System and method for removing antibiotic resistance genes in aquaculture water

Abstract

The invention discloses a system and a method for removing antibiotic resistance genes in aquaculture water, and belongs to the technical field of water environment treatment and ecological engineering. In order to solve the problems that ARGs in the existing aquaculture tail water are difficult to remove, easy to relapse and heavy in secondary pollution, a physical gradient filtering unit, an anaerobic targeting blocking unit and an aerobic phycomycete symbiotic purification unit are sequentially constructed in the system. The physical unit is filled with pelelith, quartz sand, magnetite and zeolite; pH response type intelligent microcapsules are introduced into the anaerobic unit, and zinc oxide nanoparticles and tea polyphenol are coated with a sodium alginate / chitosan compound; the aerobic unit strengthens a microalgae-bacteria symbiotic system through a plant growth regulator, deeply mineralizes residual antibiotics and adsorbs free DNA; and the tail end is coupled with a high-temperature anaerobic digestion device to realize thorough inactivation of enriched ARGs in the algae mud. The method is scientific in technological process and low in operation cost, and deep purification and resource utilization of the aquaculture tail water are achieved.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary technical field of water environment pollution control, ecological restoration, and agricultural waste resource utilization. Specifically, it relates to a system and method for removing antibiotic resistance genes (ARGs) from aquaculture water. This invention is mainly applied to the advanced treatment of wastewater from intensive freshwater or marine aquaculture, and is particularly suitable for the precise purification of aquaculture wastewater with high antibiotic residue concentrations, complex drug-resistant bacterial communities, and a high risk of gene transfer. The aim is to block the transmission chain of ARGs in the aquatic environment and ensure aquatic ecological security. Background Technology

[0002] With the increasing global population growth and rising demand for protein, aquaculture has become one of the fastest-growing sectors of food production. However, antibiotics (such as tetracyclines, sulfonamides, and macrolides) are used extensively in aquaculture to prevent fish and shrimp diseases and promote growth. Because aquatic animals have extremely low absorption and utilization rates of antibiotics (typically only 20%-30%), most antibiotics enter the water and sediment in the form of the original drug or active metabolites through excrement. This continuous exposure to low doses of antibiotics induces antibiotic resistance in environmental microorganisms through natural selection pressure, leading to the generation and accumulation of antibiotic resistance genes (ARGs).

[0003] ARGs are currently listed as a new type of environmental pollutant by the World Health Organization (WHO). Their harm lies not only in causing antibiotic resistance in pathogens, rendering them untreatable for humans, but also in their unique environmental behavior of horizontal gene transfer (HGT). ARGs can freely move between different bacterial species through plasmid conjugation, transformation, and transduction, and can even transfer from non-pathogenic environmental bacteria to human pathogens. Aquaculture environments, due to their high-density bacterial populations, abundant nutrients, and continuous antibiotic pressure, are recognized as hotspots for ARG HGT.

[0004] Existing aquaculture wastewater treatment technologies have significant limitations in removing ARGs: Traditional biological methods (such as activated sludge and biofilm processes): Although they have good removal effects on organic matter (COD) and nitrogen and phosphorus, studies have shown that traditional biological treatment units are often incubators for antibiotic resistance genes (ARGs). Due to the high aggregation of microorganisms and the failure of antibiotic residues in the influent to be degraded in time, the conjugation and transfer of antibiotic resistance plasmids between microbial communities are promoted, resulting in the relative abundance of ARGs in the effluent being even higher than that in the influent.

[0005] Physical adsorption and membrane separation: While technologies such as activated carbon adsorption and reverse osmosis membranes can retain bacteria and DNA, they do not achieve the degradation of pollutants. High concentrations of ARGs in the concentrate, if not properly treated, will cause even more serious point source pollution. Furthermore, the high viscosity and high organic load of aquaculture wastewater make it highly susceptible to biofouling of membrane modules, resulting in high operating and maintenance costs.

