Method for reducing ARGs in pig wastewater by nano-copper oxide and three-phase algal-bacterial symbiotic body

Abstract

The present application belongs to the technical field of wastewater biological treatment, and specifically relates to a method for strengthening tetracycline resistance gene reduction in pig wastewater by using nano copper oxide and three-phase algal-bacterial symbiont. The present application comprises the following steps: preparing high-purity nano copper oxide with a certain particle size, constructing three-phase algal-bacterial symbiont of Chlorella sp.-endophytic bacteria S395-2-Spiral Sporae Filum Group, mixing the pretreated pig wastewater with a certain amount of nano copper oxide and three-phase algal-bacterial symbiont, and carrying out directional culture in a tank-type photobioreactor for 8-12 days to achieve efficient reduction of various antibiotic resistance genes such as tetracycline, sulfonamide and macrolide. The present application utilizes the synergistic effect of nano copper oxide and three-phase algal-bacterial symbiont, significantly improves the stability of the symbiont, the amount of extracellular polymeric substance secretion and the stress resistance, and the total reduction rate of ARGs is greater than or equal to 92, which is 5%-8% higher than that of the traditional plant hormone regulation technology; the recovered symbiont can be recycled and utilized, and is suitable for large-scale pig wastewater ARGs treatment.

Description

Technical Field

[0001] This invention belongs to the field of wastewater biological treatment technology, specifically relating to a method for enhancing the reduction of antibiotic resistance genes (ARGs) in pig farm wastewater through nano-copper oxide synergistic triphasic algae-bacteria symbiosis. Background Technology

[0002] 1. Current Status and Hazards of ARGs Pollution in Pig Farm Wastewater my country is the world's largest pig-producing country, and the overuse of antibiotics in large-scale pig farms is a prominent problem. Statistics show that the annual antibiotic usage in my country's pig industry exceeds 100,000 tons, of which 60%-80% is directly discharged into wastewater through feces and urine without being metabolized by the animals. This results in pig wastewater becoming a high accumulation reservoir of antibiotic resistance genes (ARGs). At least 149 ARGs, including sulfonamides (sul1, sul2), tetracyclines (tetG, tetW, tetX), and macrolides (ermB, ermF), have been detected in pig wastewater, with typical ARG abundance reaching 10. 6 -10 8 copies·mL -1 It is 100-1000 times larger than natural water bodies.

[0003] ARGs, as emerging environmental pollutants, can spread through bacterial host proliferation and horizontal gene transfer, ultimately threatening human health through the food chain; meanwhile, pig farm wastewater also contains heavy metals (Hg). 2+ Cu 2+ Zn 2+ High concentrations of COD, nitrogen, phosphorus, and residual antibiotics create a complex pollution environment, further exacerbating the risk of ARGs transmission. Traditional wastewater treatment technologies struggle to achieve efficient and stable removal of ARGs, making it a pressing technical challenge in the field of environmental governance.

[0004] 2. Deficiencies of existing ARGs reduction technologies (1) Traditional physical-chemical method While physical methods (adsorption, membrane filtration) and chemical methods (advanced oxidation, disinfection) can quickly remove some ARGs, they have significant drawbacks: adsorption methods have limited adsorption capacity, are prone to saturation and difficult to regenerate; membrane filtration is costly and prone to clogging; advanced oxidation is energy-intensive and prone to producing toxic byproducts; and physical and chemical methods are difficult to inhibit the regeneration of resistant bacteria (ARBs), so ARGs are prone to rebound after treatment, and they are also prone to disrupting the ecological balance of aquatic bodies, resulting in a high risk of secondary pollution.

[0005] (2) Traditional biological method Biological methods such as constructed wetlands and activated sludge processes are low-cost and environmentally friendly, but they have low efficiency in reducing ARGs (only 30%-50%), extremely poor removal efficiency for macrolide ARGs (less than 10%), weak resistance to heavy metals and antibiotic stress, and poor stability in treating highly polluted wastewater.

