Seawater culture pond environment regulation and control based on bottom nourishing and mixed microbial agents

Abstract

The invention relates to a seawater stichopus japonicus culture pond environment regulation and control method based on a bottom nourishing device and a mixed microbial agent and application. According to the method, the seawater stichopus japonicus culture pond is subjected to bottom nourishing operation, meanwhile, mixed microbial agents of rhodobacter sphaeroides, phosphorus phagocytosis bacillus, moraxella alpha and enterococcus faecalis are splashed, the water quality of the stichopus japonicus culture pond is improved, meanwhile, the immune function of stichopus japonicus is enhanced, and remarkable environmental and economic benefits are achieved.

Description

Technical Field

[0001] This invention belongs to the fields of microbial engineering technology and environmental remediation technology, specifically relating to a method for environmental control of sea cucumber farming ponds based on a bottom-feeding device and its application. Background Technology

[0002] With the continuous development of the marine aquaculture industry, pond culture has become an important method for sea cucumber farming. The dissolved oxygen and temperature in pond seawater change with the seasons, leading to an increase in harmful substances in the pond water environment during the thawing period and the high temperatures of summer, which can easily trigger outbreaks of sea cucumber diseases. A good pond water quality environment can not only absorb harmful substances in the water but also improve the disease resistance of sea cucumbers, creating a relatively stable growth environment, which is a key technical means to ensure successful farming.

[0003] However, traditional bottom aeration systems are costly, energy-intensive, and pose certain electrical safety hazards. Furthermore, the excrement and dead aquatic plants produced by sea cucumbers accumulate at the pond bottom through adsorption, complexation, and sedimentation, resulting in a hard bottom, insufficient dissolved oxygen, and difficulty in decomposing harmful substances such as nitrite, ammonia nitrogen, and sulfides, which can easily lead to the outbreak of diseases like sea cucumber skin peeling. Therefore, improving the micro-ecological environment of seawater aquaculture ponds is essential for the healthy development of sea cucumber pond farming and an effective means to ensure the quality and safety of aquatic products. In view of this, this invention is proposed. Summary of the Invention

[0004] To address the aforementioned technical problems in existing technologies, this invention seeks a novel approach to environmental control technology for sea cucumber farming ponds, and thus proposes this invention.

[0005] This invention includes at least the following three objectives:

[0006] 1) Seeking a mixed microbial agent for environmental control in sea cucumber farming ponds;

[0007] 2) Seeking a bottom-feeding device for environmental control in sea cucumber farming ponds;

[0008] 3) Seek a method for environmental control in sea cucumber farming ponds.

[0009] To achieve the above objectives, the present invention specifically proposes the following technical solution:

[0010] This invention first provides a mixed microbial agent for environmental control in sea cucumber farming ponds, the mixed microbial agent comprising Rhodobacter sphaeroides, Fictibacillus phosphorivorans, Alteromonas abrolhosensis, and Enterococcus faecalis.

[0011] Furthermore, the *Enterococcus faecalis* has the accession number CICC 23658, the *Pseudomonas phosphatae* has the accession number CGMCC No. 36465, the *Altomorella* has the accession number CGMCC No. 36464, and the *Rhodotorula globulus* has the accession number ATCC 17023.

[0012] Furthermore, the concentration ratio of Rhodopseudomonas aeruginosa, Bacillus phosphatidylcholine, Altomoraxella catarrhalis, and Enterococcus faecalis in the mixed bacterial agent is 100-10:10-1:10-1:10-1.

[0013] Preferably, the concentration of Rhodopseudomonas spp. in the mixed bacterial agent is 10. 7 -10 9 cfu / ml, the concentration of the phosphorus-eating Pseudomonas aeruginosa was 10. 6 -10 8 cfu / ml, the concentration of Altomoraxella bacillus was 10. 6 -10 8 cfu / ml, the concentration of Enterococcus faecalis is 10. 6 -10 8 cfu / ml; more preferably, the concentration of Rhodopseudomonas spp. in the mixed bacterial agent is 10. 8 -10 9 cfu / ml, concentration of Bacillus phosphatidylcholine was 10. 7 -10 8 cfu / ml, Altomoraxella concentration was 10 7 -10 8 cfu / ml, Enterococcus faecalis concentration 10 7 -10 8 cfu / ml.

[0014] In some aspects, the specific preparation steps of the mixed bacterial agent are as follows: Take 10 8 -10 9 CFU / ml concentration of Rhodotorula glutinis stock solution, 10 7 -10 8 cfu / ml concentration of Lactobacillus salicylic acid, 10 7 -108 Altomorella at a concentration of CFU / ml and 10 7 -10 8 The four ingredients were mixed together, and then sterile seawater was added. The mixture was stored at 4°C for later use.

[0015] In some methods, the preparation method of the *Rhodotorula globulus* is as follows:

[0016] Step 1: Dissolve the freeze-dried Rhodospirillum lyophilized powder in sterile distilled water, streak the bacteria on Rhodospirillum complete vitamin medium (RCV medium) to activate it, and incubate under light in an incubator until round single colonies appear on the plate;

[0017] Step 2: Pick a single colony from the RCV solid medium and inoculate it into the RCV liquid medium. Shake and culture until the bacterial strain proliferates to the logarithmic growth phase.

[0018] Step 3: Centrifuge to collect bacterial cells, resuspend the bacterial cells in sterile seawater, and adjust the bacterial concentration to 10. 7 -10 9 cfu / ml, for later use.

[0019] Preferably, the preparation method is as follows:

[0020] Step 1: Dissolve the freeze-dried Rhodospirillum lyophilized powder in 1 ml of sterile distilled water. Activate the bacterial culture by streaking it onto Rhodospirillum complete vitamin medium (RCV medium). Incubate at 37 ℃ under light for 1-2 days until round single colonies appear on the plate.

[0021] Step 2: Pick a single colony from the RCV solid medium and inoculate it into the RCV liquid medium at pH 8.0. Incubate at 37±1 ℃ with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0022] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 7 -10 9 cfu / ml (preferably 10) 8 -10 9 (cfu / ml), for later use.

