Method for removing antibiotics from seawater aquaculture wastewater based on oyster shell composite filler

Abstract

The present application belongs to the technical field of wastewater treatment, and particularly relates to a seawater breeding wastewater antibiotic removal method based on oyster shell composite filler. Fine gravel, oyster shell and functional mineral are compounded in a fixed volume ratio of 2:1:1, and are laid in a structure with a limited particle size range and homogeneity without stratification. A salinity-directed salt-tolerant domestication process and a tidal intermittent operation mode of 48h hydraulic retention + 24h emptying and reoxygenation are matched, and salt-tolerant functional bacterial flora of Proteobacteria, Paracoccus and Shigella are enriched. The present application can adapt to a wide range of salinity conditions of 10‰-30‰, relieve high salt inhibition and ion competition, simultaneously and efficiently remove three typical antibiotics of conventional pollutants, sulfamethoxazole, trimethoprim and enrofloxacin, and reduce resistance genes. The sulfamethoxazole removal rate can reach 93.94% under the condition of 30‰ high salinity, the system runs stably and has strong adaptability, and has certain engineering application value and large-scale popularization potential in the field of seawater breeding wastewater treatment.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, and in particular relates to a method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler. Background Technology

[0002] Marine aquaculture is a crucial pillar of my country's aquaculture industry, with domestic production reaching 23.956 million tons in 2023. However, with the increasing intensification of aquaculture, the problem of antibiotic overuse in aquaculture waters has become prominent. Substances such as sulfamethoxazole, enrofloxacin, and trimethoprim persist, causing not only chemical pollution of the waters but also inducing antibiotic resistance genes, posing long-term safety risks to nearshore marine ecosystems.

[0003] Constructed wetland ecological treatment technology is widely used in the treatment of aquaculture wastewater due to its advantages such as economy, environmental friendliness, and ease of operation and maintenance. This technology relies on substrate adsorption and the synergistic effect of microorganisms to effectively degrade nitrogen, phosphorus, and antibiotic pollutants in water. Currently, oyster shells are often used as substrates for improving constructed wetlands due to their excellent physicochemical properties. However, most existing related technologies are developed around low-salinity freshwater environments, and enhanced treatment technologies specifically developed for the high-salinity and high-ionic-strength characteristics of marine aquaculture wastewater are still relatively scarce.

[0004] Unlike freshwater aquaculture wastewater, marine aquaculture wastewater exhibits a wide range of salinity fluctuations, with significant differences in salinity across different aquaculture species. Salinity is a key factor determining the treatment efficiency of constructed wetlands for marine water. The osmotic pressure generated by high-salinity environments severely inhibits the activity of nitrifying microorganisms, hindering nitrogen conversion in the water. Simultaneously, the large number of anions and cations in the water compete with antibiotic molecules for adsorption sites on the packing material. Combined with changes in the ionic form of antibiotics themselves, this further reduces the adsorption and retention efficiency of the packing material for antibiotics. Currently, the industry lacks sufficient understanding of the purification patterns and microbial response characteristics of constructed wetlands under different salinity gradients, and lacks mature technologies that can adapt to a wide salinity range and simultaneously remove multiple types of antibiotics and resistance genes.

[0005] Therefore, this invention focuses on marine aquaculture as its core application, selecting three frequently detected antibiotics—SMX, TMP, and ENR—as target substances. First, multiple packing systems were screened under freshwater conditions to identify two composite packing systems with outstanding overall performance. Then, comparative experiments were conducted at three typical seawater salinities of 10‰, 20‰, and 30‰ to examine the removal effects of different packing combinations on conventional pollutants and compound antibiotics in marine aquaculture wastewater. Furthermore, metagenomic sequencing was used to analyze the evolution of microbial community structure and the distribution patterns of resistance genes under salinity gradient changes, revealing the microbial mechanism of antibiotic degradation in salinity-stressed environments and filling the gaps in related technologies and theories under high-salinity seawater conditions.

[0006] Chinese Patent 202311090298.5 discloses a method for removing antibiotics from marine aquaculture wastewater using artificial wetlands. This method uses pure oyster shells as a single filler to construct a marine artificial wetland, which is only suitable for a single fixed salinity. It does not introduce zeolite or pyrite for filler compounding and parallel comparison, and cannot evaluate the application differences of different functional minerals in multi-salinity environments. This scheme only targets conventional pollutants such as nitrogen and phosphorus, and does not optimize the process for three types of compound antibiotics such as SMX, TMP, and ENR. It also does not set up multi-level salinity gradient tests, and does not explore problems such as adsorption competition and microbial activity inhibition caused by salinity.

[0007] Chinese patent 202211618426.4 discloses a rapid start-up method for a moving bed biofilm reactor for marine aquaculture wastewater. Although it uses oyster shell composite packing to treat marine aquaculture wastewater, pyrite is not added to the packing components, making it impossible to construct a sulfur autotrophic denitrification system, resulting in insufficient stability of nitrate nitrogen removal under high salinity conditions. This scheme only conducts experiments for a single salinity level, does not cover multiple salinity ranges, does not simultaneously track antibiotic resistance genes, and does not utilize metagenomic technology to analyze the enrichment patterns of typical salt-tolerant functional bacteria such as Paracoccus and Schistosoma, showing significant deficiencies in both treatment dimensions and depth of mechanism research.

[0008] Chinese Patent 202211355600.0 discloses a method for removing typical antibiotics from aquaculture wastewater using an artificial wetland system. It uses oyster shells combined with activated carbon as the main filler material, without adopting a modified compound scheme of zeolite and pyrite. Furthermore, the system adopts a continuous water intake operation mode and does not adopt a tidal intermittent operation system of 48h hydraulic retention + 24h drainage and settling. Its treatment targets are mainly conventional pollutants and single quinolone antibiotics, making it difficult to achieve synergistic purification of three types of antibiotics: SMX, TMP, and ENR. At the same time, it does not have a wide range of salinity adaptation design, which limits the applicable scenarios of the technology.

[0009] Currently, most mainstream oyster shell-based artificial wetlands for seawater use a single filler or a simple compounding method, without designing a 10‰ to 30‰ gradient adaptation scheme to address the large fluctuations in seawater salinity, making it difficult to operate stably within a wide salinity range. Traditional technologies have a single treatment target, only able to treat conventional nutrients or a single type of antibiotic, and cannot achieve the synergistic removal of three typical antibiotics and resistance genes. At the same time, existing technologies lack parallel screening designs with multiple fillers and have not incorporated microbiome technology to elucidate the evolution of bacterial communities and the mechanism of pollutant removal under salinity stress.

[0010] In response to the characteristics of high salinity, variable salinity, and complex compound pollution in marine aquaculture wastewater, and to address the many shortcomings of existing technologies, it is urgent to develop an artificial wetland treatment technology that features a rationally matched packing material, adaptability to multiple salinity levels, and a proprietary operating process, capable of simultaneously purifying conventional pollutants, compound antibiotics, and controlling resistance genes. Summary of the Invention

[0011] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is to provide a method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite packing. This method is adaptable to a wide range of salinity gradients from 10‰ to 30‰, effectively alleviates the problems of microbial activity inhibition and ion adsorption competition caused by high salinity, and simultaneously and efficiently removes conventional pollutants, three typical antibiotics (sulfamethoxazole, trimethoprim, and enrofloxacin), while reducing antibiotic resistance genes. The system exhibits strong operational stability, excellent salt tolerance, scientific packing material compatibility, and highly targeted process. It can also reveal the evolution of microbial communities and the mechanism of pollutant removal under salinity stress.

