Water-regulating microbial preparation for breeding largemouth bass seedlings in land-based round pond and application of water-regulating microbial preparation

Abstract

The invention relates to a water regulating microbial preparation for breeding largemouth bass seedlings in a land-based round pond and application, and belongs to the technical field of aquaculture. The preparation aims to solve the problem of combined pollution caused by synchronous exceeding of ammonia nitrogen, nitrite and hydrogen sulfide in a water body due to accumulation of residual feed and excrement in a land-based round pool high-density breeding environment. According to the scheme, the microbial agent is prepared by physically mixing bacillus standard bacterial powder, photosynthetic bacteria standard bacterial powder, compound nitrifying bacteria standard bacterial powder and thiobacillus standard bacterial powder according to specific parts by weight, wherein the bacillus standard bacterial powder is prepared by mixing bacillus subtilis and bacillus licheniformis, and the thiobacillus standard bacterial powder is prepared by mixing bacillus subtilis and bacillus licheniformis. The composite nitrifying bacteria standard bacterial powder is formed by mixing nitrite bacteria and nitrifying bacteria, and the thiobacillus thiooxidans standard bacterial powder is formed by mixing thiobacillus thiooxidans and thiobacillus thioparus. The preparation is used for synchronously degrading ammonia nitrogen, nitrite and hydrogen sulfide in aquaculture water, so that normal development of gonads of largemouth bass parents is guaranteed, and the hatching rate of fertilized eggs and the survival rate of early-stage seedlings are increased.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology. More specifically, this invention relates to a water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds and its application. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Land-based circular pond breeding technology is key to achieving efficient and controllable fry production. However, during the parent stock rearing, spawning and hatching, and early fry rearing stages, factors such as limited water space, vigorous metabolism of parent and fry, and feeding of high-protein feed easily lead to the accumulation of uneaten feed and feces at the bottom of the pond. Under the anaerobic decomposition of microorganisms, ammonia nitrogen (NH3-N) and nitrite (NO2) in the water increase significantly. - The concentrations of harmful substances such as nitrogen (N) and hydrogen sulfide (H2S) are rising rapidly, resulting in complex pollution.

[0003] The deterioration of water quality during this breeding stage is particularly severe: First, ammonia nitrogen and nitrite have a strong toxic effect on the gonadal development of parent fish, the hatching rate of fertilized eggs, and the survival and growth of early fry; second, highly toxic hydrogen sulfide not only directly harms the fish but also significantly inhibits the activity of beneficial bacteria such as nitrifying bacteria in the water, hindering the normal nitrogen cycle and leading to further accumulation of ammonia nitrogen and nitrite. This "sulfur-nitrogen" compound pollution has become a bottleneck problem restricting the efficient breeding of largemouth bass fry in land-based circular ponds.

[0004] In existing technologies, water conditioning agents used for adult fish farming often focus on degrading large molecular organic matter, or fail to fully consider the inhibitory effect of hydrogen sulfide on the nitrification system. Directly applying these agents to the more environmentally sensitive and water-quality-demanding breeding stage results in unstable effects and may even pose potential risks to fish eggs and delicate fry due to the complexity of the microbial strains or uncontrollable metabolic products. Therefore, there is an urgent need for a microecological agent specifically designed for the breeding stage of largemouth bass that can simultaneously and safely degrade ammonia nitrogen, nitrite, and hydrogen sulfide. Summary of the Invention

[0005] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages described below.

[0006] This invention provides a water-conditioning microbial preparation and its application for breeding largemouth bass fry in land-based circular ponds. It addresses the problem of "sulfur-nitrogen compound pollution" that occurs in the closed, high-density breeding environment of land-based circular ponds, where the accumulation of uneaten feed and feces leads to the simultaneous production and accumulation of three main harmful substances: ammonia nitrogen, nitrite, and hydrogen sulfide. This compound pollution not only directly harms the parent fish, eggs, and early fry individually, but more importantly, the highly toxic hydrogen sulfide severely inhibits the activity of beneficial microorganisms such as nitrifying bacteria in the water, thus hindering the normal nitrogen cycle. This leads to further accumulation of ammonia nitrogen and nitrite, creating a vicious cycle of continuous water quality deterioration, ultimately severely restricting the gonadal development of the parent fish, the hatching of fertilized eggs, and the survival of early fry.

[0007] This invention provides a water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds, which is composed of the following standardized bacterial powders in parts by weight through physical mixing: 40-50 parts of Bacillus standard bacterial powder, 30-40 parts of photosynthetic bacterial standard bacterial powder, 10-15 parts of compound nitrifying bacterial standard bacterial powder and 5-10 parts of sulfur-oxidizing bacterial standard bacterial powder. The Bacillus standard powder is composed of Bacillus subtilis (Bacillus subtilis) Bacillus subtilis Standard bacterial powder and Bacillus licheniformis ( Bacillus licheniformis The standard bacterial powder is mixed in a 1:1 weight ratio; The compound nitrifying bacteria standard powder is made by mixing nitrite-oxidizing bacteria standard powder and nitrifying bacteria standard powder in a weight ratio of (60-70):(30-40); The sulfur-oxidizing bacteria standard powder is composed of *Thiobacillus thiooxidans* (… Acidithiobacillus thiooxidans Standard bacterial powder and Thiobacillus thiocyanate ( Thiobacillus thioparus The standard bacterial powder is mixed in a 1:1 weight ratio.

[0008] Preferably, the Bacillus subtilis standard powder, Bacillus licheniformis standard powder, photosynthetic bacteria standard powder, nitrifying bacteria standard powder, nitrifying bacteria standard powder, Thiobacillus thiooxidans standard powder, and Thiobacillus thiocyanate standard powder are all powdered products obtained by immobilization on an adsorption carrier and drying, and their moisture content is all less than 10%. The effective viable count of each standard powder is stably controlled at (0.8±0.2)×10⁻⁶. 9 Within the range of CFU / g.

[0009] The water conditioning microbial preparation provided by this invention comprises the following steps: S1. Raw material rehydration: Use raw materials with an effective viable bacteria count higher than 1.0 × 10⁻⁶. 9Bacillus subtilis powder, Bacillus licheniformis powder, photosynthetic bacteria powder, nitrifying bacteria powder, nitrifying bacteria powder, Thiobacillus thiooxidans powder, and Thiobacillus thiocyanate powder, each with a CFU / g content, were rehydrated at a weight ratio of 1:(8-12) of bacterial powder to sterile water and stirred for 10-30 minutes to prepare a uniform bacterial solution. S2. Adsorption and immobilization: The bacterial solutions of each strain obtained in step S1 are stirred and adsorbed with sterile, dry composite carriers at a bacterial solution to carrier weight ratio of 1:(0.8-1.2) for 30-60 minutes to ensure that the bacterial solution fully wets the carrier; the composite carrier is made of wheat bran and zeolite powder mixed at a weight ratio of (6-8):(2-4); S3. Low-temperature drying: The adsorbed wet bacterial agents are dried at a low-temperature airflow temperature of 35-40℃ until the moisture content is less than 10%, and standardized intermediate bacterial powders of each bacterial strain are obtained. S4. Standardization and blending of intermediate microbial powder: S4.1 Perform viable cell counts on each batch of standardized intermediate bacterial powder. If the viable cell count is not within (0.8±0.2)×10⁻⁶, the count is considered incomplete. 9 Within the CFU / g range, adjustments are made by supplementing with a growth medium or high-viable-count intermediate bacterial powder until the effective viable count of each standard bacterial powder is stably controlled at (0.8±0.2)×10⁻⁶. 9 Within the range of CFU / g; S4.2 Primary mixing: Bacillus subtilis standard powder and Bacillus licheniformis standard powder are mixed at a weight ratio of 1:1 to obtain Bacillus standard powder; Thiobacillus thiooxidans standard powder and Thiobacillus thiocyanate standard powder are mixed at a weight ratio of 1:1 to obtain sulfur-oxidizing bacteria standard powder; Nitrifying bacteria standard powder and nitrifying bacteria standard powder are mixed at a weight ratio of (60-70):(30-40) to obtain compound nitrifying bacteria standard powder. S5. Final product mixing: The Bacillus standard powder, photosynthetic bacteria standard powder, compound nitrifying bacteria standard powder and sulfur bacteria standard powder obtained in step S4.2 are physically mixed at room temperature according to the weight parts described in claim 1, and then packaged to obtain the water conditioning microbial preparation.