[0006] Advanced oxidation technologies (AOPs): Ozone, Fenton oxidation, etc., can effectively destroy DNA structure, but due to the huge volume of aquaculture wastewater, the consumption of chemical agents is large, resulting in poor economic efficiency. More importantly, while strong oxidants kill bacteria, they may increase the permeability of bacterial cell membranes, releasing large amounts of intracellular DNA. If oxidation is incomplete, these free DNAs (eARGs) still have transformative activity, and the oxidation process may produce byproducts with carcinogenic, mutagenic, and teratogenic effects.

[0007] Single ecological methods: Constructed wetlands or simple algae ponds, although eco-friendly, require a large area, are significantly affected by seasonal climate, and have a single removal mechanism for ARGs, mainly relying on natural sedimentation and solar photolysis, resulting in extremely unstable removal efficiency and difficulty in meeting increasingly stringent emission standards.

[0008] In recent years, utilizing the antibacterial properties of nanomaterials (such as nano-silver and nano-zinc oxide) and the plasmid elimination properties of plant extracts (such as tea polyphenols) has become a research hotspot. However, directly adding nanomaterials to water bodies faces problems such as easy aggregation and inactivation, difficulty in recovery, and potential ecotoxicity; while natural organic matter such as tea polyphenols is easily oxidized and decomposed in oxygen-rich water, making it difficult to maintain an effective inhibitory concentration. Therefore, how to construct an ARGs removal system that can exist stably, release in a targeted manner, and utilize multiple mechanisms in a synergistic manner is a technical challenge that urgently needs to be solved. Summary of the Invention

[0009] Technical problems to be solved With the rapid development of intensive aquaculture, the widespread use of antibiotics has led to increasingly serious pollution of antibiotic resistance genes (ARGs) in aquaculture wastewater. Existing technologies face the following significant challenges in treating wastewater containing ARGs: Incomplete removal and secondary pollution: Traditional physical adsorption or membrane separation only achieves phase transfer of pollutants without degradation; although advanced oxidation methods can kill drug-resistant bacteria, they often lead to cell rupture and release of extracellular free DNA (eARGs) with transforming activity, causing potential genetic pollution to spread.

[0010] Incubator effect of biological treatment units: In conventional activated sludge or biofilm processes, high-density microbial aggregation and sub-lethal concentration pressure of residual antibiotics can easily induce horizontal gene transfer (HGT) of ARGs, especially through plasmid conjugation transfer, resulting in an increase in the abundance of ARGs in the effluent instead of a decrease.

[0011] Poor targeting and high operating costs: Existing methods for adding bactericides or nanomaterials lack targeting, are easily consumed by non-target organic matter in the water, and have broad-spectrum toxicity to the environmental microbial community with continuous addition, resulting in high operating costs and questionable ecological safety.

[0012] To address the aforementioned problems, this invention provides an ecosystem and method for removing antibiotic resistance genes from aquaculture water based on multiphase flow hierarchical synergistic regulation. This invention aims to construct a complete control system encompassing intracellular clearance, extracellular adsorption, transfer blocking, and terminal inactivation through physical cascade interception, intelligent targeted blocking in anaerobic microenvironments, and deep mineralization via aerobic algae-bacteria symbiosis, thereby achieving deep purification and resource recovery of aquaculture wastewater.

[0013] (2) Technical solution To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A system for removing antibiotic resistance genes from aquaculture water, characterized in that the system comprises, in sequence along the water flow direction, a physical filtration unit, an anaerobic treatment unit, and an aerobic algae and bacteria treatment unit; The physical filtration unit is constructed as a multi-media deep bed filtration device, which is filled with a volcanic rock layer, a quartz sand layer, a magnetite filter media layer and a zeolite layer with decreasing particle size gradient along the water flow direction. The magnetite filter media layer is used to create a micro magnetic field environment to stimulate microbial enzyme activity, and the particle size gradient is used to form graded hydraulic shear force to intercept suspended particles and attached ARGs of different sizes. The anaerobic treatment unit is configured as a baffled or upflow reactor, and its reaction zone is vertically divided into an upper slow-release control zone and a lower functional biochemical zone. The slow-release control zone is filled with pH-responsive slow-release microcapsules, which have the characteristic of swelling and releasing active ingredients in an acidic microenvironment. The wall material is a polyelectrolyte complex, and the core is coated with zinc oxide nanoparticles and tea polyphenol complex. The aerobic algae and bacteria treatment unit is a photobioreactor, inoculated with a symbiotic community of functional microalgae and aerobic bacteria acclimated by plant growth regulators. The plant growth regulators are used to regulate the permeability of the microalgal cell walls and the secretion of extracellular polymeric substances (EPS) to enhance the uptake and degradation of antibiotics and the adsorption and flocculation of free ARGs.

