Methods for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients.

Abstract

This invention discloses a method for chemostatic culture of Daphnia mongolica and synergistic enhancement of multiple indicators of nutrients. It can realize large-scale, high-density, chemostatic continuous culture of Daphnia mongolica, and through intelligent control synergistic enhancement strategy, simultaneously and significantly increase the content of astaxanthin, highly unsaturated fatty acids and amino acids in Daphnia mongolica.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically, it relates to a method for the chemostatification culture of Daphnia mongolica and the synergistic enhancement of multiple indicators by nutrients. Background Technology

[0002] Moina mongolica Daday is a saltwater cladoceran zooplankton. This species is characterized by its small size, strong adaptability, wide tolerance to salinity and alkalinity, rapid reproduction, and rich nutrition, making it a high-quality live food organism for the cultivation of fish, shrimp, and crab fry.

[0003] In aquaculture, the nutritional value of live feed directly affects the growth rate, survival rate, disease resistance, and production cost of seedlings. However, existing Daphnia mongholica cultivation techniques and nutritional values ​​have the following shortcomings:

[0004] Low culture density and unstable harvesting: Although there are reports that the culture density of *Daphnia mongolica* has reached 15,000-20,000 individuals / L, the cultivation and harvesting process involves harvesting all individuals at once or harvesting 15-20% of the individuals with liquid for 1-2 days, and then replenishing with new liquid. The long cultivation period makes it impossible to achieve daily culture-harvesting saturation, thus failing to guarantee stable harvesting and ensuring a timely and quantitative supply of aquatic seedlings to meet their feeding needs. There are also reports that the culture density of *Daphnia mongolica* has reached 18,000-23,000 individuals / L, but the expansion period still requires 6-9 days, and the harvesting method is also to harvest all individuals at once. Harvesting is not allowed during the cultivation period, which fails to achieve saturation synchronization between production and harvesting and cannot guarantee the daily feeding needs of aquatic seedlings. However, in actual production, the cultivation technology has low control, water quality and feed balance are difficult to maintain, and the accumulation of uneaten feed, feces and metabolites under high-density cultivation is serious, which restricts population growth, has a short maintenance time, and these cultivation methods are difficult to maintain stable production, making it difficult to achieve true high-density cultivation and harvesting stability.

[0005] Current technologies only focus on basic feed feeding and lack synergistic enhancement programs targeting key nutritional indicators such as fatty acids (especially eicosapentaenoic acid (ARA), eicosapentaenoic acid (EPA), total polyunsaturated fatty acids ∑PUFA), amino acids, and astaxanthin.

[0006] Insufficient astaxanthin content: The astaxanthin content in *Daphnia mongolica* is generally low, making it difficult to meet the nutritional requirements of farmed organisms for astaxanthin. Astaxanthin, as a powerful antioxidant, plays an important role in improving the body color, enhancing antioxidant function, and strengthening the stress resistance of farmed organisms.

[0007] Highly unsaturated fatty acid deficiency: The content of EPA and total polyunsaturated fatty acids (∑PUFA) in the body of *Daphnia mongolica* is low, which is insufficient to meet the essential fatty acid requirements of marine fish fry.

[0008] Limited effects of single fortification: Existing technologies mostly use single nutrient fortification (fortifying only astaxanthin or only fatty acids), ignoring the synergistic effect between different nutrients.

[0009] Therefore, developing a comprehensive technical method that can achieve high-density, stable chemostat culture of Daphnia mongolica and intelligently and synergistically enhance the content of astaxanthin, highly unsaturated fatty acids, and amino acids in its body has important industrial application significance. Summary of the Invention

[0010] The purpose of this invention is to provide a method for the chemostatic culture of *Zapatis mongolica* and the synergistic enhancement of multiple indicators by nutrients.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients, comprising the following steps:

[0013] The first step is the construction of a high-density intelligent chemostat culture system.

[0014] The high-density intelligent chemostat culture system includes a chemostat culture unit, a constant flow liquid inlet system located on the upper part of one side of the chemostat culture unit, an automatic feeding system located on the upper part of the other side of the chemostat culture unit, an overflow harvesting system located at the bottom of one side of the chemostat culture unit, and is equipped with an intelligent environmental monitoring system.

[0015] The bottom of the constant temperature incubator is equipped with a heating and temperature control device;

[0016] The automatic feeding system consists of a feed storage tank, a timer controller, a metering pump, and a disperser connected in sequence, with the disperser located above the chemiluminescent incubator.

[0017] The overflow harvesting system includes a first overflow port located at the bottom of one side of the chemiluminescence incubator;

[0018] The intelligent environmental monitoring system includes a PLC controller located outside the chemiluminescent incubator, a lighting device located directly above the outside of the chemiluminescent incubator, an environmental monitoring probe located on one side inside the chemiluminescent incubator, a turbidity sensor located on one side inside the chemiluminescent incubator, and an oxygenation device located at the bottom inside the chemiluminescent incubator. The environmental monitoring probe, turbidity sensor, oxygenation device, and lighting device are connected to the PLC controller.

[0019] The heating and temperature control device is connected to the PLC controller;

[0020] The timing controller is connected to the PLC controller;

[0021] The second step is to enhance the preparation of the container.

[0022] The enhanced container is equipped with a lighting system at the top, a heating device on one side of the bottom, a second overflow port on the other side of the bottom, and an oxygenation system. The oxygenation system includes an oxygenation device located outside the enhanced container, which is connected to an air diffuser stone via a pipeline. The air diffuser stone is located at the bottom of the enhanced container.

[0023] The third step involves a synergistic nutritional fortifier made from the following components in parts by weight: 100-200 parts astaxanthin, 50-100 parts DHA algal oil microcapsules, 50-100 parts EPA algal oil microcapsules, 10-30 parts magnesium vitamin C phosphate, 5-15 parts vitamin E acetate, 20-50 parts soybean lecithin, 30-60 parts β-cyclodextrin, 20-40 parts concentrated Chlorella vulgaris, 10-30 parts Rhodopseudomonas palustris, and purified water to bring the total to 1000 parts.

[0024] The above components are mixed in proportion, purified water is added, and the mixture is emulsified by high-speed shearing (10,000~15,000 rpm, 10~20 minutes) to prepare a stable synergistic nutrient fortifier.

[0025] The fourth step is the training phase.

[0026] Fresh, treated seawater is added to the chemostat culture vessel. The parameters of the high-density intelligent chemostat culture system are controlled, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen. Genetically stable purified germplasm is inoculated into the chemostat culture vessel at an inoculation density of 500-1000 germplasms / L (preferably 800 or 850 germplasms / L). The temperature is controlled by a heating temperature control device.

[0027] Treated fresh seawater is continuously added to the chemostat culture vessel using a constant flow inlet system at a rate controlled at 4–10 L / h (preferably 5 or 8 L / h) to achieve a dilution rate D = 0.05–0.20 h. -1 (Preferred values ​​are 0.05 and 0.08 h) -1 This allows the population of Daphnia mongholica to be in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 15,000 to 30,000 individuals / L;

[0028] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 25~28℃; b) Salinity: 2~30‰; c) pH: 7.0~8.5; d) Dissolved oxygen: 5~8 mg / L; e) Light intensity: 5000~7000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0029] The automatic feeding system controls the continuous and quantitative feeding of feed. This system uses a timer controller and a metering pump to control the time and flow rate, achieving continuous and quantitative feeding. The feed is stored in a feed tank, passes through the timer controller and metering pump, and is finally evenly sprayed into the chemostat culture vessel via a disperser. The feed tank has three independent compartments for basic feed, supplementary feed, and water quality control feed, respectively. Basic feed is fed first, and the feeding amount is automatically adjusted by the timer controller via a turbidity sensor located in the seawater, maintaining the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation, and then water quality control feed is added every 24 hours based on the water quality.

[0030] During the cultivation period, the PLC controller automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device controls the seawater temperature, which is displayed on the PLC controller's screen. The environmental monitoring probe detects the temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values, which are displayed on the PLC controller's screen. The turbidity sensor measures the seawater turbidity, which is displayed on the PLC controller's screen. The PLC controller controls the lighting device, environmental monitoring probe, turbidity sensor, oxygenation device, and heating and temperature control device, thereby regulating each parameter.

[0031] When the density of Daphnia reaches 25,000 to 28,000 cells / L (preferably 28,000 cells / L), the harvesting procedure is initiated. Daphnia mongholica is continuously harvested from the first overflow outlet, rinsed with clean seawater, and the harvest rate is 10% to 50% (preferably 10% or 25%). The Daphnia mongholica is then added to the enhanced container.

[0032] Fifth step: Intelligent collaborative enhancement processing

[0033] The enhanced container contains treated fresh seawater with a pH of 7.5-8.5 (preferably 8.3). The density of *Daphnia mongolica* is controlled at 20,000-40,000 cells / mL (preferably 28,000 or 35,000 cells / mL). The temperature is controlled at 25-28°C (preferably 26°C) using a heating device. During the enhanced period, the light intensity is controlled at 3,000-5,000 lx (preferably 4,000 lx) using a light system. The dissolved oxygen in the seawater is controlled at ≥5 mg / L using an oxygenation system. The synergistic nutrient enhancer is added to the enhanced container at a ratio of 0.5-2.0 mL (preferably 1.2 or 1.5 mL) per liter of seawater. The enhanced time is 1-7 hours (preferably 1, 3, 5, or 7 hours). Samples are taken to detect the astaxanthin, fatty acid, and amino acid content in *Daphnia mongolica*.

[0034] After the enhancement is completed, the harvesting process is started, and Daphnia mongholica is continuously harvested from the second overflow outlet. It is then rinsed with clean seawater to obtain live Daphnia mongholica food that has been enhanced with synergistic nutrition.

[0035] The overflow harvesting system includes a first multi-stage collector located on the first overflow outlet.

[0036] The side of the chemiluminescence incubator is equipped with an insulation layer.

[0037] The chemiluminescence constant incubator is selected from culture tanks and culture pools.

[0038] The culture tank is made of transparent plexiglass or plastic, and has a working volume of 500~2000L.

[0039] The culture tank is a cement culture tank, circular or rectangular in shape, with a unit working volume of 30~500m³. 3 .

[0040] The heating and temperature control device is either an integrated heating and temperature control heating rod or a stainless steel heating tube. When a stainless steel heating tube is selected, the stainless steel heating tube is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0041] The constant flow liquid inlet system is a peristaltic pump.

[0042] The first overflow port is equipped with a valve, which is used to control the flow rate.

[0043] The first multi-layer graded collector is made of a pipe that matches the first overflow port and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the pipe, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing in size sequentially.

[0044] The first screen mesh bag has a pore size of 250 micrometers.

[0045] The second sieve mesh bag has an aperture of 180 micrometers.

[0046] The third sieve mesh bag has a pore size of 150 micrometers.

[0047] The second overflow port is equipped with a second multi-layer graded collector.

[0048] The strengthening container is a strengthening cylinder with a working volume of 100~1000L.

[0049] The reinforced cylinder is selected from cylinders, cubes, and cones.

[0050] The reinforced cylinder is made of transparent plexiglass or plastic.

[0051] The heating device is an integrated heating and temperature control heating rod.

[0052] The lighting system is a plant tissue culture lamp.

[0053] The second overflow port is equipped with a valve, which is used to control the flow rate.

[0054] The second multi-layer graded collector is made of a pipe that matches the second overflow port and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the pipe, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing in size sequentially.

[0055] The first screen mesh bag has a pore size of 250 micrometers.

[0056] The second sieve mesh bag has an aperture of 180 micrometers.

[0057] The third sieve mesh bag has a pore size of 150 micrometers.

[0058] The preparation of the concentrated Chlorella: Chlorella is centrifuged to obtain concentrated Chlorella. The concentrated Chlorella has a high algal cell concentration, is convenient to transport and store, and is easy to use.

