Phycomycete co-culture system for treating aquaculture wastewater as well as preparation method and application of phycomycete co-culture system

Abstract

The invention relates to the technical field of wastewater treatment, and particularly discloses a phycomycete co-culture system for treating aquaculture wastewater as well as a preparation method and application of the phycomycete co-culture system. The phycomycete co-culture system comprises a quinolone degrading bacterial agent, resistant microalgae, a signal inducer, a porous fixed carrier, a nutrition stimulant and a domestication culture medium; the quinolone degrading bacterial agent is a compound bacterial agent of pseudomonas and acinetobacter, and the resistant microalgae is a compound algae species of chlorella and scenedesmus quadricauda; according to the system, phycomycete symbiotic particles are prepared through gradient domestication culture and immobilization; when in use, the system is put into wastewater, and treatment is carried out under the conditions of illumination and intermittent aeration; through the synergistic effect of functional bacteria and algae compounding, signal induction, composite carrier fixation and nutrition maintenance, the tolerance and degradation efficiency of the system to quinolone antibiotics are remarkably improved, meanwhile, efficient removal of ammonia nitrogen, total phosphorus and COD is achieved, and the algae and algae co-culture system is easy to recycle and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, specifically to an algae-bacteria co-culture system for treating aquaculture wastewater, its preparation method, and its application. Background Technology

[0002] With the widespread adoption of intensive aquaculture, its wastewater has become a major source of pollution. This wastewater typically contains high concentrations of ammonia nitrogen, phosphates, and organic matter, and commonly retains quinolone antibiotics. Direct discharge of such wastewater can easily lead to eutrophication of receiving water bodies. More seriously, antibiotics and their metabolites may promote the spread of resistance genes in the environment, posing a potential threat to ecological security and human health.

[0003] Currently, the treatment of this type of wastewater mainly relies on biological processes such as activated sludge. However, these methods have limited efficiency in removing nitrogen and phosphorus, and the microorganisms in the system are easily inhibited by antibiotics, leading to unstable treatment results or even system collapse. In recent years, wastewater treatment technologies based on microalgae have attracted attention due to their ability to efficiently absorb nitrogen and phosphorus and fix carbon dioxide. In particular, the co-culture system of microalgae and bacteria can theoretically achieve mutualistic symbiosis through gas exchange, thereby improving pollutant removal efficiency.

[0004] However, when applied to the treatment of aquaculture wastewater containing antibiotics, existing algae-bacteria co-culture technology faces three prominent challenges: First, common bacterial strains and algae species have poor tolerance to quinolone antibiotics, making it difficult to establish a stable symbiotic system under high antibiotic pressure; second, most studies use simple mixed culture, resulting in low efficiency of physical contact and material transfer between algae and bacteria, and a lack of effective means to regulate their symbiotic relationship, leading to insufficient ability to synergistically degrade antibiotics; and third, the biomass produced by the system is difficult to separate efficiently, resulting in high costs for subsequent treatment or resource utilization. Summary of the Invention

[0005] To address the problems of poor tolerance of existing algae-bacteria co-culture systems to quinolone antibiotics in aquaculture wastewater, low synergistic efficiency of bacteria and algae, difficulty in biomass recovery, and lack of targeted degradation capabilities for characteristic pollutants such as quinolones, this invention provides an algae-bacteria co-culture system for treating aquaculture wastewater, its preparation method, and its application.

[0006] In a first aspect, the present invention provides an algae-bacterial co-culture system for treating aquaculture wastewater, the algae-bacterial co-culture system comprising the following raw materials in parts by weight: 3-5 parts of quinolone-degrading bacterial agent, 6-8 parts of resistant microalgae, 0.3-0.5 parts of signal inducer, 30-50 parts of porous immobilization carrier, 1-2 parts of nutrient stimulant, and 600-800 parts of acclimatization culture medium.

[0007] Preferably, the quinolone-degrading bacterial agent is a compound of Pseudomonas and Acinetobacter at a live bacteria ratio of 1-3:1.

[0008] Preferably, the Pseudomonas is *Pseudomonas putida* (…). Pseudomonas putida Acinetobacter is Acinetobacter calcium acetate ( Acinetobacter calcoaceticus ).

[0009] Preferably, the resistant microalgae is a compound of Chlorella and Scenedesmus tetracauda at a biomass dry weight ratio of 1-2:1.

[0010] Preferably, the Chlorella is common Chlorella ( Chlorella vulgaris ), *Scenedesmus tetracaudus* is the common *Scenedesmus tetracaudus* ( Scenedesmus quadricauda ).

[0011] Preferably, the signal inducer is composed of sodium gluconate and algal oligosaccharides in a mass ratio of 1:10-20.