[0006] (3) Current status of algae-bacteria symbiosis technology Algal-bacterial symbiosis has become a hot topic in ARGs reduction research due to the synergistic advantages of microalgae photosynthetic oxygen supply, bacterial degradation of organic matter, and fungal fixation. Existing research mostly focuses on biphasic algal-bacterial symbionts (microalgae-bacteria, microalgae-fungi), but these studies suffer from poor spheroidization stability, long spheroidization cycles, weak stress resistance, and low extracellular polymeric substance (EPS) secretion (only 20-30 mg / g). -1 Due to issues such as these, the adsorption and degradation capacity of ARGs is limited.

[0007] Although some studies in recent years have used plant hormones (GA3, GR24, 5-DS) to regulate the performance of triphasic algae-bacteria-fungi symbionts (microalgae-bacteria-fungi) and improve the ARGs reduction rate to a certain extent, there are still significant drawbacks: ① The effect of hormones is easily affected by fluctuations in temperature, light, and water quality, and the stability is insufficient; ② Hormones are expensive and have poor economic efficiency for large-scale application; ③ The adaptability to wastewater with high concentrations of ARGs is limited, and the reduction rate of macrolide ARGs is still less than 85%; ④ Some hormones pose environmental residue risks and do not conform to the concept of green governance.

[0008] 3. Application potential and current shortcomings of nano-copper oxide Nano-CuO is a novel inorganic nanomaterial with strong oxidizing properties, high specific surface area, excellent adsorption performance, good biocompatibility, and catalytic activity. It can enhance pollutant removal through the following mechanisms: ① strong oxidizing properties directly oxidize and degrade the nucleic acid structure of ARGs; ② high specific surface area adsorbs and enriches free ARGs in wastewater; ③ low concentration stimulates microbial metabolism and increases EPS secretion; ④ enhances microbial resistance and improves survival rate in extreme environments.

[0009] Currently, nano-copper oxide has been used for the removal of heavy metals and organic pollutants, but there is no research on its combined use with triphasic algae-bacteria symbionts to reduce ARGs in pig farm wastewater. Furthermore, there are no reports on targeted synergistic ratios, process parameters, and mechanisms of action. There are significant gaps in existing technologies, and there is an urgent need to develop efficient, stable, and economical synergistic treatment technologies. Summary of the Invention

[0010] The purpose of this invention is to provide a method for enhancing the reduction of antibiotic resistance genes in pig wastewater by using nano-copper oxide in conjunction with a triphasic algae-bacteria symbiotic system, in order to overcome the shortcomings of existing technologies such as low ARG reduction efficiency, poor stability, high cost, and high risk of secondary pollution.

[0011] This invention provides a method for enhancing antibiotic resistance gene reduction in swine wastewater using a nano-copper oxide synergistic triphasic algae-bacteria symbiotic system. Through the synergistic effect of nano-copper oxide and the Chlorella-endophytic bacteria S395-2-Helicobacter pylori triphasic algae-bacteria symbiotic system, it solves the problems of weak stress resistance, insufficient EPS secretion, and low removal rate of macrolide ARGs in traditional technologies. This achieves efficient, stable, and green removal of ARGs from swine wastewater, while also possessing resource value. Specifically, it includes the following steps: (1) Preparation of nano-copper oxide; the particle size of nano-copper oxide is 50-100 nm, and the specific surface area is ≥60 m². 2 ·g -1 Purity ≥ 98%; (2) Preparation of a triphasic algal-bacterial symbiotic; In the triphasic algal-bacterial symbiotic, Chlorella: Endophytic bacteria S395-2: Polyspora spiralis = 1: (5-15): (0.02-0.10) (cell number / CFU / spore number); (3) Pretreatment of pig farm wastewater; control of heavy metals, residual antibiotics and conventional pollutants to meet standards; (4) Construction of a nano-copper oxide synergistic treatment system: Add 5-15 mg / L nano-copper oxide and 60-120 mg / L triphase algae-bacteria symbiotic to the pretreated pig wastewater in the following proportions and transfer it to a tank photobioreactor; (5) Synergistic reduction culture; at a temperature of 25±2℃ and a light intensity of 150~250μmol·m -2 ·s -1 Under conditions of 12h light-dark alternation and an aeration rate of 0.5–1.0 L / min, culture for 8–12 days to achieve efficient reduction of antibiotic resistance genes.

