Composite microbial preparation for improving water quality of mariculture pond

Abstract

The invention relates to the technical field of biology, in particular to a compound microbial preparation or fertilizer for improving water quality of a mariculture pond. The composite microbial preparation comprises the following functional components in concentration: 10 < 6 >-10 < 8 > cfu / ml of bifidobacterium thermoactinomycetes, 10 < 7 >-10 < 9 > cfu / ml of rhodopseudomonas palustris, 0.03-0.06 mg / ml of dwarf vallisneria natans extract and 0.01-0.03 mg / ml of acorus calamus extract. The compound microbial preparation is clear in components and scientific in compatibility, and all the components are mutually beneficial, mutualistic and synergistic; on the premise of ensuring no pollution, no residue and no drug resistance, the content of ammonia nitrogen, phosphorus and organic pollutants in the water body of the mariculture pond can be effectively reduced; meanwhile, heavy metals, antibiotics and vibrio harveyi in the pond water body can be effectively removed, and an obvious improvement effect on the mariculture pond water body is achieved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a composite microbial feed or preparation for improving the water quality of marine aquaculture ponds. Background Art

[0002] Pond farming is one of the important ways to increase the number of aquatic animals in my country. However, the traditional intensive pond farming model often leads to the continuous accumulation of organic matter such as leftover bait, feces and fertilizers in the pond, resulting in too low dissolved oxygen content in the water during the hot season, and too high content of ammonia nitrogen and nitrite, leading to problems such as deterioration of aquaculture water quality, excessive pond bioload and ecological imbalance in the aquaculture environment, which seriously affect aquaculture income and the sustainable development of the aquaculture industry. Due to the lack of industry norms and management standards, most of the composite microbial preparations currently on the market for improving the water quality of marine aquaculture ponds are only a single component or a mechanical combination of multiple components, rather than a reasonable combination that has been scientifically screened, and cannot achieve the expected results of pond aquaculture practitioners.

[0003] As a new type of highly efficient, green and environmentally friendly water quality improver and antibiotic substitute, microbial preparations can comprehensively improve the water quality of marine aquaculture ponds by supplementing exogenous probiotics. Actinomycetes have multiple functions in improving water quality, especially in aquaculture water. Bifidobacterium thermophilic actinomycetes have a strong ability to decompose organic matter and can degrade substances such as lignin, cellulose, and chitin. It has a good effect on the degradation of ammonia nitrogen in aquaculture water, can reduce the ammonia nitrogen content, and can also balance the pH value of the water body to keep it within the range suitable for the growth of aquatic organisms, while increasing dissolved oxygen to provide more sufficient oxygen for aquatic organisms. Photosynthetic bacteria can use organic matter in aquaculture water as a carbon source for photosynthesis, which plays a role in purifying water quality and inhibiting the growth and reproduction of pathogenic microorganisms. Among them, Rhodopseudomonas palustris contains rich proteins, vitamins and bioactive substances. In aquaculture, Rhodopseudomonas palustris can degrade toxic substances such as nitrites and sulfides in water bodies, and release disease-resistant enzymes, which can not only promote the proliferation of beneficial microorganisms, but also inhibit the growth of many pathogenic bacteria. At the same time, Rhodopseudomonas palustris has strong adaptability and can tolerate and decompose high concentrations of organic matter in aquaculture environments, producing active factors that are beneficial to the growth of aquatic animals and weakening the pathogenicity of pathogens. Extracts of Vallisneria dwarfi and Acorus calamus can absorb pollutants such as nitrogen, phosphorus, and organic matter in aquaculture water, increase the oxygen content of the water, and have a strong water purification function.

[0004] So far, there is no prior art report on the application of thermophilic bifidobacteria, Rhodopseudomonas palustris combined with Vallisneria dwarfi and Acorus calamus extracts to improve the water quality of marine aquaculture ponds. Summary of the invention

[0005] In view of this, in order to solve the above-mentioned technical problems existing in the prior art, the present application provides a composite microbial preparation for improving the water quality of marine aquaculture ponds. The microbial preparation has the advantages of clear ingredients, green environmental protection, low cost, and synergistic enhancement of functional components. It can effectively improve the water quality of marine aquaculture ponds and is suitable for large-scale production and promotion.