[0012] This invention belongs to the field of packing material shaping, salt-tolerant bacterial community reconstruction, and anti-inhibition enhanced purification technology under high salt stress conditions. Existing packing materials and supporting processes used in freshwater constructed wetlands are only suitable for salt-free environments and cannot adapt to the high salt conditions of 10‰–30‰ in marine aquaculture wastewater. The strong osmotic pressure generated by the high salt environment significantly inhibits the activity of conventional microorganisms, and the high content of phosphate and nitrate ions in the water also competes with antibiotics for adsorption sites on the packing material, resulting in decreased antibiotic removal efficiency and system instability.

[0013] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite packing material, comprising the following steps:

[0014] S1. Selection of composite fillers: Two types of composite fillers suitable for high-salinity and complexly polluted seawater environments are selected: fine gravel-oyster shell-zeolite and fine gravel-oyster shell-pyrite. Both composite fillers are homogeneously mixed from fine gravel, oyster shell, and functional mineral additives in a volume ratio of 2:1:1, without layered structure. The particle size of each component is as follows: fine gravel 5-10mm, oyster shell 1-2mm, zeolite 3-5mm, and pyrite 1-2mm.

[0015] S2. Preparation of graded salinity marine aquaculture wastewater: Using freshwater synthetic aquaculture wastewater as a base, sea salt is added to prepare marine aquaculture wastewater with different salinities.

[0016] S3. Construct salt-tolerant artificial wetland devices: Construct multiple sets of artificial wetland devices, each set having a water storage layer, a fine gravel layer, a filler layer, and a drainage layer from top to bottom; Homogenize the above-mentioned composite filler into the filler layer respectively, and the device is suitable for seawater aquaculture wastewater with a salinity of 10‰ to 30‰.

[0017] S4. Salinity-specific acclimatization of microorganisms: Activated sludge with matching salinity is introduced into the device with the corresponding salinity and continuously acclimatized for 30 days in a constant temperature environment of 23.0±0.5℃ to enrich salt-tolerant microorganisms and form a biofilm adapted to the high-salt environment.

[0018] S5. Intermittent dry and wet alternation operation: The system adopts an intermittent cycle mode of quantitative water pumping, static retention, and emptying and settling for continuous operation.

[0019] In the above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler, in step S2, 10.0g, 20.0g, and 30.0g of sea salt are added to each liter of freshwater synthetic aquaculture wastewater, respectively. After stirring and dissolving, marine aquaculture wastewater with salinity of 10‰, 20‰, and 30‰ is obtained accordingly.

[0020] In the above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler, in step S3, the artificial wetland device has the following specifications: inner diameter 20cm, total height 50cm, wall thickness 0.5cm, and an outlet is set at the bottom 2cm from the bottom surface; wherein the water storage layer is 5cm high, the fine gravel layer is 5cm high and uses gravel with a particle size of 0.5-1.0cm, the filler layer is 30cm high, and the drainage layer is 5cm high and uses gravel with a particle size of 1.0-2.0cm.

[0021] The above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler operates under a constant temperature of 23.0±0.5℃ throughout step S5. The single-cycle operation process is as follows: 4.25L of marine aquaculture wastewater is pumped in using a peristaltic pump at a flow rate of 23.67mL / min for 3 hours; after the water intake is completed, the water is statically hydraulically retained for 48 hours, and then the wastewater is drained and left to stand for 24 hours. This constitutes one complete operating cycle, and the cycle is repeated 10 times.

[0022] In the above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite packing material, the salt-tolerant biofilm obtained in step S4 is attached to the surface of the composite packing material, and simultaneously degrades conventional pollutants and residual antibiotics in the wastewater during the wastewater treatment process.

[0023] The above-mentioned method for antibiotic removal from marine aquaculture wastewater based on oyster shell composite packing material utilizes the fine gravel-oyster shell-pyrite composite packing material to alleviate the inhibitory effect of nitrate nitrogen removal under high salinity conditions through pyrite-mediated sulfur autotrophic denitrification, thereby reducing the adsorption competition between ions in the water and antibiotic molecules on the surface of the packing material.

[0024] The above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler enhances the retention effect of antibiotic molecules in a high-salt environment and helps improve the stability of antibiotic removal during the wastewater treatment process.

[0025] The above-mentioned method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler simultaneously reduces the abundance of antibiotic resistance genes in marine aquaculture wastewater during the wastewater treatment process, thereby reducing the ecological risks of resistance gene enrichment and horizontal spread.

[0026] The advantages of this invention's method for antibiotic removal from marine aquaculture wastewater based on oyster shell composite packing material are as follows: This invention abandons the approach of simply screening packing materials in freshwater systems, and instead conducts high-salt adaptation and modification of the selected packing materials under freshwater conditions, along with a complete process reconstruction. This invention uses a fixed volume ratio of fine gravel, oyster shells, and functional minerals in a 2:1:1 mixture, combined with a limited particle size range and a homogeneous, non-layered laying structure, which can better adapt to the hydraulic characteristics and adsorption environment of high-salt wastewater, improving the packing material system's adaptability to seawater conditions. Simultaneously, this invention conducts phased salt tolerance acclimatization based on different salinity gradients, gradually optimizing the microbial community structure, weakening the incompatibility of native freshwater microorganisms, and selectively enriching salt-tolerant functional microorganisms such as Proteobacteria, Paracoccus, and Schistosoma, constructing a more stable microbial community adapted to the seawater environment. By leveraging the salt tolerance characteristics of pyrite sulfur autotrophic denitrification and the adsorption compensation advantages of zeolite, the adverse effects of high-salt osmotic pressure stress and ion adsorption competition can be mitigated to some extent, which is beneficial to improving the overall purification stability of wide-salinity marine aquaculture wastewater.

[0027] This invention optimizes the filler ratio, particle size, and laying structure to enable the composite filler system to better adapt to seawater salinity fluctuations of 10‰ to 30‰, overcoming the shortcomings of traditional freshwater fillers that are difficult to adapt to high-salinity environments. By reconstructing a salt-tolerant microbial community based on a gradient salinity acclimatization method, the system's tolerance to high salinity osmotic pressure is enhanced, ensuring the continuous and stable operation of nitrification and denitrification. Simultaneously, the synergistic effect of functional minerals effectively alleviates the adsorption competition between water ions and antibiotics, reducing the inhibitory effect of high-salinity environments on antibiotic removal.