[0059] The preparation of the genetically stable purified germplasm includes the following steps:

[0060] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 60-100 cells / L (preferably 80 cells / L). Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1, reaching 25-30‰ (preferably 28‰). The light intensity is 5000-7000 lx (preferably 60000 lx), the photoperiod is L:D = 12h:12h, and the temperature is maintained at 25-28℃ (preferably 26℃). Feed daily with cultured *Chlorella vulgaris* (3 million cells / mL) until *Daphnia mongolica* grows and reproduces normally.

[0061] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0062] The preparation of the treated fresh seawater involves the following steps: seawater is sequentially filtered through sand (pore size 0.1mm~0.2mm), disinfected (using bleaching powder or strong chlorine to achieve an effective chlorine concentration of 20~30 ppm for 24~48 hours, using sodium thiosulfate, and the residual chlorine in the neutralized and reduced water is detected using starch-potassium iodide test paper), temperature adjusted (controlled temperature at 25~28℃), and oxygenated (dissolved oxygen at 5~8 mg / L) to obtain treated fresh seawater.

[0063] The automatic feeding system controls the continuous quantitative feeding of feed: by measuring the density of Daphnia mongolica in seawater and combining it with the volume of seawater in the chemostat culture device, the total weight (wet weight) of Daphnia mongolica is calculated, the theoretical value of the daily feed intake of Daphnia mongolica is calculated, and then the theoretical value is dynamically adjusted according to the water quality and the intestinal fullness of Daphnia mongolica observed under a microscope to determine the actual daily feed intake.

[0064] The basic feed is cultured Chlorella as the main feed, with a cell density of 30-50 million cells / mL (preferably 40 million cells / mL), and the feeding amount of basic feed is 3-5 million cells / mL (preferably 3-4 million cells / mL).

[0065] The supplementary feed is yeast, and the feeding amount is 20~100 mg / L (preferably 80 or 60 mg / L).

[0066] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 10~50 mg / L (preferably 30 or 20 mg / L), which is used to improve water quality and provide additional nutrition.

[0067] During the cultivation phase, the seawater in the chemostat culture vessel was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate D = 0.20~0.25 h. -1 (Preferred values ​​are 0.15 and 0.2 h) -1 ), controlled by dilution.

[0068] During the cultivation phase, the number of Daphnia mongolica in the water sample is measured daily, with at least three samplings per session (generally five). The average value is taken, and the total number of Daphnia mongolica in 1000 ml of water is calculated, which is the Daphnia density.

[0069] The synergistic nutritional fortifier is made from the following components in parts by weight: 150 parts astaxanthin, 80 parts DHA algal oil microcapsules, 80 parts EPA algal oil microcapsules, 20 parts magnesium vitamin C phosphate, 10 parts vitamin E acetate, 30 parts soybean lecithin, 45 parts β-cyclodextrin, 25 parts concentrated chlorella, 15 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

[0070] The synergistic nutritional fortifier is made from the following components in parts by weight: 120 parts astaxanthin, 65 parts DHA algal oil microcapsules, 90 parts EPA algal oil microcapsules, 20 parts magnesium vitamin C phosphate, 10 parts vitamin E acetate, 50 parts soybean lecithin, 45 parts β-cyclodextrin, 40 parts concentrated Chlorella vulgaris, 30 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

[0071] The synergistic nutritional fortifier is made from the following components in parts by weight: 175 parts astaxanthin, 85 parts DHA algal oil microcapsules, 85 parts EPA algal oil microcapsules, 15 parts magnesium vitamin C phosphate, 8 parts vitamin E acetate, 45 parts soybean lecithin, 40 parts β-cyclodextrin, 30 parts concentrated chlorella, 25 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

[0072] The synergistic nutritional fortifier is made from the following components in parts by weight: 135 parts astaxanthin, 68 parts DHA algal oil microcapsules, 68 parts EPA algal oil microcapsules, 18 parts magnesium vitamin C phosphate, 8.5 parts vitamin E acetate, 25.5 parts soybean lecithin, 38.5 parts β-cyclodextrin, 21.5 parts concentrated chlorella, 12.5 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

[0073] Preferred parameters for the high-density intelligent chemostat culture system are as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 7.5; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 6000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0074] Preferred parameters for the high-density intelligent chemostat culture system are as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 8.1; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 5000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0075] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0076] The present invention provides a method for chemostatic culture of Daphnia mongolica and synergistic enhancement of multiple indicators of nutrients, which can realize large-scale, high-density, chemostatic continuous culture of Daphnia mongolica, and simultaneously and significantly increase the content of astaxanthin, highly unsaturated fatty acids and amino acids in Daphnia mongolica through intelligent control synergistic enhancement strategy.

[0077] The present invention provides a method for chemostatic culture of Daphnia mongolica and synergistic enhancement of multiple indicators by nutrients, which significantly improves the culture density. The present invention uses a chemostatic culture system and culture technology, and the stable culture density of Daphnia mongolica can reach 25,000~28,000 cells / L, which is significantly higher than the technical level reported in the prior art.

[0078] The present invention provides a method for the synergistic enhancement of multiple indicators of Daphnia mongholica through culture and nutrient synergistic enhancement. Through intelligent control of synergistic enhancement treatment, it achieves synergistic nutrient enhancement and enrichment of fatty acids, amino acids and astaxanthin.

[0079] The present invention provides a method for the chemoautotrophic culture of naked-bellied Daphnia mongholica and the synergistic enhancement of multiple indicators by nutrients. Through intelligent and precise control by online sensors and a monitoring and control system, it can realize real-time monitoring and automatic adjustment of parameters such as temperature, pH, dissolved oxygen, and fortifier concentration. The batch-to-batch difference is less than 9%, which reduces the use of labor and lowers production costs.

[0080] The present invention provides a method for the chemostatinization culture of *Daphnia mongolica* and the synergistic enhancement of multiple indicators through nutrients, resulting in significantly improved aquaculture effects. This method is highly applicable and can be widely used for feeding economically important aquatic fish, shrimp, and crabs such as filefish, grouper, seahorse, large yellow croaker, sea bass, mud crab, river crab, whiteleg shrimp, and tiger prawn, as well as native saline-alkali fish such as *Gymnocypris qinghai Lake* fry. It can significantly improve the body color vibrancy, antioxidant capacity, and growth performance of cultured organisms, increasing fry survival rate by over 20% and growth rate by 15-25%.

[0081] This invention utilizes a high-density intelligent chemostat culture system to control the dilution rate D = 0.05~0.20 h. -1 This method aims to maintain the *Daphnia mongolica* population in a dynamic equilibrium state during its exponential growth phase, with a culture density of 25,000–28,000 cells / L. A synergistic nutrient fortifier is prepared, and an intelligent fortification strategy is employed to synergistically enhance the harvested *Daphnia mongolica*. After treatment using this method, a stable culture density of 25,000–28,000 cells / L can be achieved, significantly exceeding the levels reported in previous studies. Using the synergistic nutrient fortifier, the astaxanthin content in *Daphnia mongolica* increases by 14.47–87.11 times, ARA by 0.48–1.06 times, EPA by 0.89–1.86 times, total polyunsaturated fatty acids (∑PUFA) by 0.73–1.39 times, total amino acids by 8.4%–17.2%, total umami amino acids by 7.7%–15.6%, and total essential amino acids for fish by 7.7%–17.9%. This invention enables high-density chemostatic continuous culture of Daphnia mongolica and intelligent synergistic enhancement of multiple nutrients, significantly improving production scale and total output, and enhancing its comprehensive nutritional value as live feed, making it suitable for large-scale industrial production. Attached Figure Description

[0082] Figure 1 This is a schematic diagram of a chemostat incubator.

[0083] Figure 2 A structural diagram to reinforce the container.

[0084] Among them, 1 is a chemostatic incubator, 101 is a heating and temperature control device, and 102 is a heat preservation layer; 2 is a constant flow liquid feeding system, 3 is an automatic feeding system, 301 is a feed storage tank, 302 is a timer controller, 303 is a metering pump, and 304 is a disperser; 4 is an overflow harvesting system, 401 is a first overflow port, and 402 is a first multi-layer graded collector; 5 is an intelligent environmental monitoring system, 501 is a PLC controller, 502 is an environmental monitoring probe, 503 is a turbidity sensor, 504 is an oxygenation device, and 505 is a lighting device; 6 is a reinforced container, 601 is a heating device, 602 is a second overflow port, 603 is a second multi-layer graded collector, 604 is an oxygenation device, 605 is a lighting system, 606 is an air diffuser, and 607 is a pipeline. Detailed Implementation

[0085] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0086] A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients includes the following steps:

[0087] The first step is the construction of a high-density intelligent chemostat culture system.

[0088] The high-density intelligent chemostat culture system includes a chemostat culture unit 1, with the structure as follows: Figure 1 As shown, Figure 1 This is a schematic diagram of a chemostat culture device. It includes a constant flow liquid inlet system 2 located on the upper part of one side of the chemostat culture device 1, an automatic feeding system 3 located on the upper part of the other side of the chemostat culture device 1, an overflow harvesting system 4 located at the bottom of one side of the chemostat culture device 1, and an intelligent environmental monitoring system 5.

[0089] The bottom of the constant chemistry incubator 1 is equipped with a heating and temperature control device 101.

[0090] The automatic feeding system 3 consists of a feed storage tank 301, a timer controller 302, a metering pump 303, and a disperser 304 connected in sequence. The disperser 304 is located above the constant chemistry incubator 1.

[0091] The overflow harvesting system 4 includes a first overflow port 401 located at the bottom of one side of the chemostat 1, and a first multi-layer graded collector 402 located on the first overflow port 401.

[0092] The intelligent environmental monitoring system 5 includes a PLC controller 501 located outside the chemiluminescence incubator 1, a lighting device 505 (multiple devices, such as lamps) located directly above the outside of the chemiluminescence incubator 1, an environmental monitoring probe 502 located on one side inside the chemiluminescence incubator 1, a turbidity sensor 503 located on one side inside the chemiluminescence incubator 1, and an oxygenation device 504 (specifically, an aerator) located at the bottom inside the chemiluminescence incubator 1. The environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the lighting device 505 are connected to the PLC controller 501.

[0093] The heating and temperature control device 101 is connected to the PLC controller 501.

[0094] The timing controller 302 is connected to the PLC controller 501.

[0095] The side of the constant chemistry incubator 1 is provided with a heat insulation layer 102.

[0096] The constant chemistry incubator 1 is selected from culture tanks and culture pools.

[0097] The culture tank is made of transparent plexiglass or plastic, and has a working volume of 500~2000L.

[0098] The culture tank is a cement culture tank, circular or rectangular in shape, with a unit working volume of 30~500m³. 3 .

[0099] The heating and temperature control device 101 is an integrated heating and temperature control heating rod or a stainless steel heating tube. When a stainless steel heating tube is selected, the stainless steel heating tube is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0100] The constant flow liquid inlet system 2 is a peristaltic pump.

[0101] The first overflow port 401 is equipped with a valve, which is used to control the flow rate.

[0102] The first multi-layer graded collector 402 is made of a tube that matches the first overflow port 401 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube. The aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreases sequentially.

[0103] The first screen mesh bag has a pore size of 250 micrometers.

[0104] The second sieve mesh bag has an aperture of 180 micrometers.

[0105] The third sieve mesh bag has a pore size of 150 micrometers.

[0106] The second step is to enhance the preparation of the container.

[0107] Structure as Figure 2 As shown, Figure 2 This is a schematic diagram of the reinforced container structure. The reinforced container 6 has a lighting system 605 at its top, a heating device 601 on one side of its bottom, a second overflow port 602 on the other side of its bottom, and a second multi-layer graded collector 603 located on the second overflow port 602. It is also equipped with an oxygenation system, which includes an oxygenation device 604 (specifically an aerator) located outside the reinforced container 6. The oxygenation device 604 is connected to an aeration stone 606 via a pipe 607, and the aeration stone 606 is located at the bottom of the reinforced container 6.

[0108] The strengthening container 6 is a strengthening cylinder with a working volume of 100~1000L.