[0014] Preferably, the system further includes a high-temperature anaerobic digestion device located downstream of the aerobic algae and bacteria treatment unit. The device is connected to the sludge discharge port of the aerobic algae and bacteria treatment unit and is used to perform hot hydrolysis and anaerobic digestion of the remaining algae and bacteria sludge enriched with ARGs in a high-temperature environment of 53℃-57℃.

[0015] Preferably, the pH-responsive sustained-release microcapsules use a sodium alginate-chitosan polyelectrolyte complex as the wall material, utilizing the amino protonation properties of chitosan to respond to pH changes; the mass ratio of tea polyphenols to zinc oxide nanoparticles in the core is 1:(0.5-0.8); the microcapsules are configured to: accelerate the release of zinc oxide and tea polyphenols when local acidification of the anaerobic unit causes pH < 6.5; and maintain a low release or no release state when pH > 7.0.

[0016] Preferably, the anaerobic treatment unit is also equipped with an intelligent pH feedback control system, which includes an online pH sensor and an alkali dosing device; when the pH value of the slow-release control zone is detected to be lower than a preset safety threshold (such as pH 6.0), the alkali dosing is automatically triggered to prevent the explosive release of microcapsules and microbial poisoning caused by excessive acidification.

[0017] Preferably, the plant growth regulator added in the aerobic algae treatment unit is selected from indoleacetic acid (IAA) or 6-benzylaminopurine (6-BA), and its working concentration is maintained at 0.5-5 mg / L.

[0018] This invention also provides a method for removing antibiotic resistance genes from aquaculture water using the above-mentioned system, comprising the following steps: Step S1: Multi-stage physical interception and magnetic biological effect activation. The aquaculture wastewater is introduced into the physical filtration unit, where the multi-stage porous structure removes suspended solids and drug-resistant bacteria attached to them; at the same time, the micro-magnetic field effect of the magnetite filter media layer is used to enhance the dehydrogenase activity of microorganisms in the water, pre-activating them for subsequent biochemical treatment. Step S2: Anaerobic acid production coupled with targeted blocking. Filtered water enters the anaerobic treatment unit, where functional anaerobic bacteria hydrolyze and acidify, lowering the local pH and triggering the release of tea polyphenols and zinc oxide from pH-responsive microcapsules. Zinc oxide nanoparticles disrupt the cell walls of drug-resistant bacteria, increasing permeability, and synergistically, allow tea polyphenols to enter the cell and inhibit the expression of plasmid conjugation transfer genes (tra, trb), thus blocking the horizontal transfer of ARGs at the source. Step S3: Hormone-enhanced algal-bacterial symbiotic purification. Anaerobic effluent enters the aerobic algal-bacterial treatment unit. Under the action of plant growth regulators, the photosynthetic oxygen release and mixed nutrient metabolism of functional microalgae are enhanced, deeply degrading residual antibiotics; at the same time, microalgae are induced to secrete excessive extracellular polymers (EPS), and the free DNA (eARGs) in the water are adsorbed and removed by the net-trapping and bridging effect of EPS. Step S4: Sludge-water separation and pyrolysis inactivation. The treated supernatant meets the discharge standards; the algal flocs containing ARGs enrichment are transported to a high-temperature anaerobic digester, where high-temperature enzymatic hydrolysis completely destroys the DNA structure, eliminates biosafety risks, and recovers biogas energy.