[0028] In terms of treatment performance, this invention can simultaneously remove conventional pollutants such as nitrogen and phosphorus, as well as three typical antibiotics—sulfamethoxazole, trimethoprim, and enrofloxacin—from marine aquaculture wastewater. It also, to a certain extent, inhibits the accumulation and diffusion of antibiotic resistance genes, achieving comprehensive purification of aquaculture wastewater. The entire system can continuously enrich salt-tolerant dominant functional bacteria during operation, maintaining the stability of the bacterial community structure under high-salt conditions, alleviating the problem of bacterial community degradation caused by high-salt stress, and facilitating long-term stable operation of the device. Furthermore, this invention is adapted and improved based on a mature freshwater packing system, resulting in good technical compatibility. The overall process is simple to operate, operates under mild conditions, requires no complex packing modification processes or additional reagent additions, and has relatively controllable operating costs. It possesses certain engineering application value and potential for large-scale promotion in the field of marine aquaculture wastewater treatment. Attached Figure Description

[0029] Figure 1 A schematic diagram of the structure of an artificial wetland simulation device using two types of oyster shell composite filler under different salinity gradients;

[0030] Figure 2 This is a comparison chart of the effluent physicochemical parameters of two types of composite packing devices under different salinity conditions in an embodiment of the present invention;

[0031] Figure 3 In this embodiment of the invention, two types of composite packing devices are used to control ammonia nitrogen (NH4) in marine aquaculture wastewater under different salinity conditions. + Comparison chart of removal effects of -N);

[0032] Figure 4 In this embodiment of the invention, two types of composite packing devices are used to control nitrate nitrogen (NO3) in marine aquaculture wastewater under different salinity conditions. - Comparison chart of removal effects of -N);

[0033] Figure 5 In this embodiment of the invention, two types of composite packing devices are used to control nitrite nitrogen (NO2) in marine aquaculture wastewater under different salinity conditions. - The effluent concentration variation curve of -N);

[0034] Figure 6 This is a comparison diagram of the removal effects of two types of composite packing devices on total nitrogen (TN) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0035] Figure 7 This is a comparison diagram of the removal effects of two types of composite packing devices on chemical oxygen demand (COD) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0036] Figure 8 This is a comparison diagram of the removal effects of two types of composite packing devices on total phosphorus (TP) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0037] Figure 9 This is a comparison diagram of the removal effects of two types of composite packing devices on sulfamethoxazole (SMX) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0038] Figure 10 This is an embodiment of the invention showing the dynamic change curves of the effluent concentration of typical antibiotics in mariculture wastewater under different salinity conditions for two types of composite packing devices.

[0039] Figure 11 This is a comparison diagram of the removal effects of two types of composite packing devices on trimethoprim (TMP) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0040] Figure 12This is a comparison diagram of the removal effects of two types of composite packing devices on enrofloxacin (ENR) in marine aquaculture wastewater under different salinity conditions in an embodiment of the present invention.

[0041] Figure 13 The relative abundance distribution of phylum-level microbial communities in two types of composite packing devices under different salinity conditions;

[0042] Figure 14 The relative abundance distribution of horizontal microbial communities in two types of composite packing devices under different salinity conditions;

[0043] Figure 15 This is a heatmap showing the abundance of target antibiotic resistance genes (ARGs) in two types of composite packing devices under different salinity conditions, as described in an embodiment of the present invention. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] In this invention, unless otherwise stated, directional terms such as "upper" and "lower" generally refer to the upper and lower positions of the device in its actual use or operating state, specifically the drawing directions in the accompanying drawings; while "inner" and "outer" refer to the outline of the device. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc., are used merely as illustrative purposes and do not impose numerical requirements or establish an order. The term "multiple" means "two or more".

[0046] To verify the treatment effect of this invention on mariculture wastewater with different salinity gradients, the performance differences between the two types of composite packing materials, and the application effects of salt-tolerant microbial acclimatization and tidal intermittent operation processes, this invention sets up multiple sets of examples with different salinities and one set of comparative examples using traditional processes. All experimental groups and comparative examples strictly followed the single variable principle. Except for the preset experimental variables, the basic parameters such as device structure, packing particle size, material ratio, operating temperature, hydraulic cycle, and influent pollutant concentration were kept consistent to ensure the comparability of experimental results.

[0047] General basic conditions for the experiment:

[0048] like Figure 1 As shown, the general test conditions for all embodiments and comparative examples of the present invention are uniform as follows, and remain unchanged throughout unless otherwise specified:

[0049] 1. Experimental apparatus: An artificial wetland simulation device made of impermeable acrylic material is used, with an inner diameter of 20cm, a total height of 50cm, and a wall thickness of 0.5cm. A standardized water outlet is set at the bottom 2cm from the bottom surface. The device is divided into four layers from top to bottom: a 5cm water storage layer, a 5cm fine gravel layer (fine gravel particle size 0.5~1.0cm), a 30cm filler layer, and a 5cm drainage layer (gravel particle size 1.0~2.0cm).

[0050] 2. Filler Layer System: Two types of ternary composite fillers are used, both homogeneously mixed and laid without stratification according to a volume ratio of fine gravel, oyster shell, and functional mineral additives of 2:1:1. The particle size parameters are uniform: fine gravel 5-10mm, oyster shell 1-2mm, zeolite 3-5mm, and pyrite 1-2mm. The two types of composite fillers are: fine gravel-oyster shell-zeolite (CW-F) and fine gravel-oyster shell-pyrite (CW-H).

[0051] 3. Ambient temperature: The temperature is kept constant at 23.0±0.5℃ throughout the process to eliminate the influence of temperature on microbial activity and pollutant degradation process.

[0052] 4. Inlet water substrate: Standardized freshwater synthetic aquaculture wastewater is used as the substrate, and commercially available sea salt is added to prepare simulated seawater aquaculture wastewater with different salinities.

[0053] 5. Target pollutants: Add typical high-frequency antibiotics used in aquaculture, namely sulfamethoxazole (SMX), trimethoprim (TMP), and enrofloxacin (ENR). See Table 2 for relevant parameters.

[0054] 6. Operating process: The system adopts an intermittent operation mode with alternating wet and dry conditions. The duration of a single operating cycle is 75 hours. The specific process is as follows: water is pumped in at a flow rate of 23.67 mL / min for 3 hours, with 4.25 L of wastewater pumped in at a time; after the water is pumped in, the system remains in a static hydraulic state for 48 hours; then the wastewater is discharged, and the device is left to stand still for reoxygenation for 24 hours. The entire system continuously circulates for 10 cycles.

[0055] 7. Detection Methods: After each operating cycle, influent and effluent water samples, as well as biofilm samples from the packing material surface, are collected uniformly. Physicochemical indicators, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen, COD, total phosphorus, and other common pollutants, as well as the concentrations of three types of antibiotics, are tested. Metagenomic sequencing is used to analyze the microbial community structure and the abundance of antibiotic resistance genes.

[0056] To simulate the characteristics of wastewater with different salinity levels in marine aquaculture, this experiment set up three salinity gradients of 10‰, 20‰, and 30‰, corresponding to the device numbers CW-F1, CW-F2, CW-F3 and CW-H1, CW-H2, CW-H3. Each device had two parallel samples, and a total of 12 artificial wetland simulation devices were set up in the entire experiment. The composition of the packing layer of each device is detailed in Table 1.

[0057] Table 1 Correspondence between test apparatus packing material and influent salinity

[0058] Device number Packing layer composition Inlet water salinity CW-F1 Fine gravel (5-10mm) + oyster shells (1-2mm) + zeolite (3-5mm) 10‰ CW-F2 Fine gravel (5-10mm) + oyster shells (1-2mm) + zeolite (3-5mm) 20‰ CW-F3 Fine gravel (5-10mm) + oyster shells (1-2mm) + zeolite (3-5mm) 30‰ CW-H1 Fine gravel (5-10mm) + oyster shells (1-2mm) + pyrite (1-2mm) 10‰ CW-H2 Fine gravel (5-10mm) + oyster shells (1-2mm) + pyrite (1-2mm) 20‰ CW-H3 Fine gravel (5-10mm) + oyster shells (1-2mm) + pyrite (1-2mm) 30‰

[0059] Preparation of test influent: 10.0g, 20.0g, and 30.0g of sea salt were added to each liter of freshwater synthetic aquaculture wastewater, respectively, and stirred thoroughly until completely dissolved to prepare test influent with salinities of 10‰, 20‰, and 30‰.

