A composite microbial repair agent and its application and a repair method for sand used for snail breeding

Abstract

The present invention discloses a composite microbial repair agent and its application and a repair method for sand used for snail breeding. The composite microbial repair agent comprises an oxidant and a microbial preparation; the oxidant is a chlorine preparation; the microbial preparation comprises Pseudomonas, Corbettella, Bacillus marineus, Halomonas seawater, Bacillus flexus, and Bacillus subtilis denitrifying. The composite microbial repair agent has an excellent improvement effect on sand used for snail breeding, and can significantly reduce the content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens in the sand used for snail breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial soil remediation, and more specifically, relates to a composite microbial remediation agent and application thereof and a method for remediating sand for snail breeding. Background Art

[0002] Dongfeng conch, also known as sea pig conch, sweet conch, southern wind conch, yellow conch, and flower conch, belongs to the phylum Mollusca, class Gastropoda, subclass Prosobranchia, order Neogastropoda, family Buccinidae, and genus Babylonia. It is the main aquaculture shellfish species in my country. Dongfeng conch has the characteristics of fast growth, short breeding cycle, crisp meat, delicious taste, and has extremely high nutritional value and medicinal value.

[0003] However, the current farming technology of Dongfeng snails is not perfect, and there are still a series of problems: during the long farming period of half a year to a year, a large amount of residual bait, metabolites and other organic matter accumulates in the sand layer of Dongfeng snail farming, which consumes a lot of oxygen during decomposition, resulting in long-term hypoxia in the sand layer, excessive concentrations of methane, ammonia nitrogen, nitrite, hydrogen sulfide, etc., deterioration of sand quality, increased acidity, black and smelly, and large-scale reproduction of pathogens. It is time-consuming and laborious to clean up the sand after farming, and reuse can easily cause a new batch of snail seedlings to die in large numbers due to the poor bottom sand layer and inability to adapt to the environment. The existence of these problems seriously affects the reuse of sand and restricts the healthy development of Dongfeng snail farming.

[0004] Patent publication number CN115557627A discloses a sea cucumber aquaculture water quality repair agent and a preparation method thereof. The repair agent is composed of a bacterial agent, a nutrient solution and a water treatment liquid in a mass ratio of 2-5:1:1-3. The bacterial agent is composed of Bacillus subtilis, Bacillus cereus, Bacillus flexus and septic tank-specific bacteria in a mass ratio of 2-3:1-2:1-2:1. The septic tank-specific bacteria are composed of Pseudomonas, actinomycetes, yeasts and Streptomyces tenuifolius in a mass ratio of 1:2-4:1-3:1. The nutrient solution is composed of silkworm pupa powder and sodium chloride in a mass ratio of 1:4-6. The water treatment liquid is composed of potassium permanganate and peramide in a mass ratio of 1-2:1. However, the repair agent is for sea cucumbers and is a water quality treatment, and is not suitable for sand used in Dongfeng snail farming. Summary of the invention

[0005] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a composite microbial remediation agent, which has an excellent effect on improving the sand used for snail breeding and can significantly reduce the content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens in the sand used for snail breeding.

[0006] The second object of the present invention is to provide an application of a composite microbial remediation agent in improving and repairing sand for snail breeding.

[0007] The third object of the present invention is to provide a method for repairing sand for snail breeding.

[0008] The fourth object of the present invention is to provide a method for cultivating snails.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] A composite microbial remediation agent comprises an oxidant and a microbial preparation; the oxidant is a chlorine preparation; the microbial preparation comprises Pseudomonas, Corbettella, Bacillus marineus, Halomonas seawater, Bacillus flexus and denitrifying Bacillus subtilis.

[0011] Preferably, the ratio of the colony counts of Pseudomonas, Corbettella marine, Bacillus marine, Halomonas marine, Bacillus flexus and Bacillus subtilis denitrifying is 4-6:4-8:1-2:2-4:2-4:2-5.

[0012] Preferably, the colony count in the microbial preparation is 2.0×10 4 ~4.0×10 4 CFU / mL.

