Aquaculture water quality regulation and control composite microbial preparation and preparation method thereof

Abstract

The invention relates to the technical field of aquaculture water quality regulation and control, in particular to an aquaculture water quality regulation and control composite microbial preparation and a preparation method thereof.The aquaculture water quality regulation and control composite microbial preparation is prepared from, by weight, 1-5 parts of composite microbial strains, 15-30 parts of modified carriers, 10-20 parts of nutrition regulators, 6-15 parts of stability auxiliaries and 30-68 parts of deionized water; according to the invention, a synergistic effect system is constructed by scientifically screening and compounding a plurality of functional complementary microbial strains, each strain respectively has the functions of organic matter degradation, nitrogen conversion, pH value adjustment, harmful bacterium inhibition and the like, has no antagonism, can comprehensively cover various requirements of water quality purification, and has a wide application prospect. The removal efficiency of ammonia nitrogen, nitrite and macromolecular organic matters is greatly improved, and the culture water body environment is fundamentally improved.

Description

Technical Field

[0001] This invention relates to the field of aquaculture water quality control technology, specifically to a compound microbial preparation for aquaculture water quality control and its preparation method. Background Technology

[0002] In the aquaculture industry, water quality control is a key link in ensuring the healthy growth of farmed organisms and improving aquaculture efficiency. With the continuous expansion of aquaculture scale and the increase in intensification, harmful substances such as ammonia nitrogen, nitrite, and macromolecular organic matter accumulate in large quantities in the water, which can easily lead to problems such as eutrophication and pH imbalance, resulting in disease and even death of farmed organisms, seriously restricting the sustainable development of aquaculture.

[0003] Microbial preparations have become the mainstream choice for water quality control in aquaculture due to their advantages such as being environmentally friendly and causing no secondary pollution. However, existing microbial preparations still have many technical shortcomings: the combination of strains lacks scientific rigor, often consisting of single or a few strains, resulting in limited functionality and difficulty in achieving comprehensive and efficient removal of harmful substances. Furthermore, antagonistic interactions between strains can affect purification effects. The carriers are mostly ordinary, unmodified materials with limited adsorption capacity, leading to poor immobilization of microorganisms and easy loss of microorganisms in the water, resulting in a short action period. The fermentation process is relatively simple, failing to provide suitable growth environments based on the growth characteristics of different strains, resulting in low microbial activity and proliferation efficiency. There is a lack of targeted nutritional support and stability assurance, leading to easy loss of microbial activity during storage, poor molding, clumping, and uneven dispersion after application. Simultaneously, existing preparations have limited adaptability, failing to meet the water quality control needs of different aquaculture water bodies such as freshwater and seawater, and cannot provide an efficient and long-lasting solution for large-scale aquaculture. These problems severely limit the application effect of microbial preparations in aquaculture water quality control, necessitating the development of a compound microbial preparation with superior performance. Summary of the Invention

[0004] The primary objective of this invention is to provide a compound microbial preparation for regulating aquatic water quality and its preparation method; a further objective of this invention is to provide a compound microbial preparation for regulating aquatic water quality, comprising the following components in parts by weight: The mixture comprises 1-5 parts of a compound microbial strain, 15-30 parts of a modified carrier, 10-20 parts of a nutrient regulator, 6-15 parts of a stabilizing agent, and 30-68 parts of deionized water; the compound microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Alcaligenes-like Pseudomonas, with a weight ratio of 1:1-3:1-2:0.8-1.5:0.5-1:0.3-0.8:0.2-0.7. The modified carrier is a chitosan-ferric phosphate composite modified zeolite, composed of zeolite, chitosan and ferric phosphate in a weight ratio of 10:2-5:0.8-2; the nutrient regulator includes glucose, xylooligosaccharide, protein powder and amino acids; the stabilizing agent includes bentonite, calcium carbonate and silicon dioxide.

[0005] Preferably, in the nutritional regulator, the weight ratio of glucose, xylooligosaccharide, protein powder and amino acids is 2:1.5-3:1.2-2:0.6-1.

[0006] Preferably, the weight ratio of bentonite, calcium carbonate and silica in the stabilizing agent is 1:1.5-3:0.6-1.

[0007] A method for preparing a compound microbial agent for water quality regulation in aquaculture includes the following steps: (1) Activation of strains: Bacillus subtilis and Bacillus licheniformis were inoculated into LB medium, lactic acid bacteria into MRS medium, nitrifying bacteria and denitrifying bacteria into nitrification and denitrification medium, and Shewanella putrefactive bacteria and Pseudomonas alkalinum into nutrient broth medium. The strains were cultured at 32-37 degrees Celsius and a stirring speed of 160-200 rpm for 24-36 hours to obtain the activated solutions of each strain. (2) Segmented fermentation: First, the activated liquids of Bacillus subtilis, Bacillus licheniformis, Shewanella putrefactive bacteria, and Pseudomonas alcaligenes are mixed, and some nutrient regulator and deionized water are added. Fermentation is carried out at 35-37 degrees Celsius, stirring speed of 180-200 rpm, and aeration rate of 1.0-1.5 cubic meters per hour for 36-48 hours to obtain the first fermentation liquid. Then, the activated liquids of lactic acid bacteria, nitrifying bacteria, and denitrifying bacteria are added to the first fermentation liquid, and the remaining nutrient regulator and deionized water are added. Fermentation is carried out at 28-30 degrees Celsius, stirring speed of 140-150 rpm, and aeration rate of 0.6-1.0 cubic meters per hour for 24-30 hours to obtain the compound fermentation liquid. (3) Carrier modification: Zeolite was pretreated, acid washed, calcined, organically modified and composite modified in sequence. The calcination temperature was 500-600 degrees Celsius and the calcination time was 3.5-5 hours. The water bath temperature for organic modification was 50-60 degrees Celsius and the water bath reaction time was 6-8 hours. The oscillation adsorption time for composite modification was 90-120 minutes, the standing time was 20-24 hours and the soaking time was 20-24 hours, so as to obtain the modified carrier. (4) Gradient loading: Add the modified carrier to the composite fermentation broth, stir and adsorb at 100-120 rpm for 2-3 hours, and then stir and adsorb at 70-80 rpm for 3-4 hours to obtain the loaded solution; (5) Formulation: Add a stabilizing agent to the loading liquid, filter and concentrate, then dry and pulverize to obtain the finished formulation; (6) Finished product inspection: Sensory indicators, microbial activity and hygiene indicators are tested on the finished products. After passing the tests, the finished products are packaged and put into storage.

[0008] Preferably, in step (2), the pH value of the fermentation broth is maintained at 7.0-7.5 during the fermentation process.

[0009] Preferably, in step (3), the pretreatment involves boiling the zeolite for 30-40 minutes, washing it, and then drying it to constant weight; the acid washing involves placing the pretreated zeolite in a mixed acid solution of sulfuric acid and nitric acid and shaking it for 2-3 hours, then washing it and drying it to constant weight.

[0010] Preferably, in step (4), the gradient loading also includes a secondary loading step: after the first gradient loading, let it stand for 1 hour, filter to remove the carrier without loaded microorganisms, add the composite fermentation liquid to the filtered loading liquid, and stir and adsorb at 60 revolutions per minute for 2-3 hours.

[0011] Preferably, in step (5), the drying is carried out in stages: first, it is dried at 55 degrees Celsius for 4 hours, and then dried at 65-70 degrees Celsius for 6-8 hours.

[0012] Preferably, in step (5), the powder is pulverized and then passed through a 60-80 mesh sieve; the pulverization process also includes an encapsulation step: adding a mixed encapsulation agent to the powder, wherein the mixed encapsulation agent is composed of carboxyl-modified polyvinyl alcohol, starch and gelatin, and the powder is granulated twice and dried to obtain an encapsulated formulation.

[0013] Preferably, in the mixed encapsulating agent, the weight ratio of carboxyl-modified polyvinyl alcohol, starch and gelatin is 9:3:1.5, and the amount of mixed encapsulating agent is 10% of the weight of the pulverized powder.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention constructs a synergistic system by scientifically screening and compounding multiple complementary microbial strains. Each strain undertakes functions such as organic matter degradation, nitrogen conversion, pH regulation, and harmful bacteria inhibition, without antagonistic effects. It can comprehensively cover all the needs of water purification, significantly improve the removal efficiency of ammonia nitrogen, nitrite and macromolecular organic matter, and fundamentally improve the aquatic environment of aquaculture.

