Functional microbial agent for purifying breeding sewage and preparation method of functional microbial agent

Abstract

The invention discloses a functional microbial agent for purifying breeding sewage and a preparation method of the functional microbial agent, and belongs to the technical field of functional microbial materials. The microbial agent is composed of a core functional unit and a porous carrier according to a mass ratio of 3: 1 to 5: 1, the core functional unit comprises a symbiotic engineering flora and a compound enzyme system in a volume ratio of 7: 1, and the symbiotic engineering flora is compounded by sphingomonas aeruginosa, paenibacillus splendidus and acinetobacter hemolyticus according to a ratio of 1: 1: 1; the porous carrier is a low-cost porous carbon material prepared on the basis of an animal waste blood / iron complex. Through systematic cooperation of a specific functional flora, a compound enzyme system and a special porous carrier, synergism of adsorption protection and biodegradation is achieved, the stress resistance and purification efficiency of the microbial agent in an antibiotic and heavy metal combined pollution environment are remarkably improved, and the industrial problem that an aquaculture sewage biological treatment system is prone to collapse is particularly solved; meanwhile, waste is treated by waste, and the method is low in cost and suitable for large-scale application.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial materials technology, specifically relating to a functional microbial agent for purifying aquaculture wastewater and its manufacturing method. Background Technology

[0002] In the large-scale development of animal husbandry, unified management has reduced costs and improved economic efficiency. However, the serious environmental pollution caused by the large-scale centralized discharge of wastewater urgently needs to be addressed. Domestically and internationally, wastewater treatment methods mainly fall into two categories: resource utilization and compliance with discharge standards. These can be further categorized into three models: returning wastewater to the field, natural treatment, and industrial treatment, each with its own advantages and disadvantages. The goal of wastewater treatment in my country is to control and reduce the harm caused by wastewater, promote comprehensive utilization, and ultimately achieve the objectives of reduction, resource recovery, harmlessness, and compliance with discharge standards. This aims to reduce the impact on crop soil quality and crop quality, and lower the potential pollution risks to water bodies, soil environments, and the climate environment.

[0003] Currently, the treatment of aquaculture wastewater using microorganisms is a research hotspot. This method offers numerous benefits, primarily in the following aspects: Reducing pollutant emissions: Aquaculture wastewater contains large amounts of organic matter, nitrogen, phosphorus, and other pollutants. Direct discharge without treatment can lead to eutrophication, causing algal blooms and disrupting the aquatic ecosystem. Microorganisms can decompose organic matter in wastewater into carbon dioxide and water, and convert ammonia nitrogen into harmless substances like nitrogen gas, reducing chemical oxygen demand (COD), biochemical oxygen demand (BOD), total nitrogen, and total phosphorus levels, thus minimizing environmental pollution. For example, aerobic microorganisms such as Bacillus can rapidly decompose large organic molecules like proteins and starches in wastewater, significantly reducing COD. The synergistic effect of nitrifying and denitrifying bacteria can effectively remove ammonia nitrogen, reducing the risk of eutrophication.

[0004] No secondary pollution (or minimal pollution): Compared to some chemical treatment methods (such as adding large amounts of chemical agents for sedimentation or disinfection), microbial treatment of aquaculture wastewater generally does not produce secondary pollution. The use of chemical agents may leave harmful residues, posing potential hazards to the soil, water bodies, and other environmental elements. In contrast, during microbial treatment, microorganisms use pollutants as substrates for growth, reproduction, and metabolic activities, and the final products are mostly harmless or low-harmful substances, without introducing new toxic or harmful components. For example, when microorganisms degrade antibiotic residues in aquaculture wastewater, they break them down into small molecules, non-toxic or low-toxic substances, rather than simply transferring or altering the form of antibiotics as some chemical methods do, without truly eliminating the harm.

[0005] The applicant has conducted some preliminary research, such as in CN202411186314.5, where a method for purifying mixed wastewater from livestock farming was disclosed. This method includes solid-liquid separation and sedimentation, flotation, anaerobic treatment, aerobic treatment, flocculation, separation, and disinfection. However, subsequent application revealed that while this process is effective, it is complex and also suffers from the following problems: Weak resistance to adverse conditions: Sulfonamides and tetracyclines, often remaining in aquaculture water, inhibit many functional microorganisms. Prolonged operation leads to the "inactivation" of microbial agents, resulting in poor reusability and increased costs. Simultaneously, heavy metals in wastewater, such as copper and zinc, can also be toxic to microorganisms, inhibiting their metabolic activity and affecting treatment efficiency. Especially in high-salt, low-temperature, or environments with large pH fluctuations, the stability of the microbial community is poor, easily causing system collapse. Furthermore, when multiple pollutants coexist, single microbial species struggle to achieve synergistic degradation, leading to unstable purification effects. Therefore, there is an urgent need to develop composite functional microbial communities with resistance to antibiotic interference, tolerance to heavy metal stress, and strong environmental adaptability to improve the practicality and long-term effectiveness of microbial treatment technologies. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a functional microbial agent for purifying aquaculture wastewater and its manufacturing method. By optimizing the screening of functional microorganisms and the selection of porous carriers, the agent's stress resistance and purification capacity are significantly improved. This agent exhibits strong adaptability in complex aquaculture environments, effectively resisting the inhibitory effects of antibiotic residues and heavy metals on microbial activity, maintaining a highly efficient and stable purification effect.