[0012] Furthermore: In step (2), the sphericity of the triphasic algae-bacteria symbiont is ≥98%, the sedimentation rate is ≥0.5cm / min, the extracellular polymer content is 750-900mg / g (dry weight), the polysaccharide content is ≥45%, and the protein content is ≥35%.

[0013] In step (4), the preferred dosage of nano copper oxide is 8-12 mg / L, and the most preferred dosage is 10 mg / L; the preferred dosage of the triphase algae-bacteria symbiotic is 80-110 mg / L, and the most preferred dosage is 100 mg / L.

[0014] In step (5), the antibiotic resistance genes that can be reduced include tetracyclines, sulfonamides, and macrolides, with a single-class reduction rate of ≥90% and a total reduction rate of ≥92%.

[0015] In step (5), after 10 days of co-culture, the biomass retention rate of the triphasic algae-bacteria symbiotic organism is ≥90%, the survival rate is ≥93%, and the photosynthetic efficiency is stable at 0.78-0.85.

[0016] Furthermore, the specific procedures are as follows: (1) Preparation of nano-CuO High-purity, controllable-particle-size nano-copper oxide was prepared using a hydrothermal synthesis method. Specific steps include: ① Prepare a 0.1 mol / L copper chloride (CuCl2) solution and a 0.2 mol / L sodium hydroxide (NaOH) solution for later use; ② Slowly add copper chloride solution to sodium hydroxide solution at a volume ratio of 1:2, and stir magnetically for 30 minutes to form a blue copper hydroxide suspension; ③ Transfer the suspension to a polytetrafluoroethylene-lined reactor, react at a constant temperature of 120℃ for 4 hours, and then allow it to cool naturally to room temperature; ④ Centrifuge (8000 r / min, 10 min), collect the black precipitate, and wash with deionized water 3-5 times until the pH of the washing solution is neutral; ⑤ Vacuum dry at 60℃ for 12 hours, grind through a 200-mesh standard sieve to obtain particles with a diameter of 50-100 nm, purity ≥98%, and specific surface area ≥60 m². 2 ·g -1 The nano-copper oxide should be stored in a light-proof, sealed container for future use.

[0017] (2) Preparation of triphase algae-bacteria symbiosis A triphasic algal-bacterial symbiosis was constructed using *Chlorella* sp., endophytic bacteria S395-2 (Enterobacter sp.), and *Clonostachys rosea*. All strains were deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers: *Chlorella* CGMCC No. 202403, endophytic bacteria S395-2 CGMCC No. 202401, and *Clonostachys rosea* CGMCC No. 202402. Specific steps were as follows: ① Activation and pre-culture of bacterial strains Chlorella activation: Inoculated into BG11 medium (composition: NaNO3 1.5g / L, K2HPO4·3H2O 0.04g / L, MgSO4·7H2O 0.075g / L, citric acid 0.006g / L, ferric ammonium citrate 0.006g / L, Na2EDTA 0.001g / L, Na2CO3 0.02g / L, the remainder being deionized water, pH 7.1); culture conditions: temperature 25±1℃, light intensity 200μmol·m -2 ·s -1 The cells were cultured for 5 days with 12 hours of alternating light and dark, and an oscillation rate of 150 r / min, until the logarithmic growth phase was reached, at which point the biomass reached 2.0 × 10⁻⁶. 6 cell·mL-1 ,spare; Activation of endophytic bacteria S395-2: Inoculated into LB medium (composition: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0); culture conditions: temperature 30 ± 1℃, shaking rate 180 r / min, cultured for 14 h until the logarithmic growth phase, reaching a concentration of 2.0 × 10⁻⁶. 7 CFU·mL -1 ,spare; Activation of *Agrospora spiralis*: Inoculated onto PDA medium (composition: potato 200 g / L, glucose 20 g / L, agar 20 g / L, pH natural); culture conditions: temperature 28 ± 1℃, culture for 6 days, until the mycelium has fully colonized the plate, spores are collected and diluted with sterile water to prepare 1.0 × 10⁻⁶ spores. 5 spores·mL -1 Spore suspension, for later use.