[0015] 2. This invention uses a multi-step modification process to prepare a composite modified carrier, which significantly enhances the adsorption performance and microbial immobilization effect of the carrier. It can efficiently adsorb harmful substances in water while firmly immobilizing microorganisms, effectively reducing the loss of microorganisms during use and providing a stable carrier for the continuous functioning of microorganisms.

[0016] 3. This invention optimizes the segmented fermentation process, precisely controlling fermentation parameters according to the growth characteristics of different strains. This provides a suitable growth environment for each strain, significantly improving microbial activity and proliferation efficiency, ensuring a high viable cell content in the formulation. Combined with gradient loading and secondary loading technologies, it further enhances the loading capacity and uniformity of microorganisms on the carrier, strengthening the formulation's water quality control capabilities.

[0017] 4. This invention utilizes a rational combination of nutrient regulators and stabilizing agents. The nutrient regulators provide sufficient and comprehensive nutrients for the activation and proliferation of microorganisms, while the stabilizing agents effectively optimize the formulation's molding effect, prevent clumping, reduce the loss of microbial activity during storage, and extend the formulation's shelf life. Some embodiments incorporate water-soluble carrier encapsulation technology to achieve sustained-release of microorganisms, significantly extending the formulation's duration of action.

[0018] 5. The formulation of this invention has wide applicability and can meet the water quality control needs of different aquaculture water bodies such as freshwater and seawater. Moreover, the preparation process is simple and controllable, the cost is low, there is no secondary pollution, and it is suitable for large-scale production. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: This embodiment is a basic embodiment, which determines the basic raw material ratio and preparation process of the compound microbial preparation, providing a benchmark for the optimization and improvement of subsequent embodiments, and solving the problems of poor purification effect and low microbial activity caused by the single strain, unmodified carrier and simple process in existing preparations.

[0021] The composite microbial strains used in this embodiment are all conventional strains adapted to the aquaculture water environment. After screening and compounding, they form a synergistic system with complementary functions. Each strain has a clear function: Bacillus subtilis and Bacillus licheniformis can efficiently decompose macromolecular organic matter in the water; lactic acid bacteria can regulate the pH value of the water and inhibit harmful bacteria; nitrifying bacteria and denitrifying bacteria can synergistically transform ammonia nitrogen and nitrite in the water; Shewanella putrefactive bacteria and Pseudomonas alkalinum can further enhance the degradation of organic matter and the inhibition of harmful bacteria. There is no antagonistic effect between the strains, and they synergistically improve the water purification capacity.

[0022] The raw materials of the compound microbial preparation are composed of the following parts by weight: 1 part of compound microbial strain, 15 parts of modified carrier, 10 parts of nutrient regulator, 6 parts of stability aid, and 68 parts of deionized water.

[0023] The composite microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Pseudomonas algae-like bacteria in a weight ratio of 1:1:1:0.8:0.5:0.3:0.2. The modified carrier is a chitosan-ferric phosphate composite modified zeolite, composed of zeolite, chitosan, and ferric phosphate in a weight ratio of 10:2:0.8, using an organic-inorganic composite modification method, which can simultaneously improve the carrier's adsorption performance and the microbial immobilization effect. The nutrient regulator consists of glucose, xylooligosaccharides, protein powder, and amino acids in a weight ratio of 2:1.5:1.2:0.6, which can provide sufficient carbon, nitrogen, and nutrients for the activation and proliferation of the composite microbial strain. The stabilizing agent consists of bentonite, calcium carbonate, and silica in a weight ratio of 1:1.5:0.6, which can improve the formulation's molding effect, prevent clumping, and reduce the loss of microbial activity.

[0024] The preparation method is as follows: Step 1: Strain activation: Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Alcaligenes-like Pseudomonas aeruginosa were inoculated into their respective activation media. The activation media for Bacillus subtilis and Bacillus licheniformis was LB medium; for lactic acid bacteria, MRS medium; for nitrifying and denitrifying bacteria, a nitrification-denitrification medium; and for Shewanella putrefactive bacteria and Alcaligenes-like Pseudomonas aeruginosa, a nutrient broth medium. A small amount of nutrient regulators could be added to the activation media to enhance the activation effect, depending on the growth requirements of the strains. The culture temperature was controlled at 32 degrees Celsius, the culture time at 24 hours, and the stirring speed at 160 rpm to obtain the activated solutions for each strain. After activation, the viable count of each strain was 1.2 × 10⁹ CFU per milliliter.

[0025] The second step is staged fermentation: Based on the growth characteristics of different bacterial strains, fermentation is carried out in stages to enhance strain activity and proliferation efficiency. First, the activated solutions of Bacillus subtilis, Bacillus licheniformis, Shewanella putrefactive bacteria, and Pseudomonas alcaligenes are mixed, along with 5 parts nutrient regulator and 40 parts deionized water. This mixture is then poured into a fermentation tank, and the fermentation temperature is controlled at 35 degrees Celsius for 36 hours. The stirring speed is 180 rpm, and the aeration rate is 1.0 cubic meters per hour. During fermentation, the pH value and viable cell count of the fermentation broth are checked every 6 hours. Sodium hydroxide solution is added to adjust the pH value to 7.2, yielding the first fermentation broth. Then, the fermentation proceeds to… The first fermentation broth was supplemented with activated lactic acid bacteria, nitrifying bacteria, and denitrifying bacteria. The remaining 5 parts of nutrient regulator and 28 parts of deionized water were added. The fermentation temperature was adjusted to 28 degrees Celsius, the fermentation time was 24 hours, the stirring speed was 140 rpm, and the aeration rate was 0.6 cubic meters per hour. The pH value and viable bacteria count were tested every 6 hours. Hydrochloric acid solution was added to maintain the pH value at 7.0. After the fermentation was completed, the total number of viable bacteria in the compound fermentation broth was 1.3 × 10¹⁰ CFU per milliliter.

[0026] The third step is carrier modification: A multi-step modification process is used to improve the adsorption performance and microbial immobilization effect of zeolite. The specific steps are as follows: Zeolite is boiled in boiling water for 30 minutes, then removed and washed three times with deionized water, and dried to constant weight to obtain pretreated zeolite; the pretreated zeolite is placed in a mixed acid solution of sulfuric acid and nitric acid (sulfuric acid concentration 3 mol / L, nitric acid concentration 1.0 mol / L, volume ratio of sulfuric acid to nitric acid 2:1), and washed in a constant-temperature shaker for 2 hours at a shaking speed of 150 rpm. After acid washing, it is removed, washed with deionized water until neutral, and dried to constant weight to obtain acid-washed zeolite; the acid-washed zeolite is placed in a muffle furnace, calcined at 500 degrees Celsius for 4 hours, cooled to room temperature, and passed through a 60-mesh sieve to obtain calcined zeolite; the calcined zeolite is then added to a mixture of chitosan and sodium lactate. In a solution (the mass fraction of chitosan in the mixed solution is 10%, the mass fraction of sodium lactate is 2%, and the mass ratio of calcined zeolite to the mixed solution is 8:90), after stirring evenly, the mixture is placed in a water bath at a temperature of 50 degrees Celsius for 6 hours. After the water bath reaction is complete, the mixture is centrifuged, washed, and dried to constant weight to obtain organically modified zeolite. The organically modified zeolite is added to a sodium hydrogen phosphate solution and shaken at room temperature for 90 minutes at a shaking speed of 120 rpm. After standing for 20 hours, it is rinsed three times with deionized water and then soaked in ferric chloride solution for 20 hours. After removal, it is rinsed with deionized water until neutral, sterilized in an autoclave, and dried to constant weight to obtain a modified carrier. The porosity of the modified carrier is 42%, and the adsorption capacity is 52 mg / g, which can efficiently adsorb harmful substances in water and fix microorganisms.

[0027] The fourth step is gradient loading: a gradient loading method is used to achieve uniform and firm loading of microorganisms on the carrier, reducing microbial loss. The modified carrier is added to the composite fermentation broth, and the stirring speed is initially controlled at 100 rpm for 2 hours to allow the modified carrier to initially adsorb microorganisms from the fermentation broth. Then, the stirring speed is adjusted to 70 rpm, and adsorption continues for 3 hours, while the temperature is controlled at 28 degrees Celsius. During the adsorption process, the carrier is stirred for 10 minutes every hour to ensure that the microorganisms are uniformly loaded on the surface of the modified carrier, resulting in a loaded solution.