[0007] Furthermore, the preparation process of this microbial agent is simple and efficient, making it suitable for large-scale production applications. By loading functional microorganisms onto a porous carrier, not only is the adhesion and survival rate of the microorganisms improved, but their duration of action in the wastewater treatment process is also enhanced. This design allows the microbial agent to significantly reduce operating costs in practical applications while improving the overall efficiency of wastewater treatment, providing an economical, environmentally friendly, and efficient solution for aquaculture wastewater treatment.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A functional microbial agent for purifying aquaculture wastewater comprises a core functional unit and a porous carrier; the mass ratio of the porous carrier to the core functional unit is 3:1 to 5:1; the core functional unit contains symbiotic engineered microbial communities and a complex enzyme system, with a volume ratio of 7:1; the porous carrier is a low-cost porous carbon microbial carrier based on blood meal / iron complex.

[0009] Furthermore, the symbiotic engineered microbial community includes Kestrel (Sphingomonas sphingosine monocytogenes). Sphingomonas kaistensis ), Bacillus splendidus ( Paenibacillus lautus ) and Acinetobacter hemolyticus ( Acinetobacter haemolyticus The volume ratio of the three components is 1:1:1.

[0010] Furthermore, the strain number of the *Kestrel* sphingosine monocytogenes is CGMCC 1.10197; the strain number of the *Bacillus spleniculina* is CGMCC 1.10293; and the strain number of the *Acinetobacter hemolyticus* is CGMCC 1.12996.

[0011] Furthermore, the original preservation date of the *Kestrel* strain was August 18, 2009; the original preservation date of the *Bacillus scintillans* strain was November 5, 2009; and the original preservation date of the *Acinetobacter hemolyticus* strain was September 30, 2014. All three strains were purchased from the China General Microbiological Culture Collection Center. They can be purchased through commercial channels without the need for biological preservation.

[0012] Furthermore, the complex enzyme system consists of protease, amylase, lipase, and alkaline phosphatase.

[0013] A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18-24 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1:1 and then inoculated into fermentation medium at an inoculation amount of 5%-8%. Fermentation was carried out at 30℃ and 200 r / min for 48-72 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL is the fermentation broth of the symbiotic engineered microbial community; the fermentation medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and 1 g / L ammonium sulfate, with the pH adjusted to around 7.0.

[0014] (2) Dissolve protease, amylase, lipase and alkaline phosphatase in phosphate buffer with pH=7.0, mix them in the ratio of enzyme activity units 1:1:1:0.5, and prepare a compound enzyme solution. (3) Mix the above-mentioned symbiotic engineered microbial fermentation broth and compound enzyme solution at a volume ratio of 7:1, and add 1% of the total mass of the mixture as an anti-stress protectant to obtain the liquid core functional unit. (4) Preparation of low-cost porous carbon microbial carriers based on blood meal / iron complex: a. Prepare blood powder from farmed animal waste, disperse it in water, and stir to form a uniform suspension A; dissolve ferric chloride in water to prepare solution B; dissolve tannic acid in water to form tannic acid solution C; dissolve polyethylene glycol in water to form polyethylene glycol solution D; b. While stirring, slowly pour solutions B, C and D into suspension A and continue stirring for 2-4 hours. Observe that the solution gradually forms a dark flocculent precipitate or gel. This process is due to the complexation and cross-linking of iron ions with proteins and tannic acid in blood meal. c. Allow the mixed slurry to settle directly or filter it simply, collect the precipitate to obtain the precursor; d. Place the precursor powder in a ceramic boat and heat it to 500-700℃ at a rate of 3-5℃ / min under an inert atmosphere, and hold it for 1-2 hours; after cooling, black porous carbon material is obtained, which is then crushed and sieved to obtain porous carrier particles with a particle size of 0.1-0.5mm. (5) The prepared porous carbon carrier is subjected to uniform adsorption under negative pressure according to the mass ratio of carrier: liquid core functional unit = 3-5:1, and then dried at low temperature and ventilation at 25-32℃ until the water content is less than 10% to obtain the final bacterial agent product.

[0015] Furthermore, the stress protectant includes proline, betaine, and vitamin C in a mass ratio of 2:2:1.

[0016] Furthermore, in step a, the animal waste blood includes one or more of pig blood, chicken blood, and duck blood; the solid-liquid ratio of blood powder to water is 1g:20mL; the solid-liquid ratio of ferric chloride to water is 1g:10mL; the solid-liquid ratio of tannic acid to water is 1g:25mL; and the mass concentration of polyethylene glycol solution D is 5%.