[0060] This study selected three types of antibiotics that are frequently detected in aquaculture as target pollutants: sulfamethoxazole (SMX), trimethoprim (TMP), and enrofloxacin (ENR). The physicochemical properties of these three compounds are detailed in Table 2.

[0061] Table 2 Physicochemical parameters of the target antibiotic

[0062] SMX 723-46-6 <![CDATA[C 10 H 11 N3O2S]]> 2.10，5.30 0.90 8.10-59.36 TMP 738-70-5 <![CDATA[C 14 H 18 N4O3]]> 3.23，6.79 0.91 18.84-104.57 ENR 93106-60-6 <![CDATA[C 19 H 22 FN3O3]]> 5.94，8.70 0.70 0.10-50.24

[0063] Microbial domestication and system operation:

[0064] After the device is filled, activated sludge of the same salinity is introduced into the constructed wetland device with the corresponding salinity. It is continuously acclimatized for 30 days in a constant temperature environment of 23.0±0.5℃ to complete the enrichment of salt-tolerant functional microorganisms and form a stable biofilm on the surface of the packing material that can adapt to the high-salt environment.

[0065] The entire system operates continuously in an intermittent dry-wet alternation mode. The operation procedure for a single operating cycle is as follows:

[0066] Water intake stage: A peristaltic pump is used to pump 4.25L of synthetic wastewater with the corresponding salinity into the device at a constant flow rate of 23.67mL / min. The water intake time for a single cycle is about 3 hours.

[0067] Static retention stage: After the water intake is completed, the wastewater will remain in the packing layer for 48 hours to ensure that pollutants are fully adsorbed and biochemically degraded;

[0068] Drainage and reoxygenation stage: After the retention period, the wastewater in the device is completely drained, and the device is left to stand for 24 hours to complete the reoxygenation of the packing layer and restore the activity of microorganisms.

[0069] The above steps constitute one complete operating cycle, and all devices operate continuously for 10 cycles.

[0070] Sample collection, preservation, and testing methods:

[0071] 1. Water Sampling: Before each operating cycle, an influent sample is collected from the influent tank; before the hydraulic retention ends and wastewater is discharged, an effluent sample is collected from the outlet. The water samples are used to test the physicochemical properties of the water, as well as the concentrations of common pollutants and antibiotics.

[0072] 2. Biological sample collection: After all 10 operation cycles are completed, dismantle each set of devices, thoroughly mix the packing material in the packing layer, elute the biofilm on the surface of the packing material with sterile physiological saline, collect the eluent and centrifuge it to obtain bacterial precipitate, which will be used for subsequent metagenomic sequencing analysis.

[0073] 3. Sample preservation: All collected samples were immediately placed in sterile sealed bags, transported to the laboratory under dry ice conditions, and frozen in a -80°C freezer.

[0074] 4. Detection and sequencing methods

[0075] (1) Physicochemical indicators of water body: water temperature (WT), pH, dissolved oxygen (DO), conductivity (EC), dissolved organic matter (TDS), and salinity (SAL) were measured.

[0076] (2) Conventional pollutants: The concentrations of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen, total phosphorus, and COD were determined by the national standard spectrophotometric method;

[0077] (3) Antibiotics: The concentrations of SMX, TMP, and ENR were detected by liquid chromatography-mass spectrometry.

[0078] (4) Metagenomic sequencing: Total genomic DNA was extracted from matrix samples using the PFMag-BindSoilDNAKit. DNA purity, concentration and integrity were determined using a NanoDrop2000 ultra-micro spectrophotometer, a Qubit4.0 fluorescence quantitative quantitation system and 1% agarose gel electrophoresis. Qualified DNA samples were fragmented to 400bp fragments using a Covaris M220 ultrasonic disruptor. Libraries were constructed using the NEXTFLEX® RapidDNA-SeqKit. Finally, paired-end sequencing (PE150) was performed using the Illumina NovaSeq6000 sequencing platform.

[0079] The present application will be specifically described below through specific embodiments. The following embodiments are only some embodiments of the present application and are not intended to limit the present application.

[0080] Example 1 (Treatment of low salinity (10‰) marine aquaculture wastewater)

[0081] This embodiment provides a method for removing antibiotics from low-salinity seawater aquaculture wastewater based on oyster shell composite filler, which is suitable for seawater salinity conditions of 10‰.

[0082] This embodiment uses the above-mentioned general experimental conditions, only limiting the influent salinity to 10‰. The specific differentiated operation is as follows: 10.0g of sea salt is precisely added to each liter of freshwater synthetic aquaculture wastewater and stirred thoroughly until completely dissolved to prepare simulated seawater aquaculture wastewater with a salinity of 10‰. After the device is filled with CW-F and CW-H composite packing materials, activated sludge with a salinity adapted to 10‰ is introduced. Under constant temperature conditions, it is continuously acclimated for 30 days to enrich low-salt and salt-tolerant functional microorganisms and form a stable biofilm on the surface of the packing materials.

[0083] After the system was acclimatized, it was continuously operated for 10 cycles according to a general intermittent process. The experimental results showed that under 10‰ low salinity conditions, the microbial community richness and diversity of both types of composite packing systems were good, and the nitrification and denitrification functions of the system operated stably. Among them, the CW-H pyrite packing system, with the help of sulfur autotrophic denitrification, had a better removal effect on nitrate nitrogen and total nitrogen than the CW-F zeolite packing system. Both systems could stably maintain a removal rate of enrofloxacin (ENR) above 85%, and also had a good removal effect on sulfamethoxazole (SMX). Under this salinity condition, the inhibitory effect caused by osmotic pressure and ion competition was weak, the microbial community structure remained stable, the antibiotic residue level in the water was low, and the risk of antibiotic resistance gene enrichment was relatively small, which can effectively achieve the synergistic purification of conventional pollutants and compound antibiotics in low-salinity marine aquaculture wastewater.

[0084] Example 2 (Treatment of marine aquaculture wastewater with a salinity of 20‰)

[0085] This embodiment is used to verify the adaptability and purification performance of the technical solution of the present invention under the condition of 20‰ medium-salt seawater aquaculture wastewater.

[0086] This embodiment uses all the general experimental conditions. The only difference is the influent salinity and microbial acclimatization adaptation: 20.0g of sea salt is added to each liter of freshwater synthetic aquaculture wastewater to prepare 20‰ medium-salt simulated seawater aquaculture wastewater; correspondingly, activated sludge with 20‰ salinity is introduced and acclimatized at a constant temperature for 30 days to complete the targeted enrichment of salt-tolerant functional bacteria under medium-salt stress.

[0087] The experimental results showed that under 20‰ salinity stress, the diversity of the microbial community was slightly lower than that under low salinity conditions. The microbial community within the system underwent natural structural succession, with salt-tolerant functional bacteria such as Proteobacteria, Paracoccus, and Schistosoma gradually accumulating and stabilizing. The CW-H pyrite composite packing system could alleviate the osmotic pressure impact and ion adsorption competition caused by the saline environment to a certain extent. Its denitrification, phosphorus removal, and organic matter degradation performance remained relatively stable. It exhibited good removal effects on three typical antibiotics: SMX, TMP, and ENR. The effluent quality was stable, and no pollutant accumulation was observed. This experimental condition demonstrates that this technical solution is applicable to common nearshore salinity marine aquaculture wastewater treatment scenarios, and its overall operation is relatively stable.