[0109] The reinforced cylinder is selected from cylinders, cubes, and cones.

[0110] The reinforced cylinder is made of transparent plexiglass or plastic.

[0111] The heating device 601 is an integrated heating and temperature control heating rod.

[0112] The lighting system 605 is a plant tissue culture lamp.

[0113] The second overflow port 602 is equipped with a valve, which is used to control the flow rate.

[0114] The second multi-layer graded collector 603 is made of a tube that matches the second overflow port 602 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing sequentially.

[0115] The first screen mesh bag has a pore size of 250 micrometers.

[0116] The second sieve mesh bag has an aperture of 180 micrometers.

[0117] The third sieve mesh bag has a pore size of 150 micrometers.

[0118] The third step is the preparation of synergistic nutritional fortifiers.

[0119] The synergistic nutritional fortifier is made from the components listed in Table 1:

[0120] Table 1

[0121]

[0122] Concentrated Chlorella: Chlorella is centrifuged to obtain concentrated Chlorella. Concentrated Chlorella has a high concentration of algal cells, is easy to transport and store, and is convenient to use.

[0123] Mix the above components in proportion, add purified water, and emulsify by high-speed shearing (10,000~15,000 rpm, 10~20 minutes) to prepare a stable synergistic nutrient fortifier.

[0124] Step 4: Seed source domestication

[0125] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 80 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1%, reaching 25-30‰. The light intensity is 5000-7000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 25-28℃. Feed daily with cultured *Chlorella* sp. at a rate of 3 million cells / mL until *Daphnia mongolica* grows and reproduces normally.

[0126] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0127] Step 5, Cultivation Stage

[0128] Preparation of treated fresh seawater: Seawater is sequentially subjected to sand filtration (pore size 0.1mm~0.2mm), disinfection (using bleaching powder or strong chlorine disinfectant, with an effective chlorine concentration of 20~30 ppm, disinfection time of 24~48 hours, using sodium thiosulfate, and detecting residual chlorine in the neutralized and reduced water using starch-potassium iodide test paper), temperature adjustment (controlling the temperature at 25~28℃), and oxygenation (dissolved oxygen 5~8 mg / L) to obtain treated fresh seawater.

[0129] Fresh, treated seawater was added to the chemostat culture vessel 1. The parameters of the high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen, were controlled. Genetically stable purified germplasm was inoculated into the chemostat culture vessel 1 at a density of 500-1000 germplasms / L. The temperature was controlled by a heating and temperature control device 101, and a heat preservation layer 102 was also provided for heat preservation.

[0130] Treated fresh seawater is continuously added to the chemostat culture vessel 1 via a constant flow inlet system 2 at a rate controlled at 4–10 L / h, resulting in a dilution rate D of 0.05–0.20 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 15,000 to 30,000 individuals / L;

[0131] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 25~28℃; b) Salinity: 2~30‰; c) pH: 7.0~8.5; d) Dissolved oxygen: 5~8 mg / L; e) Light intensity: 5000~7000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0132] The automatic feeding system 3 controls the continuous and quantitative feeding of feed. By measuring the density of *Daphnia mongolica* in seawater and combining it with the volume of seawater in the chemostat culture vessel 1, the total weight (wet weight) of *Daphnia mongolica* is calculated, and the theoretical daily feed intake of *Daphnia mongolica* is calculated. Then, based on water quality and microscopic observation of the intestinal fullness of *Daphnia mongolica*, the theoretical value is dynamically adjusted to determine the actual daily feed intake. The automatic feeding system 3 controls the time and flow rate through the timer controller 302 and the metering pump 303 to achieve continuous and quantitative feeding. The feed is stored in the feed storage tank 301 and is sequentially fed through the timer controller. The feed is evenly sprayed into the constant chemistry culture vessel 1 through the dispersant 304 and the metering pump 303. The feed storage tank 301 is equipped with three independent spaces to hold basic feed, supplementary feed, and water quality control feed, respectively. First, the basic feed is fed. The feeding amount is automatically adjusted by the timer controller 302, which is located in the seawater and controlled by the turbidity sensor 503. The supplementary feed is automatically adjusted to maintain the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation. Then, water quality control feed is added every 24 hours according to the water quality.

[0133] The basic feed is mainly cultured Chlorella, with a cell density of 30-50 million cells / mL, and the feeding amount of basic feed is 3-5 million cells / mL.

[0134] The supplementary feed is yeast, and the feeding amount is 20~100 mg / L.

[0135] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 10~50 mg / L, which is used to improve water quality and provide additional nutrition.

[0136] During the cultivation period, the PLC controller 501 automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device 101 controls the seawater temperature and displays it on the display screen of the PLC controller 501. The temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values ​​monitored by the environmental monitoring probe 502 are displayed on the display screen of the PLC controller 501. The turbidity of the seawater measured by the turbidity sensor 503 is displayed on the display screen of the PLC controller 501. The PLC controller 501 controls the lighting device 505, the environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the heating and temperature control device 101, thereby regulating various parameters.

[0137] During the cultivation phase, the seawater in chemostat 1 was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate D = 0.20~0.25 h. -1 Controlled by dilution method;

[0138] During the cultivation phase, the number of *Daphnia mongolica* cells in the water sample is measured daily, with at least three samplings per batch (generally five). The average value is taken, and the total number of *Daphnia mongolica* cells in 1000 ml of water is calculated, which is the daphnia density. For example, five 10 ml centrifuge tubes are used to sample, and the number of *Daphnia mongolica* cells is counted under a stereomicroscope. After obtaining the average value, the daphnia density is calculated. When the daphnia density reaches 25,000~28,000 cells / L, the harvesting procedure is initiated. *Daphnia mongolica* cells are continuously harvested from the first overflow outlet 401, rinsed with clean seawater, with a recovery rate of 20%~50%, and added to the enhancement container 6.

[0139] Alternatively, depending on production needs, individuals of different sizes of *Daphnia mongolica* can be collected by grading and screening in the first multi-layer grading collector 402, and then separately fortified in different fortification containers before being fed to aquatic animals that need to consume *Daphnia mongolica* of different sizes. Alternatively, individuals of all sizes of *Daphnia mongolica* can be collected together in the first multi-layer grading collector 402, placed in the same fortification container for fortification, and then graded and screened to collect individuals of different sizes before being fed to aquatic species that need to consume different sizes.

[0140] The first overflow port 401 is equipped with a valve to control the flow rate. The Mongolian naked belly daphne enters the first multi-layer classifier 402 in sequence into the first screen silk mesh bag, then into the second screen silk mesh bag, and finally into the third screen silk mesh bag.

[0141] The first sieve mesh bag of this invention has a pore size of 250 micrometers, which can collect individuals of *Daphnia mongolica* larger than 250 micrometers; the second sieve mesh bag has a pore size of 180 micrometers, which can collect individuals of *Daphnia mongolica* with a size of 180-249 micrometers; and the third sieve mesh bag has a pore size of 150 micrometers, which can collect individuals of *Daphnia mongolica* with a size of 150-179 micrometers. By using sieve mesh bags with different pore sizes for graded harvesting, individuals of different sizes of *Daphnia mongolica* can be graded and harvested for feeding to aquatic species that require different sizes of feed.

[0142] Step 6: Intelligent Collaborative Enhancement Processing

[0143] The enhanced container 6 contains treated fresh seawater with a pH of 7.5-8.5, controlling the density of *Daphnia mongolica* at 20,000-40,000 cells / mL. The temperature is controlled at 25-28℃ by the heating device 601. During the enhanced period, the light intensity is controlled at 3,000-5,000 lx by the light system 605, and the dissolved oxygen in the seawater is controlled at ≥5 mg / L by the oxygenation system. The synergistic nutrient enhancer is added to the enhanced container 6 at a ratio of 0.5-2.0 mL per liter of seawater. The enhanced time is 1-7 hours. Samples are taken to detect the content of astaxanthin, fatty acids, and amino acids in *Daphnia mongolica*.

[0144] After the fortification was completed, the harvesting process was initiated, and *Daphnia mongolica* were continuously harvested from the second overflow outlet 602. The harvested fish were rinsed with clean seawater (this is to prevent residues from being carried into the aquaculture nursery and polluting the seawater there. Aquaculture nursery or aquaculture requires very high seawater quality; these nutrients are highly nutrient-rich and can easily spoil the water quality if they enter the nursery or aquaculture ponds). This yielded live feed fortified *Daphnia mongolica*.

[0145] Step 7, Feeding after fortification: Live feed for Daphnia mongholicus that has been fortified with synergistic nutrition can be fed directly. It is advisable to finish feeding within 2 hours after harvesting to maintain the best nutritional quality.

[0146] Step 8, Preservation: When short-term preservation is required, place the live feed of Daphnia mongholicus that has been synergistically fortified with nutrients in a refrigerated environment at 4-8℃, add Chlorella, and feed at a rate of 3-5 million Chlorella cells / mL to maintain nutrition. It can be preserved for about 24 hours with a survival rate of ≥85%.

[0147] After being enhanced by the intelligent collaborative enhancement treatment of container 6, the astaxanthin content in Daphnia mongholica was significantly increased, as detailed below:

[0148] After treatment with the intelligent synergistic fortification container 6, at a fortification time of 1 hour and a dosage of 0.5 mL / L of synergistic nutrient fortifier, the astaxanthin content in the live feed of *Daphnia mongolica* treated with synergistic nutrient fortification container 6 was approximately 14.47 times higher than that in the control group (without intelligent synergistic fortification container 6). At a dosage of 0.5 mL / L and a fortification time of 5 hours, the astaxanthin content in the live feed of *Daphnia mongolica* treated with synergistic nutrient fortification container 6 was approximately 27.97 times higher than that in the control group (without intelligent synergistic fortification container 6), significantly superior to that of the untreated *Daphnia mongolica*.

[0149] After treatment with the intelligent synergistic fortification container 6, at a fortification time of 1 hour and a dosage of 1.5 mL / L of synergistic nutrient fortifier, the astaxanthin content in the live feed of *Daphnia mongolica* treated with synergistic nutrient fortification was approximately 41.97 times higher than that in the control group (without intelligent synergistic fortification container 6). At a dosage of 1.5 mL / L and a fortification time of 5 hours, the astaxanthin content in the live feed of *Daphnia mongolica* treated with synergistic nutrient fortification was approximately 87.11 times higher than that in the control group (without intelligent synergistic fortification container 6), significantly superior to that in the untreated *Daphnia mongolica*.

[0150] Table 2. Astaxanthin content in *Daphnia mongolica* (n=3) (mg·Kg) -1 , wet weight)

[0151]

[0152] Note: If the superscript letters of data in the same row are the same, it means there is no significant difference between groups (P<0.05). If the superscript letters of data in the same row are different, it means there is a significant difference between groups (P<0.05).

[0153] Control group 1: The feed consisted of basal feed, which was yeast. No supplementary feed or water quality control feed was given. The control group did not undergo intelligent synergistic enhancement treatment. All other steps were the same as those described above.

[0154] Control group 2: The diet consisted of basic feed (Chlorella vulgaris), without supplemental feed or water quality control feed, and no intelligent synergistic enhancement treatment was performed. All other steps were the same as those described above.

[0155] Control group 3: The feeding process was the same as described above, with yeast as the base feed, Chlorella as the supplementary feed, and photosynthetic bacteria as the water quality control feed. All other steps were the same as described above.

[0156] Compared with control groups 1, 2, and 3, the live feed of *Daphnia mongolica* obtained by the method of this invention, after synergistic nutritional fortification, showed a synergistic increase in the key nutrients, highly unsaturated fatty acids and amino acids. The results are shown in Tables 3 and 4.

[0157] Table 3 Fatty acid content in *Daphnia mongholica* (n = 3) (expressed as a percentage of total fatty acids, %)

[0158]

[0159] Note: If the superscript letters of data in the same row are the same, it means there is no significant difference between groups (P<0.05). If the superscript letters of data in the same row are different, it means there is a significant difference between groups (P<0.05).

