A rhizosphere regulation based vertical flow constructed wetland system for removal of tetracycline antibiotics

Abstract

The application discloses a rhizosphere regulation-based vertical flow constructed wetland system for removing tetracycline antibiotics, and relates to the technical field of environmental science. The system comprises a bottom coarse gravel, a water body purification main substrate layer and a fine sand layer filled on the upper surface of the bottom coarse gravel in sequence, and further comprises large fibrous root type wetland plants planted on the upper layer; wherein the water body purification main substrate layer is a mixture of modified biochar and fine gravel, a double-layer root net is laid or not laid in the plant rhizosphere area 100 mm below the upper layer of the water body purification main substrate layer, activated sludge in a mariculture tail water pool is introduced into the plant rhizosphere area, and the mass ratio of C element and N element in the mariculture tail water is regulated to be 3:1 or 6:1. The regulation method has the advantages of ecological friendliness, convenient operation, remarkable effect and the like, and provides an ecological regulation path scheme for optimizing the purification performance of the constructed wetland on the tetracycline antibiotics in the mariculture tail water.

Description

Technical Field

[0001] This invention relates to the field of environmental science and technology, and more specifically, to a rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics. Background Technology

[0002] With the booming development of mariculture, the pollution of marine ecosystems by aquaculture wastewater has become increasingly prominent. Simultaneously, the necessary use of antibiotics in veterinary medicines leads to the continuous detection of antibiotic residues in the environment. The long-term residual stress of antibiotics in water bodies also induces the emergence of a large number of antibiotic-resistant bacteria (ARGs) and drug-resistant strains, posing significant potential risks and threats to human health and the natural environment. Among various types of antibiotics, tetracycline antibiotics (TCs) are widely used, especially in developing countries, for medical treatment due to their broad-spectrum antibacterial properties and relatively affordable price, as well as in veterinary medicine and growth promoters in livestock and aquaculture. It is noteworthy that compared to other types of antibiotics, TCs are more persistent, highly adsorbed, and difficult to degrade in soil and sediments, thus accumulating more easily in the environment.

[0003] Constructed wetlands (CWs), as a mature aquatic ecosystem restoration technology, have been widely used for the effective removal of various types of organic pollutants. Existing studies have shown that CWs have good purification effects on quinolones and sulfonamide antibiotics, but their removal efficiency for total pollutants (TCs) is relatively low, and there are significant differences in removal rates for different TC species. Further optimization and improvement are needed to achieve efficient and simultaneous removal of different TC species using CW technology.

[0004] In current research on CWs (Chemical Water Treatments) technology, the matrix packing material is generally considered to play the most important role in pollutant removal, while the role of wetland plants is often underestimated. The rhizosphere region of wetland plants is the area of ​​most intensive interaction between plants, rhizosphere microorganisms, the matrix, and target organic pollutants. Numerous biochemical reactions take place here. The ROL (Residual Oil Flow) and RE (Residual Oil Flow) secretion of wetland plant roots, as well as their functions in pollutant retention and rhizosphere microbial loading through dense root networks, significantly influence the rhizosphere microenvironment. Through scientific and effective ecological regulation of the rhizosphere microenvironment of wetland plants, the removal efficiency of various types of pollutants, such as antibiotics and heavy metals, from mariculture wastewater can be enhanced. Utilizing ecologically based regulation methods, the potential advantages of wetland plants can be further explored, improving the purification effect of mariculture wastewater while reducing disturbance to the ecological environment.

[0005] Therefore, how to develop a rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a rhizosphere-based vertical flow constructed wetland system for removing tetracycline antibiotics. It can effectively and simultaneously remove four frequently detected TCs in the environment: HCl-DOX, HCl-TC, OTC, and HCl-CTC. Simultaneously, by optimizing the rhizosphere, it stimulates the performance of wetland plants, reducing characteristic ARG residues, which is of great significance for improving the purification effect of marine aquaculture wastewater.

[0007] A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics includes: a bottom coarse gravel layer, and a water purification main matrix layer and a fine sand layer sequentially filled on the surface of the bottom coarse gravel layer.