[0017] Furthermore, in step b, the volume ratio of solution B, solution C, solution D and suspension A is 1:1:1:4.

[0018] Furthermore, in step d, the inert atmosphere is nitrogen or argon.

[0019] The method of using the microbial agent in this application is as follows: To ensure the bacterial agent operates at its optimal activity, it is recommended to perform a simple activation before use: Preparation of activation solution: Use the untreated or diluted aquaculture wastewater, add 1%-2% brown sugar or glucose as a carbon source for rapid start-up to obtain the activation solution.

[0020] Activation procedure: Mix this product at a ratio of 1 part bacterial agent to 10-20 parts activation solution (weight-volume ratio, g:L), place at room temperature (25-35℃), and gently aerate or stir for 24-48 hours.

[0021] Observation: The activation solution gradually becomes turbid, and a biofilm forms on the carrier surface, indicating that the microorganisms have been activated and proliferated. After activation, the entire system (including the carrier and the liquid) can be put into the treatment system.

[0022] On-site application methods: Dosage: Initial addition (biofilm formation start-up): The recommended dosage of microbial agent is 1.0-1.5 grams per cubic meter of wastewater.

[0023] Routine replenishment and maintenance: After the system is running stably, replenish once a month, with the replenishment amount being 10%-20% of the initial dosage, to maintain the population dominance of microorganisms in the system. If the water quality fluctuates greatly or is subjected to shocks, the replenishment amount and frequency can be increased as appropriate.

[0024] Operating steps: Evenly distribute: Evenly distribute the required amount of bacterial agent system (after activation) near the inlet of the sewage tank or aeration equipment so that the bacterial agent can be quickly dispersed throughout the water body.

[0025] Oxygenation Guarantee: The core functional bacteria in this bacterial agent are aerobic or facultative anaerobic bacteria, so it is essential to ensure sufficient dissolved oxygen (DO>2.0 mg / L) in the water. Turn on the aerator, agitator, or utilize natural drop to increase oxygenation.

[0026] Effluent monitoring: After 5-10 days of purification treatment, test the COD, ammonia nitrogen, total phosphorus and other indicators at the effluent outlet. A significant improvement in treatment effect can generally be observed within 5-10 days.

[0027] Compared with the prior art, the present invention has the following significant advantages: (1) This invention creatively utilizes blood meal derived from livestock waste to prepare a porous carbon carrier. This carrier not only has a high specific surface area and rich pore structure, providing an ideal attachment habitat for microorganisms; its surface is rich in metal elements such as iron and functional groups, which can preferentially adsorb antibiotic molecules and some heavy metal ions in sewage, just like building a "protective fortress" for functional microorganisms, significantly reducing the direct stress of toxic substances on microorganisms, and greatly improving the environmental resistance and long-term stability of the bacterial agent. (2) The specific symbiotic engineered microbial community and the complex enzyme system screened in this invention work synergistically. The complex enzyme system first rapidly hydrolyzes macromolecular organic matter (proteins, starches, fats, etc.) in wastewater, providing easily degradable carbon and nitrogen sources for the functional microbial community; then, the functional microbial community uses these substrates for growth and metabolism, efficiently completing the nitrification / denitrification removal of ammonia nitrogen and the conversion and absorption of phosphorus. In particular, the three selected strains have a unique synergistic degradation ability against typical aquaculture antibiotics such as sulfonamides, achieving efficient removal of characteristic pollutants; (3) The essence of this invention lies in the system-level cooperation between porous carriers and functional microorganisms. The carrier is responsible for "adsorption and protection", and the microorganisms are responsible for "degradation and purification". The two complement each other. This design enables the bacterial agent to quickly form a highly active biofilm system after being added to the wastewater, which can efficiently, synergistically and stably purify the complex components of aquaculture wastewater, especially solving the industry problem that biological treatment systems are prone to collapse in the presence of antibiotics; (4) In summary, this invention uses animal blood from livestock farms as the main raw material, realizing the resource utilization of waste and significantly reducing the cost of the carrier. The entire preparation process is green and environmentally friendly, providing an economical and feasible new strategy of "treating waste with waste" for livestock wastewater treatment. Attached Figure Description

[0028] Figure 1 The diagram shows the antagonistic experimental results of Kestrel sphingosine monocytogenes, Bacillus splenium, and Acinetobacter hemolyticus in this invention, where (a) is Kestrel sphingosine monocytogenes, (b) is Bacillus splenium, and (c) is Acinetobacter hemolyticus. Figure 2 These are scanning electron microscope images of the porous carbon support of the present invention, wherein (a) is a surface structure diagram and (bc) is an internal structure diagram; Figure 3 The antibiotic degradation effect of different strain combinations under heavy metal stress. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0030] Example 1 A functional microbial agent for purifying aquaculture wastewater comprises a core functional unit and a porous carrier; the mass ratio of the porous carrier to the core functional unit is 5:1; the core functional unit contains a symbiotic engineered microbial community and a complex enzyme system, with a volume ratio of 7:1; the porous carrier is a low-cost porous carbon microbial carrier based on blood meal / iron complex.