[0023] In some aspects, the preparation method of the phosphorus-eating Phanerozoellates is as follows:

[0024] Step 1: After slowing down the cryopreservation solution of the Bacillus phosphatidylis, it is activated by streaking on seawater 2216E medium and placed in an incubator for light culture until round single colonies appear on the plate;

[0025] Step 2: Pick a single colony from the seawater 2216E medium and inoculate it into the seawater 2216E liquid medium. Shake and culture until the bacterial strain proliferates to the logarithmic growth phase.

[0026] Step 3: Centrifuge to collect bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10. 6 -10 8 cfu / ml, for later use.

[0027] Preferably, the preparation method is as follows:

[0028] Step 1: After slowing down the cryopreservation solution of the Phosphophyllobacterium phoensis, it is activated by streaking on seawater 2216E medium and placed in an incubator at 37 ℃ under light for 1-2 days until round single colonies appear on the plate;

[0029] Step 2: Pick a single colony from the seawater 2216E solid medium and inoculate it into 2216E liquid medium at pH 7.0. Incubate at 37±1 ℃ with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0030] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 6 -10 8 cfu / ml (preferably 10) 7 -10 8 (cfu / ml), for later use.

[0031] In some respects, the method for preparing the *Altorolazoides* stock solution is as follows:

[0032] Step 1: After slowing down the cryopreservation solution of Altorolaella, it is activated by streaking on seawater 2216E solid medium and placed in an incubator for light cultivation until round single colonies appear on the plate;

[0033] Step 2: Pick a single colony from the 2216E solid culture medium of seawater, inoculate it into the 2216E liquid culture medium, and shake it until the bacterial strain proliferates to the logarithmic growth phase;

[0034] Step 3: Centrifuge to collect bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10. 6 -10 8 cfu / ml, for later use.

[0035] Preferably, the preparation method is as follows:

[0036] Step 1: After slowing down the cryopreservation solution of Altorolaella, streak it on seawater 2216E medium for activation, and place it in an incubator at 37 ℃ under light for 1-2 days until round single colonies appear on the plate;

[0037] Step 2: Pick a single colony from the seawater 2216E solid medium and inoculate it into 2216E liquid medium at pH 7.0. Incubate at 37±1 ℃ with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0038] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 6 -10 8 cfu / ml (preferably 10) 7 -10 8 (cfu / ml), for later use.

[0039] In some aspects, the method for preparing the *Enterococcus faecalis* is as follows:

[0040] Step 1: Dissolve the freeze-dried Enterococcus faecalis powder in sterile distilled water, activate the inoculum by streaking it on Brain Heart Extract Agar (BHI medium), and incubate under light until round single colonies appear on the plate;

[0041] Step 2: Pick a single colony from BHI agar medium and inoculate it into BHI broth medium. Shake and culture until the bacterial strain proliferates to the logarithmic growth phase.

[0042] Step 3: Centrifuge to collect bacterial cells, resuspend the bacterial cells in sterile seawater, and adjust the bacterial concentration to 10. 6 -10 8 cfu / ml, for later use.

[0043] Preferably, the preparation is as follows:

[0044] Step 1: Dissolve the freeze-dried Enterococcus faecalis powder in 1 ml of sterile distilled water. Activate the bacteria by streaking them onto Brain Heart Extract Agar (BHI medium). Incubate at 37 ℃ under light for 1-2 days until round single colonies appear on the plates.

[0045] Step 2: Pick a single colony from BHI agar medium and inoculate it into BHI broth medium at pH 8.0. Incubate at 37±1℃ with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0046] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 6 -10 8 cfu / ml (preferably 10) 7 -10 8 (cfu / ml), for later use.

[0047] This invention also provides a method for environmental control in sea cucumber farming ponds, comprising the following steps:

[0048] 1) Perform bottom-nourishing operations on sea cucumber farming ponds;

[0049] 2) After soaking the bottom, spray the aforementioned mixed bacterial agent into the water.

[0050] Furthermore, in step 1), the bottom-nourishing operation is performed using a bottom-nourishing device;

[0051] In some aspects, the bottom-feeding device comprises a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe; further, the bottom-feeding device comprises a pumping device (e.g., a water pump), an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe; the pumping device and the engine are located on the upper end of the float; the pumping device is connected to the drain pipe and the inlet pipe; the end of the drain pipe is connected to the horizontal water pipe, and the horizontal water pipe is vertically connected to the bottom-feeding pipe; the bottom-feeding pipe is perpendicular to the water surface and extends to the bottom.

[0052] More preferably, the pumping device and the engine are placed on the float, which ensures the entire equipment floats on the water surface. The pumping device is connected to two drain pipes and one inlet pipe, used for draining and drawing water respectively. The ends of the two drain pipes are connected to the two ends of the horizontal water pipe, which is horizontally positioned above the float and vertically connected to multiple bottom-feeding pipes, thereby distributing water from the drain pipes to the bottom-feeding pipes through the horizontal water pipes. The bottom-feeding pipes are perpendicular to the water surface and extend underwater to achieve bottom feeding. It is understood that a support plate (preferably a wooden board) can be installed on the float to better support the pumping device and the engine; however, considering that the float also serves a supporting function, a support plate is not a necessary structure.

[0053] More preferably, the bottom tube can be made of plastic, the number of bottom tubes is 10-16, the length of the bottom tube is 2-4 meters, and the diameter is 1-1.5 inches; the horsepower of the engine is 20-25 hp.

[0054] In some respects, the bottom-feeding device is assembled as follows: the pumping device is fixed to a floating buoy with an engine, the pumping device is connected to two drain pipes and one inlet pipe, the ends of the two drain pipes are then connected to both ends of a horizontal water pipe, and the lower end of the horizontal water pipe is connected to a corresponding bottom-feeding pipe according to the size of the pond for underwater bottom feeding.

[0055] Furthermore, in step 2), the concentration of the mixed bacterial agent is 0.5-1 ppm, and the dosage is 1 mL of mixed bacterial agent per cubic meter of water.

[0056] Furthermore, the bottom-nourishing and sprinkling operations are performed according to the water quality conditions using the bottom-nourishing device, and have the following characteristics:

[0057] After the ice melts in spring, when the water temperature rises to around 10°C, use the bottom-nourishing device to perform a thorough and deep bottom nourishing process, ensuring that the end of the bottom-nourishing tube is more than 10cm away from the bottom of the pool; preferably, nourish the bottom 3-4 times a week.