[0019] (3) Beneficial effects Compared with the prior art, the present invention has the following significant advantages: Mechanism innovation enables precise targeting of ARGs both internally and externally: This invention innovatively constructs a synergistic mechanism of ZnO cell disruption and tea polyphenol gene silencing. Zinc oxide nanoparticles disrupt bacterial cell membranes, opening channels for tea polyphenols to enter the cell; tea polyphenols specifically inhibit the expression of DNA gyrase and conjugation transfer genes. This combination of physical damage and molecular inhibition not only kills drug-resistant bacteria (intracellular ARGs) but, more importantly, blocks the horizontal diffusion pathway of drug-resistant plasmids.

[0020] Intelligent response resolves the conflict between drug toxicity and cost: Utilizing pH-responsive microencapsulation technology, and taking advantage of the metabolically active acid-producing characteristics of anaerobic biochemical reactions, intelligent drug delivery is achieved where active bacterial communities are present. This avoids the waste of drugs caused by indiscriminate dosing and prevents the toxicity of excessive metal ions to functional microorganisms, significantly reducing operating costs.

[0021] Ecological enhancement overcomes the challenge of removing cell-free DNA (eARGs): Plant growth regulators (IAA / CKs) are introduced to regulate the algae-microbe symbiotic system. IAA not only promotes microalgae growth and antibiotic degradation, but more importantly, significantly increases the secretion of extracellular polymeric substances (EPS) by microalgae. High concentrations of EPS act like a biological web, efficiently adsorbing and immobilizing extremely difficult-to-remove cell-free DNA fragments in the water, transferring them from the liquid phase to the solid sludge, thus greatly reducing the risk of genetic pollution in the effluent.

[0022] The entire closed-loop system achieves the unity of pollution control and resource utilization: it integrates four stages: physical interception, chemical blocking, biodegradation, and pyrolysis inactivation. The high-temperature anaerobic digestion at the end not only completely solves the secondary pollution problem of ARGs-enriched sludge (high-temperature hydrolysis of DNA), but also produces biogas energy that is recycled for system heating, embodying the concept of a green, low-carbon, and circular economy. Attached Figure Description

[0023] Appendix Figure 1This is a schematic diagram of the overall process flow and structure of the system for removing antibiotic resistance genes from aquaculture water provided in an embodiment of the present invention. In the diagram: 1-Inlet, 2-Physical filtration unit, 21-Volcanic rock layer, 22-Quartz sand layer, 23-Magnetite filter media layer, 24-Zeolite layer, 3-Anaerobic treatment unit, 31-Slow-release microcapsule layer, 32-Functional anaerobic bacteria layer, 33-pH monitoring and control device, 4-Aerobic algae and bacteria treatment unit, 41-Functional microalgae, 42-Aerobic bacteria, 43-Aeration device, 5-Outlet, 6-High-temperature anaerobic digestion device.

[0024] Appendix Figure 2 This is a schematic diagram of the microstructure and release mechanism of the pH-responsive sustained-release microcapsules described in this invention. The diagram shows the process under acidic conditions (pH < 6.5), where protonation of chitosan segments leads to an increase in capsule pore size, accelerating the release of the core material (ZnO NPs and tea polyphenols); and under neutral conditions (pH > 7.0), the capsule structure is dense, resulting in restricted release.

[0025] Appendix Figure 3 This is a bar chart comparing the removal effects of different processing units on various ARGs (tetA, sul1, ermB) and horizontal transfer indicator genes (intI1) in embodiments of the present invention.

[0026] Appendix Figure 4 This is a comparison chart of the growth curves and extracellular polymeric substances (EPS) secreted by microalgae before and after the addition of plant growth regulator (IAA) in an embodiment of the present invention. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the 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.

[0028] (1) Example of core material preparation and pretreatment Example A: Preparation of pH-responsive sustained-release microcapsules This embodiment prepares pH-responsive smart microcapsules for anaerobic treatment units, and the specific steps are as follows: Preparation of core material suspension: Weigh 5.0 g of 98% pure tea polyphenol (TP) powder and dissolve it in 100 mL of deionized water; add 3.0 g of zinc oxide nanoparticles (ZnO NPs) with an average particle size of 30 nm. Disperse the ZnO uniformly in the tea polyphenol solution using an ultrasonic cell disruptor at 400 W for 30 min to obtain the core material suspension.