[0160] Table 4. Amino acid content in *Daphnia mongholica* (n=3) (g / 100g wet sample)

[0161]

[0162] Note: If the superscript letters of data in the same row are the same, it means there is no significant difference between groups (P<0.05). If the superscript letters of data in the same row are different, it means there is a significant difference between groups (P<0.05).

[0163] Example 1

[0164] A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients includes the following steps:

[0165] The first step is the construction of a high-density intelligent chemostat culture system.

[0166] The high-density intelligent chemostat culture system includes a chemostat culture device 1, a constant flow liquid inlet system 2 located on the upper side of one side of the chemostat culture device 1, an automatic feeding system 3 located on the upper side of the other side of the chemostat culture device 1, an overflow harvesting system 4 located at the bottom of one side of the chemostat culture device 1, and an intelligent environmental monitoring system 5.

[0167] The bottom of the constant chemistry incubator 1 is equipped with a heating and temperature control device 101.

[0168] The automatic feeding system 3 consists of a feed storage tank 301, a timer controller 302, a metering pump 303, and a disperser 304 connected in sequence. The disperser 304 is located above the constant chemistry incubator 1.

[0169] The overflow harvesting system 4 includes a first overflow port 401 located at the bottom of one side of the constant chemistry incubator 1.

[0170] The intelligent environmental monitoring system 5 includes a PLC controller 501 located outside the chemiluminescence incubator 1, a lighting device 505 (multiple devices, such as lamps) located directly above the outside of the chemiluminescence incubator 1, an environmental monitoring probe 502 located on one side inside the chemiluminescence incubator 1, a turbidity sensor 503 located on one side inside the chemiluminescence incubator 1, and an oxygenation device 504 (specifically, an aerator) located at the bottom inside the chemiluminescence incubator 1. The environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the lighting device 505 are connected to the PLC controller 501.

[0171] The heating and temperature control device 101 is connected to the PLC controller 501.

[0172] The timing controller 302 is connected to the PLC controller 501.

[0173] The side of the constant chemistry incubator 1 is provided with a heat insulation layer 102.

[0174] The chemiluminescence constant culture vessel 1 is selected from a culture tank.

[0175] The culture tank is made of plastic and has a working volume of 1200L.

[0176] The heating and temperature control device 101 is a stainless steel heating tube. When a stainless steel heating tube is selected, it is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0177] The constant flow liquid inlet system 2 is a peristaltic pump.

[0178] The first overflow port 401 is equipped with a valve, which is used to control the flow rate.

[0179] The second step is to enhance the preparation of the container.

[0180] The enhanced container 6 is equipped with a lighting system 605 on the top, a heating device 601 on one side of the bottom, a second overflow port 602 on the other side of the bottom, and a second multi-layer graded collector 603 on the second overflow port 602. It is also equipped with an oxygenation system, which includes an oxygenation device 604 (specifically an aerator) located outside the enhanced container 6. The oxygenation device 604 is connected to an air diffuser 606 through a pipe 607. The air diffuser 606 is located at the bottom of the enhanced container 6.

[0181] The strengthening container 6 is a strengthening cylinder with a working volume of 250L.

[0182] The reinforced cylinder is a cube.

[0183] The reinforced cylinder is made of plastic.

[0184] The heating device 601 is an integrated heating and temperature control heating rod.

[0185] The lighting system 605 is a plant tissue culture lamp.

[0186] The second overflow port 602 is equipped with a valve, which is used to control the flow rate.

[0187] The second multi-layer graded collector 603 is made of a tube that matches the second overflow port 602 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing sequentially.

[0188] The first screen mesh bag has a pore size of 250 micrometers.

[0189] The second sieve mesh bag has an aperture of 180 micrometers.

[0190] The third sieve mesh bag has a pore size of 150 micrometers.

[0191] The third step is the preparation of synergistic nutritional fortifiers.

[0192] The synergistic nutritional fortifier is made from the components listed in Table 5:

[0193] Table 5

[0194]

[0195] The total amount of the synergistic nutrient fortifier is 1000g. The above components are mixed in proportion, purified water is added, and the mixture is emulsified by high-speed shearing (12000 rpm, 15 minutes) to produce a stable synergistic nutrient fortifier.

[0196] Step 4: Seed source domestication

[0197] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 80 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1% to reach 28‰. The light intensity is 6000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 26℃. Feed daily with cultured *Chlorella vulgaris* at a rate of 3 million cells / mL. Cultivate for 5 days until *Daphnia mongolica* grows and reproduces normally.

[0198] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0199] Step 5, Cultivation Stage

[0200] Preparation of treated fresh seawater: Seawater is sequentially treated by sand filtration (pore size 0.1 mm), disinfection (using bleaching powder, achieving an effective chlorine concentration of 25 ppm, disinfection time of 24 hours, using sodium thiosulfate, and detecting residual chlorine in the neutralized and reduced water using starch-potassium iodide test paper), temperature adjustment (controlling the temperature at 26℃), and oxygenation (dissolved oxygen 7 mg / L) to obtain treated fresh seawater.

[0201] Add 1000L of treated fresh seawater to the chemostat culture vessel 1, control the parameters of the high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen, inoculate the genetically stable purified germplasm into the chemostat culture vessel 1 at an inoculation density of 800 germplasms / L, control the temperature using a heating and temperature control device 101, and simultaneously provide a heat preservation layer 102 for heat preservation;

[0202] Treated fresh seawater was continuously added to the chemostat culture vessel 1 via a constant flow inlet system 2 at a rate controlled at 5 L / h, resulting in a dilution rate D = 0.05 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 25,000~28,000 individuals / L;

[0203] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 7.5; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 6000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0204] The automatic feeding system 3 controls the continuous and quantitative feeding of feed. By measuring the density of *Daphnia mongolica* in seawater and combining it with the volume of seawater in the chemostat culture vessel 1, the total weight (wet weight) of *Daphnia mongolica* is calculated, and the theoretical daily feed intake of *Daphnia mongolica* is calculated. Then, based on water quality and microscopic observation of the intestinal fullness of *Daphnia mongolica*, the theoretical value is dynamically adjusted to determine the actual daily feed intake. The automatic feeding system 3 controls the time and flow rate through the timer controller 302 and the metering pump 303 to achieve continuous and quantitative feeding. The feed is stored in the feed storage tank 301 and is sequentially fed through the timer controller. The feed is evenly sprayed into the constant chemistry culture vessel 1 through the dispersant 304 and the metering pump 303. The feed storage tank 301 is equipped with three independent spaces to hold basic feed, supplementary feed, and water quality control feed, respectively. First, the basic feed is fed. The feeding amount is automatically adjusted by the timer controller 302, which is located in the seawater and controlled by the turbidity sensor 503. The supplementary feed is automatically adjusted to maintain the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation. Then, water quality control feed is added every 24 hours according to the water quality.

[0205] The basic feed consists of cultured Chlorella as the main feed, with a cell density of 40 million cells / mL and a feeding amount of 3 million cells / mL.

[0206] The supplementary feed is yeast, and the feeding amount is 80 mg / L.

[0207] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 30 mg / L, which is used to improve water quality and provide additional nutrition.

[0208] During the cultivation period, the PLC controller 501 automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device 101 controls the seawater temperature and displays it on the display screen of the PLC controller 501. The temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values ​​monitored by the environmental monitoring probe 502 are displayed on the display screen of the PLC controller 501. The turbidity of the seawater measured by the turbidity sensor 503 is displayed on the display screen of the PLC controller 501. The PLC controller 501 controls the lighting device 505, the environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the heating and temperature control device 101, thereby regulating various parameters.

[0209] During the cultivation phase, the seawater in chemostat 1 was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate of D = 0.15 h. -1 Controlled by dilution method;

[0210] During the cultivation phase, the number of *Daphnia mongolica* cells in the water samples is measured daily, with at least three samplings per batch (generally five). The average value is taken, and the total number of *Daphnia mongolica* cells per 1000 ml of water is calculated, which is the daphnia density. For example, five 10 ml centrifuge tubes are used to sample, and the number of *Daphnia mongolica* cells is counted under a stereomicroscope. After obtaining the average value, the daphnia density is calculated. When the daphnia density reaches 28,000 cells / L, the harvesting procedure is initiated. *Daphnia mongolica* cells are continuously harvested from the first overflow outlet 401, rinsed with clean seawater, with a recovery rate of 25%, and added to the enhancement container 6.

[0211] All individuals of different sizes of *Daphnia mongolica* were collected from the first overflow outlet 401.

[0212] Step 6: Intelligent Collaborative Enhancement Processing

[0213] The enhanced container 6 contained 200L of treated fresh seawater with a pH of 8.3, and the density of *Daphnia mongolica* was controlled at 35,000 cells / mL. The temperature was controlled at 26℃ by the heating device 601. During the enhanced period, the light intensity was controlled at 4000 lx by the light system 605, and the dissolved oxygen in the seawater was controlled at ≥5 mg / L by the oxygenation system. The synergistic nutrient enhancer was added to the enhanced container 6 at a ratio of 1.5mL per liter of seawater. The enhanced period was 5 hours. Samples were taken to test the content of astaxanthin, fatty acids, and amino acids in *Daphnia mongolica*.

[0214] After the enhancement was completed, the harvesting process was started, and Daphnia mongholica was continuously harvested from the second overflow outlet 602. The Daphnia mongholica was rinsed with clean seawater to obtain live food for Daphnia mongholica that had been enhanced with synergistic nutrition.

[0215] During harvesting, a second multi-layer grading collector 603 with different sieves and silk screens is used for grading and harvesting, and then the products are fed to aquatic species that need to consume different sizes of food.

[0216] Step 7, Feeding after fortification: Live feed for Daphnia mongholicus that has been fortified with synergistic nutrition can be fed directly. It is advisable to finish feeding within 2 hours after harvesting to maintain the best nutritional quality.

[0217] Step 8, Preservation: When short-term preservation is required, place the live feed of Daphnia mongholicus that has been synergistically fortified with nutrients in a refrigerated environment at 4-8℃, add Chlorella, and feed at a rate of 3-5 million Chlorella cells / mL to maintain nutrition. It can be preserved for about 24 hours with a survival rate of ≥85%.

[0218] The results of testing the live feed of *Daphnia mongolica* obtained in this embodiment after synergistic nutritional fortification are shown in Table 6:

[0219] Table 6

[0220]

[0221] Live feed of Daphnia mongholicus, after being fortified with synergistic nutrition, exhibits a bright orange-red color, and under a microscope, the intestines are found to be filled with synergistic nutritional fortifiers.

[0222] Aquaculture Application Trial: The live feed of *Daphnia mongolica* obtained above with synergistic nutritional fortification was fed to juvenile *Litopenaeus vannamei* (body length 0.9-1.2 cm). Three treatment groups were set up: Control Group 1: The feed consisted of basal feed (yeast), without supplementary feed or water quality control feed, and without intelligent synergistic fortification treatment, all other steps were the same as above. Control Group 2: The feed consisted of basal feed (Chlorella vulgaris) + supplementary feed, without water quality control feed, and without intelligent synergistic fortification treatment, all other steps were the same as above. The live feed of *Daphnia mongolica* obtained by the method of this invention was used as the experimental group. After 15 days of culture, the results were as follows: The survival rate of Control Group 1 was 72.5%, and the body color score was 2.2 / 5; the survival rate of Control Group 2 was 83.2%, and the body color score was 3.8 / 5; the survival rate of the experimental group was 94.6%, and the body color score was 4.7 / 5. The survival rate of the experimental group was 30.48% higher than that of control group 1 and 13.70% higher than that of control group 2. The astaxanthin content in the muscle of control group 1 was 9.14 mg / kg, the astaxanthin content in the muscle of control group 2 was 12.08 mg / kg, and the astaxanthin content in the muscle of the experimental group was 56.68 mg / kg. The shrimp in the experimental group had brighter body color, and the astaxanthin content in the muscle of the experimental group was 5.2 times that of control group 1; the astaxanthin content in the muscle of the experimental group was 3.7 times that of control group 2.