[0006] Therefore, this application includes at least the following objectives:

[0007] The first objective: to seek a more effective composite microbial preparation for improving the water quality of marine aquaculture ponds;

[0008] The second purpose is to find a new method to improve the water quality of marine aquaculture ponds.

[0009] To achieve the above objectives, this application specifically provides the following technical solutions:

[0010] The present application first provides a composite microbial preparation for improving the water quality of marine aquaculture ponds, wherein the functional components of the composite microbial preparation include Thermoactinomyces dichotomicus, Rhodopseudomonas palustris, Vallisneria dwarfi extract, and Acorus calamus extract. The concentrations of the functional components of the composite microbial preparation are 10 6 -10 8 cfu / ml, Rhodopseudomonas palustris 10 7 -10 9 cfu / ml, dwarf vallisneria extract 0.03-0.06mg / ml, calamus extract 0.01-0.03mg / ml.

[0011] In some optimal technical solutions: the concentrations of the functional components of the composite microbial preparation are respectively 10 7 cfu / ml, Rhodopseudomonas palustris 10 8 cfu / ml, dwarf vallisneria extract 0.05mg / ml, calamus extract 0.02mg / ml.

[0012] Furthermore, the deposit number of the bifidophilic actinomycetes is ATCC 49854;

[0013] Furthermore, the deposit number of the Rhodopseudomonas palustris is CICC 23812.

[0014] In some embodiments, the method for preparing the bifidophilic actinomycetes comprises:

[0015] Step 1: dissolving the bifidobacterium thermophilic actinomycete freeze-dried powder in distilled water, streaking the strain on a solid culture medium for activation, and culturing in an incubator at 50-55° C. for 2-4 days until a single pale yellow colony with neat edges appears on the plate; preferably, the solid culture medium is a nutrient broth agar medium;

[0016] Step 2: Pick a single colony and inoculate it into a nutrient broth liquid culture medium at a culture temperature of 50-55°C. Shake and culture in an incubator for 2-3 days until the bacteria proliferate to the logarithmic growth phase;

[0017] Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

[0018] In some embodiments, the method for preparing the Rhodopseudomonas palustris comprises:

[0019] Step 1: fully dissolve the freeze-dried powder of Rhodopseudomonas palustris in distilled water, streak the strain on a solid culture medium to activate it, and culture it in an anaerobic incubator for 3-5 days under the conditions of an illumination of 2000-3000 lux and a temperature of 28-30°C until a single red colony appears on the plate; preferably, the components of the Rhodopseudomonas palustris culture medium are: tryptone 10-15.0 g / L, soytone 3-5.0 g / L, sodium chloride 3-5.0 g / L, agar 15-20 g / L, pH 7.5-8.0;

[0020] Step 2: Pick a single red colony and inoculate it into a liquid culture medium of Rhodopseudomonas palustris, with a culture temperature of 28-30°C and a light intensity of 2000-3000 lux, and culture it in an anaerobic incubator for 3-5 days until the bacteria proliferate to the logarithmic growth phase and the bacterial solution turns dark red;

[0021] Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

[0022] In some embodiments, the preparation method of the dwarf Vallisneria extract and the calamus extract is: wash the dwarf Vallisneria and the calamus with distilled water respectively, ventilate and dry in the shade and then crush them, add 75% ethanol solution, extract for about 1.5-2h under the action of ultrasound with an operating frequency of 50-60KHZ and a power of 100-150W, filter to remove impurities, and reduce the pressure to concentrate the extract and spray dry it to obtain the dwarf Vallisneria and calamus extracts.

[0023] Furthermore, the functional components in the composite microbial preparation are mutually beneficial and synergistic: bifidobacterium thermophilic actinomycetes have a nitrogen-fixing effect and can obtain amino acids, nitrogen, etc. from Rhodopseudomonas palustris as a matrix to inhibit the proliferation of pathogenic bacteria in the water body; at the same time, bifidobacterium thermophilic actinomycetes mainly decompose organic matter, while Rhodopseudomonas palustris can use photosynthesis to convert inorganic matter into organic matter; the two are complementary in terms of decomposition and conversion of substances, and can improve water quality more comprehensively; the extracts of dwarf Vallisneria and calamus can provide the functional bacteria with specific substrates and carbon sources required for growth, thereby improving their survival rate; and after mixing with bifidobacterium thermophilic actinomycetes and Rhodopseudomonas palustris and fermenting, the indigestible macromolecules in the extracts of dwarf Vallisneria and calamus can be decomposed into small molecules, and the effective ingredients can be fully released, which is more conducive to the improvement of water quality.