[0012] Preferably, the preparation of the algal oligosaccharide includes the following steps: A1. Take Chlorella powder and resuspend it in PBS buffer at a mass ratio of 1:10-20 to prepare an algal suspension. A2. The algal suspension was subjected to ultrasonic disruption under the following conditions: ultrasonic power 200-500W, working time 2-5s, interval time 3-8s, and total duration 10-30min. A3. Add a complex enzyme to the crushed system for enzymatic hydrolysis. The complex enzyme contains cellulase, pectinase and β-glucanase. The enzymatic hydrolysis conditions are: pH 4.5-5.5, temperature 45-55℃, and time 6-12h. A4. After the enzymatic hydrolysis is complete, heat to 90-100℃ and maintain for 10-15 minutes to inactivate the enzyme. Then centrifuge at 3000-5000 rpm to remove the residue and collect the supernatant. A5. The supernatant is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa, the filtered components are collected, and the concentration is increased to a soluble solids content of 20-50% (w / v). Then, it is placed in a freeze dryer at -50°C and dried for 48-72 hours to obtain a brownish-yellow powder, which is the algal oligosaccharide.

[0013] Preferably, in step A3, the amount of the compound enzyme added accounts for 0.5-2.0% of the weight of Chlorella powder, wherein the ratio of enzyme activity units of cellulase, pectinase and β-glucanase is 2-3:1-2:1.

[0014] Preferably, the preparation of the porous immobilization carrier includes the following steps: mixing sodium alginate solution, chitosan solution and coconut shell powder activated carbon to obtain a composite gel solution; dripping the composite gel solution into calcium chloride solution to solidify it and form gel microspheres; pre-freezing and vacuum freeze-drying the gel microspheres to obtain the porous immobilization carrier.

[0015] Preferably, the preparation of the porous immobilization carrier includes the following specific steps: B1. Mix sodium alginate solution with a mass concentration of 2-4%, chitosan solution with a mass concentration of 1-2%, and coconut shell powder activated carbon with a particle size of 150-200 mesh at a volume mass ratio of 2-4 mL: 1 mL: 0.5-2 g to obtain a composite gel solution. B2. The composite gel solution is dropped into a 2-5% calcium chloride solution at a rate of 0.5-1 mL / s to solidify (the volume ratio of the composite gel solution to the calcium chloride solution is 1:10-20) to form gel microspheres. B3. Pre-freeze the gel microspheres in an environment of -20℃ to -40℃ for 2-6 hours, and then freeze-dry them at -50℃ and a vacuum degree of 8-10Pa for 24-48 hours to obtain a porous fixed carrier.

[0016] Preferably, the nutrient stimulant is an ammonium molybdate-vitamin B12 aqueous solution, wherein the concentration of ammonium molybdate is 0.05-0.1 mg / L and the concentration of vitamin B12 is 0.01-0.05 mg / L.

[0017] Preferably, the acclimatization culture medium comprises: glucose 10-12 g / L, ammonium chloride 1-1.6 g / L, potassium dihydrogen phosphate 0.4-0.6 g / L, magnesium sulfate 0.2-0.4 g / L, calcium chloride 0.1-0.3 g / L, ferrous sulfate heptahydrate 0.01-0.03 g / L, and quinolone antibiotics 5-15 μg / L; the pH is adjusted to 6.8-7.2.

[0018] Preferably, the quinolone antibiotic is selected from one or more of enrofloxacin, ciprofloxacin, levofloxacin, and norfloxacin.

[0019] Secondly, the present invention provides a method for preparing an algae-bacteria co-culture system for treating aquaculture wastewater as described above, comprising the following steps: S1. Algae-bacteria co-culture and domestication: Quinolone-degrading bacterial agent and resistant microalgae are inoculated into domestication culture medium and domestication culture is carried out with gradient concentrations to obtain algae-bacteria mixture; S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture, signal inducer and nutrient stimulant are loaded onto a porous immobilization carrier to obtain algae-bacteria symbiotic particles, which are then dried to obtain algae-bacteria co-culture system.

[0020] Preferably, the algae-bacteria co-culture and domestication process in step S1 specifically includes: S1.1 Co-culture of bacteria and algae and gradient acclimatization: Throughout the acclimatization process, the dissolved oxygen (DO) concentration was controlled at 2.5-3.5 mg / L, the light intensity at 800-1200 lux, and the light-dark cycle at 12 hours of light and 12 hours of darkness. Quinolone-degrading bacteria and resistant microalgae were inoculated into the acclimatization medium at a ratio of total viable bacteria to total algal cells of 10:1, with the initial concentration controlled at 1×10⁻⁶. 7 CFU / mL and 1×10 6 cells / mL, then start the quinolone antibiotic concentration from 5-15 μg / L, and increase it by 10-20 μg / L every 3-5 days until it reaches 150 μg / L; S1.2 Collection of bacterial-algae mixture: After acclimatization, collect the bacterial-algae mixture by centrifugation at 6000-10000 rpm for 10-20 min.

[0021] Preferably, the immobilization preparation process of the algae-bacteria co-culture system in step S2 specifically includes: A mixture of bacteria and algae, a signal inducer, and a nutrient stimulant were loaded onto a porous immobilization carrier. The loading conditions were 25-30℃ and 120-150 rpm for 20-28 hours of oscillation and adsorption to obtain algae-bacterial symbiotic particles. Subsequently, the particles were dried at 25-35℃ with ventilation until the moisture content was 30-40%, thus obtaining an algae-bacterial co-culture system.