[0008] It also includes large fibrous-rooted wetland plants planted on the upper layer, with the rhizosphere of these plants located in the main substrate layer for water purification. The main substrate layer for water purification is a mixture of modified biochar and fine gravel. A double-layer root net is laid or not laid in the plant rhizosphere region 100 mm below the surface of the main substrate layer for water purification. Activated sludge from the mariculture tailwater pond is introduced into the plant rhizosphere region. The wet sludge density is 25 g / L. The mass ratio of C and N elements in the mariculture tailwater influent is controlled to be 3:1 or 6:1. The volume ratio of the modified biochar to fine gravel is 1:(2.85-3.15).

[0009] Furthermore, the modified biochar is sodium hydroxide-modified straw biochar with a particle size of 2-8 mm.

[0010] Furthermore, the preparation method of the sodium hydroxide modified straw biochar includes the following steps: using crop straw as raw material, under N2 protection, the temperature is raised to 450±25℃ at a heating rate of 10 ℃ / min, and pyrolyzed for 2 hours. The pyrolyzed straw biochar is passed through a 2 mm aperture sieve, and the straw biochar on the sieve is retained. The straw biochar on the sieve is then passed through an 8 mm aperture sieve, and the straw biochar under the sieve is retained. The straw biochar on the sieve is further crushed, and the sieving is repeated. Finally, straw biochar with a particle size of 2-8 mm is retained. The sieved straw biochar is soaked in clean water and rinsed repeatedly 3-5 times until no obvious ash is visible in the washing water. Then, it is cleaned with an ultrasonic cleaner with an ultrasonic power of 700W for 10 minutes and dried in an oven to constant weight. Finally, it is fully soaked in 1.0 mol / L NaOH alkaline solution for 24 hours, and stirred evenly once every 4 hours during the period. After soaking, it is dried again to constant weight to obtain sodium hydroxide modified straw biochar.

[0011] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: Avoiding excessively small particle size of straw biochar results in a low permeability coefficient, making it prone to clogging. Conversely, excessively large particle size reduces pollutant removal efficiency. Therefore, to prevent clogging and ensure efficient pollutant removal, a suitable particle size range and appropriate ratio are necessary to achieve optimal technical results. Furthermore, straw biochar, as a supplementary carbon source for constructed wetland substrates, can promote microbial denitrification and plant phosphorus removal, enhancing the effective removal of pollutants such as N and P from marine aquaculture wastewater. Compared to unmodified biochar, modified straw biochar increases micropore volume and specific surface area, and increases the number of functional groups, further improving its adsorption and ion exchange capacity. This facilitates research on modified biochar as a substrate filler in constructed wetlands for the effective purification of marine aquaculture wastewater.

[0012] Furthermore, the bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

[0013] Furthermore, the fine gravel is slightly weathered basalt gravel with a particle size of 2-10 mm.

[0014] The beneficial effects of adopting the above-mentioned further technical solutions are: meeting the permeability requirements of constructed wetland substrates while obtaining a larger contact area with effluent, thus improving the purification effect; and preventing biochar powder generated during construction from clogging the constructed wetland system due to water flow, as the biochar powder in the gravel can be biodegraded to alleviate clogging.

[0015] Furthermore, the fine sand layer has a particle size of 0.5-2 mm.

[0016] The beneficial effects of adopting the above-mentioned further technical solutions are: promoting the growth of wetland plants, reducing the impact of downstream water flow on plant roots, reducing the floating of modified biochar in the main substrate layer caused by direct water erosion, inducing redundant development of wetland plant roots, and forming a more vigorous and dense network of fibrous roots.

[0017] Furthermore, the root mesh aperture is 1-2 mm.

[0018] Furthermore, sucrose was used to regulate the mass ratio of carbon and nitrogen elements in the effluent from marine aquaculture.

[0019] Furthermore, the large, fibrous-rooted wetland plant is reed, and the reed's height is 300-320 mm, with a planting density of 200-250 plants / m². 2 .