[0024] The present application also provides the use of the aforementioned bioactive preparation in improving the water quality of marine aquaculture ponds.

[0025] The present application also provides a method for preparing the aforementioned composite microbial preparation, which comprises the step of fully mixing the bifidophilic actinomycetes, Rhodopseudomonas palustris, Vallisneria dwarfi extract and Acorus calamus extract.

[0026] The present application also provides a method for improving water quality in a marine aquaculture pond, the method comprising adding any of the aforementioned composite microbial preparations into a marine aquaculture pond;

[0027] In some preferred embodiments, the amount of each input is 3-5 ml / m 3 , invested every 6-7 days.

[0028] Advantages of this application:

[0029] 1) This application is the first to jointly apply bifidophilic actinomycetes, Rhodopseudomonas palustris, dwarf Vallisneria extract, and calamus extract to improve the water quality of marine aquaculture ponds. The components of this composite microbial preparation complement each other, coexist in a mutually beneficial manner, and have a significant synergistic effect. Bifidophilic actinomycetes can obtain amino acids, nitrogen, etc. from Rhodopseudomonas palustris as a matrix to produce antibiotics, vitamins, and enzymes, etc., to inhibit the proliferation of pathogens in the water body. At the same time, bifidophilic actinomycetes mainly decompose organic matter, while Rhodopseudomonas palustris can use photosynthesis to convert inorganic matter into organic matter. The two are complementary in decomposing and converting substances, and can improve water quality more comprehensively.

[0030] 2) Vallisneria dwarfi and Acorus calamus are widely available, and have the advantages of low cost, pure natural, low toxicity, etc., and will not cause environmental safety problems. This application is the first to use them as additional components to improve the water quality of marine aquaculture. Vallisneria dwarfi and Acorus calamus extracts can provide functional bacteria with the specific substrate and carbon source required for growth, thereby improving their survival rate. After microbial fermentation of the two probiotics of this application, the indigestible macromolecules in the Vallisneria dwarfi and Acorus calamus extracts can be decomposed into small molecules, and the effective ingredients can be fully released, which is more conducive to the improvement of water quality.

[0031] 3) The composite microbial preparation has clear ingredients, low cost, simple preparation and convenient storage. It is pollution-free, non-resistant and non-toxic. It will not cause aquatic product safety risks and environmental safety problems, and is suitable for large-scale production and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the total nitrogen content of the aquaculture pond water;

[0034] Figure 2 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the ammonia nitrogen content in the water of aquaculture ponds;

[0035] Figure 3 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the nitrite content in the water of aquaculture ponds;

[0036] Figure 4 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the total phosphorus content in the water of aquaculture ponds;

[0037] Figure 5 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the phosphate content in the water of aquaculture ponds;

[0038] Figure 6 , a schematic diagram showing the effect of the composite microbial preparation of the present application on the chemical oxygen demand of aquaculture pond water;

[0039] Figure 7 , Schematic diagram of the effect of the composite microbial preparation of the present application on the heavy metal content in the water of aquaculture ponds;

[0040] Figure 8, a schematic diagram showing the effect of the composite microbial preparation of the present application on the antibiotic content in aquaculture pond water;

[0041] Fig. 9 , Schematic diagram of the effect of the composite microbial preparation of the present application on the content of pathogenic Vibrio in aquaculture pond water. DETAILED DESCRIPTION

[0042] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0043] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0044] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited 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 of only these embodiments.

[0045] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0046] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0047] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0048] The core solution of the present application is such a composite microbial preparation, which can be used to improve the water quality of marine aquaculture ponds. The functional components of the composite microbial preparation include bifidophilic actinomycetes, Rhodopseudomonas palustris, dwarf Vallisneria extract, and calamus extract.

[0049] In some embodiments of the present application, the concentrations of the functional components of the composite microbial preparation are respectively 10 6 -10 8 cfu / ml, Rhodopseudomonas palustris 10 7 -10 9 cfu / ml, dwarf vallisneria extract 0.03-0.06mg / ml, calamus extract 0.01-0.03mg / ml.