[0018] ② Construction of a triphasic algae-bacteria symbiotic system Chlorella: Endophytic bacteria S395-2: Polyspora spiralis = 1:10:0.05 (cells / CFU / spores), inoculated into modified BG11 medium (with 0.1 g / L glucose added, otherwise the same as BG11); culture conditions: temperature 25±1℃, light intensity 200 μmol·m -2 ·s -1 The mixture was cultured for 3 days with alternating light and dark conditions for 12 hours and an oscillation rate of 110 r / min. This resulted in the formation of a triphase algae-bacterial symbiotic organism with a diameter of 3-4 mm, a dark green color, a smooth surface, a ball formation rate of ≥98%, and a sedimentation rate of ≥0.5 cm / min. This organism was then ready for use.

[0019] (3) Pretreatment of pig farm wastewater Wastewater from anaerobic fermentation at large-scale pig farms is collected and pretreated to remove impurities and regulate water quality. Specific steps include: ① Bar screen filtration: A 20-mesh mechanical bar screen is used to remove large suspended impurities such as pig hair and feed residue; ② Settling: Transfer to a sedimentation tank and let stand at 25℃ for 24 hours to remove mud, sand and suspended solids. The supernatant is then ready for use. ③ Water quality control: Monitor the supernatant indicators and control: heavy metals (Hg) 2+ ≤1mg / L, Cu 2+ ≤10mg / L, Zn 2+ ≤10mg / L; residual antibiotics oxytetracycline (OTC), ciprofloxacin (CPFX), and sulfadiazine (SM2) are all ≤1mg / L; COD 1200-1600mg / L, TN 300-360mg / L, TP 40-50mg / L; initial ARGs abundance 1.0×10 6 -1.0×108 copies·mL -1 If the concentration exceeds the standard, dilute with distilled water or adjust with activated carbon adsorption, and keep on hand.

[0020] (4) Construction of a synergistic treatment system for nano-copper oxide Nano-copper oxide and a three-phase algae-bacteria symbiotic system were simultaneously added to the pretreated pig farm wastewater to construct a synergistic treatment system. Nano copper oxide dosage: 5-15 mg / L (preferably 8-12 mg / L, most preferably 10 mg / L); Triphasic algae-bacteria symbiotic dosage: 60-120 mg / L (preferably 80-110 mg / L, most preferably 100 mg / L). After mixing evenly, the mixture is transferred to a tank-type photobioreactor (effective volume 50-500L, reflectivity of the inner wall reflective layer ≥90%, aeration heads with 15μm pore size evenly distributed at the bottom, and surrounded by 400-700nm LED light source).

[0021] (5) Synergistic reduction culture Controlling reactor culture conditions to directionally enhance ARG reduction: Temperature: 25±2℃ (preferably 25±1℃); Light intensity: 200 μmol·m -2 ·s -1 ; Light-to-dark ratio: 12 hours of light / 12 hours of darkness; Aeration rate: 0.8 L / min (aeration gas is air, containing 0.03%-0.05% CO2 by volume); Culture period: 10 days; Monitoring: Sampling was conducted on days 0, 1, 3, 7, and 10 to detect ARGs abundance, COD, TN, TP, symbiotic photosynthetic efficiency (Fv / Fm), and survival rate.