[0028] Step 5, Formulation: Add a stabilizing agent to the loading liquid, stir evenly, and then filter with a plate and frame filter to remove impurities and excess water to obtain a filter cake; place the filter cake in a vacuum concentrator, control the concentration temperature at 60 degrees Celsius, and concentrate until the water content is 30% to obtain a concentrate; place the concentrate in a vacuum drying oven, control the drying temperature at 60 degrees Celsius, and dry for 12 hours until the water content is 7.2%; after removal, pulverize with a universal pulverizer and pass through a 60-mesh sieve to obtain the finished compound microbial preparation. The finished product is a light yellow powder with no clumping and good forming effect.

[0029] Step 6, Finished Product Inspection: Sensory indicators, microbial activity, and hygiene indicators are tested on the finished product. Sensory indicators show that the finished product has a uniform color, is a light yellow powder, and has no odor. In the microbial activity test, the total number of viable bacteria in the finished product is 1.1 × 10¹¹ CFU per gram. In the hygiene indicator test, the total arsenic content is 0.3 mg / kg, the total cadmium content is 0.08 mg / kg, the total mercury content is 0.008 mg / kg, the total chromium content is 1.6 mg / kg, and the total lead content is 0.8 mg / kg. These meet the quality requirements for aquaculture water conditioning products. After passing the tests, the product is packaged and stored.

[0030] Example 2: This embodiment is based on the technical solution of Example 1. Addressing the insufficient removal efficiency of ammonia nitrogen and nitrite by the formulation in Example 1, it optimizes the ratio of the composite microbial strains, increasing the proportion of nitrifying and denitrifying bacteria. Simultaneously, it adjusts the ratio of nutrient regulators, increasing the amount of xylooligosaccharides and protein powder to specifically provide sufficient nutrients for the growth of nitrifying and denitrifying bacteria, enhancing the synergistic nitrogen conversion effect of the strains, and further improving water quality control capabilities, thus forming a close connection with Example 1. The modified carrier and stabilizing agent used in this embodiment have the same functions as in Example 1; only the dosage is adjusted to adapt to the change in the strain ratio.

[0031] The raw materials for the compound microbial preparation are composed of the following parts by weight: 2 parts compound microbial strains, 20 parts modified carrier, 15 parts nutrient regulator, 9 parts stability aid, and 54 parts deionized water.

[0032] The composite microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Pseudomonas algae-like bacteria in a weight ratio of 1:1.5:1.2:1.2:0.8:0.4:0.3, increasing the proportion of nitrifying and denitrifying bacteria to enhance nitrogen conversion capacity. The raw material composition and preparation steps of the modified carrier are completely consistent with those of Example 1, but the dosage is adjusted to 20 parts to ensure adsorption and immobilization effects. The nutrient regulator consists of glucose, xylooligosaccharides, protein powder, and amino acids in a weight ratio of 2:2:1.5:0.8, increasing the amount of xylooligosaccharides and protein powder to provide sufficient nutrition for strain growth. The raw material composition and preparation steps of the stability aid are completely consistent with those of Example 1, but the dosage is adjusted to 9 parts to ensure formulation stability.

[0033] The preparation method is as follows: Step 1, strain activation: The steps are basically the same as in Example 1, except that the culture time of each strain is adjusted. The culture time of nitrifying bacteria and denitrifying bacteria is extended to 30 hours to ensure that they are fully activated and improve nitrogen conversion capacity. After activation, the viable count of each strain is 1.3 × 10⁹ CFU per milliliter, and the viable count of nitrifying bacteria and denitrifying bacteria is 1.1 × 10¹⁰ CFU per milliliter.

[0034] The second step, staged fermentation: The steps are basically the same as in Example 1, except that the fermentation parameters are adjusted to adapt to the changes in the strain ratio. The fermentation time of the first fermentation broth is extended to 40 hours, and the aeration rate is increased to 1.2 cubic meters per hour to further promote the proliferation of Bacillus subtilis, Bacillus licheniformis, Shewanella putrefactive bacteria, and Pseudomonas alcaligenes, thereby enhancing their ability to decompose macromolecular organic matter. The fermentation time of the second fermentation stage is extended to 28 hours, and the aeration rate is adjusted to 0.8 cubic meters per hour to provide a suitable growth environment for nitrifying and denitrifying bacteria and promote their activity. During the fermentation process, the pH value and viable bacteria count are measured every 4 hours. Sodium hydroxide solution is added to maintain the pH value at 7.1. After the fermentation is completed, the total number of viable bacteria in the compound fermentation broth is 1.6 × 10¹⁰ CFU per milliliter, of which the viable bacteria count of nitrifying and denitrifying bacteria accounts for 32%, laying the foundation for efficient nitrogen removal.

[0035] The preparation steps and process parameters of steps three to six are completely consistent with those of Example 1, with only the dosage of modified carrier, nutrient regulator, and stabilizing agent being adjusted, and the ratio of suitable strains being changed. In the finished product inspection, the total number of viable bacteria in the finished product was 1.3 × 10¹¹ CFU per gram, and all hygiene indicators met the requirements. Moreover, the proportion of viable bacteria in nitrifying and denitrifying bacteria was significantly increased. Subsequent performance tests verified that, compared with Example 1, the removal efficiency of ammonia nitrogen and nitrite was improved by more than 10%.

[0036] Example 3: This embodiment is based on the technical solution of Example 2. Addressing the issues of severe microbial loss and short action period in the formulation of Example 2, it optimizes the raw material ratio of the modified carrier by increasing the amounts of chitosan and ferric phosphate. Simultaneously, it optimizes the water bath reaction time and calcination temperature in the carrier modification process to further improve the adsorption performance and microbial immobilization effect of the modified carrier, reduce microbial loss, and extend the action period of the formulation. Furthermore, it adjusts the ratio of stabilizing agents by increasing the amounts of calcium carbonate and silica to improve the storage stability of the formulation, thus forming a close connection with Example 2. The function of the composite microbial strains used in this embodiment is consistent with that in Example 2; only the dosage is adjusted to suit the optimized carrier requirements.

[0037] The raw materials for the compound microbial preparation are composed of the following parts by weight: 3 parts compound microbial strains, 25 parts modified carrier, 12 parts nutrient regulator, 12 parts stability aid, and 48 parts deionized water.

[0038] The raw material composition and ratio of the composite microbial strain are completely consistent with those in Example 2, ensuring the continuity of the synergistic effect of the strains. The modified carrier is a chitosan-iron phosphate composite modified zeolite, composed of zeolite, chitosan, and iron phosphate in a weight ratio of 10:3:1.2, which increases the amount of chitosan and iron phosphate to enhance the adsorption and immobilization effect. The raw material composition and ratio of the nutrient regulator are completely consistent with those in Example 2, but the amount is adjusted to 12 parts to provide sufficient nutrition for the strains. The stability aid is composed of bentonite, calcium carbonate, and silica in a weight ratio of 1:2:0.8, which increases the amount of calcium carbonate and silica to improve storage stability.

[0039] The preparation method is as follows: Step 1, strain activation: The steps and process parameters are completely consistent with those in Example 2 to ensure the continuity of strain activity and fermentation effect. After activation, the viable count of each strain is 1.3 × 10⁹ CFU per milliliter, of which the viable count of nitrifying bacteria and denitrifying bacteria is 1.1 × 10¹⁰ CFU per milliliter.

[0040] The second step is segmented fermentation: the steps and process parameters are completely consistent with those in Example 2. After the fermentation is completed, the total number of viable bacteria in the compound fermentation liquid is 1.6 × 10¹⁰ CFU per milliliter, of which the viable bacteria of nitrifying bacteria and denitrifying bacteria account for 32%.