[0022] Thirdly, the present invention provides an application of the algae-bacteria co-culture system described above in the treatment of aquaculture wastewater, wherein the method for treating aquaculture wastewater includes the following steps: Add the algae-bacteria co-culture system to the aquaculture wastewater to be treated at a mass-volume ratio of 3-5g:20L; after batch treatment for 48-96 hours under light and intermittent aeration conditions, filter and recover the algae-bacteria co-culture system.

[0023] Preferably, the lighting conditions include: a light intensity of 2000-5000 lux, and a light-dark cycle of 12 hours of light and 12 hours of darkness; the aeration conditions include: a cycle of 1-2 hours of aeration followed by 1-1.5 hours of settling.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention constructs a targeted degradation functional system with clear division of labor and synergistic stress resistance. Each biological component is complementary and indispensable. This invention is not a simple mixture of bacteria and algae, but rather a highly efficient degradation chain formed by the specific combination of *Pseudomonas putida* (which mainly targets the initial ring-opening of organic matter and quinolones) and *Acinetobacter calcitrinum* (which is good at utilizing intermediate products and is resistant to environmental pressure). At the same time, the combination of *Chlorella vulgaris* (which grows quickly and secretes polysaccharides) and *Scenedesmus tetracaulis* (which has a stable structure and strong tolerance) not only provides stable photosynthetic oxygen production and carbon sink, but also the extracellular polysaccharides secreted by them can form a synergistic protective matrix with the metabolic products of the degrading bacteria, further enhancing the stress resistance of the entire system, while providing a continuous carbon source for the bacterial community.

[0025] (2) This invention introduces a signal inducer, realizing a leap from passive physical coexistence to active chemical and biological regulation of algal symbiosis. This invention combines sodium gluconate (bacterial quorum sensing signal) with homologous algal oligosaccharides (microalgal recognition signal). This inducer simultaneously activates the bacterial quorum sensing system and the surface recognition and response of microalgae, actively guiding and stabilizing the symbiotic assembly of bacteria and algae at the carrier interface.

[0026] (3) This invention uses a high-performance porous immobilization carrier adapted to bacterial-algae symbiosis: a sodium alginate-chitosan-biochar composite carrier prepared by a specific ratio and freeze-drying process, which has an internal micron-level pore structure and a surface millimeter-level network hierarchical structure. This structure provides the most suitable colonization microenvironment for both bacteria and algae, realizing the spatially ordered distribution and close symbiosis of bacteria and algae; the carrier also has good biocompatibility and mechanical strength, which ensures the long-term stable operation of the system.

[0027] (4) This invention designs a nutrient stimulant to overcome the metabolic bottleneck under antibiotic stress and ensure the continuous and efficient operation of the system. This invention found that in antibiotic-stressed wastewater, the bottleneck of the bacterial-algae co-culture system is often not the lack of basic nutrients (nitrogen and phosphorus), but the metabolic decline caused by the depletion of key trace elements and coenzymes. To this end, this invention specifically formulated a nutrient stimulant with ammonium molybdate and vitamin B12 as its core. Molybdenum is an essential element for key enzyme cofactors such as nitrate reductase, which significantly enhances the denitrification metabolic capacity of the system; vitamin B12, as a coenzyme involved in important reactions such as molecular rearrangement and methyl transfer, effectively maintains the activity of carbon skeleton reconstruction, stress-resistance substance synthesis, and quinolone co-metabolism pathways of bacteria and algae under quinolone antibiotic stress.

[0028] (5) This invention achieves the dual goals of deep purification of pollutants and resource recovery: the system can simultaneously and efficiently remove antibiotics, ammonia nitrogen, total phosphorus and COD from aquaculture wastewater. Its unique carrier design and granular form allow the treated algae and bacteria biomass to be efficiently recovered through a simple screen. The recovered biomass is rich in protein and polysaccharides and can be used as a potential feed additive or soil conditioner raw material, realizing the circular economy concept of "treating waste with waste and turning waste into treasure". Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Pseudomonas putida ( Pseudomonas putida Purchased from Hangzhou Branch of Wuhan Gray Algae Biotechnology Co., Ltd., item number: HZB112046; Acinetobacter calcium acetate ( Acinetobacter calcoaceticus Purchased from Hangzhou Branch of Wuhan Gray Algae Biotechnology Co., Ltd., item number: HZB112629; Common Chlorella ( Chlorella vulgaris Purchased from Hangzhou Branch of Wuhan Gray Algae Biotechnology Co., Ltd., item number: HZB466537; Common Four-tailed Scenedesmus ( Scenedesmus quadricauda Purchased from Hangzhou Branch of Wuhan Gray Algae Biotechnology Co., Ltd., item number: HZB470429; Chlorella powder was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd. PBS buffer was purchased from Beyotime Biotechnology, product number: ST447; Coconut shell powder activated carbon was purchased from Sevenfold Activated Carbon (Ningxia) Co., Ltd.

