A complex microbial agent for in-situ purification of a breeding water body and a preparation method and use method thereof

Abstract

The present application belongs to the technical field of water pollution treatment, and relates to a compound microbial agent for in-situ purification of aquaculture water as well as a preparation method and a use method thereof. The present application provides a compound microbial agent for in-situ purification of aquaculture water, which is characterized by comprising hydrolytic oxidation functional bacteria, nitrosation functional bacteria, nitrification functional bacteria, anaerobic ammonia oxidation functional bacteria, sulfur autotrophic denitrification functional bacteria, biological reinforcement auxiliary functional bacteria and auxiliary materials. The compound microbial agent can realize in-situ purification of water quality in the aquaculture industry, and can realize in-situ conversion of ammonia in the water body into nitrogen and precipitation of phosphate compounds in the form of compounds under the condition of greatly reducing the dependence on carbon sources, and is a green aquaculture agent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of water pollution treatment, and particularly relates to a composite microbial agent for in-situ purification of aquaculture water as well as a preparation method and use method thereof. BACKGROUND

[0002] With the gradual deterioration of the ecological environment, the problem of reducing pollution of aquaculture water is gradually valued in the process of large-scale and high-density aquaculture. However, due to the need to maintain a certain temperature and dissolved oxygen in the process of water products, the dissolved oxygen is consumed in large quantities by the decay of residual food and organic matter in the excretion of fish and shrimp, and the remaining nitrogen, phosphorus and sulfur pollutants can only be oxidized into oxidized substances. This state easily leads to the continuous increase of inorganic nitrogen and phosphorus in water, and further deteriorates the water quality, which requires additional addition of a large amount of hormones and antibiotics to improve the survival rate of water products. The conventional sewage treatment facilities need to be used to treat the aquaculture wastewater independently, and a large amount of organic matter needs to be added in the treatment process. Although some microbial agents have been developed for the pollutants generated in the process of aquaculture, the microbial agents have single function, and the coordinated strengthening effect of non-functional microorganisms is ignored in the process of development of the microbial agents, resulting in large amount of use and high frequency of addition of the microbial agents in the use process (for example, the patents with the application numbers 201610105657.3 and 200910077356.4). In addition, a large amount of reagents need to be added in the process of addition of the functional bacteria, for example, a large amount of carbon source needs to be added in the process of addition of denitrifying bacteria, and the treatment efficiency is low. At present, there is a lack of a high-efficiency product for treating polluted aquaculture water. SUMMARY

[0003] The present application aims to provide a composite microbial agent for in-situ purification of aquaculture water as well as a preparation method and use method thereof. The composite microbial agent can realize in-situ purification of water quality in the aquaculture industry, and can realize in-situ precipitation of ammonia nitrogen to nitrogen and phosphate compounds in water under the condition of greatly reducing carbon source, and is a green aquaculture agent.

[0004] The present application provides a composite microbial agent for in-situ purification of aquaculture water, which comprises hydrolytic oxidation functional bacteria, nitrosation functional bacteria, nitrification functional bacteria, anaerobic ammonia oxidation functional bacteria, sulfur autotrophic denitrification functional bacteria, biological strengthening auxiliary functional bacteria and auxiliary materials.

[0005] The hydrolytic oxidation functional bacteria comprise Chlorobium.

[0006] The nitrosation functional bacteria comprise Nitrosomonas bacteria and / or Nitrosospina bacteria.

[0007] The nitrification functional bacteria comprise Nitrospina bacteria.

[0008] The anaerobic ammonia oxidizing functional bacteria include the genus Brocadia and / or the genus Kuenenia.

[0009] The sulfur autotrophic denitrifying bacteria include two or more of the following: Sulfurimonas, Thiomonas, and Thiobacillus.

[0010] The bio-enhancing auxiliary bacteria include two or more of the following: Pseudomonas, Shinella, Thaurea, and Flavobacterium.

[0011] The excipients include calcium carbonate and ferrous chloride.

[0012] Preferably, when the compound bacterial agent is used to treat seawater aquaculture water, the anaerobic ammonia oxidizing functional bacteria are Candidatus Brocadia; when the compound bacterial agent is used to treat freshwater aquaculture water, the anaerobic ammonia oxidizing functional bacteria are Candidatus Kuenenia.

[0013] Preferably, the composition comprises the following components in parts by weight: 15-25 parts of hydrolytic oxidation functional bacteria, 15-25 parts of nitrifying functional bacteria, 8-12 parts of nitrifying functional bacteria, 15-25 parts of anaerobic ammonia oxidation functional bacteria, 8-12 parts of sulfur autotrophic denitrification functional bacteria, 8-12 parts of bio-enhancing auxiliary functional bacteria, and 6-16 parts of excipients; wherein the excipients include 3-8 parts of calcium carbonate and 3-8 parts of ferrous chloride.

[0014] Preferably, the sources of the anaerobic ammonia oxidizing functional bacteria and the sulfur autotrophic denitrifying functional bacteria include domestication, with a moisture content of 25-35%, respectively; the concentration of the anaerobic ammonia oxidizing functional bacteria is 6 × 10⁻⁶. 8 ~8×10 8 CFU / mL; concentration of sulfur-autotrophic denitrifying bacteria was 1×10⁻⁶. 8 ~2×10 8 CFU / mL; the bacterial concentrations of hydrolytic oxidizing bacteria, nitrifying bacteria, and nitrifying bacteria were 2×10⁻⁶. 9 ~3×10 9 CFU / mL; the concentration of bio-enhanced auxiliary bacteria was 1×10⁻⁶. 8 ~2×10 8 CFU / mL; calcium carbonate and ferrous chloride are added in solution form, with the concentration of calcium carbonate being 70-90 mg / L and the concentration of ferrous chloride being 0.5 mol / L.

[0015] Preferably, the NCBI classification numbers are as follows: Chlorobium (1091); Nitrosomonas (914); Nitrospisina (35798); Nitrospina (35800); Candidatus Brocadia (380240); and Candidatus Brocadia (380240). The NCBI classification numbers for the following species are as follows: Kuenenia (380738), Sulfurimonas (202746), Thiomonas (32012), Thiobacillus (919), Pseudomonas (286), Shinella (323620), Thaurera (33057), and Flavobacterium (237).

[0016] Preferably, the Chlorobium phylum includes one or more of the following: Chlorobium phaeovibrioides (NCBI classification number 1094); Chlorobium phaeobacteroides (NCBI classification number 1096); and Chlorobium limicola (NCBI classification number 1092).

[0017] The genus *Nitrosomonas* includes one or more of the following: *Nitrosomonasureae* (NCBI classification number 44577); *Nitrosomonas nitrosa* (NCBI classification number 52442); and *Nitrosomonas europaea* (NCBI classification number 915).

[0018] The genus Nitrosospina includes one or more of the following: Nitrosospira multiformis (NCBI classification number 1231) and Nitrosospira briensis (NCBI classification number 35799).