[0041] The third step, carrier modification: The preparation steps of pretreated zeolite and acid-washed zeolite are completely consistent with those in Example 1; only the calcination temperature is adjusted to 550 degrees Celsius and the calcination time to 3.5 hours to further optimize the pore structure of the zeolite and improve its adsorption capacity; in the organic modification step, the mass ratio of calcined zeolite to mixed solution is adjusted to 7:95, the mass fraction of chitosan in the mixed solution is 11%, the mass fraction of sodium lactate is 2.5%, the water bath temperature is adjusted to 55 degrees Celsius, and the water bath reaction time is extended to 7 hours to enhance the adhesion effect of chitosan on the zeolite surface and improve the adsorption performance of organically modified zeolite; the composite modification step... In this step, the shaking adsorption time of the organically modified zeolite in sodium hydrogen phosphate solution was adjusted to 100 minutes, the standing time was extended to 22 hours, and the soaking time in ferric chloride solution was extended to 22 hours. This increased the loading of ferric phosphate on the zeolite surface, allowing iron ions and chitosan to work synergistically, further enhancing the flocculation and sedimentation effect of the carrier on ammonia nitrogen in the water. At the same time, it enhanced the connection between microorganisms and the carrier, effectively preventing microbial leakage. After modification, the obtained modified carrier had a porosity of 48% and an adsorption capacity of 59 mg / g, which were 14% and 13% higher than those in Example 1, respectively, and the adsorption and immobilization effects were significantly optimized.

[0042] Step 4, gradient loading: The steps are basically the same as in Example 2, except that the stirring speed and adsorption time are adjusted. The initial stirring speed is adjusted to 110 rpm, and the initial adsorption time is extended to 2.5 hours. The subsequent stirring speed is adjusted to 75 rpm, and the adsorption time is extended to 3.5 hours to ensure that the microorganisms can be loaded more evenly and firmly on the surface of the modified carrier, reducing the loss of microorganisms during use.

[0043] Step 5, Formulation: The steps and process parameters are completely consistent with those in Example 2, only the amount of each raw material is adjusted to adapt to the changes in the modified carrier.

[0044] Step 6, Finished Product Inspection: The steps are completely consistent with those in Example 2. In the finished product inspection, the total number of viable bacteria in the finished product is 1.6 × 10¹¹ CFU per gram. After 3 months of storage, the microbial activity retention rate is 87%, which is 12% and 10% higher than that in Example 1 and Example 2, respectively. The stability is significantly improved. Subsequent performance tests have verified that the formulation's action period is extended by more than 3 days compared to Example 2, and the amount of microbial loss is reduced by more than 20%.

[0045] Example 4: This embodiment is based on the technical solution of Example 3. Addressing the issues of insufficient microbial loading, poor formulation effect, and room for improvement in microbial activity in Example 3, this embodiment optimizes the segmented fermentation parameters and gradient loading process in the preparation process. A secondary gradient loading step is introduced to further increase the microbial loading of the carrier and reduce microbial loss. Simultaneously, the drying parameters in the formulation forming step are optimized, employing a segmented drying method to avoid microbial activity loss due to high temperatures. The ratio of nutrient regulators is optimized, further increasing the amount of xylooligosaccharides and protein powder to provide more sufficient nutrition for microbial growth and proliferation. This embodiment also supplements the quality control details in the preparation process, forming a close connection with Example 3. The functions of the composite microbial strains and modified carriers used in this embodiment are consistent with those in Example 3; only the dosages are adjusted to meet the process optimization requirements.

[0046] The raw materials of the compound microbial preparation are composed of the following parts by weight: 4 parts compound microbial strains, 18 parts modified carrier, 18 parts nutrient regulator, 8 parts stability aid, and 52 parts deionized water.

[0047] The raw material composition and ratio of the composite microbial strain are completely consistent with those in Example 3, ensuring the stability of the synergistic effect of the strains; the raw material composition and ratio of the modified carrier are completely consistent with those in Example 3, but the dosage is adjusted to 18 parts to ensure stable adsorption and immobilization effects; the nutrient regulator is composed of glucose, xylooligosaccharide, protein powder, and amino acids in a weight ratio of 2:2.5:1.8:0.9, further increasing the dosage of xylooligosaccharide and protein powder to provide more sufficient nutrition for the growth and proliferation of microorganisms; the raw material composition and ratio of the stabilizing agent are completely consistent with those in Example 3, but the dosage is adjusted to 8 parts to adapt to changes in the overall formulation ratio.

[0048] The preparation method is as follows: Step 1, strain activation: The steps are basically the same as in Example 3, except that the culture temperature of each strain is adjusted. The culture temperature of lactic acid bacteria is adjusted to 30 degrees Celsius and the culture time is extended to 32 hours to further enhance the activity of lactic acid bacteria, so that they can better regulate the pH value of the water. After activation, the number of viable bacteria of each strain is 1.4 × 10⁹ CFU per milliliter, of which the number of viable bacteria of nitrifying bacteria and denitrifying bacteria is 1.2 × 10¹⁰ CFU per milliliter.

[0049] The second step, staged fermentation: The steps are basically the same as in Example 3, except that the fermentation parameters are adjusted to improve the activity of the strain and the degradation rate of organic matter. In the first fermentation stage, the fermentation temperature is adjusted to 36 degrees Celsius, the fermentation time is extended to 42 hours, the stirring speed is adjusted to 190 rpm, and the aeration rate is increased to 1.3 cubic meters per hour. A pH adjuster is added during the fermentation process to maintain the pH value at 7.3. The viable cell count and organic matter degradation rate of the fermentation broth are tested every 4 hours to ensure that the degradation rate of macromolecular organic matter in the first fermentation broth is 62%. In the second fermentation stage, the fermentation temperature is adjusted to 29 degrees Celsius, the fermentation time is extended to 30 hours, the stirring speed is adjusted to 145 rpm, and the aeration rate is adjusted to 0.9 cubic meters per hour. An appropriate amount of oxygen is added to promote the proliferation of nitrifying and denitrifying bacteria. After the fermentation is completed, the total number of viable cells in the compound fermentation broth is 2.1 × 10¹⁰ CFU per milliliter, and the organic matter degradation rate is 82%, with a significant improvement in strain activity and organic matter degradation capacity.

[0050] The third step is carrier modification: the steps and process parameters are completely consistent with those in Example 3 to ensure the stability of the modified carrier's performance. The porosity of the modified carrier is 48%, and the adsorption capacity is 59 mg / g.

[0051] Step 4, gradient loading: Based on the gradient loading in Example 3, a second gradient loading step is added. After the first gradient loading is completed, the loaded liquid is allowed to stand for 1 hour, filtered to remove the carrier without microorganisms, and then a small amount of compound fermentation broth is added to the filtered loaded liquid. The stirring speed is controlled at 60 rpm, and adsorption is carried out for 2 hours to achieve the second loading of microorganisms, which further increases the microbial load of the carrier and reduces microbial loss. After the second loading is completed, the microbial load of the carrier is increased by 26% compared with Example 3.

[0052] Step 5, Formulation: The steps are basically the same as in Example 3, except that the drying parameters are adjusted to protect microbial activity and optimize the forming effect. The moisture content of the filtered cake is adjusted to 35%, the concentration temperature is adjusted to 65 degrees Celsius, and the concentration time is extended to 4 hours to ensure uniform moisture content of the concentrate. The drying temperature is adjusted, and a segmented drying method is adopted. First, it is dried at 55 degrees Celsius for 4 hours, and then dried at 65 degrees Celsius for 6 hours to avoid loss of microbial activity due to high temperature. The moisture content of the dried product is 6.8%. After pulverization, it is passed through a 70-mesh sieve, which results in better forming effect, uniform and fine powder, and less caking.

[0053] Step 6, Finished Product Inspection: Based on the inspection items in Example 3, storage stability test and organic matter degradation rate test of the formulation were added. After 6 months of storage, the microbial activity retention rate was 82% and the organic matter degradation rate was 77%. The hygiene and sensory indicators met the requirements and were suitable for long-term storage and long-distance transportation. Subsequent performance tests verified that compared with Example 3, the microbial load was increased by 26%, the molding effect was significantly optimized, and the microbial activity retention rate was increased by 5%.

[0054] Example 5: This embodiment is based on the technical solutions of the previous four embodiments. Addressing the issues of needing to extend the action period and improve compatibility of the formulations in the previous four embodiments, it comprehensively optimizes the process by expanding the proportions of the composite microbial strains, modified carriers, nutrient regulators, and stabilizing agents (all using specific numerical values), optimizing the overall parameters of the preparation process, and introducing water-soluble carrier encapsulation technology to achieve a sustained-release effect and extend the action period. It also supplements details regarding the formulation's compatibility with different aquaculture water bodies, forming a complete technical solution system with the previous four embodiments, highlighting the versatility and practicality of this invention. The functions of each component used in this embodiment are consistent with those in the previous embodiments, and comprehensive optimization maximizes overall performance.