[0033] The aquaculture wastewater was taken from the sewage outlet of a large-scale freshwater fish and shrimp integrated aquaculture pond. The pH was 6.8, COD was 350 mg / L, ammonia nitrogen was 50 mg / L, total phosphorus was 12 mg / L, and enrofloxacin was 150 μg / L.

[0034] Examples 1-3 provide an algae-bacterial co-culture system for treating aquaculture wastewater and its preparation method.

[0035] Example 1 An algae-bacteria co-culture system for treating aquaculture wastewater and its preparation method: An algae-bacteria co-culture system for treating aquaculture wastewater comprises the following raw materials in parts by weight: 3 parts quinolone-degrading bacterial agent, 6 parts resistant microalgae, 0.3 parts signal inducer, 30 parts porous immobilization carrier, 1 part nutrient stimulant, and 600 parts acclimatization culture medium.

[0036] The quinolone-degrading bacterial agent is a compound of *Pseudomonas putida* and *Acinetobacter calcareae* at a live bacteria ratio of 1:1.

[0037] The resistant microalgae are a mixture of Chlorella and Scenedesmus tetraphylla at a biomass dry weight ratio of 1:1.

[0038] The signal inducer is composed of sodium gluconate and algal oligosaccharide in a mass ratio of 1:10; The preparation of algal oligosaccharides includes the following steps: A1. Take Chlorella powder at a mass ratio of 1:10 and resuspend it in PBS buffer to prepare an algal suspension. A2. The algal suspension was subjected to ultrasonic disruption under the following conditions: ultrasonic power 200W, working time 2s, interval time 3s, and total duration 10min. A3. Add a compound enzyme to the crushed system for enzymatic hydrolysis. The compound enzyme contains cellulase, pectinase and β-glucanase. The amount of compound enzyme added accounts for 0.5% of the weight of Chlorella powder. The ratio of enzyme activity units of cellulase, pectinase and β-glucanase is 2:1:1. The enzymatic hydrolysis conditions are: pH 4.5, temperature 45℃, time 6h. A4. After the enzymatic hydrolysis is complete, heat to 90℃ and hold for 10 minutes to inactivate the enzyme. Then centrifuge at 3000 rpm to remove the residue and collect the supernatant. A5. The supernatant is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa. The filtered components are collected and concentrated to a soluble solids content of 20% (w / v). Then, it is placed in a freeze dryer at -50°C and dried for 48 hours to obtain a brownish-yellow powder, which is the algal oligosaccharide.

[0039] The preparation of porous immobilization carriers includes the following specific steps: B1. A 2% sodium alginate solution, a 1% chitosan solution, and coconut shell powder activated carbon with a particle size of 150 mesh are mixed at a volume-to-mass ratio of 2 mL: 1 mL: 0.5 g to obtain a composite gel solution. B2. The composite gel solution is dropped into a 2% calcium chloride solution at a rate of 0.5 mL / s to solidify (the volume ratio of the composite gel solution to the calcium chloride solution is 1:10) to form gel spheres. B3. Pre-freeze the gel microspheres at -20℃ for 2 hours, and then freeze-dry them at -50℃ and 8Pa vacuum for 24 hours to obtain a porous immobilizing carrier.

[0040] The nutrient stimulant is an aqueous solution of ammonium molybdate and vitamin B12, wherein the concentration of ammonium molybdate is 0.05 mg / L and the concentration of vitamin B12 is 0.01 mg / L.

[0041] The acclimatization culture medium consisted of: glucose 10 g / L, ammonium chloride 1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.1 g / L, ferrous sulfate heptahydrate 0.01 g / L, and enrofloxacin (a quinolone antibiotic) 5 μg / L; the pH was adjusted to 6.8.

[0042] A method for preparing an algae-bacteria co-culture system for treating aquaculture wastewater includes the following steps: S1. Preparation and domestication of algae-bacteria symbiotic particles: S1.1 Co-culture of bacteria and algae and gradient acclimatization: Throughout the acclimatization process, the DO concentration was controlled at 2.5 mg / L, the light intensity at 800 lux, and the light-dark cycle at 12 h of light and 12 h of darkness. Quinolone-degrading bacteria and resistant microalgae were inoculated into the acclimatization medium at a ratio of total viable bacteria to total algal cells of 10:1, with the initial concentration controlled at 1×10⁻⁶. 7 CFU / mL and 1×10 6 cells / mL, and then the concentration of enrofloxacin in the acclimatization medium was increased from 5 μg / L to 10 μg / L every 3 days until it reached 150 μg / L; S1.2 Collection of bacterial-algae mixture: After acclimatization, collect the bacterial-algae mixture by centrifugation at 6000 rpm for 10 min.

[0043] S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture, signal inducer and nutrient stimulant are loaded together in a porous immobilization carrier. The loading conditions are 25℃, 120rpm oscillation adsorption for 20h to obtain algae-bacteria symbiotic particles. Then, they are dried at 25℃ with ventilation until the moisture content is 30% to obtain algae-bacteria co-culture system.