[0029] Preparation of wall material matrix: Weigh 2.0g of low-viscosity sodium alginate (SA) and dissolve it in 100mL of 60℃ warm water, stirring mechanically until completely dissolved; separately weigh 1.0g of chitosan (CS, degree of deacetylation 90%) and dissolve it in 100mL of 1% (v / v) acetic acid solution. Mix the two solutions at a volume ratio of 1:1, allow to stand to defoam, and obtain the polyelectrolyte wall material solution.

[0030] Microcapsule Forming (Sharp-Orifice Coagulation Bath Method): The core material suspension and wall material solution are mixed uniformly at a volume ratio of 1:3. Using a high-voltage electrostatic microcapsule granulator, the mixture is dripped through a 0.4 mm orifice nozzle into a coagulation bath containing 2% CaCl₂ and pH 5.0 acetate buffer. The droplets undergo CaCl₂-SA crosslinking and CS-SA polyelectrolyte complexation reactions in the coagulation bath, with a curing time of 2 hours.

[0031] Post-processing: The collected microcapsules were filtered, washed three times with deionized water to remove residual reagents on the surface, and finally freeze-dried under vacuum at -40°C for 24 hours to obtain the finished microcapsules.

[0032] Performance characterization: The average particle size of the microcapsules was determined to be 1.5-2.0 mm. In a simulated anaerobic acidification solution at pH 6.0, the cumulative release rate reached 78.5% after 48 hours; while in a neutral environment at pH 7.2, the release rate was only 15.2% during the same period, demonstrating significant acid-sensitive response characteristics.

[0033] Example B: Domestication of Hormone-Enhanced Functional Algae The specific steps for preparing the inoculum agent for the aerobic algae and bacteria treatment unit in this embodiment are as follows: Microalgae propagation: Selected *Crescentia serrata* ( Raphidocelis subcapitata Scenedesmus obliqueis ( Scenedesmus obliquus ) and common Chlorella ( Chlorella vulgaris As the starting algal strain, cells were inoculated into BG11 medium at a cell count ratio of 1:1:1. Culture conditions: light intensity 3000 lux, light-dark ratio 12h:12h, temperature 251℃.

[0034] Hormone-induced acclimatization: After the algal solution entered the logarithmic growth phase, the plant growth regulator indoleacetic acid (IAA) was added to the culture medium at an initial concentration of 0.5 mg / L, and replenished every 3 days, with the concentration gradually increased to 2.0 mg / L. After three consecutive subcultures, dominant algal strains exhibiting significantly increased extracellular polymeric substance (EPS) secretion under IAA induction (more than 80% higher than before acclimatization) were selected.

[0035] Microbial agent compounding: The acclimated mixed microalgae liquid is mixed with aerobic denitrifying bacteria (mainly Bacillus subtilis) screened from activated sludge at a biomass (dry weight) ratio of 3:1, and used as inoculum after 24 hours of pre-acclimation.

[0036] (2) System Construction Implementation Example Example 1: System construction for removing ARGs from aquaculture water Reference manual attached Figure 1 A pilot-scale system with a daily processing capacity of 5 m³ / d was constructed, and the system was connected sequentially along the water flow direction: Physical filtration unit: Structure: Vertical flow multi-media deep bed filter, total height 2.0m, diameter 0.6m.

[0037] Packing material gradation: Filled sequentially from top to bottom: First layer: Volcanic rock layer (particle size 3-5cm, thickness 40cm), used to trap large particles of uneaten bait and feces; The second layer: a quartz sand layer (particle size 2-4cm, thickness 40cm), used to intercept fine suspended matter; The third layer: magnetite filter media layer (particle size 1-2cm, thickness 40cm), which creates a micro magnetic field environment (magnetic field strength about 10-50 mT) to stimulate microbial enzyme activity; Fourth layer: Zeolite layer (particle size 0.5-1cm, thickness 40cm), adsorbs ammonia nitrogen.

[0038] A stainless steel screen with a 2mm aperture is installed between the layers to prevent mixing.