[0010] Preferably, during the mixing process of the composite carrier in step S2, 5%-8% of sorbitol powder, accounting for the total weight of the composite carrier, is added simultaneously; the sorbitol powder is uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

[0011] Preferably, the bran of the composite carrier in step S2 is pre-hydrolyzed; the specific steps of the hydrolysis treatment are as follows: the bran is mixed with dilute sulfuric acid with a mass concentration of 3%-5% at a weight ratio of 1:(5-8), and stirred and reacted at a temperature of 100℃-105℃ for 30-40 minutes; after the reaction is completed, the mixture is filtered, and the pH of the filter residue is adjusted to 6.0-7.0 with sodium hydroxide solution, and then mixed with zeolite powder in proportion to form a composite carrier.

[0012] Preferably, during the mixing process of the composite carrier in step S2, 0.5%-1.5% of L-ascorbic acid and 0.2%-0.8% of sodium citrate, accounting for the total weight of the composite carrier, are also added; the L-ascorbic acid and sodium citrate are added simultaneously with the sorbitol powder and are uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

[0013] The present invention also provides the application of the above-mentioned water conditioning microbial preparation in the breeding of largemouth bass fry in land-based circular ponds. The application is used to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water body where largemouth bass fry are bred, to ensure the development of parent gonads, improve the hatching rate of fertilized eggs and the early survival rate of fry.

[0014] Preferably, the specific application method is as follows: Apply according to the following standards at different growth stages when breeding largemouth bass fry in land-based circular ponds. Before application, the water must meet the following conditions: dissolved oxygen ≥ 6 mg / L and pH 7.5-8.5: Parent rearing period: Apply once every 15 days, with a routine application rate of 0.8-1.0 g / m³ of water; 30-45 days before spawning and until the critical period of spawning, the initial application rate is adjusted to 1.5-2.0 g / m³ of water, and thereafter maintained at the routine rate. During the spawning and hatching period: apply once every 20 days, at a rate of 0.5-0.8 grams per cubic meter of water. During the fry's initial feeding period (0-15 days old): Apply once every 25 days, with a standard application rate of 0.3-0.5 g / m³ of water. Within 3 days after the fry first appear to be swimming in groups and actively feeding on exogenous food in the water, the initial application rate should be adjusted to 0.6-1.0 g / m³ of water, and then maintained at the standard rate thereafter. Fry rearing period (15-30 days old): Apply once every 20 days, at a rate of 0.5-0.8 grams per cubic meter of water; When applying, the preparation should be mixed with pool water at a weight ratio of 1:(10-15), and 1%-2% of the weight of brown sugar should be added. The mixture should be aerated and activated at 25-30℃ for 6-8 hours.

[0015] Preferably, when activating the formulation with pool water, 0.5%-2% of vitamin B by weight of the formulation is added. 12 powder; The vitamin B 12 The powder, brown sugar, preparation, and pool water are activated together for 6-8 hours; After activation, evenly sprinkle the solution throughout the pool between 5 PM and 7 PM daily. After sprinkling, turn off the circulation pump for 1-2 hours and continue oxygenation for more than 12 hours to maintain dissolved oxygen in the water > 4 mg / L.

[0016] The present invention has at least the following beneficial effects: First, a synergistic microecological preparation was constructed by carefully selecting and combining specific functional microbial communities. This preparation contains Bacillus, which decomposes large organic molecules in the water, such as uneaten feed and feces, reducing the production of ammonia and hydrogen sulfide at the source. Photosynthetic bacteria can directly utilize small molecules such as ammonia nitrogen as nutrients, directly reducing their concentration. Compound nitrifying bacteria are specifically responsible for converting highly toxic ammonia nitrogen into nitrite, and further converting nitrite into less toxic nitrate, thus opening up and facilitating the key pathway of the nitrogen cycle. The specially introduced sulfur-oxidizing bacteria specifically target hydrogen sulfide, oxidizing it into non-toxic sulfate. This eliminates the direct toxicity of hydrogen sulfide and also removes the inhibition of nitrifying bacteria by hydrogen sulfide, clearing the way for nitrification. This systematic combination of multiple microbial species constitutes a microecological network capable of simultaneously and synergistically degrading ammonia nitrogen, nitrite, and hydrogen sulfide, fundamentally breaking the vicious cycle of "sulfur-nitrogen compound pollution."

[0017] Secondly, in terms of the preparation process, the bacterial solution is immobilized using an adsorption carrier, providing a physical protective barrier for the bacteria and preventing mass death and loss during drying and storage. Furthermore, standardized viable cell counts ensure the accurate, stable, and reliable quantity of various functional bacteria in each batch, thus guaranteeing the reproducibility of the formulation's effects. In addition, optimizing the carrier composition, such as acid hydrolysis of wheat bran, increases the carrier's porosity and adsorption capacity, while providing an initial carbon source for the immobilized bacteria, facilitating rapid recovery and colonization after introduction into water. The addition of protective agents such as sorbitol, L-ascorbic acid, and sodium citrate during preparation significantly enhances the bacteria's resistance to stresses such as drying, oxidation, and storage by forming a protective layer around the bacteria, scavenging free radicals, and stabilizing the microenvironment. This maximizes the preservation of bacterial activity, extends the product's shelf life, and ensures that the product continues to provide highly active bacterial agents during use.

[0018] Finally, regarding the application method, vitamin B is added during the activation process. 12This provides important cofactors for the enzyme systems and metabolic processes of microorganisms, especially nitrifying and photosynthetic bacteria, playing a "nutrient-initiating" role and accelerating the transition of the bacterial community from a dormant state to a highly active working state. Spraying at dusk avoids the damaging effects of strong ultraviolet radiation during the day. Simultaneously, temporarily shutting off the water circulation pump after spraying provides a valuable window of opportunity for microorganisms to settle, attach, and colonize on the bottom and in the water, allowing them to more effectively establish dominant bacterial communities in areas rich in pollutants, thus performing water purification functions more efficiently.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0020] Figure 1 A schematic diagram of the process of pouring the preparation from the mineral water bottle into the activated liquid after activation with pool water into a bucket; Figure 2 A schematic diagram showing the process of evenly spreading the activation solution into the land-based circular pool. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials mentioned are commercially available unless otherwise specified. All bacterial strains used in this invention are common commercial strains in the field and can be purchased through commercial channels. For example, Bacillus subtilis powder provided by Sichuan Shengluyuan Bioengineering Co., Ltd.; Bacillus licheniformis powder and photosynthetic bacteria powder provided by Shandong Baolai Lailai Bioengineering Co., Ltd.; nitrifying bacteria powder and nitrifying bacteria powder provided by Qingdao Genyuan Biotechnology Group Co., Ltd.; and Thiobacillus sulfoxidans powder and Thiobacillus sulfide-excreting powder provided by Qingdao Haibo Biotechnology Co., Ltd. The photosynthetic bacteria are Rhodopseudomonas palustris (…). Rhodopseudomonas palustris Nitrifying bacteria are *Nitrosomonas cerevisiae* (European nitrosomonas). Nitrosomonas europaea The nitrifying bacteria are *Nitrospirillum moscoparia* (…). Nitrospira moscoviensis ).

[0023] Example 1 This embodiment provides a water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds, which is physically mixed from the following standardized bacterial powders in parts by weight: 40 parts by weight of Bacillus standard powder; 30 parts by weight of standard photosynthetic bacteria powder; 10 parts by weight of compound nitrifying bacteria standard powder; Five parts by weight of standard sulfur-forming bacteria powder.

