Microbial complex bacterial population for treating aquaculture tail water and application thereof

Abstract

The application discloses a kind of microorganism complex bacterial population for treating aquaculture tail water and application thereof, and the microorganism complex bacterial population includes Aeromonas veronii, shewanella, paralutealidium and acinetobacter, and the ratio of viable bacterial count of Aeromonas veronii, shewanella, paralutealidium and acinetobacter is (1-3):(2-3.5):(0.5-1.5):(1.5-2.5). The strains of the application can effectively degrade tricaine mesylate pollutants, total phosphorus, total nitrogen and COD in aquaculture tail water through synergistic effect, and have excellent properties of low-temperature tolerance and high-salt tolerance.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a microbial complex for treating aquaculture wastewater and its application. Background Technology

[0002] The intensive and large-scale development of aquaculture, while ensuring product supply, has also generated a large amount of complex aquaculture wastewater. This wastewater not only contains high concentrations of conventional nutrient pollutants such as ammonia nitrogen, nitrite, nitrate, phosphate, and organic oxygen-consuming substances, but also increasingly highlights the pollution problems caused by specific chemical substances introduced through aquaculture operations, with residues of fish anesthetics being the most typical. Tricaine mesylate, a widely used fish anesthetic globally, is discharged into the environment with wastewater after fishing, sorting, vaccination, and transportation, becoming a new type of water pollutant of great concern. The persistent presence of tricaine mesylate and its metabolites in natural water bodies may have negative effects such as neurotoxicity and growth inhibition on non-target aquatic organisms, and may pose potential ecological risks through the food chain. Currently, the treatment technologies for aquaculture wastewater mainly focus on removing conventional pollutants such as nitrogen and phosphorus. The physical, chemical, and conventional biological methods used are not very effective against tricaine mesylate, which has a complex composition and stable structure. These methods have multiple limitations, such as low removal efficiency, high treatment costs, easy generation of secondary pollution, or difficulty in achieving simultaneous deep purification.

[0003] In the field of biological treatment, although microbial degradation is considered an economical and environmentally friendly approach to removing organic pollutants, existing technologies face significant bottlenecks when treating aquaculture wastewater containing specific drug residues such as tricaine mesylate. There is a lack of target pollutant-degrading strains, and their functions are often limited. The microbial communities in ordinary activated sludge or common commercial microbial agents are usually not domesticated or selected for degrading synthetic organic compounds like tricaine mesylate, resulting in limited recognition and metabolic capabilities for these pollutants, low and unstable removal rates, and poor environmental adaptability. Therefore, developing a composite microbial community capable of specifically and efficiently degrading tricaine mesylate while simultaneously achieving deep nitrogen, phosphorus, and COD reduction is of urgent practical need and significant technological value for solving the problems of specific drug residues and complex pollution in aquaculture wastewater and protecting aquatic environmental safety. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a microbial composite flora for treating aquaculture wastewater and its application. Through synergistic effects among the strains, it can effectively degrade tricaine mesylate pollutants in aquaculture wastewater and has excellent properties such as low temperature resistance and high salt resistance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A microbial complex, comprising:

[0007] Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter sp.

[0008] Aeromonas bivalvium was deposited at the China General Microbiological Culture Collection Center on March 29, 2024, with accession number CGMCC No. 30186;

[0009] Shewanella baltica was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 29, 2024, with accession number CGMCC No. 30187.

[0010] Bacillus paralicheniformis was deposited at the China General Microbiological Culture Collection Center on March 29, 2024, with accession number CGMCC No. 30188;

[0011] The ratio of viable counts of Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter sp. was (1-3):(2-3.5):(0.5-1.5):(1.5-2.5).

[0012] Preferably, the ratio of viable counts of the aforementioned Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter p. is 3:2:0.5:2.5.

[0013] A microbial compound agent comprising the aforementioned microbial compound flora.

[0014] The above-mentioned microbial complex or microbial complex agent is used in the degradation of tricaine mesylate in aquaculture wastewater. The concentration of tricaine mesylate in the aquaculture wastewater is 50-400 mg / L, the concentration of NaCl is 5-35 g / L, and the temperature is 10-35 ℃.