[0050] In some best embodiments: the concentrations of the functional components of the composite microbial preparation are respectively 10 7 cfu / ml, Rhodopseudomonas palustris 10 8 cfu / ml, dwarf vallisneria extract 0.05mg / ml, calamus extract 0.02mg / ml.

[0051] In some embodiments of the present application, the deposit number of the bifidophilic actinomycetes is ATCC 49854;

[0052] In some embodiments of the present application, the deposit number of the Rhodopseudomonas palustris is CICC 23812.

[0053] In some embodiments of the present application, the method for preparing the bifidophilic actinomycetes comprises:

[0054] Step 1: dissolving the bifidobacterium thermophilic actinomycete freeze-dried powder in distilled water, streaking the strain on a solid culture medium for activation, and culturing in an incubator at 50-55° C. for 2-4 days until a single pale yellow colony with neat edges appears on the plate; preferably, the solid culture medium is a nutrient broth agar medium;

[0055] Step 2: Pick a single colony and inoculate it into a nutrient broth liquid culture medium at a culture temperature of 50-55°C. Shake and culture in an incubator for 2-3 days until the bacteria proliferate to the logarithmic growth phase;

[0056] Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

[0057] In some embodiments of the present application, the method for preparing Rhodopseudomonas palustris comprises:

[0058] Step 1: fully dissolve the freeze-dried powder of Rhodopseudomonas palustris in distilled water, streak the strain on a solid culture medium to activate it, and culture it in an anaerobic incubator for 3-5 days under the conditions of an illumination of 2000-3000 lux and a temperature of 28-30°C until a single red colony appears on the plate; preferably, the components of the Rhodopseudomonas palustris culture medium are: tryptone 10-15.0 g / L, soytone 3-5.0 g / L, sodium chloride 3-5.0 g / L, agar 15-20 g / L, pH 7.5-8.0;

[0059] Step 2: Pick a single red colony and inoculate it into a liquid culture medium of Rhodopseudomonas palustris, with a culture temperature of 28-30°C and a light intensity of 2000-3000 lux, and culture it in an anaerobic incubator for 3-5 days until the bacteria proliferate to the logarithmic growth phase and the bacterial solution turns dark red;

[0060] Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

[0061] In some embodiments of the present application, the preparation method of the dwarf Vallisneria extract and the calamus extract is: respectively clean the dwarf Vallisneria and the calamus with distilled water, ventilate and dry in the shade and then crush them, add 75% ethanol solution, extract for about 1.5-2h under the action of ultrasound with an operating frequency of 50-60KHZ and a power of 100-150W, filter to remove impurities, and reduce the pressure to concentrate the extract and spray dry it to obtain the dwarf Vallisneria and calamus extracts.

[0062] In some embodiments of the present application, the functional components in the composite microbial preparation are mutually beneficial and synergistic: Bifidobacterium thermophilic actinomycetes have a nitrogen fixation effect and can obtain amino acids, nitrogen, etc. from Rhodopseudomonas palustris as a matrix to inhibit the proliferation of pathogenic bacteria in the water body; at the same time, Bifidobacterium thermophilic actinomycetes mainly decompose organic matter, while Rhodopseudomonas palustris can use photosynthesis to convert inorganic matter into organic matter; the two are complementary in decomposing and converting substances, and can improve water quality more comprehensively; dwarf Vallisneria and calamus extracts can provide functional bacteria with specific substrates and carbon sources required for growth, thereby improving their survival rate; and after mixing with bifidobacterium thermophilic actinomycetes and Rhodopseudomonas palustris and fermenting, the indigestible macromolecules in the extracts of dwarf Vallisneria and calamus can be decomposed into small molecules, and the effective ingredients can be fully released, which is more conducive to the improvement of water quality.

[0063] The microecological preparation of the present application can be used to improve the water quality of marine aquaculture ponds, and usually the composite microbial preparation needs to be put into the marine aquaculture pond;

[0064] In some preferred embodiments, during the culture cycle, the amount of each input is 3-5 ml / m 3 , invested every 6-7 days.

[0065] The implementation scheme of the present application is described in detail below in conjunction with the examples. The present application includes but is not limited to the following implementation schemes. Any changes made to the present application based on the prior art that do not deviate from the essence of the present application still fall within the scope of protection of the present application.