[0019] The genus *Nitrospina* includes: *Nitrospinawatsonii*, NCBI classification number 1323948; and / or *Nitrospina gracilis*, NCBI classification number 35801;

[0020] The genus *Candidatus Brocadia* includes one or more of the following: *Candidatus Brocadia sinica* (NCBI classification number 795830); *Candidatus Brocadia sapporoensis* (NCBI classification number 392547); and *Candidatus Brocadia fulgida* (NCBI classification number 380242).

[0021] The genus Candidatus Kuenenia includes: Candidatus Kuenenia stuttgartiensis (NCBI classification number 174633); and / or Candidatus Kuenenia hertensis (NCBI classification number 2996012).

[0022] The genus *Sulfurimonas* mentioned above includes one or more of the following: *Sulfurimonas autotrophica* (NCBI classification number 202747); *Sulfurimonas crateris* (NCBI classification number 2574727); *Sulfurimonas denitrificans* (NCBI classification number 39766); and *Sulfurimonas gotlandica* (NCBI classification number 1176482).

[0023] The genus Thiomonas includes one or more of the following: *Thiomonas arsenitoxydans* (NCBI classification number 426114); *Thiomonas intermedia* (NCBI classification number 926); *Thiomonas delicata* (NCBI classification number 364030); and *Thiomonas bhubaneswarensis* (NCBI classification number 339866).

[0024] The genus Thiobacillus includes: Thiobacillus denitrificans, NCBI classification number 36861; and / or Thiobacillus thioparus, NCBI classification number 931;

[0025] The genus *Pseudomonas* includes: *Pseudomonas yamanorum*, NCBI classification number 515393; *Pseudomonas umsongensis*, NCBI classification number 198618; and *Pseudomonas viridiflava*, NCBI classification number 33069.

[0026] The genus *Shinella* includes: *Shinella zoogloeoides*, NCBI classification number 352475; and *Shinella sumterensis*, NCBI classification number 1967501.

[0027] The Thaurae genus includes: Thaurae aminoaromatica (NCBI classification number 164330) and Thaurae aromatica (NCBI classification number 59405).

[0028] The genus Flavobacterium includes: Flavobacterium psychrophilum (NCBI classification number 96345) and Flavobacterium columnnae (NCBI classification number 996).

[0029] The present invention also provides a method for preparing the compound microbial agent described in the above technical solution, comprising the following steps:

[0030] The compound microbial agent is obtained by mixing hydrolytic oxidation bacteria, nitrification bacteria, nitrification bacteria, anaerobic ammonia oxidation bacteria, sulfur autotrophic denitrification bacteria, bio-enhanced auxiliary bacteria and excipients.

[0031] The present invention also provides the application of the compound microbial agent described in the above technical solution or the compound microbial agent prepared by the preparation method described in the above technical solution in purifying aquatic aquaculture water.

[0032] This invention also provides a method for in-situ purification of aquaculture water using the compound microbial agent prepared based on the compound microbial agent described in the above technical solution or the preparation method described in the above technical solution, comprising the following steps:

[0033] In the early stages of aquaculture, compound microbial agents are added to the biological bed in the aquaculture pond for aquaculture.

[0034] Preferably, the amount of the compound microbial agent added is 8-12 g / m³. 3 Before being added, the compound microbial agent also undergoes a nutrient solution reactivation treatment, wherein the nutrient solution includes ammonia nitrogen and sulfides; the dissolved oxygen in the reactivation treatment is 0.5 mg / L, the temperature is 30-35℃, the pH value is 7.5-8.5, and the time is 1-3 days.

[0035] This invention provides a compound microbial agent for in-situ purification of aquaculture water. Currently, high-density aquaculture suffers from water quality deterioration due to residual feed and animal feces, necessitating additional wastewater treatment facilities and increasing aquaculture costs. This invention discloses a compound microbial agent for in-situ purification of aquaculture water. Using this compound microbial agent, in-situ green treatment of nitrogen and phosphorus in aquaculture wastewater can be achieved, improving both water quality and the quality of aquatic products, while also being cost-effective. In short, this invention provides a wastewater treatment microbial agent for the aquaculture industry, primarily capable of removing nitrogen and phosphorus.

[0036] Unlike the current mainstream off-site treatment method for aquaculture water (Energy and Environmental Protection Journal, 2023, 37(6): 64-78, "Research Progress on Deep Denitrification of Aquaculture Wastewater" summarizes the current mainstream aquaculture water purification methods), this invention adopts an in-situ treatment method, which has the advantages of not needing to build external treatment facilities, not needing to re-plan the pipeline route of the aquaculture pond, not needing professional debugging personnel, and not needing to add carbon sources, which can greatly reduce the treatment cost of aquaculture water. Detailed Implementation

[0037] This invention provides a compound bacterial agent for in-situ purification of aquaculture water (including aquaculture wastewater), comprising hydrolytic oxidation functional bacteria, nitrification functional bacteria, nitrification functional bacteria, anaerobic ammonia oxidation functional bacteria, sulfur autotrophic denitrification functional bacteria, bio-enhancing auxiliary functional bacteria, and excipients.

[0038] The hydrolytic and oxidizing bacteria include those belonging to the Chlorobium phylum.

[0039] The nitrifying functional bacteria include Nitrosomonas and / or Nitrosospina.

[0040] The nitrifying bacteria include Nitrospina species.

[0041] The anaerobic ammonia oxidizing functional bacteria include Candidatus Brocadia and / or Candidatus Kuenenia.

[0042] The sulfur autotrophic denitrifying bacteria include two or more of the following: Sulfurimonas, Thiomonas, and Thiobacillus.

[0043] The bio-enhancing auxiliary bacteria include two or more of the following: Pseudomonas, Shinella, Thaurea, and Flavobacterium.

[0044] The excipients include calcium carbonate and ferrous chloride.

[0045] The compound microbial agent of the present invention preferably comprises 15-25 parts of hydrolytic oxidation functional bacteria, more preferably 20 parts. In the present invention, the concentration of the hydrolytic oxidation functional bacteria is preferably 2 × 10⁻⁶. 9 ~3×10 9CFU / mL. In this invention, the hydrolytic oxidizing bacteria include Chlorobium. In this invention, the NCBI classification number of Chlorobium is preferably 1091. In this invention, the Chlorobium preferably includes one or more of the following: Chlorobium phaeovibrioides (NCBI classification number 1094); Chlorobium phaeobacteroides (NCBI classification number 1096); and Chlorobium limicola (NCBI classification number 1092).

