A composite microbial agent and its application

Abstract

The present invention discloses a compound microbial agent and its application, belonging to the technical field of microbiology. The compound microbial agent includes Bacillus sp. BS-K1 and Pseudomonas sp. CN-B1. The preservation number of the Bacillus is CCTCC NO: M 20242022; the preservation number of the Pseudomonas is CCTCC NO: M 20242021. The compound microbial agent provided by the present invention combines probiotics with antibacterial effects ( Bacillus sp. BS-K1) and denitrifying bacteria ( Pseudomonas sp. CN-B1) to obtain a compound microbial agent for aquaculture. When applied to aquaculture water bodies, it has excellent denitrification ability and inhibitory effects on related pathogenic bacteria in the water, can improve the removal efficiency of ammonia nitrogen and nitrite nitrogen in the aquaculture water, reduce the number of related pathogenic bacteria, and ensure the safety of the aquaculture environment.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a composite bacterium agent and its application. Background Art

[0002] Aquaculture, also known as fish farming, has made important contributions to solving the problem of urban and rural residents in China having difficulty accessing fish, increasing the supply of high-quality animal protein, improving the national nutrition and health level, and ensuring food safety in China. Aquaculture is an important part of agricultural and sideline production. With the progress of technology and the change of market demand, traditional aquaculture has gradually developed towards intensification. Highly intensive aquaculture refers to a farming mode that realizes high yields and high economic benefits through means such as high-density farming, efficient feed feeding, water quality management, and disease prevention and control in a limited space.

[0003] However, in highly intensive aquaculture, a large amount of unconsumed feed, excrement, animal carcasses, etc. are discharged into the water, resulting in an increase in the concentrations of ammonia nitrogen and nitrite nitrogen in the aquaculture water body. At the same time, the presence of pathogenic bacteria in the water body has a serious harmful effect on both the ecological environment and the aquaculture products. Regarding the problem of improving water quality, currently, physical treatment methods such as sedimentation, filtration, and air flotation, chemical treatment methods such as coagulation sedimentation and redox, and biological treatment methods that use microorganisms to degrade organic matter and remove nitrogen and phosphorus are adopted. Biological treatment has significant advantages in treating aquaculture wastewater, including environmental friendliness, economic efficiency, good treatment effect, strong sustainability, simple operation, and versatility. These advantages make biological treatment one of the preferred methods in the current and future wastewater treatment fields.

[0004] . The current biological treatment using a single strain also has deficiencies in water quality improvement. It often has a single function and does not have the characteristics of multiple functions; it is easily restricted by the water environment during the proliferation of the applied water body and has certain limitations; at the same time, the effect of the composite bacterium agents on the market is poor, and the improvement of the removal effect of ammonia nitrogen and nitrite nitrogen in the water body is not obvious. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite bacterium agent that can improve the removal effect of ammonia nitrogen and nitrite nitrogen in the aquaculture water body and reduce the number of related pathogenic bacteria; another purpose of the present invention is to provide an application of the composite bacterium agent.

[0006] The present invention discloses a composite bacterium agent, which includes Bacillus ( Bacillus sp . BS-K1) and Pseudomonas ( Pseudomonas sp. CN--B1), the Bacillus was deposited at the China Center for Type Culture Collection on September 23, 2024, with the deposit number CCTCC NO: M 20242022; the Pseudomonas was deposited at the China Center for Type Culture Collection on September 23, 2024, with the deposit number CCTCC NO: M 20242021.

[0007] The composite bacterial agent includes Bacillus ( Bacillus sp . BS-K1) and Pseudomonas ( Pseudomonas sp . CN--B1) and a microbial culture. The Acinetobacter Bacillus ( Bacillus sp. BS-K1) and Pseudomonas ( Pseudomonas sp. CN--B1) are used as the active ingredients of the bacterial preparation.

[0008] Bacillus is a probiotic widely used in aquaculture, which can regulate water quality and inhibit the growth of pathogenic bacteria. The denitrifying bacterium Pseudomonas can convert organic pollutants in sewage into harmless substances such as carbon dioxide and water, and at the same time can convert nitrogen in sewage into harmless nitrogen gas, thereby removing these pollutants.