[0024] The Bacillus standard powder is composed of Bacillus subtilis (Bacillus subtilis) Bacillus subtilis Standard bacterial powder and Bacillus licheniformis ( Bacillus licheniformis The standard bacterial powder is prepared by mixing nitrifying bacteria standard powder and nitrifying bacteria standard powder in a weight ratio of 1:1; the compound nitrifying bacteria standard powder is prepared by mixing nitrite-oxidizing bacteria standard powder and nitrifying bacteria standard powder in a weight ratio of 60:40; the sulfur-oxidizing bacteria standard powder is prepared by mixing thiobacillus thiooxidans (… Acidithiobacillus thiooxidans Standard bacterial powder and Thiobacillus thiocyanate ( Thiobacillus thioparus The standard bacterial powder is mixed in a 1:1 weight ratio.

[0025] Each standard bacterial powder was prepared by the following adsorption and immobilization method: Raw material rehydration: Use commercially available raw materials with an effective viable bacteria count higher than 1.0 × 10⁻⁶. 9 Each single bacterial strain powder (Bacillus subtilis, Bacillus licheniformis, photosynthetic bacteria, nitrifying bacteria, nitrifying bacteria, Thiobacillus thiooxidans, Thiobacillus thiocyanate) at CFU / g was rehydrated with sterile water at a weight ratio of 1:10 and stirred for 20 minutes to prepare a homogeneous bacterial solution. The rehydration of the raw materials was specifically performed by placing them on a magnetic stirrer or a low-speed mechanical stirrer and stirring at a speed of 200-500 rpm.

[0026] Adsorption and immobilization: The above-mentioned bacterial solutions and adsorption carriers (150-mesh zeolite powder) were mixed and stirred at a weight ratio of 1:1 for 30 minutes to ensure that the bacterial solutions were fully adsorbed onto the carriers. Specifically, the stirring for adsorption and immobilization was performed in a mixer at a speed of 200-400 rpm.

[0027] Drying: The adsorbed wet bacterial agent was dried by forced air at 35°C until the moisture content was below 10%, obtaining standardized bacterial powders for each bacterial species. No precise adjustment of viable cell counts was performed during this process.

[0028] The Bacillus subtilis standard bacterial powder, Bacillus licheniformis standard bacterial powder, photosynthetic bacteria standard bacterial powder, nitrifying bacteria standard bacterial powder, nitrifying bacteria standard bacterial powder, Thiobacillus thiooxidans standard bacterial powder, and Thiobacillus thiocyanate standard bacterial powder are all powdered products obtained by the above methods, and their moisture content is all below 10%. Testing showed that the effective viable bacteria count of each standard bacterial powder was approximately (0.5-1.2) × 10⁻⁶. 9 Within the range of CFU / g, its stability is poor.

[0029] During preparation, the standard bacterial powder meeting the specifications is directly taken, physically mixed evenly at room temperature according to the ratio, and then packaged to obtain the water conditioning microbial preparation. This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite, and hydrogen sulfide in the water body where largemouth bass fry are bred.

[0030] Example 2 This embodiment provides a water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds, which is physically mixed from the following standardized bacterial powders in parts by weight: 45 parts by weight of Bacillus standard powder; 35 parts by weight of standard photosynthetic bacteria powder; 12 parts by weight of compound nitrifying bacteria standard powder; Eight parts by weight of standard sulfur-forming bacteria powder.

[0031] The Bacillus standard powder is composed of Bacillus subtilis standard powder and Bacillus licheniformis standard powder mixed in a weight ratio of 1:1; the composite nitrifying bacteria standard powder is composed of nitrite-oxidizing bacteria standard powder and nitrifying bacteria standard powder mixed in a weight ratio of 65:35; and the sulfur-oxidizing bacteria standard powder is composed of Thiobacillus thiooxidans standard powder and Thiobacillus thiocyanate standard powder mixed in a weight ratio of 1:1.

[0032] Each standard bacterial powder was prepared by the following adsorption and immobilization method: Rehydration of raw materials: Same as in Example 1.

[0033] Adsorption and immobilization: Each bacterial solution and adsorption carrier (150 mesh bentonite) were mixed and stirred at a weight ratio of 1:1 for 30 minutes.

[0034] Drying: Same as in Example 1.

[0035] Each of the standard bacterial powders was obtained as a powder product using the above method, and its moisture content was less than 10%. Testing showed that the effective viable count of each standard bacterial powder was approximately (0.6-1.3) × 10⁻⁶. 9 Within the range of CFU / g, its stability is poor.

[0036] During preparation, the standard bacterial powder meeting the specifications is directly taken, physically mixed evenly at room temperature according to the ratio, and then packaged to obtain the water conditioning microbial preparation. This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite, and hydrogen sulfide in the water body where largemouth bass fry are bred.

[0037] Example 3 This embodiment provides a water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds, which is physically mixed from the following standardized bacterial powders in parts by weight: 50 parts by weight of Bacillus standard bacterial powder; 40 parts by weight of standard photosynthetic bacteria powder; 15 parts by weight of compound nitrifying bacteria standard powder; 10 parts by weight of standard sulfur bacteria powder.

[0038] The Bacillus standard powder is composed of Bacillus subtilis standard powder and Bacillus licheniformis standard powder mixed in a weight ratio of 1:1; the composite nitrifying bacteria standard powder is composed of nitrite-oxidizing bacteria standard powder and nitrifying bacteria standard powder mixed in a weight ratio of 70:30; and the sulfur-oxidizing bacteria standard powder is composed of Thiobacillus thiooxidans standard powder and Thiobacillus thiocyanate standard powder mixed in a weight ratio of 1:1.

[0039] Each standard bacterial powder was prepared by the following adsorption and immobilization method: Rehydration of raw materials: Same as in Example 1.

[0040] Adsorption and immobilization: Each bacterial solution was mixed with a conventional composite adsorption carrier (made by simple physical mixing of wheat bran and zeolite powder in a weight ratio of 1:1) at a bacterial solution to carrier weight ratio of 1:1 and stirred for 40 minutes.

[0041] Drying: The adsorbed wet bacterial agent was dried by forced air at 35°C until the moisture content was below 10%, obtaining standardized bacterial powders for each bacterial species. No precise adjustment of viable cell counts was performed during this process.

[0042] Each of the standard bacterial powders was obtained as a powder product using the above method, and its moisture content was less than 10%. Testing showed that the effective viable count of each standard bacterial powder was approximately (0.7-1.4) × 10⁻⁶. 9 Within the CFU / g range, the stability is relatively better than that of Examples 1 and 2.

[0043] During preparation, the above-mentioned standard bacterial powder meeting the specifications is directly taken, physically mixed evenly at room temperature according to the ratio, and then packaged to obtain the water conditioning microbial preparation. This preparation can be used for the breeding of largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water body where largemouth bass fry are bred, ensuring the development of parent gonads, improving the hatching rate of fertilized eggs and the early survival rate of fry.