[0046] The compound microbial agent of the present invention preferably comprises 15-25 parts of nitrite-oxidizing functional bacteria, more preferably 20 parts. In the present invention, the concentration of the nitrite-oxidizing functional bacteria is preferably 2 × 10⁻⁶. 9 ~3×10 9 CFU / mL. In this invention, the nitrifying functional bacteria include *Nitrosomonas* and / or *Nitrosospina*. In this invention, the NCBI classification number of *Nitrosomonas* is preferably 914; the NCBI classification number of *Nitrosospina* is preferably 35798. In this invention, the *Nitrosomonas* preferably includes one or more of the following: *Nitrosomonas ureae* (NCBI classification number 44577); *Nitrosomonas nitrosa* (NCBI classification number 52442); and *Nitrosomonas europaea* (NCBI classification number 915). In this invention, the genus Nitrosospina preferably includes one or more of the following: Nitrosospira multiformis (NCBI classification number 1231) and Nitrosospira briensis (NCBI classification number 35799).

[0047] The compound microbial agent of the present invention preferably comprises 8-12 parts of nitrifying functional bacteria, more preferably 10 parts. In the present invention, the concentration of the nitrifying functional bacteria is preferably 2 × 10⁻⁶. 9 ~3×10 9CFU / mL. In this invention, the nitrifying functional bacteria include *Nitrospina* species. Preferably, the NCBI classification number of *Nitrospina* is 35800. Preferably, the *Nitrospina* species includes: *Nitrospinawatsonii*, NCBI classification number 1323948; and / or *Nitrospina gracilis*, NCBI classification number 35801.

[0048] The compound microbial agent of this invention preferably comprises 15-25 parts of anaerobic ammonia-oxidizing functional bacteria, more preferably 20 parts. The source of the anaerobic ammonia-oxidizing functional bacteria of this invention preferably includes domestication, or it can be prepared by a microbial agent company. After domestication, the moisture content of the anaerobic ammonia-oxidizing functional bacteria is preferably 25-35%. In this invention, the bacterial concentration of the anaerobic ammonia-oxidizing functional bacteria is preferably 6 × 10⁻⁶. 8 ~8×10 8 CFU / mL. The anaerobic ammonia-oxidizing strain in the compound bacterial agent of this invention is an autotrophic species, and no carbon source is required during the ammonia nitrogen removal process. Its reaction formula is as follows:

[0049] NH4 + +1.32NO2 - +0.066HCO3 - +0.13H + →1.02N2+0.26NO3 - +0.066CH2O 0.5 N 0.15 +2.

[0050] 03H2O.

[0051] In this invention, the anaerobic ammonia-oxidizing functional bacteria include *Candidatus Brocadia* and / or *Candidatus Kuenenia*. Preferably, the NCBI classification number of *Candidatus Brocadia* is 380240, and the NCBI classification number of *Candidatus Kuenenia* is 380738. Preferably, the anaerobic ammonia-oxidizing bacteria genus *Candidatus Brocadia* includes one or more of the following: *Candidatus Brocadia sinica* (NCBI classification number 795830); *Candidatus Brocadia sapporoensis* (NCBI classification number 392547); and *Candidatus Brocadia fulgida* (NCBI classification number 380242). In this invention, the anaerobic ammonia-oxidizing bacteria genus (Candidatus Kuenenia) preferably includes: *Candidatus Kuenenia stuttgartiensis*, NCBI classification number 174633; and / or *Candidatus Kuenenia hertensis*, NCBI classification number 2996012. In this invention, when the compound bacterial agent is used to treat marine aquaculture water, the anaerobic ammonia-oxidizing functional bacteria are preferably *Candidatus Brocadia*; when the compound bacterial agent is used to treat freshwater aquaculture water, the anaerobic ammonia-oxidizing functional bacteria are preferably *Candidatus Kuenenia*.

[0052] The compound microbial agent of this invention preferably comprises 8-12 parts of sulfur-autotrophic denitrifying functional bacteria, more preferably 10 parts. In this invention, the sulfur-autotrophic denitrifying functional bacteria are preferably obtained through domestication, or can be prepared by a microbial agent company. After domestication, the moisture content of the sulfur-autotrophic denitrifying functional bacteria is preferably 25-35%. In this invention, the bacterial concentration of the sulfur-autotrophic denitrifying functional bacteria is preferably 1×10⁻⁶. 8 ~2×10 8CFU / mL. In this invention, the sulfur autotrophic denitrifying bacteria include two or more of the genera *Sulfurimonas*, *Thiomonas*, and *Thiobacillus*. Preferably, the NCBI classification number of *Sulfurimonas* is 202746, the NCBI classification number of *Thiomonas* is 32012, and the NCBI classification number of *Thiobacillus* is 919. In this invention, the *Sulfurimonas* species preferably includes one or more of the following: *Sulfurimonas autotrophica* (NCBI classification number 202747); *Sulfurimonas crateris* (NCBI classification number 2574727); *Sulfurimonas denitrificans* (NCBI classification number 39766); and *Sulfurimonas gotlandica* (NCBI classification number 1176482). In this invention, the *Thiomonas* genus preferably includes one or more of the following: *Thiomonas arsenitoxydans* (NCBI classification number 426114); *Thiomonas intermedia* (NCBI classification number 926); *Thiomonas delicata* (NCBI classification number 364030); and *Thiomonas bhubaneswarensis* (NCBI classification number 339866). In this invention, the *Thiobacillus* genus preferably includes: *Thiobacillus denitrificans* (NCBI classification number 36861); and / or *Thiobacillus thioparus* (NCBI classification number 931).

[0053] The five genera of bacteria in the above-mentioned compound bacterial agent of this invention (hydrolytic oxidation bacteria, nitrifying bacteria, nitrifying bacteria, anaerobic ammonia oxidation bacteria, and sulfur autotrophic denitrification bacteria) play a complementary role. Without any of these bacteria, the complete removal of nitrogen pollution cannot be achieved. Their specific functions are as follows: 1. Chlorobium reduces nitrogen compounds to ammonia through the catalytic action of enzymes such as nitrogen reductase. This is the process of reducing nitrogen from a higher oxidation state to ammonia. The released ammonia can be further converted into ammonia ions by other microorganisms through ammonification. This is the process of converting organic nitrogen into inorganic nitrogen. These processes help release nitrogen from organic matter into the environment, making it available for use by other organisms. 2. Nitrosomonas and / or Nitrosospina utilize excess dissolved oxygen in aquaculture water to convert ammonia nitrogen in wastewater into nitrite nitrogen, providing a substrate for subsequent denitrification processes. 3. Nitrospina utilizes excess dissolved oxygen in aquaculture water to convert nitrite nitrogen into nitrate nitrogen, providing a substrate for subsequent denitrification processes. IV. Brocadia and / or Kuenenia utilize ammonia nitrogen as an electron acceptor and nitrite nitrogen as an electron donor to directly convert ammonia nitrogen and nitrite nitrogen into nitrogen gas, thus achieving ammonia nitrogen removal. Since anaerobic ammonia oxidizing bacteria are autotrophic microorganisms, no carbon source needs to be added during the denitrification process. However, 11% nitrate nitrogen will also be produced during anaerobic ammonia oxidation. V. Sulfurimonas, Thiomonas, and / or Thiobacillus use reduced sulfur as an electron donor and nitrite nitrogen or nitrate nitrogen as an electron acceptor to oxidize low-valence sulfides to sulfates and reduce nitrate nitrogen or nitrite nitrogen to nitrogen gas, thus achieving nitrogen pollution removal. This process also does not require an external organic carbon source.