[0009] The present invention also discloses a composite bacterial agent culture medium for culturing the composite bacterial agent as described above. The composite bacterial agent culture medium includes: 5 g of glucose, K 2 HPO 4 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 0.2 g; 2 mL of trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, 1000 mL of distilled water.

[0010] Furthermore, the pH of the composite bacterial agent culture medium is 7.0 - 7.2.

[0011] Furthermore, the concentration of the yeast powder solution is: 0.5 g / 10 mL; the concentration of the peptone solution is: 1 g / 10 mL.

[0012] Furthermore, the trace salt solution includes: 50 g of ethylenediaminetetraacetic acid (EDTA), ZnSO 4 2.2 g, CaCl 2 5.5 g, CuSO 4 •5H 2 O 1.57 g, MnCl 2 •4H 2 O 5.06 g, FeSO 4 •7H 2O 5 g, CoCl 2 •6H 2 O 1.61 g, distilled water 1000 mL.

[0013] The present invention also discloses a preparation method of a composite bacterium agent, comprising the following steps:

[0014] Inoculate the Bacillus and culture to form a first seed liquid, inoculate the Pseudomonas and culture to form a second seed liquid;

[0015] Mix the first seed liquid and the second seed liquid evenly to form a seed liquid;

[0016] Perform high-density fermentation on the mixed seed liquid to obtain the composite bacterium agent as described above.

[0017] Further, the OD in the first seed liquid and the second seed liquid 600 is 1.0; the volume ratio of the first seed liquid to the second seed liquid during mixing is 1:1.

[0018] Further, the temperature of the high-density fermentation is 25 - 30 °C, the pH is 7.0 - 7.2, the rotation speed is 150 - 200 r / min, and the time is 20 - 26 h.

[0019] The present invention also discloses an application of the composite bacterium agent as described above in a culture water body, and the composite bacterium agent is used to degrade ammonia nitrogen and nitrite nitrogen in the culture water body and inhibit the growth of pathogenic bacteria in the culture water body.

[0020] Further, the composite bacterium agent is added into the culture water body at a dosage of 1% (v / v), and added once every 7 days.

[0021] The composite bacterium agent provided by the present invention combines a probiotic with antibacterial effect ( Bacillus sp. BS-K1) and a denitrifying bacterium ( Pseudomonas sp. CN--B1) to obtain a composite bacterium agent for aquaculture. When applied to an aquaculture water body, it has excellent denitrification ability and inhibitory effect on related pathogenic bacteria in the water body, can improve the removal efficiency of ammonia nitrogen and nitrite nitrogen in the culture water body, reduce the number of related pathogenic bacteria, and ensure the safety of the culture environment. Description of the Drawings

[0022] Figure 1 is the experimental result diagram of the antagonism between Bacillus BS-K1 and Staphylococcus aureus in Example 1 of the present invention;

[0023] Figure 2 is the experimental result diagram of the antagonism between Bacillus BS-K1 and Escherichia coli in Example 1 of the present invention;

[0024] Figure 3 It is the experimental result diagram of the 96-well plate in Example 1 of the present invention;

[0025] Figure 4 It is the phylogenetic tree of strain BS-K1;

[0026] Figure 5 It is the phylogenetic tree of strain CN-B1;

[0027] Figure 6 It is the purification effect diagram of strains BS-K1 and CN-B1 on the aquaculture water body in Example 4 of the present invention;

[0028] Figure 7 It is the purification effect diagram of the compound bacterium agent on the aquaculture water body in Example 4 of the present invention;

[0029] Figure 8 It is the result diagram of the influence of the compound bacterium agent on the change of the microbial species in the aquaculture water body in Example 5 of the present invention;

[0030] Figure 9 It is the result diagram of the influence of the compound bacterium agent on the species within the genus Staphylococcus in the aquaculture water body in Example 5 of the present invention. Detailed implementation manners

[0031] To make the technical solution of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and specific embodiments.

[0032] Culture medium

[0033] LB culture medium: peptone 10 g; yeast powder 5 g; NaCl 10 g; pH 7.0 - 7.2, distilled water 1000 mL.

[0034] Nitrification culture medium: sodium acetate 5 g; (NH 4 ) 2 SO 4 0.235 g (50 mg / L); KH 2 PO 4 0.5 g; Na 2 HPO 4 0.5 g; MgSO 4 ·7H 2 O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0 - 7.2, add 2 wt% agar to the solid culture medium.

