entire aquaculture ecosystem; 2. It causes stress reactions in aquaculture organisms. The use of chemical agents will change the water body in a short period of time. The chemical properties of the water, such as redox potential, pH, etc., these sudden changes will make it difficult for the farmed organisms to adapt and produce a stress response. The farmed organisms may experience problems such as loss of appetite, decreased immunity, slow growth, and even death in severe cases; 3. The effect is short-lasting. Although the chemical method can quickly reduce the concentration of nitrite and sulfide in the short term, it does not fundamentally solve the root cause of the problem. Over time, nitrite and sulfide in the water will accumulate again, requiring frequent use of chemical agents for treatment, which not only increases costs, but also further aggravates the damage to the water environment.

[0004] Therefore, based on the above-mentioned related technologies, it is urgent to develop a preparation method and application of a composite bacterial agent for removing nitrite and sulfide in aquaculture water. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to propose a preparation method and application of a composite bacterial agent for removing nitrite and sulfide in aquaculture water, so as to provide an eco-friendly composite bacterial agent that does not affect the aquaculture environment, achieve simultaneous desulfurization and nitrogen removal, and reduce aquaculture costs.

[0006] Based on the above objectives, the present invention provides a preparation method and application of a composite bacterial agent for removing nitrite and sulfide in aquaculture water.

[0007] A method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water comprises the following steps: Step S1. Cultivating and enriching the sample to obtain an enriched bacterial solution; Step S2. Modifying the enriched bacterial solution with alumina to obtain an inorganically embedded bacterial solution; Step S3. Modifying the enriched bacterial solution with sodium alginate to obtain a first organically embedded bacterial solution; Step S4. Modifying the enriched bacterial solution with polyvinyl alcohol to obtain a second organically embedded bacterial solution; Step S5. Modifying the enriched bacterial solution with a mixture of aluminum oxide, sodium alginate, and polyvinyl alcohol to obtain a composite embedded bacterial solution; Step S6. The enriched bacterial solution, the inorganic embedded bacterial solution, the first organic embedded bacterial solution, the second organic embedded bacterial solution and the composite embedded bacterial solution are mixed to obtain a composite bacterial agent.

[0008] Preferably, in step S6, the mass ratio of the enriched bacterial solution, the inorganic embedding bacterial solution, the first organic embedding bacterial solution, the second organic embedding bacterial solution and the composite embedding bacterial solution is 4-5:1-2:1-2:1-1.4:0.8-1.2.

[0009] Preferably, the culturing and enrichment process in step S1 is as follows: Step S101. Sampling and pre-processing: collecting samples from the bottom mud of the aquaculture pond and the soil around the pond, and performing pre-processing to obtain a sample suspension; Step S102. Prepare a culture medium: add NaNO2, KH2PO4, MgCl2, EDTA, MnCl2, FeSO4, CoCl2, NiCl2, H3BO3, (NH4)2MoO4, CuSO4, ZnSO4 and NaHCO3 into distilled water, mix well, add Na2S, and sterilize to obtain a culture medium; Step S103. Inoculation: inoculating the collected environmental sample into the culture medium; Step S104. Purification: placing in a constant temperature incubator for proliferation and repeated screening; Step S105. Enrichment: The screened bacterial strains are placed in the above culture medium for enrichment, and are collected when the OD value reaches 0.8-1.2 to obtain an enriched bacterial solution.

[0010] Preferably, the pretreatment process in step S101 is as follows: the collected sample is placed in a sterile bag and brought back to the laboratory, the collected sample is sieved through a 40-mesh sieve to remove stones and plant residues, and then 100-150 g of the sieved sample is weighed and added to a conical flask containing 400-480 mL of sterile water, and shaken on a shaker at 150-200 rpm for 30-45 minutes to fully disperse the sample to obtain a sample suspension; The usage ratio of NaNO2, KH2PO4, MgCl2, EDTA, MnCl2, FeSO4, CoCl2, NiCl2, H3BO3, (NH4)2MoO4, CuSO4, ZnSO4, NaHCO3, distilled water and Na2S in step S102 is 1.5-2.5g:0.5-1.0g:0.2-0.4g:0.05-0.1g:0.01-0.03g:0.02-0.04g:0.005-0.015g:0.005-0.015g:0.01-0.03g:0.005-0.015g:0.005-0.015g:0.01-0.03g:1.0-2.0g:1L:0.8-1.2g; Before the sterilization treatment in step S102, the pH of the culture medium is adjusted to 7.0-7.5 with 1 mol / L HCl solution. The temperature of the sterilization treatment is 120-124° C., and the time of the sterilization treatment is 20-30 min.

[0011] Preferably, the inoculation process in step S103 is as follows: on a workbench, first wipe your hands and the inoculating loop with a 75% alcohol cotton ball, light an alcohol burner, burn the inoculating loop over the flame until it is red hot, and after cooling, dip 1-2 mL of the sample suspension into the inoculating loop, quickly insert the loop into the culture medium flask, and gently draw a line on the surface of the culture medium to evenly distribute the sample suspension on the culture medium. After the inoculation is completed, immediately plug the flask with cotton and remove it from the workbench; The specific process of purification described in step S104 is as follows: place the inoculated triangular flask in a constant temperature incubator and culture it at 30-35°C for 24-48 hours to allow the bacteria to fully proliferate. When colonies grow on the surface of the culture medium in the triangular flask, pick a single colony with a sterile inoculating loop, perform four-zone line separation on a new culture medium plate, turn the plate upside down and place it in a constant temperature incubator, culture it at 30-35°C for 12-24 hours, repeat the four-zone line separation operation 2-3 times to obtain a pure single colony.

[0012] Preferably, the specific process of the enrichment in step S105 is as follows: the single colony obtained by screening is inoculated into a triangular flask containing 100-150 mL of culture medium, with an inoculation volume of 3%-5% by volume, and cultured on a shaker at 30-35° C. and 150-200 rpm; The process of collecting the bacterial solution in step S105 is as follows: 1-2 mL of the bacterial solution is taken every 2-3 hours, and the OD value is measured at a wavelength of 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, the bacterial solution is collected. At this time, the bacterial solution is transferred to a centrifuge tube and centrifuged at a speed of 4000-6000 rpm for 10-15 minutes. The supernatant is discarded, and the precipitated bacteria are resuspended with sterile saline to obtain an enriched bacterial solution.