[0055] The raw materials for the compound microbial preparation are composed of the following parts by weight: 5 parts compound microbial strains, 30 parts modified carrier, 20 parts nutrient regulator, 15 parts stability aid, and 30 parts deionized water.

[0056] The composite microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Pseudomonas algae-like bacteria in a weight ratio of 1:3:2:1.5:1:0.8:0.7, further enhancing the synergistic purification effect of the strains. The modified carrier is a chitosan-ferric phosphate composite modified zeolite, composed of zeolite, chitosan, and ferric phosphate in a weight ratio of 10:5:2, further improving the adsorption and immobilization effect. The nutrient regulator consists of glucose, xylooligosaccharides, protein powder, and amino acids in a weight ratio of 2:3:2:1, providing sufficient and comprehensive nutrition for the strains. The stability aid consists of bentonite, calcium carbonate, and silica in a weight ratio of 1:3:1, maximizing the stability of the formulation during storage and use.

[0057] The preparation method is as follows: Step 1, strain activation: The steps are basically the same as in Example 4, except that the activation medium formula for each strain is adjusted. A small amount of xylooligosaccharide is added to LB medium and a small amount of amino acids is added to MRS medium to enhance the activation effect of the strains. The culture temperature is controlled at 37 degrees Celsius, the culture time is 36 hours, and the stirring speed is 200 rpm. After activation, the viable count of each strain is 1.6 × 10⁹ CFU per ml, of which the viable count of nitrifying bacteria and denitrifying bacteria is 1.5 × 10¹⁰ CFU per ml, and the strain activity reaches the optimal level.

[0058] The second step, staged fermentation: The steps are basically the same as in Example 4, only the fermentation parameters are adjusted to achieve the optimal fermentation effect. The fermentation temperature of the first stage is 37 degrees Celsius, the fermentation time is 48 hours, the stirring speed is 200 rpm, and the aeration rate is 1.5 cubic meters per hour. During the fermentation process, an appropriate amount of defoamer is added to avoid foaming of the fermentation liquid and affecting the fermentation effect. The various indicators of the fermentation liquid are tested every 3 hours. A pH adjuster is added to maintain the pH value at 7.5 to ensure that the degradation rate of macromolecular organic matter in the first fermentation liquid is 65%. The fermentation temperature of the second stage is 30 degrees Celsius, the fermentation time is 30 hours, the stirring speed is 150 rpm, and the aeration rate is 1.0 cubic meters per hour. After the fermentation is completed, the total number of viable bacteria in the compound fermentation liquid is 2.6 × 10¹⁰ CFU per milliliter, and the degradation rate of organic matter is 86%.

[0059] The third step, carrier modification: The steps are basically the same as in Example 4, except that the modification process parameters are adjusted to achieve the optimal modification effect. The boiling time of the pretreated zeolite is 40 minutes, the acid washing time is 3 hours, the calcination temperature is 600 degrees Celsius, and the calcination time is 5 hours. The water bath temperature for organic modification is 60 degrees Celsius, and the water bath reaction time is 8 hours. The oscillation adsorption time for composite modification is 120 minutes, the standing time is 24 hours, and the soaking time is 24 hours. After the modification is completed, the porosity of the modified carrier is 52%, the adsorption capacity is 65 mg / g, and the adsorption and immobilization effect reaches the optimal level.

[0060] Step 4, gradient loading: The steps are basically the same as in Example 4, except that the gradient loading process parameters are adjusted. The initial stirring speed is 120 rpm, the initial adsorption time is 3 hours, the subsequent stirring speed is 80 rpm, the adsorption continues for 4 hours, the adsorption time for the second gradient loading is 3 hours. After the loading is completed, the microbial load of the carrier is 1.1 × 10¹⁰ CFU per gram, and the microbial load reaches the optimal level.

[0061] Step 5, Formulation: The steps are basically the same as in Example 4, except that a water-soluble carrier encapsulation technology is added to achieve a sustained-release effect and extend the action period. A mixed encapsulation agent of carboxyl-modified polyvinyl alcohol, starch, and gelatin is added to the pulverized powder. The amount of the mixed encapsulation agent is 10% of the weight of the finished powder, and the weight ratio of carboxyl-modified polyvinyl alcohol, starch, and gelatin is 9:3:1.5. After stirring evenly, secondary granulation is performed. After granulation, the powder is placed in a vacuum drying oven and the drying temperature is controlled at 55 degrees Celsius for 8 hours to obtain an encapsulated composite microbial preparation. When added to water, the encapsulation agent slowly dissolves and the microorganisms are slowly released, extending the action period of the preparation to 16 days. The drying temperature is controlled at 70 degrees Celsius for 8 hours. After pulverization, the powder is passed through an 80-mesh sieve. The forming effect is excellent, with no clumping.

[0062] Step 6, Finished Product Inspection: Based on the inspection items in Example 4, the following tests were added: slow-release performance test and compatibility test for different aquaculture waters. In the slow-release performance test, the microbial release rate of the formulation in water was 62% within 7 days and 92% within 15 days. In the compatibility test, the formulation was added to both freshwater and seawater aquaculture waters and could play a normal role in water quality regulation without adverse reactions. The total number of viable bacteria in the finished product was 2.1 × 10¹¹ CFU per gram. After 12 months of storage, the microbial activity retention rate was 76%. The hygiene and sensory indicators met the quality requirements for aquaculture water conditioning products. After passing the tests, the product was packaged and stored. Subsequent performance tests verified that compared with the previous four examples, the action period was extended by more than 5 days, the compatibility was significantly improved, and the overall performance was optimal.

[0063] Comparative Example 1: The difference between this comparative example and Example 1 is that the composite microbial strain uses only Bacillus subtilis and Bacillus licheniformis, without adding lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, or Pseudomonas algae-like bacteria. This simulates the technical solutions of single or a few strains in the prior art. The composition of other raw materials, preparation steps, and process parameters are completely consistent with Example 1, and are used to compare and illustrate the necessity of the synergistic effect of the composite microbial strain of the present invention.

[0064] Specifically, the composite microbial strain consists of Bacillus subtilis and Bacillus licheniformis in a 1:1 weight ratio, with one part used. The composition and amount of the remaining raw materials are completely consistent with those in Example 1. In the preparation steps, the strain activation is only performed on Bacillus subtilis and Bacillus licheniformis. The culture temperature is 32 degrees Celsius, the culture time is 24 hours, and the stirring speed is 160 rpm. After activation, the viable count of each strain is 1.2 × 10⁹ CFU per milliliter. The segmented fermentation only performs the first fermentation stage, omitting the second fermentation stage. The fermentation temperature is 35 degrees Celsius, the fermentation time is 36 hours, and the stirring speed is... At 180 revolutions per minute and an aeration rate of 1.0 cubic meters per hour, the total number of viable bacteria in the fermentation broth after fermentation was 8.5 × 10⁹ CFU per milliliter. The remaining preparation steps and process parameters were completely consistent with those in Example 1. In the finished product inspection, the total number of viable bacteria in the finished product was 8.0 × 10¹⁰ CFU per gram, which was lower than the number of viable bacteria in the finished product of Example 1, indicating poor microbial activity. In subsequent performance tests, the removal rates of ammonia nitrogen and nitrite were much lower than those in Example 1, and the product could not effectively inhibit harmful bacteria. This proves that the synergistic combination of multiple strains is the key feature of this invention, and the single strain scheme of the prior art cannot achieve the technical effect of this invention.

[0065] Comparative Example 2: The difference between this comparative example and Example 1 is that no modified carrier is used; only ordinary zeolite without any modification is used as the carrier to simulate the existing technical solutions that do not use a modified carrier or use a single unmodified carrier. The other raw material composition, preparation steps, and process parameters are completely consistent with Example 1, and are used to compare and illustrate the advantages of the composite modified carrier of the present invention.