[0015] A method for degrading tricaine mesylate in aquaculture wastewater using a microbial compound agent includes the following steps: adding the compound agent to the aquaculture wastewater at a dosage of 0.1%-1.0% w / v by mass and stirring until homogeneous, then proceeding with the degradation.

[0016] The preparation method of microbial compound inoculant includes the following steps:

[0017] S1. Pre-adaptation cultures were performed on Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter p. to obtain seed cultures for each strain.

[0018] S2. The seed culture of each strain is co-cultured in multiple stages to form a synergistic mycelial sludge;

[0019] S3. The synergistic bacterial mud is mixed with the carrier suspension, cross-linked and solidified to form gel spheres, and then post-processed to obtain an embedded immobilized microbial composite agent.

[0020] Preferably, the specific method for pre-adaptation culture in step S1 is as follows:

[0021] (1) Inoculate Aeromonas bivalve and Bacillus paralichrysogenum into LB liquid medium and culture at 25-35℃ and 110-130 rpm for 24-48 h with shaking.

[0022] The specific components of LB liquid medium are: 50 mg / L NH4Cl, 30 mg / L NaNO2, 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2;

[0023] (2) Inoculate Shewanella into 2216E liquid medium, anaerobic, and incubate at 25-35℃ for 36-60 h;

[0024] The specific components of 2216E liquid culture medium are: 5 mM NaNO3, 0.5 mM FeCl3, 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0025] (3) Inoculate Acinetobacter into phosphorus-containing medium and culture for 10-15 h. Then, centrifuge and transfer to phosphorus-free medium for starvation culture for 5-7 h, and then transfer to phosphorus-containing medium for 10-15 h.

[0026] The specific components of the phosphorus-containing culture medium are: 10 g / L glucose, 5.0 g / L calcium phosphate, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, and 0.03 g / L manganese sulfate.

[0027] The specific components of the phosphorus-free culture medium are: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, and 0.03 g / L manganese sulfate.

[0028] Preferably, the specific method for multi-stage co-cultivation in step S2 is as follows:

[0029] (1) Mix the seed culture of pre-adapted Aeromonas bivalve, Bacillus paralichrysogenum and Acinetobacter bacillus, inoculate it into co-culture medium, and carry out the first stage of aerobic co-culture at 160-200 rpm for 16-20 h to form a preliminary bacterial community aggregate;

[0030] (2) Add the seed liquid of Shewanella after pre-adaptation culture to the initial bacterial community aggregate, and carry out the second stage of microaerobic co-culture at a speed of 60-100 rpm for 10-15 hours to form a stable synergistic bacterial sludge.

[0031] Preferably, in step S3 above, the method for preparing the carrier suspension is as follows: take porous diatomaceous earth and biochar powder, mix them evenly, and disperse them in sodium alginate solution to form a suspension; the mass ratio of porous diatomaceous earth, biochar powder and sodium alginate is 8-12:1-2:1-3.

[0032] Preferably, in step S3 above, the mass ratio of the synergistic bacterial sludge to the carrier suspension is 1-2:5.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: In the compound microbial community of the present invention, the strains can effectively degrade tricaine mesylate pollutants in aquaculture tailwater through synergistic effects, and also have excellent removal effects on total phosphorus, total nitrogen and COD, and have excellent performance in low temperature resistance and high salt resistance. Attached Figure Description

[0034] Figure 1 The graph shows the degradation effect of the compound bacterial agent at different initial concentrations of tricaine mesylate.

[0035] Figure 2 These are graphs showing the degradation effect of the compound microbial agent at different temperatures;

[0036] Figure 3 These are graphs showing the degradation effect of the compound bacterial agent under different NaCl concentrations;

[0037] Figure 4 This is a graph showing the effect of the preparation method of the compound microbial agent on the degradation effect;

[0038] Figure 5 This is a diagram showing the treatment effect of compound microbial agents on actual aquaculture wastewater. Detailed Implementation

[0039] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0040] Example 1

[0041] A microbial complex comprising Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter sp.

[0042] Aeromonas bivalvium was deposited at the China General Microbiological Culture Collection Center on March 29, 2024, with accession number CGMCC No. 30186; it has been disclosed in the invention patent application with application number CN202411174334.0.

[0043] Shewanella baltica was deposited on March 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30187; it has been disclosed in the invention patent application CN202411517370.2.