[0044] Example 4 This embodiment provides a water conditioning microbial preparation for breeding largemouth bass fry in a land-based circular pond. The proportions of each bacterial powder are the same as in Example 3, but the preparation method includes the following steps: S1. Raw material rehydration: Use commercially available raw materials with an effective viable bacteria count higher than 1.0 × 10⁻⁶. 9Bacillus subtilis powder, Bacillus licheniformis powder, photosynthetic bacteria powder, nitrifying bacteria powder, nitrifying bacteria powder, Thiobacillus thiooxidans powder, and Thiobacillus thiocyanate powder, each with a CFU / g content, were rehydrated with sterile water at a weight ratio of 1:10 and stirred for 20 minutes to prepare a uniform bacterial solution. S2. Adsorption and immobilization: The bacterial solutions of each strain obtained in step S1 are respectively mixed with sterile and dry composite carriers at a weight ratio of 1:1 for adsorption and stirring for 45 minutes to allow the bacterial solution to fully wet the carrier; the composite carrier is composed of wheat bran and zeolite powder mixed at a weight ratio of 7:3. S3. Low-temperature drying: The adsorbed wet bacterial agents are dried at 38°C using low-temperature airflow until the moisture content is less than 10%, thus obtaining standardized intermediate bacterial powders for each bacterial strain. S4. Standardization and blending of intermediate microbial powder: S4.1 Perform viable cell counts on each batch of standardized intermediate bacterial powder. If the viable cell count is not within (0.8±0.2)×10⁻⁶, the count is considered incomplete. 9 Within the CFU / g range, adjustments are made by supplementing with a growth medium or high-viable-count intermediate bacterial powder until the effective viable count of each standard bacterial powder is stably controlled at (0.8±0.2)×10⁻⁶. 9 Within the range of CFU / g; S4.2 Primary mixing: Bacillus subtilis standard powder and Bacillus licheniformis standard powder are mixed at a weight ratio of 1:1 to prepare Bacillus standard powder; Thiobacillus thiooxidans standard powder and Thiobacillus thiocyanate standard powder are mixed at a weight ratio of 1:1 to prepare sulfur-oxidizing bacteria standard powder; Nitrifying bacteria standard powder and nitrifying bacteria standard powder are mixed at a weight ratio of 70:30 to prepare compound nitrifying bacteria standard powder. S5. Final product mixing: 50 parts by weight of Bacillus standard powder, 40 parts by weight of photosynthetic bacteria standard powder, 15 parts by weight of compound nitrifying bacteria standard powder and 10 parts by weight of sulfur bacteria standard powder obtained in step S4.2 are physically mixed at room temperature, and after being uniformly mixed, they are packaged to obtain the water conditioning microbial preparation.

[0045] This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water, ensuring the development of parent gonads, improving the hatching rate of fertilized eggs and the early survival rate of fry.

[0046] Example 5 It is based on Example 4 with the addition of sorbitol.

[0047] This embodiment 5 provides a water conditioning microbial preparation for breeding largemouth bass fry in a land-based circular pond. The proportion of bacterial powder and the preparation method are the same as those in embodiment 4, but in the adsorption and immobilization step S2, 6.5% of the total weight of the composite carrier of sorbitol powder is added simultaneously; the sorbitol powder is uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

[0048] Specifically, in step S2, the composite carrier is made by mixing wheat bran and zeol powder in a weight ratio of 7:3, and sorbitol powder (accounting for 6.5% of the total weight of the composite carrier) is added during the mixing process. After being evenly dispersed, it is stirred and adsorbed with the bacterial solution in proportion. The remaining steps are the same as in Example 4.

[0049] This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water, ensuring the development of parent gonads, improving the hatching rate of fertilized eggs and the early survival rate of fry.

[0050] Example 6 It incorporates bran hydrolysis treatment based on Example 5.

[0051] This embodiment 6 provides a water conditioning microbial preparation for breeding largemouth bass fry in a land-based circular pond. The proportion of bacterial powder and the preparation method are the same as in embodiment 5. However, in step S2, the bran of the composite carrier is pre-hydrolyzed. The specific steps of the hydrolysis treatment are as follows: the bran is mixed with 4% dilute sulfuric acid at a weight ratio of 1:6.5 and stirred at 102°C for 35 minutes. After the reaction is completed, the mixture is filtered, and the pH of the filter residue is adjusted to 6.5 with sodium hydroxide solution. Then, it is mixed with zeolite powder at a weight ratio of 7:3 to form a composite carrier.

[0052] In the adsorption and immobilization step S2, the composite carrier consists of hydrolyzed wheat bran and zeol powder, and sorbitol powder accounting for 6.5% of the total weight of the composite carrier is added simultaneously. After being uniformly dispersed, it is mixed with the bacterial solution. The remaining steps are the same as in Example 5.

[0053] This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water, ensuring the development of parent gonads, improving the hatching rate of fertilized eggs and the early survival rate of fry.

[0054] Example 7 It is based on Example 6 with the addition of L-ascorbic acid and sodium citrate.

[0055] This embodiment 7 provides a water conditioning microbial preparation for breeding largemouth bass fry in a land-based circular pond. The proportion of bacterial powder and the preparation method are the same as in embodiment 6. However, during the mixing process of the composite carrier in step S2, 1.0% of L-ascorbic acid and 0.5% of sodium citrate, accounting for the total weight of the composite carrier, are added. The L-ascorbic acid and sodium citrate are added simultaneously with sorbitol powder and are uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

[0056] Specifically, in step S2, the composite carrier is composed of hydrolyzed wheat bran and zeolite powder mixed in a weight ratio of 7:3. During the mixing process, sorbitol powder (6.5% of the total weight of the composite carrier), L-ascorbic acid (1.0% of the total weight of the composite carrier), and sodium citrate (0.5% of the total weight of the composite carrier) are added simultaneously and evenly dispersed. Then, it is stirred and adsorbed with the bacterial solution in proportion. The remaining steps are the same as in Example 6.

[0057] This preparation can be used for breeding largemouth bass fry in land-based circular ponds to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in the water, ensuring the development of parent gonads, improving the hatching rate of fertilized eggs and the early survival rate of fry.

[0058] Comparative Example 1 It is basically the same as Example 3, but lacks sulfur-oxidizing bacteria.

[0059] Preparation method of Comparative Example 1: Except for the absence of sulfur-oxidizing bacteria standard powder, the types, proportions, and preparation methods of the other bacterial powders are exactly the same as in Example 3. That is, the preparation is physically mixed from 50 parts by weight of Bacillus standard powder, 40 parts by weight of photosynthetic bacteria standard powder, and 15 parts by weight of compound nitrifying bacteria standard powder.

[0060] Comparative Example 2 Preparation method: Take the bacterial solution obtained in the same step as S1 in Example 4.

[0061] The adsorption and immobilization step S2 is omitted. The bacterial solutions are directly dried at 38°C using low-temperature airflow until the moisture content is below 10%, resulting in primary dried bacterial powders for each strain.

[0062] Key Supplement: Standardization Process. For each batch of primary dried bacterial powder, viable cell counts were performed. 150-mesh zeolite powder was added as a dilution carrier to adjust the effective viable cell count of each powder to (0.8±0.2)×10⁻⁶. 9 Standardized bacterial powders for each strain were obtained within the range of CFU / g.

[0063] The water conditioning microbial preparation was then physically mixed according to the final product ratio of Example 3 to obtain the preparation.

[0064] Comparative Example 3 Preparation method: Compared with Example 7, the composite carrier in step S2 is made by mixing wheat bran and zeolite powder in a weight ratio of 7:3 after undergoing the same acid hydrolysis treatment as in Example 7, but without adding sorbitol powder, L-ascorbic acid and sodium citrate. The remaining preparation steps (including hydrolysis treatment parameters, adsorption, drying, and standardization in step S4.1, etc.) are exactly the same as in Example 7.

[0065] Comparative Example 4 The method of hydrolyzing the bran has been changed.

[0066] Preparation method: Compared with Example 7, only the hydrolysis treatment of bran in step S2 is changed. Specifically, bran and water are mixed at a weight ratio of 1:6.5 and stirred at 80°C for 60 minutes (this is a conventional mild pretreatment). After the reaction, the mixture is filtered, and the filter residue is directly mixed with zeolite powder without pH adjustment to form a composite carrier. The remaining preparation steps (including the addition of protective agents, adsorption, drying, and standardization in step S4.1) are exactly the same as in Example 7.