[0031] The symbiotic engineered microbial community includes Kestrel (Sphingomonas ketosphingosine monocytogenes) Sphingomonas kaistensis ), Bacillus splendidus ( Paenibacillus lautus ) and Acinetobacter hemolyticus ( Acinetobacter haemolyticus The volume ratio of the three components is 1:1:1.

[0032] The strain number of *Sphingomonas ketosporum* is CGMCC 1.10197; the strain number of *Bacillus spleniculina* is CGMCC 1.10293; and the strain number of *Acinetobacter hemolyticus* is CGMCC 1.12996.

[0033] The original preservation date of the *Kestrel* strain was August 18, 2009; the original preservation date of the *Bacillus scintillans* strain was November 5, 2009; and the original preservation date of the *Acinetobacter hemolyticus* strain was September 30, 2014. All three strains were purchased from the China General Microbiological Culture Collection Center. They can be purchased through commercial channels without the need for biological preservation.

[0034] Sphingomonas ketosporum, Bacillus spleniculina, and Acinetobacter hemolyticus were streaked in pairs on solid LB medium to verify whether there was antagonistic interaction between the bacteria. After 24 hours of incubation, no obvious inhibition zone was observed at the interface between the strains, indicating that there was no antagonistic interaction among the three and that they had a basis for coexistence. The antagonistic experiment diagram is shown below. Figure 1 As shown.

[0035] The complex enzyme system consists of protease, amylase, lipase and alkaline phosphatase.

[0036] A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1:1 and then inoculated into fermentation medium at an inoculation amount of 5%. Fermentation was carried out at 30℃ and 200 r / min for 48 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL is the fermentation broth of the symbiotic engineered microbial community; the fermentation medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and 1 g / L ammonium sulfate, with the pH adjusted to around 7.0. (2) Dissolve protease, amylase, lipase and alkaline phosphatase in phosphate buffer with pH=7.0, mix them in the ratio of enzyme activity units 1:1:1:0.5, and prepare a compound enzyme solution. (3) Mix the above-mentioned symbiotic engineered microbial fermentation broth and compound enzyme solution at a volume ratio of 7:1, and add 1% of the total mass of the mixture as an anti-stress protectant to obtain the liquid core functional unit. (4) Preparation of low-cost porous carbon microbial carriers based on blood meal / iron complex: a. Prepare blood powder from farmed animal waste, disperse it in water, and stir to form a uniform suspension A; dissolve ferric chloride in water to prepare solution B; dissolve tannic acid in water to form tannic acid solution C; dissolve polyethylene glycol in water to form polyethylene glycol solution D; b. While stirring, slowly pour solutions B, C and D into suspension A and continue stirring for 2 hours. Observe that the solution gradually forms a dark flocculent precipitate or gel. This process is due to the complexation and cross-linking of iron ions with proteins and tannic acid in blood meal. c. Allow the mixed slurry to settle directly or filter it simply, collect the precipitate to obtain the precursor; d. Place the precursor powder in a ceramic boat and heat it to 500-700℃ at a rate of 3-5℃ / min under an inert atmosphere (to reduce the pyrolysis temperature and save energy), and hold for 1 hour; after cooling, a black porous carbon material is obtained, which is then pulverized and sieved to obtain porous carrier particles with a particle size of 0.1-0.5mm; its surface morphology is observed using a scanning electron microscope. Figure 2 a) and internal morphology ( Figure 2 bc), such as Figure 2 As shown.

[0037] (5) The prepared porous carbon carrier is uniformly adsorbed under negative pressure according to the mass ratio of carrier: liquid core functional unit = 3:1, and then dried at low temperature and ventilation at 25-32℃ until the water content is less than 10% to obtain the final bacterial agent product.

[0038] The stress-protective agents include proline, betaine, and vitamin C, with a mass ratio of 2:2:1.

[0039] In step a, the animal waste blood includes one or more types of pig blood, the solid-liquid ratio of blood powder to water is 1g:20mL, the solid-liquid ratio of ferric chloride to water is 1g:10mL, the solid-liquid ratio of tannic acid to water is 1g:25mL, and the mass concentration of polyethylene glycol solution D is 5%.

[0040] In step b, the volume ratio of solution B, solution C, solution D and suspension A is 1:1:1:4.

[0041] In step d, the inert atmosphere is nitrogen.

[0042] Example 2 A functional microbial agent for purifying aquaculture wastewater comprises a core functional unit and a porous carrier; the mass ratio of the porous carrier to the core functional unit is 3:1; the core functional unit contains a symbiotic engineered microbial community and a complex enzyme system, with a volume ratio of 7:1; the porous carrier is a low-cost porous carbon microbial carrier based on blood meal / iron complex.