[0044] Bacillus paralicheniformis was deposited at the China General Microbiological Culture Collection Center on March 29, 2024, with accession number CGMCC No. 30188; it has been disclosed in the invention patent application with application number CN202410854072.6.

[0045] Acinetobacter is a known species that can be purchased. The Acinetobacter used in this embodiment of the invention is Acinetobacter johnsonii, with accession number CGMCC 1.8823, purchased from the China General Microbiological Culture Collection Center.

[0046] Each strain was activated and cultured in LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2) at 30 °C and 180 rpm for 24 hours before use.

[0047] Example 2

[0048] The activated bacterial cultures from Example 1 were centrifuged at 5000 rpm for 3 minutes, washed three times with PBS buffer, and diluted to 0.6 with an OD600 value. Five gradients of bacterial inoculum were set: 1%, 2%, 3%, 4%, and 5%. A water sample without bacterial inoculum was used as a blank control. Three replicates were set for each sample. Tricaine mesylate, total nitrogen, total phosphorus, and COD were measured to compare the optimal removal efficiency of water indicators at different inoculum levels. Based on the optimal results for each single bacterial inoculum, three different inoculum levels were designed, as shown in Table 1 (inoculum volume percentage, %). Then, a four-factor, three-level orthogonal experiment was designed based on Table 1, as shown in Table 2.

[0049] Table 1. Orthogonal Design Factors and Levels

[0050]

[0051] Table 2. Orthogonal Experiment Analysis Table

[0052]

[0053] The cultured bacterial solutions were placed in a centrifuge and centrifuged at 8000 rpm for 3 min. They were washed three times with PBS buffer and diluted to 0.6 with an OD600 value. The bacterial suspension was prepared according to Tables 1 and 2 and added to 200 mL of aquaculture wastewater at a pH of 6.5. The treatment temperature was 25℃ and the initial concentration of tricaine mesylate was 50 mg / L. A water sample without bacterial solution was used as a blank control. The degradation effects of tricaine mesylate, total nitrogen, total phosphorus, and COD at different ratios were measured. The best composite ratio was screened, and the results are shown in Table 3.

[0054] Table 3 Degradation effects of various pollutants in aquaculture wastewater

[0055]

[0056] As shown in Table 3, experimental group 8 showed the best removal effect of tricaine mesylate. Therefore, the optimal ratio of the compound microbial community, namely the ratio of viable counts of Aeromonas bivalvium, Shewanella baltica, Bacillus paralicheniformis, and Acinetobacter sp., is 3:2:0.5:2.5. Within the range of viable count ratio of (1-3):(2-3.5):(0.5-1.5):(1.5-2.5), the degradation rate of tricaine mesylate is above 76%, demonstrating excellent degradation effect.

[0057] Example 3: The preparation method of the compound microbial agent includes the following specific steps:

[0058] (1) Stress induction and pre-adaptation culture of strains

[0059] Four strains of bacteria were subjected to targeted enhancement culture:

[0060] Aeromonas bivalve and Bacillus paralichrysogenus were inoculated into LB liquid medium and cultured at 30°C and 120 rpm (hypoxic conditions) for 36 hours to induce their tolerance to ammonia nitrogen and nitrite stress.

[0061] Shewanella was inoculated into 2216E liquid medium, placed in an anaerobic workstation, and incubated at 30°C for 48 hours to activate its anaerobic respiratory enzyme system.

[0062] After culturing Acinetobacter in phosphorus-containing medium for 12 hours, it was centrifuged and transferred to phosphorus-free medium for starvation culture for 6 hours. This process was repeated for 2 cycles. Finally, it was transferred to phosphorus-rich medium and cultured for 12 hours to strongly induce its polyphosphate accumulation ability.

[0063] The specific components of the LB liquid culture medium are: 50 mg / L NH4Cl, 30 mg / L NaNO2, 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2;

[0064] The specific components of 2216E liquid culture medium are: 5 mM NaNO3, 0.5 mM FeCl3, 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0065] The specific components of the phosphorus-containing culture medium are: 10 g / L glucose, 5.0 g / L calcium phosphate, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, and 0.03 g / L manganese sulfate.