[0067] Example 8 This embodiment describes the application of the water-conditioning microbial preparation prepared in Example 7 in the breeding of largemouth bass fry in a land-based circular pond. The application method is as follows: I. Preparations before application Water pretreatment: One hour before application, test key water indicators to ensure dissolved oxygen ≥ 6 mg / L (using a portable dissolved oxygen meter, sampling the middle layer of water at three evenly distributed points in the center and edge of the pool, and taking the average value), and pH 7.5-8.5 (using a pH meter for simultaneous sampling and testing). If dissolved oxygen is insufficient, turn on aeration equipment (such as a microporous aerator, power 1-2 kW / acre) for 2 hours; if pH < 7.5, apply 5-10 g / m³ of quicklime 24 hours in advance to adjust; if pH > 8.5, add 0.5-1.0 g / m³ of citric acid to adjust until the indicators meet the requirements.

[0068] Formulation dosage calculation: The formulation dosage is accurately calculated based on the actual water volume of the land-based circular pond. The calculation formula is "Required formulation weight (grams) = Water volume (cubic meters) × Application rate at the corresponding stage (grams / cubic meter)". Example: A land-based circular pond has a diameter of 3m and a water depth of 1.2m, with a water volume of ≈8.5 cubic meters. If it is in the routine application stage of the parent stock cultivation period (application rate 0.8-1.0 grams / cubic meter), taking the middle value of 0.9 grams / cubic meter, the required formulation weight = 8.5 × 0.9 = 7.65 grams.

[0069] Preparation of activation materials: Prepare a clean, oil-free activation container (such as a plastic bucket or bottle), pond water (taken from the top 20cm of the aquaculture pond and filtered to remove uneaten feed, feces and other impurities), brown sugar (purity ≥98%), a small aeration pump (air output 0.5-1m³ / h), and a stirring rod (glass or plastic).

[0070] II. Graded Application Procedures Application during parent breeding period: Routine application: once every 15 days, at a rate of 0.8-1.0 g / m³. After calculating the dosage according to the above formula, take the corresponding weight of the preparation and proceed to the activation step.

[0071] Application during critical periods: When applying for the first time 30-45 days before the parent spawning (e.g., 40 days before spawning), the dosage is adjusted to 1.5-2.0 g / m³ (example water volume required = 8.5 × 1.8 = 15.3 g). Subsequently, continue to apply the usual dosage once every 15 days until the parent spawning ends.

[0072] Application during the spawning and hatching period: once every 20 days, at a dosage of 0.5-0.8 g / m³ (for example, the water body uses the midpoint of 0.65 g / m³, so the required preparation weight = 8.5 × 0.65 = 5.525 g). Maintain a slight flow of water throughout the process, with the water flow speed controlled at 0.1-0.2 m / s, to avoid water flow impacting the fish eggs.

[0073] Application during the fry's initial feeding stage (0-15 days old): Routine application: once every 25 days, at a rate of 0.3-0.5 g / m³ (for example, the water body uses the middle value of 0.4 g / m³, and the required preparation weight = 8.5 × 0.4 = 3.4 g).

[0074] Application during the critical period: The first application should be made within 3 days (e.g., the 2nd day) after the fish fry are first observed to swim in groups and actively feed on exogenous food in the water. The dosage should be adjusted to 0.6-1.0 g / m³ (the required amount for the example water body = 8.5 × 0.8 = 6.8 g). Subsequently, apply once every 25 days according to the conventional dosage.

[0075] Application during the juvenile rearing period (15-30 days old): once every 20 days, at a rate of 0.5-0.8 g / m³ (same as the standard rate during the spawning and hatching period; the example water body requires 5.525 g). Turn on the microporous aerator to maintain dissolved oxygen ≥5 mg / L.

[0076] III. Activation and Spraying Procedures: Mixing ratio: Pour the calculated formulation into the activation container, and add pretreated pool water at a formulation to pool water weight ratio of 1:(10-15) (Example: 7.65g of formulation to 76.5-114.75g of pool water, i.e. 76.5-114.75mL, take the middle value of 95mL), and stir until the formulation is completely dispersed.

[0077] Nutritional addition: Add 1%-2% of the weight of the preparation with brown sugar (example: 0.0765-0.153 grams of brown sugar for 7.65 grams of preparation, take the middle value of 0.11 grams), and stir until the brown sugar is completely dissolved.

[0078] Aeration activation: Place the aeration head of the aeration pump at the bottom of the activation container, turn on aeration (adjust the air output to form uniform, fine bubbles), and control the activation time according to the ambient temperature. At normal temperature (25-30℃): activate for 6-8 hours, stirring with a stirring rod every 1.5 hours for 5 minutes each time to ensure that the bacterial solution is evenly exposed to oxygen and nutrients.

[0079] Low temperature environment (<15℃): Place the activation container in a constant temperature environment of 25-30℃ (such as a greenhouse or constant temperature box), extend the activation time to 10-12 hours, and stir once every 1 hour.

[0080] High temperature environment (>30℃): shorten the activation time to 4-6 hours, stir once every 1 hour, and avoid exposing the container to the sun. If necessary, cover it with a light-proof cloth.

[0081] Full pool application: After activation, apply the solution between 5 PM and 7 PM daily (ideally 6 PM). See details. Figure 1-2 As shown on site. Use a watering can or spoon to evenly spray along the edge of the pool, ensuring the bacterial solution covers the entire pool; for circular pools, spray in a spiral motion from the edge to the center to avoid excessively high concentrations in some areas.

[0082] Subsequent management: Immediately after spraying, turn off the water circulation pump and keep it off for 1-2 hours (1.5 hours in this example) to provide conditions for microbial colonization; at the same time, turn on all aeration equipment and continue aeration for more than 12 hours, during which dissolved oxygen should be tested every 2 hours to ensure that dissolved oxygen is >4mg / L. If dissolved oxygen is lower than 4mg / L, turn on emergency aeration equipment (such as portable aerator) in time.

[0083] Example 9 I. Preparations before application Based on the preparations made in Example 8, additional vitamin B was prepared. 12 Powder (purity ≥ 99%), and ensure the activation container is dry and free of impurities to avoid vitamin B. 12 Invalid.

[0084] II. Graded Application Procedures The application procedure is completely consistent with that in Example 8, including the application frequency, dosage calculation, and dosage adjustment during critical periods for each growth stage.

[0085] III. Activation and Spraying Operations Mixing ratio: Same as in Example 8, pour the preparation into the activation container, add the pretreated pool water in proportion, and stir until the preparation is completely dispersed.

[0086] Nutritional fortification: Two nutrients are added simultaneously: Brown sugar: The dosage is the same as in Example 8 (1%-2% of the formulation weight), and 0.11 grams of brown sugar are added to a 65-gram formulation in Example 7.

[0087] Vitamin B 12 Powder: Add 0.5%-2% (by weight of the preparation) of vitamin B. 12 (Example: Add 0.038-0.153 grams of vitamin B to a 7.65-gram preparation) 12 (Take the median value of 0.09 grams).

[0088] Stirring order: First, add brown sugar and vitamin B. 12 Mix the powder thoroughly, then pour it into the mixture of the preparation and pool water. Stir quickly for 3 minutes with a stirring rod to ensure that the two nutrients are completely dissolved and there is no sediment.

[0089] Aeration activation: The activation conditions are the same as in Example 8, with the activation time controlled according to the ambient temperature, and the stirring frequency and duration remaining unchanged. During activation, observe the state of the bacterial solution; if uniform turbidity and no stratification or sedimentation occur, the activation is considered successful.

[0090] Full pool spraying: The spraying time and method are the same as in Example 8 (spraying evenly throughout the entire pool from 17:00 to 19:00).

[0091] Subsequent management: After spraying, turn off the circulation pump for 1-2 hours (1.5 hours in this example) and continue oxygenation for more than 12 hours. In addition to monitoring dissolved oxygen, check the pH of the water body once every 4 hours after spraying to ensure that the pH is maintained at 7.5-8.5. If the pH fluctuates by more than 0.5, adjust it in time according to the adjustment method in Example 8.

[0092] After spraying, clean the activation container promptly to prevent residual bacterial solution from breeding other bacteria and affecting the next use.

[0093] Experiment 1 This experiment aims to verify that the series of preparation methods used in this invention, such as composite carrier, hydrolysis treatment, and the addition of protective agents, can effectively maintain the activity of the strain, extend the shelf life of the product, and ensure that the formulation still has high activity when applied to water.