[0223] The activities of antioxidant enzymes SOD, CAT, and GPx in the serum of shrimp in control group 1 were 1.98 U / mg prot, 0.60 μmol / min / mg prot, and 3.07 nmol / min / mg prot, respectively. The activities of antioxidant enzymes SOD, CAT, and GPx in the serum of shrimp in control group 2 were 2.16 U / mg prot, 0.67 μmol / min / mg prot, and 3.45 nmol / min / mg prot, respectively. The activities of antioxidant enzymes SOD, CAT, and GPx in the serum of shrimp in the experimental group were 2.61 U / mg prot, 0.84 μmol / min / mg prot, and 4.22 nmol / min / mg prot, respectively. The activities of antioxidant enzymes SOD, CAT, and GPx in the serum of the experimental group were increased by 31.82%, 40.0%, and 37.46% compared with those in the serum of control group 1. The activities of antioxidant enzymes SOD, CAT, and GPx in the serum of the experimental group were increased by 20.83%, 25.37%, and 22.32% respectively compared with those in the control group 2.

[0224] Example 2

[0225] A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients includes the following steps:

[0226] The first step is the construction of a high-density intelligent chemostat culture system.

[0227] The high-density intelligent chemostat culture system includes a chemostat culture device 1, a constant flow liquid inlet system 2 located on the upper side of one side of the chemostat culture device 1, an automatic feeding system 3 located on the upper side of the other side of the chemostat culture device 1, an overflow harvesting system 4 located at the bottom of one side of the chemostat culture device 1, and an intelligent environmental monitoring system 5.

[0228] The bottom of the constant chemistry incubator 1 is equipped with a heating and temperature control device 101.

[0229] The automatic feeding system 3 consists of a feed storage tank 301, a timer controller 302, a metering pump 303, and a disperser 304 connected in sequence. The disperser 304 is located above the constant chemistry incubator 1.

[0230] The overflow harvesting system 4 includes a first overflow port 401 located at the bottom of one side of the constant chemistry incubator 1.

[0231] The intelligent environmental monitoring system 5 includes a PLC controller 501 located outside the chemiluminescence incubator 1, a lighting device 505 (multiple devices, such as lamps) located directly above the outside of the chemiluminescence incubator 1, an environmental monitoring probe 502 located on one side inside the chemiluminescence incubator 1, a turbidity sensor 503 located on one side inside the chemiluminescence incubator 1, and an oxygenation device 504 (specifically, an aerator) located at the bottom inside the chemiluminescence incubator 1. The environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the lighting device 505 are connected to the PLC controller 501.

[0232] The heating and temperature control device 101 is connected to the PLC controller 501.

[0233] The timing controller 302 is connected to the PLC controller 501.

[0234] The side of the constant chemistry incubator 1 is provided with a heat insulation layer 102.

[0235] The chemiluminescence constant culture vessel 1 is selected from a culture tank.

[0236] The culture tank is made of plastic and has a working volume of 1200L.

[0237] The heating and temperature control device 101 is a stainless steel heating tube. When a stainless steel heating tube is selected, it is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0238] The constant flow liquid inlet system 2 is a peristaltic pump.

[0239] The first overflow port 401 is equipped with a valve, which is used to control the flow rate.

[0240] The second step is to enhance the preparation of the container.

[0241] The enhanced container 6 is equipped with a lighting system 605 on the top, a heating device 601 on one side of the bottom, a second overflow port 602 on the other side of the bottom, and a second multi-layer graded collector 603 on the second overflow port 602. It is also equipped with an oxygenation system, which includes an oxygenation device 604 (specifically an aerator) located outside the enhanced container 6. The oxygenation device 604 is connected to an air diffuser 606 through a pipe 607. The air diffuser 606 is located at the bottom of the enhanced container 6.

[0242] The strengthening container 6 is a strengthening cylinder with a working volume of 250L.

[0243] The reinforced cylinder is a cube.

[0244] The reinforced cylinder is made of plastic.

[0245] The heating device 601 is an integrated heating and temperature control heating rod.

[0246] The lighting system 605 is a plant tissue culture lamp.

[0247] The second overflow port 602 is equipped with a valve, which is used to control the flow rate.

[0248] The second multi-layer graded collector 603 is made of a tube that matches the second overflow port 602 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing sequentially.

[0249] The first screen mesh bag has a pore size of 250 micrometers.

[0250] The second sieve mesh bag has an aperture of 180 micrometers.

[0251] The third sieve mesh bag has a pore size of 150 micrometers.

[0252] The third step is the preparation of synergistic nutritional fortifiers.

[0253] The synergistic nutritional fortifier is made from the components listed in Table 7:

[0254] Table 7

[0255]

[0256] The total amount of the synergistic nutrient fortifier is 1000g. The above components are mixed in proportion, purified water is added, and the mixture is emulsified by high-speed shearing (12000 rpm, 15 minutes) to produce a stable synergistic nutrient fortifier.

[0257] Step 4: Seed source domestication

[0258] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 80 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1% to reach 28‰. The light intensity is 6000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 26℃. Feed daily with cultured *Chlorella vulgaris* at a rate of 3 million cells / mL. Cultivate for 5 days until *Daphnia mongolica* grows and reproduces normally.

[0259] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0260] Step 5, Cultivation Stage

[0261] Preparation of treated fresh seawater: Seawater is sequentially treated by sand filtration (pore size 0.1 mm), disinfection (using bleaching powder, achieving an effective chlorine concentration of 25 ppm, disinfection time of 24 hours, using sodium thiosulfate, and detecting residual chlorine in the neutralized and reduced water using starch-potassium iodide test paper), temperature adjustment (controlling the temperature at 26℃), and oxygenation (dissolved oxygen 7 mg / L) to obtain treated fresh seawater.

[0262] Add 1000L of treated fresh seawater to the chemostat culture vessel 1, control the parameters of the high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen, inoculate the genetically stable purified germplasm into the chemostat culture vessel 1 at an inoculation density of 800 germplasms / L, control the temperature using a heating and temperature control device 101, and simultaneously provide a heat preservation layer 102 for heat preservation;

[0263] Treated fresh seawater was continuously added to the chemostat culture vessel 1 via a constant flow inlet system 2 at a rate controlled at 5 L / h, resulting in a dilution rate D = 0.05 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 25,000~28,000 individuals / L;

[0264] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 7.5; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 6000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0265] The automatic feeding system 3 controls the continuous and quantitative feeding of feed. By measuring the density of *Daphnia mongolica* in seawater and combining it with the volume of seawater in the chemostat culture vessel 1, the total weight (wet weight) of *Daphnia mongolica* is calculated, and the theoretical daily feed intake of *Daphnia mongolica* is calculated. Then, based on water quality and microscopic observation of the intestinal fullness of *Daphnia mongolica*, the theoretical value is dynamically adjusted to determine the actual daily feed intake. The automatic feeding system 3 controls the time and flow rate through the timer controller 302 and the metering pump 303 to achieve continuous and quantitative feeding. The feed is stored in the feed storage tank 301 and is sequentially fed through the timer controller. The feed is evenly sprayed into the constant chemistry culture vessel 1 through the dispersant 304 and the metering pump 303. The feed storage tank 301 is equipped with three independent spaces to hold basic feed, supplementary feed, and water quality control feed, respectively. First, the basic feed is fed. The feeding amount is automatically adjusted by the timer controller 302, which is located in the seawater and controlled by the turbidity sensor 503. The supplementary feed is automatically adjusted to maintain the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation. Then, water quality control feed is added every 24 hours according to the water quality.

[0266] The basic feed consists of cultured Chlorella as the main feed, with a cell density of 40 million cells / mL and a feeding amount of 3 million cells / mL.

[0267] The supplementary feed is yeast, and the feeding amount is 80 mg / L.

[0268] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 30 mg / L, which is used to improve water quality and provide additional nutrition.

[0269] During the cultivation period, the PLC controller 501 automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device 101 controls the seawater temperature and displays it on the display screen of the PLC controller 501. The temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values ​​monitored by the environmental monitoring probe 502 are displayed on the display screen of the PLC controller 501. The turbidity of the seawater measured by the turbidity sensor 503 is displayed on the display screen of the PLC controller 501. The PLC controller 501 controls the lighting device 505, the environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the heating and temperature control device 101, thereby regulating various parameters.

[0270] During the cultivation phase, the seawater in chemostat 1 was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate of D = 0.15 h. -1 Controlled by dilution method;

[0271] During the cultivation phase, the number of *Daphnia mongolica* cells in the water samples is measured daily, with at least three samplings per batch (generally five). The average value is taken, and the total number of *Daphnia mongolica* cells per 1000 ml of water is calculated, which is the daphnia density. For example, five 10 ml centrifuge tubes are used to sample, and the number of *Daphnia mongolica* cells is counted under a stereomicroscope. After obtaining the average value, the daphnia density is calculated. When the daphnia density reaches 28,000 cells / L, the harvesting procedure is initiated. *Daphnia mongolica* cells are continuously harvested from the first overflow outlet 401, rinsed with clean seawater, with a recovery rate of 25%, and added to the enhancement container 6.

[0272] According to production needs, different sizes of *Daphnia mongolica* individuals are collected by grading and screening in the first multi-layer grading collector 402, and then separately fortified in different fortification containers and fed to aquatic animals that need to consume *Daphnia mongolica* of different sizes.

[0273] The first overflow port 401 is equipped with a valve to control the flow rate. The Mongolian naked belly daphne enters the first multi-layer classifier 402 in sequence into the first screen silk mesh bag, then into the second screen silk mesh bag, and finally into the third screen silk mesh bag.

[0274] The first sieve mesh bag has a pore size of 250 micrometers, which can collect individuals of *Daphnia mongolica* larger than 250 micrometers; the second sieve mesh bag has a pore size of 180 micrometers, which can collect individuals of *Daphnia mongolica* with a size of 180-249 micrometers; and the third sieve mesh bag has a pore size of 150 micrometers, which can collect individuals of *Daphnia mongolica* with a size of 150-179 micrometers.

[0275] Step 6: Intelligent Collaborative Enhancement Processing

[0276] Individuals of *Daphnia mongolica* obtained from the previous step of graded screening were placed in different enhancement containers 6 for intelligent collaborative enhancement treatment.

[0277] The enhanced container 6 contained 200L of treated fresh seawater with a pH of 8.3, and the density of *Daphnia mongolica* was controlled at 35,000 cells / mL. The temperature was controlled at 26℃ by the heating device 601. During the enhanced period, the light intensity was controlled at 4000 lx by the light system 605, and the dissolved oxygen in the seawater was controlled at ≥5 mg / L by the oxygenation system. The synergistic nutrient enhancer was added to the enhanced container 6 at a ratio of 1.5mL per liter of seawater. The enhanced period was 5 hours. Samples were taken to test the content of astaxanthin, fatty acids, and amino acids in *Daphnia mongolica*.

[0278] After the enhancement was completed, the harvesting process was started, and Daphnia mongholica was continuously harvested from the second overflow outlet 602. The Daphnia mongholica was rinsed with clean seawater to obtain live food for Daphnia mongholica that had been enhanced with synergistic nutrition.

[0279] During harvesting, a second multi-layer grading collector 603 with different sieves and silk screens is used for grading and harvesting, and then the products are fed to aquatic species that need to consume different sizes of food.

[0280] Step 7, Feeding after fortification: Live feed for Daphnia mongholicus that has been fortified with synergistic nutrition can be fed directly. It is advisable to finish feeding within 2 hours after harvesting to maintain the best nutritional quality.

[0281] Step 8, Preservation: When short-term preservation is required, place the live feed of Daphnia mongholicus that has been synergistically fortified with nutrients in a refrigerated environment at 4-8℃, add Chlorella, and feed at a rate of 3-5 million Chlorella cells / mL to maintain nutrition. It can be preserved for about 24 hours with a survival rate of ≥85%.