[0066] The specific components of the phosphorus-free culture medium are: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, and 0.03 g / L manganese sulfate.

[0067] (2) Multi-level co-cultivation forms synergistic microbial mud

[0068] First-stage aerobic co-culture: Pre-acclimated Aeromonas bivalve, Bacillus paralichrysogenus, and Acinetobacter were inoculated into co-culture medium (5 g / L yeast extract, 10 g / L peptone, 3 g / L glycerol, pH 7.0) at a ratio of 3:0.5:2.5 viable cells, and cultured at 30°C and 180 rpm with shaking until mid-log phase (approximately 18 hours) to form a preliminary bacterial community aggregate.

[0069] Second-stage microaerobic co-culture: Pre-adapted Shewanella bacteria were inoculated into the initial bacterial aggregate. The total ratio of Aeromonas bivalve, Shewanella, Bacillus paralichrysogenus, and Acinetobacter was 3:2:0.5:2.5. The shaking speed was reduced to 80 rpm to decrease dissolved oxygen, and the culture was continued for 12 hours to promote the formation of a stable spatial symbiotic structure of the four bacteria. Afterward, the co-culture solution was centrifuged at 4°C and 8000 rpm for 10 minutes to collect the bacterial cells, which were then washed with sterile physiological saline to obtain synergistic bacterial sludge.

[0070] (3) Preparation of functionalized carriers and formulation of microbial agents

[0071] Carrier preparation: Take 50g of porous diatomaceous earth (100 mesh) and 5g of biochar powder (300 mesh) and mix them evenly. Dissolve 10g of sodium alginate in 100mL of deionized water and disperse the above solid powder in it to form a suspension.

[0072] Immobilization: The synergistic bacterial sludge and the above sodium alginate carrier suspension were mixed at a mass ratio of 1:5. The mixture was then dripped into a 4% (w / v) calcium chloride solution using a peristaltic pump to form gel spheres with a diameter of about 2-3 mm. The mixture was then cross-linked and cured for 30 minutes.

[0073] Post-treatment: Remove the solidified microspheres, gently rinse with sterile water, and then immerse them in 0.5% chitosan (dissolved in 1% acetic acid solution) for 10 minutes to enhance strength. Finally, dry the embedded microspheres at 30°C in a low-temperature, ventilated environment until the moisture content is below 20%, thus obtaining the embedded immobilized composite bacterial agent.

[0074] Example 4: Degradation effect of compound bacterial agent at different initial concentrations of tricaine mesylate

[0075] The compound bacterial agent prepared in Example 3 was inoculated at an inoculation rate of 0.5% w / v into culture media containing different initial concentrations of tricaine mesylate. The culture media components were: 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L. The initial concentrations of tricaine mesylate were 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L. After incubation at pH 7.0, 25℃, and 200 rpm for 72 h, the degradation effect was as follows: Figure 1 As shown.

[0076] Depend on Figure 1 It is known that when the initial concentration of tricaine mesylate is 50-300 mg / L, the degradation rate is above 80%, showing excellent degradation effect. When the initial concentration is 300-400 mg / L, the degradation rate is still above 71%, indicating that the compound bacterial agent of the present invention can tolerate high concentrations of tricaine mesylate contaminants.

[0077] Example 5: Degradation effect of compound microbial agent at different temperatures

[0078] The compound bacterial agent prepared in Example 3 was inoculated into culture media at different temperatures at an inoculation rate of 0.5% w / v. The culture media components were: 100 mg tricaine mesylate, 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L. The temperatures of the culture media were adjusted to 5, 10, 15, 20, 25, 30, and 35 °C, respectively. After incubation at pH 7.0 and 200 rpm for 72 h, the degradation effect was as follows: Figure 2 As shown.

[0079] Depend on Figure 2 It can be seen that the compound microbial agent can effectively degrade tricaine mesylate at temperatures ranging from 20 to 35°C, with a degradation rate of over 91%. At 10°C, the degradation rate of tricaine mesylate is 68%, indicating that the compound microbial agent can still degrade tricaine mesylate contaminants at lower temperatures, demonstrating excellent low-temperature resistance.