[0058] During the high-temperature period in summer, when the water temperature reaches above 26℃, the bottom-nourishing device is used for shallow bottom nourishing, ensuring that the end of the bottom-nourishing tube is more than 30cm away from the bottom of the pool; preferably, the bottom is nourished once a day, and the nourishing is stopped when the water becomes turbid and blocks light.

[0059] In spring, after the ice melts, pond water temperatures are low and dissolved oxygen levels are high. Deep bottom cleaning at this time allows harmful substances such as ammonia nitrogen and hydrogen sulfide in the bottom sediment to be fully exposed in the water and decomposed by the abundant oxygen, without harming the farmed animals. However, during the hot summer months, pond water temperatures are high and dissolved oxygen levels are low. In this case, the key to bottom cleaning is shallow and frequent cleaning, and the time should not be too long to prevent harmful substances in the bottom sediment from seeping out and consuming dissolved oxygen, which could easily cause oxygen deprivation and death in the farmed animals.

[0060] Therefore, in some preferred embodiments, the specific steps of moisturizing and spraying are as follows:

[0061] After the ice melts in spring, when the water temperature rises to around 10℃, perform a thorough deep bottom cleaning (at least 10cm from the bottom of the pool) 3-4 times a week. After each cleaning, apply 0.5-1 ppm of a mixed bacterial agent, using 1 mL of the mixed bacterial agent per cubic meter of water. During the high-temperature period in summer, when the water temperature reaches above 26℃, perform a shallow bottom cleaning (at least 30cm from the bottom of the pool). Stop when the water becomes turbid and requires light to be blocked. Do this once a day, and simultaneously apply 0.5-1 ppm of the mixed bacterial agent once a week, using 1 mL of the mixed bacterial agent per cubic meter of water.

[0062] This invention also provides a bottom-nurturing device for environmental control in sea cucumber farming ponds. The bottom-nurturing device consists of a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-nurturing pipe. The pumping device and the engine are located on the upper end of the float. The pumping device is connected to the drain pipe and the inlet pipe. The end of the drain pipe is connected to the horizontal water pipe, which is vertically connected to the bottom-nurturing pipe. The bottom-nurturing pipe is perpendicular to the water surface and extends underwater.

[0063] Preferably, the bottom-feeding device comprises a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe; the pumping device and the engine are located on the upper end of the float; the pumping device is connected to two drain pipes and one inlet pipe; the ends of the two drain pipes are respectively connected to the two ends of the horizontal water pipe, the horizontal water pipe is horizontally placed above the float and vertically connected to multiple bottom-feeding pipes; the bottom-feeding pipe is perpendicular to the water surface and extends underwater.

[0064] More preferably, the number of the bottom tubes is 10-16, the length of the bottom tubes is 2-4 meters, and the diameter is 1-1.5 inches.

[0065] In some embodiments, the bottom-feeding device is assembled as follows: the pumping device is fixed to a float with an engine, the pumping device is connected to two drain pipes and one inlet pipe, the ends of the two drain pipes are then connected to both ends of a horizontal water pipe, and the lower end of the horizontal water pipe is connected to a corresponding bottom-feeding pipe according to the size of the pond.

[0066] Advantages of this invention:

[0067] 1) This invention is the first to apply a mixture of four bacteria—Rhodophyta spp. (CICC 23658), Pyrophagocytophthora phosphate-eating bacteria (CGMCC No. 36465), Altomoraxella catarrhalis (CGMCC No. 36464), and Enterococcus faecalis (ATCC 17023)—as functional microorganisms to improve the environment of sea cucumber farming ponds. These four bacteria can complement and synergistically interact. Rhodophyta spp. possess flagella, are motile, reproduce rapidly, and exhibit facultative anaerobic characteristics, particularly in anaerobic light environments rich in organic matter. They have low light requirements and can still absorb and utilize toxic substances such as ammonia nitrogen, hydrogen sulfide, and organic acids effectively, even on cloudy or rainy days or in environments with low water transparency, serving as a nutrient source for photosynthesis. Through photosynthesis, they increase dissolved oxygen in the water, providing supplemental oxygen for Enterococcus faecalis and Pyrophagocytophthora phosphate-eating bacteria to decompose nitrogen and phosphorus substances and excrement in the water. Altomorella can rapidly break down large organic molecules into smaller ones, while simultaneously consuming organic matter secreted by algae, reducing oxygen consumption in the water and indirectly increasing dissolved oxygen. Enterococcus faecalis excels at decomposing and utilizing smaller molecules. Therefore, the four strains form a highly efficient coupled system, which is more efficient and faster at treating organic waste and toxic substances than a single strain. Furthermore, Enterococcus faecalis promotes the secretion of digestive enzymes in farmed animals, activates the intestinal immune system, enhances disease resistance, and helps promote nutrient absorption and utilization, while inhibiting the growth of harmful bacteria. Altomorella can produce secondary metabolites with antibacterial or antioxidant activity, and Rhodopseudomonas cocci are rich in coenzyme Q10 and 5-ALA, which can promote the growth of beneficial bacteria such as Bacillus phosphatidylinus.

[0068] 2) This invention is the first to apply a bottom-nourishing device to regulate the environment of seawater sashimi farming ponds. At the same time, different bottom-nourishing methods are selected according to the seasonal characteristics of the pond water environment. During the operation, the bottom-nourishing frequency and depth can be adjusted at any time according to the different pond conditions, which is more targeted and has a significant effect on regulating the pond environment. At the same time, it keeps the physicochemical indicators of the water environment relatively stable and will not affect the farmed animals.

[0069] 3) This invention combines physical bottom-nourishing methods with microbial agents. Deep bottom nourishing followed by the application of probiotics rapidly decomposes harmful substances and uneaten feed deposited in the bottom mud, improving the pond's bottom environment. Shallow bottom nourishing reduces the fermentation of harmful substances in the bottom mud, while the application of probiotics decomposes harmful substances and uneaten feed in the bottom mud and aquatic environment, reducing the proliferation of pathogenic bacteria in the pond during high-temperature periods. These two methods complement each other, synergistically enhancing their effectiveness.