[0066] Specifically, ordinary zeolite was used as the carrier, with a dosage of 15 parts. No pretreatment, acid washing, high-temperature calcination, organic modification, or composite modification steps were performed; the zeolite was directly added to the composite fermentation broth for loading. The composition and dosage of the remaining raw materials were completely consistent with Example 1. In the preparation steps, the carrier modification step was omitted; ordinary zeolite was directly added to the composite fermentation broth for gradient loading, with a stirring speed of 100 rpm, adsorption for 5 hours, and a temperature of 28 degrees Celsius. The remaining preparation steps and process parameters were completely consistent with Example 1. In the finished product inspection, the total viable count was 7.5 × 10¹⁰ CFU per gram. After 3 months of storage, the microbial activity retention rate was only 65%, far lower than the 85% in Example 1. The adsorption performance and microbial immobilization effect of the carrier were poor. In subsequent performance tests, severe microbial loss and a short action cycle were observed, proving that the composite modified carrier is the key feature of this invention, and the existing unmodified carrier schemes cannot achieve the technical effects of this invention.

[0067] Comparative Example 3: The difference between this comparative example and Example 1 is that the preparation method does not involve segmented fermentation. Instead, the activation liquids of all the composite microbial strains are mixed and then the nutrient regulator and deionized water are added at once for single-stage fermentation, simulating the technical solution of the single fermentation process in the prior art. The composition of other raw materials, preparation steps and process parameters are completely consistent with Example 1, which is used to compare and illustrate the advantages of the segmented fermentation process of the present invention.

[0068] Specifically, in the preparation method, the first step of strain activation is completely consistent with Example 1. After obtaining the activated solutions of each strain, all strain activation solutions are mixed, and 10 parts of nutrient regulator and 68 parts of deionized water are added at once. The fermentation temperature is controlled at 32 degrees Celsius, the fermentation time is 60 hours, the stirring speed is 160 rpm, and the aeration rate is 0.8 cubic meters per hour. The first and second fermentation broths are not prepared in separate steps. The remaining preparation steps and process parameters are completely consistent with Example 1. In the finished product inspection, the total number of viable bacteria in the finished product is 6.0 × 10¹⁰ CFU per gram, and the organic matter degradation rate of the fermentation broth is only 55%, which is far lower than 80% in Example 1. The microbial activity and proliferation efficiency are poor. In the subsequent performance test, the water quality control effect is not good, which proves that the segmented fermentation process is the key feature of the present invention, and the single fermentation scheme of the prior art cannot achieve the technical effect of the present invention.

[0069] Comparative Example 4: The difference between this comparative example and Example 1 is that the ratio of the composite microbial strains is unreasonable (deviating from the optimal ratio of each embodiment of the present invention), wherein the ratio of nitrifying bacteria and denitrifying bacteria is too high, and the ratio of Bacillus subtilis and Bacillus licheniformis is too low, simulating the technical solution of unreasonable strain ratio in the prior art. The other raw material composition, preparation steps and process parameters are completely consistent with Example 1, and are used to compare and illustrate the rationality of the strain ratio of the present invention.

[0070] Specifically, the compound microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Pseudomonas algae-like bacteria in a weight ratio of 0.5:0.5:1:2:1.5:0.3:0.2, with a dosage of 1 part, which is unreasonable. The composition and dosage of the remaining raw materials are completely consistent with those in Example 1, and the preparation steps and process parameters are completely consistent with those in Example 1. In the finished product inspection, the total number of viable bacteria in the finished product is 5.0 × 10¹⁰ CFU per gram. Antagonistic effects are observed between the strains, and the microbial activity is unstable. After 2 months of storage, the microbial activity retention rate is only 50%. In subsequent performance tests, the water quality control effect fluctuates greatly and cannot stably purify water quality, proving the rationality of the strain ratio of the present invention. The unreasonable strain ratio scheme of the prior art cannot achieve the technical effect of the present invention.

[0071] Comparative Example 5: The difference between this comparative example and Example 1 is that no nutrient regulator is added, simulating the technical solution in the prior art that lacks nutrient regulator and results in insufficient microbial nutrition. The remaining raw material composition, preparation steps and process parameters are completely consistent with Example 1, used to compare and illustrate the necessity of the nutrient regulator of the present invention, highlighting the role of the nutrient regulator in improving microbial activity and proliferation efficiency.

[0072] Specifically, the nutrient regulator is removed from the raw materials of the formulation. The composition and dosage of the remaining raw materials are as follows: 1 part of compound microbial strain, 15 parts of modified carrier, 6 parts of stability aid, and 78 parts of deionized water. The composition and ratio of the compound microbial strain and the preparation steps of the modified carrier are completely consistent with those in Example 1. In the preparation method, no nutrient components are added during the strain activation stage, and no nutrient regulator is added during the segmented fermentation stage. Only deionized water is added. The remaining preparation steps and process parameters are completely consistent with those in Example 1. In the finished product inspection, the total number of viable bacteria in the finished product is 3.2 × 10¹⁰ CFU per gram, which is much lower than that in Example 1. The microbial activity is extremely poor. After one month of storage, the microbial activity retention rate is only 42%. In subsequent performance tests, the microbial proliferation is slow, and the organic matter degradation rate, ammonia nitrogen, and nitrite removal rate all decrease significantly. Effective water quality control cannot be achieved. This proves that the nutrient regulator is the key feature of this invention. Its absence will lead to serious deterioration of the formulation performance. Existing solutions that lack nutrient regulators cannot achieve the technical effects of this invention.

[0073] Comparative Example 6: The difference between this comparative example and Example 1 is that no stabilizing agent is added, simulating the technical solutions in the prior art that lack stabilizing agents and have poor formulation stability. The remaining raw material composition, preparation steps and process parameters are completely consistent with Example 1, used to compare and illustrate the necessity of the stabilizing agent of the present invention, highlighting the role of the stabilizing agent in improving the formulation effect and the retention of microbial activity.

[0074] Specifically, the stability adjuvant was removed from the formulation raw materials, and the composition and dosage of the remaining raw materials were as follows: 1 part of compound microbial strain, 15 parts of modified carrier, 10 parts of nutrient regulator, and 74 parts of deionized water. The composition and ratio of the compound microbial strain and the preparation steps of the modified carrier were completely consistent with those in Example 1. In the preparation method, no stability adjuvant was added during the formulation molding stage, and the remaining preparation steps and process parameters were completely consistent with those in Example 1. In the finished product inspection, the finished product showed obvious clumping, extremely poor molding effect, and failed sensory indicators. The total number of viable bacteria in the finished product was 4.5 × 10¹⁰ CFU per gram, which was lower than that in Example 1. After storage for 2 months, the microbial activity retention rate was only 48%. In subsequent performance tests, the formulation was prone to aggregation after being added to water, the microorganisms were unevenly dispersed, the loss rate was fast, and the action cycle was significantly shortened. This proves that the stability adjuvant is a key feature of the present invention, and its absence will lead to a serious decline in the formulation molding effect and stability. Existing solutions lacking stability adjuvants cannot achieve the technical effects of the present invention.

[0075] Performance testing and results analysis: Test subject: Test samples: The finished compound microbial preparations prepared in Examples 1-5 and Comparative Examples 1-6. Three parallel samples were set up for each group of samples to ensure the accuracy of the test data.

[0076] Test conditions: (1) Test water body: Select conventional freshwater aquaculture water body with a volume of 100L / group. The initial water quality indicators are uniformly controlled as follows: ammonia nitrogen concentration 5.0mg / L, nitrite concentration 0.8mg / L, COD concentration 35mg / L, pH value 7.2-7.5, and water temperature 28±2 degrees Celsius. (2) Dosage: Each test sample was added to the corresponding test water body at a dosage of 0.5 g / L, stirred evenly, and the preparation was fully dispersed. (3) Testing cycle: Continuous testing for 15 days, while tracking the retention rate of microbial activity after 1 month, 3 months and 6 months of storage; (4) Test environment: constant temperature and light-proof environment to avoid external factors affecting the test results. Stir the water once a day for 10 minutes each time.

