A method for mixed crab and shellfish farming

Abstract

This method for mixed crab and shellfish aquaculture includes the following steps: S1 Water purification: Select a pond and add a compound microalgae ecological preparation to the aquaculture water. The compound microalgae ecological preparation contains microalgae to purify the water. S2 Seedling stocking: Place healthy crab seedlings and shellfish seedlings into the aquaculture water purified in step S1 and feed them for aquaculture. S21 Disease prevention and control: During the aquaculture period in step S2, add a copper sulfate and zinc sulfate mixture to the aquaculture water. The method is characterized in that: in step S21, the copper sulfate content in the copper sulfate and zinc sulfate mixture is 0.4–1 g / mL, and the zinc sulfate content is 0.8–2 g / mL. The dosage of the copper sulfate and zinc sulfate mixture is 0.03–0.045 mL per cubic meter of aquaculture water. At this dosage, the copper ion concentration in the aquaculture water will not inhibit the growth of microalgae and can significantly promote the synthesis of chlorophyll a in microalgae, thereby increasing the growth rate of microalgae.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal farming, and more particularly to a method for mixed crab and shellfish farming. Background Technology

[0002] The mud crab and the hard-shelled clam are two common aquatic economic animals. In the waters they live in, environmental factors such as water pollution and changes in water temperature lead to water quality deterioration and the breeding and spread of parasites. At the same time, mud crabs and hard-shelled clams usually feed on other organisms. If these organisms are infected with parasites, the mud crabs will also become infected by preying on these organisms. Mud crabs and hard-shelled clams also come into contact with other parasite host animals, such as aquatic insects or other aquatic organisms infected with parasites, and thus become infected.

[0003] To address the aforementioned technical problems, existing technologies, such as the Chinese invention application CN201210119189.7 (publication number CN103371114A) entitled "A Method for Raising River Crabs," discloses planting Elodea and Hydrilla verticillata at the bottom of the pond to improve water quality and immersing the crabs in a 5 PPM copper sulfate solution for 10-20 minutes to treat crab larvae disease. However, this method requires removing the diseased crabs from the pond. Since crabs are benthic animals, they sometimes hide in burrows in the soil at the bottom of the pond, making them difficult to spot and causing the diseased crabs to be missed. Releasing healthy crabs back into the pond can lead to secondary infections. Furthermore, this method cannot remove crab larvae in the form of planktonic organisms from the pond water. Generally, removing crab larvae from the pond water requires pond cleaning and disinfection before stocking. If copper sulfate solution is directly added to the pond water during the growth period of juvenile crabs, it will cause damage to the cell membranes and cell death of algae used to purify the water, thereby affecting the water quality.

[0004] Therefore, providing a cultivation method that can remove crab larvae from the water during the cultivation of mud crabs without damaging algal cells is an urgent problem to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for mixed crab and shellfish farming that can remove crab larvae from the water during the farming of mud crabs without damaging algal cells, in light of the above-mentioned existing technology.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: the method for mixed crab and shellfish farming includes the following steps:

[0007] S1 Water Purification: Select a pond and introduce water into it as aquaculture water. Add a compound microalgae ecological preparation to the aquaculture water. The compound microalgae ecological preparation contains microalgae to purify the water.

[0008] S2 Seedling stocking: Healthy crab seedlings and shellfish seedlings are placed into the aquaculture water purified in step S1 and fed with feed for aquaculture.

[0009] S21 Disease Prevention and Control: During the breeding period in step S2, add a mixture of copper sulfate and zinc sulfate to the breeding water. The mixture of copper sulfate and zinc sulfate contains copper sulfate and zinc sulfate.

[0010] The feature is that: in step S21, the content of copper sulfate in the copper sulfate and zinc sulfate mixture is 0.4-1 g / mL and the content of zinc sulfate is 0.8-2 g / mL, and the dosage of the copper sulfate and zinc sulfate mixture is 0.03-0.045 mL of copper sulfate and zinc sulfate mixture per cubic meter of aquaculture water.

[0011] Furthermore, the preparation method of the copper sulfate and zinc sulfate mixture includes the following steps:

[0012] 1) Weigh 2-3 parts of copper sulfate powder and add 3-5 parts of distilled water. Stir and mix thoroughly for 5-10 minutes. Then add 4-6 parts of zinc sulfate powder and continue stirring for 5-10 minutes until the copper sulfate powder and zinc sulfate powder are fully dissolved and mixed to obtain a copper sulfate and zinc sulfate mixture; one part is 10g.

[0013] 2) Pour the copper sulfate and zinc sulfate mixture obtained in step 2) into a sterilized container, seal it, and store it in a cool, dry place.

[0014] To further reduce the harmful effects of copper and zinc ions on microalgae, the feed in step S2 further includes a multi-nutrient compound preparation containing protein powder and bone meal. The bone meal contains tricalcium phosphate, bone glue, and fat, as well as water-soluble compounds of nitrogen, manganese, iron, and zinc, and collagen.

[0015] Among them, the amino acids and phosphates contained in the protein powder and bone meal in the multi-nutrient compound preparation can form stable complexes with copper and zinc. The formation of complexes will change the form of copper and zinc ions in water, causing them to change from free ion state to complex state. The complexes need a longer time to decompose or dissociate to release metal ions, and the stability of the complexes will also affect their decomposition rate, thus affecting the release of metal ions.

[0016] Protein powder also provides microalgae with essential amino acids to support the synthesis of proteins required for cell growth and division. At the same time, proteins participate in the metabolic processes of microalgae, which helps energy conversion and biosynthetic reactions.

[0017] Bone meal contains minerals such as tricalcium phosphate, which are essential for the growth of microalgae and help maintain the balance of the intracellular and extracellular environment. Trace elements such as manganese, iron, and zinc play important roles in the metabolism of microalgae, such as participating in enzyme activation and energy metabolism.

[0018] To further protect the microalgae cells, the multi-nutrient compound preparation also contains red palm oil, anthocyanins, and carnosine powder.

[0019] Red palm oil contains saturated and unsaturated fatty acids, as well as antioxidants such as provitamin A and vitamin E. Fatty acids provide energy for microalgae, supporting their energy needs during growth and reproduction.

[0020] Antioxidants such as provitamin A and vitamin E help protect microalgal cells from oxidative damage; anthocyanins are a powerful natural antioxidant.

[0021] Anthocyanins can scavenge free radicals and protect microalgal cells from oxidative stress, thereby maintaining normal cell growth and reproduction. Carnosine powder can inhibit lipid oxidation caused by free radicals and protect cell membranes from oxidative damage, thereby maintaining the health of microalgal cells.

[0022] Furthermore, the components and dosage of the multi-nutrient compound preparation applied per cubic meter of aquaculture water are as follows: 300-500 parts carnosine powder, 25-50 parts protein powder, 10-25 parts bone meal, 5-15 parts red palm oil, and 5-10 parts anthocyanins.

[0023] Furthermore, the preparation method of the multi-nutrient compound preparation includes the following steps:

[0024] 3) Weigh out 300-500 parts of carnosine powder, 25-50 parts of protein powder, 10-25 parts of bone meal, 5-15 parts of red palm oil, and 5-10 parts of anthocyanins, and grind them to obtain a multi-nutrient compound preparation powder.

[0025] 4) Add the multi-nutrient compound preparation powder from step 3) to 300-400 parts of water and stir thoroughly to obtain a multi-nutrient compound preparation suspension; one part is 1g;

[0026] 5) Allow the suspension of the multi-nutrient compound preparation from step 4) to stand and separate into layers, take out the supernatant after separation to obtain the multi-nutrient compound preparation, and seal and store the multi-nutrient compound preparation.