[0013] Preferably, the oxidant is selected from one or more of 1-bromo-3-chloro-5,5-dimethylhydantoin, trichloroisocyanuric acid or sodium hypochlorite.

[0014] Preferably, the composite microbial remediation agent also includes a regulator, which includes, by weight: 5 to 10 parts of tetrakis hydroxymethyl phosphonium sulfate, 3 to 5 parts of sodium acetate, 2 to 6 parts of oxalic acid, 1 to 3 parts of α-hydroxy acid, and 4 to 8 parts of humic acid.

[0015] Preferably, the mass ratio of the oxidant, the regulator and the microbial preparation is 1.5-3:2-3:2-5.

[0016] The invention provides a composite microbial repair agent, which has excellent improvement and repair effects on sand used for snail breeding.

[0017] Specifically, the present invention adopts chlorine preparation as oxidant. Chlorine preparation has high efficiency and broad-spectrum disinfection effect, and has killing effect on bacteria, viruses, fungi, spores, etc., and the use of chlorine preparation has certain safety.

[0018] In the present invention, the microbial preparation includes Poseidonomonas, Corbettella, Bacillus marineus, Halomonas marinum, Bacillus flexus and Bacillus subtilis denitrifying. These beneficial bacteria are all marine bacteria. The inventors have found through research that the antibacterial substances produced by the above-mentioned specific combination of microbial preparations can effectively inhibit the occurrence of harmful pathogens, improve the immune function of snails, regulate the balance of microecology, and degrade the content of ammonia nitrogen, nitrite, hydrogen sulfide and other substances in the sand used for snail breeding.

[0019] In the present invention, the composite microbial remediation agent also includes a regulator, which includes tetrakis hydroxymethyl phosphite, sodium acetate, oxalic acid, α-hydroxy acid and humic acid. The above-mentioned specific regulator can play an excellent role in complex adsorption, oxidation and sterilization, bleaching and acidification, and can effectively remove harmful substances remaining in the sand used for snail breeding.

[0020] The composite microbial remediation agent provided by the present invention adopts a chlorine preparation, a microbial preparation of a specific composition, and a regulator of a specific composition. When the above three compositions are used in combination, the effect of improving and repairing the sand for snail breeding can be synergistically improved. The content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens in the repaired sand for snail breeding are extremely low, and it has the advantages of being recyclable, clean and white, etc., which better meets the demand for sand for snail breeding.

[0021] Furthermore, the present invention seeks to protect the use of the above-mentioned composite microbial remediation agent in improving and repairing sand for snail breeding.

[0022] Further preferably, the snail breeding sand is sand for Dongfeng snail breeding.

[0023] Specifically, the babylonia is of the genus Babylonia (Babylonia), including but not limited to Babylonia areolata, Babylonia lutosa, Babylonia formosae, etc.

[0024] Furthermore, the present invention seeks to protect a method for preparing the composite microbial remediation agent, comprising:

[0025] Tetrakis hydroxymethyl phosphonium sulfate, sodium acetate, oxalic acid, α-hydroxy acid and humic acid are mixed evenly to prepare a regulator. Pseudomonas, Corbettella, Bacillus marineus, Halomonas seawater, Bacillus flexus and Bacillus subtilis denitrifying are mixed evenly to prepare a microbial preparation.

[0026] Preferably, the above-mentioned regulators can be mixed under normal temperature and pressure conditions, and the mixing time can be 10 to 30 minutes.

[0027] Preferably, the above-mentioned microbial preparation can be mixed under the conditions of pH of 7.8-8.2 and air temperature of 29-31° C., and the mixing time can be 10-30 minutes.

[0028] Furthermore, the present invention seeks to protect a method for repairing sand for snail breeding, using the above-mentioned composite microbial repair agent to repair the sand for snail breeding, and the repair method comprises the following steps:

[0029] S1. Using oxidants to disinfect the sand used for snail farming, and then using antidotes to detoxify;

[0030] S2. Dry the detoxified snail culture sand to make it mineralized, or

[0031] The disinfected snail breeding sand is dried to mineralize it, and then a regulator is sprayed on the snail breeding sand to improve it, and then neutralized after improvement;

[0032] S3. The mineralized or neutralized snail farming sand is treated with microbial preparations.