[0066] Screening example: Screening of components of composite microbial preparations for improving water quality in marine aquaculture ponds

[0067] In the early stage of this application, the removal function of pollutants in marine aquaculture ponds was predicted and verified by 7 functional strains (Bacillus licheniformis, Lactobacillus acidophilus, Nitrifying Bacteria, Marine Red Yeast, Clostridium, Bifidobacterium thermophilic actinomycetes, and Rhodopseudomonas palustris) and 6 water purification plants (Phragmites australis, Water Plantain, Foxtail Algae, Canna, Dwarf Vallisneria, and Acorus) (see Table 1 for details). After gradient screening and component compatibility orthogonal tests, the dominant bacterial components Bifidobacterium thermophilic actinomycetes and Rhodopseudomonas palustris, as well as Dwarf Vallisneria extract and Acorus calamus extract were finally determined, and the components were then mixed and evaluated.

[0068] Table 1 Removal efficiency of each functional component on pollutants in marine aquaculture pond water (%)

[0069]

[0070] Example

[0071] In order to further evaluate the effects of the screened components and whether their mixture is more conducive to the improvement of aquaculture seawater quality, this application further studies the mixing of components under different ratios to screen microbial preparations with more reasonable compatibility and more significant synergistic effects.

[0072] Example 1

[0073] In this embodiment, the concentrations of the functional components of the composite microbial preparation are set to 10 7 cfu / ml, Rhodopseudomonas palustris 10 9 cfu / ml, dwarf vallisneria extract 0.04mg / ml, calamus extract 0.03mg / ml.

[0074] 1) The preparation method of bifidophilic actinomycetes is:

[0075] Step 1: Dissolve the lyophilized powder of bifidobacterium thermophilum (ATCC 49854) purchased by the laboratory in 1 ml of sterile distilled water, and activate the strain by streaking on a solid culture medium. The solid plate is cultured in an incubator at 55°C for 2-4 days until a single pale yellow colony with neat edges appears on the plate. The culture medium for bifidobacterium thermophilum is a nutrient broth agar medium.

[0076] Step 2: Pick a single colony and inoculate it into nutrient broth liquid culture medium at a culture temperature of 55°C. Incubate with shaking in an incubator for 2-3 days until the bacteria proliferate to the logarithmic growth phase.

[0077] Step 3: Centrifuge at 6000 rpm for 10 min, collect the bacteria, resuspend the bacteria in sterile seawater, adjust the concentration of the bacterial solution by McFarland turbidimetry, and set aside.

[0078] 2) The preparation method of Rhodopseudomonas palustris is:

[0079] Step 1: Dissolve the lyophilized powder of Rhodopseudomonas palustris (CICC 23812) purchased by the laboratory in 1 ml of sterile distilled water, and activate the strain by streaking on the solid culture medium. The solid plate is cultured in an anaerobic incubator at an illumination of 2000-3000 lux and a temperature of 30°C for 3-5 days until a single red colony appears on the plate. The components of the Rhodopseudomonas palustris culture medium are: tryptone 15.0 g / L, soytone 5.0 g / L, sodium chloride 5.0 g / L, agar 20 g / L, pH 7.5-8.0.

[0080] Step 2: Pick a single red colony and inoculate it into the liquid culture medium of Rhodopseudomonas palustris. The culture temperature is 30°C and the light intensity is 2000-3000 lux. Incubate it in an anaerobic incubator for 3-5 days until the bacteria proliferate to the logarithmic growth phase and the bacterial solution turns dark red.

[0081] Step 3: Centrifuge at 6000 rpm for 10 min, collect the bacteria, resuspend the bacteria in sterile seawater, adjust the concentration of the bacterial solution by McFarland turbidimetry, and set aside.

[0082] 3) The preparation method of dwarf Vallisneria extract and calamus extract is as follows: wash dwarf Vallisneria and calamus with distilled water respectively, ventilate and dry in the shade, and then crush them with a homogenizer, add 75% ethanol solution (solid-liquid ratio is 1g / 25ml), extract for 1.5h under ultrasonic action with an operating frequency of 60KHZ and a power of 150W, filter with sterile gauze to remove impurities, concentrate the extract under reduced pressure, and spray dry to obtain dwarf Vallisneria and calamus extracts.

[0083] 4) The method of compound microbial preparation is as follows: Thermobifidobacteria, Rhodopseudomonas palustris, Vallisneria dwarfi extract and Acorus calamus extract are mixed together according to the added concentrations, stirred evenly and set aside.