[0054] Experiments have shown that the absence of any one of the five types of bacteria described in this invention will prevent the complete purification of polluted water. The lack of hydrolytic acidifying bacteria will prevent the release of organic nitrogen from the water, resulting in incomplete nitrogen removal. The lack of nitrifying bacteria will prevent the provision of a reaction substrate for anaerobic ammonia oxidizing bacteria, thus hindering nitrogen removal. The lack of anaerobic ammonia oxidizing bacteria will prevent nitrogen removal without an external carbon source. The lack of nitrifying bacteria will prevent the conversion of ammonia nitrogen remaining from anaerobic ammonia oxidation into nitrate nitrogen for removal by denitrifying bacteria. The lack of sulfur-autotrophic denitrifying bacteria will prevent the use of sulfur in the water for denitrification to remove nitrate and nitrite nitrogen, leading to the accumulation of sulfur and nitrate nitrogen in the water.

[0055] Since this invention does not require the addition of a carbon source, anaerobic ammonia oxidation is the main reaction for nitrogen removal. However, anaerobic ammonia oxidation itself produces nitrate nitrogen, which removes 11% of the ammonia nitrogen. This nitrate needs to be removed through denitrification. Simultaneously, because there is insufficient carbon source in the water, traditional heterotrophic denitrification cannot be used. However, the water contains some reducing sulfur, which can serve as an electron donor for sulfur autotrophic denitrification, enabling the denitrification process while simultaneously removing sulfur and nitrate nitrogen from the water.

[0056] The compound microbial agent of the present invention preferably comprises 8-12 parts of bio-enhanced auxiliary functional bacteria, more preferably 10 parts. In the present invention, the concentration of the bio-enhanced auxiliary functional bacteria is preferably 1×10⁻⁶. 8 ~2×10 8CFU / mL. In this invention, the bio-enhancing auxiliary bacteria include two or more of the genera *Pseudomonas*, *Shinella*, *Thauera*, and *Flavobacterium*. In this invention, the bio-enhancing auxiliary bacteria can improve the aquatic environment. *Pseudomonas*, *Shinella*, *Thauera*, and *Flavobacterium* microorganisms enhance the growth of extracellular polymers and filamentous bacteria of *Chlorobium phaeovibrioides*, *Chlorobium phaeobacteroides*, and *Chlorobium limicola*, promoting microbial aggregation and enhancing the pollutant transformation efficiency of the functional microorganisms. In this invention, the preferred NCBI classification numbers for *Pseudomonas* are 286, for *Shinella* 323620, for *Thauera* 33057, and for *Flavobacterium* 237. Preferably, the *Pseudomonas* genus in this invention includes: *Pseudomonas yamanorum* (NCBI classification number 515393), *Pseudomonas umsongensis* (NCBI classification number 198618), and *Pseudomonas viridiflava* (NCBI classification number 33069). In this invention, the *Shinella* genus preferably includes: *Shinella zoogloeoides*, NCBI classification number 352475; and *Shinella sumterensis*, NCBI classification number 1967501. In this invention, the *Thauera* genus preferably includes: *Thauera aminoaromatica*, NCBI classification number 164330; and *Thauera aromatica*, NCBI classification number 59405. In this invention, the *Flavobacterium* genus preferably includes: *Flavobacterium psychrophilum*, NCBI classification number 96345; and *Flavobacterium columnnare*, NCBI classification number 996.

[0057] The present invention does not have any special limitation on the source of the above-mentioned bacteria; conventional commercially available products can be used.

[0058] The compound microbial agent of this invention preferably comprises 6-16 parts of excipients, more preferably 10 parts. In this invention, the excipients preferably comprise 3-8 parts of calcium carbonate and 3-8 parts of ferrous chloride, more preferably 5 parts of calcium carbonate and 5 parts of ferrous chloride. In this invention, calcium carbonate and ferrous chloride are preferably added in solution form, with the concentration of calcium carbonate preferably being 70-90 mg / L and the concentration of ferrous chloride preferably being 0.5 mol / L. This invention does not specifically limit the source of calcium carbonate and ferrous chloride; they are conventional commercially available chemical agents known to those skilled in the art, in powder form, used for phosphorus removal from wastewater. In this invention, calcium carbonate can react with phosphate to form hydroxyapatite precipitate, thereby achieving phosphorus removal. Phosphorus removal by calcium carbonate is usually carried out under alkaline conditions. The iron ions in ferrous chloride can form insoluble iron-phosphorus precipitates with phosphorus, thereby achieving phosphorus removal. Phosphorus removal by ferrous chloride is usually carried out under neutral to weakly alkaline conditions (6.0 ≤ pH ≤ 8.0).

[0059] The weight ratio of each component in the compound microbial agent of the present invention can be appropriately adjusted at different stages of aquaculture and when there are significant changes in the composition of water pollution.

[0060] The present invention also provides a method for preparing the compound microbial agent described in the above technical solution, comprising the following steps:

[0061] The compound microbial agent is obtained by mixing hydrolytic oxidation bacteria, nitrification bacteria, nitrification bacteria, anaerobic ammonia oxidation bacteria, sulfur autotrophic denitrification bacteria, bio-enhanced auxiliary bacteria and excipients.

[0062] The present invention also provides the application of the compound microbial agent described in the above technical solution or the compound microbial agent prepared by the preparation method described in the above technical solution in purifying aquatic aquaculture water.

[0063] This invention also provides a method for in-situ purification of aquaculture water using the compound microbial agent prepared based on the compound microbial agent described in the above technical solution or the preparation method described in the above technical solution, comprising the following steps:

[0064] In the early stages of aquaculture, compound microbial agents are added to the biological bed in the aquaculture pond for aquaculture.