[0094] 1.1 Test Sample Example 3: The formulation prepared in this example.

[0095] Example 7: The formulation prepared in this example.

[0096] Comparative Example 2: Adsorption-free immobilization process.

[0097] Comparative Example 3: The composition of the composite carrier was changed (sorbitol and additives were missing).

[0098] Comparative Example 4: Changes in the method of wheat bran hydrolysis.

[0099] 1.2 Test Methods Shelf life simulation: The above samples were placed in a 37°C incubator for accelerated storage for 30 days (simulating approximately 6 months of room temperature storage). Samples were taken at 0, 15, and 30 days of storage to determine the total viable count (CFU / g). The rate of decline in viable count after 30 days of storage was calculated.

[0100] Viable count decline rate (%) = [(Initial viable count - Viable count after 30 days) / Initial viable count] × 100%; Simulated Viability Test: Take samples after 30 days of accelerated storage and activate them according to the method in Example 8 (without adding Vitamin B). 12 After activation, the total number of viable bacteria (CFU / mL) in the activation solution was determined by plate counting to assess the activity of the starting agent in actual use after storage.

[0101] The experimental results are shown in Table 1.

[0102] Table 1: As shown in Table 1, Comparative Example 2 (without immobilization) exhibited the most significant decrease in viable cell count and the lowest application activity, highlighting the necessity of the adsorption-immobilization process for protecting the bacterial cells and maintaining product stability. Comparative Example 3 (with altered carrier composition) showed significantly worse stability and application activity than Example 7, indicating that the addition of protective agents such as sorbitol played a crucial role in helping the bacterial cells resist drying and storage stress. Comparative Example 4 (with altered hydrolysis method) showed effects between Comparative Example 3 and Example 7, demonstrating that optimized acid hydrolysis treatment better modifies the carrier structure, providing optimal protection for its adsorption performance and bacterial cell protection. This invention, through the synergistic effects of adsorption-immobilization, optimized carrier hydrolysis, and the addition of composite protective agents, significantly extends the product's shelf life and ensures that the product continues to provide highly active bacterial agents during use.

[0103] Experiment 2 This experiment aims to verify the effects of formulations with different compositions on water quality and aquaculture results in actual aquaculture applications, and to examine the efficacy enhancement effect of the application method of the present invention (Example 9) on formulations of different quality.

[0104] 2.1 Test Grouping The experiment was set up with the following groups: Group A: The formulation prepared in Example 7 was administered according to the method of Example 9 (including vitamin B1). 12 (Activation and evening spraying).

[0105] Group B: The formulation prepared in Example 7 was administered according to the method described in Example 8 (without Vitamin B1). 12 activation).

[0106] Group C: Formulations prepared using different examples (Examples 3, 4, 5, and 6) were all administered according to the method of Example 9.

[0107] Group D: The formulation prepared in Comparative Example 1 (lacking sulfur-oxidizing bacteria) was applied according to the method of Example 9.

[0108] Group E: Blank control group, which received no water conditioning microbial agents and only underwent routine management.

[0109] Group F: Commercially available control group, using commercially available conventional water conditioner (e.g., a certain brand of compound microbial water conditioner), applied according to the recommended method in its instructions, at a uniform dose of 1.0 g / m³, once every 10 days, and activated according to the instructions (without grading or temperature control).

[0110] 2.2 Test Conditions and Methods 2.2.1 Test Site and Facilities Location: Land-based circular pond aquaculture workshop of a certain aquaculture technology center; Test pools: Circular pools of uniform specifications (3m in diameter, 1.2m in depth, and approximately 8.5 cubic meters of water), with 4 replicate pools in each group; Supporting equipment: microporous oxygenator (1-2kW / acre), portable dissolved oxygen meter (accuracy ±0.1mg / L), pH meter (accuracy ±0.01), and small aeration pump (for formulation activation).

[0111] 2.2.2 Seedling and Breeding Management Parent stocking: 6 2-3 year old largemouth bass parents (female to male ratio 1:1, weight 1.8-2.2 kg / parent) are stocked in each pond at a density of 0.7 parents / cubic meter. Diseased or weak individuals are quarantined before stocking. Feeding: Use high-protein compound feed (45% crude protein content) twice a day (8:00 and 16:00), with the amount of feed being 3%-5% of the fish's body weight; Basic environment: water temperature controlled at 22-25℃, dissolved oxygen ≥5mg / L (routine management), and environmental conditions are completely consistent for each group.

[0112] 2.2.3 Formulation administration (groups A, B, C, D, and F only) Strictly follow the tiered and phased implementation guidelines, with specific parameters as follows: Parental rearing period: Apply once every 15 days, with a standard application rate of 0.8-1.0 g / m³; when applying for the first time 30-45 days before spawning, double the dose to 1.5-2.0 g / m³, and maintain the standard dose thereafter. During the egg-laying and hatching period: Apply once every 20 days at a rate of 0.5-0.8 grams per cubic meter, maintaining a constant flow of water at a rate of 0.1-0.2 m / s throughout the process; During the fry's initial feeding period (0-15 days old): apply once every 25 days, with a standard application rate of 0.3-0.5 g / m³; within 3 days after the fry first start feeding on exogenous feed in groups, double the initial application rate to 0.6-1.0 g / m³, and maintain the standard application rate thereafter. During the juvenile rearing period (15-30 days old): Apply once every 20 days at a rate of 0.5-0.8 g / m³, and maintain dissolved oxygen at ≥5 mg / L by opening micropores for oxygenation; Activation requirements: Groups A, C, and D require the addition of 0.5%-2% (by weight) of vitamin B. 12 Powder (added simultaneously with brown sugar), aerated and activated at different temperatures (25-30℃ for 6-8 hours, <15℃ for 10-12 hours, >30℃ for 4-6 hours); the spraying time for all groups is uniformly from 17:00 to 19:00, after spraying the circulation pump is turned off for 1-2 hours, and oxygenation is continued for more than 12 hours.

[0113] 2.2.4 Indicator Testing (1) Water quality indicators Sampling: Collect water samples every 5 days (3 points at the center and edge of the pool, and a mixture of 20cm at the surface, 60cm at the middle, and 100cm at the bottom). During key stages such as parent breeding and spawning / hatching, additional tests are conducted 1 day before and 3 days after application. Detection indicators and methods: Ammonia nitrogen (NH3-N): Nessler's reagent spectrophotometric method (HJ535-2009); Nitrite (NO2) - -N): N-(1-naphthyl)-ethylenediamine spectrophotometry (GB / T7493-1987); Hydrogen sulfide (H2S): Methylene blue spectrophotometry (GB / T16489-1996).

[0114] (2) Biological indicators Parental gonadal maturity rate: During the peak egg-laying period, 10 parental animals were randomly selected from each group and assessed by the abdominal compression method (mature eggs / sperm can be squeezed out). Fertilized egg hatching rate: Collect fertilized eggs from each group, take 3 portions from each group (10,000 eggs per portion), and incubate them under a constant temperature of 25℃. Calculate "number of hatched larvae / total number of fertilized eggs × 100%"; Seedling survival rate: After hatching, the fry are raised to 30 days of age, and the survival rate is calculated as "number of surviving seedlings / initial number of seedlings × 100%" (initial stocking was 5000 fry / pond).

[0115] 2.3 The test results are shown in Table 2.

[0116] Table 2: Comparison of water quality and biological indicators between Group A and Group B In Table 2, group A (containing vitamin B) 12 The activated group (group B) outperformed group B (conventional activated group) in all water quality indicators (ammonia nitrogen, nitrite) and biological indicators (hatching rate, survival rate). This was due to the superior performance of vitamin B. 12 As a metabolic cofactor for nitrifying bacteria and photosynthetic bacteria, it can accelerate the transformation of bacterial cells from a dormant state to an active state, thereby improving the efficiency of pollutant degradation.