[0070] 4) This pond environmental control technology has the advantages of simple equipment assembly, low cost, easy operation, and clearly defined mixed bacterial agent composition. It can be adjusted at any time during application, has good stability, and will not produce secondary pollution. It can significantly improve the micro-ecological environment of marine aquaculture ponds and is suitable for large-scale use and promotion. Attached Figure Description

[0071] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0072] Figure 1 The diagram shows the assembled effect of the bottom-feeding device of the present invention, including front view a, side view b and top view c;

[0073] Figure 2 This is a diagram showing the bottom-feeding device of the present invention in operation at sea.

[0074] Figure 3 This is a diagram illustrating the morphological characteristics of the mixed bacterial solution of the present invention; wherein, Figure 3 Image a shows the morphological characteristics of Rhodopseudomonas cocci. Figure 3 b is a diagram showing the morphological characteristics of Enterococcus faecalis. Figure 3 c is a diagram showing the morphological characteristics of Altomorella. Figure 3 d is a morphological feature diagram of Bacillus phosphatidylis;

[0075] Figure 4 This refers to the bottom-nourishing effect at a distance of 10cm from the bottom of the pool in Example 3;

[0076] Figure 5This is a schematic diagram illustrating the effect of the present invention on the ammonia nitrogen content in seawater aquaculture ponds;

[0077] Figure 6 This is a schematic diagram illustrating the effect of the present invention on the nitrite content of marine aquaculture ponds;

[0078] Figure 7 This is a schematic diagram illustrating the effect of the present invention on the sulfide content of seawater aquaculture ponds;

[0079] Figure 8 A schematic diagram illustrating the effect of this invention on the suspended solids content in seawater aquaculture ponds;

[0080] Figure 9 A schematic diagram illustrating the effect of this invention on the immune enzyme activity of sea cucumbers in marine aquaculture ponds, wherein... Figure 9 a represents the superoxide dismutase group. Figure 9 b is the lysozyme group. Figure 9 c represents the acid phosphatase group. Figure 9 d represents the alkaline phosphatase group. Detailed Implementation

[0081] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0082] Definitions of some terms

[0083] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0084] As used in this invention, the indefinite or definite articles used when referring to singular nouns, such as “a” or “a kind”, “the”, include the plural form of the noun.

[0085] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0086] The term "approximately" in this invention refers to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0087] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0088] The embodiments of the present invention will be described in detail below with reference to examples. The present invention includes, but is not limited to, the following embodiments. Any modifications made to the present invention based on existing technology that do not depart from the essential content of the present invention are still within the protection scope of the present invention.

[0089] In the following embodiments of the present invention, *Rhodobacter sphaeroides* was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with accession number ATCC 17023; *Fictibacillus phosphorivorans* was isolated by the applicant from the attachment material of sea cucumber farming cages in Xingcheng, and the applicant deposited the strain at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 36465, accession date: November 3, 2025; *Alteromonas abrolhosensis* was isolated by the applicant from nearshore seawater of Heishijiao, Dalian, and the applicant deposited the strain at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 36464, accession date: November 3, 2025; *Enterococcus faecalis* was purchased from the China Industrial Microbiological Culture Collection Center, accession number CICC. 23658.

[0090] Example 1: Development and assembly of the bottom-feeding device

[0091] During their long-term research on improving the marine aquaculture environment, the inventors discovered that during the low-temperature spring season, repeatedly stirring the pond bottom mud after the ice melts using physical methods ensures that all the bottom mud is fully exposed to the water. At this time, the water temperature is low and the dissolved oxygen content is high, which can decompose harmful substances such as ammonia nitrogen, sulfides, and nitrites buried in the bottom mud without affecting the farmed organisms in the pond. Subsequently, sprinkling a certain amount of mixed bacterial solution into the pond may help decompose harmful substances in the water and improve the disease resistance of farmed animals. However, during the high-temperature summer season, the water temperature is high and the dissolved oxygen content is low. Deep stirring of the bottom mud at this time requires a large amount of oxygen to decompose harmful substances, easily causing significant oxygen deficiency in the water and damaging farmed organisms. Therefore, during the high-temperature period, shallow stirring of the bottom should be done daily, only stirring the water to make it turbid, ensuring that sunlight cannot penetrate the water layer to reach the bottom and prevent the fermentation and overflow of harmful substances. Sprinkling a certain amount of mixed bacterial agent at this time can help decompose harmful substances in the water without consuming excessive oxygen.

[0092] Based on this, the inventor designed a novel bottom-feeding device suitable for sea cucumber farming environments. The basic structural components of this device are as follows: Figure 1 As shown, the bottom-feeding device consists of a pumping unit, an engine, a float, drain pipes, inlet pipes, horizontal water pipes, and bottom-feeding pipes. The pumping unit and engine are mounted on the float, which ensures the entire device floats on the water surface. The pumping unit connects two drain pipes and one inlet pipe, used for pumping water out and inlet, respectively. The ends of the two drain pipes are connected to the two ends of the horizontal water pipes, which are horizontally positioned above the float and vertically connected to multiple bottom-feeding pipes, thus distributing water from the drain pipes to the bottom-feeding pipes via the horizontal water pipes. The bottom-feeding pipes are perpendicular to the water surface and extend underwater to achieve bottom feeding. It is understood that a support plate (preferably a wooden board) could be installed on the float to better support the pumping unit and engine; however, considering that the float also serves a supporting function, a support plate is not a necessary structure. The bottom-feeding pipes can be made of plastic, with 10-16 pipes, each 2-4 meters long and 1-1.5 inches in diameter; the engine has a horsepower of 20-25 hp.

[0093] The specific assembly includes the following steps: 1) Install a diesel engine of approximately 22 horsepower on the water pump and fix the water pump on a wooden float with the engine measuring 3*8 meters; 2) The water pump consists of two drain pipes and one inlet pipe, with one end of the drain pipes connected to the same horizontal water pipe; 3) The lower end of the horizontal water pipe can be connected to 16 plastic bottom-feeding pipes, each 3 meters long and 1 inch in diameter, for underwater bottom feeding (see the actual usage diagram). Figure 2 (As shown).