[0013] Preferably, the preparation process of the inorganic embedded bacterial solution is as follows: Step B1. Alumina and hydrochloric acid solution were mixed and stirred at room temperature for 2-3 hours. After the reaction was completed, the alumina was repeatedly rinsed with deionized water until the rinse was neutral. The alumina was then dried in an oven at 80-100°C to constant weight to obtain activated alumina. Step B2. Grind the activated alumina into a powder, pass it through a 100-200 mesh sieve, mix the enriched bacterial solution with the activated alumina powder, add sterile water, and stir evenly to form a uniform paste. The paste is placed in a mold, formed at 0.5-1 MPa, and then dried at room temperature for 24-48 hours to obtain an inorganically embedded bacterial solution. The concentration of the hydrochloric acid solution in step B1 is 3-5 mol / L; The ratio of aluminum oxide to hydrochloric acid solution in step B1 is 1-1.4 g: 5-8 mL; The usage ratio of the enriched bacterial solution, activated alumina and sterile water in step B2 is 1-1.3 g: 3.5-5 g: 8-10 mL.

[0014] Preferably, the preparation process of the first organic embedded bacterial solution is as follows: Sodium alginate is added to sterile water, stirred at 60-70°C, and cooled for later use to obtain a sodium alginate solution. The enriched bacterial solution and the sodium alginate solution are mixed evenly, and the resulting mixture is added dropwise to a calcium chloride solution using a syringe. The mixture is cross-linked and cured for 1-2 hours to obtain a first organically embedded bacterial solution. The dosage ratio of the sodium alginate and sterile water is 1-2 g:100 mL; The volume ratio of the enriched bacterial solution to the sodium alginate solution is 1-2:5-8; The concentration of the calcium chloride solution is 0.1-0.2 mol / L.

[0015] Preferably, the preparation process of the second organic embedded bacterial solution is as follows: Add polyvinyl alcohol to sterile water, heat to 90-95°C and stir evenly to completely dissolve the polyvinyl alcohol, cool to room temperature for later use to obtain a polyvinyl alcohol solution, mix the enriched bacterial solution and the polyvinyl alcohol solution evenly, pour the resulting mixture into a mold, and air-dry at room temperature for 24-48 hours to obtain a second organically embedded bacterial solution; The dosage ratio of the polyvinyl alcohol and sterile water is 0.5-1g:100mL; The volume ratio of the enriched bacterial solution to the polyvinyl alcohol solution is 1-2:5-8; The preparation process of the composite embedded bacterial solution is as follows: Activated alumina, sodium alginate, polyvinyl alcohol and sterile water were mixed in a dosage ratio of 2-3g:1-2g:0.5-1g:120-140mL, heated to 60-70°C and stirred evenly. The enriched bacterial solution and the obtained mixed solution were mixed in a volume ratio of 1-2:5-8. After stirring evenly, the mixed solution was added dropwise to a calcium chloride solution with a concentration of 0.1-0.2mol / L using a syringe, and cross-linked and cured for 1-2h to obtain a composite embedded bacterial solution.

[0016] The invention discloses an application of a composite bacterial agent for removing nitrite and sulfide in aquaculture water. The composite bacterial agent can be used to remove nitrite and sulfide in aquaculture water.

[0017] Beneficial effects of the present invention: The present invention provides a method for preparing and applying a composite bacterial agent for removing nitrite and sulfide from aquaculture water. The present invention prepares the composite bacterial agent by sampling from the bottom mud and surrounding soil of the aquaculture pond, and undergoing a series of unique processes such as cultivation, enrichment, and separate and mixed embedding of multiple materials. Compared with the existing technology, the present composite bacterial agent can simultaneously and efficiently remove nitrite and sulfide from aquaculture water, achieving simultaneous desulfurization and nitrogen removal. The bacterial solutions with different embedding methods work together to enhance the stability and activity of the strains in complex water environments and prolong the action time. At the same time, the composite bacterial agent is an eco-friendly product that will not damage the aquaculture ecological environment like chemical methods, and also avoids problems such as dissolved oxygen limitation in biological methods, effectively reducing aquaculture costs and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a graph showing changes in nitrite and sulfide over time under aerobic conditions; Figure 2 This is a graph showing changes in nitrite and sulfide over time under anaerobic conditions; Figure 3 This is the curve of nitrite changes in the aquaculture pond; Figure 4 This is a curve chart of sulfide changes in the aquaculture pond. 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 with reference to specific embodiments.

