Complex microbial inoculant and application thereof

Abstract

The invention discloses a complex microbial inoculant and application thereof, and belongs to the technical field of microorganisms. The composite microbial agent comprises bacillus (bacillus sp.BS-K1) and pseudomonas sp.CN-B1), and the preservation number of the bacillus is CCTCC (China Center for Type Culture Collection) NO: M 20242022, and the preservation number of the pseudomonas is CCTCC NO: M 20242022. The preservation number of the pseudomonas is CCTCC (China Center for Type Culture Collection) NO: M 20242021. According to the complex microbial inoculant provided by the invention, probiotics (Bacillus sp.BS-K1) and denitrifying bacteria (Pseudomonas sp.CN-B1) which have antibacterial effects are compounded to obtain the complex microbial inoculant for aquaculture, and when the complex microbial inoculant is applied to an aquaculture water body, the complex microbial inoculant has excellent denitrification capability and an inhibition effect on related pathogenic bacteria in the water body; the removal efficiency of ammonia nitrogen and nitrite nitrogen in the culture water body can be improved, the number of related pathogenic bacteria is reduced, and the safety of the culture environment is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite bacterial agent and application thereof. Background Art

[0002] Aquaculture, also known as aquaculture, has made important contributions to solving the problem of fish consumption for urban and rural residents in my country, increasing the supply of high-quality animal protein, improving the nutritional health level of the whole people, and ensuring food safety in my country. Aquaculture is an important part of agricultural and sideline production. With the advancement of technology and changes in market demand, traditional aquaculture has gradually developed in an intensive direction. Highly intensive aquaculture refers to a breeding model that achieves high yields and high economic benefits in a limited space through high-density breeding, efficient feed feeding, water quality management, and disease prevention and control.

[0003] However, due to highly intensive aquaculture, a large amount of unconsumed feed, excrement, animal carcasses, etc. are discharged into the water, causing the concentration of ammonia nitrogen and nitrite nitrogen in aquaculture water to rise. At the same time, the presence of pathogenic bacteria in the water has serious harmful effects on the ecological environment and aquaculture products. In order to improve the water quality, there are currently physical treatment methods such as sedimentation, filtration, and flotation, chemical treatment methods such as coagulation and sedimentation, oxidation-reduction, and biological treatment methods that use microorganisms to degrade organic matter and remove nitrogen and phosphorus. 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 field.

[0004] The current biological treatment using a single bacterial species also has shortcomings in improving water quality. It often has a single function and lacks multifunctional characteristics. It is easily restricted by the water environment during its proliferation in the applied water body and has certain limitations. At the same time, the composite bacterial agents on the market have poor effects and do not significantly improve the removal effect of ammonia nitrogen and nitrite nitrogen in water bodies. Summary of the invention

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

[0006] The present invention discloses a composite bacterial agent, comprising 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.

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

[0008] Bacillus is a probiotic widely used in aquaculture. It has the functions of regulating water quality and inhibiting the growth of pathogenic bacteria. The denitrifying bacteria Pseudomonas can convert organic pollutants in sewage into harmless substances such as carbon dioxide and water, and can also 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, wherein the composite bacterial agent culture medium comprises: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, and 1000 mL 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), 2.2 g of ZnSO4, 5.5 g of CaCl2, 1.57 g of CuSO4•5H2O, 5.06 g of MnCl2•4H2O, 5 g of FeSO4•7H2O, 1.61 g of CoCl2•6H2O, and 1000 mL of distilled water.

[0013] The present invention also discloses a method for preparing a composite bacterial agent, comprising the following steps: Inoculating the Bacillus and culturing to form a first seed solution, inoculating the Pseudomonas and culturing to form a second seed solution; 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 described above.

[0014] Furthermore, 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.

[0015] Furthermore, the high-density fermentation temperature is 25-30°C, pH 7.0-7.2, the rotation speed is 150-200 r / min, and the time is 20-26 h.

[0016] The present invention also discloses an application of the composite bacterial agent as described above in aquaculture water, wherein the composite bacterial agent is used to degrade ammonia nitrogen and nitrite nitrogen in the aquaculture water and inhibit the growth of pathogenic bacteria in the aquaculture water.

[0017] Furthermore, the composite bacterial agent is added into the aquaculture water at a dosage of 1% (v / v), once every 7 days.