[0035] Trace salt solution: ethylenediaminetetraacetic acid (EDTA) 50 g, ZnSO 4 2.2 g, CaCl 2 5.5 g, CuSO4 • 5H 2 O 1.57 g, MnCl 2 • 4H 2 O 5.06 g, FeSO 4 • 7H 2 O 5 g, CoCl 2 • 6H 2 O 1.61 g, distilled water 1000 mL, pH 7.0 - 7.2.

[0036] Seed culture medium: glucose 5 g, K 2 HPO 4 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 0.2 g; trace salt solution 2 mL, 1% (v / v) yeast extract solution, 1% (v / v) peptone solution, distilled water 1000 mL, pH 7.0 - 7.2. Among them: yeast extract solution concentration: 0.5 g / 10 mL; peptone solution concentration: 1 g / 10 mL.

[0037] Add 2 wt% agar to the solid medium.

[0038] Example 1

[0039] Isolation and screening of Bacillus ( Bacillus sp. BS - K1) and Pseudomonas ( Pseudomonas sp . CN - B1):

[0040] Take 1 mL of water sample from the aquaculture base and add it to a 250 mL Erlenmeyer flask containing 100 mL of sterilized nitrification medium, and culture it at 30 °C and 180 r / min for 24 h.

[0041] Dilute the enriched bacterial solution by gradient dilution method to a concentration of 10 -8 Concentration, evenly spread it on the LB solid medium plate and culture it, and culture it in a biochemical incubator at 30 °C for 24 h. After the colonies are formed, select different colonies with different morphological colors from the LB solid medium for isolation.

[0042] Inoculate the single colony into the LB solid medium for streaking, and culture it in a strain incubator for 24 h at a temperature of 30 °C.

[0043] Verify the inhibitory ability of the obtained strains against common pathogenic bacteria in water by antagonistic experiment:

[0044] Separate the culture solutions of common pathogenic bacteria (Staphylococcus aureus and Escherichia coli) in the aquaculture water body and mix them with the LB medium that has been sterilized but not yet solidified and has a slightly lower temperature (when it can be tolerated by touching with the hand), then pour the mixture onto a petri dish. After solidification, use forceps to place a sterilized 6-mm filter paper in the middle of the medium, and use a sterilized bamboo stick to dip the culture of the purified strain to moisten the filter paper to complete inoculation. Place the medium in an incubator at the temperature recorded during sampling (30 °C) for incubation. If an inhibition zone appears around the middle colony during the incubation process, it indicates that there is an antagonistic effect between the two strains. Select the strain BS-K1 with the best antagonistic effect against Staphylococcus aureus and Escherichia coli.

[0045] As Figures 1 - 2 shown, the strain BS-K1 produced clear zones on the LB solid plates supplemented with the culture solutions of Staphylococcus aureus and Escherichia coli, indicating that the strain BS-K1 has an antagonistic effect against Staphylococcus aureus and Escherichia coli.

[0046] The 96-well plate colorimetric method was used to explore the denitrification ability of the strain by adding ammonia nitrogen and nitrite nitrogen colorimetric reagents:

[0047] The purified strain was inoculated into a 96-well plate containing nitrification medium at an inoculation amount of 1% (v / v), and cultured for 48 h under the conditions of 30 °C and 180 r / min. Then, ammonia nitrogen and nitrite nitrogen colorimetric reagents were added. Strains with better denitrification effects were selected according to the depth of the color (the lighter the color, the better the denitrification effect). The results are as Figure 3 shown. The one with the lightest color in the nitrification medium was selected and named CN--B1.

[0048] Example 2

[0049] Identification of Bacillus ( Bacillus sp. BS-K1) and Pseudomonas ( Pseudomonas sp . CN--B1):

[0050] Single colonies were selected for colony PCR, and the primers were 27F (5’-agagtttgatcctggctag -3’) and 1495R (5’-CTACGGCTACCTTGT TACGA-3’). The products were purified and sequenced. The sequences retrieved from GenBank through BLAST were aligned, and the 16S rDNA sequences with relatively high similarity to this sequence were screened out. A phylogenetic tree was constructed using MEGA7.0 to find the strains with the closest homology.