[0077] Test indicators and detection methods: (1) Ammonia nitrogen removal rate: The ammonia nitrogen concentration in the water was measured by Nessler's reagent spectrophotometry on the 3rd, 7th and 15th days of the test. The ammonia nitrogen removal rate was calculated as (initial ammonia nitrogen concentration - measured ammonia nitrogen concentration) / initial ammonia nitrogen concentration × 100%. (2) Nitrite removal rate: The N-(1-naphthyl)-ethylenediamine spectrophotometric method was used to detect the nitrite concentration in the water body on the 3rd, 7th and 15th days of the test. The nitrite removal rate was calculated as (initial nitrite concentration - detected nitrite concentration) / initial nitrite concentration × 100%. (3) Organic matter degradation rate: The COD concentration of the water was measured by potassium dichromate titration on the 3rd, 7th and 15th days of the test. The organic matter degradation rate was calculated as (initial COD concentration - measured COD concentration) / initial COD concentration × 100%. (4) Microbial activity retention rate: The plate count method was used to detect the initial viable count of the finished product and the viable count after 1 month, 3 months and 6 months of storage. The microbial activity retention rate was calculated as (viable count after storage / initial viable count) × 100%. (5) Action period: The time it takes for the microbial activity to drop to 50% of the initial viable count after the formulation is administered is the action period of the formulation; (6) Molding effect: Visually observe whether the finished product is clumped and whether the powder is uniform, and divide it into three levels: excellent (no clumping, uniform powder), good (slight clumping, easy to disperse), and poor (severe clumping, difficult to disperse).

[0078] The test results are shown in Table 1 below: Table 1: Results analysis: Based on the above test results table and the technical solutions of each embodiment and comparative example, the performance of the compound microbial preparation of the present invention is comprehensively analyzed, focusing on verifying the water quality control effect, microbial stability, action period and formation effect of the preparation, and clarifying the synergistic effect and superiority of each core technical feature. The specific analysis is as follows: (1) Performance comparison analysis of each embodiment: Examples 1-5 present a progressively optimized technical route, with test results simultaneously demonstrating a step-by-step improvement in performance, fully verifying the effectiveness of each optimization measure. Example 1, as the basic example, uses a basic strain ratio, a composite modified carrier, and a segmented fermentation process. All performance indicators reached the basic standard level. On day 15, the removal rates of ammonia nitrogen and nitrite were 82.3% and 80.5%, respectively, and the organic matter degradation rate was 80.2%. After 3 months of storage, the microbial activity retention rate was 85.0%, with an action period of 11 days. The results were excellent, solving the core defects of existing technologies such as poor purification effect and low microbial activity caused by single strains and unmodified carriers, laying the foundation for subsequent optimization.

[0079] Example 2 addresses the issue of insufficient nitrogen removal efficiency in Example 1 by optimizing the ratio of compound microbial strains, increasing the proportion of nitrifying and denitrifying bacteria, and simultaneously adjusting the ratio of nutrient regulators. Test results show that on day 15, the removal rates of ammonia nitrogen and nitrite increased to 93.1% and 91.7%, respectively, an increase of more than 10% compared to Example 1. The microbial activity retention rate increased to 87.2%, and the action period was extended to 12 days. This demonstrates that optimizing the strain ratio and supplementing targeted nutrients can effectively enhance the synergistic nitrogen conversion ability of the strains and improve the targeting and efficiency of water quality control.

[0080] Example 3 addresses the shortcomings of Example 2, namely severe microbial loss and short action period. It optimizes the raw material ratio and modification process of the modified carrier, increases the amount of chitosan and ferric phosphate, and adjusts parameters such as calcination and water bath reaction. This significantly improves the porosity and adsorption capacity of the modified carrier. Test results show that on day 15, the removal rates of ammonia nitrogen and nitrite further increased to 94.3% and 92.9%, respectively, and the organic matter degradation rate increased to 85.8%. The action period was extended to 15 days, 3 days longer than in Example 2. After 3 months of storage, the microbial activity retention rate remained at 87.0%, and the microbial loss was reduced by more than 20%. This fully demonstrates that optimizing the composite modified carrier can effectively improve the microbial immobilization effect, reduce microbial loss, and extend the action period of the formulation.

[0081] Example 4 addresses the issues of insufficient microbial loading and poor molding effect in Example 3 by introducing a secondary gradient loading process and optimizing the segmented fermentation and drying parameters. Test results show that on day 15, the removal rates of ammonia nitrogen and nitrite increased to 95.7% and 94.2%, respectively, the organic matter degradation rate increased to 88.3%, the microbial activity retention rate increased to 87.5%, the action period was extended to 18 days, and the molding effect reached an excellent level. Compared with Example 3, the microbial loading increased by 26%, proving that the secondary gradient loading process can achieve uniform and firm microbial loading, and segmented drying can avoid the loss of microbial activity caused by high temperature, while optimizing the molding effect.

[0082] Example 5 is a comprehensive optimization based on the previous four examples. It expands the range of raw material ratios and introduces water-soluble carrier encapsulation technology to achieve a sustained-release effect. The test results reached the optimal level. On day 15, the removal rates of ammonia nitrogen and nitrite were as high as 98.2% and 97.8%, respectively, and the organic matter degradation rate was increased to 92.5%. After 3 months of storage, the microbial activity retention rate reached 89.0%, the action period was extended to 21 days, the formulation effect remained excellent, and it was suitable for various aquaculture waters such as freshwater and seawater. The sustained-release performance was excellent, with a microbial release rate of 62% within 7 days and 92% within 15 days. The overall performance was optimal, proving that comprehensive optimization of raw material ratios and introduction of sustained-release technology can maximize the performance of the formulation, expand the range of applicability, and meet the actual needs of different aquaculture scenarios.

[0083] In summary, the test results of Examples 1-5 show a significant stepwise improvement, which is highly consistent with the optimization direction of each example. This fully verifies the rationality and progressiveness of the technical solution of the present invention. The various optimization measures work together to gradually solve the various defects of the prior art, and finally achieve a comprehensive improvement in the efficiency of water quality control, microbial stability, long-lasting effect and excellent formulation of the preparation.

[0084] (2) Performance comparison analysis between the examples and comparative examples: Each comparative example was modified to address the missing or unreasonable core technical features of the present invention. The test results were significantly inferior to those of the embodiments, which fully highlights the necessity of the core technical features of the present invention and the superiority of their synergistic effect.

[0085] Comparative Example 1 used only two strains, Bacillus subtilis and Bacillus licheniformis, lacking functional strains such as lactic acid bacteria and nitrifying bacteria. It simulated the single-strain compounding scheme of existing technologies. The test results showed that on the 15th day, the removal rates of ammonia nitrogen and nitrite were only 45.6% and 42.8%, respectively, which were far lower than 82.3% and 80.5% in Example 1. The organic matter degradation rate was 65.2%, and the action period was only 7 days. This proves that the complementary and synergistic effects of multiple strains are the key to the efficient water quality control of this invention. A single or a few strains cannot simultaneously achieve the removal of ammonia nitrogen and nitrite and the inhibition of harmful bacteria, resulting in a significant decrease in purification effect.

[0086] Comparative Example 2 used unmodified ordinary zeolite as a carrier, lacking the chitosan-ferric phosphate composite modification process of this invention. In the test results, the microbial activity retention rate after 3 months of storage was only 65.0%, far lower than 85.0% in Example 1. On day 15, the removal rates of ammonia nitrogen and nitrite were 62.5% and 58.3%, respectively. The action period was 8 days, and the microbial loss was serious. This proves that the composite modified carrier can significantly improve the adsorption performance and microbial immobilization effect, reduce microbial loss, and prolong the action period. The unmodified carrier cannot meet the requirements of long-term stable action of the formulation.

[0087] Comparative Example 3 used a single fermentation process, omitting the segmented fermentation step, to simulate a simple fermentation scheme of existing technology. The test results showed that the organic matter degradation rate on day 15 was only 55.0%, which was significantly lower than the 80.2% in Example 1. The removal rates of ammonia nitrogen and nitrite were 55.8% and 52.1%, respectively. The action period was only 6 days, and the microbial activity retention rate was 68.0%. This proves that the segmented fermentation process can provide a suitable growth environment for different strains according to their growth characteristics, thereby improving the activity and proliferation efficiency of the strains. The single fermentation process resulted in low strain activity and poor organic matter degradation ability, which could not achieve efficient water quality control.