[0020] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: This system uses reeds, a large wetland plant with well-developed root systems and short growth cycles, which can improve the thorough and effective removal of pollutants such as TCs from the effluent by plants and rhizosphere microbial communities in the system. The vertical growth space stress exerted by the root network on the wetland plants can induce redundant development of plant roots, forming a denser fibrous root network structure, enhancing the secretion of oxygen and root exudates by the roots, promoting the colonization of rhizosphere microbial communities, and improving the purification of marine aquaculture effluent.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The vertical flow constructed wetland system has a high potential for removing tetracycline antibiotics, and has a strong removal effect on tetracycline hydrochloride (HCl-TC), chlortetracycline hydrochloride (HCl-CTC), doxycycline hydrochloride (HCl-DOX) and oxytetracycline (OTC), with an overall removal rate of over 92%.

[0022] The vertical flow constructed wetland purification system achieves removal rates of 91.67%-97.32%, 88.14%-93.94%, 96.86%-99.57%, and 89.80%-95.96% respectively for HCl-TC, HCl-CTC, HCl-DOX, and OTC in mariculture tailwater containing 250 μg / L of each. This constructed wetland purification system can achieve efficient simultaneous removal of the four TCs, all of which have high and stable removal potential. Among them, it has the strongest removal performance for DOX, which is the most widely used in mariculture veterinary drugs. The average removal rate of HCl-DOX can reach 99.07%, which can basically achieve complete removal. The average removal rate of HCl-CTC can also reach more than 92%.

[0023] 1) By setting up a root network in the matrix layer and regulating the organic load of the influent, the rhizosphere microenvironment of large fibrous-rooted wetland plants can be controlled, promoting the redundant development of the root system and generating changes in the rhizosphere microenvironment of wetland plants. Combined with the functional effects of modified biochar, it can achieve efficient and simultaneous removal of four types of TCs, and improve the purification effect of artificial wetlands on marine aquaculture tailwater with high concentrations of TCs.

[0024] 2) It can also reduce the residue of tetracycline ARGs in the constructed wetland system environment. tet(A) , tet(M) and intI1 The residual levels were lower in the upper matrix, plant stems and leaves, and root samples.

[0025] 3) This invention also provides the differences in root structure, root oxygen secretion capacity, and types and quantities of root exudates of wetland plants after the application of root network and influent organic load regulation, which can effectively achieve the performance optimization and transformation of the rhizosphere microenvironment of wetland plants.

[0026] This invention discloses a vertical flow constructed wetland system capable of efficiently degrading tetracycline antibiotics. Through a rhizosphere regulation and optimization method involving the addition of a root network and control of influent organic load, the system optimizes the root architecture, ROL (Residual Oxygen Level), RE (Residual Organic Load), and microbial community structure and composition within the constructed wetland's rhizosphere microenvironment. This achieves efficient removal of tetracycline antibiotics while effectively reducing the residue of antibiotic resistance genes within the system. The regulation method of this invention has advantages such as being eco-friendly, easy to operate, and highly effective, providing an ecologically based regulatory pathway for optimizing the purification performance of tetracycline antibiotics in marine aquaculture wastewater from constructed wetlands. Attached Figure Description

[0027] Figure 1 The figures are comparative diagrams of the constructed wetland system structure under different control effects in each embodiment. (A) is a schematic diagram of the constructed wetland system structure in Embodiment 1, (B) is a schematic diagram of the constructed wetland system structure in Embodiment 2, (C) is a schematic diagram of the constructed wetland system structure in Embodiment 3, and (D) is a schematic diagram of the constructed wetland system structure in Embodiment 4.

[0028] Figure 2 The figures show the differences in root growth of artificial wetland plants after different regulatory effects in the examples. Figure A shows the root growth of plants in column A in Example 1, Figure B shows the root growth of plants in column B in Example 2, Figure C shows the root growth of plants in column C in Example 3, and Figure D shows the root growth of plants in column D in Example 4.

[0029] Figure 3 The figures show the differences in oxygen secretion from the roots of artificial wetland plants after different regulatory effects in the examples. Figure A shows the oxygen secretion from the roots of column A in Example 1, Figure B shows the oxygen secretion from the roots of column B in Example 2, Figure C shows the oxygen secretion from the roots of column C in Example 3, and Figure D shows the oxygen secretion from the roots of column D in Example 4.

[0030] Figure 4 The figures show the differences in the composition of root exudates of artificial wetland plants after different regulatory effects in the examples. Figure A shows the composition of root exudates of plants in column A in Example 1, Figure B shows the composition of root exudates of plants in column B in Example 2, Figure C shows the composition of root exudates of plants in column C in Example 3, and Figure D shows the composition of root exudates of plants in column D in Example 4.