[0044] Example 2 An algae-bacteria co-culture system for treating aquaculture wastewater and its preparation method: An algae-bacteria co-culture system for treating aquaculture wastewater comprises the following raw materials in parts by weight: 4 parts quinolone-degrading bacterial agent, 7 parts resistant microalgae, 0.4 parts signal inducer, 40 parts porous immobilization carrier, 1.5 parts nutrient stimulant, and 700 parts acclimatization culture medium.

[0045] The quinolone-degrading bacterial agent is a compound of *Pseudomonas putida* and *Acinetobacter calcareae* at a live bacteria ratio of 2:1.

[0046] The resistant microalgae were formulated by combining Chlorella vulgaris and Scenedesmus tetracauda at a biomass dry weight ratio of 1.5:1.

[0047] The signal inducer is composed of sodium gluconate and algal oligosaccharide in a mass ratio of 1:15; The preparation of algal oligosaccharides includes the following steps: A1. Take Chlorella powder at a mass ratio of 1:15 and resuspend it in PBS buffer to prepare an algal suspension. A2. The algal suspension was subjected to ultrasonic disruption under the following conditions: ultrasonic power 350W, working time 4s, interval time 5s, and total duration 20min. A3. Add a compound enzyme to the crushed system for enzymatic hydrolysis. The compound enzyme contains cellulase, pectinase and β-glucanase. The amount of compound enzyme added accounts for 1.5% of the weight of Chlorella powder. The ratio of enzyme activity units of cellulase, pectinase and β-glucanase is 2.5:1.5:1. The enzymatic hydrolysis conditions are: pH 5.0, temperature 50℃, time 9h. A4. After the enzymatic hydrolysis is completed, the temperature is raised to 95℃ and held for 12 minutes to inactivate the enzyme. Then, the residue is removed by centrifugation at 4000 rpm, and the supernatant is collected. A5. The supernatant is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa, the filtered components are collected, and the concentration is increased to a soluble solids content of 35% (w / v). Then, it is dried in a freeze dryer at -50°C for 60 hours to obtain a brownish-yellow oligosaccharide powder, which is the algal oligosaccharide.

[0048] The preparation of porous immobilization carriers includes the following specific steps: B1. A 3% sodium alginate solution, a 1.5% chitosan solution, and coconut shell powder activated carbon with a particle size of 180 mesh were mixed at a volume-mass ratio of 3 mL: 1 mL: 1.5 g to obtain a composite gel solution. B2. The composite gel solution is dropped into a 3% calcium chloride solution at a rate of 0.75 mL / s to solidify (the volume ratio of the composite gel solution to the calcium chloride solution is 1:15) to form gel spheres. B3. Pre-freeze the gel microspheres at -30℃ for 4 hours, and then freeze-dry them at -50℃ and a vacuum of 9Pa for 36 hours to obtain a porous immobilizing carrier.

[0049] The nutrient stimulant is an aqueous solution of ammonium molybdate and vitamin B12, wherein the concentration of ammonium molybdate is 0.075 mg / L and the concentration of vitamin B12 is 0.03 mg / L.

[0050] The acclimatization culture medium consisted of: glucose 11 g / L, ammonium chloride 1.3 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.3 g / L, calcium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.02 g / L, and enrofloxacin (a quinolone antibiotic) 10 μg / L; the pH was adjusted to 7.0.

[0051] A method for preparing an algae-bacteria co-culture system for treating aquaculture wastewater includes the following steps: S1.1 Co-culture of bacteria and algae and gradient acclimatization: Throughout the acclimatization process, the DO concentration was controlled at 2 mg / L, the light intensity at 1000 lux, and the light-dark cycle at 12 h of light and 12 h of darkness. Quinolone-degrading bacteria and resistant microalgae were inoculated into the acclimatization medium at a ratio of total viable bacteria to total algal cells of 10:1, with the initial concentration controlled at 1×10⁻⁶. 7 CFU / mL and 1×10 6 cells / mL; then the concentration of enrofloxacin in the acclimatization medium was increased from 10 μg / L to 150 μg / L every 4 days, starting with 10 μg / L. S1.2 Collection of bacterial-algae mixture: After acclimatization, collect the bacterial-algae mixture by centrifugation at 8000 rpm for 15 min.

[0052] S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture, signal inducer and nutrient stimulant are loaded together in a porous immobilization carrier. The loading conditions are 27℃, 130rpm oscillation adsorption for 24h to obtain algae-bacteria symbiotic particles. Then, the particles are dried at 30℃ with ventilation until the moisture content is 35% to obtain the algae-bacteria co-culture system.

[0053] Example 3 Algae-bacteria co-culture system for treating aquaculture wastewater and its preparation method: An algae-bacteria co-culture system for treating aquaculture wastewater comprises the following raw materials in parts by weight: 5 parts quinolone-degrading bacterial agent, 8 parts resistant microalgae, 0.5 parts signal inducer, 50 parts porous immobilization carrier, 2 parts nutrient stimulant, and 800 parts acclimatization culture medium.