[0094] Example 2: Development and preparation of mixed microbial agents

[0095] Based on past research experience, the inventors analyzed that *Rhodotorula foetida* is an important member of the pond ecosystem, not only improving and purifying the water quality of seawater aquaculture ponds, but also requiring less light intensity and functioning even on cloudy days or when water transparency is low. *Phanerophyte Pyrophaga sinensis* can rapidly decompose organic matter such as nitrogen and phosphorus in uneaten feed, feces, and animal and plant carcasses, reducing the production of toxic substances such as ammonia nitrogen and nitrite. *Altomoraxella* can decompose large-molecule organic pollutants such as organic matter secreted by algae and animal and plant carcasses into smaller molecules, reducing oxygen consumption and indirectly increasing dissolved oxygen in the water. *Enterococcus faecalis* has a rapid reproduction rate, achieving high bacterial density in a short time, decomposing small molecules in the water, and quickly exerting water purification and antibacterial effects. In the intestines, *Enterococcus faecalis* plays an important role in providing nutrition and improving intestinal function for farmed animals. By competing for attachment sites and nutrients, it directly inhibits pathogenic bacteria in the intestines, effectively enhancing the disease resistance of farmed animals. *Altomoraxella* can produce antibacterial or antioxidant secondary metabolites, which help improve the immune function of sea cucumbers. The combined application of Rhodobacter sphaeroides, Fictibacillus phosphorivorans, Alteromonas abrolhosensis, and Enterococcus faecalis exhibits high biological efficacy. Rhodobacter sphaeroides provides oxygen for Fictibacillus phosphorivorans and Enterococcus faecalis to decompose harmful substances through photosynthesis, without consuming dissolved oxygen in the water or producing secondary pollutants. Alteromonas decomposes large organic molecules into smaller molecules, which is more conducive to the decomposition and utilization by Enterococcus faecalis, while also helping to improve the immune function of sea cucumbers. The combined action of these four probiotics can specifically regulate the buffering capacity and self-purification capacity of the pond's micro-ecosystem, while simultaneously increasing the abundance of intestinal flora in farmed animals and enhancing their disease resistance. Therefore, the inventors have prepared a mixed bacterial agent containing Rhodobacter sphaeroides, Fictibacillus phosphorivorans, Alteromonas abrolhosensis, and Enterococcus faecalis.

[0096] The specific preparation method of this microbial agent includes the following aspects:

[0097] The preparation of the stock solution of Rhodopseudomonas spp. is as follows:

[0098] Step 1: Dissolve the lyophilized powder of Rhodospirillum spp. (ATCC 17023) purchased from Ningbo Mingzhou Biotechnology Co., Ltd. in 1 ml of sterile distilled water. Activate the bacteria by streaking on Rhodospirillum complete vitamin medium (RCV medium) and incubate at 37 ℃ under light for 48 h until round single colonies appear on the plate.

[0099] Step 2: Pick a single colony from the RCV solid medium and inoculate it into the RCV liquid medium at pH 8.0. Incubate at 37°C with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0100] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 9 cfu / ml, for later use (microscopic examination results as follows) Figure 3 a).

[0101] The preparation of the Bacillus phosphatidylcholine stock solution is as follows:

[0102] Step 1: Add sterile distilled water to the collected net cage attachment material and shake well. Take the mixture and inoculate it into seawater 2216E liquid culture medium. Add 0.5% lecithin and 1% glucose to the culture medium and culture it in a shaker. Screen for dominant strains that can efficiently decompose organic phosphorus.

[0103] Step 2: Activate the enriched bacterial solution by streaking it on 2216E solid medium and incubate it under light until single colonies of the target bacteria appear on the plate;

[0104] Step 3: Screen and select dominant single colonies, perform 16S sequencing, and identify them as *Fictibacillus phosphorivorans* (and deposit them in microbial culture, accession number CGMCC No. 36465); inoculate the dominant bacteria into seawater 2216E liquid medium and culture with shaking until the bacteria proliferate to the logarithmic growth phase;

[0105] Step 4: Collect bacterial cells by centrifugation, resuspend the bacterial cells in sterile seawater, and adjust the bacterial concentration to 10. 6 -10 8 cfu / ml, for later use.

[0106] The preparation method of the *Altorazine* stock solution is as follows:

[0107] Step 1: Inoculate the collected seawater sample into Seawater 2216E liquid culture medium, add 0.1% potassium nitrate and 0.5% sodium acetate to the culture medium, and culture in a shaker to screen out strains with superior denitrification performance.

[0108] Step 2: Activate the enriched bacterial solution by streaking it on 2216E solid medium and incubate it under light until single colonies of the target bacteria appear on the plate;

[0109] Step 3: Screen and select single colonies for 16S sequencing, which identified them as Alteromonas abrolhosensis (and deposited them in a biological archive with accession number CGMCC No. 36464); inoculate the dominant bacterium into seawater 2216E liquid medium and culture with shaking until the bacterial strain proliferates to the logarithmic growth phase;

[0110] Step 4: Collect bacterial cells by centrifugation, resuspend the bacterial cells in sterile seawater, and adjust the bacterial concentration to 10. 6 -10 8 cfu / ml, for later use.

[0111] The preparation of Enterococcus faecalis stock solution is as follows:

[0112] Step 1: Dissolve the freeze-dried Enterococcus faecalis (CICC 23658) purchased from the China Industrial Microbial Culture Collection Center in 1 ml of sterile distilled water. Activate the culture by streaking it onto Brain Heart Extract Agar (BHI medium) and incubate it in an incubator at 37 ℃ under light for 48 h until round single colonies appear on the plate.

[0113] Step 2: Pick a single colony from BHI agar medium and inoculate it into BHI broth medium at pH 8.0. Incubate at 37°C with shaking at 150 r / min until the bacterial strain proliferates to the logarithmic growth phase.

[0114] Step 3: Centrifuge at 6000 rpm for 15 min, collect the bacterial cells, resuspend the cells in sterile seawater, and adjust the bacterial concentration to 10 using the McFarland turbidimetric method. 8 cfu / ml, for later use (microscopic examination results as follows) Figure 3 b).

[0115] The preparation method of 0.5 ppm mixed bacterial agent is as follows:

[0116] Step 1: Take 0.025 ml of Rhodopseudomonas spp. stock solution (10 9 (cfu / ml).

[0117] Step 2: Take 0.025 ml of Enterococcus faecalis stock solution (10 8 (cfu / ml).

[0118] Step 3: Take 0.025 ml of Bacillus phosphatidylcholine stock solution (10 8 (cfu / ml).