[0088] Example 3 (Treatment of high salinity (30‰) marine aquaculture wastewater)

[0089] This embodiment is used to verify the anti-interference ability, pollutant enhanced removal effect and resistance gene control ability of the technical solution of the present invention under extreme seawater conditions of 30‰ high salinity, and to evaluate the wide range of salinity adaptation limit performance of the technical solution.

[0090] The general parameters remain unchanged in this embodiment, and the differentiated operations are as follows: 30.0g of sea salt is added to each liter of freshwater synthetic aquaculture wastewater to prepare 30‰ high-salt simulated seawater aquaculture wastewater; 30‰ high-salt dedicated activated sludge is used and acclimatized at a constant temperature for 30 days to enhance the high-salt tolerance of the bacterial community and construct a stable biofilm adapted to the extreme high-salt environment.

[0091] Experimental results showed that under 30‰ high-salt stress, microbial community diversity decreased, with Proteobacteria gradually becoming the dominant phylum, achieving a community structure succession adapted to the high-salt environment. The ion competition brought about by the high-salt environment inhibited the removal of TMP primarily through adsorption; both types of packing systems achieved overall TMP removal rates below 50%. In comparison, the CW-H pyrite packing system exhibited better adaptability to high-salt environments, mitigating the adverse effects of high-salt conditions to some extent. Its SMX removal rate reached 93.94%, superior to the CW-F zeolite system's 80.65% (p < 0.05). Simultaneously, the CW-H system can efficiently degrade antibiotics in water, reducing environmental selection pressure and helping to alleviate the accumulation and spread of antibiotic resistance genes, thus improving the problems of poor purification effects and high ecological risks associated with traditional processes under high-salt conditions.

[0092] Comparative examples (traditional freshwater filler, no salt tolerance acclimatization, single process control).

[0093] This comparative example employs a complete set of traditional freshwater constructed wetland technology systems to contrast with the core innovations of this invention, showcasing the technical characteristics of the invention's composite filler compounding, gradient salt tolerance acclimatization, and tidal intermittent process. This comparative example uniformly uses the 30‰ high-salt influent condition of Example 3 to ensure unique and comparable experimental variables.

[0094] The specific settings for the comparative example are as follows: a single oyster shell packing material is used, without the addition of zeolite and pyrite functional minerals, and a 2:1:1 homogenization ratio is not set; the tidal wet-dry alternation process is abandoned, and the traditional continuous water influent operation mode is adopted; no specific salinity-oriented salt tolerance acclimatization is carried out, and conventional freshwater activated sludge is directly inoculated; the other parameters such as device size, ambient temperature, and influent pollutant concentration are kept consistent with those in Example 3.

[0095] Experimental results show that traditional freshwater constructed wetland processes are poorly adapted to the 30‰ high-salinity marine aquaculture wastewater condition. The osmotic pressure generated by the high-salinity environment significantly inhibits the activity of native freshwater microorganisms, resulting in a significant reduction in the system's nitrification and denitrification functions, and a substantial decrease in nitrogen and phosphorus removal performance. Simultaneously, the large number of anions in the water competes with antibiotics for adsorption sites on the packing surface, significantly weakening the removal of SMX and ENR, and making TMP difficult to remove effectively. Furthermore, freshwater microbial communities are prone to reduced activity and the death of some bacteria under high-salinity stress, leading to an imbalance in the microbial community structure, a decrease in microbial diversity, and consequently, the accumulation of antibiotic resistance genes. Traditional freshwater processes are insufficient to meet the purification requirements of high-salinity marine aquaculture wastewater and pose certain ecological risks, further demonstrating the applicability of the technical solution of this invention in the treatment of high-salinity marine aquaculture wastewater.

[0096] The experimental results of the 10‰, 20‰, and 30‰ gradient salinity examples and the comparative examples of traditional processes show that the technical solution of the present invention, which combines the fixed ratio of ternary composite packing, the domestication of salt-tolerant bacterial communities in a gradient manner, and the tidal dry-wet alternation operation process, can improve the problems of traditional freshwater artificial wetlands that are difficult to adapt to the wide salinity environment of seawater, the reduced microbial activity under high salinity conditions, the prominent competition for ion adsorption, the fluctuation of antibiotic removal effect, and the enrichment of resistance genes.

[0097] Core mechanism of this invention

[0098] 1. This invention employs two types of ternary composite fillers, one composed of fine gravel and oyster shells, and the other of zeolite and pyrite. Each component functions complement each other and works synergistically. Oyster shells buffer water pH, assist in phosphorus removal, and provide basic adsorption sites; zeolite enhances adsorption capacity and alleviates adsorption competition between ions and antibiotics; pyrite continuously releases sulfur to construct a salt-tolerant, sulfur-autotrophic denitrification system, stably achieving nitrate nitrogen removal in high-salt environments while simultaneously consuming anions in the water, further weakening the impact of adsorption competition. The fillers are homogeneously laid out in a fixed ratio with limited particle size to ensure stable hydraulic conditions and fully leverage the synergistic purification efficiency of each component.

[0099] 2. This invention utilizes a gradient salinity acclimation method to cultivate salt-tolerant microbial communities. After long-term acclimation with activated sludge at different salinities, the microbial community undergoes orderly succession as salinity increases, with Proteobacteria becoming the dominant phylum, and salt-tolerant functional bacteria such as Paracoccus and Schistosoma continuously accumulating. The pyrite system is more conducive to the colonization and proliferation of functional bacteria, enabling the construction of a structurally stable high-salt-adapted microbial community. This effectively resists the inhibition of microbial activity by high salinity osmotic pressure, ensuring the stable progress of nitrification, denitrification, and pollutant degradation processes. Combined with an intermittent tidal dry-wet alternating operation mode, it can continuously replenish dissolved oxygen in the packing layer, restore microbial activity, extend the pollutant reaction time, and improve the operational stability of the entire unit across a wide salinity range.

[0100] 3. This invention relies on the dual effects of adsorption by the packing material and microbial degradation to simultaneously remove three typical antibiotics (sulfamethoxazole, trimethoprim, and enrofloxacin) along with conventional pollutants such as nitrogen, phosphorus, and COD. High salinity and antibiotic residues create dual selective pressure, easily leading to the accumulation and spread of resistance genes. This invention can effectively degrade antibiotics in water, reduce environmental selective pressure, and simultaneously optimize the microbial community structure, reducing the accumulation of host bacteria for resistance genes. Compared to zeolite composite systems, pyrite composite packing material is more adaptable to high-salinity environments, effectively controlling the accumulation and spread of antibiotic resistance genes while enhancing pollutant removal, thus balancing water treatment efficiency and ecological safety.

[0101] Experimental Results and Analysis

[0102] 1. Physicochemical indicators of water bodies:

[0103] Figure 2 This reflects the changes in the effluent physicochemical parameters of two types of packing systems, CW-F and CW-H, under different salinity conditions. Among them, Figure 2 (a) represents the pH value. Figure 2 (b) represents electrical conductivity. Figure 2 (c) represents the total dissolved solids. Figure 2 (d) represents salinity. Figure 2 (e) represents the water temperature. Figure 2 (f) represents dissolved oxygen; the horizontal axis represents the numbers of different experimental devices, and the vertical axis represents the measured values ​​of the corresponding physicochemical parameters. The experiment shows that this process can maintain a relatively stable water environment within a salinity range of 10‰–30‰: the effluent pH remains in the neutral-to-alkaline range of 7.2–7.8; the slow-release calcium salts from oyster shells can buffer pH fluctuations caused by high salinity, creating suitable conditions for the biodegradation of sulfonamide antibiotics; conductivity, total dissolved solids, and salinity increase with increasing influent salinity. The ion load in the effluent from the pyrite group is slightly lower than that from the zeolite group, indicating that sulfur autotrophic denitrification can consume some anions in the water, mitigating the adsorption competition between ions and antibiotics to some extent. The water temperature remained stable throughout the experiment, eliminating the interference of temperature on the results; relying on the tidal alternating wet and dry operation mode, the dissolved oxygen content in the device remained stable, and the pyrite system can still meet the dissolved oxygen requirements for nitrification, denitrification, and antibiotic degradation under high salinity conditions. Overall, the combination of composite packing material and tidal operation technology can improve the physicochemical state of water in high-salt environments, create a suitable water environment for the growth of salt-tolerant microorganisms and the degradation of pollutants, and play a positive role in the synergistic treatment of conventional pollutants and antibiotics in marine aquaculture wastewater.