[0033] In the present invention, since the sand is soaked in water for a long time, mineralization can change the sand environment, promote the circulation of substances such as ammonia nitrogen, and change the original harmful bacterial community of the sand.

[0034] Preferably, in the step S1, 15 to 30 grams of oxidant is soaked per cubic meter of sand for snail breeding; or in the step S2, 20 to 30 grams of regulator is sprayed per cubic meter of sand for snail breeding; or in the step S3, 20 to 50 grams of microbial preparation is sprayed per cubic meter of sand for snail breeding.

[0035] Preferably, in the step S1, the time for soaking the snail breeding sand with the oxidant is 1 to 7 days; or in the step S2, the time for improving the snail breeding sand by spraying the regulator is 2 to 5 days; or in the step S3, the time for treating the snail breeding sand by spraying the microbial preparation is 2 to 6 days.

[0036] Preferably, in step S1, aeration is performed during the disinfection process, and aeration during the disinfection process can make the oxidant mix evenly.

[0037] Preferably, the detoxifying agent may be a substance conventionally used by those skilled in the art for detoxifying chlorine preparations, such as sodium thiosulfate.

[0038] Preferably, a conventional buffer solution in the art, such as tris(hydroxymethyl)aminomethane solution, can be used for neutralization.

[0039] Furthermore, the present invention seeks to protect a method for cultivating snails, in which snails are cultivated using snail cultivation sand repaired by the above-mentioned repair method.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a composite microbial remediation agent, which has an excellent effect on improving sand for snail breeding, can significantly reduce the contents of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens in the sand for snail breeding, and has the advantages of being recyclable, clean and white, and is more in line with the demand for sand for snail breeding.

[0042] Instruction Manual

[0043] Figure 1 Schematic diagram of the pre-repaired sand in Example 1.

[0044] Figure 2 This is a schematic diagram of Example 4, in which the sand repaired in Example 1 is laid in the breeding pond to continue breeding. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Example 1 Method for repairing sand used for breeding of babylonica

[0047] (1) Install a row of porous water-permeable baffles on the bottom of the pool, then lay a layer of 80-mesh gauze on the baffles, and then lay pre-repaired sand to create an empty space (such as Figure 1 As shown, the sand was used to culture Babylonia squarella for 10 months. The concentrations of harmful Vibrio, ammonia nitrogen, nitrite, hydrogen sulfide and other substances in the sand exceeded the standard. The sand was acidic, black and smelly. The sand was repeatedly washed and stirred with seawater until the water was clear, without sewage or impurities.

[0048] (2) Disinfection: The sand after dredging is disinfected by soaking in an oxidant (the oxidant is trichloroisocyanuric acid, source: Guangzhou Jingbo Biotechnology Co., Ltd., trade name "Strong Chloroquine", 30 grams of oxidant is used for each cubic meter of pre-repaired sand). The total aeration disinfection time is 6 days; then, sodium thiosulfate of the same weight as the oxidant is used to detoxify the sand.

[0049] (3) Mineralization: The detoxified sand in step (2) is dried at 75°C for 12 hours. After drying, the sand is taken out.

[0050] (4) Conditioning: Collect the mineralized sand in step (3) and pile it in a cool place. Spread it flat and evenly spray it with a conditioning agent for improvement (25 grams of conditioning agent per cubic meter of pre-repaired sand) for 4 days.

[0051] The regulator includes, by weight: 8 parts of tetrakis hydroxymethyl phosphonium sulfate, 5 parts of sodium acetate, 4 parts of oxalic acid, 2 parts of α-hydroxy acid, and 6 parts of humic acid; the preparation method of the regulator is: under normal temperature and pressure conditions, the above components are added into a solid material mixer and mixed for 20 minutes, and after being fully stirred, the regulator is prepared.