[0031] Figure 5 To compare the treatment effects of TCs in marine aquaculture effluent under different control conditions in the examples, (a) shows the comparison of TCs concentration in the effluent of constructed wetlands in Examples 1-4, and (b) shows the comparison of TCs removal rates in the effluent of constructed wetlands in Examples 1-4. In the figures, A is column A of Example 1, B is column B of Example 2, C is column C of Example 3, and D is column D of Example 4. Figure 6 (The letters in the middle refer to the same thing). u indicates water from the upper layer, and d indicates water from the lower layer.

[0032] Figure 6 Figure 1 shows the comparison of ARGs residual abundance in the constructed wetland system under different control conditions in the examples. (a) Figure 2 shows the comparison of ARGs residual abundance in the substrate layer of the constructed wetland in Examples 1-4, where u is the upper substrate and d is the lower substrate. (b) Figure 3 shows the comparison of ARGs residual abundance in the plants of the constructed wetland in Examples 1-4, where L is the stem and leaf tissue and R is the root tissue.

[0033] Figure 7 The physical diagrams of the root network set in Examples 3 and 4 are shown. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only 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 are within the scope of protection of the present invention.

[0035] In Examples 1-4 of this invention, the modified biochar is sodium hydroxide-modified straw biochar with a particle size of 2-8 mm.

[0036] The preparation method of sodium hydroxide modified straw biochar includes the following steps: using crop straw as raw material, under N2 protection, the temperature is raised to 450±25℃ at a heating rate of 10 ℃ / min, and pyrolyzed for 2 hours. The pyrolyzed straw biochar is passed through a 2 mm sieve, and the straw biochar on the sieve is retained. The straw biochar on the sieve is then passed through an 8 mm sieve, and the straw biochar under the sieve is retained. The straw biochar on the sieve is further crushed, and the sieving is repeated. Finally, straw biochar with a particle size of 2-8 mm is retained. The sieved straw biochar is soaked in clean water and rinsed repeatedly 3 times until no obvious ash is visible in the washing water. Then, it is cleaned with an ultrasonic cleaner with an ultrasonic power of 700W for 10 minutes. It is then dried in an oven to constant weight. Finally, it is fully soaked in 1.0 mol / L NaOH alkaline solution for 24 hours, and stirred evenly once every 4 hours during the soaking period. After soaking, it is dried again to constant weight to obtain sodium hydroxide modified straw biochar.

[0037] Example 1 A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics, named Column A, consists of a constructed wetland substrate column with a height of 650 mm and an inner diameter of 210 mm, made of plexiglass. The constructed wetland substrate column includes: a bottom layer of coarse gravel, and a water purification main substrate layer and a fine sand layer sequentially filled on the surface of the bottom coarse gravel. The main substrate layer for water purification is a mixture of modified biochar and fine gravel, with a volume ratio of 1:3. Activated sludge from the mariculture tailwater pond is introduced into the plant rhizosphere area, with a wet sludge density of 25 g / L. Sucrose is used to regulate the mass ratio of C and N elements in the mariculture tailwater influent to 3:1. The total concentration of TCs in the influent is 1 mg / L, and the COD concentration is 105 mg / L.

[0038] The bottom coarse gravel layer is 100 mm high, the main water purification matrix layer is 450 mm high, and the fine sand layer is 60 mm high.

[0039] The bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

[0040] The fine gravel is basalt crushed stone with a particle size of 2-10 mm.

[0041] The fine sand layer has a particle size of 0.5-2 mm.

[0042] It also includes: an ecosystem, which includes reeds, with the reed rhizosphere area set in the main substrate layer for water purification, and the reeds being 300 mm tall, with 4 plants planted.

[0043] The vertical flow constructed wetland system can remove HCl-TC, HCl-CTC, HCl-DOX and OTC from mariculture wastewater containing 250 μg / L of each at a rate of 95.70%, 92.57%, 98.22% and 93.51%, respectively.