[0104] 2. Purification effect of conventional pollutants

[0105] Figures 3 to 8The results reflect the purification effects of the CW-F zeolite-oyster shell system and the CW-H pyrite-oyster shell system on ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen, COD, and total phosphorus in marine aquaculture wastewater under different salinity stress conditions. Overall, the experimental results show that increased salinity has a certain inhibitory effect on the purification performance of conventional pollutants in constructed wetland systems. As the salinity increases from 10‰ to 30‰, the treatment effect of the CW-F system on various pollutants decreases to varying degrees, with increased effluent pollutant concentration and decreased removal stability. In contrast, the CW-H pyrite composite system is more adaptable to salinity fluctuations, and its overall effluent quality and removal stability are superior to the CW-F system under the same conditions, with the performance difference being more pronounced in high-salinity environments (p < 0.05). Under low salinity conditions, both systems showed relatively good pollutant removal effects, but the CW-H system has shown certain performance advantages. As salinity gradually increases, the osmotic pressure stress caused by high salinity will inhibit the activity of nitrification, heterotrophic denitrification and heterotrophic degradation microorganisms, while exacerbating the ion competition effect in the water body, resulting in significant fluctuations in the removal effects of nitrogen, phosphorus and organic matter in the CW-F system.

[0106] The CW-H system, relying on pyrite-mediated sulfur autotrophic denitrification, exhibits good tolerance to high-salt environments. It can stably complete the nitrate reduction process while reducing dependence on heterotrophic bacteria, minimizing the accumulation of intermediate products such as nitrite and maintaining a complete nitrogen conversion pathway. Simultaneously, this invention can stably enrich salt-tolerant functional microorganisms such as *Paracoccus* and *Schönleinii* through gradient salt tolerance acclimation, effectively improving the problems of insufficient microbial activity and community structure imbalance under high-salt conditions, ensuring the stability of the system's nitrification function and organic matter degradation capacity. Regarding phosphorus removal, the CW-H system combines the calcium-based precipitation effect of oyster shells with the pyrite-assisted iron-phosphorus precipitation effect, and relies on stable microbial assimilation, which can, to some extent, offset the adverse effects of high-salt ion competition, maintaining a good total phosphorus removal effect. Overall, compared with traditional zeolite composite packing systems, the pyrite-oyster shell composite packing system used in this invention is better adapted to a wide salinity range of 10‰ to 30‰, effectively mitigating the negative interference of high salt stress on denitrification, phosphorus removal and organic matter degradation processes, with more stable operation performance and better application adaptability in the purification of conventional pollutants in marine aquaculture wastewater.

[0107] 3. Typical antibiotic removal effect

[0108] Figures 9 to 12 This study demonstrates the removal efficiency and effluent concentration changes of sulfamethoxazole (SMX), trimethoprim (TMP), and enrofloxacin (ENR) by two packing systems, CW-F and CW-H, under different salinity conditions. The results show that increased salinity inhibits the removal of these three antibiotics to varying degrees, and the three substances exhibit different sensitivities to salinity changes. Figure 10 The horizontal axis represents the number of tests, and the vertical axis represents the antibiotic concentration in the effluent, in mg / L. The low salinity, medium salinity, and high salinity marked in the figure correspond to influent salinity of 10‰, 20‰, and 30‰, respectively. In the legend, CW-F represents a fine gravel-oyster shell-zeolite packing device, CW-H represents a fine gravel-oyster shell-pyrite packing device, and SMX, TMP, and ENR correspond to sulfamethoxazole, trimethoprim, and enrofloxacin, respectively. The error bars in the figure represent the standard deviation of the measured values.

[0109] ENR can be removed through a combination of adsorption by packing material, photolysis, and microbial degradation. It exhibits good adaptability to salinity, with both systems maintaining a removal rate above 85% overall, showing minimal impact from salinity changes. SMX removal relies on a combination of adsorption and biodegradation. Increased salinity gradually reduces the removal efficiency, but under the same high-salinity conditions, the CW-H system achieves a SMX removal rate of 93.94%, higher than the 80.65% of the CW-F system (p<0.05), demonstrating a certain enhancement effect. TMP removal mainly depends on the adsorption of packing material. Anions such as phosphate and nitrate in the water compete with it for adsorption sites, resulting in an overall TMP removal rate of less than 50% for all systems. Increased salinity further exacerbates this inhibitory effect.

[0110] Comparing the two packing systems, it was found that as the salinity increased from 10‰ to 30‰, the treatment effect of the CW-F system on all three types of antibiotics declined, with an increase in effluent concentration and larger data fluctuations. The CW-H pyrite-oyster shell system was less affected by salinity, maintaining a relatively stable removal effect across various salinity gradients. This is because the combination of pyrite and oyster shell enriches the adsorption sites of the packing material, mitigating the adverse effects of ion competition to some extent. Simultaneously, the functional bacterial community obtained through gradient salt tolerance acclimation can maintain corresponding degradation activity in high-salt environments. Furthermore, the pyrite-mediated sulfur autotrophic denitrification process exhibits good salt tolerance, stably reducing nitrate levels in the water, indirectly mitigating ion competition, and further optimizing the antibiotic removal environment.

[0111] Overall, within a salinity range of 10‰ to 30‰, the pyrite-oyster shell composite packing system can mitigate the negative impact of high-salinity environments on the removal of three typical antibiotics to a certain extent. Its overall treatment effect and operational stability are superior to those of the zeolite-oyster shell system, which helps to achieve stable removal of antibiotics from marine aquaculture wastewater.

[0112] 4. Microbial community and resistance gene analysis

[0113] (1) Microbial diversity analysis

[0114] As shown in Table 3, salinity has a significant impact on the alpha diversity of microbial communities within constructed wetland devices. Overall, the species richness and diversity of microbial communities were optimal under a low salinity condition of 10‰ (p < 0.05). As environmental salinity gradually increased, the overall richness and diversity of microbial communities showed a downward trend. Among them, the high salinity stress condition of 30‰ had the most significant inhibitory effect on the microbial community structure, resulting in a significant reduction in community diversity and species richness.