[0051] The results of 16S rDNA sequence determination were as follows:

[0052] Determination Bacillus sp.16S rDNA sequence of BS-K1, the measured result SEQ ID No.1:

[0053] GGGGTGCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGAAGTCC。

[0054] Determination Pseudomonas sp. The 16S rDNA sequence of CN--B1, and the determination result is SEQ ID ID No.2:

[0055] GCGTATACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTGGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGA。

[0056] The obtained results were compared with the 16S rDNA gene sequences of the type strains in the NCBI database for homology analysis and identification. Bacillus sp . BS-K1 was identified as Bacillus subtilis, Gram-positive. The phylogenetic tree is shown in Figure 4 Figure []. Pseudomonas sp . CN--B1 was identified as Pseudomonas juntendi, Gram-negative. The phylogenetic tree is shown in Figure 5 Figure [].

[0057] Example 3

[0058] Preparation of the compound microbial agent:

[0059] The screened strains Bacillus sp. BS-K1 and Pseudomonas sp . CN--B1 were separately inoculated into LB medium and cultured at 30 °C, pH 7.0 - 7.2, and a rotation speed of 180 r / min to obtain seed solutions. The OD of the seed solutions of the two strains was controlled at 1.0. Then, they were mixed in a 1:1 ratio to obtain the seed solution of the compound microbial agent. 600

[0060] The seed solution of the compound microbial agent was transferred to a fermenter for high-density fermentation. During the scale-up culture, the ratio of the volume of the seed solution to the volume of the seed medium in the fermenter was 1:100. The fermentation temperature was 25 - 30 °C, pH 7.0 - 7.2, rotation speed 180 r / min, and fermentation time 24 h.

[0061] Example 4

[0062] Application of the compound microbial agent in aquaculture water:

[0063] Ammonia nitrogen, nitrate, and pH are important indicators that need to be focused on during aquaculture and have a great impact on the aquaculture process. In this experiment, the site was the Yixing Aquaculture Base. The initial concentrations of ammonia nitrogen and nitrite nitrogen were in the range of 4.1 - 4.4 mg / L and 1.4 - 1.9 mg / L, respectively, and the pH was in the range of 6.6 - 6.9. The prepared compound microbial agent, the single Bacillus sp . BS-K1 microbial agent, and the single Pseudomonas sp. CN--B1 bacterial solution were added to the water body through on-site microbial agent dosing equipment at a dosing amount of 1% (v / v), and the dosing was carried out once every 7 days.

[0064] As shown in Figure 6 , the single Bacillus sp . BS-K1 microbial agent and the single Pseudomonas sp.The bacterial agent of CN--B1. It can be seen that the single denitrifying bacterium CN--B1 has a good denitrification effect on ammonia nitrogen and nitrite nitrogen in water. The removal rates of ammonia nitrogen and nitrite nitrogen can reach 42.0% and 40.1% respectively, while the removal rates of ammonia nitrogen and nitrite nitrogen of Bacillus sp. BS-K1 in water are 26.1% and 25.2% respectively.

[0065] As Figure 7 shown, after adding the compound bacterial agent, both ammonia nitrogen and nitrite showed a downward trend. The ammonia nitrogen concentration at equilibrium can be as low as 2.0 mg / L, and the nitrite nitrogen can be as low as 0.9 mg / L. The removal rates of ammonia nitrogen and nitrite nitrogen increased to 55.5% and 52.6% respectively. At the same time, it can be seen that the pH value also changed, fluctuating within the range of 6.9 - 7.3. During the aquaculture process, the phenomenon of unstable and decreasing pH often occurs, affecting the aquaculture process. Therefore, from the results, the addition of the probiotic compound bacterial agent can promote the removal of ammonia nitrogen and nitrite nitrogen in water, and at the same time can effectively solve the problem of decreasing pH, and also has a promoting effect on stabilizing the pH in water.

[0066] Example 5

[0067] The influence of the compound bacterial agent on the microbial diversity in aquaculture water:

[0068] The application site was selected as an aquaculture base in Yixing City, Jiangsu Province, and the experimental period was 7 days. Experimental group A: The prepared probiotic compound bacterial agent was added to the aquaculture water at a dosage of 1% (v / v), and water samples were taken after seven days. Experimental group B: Aquaculture water samples without adding any bacterial agent products. Then, the water samples of the two groups were filtered through a 0.45 μm filter membrane to obtain a filter membrane enriched with microorganisms, and then sent to a relevant sequencing company for metagenomic sequencing to explore the microbial diversity and community characteristics in the water of the two experimental groups.