[0065] In this invention, the biological bed serves to immobilize the compound microbial agent. The source of the biological bed is not specifically limited; any biological bed prepared from fibrous materials, such as Suzhou Sujing biological packing material, is acceptable. Recommended models are: SJ-III-55A, SJ-III-55B, SJ-III-80C, SJ-II-80A, or SJ-II-80B. The aquaculture described in this invention is preferably carried out in aquaculture cages (single aquaculture ponds containing aquatic organisms in intensive aquaculture). Before adding the biological bed, this invention preferably attaches the compound microbial agent to the biological bed first, which can reduce the loss of the agent in the aquaculture cage. The compound microbial agent of this invention will eventually attach and grow on the biological bed and continuously self-proliferate; different aquatic organisms will not affect the effectiveness of the agent. In this invention, the preferred dosage of the compound microbial agent is 8–12 g / m³. 3 More preferably 10g / m 3 Before adding the compound microbial agent, it is preferable to further include a nutrient solution reactivation treatment, wherein the nutrient solution comprises ammonia nitrogen and sulfide; the dissolved oxygen in the reactivation treatment is preferably 0.5 mg / L, the temperature is preferably 30-35℃, the pH value is preferably 7.5-8.5, and the time is preferably 1-3 days. In this invention, the concentration of ammonia nitrogen in the nutrient solution is preferably 80-120 mg / L, more preferably 100 mg / L. In this invention, the concentration of sulfide in the nutrient solution is preferably 15-30 mg / L, more preferably 20 mg / L. In this invention, the ammonia nitrogen and sulfide are preferably derived from ammonium bicarbonate (NH4HCO3) and sodium sulfide nonahydrate (Na2S·9H2O), respectively.

[0066] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a compound bacterial agent for in-situ purification of aquaculture water, its preparation method, and its usage method, is provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] Preparation method:

[0069] I. Preparation of Sulfur Autotrophic Denitrification Functional Bacterial Agent

[0070] Reactor: An anaerobic reactor with an effective volume of 3L was used for acclimatization. The reactor is equipped with a water bath insulation system and a stirring device at the top. Water is fed into the reactor using a peristaltic pump.

[0071] Culture medium preparation: Nitrates (NO3) are provided by adding sodium nitrate (NaNO3) and sodium sulfide (Na2S·9H2O). - -N) and S 2-The addition of NaHCO3 provides alkalinity and inorganic carbon source; the addition of 136 mg / L CaCl2, 200 mg / L MgCl2, 70 mg / L KH2PO4, 1 mg / L trace element I, and 1.25 mg / L trace element II provides nutrients such as Ca, Mg, and Fe.

[0072] Trace element I: 5g / LEDTA·2Na, 5g / LFeSO4·7H2O

[0073] Trace elements II: 15g / L EDTA·2Na, 0.43g / LFeSO4·7H2O, 0.24g / L CoCl2·6H2O, 0.99g / L MnCl2·4H2O, 0.25g / L CuSO4·5H2O, 0.22g / L Na2M O O4·2H2O, 0.19g / LNiCl2·6H2O, 0.014g / LH3BO3.

[0074] Sludge inoculation: During the start-up phase of sulfur autotrophic denitrifying bacteria enrichment, ordinary activated sludge from the sludge thickening tank of the municipal wastewater treatment plant is obtained and inoculated into the reactor at a rate of 1800–2200 mg MLSS / L.

[0075] Controlling NO3 in culture medium - -N and S 2- The concentrations were 90–105 mg / L and 95–115 mg / L, respectively, with an S / N ratio controlled at 0.43–0.47. Continuous water infusion was used for cultivation, with a hydraulic retention time controlled at 7–9 h, a temperature controlled at 30–32 °C, a pH controlled at 8–10, and an acclimatization period of 9–12 days. When the effluent NO3… - -N and S 2-When the concentration is stabilized at 0–15 mg / L, bacterial solution A with sulfur autotrophic denitrification function can be obtained. Bacterial solution A is then dehydrated to a water content of 25%–35% to obtain sulfur autotrophic denitrifying bacterial solution B. The sulfur autotrophic denitrifying bacterial solutions obtained by this preparation method were tested by Meiji Biotechnology. At the genus level, *Thiobacillus* (NCB: 919) was the most abundant bacterium (accounting for 11.02%), while *Sulfurimonas* (NCB: 202746) and *Thiomonas* (NCB: 32012) were also detected as dominant bacteria. Further testing at the species level revealed *Thiobacillus denitrificans* (NCB: 36861) and *Thiobacillus thioparus* (NCB: 931) within the *Thiobacillus* genus. The following species were detected in the genus *Sulfurimonas*: *Sulfurimonas autotrophica* (NCB: 202747), *Sulfurimonascrateris* (NCB: 2574727), and *Sulfurimonas denitrificans* (NCB: 39766). The following species were detected in the genus *Thiomonas*: *Thiomonas arsenitoxydans* (NCB: 426114), *Thiomonas intermedia* (NCB: 926), and *Thiomonas bhubaneswarensis* (NCB: 339866).

[0076] II. Preparation of Anaerobic Ammonia Oxidation Functional Bacterial Agents

[0077] Reactor: An anaerobic reactor with an effective volume of 4L was used for acclimatization. The reactor is equipped with a water bath insulation system and a stirring device at the top. Water is fed into the reactor using a peristaltic pump.

[0078] Culture medium preparation: Nitrite (NO2) was provided by adding sodium nitrite (NaNO2), ammonium bicarbonate (NH4HCO3), and sodium acetate (CH3COONa). - -N), ammonia nitrogen (NH4) + -N) and COD; by adding 180mg / L CaCl2·2H2O, 300mg / L MgSO4·7H2O, 27mg / L KH2PO4, 0.5mg / L trace element I, and 0.5mg / L trace element II, it provides nutrients such as Ca, Mg, and Fe;

[0079] Trace element I: 5g / LEDTA·2Na, 5g / LFeSO4·7H2O.

[0080] Trace elements II: 15g / L EDTA·2Na, 0.43g / LFeSO4·7H2O, 0.24g / L CoCl2·6H2O, 0.99g / L MnCl2·4H2O, 0.25g / L CuSO4·5H2O, 0.22g / L Na2M O O4·2H2O, 0.19g / LNiCl2·6H2O, 0.014g / LH3BO3.

[0081] In the initial stage of anaerobic ammonia-oxidizing functional bacteria enrichment and acclimatization (25–30 days), denitrifying activated sludge was obtained from a municipal wastewater treatment plant, and the amount of sludge inoculated into the reactor was 1800–2200 mg MLSS / L. The COD:N ratio was controlled at 75–85:1, the temperature at 32–36℃, the pH at 6.5–7.5, the hydraulic retention time at 6–8 h, and the NH4+ level in the culture medium. + -N and NO2 - The -N concentrations were 18–22 mg / L and 18–22 mg / L, respectively, at which point the influent NH4+ + -N and NO2 - The -N concentration ratio can be controlled at around 1.

[0082] Mid-stage of anaerobic ammonia-oxidizing functional bacteria enrichment and acclimatization (38–42 days), waiting for NH4... + -N and NO2 - When the NH4+ removal rate stabilizes at around 45% to 60% and the sludge color lightens, the NH4+ in the culture medium can be removed. + -N and NO2 - When the -N concentration increases to 65–75 mg / L and 78–90 mg / L, the influent NH4+ concentration increases. + -N and NO2 - The -N concentration ratio should be controlled between 1.1 and 1.3. The COD:N ratio should be controlled at 35–45:1, the temperature at 32–36℃, the pH at 7.5–8.0, and the hydraulic retention time at 6–8 hours.