[0117] Table 3: Comparison of the effects of different formulations under the application method in Example 9 In Table 3, the comparative example 1, which lacked sulfur-oxidizing bacteria, showed a comprehensive deterioration in water quality and reproductive indicators, with hydrogen sulfide concentration reaching as high as 0.07 mg / L, and peak values ​​of ammonia nitrogen and nitrite being more than twice that of Example 7.

[0118] It is evident that hydrogen sulfide is not only highly toxic to fish, but also strongly inhibits the cytochrome activity of nitrifying bacteria, thus thwarting a key link in the nitrogen cycle of water. In the bacterial strain of this invention, sulfur-oxidizing bacteria (Thiobacillus thiooxidans and Thiobacillus thiopureum) oxidize highly toxic H2S into non-toxic sulfate, eliminating direct toxicity and clearing the way for nitrifying bacteria. This allows the combined nitrifying bacteria (nitrite-oxidizing and nitrifying bacteria) to unimpededly convert ammonia nitrogen into nitrite, and further into non-toxic nitrate. Bacillus reduces pollution sources by decomposing large organic molecules, and photosynthetic bacteria can also utilize small molecules such as ammonia nitrogen. In Comparative Example 1 (lacking sulfur-oxidizing bacteria), the peak hydrogen sulfide concentration reached 0.07 mg / L, and the peak concentrations of ammonia nitrogen and nitrite were more than twice that of Example 7, with a seedling survival rate of only 66%. This is because hydrogen sulfide not only directly poisons the seedlings but also inhibits the activity of nitrifying bacteria and blocks the nitrogen cycle. Even with graded application, the lack of key bacterial groups still leads to a vicious cycle of "sulfur-nitrogen compound pollution."

[0119] Table 3 shows that from Example 3 (basic process) to Example 7 (fully optimized process), the water quality indicators and breeding results showed a stable and significant gradual improvement.

[0120] It is evident that by standardizing the number of live bacteria, the accurate dosage and reliability of functional microbiota in each batch of products are ensured.

[0121] Example 5 (Addition of Sorbitol): Sorbitol, as an excellent stress protectant, forms a glassy protective layer on the surface of the bacteria during drying and storage, effectively reducing the mortality of live bacteria. This is directly related to the improved shelf life and application viability in Experiment 1, ensuring that a sufficient amount of highly viable bacteria are still introduced into the water body at the time of use.

[0122] Example 6 (Bran Acid Hydrolysis): Dilute sulfuric acid hydrolyzes bran at high temperature, causing partial breakdown of its fibrous structure, producing more pores and sugars that can be utilized by bacteria. This not only significantly increases the adsorption capacity of the carrier but also provides an initial "carbon and energy source" buffer for the immobilized bacterial community, which helps the community survive before being introduced into the water and recover quickly after introduction.

[0123] Example 7 (with the addition of L-ascorbic acid and sodium citrate): L-ascorbic acid is an antioxidant that can scavenge free radicals generated during drying and storage, protecting the bacterial cell membrane and DNA. Sodium citrate, as a chelating agent and pH buffer, can improve the bacterial microenvironment. Together with sorbitol, they form a complex protective system, providing more comprehensive protection and thus achieving optimal application results.

[0124] Experiment 3 I. Toxicological Tests Acute toxicity tests (LD50) were conducted on nitrifying bacteria (nitrite-oxidizing bacteria, nitrifying bacteria) and sulfur-oxidizing bacteria (thiobacillus thiooxidans, thiobacillus thiocyanate) in the formulation. 50 The pathogenicity test was conducted, and the test strains were all taken from the standard bacterial powder used in Examples 1-7. After verification, no contamination by other bacteria was found.

[0125] 1.1 Experimental Materials and Instruments Test strain: Nitrifying bacteria compound powder (live count 1.2 × 10⁻⁶) 9 CFU / g), sulfur-containing bacteria compound powder (live count 0.9×10⁻⁶) 9 (CFU / g), all referring to the batches of raw materials used in the examples; Experimental organisms: Healthy largemouth bass fry (body length 3±0.5cm, weight 0.5±0.1g), with a survival rate ≥95% after 7 days of temporary rearing, and fasted 24 hours before the experiment; Culture media and reagents: Nitrifying bacteria culture medium (peptone 5 g / L, sodium nitrite 1 g / L), sulfurizing bacteria culture medium (sodium thiosulfate 10 g / L, ammonium sulfate 2 g / L), sterile physiological saline, blood agar plates; Instruments: Biochemical incubator (36±1℃), electronic balance (0.001g), stereomicroscope, sterile operating table.

[0126] 1.2 Acute toxicity test (LD50) 50 (Measurement) The experimental design was as follows: Oral gavage was used. 1) Preparation of test substances: The two bacterial powders were diluted with sterile physiological saline to five concentration gradients (nitrifying bacteria: 1.0 × 10⁻⁶). 7 5.0×10 7 1.0×10 8 5.0×10 8 1.0×10 9 CFU / mL; sulfur bacteria: same concentration gradient), with 3 parallel groups per gradient, 10 fish fry per group, and a physiological saline blank control group; 2) Experimental procedure: The test substance was administered by gavage at a dose of 10 μL / g body weight once a day for 3 consecutive days. The mortality of the fish fry was observed within 7 days, and the symptoms of poisoning (such as abnormal activity, congestion of the body surface, etc.) were recorded. 3) Result Calculation: LD was calculated using the Bliss method. 50 If the mortality rate in the highest dose group is <50%, then LD is determined. 50 >1.0×10 10 CFU / kg body weight.

[0127] Experimental results: No mortality or abnormal symptoms were observed in largemouth bass fry at any concentration group. The survival rate of the blank control group was 100%. The LD50 of nitrifying bacteria and sulfur bacteria was [not specified]. 50 All > 1.0 × 10 10 CFU / kg body weight meets the requirements.

[0128] 1.3 Pathogenicity test Including broken skin irritation test, hemolysis test and challenge test, to comprehensively verify the pathogenicity of the strain: 1) Broken skin irritation test: Three healthy rabbits were selected, and "#"-shaped wounds were made on their backs after hair removal. A dressing containing 1.0×10⁻⁶ mg / L was applied. 9 Gauze containing CFU / mL bacterial suspension was removed after 4 hours, and the reaction was observed at 1 hour, 24 hours, and 48 hours. The results showed that the rabbit skin showed no erythema or edema, and the irritation index was 0, indicating no irritation. 2) Hemolysis test: Inoculate the bacterial culture into blood agar plates and incubate at 36±1℃ for 24h. If there is no hemolysis zone around the colony, consistent with the negative control, the hemolysis test is considered negative. 3) Challenge test: Largemouth bass fry were injected intraperitoneally with 1.0 × 10⁻⁶ ppm. 8 (CFU / tail), observed for 14 days, no disease or death, and no pathological changes were found in the liver, pancreas, kidneys and other tissues after autopsy, proving that it is non-pathogenic.

[0129] II. Heavy Metal Detection Test The final formulations of Examples 1-7 were tested for mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr), and arsenic (As).

[0130] 2.1 Test Materials and Methods Test samples: Finished formulations of Examples 1-7 (1 part each), each sample amount ≥50g; Pretreatment method: Microwave digestion was used. The sample was digested with nitric acid and hydrogen peroxide (4:1) and then the volume was adjusted to 25 mL. Detection instrument: Inductively coupled plasma mass spectrometry (ICP-MS), detection limit ≤0.001 mg / kg; Quality control: Conduct recovery rate verification, with a recovery rate range of 90%-110%.

[0131] 2.2 Test Results and Judgment The heavy metal content of all samples was below the limit standards (Hg≤5mg / kg, Cd≤10mg / kg, Pb≤100mg / kg, Cr≤150mg / kg, As≤75mg / kg). The specific test results are shown in Table 4 below: The experimental results show that the acute toxicity LD50 of the nitrifying and sulfurizing bacteria in the formulation of this invention is [not specified]. 50 >1.0×10 10 The CFU / kg body weight test showed no irritation, no hemolytic reaction, and no pathogenic effect, meeting safety requirements. The heavy metal content of the finished formulations in Examples 1-7 was all below the limit standard, with no risk of heavy metal pollution.