[0039] Anaerobic treatment unit: Structure: Baffled anaerobic reactor (ABR), effective volume 2.0m³.

[0040] Zoning: The reactor is vertically divided into a lower functional biochemical zone and an upper slow-release regulation zone.

[0041] Filling: Anaerobic granular sludge is inoculated in the functional biochemical zone; a polyethylene mesh cage is set in the slow-release control zone, and the inside is filled with pH-responsive microcapsules prepared in Example A, with a filling density of 100 g / m.

[0042] Intelligent control system: Equipped with an online pH sensor and alkali metering pump. The control logic is set to automatically start the metering pump to add saturated lime water when the pH at the monitoring point is less than 6.0, until the pH rises back to 6.8, preventing excessive acidification that could lead to explosive release of microcapsules and microbial acidosis.

[0043] Aerobic algae and bacteria treatment unit: Structure: racetrack-shaped photobioreactor with an effective volume of 4.0 m³ and a water depth of 0.8 m.

[0044] Configuration: A microporous aeration pipe is laid at the bottom to control dissolved oxygen (DO) at 3.0-5.0 mg / L; an LED supplemental light is installed at the top.

[0045] Inoculation: The hormone-enhanced functional algae culture prepared in Example B was inoculated at an initial inoculation concentration of MLSS = 1500 mg / L.

[0046] Dosing device: Equipped with a micro-flow pump for periodically adding IAA mother liquor.

[0047] High-temperature anaerobic digestion apparatus: Connection: The sludge inlet is connected to the bottom of the sedimentation zone of the aerobic unit via a sludge pump.

[0048] Parameters: Equipped with an electric heating jacket, the temperature control system maintains the reaction temperature at 551℃.

[0049] (3) Application and Effect Verification Examples Example 2: System operation and ARGs removal effect test The system constructed in Example 1 was used to treat the wastewater from an intensive Litopenaeus vannamei farming base in Jiangsu Province.

[0050] Operating parameters: Hydraulic load of physical filtration unit: 1.0 m / (mh); Hydraulic retention time (HRT) of anaerobic unit: 24 hours; HRT of aerobic unit: 48 hours; Plant growth regulator dosing: IAA is pulsedly added to aerobic unit every 5 days to achieve an instantaneous concentration of 1.5 mg / L.

[0051] Influent water quality characteristics: COD 120-180 mg / L, ammonia nitrogen 5-10 mg / L, tetracycline resistance gene (tetA) abundance copies / mL, abundance of sulfonamide resistance gene (sul1) copies / mL, abundance of integrase gene (intI1) copies / mL.

[0052] Treatment effect (average value after 60 days of continuous stable operation): Results analysis: This system achieved a five-order-of-magnitude (Log Removal Value > 5) removal efficiency for resistant genes (ARGs), significantly outperforming traditional activated sludge processes (typically 1-2 log). In particular, the indicator gene intI1, representing horizontal gene transfer, was significantly reduced, confirming that the tea polyphenol-ZnO complex effectively inhibited conjugation and transfer processes in the anaerobic unit.

[0053] Example 3: Comparative experiment on the removal of cell-free DNA (eARGs) by plant growth regulators (IAA). To verify the mechanism by which plant growth regulators enhance the adsorption of free DNA by EPS, two parallel experiments were set up.

[0054] Experimental group: Operated according to Example 2, with IAA (1.5 mg / L) added periodically.

[0055] Control group: No IAA was added, and all other conditions were exactly the same.

[0056] Comparison of experimental results: Conclusion: Experimental data strongly demonstrate that the addition of IAA not only promotes algal growth, but more importantly, it acts as a signaling molecule to induce microalgae to secrete highly viscous EPS. These EPS, rich in functional groups, act like bio-glue, efficiently capturing free DNA (eARGs) released from cell lysis in the water and transferring them from the liquid phase to the solid sludge, thus solving the problem of sterilization without gene removal in traditional processes.

[0057] Example 4: Impact Load Test On the 30th day of system operation, to simulate an outbreak of antibiotic pollution, a high concentration of oxytetracycline (5 mg / L, about 100 times the normal value) was added to the influent in a single dose.