[0021] Example 1: A method for preparing a composite bacterial agent for removing nitrite and sulfide from aquaculture water, comprising the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the samples in sterile bags and bring them back to the laboratory. Pass the samples through a 40-mesh sieve to remove stones and plant debris. Then, weigh 100 g of the sieved sample and add it to a flask containing 400 mL of sterile water. Shake the flask at 150 rpm for 30 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 1.5 g NaNO2, 0.5 g KH2PO4, 0.2 g MgCl2, 0.05 g EDTA, 0.01 g MnCl2, 0.02 g FeSO4, 0.005 g CoCl2, 0.005 g NiCl2, 0.01 g H3BO3, 0.005 g (NH4)2MoO2, 0.005 g CuSO4, 0.01 g ZnSO4, and 1.0 g NaHCO3 to 1 L of distilled water and mix thoroughly. Add 0.8 g Na2S, adjust the pH of the culture medium to 7.0 with 1 mol / L HCl solution, and sterilize at 120°C for 20 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, sip 1 mL of the sample suspension from the loop and quickly insert it into the culture flask. Gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 30°C for 24 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, use a sterile inoculating loop to pick a single colony and perform a four-zone streak on a new culture medium plate. Invert the plate and place it in a constant-temperature incubator. Incubate at 30°C for 12 hours. Repeat the four-zone streak twice to obtain a pure single colony. S5. Inoculate the selected single colony into a flask containing 100 mL of culture medium at a 3% volume ratio. Incubate on a shaker at 30°C and 150 rpm. Take 1 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, collect the culture medium. At this time, transfer the culture medium to a centrifuge tube and centrifuge at 4000 rpm for 10 minutes. Discard the supernatant and resuspend the precipitated cells in sterile saline to obtain the enriched culture medium. S6. 1 g of alumina was mixed with 5 mL of 3 mol / L hydrochloric acid solution and stirred at room temperature for 2 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral. The alumina was then dried in an oven at 80°C to constant weight to obtain activated alumina. S7. Grind 3.5g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1g of the enriched bacterial solution with the activated alumina powder, add 8mL of sterile water, stir well to make a uniform paste mixture, place the paste mixture in a mold, shape it under 0.5MPa, and then dry it at room temperature for 24h to obtain an inorganic embedded bacterial solution; S8. Add 1 g of sodium alginate to 100 mL of sterile water, stir at 60°C, and cool to obtain a sodium alginate solution. Mix 1 mL of the enriched bacterial solution with 5 mL of the sodium alginate solution. Add the resulting mixture dropwise to a 0.1 mol / L calcium chloride solution using a syringe and allow to crosslink and cure for 1 hour to obtain a first organically embedded bacterial solution. S9. Add 0.5 g of polyvinyl alcohol to 100 mL of sterile water, heat to 90°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 1 mL of the enriched bacterial solution with 5 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 24 hours to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 2 g:1 g:0.5 g:120 mL, heated to 60°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 1:5 and stirred evenly. The mixture was then added dropwise to a 0.1 mol / L calcium chloride solution using a syringe and cross-linked for 1 hour to obtain a composite embedded bacterial solution. S11. Mix 4 g of the enriched bacterial solution, 1 g of the inorganic embedded bacterial solution, 1 g of the first organic embedded bacterial solution, 1 g of the second organic embedded bacterial solution, and 0.8 g of the composite embedded bacterial solution to obtain a composite bacterial agent.

[0022] Example 2: A method for preparing a composite bacterial agent for removing nitrite and sulfide from aquaculture water, comprising the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the sample in a sterile bag and bring it back to the laboratory. Pass the sample through a 40-mesh sieve to remove stones and plant debris. Then weigh 110 g of the sieved sample and add it to a flask containing 420 mL of sterile water. Oscillate on a shaker at 160 rpm for 32 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 1.7 g NaNO2, 0.6 g KH2PO4, 0.25 g MgCl2, 0.06 g EDTA, 0.015 g MnCl2, 0.025 g FeSO4, 0.007 g CoCl2, 0.007 g NiCl2, 0.015 g H3BO3, 0.007 g (NH4)2MoO4, 0.007 g CuSO4, 0.015 g ZnSO4, and 1.2 g NaHCO3 to 1 L of distilled water and mix thoroughly. Add 0.9 g Na2S and adjust the pH of the culture medium to 7.2 with 1 mol / L HCl solution. Sterilize at 121°C for 22 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, scoop 1.2 mL of the sample suspension from the loop and quickly insert it into the culture flask. Gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 31°C for 30 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, pick a single colony with a sterile inoculating loop and isolate it by streaking onto a new culture medium plate. Invert the plate and place it in a constant-temperature incubator at 31°C for 16 hours. Repeat the streaking operation three times to obtain a pure single colony. S5. Inoculate a single colony obtained by screening into a flask containing 110 mL of culture medium at a 4% volume ratio. Incubate on a shaker at 31°C and 160 rpm. Take 1.5 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. Collect the culture medium when the OD value reaches 0.8-1.2. At this point, transfer the culture medium to a centrifuge tube and centrifuge at 4400 rpm for 11 minutes. Discard the supernatant and resuspend the precipitated cells in sterile saline to obtain the enriched culture medium. S6. 1.1 g of alumina was mixed with 6 mL of 4 mol / L hydrochloric acid solution and stirred at room temperature for 2.5 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral. The alumina was then dried in an oven at 85°C to constant weight to obtain activated alumina. S7. Grind 3.8g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1.1g of the enriched bacterial solution with the activated alumina powder, add 9mL of sterile water, stir well to form a uniform paste mixture, place the paste mixture in a mold, shape it at 0.6MPa, and then dry it at room temperature for 30h to obtain an inorganic embedded bacterial solution; S8. Add 1.2 g of sodium alginate to 100 mL of sterile water, stir at 62°C, and cool to obtain a sodium alginate solution. Mix 1.2 mL of the enriched bacterial suspension and 5.5 mL of the sodium alginate solution. Add the resulting mixture dropwise to a 0.15 mol / L calcium chloride solution using a syringe and allow to crosslink and cure for 1.5 h to obtain a first organically embedded bacterial solution. S9. Add 0.6 g of polyvinyl alcohol to 100 mL of sterile water, heat to 91°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 1.2 mL of the enriched bacterial solution with 6 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 30 h to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 2.2 g:1.2 g:0.6 g:125 mL, heated to 62°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 1.2:5.5, stirred evenly, and the mixture was added dropwise to a 0.15 mol / L calcium chloride solution using a syringe. The mixture was cross-linked and cured for 1.5 h to obtain a composite embedded bacterial solution. S11. 4.2 g of the enriched bacterial solution, 1.2 g of the inorganic embedded bacterial solution, 1.2 g of the first organic embedded bacterial solution, 1.1 g of the second organic embedded bacterial solution, and 0.9 g of the composite embedded bacterial solution were mixed to obtain a composite bacterial agent.