[0022] (6) Wastewater discharge and symbiotic recycling and resource utilization ① Emission standards: After the cultivation is completed, the detection indicators must meet the following requirements: total ARGs reduction rate ≥92%, COD ≤100mg / L, TN ≤15mg / L, TP ≤0.5mg / L, and heavy metals must comply with the "Emission Standard of Pollutants for Livestock and Poultry Breeding Industry" (GB 18596-2001) before emissions can be made; ② Symbiotic recovery: The filter is made using a plate and frame filter press (150 mesh filter cloth) and rinsed three times with sterile water to remove surface contaminants; ③ Resource utilization: Vacuum dried at 60℃ to a moisture content of ≤10%, the dried symbiont has a protein content of ≥32%, an oil content of ≥24%, and heavy metals that meet the "Feed Hygiene Standard" (GB 13078-2021). It can be used directly for feed production or for extracting protein and oil.

[0023] Furthermore: The EPS content of the triphasic algae-bacteria symbiotic organism reaches 750-900 mg / g (dry weight), of which polysaccharides account for ≥45% and proteins account for ≥35%.

[0024] Eligible ARGs include: tetracyclines (tetG, tetW, tetX), sulfonamides (sul1, sul2), and macrolides (ermB, ermF). The reduction rate of a single ARG is ≥90%, and the total reduction rate is ≥92%.

[0025] After 10 days of co-culture, the biomass retention rate of the symbiont was ≥90%, the survival rate was ≥93%, and the photosynthetic efficiency was stable at 0.78-0.85.

[0026] Compared with the prior art, the present invention has the following outstanding advantages: 1. Synergistic effect, significantly improved ARGs reduction efficiency: Nano-copper oxide directly degrades ARGs nucleic acids with strong oxidizing properties, and high specific surface area adsorbs and enriches free ARGs; low concentration stimulates symbiont metabolism, increasing EPS secretion by 30%-50% (to 750-900mg / g), enhancing ARGs adsorption and fixation; total ARGs reduction rate ≥92%, macrolide ARGs reduction rate ≥90%, which is 5%-8% higher than GR24 and 5-DS hormone regulation technology, solving the core pain point of low macrolide removal rate in traditional technology; 2. Strong resistance to stress and excellent treatment stability: Nano-copper oxide enhances the toughness of the symbiont cell wall and improves its tolerance to heavy metals and residual antibiotics; after 10 days of continuous culture, the symbiont biomass retention rate is ≥90%, the survival rate is ≥93%, and the photosynthetic efficiency is stable at 0.78-0.85, unaffected by fluctuations in water quality and light, and its stability is significantly better than that of hormone regulation systems. 3. Green and residue-free with low risk of secondary pollution: The amount of nano copper oxide used is low (only 10mg / L), with good biocompatibility. It can be recycled with the symbiont and leaves no residue. It does not require expensive plant hormones, avoiding the risk of hormone residues and conforming to the concept of green environmental protection. 4. Resource utilization with significant economic benefits: The recovered symbiont has high protein and oil content, meeting feed standards and realizing the integration of "pollution control and resource utilization"; the preparation process of nano copper oxide is simple and low-cost, requiring no complex equipment, and the cost of large-scale application is 40%-50% lower than that of hormone technology, making it economically outstanding. 5. Wide range of applications and high promotion value: It is suitable for pig farm wastewater with different concentrations of ARGs, heavy metals and residual antibiotics. It can be applied on a large scale to the treatment of wastewater from various pig farms. It can also be extended to the treatment of ARGs pollution in livestock and poultry breeding wastewater and aquaculture wastewater. It has broad application prospects. It is especially suitable for the deep purification treatment of pig farm wastewater containing high abundance of tetracycline, sulfonamide and macrolide antibiotic resistance genes. Attached Figure Description

[0027] Figure 1 This is a process flow diagram of the present invention for reducing ARGs in pig farm wastewater using nano-copper oxide synergistic triphase algae-bacteria symbiosis.

[0028] Figure 2 The image shows the particle size distribution and scanning electron microscope (SEM) image of nano-copper oxide.