[0027] To maintain water cleanliness, the compound microalgae ecological preparation preferably contains Chlorella, Phaeodactylum tricornutum, Pavlova, red algae, and protozoa. Among these, Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae in the compound microalgae ecological preparation can absorb organic matter and nutrients in the water, regulate the nutrient concentration, prevent eutrophication, and maintain water cleanliness. Furthermore, the various microalgae and protozoa can compete with other microorganisms in the water for survival resources, inhibit the growth of harmful microorganisms, reduce the number of pathogenic microorganisms, and play a certain role in disease prevention, while also inhibiting aquatic plant and animal diseases.

[0028] Furthermore, the preparation method of the compound microalgae ecological agent includes the following steps:

[0029] 6) Prepare algae and protozoa: Chlorella solution with a mass concentration of 3-5 g / L, Phaeodactylum tricornutum solution with a mass concentration of 2-4 g / L, Pavlova solution with a mass concentration of 0.2-1 g / L, and red algae solution with a mass concentration of 0.2-1 g / L; and protozoa solution with a distribution density of 5-10 individuals / mL.

[0030] 7) Cultivating microalgae and protozoa: Chlorella, Brown's Finger Algae, Pavlova, and Red Algae were extracted from the Chlorella solution, Brown's Finger Algae solution, Pavlova solution, and Red Algae solution in step 6) and cultured separately in their respective algal culture media. The algal culture media contained the following components and were mixed with 1000 mL of distilled water: 100 g potassium nitrate powder, 20 g disodium EDTA powder, 10 g potassium hydrogen phosphate powder, 6 mg vitamin B1 powder, 50 μg vitamin B12 powder, 2.5 g ferric sulfate heptahydrate powder, and 0.25 g manganese sulfate powder.

[0031] Protozoa are extracted from the protozoan fluid in step 6) and placed in a protozoan culture medium for culture. The protozoan culture medium contains the following components and is mixed with 800 mL of distilled water: 15 g glucose powder, 8 g peptone powder, 5 g yeast extract powder, 4 g sodium chloride powder, and 2 mL of 1×PBS solution.

[0032] 8) Monitoring growth: Place the algal culture medium containing microalgae from step 7) in a shaking incubator and shake it. Set the temperature to 20–30°C and the light intensity to 100–200 μmol / m². 2 / s, with a photoperiod of 16-18 hours of light and 8-6 hours of darkness, and a pH value set at 6.0-9.0, when the cell number of each microalga reaches 10 6 Algal culture medium was obtained at CFU / mL;

[0033] The protozoan culture medium containing protozoa from step 7) is placed in a shaking incubator and shaken. The temperature is set to 15–25°C, the pH to 6.5–8.0, the light intensity to 1000–5000 lux, and oxygen is provided for continuous culture for 3–5 days. When the number of protozoa reaches 10… 3 CFU / mL was used to obtain the protozoan culture medium;

[0034] 9) Mixing microalgae and protozoa: Pour 500 mL of the algae culture medium from step 8) and 100 mL of the protozoa culture medium into a mixing container. At the same time, add 10-15 mL of lactic acid bacteria solution, 7-10 mL of phosphate adsorbent, 3-5 g of chrysanthemum powder, 3-5 g of sophora root powder, 5-10 mL of glycerol, 4-6 g of chitosan powder, and 3-5 mL of Tween 80 to the mixing container. Stir with a stirring rod for 5-10 minutes to mix thoroughly and evenly. After standing for 4-5 hours, remove the supernatant to obtain the compound microalgae ecological preparation.

[0035] 10) Colony harvesting and preparation of formulation: The compound microalgae ecological formulation from step 9) is bottled and sealed in a cool place.

[0036] To increase the yield of mud crabs and hard-shelled clams, the specific usage and dosage of the compound microalgae ecological preparation, copper sulfate and zinc sulfate mixture, and multi-nutrient compound preparation are as follows: The compound microalgae ecological preparation is applied once every 20-30 days before the seedling stocking in step S2 and during the breeding period, with each application being 0.11-0.15 mL / m³. 3 During the breeding period in step S2, a multi-nutrient compound preparation is administered once every 15 to 20 days, with each administration being 0.2 to 0.3 mL / m³. 3 During the breeding period in step S2, a mixture of copper sulfate and zinc sulfate is added once every 20 to 30 days, with each addition being 0.03 to 0.06 mL / m³. 3 This technical solution can comprehensively improve the average carapace width, weight, and plumpness of mud crabs and hard-shelled clams.

[0037] Compared with the prior art, the advantages of the present invention are as follows:

[0038] 1. The copper sulfate in the copper sulfate and zinc sulfate mixture can quickly penetrate the body wall of parasites such as crab worms and mud worms, destroy their cell structure, and cause the parasites to die; moreover, copper sulfate has a certain astringent effect, which can help injured tissues heal.

[0039] 2. When 0.03–0.045 mL of copper sulfate and zinc sulfate mixture is added per cubic meter of aquaculture water, the copper ion concentration in the aquaculture water will not inhibit the growth of microalgae. At the same time, since copper is a cofactor or activator of metabolic enzymes such as superoxide dismutase and cytochrome oxidase, and participates in key processes such as photosynthesis, respiration, and antioxidant defense, and can significantly promote the synthesis of chlorophyll a in microalgae, thereby increasing the growth rate of microalgae, at this dosage, copper sulfate and zinc sulfate mixture can be directly applied to the aquaculture water instead of removing mud crabs and hard-shelled clams to soak in the solution, saving time.

[0040] 3. Zinc in zinc sulfate is a component or activator of carbonic anhydrase, RNA polymerase, etc., which is essential for life activities such as protein synthesis, DNA replication and repair. It helps maintain the health of the shells of mud crabs and hard-shelled clams and prevents secondary infection. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the embodiments.

[0042] Example 1: Effects of different dosages of copper sulfate and zinc sulfate mixture on the growth of Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae.

[0043] I. Preparation of a mixture of copper sulfate and zinc sulfate

[0044] The preparation method of the copper sulfate and zinc sulfate mixture in this embodiment includes the following steps:

[0045] 1) Weigh 25g of copper sulfate powder and add 40g of distilled water. Stir thoroughly for 5-10 minutes, then add 50g of zinc sulfate powder and continue stirring for 5-10 minutes until the copper sulfate powder and zinc sulfate powder are fully dissolved and mixed to obtain a copper sulfate and zinc sulfate mixture (one portion is 10g).

[0046] 2) Pour the copper sulfate and zinc sulfate mixture obtained in step 1) into a sterilized container, seal it, and store it in a cool, dry place.

[0047] II. 0.06 mL / m 3 Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0048] Experimental equipment: 24 x 1m 3 The following components were used in this invention: a square plastic bucket, 30 tons of seawater, 4 bottles of microalgae culture medium, 24 incandescent lamps, 1 aerator pump + 24 air heads, 4 bottles of algal solution (Chlorella vulgaris, Phaeodactylum tricornutum, Pavlova spp., and red algae), and 0.5L of a copper sulfate and zinc sulfate mixture solution. All components in this invention were commercially available.

[0049] Experimental steps: The experiment first uses a volume of 1m³3 Plastic buckets were used to cultivate Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae. During the cultivation, incandescent lamps and aerators were used for supplemental lighting and oxygenation. Three parallel experimental groups and a control group were set up for each type of microalgae (Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae). Stress experiments were conducted on each type of microalgae using a mixture of 0.06 mL of copper sulfate and zinc sulfate. The experimental results are shown in Table 1.

[0050] Table 10. Effects of a 0.06 mL dose of a copper sulfate and zinc sulfate mixture on various microalgae.