[0023] Example 3: A method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water comprises the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the sample in a sterile bag and bring it back to the laboratory. Pass the sample through a 40-mesh sieve to remove stones and plant debris. Then weigh 120 g of the sieved sample and add it to a flask containing 440 mL of sterile water. Shake on a shaker at 170 rpm for 35 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 1.9 g NaNO2, 0.7 g KH2PO4, 0.3 g MgCl2, 0.07 g EDTA, 0.02 g MnCl2, 0.03 g FeSO4, 0.009 g CoCl2, 0.009 g NiCl2, 0.02 g H3BO3, 0.009 g (NH4)2MoO4, 0.009 g CuSO4, 0.02 g ZnSO4, and 1.4 g NaHCO3 to 1 L of distilled water and mix thoroughly. Add 1.0 g Na2S and adjust the pH of the culture medium to 7.4 with 1 mol / L HCl solution. Sterilize at 122°C for 24 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, scoop 1.4 mL of the sample suspension from the loop and quickly insert it into the culture flask. Gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 32°C for 32 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, use a sterile inoculating loop to pick a single colony and perform a four-zone streak on a new culture medium plate. Invert the plate and place it in a constant-temperature incubator at 32°C for 18 hours. Repeat the four-zone streak twice to obtain a pure single colony. S5. Inoculate a single colony obtained by screening into a flask containing 120 mL of culture medium at a 5% volume ratio. Incubate on a shaker at 32°C and 170 rpm. Take 2 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, collect the culture medium. At this time, transfer the culture medium to a centrifuge tube and centrifuge at 4800 rpm for 12 minutes. Discard the supernatant and resuspend the precipitated cells in sterile saline to obtain the enriched culture medium. S6. 1.2 g of alumina was mixed with 6.5 mL of 5 mol / L hydrochloric acid solution and stirred at room temperature for 3 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral. The alumina was then dried in an oven at 90°C to constant weight to obtain activated alumina. S7. Grind 4g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1.2g of the enriched bacterial solution with the activated alumina powder, add 10mL of sterile water, stir well to form a uniform paste mixture, place the paste mixture in a mold, shape it under 0.7MPa, and then dry it at room temperature for 36h to obtain an inorganic embedded bacterial solution; S8. Add 1.4 g of sodium alginate to 100 mL of sterile water, stir at 64°C, and cool to obtain a sodium alginate solution. Mix 1.4 mL of the enriched bacterial solution and 6 mL of the sodium alginate solution. Add the resulting mixture dropwise to a 0.2 mol / L calcium chloride solution using a syringe and allow to crosslink and cure for 2 h to obtain a first organically embedded bacterial solution. S9. Add 0.7 g of polyvinyl alcohol to 100 mL of sterile water, heat to 92°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 1.4 mL of the enriched bacterial solution with 7 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 36 hours to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 2.4 g:1.4 g:0.7 g:130 mL, heated to 64°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 1.4:6 and stirred evenly. The mixture was then added dropwise to a 0.2 mol / L calcium chloride solution using a syringe and cross-linked for 2 h to obtain a composite embedded bacterial solution. S11. 4.4 g of the enriched bacterial solution, 1.4 g of the inorganic embedded bacterial solution, 1.4 g of the first organic embedded bacterial solution, 1.2 g of the second organic embedded bacterial solution, and 1.0 g of the composite embedded bacterial solution were mixed to obtain a composite bacterial agent.

[0024] Example 4: A method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water comprises the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the samples in sterile bags and bring them back to the laboratory. Pass the samples through a 40-mesh sieve to remove stones and plant debris. Then, weigh 130 g of the sieved sample and add it to a flask containing 460 mL of sterile water. Shake on a shaker at 180 rpm for 40 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 2.1 g NaNO2, 0.8 g KH2PO4, 0.35 g MgCl2, 0.08 g EDTA, 0.025 g MnCl2, 0.035 g FeSO4, 0.012 g CoCl2, 0.012 g NiCl2, 0.025 g H3BO3, 0.012 g (NH4)2MoO4, 0.012 g CuSO4, 0.022 g ZnSO4, and 1.6 g NaHCO3 to 1 L of distilled water, mix thoroughly, add 1.1 g Na2S, adjust the pH of the culture medium to 7.5 with 1 mol / L HCl solution, and sterilize at 123°C for 26 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, scoop 1.6 mL of the sample suspension from the loop and quickly insert it into the culture flask. Gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 33°C for 36 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, pick a single colony with a sterile inoculating loop and perform a four-zone streak on a new culture medium plate. Invert the plate and place it in a constant-temperature incubator at 33°C for 20 hours. Repeat the four-zone streak three times to obtain a pure single colony. S5. Inoculate a single colony obtained by screening into a flask containing 130 mL of culture medium at a 3% volume ratio. Incubate on a shaker at 33°C and 180 rpm. Take 1 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, collect the culture medium. At this point, transfer the culture medium to a centrifuge tube and centrifuge at 5200 rpm for 13 minutes. Discard the supernatant and resuspend the pellet in sterile saline to obtain the enriched culture medium. S6. 1.3 g of alumina was mixed with 7 mL of 3 mol / L hydrochloric acid solution and stirred at room temperature for 2 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral. The alumina was then dried in an oven at 95°C to constant weight to obtain activated alumina. S7. Grind 4.2g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1.2g of the enriched bacterial solution with the activated alumina powder, add 8mL of sterile water, stir well to form a uniform paste mixture, place the paste mixture in a mold, shape it at 0.8MPa, and then dry it at room temperature for 42h to obtain an inorganic embedded bacterial solution; S8. Add 1.6 g of sodium alginate to 100 mL of sterile water, stir uniformly at 66°C, and cool for later use to obtain a sodium alginate solution. 1.6 mL of the enriched bacterial suspension and 6.5 mL of the sodium alginate solution are then mixed uniformly. The resulting mixture is then added dropwise to a 0.1 mol / L calcium chloride solution using a syringe and cross-linked for 1 hour to obtain a first organically embedded bacterial solution. S9. Add 0.8 g of polyvinyl alcohol to 100 mL of sterile water, heat to 93°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 1.6 mL of the enriched bacterial solution with 6 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 42 hours to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 2.6 g:1.6 g:0.8 g:135 mL, heated to 66°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 1.6:6.5, stirred evenly, and the mixture was added dropwise to a 0.1 mol / L calcium chloride solution using a syringe. The mixture was cross-linked and cured for 1 hour to obtain a composite embedded bacterial solution. S11. 4.6 g of the enriched bacterial solution, 1.6 g of the inorganic embedded bacterial solution, 1.6 g of the first organic embedded bacterial solution, 1.3 g of the second organic embedded bacterial solution, and 1.1 g of the composite embedded bacterial solution were mixed to obtain a composite bacterial agent.