[0043] The symbiotic engineered microbial community includes Kestrel (Sphingomonas ketosphingosine monocytogenes) Sphingomonas kaistensis ), Bacillus splendidus ( Paenibacillus lautus ) and Acinetobacter hemolyticus ( Acinetobacter haemolyticus The volume ratio of the three components is 1:1:1.

[0044] The strain number of *Sphingomonas ketosporum* is CGMCC 1.10197; the strain number of *Bacillus spleniculina* is CGMCC 1.10293; and the strain number of *Acinetobacter hemolyticus* is CGMCC 1.12996.

[0045] The original preservation date of the *Kestrel* strain was August 18, 2009; the original preservation date of the *Bacillus scintillans* strain was November 5, 2009; and the original preservation date of the *Acinetobacter hemolyticus* strain was September 30, 2014. All three strains were purchased from the China General Microbiological Culture Collection Center. They can be purchased through commercial channels without the need for biological preservation.

[0046] The complex enzyme system consists of protease, amylase, lipase and alkaline phosphatase.

[0047] A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 24 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1:1 and then inoculated into fermentation medium at an inoculation amount of 8%. Fermentation was carried out at 30℃ and 200 r / min for 72 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms; (2) Dissolve protease, amylase, lipase and alkaline phosphatase in phosphate buffer with pH=7.0, mix them in the ratio of enzyme activity units 1:1:1:0.5, and prepare a compound enzyme solution. (3) Mix the above-mentioned symbiotic engineered microbial fermentation broth and compound enzyme solution at a volume ratio of 7:1, and add 1% of the total mass of the mixture as an anti-stress protectant to obtain the liquid core functional unit. (4) Preparation of low-cost porous carbon microbial carriers based on blood meal / iron complex: a. Prepare blood powder from farmed animal waste, disperse it in water, and stir to form a uniform suspension A; dissolve ferric chloride in water to prepare solution B; dissolve tannic acid in water to form tannic acid solution C; dissolve polyethylene glycol in water to form polyethylene glycol solution D; b. While stirring, slowly pour solutions B, C and D into suspension A and continue stirring for 4 hours. Observe that the solution gradually forms a dark flocculent precipitate or gel. This process is due to the complexation and cross-linking of iron ions with proteins and tannic acid in blood meal. c. Allow the mixed slurry to settle directly or filter it simply, collect the precipitate to obtain the precursor; d. Place the precursor powder in a ceramic boat and heat it to 500-700℃ at a rate of 3-5℃ / min under an inert atmosphere (to reduce the pyrolysis temperature and save energy), and hold it at that temperature for 2 hours. After cooling, black porous carbon material is obtained. Crush and sieve it to obtain porous carrier particles with a particle size of 0.1-0.5mm. (5) The prepared porous carbon carrier is subjected to uniform adsorption under negative pressure at a mass ratio of carrier: liquid core functional unit = 5:1, and then dried at low temperature and ventilation at 25-32℃ until the water content is less than 10% to obtain the final bacterial agent product.

[0048] The stress-protective agents include proline, betaine, and vitamin C, with a mass ratio of 2:2:1.

[0049] In step a, the animal waste blood includes chicken blood, and the solid-liquid ratio of blood powder to water is 1g:20mL; the solid-liquid ratio of ferric chloride to water is 1g:10mL; the solid-liquid ratio of tannic acid to water is 1g:25mL; and the mass concentration of polyethylene glycol solution D is 5%.

[0050] In step b, the volume ratio of solution B, solution C, solution D and suspension A is 1:1:1:4.

[0051] In step d, the inert atmosphere is argon.

[0052] Comparative Example 1 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, only Kestrel sphingosine monocytogenes is used in the symbiotic engineered microbial fermentation broth, i.e., in preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingosine monocytogenes* was inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain the seed culture of the strain; then it was inoculated into fermentation medium at an inoculation amount of 5% and fermented at 30℃ and 200 r / min for 48 h to obtain a high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0053] Comparative Example 2 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, in the symbiotic engineered microbial fermentation broth, only Bacillus Brilliantus is used, that is, in preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) Inoculate *Bacillus splendens* into LB liquid medium and culture with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain the seed culture of the strain; then inoculate it into fermentation medium at an inoculation amount of 5% and ferment at 30℃ and 200 r / min for 48 h to obtain a high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0054] Comparative Example 3 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, in the symbiotic engineered microbial fermentation broth, only Acinetobacter hemolyticus is used, that is, in preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) Acinetobacter hemolyticus was inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain the seed culture of the strain; then it was inoculated into fermentation medium at an inoculation amount of 5% and fermented at 30℃ and 200 r / min for 48 h to obtain a high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0055] Comparative Example 4 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, in the symbiotic engineered microbial fermentation broth, only Kestrel-Sphingomonas and Bacillus spleniculatus are used. In preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas ketosporine* and *Bacillus splendens* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1 and then inoculated into fermentation medium at an inoculation amount of 5%. Fermentation was carried out at 30℃ and 200 r / min for 48 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0056] Comparative Example 5 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, in the symbiotic engineered microbial fermentation broth, only Kestrel sphingosine monocytogenes and Acinetobacter hemolyticus are used. In preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas kastrozole* and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1 and then inoculated into fermentation medium at an inoculation amount of 5%. Fermentation was carried out at 30℃ and 200 r / min for 48 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0057] Comparative Example 6 In this comparative example, except for the change in the core microbial composition, the other raw materials and preparation processes are the same as in Example 1. That is, in the symbiotic engineered microbial fermentation broth, only Bacillus scintillans and Acinetobacter hemolyticus are used. In preparation step (1): A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Bacillus spleniculina* and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1 and then inoculated into fermentation medium at an inoculation amount of 5%. Fermentation was carried out at 30℃ and 200 r / min for 48 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms.