[0052] (5) Neutralization: neutralizing the sand adjusted in step (4) using a buffer solution (tris(hydroxymethyl)aminomethane solution, pH 7.5-8.0);

[0053] (6) Improvement: The sand neutralized in step (5) is spread evenly and sprayed evenly with the microbial preparation (50 g of microbial preparation is used per cubic meter of pre-repaired sand). The sand is remediated after 5 days.

[0054] Among them, the microbial preparations include Pseudomonas (source: China Marine Microbial Culture Collection Administration Center, collection number 1K03613), Corbettella (source: China Marine Microbial Culture Collection Administration Center, collection number 1A00811), Bacillus marineus (source: General Microbiological Center of China Microbial Culture Collection Administration Committee, collection number CGMCC No.25763), Halomonas marineus (source: China Marine Microbial Culture Collection Administration Center, collection number 1A00416), Bacillus flexus (source: Guangdong Provincial Microbial Culture Collection Center, collection number GDMCC No.6277), and Bacillus subtilis denitrifying (source: Guangdong Provincial Microbial Culture Collection Center, collection number GDMCC No:62676); the ratio of the colony counts of the above-mentioned species is 4:6:2:4:3:4, respectively, and the colony count of the microbial preparation is 3.0×10 4 The preparation method of the microbial preparation is as follows: put the above components into a solid material mixer, mix and stir for 20 minutes under the conditions of pH 7.8-8.2 and temperature 29-31°C to prepare the microbial preparation.

[0055] Example 2 Method for repairing sand used for breeding of babylonica

[0056] The difference between this embodiment and embodiment 1 is that:

[0057] The regulator includes, by weight: 10 parts of tetrakis hydroxymethyl phosphonium sulfate, 5 parts of sodium acetate, 5 parts of oxalic acid, 3 parts of α-hydroxy acid and 5 parts of humic acid;

[0058] Among the microbial preparations, the ratios of the colony counts of Poseidononas, Corbettella, Bacillus marineus, Halomonas marinum, Bacillus flexus and Bacillus subtilis denitrifying were 6:8:2:2:4:5, respectively.

[0059] Example 3: Method for repairing sand used for breeding of babylonica

[0060] The difference between this embodiment and embodiment 1 is that:

[0061] The regulator includes, by weight: 5 parts of tetrakis hydroxymethyl phosphonium sulfate, 3 parts of sodium acetate, 4 parts of oxalic acid, 1 part of α-hydroxy acid and 4 parts of humic acid;

[0062] Among the microbial preparations, the ratios of the colony counts of Poseidononas, Corbettella, Bacillus marineus, Halomonas marinum, Bacillus curvatus and Bacillus subtilis denitrifying were 4:4:1:2:2:2, respectively.

[0063] Example 4 Babylonia concha farming

[0064] The experiment used an indoor cement pool of 2.5m×6.0m×0.5m as the breeding pool for Babylonia snails. 2 ) The bottom of the pool is laid with a bottom plate, a gauze and the sand repaired in Example 1 (such as Figure 2 The sand layer thickness is kept at 5 cm. The seawater is extracted from the sand filter well and directly enters the breeding pond after passing through the filter pool. The seedlings are from the young snails with a shell length of 5 to 10 mm bred by the parent snails of the square-headed snails cultivated in the Yandun breeding farm in Wenchang. The breeding density in each pond is 800 grains / m 2 .

[0065] During the experiment, white squid was fed throughout the experiment, and the feeding amount accounted for about 7% of the body weight of the mud snails. It was best to have no food left within 20 minutes after feeding. The water was changed 30 minutes after feeding, and the amount of water changed was enough not to expose the sand layer. Then the water was cultured in a flowing water, and the flow rate was controlled at 10 to 15 flows per day.

[0066] Example 5 Babylonia snail farming

[0067] The difference between this embodiment and embodiment 4 is that the sand repaired in embodiment 2 is used.

[0068] Example 6 Babylonia concha farming

[0069] The difference between this embodiment and embodiment 4 is that the sand repaired in embodiment 3 is used.