[0025] Example 5: A method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water comprises the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the samples in sterile bags and bring them back to the laboratory. Sieve the samples through a 40-mesh sieve to remove stones and plant debris. Weigh 140 g of the sieved sample and add it to a flask containing 470 mL of sterile water. Shake on a shaker at 190 rpm for 42 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 2.3 g NaNO2, 0.9 g KH2PO4, 0.38 g MgCl2, 0.09 g EDTA, 0.028 g MnCl2, 0.038 g FeSO4, 0.013 g CoCl2, 0.013 g NiCl2, 0.028 g H3BO3, 0.013 g (NH4)2MoO4, 0.013 g CuSO4, 0.025 g ZnSO4, and 1.8 g NaHCO3 to 1 L of distilled water, mix thoroughly, add 1.0 g Na2S, adjust the pH of the culture medium to 7.0 with 1 mol / L HCl solution, and sterilize at 124°C for 28 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, scoop 1.8 mL of the sample suspension into the culture flask. Quickly insert the inoculating loop into the culture flask and gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 34°C for 42 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, use a sterile inoculating loop to pick a single colony and perform a four-zone streak on a new culture medium plate. Invert the plate and place it in a constant-temperature incubator at 34°C for 22 hours. Repeat the four-zone streak twice to obtain a pure single colony. S5. Inoculate a single colony obtained by screening into a flask containing 140 mL of culture medium at a 4% volume ratio. Incubate on a shaker at 34°C and 190 rpm. Take 1.5 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, collect the culture medium. At this time, transfer the culture medium to a centrifuge tube and centrifuge at 5600 rpm for 14 minutes. Discard the supernatant and resuspend the precipitated cells in sterile saline to obtain the enriched culture medium. S6. 1.3 g of alumina was mixed with 7.5 mL of 4 mol / L hydrochloric acid solution and stirred at room temperature for 2.5 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral. The alumina was then dried in an oven at 98°C to constant weight to obtain activated alumina. S7. Grind 4.5g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1.1g of the enriched bacterial solution with the activated alumina powder, add 9mL of sterile water, stir well to form a uniform paste mixture, place the paste mixture in a mold, shape it at 0.9MPa, and then dry it at room temperature for 45h to obtain an inorganic embedded bacterial solution; S8. Add 1.8 g of sodium alginate to 100 mL of sterile water, stir at 68°C, and cool to obtain a sodium alginate solution. Mix 1.8 mL of the enriched bacterial solution and 7 mL of the sodium alginate solution. Add the resulting mixture dropwise to a 0.15 mol / L calcium chloride solution using a syringe and allow to crosslink and cure for 1.5 h to obtain a first organically embedded bacterial solution. S9. Add 0.9 g of polyvinyl alcohol to 100 mL of sterile water, heat to 94°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 1.8 mL of the enriched bacterial solution with 7 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 46 hours to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 2.8 g:1.8 g:0.9 g:138 mL, heated to 68°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 1.8:7 and stirred evenly. The mixture was then added dropwise to a 0.15 mol / L calcium chloride solution using a syringe and cross-linked for 1.5 h to obtain a composite embedded bacterial solution. S11. 4.8 g of the enriched bacterial solution, 1.8 g of the inorganic embedded bacterial solution, 1.8 g of the first organic embedded bacterial solution, 1.4 g of the second organic embedded bacterial solution, and 1.2 g of the composite embedded bacterial solution were mixed to obtain a composite bacterial agent.

[0026] Example 6: A method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water comprises the following steps: S1. Collect samples from the bottom mud and surrounding soil of the aquaculture pond. Place the samples in sterile bags and bring them back to the laboratory. Pass the samples through a 40-mesh sieve to remove stones and plant debris. Then, weigh 150 g of the sieved sample and add it to a flask containing 480 mL of sterile water. Shake on a shaker at 200 rpm for 45 minutes to fully disperse the sample to obtain a sample suspension. S2. Add 2.5 g NaNO2, 1.0 g KH2PO4, 0.4 g MgCl2, 0.1 g EDTA, 0.03 g MnCl2, 0.04 g FeSO4, 0.015 g CoCl2, 0.015 g NiCl2, 0.03 g H3BO3, 0.015 g (NH4)2MoO4, 0.015 g CuSO4, 0.03 g ZnSO4, and 2.0 g NaHCO3 to 1 L of distilled water, mix thoroughly, add 1.2 g Na2S, adjust the pH of the culture medium to 7.5 with 1 mol / L HCl solution, and sterilize at 124°C for 30 min to obtain a culture medium. S3. On the workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball. Light an alcohol burner and hold the inoculating loop over the flame until red hot. After cooling, sip 2 mL of the sample suspension from the loop and quickly insert it into the culture flask. Gently draw a line across the surface of the culture medium to evenly distribute the sample suspension. Once inoculation is complete, immediately plug the flask with cotton and remove it from the workbench. S4. Place the inoculated flask in a constant-temperature incubator at 35°C for 48 hours to allow the bacteria to fully proliferate. Once colonies form on the surface of the culture medium in the flask, pick a single colony with a sterile inoculating loop and perform a four-zone streak on a new culture medium plate. Invert the plate and place it in a constant-temperature incubator at 35°C for 24 hours. Repeat the four-zone streak three times to obtain a pure single colony. S5. Inoculate a single colony obtained by screening into a flask containing 150 mL of culture medium at a 5% volume ratio. Incubate on a shaker at 35°C and 200 rpm. Take 2 mL of the culture medium every 2-3 hours and measure the OD value at 600 nm using a spectrophotometer. Collect the culture medium when the OD value reaches 0.8-1.2. At this point, transfer the culture medium to a centrifuge tube and centrifuge at 6000 rpm for 15 minutes. Discard the supernatant and resuspend the precipitated cells in sterile saline to obtain the enriched culture medium. S6. 1.4 g of alumina was mixed with 8 mL of 5 mol / L hydrochloric acid solution and stirred at room temperature for 3 h. After the reaction, the alumina was repeatedly rinsed with deionized water until the rinse solution was neutral and then dried in an oven at 100°C to constant weight to obtain activated alumina. S7. Grind 5g of activated alumina into powder, pass through a 100-200 mesh sieve, mix 1.3g of the enriched bacterial solution with the activated alumina powder, add 10mL of sterile water, stir well to form a uniform paste mixture, place the paste mixture in a mold, shape it at 1MPa, and then dry it at room temperature for 48h to obtain an inorganic embedded bacterial solution; S8. Add 2 g of sodium alginate to 100 mL of sterile water, stir at 70°C, and cool to obtain a sodium alginate solution. Mix 2 mL of the enriched bacterial suspension and 8 mL of the sodium alginate solution. Add the resulting mixture dropwise to a 0.2 mol / L calcium chloride solution using a syringe and allow to crosslink and cure for 2 h to obtain a first organically embedded bacterial solution. S9. Add 1 g of polyvinyl alcohol to 100 mL of sterile water, heat to 95°C, and stir to completely dissolve the polyvinyl alcohol. Cool to room temperature and set aside to obtain a polyvinyl alcohol solution. Mix 2 mL of the enriched bacterial solution with 8 mL of the polyvinyl alcohol solution. Pour the resulting mixture into a mold and air-dry at room temperature for 48 hours to obtain a second organic-encapsulated bacterial solution. S10. Activated alumina, sodium alginate, polyvinyl alcohol, and sterile water were mixed in a volume ratio of 3 g:2 g:1 g:140 mL, heated to 70°C, and stirred evenly. The enriched bacterial solution and the resulting mixture were mixed in a volume ratio of 2:8 and stirred evenly. The mixture was then added dropwise to a 0.2 mol / L calcium chloride solution using a syringe and cross-linked for 2 h to obtain a composite embedded bacterial solution. S11. Mix 5 g of the enriched bacterial solution, 2 g of the inorganic embedded bacterial solution, 2 g of the first organic embedded bacterial solution, 1.3 g of the second organic embedded bacterial solution, and 1.0 g of the composite embedded bacterial solution to obtain a composite bacterial agent.