[0044] Example 2 A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics, named Column B, was developed based on Example 1. Sucrose was used to regulate the mass ratio of C to N elements in the effluent from seawater aquaculture to 6:1, while all other experimental parameters remained unchanged. The constructed wetland substrate column is 650 mm high and has an inner diameter of 210 mm, made of plexiglass. The constructed wetland substrate column consists of a bottom layer of coarse gravel, and a water purification main substrate layer and a fine sand layer sequentially filled on the surface of the bottom coarse gravel. The main substrate layer for water purification is a mixture of modified biochar and fine gravel; the volume ratio of modified biochar to fine gravel is 1:3. Activated sludge from the effluent pond of seawater aquaculture is introduced into the plant rhizosphere area, with a wet sludge density of 25 g / L.

[0045] The bottom coarse gravel layer is 100 mm high, the main water purification matrix layer is 450 mm high, and the fine sand layer is 60 mm high.

[0046] The bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

[0047] The fine gravel is basalt crushed stone with a particle size of 2-10 mm.

[0048] The fine sand layer has a particle size of 0.5-2 mm.

[0049] It also includes: an ecosystem, which includes reeds, with the reed rhizosphere area set in the main substrate layer for water purification, and the reeds being 300 mm tall, with 4 plants planted.

[0050] The vertical flow constructed wetland system can remove HCl-TC, HCl-CTC, HCl-DOX and OTC from mariculture wastewater containing 250 μg / L of each at a rate of 96.40%, 92.79%, 98.88% and 95.07%, respectively.

[0051] Example 3 A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics, named C-column, was developed based on Example 1. A double-layered, cross-laid HDPE root net (3 mm pore size; vertical pore size 1.5 mm after double-layer cross-layout) was installed in the plant rhizosphere region 100 mm below the surface of the main water purification substrate layer. All other experimental parameters remained unchanged. The constructed wetland substrate column was 650 mm high and 210 mm in inner diameter, made of plexiglass. The constructed wetland substrate column consisted of a bottom layer of coarse gravel, and a main water purification substrate layer and a fine sand layer sequentially filled on the surface of the bottom coarse gravel. The main substrate layer for water purification consists of a mixture of modified biochar and fine gravel, with a volume ratio of 1:3. Activated sludge from the mariculture tailwater pond is introduced into the rhizosphere region of the plants, with a wet sludge density of 25 g / L. Sucrose is used to adjust the mass ratio of C to N elements in the mariculture tailwater influent to 3:1.

[0052] The bottom coarse gravel layer is 100 mm high, the main water purification matrix layer is 450 mm high, and the fine sand layer is 60 mm high.

[0053] The bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

[0054] The fine gravel is basalt crushed stone with a particle size of 2-10 mm.

[0055] The fine sand layer has a particle size of 0.5-2 mm.

[0056] It also includes: an ecosystem, which includes reeds, with the reed rhizosphere area set in the main substrate layer for water purification, and the reeds being 300 mm tall, with 4 plants planted.

[0057] The vertical flow constructed wetland system can remove HCl-TC, HCl-CTC, HCl-DOX and OTC from mariculture wastewater containing 250 μg / L of each at a rate of 96.07%, 92.88%, 98.80% and 94.03%, respectively.

[0058] Example 4 A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics, named D-column, was developed based on Example 3. Sucrose was used to regulate the mass ratio of C to N elements in the effluent from seawater aquaculture to 6:1, while all other experimental parameters remained unchanged. The constructed wetland matrix column is 650 mm high and has an inner diameter of 210 mm, made of plexiglass. The constructed wetland matrix column consists of a bottom layer of coarse gravel, and a water purification main matrix layer and a fine sand layer sequentially filled on the surface of the bottom coarse gravel. The main substrate layer for water purification is a mixture of modified biochar and fine gravel, with a volume ratio of 1:3. Activated sludge from the effluent pond of seawater aquaculture is introduced into the rhizosphere region, with a wet sludge density of 25 g / L. A double-layered, cross-laid HDPE root mesh (3 mm pore size; vertical pore size 1.5 mm after double-layer cross-laying) is installed in the rhizosphere region 100 mm below the surface of the main substrate layer for water purification.

[0059] The bottom coarse gravel layer is 100 mm high, the main water purification matrix layer is 450 mm high, and the fine sand layer is 60 mm high.