[0069] It can be Figure 8 seen that the microbial diversity of the two groups has changed. Compared with group B, the numbers of Sphingobacterium spp. ( Novosphingobium ) and Pseudomonas spp. ( pseudomonas ) in group A increased significantly. Among them, Sphingobacterium spp. is a type with the ability to degrade organic pollutants, and these bacteria show potential application value in environmental pollution control and ecological restoration. The increase in the number of Pseudomonas spp. indicates that the Pseudomonas in the compound bacterial agent can grow rapidly in water, playing an effect of improving nitrogen removal. Thus, it can be seen that the addition of the compound bacterial agent can effectively improve the microbial community structure in aquaculture water, thereby improving the purification effect of water.

[0070] As Figure 9 shown, the Staphylococcus spp. ( staphylococcusThe abundance of () also changed significantly. The relative abundance in Group A was 0.00024, and the relative abundance in Group B was 0.032. The relative abundance in Group A decreased by 99.2% compared to Group B, and from Figure 9 It seems that among the Staphylococcus genus in Group A, the proportion of Staphylococcus aureus ( Staphylococcus aureus ) was 25.0%, and the proportion of Staphylococcus aureus in Group B was 15%. By comparison, it was found that in Group A with the addition of the probiotic complex agent, the abundance of the pathogenic bacterium Staphylococcus aureus decreased by 98.7%, showing good antibacterial properties.

[0071] Therefore, the addition of the complex agent has a great impact on the richness of the bacterial community in the water body, can effectively increase the number of denitrifying microorganisms in the water body, improve the denitrification efficiency, and at the same time can inhibit the growth of pathogenic bacteria in the water body.

[0072] The complex agent provided in the present invention has the ability to degrade ammonia nitrogen and nitrite in the water body and inhibit the growth of Staphylococcus aureus, and can play a role in regulating the pH in the water body. Compared with single-function products on the market, this product has a wider application.

[0073] The complex agent of the present invention has significantly improved the removal effects of ammonia nitrogen and nitrite nitrogen in the aquaculture system, which can be increased to 55.5% and 52.6% respectively.

[0074] The inhibition rate of the complex agent in the present invention against the pathogenic bacterium Staphylococcus aureus in the aquaculture water body can reach 98.7%, and can effectively improve the microbial community structure in the water body.

[0075] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A composite bacterial agent, characterized in that: Including Bacillus ( Bacillus sp .) BS-K1 and Pseudomonas ( Pseudomonas sp .) CN--B1, the Bacillus was deposited in the China Center for Type Culture Collection on September 23, 2024, with the deposit number CCTCC NO: M 20242022; the Pseudomonas was deposited in the China Center for Type Culture Collection on September 23, 2024, with the deposit number CCTCC NO: M 20242021.

2. A method for preparing a composite bacterial agent, characterized in that: The following steps are involved: Inoculation with Bacillus sp. Bacillus sp .) BS-K1, and cultured to form the first seed solution, inoculated with Pseudomonas ( Pseudomonas sp .) CN--B1, and culture to form the second seed liquid; uniformly mixing the first seed solution and the second seed solution to form a seed solution; The mixed seed liquid is subjected to high-density fermentation to obtain the composite bacterial agent as claimed in claim 1.

3. The method for preparing the composite bacterial agent according to claim 2, characterized in that: The OD of the first seed solution and the second seed solution 600 is 1.0; when mixing, the volume ratio of the first seed liquid to the second seed liquid is 1:

1.

4. The method for preparing the composite bacterial agent according to claim 3, characterized in that: The high-density fermentation has a temperature of 25-30°C, a pH of 7.0-7.2, a rotation speed of 150-200 r / min, and a time of 20-26 h.

5. An application of the composite bacterial agent as claimed in claim 1 in aquaculture water, characterized in that: Used to degrade ammonia nitrogen and nitrite nitrogen in aquaculture water and inhibit the growth of Escherichia coli and Staphylococcus aureus in aquaculture water.

6. The use of the composite bacterial agent in aquaculture water according to claim 5, characterized in that: Add the compound bacterial agent into the aquaculture water at a dosage of 1% by volume, once every 7 days.

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