[0054] The quinolone-degrading bacterial agent is a compound of *Pseudomonas putida* and *Acinetobacter calcium acetate* in a live bacteria ratio of 3:1.

[0055] The resistant microalgae were formulated by combining Chlorella vulgaris and Scenedesmus tetracauda at a biomass dry weight ratio of 2:1.

[0056] The signal inducer is composed of sodium gluconate and algal oligosaccharides in a mass ratio of 1:20. The preparation of the algal oligosaccharides includes the following steps: A1. Take Chlorella powder at a mass ratio of 1:20 and resuspend it in PBS buffer to prepare an algal suspension. A2. The algal suspension was subjected to ultrasonic disruption under the following conditions: ultrasonic power 500W, working time 5s, interval time 8s, and total duration 30min. A3. Add a compound enzyme to the crushed system for enzymatic hydrolysis. The compound enzyme contains cellulase, pectinase and β-glucanase. The amount of compound enzyme added accounts for 2.0% of the weight of Chlorella powder. The ratio of enzyme activity units of cellulase, pectinase and β-glucanase is 3:2:1. The enzymatic hydrolysis conditions are: pH 5.5, temperature 55℃, time 12h. A4. After the enzymatic hydrolysis is completed, heat to 100℃ and hold for 15 minutes to inactivate the enzyme. Then centrifuge at 5000 rpm to remove the residue and collect the supernatant. A5. The supernatant is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa, the filtered components are collected, and the concentration is increased to a soluble solids content of 50% (w / v). Then, it is dried in a freeze dryer at -50°C for 72 hours to obtain a brownish-yellow oligosaccharide powder, which is the algal oligosaccharide.

[0057] The preparation of porous immobilization carriers includes the following specific steps: B1. A 4% sodium alginate solution, a 2% chitosan solution, and coconut shell powder activated carbon with a particle size of 200 mesh were mixed at a volume-mass ratio of 4 mL: 1 mL: 2 g to obtain a composite gel solution. B2. The composite gel solution is dropped into a 5% calcium chloride solution at a rate of 1 mL / s to solidify (the volume ratio of the composite gel solution to the calcium chloride solution is 1:20) to form gel spheres. B3. Pre-freeze the gel microspheres at -40℃ for 6 hours, and then freeze-dry them at -50℃ and 10Pa vacuum for 48 hours to obtain a porous immobilizing carrier.

[0058] The nutrient stimulant is an aqueous solution of ammonium molybdate and vitamin B12, wherein the concentration of ammonium molybdate is 0.1 mg / L and the concentration of vitamin B12 is 0.05 mg / L.

[0059] The acclimatization culture medium consisted of: glucose 12 g / L, ammonium chloride 1.6 g / L, potassium dihydrogen phosphate 0.6 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, and enrofloxacin (a quinolone antibiotic) 15 μg / L; the pH was adjusted to 7.2.

[0060] A method for preparing an algae-bacteria co-culture system for treating aquaculture wastewater includes the following steps: S1. Preparation and domestication of algae-bacteria symbiotic particles: S1.1 Co-culture of bacteria and algae and gradient acclimatization: Throughout the acclimatization process, the DO concentration was controlled at 3.5 mg / L, the light intensity at 1200 lux, and the light-dark cycle at 12 h of light and 12 h of darkness. Quinolone-degrading bacteria and resistant microalgae were inoculated into the acclimatization medium at a ratio of total viable bacteria to total algal cells of 10:1, with the initial concentration controlled at 1×10⁻⁶. 7 CFU / mL and 1×10 6 cells / mL; then the concentration of enrofloxacin in the acclimatization medium was increased from 15 μg / L to 20 μg / L every 5 days until it reached 150 μg / L. S1.2 Collection of bacterial-algae mixture: After acclimatization, collect the bacterial-algae mixture by centrifugation at 10,000 rpm for 20 min.

[0061] S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture, signal inducer and nutrient stimulant are loaded together in a porous immobilization carrier. The loading conditions are 30℃, 150rpm oscillation adsorption for 28h to obtain algae-bacteria symbiotic particles. Then, they are dried at 35℃ with ventilation until the moisture content is 40% to obtain algae-bacteria co-culture system.

[0062] Comparative Example 1 The only difference between this comparative example and Example 2 is that the quinolone-degrading bacterial agent consists only of *Pseudomonas putida* and does not contain *Acinetobacter calcareae*. All other components of the system, preparation methods, and steps and parameters of the usage methods are consistent with those of Example 2.

[0063] Comparative Example 2 The only difference between this comparative example and Example 2 is that the resistant microalgae consist only of Chlorella and do not contain Scenedesmus tetracaulis; all other components of the system, preparation methods, and steps and parameters of the usage methods are consistent with those of Example 2.