[0027] Comparative Example 1: Compared with Example 1, this comparative example only replaced the "inorganic embedded bacterial solution" with the "enriched bacterial solution", and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0028] Comparative Example 2: Compared with Example 1, this comparative example only replaced the "first organically embedded bacterial solution" with the "enriched bacterial solution", and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0029] Comparative Example 3: Compared with Example 1, this comparative example only replaced the "second organically embedded bacterial solution" with the "enriched bacterial solution", and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0030] Comparative Example 4: Compared with Example 1, this comparative example only replaced the "composite embedded bacterial solution" with the "enriched bacterial solution", and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0031] Comparative Example 5: Compared with Example 1, this comparative example only adjusted the amount of the "composite embedding bacterial solution" from "5 g" to "5.8 g" and the amount of the "composite embedding bacterial solution" from "1.0 g" to "0.2 g". The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0032] Performance test: The composite bacterial agents prepared in Examples 1 to 6 and Comparative Examples 1 to 5 were subjected to the following performance tests: Under laboratory conditions, 1L of culture medium was prepared and inoculated with 50mL of enriched bacterial solution. The OD600 value of the bacterial solution was 1.0, the concentration of nitrite in the culture solution was 10mg / L, and S 2- The concentration is 3.5 mg / L, the dissolved oxygen is maintained at 4-7 mg / L, the temperature is 30°C, and the concentrations of nitrite and sulfide in the solution at 0h, 2h, 4h, 6h and 8h are measured. The results are as follows Figure 1 As shown, keeping the above conditions unchanged, when the dissolved oxygen is less than 0.5 mg / L, the concentrations of nitrite and sulfide change with time as shown in Figure 2 As shown, the results indicate that the strain can simultaneously degrade nitrite and sulfide under both aerobic and anaerobic conditions; The composite bacterial agent prepared in Example 1 was applied in a small shrimp culture pond. The environmental conditions are shown in Table 3. The dosage of the composite bacterial agent was 200 mg / mu / m. The concentrations of nitrite and sulfide were shown in Table 3. Figure 3 and Figure 4 ,The results showed that the strain had good effects of removing nitrite and desulfurization in practical application; Nitrite removal rate test: Test method: Take 10L of simulated aquaculture water with an initial nitrite concentration of 20mg / L. Add 100g of the composite bacterial agent prepared in each example and comparative example respectively. Under the conditions of temperature 30°C and dissolved oxygen 5mg / L, aerate and stir continuously. Take 100mL of water sample every 12 hours and measure the nitrite content by spectrophotometry. The calculation formula is as follows: Nitrite removal rate (%) = 00%; Sulfide removal rate test: Test method: Take 10L of simulated aquaculture water with an initial sulfide concentration of 15mg / L. After adding the composite bacterial agent, the conditions are the same as those for the nitrite removal rate test. Take 100mL of water sample every 12 hours and determine the sulfide content using the iodine titration method. The calculation formula is as follows: Sulfide removal rate (%) = 00%; Stability test: Test Method: The composite inoculant was stored at 4°C, 25°C, and 40°C for 7 days. A certain amount of the inoculant was taken every two days and its removal capacity in simulated aquaculture water was measured according to the above-mentioned nitrite and sulfide removal rate test methods to evaluate its stability.