[0051]

[0052] Experimental results showed that excessive amounts of the copper sulfate and zinc sulfate mixture inhibited microalgal growth or caused microalgal death. High doses of the copper sulfate and zinc sulfate mixture had significant toxic effects on microalgal cells. Copper sulfate, a heavy metal salt, at high concentrations, can damage cell membrane structures, leading to leakage of cell contents and ultimately cell death. Simultaneously, copper ions can bind to intracellular proteins and enzymes, inhibiting their normal functions, including key metabolic processes such as photosynthesis and respiration. While zinc sulfate is an essential trace element for many organisms, excessive amounts can also cause similar toxic effects, such as disrupting intracellular ion balance and damaging enzyme activity. Different species of microalgae exhibit varying sensitivities to high doses of the copper sulfate and zinc sulfate mixture due to differences in their physiological characteristics and cell structures. However, regardless of the species, all microalgae exposed to excessively high concentrations of the mixture will experience similar toxic reactions, ultimately leading to cell death.

[0053] III. 0.02 mL / m 3 Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0054] Experimental equipment: 24 x 1m 3 Square plastic buckets, 30 tons of seawater, 4 bottles of microalgae culture medium, 24 incandescent lamps, 1 aerator pump + 24 air heads, 4 bottles of algae solution (Chlorella vulgaris, Phaeodactylum tricornutum, Pavlova spp., and red algae), and 0.5L of copper sulfate and zinc sulfate mixture solution;

[0055] Experimental steps: The experiment first uses a volume of 1m³ 3 Plastic buckets were used to cultivate Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae. During the cultivation, incandescent lamps and aerators were used for supplemental lighting and oxygenation. Three parallel experimental groups and a control group were set up for each type of microalgae (Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae). Stress experiments were conducted on each type of microalgae using a mixture of 0.02 mL of copper sulfate and zinc sulfate. The experimental results are shown in Table 2.

[0056] Table 2. Stress experiments on various microalgae with a copper sulfate and zinc sulfate mixture at a dose of 0.02 mL.

[0057]

[0058] Experimental results showed that at low doses, the combined copper sulfate and zinc sulfate had little or no effect on the growth and metabolism of microalgae. The effects of copper sulfate and zinc sulfate on microalgae are generally concentration-dependent. At low concentrations, these chemicals are insufficient to significantly affect the physiological functions of microalgae. For example, as an algaecide, the algicidal effect of copper sulfate often increases with increasing concentration. At low doses, the toxicity of copper ions is insufficient to interfere with algal photosynthesis or other key metabolic processes. Copper sulfate mainly interferes with the photosynthetic reaction system of algae through the toxicity of copper ions, preventing them from performing photosynthesis normally and causing them to gradually die. However, at low doses, the concentration of copper ions is insufficient to cause fatal damage to algal cells, thus the effect is small or nonexistent. Although zinc sulfate also has some toxicity, its main function is not to directly kill algae, but rather to affect aquatic organisms through other mechanisms (such as regulating osmotic pressure and astringing the epidermis). At low doses, these effects are not significant. Microalgae exhibit a certain degree of tolerance to environmental stress within a specific range. At low doses, they can cope with these stresses through their own physiological regulatory mechanisms, thereby maintaining normal growth and metabolic activities. Meanwhile, the toxicity of copper sulfate and zinc sulfate is affected by various environmental factors, including water temperature, pH, hardness, and organic matter content. Under specific environmental conditions (such as low temperature, high pH, ​​or high hardness water), the toxicity of these chemicals decreases, thus reducing their impact on microalgae.

[0059] IV. 0.03~0.05mL / m 3 Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0060] 4.10.04mL / m 3 Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0061] Experimental equipment: 24 x 1m 3 Square plastic buckets, 30 tons of seawater, 4 bottles of microalgae culture medium, 24 incandescent lamps, 1 aerator pump + 24 air heads, 4 bottles of algae solution (Chlorella vulgaris, Phaeodactylum tricornutum, Pavlova spp., and red algae), and 0.5L of copper sulfate and zinc sulfate mixture solution;

[0062] The experiment first used a volume of 1m³ 3Plastic buckets were used to cultivate Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae. During the cultivation, incandescent lamps and aerators were used to supplement light and oxygen. Three parallel experimental groups and a control group were set up for each type of microalgae (Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae). Stress experiments were conducted on each type of microalgae using a mixture of 0.04 mL of copper sulfate and zinc sulfate. The experimental results are shown in Table 3.

[0063] Table 3. Stress experiments on various microalgae with a copper sulfate and zinc sulfate mixture at a dose of 0.04 mL.

[0064]

[0065] 4.20.03mL / m 3 Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0066] Experimental equipment: 24 x 1m 3 Square plastic buckets, 30 tons of seawater, 4 bottles of microalgae culture medium, 24 incandescent lamps, 1 aerator pump + 24 air heads, 4 bottles of algae solution (Chlorella vulgaris, Phaeodactylum tricornutum, Pavlova spp., and red algae), and 0.5L of copper sulfate and zinc sulfate mixture solution;

[0067] The experiment first used a volume of 1m³ 3 Plastic buckets were used to cultivate Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae. During the cultivation, incandescent lamps and aerators were used to supplement light and oxygen. Three parallel experimental groups and a control group were set up for each type of microalgae (Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae). Stress experiments were conducted on each type of microalgae using a mixture of 0.03 mL of copper sulfate and zinc sulfate. The experimental results are shown in Table 4.

[0068] Table 4. Stress experiments on various microalgae with a copper sulfate and zinc sulfate mixture at a dose of 0.03 mL.

[0069]

[0070] The results showed that *Chlorella vulgaris* exhibited the highest cell number and density under treatment with a 0.03 mL dose of the copper sulfate and zinc sulfate mixture, followed by *Phaeodactylum tricornutum*, *Phaeodactylum pulmonale*, and red algae. *Chlorella vulgaris* also showed the highest chlorophyll a content and growth rate, indicating that this dose had a good promoting effect on the growth and metabolism of *Chlorella vulgaris*. The cell number, cell density, chlorophyll a content, and growth rate of all microalgae were increased, demonstrating that the 0.03 mL dose of the copper sulfate and zinc sulfate mixture had a positive effect on promoting microalgal growth. The data indicate that the 0.03 mL dose of the copper sulfate and zinc sulfate mixture can effectively improve the growth performance of microalgae, especially showing the most significant effect in *Chlorella vulgaris*.

[0071] 4.30.05mL / m 3Stress experiments on various microalgae caused by a mixture of copper sulfate and zinc sulfate at different dosages.

[0072] Experimental equipment: 24 x 1m 3 Square plastic buckets, 30 tons of seawater, 4 bottles of microalgae culture medium, 24 incandescent lamps, 1 aerator pump + 24 air heads, 4 bottles of algae solution (Chlorella vulgaris, Phaeodactylum tricornutum, Pavlova spp., and red algae), and 0.5L of copper sulfate and zinc sulfate mixture solution;

[0073] The experiment first used a volume of 1m³ 3 Plastic buckets were used to cultivate Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae. During the cultivation, incandescent lamps and aerators were used to supplement light and oxygen. Three parallel experimental groups and a control group were set up for each type of microalgae (Chlorella, Phaeodactylum tricornutum, Pavlova, and red algae). Stress experiments were conducted on each type of microalgae using a mixture of 0.05 mL of copper sulfate and zinc sulfate. The experimental results are shown in Table 3.

[0074] Table 5. Stress experiments on various microalgae with a copper sulfate and zinc sulfate mixture at a dose of 0.05 mL.

[0075]

[0076] The results showed that a 0.05 mL dose of the copper sulfate and zinc sulfate mixture further increased the cell number, cell density, chlorophyll a content, and growth rate of various microalgae. *Chlorella vulgaris* still exhibited the best growth performance, suggesting that a 0.05 mL dose may be a better choice for *Chlorella vulgaris*. Various indicators of *Phaeodactylum tricornutum*, *Pavlova*, and red algae also showed improvement, indicating that this dose also had a certain promoting effect on these three microalgae. Compared to a 0.03 mL dose, a 0.05 mL dose of the copper sulfate and zinc sulfate mixture was more effective in promoting microalgal growth, especially in increasing cell number and growth rate. When the dose of the copper sulfate and zinc sulfate mixture was increased to 0.05 mL, the growth and metabolic activities of all microalgal species were further enhanced, and cell number, cell density, chlorophyll a content, and growth rate all reached higher levels, showing a good response of microalgae to nutrients at this dose. However, monitoring is necessary to avoid potential toxic effects from further increases in dosage.