[0282] The results of testing the live feed of *Daphnia mongolica* obtained in this embodiment after synergistic nutritional fortification are shown in Table 8:

[0283] Table 8

[0284]

[0285] Live feed of Daphnia mongholicus, after being fortified with synergistic nutrition, exhibits a bright orange-red color, and under a microscope, the intestines are found to be filled with synergistic nutritional fortifiers.

[0286] Example 3

[0287] A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients includes the following steps:

[0288] The first step is the construction of a high-density intelligent chemostat culture system.

[0289] The high-density intelligent chemostat culture system includes a chemostat culture device 1, a constant flow liquid inlet system 2 located on the upper side of one side of the chemostat culture device 1, an automatic feeding system 3 located on the upper side of the other side of the chemostat culture device 1, an overflow harvesting system 4 located at the bottom of one side of the chemostat culture device 1, and an intelligent environmental monitoring system 5.

[0290] The bottom of the constant chemistry incubator 1 is equipped with a heating and temperature control device 101.

[0291] The automatic feeding system 3 consists of a feed storage tank 301, a timer controller 302, a metering pump 303, and a disperser 304 connected in sequence. The disperser 304 is located above the constant chemistry incubator 1.

[0292] The overflow harvesting system 4 includes a first overflow port 401 located at the bottom of one side of the constant chemistry incubator 1.

[0293] The intelligent environmental monitoring system 5 includes a PLC controller 501 located outside the chemiluminescence incubator 1, a lighting device 505 (multiple devices, such as lamps) located directly above the outside of the chemiluminescence incubator 1, an environmental monitoring probe 502 located on one side inside the chemiluminescence incubator 1, a turbidity sensor 503 located on one side inside the chemiluminescence incubator 1, and an oxygenation device 504 (specifically, an aerator) located at the bottom inside the chemiluminescence incubator 1. The environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the lighting device 505 are connected to the PLC controller 501.

[0294] The heating and temperature control device 101 is connected to the PLC controller 501.

[0295] The timing controller 302 is connected to the PLC controller 501.

[0296] The side of the constant chemistry incubator 1 is provided with a heat insulation layer 102.

[0297] The chemiluminescence constant culture vessel 1 is selected from a culture tank.

[0298] The culture tank is made of plastic and has a working volume of 1200L.

[0299] The heating and temperature control device 101 is a stainless steel heating tube. When a stainless steel heating tube is selected, it is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0300] The constant flow liquid inlet system 2 is a peristaltic pump.

[0301] The first overflow port 401 is equipped with a valve, which is used to control the flow rate.

[0302] The second step is to enhance the preparation of the container.

[0303] The enhanced container 6 is equipped with a lighting system 605 on the top, a heating device 601 on one side of the bottom, a second overflow port 602 on the other side of the bottom, and a second multi-layer graded collector 603 on the second overflow port 602. It is also equipped with an oxygenation system, which includes an oxygenation device 604 (specifically an aerator) located outside the enhanced container 6. The oxygenation device 604 is connected to an air diffuser 606 through a pipe 607. The air diffuser 606 is located at the bottom of the enhanced container 6.

[0304] The strengthening container 6 is a strengthening cylinder with a working volume of 250L.

[0305] The reinforced cylinder is a cube.

[0306] The reinforced cylinder is made of plastic.

[0307] The heating device 601 is an integrated heating and temperature control heating rod.

[0308] The lighting system 605 is a plant tissue culture lamp.

[0309] The second overflow port 602 is equipped with a valve, which is used to control the flow rate.

[0310] The second multi-layer graded collector 603 is made of a tube that matches the second overflow port 602 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing sequentially.

[0311] The first screen mesh bag has a pore size of 250 micrometers.

[0312] The second sieve mesh bag has an aperture of 180 micrometers.

[0313] The third sieve mesh bag has a pore size of 150 micrometers.

[0314] The third step is the preparation of synergistic nutritional fortifiers.

[0315] The synergistic nutritional fortifier is made from the components listed in Table 9:

[0316] Table 9

[0317]

[0318] The total amount of the synergistic nutrient fortifier is 1000g. The above components are mixed in proportion, purified water is added, and the mixture is emulsified by high-speed shearing (12000 rpm, 15 minutes) to produce a stable synergistic nutrient fortifier.

[0319] Step 4: Seed source domestication

[0320] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 80 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1% to reach 28‰. The light intensity is 6000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 26℃. Feed daily with cultured *Chlorella vulgaris* at a rate of 3 million cells / mL. Cultivate for 5 days until *Daphnia mongolica* grows and reproduces normally.

[0321] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0322] Step 5, Cultivation Stage

[0323] Preparation of treated fresh seawater: Seawater is sequentially treated by sand filtration (pore size 0.1 mm), disinfection (using bleaching powder, achieving an effective chlorine concentration of 25 ppm, disinfection time of 24 hours, using sodium thiosulfate, and detecting residual chlorine in the neutralized and reduced water using starch-potassium iodide test paper), temperature adjustment (controlling the temperature at 26℃), and oxygenation (dissolved oxygen 7 mg / L) to obtain treated fresh seawater.

[0324] Add 1000L of treated fresh seawater to the chemostat culture vessel 1, control the parameters of the high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen, inoculate the genetically stable purified germplasm into the chemostat culture vessel 1 at an inoculation density of 800 germplasms / L, control the temperature using a heating and temperature control device 101, and simultaneously provide a heat preservation layer 102 for heat preservation;

[0325] Treated fresh seawater was continuously added to the chemostat culture vessel 1 via a constant flow inlet system 2 at a rate controlled at 5 L / h, resulting in a dilution rate D = 0.05 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 25,000~28,000 individuals / L;

[0326] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 7.5; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 6000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0327] The automatic feeding system 3 controls the continuous and quantitative feeding of feed. By measuring the density of *Daphnia mongolica* in seawater and combining it with the volume of seawater in the chemostat culture vessel 1, the total weight (wet weight) of *Daphnia mongolica* is calculated, and the theoretical daily feed intake of *Daphnia mongolica* is calculated. Then, based on water quality and microscopic observation of the intestinal fullness of *Daphnia mongolica*, the theoretical value is dynamically adjusted to determine the actual daily feed intake. The automatic feeding system 3 controls the time and flow rate through the timer controller 302 and the metering pump 303 to achieve continuous and quantitative feeding. The feed is stored in the feed storage tank 301 and is sequentially fed through the timer controller. The feed is evenly sprayed into the constant chemistry culture vessel 1 through the dispersant 304 and the metering pump 303. The feed storage tank 301 is equipped with three independent spaces to hold basic feed, supplementary feed, and water quality control feed, respectively. First, the basic feed is fed. The feeding amount is automatically adjusted by the timer controller 302, which is located in the seawater and controlled by the turbidity sensor 503. The supplementary feed is automatically adjusted to maintain the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation. Then, water quality control feed is added every 24 hours according to the water quality.

[0328] The basic feed consists of cultured Chlorella as the main feed, with a cell density of 40 million cells / mL and a feeding amount of 3 million cells / mL.

[0329] The supplementary feed is yeast, and the feeding amount is 80 mg / L.

[0330] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 30 mg / L, which is used to improve water quality and provide additional nutrition.

[0331] During the cultivation period, the PLC controller 501 automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device 101 controls the seawater temperature and displays it on the display screen of the PLC controller 501. The temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values ​​monitored by the environmental monitoring probe 502 are displayed on the display screen of the PLC controller 501. The turbidity of the seawater measured by the turbidity sensor 503 is displayed on the display screen of the PLC controller 501. The PLC controller 501 controls the lighting device 505, the environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the heating and temperature control device 101, thereby regulating various parameters.

[0332] During the cultivation phase, the seawater in chemostat 1 was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate of D = 0.15 h. -1 Controlled by dilution method;

[0333] During the cultivation phase, the number of *Daphnia mongolica* cells in the water samples is measured daily, with at least three samplings per batch (generally five). The average value is taken, and the total number of *Daphnia mongolica* cells per 1000 ml of water is calculated, which is the daphnia density. For example, five 10 ml centrifuge tubes are used to sample, and the number of *Daphnia mongolica* cells is counted under a stereomicroscope. After obtaining the average value, the daphnia density is calculated. When the daphnia density reaches 28,000 cells / L, the harvesting procedure is initiated. *Daphnia mongolica* cells are continuously harvested from the first overflow outlet 401, rinsed with clean seawater, with a recovery rate of 25%, and added to the enhancement container 6.

[0334] All individuals of different sizes of *Daphnia mongolica* were collected from the first overflow outlet 401.

[0335] Step 6: Intelligent Collaborative Enhancement Processing

[0336] The enhanced container 6 contained 200L of treated fresh seawater with a pH of 8.3, and the density of *Daphnia mongolica* was controlled at 35,000 cells / mL. The temperature was controlled at 26℃ by the heating device 601. During the enhanced period, the light intensity was controlled at 4000 lx by the light system 605, and the dissolved oxygen in the seawater was controlled at ≥5 mg / L by the oxygenation system. The synergistic nutrient enhancer was added to the enhanced container 6 at a ratio of 1.5mL per liter of seawater. The enhanced period was 5 hours. Samples were taken to test the content of astaxanthin, fatty acids, and amino acids in *Daphnia mongolica*.

[0337] After the enhancement was completed, the harvesting process was started, and Daphnia mongholica was continuously harvested from the second overflow outlet 602. The Daphnia mongholica was rinsed with clean seawater to obtain live food for Daphnia mongholica that had been enhanced with synergistic nutrition.

[0338] During harvesting, a second multi-layer grading collector 603 with different sieves and silk screens is used for grading and harvesting, and then the products are fed to aquatic species that need to consume different sizes of food.

[0339] Step 7, Feeding after fortification: Live feed for Daphnia mongholicus that has been fortified with synergistic nutrition can be fed directly. It is advisable to finish feeding within 2 hours after harvesting to maintain the best nutritional quality.

[0340] Step 8, Preservation: When short-term preservation is required, place the live feed of Daphnia mongholicus that has been synergistically fortified with nutrients in a refrigerated environment at 4-8℃, add Chlorella, and feed at a rate of 3-5 million Chlorella cells / mL to maintain nutrition. It can be preserved for about 24 hours with a survival rate of ≥85%.

[0341] The results of testing the live feed of *Daphnia mongolica* obtained in this embodiment after synergistic nutritional fortification are shown in Table 10:

[0342] Table 10

[0343]

[0344] Live feed of Daphnia mongholicus, after being fortified with synergistic nutrition, exhibits a bright orange-red color, and under a microscope, the intestines are found to be filled with synergistic nutritional fortifiers.

[0345] Example 4

[0346] A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients includes the following steps:

[0347] The first step is the construction of a high-density intelligent chemostat culture system.

[0348] The high-density intelligent chemostat culture system includes a chemostat culture device 1, a constant flow liquid inlet system 2 located on the upper side of one side of the chemostat culture device 1, an automatic feeding system 3 located on the upper side of the other side of the chemostat culture device 1, an overflow harvesting system 4 located at the bottom of one side of the chemostat culture device 1, and an intelligent environmental monitoring system 5.

[0349] The bottom of the constant chemistry incubator 1 is equipped with a heating and temperature control device 101.

[0350] The automatic feeding system 3 consists of a feed storage tank 301, a timer controller 302, a metering pump 303, and a disperser 304 connected in sequence. The disperser 304 is located above the constant chemistry incubator 1.

[0351] The overflow harvesting system 4 includes a first overflow port 401 located at the bottom of one side of the constant chemistry incubator 1.

[0352] The intelligent environmental monitoring system 5 includes a PLC controller 501 located outside the chemiluminescence incubator 1, a lighting device 505 (multiple devices, such as lamps) located directly above the outside of the chemiluminescence incubator 1, an environmental monitoring probe 502 located on one side inside the chemiluminescence incubator 1, a turbidity sensor 503 located on one side inside the chemiluminescence incubator 1, and an oxygenation device 504 (specifically, an aerator) located at the bottom inside the chemiluminescence incubator 1. The environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the lighting device 505 are connected to the PLC controller 501.