[0060] The bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

[0061] The fine gravel is basalt crushed stone with a particle size of 2-10 mm.

[0062] The fine sand layer has a particle size of 0.5-2 mm.

[0063] It also includes: an ecosystem, which includes reeds, with the reed rhizosphere area set in the main substrate layer for water purification, and the reeds being 300 mm tall, with 4 plants planted.

[0064] The vertical flow constructed wetland system can remove HCl-TC, HCl-CTC, HCl-DOX and OTC from mariculture wastewater containing 250 μg / L of each at a rate of 97.02%, 93.25%, 99.35% and 95.20% respectively.

[0065] Data testing: Based on the vertical flow constructed wetland system in Examples 1-4, two types of simulated marine aquaculture effluent with low C / N ratio and high C / N ratio were treated respectively. 250 μg / L of HCl-TC, HCl-CTC, HCl-DOX, and OTC were added to bring the total TC concentration in the influent to 1 mg / L. The salinity was measured using a salinity meter, and the salinity of the influent aquaculture effluent was adjusted and controlled within the range of 17.1%-18.1‰. Sucrose was used to adjust the COD concentration, and ammonium chloride, potassium nitrate, and potassium dihydrogen phosphate were used to regulate TN and NH4+ in the simulated effluent, respectively. + -N and TP concentration levels, intermittent operation, and downflow influent operation for 4 months, with specific parameter configurations for the constructed wetland operation are shown in Table 1. The hydraulic retention times of the mariculture tailwater were 5 days, 4 days, and 3 days, with an intermittent 2-day interval; the hydraulic loading rates were 0.046 / 0.058 / 0.077 m³, respectively. 3 / (m 2 •d) Indoor operation, room temperature 23-27 ℃. Collect 100 mL of influent and effluent from each constructed wetland at the end of each hydraulic retention time for each embodiment. Take three parallel samples. For each sample, the effluent from 100 mm below the surface of the main substrate layer is the upper effluent, used to evaluate the pollutant removal effect of the plant root zone; the effluent from 300 mm below the surface is the lower effluent, used to evaluate the TCs removal effect of the substrate packing layer. Water samples are stored at 4 ℃ and relevant physicochemical indicators are measured within 24 h.

[0066] Table 1 Configuration of Operating Parameters for Constructed Wetlands

[0067] After the entire operation cycle was completed, all plants and an appropriate amount of substrate samples were collected without damaging the original root system architecture. For plant samples, sufficient root, stem, and leaf samples were collected from the constructed wetlands of each embodiment using sterile scissors. For substrate samples, samples were collected at three sampling points at the same height as the upper and lower water outlets, arranged in an equilateral triangle pattern, and mixed into one sample. 25 g of each sample was weighed and added to 200 mL of ultrapure water. The mixture was shaken at 220 r / min for 1 h. The shaken suspension was then completely filtered through a 0.22 μm aqueous filter membrane, retaining the filter membrane and its sediment. The collected plant and substrate sediment samples were stored in 5 mL cryovials, freeze-dried, and then cryopreserved at -80℃ to inhibit the activity of the microbial community in the samples, for the detection of TCs antibiotic resistance gene abundance. The remaining plant samples were used to determine relevant experimental parameters such as root oxygen secretion, root exudates, and root architecture within 48 h.

[0068] TCs in water samples were determined using a high-resolution liquid chromatography-mass spectrometry (UHPLC-HRMS) system (Orbitrap Exploris 120, electrospray ionization (ESI) source, dynamic multiple reaction mode (DMRM)). The mobile phase consisted of buffer solution (0.1% formic acid in water) (A) and acetonitrile (B). The gradient elution program was as follows: 0 min (90% A; 10% B), 5 min (85% A; 15% B), 7 min (80% A; 20% B), 11 min (60% A; 40% B), 15 min (40% A; 60% B), 16 min (5% A; 95% B), 25 min (5% A; 95% B). The flow rate of the mobile phase was 0.3 mL / min. Mass spectrometry conditions in positive ion mode: drying gas temperature and flow rate of 325 °C and 6 mL / min; nebulizer pressure of 45 psi; sheath gas temperature and flow rate of 350 °C and 11 L / min; capillary voltage and nozzle voltage of 3500 V and 0 V, respectively. The chromatographic column was a Thermo Scientific™ Acclaim™ 120 C18 (150 mm × 2.1 mm, 2.2 μm). Data acquisition and analysis were performed in positive ion mode, and analyte quantification was based on an external standard curve.