[0018] The composite bacterial agent provided by the present invention comprises probiotics ( Bacillus sp. BS-K1) and denitrifying bacteria ( Pseudomonas sp. CN--B1) is compounded to obtain a composite bacterial agent for aquaculture. When applied to aquaculture water, it has excellent denitrification ability and inhibitory effect on related pathogenic bacteria in the water. It can improve the removal efficiency of ammonia nitrogen and nitrite nitrogen in aquaculture water, reduce the number of related pathogenic bacteria, and ensure the safety of the aquaculture environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the results of an antagonistic experiment between Bacillus BS-K1 and Staphylococcus aureus in Example 1 of the present invention; Figure 2 This is a graph showing the results of an antagonistic experiment between Bacillus BS-K1 and Escherichia coli in Example 1 of the present invention; Figure 3 96-well plate experimental result diagram of Example 1 of the present invention; Figure 4 is the phylogenetic tree of strain BS-K1; Figure 5 is the phylogenetic tree of strain CN--B1; Figure 6 This is a diagram showing the purification effect of the strains BS-K1 and CN--B1 on aquaculture water in Example 4 of the present invention; Figure 7 This is a diagram showing the purification effect of the composite bacterial agent on aquaculture water in Example 4 of the present invention; Figure 8 This is a graph showing the effect of the composite bacterial agent in Example 5 of the present invention on the changes in the types of microorganisms in aquaculture water; Fig. 9 This is a graph showing the effect of the composite bacterial agent in Example 5 of the present invention on species of the genus Staphylococcus in aquaculture water. DETAILED DESCRIPTION

[0020] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Culture medium LB medium: peptone 10 g; yeast powder 5 g; NaCl 10 g; pH 7.0·7.2, distilled water 1000 mL.

[0022] Nitration medium: sodium acetate 5 g; (NH4)2SO4 0.235 g (50 mg / L); KH2PO4 0.5 g; Na2HPO4 0.5 g; MgSO4·7H2O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0-7.2, solid medium plus 2 wt% agar.

[0023] Trace salt solution: ethylenediaminetetraacetic acid (EDTA) 50 g, ZnSO4 2.2 g, CaCl2 5.5 g, CuSO4•5H2O1.57 g, MnCl2•4H2O 5.06 g, FeSO4•7H2O 5 g, CoCl2•6H2O 1.61 g, distilled water 1000 mL, pH 7.0-7.2.

[0024] Seed culture medium: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, 1000 mL distilled water, pH 7.0-7.2. Among them: yeast powder solution concentration: 0.5 g / 10 mL; peptone solution concentration: 1 g / 10 mL.

[0025] Solid medium was supplemented with 2 wt% agar.

[0026] Example 1 Bacillus ( Bacillus sp. BS-K1) and Pseudomonas ( Pseudomonas sp . CN--B1) separation and screening: Take 1 mL of water sample from the breeding 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.

[0027] The enriched bacterial suspension was diluted to 10 -8 The concentration was evenly spread on LB solid medium plates and cultured in a biochemical incubator at 30°C for 24 h. After the colonies were formed, different colonies with different morphologies and colors were selected from the LB solid medium for separation.

[0028] A single colony was inoculated into LB solid medium for streaking and cultured in a bacterial incubator for 24 h at 30°C.

[0029] Antagonism experiments were used to verify the inhibitory ability of the obtained strains against common pathogens in water: The culture solution of common pathogenic bacteria (Staphylococcus aureus and Escherichia coli) in aquaculture water was mixed with the LB medium that had not solidified after sterilization but was slightly lower in temperature (when it was tolerable to touch by hand) and poured into a plate. After solidification, a sterilized 6 mm filter paper was placed in the middle of the culture medium with tweezers. The culture of the purified strain was dipped in a sterilized bamboo stick to wet the filter paper to complete the inoculation. The culture medium was placed at the temperature recorded when sampling (30°C) for cultivation. If an inhibition zone is generated around the middle colony during the culture process, it indicates that there is an antagonistic effect between the two strains. The strain BS-K1 with the best antagonistic effect on Staphylococcus aureus and Escherichia coli was selected.

[0030] like Figure 1-Figure 2 As shown, strain BS-K1 produced a transparent zone on the LB solid plate added with culture solution of Staphylococcus aureus and Escherichia coli, indicating that strain BS-K1 has antagonistic effect on Staphylococcus aureus and Escherichia coli.