[0084] 5) Application method of compound microbial preparations: long-term use during the breeding cycle, with a dosage of 4-5 ml / m 3 , usually used once every 5-7 days.

[0085] Example 2

[0086] The preparation process and application method of the composite microbial preparation in this embodiment are basically the same as those in Example 1, except that the concentrations of the functional components are: 10 6 cfu / ml, Rhodopseudomonas palustris 10 7 cfu / ml, dwarf vallisneria extract 0.03mg / ml, calamus extract 0.01mg / ml.

[0087] Example 3

[0088] The preparation process and application method of the composite microbial preparation in this embodiment are basically the same as those in Example 1. The difference is that the concentrations of the functional components are: Bifidobacterium thermophilum 10 8 cfu / ml, Rhodopseudomonas palustris 10 8 cfu / ml, dwarf vallisneria extract 0.06mg / ml, calamus extract 0.02mg / ml.

[0089] Example 4

[0090] The application site of this application is in the Linghai Sea Cucumber Farming Area in Jinzhou City, Liaoning Province. Seawater aquaculture ponds are selected for the application test of the composite microbial preparation. Each aquaculture pond has an area of ​​4000-5000m 2 , water depth 2-2.5m, pond bottom is muddy. Pond A is used as a control example, no composite microbial preparation is applied. Pond B is used as an actinomycete comparison example, only Bifidobacterium thermophilic actinomycetes 10 8 cfu / ml and 0.06 mg / ml of Vallisneria dwarf extract and 0.03 mg / ml of Acorus calamus extract. Pond C was used as a photosynthetic bacteria control, with only Rhodopseudomonas palustris added at 10 9 cfu / ml and 0.06mg / ml of Vallisneria dwarf extract and 0.03mg / ml of Acorus calamus extract. Ponds D, E and F were groups of Example 1, 2 and 3 respectively. Each treatment group was administrated once every 7 days, and the dosage was 5ml / m 3 The control group did not use the compound microbial preparation, and other aquaculture management was the same as the treatment group. The experiment lasted for 21 days, and pond water samples were taken every 7 days for physical and chemical index testing (total nitrogen, ammonia nitrogen, nitrite, total phosphorus, phosphate, chemical oxygen demand).

[0091] After the pond test, 500L of water samples were taken from each treatment group and placed in a cylindrical aquarium (100cm in diameter and 90cm in height) for pollutant infection test. A mixture of heavy metals, antibiotics, and pathogenic Vibrio was added to each aquarium. The heavy metal mixed solution included lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg), and the concentration of each component added to the water was 100μg / L. The antibiotic mixed solution included oxytetracycline (OTC), sulfadiazine (SD), tetracycline (TCS), and lincomycin (LIN), and the concentration of each component added to the water was 100μg / L. The pathogenic Vibrio was Vibrio harveyi, and the concentration added to the water was 10 9 cfu / L. The test lasted for 72 hours, and water samples were taken every 24 hours to determine the content of composite pollutants. The heavy metal content was determined by atomic absorption spectrometry, the antibiotic content was determined by high performance liquid chromatography-electrospray ionization tandem mass spectrometry, and the number of pathogenic Vibrio was determined by plate coating method. The heavy metal and antibiotic solutions used were provided by the Molecular Biology Laboratory of Liaoning Marine Fisheries Science Research Institute. The Vibrio harveyi (MCCC1A00232) used was purchased from the Marine Microbial Culture Collection Management Center.

[0092] The effects of specific composite microbial preparations on the ammonia nitrogen, nitrite, phosphorus content and chemical oxygen demand in aquaculture pond water. Figure 1-6 As shown. High concentrations of ammonia nitrogen and nitrite pollutants in pond water will reduce the dissolved oxygen content in the water, causing eutrophication of the water, inhibiting the growth and development of farmed animals, and leading to diseases and death of farmed animals. Excessive phosphorus pollutants will cause a large number of harmful algae to float on the surface of the aquaculture water, causing red tides or algal blooms, which will lead to hypoxia in the water and reduce the immune function and feeding ability of farmed animals. Chemical oxygen demand is a chemical method to measure the amount of reducing substances that need to be oxidized in the water. If the value is too high, it means that the content of organic pollutants in the pond water is too high, the oxygen transport capacity is weak, and the respiratory process of farmed animals is inhibited.