[0058] Comparative Example 7 In this comparative example, except for the use of ordinary biochar, all other raw materials and preparation processes are the same as in Example 1, namely: A method for manufacturing a functional microbial agent for purifying aquaculture wastewater includes the following preparation steps: (1) *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1:1 and then inoculated into fermentation medium at an inoculation amount of 5%. Fermentation was carried out at 30℃ and 200 r / min for 48 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL is the fermentation broth of the symbiotic engineered microbial community; the fermentation medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and 1 g / L ammonium sulfate, with the pH adjusted to around 7.0. (2) Dissolve protease, amylase, lipase and alkaline phosphatase in phosphate buffer with pH=7.0, mix them in the ratio of enzyme activity units 1:1:1:0.5, and prepare a compound enzyme solution. (3) Mix the above-mentioned symbiotic engineered microbial fermentation broth and compound enzyme solution at a volume ratio of 7:1, and add 1% of the total mass of the mixture as an anti-stress protectant to obtain the liquid core functional unit. (4) The porous carbon carrier is uniformly adsorbed under negative pressure according to the mass ratio of carrier to liquid core functional unit = 5:1, and then dried at low temperature and ventilation at 25-32℃ until the moisture content is less than 10% to obtain the final bacterial agent product.

[0059] The porous carbon carrier is the biochar product in reference application number: CN2014104705961, and the activated carbon carrier is coal-based columnar activated carbon, which is a solid cylindrical activated carbon made by shaping 6-mesh coal-based activated carbon.

[0060] Performance testing Tests on heavy metal tolerance and antibiotic degradation ability of different strains: *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB medium and cultured at 30°C with shaking for 48 h. The bacterial suspension was centrifuged at 8000 r / min for 5 min, and the bacterial pellet was collected, washed twice with sterile inorganic salt solution, and the OD was adjusted. 600 The concentration was 1.5, resulting in three bacterial suspensions.

[0061] The bacterial suspensions were mixed according to the volume ratios in Table 1 to obtain a composite bacterial suspension. The degradation effects of different strain combinations on antibiotics were tested.

[0062] Table 1. Composition of bacterial suspension (v:v) In 100 mL of inorganic salt culture medium containing 100 mg / L ciprofloxacin, 50 mg / L sulfamethoxazole, and 30 mg / L chloramphenicol, an equal volume of 3 mL of a compound bacterial suspension was inoculated. The mixture was then cultured at 30℃ and 200 r / min for 24 h with shaking. The antibiotic content in the samples was determined by HPLC, and the degradation rate of different antibiotics by the strains was calculated as follows: Degradation rate (%) = (C0 - C) / C0 × 100%; where C0 is the concentration of antibiotics in the sample at 0 h, and C is the concentration of antibiotics in the sample at the end of culture. Each experimental group was repeated three times, and the results were averaged.

[0063] Basic inorganic salt liquid medium (MSM): (NH4)2SO4 1.5 g, KH2PO4 0.5 g, K2HPO4 1.5 g, MgSO4 0.2 g, NaCl 0.5 g, add water to 1L, and adjust pH to 7.0.

[0064] Table 2. Degradation effect of bacterial suspensions from different experimental groups on antibiotics. Heavy metal tolerance: The heavy metal ions used were provided by four compounds: CuCl2, ZnSO4·7H2O, K2Cr2O7, and Pb(NO3)2. Zn²⁺, Cu²⁺, Pb²⁺, and Cr(VI) were added to a basic inorganic salt medium at equal concentrations to prepare mixed solutions with a total concentration of 200 mg / L. Taking ciprofloxacin degradation as an example, the inoculum size was 3%. After 24 hours of shaking culture, the degradation rate of sulfamethoxazole was measured. Each group had three replicates, and the average value was taken. The results are as follows: Figure 3 As shown.