[0083] The later stage of anaerobic ammonia-oxidizing functional bacteria enrichment and acclimatization (lasting 30-35 days), waiting for NH4... + -N and NO2 - When the -N removal rate is consistently around 50%, the NH4+ in the culture medium can be removed. + -N and NO2 - When the -N concentration increases to 80–100 mg / L and 100–120 mg / L, the influent NH4+ concentration... + -N and NO2 -The -N concentration ratio should be controlled between 1.1 and 1.3. Stop adding COD, maintain a temperature of 32–36℃, a pH of 8.0–8.5, and a hydraulic retention time of 6–8 hours. Wait for NH4+ to... + When the nitrogen removal rate stabilizes above 90% and the sludge turns red, the anaerobic ammonia oxidizing bacteria have matured. A bacterial solution A with anaerobic ammonia oxidation function can then be obtained. Further dehydration of bacterial solution A to a moisture content of 25%–35% yields anaerobic ammonia oxidizing bacterial solution B.

[0084] The anaerobic ammonia-oxidizing bacterial culture obtained by this preparation method was tested by Meiji Biotechnology. At the genus level, *Brocadia* (NCB: 380240) was the most abundant bacterium (accounting for 4.17%), and *Kuenenia* (NCB: 380738) was also detected. However, since the culture medium was freshwater, the highest proportion of *Kuenenia* was only 0.51%. Further testing at the species level revealed *Candidatus Brocadia sinica* (NCB: 795830), *Candidatus Brocadiasapporoensis* (NCB: 392547), and *Candidatus Brocadia fulgida* (NCB: 380242) within the *Brocadia* genus. Within the *Kuenenia* genus, *Candidatus Kuenenia stuttgartiensis* (NCB: 174633) and *Candidatus Kuenenia hertensis* (NCB: 2996012) were detected.

[0085] The aforementioned hydrolytic oxidizing bacteria, nitrifying bacteria, and nitrite-oxidizing bacteria have established market channels, and the public can directly contact the relevant bacterial agent companies to purchase mature bacterial solutions. Alternatively, the bacterial agents can be separated, purified, and expanded by the bacterial agent preparation companies based on their NCBI numbers, thereby preparing the corresponding bacterial solutions. The hydrolytic oxidizing bacteria were prepared by expanding *Chlorobium phaeovibrioides*, *Chlorobium phaeobacteroides*, and *Chlorobium limicola* by the bacterial agent preparation company. The nitrifying bacteria were prepared by expanding *Nitrospinawatsonii* and *Nitrospina gracilis* from the *Nitrospina* genus by the bacterial agent preparation company. The nitrite-oxidizing bacteria were prepared by expanding *Nitrosomonas ureae*, *Nitrosomonas nitrosa*, *Nitrosomonas europaea*, as well as *Nitrosospira multiformis* and *Nitrosospirabriensis* by the bacterial agent preparation company. The concentration of the prepared bacterial agents was 2 × 10⁻⁶.9 ~3×10 9 CFU / mL, and the concentration of the above-mentioned bacterial strains in the bacterial solution must be the same during the preparation of the bacterial agent.

[0086] The bio-enhanced auxiliary functional bacteria agent was prepared by expanding the culture of Pseudomonas, Shinella, Thaurasa, and Flavobacterium by the bacterial agent preparation company, with a bacterial count of 1×10⁻⁶. 8 ~2×10 8 The concentration of the four bacterial strains was the same in the bacterial solution (CFU / mL). The excipients were prepared with calcium carbonate at a concentration of 80 mg / L and ferrous chloride at a concentration of 0.5 mol / L.

[0087] The key point of this invention is to achieve in-situ purification of aquaculture water using a rationally combined bacterial agent. The compound bacterial agent is obtained by mixing hydrolytic oxidation bacteria, nitrifying bacteria, nitrifying bacteria, anaerobic ammonia oxidation bacteria, sulfur autotrophic denitrification bacteria, bio-enhancing auxiliary bacteria, and excipients. The compound bacterial agent contains 15 parts hydrolytic oxidation bacteria, 15 parts nitrifying bacteria, 8 parts nitrifying bacteria, 25 parts anaerobic ammonia oxidation bacteria, 12 parts sulfur autotrophic denitrification bacteria, 12 parts bio-enhancing auxiliary bacteria, and 13 parts excipients; the excipients include 7 parts calcium carbonate and 6 parts ferrous chloride.

[0088] Example 2

[0089] The sulfur-autotrophic denitrifying bacteria inoculum was prepared by a company that expanded and cultured *Thiobacillus denitrificans*, *Thiobacillus thioparus*, *Sulfurimonas autotrophica*, *Sulfurimonas crateris*, *Sulfurimonas denitrificans*, as well as *Thiomonas arsenitoxydans*, *Thiomonas intermedia*, and *Thiomonas bhubaneswarensis*. The prepared inoculum contained 1 × 10⁻⁶ bacteria. 8 ~2×10 8 CFU / mL, and the concentration of the above-mentioned bacterial strains in the bacterial solution must be the same during the preparation of the bacterial agent.

[0090] The anaerobic ammonia-oxidizing bacterial agent was prepared by a company that expanded and cultured *Candidatus Brocadia sinica*, *Candidatus Brocadia sapporoensis*, *Candidatus Brocadia fulgida*, *Candidatus Kuenenia stuttgartiensis*, and *Candidatus Kuenenia hertensis*. The prepared agent contained 6 × 10⁻⁶ bacteria. 8 ~8×10 8 CFU / mL, and the concentration of the above-mentioned bacterial strains in the bacterial solution must be the same during the preparation of the bacterial agent.

[0091] The hydrolytic oxidation inoculant was prepared by expanding *Chlorobium phaeovibrioides*, *Chlorobium phaeobacteroides*, and *Chlorobium limicola* using the inoculant preparation company. The nitrifying inoculant was prepared by expanding *Nitrospinawatsonii* and *Nitrospinawa gracilis* from the *Nitrospina* genus using the inoculant preparation company. The nitrite-oxidizing inoculant was prepared by expanding *Nitrosomonas ureae*, *Nitrosomonas nitrosa*, *Nitrosomonas europaea*, as well as *Nitrosospira multiformis* and *Nitrosospirabriensis* using the inoculant preparation company. The concentration of the prepared inoculants was 2 × 10⁻⁶. 9 ~3×10 9 CFU / mL, and the concentration of the above-mentioned bacterial strains in the bacterial solution must be the same during the preparation of the bacterial agent.