[0119] Step 4: Take 0.025 ml of Altomoraxella stock solution (10 8 (cfu / ml).

[0120] Step 5: Mix the two together, then add 0.4 ml of sterile seawater to make the total volume 0.5 ml, and store at 4℃ for later use.

[0121] The preparation method of 1 ppm mixed bacterial agent is as follows:

[0122] Step 1: Take 0.05 ml of Rhodopseudomonas spp. stock solution (10 9 (cfu / ml).

[0123] Step 2: Take 0.05 ml of Enterococcus faecalis stock solution (10 8 (cfu / ml).

[0124] Step 3: Take 0.05 ml of Bacillus phosphatidylcholine stock solution (10 8 (cfu / ml).

[0125] Step 4: Take 0.05 ml of Altomorella stock solution (10 8 (cfu / ml).

[0126] Step 5: Mix the two together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml, and store at 4℃ for later use.

[0127] This mixed microbial agent is suitable for long-term use throughout the aquaculture cycle, with an optimal water temperature of 10–30℃. After bottom preparation during the thawing period (when water temperature is above 10℃), it should be evenly sprinkled into the pond at a dosage of 1 ml / m² each time. 3 During the high-temperature period in summer (water temperature above 26℃), spray once a week, with a dosage of 1ml / m³ each time. 3 .

[0128] Example 3: Summer Seawater Aquaculture Pond Experiment

[0129] An application experiment of the bottom-feeding device was conducted in a seawater aquaculture pond in the Linghai sea cucumber farming area of ​​Jinzhou City, Liaoning Province. The pond area was 100 mu (approximately 6.7 hectares), with a water depth of 2-2.5 m and a muddy-sandy bottom. After the water temperature reached 26℃, the bottom-feeding pipe was set at three different heights from the bottom of the pond, and two bottom-feeding frequencies were selected for optimization. The control group consisted of ponds without bottom-feeding. A mixed bacterial solution of 1 ppm was sprayed. The results showed that after bottom-feeding at a distance of 10 cm from the bottom, deep grooves appeared on the pond bottom, and the sea cucumbers died in these grooves (e.g., ...). Figure 4As shown in Table 1), after bottom cleaning at a distance of 20cm from the bottom of the pond, the dissolved oxygen content at the bottom dropped rapidly, with some points falling below 4mg / L, making sea cucumbers highly susceptible to oxygen deficiency. After bottom cleaning at a distance of 30cm from the bottom, the dissolved oxygen at the bottom of the pond remained above 6mg / L, and the content of toxic substances such as ammonia nitrogen and sulfides in the water decreased significantly. In addition, the ammonia nitrogen and sulfide content in ponds cleaned once a day was significantly lower than that in ponds cleaned every other day. Therefore, during the high-temperature period in summer, bottom cleaning should be carried out at a distance of more than 30cm from the bottom of the pond, with a frequency of once a day (as shown in Table 1).

[0130] Table 1. Effects of different bottom depths on the content of toxic substances in water during the high-temperature period in summer.

[0131]

[0132] Example 4: Spring Seawater Aquaculture Pond Experiment

[0133] The difference from Example 3 is that a study on bottom-feeding regulation was conducted on the sea cucumber farming pond where the water temperature reached above 10°C after the ice melted in spring. The bottom-feeding pipe was set at three different heights from the bottom of the pond, and two bottom-feeding frequencies were selected for optimization and screening. The mixed bacterial agent was sprayed at 1 ppm.

[0134] Experimental results showed that no deep trenches appeared on the bottom of the pool at different bottom heights, and the effect on dissolved oxygen content in the water was not significant. The content of toxic substances such as ammonia nitrogen and sulfide decreased the most when the bottom was moistened at 10 cm from the bottom. The frequency of bottom moistening had no significant effect on the content of ammonia nitrogen, sulfide and dissolved oxygen in the water (see Table 2 for details).

[0135] Table 2. Effects of different bottom depths on the content of toxic substances in water after ice melts in spring.

[0136]

[0137] Example 5: Comparison of the regulatory effects of different mixed bacterial agents combined with the substrate

[0138] This embodiment further compares the regulatory effects of bottom-feeding operations combined with different types of mixed bacterial agents. The experimental location and water temperature conditions were basically the same as in Example 3, and the fixed bottom-feeding tube was selected at a distance of 30cm from the bottom of the pond. The four bacterial strains were divided into two categories according to their water quality improvement function and multifunctional probiotics that also promote growth and regulate ecology. Rhodopseudomonas sphaeroides and Pyrophagocytosylvatica belonged to the former, while Altomoraxella catarrhalis and Enterococcus faecalis belonged to the latter. The effects of various bacterial solutions were compared in this way.

[0139] The inventors prepared the following groups of different mixed bacterial solutions (where: A represents Rhodotorula globulus, B represents Pyrophagocytic Bacillus, C represents Altomorella, and D represents Enterococcus faecalis):

[0140] Preparation of Group 1, 1 ppm A+C mixed bacterial agent:

[0141] Step 1: Take 0.1 ml of Rhodopseudomonas spp. stock solution (10 9 (cfu / ml).

[0142] Step 2: Take 0.1 ml of Altomorella stock solution (10 8 (cfu / ml).

[0143] Step 3: Mix the two together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml, and store at 4°C for later use.

[0144] Group 2, Preparation of 1 ppm A+D mixed bacterial agent:

[0145] Step 1: Take 0.1 ml of Rhodopseudomonas spp. stock solution (10 9 (cfu / ml).

[0146] Step 2: Take 0.1 ml of Enterococcus faecalis stock solution (10 8 Step 3: Mix the two together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml, and store at 4°C for later use.

[0147] Group 3, Preparation of 1 ppm B+C mixed bacterial agent:

[0148] Step 1: Take 0.1 ml of Bacillus phosphatidylcholine stock solution (10 9 (cfu / ml).

[0149] Step 2: Take 0.1 ml of Altomorella stock solution (10 8 (cfu / ml).

[0150] Step 3: Mix the two together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml, and store at 4°C for later use.

[0151] Group 4, Preparation of 1 ppm B+D mixed bacterial agent:

[0152] Step 1: Take 0.1 ml of Bacillus phosphatidylcholine stock solution (10 9 (cfu / ml).