[0065] From Table 2 and Figure 3 We can see that the combined bacterial flora in Example 1 showed significantly better degradation effects on antibiotics than the other comparative examples. This indicates that the synergistic effect of the three strains can significantly improve degradation efficiency, while the absence of any one strain weakens the effect. The combination of *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* exhibited high activity in degrading ciprofloxacin, sulfamethoxazole, and chloramphenicol, with degradation rates reaching 80.2%, 78.6%, and 83.7%, respectively. In contrast, the degradation ability of a single strain or a combination of two strains was significantly reduced, indicating a functional complementarity and synergistic effect among the three.

[0066] Further analysis of the heavy metal tolerance experiment results revealed that the composite bacterial community in Example 1 maintained a high antibiotic degradation capacity even under high concentrations of heavy metals. This indicates that the composite system composed of the three strains not only possesses strong environmental stress tolerance but also maintains stable degradation activity under conditions of heavy metal coexistence.

[0067] Real-world application testing: Wastewater from the experimental farm: Wastewater from a pig farm with a scale of 2000 pigs, with a daily wastewater discharge of 60-70 cubic meters. 3 It contains high concentrations of oxytetracycline, sulfonamide antibiotics, and heavy metal residues.

[0068] On-site application methods: Dosage: Initial addition (biofilm formation start-up): The recommended dosage of microbial agent is 1.0 g / m³ of wastewater.

[0069] Operating steps: Evenly distribute: Evenly distribute the required amount of bacterial agent system (after activation) near the inlet of the sewage tank or aeration equipment so that the bacterial agent can be quickly dispersed throughout the water body.

[0070] Oxygenation Guarantee: The core functional bacteria in this bacterial agent are aerobic or facultative anaerobic bacteria, so it is essential to ensure sufficient dissolved oxygen (DO>2.0 mg / L) in the water. Turn on the aerator, agitator, or utilize natural drop to increase oxygenation.

[0071] Effluent monitoring: Five days after purification treatment, COD, ammonia nitrogen, total phosphorus, and other indicators were tested at the effluent outlet. The removal rate was calculated by measuring various indicators of the water body before and after treatment according to national standard methods. The testing methods are as follows: COD, BOD: GB / T 11914-89 Potassium dichromate method, GB11914-89, Dilution and inoculation method.

[0072] Ammonia nitrogen and total phosphorus: "Technical Specifications for Environmental Monitoring" and "Methods for Monitoring and Analysis of Water and Wastewater".

[0073] The indicators for determining metal elements in wastewater include Cu, Zn, Mn, Cd, and Cr. 6+ And As.

[0074] Determination methods: Cu was determined by sodium diethyldithiocarbamate spectrophotometry; Zn by dithizone spectrophotometry; Mn by flame atomic absorption spectrophotometry; Cd by dithizone spectrophotometry; Cr... 6+ The diphenylcarbazide spectrophotometric method was used; the arsenic spot method was used for As.

[0075] The test results are shown in Table 3: Table 3. Test Results Table 4. Test Results The test results in Tables 3 and 4 show that Examples 1 and 2 exhibit significant advantages in treating aquaculture wastewater. In Examples 1 and 2, the main indicators of the wastewater, such as COD, ammonia nitrogen, total phosphorus, and BOD, all decreased significantly. In particular, the concentrations of antibiotic residues, such as sulfamethoxazole and oxytetracycline, were effectively reduced to below the detection limit, indicating that the composite microbial community has a highly efficient degradation capacity for these pollutants. Simultaneously, heavy metals such as Cu, Zn, Mn, Cd, and Cr were also significantly reduced. 6+ The removal of heavy metal ions (As) was also very significant, with concentrations far lower than those in the original wastewater, achieving a relatively ideal purification effect. This is mainly attributed to the adsorption of heavy metal ions by the porous carrier, as well as the tolerance and transformation of the microorganisms themselves.

[0076] In contrast, Comparative Examples 1 to 7 performed significantly worse. Comparative Examples 1 to 3, each using a single strain, showed a significant decrease in their ability to remove antibiotics and heavy metals, indicating that single strains are insufficient to handle complex contaminated environments. Comparative Examples 4 to 6, employing a combination of two strains, improved the treatment effect to some extent, but still could not compare with Example 1, further validating the importance of the synergistic effect of the three strains. Comparative Example 7, using ordinary biochar as a carrier, resulted in an unsatisfactory overall treatment effect, particularly poor performance in the removal rates of antibiotics and heavy metals, demonstrating that the choice of carrier material has a significant impact on the performance of the bacterial agent.

[0077] Comprehensive analysis shows that the adaptability and degradation capacity of the composite microbial system of this invention under complex pollutant environments have been fully verified. It not only efficiently degrades various antibiotic residues but also maintains stable performance under high concentrations of heavy metals, providing a feasible technical solution for aquaculture wastewater treatment. Furthermore, practical application tests have revealed that this microbial agent system is easy to operate, highly adaptable, and can be quickly started and effective on-site, demonstrating high practical value. Future research can further optimize the microbial agent formulation and process parameters to improve its applicability under different water quality conditions.