[0031] 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: The purified strain was inoculated into a 96-well plate containing nitrification medium at a 1% (v / v) inoculation rate and cultured for 48 h at 30°C and 180 rpm. Ammonia nitrogen and nitrite nitrogen colorimetric reagents were then added. The strains with better denitrification effects were selected based on the color depth (the lighter the color, the better the denitrification effect). The results are shown in Figure 2. Figure 3 As shown, the lightest color in the nitrification medium was selected and named CN--B1.

[0032] Example 2 Bacillus ( Bacillus sp. BS-K1) and Pseudomonas ( Pseudomonas sp . CN--B1) identification: Single colonies were selected for colony PCR, with primers 27F (5'-agagtttgatcctggctag -3') and 1495R (5'-CTACGGCTACCTTGT TACGA-3'), and the products were purified and sequenced. The sequences obtained were retrieved in GenBank by BLAST and compared, and the 16S rDNA sequences with high similarity to the sequence were screened out. The phylogenetic tree was constructed using MEGA7.0 to find the strains closest to its homology.

[0033] The results of 16SrDNA sequencing are: Determination Bacillus sp. 16SrDNA sequence of BS-K1, determination result SEQ ID No.1: GGGGTGCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGAAGTCC。

[0034] Determination Pseudomonas sp. The 16SrDNA sequence of CN--B1, the result of determination is SEQ ID ID No.2: GCGTATACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTGGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGA。

[0035] The results were compared with the 16S rDNA gene sequence of the model strain in the NCBI database to identify Bacillus sp . BS-K1 is Bacillus subtilis, Gram-positive, developmental tree Figure 4 shown. Pseudomonas sp . CN--B1 was identified as Pseudomonas juntendi, Gram-negative, and the developmental tree is as follows Figure 5 shown.

[0036] Example 3 Preparation of compound bacterial agent: The selected strains Bacillus sp. BS-K1 and Pseudomonas sp CN--B1 was inoculated into LB medium at 30℃, pH 7.0-7.2, and a speed of 180 r / min. It was used as seed liquid to control the OD of the seed liquid of the two strains. 600 is 1.0, and then mixed in a ratio of 1:1 to obtain a seed solution of the composite bacterial agent.

[0037] The seed liquid of the composite bacterial agent was transferred to the fermentation tank for high-density fermentation. During the expansion, the ratio of the seed liquid to the seed culture medium in the fermentation tank was 1:100. The fermentation temperature was 25-30°C, pH 7.0-7.2, the rotation speed was 180 r / min, and the fermentation time was 24 h.

[0038] Example 4 Application of compound bacterial agents in aquaculture water: Ammonia nitrogen, nitrate and pH are the indicators that need to be paid attention to in the process of aquaculture, which have a great impact on the aquaculture process. The experimental site is in the Yixing aquaculture base. The initial concentrations of ammonia nitrogen and nitrite nitrogen are in the range of 4.1-4.4 mg / L and 1.4-1.9 mg / L, and the pH is in the range of 6.6-6.9. Bacillus sp . BS-K1 bacterial agent, single Pseudomonas sp. The CN--B1 bacterial solution is added to the water body at a dosage of 1% (v / v) through on-site bacterial agent dosing equipment, once every 7 days.

[0039] like Figure 6 As shown, add a single Bacillus sp . BS-K1 microbial agent and single Pseudomonas sp.CN--B1 bacterial agent, it can be seen that the single denitrifying bacteria CN--B1 has a good denitrification effect on ammonia nitrogen and nitrite nitrogen in water bodies, and the removal rates of ammonia nitrogen and nitrite nitrogen can reach 42.0% and 40.1%, while the removal rates of ammonia nitrogen and nitrite nitrogen in water bodies by Bacillus BS-K1 are 26.1% and 25.2%, respectively.

[0040] like Figure 7 As shown in the figure, after adding the compound bacteria, ammonia nitrogen and nitrite showed a downward trend. The ammonia nitrogen concentration can be as low as 2.0 mg / L when stable, and 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 has also changed, fluctuating in the range of 6.9-7.3. In the breeding process, the phenomenon of unstable and declining pH often occurs, which affects the breeding process. Therefore, from the results, the addition of probiotic compound bacteria can promote the removal of ammonia nitrogen and nitrite nitrogen in the water body, and can effectively solve the problem of pH decline, and also promote the stabilization of pH in the water body.