Claims

1. A water conditioning microbial preparation for breeding largemouth bass fry in land-based circular ponds, characterized in that, It is made by physically mixing the following standardized bacterial powders in parts by weight: 40-50 parts of Bacillus standard bacterial powder, 30-40 parts of photosynthetic bacterial standard bacterial powder, 10-15 parts of compound nitrifying bacterial standard bacterial powder and 5-10 parts of sulfur-oxidizing bacterial standard bacterial powder; The Bacillus standard powder is composed of Bacillus subtilis (Bacillus subtilis) Bacillus subtilis Standard bacterial powder and Bacillus licheniformis ( Bacillus licheniformis The standard bacterial powder is mixed in a 1:1 weight ratio; The compound nitrifying bacteria standard powder is made by mixing nitrite-oxidizing bacteria standard powder and nitrifying bacteria standard powder in a weight ratio of (60-70):(30-40); The sulfur-oxidizing bacteria standard powder is composed of *Thiobacillus thiooxidans* (… Acidithiobacillus thiooxidans Standard bacterial powder and Thiobacillus thiocyanate ( Thiobacillus thioparus The standard bacterial powder is mixed in a 1:1 weight ratio.

2. The water conditioning microbial preparation according to claim 1, characterized in that, The Bacillus subtilis standard powder, Bacillus licheniformis standard powder, photosynthetic bacteria standard powder, nitrifying bacteria standard powder, nitrifying bacteria standard powder, Thiobacillus thiooxidans standard powder, and Thiobacillus thiocyanate standard powder are all powdered products obtained by immobilization on an adsorption carrier and drying, and their moisture content is all less than 10%. The effective viable count of each standard powder is stably controlled at (0.8±0.2)×10⁻⁶. 9 Within the range of CFU / g.

3. The water conditioning microbial preparation according to claim 2, characterized in that, Its preparation method includes the following steps: S1. Raw material rehydration: Use raw materials with an effective viable bacteria count higher than 1.0 × 10⁻⁶. 9 Bacillus subtilis powder, Bacillus licheniformis powder, photosynthetic bacteria powder, nitrifying bacteria powder, nitrifying bacteria powder, Thiobacillus thiooxidans powder, and Thiobacillus thiocyanate powder, each with a CFU / g content, were rehydrated at a weight ratio of 1:(8-12) of bacterial powder to sterile water and stirred for 10-30 minutes to prepare a uniform bacterial solution. S2. Adsorption and immobilization: The bacterial solutions of each strain obtained in step S1 are stirred and adsorbed with sterile, dry composite carriers at a bacterial solution to carrier weight ratio of 1:(0.8-1.2) for 30-60 minutes to ensure that the bacterial solution fully wets the carrier; the composite carrier is made of wheat bran and zeolite powder mixed at a weight ratio of (6-8):(2-4); S3. Low-temperature drying: The adsorbed wet bacterial agents are dried at a low-temperature airflow temperature of 35-40℃ until the moisture content is less than 10%, and standardized intermediate bacterial powders of each bacterial strain are obtained. S4. Standardization and blending of intermediate microbial powder: S4.1 Perform viable cell counts on each batch of standardized intermediate bacterial powder. If the viable cell count is not within (0.8±0.2)×10⁻⁶, the count is considered incomplete. 9 Within the CFU / g range, adjustments are made by supplementing with a growth medium or high-viable-count intermediate bacterial powder until the effective viable count of each standard bacterial powder is stably controlled at (0.8±0.2)×10⁻⁶. 9 Within the range of CFU / g; S4.2 Primary mixing: Bacillus subtilis standard powder and Bacillus licheniformis standard powder are mixed at a weight ratio of 1:1 to obtain Bacillus standard powder; Thiobacillus thiooxidans standard powder and Thiobacillus thiocyanate standard powder are mixed at a weight ratio of 1:1 to obtain sulfur-oxidizing bacteria standard powder; Nitrifying bacteria standard powder and nitrifying bacteria standard powder are mixed at a weight ratio of (60-70):(30-40) to obtain compound nitrifying bacteria standard powder. S5. Final product mixing: The Bacillus standard powder, photosynthetic bacteria standard powder, compound nitrifying bacteria standard powder and sulfur bacteria standard powder obtained in step S4.2 are physically mixed at room temperature according to the weight parts described in claim 1, and then packaged to obtain the water conditioning microbial preparation.

4. The water conditioning microbial preparation according to claim 3, characterized in that, During the mixing process of the composite carrier in step S2, 5%-8% of sorbitol powder, accounting for the total weight of the composite carrier, is added simultaneously; the sorbitol powder is uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

5. The water conditioning microbial preparation according to claim 4, characterized in that, The bran of the composite carrier in step S2 is pre-hydrolyzed; the specific steps of the hydrolysis treatment are as follows: the bran is mixed with dilute sulfuric acid with a mass concentration of 3%-5% at a weight ratio of 1:(5-8), and stirred and reacted at a temperature of 100℃-105℃ for 30-40 minutes; after the reaction is completed, the mixture is filtered, and the pH of the filter residue is adjusted to 6.0-7.0 with sodium hydroxide solution, and then mixed with zeolite powder in proportion to form a composite carrier.

6. The water conditioning microbial preparation according to claim 4 or 5, characterized in that, In the process of mixing the composite carrier in step S2, 0.5%-1.5% of L-ascorbic acid and 0.2%-0.8% of sodium citrate, accounting for the total weight of the composite carrier, are also added; the L-ascorbic acid and sodium citrate are added at the same time as sorbitol powder and are uniformly dispersed in the composite carrier before the carrier and bacterial solution are mixed.

7. The application of a water-conditioning microbial preparation as described in any one of claims 1-5 in the breeding of largemouth bass fry in a land-based circular pond, characterized in that, The application is used to control the simultaneous accumulation of ammonia nitrogen, nitrite and hydrogen sulfide in water bodies used for breeding largemouth bass fry.

8. The application according to claim 7, characterized in that, The specific application method is as follows: Apply the solution according to the following standards at different growth stages when breeding largemouth bass fry in land-based circular ponds. Before application, the water must meet the following conditions: dissolved oxygen ≥ 6 mg / L and pH 7.5-8.

5. Parent rearing period: Apply once every 15 days, with a routine application rate of 0.8-1.0 g / m³ of water; 30-45 days before spawning and until the critical period of spawning, the initial application rate is adjusted to 1.5-2.0 g / m³ of water, and thereafter maintained at the routine rate. During the spawning and hatching period: apply once every 20 days, at a rate of 0.5-0.8 grams per cubic meter of water. During the fry's feeding period: Apply once every 25 days, with a standard application rate of 0.3-0.5 g / m³ of water. Within 3 days after the fry first appear to be swimming in groups and actively feeding on exogenous food in the water, adjust the initial application rate to 0.6-1.0 g / m³ of water, and maintain the standard application rate thereafter. During the juvenile rearing period: apply once every 20 days, at a rate of 0.5-0.8 grams per cubic meter of water. When applying, the preparation should be mixed with pool water at a weight ratio of 1:(10-15), and 1%-2% of the weight of brown sugar should be added. The mixture should be aerated and activated at 25-30℃ for 6-8 hours.

9. The application according to claim 8, characterized in that, When activating the formulation with pool water, add 0.5%-2% vitamin B by weight of the formulation. 12 powder; The vitamin B 12 The powder, brown sugar, preparation, and pool water are activated together for 6-8 hours; After activation, evenly sprinkle the solution throughout the pool between 5 PM and 7 PM daily. After sprinkling, turn off the circulation pump for 1-2 hours and continue oxygenation for more than 12 hours to maintain dissolved oxygen in the water > 4 mg / L.

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