[0058] Phenomenon: The pH of the anaerobic unit dropped from 7.1 to 6.3 within 6 hours (microorganisms were inhibited from producing acid).

[0059] Response: Microcapsules sense pH changes, zinc ions The release rate automatically spiked from 0.05 mg / (Lh) to 0.25 mg / (Lh).

[0060] Results: Despite the dramatic increase in influent antibiotic concentration, effluent ARGs did not show explosive growth (only fluctuated briefly before recovering), indicating that the system's pH response mechanism successfully achieved on-demand drug delivery, providing the maximum dose of inhibitor at the moment when the microbial community was most vulnerable (most susceptible to stress-induced horizontal shifts).

[0061] In summary, this invention addresses the industry challenge of deeply removing antibiotic resistance genes from aquaculture wastewater through a system integration of physical graded interception, anaerobic intelligent targeted blocking, aerobic hormone-enhanced symbiosis, and end-of-pipe high-temperature pyrolysis. In particular, the innovative technologies of pH-responsive microcapsules and plant growth regulators achieve technological breakthroughs by blocking HGT at the source and adsorbing eARGs at the end, respectively, demonstrating significant potential for widespread application.

Claims

1. A system for removing antibiotic resistance genes from aquaculture water, characterized in that, The system is connected in sequence along the water flow direction, including a physical filtration unit (2), an anaerobic treatment unit (3), and an aerobic algae and bacteria treatment unit (4). The physical filtration unit (2) is a multi-media deep bed filtration device. Its interior is filled with a volcanic rock layer (21), a quartz sand layer (22), a magnetite filter media layer (23), and a zeolite layer (24) with decreasing particle size gradients along the water flow direction, in order to construct a retention environment with different hydraulic shear force gradients. The anaerobic treatment unit (3) is a baffled or upflow anaerobic reactor, and its reaction zone is divided vertically into an upper slow-release control zone and a lower functional biochemical zone. The slow-release control zone is filled with a pH-responsive slow-release microcapsule layer (31). The microcapsules use sodium alginate-chitosan polyelectrolyte complex as the wall material and the core is coated with zinc oxide nanoparticles and tea polyphenol complex. The functional biochemical zone is inoculated with a functional anaerobic bacteria layer (32), which includes denitrifying phosphorus-removing bacteria, denitrifying thiobacilli and methanogenic bacteria. The aerobic algae and bacteria treatment unit (4) is a photobioreactor, which is inoculated with a symbiotic community of functional microalgae (41) and aerobic bacteria (42) that have been domesticated by plant growth regulators, and is equipped with a microporous aeration system to maintain the dissolved oxygen concentration.

2. The system according to claim 1, characterized in that, The particle size and filling parameters of each filter layer in the physical filtration unit (2) are set as follows: volcanic rock layer with a particle size of 3-5cm and a porosity of 40%-50%; quartz sand layer with a particle size of 2-4cm, mainly used to intercept large suspended particles; magnetite filter layer with a particle size of 1-2cm, which has a weak magnetic field effect to stimulate microbial enzyme activity; zeolite layer with a particle size of 0.5-1cm, used for specific adsorption of ammonia nitrogen; the thickness ratio of each layer is 1:1:1:1, and anti-mixing screens are provided between the layers.

3. The system according to claim 1, characterized in that, The anaerobic treatment unit (3) is also equipped with an intelligent pH feedback control system (33), which includes an online pH sensor, a logic controller and an alkali metering pump. The logic controller is configured to: when the pH value of the slow-release control zone is detected to be lower than 6.5, trigger the alkali metering pump to add a small amount of saturated quicklime solution to the zone until the pH rises back to the range of 6.8-7.2, so as to adjust the swelling degree and release rate of the pH-responsive slow-release microcapsules and prevent microbial toxicity caused by excessive release of active ingredients.