[0029] Figure 3 This is a SEM image of a triphasic algal-bacterial symbiotic organism.

[0030] Figure 4 The curves showing the ARGs reduction rate at different culture times in Example 1 represent the removal efficiency of the treatment system for three typical antibiotic resistance genes.

[0031] Figure 5 The curves showing the ARGs reduction rate at different incubation times in Example 1 represent the removal efficiency of the system for ARGs, COD, TN, and TP at different incubation times.

[0032] Figure 6 This is a schematic diagram illustrating the synergistic mechanism between nano-copper oxide and the triphasic algae symbiotic organism in Example 1.

[0033] Numbering in the diagram: 1-Grid; 2-Sedimentation tank; 3-Tank photobioreactor; 4-LED light source; 5-Aeration device; 6-Tail gas collection device; 7-Plate and frame filter press; 8-Drying device; 9-Finished feed. Detailed Implementation

[0034] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Example 1

[0035] (1) Preparation of nano copper oxide ① Prepare 100 mL of 0.1 mol / L CuCl2 solution and 200 mL of 0.2 mol / L NaOH solution; ② Add the mixture dropwise and stir for 30 minutes to form a blue suspension; ③ Perform hydrothermal reaction at 120℃ for 4 hours, then cool and centrifuge; ④ Wash with deionized water 4 times, dry at 60℃ for 12 hours, and pass through a 200-mesh sieve; A particle size of 70 nm, a purity of 98.5%, and a specific surface area of ​​68 m² were obtained. 2 ·g -1 Nano copper oxide.

[0036] (2) Preparation of triphase algae-bacteria symbiosis ① Chlorella, S395-2, and Polyspora spiralis were activated to the target concentrations, respectively; ② Inoculate with modified BG11 medium at a ratio of 1:10:0.05; ③ 25℃, 200μmol·m -2 ·s -1 Cultured for 3 days in 12 hours of light and dark at 110 rpm; The pellet formation rate was 98.2%, the diameter was 3.5 mm, and the settling rate was 0.55 cm / min.

[0037] (3) Pretreatment of pig farm wastewater Wastewater from a large-scale pig farm in Jiaxing was collected, filtered through a bar screen, and settled for 24 hours. Water quality indicators: COD 1400mg / L, TN 330mg / L, TP 45mg / L; Cu 2+ 8 mg / L, OTC 0.8 mg / L; ARGs abundance 5.0 × 10⁸ 7 copies / mL.

[0038] (4) Construction of collaborative processing system Add 10 mg / L of nano-copper oxide and 100 mg / L of triphase algae-bacteria symbiosis, and transfer to a 100L tank-type photobioreactor.

[0039] (5) Collaborative training 25℃, 200μmol·m -2 ·s -1 12 hours of light and dark, aeration at 0.8 L / min, cultured for 10 days.

[0040] (6) Test results ARGs reduction: tetG 96.3%, sul1 95.5%, ermB 93.2%, total reduction rate 94.1%; Common pollutants: COD removal rate 93.5%, TN 86.2%, TP 89.1%; Symbiont: EPS content 820mg / g (polysaccharide 48%, protein 36%), biomass retention rate 91.5%, photosynthetic efficiency 0.82; Resource utilization: The dried symbiont contains 33% protein and 24.5% oil, which meets feed standards. Example 2

[0041] Consistent with Example 1, except that the dosage of nano-copper oxide was adjusted to 8 mg / L and cultured for 10 days.

[0042] Results: The total reduction rate of ARGs was 92.8%, tetG 95.1%, sul1 94.3%, and ermB 91.7%; EPS content was 780 mg / g, and biomass retention rate was 90.2%. Example 3

[0043] Consistent with Example 1, except that the dosage of nano-copper oxide was adjusted to 12 mg / L and cultured for 10 days.

[0044] Results: The total reduction rate of ARGs was 93.8%, tetG 95.8%, sul1 95.0%, and ermB 92.5%; EPS content was 850 mg / g, and biomass retention rate was 92.0%.