[0077] 4.4 Discussion of Experimental Results

[0078] The experimental results from sections 4.1 to 4.3 of this embodiment show that when the dosage of the copper sulfate and zinc sulfate mixture is within a suitable range, i.e., 0.03–0.05 mL / m 3Copper and zinc sulfate can promote the growth, metabolism, and reproduction of microalgae. Copper is a cofactor or activator of many enzymes (such as superoxide dismutase and cytochrome oxidase), participating in key processes such as photosynthesis, respiration, and antioxidant defense. Zinc is a component or activator of many enzymes (such as carbonic anhydrase and RNA polymerase), crucial for life activities such as protein synthesis, DNA replication, and repair. When the dosage of copper sulfate and zinc sulfate is within an appropriate range, it can effectively promote the activity of related enzymes and the smoothness of metabolic pathways within microalgal cells, thereby improving physiological indicators such as growth rate, photosynthetic efficiency, biomass accumulation, and stress resistance. In addition, appropriate micronutrient concentrations also help maintain ion balance and osmotic pressure stability within microalgal cells, protecting cells from damage caused by external environmental stresses.

[0079] In summary, the effects of the copper sulfate and zinc sulfate mixture on microalgal growth and metabolism depend on the dosage. Excessive dosage can lead to toxicity and kill microalgae; excessively low dosage has no significant effect; while appropriate dosage can significantly promote microalgal growth and metabolic activity.

[0080] Through multiple experiments with different dosages, results were obtained for different dosage ranges, revealing that microalgae thrive at dosages ranging from 0.03 to 0.045 mL / m³. 3 The combination of copper sulfate and zinc sulfate can promote the growth and metabolism of microalgae. This analysis aims to scientifically explore the potential stress effects of this combination on microalgae at different concentration gradients, and the specific manifestations of these effects in microalgal physiological metabolism, photosynthetic efficiency, and cell integrity, thereby providing a solid experimental basis for the stress experiments of copper sulfate and zinc sulfate combination on microalgae.

[0081] Example 2: Effects of different contents of compound microalgae ecological preparation, multi-nutrient compound preparation, and copper sulfate and zinc sulfate mixture on the growth performance and economic value of mud crab and hard-shelled clam during actual aquaculture.

[0082] I. Preparation of Compound Microalgae Ecological Preparations, Multi-nutrient Compound Preparations, and Copper Sulfate and Zinc Sulfate Mixtures

[0083] 1.1 Sources of Experimental Materials: Various microalgae and protozoa were purchased from DIC Corporation (Japan), Yunnan Green A Biotechnology Co., Ltd., and Fuqing Xindaze Spirulina Co., Ltd., respectively. Carnosine powder was purchased from Hubei Weideli Chemical Reagent Co., Ltd., protein powder from Xiamen Yangzhen Health Food Co., Ltd., bone meal from Shandong Maina New Materials Co., Ltd., red palm oil from Jinan Ausli Chemical Co., Ltd., and anthocyanins (antioxidant) from Hebei Runbu Biotechnology Co., Ltd.

[0084] 1.2 Experimental Procedure

[0085] The preparation method of the multi-nutrient compound preparation in this embodiment includes the following steps:

[0086] 3) Weigh out 400 parts of carnosine powder, 37.5 parts of protein powder, 17.5 parts of bone meal, 10 parts of red palm oil, and 7.5 parts of anthocyanins, grind them to obtain a multi-nutrient compound preparation powder.

[0087] 4) Add the multi-nutrient compound preparation powder from step 3) of this embodiment to 350 parts of water and stir thoroughly to obtain a multi-nutrient compound preparation suspension; one part is 1g;

[0088] 5) Allow the suspension of the multi-nutrient compound preparation from step 4) of this embodiment to stand and separate into layers. Take out the supernatant after separation to obtain the multi-nutrient compound preparation. Seal and store the multi-nutrient compound preparation.

[0089] The preparation method of the compound microalgae ecological agent in this embodiment includes the following steps:

[0090] 6) Prepare algae and protozoa: Chlorella solution with a mass concentration of 4 g / L, Phaeodactylum tricornutum solution with a mass concentration of 3 g / L, Phaeodactylum pulchella solution with a mass concentration of 0.6 g / L, red algae solution with a mass concentration of 0.6 g / L, and protozoa solution with a distribution density of 7.5 protozoa / mL.

[0091] 7) Cultivating microalgae and protozoa: Chlorella, Brown's Finger Algae, Pavlova, and Red Algae were extracted from the Chlorella, Brown's Finger Algae, Pavlova, and Red Algae solutions from step 6) and cultured separately in their respective algal culture media. The algal culture media contained the following components and were mixed with 1000 mL of distilled water: 100 g potassium nitrate powder, 20 g disodium EDTA powder, 10 g potassium hydrogen phosphate powder, 6 mg vitamin B1 powder, 50 μg vitamin B12 powder, 2.5 g ferric sulfate heptahydrate powder, and 0.25 g manganese sulfate powder.

[0092] Protozoa were extracted from the protozoan fluid in step 6) and placed in a protozoan culture medium for culture. The protozoan culture medium contained the following components and was mixed with 800 mL of distilled water: 15 g glucose powder, 8 g peptone powder, 5 g yeast extract powder, 4 g sodium chloride powder, and 2 mL of 1×PBS solution.

[0093] 8) Monitoring growth: Place the algal culture medium containing microalgae from step 7) in a shaking incubator and shake it. Set the temperature to 25℃ and the light intensity to 150 μmol / m². 2 / s, with a photoperiod of 17 hours of light and 7 hours of darkness, and a pH set at 7.5, when the cell count of each microalgae reaches 10 6 Algal culture medium was obtained at CFU / mL;

[0094] Place the protozoan-containing culture medium from step 7) in a shaking incubator and shake it. Set the temperature to 20℃, pH to 7, light intensity to 3000 lux, and provide oxygen. Incubate continuously for 4 days until the number of protozoa reaches 10. 3 CFU / mL was used to obtain the protozoan culture medium;

[0095] 9) Mixing microalgae and protozoa: Pour 500 mL of the algae culture medium from step 8) and 100 mL of the protozoa culture medium into a mixing container. At the same time, add 12.5 mL of lactic acid bacteria solution, 8.5 mL of phosphate adsorbent, 4 g of chrysanthemum powder, 4 g of sophora flavescens powder, 7.5 mL of glycerol, 5 g of chitosan powder, and 4 mL of Tween-80 solution to the mixing container. Stir with a stir bar for 7.5 minutes to mix thoroughly and evenly. After standing for 4.5 hours, remove the supernatant to obtain the compound microalgae ecological preparation.

[0096] 10) Colony harvesting and preparation of formulation: The compound microalgae ecological formulation from step 9) is bottled and sealed in a cool place.

[0097] The composition and preparation method of the copper sulfate and zinc sulfate mixture in this embodiment are the same as those in the first part of Example 1.

[0098] II. Experimental Group 1: 0.08 mL / m 3 Compound microalgae ecological preparation, 0.1 mL / m 3 Multi-nutrient compound preparation with 0.02mL / m 3 Analysis of the effects of copper sulfate and zinc sulfate mixture on the growth and yield of mud crab and hard-shelled clam.