[0353] The heating and temperature control device 101 is connected to the PLC controller 501.

[0354] The timing controller 302 is connected to the PLC controller 501.

[0355] The side of the constant chemistry incubator 1 is provided with a heat insulation layer 102 (that is, a foam module heat insulation layer is placed in the cement mortar inside the cement pool wall).

[0356] The chemostat culture vessel 1 is selected from the culture tank.

[0357] The cultivation tank is a cement cultivation tank, made of concrete, rectangular in shape, with a unit working volume of 72m³. 3 Stainless steel heating pipes were laid around the bottom of the cement pool.

[0358] The heating and temperature control device 101 is a stainless steel heating tube. When a stainless steel heating tube is selected, it is connected to the boiler. The water temperature is increased through boiler heating and heat conduction by the stainless steel heating tube. When the preset water temperature is reached, the temperature control system automatically stops heating.

[0359] The constant flow liquid inlet system 2 is a peristaltic pump.

[0360] The first overflow port 401 is equipped with a valve, which is used to control the flow rate.

[0361] The second step is to enhance the preparation of the container.

[0362] The enhanced container 6 is equipped with a lighting system 605 on the top, a heating device 601 on one side of the bottom, a second overflow port 602 on the other side of the bottom, and a second multi-layer graded collector 603 on the second overflow port 602. It is also equipped with an oxygenation system, which includes an oxygenation device 604 (specifically an aerator) located outside the enhanced container 6. The oxygenation device 604 is connected to an air diffuser 606 through a pipe 607. The air diffuser 606 is located at the bottom of the enhanced container 6.

[0363] The strengthening container 6 is a strengthening cylinder with a working volume of 1000L, and there are 8 of them.

[0364] The reinforced cylinder is a cube.

[0365] The reinforced cylinder is made of plastic.

[0366] The heating device 601 is an integrated heating and temperature control heating rod.

[0367] The lighting system 605 is a plant tissue culture lamp.

[0368] The second overflow port 602 is equipped with a valve, which is used to control the flow rate.

[0369] The second multi-layer graded collector 603 is made of a tube that matches the second overflow port 602 and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the tube, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing sequentially.

[0370] The first screen mesh bag has a pore size of 250 micrometers.

[0371] The second sieve mesh bag has an aperture of 180 micrometers.

[0372] The third sieve mesh bag has a pore size of 150 micrometers.

[0373] The third step is the preparation of synergistic nutritional fortifiers.

[0374] The synergistic nutritional fortifier is made from the components listed in Table 11:

[0375] Table 11

[0376]

[0377] The total amount of the synergistic nutrient fortifier is 1000g. The above components are mixed in proportion, purified water is added, and the mixture is emulsified by high-speed shearing (12000 rpm, 15 minutes) to produce a stable synergistic nutrient fortifier.

[0378] Step 4: Seed source domestication

[0379] Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 80 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1% to reach 28‰. The light intensity is 6000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 26℃. Feed daily with cultured *Chlorella vulgaris* at a rate of 3 million cells / mL. Cultivate for 5 days until *Daphnia mongolica* grows and reproduces normally.

[0380] After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

[0381] Step 5, Cultivation Stage

[0382] Preparation of treated fresh seawater: Seawater is sequentially treated by sand filtration (pore size 0.1 mm), disinfection (using bleaching powder, achieving an effective chlorine concentration of 25 ppm, disinfection time of 24 hours, using sodium thiosulfate, and detecting residual chlorine in the neutralized and reduced water using starch-potassium iodide test paper), temperature adjustment (controlling the temperature at 26℃), and oxygenation (dissolved oxygen 7 mg / L) to obtain treated fresh seawater.

[0383] Add 60 mg of [acid] to incubator 1. 3The treated fresh seawater is used to control the parameters of a high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen. Genetically stable purified germplasm is inoculated into chemostat culture vessel 1 at an inoculation density of 850 germplasms / L. The temperature is controlled by a heating and temperature control device 101, and a heat preservation layer 102 is also provided for heat preservation.

[0384] Treated fresh seawater was continuously added to the chemostat culture vessel 1 via a constant flow inlet system 2 at a rate controlled at 8 L / h, resulting in a dilution rate D = 0.08 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 25,000~28,000 individuals / L;

[0385] The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 26℃; b) Salinity: 28‰; c) pH: 8.1; d) Dissolved oxygen: 7 mg / L; e) Light intensity: 5000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L.

[0386] The automatic feeding system 3 controls the continuous and quantitative feeding of feed. By measuring the density of *Daphnia mongolica* in seawater and combining it with the volume of seawater in the chemostat culture vessel 1, the total weight (wet weight) of *Daphnia mongolica* is calculated, and the theoretical daily feed intake of *Daphnia mongolica* is calculated. Then, based on water quality and microscopic observation of the intestinal fullness of *Daphnia mongolica*, the theoretical value is dynamically adjusted to determine the actual daily feed intake. The automatic feeding system 3 controls the time and flow rate through the timer controller 302 and the metering pump 303 to achieve continuous and quantitative feeding. The feed is stored in the feed storage tank 301 and is sequentially fed through the timer controller. The feed is evenly sprayed into the constant chemistry culture vessel 1 through the dispersant 304 and the metering pump 303. The feed storage tank 301 is equipped with three independent spaces to hold basic feed, supplementary feed, and water quality control feed, respectively. First, the basic feed is fed. The feeding amount is automatically adjusted by the timer controller 302, which is located in the seawater and controlled by the turbidity sensor 503. The supplementary feed is automatically adjusted to maintain the turbidity of the seawater between 0.3 and 0.5 (OD680). Supplementary feed is added every 12 hours during the middle stage of cultivation. Then, water quality control feed is added every 24 hours according to the water quality.

[0387] The basic feed consists of cultured Chlorella as the main feed, with a cell density of 40 million cells / mL and a feeding amount of 4 million cells / mL.

[0388] The supplementary feed is yeast, and the feeding amount is 60 mg / L.

[0389] The water quality control feed is photosynthetic bacteria (PSB), and the feeding amount is 20 mg / L, which is used to improve water quality and provide additional nutrition.

[0390] During the cultivation period, the PLC controller 501 automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device 101 controls the seawater temperature and displays it on the display screen of the PLC controller 501. The temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values ​​monitored by the environmental monitoring probe 502 are displayed on the display screen of the PLC controller 501. The turbidity of the seawater measured by the turbidity sensor 503 is displayed on the display screen of the PLC controller 501. The PLC controller 501 controls the lighting device 505, the environmental monitoring probe 502, the turbidity sensor 503, the oxygenation device 504, and the heating and temperature control device 101, thereby regulating various parameters.

[0391] During the cultivation phase, the seawater in chemostat 1 was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate of D = 0.2 h. -1 Controlled by dilution method;

[0392] During the cultivation phase, the number of *Daphnia mongolica* cells in the water samples is measured daily, with at least three samplings per batch (generally five). The average value is taken, and the total number of *Daphnia mongolica* cells per 1000 ml of water is calculated, which is the daphnia density. For example, five 10 ml centrifuge tubes are used to sample, and the number of *Daphnia mongolica* cells is counted under a stereomicroscope. After obtaining the average value, the daphnia density is calculated. When the daphnia density reaches 28,000 cells / L, the harvesting procedure is initiated. *Daphnia mongolica* cells are continuously harvested from the first overflow outlet 401, rinsed with clean seawater, with a harvest rate of 10%, and added to the enhancement container 6.

[0393] All individuals of different sizes of *Daphnia mongolica* were collected from the first overflow outlet 401.

[0394] After 45 days of continuous culture, the density of *Daphnia mongolica* remained stable at 25,000–28,000 cells / L, and the total daily water volume collected was 6 m³. 3 Daily output reaches 150-168 million units, with a total cumulative output of 6.75-7.56 billion units during the production period.

[0395] Step 6: Intelligent Collaborative Enhancement Processing

[0396] The enhanced container 6 contained 800L of treated fresh seawater with a pH of 8.3, and the density of *Daphnia mongolica* was controlled at 28,000 cells / mL. The temperature was controlled at 26℃ by the heating device 601. During the enhanced period, the light intensity was controlled at 4000 lx by the light system 605, and the dissolved oxygen in the seawater was controlled at ≥5 mg / L by the oxygenation system. The synergistic nutrient enhancer was added to the enhanced container 6 at a ratio of 1.2mL per liter of seawater. The enhanced period was 5 hours. Samples were taken to test the content of astaxanthin, fatty acids, and amino acids in *Daphnia mongolica*.

[0397] After the enhancement was completed, the harvesting process was started, and Daphnia mongholica was continuously harvested from the second overflow outlet 602. The Daphnia mongholica was rinsed with clean seawater to obtain live food for Daphnia mongholica that had been enhanced with synergistic nutrition.

[0398] During harvesting, a second multi-layer grading collector 603 with different sieves and silk screens is used for grading and harvesting, and then the products are fed to aquatic species that need to consume different sizes of food.

[0399] Step 7, Feeding after fortification: Live feed for Daphnia mongholicus that has been fortified with synergistic nutrition can be fed directly. It is advisable to finish feeding within 2 hours after harvesting to maintain the best nutritional quality.

[0400] Step 8, Preservation: When short-term preservation is required, place the live feed of Daphnia mongholicus that has been synergistically fortified with nutrients in a refrigerated environment at 4-8℃, add Chlorella, and feed at a rate of 3-5 million Chlorella cells / mL to maintain nutrition. It can be preserved for about 24 hours with a survival rate of ≥85%.

[0401] The results of testing the live feed of *Daphnia mongolica* obtained in this embodiment after synergistic nutritional fortification are shown in Table 12:

[0402] Table 12

[0403]

[0404] Live feed fortified with synergistic nutrients resulted in vibrant body color, and microscopic examination revealed the intestines saturated with synergistic nutrient fortifiers. The results indicate that the method of this invention not only significantly increased the astaxanthin content in *Daphnia mongolica*, but also simultaneously improved the content of fatty acids and amino acids, achieving the synergistic enrichment of multiple nutrients.

[0405] Aquaculture Application Trial: The live feed of *Daphnia mongolica* obtained above and synergistically fortified was fed to megalopa larvae of *Scylla paramamosain*. Three treatment groups were set up: Control Group 1: The feed consisted of basal feed (yeast), without supplemental feed or water quality control feed, and without intelligent synergistic fortification treatment, all other steps were the same as above. Control Group 2: The feed consisted of basal feed (Chlorella) + supplemental feed, without water quality control feed, and without intelligent synergistic fortification treatment, all other steps were the same as above. The live feed fortified with synergistic nutrition obtained using the method of this invention was used as the experimental group. After 6 days of rearing, the results were as follows: Control group 1 had a survival rate of 75.3%, a metamorphosis rate of 48.3%, and a body color score of 3.1 / 5; Control group 2 had a survival rate of 84.7%, a metamorphosis rate of 64.6%, and a body color score of 4.1 / 5; the experimental group had a survival rate of 97.5%, a metamorphosis rate of 83.1%, and a body color score of 4.8 / 5. The experimental group showed a 29.48% increase in survival rate and a 72.0% increase in metamorphosis rate compared to control group 1, and a 15.11% increase in survival rate and a 28.6% increase in metamorphosis rate compared to control group 2.

[0406] After feeding on Daphnia mongolica in control groups 1 and 2, the body color of the Daphnia mongolica larvae in the blue crab megalopa feeding group was grayish-white, their swimming behavior was not active, and they showed signs of sinking to the bottom. However, the Daphnia mongolica larvae in the experimental group showed the yellowish-brown color of natural megalopa larvae, their swimming was active, they fed vigorously, and they swam around in the water and on the surface without showing signs of sinking to the bottom.