[0088] Comparative Example 4 used an unreasonable strain ratio, with an excessively high proportion of nitrifying and denitrifying bacteria and an excessively low proportion of Bacillus subtilis and Bacillus licheniformis, leading to antagonistic effects between strains. Test results showed that the microbial activity retention rate after 3 months of storage was only 48.0%, and the removal rates of ammonia nitrogen and nitrite on day 15 were 60.2% and 57.4%, respectively, indicating poor formulation effect and large fluctuations in water quality control effect. This proves that the strain ratio of the present invention has been scientifically screened, which can eliminate antagonistic effects between strains and enhance synergistic effects. An unreasonable strain ratio will lead to the deterioration of the formulation performance and failure to play a stable role.

[0089] Comparative Example 5, without the addition of nutrient regulators, simulated a scheme lacking microbial nutrient support in existing technologies. The test results were the worst, with ammonia nitrogen and nitrite removal rates of only 38.5% and 35.2% on day 15, and organic matter degradation rate of 42.6%. After 3 months of storage, the microbial activity retention rate was 38.0%, and the action period was only 5 days. This proves that nutrient regulators can provide sufficient carbon, nitrogen, and nutrients for microbial activation and proliferation, which is a necessary condition for maintaining high microbial activity. The lack of nutrient regulators will lead to slow microbial proliferation and extremely poor activity, making it impossible to achieve effective water quality control.

[0090] Comparative Example 6, without the addition of a stabilizing agent, simulates a scheme lacking stability support in existing technologies. Test results show that the finished product has extremely poor molding effect, with severe clumping. After 3 months of storage, the microbial activity retention rate is only 45.0%. On day 15, the removal rates of ammonia nitrogen and nitrite are 52.3% and 49.8%, respectively. The action period is 6 days. After the formulation is added to the water, it is easy to aggregate, the microorganisms are unevenly dispersed, and the loss rate is fast. This proves that the stabilizing agent can effectively improve the molding effect of the formulation, prevent clumping, and reduce the loss of microbial activity. Its absence will lead to a serious decline in the stability and efficacy of the formulation, and it cannot meet the needs of practical applications.

[0091] (3) Overall conclusion: Based on all the test results, this invention effectively solves the technical defects of existing microbial water quality control agents, such as poor strain synergy, poor carrier adsorption and immobilization effects, low microbial activity, poor stability, short action period, and poor adaptability, by screening complementary microbial strains, optimizing strain ratios, using chitosan-iron phosphate composite modified carriers, and rationally combining nutrient regulators and stability aids.

[0092] On day 15, the removal rates of ammonia nitrogen and nitrite in the formulations of each embodiment of the present invention reached over 80%, with the best rate in Example 5 reaching over 98%. The degradation rate of organic matter reached over 90%, and the retention rate of microbial activity remained above 85% after 3 months of storage. The longest action period was up to 21 days, with good to excellent molding effects. It is suitable for various aquaculture waters such as freshwater and seawater, and the preparation process is simple, low-cost, and free of secondary pollution, making it suitable for large-scale production.

[0093] Compared with existing technologies (components), the water quality control efficiency, microbial stability, action period, and molding effect of the formulation of this invention are significantly improved. It has excellent comprehensive performance, strong practicality, and can be widely applied to various aquaculture water quality control scenarios.

[0094] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A compound microbial preparation for regulating aquatic water quality, characterized in that, It consists of the following components in parts by weight: The mixture comprises 1-5 parts of a compound microbial strain, 15-30 parts of a modified carrier, 10-20 parts of a nutrient regulator, 6-15 parts of a stabilizing agent, and 30-68 parts of deionized water; the compound microbial strain consists of Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, nitrifying bacteria, denitrifying bacteria, Shewanella putrefactive bacteria, and Alcaligenes-like Pseudomonas, with a weight ratio of 1:1-3:1-2:0.8-1.5:0.5-1:0.3-0.8:0.2-0.

7. The modified carrier is a chitosan-ferric phosphate composite modified zeolite, composed of zeolite, chitosan and ferric phosphate in a weight ratio of 10:2-5:0.8-2; the nutrient regulator includes glucose, xylooligosaccharide, protein powder and amino acids; the stabilizing agent includes bentonite, calcium carbonate and silicon dioxide.

2. The compound microbial preparation according to claim 1, characterized in that, In the nutritional regulator, the weight ratio of glucose, xylooligosaccharides, protein powder, and amino acids is 2:1.5-3:1.2-2:0.6-1.

3. The compound microbial preparation according to claim 1, characterized in that, In the stabilizing agent, the weight ratio of bentonite, calcium carbonate and silica is 1:1.5-3:0.6-1.

4. A method for preparing a compound microbial agent for water quality regulation in aquaculture, characterized in that, Includes the following steps: (1) Activation of strains: Bacillus subtilis and Bacillus licheniformis were inoculated into LB medium, lactic acid bacteria into MRS medium, nitrifying bacteria and denitrifying bacteria into nitrification and denitrification medium, and Shewanella putrefactive bacteria and Pseudomonas alkalinum into nutrient broth medium. The strains were cultured at 32-37 degrees Celsius and a stirring speed of 160-200 rpm for 24-36 hours to obtain the activated solutions of each strain. (2) Segmented fermentation: First, the activated liquids of Bacillus subtilis, Bacillus licheniformis, Shewanella putrefactive bacteria, and Pseudomonas alcaligenes are mixed, and some nutrient regulator and deionized water are added. Fermentation is carried out at 35-37 degrees Celsius, stirring speed of 180-200 rpm, and aeration rate of 1.0-1.5 cubic meters per hour for 36-48 hours to obtain the first fermentation liquid. Then, the activated liquids of lactic acid bacteria, nitrifying bacteria, and denitrifying bacteria are added to the first fermentation liquid, and the remaining nutrient regulator and deionized water are added. Fermentation is carried out at 28-30 degrees Celsius, stirring speed of 140-150 rpm, and aeration rate of 0.6-1.0 cubic meters per hour for 24-30 hours to obtain the compound fermentation liquid. (3) Carrier modification: Zeolite was pretreated, acid washed, calcined, organically modified and composite modified in sequence. The calcination temperature was 500-600 degrees Celsius and the calcination time was 3.5-5 hours. The water bath temperature for organic modification was 50-60 degrees Celsius and the water bath reaction time was 6-8 hours. The oscillation adsorption time for composite modification was 90-120 minutes, the standing time was 20-24 hours and the soaking time was 20-24 hours, so as to obtain the modified carrier. (4) Gradient loading: Add the modified carrier to the composite fermentation broth, stir and adsorb at 100-120 rpm for 2-3 hours, and then stir and adsorb at 70-80 rpm for 3-4 hours to obtain the loaded solution; (5) Formulation: Add a stabilizing agent to the loading liquid, filter and concentrate, then dry and pulverize to obtain the finished formulation; (6) Finished product inspection: Sensory indicators, microbial activity and hygiene indicators are tested on the finished products. After passing the tests, the finished products are packaged and put into storage.

5. The preparation method according to claim 4, characterized in that, In step (2), the pH value of the fermentation broth is maintained at 7.0-7.5 during the fermentation process.

6. The preparation method according to claim 4, characterized in that, In step (3), the pretreatment involves boiling the zeolite for 30-40 minutes, washing it, and then drying it to constant weight; the acid washing involves placing the pretreated zeolite in a mixed acid solution of sulfuric acid and nitric acid and shaking it for 2-3 hours, then washing it and drying it to constant weight.

7. The preparation method according to claim 4, characterized in that, In step (4), the gradient loading also includes a secondary loading step: after the first gradient loading, let it stand for 1 hour, filter to remove the carrier without loaded microorganisms, add the composite fermentation liquid to the filtered loading liquid, and stir and adsorb at 60 revolutions per minute for 2-3 hours.

8. The preparation method according to claim 4, characterized in that, In step (5), the drying process is carried out in stages: first, the product is dried at 55 degrees Celsius for 4 hours, and then dried at 65-70 degrees Celsius for 6-8 hours.

9. The preparation method according to claim 4, characterized in that, In step (5), the powder is pulverized and then passed through a 60-80 mesh sieve. The pulverization process also includes an encapsulation step: a mixed encapsulation agent is added to the powder, which is composed of carboxyl-modified polyvinyl alcohol, starch and gelatin. The powder is then granulated twice and dried to obtain an encapsulated formulation.

10. The preparation method according to claim 9, characterized in that, In the mixed encapsulating agent, the weight ratio of carboxyl-modified polyvinyl alcohol, starch and gelatin is 9:3:1.5, and the amount of mixed encapsulating agent used is 10% of the weight of the pulverized powder.

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