[0078] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A functional microbial agent for purifying aquaculture wastewater, characterized in that, It consists of a core functional unit and a porous carrier; the mass ratio of the porous carrier to the core functional unit is 3:1 to 5:1; the core functional unit contains a symbiotic engineered microbial community and a complex enzyme system, with a volume ratio of 7:1; the porous carrier is a low-cost porous carbon microbial carrier based on blood meal / iron complex.

2. The functional microbial agent for purifying aquaculture wastewater according to claim 1, characterized in that, The symbiotic engineered microbial community includes Kestrel-Sphingomonas (Kestrel-Sphingomonas) Sphingomonas kaistensis ), Bacillus splendidus ( Paenibacillus lautus ) and Acinetobacter hemolyticus ( Acinetobacter haemolyticus The volume ratio of the three components is 1:1:

1.

3. The functional microbial agent for purifying aquaculture wastewater according to claim 1, characterized in that, The strain number of *Sphingomonas ketosporum* is CGMCC 1.10197; the strain number of *Bacillus spleniculina* is CGMCC 1.10293; and the strain number of *Acinetobacter hemolyticus* is CGMCC 1.12996.

4. The functional microbial agent for purifying aquaculture wastewater according to claim 1, characterized in that, The complex enzyme system consists of protease, amylase, lipase and alkaline phosphatase.

5. A method for manufacturing a functional microbial agent for purifying aquaculture wastewater as described in any one of claims 1-4, characterized in that, The preparation steps include the following: (1) *Sphingomonas ketosporine*, *Bacillus spleniculina*, and *Acinetobacter hemolyticus* were inoculated into LB liquid medium and cultured with shaking at 30-32℃ and 180-200 r / min for 18-24 h to obtain seed culture of each strain. The seed culture of each strain was mixed at a volume ratio of 1:1:1 and then inoculated into fermentation medium at an inoculation amount of 5%-8%. Fermentation was carried out at 30℃ and 200 r / min for 48-72 h to obtain high-density fermentation broth with a viable count >10. 9 CFU / mL refers to the fermentation broth of symbiotic engineered microorganisms; (2) Dissolve protease, amylase, lipase and alkaline phosphatase in phosphate buffer with pH=7.0, mix them in the ratio of enzyme activity units 1:1:1:0.5, and prepare a compound enzyme solution. (3) Mix the above-mentioned symbiotic engineered microbial fermentation broth and compound enzyme solution at a volume ratio of 7:1, and add 1% of the total mass of the mixture as an anti-stress protectant to obtain the liquid core functional unit. (4) Preparation of low-cost porous carbon microbial carriers based on blood meal / iron complex: a. Prepare blood powder from farmed animal waste, disperse it in water, and stir to form a uniform suspension A; dissolve ferric chloride in water to prepare solution B; dissolve tannic acid in water to form tannic acid solution C; dissolve polyethylene glycol in water to form polyethylene glycol solution D; b. While stirring, slowly pour solutions B, C and D into suspension A and continue stirring for 2-4 hours. Observe that the solution gradually forms a dark flocculent precipitate or gel. This process is due to the complexation and cross-linking of iron ions with proteins and tannic acid in blood meal. c. Allow the mixed slurry to settle directly or filter it simply, collect the precipitate to obtain the precursor; d. Place the precursor powder in a ceramic boat and heat it to 500-700℃ at a rate of 3-5℃ / min under an inert atmosphere, and hold it for 1-2 hours; after cooling, black porous carbon material is obtained, which is then crushed and sieved to obtain porous carrier particles with a particle size of 0.1-0.5mm. (5) The prepared porous carbon carrier is subjected to uniform adsorption under negative pressure according to the mass ratio of carrier: liquid core functional unit = 3-5:1, and then dried at low temperature and ventilation at 25-32℃ until the water content is less than 10% to obtain the final bacterial agent product.

6. The method for manufacturing the functional microbial agent for purifying aquaculture wastewater according to claim 5, characterized in that, The stress-protective agents include proline, betaine, and vitamin C, with a mass ratio of 2:2:

1.

7. The method for manufacturing the functional microbial agent for purifying aquaculture wastewater according to claim 5, characterized in that, In step a, the animal waste blood includes one or more of pig blood, chicken blood, and duck blood. The solid-liquid ratio of blood powder to water is 1g:20mL; the solid-liquid ratio of ferric chloride to water is 1g:10mL; the solid-liquid ratio of tannic acid to water is 1g:25mL; and the mass concentration of polyethylene glycol solution D is 5%.

8. The method for manufacturing the functional microbial agent for purifying aquaculture wastewater according to claim 5, characterized in that, In step b, the volume ratio of solution B, solution C, solution D and suspension A is 1:1:1:

4.

9. The method for manufacturing the functional microbial agent for purifying aquaculture wastewater according to claim 5, characterized in that, In step d, the inert atmosphere is nitrogen or argon.

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