[0099] 2.1 Experimental Site: From April to October 2021, the culture of mud crabs and hard-shelled clams was carried out at Dongying Yuwo Biotechnology Co., Ltd. in Shandong Province, with good results. The culture pond was rectangular in shape, with an area of ​​10 mu (approximately 6,667 square meters). In the middle area of ​​the pond, a net was used to separate the mud crab culture area from the hard-shelled clam culture area. Hard-shelled clams were cultured inside the net, and mud crabs were cultured outside the net.

[0100] 2.2 Experimental Procedure:

[0101] 12) Apply the compound microalgae ecological preparation once every 20 days before stocking and again every 25 days during the cultivation period, with each application being 0.08 mL / m². 3 It is used to decompose ammonia nitrogen, nitrite and sulfide in aquaculture water, while inhibiting the growth and reproduction of pathogenic microorganisms;

[0102] 13) During the breeding period, administer a multi-nutrient compound preparation once every 18 days, with each administration being 0.1 mL / m³. 3 It is used to provide nutrients such as protein, fat, amino acids, calcium, and phosphorus to promote the growth of mud crabs and hard-shelled clams and improve their resistance to adverse conditions.

[0103] 14) During the breeding period, administer a mixture of copper sulfate and zinc sulfate once every 25 days, with each administration being 0.02 mL / m³. 3 It is used to remove parasites such as crab larvae from the body of mud crabs and mud worms from the body of hard-shelled clams.

[0104] After the breeding period, growth indicators and yields of mud crabs and hard-shelled clams were calculated.

[0105] 2.3 Experimental Results:

[0106] After the aquaculture period ended in mid-October, 5300 kg of hard-shelled clams were harvested, and 234 kg of mud crabs were harvested on October 16; other relevant indicators are shown in Table 6.

[0107] Table 6 0.08 mL / m 3 Compound microalgae ecological preparation, 0.1 mL / m 3 Multi-nutrient compound preparation with 0.02mL / m 3 Effects of copper sulfate and zinc sulfate mixture on the growth and economic benefits of mud crab and hard-shelled clam

[0108]

[0109]

[0110] III. Experimental Group 2: 0.25 mL / m 3 Compound microalgae ecological preparation, 0.5 mL / m 3 Multi-nutrient compound preparation with 0.08 mL / m 3 Analysis of the effects of copper sulfate and zinc sulfate mixture on the growth and yield of mud crab and hard-shelled clam.

[0111] 3.1 Experimental Site: From April to October 2021, the culture of mud crabs and hard-shelled clams was carried out at Dongying Yuwo Biotechnology Co., Ltd. in Shandong Province, with good results. The culture pond was rectangular in shape, with an area of ​​10 mu (approximately 6,667 square meters). In the middle area of ​​the pond, a net was used to separate the mud crab culture area from the hard-shelled clam culture area. Hard-shelled clams were cultured inside the net, and mud crabs were cultured outside the net.

[0112] 3.2 Experimental Procedure:

[0113] 15) Apply the compound microalgae ecological preparation once every 20-30 days before stocking and during the cultivation period, with each application being 0.25 mL / m². 3 It is used to decompose ammonia nitrogen, nitrite and sulfide in aquaculture water, while inhibiting the growth and reproduction of pathogenic microorganisms;

[0114] 16) During the breeding period, administer a multi-nutrient compound preparation once every 15-20 days, with each administration being 0.5 mL / m³.3 It is used to provide nutrients such as protein, fat, amino acids, calcium, and phosphorus to promote the growth of mud crabs and hard-shelled clams and improve their resistance to adverse conditions.

[0115] 17) During the breeding period, administer a mixture of copper sulfate and zinc sulfate every 20-30 days, with each administration being 0.08 mL / m³. 3 It is used to remove parasites such as crab larvae from the body of mud crabs and mud worms from the body of hard-shelled clams.

[0116] After the breeding period, growth indicators and yields of mud crabs and hard-shelled clams were calculated.

[0117] 3.3 Experimental results: 5100 kg of hard-shelled clams were harvested after the aquaculture period ended in mid-October, and 243 kg of mud crabs were harvested on October 16; other relevant indicators are shown in Table 7.

[0118] Table 7 0.25mL / m 3 Compound microalgae ecological preparation, 0.5 mL / m 3 Multi-nutrient compound preparation with 0.08 mL / m 3 Effects of copper sulfate and zinc sulfate mixture on the growth and economic benefits of mud crab and hard-shelled clam

[0119] Blue crab hard-shelled clams Average total nail width / cm 8.38 4.1 Average body weight / g 270 37 <![CDATA[Condition factor g / cm 3 > 0.13 0.006 Production / kg 243 5100 Revenue / yuan 44530 35730 Cost / yuan 32000 30000 Profit / Yuan 12530 5730 Profit per mu (yuan) 1253 573

[0120] IV. Experimental Group 3: 0.11 mL / m 3 Compound microalgae ecological preparation, 0.2 mL / m 3 Multi-nutrient compound preparation with 0.03 mL / m 3 Analysis of the effects of copper sulfate and zinc sulfate mixture on the growth and yield of mud crab and hard-shelled clam.

[0121] 4.1 Experimental Site: From April to October 2021, the culture of mud crabs and hard-shelled clams was carried out at Dongying Yuwo Biotechnology Co., Ltd. in Shandong Province, with good results. The culture pond was rectangular in shape, with an area of ​​10 mu (approximately 6,667 square meters). In the middle area of ​​the pond, a net was used to separate the mud crab culture area from the hard-shelled clam culture area. Hard-shelled clams were cultured inside the net, and mud crabs were cultured outside the net.

[0122] 4.2 Experimental Procedure:

[0123] 18) Apply the compound microalgae ecological preparation once every 20 days before stocking and again every 25 days during the cultivation period, with each application being 0.11 mL / m². 3 It is used to decompose ammonia nitrogen, nitrite and sulfide in aquaculture water, while inhibiting the growth and reproduction of pathogenic microorganisms;

[0124] 19) During the breeding period, administer a multi-nutrient compound preparation once every 18 days, with each administration being 0.2 mL / m². 3It is used to provide nutrients such as protein, fat, amino acids, calcium, and phosphorus to promote the growth of mud crabs and hard-shelled clams and improve their resistance to adverse conditions.

[0125] 20) During the breeding period, administer a mixture of copper sulfate and zinc sulfate once every 25 days, with each administration being 0.03 mL / m³. 3 It is used to remove parasites such as crab larvae from the body of mud crabs and mud worms from the body of hard-shelled clams.

[0126] After the breeding period, growth indicators and yields of mud crabs and hard-shelled clams were calculated.

[0127] 4.3 Experimental Results:

[0128] After the aquaculture period ended in mid-October, 9,500 kg of hard-shelled clams were harvested, and 463 kg of mud crabs were harvested on October 16; other relevant indicators are shown in Table 8.

[0129] Table 8 0.11 mL / m 3 Compound microalgae ecological preparation, 0.2 mL / m 3 Multi-nutrient compound preparation with 0.03 mL / m 3 Effects of copper sulfate and zinc sulfate mixture on the growth and economic benefits of mud crab and hard-shelled clam

[0130] Blue crab hard-shelled clams Average total nail width / cm 12.1 6.4 Average body weight / g 382 70 <![CDATA[Condition factor g / cm 3 > 0.203 0.012 Production / kg 463 9500 Revenue / yuan 83340 57850 Cost / yuan 32000 30000 Profit / Yuan 51340 27850 Profit per mu (yuan) 5134 2785

[0131] V. Experimental Group 4: 0.15 mL / m 3 Compound microalgae ecological preparation, 0.3 mL / m 3 Multi-nutrient compound preparation with 0.06 mL / m 3 Analysis of the effects of copper sulfate and zinc sulfate mixture on the growth and yield of mud crab and hard-shelled clam.