[0092] The bio-enhanced auxiliary functional bacteria agent was prepared by a bacterial agent preparation company through the expansion and cultivation of Pseudomonas, Shinella, Thaurasa, and Flavobacterium, with a bacterial concentration of 1×10⁻⁶. 8 ~2×10 8 The concentration of the four bacterial strains in the bacterial solution is the same (CFU / mL). The excipients are prepared by calcium carbonate solution at a concentration of 80 mg / L and ferrous chloride solution at a concentration of 0.5 mol / L. Both calcium carbonate and ferrous chloride concentrations refer to the mother liquor concentrations, and the solvent is pure water.

[0093] The compound microbial agent is obtained by mixing hydrolytic oxidizing bacteria, nitrifying bacteria, nitrifying bacteria, anaerobic ammonia oxidizing bacteria, sulfur autotrophic denitrifying bacteria, bio-enhancing auxiliary bacteria, and excipients. The compound microbial agent contains 25 parts hydrolytic oxidizing bacteria, 25 parts nitrifying bacteria, 12 parts nitrifying bacteria, 15 parts anaerobic ammonia oxidizing bacteria, 8 parts sulfur autotrophic denitrifying bacteria, 8 parts bio-enhancing auxiliary bacteria, and 7 parts excipients, including 3 parts calcium carbonate and 4 parts ferrous chloride.

[0094] Example 3

[0095] Three shrimp farming ponds at a shrimp farming base in Nantong City were selected for the experiment. Each pond was 1.2m deep, 10m wide, and 40m long, with a water volume of approximately 400m³. 3 The water quality of the three ponds is shown in Table 1:

[0096] Table 1. Pond Water Quality

[0097]

[0098] This experiment used the composite microbial agent prepared in Example 1 of this invention to treat pollutants in situ. The microbial agent was first reactivated using a nutrient solution containing 100 mg / L ammonia nitrogen and 20 mg / L sulfide. During the reactivation period, dissolved oxygen was controlled at 0.5 mg / L, temperature at 30–35℃, and pH at 7.5–8.5. The reactivation time was 28 hours. A biological bed made of fiber material (Suzhou Sujing biological packing material, SJ-III-55A) was then placed in the reactivated composite microbial agent, allowing the agent to attach and grow on the biological bed. This stage lasted approximately 24 hours. The dosage of the composite microbial agent was controlled at 4 kg ± 80 g (10 ± 2 g / m³) in all three ponds. 3 Based on the size and structure of the pond, four biological bed placement points are set up for each pond.

[0099] Normal aquaculture operations were maintained during the experiment, and no carbon source or compound microbial agent was added to the pond.

[0100] Forty hours after the biological bed was placed in the aquaculture ponds, the ammonia nitrogen concentration in all three ponds dropped to below 1 mg / L, and the total nitrogen concentration dropped to below 3 mg / L. The biological bed in the purified ponds could be removed and placed in other ponds for continued use. These three groups of ponds independently selected the timing and duration of biological bed addition based on water quality conditions, maintaining pollutant concentrations at low levels throughout the six-month trial. The hatching rate of shrimp larvae in the three experimental ponds (average hatching rate 78%) was significantly higher than in other ponds without this compound microbial agent (average hatching rate 46%), demonstrating that the use of this compound microbial agent greatly improved the survival rate of aquatic organisms.

[0101] Example 4

[0102] This experiment used the compound bacterial agent prepared in Example 2 of this invention to treat pollutants in situ. The pond location selected for the experiment was the same as in Example 1, and the water quality was not significantly different from that in Example 1.

[0103] Table 2 Pond Water Quality

[0104]

[0105] The specific implementation was consistent with Example 1. 38 hours after the biological bed was placed in the aquaculture ponds, the ammonia nitrogen concentration in all three ponds dropped below 1 mg / L, and the total nitrogen concentration dropped below 3 mg / L. Furthermore, the pollutant concentrations remained at a low level for an extended period. The hatching rate of shrimp larvae in the three ponds tested reached 79%, which was higher than that in ponds without the addition of the bacterial agent of this invention.

[0106] Comparative Example 1

[0107] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0108] The difference between this comparison and Example 1 is that no hydrolytic oxidizing bacteria agent was added. The remaining amounts of bacteria agents were the same as in Example 1.

[0109] Comparative Example 2

[0110] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0111] The difference between this comparison and Example 1 is that no nitrifying bacteria agent was added. The mixing amounts of the remaining bacteria agents are the same as in Example 1.

[0112] Comparative Example 3

[0113] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0114] The difference between this comparison and Example 1 is that no nitrifying bacteria agent was added. The mixing amounts of the remaining bacteria agents are the same as in Example 1.

[0115] Comparative Example 4

[0116] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0117] The difference between this comparison and Example 2 is that no sulfur-autotrophic denitrifying bacteria were added. The mixing amounts of the remaining bacteria were the same as in Example 2.

[0118] Comparative Example 5

[0119] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0120] The difference between this comparison and Example 2 is that no anaerobic ammonia-oxidizing bacteria were added. The mixing amounts of the remaining bacteria were the same as in Example 2.

[0121] Comparative Example 6

[0122] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0123] The difference between this comparative example and Example 2 is that no hydrolyzing acidifying bacterial agent was added. The mixing amounts of the remaining bacterial agents are the same as in Example 2.

[0124] Comparative Example 7

[0125] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0126] The difference between this comparison and Example 2 is that no nitrifying bacteria agent was added. The mixing amounts of the remaining bacteria agents are the same as in Example 2.

[0127] Comparative Example 8

[0128] A method for preparing a compound bacterial agent for in-situ purification of aquaculture water.

[0129] The difference between this comparison and Example 2 is that no nitrifying bacteria agent was added. The mixing amounts of the remaining bacteria agents are the same as in Example 2.

[0130] Ponds with conditions identical to those in Example 1 were selected, and Comparative Examples 1-3 were tested according to the operating procedures of Example 1. The results showed that Comparative Examples 1-3 could not achieve long-term water quality purification in the aquaculture water, and the final hatching rate was not significantly different from that of ponds without added bacterial agents.

[0131] Ponds with conditions identical to those in Example 2 were selected, and Comparative Examples 4-8 were tested according to the operating procedures of Example 2. The results showed that Comparative Examples 4-8 could not achieve long-term water quality purification in the aquaculture water, and the final hatching rate was not significantly different from that of ponds without added bacterial agents.

[0132] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound bacterial agent for in-situ purification of aquaculture water, characterized in that, The product comprises the following components in parts by weight: 15-25 parts of hydrolytic oxidation functional bacteria, 15-25 parts of nitrite functional bacteria, 8-12 parts of nitrification functional bacteria, 15-25 parts of anaerobic ammonia oxidation functional bacteria, 8-12 parts of sulfur autotrophic denitrification functional bacteria, 8-12 parts of bio-enhancing auxiliary functional bacteria, and 6-16 parts of excipients; the excipients include 3-8 parts of calcium carbonate and 3-8 parts of ferrous chloride. The hydrolytic and oxidizing bacteria include Chlorobium; The nitrifying functional bacteria include Nitrosomonas and / or Nitrosospira. The nitrifying bacteria include Nitrospina species. The anaerobic ammonia oxidizing bacteria include Candidatus Brocadia and / or Candidatus Kuenenia. The sulfur autotrophic denitrifying bacteria include two or more of the following: Sulfurimonas, Thiomonas, and Thiobacillus. The bio-enhancing auxiliary bacteria include two or more of the following: Pseudomonas, Shinella, Thaurea, and Flavobacterium.