[0080] Example 6: Degradation effect of compound bacterial agent at different NaCl concentrations

[0081] The compound bacterial agent prepared in Example 3 was inoculated into culture media with different NaCl concentrations at an inoculation rate of 0.5% w / v. The culture media composition was: 100 mg tricaine mesylate, 10.0 g peptone, 3.0 g beef extract, and distilled water to a final volume of 1 L. The NaCl concentrations were 5, 10, 15, 20, 25, 30, 35, and 40 g / L, respectively. After incubation at pH 7.0, 25℃, and 200 rpm for 72 h, the degradation effect was as follows: Figure 3 As shown.

[0082] Depend on Figure 3 It can be seen that the degradation rate of tricaine mesylate can reach more than 82% at a salinity of 5-25 g / L, and the degradation rate of tricaine mesylate can reach more than 65% at a high salinity of 30-35 g / L, indicating that the compound bacterial agent has excellent salt tolerance.

[0083] Example 7: The effect of the preparation method of the compound microbial agent on the degradation effect

[0084] To verify the superiority of this preparation method, two sets of experiments were set up for comparison: an experimental group (the encapsulated bacterial agent prepared in Example 3) and a control group (the four strains obtained in step (1) of Example 3 were simply mixed and adsorbed onto zeolite powder to obtain a traditional solid bacterial agent). The compound bacterial agents prepared in the experimental group and the control group were inoculated into the culture medium at an inoculation rate of 0.5% w / v. The culture medium components were: 100 mg tricaine mesylate, 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L. After incubation at pH 7.0, 25℃, and 200 rpm for 72 h, the degradation effect was as follows: Figure 4 As shown.

[0085] according to Figure 4 It can be seen that the experimental group has a high degradation rate of tricaine mesylate and can efficiently remove COD, TP and TN, and the synergistic effect among the strains is significant.

[0086] Example 8: Stability of the compound microbial agent

[0087] The strains from the experimental and control groups in Example 6 were inoculated into culture media. The culture media consisted of 100 mg tricaine mesylate, 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L. The culture media were stored at pH 7.0 and 4°C for 90 days. The viability of the strains was then tested, and the results are shown in Table 4.

[0088] Table 4 Viability of Compound Microbial Agent

[0089]

[0090] As shown in Table 4, the compound microbial agent of the present invention has excellent storage stability.

[0091] Example 9: The effect of compound bacterial agent on the treatment of wastewater from actual aquaculture.

[0092] The compound bacterial agent was prepared according to the method in Example 3. The water being treated was the aquaculture wastewater from a certain aquaculture farm. A laboratory-simulated biochemical system aeration treatment was used, with a treatment volume of 5 L and a compound bacterial agent dosage of 0.5% w / v. Aeration was performed to maintain dissolved oxygen above 2 mg / L, and the wastewater pH was 6.0–7.5. The treatment temperatures were controlled at 10℃ and 25℃, with 1 L of water being influent and effluent per day for 48 hours. Samples were taken to test the treatment effect of the compound bacterial agent at the two temperatures. The results are shown in [Figure 1]. Figure 5 .

[0093] according to Figure 5 It can be seen that at a temperature of 25℃, the degradation rate of tricaine mesylate, COD, TP, and TN by the compound microbial agent is all above 90%. At a low temperature of 10℃, the degradation rate of tricaine mesylate by the compound microbial agent can reach 60%, and the degradation rates of COD, TP, and TN are all between 50% and 65%. This shows that the compound microbial agent can effectively remove tricaine mesylate, COD, TP, and TN pollutants from aquaculture wastewater, and still has a certain degradation effect at lower temperatures.

[0094] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A microbial complex for treating aquaculture wastewater, characterized in that, include: Aeromonas bivalve ( Aeromonas bivalvium Shewanella ( Shewanella baltica ), Bacillus paralicheniformis ( Bacillus paralicheniformis ) and Acinetobacter ( Acinetobacter sp. ); The bivalve aeromonas ( Aeromonas bivalvium It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 29, 2024, with accession number CGMCC No. 30186; The Shewanella ( Shewanella baltica It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 29, 2024, with accession number CGMCC No. 30187; The Bacillus paralichrysogenus ( Bacillus paralicheniformis It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 29, 2024, with accession number CGMCC No. 30188; The Acinetobacter ( Acinetobacter sp. Acinetobacter johnsonii ( ) Acinetobacter johnsonii The accession number is CGMCC 1.8823. The bivalve aeromonas ( Aeromonas bivalvium Shewanella ( Shewanella baltica ), Bacillus paralicheniformis ( Bacillus paralicheniformis ) and Acinetobacter johnsonii ( Acinetobacter johnsonii The ratio of viable bacteria in the group was (1-3):(2-3.5):(0.5-1.5):(1.5-2.5).