[0093] The results show that before the application of the composite microbial preparation, there was no significant difference in the physical and chemical indicators of the pond water in each treatment group. As the experiment progressed, except for the control group, each treatment group was able to effectively improve the physical and chemical indicators of the aquaculture pond water, and compared with the actinomycete control group and the photosynthetic bacteria control group, Examples 1, 2, and 3 had more obvious improvement effects on the aquaculture water. Among them, Example 1 had the best removal effect on total nitrogen and total phosphorus, and the chemical oxygen demand of the water was the lowest. Example 2 had the best removal effect on ammonia nitrogen. Example 3 had the best removal effect on nitrite and phosphate. It shows that the composite microbial preparation can significantly reduce the content of ammonia nitrogen, phosphorus and organic pollutants in pond water.

[0094] Effects of microbial preparations in each treatment group on the contents of heavy metals, antibiotics, and pathogenic Vibrio in the water of marine aquaculture ponds Figure 7-9As shown. Heavy metal ions will produce a large number of free radicals in the aquaculture water, interfere with cell metabolism, accumulate in the body of farmed animals, and then enter the food chain cycle, posing a potential threat to human health. Antibiotics remaining in the aquaculture process will spread to the aquatic environment, destroying the balance of microbial communities and the diversity of phytoplankton and zooplankton by selecting antibiotic-resistant species. Pathogenic Vibrio can cause pathogenic effects on aquatic animals by secreting hemolysins, enterotoxins, etc., and can infect shellfish, shrimps and fish in a short period of time and cause a large number of deaths. The results show that, except for the control group, each treatment group can effectively reduce the content of heavy metals, antibiotics, and pathogenic Vibrio, and compared with the actinomycete control and photosynthetic bacteria control, Examples 1, 2, and 3 can more significantly reduce the content of heavy metals, antibiotics, and pathogenic Vibrio. Among them, Example 1 has the best removal effect on lead, arsenic, and tetracycline, Example 2 has the best removal effect on cadmium and oxytetracycline, and Example 3 has the best removal effect on mercury, sulfadiazine, lincomycin, and Vibrio harveyi.

[0095] It can be seen from the above test results that the components in the composite microecological agent of the present application can mutually benefit and coexist, and have synergistic synergy, which can effectively reduce the content of ammonia nitrogen, phosphorus and organic pollutants in the water of seawater aquaculture ponds. At the same time, heavy metals, antibiotics and pathogenic vibrio in the pond water can be effectively removed, and the water of seawater aquaculture ponds has a significant improvement effect. According to analysis, bifidobacterium thermophilic actinomycetes are aerobic bacteria with nitrogen fixation. It can obtain amino acids, nitrogen and the like from Rhodopseudomonas palustris as a matrix, produce antibiotics, vitamins and enzymes, etc., and inhibit the proliferation of pathogenic bacteria in the water body; at the same time, bifidobacterium thermophilic actinomycetes mainly decompose organic matter, while Rhodopseudomonas palustris can use photosynthesis to convert inorganic matter into organic matter. The two are complementary in decomposition and conversion of substances, and can improve water quality more comprehensively. Dwarf Vallisneria and Acorus calamus extracts can provide functional bacteria with the specific substrate and carbon source required for growth, thereby improving their survival rate. After the microbial fermentation of probiotics, the indigestible macromolecules in the extracts of Vallisneria dwarfi and Acorus calamus can be decomposed into small molecules, and the effective ingredients can be fully released, which is more conducive to the improvement of water quality. The action processes of the various components of this composite microbial preparation are coupled and synergistic, which can comprehensively improve the aquaculture water.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A composite microbial preparation for improving the water quality of marine aquaculture ponds, characterized in that: The functional components of the composite microbial preparation include Thermoactinomyces dichotomicus, Rhodopseudomonas palustris, Vallisneria dwarfi extract and Acorus calamus extract.

2. The composite microbial preparation according to claim 1, characterized in that: The deposit number of the bifidophilic actinomycetes is ATCC 49854; the deposit number of the Rhodopseudomonas palustris is CICC 23812.

3. The composite microbial preparation according to any one of claims 1-2, characterized in that: The concentration of bifidobacterium thermophilic actinomycetes in the composite microbial preparation is 10 6 -10 8 cfu / ml, the concentration of Rhodopseudomonas palustris was 10 7 -10 9 cfu / ml, the concentration of dwarf vallisneria extract was 0.03-0.06mg / ml, and the concentration of calamus extract was 0.01-0.03mg / ml.