[0132] 5.1 Experimental Site: From April to October 2021, the culture of mud crabs and hard-shelled clams was carried out at Dongying Yuwo Biotechnology Co., Ltd. in Shandong Province, with good results. The culture pond was rectangular in shape, with an area of ​​10 mu (approximately 6,667 square meters). In the middle area of ​​the pond, a net was used to separate the mud crab culture area from the hard-shelled clam culture area. Hard-shelled clams were cultured inside the net, and mud crabs were cultured outside the net.

[0133] 5.2 Experimental Procedure:

[0134] 21) Apply the compound microalgae ecological preparation once every 25 days before stocking and again during the cultivation period, with each application being 0.15 mL / m². 3 It is used to decompose ammonia nitrogen, nitrite and sulfide in aquaculture water, while inhibiting the growth and reproduction of pathogenic microorganisms;

[0135] 22) During the breeding period, administer a multi-nutrient compound preparation once every 18 days, with each administration being 0.3 mL / m².3 It is used to provide nutrients such as protein, fat, amino acids, calcium, and phosphorus to promote the growth of mud crabs and hard-shelled clams and improve their resistance to adverse conditions.

[0136] 23) During the breeding period, administer a mixture of copper sulfate and zinc sulfate once every 25 days, with each administration being 0.06 mL / m³. 3 It is used to remove parasites such as crab larvae from the body of mud crabs and mud worms from the body of hard-shelled clams.

[0137] After the breeding period, growth indicators and yields of mud crabs and hard-shelled clams were calculated.

[0138] 5.3 Experimental Results:

[0139] After the aquaculture period ended in mid-October, 11,200 kg of hard-shelled clams were harvested, and 478 kg of mud crabs were harvested on October 16; other relevant indicators are shown in Table 9.

[0140] Table 9 0.15mL / m 3 Compound microalgae ecological preparation, 0.3 mL / m 3 Multi-nutrient compound preparation with 0.06 mL / m 3 Effects of copper sulfate and zinc sulfate mixture on the growth and economic benefits of mud crab and hard-shelled clam

[0141] Blue crab hard-shelled clams Average body length / cm 12.4 6.7 Average body weight / g 392 73 <![CDATA[Condition factor g / cm 3 > 0.218 0.013 Production / kg 478 11200 Revenue / yuan 86040 59350 Cost / yuan 32000 30000 Profit / Yuan 54540 30350 Profit per mu (yuan) 5454 3035

[0142] VI. Experimental Group 5: 0.13 mL / m 3 Compound microalgae ecological preparation, 0.25 mL / m 3 Multi-nutrient compound preparation with 0.04 mL / m 3 Analysis of the effects of copper sulfate and zinc sulfate mixture on the growth and yield of mud crab and hard-shelled clam.

[0143] 6.1 Experimental Site: From April to October 2021, the culture of mud crabs and hard-shelled clams was carried out at Dongying Yuwo Biotechnology Co., Ltd. in Shandong Province, with good results. The culture pond was rectangular in shape, with an area of ​​10 mu (approximately 6,667 square meters). In the middle area of ​​the pond, a net was used to separate the mud crab culture area from the hard-shelled clam culture area. Hard-shelled clams were cultured inside the net, and mud crabs were cultured outside the net.

[0144] 6.2 Experimental Procedure:

[0145] 24) Apply the compound microalgae ecological preparation once every 25 days before stocking and again during the cultivation period, with each application being 0.13 mL / m². 3 It is used to decompose ammonia nitrogen, nitrite and sulfide in aquaculture water, while inhibiting the growth and reproduction of pathogenic microorganisms;

[0146] 25) During the breeding period, administer a multi-nutrient compound preparation once every 18 days, with each administration being 0.25 mL / m². 3 It is used to provide nutrients such as protein, fat, amino acids, calcium, and phosphorus to promote the growth of mud crabs and hard-shelled clams and improve their resistance to adverse conditions.

[0147] 26) During the breeding period, administer a mixture of copper sulfate and zinc sulfate once every 25 days, with each administration being 0.04 mL / m³. 3 It is used to remove parasites such as crab larvae from the body of mud crabs and mud worms from the body of hard-shelled clams.

[0148] After the breeding period, growth indicators and yields of mud crabs and hard-shelled clams were calculated.

[0149] 6.3 Experimental Results:

[0150] After the aquaculture period ended in mid-October, 11,350 kg of hard-shelled clams were harvested, and 482 kg of mud crabs were harvested on October 16; other relevant indicators are shown in Table 10.

[0151] Table 10 0.13 mL / m 3 Compound microalgae ecological preparation, 0.25 mL / m 3 Multi-nutrient compound preparation with 0.04 mL / m 3 Effects of copper sulfate and zinc sulfate mixture on the growth and economic benefits of mud crab and hard-shelled clam

[0152] Blue crab hard-shelled clams Average body length / cm 12.5 6.8 Average body weight / g 396 76 fatness g / cm3 0.22 0.014 Production / kg 482 11350 Revenue / yuan 86620 60130 Cost / yuan 32000 30000 Profit / Yuan 54620 30130 Profit per mu (yuan) 5460 3013

[0153] A comprehensive analysis of the effects of different doses of compound microalgae ecological preparations, multi-nutrient compound preparations, and copper sulfate and zinc sulfate mixtures on the growth and yield of mud crabs and hard-shelled clams in the five experimental groups of this embodiment yields the following conclusions:

[0154] In experimental groups 3-5, the growth performance and yield of *Scylla serrata* and *Clams scutellariae* were superior to those in experimental groups 1 and 2, indicating that the optimal dosage of the compound microalgae ecological preparation was 0.11-0.15 mL / m³. 3 The dosage of the multi-nutrient compound preparation is 0.2–0.3 mL / m² per administration. 3 The dosage of the copper sulfate and zinc sulfate mixture is 0.03–0.06 mL / m² each time. 3 Within a certain range, the growth of mud crabs and hard-shelled clams can be promoted, while growth is slower below or above this range.

[0155] Example 3: First endpoint value of the time interval between the administration of the compound microalgae ecological preparation, the multi-nutrient compound preparation, and the copper sulfate and zinc sulfate mixture.

[0156] The time intervals for the administration of experimental steps 1-5 in Example 2 can also be changed to the following time intervals.

[0157] Apply the compound microalgae ecological preparation once every 20 days before stocking and again during the breeding period.

[0158] During the breeding period, administer a multi-nutrient compound preparation once every 15 days.

[0159] During the breeding period, a mixture of copper sulfate and zinc sulfate is applied once every 20 days.

[0160] Example 4: Second endpoint value of the time interval between the administration of the compound microalgae ecological preparation, the multi-nutrient compound preparation, and the copper sulfate and zinc sulfate mixture.

[0161] The time intervals for the administration of experimental steps 1-5 in Example 2 can also be changed to the following time intervals.

[0162] Apply the compound microalgae ecological preparation once every 30 days before stocking and again during the breeding period;

[0163] During the breeding period, administer a multi-nutrient compound preparation once every 20 days.

[0164] During the breeding period, a mixture of copper sulfate and zinc sulfate is applied once every 30 days.

[0165] Example 5: Preparation method of copper sulfate and zinc sulfate mixture; Step parameters; First endpoint value

[0166] The preparation method of the copper sulfate and zinc sulfate mixture in Example 1 can also be modified to the following steps:

[0167] 1) Weigh 2 parts of copper sulfate powder and add 3 parts of distilled water. Stir thoroughly for 5-10 minutes. Then add 5 parts of zinc sulfate powder and continue stirring for 5-10 minutes until the copper sulfate powder and zinc sulfate powder are fully dissolved and mixed to obtain a copper sulfate and zinc sulfate mixture (one part is 10g).

[0168] 2) Pour the copper sulfate and zinc sulfate mixture obtained in step 1) into a sterilized container, seal it, and store it in a cool, dry place.