2. The microbial complex according to claim 1, characterized in that, The bivalve aeromonas ( Aeromonas bivalvium Shewanella ( Shewanella baltica ), Bacillus paralicheniformis ( Bacillus paralicheniformis ) and Acinetobacter johnsonii ( Acinetobacter johnsonii The ratio of viable bacteria in the samples was 3:2:0.5:2.

5.

3. A microbial compound inoculant, characterized in that, Includes the microbial complex described in claim 1.

4. The application of the microbial complex flora as described in claim 1 or the microbial complex agent as described in claim 3 in the degradation of tricaine mesylate in aquaculture wastewater, characterized in that, The concentration of tricaine mesylate in the aquaculture wastewater is 50-400 mg / L, the concentration of NaCl is 5-35 g / L, and the temperature is 10-35 ℃.

5. A method for degrading tricaine mesylate in aquaculture wastewater using the microbial compound agent according to claim 3, characterized in that, The specific steps are as follows: add the compound microbial agent to the aquaculture wastewater at a dosage of 0.1%-1.0% w / v by mass and volume, stir evenly, and allow it to degrade.

6. The method for preparing the microbial compound inoculant according to claim 3, characterized in that, Includes the following steps: S1, respectively targeting Aeromonas bivalve ( Aeromonas bivalvium Shewanella ( Shewanella baltica ), Bacillus paralicheniformis ( Bacillus paralicheniformis ) and Acinetobacter johnsonii ( Acinetobacter johnsonii Pre-adaptation culture was carried out to obtain seed culture for each strain; S2. The seed culture of each strain is co-cultured in multiple stages to form a synergistic mycelial sludge; S3. The synergistic bacterial mud is mixed with the carrier suspension, cross-linked and solidified to form gel spheres, and then post-processed to obtain an embedded immobilized microbial composite agent.

7. The method for preparing the microbial compound inoculant according to claim 6, characterized in that, In step S1, the specific method for pre-adaptation culture is as follows: (1) Inoculate Aeromonas bivalve and Bacillus paralichrysogenus into LB liquid medium and culture at 25-35℃ and 110-130 rpm for 24-48 h with shaking. (2) Inoculate Shewanella into 2216E liquid medium, anaerobic, and incubate at 25-35℃ for 36-60 h; (3) Inoculate Acinetobacter johnsonii into phosphorus-containing medium and culture for 10-15 h. Then, centrifuge and transfer to phosphorus-free medium for starvation culture for 5-7 h, and then transfer to phosphorus-containing medium for 10-15 h.

8. The method for preparing the microbial compound inoculant according to claim 6, characterized in that, In step S2, the specific method for multi-level co-cultivation is as follows: (1) Mix the seed cultures of Aeromonas bivalve, Bacillus paralichrysogenus and Acinetobacter johnsonii after pre-adaptation culture, inoculate them into co-culture medium, and carry out the first-stage aerobic co-culture to form a preliminary bacterial community aggregate; (2) Add the seed liquid of Shewanella after pre-adaptation culture to the initial bacterial community aggregate and carry out the second stage of microaerobic co-culture to form a stable synergistic bacterial sludge.

9. The method for preparing the microbial compound inoculant according to claim 8, characterized in that, In step (1), the rotation speed is 160-200 rpm and the culture time is 16-20h; In step (2), the rotation speed is 60-100 rpm and the culture time is 10-15h.

10. The method for preparing the microbial compound inoculant according to claim 6, characterized in that, In step S3 The method for preparing the carrier suspension is as follows: take porous diatomaceous earth and biochar powder, mix them evenly, and disperse them in sodium alginate solution to form a suspension; The mass ratio of porous diatomaceous earth, biochar powder, and sodium alginate is 8-12:1-2:1-3; The mass ratio of synergistic bacterial sludge to carrier suspension is 1-2:5.

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