[0070] Example 7 Babylonia snail farming

[0071] The difference between this embodiment and embodiment 4 is that in the sand repaired in embodiment 1, no conditioning agent is used to treat the sand.

[0072] Example 8 Babylonia snail farming

[0073] The difference between this embodiment and embodiment 4 is that in the sand repaired in embodiment 1, the conditioning agent only includes humic acid.

[0074] Example 9: Babylonia snail farming

[0075] The difference between this embodiment and embodiment 4 is that in the sand repaired in embodiment 1, the conditioning agent only includes sodium acetate.

[0076] Comparative Example 1

[0077] The difference between this comparative example and Example 4 is that in the sand repaired in Example 1, no oxidant is used to treat the sand.

[0078] Comparative Example 2

[0079] The difference between this comparative example and Example 4 is that in the sand repaired in Example 1, no microbial preparation is used to treat the sand.

[0080] Comparative Example 3

[0081] The difference between this comparative example and Example 4 is that in the sand repaired in Example 1, no oxidant is used to treat the sand; no regulator is used to treat the sand; and no microbial agent is used to treat the sand.

[0082] Comparative Example 4

[0083] The difference between this comparative example and Example 4 is that in the sand repaired in Example 1, the microbial preparation only includes Bacillus flexus.

[0084] Comparative Example 5

[0085] The difference between this comparative example and Example 4 is that in the sand repaired in Example 1, the microbial preparation only includes denitrifying Bacillus subtilis.

[0086] Test Example 1

[0087] The testing method of the present invention is as follows:

[0088] (1) Detection of Vibrio content and total bacteria: Take water near the sand layer at the drainage outlet of the culture pond in Examples 4 to 9 and Comparative Examples 1 to 5, and detect the Vibrio and bacteria content.

[0089] Vibrio detection: TCBS culture method was used, and water samples were taken on site and diluted with sterile seawater to 1, 10 -1 , 10 -2 100 μl of each gradient was spread on TCBS agar plates, and each gradient was replicated three times. The inoculated plates were incubated at 28°C for 48 h, and the colonies with an average colony count of 30 to 300 were selected as valid colonies, and the number of colony forming units (CFU) was counted.

[0090] Bacteria detection: According to the national standard GB 17378.7-2007 "Ocean Monitoring Specifications" Part 7, the plate count method was used, using 2216E agar medium, and the water samples were diluted to 1, 10-1 , 10 -2 100 μl of each gradient was applied to the plate, and 3 parallels were made for each gradient. The inoculated plates were incubated at 25°C for 7 days, and the average colony count was 30-300 as the effective colony, and the CFU number was counted.

[0091] (2) Ammonia nitrogen content detection: Use Amtax NA8000 ammonia nitrogen detector (Hach Company) for detection.

[0092] (3) Nitrite content detection: Detection was performed using Shimadzu UV-Vis spectrophotometer UV-2450 / 2550 (Shanghai Na-Ning Industrial Co., Ltd.).

[0093] (4) Hydrogen sulfide content detection: using hydrogen sulfide analyzer GPro500 (Mettler Toledo International Ltd.).

[0094] The test results of each group of embodiments and comparative examples are shown in Table 1 below.

[0095] Table 1

[0096]

[0097] It can be seen from the above examples that the composite microbial remediation agent of the present invention has excellent improvement and repair effects on the sand used for snail breeding. The repaired sand for snail breeding significantly reduces the content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens, which is more in line with the demand for sand for snail breeding. The ammonia nitrogen content in the repaired sand for snail breeding is ≤0.60mg / L, the nitrite content is ≤0.150mg / L, the hydrogen sulfide content is ≤33mg / L, and the Vibrio content is ≤1700CFU·mL -1 , total bacterial content ≤5830CFU·mL -1 Preferably, the ammonia nitrogen content in the repaired snail breeding sand is less than 0.02 mg / L, the nitrite content is less than 0.003 mg / L, there is no hydrogen sulfide and Vibrio, and the total bacterial content is less than 36 CFU·mL -1 , and has the advantages of being clean and white.