[0115] Table 3 Microbial Alpha Diversity Index

[0116] CW-F1 <![CDATA[6.49±0.16 a ]]> 0.0110±0.0027ª 34117.00±1650.85ª 1.0000±0.0000 CW-F2 <![CDATA[6.16±0.02 ᵇ ]]> <![CDATA[0.0127±0.0004 ᵇ ]]> <![CDATA[28018.67±82.25 ᵇ ]]> 1.0000±0.0000 CW-F3 <![CDATA[5.85±0.08 ᶜ ]]> <![CDATA[0.0226±0.0027 ᶜ ]]> <![CDATA[28854.00±269.02 ᵇ ]]> 1.0000±0.0000 CW-H1 <![CDATA[6.69±0.04 a ]]> <![CDATA[0.0101±0.0005 a ]]> <![CDATA[35991.00±405.18 a ]]> 1.0000±0.0000 CW-H2 <![CDATA[6.16±0.02 ᵇ ]]> <![CDATA[0.0127±0.0004 ᵇ ]]> <![CDATA[28018.67±82.25 ᵇ ]]> 1.0000±0.0000 CW-H3 <![CDATA[5.85±0.04 ᶜ ]]> <![CDATA[0.0175±0.0006 ᶜ ]]> <![CDATA[29087.00±53.73 ᵇ ]]> 1.0000±0.0000

[0117] (2) Phylogenetic horizontal community composition

[0118] At the phylum level, the microbial community structure of the two types of packing devices differs significantly under different salinity conditions, and the dominant phylum undergoes regular succession as salinity increases. Figure 13 The relative abundance distribution of microorganisms at the phylum level in the CW-F and CW-H packing systems under different salinity conditions is shown. The experimental results indicate that salinity is a key environmental factor affecting the structure of microbial communities; compared with the zeolite-oyster shell packing system, the pyrite-oyster shell packing system is more conducive to the enrichment of salt-tolerant functional bacteria and the maintenance of community structure stability.

[0119] Figure 13 It contains three sets of subgraphs, among which Figure 13 (a) shows the community distribution under low salinity (10‰) conditions. Figure 13 (b) shows the community distribution under medium salinity (20‰) conditions. Figure 13 (c) shows the community distribution under high salinity (30‰) conditions; the horizontal axis represents the experimental setup number, CW-F represents the fine gravel-oyster shell-zeolite packing setup, and CW-H represents the fine gravel-oyster shell-pyrite packing setup; the vertical axis represents the relative abundance of microbial phyla, in %. In the legend, Pseudomonadota represents Pseudomonas, Actinomycetota represents Actinomycetes, Chloroflexota represents Chloroflexota, Bacteroidota represents Bacteroidetes, and the other legends correspond to other microbial phyla.

[0120] Proteobacteria were the dominant phylum in all experimental groups, with their relative abundance gradually increasing with salinity. Under high-salt conditions, the proportion of Proteobacteria in the CW-F3 and CW-H3 devices reached 70.44% and 64.84%, respectively, fully demonstrating the excellent adaptability of this phylum to high-salt environments. Under low-salt conditions, the bacterial community structures of the two groups showed high similarity and good community diversity. As salinity increased, the bacterial community gradually evolved towards salt-tolerant groups, and the dominance of Proteobacteria continued to strengthen. Furthermore, the community succession process in the CW-H system was more stable, and the enrichment effect of functional bacterial communities was more prominent.

[0121] The mechanism of action involves Proteobacteria, which contain a variety of salt-tolerant microorganisms capable of simultaneously completing nitrification, denitrification, and organic pollutant degradation processes. These microorganisms are the core functional group in high-salt wastewater treatment. The pyrite-oyster shell composite packing material used in this invention continuously supplies sulfur while improving the packing material's adhesion environment, effectively promoting the proliferation of sulfur-autotrophic denitrifying microorganisms within the Proteobacteria phylum. Combined with gradient salt tolerance acclimation methods, salt-tolerant functional bacteria can be further selectively screened and enriched, constructing a stable microbial community adapted to high-salt environments, ensuring the system maintains stable pollutant degradation capabilities across a wide salinity range.

[0122] (3) Belongs to the horizontal community composition:

[0123] At the same level, the relative abundance distribution of microbial communities in the two types of packing systems under different salinity conditions is as follows: Figure 14 As shown in the figure. The experimental results indicate that salinity changes are an important factor in altering the microbial community structure of constructed wetlands, and the pyrite-oyster shell filler system is more suitable for the enrichment and stable colonization of salt-tolerant functional bacteria.

[0124] Figure 14 It contains three sets of subgraphs, among which Figure 14 (a) For low salinity (10‰) working conditions, Figure 14 (b) For medium salinity (20‰) conditions, Figure 14 (c) Corresponding to high salinity (30‰) conditions; the horizontal axis represents the experimental apparatus number, CW-F represents the fine gravel-oyster shell-zeolite packing apparatus, and CW-H represents the fine gravel-oyster shell-pyrite packing apparatus; the vertical axis represents the relative abundance of microbial genera, in %; in the legend, Paracoccus is Paracoccus, Brevundimonas is Brevundimonas, Aquaticola is Aquatic Bacteria, and the rest of the legends correspond to other microbial genera.

[0125] Overall microbial community analysis showed that *Paragonimus* was the dominant genus in both sets of devices, and its relative abundance gradually increased with increasing salinity. Under the same salinity conditions, the abundance of *Paragonimus* in the CW-H system was consistently higher than that in the CW-F system. In low-salinity (10‰) conditions, the microbial community structure of the two types of packing devices showed little difference, and the microbial diversity was good. As salinity increased, the community gradually evolved towards salt-tolerant functional bacteria, with *Paragonimus* and *Schefflera* species showing significant enrichment. This provides a crucial microbial basis for the stable denitrification, phosphorus removal, and antibiotic removal achieved by the CW-F3 and CW-H3 devices under high-salinity conditions.

[0126] Analysis shows that *Paracococcus* is a core functional bacterium involved in sulfur autotrophic denitrification in high-salt environments, playing a crucial role in the removal of nitrate nitrogen from water. The CW-H pyrite-oyster shell system continuously releases sulfur sources, providing a material basis for the proliferation of *Paracococcus*. Combined with a gradient salt tolerance acclimation method, this system can further selectively enrich salt-tolerant denitrification functional bacteria, forming a stable microbial community adapted to high-salt environments, ensuring the system has stable nitrogen removal capacity under high-salt conditions.

[0127] (4) Analysis of antibiotic resistance genes:

[0128] This study detected and analyzed antibiotic resistance genes within the device. Figure 15 This is a heatmap showing the abundance of target antibiotic resistance genes in two composite packing systems, CW-F and CW-H, under different salinity conditions. The results show that all 11 target resistance genes selected in this study were detected in all groups of devices, with significant differences in the absolute abundance among different experimental groups. Overall, the abundance of resistance genes generally increased with increasing water salinity, and the enrichment level of resistance genes under high salinity conditions was significantly higher than that under low salinity conditions. Under the same salinity conditions, the variation patterns of resistance genes in the CW-F and CW-H groups were basically consistent, indicating that salinity is the main environmental factor driving the distribution of resistance genes, and the type of packing material has a relatively limited impact on the overall distribution characteristics of resistance genes.

[0129] Figure 15 In the diagram, the vertical axis represents the experimental apparatus number, where CW-F represents the fine gravel-oyster shell-zeolite packing apparatus, and CW-H represents the fine gravel-oyster shell-pyrite packing apparatus; the suffixes 1, 2, and 3 correspond to influent salinity of 10‰, 20‰, and 30‰, respectively, and CW-F and CW-H represent the average abundance of the corresponding systems at different salinities; the horizontal axis represents the target antibiotic resistance genes, including sulfonamide resistance genes (sul1, sul2, sul3), dihydrofolate reductase genes (dfrA1, dfrA3, dfrA5, dfrA26), quinolone resistance genes (QnrVC1, oqxA, oqxB), and multidrug resistance genes (mfpA); the color intensity in the diagram indicates the relative abundance of resistance genes, and the color scale on the right is the abundance scale.