[0045] Comparative Example 1 (GR24 hormone regulation, Patent 2 method (Triphasic algae-bacteria symbiosis based on the combined use of plant hormones and its construction method and application; CN 122214234 A)) Add 10 -9 M GR24 was used to regulate the triphase algae-bacteria symbiosis, and the wastewater was treated in the same way as in Example 1. The mixture was cultured for 10 days.

[0046] Results: The total reduction rate of ARGs was 87.3%, and the ermB content was 82.5%; the EPS content was 680 mg / g, and the biomass retention rate was 84.6%.

[0047] Comparative Example 2 (5-DS hormone regulation, Patent 3 method (a method for enhancing ARGs reduction in pig wastewater based on 5-DS regulation of algal-bacterial symbiotic EPS; CN 122212390 A)) Add 10 -11 The wastewater was treated with M 5-DS in the same manner as in Example 1 and cultured for 14 days.

[0048] Results: The total reduction rate of ARGs was 88.5%, and the ermB content was 83.7%; the EPS content was 710 mg / g, and the biomass retention rate was 86.1%.

[0049] Comparative Example 3 (Triphasic algae-bacteria symbiosis only, without nano-copper oxide) The wastewater was treated in the same way as in Example 1 and cultured for 10 days.

[0050] Results: The total reduction rate of ARGs was 81.2%, and the ermB content was 75.3%; the EPS content was 590 mg / g, and the biomass retention rate was 78.5%.

Claims

1. A method for enhancing antibiotic resistance gene reduction in pig farm wastewater using nano-copper oxide synergistic with a triphasic algae-bacteria symbiotic system, characterized in that, The efficient, stable, and green removal of ARGs from pig farm wastewater is achieved through the synergistic effect of nano-copper oxide and the Chlorella-endophytic bacteria S395-2-Helicobacter pylori triphasic algae-bacteria symbiosis. The specific steps include: (1) Preparation of nano-copper oxide; the particle size of nano-copper oxide is 50-100 nm, and the specific surface area is ≥60 m². 2 ·g -1 Purity ≥ 98%; (2) Preparation of a triphasic algal-bacterial symbiotic; In the triphasic algal-bacterial symbiotic, Chlorella: Endophytic bacteria S395-2: Polyspora spiralis = (1): (5-15): (0.02-0.10) (cell number / CFU / spore number); (3) Pretreatment of pig farm wastewater; control of heavy metals, residual antibiotics and conventional pollutants to meet standards; (4) Construction of a nano-copper oxide synergistic treatment system: Add 5-15 mg / L nano-copper oxide and 60-120 mg / L triphase algae-bacteria symbiotic to the pretreated pig wastewater in the following proportions and transfer it to a tank photobioreactor; (5) Synergistic reduction culture; at a temperature of 25±2℃ and a light intensity of 150~250μmol·m -2 ·s -1 Under conditions of 12h light-dark alternation and an aeration rate of 0.5–1.0 L / min, culture for 8–12 days can achieve efficient reduction of antibiotic resistance genes.

2. The method according to claim 1, characterized in that, In step (2), the extracellular polymeric content of the triphasic algae symbiont is 750-900 mg / g, dry weight, with polysaccharide content ≥45% and protein content ≥35%.

3. The method according to claim 1, characterized in that, In step (4), the dosage of nano-copper oxide is 8-12 mg / L; the dosage of the triphasic algae-bacteria symbiotic is 80-110 mg / L.

4. The method according to claim 1, characterized in that, In step (5), the antibiotic resistance genes that can be reduced include tetracyclines, sulfonamides, and macrolides, with a single-class reduction rate of ≥90% and a total reduction rate of ≥92%.

5. The method according to claim 1, characterized in that, In step (5), after 10 days of co-culture, the biomass retention rate of the triphasic algae-bacteria symbiotic organism is ≥90%, the survival rate is ≥93%, and the photosynthetic efficiency is stable at 0.78-0.85.

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