[0064] Comparative Example 3 The only difference between this comparative example and Example 2 is that the signal inducer consists only of sodium gluconate and does not contain algal oligosaccharides; all other components of the system, preparation methods, and steps and parameters of the usage methods are consistent with those of Example 2.

[0065] Comparative Example 4 The only difference between this comparative example and Example 2 is that in step S2, “Immobilization preparation of algae-bacteria co-culture system”, no signal inducer is added, and only the algae-bacteria mixture and nutrient stimulant are loaded onto a porous immobilization carrier; all other components of the system, preparation methods, and usage methods are consistent with those of Example 2.

[0066] Step S2, "Immobilization preparation of the algae-bacteria co-culture system," is detailed below: S2. Immobilization preparation of algae-bacteria co-culture system: The algae-bacteria mixture and nutrient stimulant were loaded together in a porous immobilization carrier. The loading conditions were 27℃ and 130rpm oscillation adsorption for 24h to obtain algae-bacteria symbiotic particles. Subsequently, the particles were dried at 30℃ with ventilation until the moisture content was 35% to obtain the algae-bacteria co-culture system.

[0067] Comparative Example 5 The only difference between this comparative example and Example 2 is that the porous immobilization carrier is replaced with a single sodium alginate carrier (chitosan and coconut shell activated carbon are removed); all other components of the system, preparation methods, and steps and parameters of the usage methods are consistent with those of Example 2.

[0068] The preparation of the porous immobilization carrier is as follows: The preparation of porous immobilization carriers includes the following specific steps: B1. A 3% sodium alginate solution is dropped into a 3% calcium chloride solution at a rate of 0.75 mL / s to solidify (the volume ratio of sodium alginate solution to calcium chloride solution is 1:15), forming gel spheres. B2. Pre-freeze the gel microspheres at -30℃ for 4 hours, and then freeze-dry them at -50℃ and 9Pa vacuum for 36 hours to obtain a porous immobilizing carrier.

[0069] Comparative Example 6 The only difference between this comparative example and Example 2 is that in step S2, “Immobilization preparation of algae-bacteria co-culture system”, no nutrient stimulant (ammonium molybdate-vitamin B12 aqueous solution) is added, and only the algae-bacteria mixture and signal inducer are loaded onto a porous immobilization carrier; all other components of the system, preparation methods and usage methods, and all steps and parameters are consistent with Example 2.

[0070] Step S2, "Immobilization preparation of the algae-bacteria co-culture system," is detailed below: S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture and signal inducer were loaded together in a porous immobilization carrier. The loading conditions were 27℃ and 130rpm oscillation adsorption for 24h to obtain algae-bacteria symbiotic particles. Subsequently, the particles were dried at 30℃ with ventilation until the moisture content was 35% to obtain the algae-bacteria co-culture system.

[0071] Comparative Analysis of Treatment Effects of Aquaculture Wastewater A method for treating aquaculture wastewater using an algae-bacteria co-culture system includes the following steps: The algae-bacterial co-culture system was added to the aquaculture wastewater to be treated at a mass-to-volume ratio of 4g:20L. After batch treatment for 72 hours under light and intermittent aeration conditions, the algae-bacterial co-culture system was recovered. The light conditions included a light intensity of 4000 lux and a light-dark cycle of 12 hours of light and 12 hours of darkness. The aeration conditions included a cycle of 2 hours of aeration followed by 1.5 hours of settling.

[0072] Using the above methods, the algae-bacteria co-culture systems used in Examples 1-3 and Comparative Examples 1-6 for treating aquaculture wastewater were selected to treat the same batch of aquaculture wastewater (aquaculture wastewater was taken from the sewage outlet of a large-scale freshwater fish and shrimp integrated aquaculture pond, pH 6.8, COD 350 mg / L, ammonia nitrogen 50 mg / L, total phosphorus 12 mg / L, enrofloxacin 150 μg / L). The core indicators were tested using the following standard methods. All experiments were conducted with 3 parallel samples, and the results were taken as mean ± standard deviation. (1) Enrofloxacin degradation rate: High performance liquid chromatography (HPLC, GB / T 20751-2006) was used to detect the concentration change of enrofloxacin and calculate the degradation rate = (initial concentration - post-treatment concentration) / initial concentration × 100%; (2) Removal rates of conventional pollutants: COD (potassium dichromate method, HJ 828-2017), ammonia nitrogen (Nessler's reagent colorimetric method, HJ 535-2009), and total phosphorus (molybdenum antimony spectrophotometric method, GB / T 11893-1989). (3) Performance of algae-bacteria symbiotic particles: bacterial and algae loading (the number of viable bacteria is detected by plate counting method, and the number of microalgae cells is detected by hemocytometer), mechanical strength (compressive strength is detected by universal testing machine).

[0073] The core indicator detection data are shown in Table 1 and Table 2.

[0074] Table 1 Comparison of pollutant removal effects

[0075] Table 2 Comparison of performance of algae-bacteria symbiotic granules

[0076] The data in Tables 1 and 2 show that the absence or degradation of any single component will lead to a significant deterioration in performance, confirming that the technical solution of the present invention is an organic whole that produces a synergistic effect of "1+1>2".