[0153] Step 2: Take 0.1 ml of Enterococcus faecalis stock solution (10 8 (cfu / ml).

[0154] Step 3: Mix the two together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml, and store at 4°C for later use.

[0155] Group 5, Preparation of 1 ppm A+B+C+D mixed bacterial agent:

[0156] Step 1: Take 0.05 ml of Rhodopseudomonas spp. stock solution (10 9 (cfu / ml).

[0157] Step 2: Take 0.05 ml of Bacillus phosphatidylcholine stock solution (10 8 (cfu / ml).

[0158] Step 3: Take 0.05 ml of Altomorella stock solution (10 8 (cfu / ml).

[0159] Step 4: Take 0.05 ml of Enterococcus faecalis stock solution (10 8 (cfu / ml).

[0160] Step 5: Mix the four ingredients together, then add 0.8 ml of sterile seawater to make a total volume of 1 ml. Store at 4°C for later use.

[0161] The spraying was carried out at the same concentration, and the measurement results after treatment are shown in Table 3. Except for the control group, the ammonia nitrogen and sulfide in the water environment of all other groups decreased significantly. However, the dissolved oxygen in the seawater of groups B+C and B+D decreased too much, which could easily cause hypoxia in farmed animals. Although the dissolved oxygen content of groups A+C and A+D was relatively sufficient, the effect on reducing ammonia nitrogen and sulfide content was not as good as that of groups A+B+C+D. It can be seen that the overall effect of using a mixed bacterial agent made of Rhodopseudomonas stolonifer, Enterococcus faecalis, Altomoraxella catarrhalis, and Pyrophagocytic Pseudomonas phosphate-rich bacteria for bottom fertilization followed by spraying is the best.

[0162] Table 3. Effects of different mixed bacterial agents on the content of toxic substances in water after bottom treatment during the high-temperature period in summer.

[0163]

[0164] Example 6: Comparison of the effects of mixed bacterial agent concentration

[0165] The experimental location and water temperature conditions in this embodiment are basically the same as in embodiment 3. The fixed bottom-feeding pipe is selected 30cm away from the bottom of the pond. A is used as a control example, and no mixed bacterial solution is applied after bottom-feeding; B is used as comparative example 1, and only a 10% concentration is sprayed after bottom-feeding. 9 CFU / ml Rhodotorula glutinis was applied at a dose of 1 ppm; C was used as control example 2, and after fertilization, only a 10% concentration was sprayed. 8 cfu / ml Enterococcus faecalis, application dose 1ppm; D was used as control example 3, after base application only sprayed with a concentration of 10. 8 cfu / ml Bacillus phosphatidylis; E was used as control example 4, and only a concentration of 10 was sprayed after the base treatment. 8cfu / ml Altomoraxella; F was used as the low-concentration group, in the mixed bacterial solution sprinkled after the base treatment, the concentration was 10 cfu / ml of Rhodotorula glutinis. 7 cfu / ml, Enterococcus faecalis 10 6 cfu / ml, Bacillus phosphatidylcholine 10 6 cfu / ml, Altomorella 10 6 The concentration was 0.5 ppm (cfu / ml); G was used as the high-concentration group, with a concentration of 10 cfu / ml of Rhodopseudomonas spp. in the mixed bacterial solution. 8 cell / ml, Enterococcus faecalis 10 7 cfu / ml, Altomorella 10 7 cfu / ml, Bacillus phosphatidylcholine 10 7 The concentration of cfu / ml was 1 ppm. The experiment lasted for 30 days, with pond water samples taken every 15 days for physicochemical index testing (suspended solids, nitrite, ammonia nitrogen, sulfides). The supernatant of the sea cucumber coelomic fluid in the pond was used for immunoenzyme activity testing.

[0166] The effects of the application of mixed bacterial solutions in each experimental group on the physicochemical indicators of water quality in seawater aquaculture ponds are as follows: Figure 5-8 As shown. Figure 5 This diagram illustrates the effect of mixed bacterial solutions on ammonia nitrogen levels in seawater aquaculture ponds (in the diagram, a and b indicate significant differences, while ab indicates no significant difference compared to a or b). Figure 6 This is a schematic diagram illustrating the effect of mixed bacterial solutions on nitrite content in seawater aquaculture ponds. Figure 7 This is a schematic diagram illustrating the effect of mixed bacterial solutions on the sulfide content in seawater aquaculture ponds. Figure 8 This diagram illustrates the impact of mixed bacterial solutions on suspended solids levels in marine aquaculture ponds. High concentrations of ammonia nitrogen pollutants in pond water reduce dissolved oxygen levels, leading to eutrophication and impacting energy flow and material exchange within the pond ecosystem. Sulfides can bind with hemoglobin in the blood of farmed animals to produce sulfohhemoglobin, reducing the blood's oxygen-carrying capacity and inhibiting metabolic activity.

[0167] The results showed that, except for a significant reduction in sulfide content, the physicochemical indicators of the pond water in the control and comparative groups did not show significant improvement after bottom nourishment. However, after applying the mixed bacterial solution, both the low-concentration and high-concentration groups showed significant improvement in various physicochemical indicators of the aquaculture pond water. Compared with the low-concentration group, the high-concentration group showed a more significant reduction in sulfide content. This indicates that the combination of bottom nourishment technology and mixed bacterial solution can significantly reduce the content of ammonia nitrogen, nitrite, sulfur pollutants, and suspended solids in pond water, with higher concentrations of mixed bacterial solution showing more significant effects.

[0168] In addition, the application of mixed bacterial solutions in each experimental group had an effect on the activity of immune enzymes in the supernatant of sea cucumber coelomic fluid in ponds, such as... Figure 9As shown, superoxide dismutase activity plays a crucial role in enhancing the defensive capabilities of phagocytes and the body's immune function. Lysosomal enzymes, composed of lysozyme, acid phosphatase, and alkaline phosphatase, exist in sea cucumber phagocytes and participate in the degradation of exogenous substances. The results showed that the control group and the four comparative groups did not significantly enhance the activity of immune enzymes in sea cucumbers. However, spraying the mixed bacterial solution significantly activated the activities of the other three immune enzymes in sea cucumbers within a short period. Although the short-term enhancement of lysozyme activity was not significant, long-term application of the mixed bacterial solution significantly enhanced lysozyme activity. This indicates that spraying the mixed bacterial solution can improve the phagocytic and pathogen-eliminating ability of sea cucumber coelomic cells and promote the digestion and absorption of nutrients, with the high-concentration group showing a better enhancing effect than the low-concentration group.