2. The compound microbial agent according to claim 1, characterized in that, When the compound bacterial agent is used to treat marine aquaculture water, the anaerobic ammonia-oxidizing functional bacteria are Candidatus Brocadia; when the compound bacterial agent is used to treat freshwater aquaculture water, the anaerobic ammonia-oxidizing functional bacteria are Candidatus Kuenenia.

3. The compound microbial agent according to claim 1, characterized in that, The sources of anaerobic ammonia oxidizing functional bacteria and sulfur autotrophic denitrifying functional bacteria include domestication, with water content of 70-90% respectively; the bacterial concentration of anaerobic ammonia oxidizing functional bacteria is 6×10⁸~8×10⁸ CFU / mL; the bacterial concentration of sulfur autotrophic denitrifying functional bacteria is 1×10⁸~2×10⁸ CFU / mL; the bacterial concentrations of hydrolytic oxidation functional bacteria, nitrite functional bacteria and nitrifying functional bacteria are 2×10⁹~3×10⁹ CFU / mL respectively; the bacterial concentration of biofortification auxiliary functional bacteria is 1×10⁸~2×10⁸ CFU / mL; calcium carbonate and ferrous chloride are added in solution form, with the concentration of calcium carbonate being 70-90 mg / L and the concentration of ferrous chloride being 0.5 mol / L.

4. The compound microbial agent according to claim 1, characterized in that, The NCBI classification number for Chlorobium (a type of green bacteria) is 1091; the NCBI classification number for Nitrosomonas (a type of nitrosomonas) is 914; the NCBI classification number for Nitrosospira (a type of nitrosogenic bacteria) is 35798; the NCBI classification number for Nitrospina (a type of nitrifying bacteria) is 35800; and the NCBI classification number for Candidatus (an anaerobic ammonia-oxidizing bacteria) is... The NCBI classification number for Brocadia is 380240; the NCBI classification number for Candidatus Kuenenia is 380738; the NCBI classification number for Sulfurimonas is 202746; the NCBI classification number for Thiomonas is 32012; the NCBI classification number for Thiobacillus is 919; the NCBI classification number for Pseudomonas is 286; the NCBI classification number for Shinella is 323620; the NCBI classification number for Thaurera is 33057; and the NCBI classification number for Flavobacterium is 237.

5. The compound microbial agent according to claim 4, characterized in that, The Chlorobium phylum includes one or more of the following: Chlorobium phaeovibrioides (NCBI classification number 1094), Chlorobium phaeobacteroides (NCBI classification number 1096), and Chlorobium limicola (NCBI classification number 1092). The genus Nitrosomonas includes one or more of the following: Nitrosomonas ureae (NCBI classification number 44577); Nitrosomonas nitrosa (NCBI classification number 52442); and Nitrosomonas europaea (NCBI classification number 915). The genus Nitrosospira includes: Nitrosospira multiformis, NCBI classification number 1231; and / or Nitrosospira briensis, NCBI classification number 35799; The genus *Nitrospina* includes: *Nitrospina watsonii*, NCBI classification number 1323948; and / or *Nitrospina gracilis*, NCBI classification number 35801; The genus Candidatus Brocadia, which contains anaerobic ammonia-oxidizing bacteria, includes one or more of the following: Candidatus Brocadia sinica (NCBI classification number 795830), Candidatus Brocadiasapporoensis (NCBI classification number 392547), and Candidatus Brocadiafulgida (NCBI classification number 380242). The genus Candidatus Kuenenia of anaerobic ammonia oxidizing bacteria includes: Candidatus Kuenenia stuttgartensis, NCBI classification number 174633; and / or Candidatus Kuenenia hertensis, NCBI classification number 2996012; The *Sulfurimonas* genus mentioned in Xiamen includes one or more of the following: *Sulfurimonas autotrophica* (NCBI classification number 202747); *Sulfurimonas crateris* (NCBI classification number 2574727); *Sulfurimonas denitrificans* (NCBI classification number 39766); and *Sulfurimonas gotlandica* (NCBI classification number 1176482). The genus Thiomonas includes one or more of the following: Thiomonas arsenitoxydans (NCBI classification number 426114); Thiomonas intermedia (NCBI classification number 926); Thiomonas delicata (NCBI classification number 364030); and Thiomonas bhubaneswarensis (NCBI classification number 339866). The genus Thiobacillus includes: Thiobacillus denitrificans, NCBI classification number 36861; and / or Thiobacillus thioparus, NCBI classification number 931; The genus Pseudomonas includes one or more of the following: Pseudomonas yamanorum (NCBI classification number 515393); Pseudomonas umsongensis (NCBI classification number 198618); and Pseudomonas viridiflava (NCBI classification number 33069). The genus *Shinella* includes: *Shinella zoogloeoides*, NCBI classification number 352475; and / or *Shinella sumterensis*, NCBI classification number 1967501; The Thaurae genus includes: Thaurae aminoaromatica, NCBI classification number 164330; and / or Thaurae aromatica, NCBI classification number 59405. The Flavobacterium genus includes: Flavobacterium psychrophilum, NCBI classification number 96345; and / or Flavobacterium columnare, NCBI classification number 996.

6. A method for preparing the compound microbial agent according to any one of claims 1 to 5, comprising the following steps: The compound microbial agent is obtained by mixing hydrolytic oxidation bacteria, nitrification bacteria, nitrification bacteria, anaerobic ammonia oxidation bacteria, sulfur autotrophic denitrification bacteria, bio-enhanced auxiliary bacteria and excipients.

7. The application of the compound microbial agent according to any one of claims 1 to 5 or the compound microbial agent prepared by the preparation method according to claim 6 in purifying aquatic aquaculture water.

8. A method for in-situ purification of aquaculture water using the compound microbial agent prepared based on any one of claims 1 to 5 or the preparation method described in claim 6, characterized in that, Includes the following steps: In the early stages of aquaculture, compound microbial agents are added to the biological bed in the aquaculture pond for aquaculture.

9. The method according to claim 8, characterized in that, The amount of the compound microbial agent added is 8~12g / m3; before adding the compound microbial agent, a nutrient solution reactivation treatment is also included, wherein the nutrient solution includes ammonia nitrogen and sulfides; the dissolved oxygen in the reactivation treatment is 0.5mg / L, the temperature is 30~35℃, the pH value is 7.5~8.5, and the time is 1~3 days.

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