[0169] Example 6: Preparation method of copper sulfate and zinc sulfate mixture; step parameters; second endpoint value.

[0170] The preparation method of the copper sulfate and zinc sulfate mixture in Example 1 can also be modified to the following steps:

[0171] 1) Weigh 3 parts of copper sulfate powder and add 5 parts of distilled water. Stir thoroughly for 5-10 minutes. Then add 6 parts of zinc sulfate powder and continue stirring for 5-10 minutes until the copper sulfate powder and zinc sulfate powder are fully dissolved and mixed to obtain a copper sulfate and zinc sulfate mixture (one part is 10g).

[0172] 2) Pour the copper sulfate and zinc sulfate mixture obtained in step 1) into a sterilized container, seal it, and store it in a cool, dry place.

[0173] Example 7: Preparation method of multi-nutrient compound preparation; step parameters; left endpoint value

[0174] The preparation method of the multi-nutrient compound preparation in Example 2 can also be modified to the following steps:

[0175] 3) Weigh out 300 parts of carnosine powder, 25 parts of protein powder, 10 parts of bone meal, 5 parts of red palm oil, and 5 parts of anthocyanins, and grind them to obtain a multi-nutrient compound powder; (one part is 1g).

[0176] 4) Add the multi-nutrient compound preparation powder from step 3) of this embodiment to 300 parts of water and stir thoroughly to obtain a multi-nutrient compound preparation suspension.

[0177] 5) Allow the suspension of the multi-nutrient compound preparation from step 4) of this embodiment to stand and separate into layers. Take out the supernatant after separation to obtain the multi-nutrient compound preparation. Seal and store the multi-nutrient compound preparation.

[0178] Example 8: Preparation method of multi-nutrient compound preparation, step parameters, right endpoint value

[0179] The preparation method of the multi-nutrient compound preparation in Example 2 can also be modified to the following steps:

[0180] 3) Weigh out 500 parts of carnosine powder, 50 parts of protein powder, 25 parts of bone meal, 15 parts of red palm oil, and 10 parts of anthocyanins, grind them to obtain a multi-nutrient compound preparation powder.

[0181] 4) Add the multi-nutrient compound preparation powder from step 3) of this embodiment to 400 parts of water and stir thoroughly to obtain a multi-nutrient compound preparation suspension; one part is 1g;

[0182] 5) Allow the suspension of the multi-nutrient compound preparation from step 4) of this embodiment to stand and separate into layers. Take out the supernatant after separation to obtain the multi-nutrient compound preparation. Seal and store the multi-nutrient compound preparation.

[0183] Example 9: Preparation method of compound microalgae ecological preparation - Step parameters - First endpoint value

[0184] The preparation method of the compound microalgae ecological agent in Example 2 can also be modified to the following steps:

[0185] 6) Prepare algae and protozoa: Chlorella solution with a mass concentration of 3 g / L, Phaeodactylum tricornutum solution with a mass concentration of 2 g / L, Phaeodactylum pulchella solution with a mass concentration of 0.2 g / L, red algae solution with a mass concentration of 0.2 g / L, and protozoa solution with a distribution density of 5 protozoa / mL.

[0186] 7) Cultivating microalgae and protozoa: Chlorella, Brown's Finger Algae, Pavlova, and Red Algae were extracted from the Chlorella solution, Brown's Finger Algae solution, Pavlova solution, and Red Algae solution in step 6) and cultured separately in their respective algal culture media. The algal culture media contained the following components and were mixed with 1000 mL of distilled water: 100 g potassium nitrate powder, 20 g disodium EDTA powder, 10 g potassium hydrogen phosphate powder, 6 mg vitamin B1 powder, 50 μg vitamin B12 powder, 2.5 g ferric sulfate heptahydrate powder, and 0.25 g manganese sulfate powder.

[0187] Protozoa are extracted from the protozoan fluid in step 6) and placed in a protozoan culture medium for culture. The protozoan culture medium contains the following components and is mixed with 800 mL of distilled water: 15 g glucose powder, 8 g peptone powder, 5 g yeast extract powder, 4 g sodium chloride powder, and 2 mL of 1×PBS solution.

[0188] 8) Monitoring growth: Place the algal culture medium containing microalgae from step 7) in a shaking incubator and shake it. Set the temperature to 20℃ and the light intensity to 100 μmol / m². 2 / s, with a photoperiod of 16 hours of light and 8 hours of darkness, and a pH set at 6.0, when the cell count of each microalgae reaches 10 6 Algal culture medium was obtained at CFU / mL;

[0189] The protozoan culture medium containing protozoa from step 7) was placed in a shaking incubator and shaken. The temperature was set to 15°C, the pH to 6.5, the light intensity to 1000 lux, and oxygen was provided for incubation. The culture was continued for 3 days until the number of protozoa reached 10. 3 CFU / mL was used to obtain the protozoan culture medium;

[0190] 9) Mixing microalgae and protozoa: Pour 500 mL of the algae culture medium from step 8) and 100 mL of the protozoa culture medium into a mixing container. At the same time, add 10 mL of lactic acid bacteria solution, 7 mL of phosphate adsorbent, 3 g of chrysanthemum powder, 3 g of sophora root powder, 5 mL of glycerol, 4 g of chitosan powder, and 3 mL of Tween 80 solution to the mixing container. Stir with a stirring rod for 5 minutes to mix thoroughly and evenly. After standing for 4 hours, remove the supernatant to obtain the compound microalgae ecological preparation.

[0191] 10) Colony harvesting and preparation of formulation: The compound microalgae ecological formulation from step 9) is bottled and sealed in a cool place.

[0192] Example 9: Preparation method of compound microalgae ecological preparation - Step parameters - Second endpoint value

[0193] The preparation method of the compound microalgae ecological agent in Example 2 can also be modified to the following steps:

[0194] 6) Prepare algae and protozoa: Chlorella solution with a mass concentration of 5 g / L, Phaeodactylum tricornutum solution with a mass concentration of 4 g / L, Phaeodactylum pulchella solution with a mass concentration of 1 g / L, red algae solution with a mass concentration of 1 g / L, and protozoa solution with a distribution density of 10 protozoa / mL.

[0195] 7) Cultivating microalgae and protozoa: Chlorella, Brown's Finger Algae, Pavlova, and Red Algae were extracted from the Chlorella solution, Brown's Finger Algae solution, Pavlova solution, and Red Algae solution in step 6) and cultured separately in their respective algal culture media. The algal culture media contained the following components and were mixed with 1000 mL of distilled water: 100 g potassium nitrate powder, 20 g disodium EDTA powder, 10 g potassium hydrogen phosphate powder, 6 mg vitamin B1 powder, 50 μg vitamin B12 powder, 2.5 g ferric sulfate heptahydrate powder, and 0.25 g manganese sulfate powder.

[0196] Protozoa are extracted from the protozoan fluid in step 6) and placed in a protozoan culture medium for culture. The protozoan culture medium contains the following components and is mixed with 800 mL of distilled water: 15 g glucose powder, 8 g peptone powder, 5 g yeast extract powder, 4 g sodium chloride powder, and 2 mL of 1×PBS solution.