[0130] Under the same salinity conditions, the abundance of resistance genes in the CW-H pyrite-oyster shell system was generally slightly lower than that in the CW-F zeolite-oyster shell system, and this difference was more pronounced in high-salinity environments. High-salinity stress combined with antibiotic residues in water creates a dual environmental selection pressure, easily inducing the expression and horizontal transfer of microbial resistance genes, thereby increasing the accumulation and spread potential of resistance genes. The pyrite composite packing system used in this invention can degrade residual antibiotics in water to a certain extent, reducing environmental selection pressure; simultaneously, this system can optimize the microbial community structure within the device, moderately reducing the enrichment of host microorganisms for resistance genes, thus helping to alleviate the accumulation and spread of resistance genes.

[0131] In summary, the pyrite-oyster shell composite filler system of the present invention can optimize the microbial community structure and enrich salt-tolerant microorganisms within a wide salinity range of 10‰ to 30‰, providing certain microbial support and ecological security for the stable purification treatment of marine aquaculture wastewater with different salinities.

[0132] Based on a comprehensive analysis of all experimental results, this invention addresses the shortcomings of existing freshwater constructed wetland technologies by optimizing and improving the packing material combination, microbial acclimatization, and operation methods. Traditional freshwater constructed wetlands have a limited applicable salinity range. When applied to marine aquaculture wastewater with varying salinity, they are prone to problems such as suppressed microbial activity, intensified competition for ion adsorption, fluctuating pollutant treatment effects, and enrichment of resistance genes, making it difficult to meet the treatment requirements of marine aquaculture effluent.

[0133] This invention formulates two types of ternary composite packing materials: fine gravel-oyster shell-pyrite and fine gravel-oyster shell-zeolite. These materials are homogeneously mixed in a fixed ratio. Based on the synergistic effect of calcium-based materials and functional minerals, this invention overcomes the limitations of single oyster shell packing materials, which have relatively limited functionality and weak adaptability to high-salt environments. The pyrite composite packing material can exert sulfur autotrophic denitrification, mitigating the osmotic pressure effects and ion competition interference caused by high salinity to a certain extent. Under high-salt conditions, its treatment effect on nitrogen and phosphorus pollutants is superior to that of the zeolite packing system. This system achieves a removal rate of 93.94% for sulfamethoxazole (SMX), an improvement of over 13% compared to traditional processes, thus helping to address the problem of decreased antibiotic removal efficiency under high-salt environments.

[0134] Meanwhile, this invention employs a salinity-specific acclimatization combined with an intermittent operation mode of alternating wet and dry periods. This gradually enriches salt-tolerant functional microorganisms such as Proteobacteria, Paracoccus, and Schistosoma, optimizing the community structure and enhancing the adaptability of the microbial community to the salinity range of 10‰–30‰. This alleviates problems such as reduced microbial diversity and weakened denitrification performance in high-salt environments encountered by traditional processes. Furthermore, the pyrite composite packing system can degrade antibiotics in water, reducing environmental selection pressure and helping to slow the accumulation and diffusion of resistance genes, thus balancing water treatment effectiveness and ecological safety.

[0135] The packing material used in this invention is readily available, the overall process is simple to operate, and the operating cost is moderate. It can adapt to the salinity fluctuations of marine aquaculture wastewater, and can simultaneously treat conventional pollutants and typical antibiotics, while reducing the potential risks posed by resistance genes. Compared to traditional freshwater constructed wetlands, this solution improves salinity adaptability and comprehensive treatment effectiveness, providing a viable technical approach for the harmless treatment of marine aquaculture wastewater.

[0136] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should be protected by the present invention.

Claims

1. A method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite packing material, characterized in that, Includes the following steps: S1. Selection of composite fillers: Two types of composite fillers suitable for high-salinity and complexly polluted seawater environments are selected: fine gravel-oyster shell-zeolite and fine gravel-oyster shell-pyrite. Both composite fillers are homogeneously mixed from fine gravel, oyster shell, and functional mineral additives in a volume ratio of 2:1:1, without layered structure. The particle size of each component is as follows: fine gravel 5-10mm, oyster shell 1-2mm, zeolite 3-5mm, and pyrite 1-2mm. S2. Preparation of graded salinity marine aquaculture wastewater: Using freshwater synthetic aquaculture wastewater as a base, sea salt is added to prepare marine aquaculture wastewater with different salinities. S3. Construct salt-tolerant artificial wetland devices: Construct multiple sets of artificial wetland devices, each set having a water storage layer, a fine gravel layer, a filler layer, and a drainage layer from top to bottom; Homogenize the above-mentioned composite filler into the filler layer respectively, and the device is suitable for seawater aquaculture wastewater with a salinity of 10‰ to 30‰. S4. Salinity-specific acclimatization of microorganisms: Activated sludge with matching salinity is introduced into the device with the corresponding salinity and continuously acclimatized for 30 days in a constant temperature environment of 23.0±0.5℃ to enrich salt-tolerant microorganisms and form a biofilm adapted to the high-salt environment. S5. Intermittent dry-wet alternating operation: Continuous operation is carried out using an intermittent cycle mode of metered water pumping, static retention, and emptying and settling.

2. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite packing material according to claim 1, characterized in that: In step S2, 10.0g, 20.0g, and 30.0g of sea salt are added to each liter of freshwater synthetic aquaculture wastewater, respectively. After stirring and dissolving, seawater aquaculture wastewater with salinity of 10‰, 20‰, and 30‰ is obtained.

3. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1, characterized in that: In step S3, the artificial wetland device has the following specifications: inner diameter 20cm, total height 50cm, wall thickness 0.5cm, and an outlet is set at the bottom 2cm from the bottom surface; wherein the water storage layer is 5cm high, the fine gravel layer is 5cm high and uses gravel with a particle size of 0.5-1.0cm, the filler layer is 30cm high, and the drainage layer is 5cm high and uses gravel with a particle size of 1.0-2.0cm.

4. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1, characterized in that: Step S5 is operated under a constant temperature of 23.0±0.5℃ throughout. The single-cycle operation process is as follows: 4.25L of seawater aquaculture wastewater is pumped in using a peristaltic pump at a flow rate of 23.67mL / min for 3 hours. After the water is pumped in, the water is left to stand for 48 hours. Then the wastewater is drained and left to stand for 24 hours. This is one complete operation cycle, and the cycle is repeated 10 times.

5. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1, characterized in that: The salt-tolerant biofilm obtained in step S4 adheres to the surface of the composite packing material and simultaneously degrades conventional pollutants and residual antibiotics in the wastewater during the wastewater treatment process.

6. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1, characterized in that: in During wastewater treatment, the fine gravel-oyster shell-pyrite composite packing material alleviates the inhibition effect of nitrate nitrogen removal under high salinity conditions through pyrite-mediated sulfur autotrophic denitrification, and weakens the adsorption competition between ions and antibiotic molecules in the water on the packing surface.

7. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1, characterized in that: in During wastewater treatment, the fine gravel-oyster shell-zeolite composite packing enhances the retention effect of antibiotic molecules in high-salt environments through the adsorption and enrichment effect of zeolite, thereby helping to improve the stability of antibiotic removal.

8. The method for removing antibiotics from marine aquaculture wastewater based on oyster shell composite filler according to claim 1 or 6, characterized in that: The method simultaneously reduces the abundance of antibiotic resistance genes in mariculture wastewater during wastewater treatment, thereby reducing the ecological risks of resistance gene enrichment and horizontal spread.

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