[0169] The experimental results above show that the bottom-nourishing technology and the application of the mixed bacterial agent in this invention have a synergistic effect and complement each other. The functional components in the mixed bacterial agent work together to enhance each other's effects, which can effectively reduce the content of ammonia nitrogen, nitrite, sulfur pollutants and suspended solids in the water of seawater aquaculture ponds, while enhancing the immune enzyme activity of sea cucumbers and improving their disease resistance.

[0170] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A mixed microbial agent for environmental control in sea cucumber farming ponds, characterized in that, The mixed bacterial agent comprises Rhodobacter sphaeroides, Fictibacillus phosphorivorans, Alteromonas abrolhosensis, and Enterococcus faecalis; preferably, the Enterococcus faecalis has the accession number CICC 23658, the Fictibacillus phosphorivorans has the accession number CGMCC No. 36465, the Alteromonas abrolhosensis has the accession number CGMCC No. 36464, and the Rhodobacter sphaeroides has the accession number ATCC 17023.

2. The mixed microbial agent according to claim 1, characterized in that, The concentration ratio of Rhodopseudomonas aeruginosa, Pseudomonas phosphatidylcholine, Altomoraxella catarrhalis, and Enterococcus faecalis in the mixed bacterial agent is 100-10:10-1:10-1:10-1.

3. The mixed microbial agent according to claim 3, characterized in that, The concentration of Rhodotorula glutinis in the mixed bacterial agent is 10. 8 -10 9 cfu / ml, concentration of Bacillus phosphatidylcholine was 10. 7 -10 8 cfu / ml, Altomoraxella concentration was 10 7 -10 8 cfu / ml, Enterococcus faecalis concentration 10 7 -10 8 cfu / ml.

4. The mixed microbial agent according to any one of claims 1-3, characterized in that, The preparation steps of the mixed bacterial agent are as follows: take the stock solution of Rhodotorula foetida, the stock solution of Pseudomonas phosphatidylinum, the stock solution of Altomoraxella catarrhalis and the stock solution of Enterococcus faecalis, mix the four together, add sterile seawater, and store at 4°C for later use.

5. A method for environmental control in a sea cucumber aquaculture pond, characterized in that, The method includes the following steps: 1) Perform bottom-nourishing operations on sea cucumber farming ponds; 2) After soaking the bottom, spray the mixed bacterial agent as described in any one of claims 1-4 onto the water body.

6. The environmental control method according to claim 5, characterized in that, The bottom-feeding operation is performed using a bottom-feeding device; the bottom-feeding device consists of a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe; the pumping device and the engine are located on the upper end of the float; the pumping device is connected to the drain pipe and the inlet pipe; the end of the drain pipe is connected to the horizontal water pipe, and the horizontal water pipe is vertically connected to the bottom-feeding pipe; the bottom-feeding pipe is perpendicular to the water surface and extends underwater. Preferably, the bottom-feeding device comprises a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe; the pumping device and the engine are located on the upper end of the float; the pumping device is connected to two drain pipes and one inlet pipe; the ends of the two drain pipes are respectively connected to the two ends of the horizontal water pipe, the horizontal water pipe is horizontally positioned above the float and vertically connected to multiple bottom-feeding pipes; the bottom-feeding pipe is perpendicular to the water surface and extends underwater; More preferably, the number of the bottom tubes is 10-16, the length of the bottom tubes is 2-4 meters, and the diameter is 1-1.5 inches.

7. The environmental control method according to claim 5, characterized in that, The concentration of the mixed bacterial agent is 0.5-1 ppm, and the dosage is 1 mL of the mixed bacterial agent per cubic meter of water.

8. The environmental control method according to any one of claims 5-6, characterized in that, The bottom nourishing operation is performed using the bottom nourishing device according to the water quality conditions. Preferred: After the ice melts in spring, when the seawater temperature rises to about 10°C, the bottom-nourishing device is used to carry out comprehensive and deep bottom nourishment, ensuring that the end of the bottom-nourishing tube is more than 10cm away from the bottom of the pool; preferably, the bottom nourishment frequency is 3-4 times per week; after each bottom nourishment, 0.5-1 ppm of the mixed bacterial agent is sprayed, and the dosage is 1 mL of the mixed bacterial agent per cubic meter of water. During the high-temperature period in summer, when the seawater temperature reaches above 26℃, the bottom-nourishing device is used for shallow bottom nourishing, ensuring that the end of the bottom-nourishing tube is more than 30cm away from the bottom of the pool; preferably, the bottom nourishing frequency is once a day, and it is stopped when the water becomes turbid and the light is blocked; after each bottom nourishing, 0.5-1 ppm of the mixed bacterial agent is sprinkled, and the dosage is 1 mL of the mixed bacterial agent per cubic meter of water.

9. A bottom-feeding device for environmental control in seawater sea cucumber farming ponds, characterized in that, The bottom-feeding device consists of a pumping device, an engine, a float, a drain pipe, an inlet pipe, a horizontal water pipe, and a bottom-feeding pipe. The pumping device and the engine are located on the upper end of the float. The pumping device is connected to two drain pipes and one inlet pipe. The ends of the two drain pipes are respectively connected to the two ends of the horizontal water pipe. The horizontal water pipe is placed horizontally above the float and vertically connected to multiple bottom-feeding pipes. The bottom-feeding pipes are perpendicular to the water surface and extend underwater. Preferably, the number of bottom-feeding pipes is 10-16, and the length of the bottom-feeding pipe is 2-4 meters and the diameter is 1-1.5 inches.

10. The bottom-feeding device according to claim 9, characterized in that, The bottom-feeding device is assembled as follows: the pumping device is fixed on a floating buoy with an engine, the pumping device is connected to two drain pipes and one inlet pipe, the ends of the two drain pipes are then connected to both ends of a horizontal water pipe, and the lower end of the horizontal water pipe is connected to a corresponding bottom-feeding pipe according to the size of the pond.

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