[0197] 8) Monitoring growth: Place the algal culture medium containing microalgae from step 7) in a shaking incubator and shake it. Set the temperature to 30℃ and the light intensity to 200 μmol / m². 2 / s, with a photoperiod of 18 hours of light and 6 hours of darkness, and a pH set at 9.0, when the cell count of each microalgae reaches 10 6 Algal culture medium was obtained at CFU / mL;

[0198] The protozoan culture medium containing protozoa from step 7) was placed in a shaking incubator and shaken. The temperature was set to 25°C, the pH to 8.0, the light intensity to 5000 lux, and oxygen was provided for continuous culture for 5 days. When the number of protozoa reached 10... 3 CFU / mL was used to obtain the protozoan culture medium;

[0199] 9) Mixing microalgae and protozoa: Pour 500 mL of the algae culture medium from step 8) and 100 mL of the protozoa culture medium into a mixing container. At the same time, add 15 mL of lactic acid bacteria solution, 10 mL of phosphate adsorbent, 5 g of chrysanthemum powder, 5 g of sophora flavescens powder, 10 mL of glycerol, 6 g of chitosan powder, and 5 mL of Tween 80 solution to the mixing container. Stir with a stirring rod for 10 minutes to mix thoroughly and evenly. After standing for 5 hours, remove the supernatant to obtain the compound microalgae ecological preparation.

[0200] 10) Colony harvesting and preparation of formulation: The compound microalgae ecological formulation from step 9) is bottled and sealed in a cool place.

Claims

1. A method for mixed crab and shellfish farming, comprising the following steps: S1 Water Purification: Select a pond and introduce water into it as aquaculture water. Add a compound microalgae ecological preparation to the aquaculture water. The compound microalgae ecological preparation contains microalgae to purify the water. S2 Seedling stocking: Healthy crab seedlings and shellfish seedlings are placed into the aquaculture water purified in step S1 and fed with feed for aquaculture. S21 Disease Prevention and Control: During the breeding period in step S2, add a mixture of copper sulfate and zinc sulfate to the breeding water. The mixture of copper sulfate and zinc sulfate contains copper sulfate and zinc sulfate. Its features are: In step S21, the content of copper sulfate in the copper sulfate and zinc sulfate mixture is 0.4~1g / mL and the content of zinc sulfate is 0.8~2g / mL. The dosage of the copper sulfate and zinc sulfate mixture is 0.03~0.045mL of copper sulfate and zinc sulfate mixture per cubic meter of aquaculture water. The feed in step S2 includes a multi-nutrient compound preparation containing protein powder, bone meal, red palm oil, anthocyanins and carnosine powder. The bone meal contains tricalcium phosphate, bone glue and fat, as well as water-soluble compounds of nitrogen, manganese, iron and zinc elements and collagen.

2. The method for mixed crab and shellfish farming according to claim 1, characterized in that, The preparation method of the copper sulfate and zinc sulfate mixture includes the following steps: 1) Weigh 2-3 parts of copper sulfate powder and add 3-5 parts of distilled water. Stir and mix thoroughly for 5-10 minutes. Then add 4-6 parts of zinc sulfate powder and continue stirring for 5-10 minutes until the copper sulfate powder and zinc sulfate powder are fully dissolved and mixed to obtain a copper sulfate and zinc sulfate mixture. 2) Pour the copper sulfate and zinc sulfate mixture obtained in step 1) into a sterilized container, seal it, and store it in a cool, dry place.

3. The method for mixed crab and shellfish farming according to claim 1, characterized in that, The components and dosage of the multi-nutrient compound preparation applied per cubic meter of aquaculture water are as follows: 300-500 parts carnosine powder, 25-50 parts protein powder, 10-25 parts bone meal, 5-15 parts red palm oil, and 5-10 parts anthocyanins.

4. The method for mixed crab and shellfish farming according to claim 3, characterized in that, The preparation method of the multi-nutrient compound preparation includes the following steps: 3) Weigh out 300-500 parts of carnosine powder, 25-50 parts of protein powder, 10-25 parts of bone meal, 5-15 parts of red palm oil, and 5-10 parts of anthocyanins, and grind them to obtain a multi-nutrient compound preparation powder. 4) Add the multi-nutrient compound preparation powder from step 3) to 300-400 parts of water and stir thoroughly to obtain a multi-nutrient compound preparation suspension; 5) Allow the suspension of the multi-nutrient compound preparation from step 4) to stand and separate into layers, take out the supernatant after separation to obtain the multi-nutrient compound preparation, and seal and store the multi-nutrient compound preparation.

5. The method for mixed crab and shellfish farming according to claim 1, characterized in that: The compound microalgae ecological preparation contains Chlorella, Brown finger algae, Pavlova, red algae, and protozoa.

6. The method for mixed crab and shellfish farming according to claim 5, characterized in that: The preparation method of the compound microalgae ecological agent includes the following steps: 6) Prepare algae and protozoa: Chlorella solution with a mass concentration of 3-5 g / L, Phaeodactylum tricornutum solution with a mass concentration of 2-4 g / L, Pavlova solution with a mass concentration of 0.2-1 g / L, and red algae solution with a mass concentration of 0.2-1 g / L; and protozoa solution with a distribution density of 5-10 individuals / mL. 7) Cultivating microalgae and protozoa: Chlorella, Brown's Finger Algae, Pavlova, and Red Algae were extracted from the Chlorella solution, Brown's Finger Algae solution, Pavlova solution, and Red Algae solution in step 6) and cultured separately in their respective algal culture media. The algal culture media contained the following components and were mixed with 1000 mL of distilled water: 100 g potassium nitrate powder, 20 g disodium EDTA powder, 10 g potassium hydrogen phosphate powder, 6 mg vitamin B1 powder, 50 μg vitamin B12 powder, 2.5 g ferric sulfate heptahydrate powder, and 0.25 g manganese sulfate powder. Protozoa are extracted from the protozoan fluid in step 6) and placed in a protozoan culture medium for culture. The protozoan culture medium contains the following components and is mixed with 800 mL of distilled water: 15 g glucose powder, 8 g peptone powder, 5 g yeast extract powder, 4 g sodium chloride powder, and 2 mL of 1×PBS solution. 8) Monitoring Growth: Place the algal culture medium containing microalgae from step 7) in a shaking incubator and shake it. Set the temperature to 20-30°C, the light intensity to 100-200 μmol / m² / s, the photoperiod to 16-18 hours of light and 8-6 hours of darkness, and the pH to 6.0-9.

0. When the cell count of each type of microalgae reaches 10... 6 CFU / mL was used to obtain algal culture medium; The protozoan-containing culture medium from step 7) is placed in a shaking incubator and shaken. The temperature is set to 15-25℃, the pH to 6.5-8.0, the light intensity to 1000-5000 lux, and oxygen is provided for continuous culture for 3-5 days. When the number of protozoa reaches 10... 3 CFU / mL was used to obtain the protozoan culture medium; 9) Mixing microalgae and protozoa: Pour 500 mL of the algae culture medium from step 8) and 100 mL of the protozoa culture medium into a mixing container. At the same time, add 10-15 mL of lactic acid bacteria solution, 7-10 mL of phosphate adsorbent, 3-5 g of chrysanthemum powder, 3-5 g of sophora root powder, 5-10 mL of glycerol, 4-6 g of chitosan powder, and 3-5 mL of Tween 80 solution to the mixing container. Stir with a stirring rod for 5-10 minutes to mix thoroughly and evenly. After standing for 4-5 hours, remove the supernatant to obtain the compound microalgae ecological preparation. 10) Colony harvesting and preparation of formulation: The compound microalgae ecological formulation from step 9) is bottled and sealed in a cool place.

7. The method for mixed crab and shellfish farming according to claim 5 or 6, characterized in that, The specific usage and dosage of the compound microalgae ecological preparation, the copper sulfate and zinc sulfate mixture, and the multi-nutrient compound preparation are as follows: Apply the compound microalgae ecological preparation once every 20-30 days before seedling stocking in step S2 and during the cultivation period, with each application being 0.11-0.15 mL / m³. 3 During the breeding period in step S2, a multi-nutrient compound preparation is administered once every 15-20 days, with each administration being 0.2-0.3 mL / m³. 3 During the breeding period in step S2, a mixture of copper sulfate and zinc sulfate is added once every 20-30 days, with each addition being 0.03-0.06 mL / m³. 3 .

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