4. The composite microbial preparation according to any one of claims 1 to 3, characterized in that: The preparation method of the bifidophilic actinomycetes comprises: Step 1: dissolving the bifidobacterium thermophilic actinomycete freeze-dried powder in distilled water, streaking the strain on a solid culture medium for activation, and culturing in an incubator at 50-55° C. for 2-4 days until a single pale yellow colony with neat edges appears on the plate; preferably, the solid culture medium is a nutrient broth agar medium; Step 2: Pick a single colony and inoculate it into a nutrient broth liquid culture medium at a culture temperature of 50-55°C. Shake and culture in an incubator for 2-3 days until the bacteria proliferate to the logarithmic growth phase; Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

5. The composite microbial preparation according to any one of claims 1 to 3, characterized in that: The preparation method of the Rhodopseudomonas palustris comprises: Step 1: fully dissolve the freeze-dried powder of Rhodopseudomonas palustris in distilled water, streak the strain on a solid culture medium to activate it, and culture it in an anaerobic incubator for 3-5 days under the conditions of an illumination of 2000-3000 lux and a temperature of 28-30°C until a single red colony appears on the plate; preferably, the components of the Rhodopseudomonas palustris culture medium are: tryptone 10-15.0 g / L, soytone 3-5.0 g / L, sodium chloride 3-5.0 g / L, agar 15-20 g / L, pH 7.5-8.0; Step 2: Pick a single red colony and inoculate it into a liquid culture medium of Rhodopseudomonas palustris, with a culture temperature of 28-30°C and a light intensity of 2000-3000 lux, and culture it in an anaerobic incubator for 3-5 days until the bacteria proliferate to the logarithmic growth phase and the bacterial solution turns dark red; Step 3: Collect the cells by centrifugation at 5000-6000 rpm, resuspend the cells in sterile seawater and set aside.

6. The composite microbial preparation according to any one of claims 1-2, characterized in that: The preparation method of the dwarf Vallisneria extract and the calamus extract is as follows: respectively clean the dwarf Vallisneria and the calamus with distilled water, ventilate and dry in the shade, and then crush them, add 75% ethanol solution, extract for about 1.5-2h under the action of ultrasound with a working frequency of 50-60KHZ and a power of 100-150W, filter to remove impurities, and reduce the pressure to concentrate the extract and spray dry it to obtain the dwarf Vallisneria and calamus extracts.

7. The composite microbial preparation according to claims 1-6, characterized in that: The functional components in the composite microbial preparation are mutually beneficial and synergistic: the bifidobacterium thermophilic actinomycetes has a nitrogen-fixing effect and can obtain amino acids, nitrogen and the like from the palustris Rhodopseudomonas as a matrix to inhibit the proliferation of pathogenic bacteria in the water body; at the same time, the bifidobacterium thermophilic actinomycetes mainly decompose organic matter, while the palustris Rhodopseudomonas can use photosynthesis to convert inorganic matter into organic matter; the two are complementary in terms of decomposition and conversion of substances, and can improve water quality more comprehensively; the extracts of dwarf Vallisneria and calamus can provide the functional bacteria with specific substrates and carbon sources required for growth, thereby improving their survival rate; and after being mixed with the bifidobacterium thermophilic actinomycetes and the palustris Rhodopseudomonas, the indigestible macromolecular substances in the extracts of dwarf Vallisneria and calamus can be decomposed into small molecular substances after fermentation, and the effective ingredients can be fully released, which is more conducive to the improvement of water quality.

8. Use of the bioactive agent according to any one of claims 1 to 7 in improving the water quality of marine aquaculture ponds.

9. The method for preparing the composite microbial preparation according to any one of claims 1 to 7, characterized in that: The method comprises the step of thoroughly mixing bifidophilic actinomycetes, Rhodopseudomonas palustris, Vallisneria dwarfi extract and Acorus calamus extract.

10. A method for improving water quality in marine aquaculture ponds, characterized in that: The method comprises adding the composite microbial preparation according to any one of claims 1 to 7 into a marine aquaculture pond; preferably, the amount of each addition is 3-5 ml / m 3 , invested every 6-7 days.

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