[0077] Specifically, Comparative Examples 1 and 2 demonstrate that specific combinations of *Pseudomonas putida* with *Acinetobacter calcitonin* and *Chlorella vulgaris* with *Scenedesmus tetracaulis* are key to constructing efficient degradation chains and stable photosynthetic systems, and that single bacterial or algal species cannot achieve comprehensive and efficient pollutant removal.

[0078] Comparative Examples 3 and 4 show that the complete signal inducer composed of sodium gluconate and algal oligosaccharides significantly improves the density and functional bioload of the biofilm on the carrier surface through cross-border synergistic regulation. Its absence will directly weaken the treatment efficiency and system stability.

[0079] The results of Comparative Example 5 are particularly outstanding. Its mechanical strength and bioburden are significantly lower than those of Example 2, and its pollutant removal effect is the worst. This directly and quantitatively proves the irreplaceable role of the sodium alginate-chitosan-activated carbon composite carrier in providing a stable colonization microenvironment and ensuring particle integrity and recyclability.

[0080] Comparative Example 6 shows that under long-term antibiotic stress, the lack of ammonium molybdate-vitamin B12 nutritional stimulant will lead to a decline in metabolic activity and a decrease in enrofloxacin degradation rate, indicating that this component is an important metabolic maintenance agent for maintaining the continuous and efficient operation of the system, rather than a common nutritional supplement.

[0081] In summary, Examples 1-3 exhibited superior and stable performance, in stark contrast to the overall inferiority of all comparative examples. This fully demonstrates that the technical system of "functional combination-signal induction-carrier fixation-nutrient maintenance" of this invention produces a synergistic effect that transcends simple superposition, effectively solving the bottlenecks of existing technologies and possessing outstanding substantive features and significant progress.

[0082] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An algae-bacteria co-cultivation system for treating aquaculture wastewater, characterized in that, The algae-bacteria co-culture system comprises the following raw materials in parts by weight: 3-5 parts quinolone-degrading bacterial agent, 6-8 parts resistant microalgae, 0.3-0.5 parts signal inducer, 30-50 parts porous immobilization carrier, 1-2 parts nutrient stimulant, and 600-800 parts acclimatization culture medium.

2. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The quinolone-degrading bacterial agent is a compound of Pseudomonas and Acinetobacter at a live bacteria ratio of 1-3:

1.

3. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The resistant microalgae are composed of Chlorella and Scenedesmus tetracauda at a biomass dry weight ratio of 1-2:

1.

4. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The signal inducer is composed of sodium gluconate and algal oligosaccharides in a mass ratio of 1:10-20.

5. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The preparation of the porous immobilization carrier includes the following steps: Sodium alginate solution, chitosan solution, and coconut shell powder activated carbon are mixed to obtain a composite gel solution; the composite gel solution is dropped into calcium chloride solution to solidify and form gel microspheres; the gel microspheres are pre-frozen and vacuum freeze-dried to obtain a porous fixed carrier.

6. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The nutrient stimulant is an aqueous solution of ammonium molybdate and vitamin B12, wherein the concentration of ammonium molybdate is 0.05-0.1 mg / L and the concentration of vitamin B12 is 0.01-0.05 mg / L.

7. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 1, characterized in that, The acclimatization culture medium comprises: glucose 10-12 g / L, ammonium chloride 1-1.6 g / L, potassium dihydrogen phosphate 0.4-0.6 g / L, magnesium sulfate 0.2-0.4 g / L, calcium chloride 0.1-0.3 g / L, ferrous sulfate heptahydrate 0.01-0.03 g / L, and quinolone antibiotics 5-15 μg / L; the pH is adjusted to 6.8-7.

2.

8. The algae-bacteria co-cultivation system for treating aquaculture wastewater according to claim 7, characterized in that, The quinolone antibiotics are selected from one or more of enrofloxacin, ciprofloxacin, levofloxacin, and norfloxacin.

9. A method for preparing an algae-bacteria co-culture system for treating aquaculture wastewater according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Algae-bacteria co-culture and domestication: Quinolone-degrading bacterial agent and resistant microalgae are inoculated into domestication culture medium and domestication culture is carried out with gradient concentrations to obtain algae-bacteria mixture; S2. Immobilization preparation of algae-bacteria co-culture system: Algae-bacteria mixture, signal inducer and nutrient stimulant are loaded onto a porous immobilization carrier to obtain algae-bacteria symbiotic particles, which are then dried to obtain algae-bacteria co-culture system.

10. The application of the algae-bacteria co-culture system according to any one of claims 1-8 in the treatment of aquaculture wastewater, characterized in that, The method for treating aquaculture wastewater includes the following steps: Add the algae-bacteria co-culture system to the aquaculture wastewater to be treated at a mass-volume ratio of 3-5g:20L; after batch treatment for 48-96 hours under light and intermittent aeration conditions, filter and recover the algae-bacteria co-culture system.

Images