[0033] Impact test on farmed organisms: Test Method: Fifty healthy, uniformly sized crucian carp were randomly divided into 11 groups of five each. Each group was placed in 11 aquariums, each containing 5 liters of water. Ten of the aquariums were treated with a different composite bacterial agent to a concentration of 10 mg / L. Ten served as a blank control group. The behavior and feeding habits of the crucian carp were observed daily. After seven days, their weight, length, and blood stress hormone levels were measured. The results are shown in Tables 1 and 2 below: Table 1 Table 2 Table 3 Data Analysis: Nitrite and sulfide removal rate: In the examples, the high removal rate of the composite bacterial agent is due to the synergistic effect of microbial metabolism and multiple embedding methods. The microorganism's own enzyme system can efficiently catalyze the conversion of nitrite to nitrate and sulfide to sulfate. The activated alumina-embedded bacteria utilize the high specific surface area and adsorption properties of activated alumina to enrich the nitrite and sulfide in the water around the bacteria, increasing the contact area between the microorganisms and the pollutants, thereby accelerating the degradation reaction, so that the nitrite and sulfide removal rates can be maintained at a high level for 24 hours and 48 hours. For example, in Example 6, the 24-hour nitrite removal rate reached 78%, and the sulfide removal rate reached 82%. This is because the activated alumina fully exerted its enrichment effect, providing more favorable reaction conditions for the microorganisms. However, due to the lack of a specific embedding bacterial solution in the comparative example, the survival and action environment of the microorganisms in the water body is relatively single. For example, in Comparative Example 1, the lack of inorganic embedding solution and the lack of the protection and enrichment of activated alumina resulted in insufficient contact between the microorganisms and pollutants. Consequently, the 24-hour nitrite removal rate was only 50%, and the sulfide removal rate was 55%. In Comparative Example 5, although various embedding solutions were used, the ratios were inappropriate, and the amount of composite embedding solution was too low. This failed to fully utilize the advantages of the mixed embedding materials, affecting the synergistic effect of the microorganisms. Its 24-hour nitrite removal rate was 62%, and the sulfide removal rate was 68%, lower than most examples.

[0034] Stability: The composite bacterial agents in the examples exhibited good stability at different temperatures. This is because the different embedding materials provide a stable microenvironment for the microorganisms. The gel structure formed by sodium alginate embedding mitigates the effects of temperature fluctuations on bacterial activity at both low temperatures (4°C) and high temperatures (40°C), maintaining the stability of the microbial enzyme system. The film-forming properties of polyvinyl alcohol make the bacterial strain less susceptible to external physical interference during storage, further enhancing stability. For example, Example 5 maintained a nitrite and sulfide removal capacity of 96% after storage at 4°C for 7 days, and 85% at 40°C. However, the stability of the comparative example was relatively poor. Comparative Example 2 lacked the first organic embedding solution (sodium alginate embedding). Without the protection of the sodium alginate gel structure during temperature fluctuations, the bacterial strain was easily affected by the enzyme system. After storage at 40°C for 7 days, its removal capacity was only 68%, significantly lower than that of the examples. Comparative Example 3 lacks the second organic embedding solution (polyvinyl alcohol embedding), and the bacteria are easily disturbed by physical factors during storage, resulting in decreased activity. After being stored at 25°C for 7 days, its removal capacity remained at 80%, which is lower than that of most examples.

[0035] Impact on farmed organisms: The positive effects of the composite microbial agent on the weight and length of crucian carp and the lower changes in stress hormone content in the examples are because the composite microbial agent is eco-friendly and will not destroy the ecological balance of the water body like chemical methods and affect the normal physiological functions of farmed organisms. At the same time, the various embedding methods maintain the stable effect of microorganisms in the water body, avoiding the adverse effects of excessive microbial reproduction or activity fluctuations on farmed organisms. For example, in Example 4, the weight of crucian carp increased by 6.5g, the body length increased by 1.5cm, and the stress hormone content increased by only 5%. This shows that the composite microbial agent effectively removes pollutants while providing a good living environment for farmed organisms; in the comparative examples, due to the unstable performance of the composite microbial agent or the lack of key embedding bacterial solution, the effect in the water body is poor or it causes certain interference to the water body ecology. In Comparative Example 1, due to the lack of inorganic embedding bacterial solution, the composite microbial agent is unstable in the water body, causing the stress hormone content of crucian carp to increase by 20%, affecting the growth and health of crucian carp, with the weight only increasing by 3g and the body length increasing by 0.8cm.

[0036] 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.

[0037] 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 method for preparing a composite bacterial agent for removing nitrite and sulfide from aquaculture water, characterized in that: The following steps are involved: Step S1. Cultivating and enriching the sample to obtain an enriched bacterial solution; Step S2. Modifying the enriched bacterial solution with alumina to obtain an inorganically embedded bacterial solution; Step S3. Modifying the enriched bacterial solution with sodium alginate to obtain a first organically embedded bacterial solution; Step S4. Modifying the enriched bacterial solution with polyvinyl alcohol to obtain a second organically embedded bacterial solution; Step S5. Modifying the enriched bacterial solution with a mixture of aluminum oxide, sodium alginate, and polyvinyl alcohol to obtain a composite embedded bacterial solution; Step S6. The enriched bacterial solution, the inorganic embedded bacterial solution, the first organic embedded bacterial solution, the second organic embedded bacterial solution and the composite embedded bacterial solution are mixed to obtain a composite bacterial agent.

2. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 1, characterized in that: In step S6, the mass ratio of the enriched bacterial solution, the inorganic embedding bacterial solution, the first organic embedding bacterial solution, the second organic embedding bacterial solution and the composite embedding bacterial solution is 4-5:1-2:1-2:1-1.4:0.8-1.

2.

3. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 1, characterized in that: The process of culturing and enrichment described in step S1 is as follows: Step S101. Sampling and pre-processing: collecting samples from the bottom mud of the aquaculture pond and the soil around the pond, and performing pre-processing to obtain a sample suspension; Step S102. Prepare a culture medium: add NaNO2, KH2PO4, MgCl2, EDTA, MnCl2, FeSO4, CoCl2, NiCl2, H3BO3, (NH4)2MoO4, CuSO4, ZnSO4 and NaHCO3 into distilled water, mix well, add Na2S, and sterilize to obtain a culture medium; Step S103. Inoculation: inoculating the collected environmental sample into the culture medium; Step S104. Purification: placing in a constant temperature incubator for proliferation and repeated screening; Step S105. Enrichment: The screened bacterial strains are placed in the above culture medium for enrichment, and are collected when the OD value reaches 0.8-1.2 to obtain an enriched bacterial solution.

4. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 3, characterized in that: The pretreatment process in step S101 is as follows: the collected sample is placed in a sterile bag and brought back to the laboratory. The collected sample is sieved through a 40-mesh sieve to remove stones and plant debris. Then, 100-150 g of the sieved sample is weighed and added to a conical flask containing 400-480 mL of sterile water. The sample is shaken on a shaker at 150-200 rpm for 30-45 minutes to fully disperse the sample to obtain a sample suspension. The usage ratio of NaNO2, KH2PO4, MgCl2, EDTA, MnCl2, FeSO4, CoCl2, NiCl2, H3BO3, (NH4)2MoO4, CuSO4, ZnSO4, NaHCO3, distilled water and Na2S in step S102 is 1.5-2.5g:0.5-1.0g:0.2-0.4g:0.05-0.1g:0.01-0.03g:0.02-0.04g:0.005-0.015g:0.005-0.015g:0.01-0.03g:0.005-0.015g:0.005-0.015g:0.01-0.03g:1.0-2.0g:1L:0.8-1.2g; Before the sterilization treatment in step S102, the pH of the culture medium is adjusted to 7.0-7.5 with 1 mol / L HCl solution. The temperature of the sterilization treatment is 120-124° C., and the time of the sterilization treatment is 20-30 min.

5. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 3, characterized in that: The inoculation process in step S103 is as follows: on a workbench, first wipe your hands and inoculating loop with a 75% alcohol cotton ball, light an alcohol burner, and burn the inoculating loop over the flame until it is red hot. After cooling, dip 1-2 mL of the sample suspension into the inoculating loop and quickly insert it into the culture medium flask. Gently draw a line on the surface of the culture medium to evenly distribute the sample suspension on the culture medium. After inoculation is completed, immediately plug the flask with cotton and remove it from the workbench. The specific process of purification described in step S104 is as follows: place the inoculated triangular flask in a constant temperature incubator and culture it at 30-35°C for 24-48 hours to allow the bacteria to fully proliferate. When colonies grow on the surface of the culture medium in the triangular flask, pick a single colony with a sterile inoculating loop, perform four-zone line separation on a new culture medium plate, turn the plate upside down and place it in a constant temperature incubator, culture it at 30-35°C for 12-24 hours, repeat the four-zone line separation operation 2-3 times to obtain a pure single colony.

6. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 3, characterized in that: The specific process of the enrichment in step S105 is as follows: the single colony obtained by screening is inoculated into a triangular flask containing 100-150 mL of culture medium at an inoculation volume of 3%-5% by volume, and the culture is shaken on a shaker at 30-35°C and 150-200 rpm; The process of collecting the bacterial solution in step S105 is as follows: 1-2 mL of the bacterial solution is taken every 2-3 hours, and the OD value is measured at a wavelength of 600 nm using a spectrophotometer. When the OD value reaches 0.8-1.2, the bacterial solution is collected. At this time, the bacterial solution is transferred to a centrifuge tube and centrifuged at a speed of 4000-6000 rpm for 10-15 minutes. The supernatant is discarded, and the precipitated bacteria are resuspended with sterile saline to obtain an enriched bacterial solution.

7. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 1, characterized in that: The preparation process of the inorganic embedded bacterial solution is as follows: Step B1. Alumina and hydrochloric acid solution were mixed and stirred at room temperature for 2-3 hours. After the reaction was completed, the alumina was repeatedly rinsed with deionized water until the rinse was neutral. The alumina was then dried in an oven at 80-100°C to constant weight to obtain activated alumina. Step B2. Grind the activated alumina into a powder, pass it through a 100-200 mesh sieve, mix the enriched bacterial solution with the activated alumina powder, add sterile water, and stir evenly to form a uniform paste. The paste is placed in a mold, formed at 0.5-1 MPa, and then dried at room temperature for 24-48 hours to obtain an inorganically embedded bacterial solution. The concentration of the hydrochloric acid solution in step B1 is 3-5 mol / L; The ratio of aluminum oxide to hydrochloric acid solution in step B1 is 1-1.4 g: 5-8 mL; The usage ratio of the enriched bacterial solution, activated alumina and sterile water in step B2 is 1-1.3 g: 3.5-5 g: 8-10 mL.

8. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 1, characterized in that: The preparation process of the first organic embedded bacterial solution is as follows: Sodium alginate is added to sterile water, stirred at 60-70°C, and cooled for later use to obtain a sodium alginate solution. The enriched bacterial solution and the sodium alginate solution are mixed evenly, and the resulting mixture is added dropwise to a calcium chloride solution using a syringe. The mixture is cross-linked and cured for 1-2 hours to obtain a first organically embedded bacterial solution. The dosage ratio of the sodium alginate and sterile water is 1-2 g:100 mL; The volume ratio of the enriched bacterial solution to the sodium alginate solution is 1-2:5-8; The concentration of the calcium chloride solution is 0.1-0.2 mol / L.

9. The method for preparing a composite bacterial agent for removing nitrite and sulfide in aquaculture water according to claim 1, characterized in that: The preparation process of the second organic embedded bacterial solution is as follows: Add polyvinyl alcohol to sterile water, heat to 90-95°C and stir evenly to completely dissolve the polyvinyl alcohol, cool to room temperature for later use to obtain a polyvinyl alcohol solution, mix the enriched bacterial solution and the polyvinyl alcohol solution evenly, pour the resulting mixture into a mold, and air-dry at room temperature for 24-48 hours to obtain a second organically embedded bacterial solution; The dosage ratio of the polyvinyl alcohol and sterile water is 0.5-1g:100mL; The volume ratio of the enriched bacterial solution to the polyvinyl alcohol solution is 1-2:5-8; The preparation process of the composite embedded bacterial solution is as follows: Activated alumina, sodium alginate, polyvinyl alcohol and sterile water were mixed in a dosage ratio of 2-3g:1-2g:0.5-1g:120-140mL, heated to 60-70°C and stirred evenly. The enriched bacterial solution and the obtained mixed solution were mixed in a volume ratio of 1-2:5-8. After stirring evenly, the mixed solution was added dropwise to a calcium chloride solution with a concentration of 0.1-0.2mol / L using a syringe, and cross-linked and cured for 1-2h to obtain a composite embedded bacterial solution.

10. Use of the composite bacterial agent for removing nitrite and sulfide in aquaculture water according to any one of claims 1 to 9, characterized in that: The composite bacterial agent can be used to remove nitrite and sulfide in aquaculture water.

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