[0407] After larvae of mud crabs fed on Daphnia mongolica, the activities of antioxidant enzymes SOD, CAT, and GPx in the muscle of control group 1 were 10.50 U / mg prot, 5.50 μmol / min / mg prot, and 33.36 nmol / min / mg prot, respectively; the activities of antioxidant enzymes SOD, CAT, and GPx in the muscle of control group 2 were 11.26 U / mg prot, 6.05 μmol / min / mg prot, and 35.10 nmol / min / mg prot, respectively; and the activities of antioxidant enzymes SOD, CAT, and GPx in the muscle of the experimental group were 14.94 U / mg prot, 7.32 μmol / min / mg prot, and 45.43 nmol / min / mg prot, respectively. The activities of antioxidant enzymes SOD, CAT, and GPx in the experimental group were increased by 42.29%, 33.09%, and 36.18% compared with those in control group 1, respectively. The activities of antioxidant enzymes SOD, CAT, and GPx in the experimental group were increased by 32.68%, 20.99%, and 29.43% respectively compared with those in the control group 2.

[0408] Example 1 of cultivation: using one effective culture water volume of 2m 3 The *Daphnia mongolica* species were cultured in plastic tanks for 70 days under constant temperature conditions. During this period, there were 65 harvests, with a culture density of 28,000 cells / mL and an average daily yield of 0.56 kg.

[0409] In Example 2, one effective culture water volume of 8m was used. 3 The fiberglass water tank was used to culture Daphnia mongolica for 45 days, during which 40 harvests were conducted. The culture density was 28,000 cells / mL, and the average daily yield was 0.54 kg.

[0410] In Example 3, one effective culture water volume of 24m³ was used. 3 The Mongolian naked-bellied daphnia was cultured in cement pools for 38 days, with a total of 32 harvests during this period. The culture density was 25,000 cells / mL, and the average daily yield was 0.46 kg.

[0411] In Example 4, one effective culture water volume of 60m was used. 3 The Mongolian naked-bellied daphnia was cultured in cement pools for 38 days, with a total of 32 harvests during this period. The culture density was 25,000 cells / mL, and the average daily yield was 0.44 kg.

[0412] As shown in Table 13:

[0413] Table 13

[0414]

[0415] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients, characterized in that... Includes the following steps: Step 1: Construction of a high-density intelligent chemostat culture system: The high-density intelligent chemostat culture system includes a chemostat culture unit, a constant flow liquid inlet system located on the upper part of one side of the chemostat culture unit, an automatic feeding system located on the upper part of the other side of the chemostat culture unit, an overflow harvesting system located at the bottom of one side of the chemostat culture unit, and is equipped with an intelligent environmental monitoring system. The bottom of the constant temperature incubator is equipped with a heating and temperature control device; The automatic feeding system consists of a feed storage tank, a timer controller, a metering pump, and a disperser connected in sequence, with the disperser located above the chemiluminescent incubator. The overflow harvesting system includes a first overflow port located at the bottom of one side of the chemiluminescence incubator; The intelligent environmental monitoring system includes a PLC controller located outside the chemiluminescent culture vessel, a lighting device located directly above the outside of the chemiluminescent culture vessel, an environmental monitoring probe located on one side inside the chemiluminescent culture vessel, a turbidity sensor located on one side inside the chemiluminescent culture vessel, and an oxygenation device located at the bottom inside the chemiluminescent culture vessel. The environmental monitoring probe, turbidity sensor, oxygenation device, and lighting device are connected to the PLC controller. The heating and temperature control device is connected to the PLC controller; The timing controller is connected to the PLC controller; The second step is to strengthen the preparation of the container: The enhanced container is equipped with a lighting system at the top, a heating device on one side of the bottom, a second overflow port on the other side of the bottom, and an oxygenation system. The oxygenation system includes an oxygenation device located outside the enhanced container, which is connected to an air diffuser stone via a pipeline. The air diffuser stone is located at the bottom of the enhanced container. The third step involves a synergistic nutritional fortifier made from the following components in parts by weight: 100-200 parts astaxanthin, 50-100 parts DHA algal oil microcapsules, 50-100 parts EPA algal oil microcapsules, 10-30 parts magnesium vitamin C phosphate, 5-15 parts vitamin E acetate, 20-50 parts soybean lecithin, 30-60 parts β-cyclodextrin, 20-40 parts concentrated Chlorella vulgaris, 10-30 parts Rhodopseudomonas palustris, and purified water to bring the total to 1000 parts. The above components are mixed in proportion, purified water is added, and high-speed shear emulsification is performed to produce a stable synergistic nutrient fortifier. Step 4, the training phase: Fresh, treated seawater was added to the chemostat culture vessel. The parameters of the high-density intelligent chemostat culture system, including temperature, salinity, pH, dissolved oxygen, light intensity, photoperiod, and ammonia nitrogen, were controlled. Genetically stable purified germplasm was inoculated into the chemostat culture vessel at a density of 500-1000 germplasms / L. The temperature was controlled by a heating and temperature control device. Treated fresh seawater was continuously added to the chemostat culture vessel using a constant flow inlet system at a rate controlled at 4–10 L / h, resulting in a dilution rate D of 0.05–0.20 h. -1 This ensures that the population of Daphnia mongholica is in a dynamic equilibrium state during the exponential growth phase, with the culture density maintained at 15,000 to 30,000 individuals / L; The parameters of the high-density intelligent chemostat culture system are set as follows: a) Temperature: 25~28℃; b) Salinity: 2~30‰; c) pH: 7.0~8.5; d) Dissolved oxygen: 5~8 mg / L; e) Light intensity: 5000~7000 lx; ​​f) Photoperiod: L∶D=12h∶12h; g) Ammonia nitrogen: <0.5 mg / L. The automatic feeding system controls the continuous and quantitative feeding of feed. This system uses a timer controller and a metering pump to control the time and flow rate, achieving continuous and quantitative feeding. The feed is stored in a feed tank, passes through the timer controller and metering pump, and is finally evenly sprayed into the chemostat culture vessel via a disperser. The feed tank has three independent compartments for basic feed, supplementary feed, and water quality control feed, respectively. Basic feed is fed first, and the feeding amount is automatically adjusted by a turbidity sensor located in the seawater, which in turn automatically regulates the timer controller to maintain the turbidity of the seawater between 0.3 and 0.

5. During the middle stage of cultivation, supplementary feed is added every 12 hours, and then water quality control feed is added every 24 hours based on the water quality. During the cultivation period, the PLC controller automatically monitors multiple parameters of the cultivation environment. The heating and temperature control device controls the seawater temperature, which is displayed on the PLC controller's screen. The environmental monitoring probe detects the temperature, salinity, pH, dissolved oxygen, and ammonia nitrogen values, which are displayed on the PLC controller's screen. The turbidity sensor measures the seawater turbidity, which is displayed on the PLC controller's screen. The PLC controller controls the lighting device, environmental monitoring probe, turbidity sensor, oxygenation device, and heating and temperature control device, thereby regulating each parameter. When the density of Daphnia reaches 25,000 to 28,000 cells / L, the harvesting procedure is initiated. Daphnia mongholica is continuously harvested from the first overflow outlet, rinsed with clean seawater, and the harvest rate is 10% to 50%. The Daphnia mongholica is then added to the enhanced container. Step 5, Intelligent Collaborative Enhancement Processing: The enhanced container contained treated fresh seawater with a pH of 7.5-8.5, and the density of *Daphnia mongolica* was controlled at 20,000-40,000 cells / mL. The temperature was controlled at 25-28℃ using a heating device. During the enhanced period, the light intensity was controlled at 3,000-5,000 lx using a light system, and the dissolved oxygen in the seawater was controlled at ≥5 mg / L using an oxygenation system. The co-nutrient enhancer was added to the enhanced container at a ratio of 0.5-2.0 mL per liter of seawater. The enhanced period was 1-7 hours. Samples were taken to test the astaxanthin, fatty acid, and amino acid content in *Daphnia mongolica*. After the enhancement is completed, the harvesting process is started, and Daphnia mongholica is continuously harvested from the second overflow outlet. It is then rinsed with clean seawater to obtain live Daphnia mongholica food that has been enhanced with synergistic nutrition.

2. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that... The overflow harvesting system includes a first multi-layer graded collector located on the first overflow outlet; The side of the chemiluminescence incubator is provided with a heat insulation layer; The second overflow port is equipped with a second multi-layer graded collector.

3. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 2, characterized in that... The first multi-layer graded collector is made of a pipe that matches the first overflow port and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the pipe, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing in size sequentially.

4. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 2, characterized in that... The second multi-layer graded collector is made of a pipe that matches the second overflow port and a first screen silk mesh bag, a second screen silk mesh bag, and a third screen silk mesh bag that are sequentially fitted onto the pipe, with the aperture of the first screen silk mesh bag, the second screen silk mesh bag, and the third screen silk mesh bag decreasing in size sequentially.

5. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 3 or 4, characterized in that... The pore size of the first screen mesh bag is 250 micrometers; The second sieve mesh bag has an aperture of 180 micrometers; The third sieve mesh bag has a pore size of 150 micrometers.

6. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that... The preparation of the genetically stable purified germplasm includes the following steps: Collect *Daphnia mongolica* and its original water from saline-alkali waters into containers. The initial culture density is 60-100 cells / L. Cultivate in the original water, adding filtered seawater daily to increase the salinity by 1% to reach 25-30‰. The light intensity is 5000-7000 lx, the photoperiod is L:D = 12h:12h, and the temperature is maintained at 25-28℃. Feed daily with cultured *Chlorella vulgaris* until *Daphnia mongolica* grows and reproduces normally. After domestication, the Mongolian naked-bellied daphnia underwent 3-5 generations of parthenogenetic purification culture to obtain genetically stable purified germplasm.

7. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that... The basic feed is mainly cultured Chlorella, with a cell density of 30-50 million cells / mL, and the feeding amount of basic feed is 3-5 million cells / mL. The supplementary feed is yeast, and the feeding amount is 20~100 mg / L; The water quality control feed is photosynthetic bacteria, and the feeding amount is 10~50 mg / L.

8. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that... During the cultivation phase, the seawater in the chemostat culture vessel was sampled daily to check for the presence of protozoa. If protozoa contaminated the culture seawater, the influent flow rate was increased to a dilution rate D = 0.20~0.25 h. -1 This is controlled by dilution.

9. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that... The synergistic nutritional fortifier is made from the following components in parts by weight: 150 parts astaxanthin, 80 parts DHA algal oil microcapsules, 80 parts EPA algal oil microcapsules, 20 parts magnesium vitamin C phosphate, 10 parts vitamin E acetate, 30 parts soybean lecithin, 45 parts β-cyclodextrin, 25 parts concentrated Chlorella vulgaris, 15 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts. Alternatively, the synergistic nutritional fortifier is made from the following components in parts by weight: 120 parts astaxanthin, 65 parts DHA algal oil microcapsules, 90 parts EPA algal oil microcapsules, 20 parts magnesium vitamin C phosphate, 10 parts vitamin E acetate, 50 parts soybean lecithin, 45 parts β-cyclodextrin, 40 parts concentrated Chlorella vulgaris, 30 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

10. The method for chemostatic culture of *Daphnia mongolica* and synergistic enhancement of multiple indicators by nutrients according to claim 1, characterized in that, The synergistic nutritional fortifier is made from the following components in parts by weight: 175 parts astaxanthin, 85 parts DHA algal oil microcapsules, 85 parts EPA algal oil microcapsules, 15 parts magnesium vitamin C phosphate, 8 parts vitamin E acetate, 45 parts soybean lecithin, 40 parts β-cyclodextrin, 30 parts concentrated Chlorella vulgaris, 25 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts. Alternatively, the synergistic nutritional fortifier is made from the following components in parts by weight: 135 parts astaxanthin, 68 parts DHA algal oil microcapsules, 68 parts EPA algal oil microcapsules, 18 parts magnesium vitamin C phosphate, 8.5 parts vitamin E acetate, 25.5 parts soybean lecithin, 38.5 parts β-cyclodextrin, 21.5 parts concentrated Chlorella vulgaris, 12.5 parts Rhodopseudomonas palustris, and purified water to make up to 1000 parts.

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