4. The system according to claim 1, characterized in that, The pH-responsive sustained-release microcapsules are characterized by the following preparation method: using a sodium alginate solution with a mass concentration of 1.5%-2.5% and a chitosan acetic acid solution with a mass concentration of 0.5%-1.0% as the matrix, the microcapsules are prepared by a sharp-pore coagulation bath method; the zinc oxide nanoparticles in the core have a particle size of 20-50 nm, the tea polyphenol purity is greater than 98%, and the mass ratio of tea polyphenols to zinc oxide nanoparticles is 1:(0.5-0.8); the cumulative release rate of the microcapsules at pH 5.0 is significantly higher than that at pH 7.

0.

5. The system according to claim 1, characterized in that, The functional microalgae (41) inoculated in the aerobic algae and bacteria treatment unit (4) are selected from one or more combinations of Crescentella asiatica, Scenedesmus obliquus, or Chlorella vulgaris; the aerobic bacteria (42) include Bacillus subtilis, photosynthetic bacteria, and nitrifying bacteria; the plant growth regulator is indoleacetic acid or 6-benzylaminopurine, and its working concentration in the reactor water is maintained at 0.5-5 mg / L.

6. The system according to claim 1, characterized in that, The system also includes a high-temperature anaerobic digestion device (6) located downstream of the aerobic algae treatment unit (4). The device is equipped with a sludge inlet connected to the sludge discharge pipe of the aerobic algae treatment unit. The high-temperature anaerobic digestion device (6) is equipped with a temperature control heating system to maintain the digestion temperature at 53℃-57℃ and is used to process regularly harvested microalgae biomass rich in antibiotic resistance genes.

7. A method for removing antibiotic resistance genes from aquaculture water using the system described in any one of claims 1-6, characterized in that, Includes the following steps: Step S1: Multi-stage physical interception. The aquaculture wastewater is introduced into the physical filtration unit, which utilizes the multi-stage porous structure of volcanic rock, quartz sand, magnetite and zeolite to not only remove suspended solids, but also to adsorb some of the attached antibiotic-resistant bacteria using the biofilm on the surface of the filter media; Step S2: Anaerobic Targeted Blocking. Filtered water enters the anaerobic treatment unit. During the upward flow, it first contacts the functional anaerobic bacteria layer, causing organic matter hydrolysis and acidification, resulting in a local pH decrease. Subsequently, the water flows into the slow-release microcapsule layer. The acidic environment triggers the protonation and swelling of the chitosan wall material of the microcapsule, releasing tea polyphenols and zinc oxide. Tea polyphenols inhibit the binding and transfer of bacterial plasmids, while zinc oxide destroys the cell walls of drug-resistant bacteria. Together, they reduce intracellular ARGs. Step S3: Aerobic Deep Purification. Anaerobic effluent enters the aerobic algae and bacteria treatment unit. Under the stimulation of light and exogenous plant growth regulators, functional microalgae provide oxygen to aerobic bacteria through photosynthesis and directly absorb and degrade small molecule antibiotics through a mixed nutrient mode. The bioflocs formed by the algae and bacteria symbiosis further adsorb and remove free ARGs. Step S4: Resource recovery and harmlessness. The treated water that meets the standards is reused or discharged; the microalgae sludge proliferated in the aerobic unit is periodically transported to the high-temperature anaerobic digester, where thermophilic bacteria and high-temperature hydrolytic enzymes destroy the algal cell walls and degrade the intracellular ARGs, and the biogas produced is used for energy recovery.

8. The method according to claim 7, characterized in that, In step S2, the initial dosage of the slow-release microcapsules is 80-100 mg / L of the effective volume of the reactor, and is periodically replenished according to the zinc ion concentration in the effluent; the hydraulic retention time of the anaerobic treatment unit is controlled at 20-28 hours.

9. The method according to claim 7, characterized in that, In step S3, the plant growth regulator is administered in a pulsed manner, once every 5-7 days, and the concentration in the water is maintained at 1.0-2.0 mg / L after administration to enhance the permeability of the microalgal cell wall and the secretion of extracellular polymers.

10. The method according to claim 7, characterized in that, In step S4, the solid retention time during the high-temperature anaerobic digestion process is controlled to be 15-20 days, and the concentration of volatile fatty acids in the digestate is controlled to be below 3000 mg / L, ensuring that the removal rate of ARGs and residual antibiotics reaches more than 99.9%.

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