[0041] Example 5 Effects of compound bacterial agents on microbial diversity in aquaculture water: The application site was selected at the 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 obtained after seven days. Experimental Group B: Aquaculture water samples without any bacterial agent products. After that, the two groups of water samples were filtered through a 0.45μm filter membrane to obtain a microbial enrichment filter membrane, which was subsequently sent to a related sequencing company for metagenomic sequencing to explore the microbial diversity and community characteristics in the water bodies of the two experimental groups.

[0042] Depend on Figure 8 It can be seen that the microbial diversity of the two groups has changed. Compared with group B, the number of Neosphingobacterium in group A ( Novosphingobium ) and Pseudomonas ( pseudomonas ) increased significantly, among which Sphingobacterium is a type of bacteria that has the ability to degrade organic pollutants. These bacteria show potential application value in environmental pollution control and ecological restoration. The increase in the number of Pseudomonas indicates that the Pseudomonas in the composite bacterial agent can grow rapidly in the water body and improve the effect of nitrogen removal. It can be seen that the addition of the composite bacterial agent can effectively improve the microbial community structure in the aquaculture water body, thereby improving the purification effect of the water body.

[0043] like Fig. 9 As shown, Staphylococcus spp. ( staphylococcusThe relative abundance of group A was 0.00024, and the relative abundance of group B was 0.032. The relative abundance of group A decreased by 99.2% compared with group B, and Fig. 9 It appears that among the Staphylococci in group A, Staphylococcus aureus ( Staphylococcus aureus ) accounted for 25.0%, and Staphylococcus aureus accounted for 15% in group B. By comparison, it was found that in group A with the addition of probiotic compound bacteria, the abundance of pathogenic Staphylococcus aureus was reduced by 98.7%, showing good antibacterial activity.

[0044] Therefore, the addition of composite bacterial agents has a great impact on the richness of bacterial flora in water bodies. It can effectively increase the number of denitrifying microorganisms in water bodies, improve denitrification efficiency, and at the same time inhibit the growth of pathogenic bacteria in water bodies.

[0045] The composite bacterial agent provided in the present invention has the ability to degrade ammonia nitrogen and nitrite in water and inhibit the growth of Staphylococcus aureus, and can also play a role in regulating the pH in water. Compared with single-function products on the market, this product has a wider application.

[0046] The composite bacterial agent of the present invention significantly improves the removal effect of ammonia nitrogen and nitrite nitrogen in the aquaculture system, which can be increased to 55.5% and 52.6% respectively.

[0047] The composite bacterial agent of the present invention can achieve an inhibition rate of 98.7% on the pathogenic bacteria Staphylococcus aureus in aquaculture water, and can effectively improve the microbial community structure in the water.

[0048] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached 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 composite bacterial agent culture medium for culturing the composite bacterial agent according to claim 1, characterized in that: The composite bacterial agent culture medium includes: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% yeast powder solution, 1% peptone solution, and 1000 mL distilled water.

3. The composite bacterial agent culture medium according to claim 2, characterized in that: The pH of the composite bacterial agent culture medium is 7.0-7.

2.

4. The composite bacterial agent culture medium according to claim 2, characterized in that: The concentration of the yeast powder solution is 0.5 g / 10 mL; the concentration of the peptone solution is 1 g / 10 mL.

5. The composite bacterial agent culture medium according to claim 2, characterized in that: The trace salt solution includes: 50 g of ethylenediaminetetraacetic acid, 2.2 g of ZnSO4, 5.5 g of CaCl2, 1.57 g of CuSO4•5H2O, 5.06 g of MnCl2•4H2O, 5 g of FeSO4•7H2O, 1.61 g of CoCl2•6H2O, and 1000 mL of distilled water.

6. A method for preparing a composite bacterial agent, characterized in that: The following steps are involved: Inoculating the Bacillus and culturing to form a first seed solution, and inoculating the Pseudomonas and culturing to form a second seed solution; 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.

7. The method for preparing the composite bacterial agent according to claim 6, 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.

8. The method for preparing the composite bacterial agent according to claim 7, characterized in that: The high-density fermentation temperature is 25-30°C, pH is 7.0-7.2, the rotation speed is 150-200 r / min, and the time is 20-26 h.

9. Use 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 pathogenic bacteria in aquaculture water.

10. The use of the composite bacterial agent in aquaculture water according to claim 9, characterized in that: Add the compound bacterial agent into the aquaculture water at a dosage of 1% by volume, once every 7 days.

Images