[0098] It can be seen from Examples 4 and 7 to 9 that, compared with a composite microbial remediation agent that does not contain a regulator, or a regulator that contains only one of the components, when the regulator in the composite microbial remediation agent adopts a specific combination, it can more significantly reduce the content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens, and has the advantages of being clean and white, which is more in line with the demand for sand for snail farming.

[0099] It can be seen from Example 4, Comparative Example 2, Comparative Example 4 and Comparative Example 5 that, compared with a composite microbial remediation agent containing no microbial preparation, or a microbial preparation containing only one of the components, when the microbial preparation in the composite microbial remediation agent adopts a specific combination, it can more significantly reduce the content of ammonia nitrogen, nitrite, hydrogen sulfide and the number of pathogens, and has the advantages of being clean and white, which is more in line with the demand for sand for snail breeding.

[0100] It can be seen from Example 4, Example 7, Comparative Example 1, Comparative Example 2 and Comparative Example 3 that the chlorine preparation, the microbial preparation and the regulator can synergistically improve the effect of improving and repairing the sand for snail breeding. The ammonia nitrogen, nitrite, hydrogen sulfide content and the number of pathogens in the repaired sand for snail breeding are extremely low. It is difficult to achieve the technical effect of the present invention by using only two of the components.

[0101] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A composite microbial remediation agent, characterized in that: It includes an oxidant and a microbial preparation; the oxidant is a chlorine preparation; the microbial preparation includes Pinemonas, Corbettella, Bacillus marine, Halomonas marine, Bacillus flexus and Bacillus subtilis denitrifying; the colony count ratio of Pinemonas, Corbettella marine, Bacillus marine, Halomonas marine, Bacillus flexus and Bacillus subtilis denitrifying is 4-6:4-8:1-2:2-4:2-4:2-5; The composite microbial remediation agent also includes a regulator, which includes, by weight: 5 to 10 parts of tetrakis hydroxymethyl phosphonium sulfate, 3 to 5 parts of sodium acetate, 2 to 6 parts of oxalic acid, 1 to 3 parts of α-hydroxy acid, and 4 to 8 parts of humic acid; The mass ratio of the oxidant, the regulator and the microbial preparation is 1.5-3:2-3:2-5.

2. The composite microbial remediation agent according to claim 1, characterized in that: In the microbial preparation, the colony count was 2.0×10 4 ~4.0×10 4 CFU / mL.

3. The composite microbial remediation agent according to claim 1, characterized in that: The oxidant is selected from one or more of 1-bromo-3-chloro-5,5-dimethylhydantoin, trichloroisocyanuric acid or sodium hypochlorite.

4. Use of the composite microbial remediation agent according to any one of claims 1 to 3 in improving and repairing sand for snail breeding.

5. A method for repairing sand for snail breeding, characterized in that: The method of repairing the sand for snail breeding by using the composite microbial repair agent according to any one of claims 1 to 3 comprises the following steps: S1. Use oxidants to disinfect the snail culture sand, and then use antidotes to detoxify; S2. The disinfected snail breeding sand is dried to mineralize it, and then a regulator is sprayed on the snail breeding sand to improve it, and then neutralized after improvement; S3. The mineralized or neutralized snail culture sand is treated with microbial preparations.

6. The repair method according to claim 5, characterized in that: Use any of the following conditions: In the step S1, 15 to 30 grams of oxidant is used to soak each cubic meter of sand for snail breeding; or In step S2, 20 to 30 grams of a regulator is sprayed per cubic meter of snail breeding sand; or In step S3, 20 to 50 grams of microbial preparation is sprayed per cubic meter of snail breeding sand.

7. The repair method according to claim 6, characterized in that: Use any of the following conditions: In the step S1, the snail breeding sand is soaked in an oxidant for 1 to 7 days; or In step S2, the time for improving the sand for snail breeding by spraying the regulator is 2 to 5 days; or In step S3, the microbial preparation is sprayed on the snail breeding sand for treatment for 2 to 6 days.

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