[0069] The target gene was detected in substrate, plant root, and plant stem and leaf samples collected after the entire regulatory experiment. DNA was extracted from the samples strictly according to the kit (Omega, cat: M5635-02), and then quantitatively analyzed by real-time fluorescence PCR. intI1 , tet(A) and tet(M) Three target genes are identified, and the primer information for each target gene is shown in Table 2.

[0070] Table 2. Primer information for the target gene

[0071] The HD-WinRHIZ0-XL plant root analysis system was used to scan and analyze the root architecture of reed plants in each embodiment after the root regulation experiment. The root oxygen secretion intensity was measured and analyzed using a Micro1100 microelectrode testing system. Three to five reed plants from each experimental group, representing the average growth status of the entire plant, were transplanted into acrylic culture boxes for root ROL strength measurement. The acrylic culture boxes were filled with 20-30 cm of fine sand to support the reed roots and simulate the actual underground root growth environment. Using a DO microelectrode, the DO concentration near the plant roots at a depth of 10 cm below the surface layer of fine sand was measured under flooded conditions. The peak value of the rhizosphere DO concentration measured by the microelectrode was divided by the detection time to reflect the current true ROL strength of the plant. The average value of the effective test results at three different points in the rhizosphere of each group was used as the standard.

[0072] After the ROL determination experiment, the root systems of the tested plants in each group were first rinsed with tap water and then with ultrapure water. They were then placed in a 1.5 L container, and 1 L of ultrapure water was added to ensure the plant roots were completely submerged. The container opening was sealed with plastic wrap to prevent atmospheric carbon compounds from dissolving and interfering with the experimental results. After 24 hours of hydroponics, 200 mL of the culture medium was collected. The culture medium was first coarsely filtered through filter paper, and then filtered through a clean 0.45 μm aqueous filter membrane. The collected liquid was pre-frozen for 12 hours, then lyophilized to a dry powder. It was then redissolved in 2 mL of ethyl acetate, and an appropriate amount of anhydrous sodium sulfate was added to remove water. The solution was then filtered again through a 0.22 μm organic phase filter membrane and stored in a 1.5 mL vial for analysis to determine the composition of root exudates of reeds after root regulation in each example.

[0073] The composition of root exudates was determined using gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: HP-5ms capillary column; carrier gas: He, flow rate 1 mL / min. -1 Programmed temperature rise: Initial temperature 50 ℃, hold for 2 min, then increase at 6 ℃·min -1 The temperature was increased to 250 °C; the injection volume was 1 μL; the mass spectrometry conditions were: electron impact source, impact voltage 70 eV, scan range M / Z 33-453, scan rate 0.2 s for the entire spectrum, and ion source temperature 200 °C. Computer-aided determination of unknown substances and their relative contents was performed, and the relative content of each substance was calculated based on the peak area of ​​each detected component in the chromatogram.

[0074] Data processing: The experimental data were recorded and summarized using Microsoft Office 2021, and analyzed using IBM SPSS Statistics 26.0 and Origin 2021 software for ANOVA one-way ANOVA, multiple comparison tests, and plotting.

[0075] Test results are as follows Figures 2 to 6 As shown.

[0076] In each embodiment, after four months of rhizosphere regulation and reaching a mature and stable growth stage, the root system architecture of the artificial wetland plants was scanned using the HD-WinRHIZ0-XL plant root analysis system. Scan images of some characteristic regions are shown below. Figure 2 As shown in the figure. Through comparison, it can be clearly found that after the root network group provides vertical growth space stress to the wetland plant roots, a denser and more vigorous fibrous root structure is induced around the main root. The rhizosphere space stress can induce the wetland plant roots to produce the characteristic of redundant root development. Among them, the plants in column C have the largest number of fibrous roots and the highest density, and have a stronger function in intercepting pollutants in the tailwater and carrying microbial communities.

[0077] The root oxygen secretion of artificial wetland plants after rhizosphere regulation was measured using the Micro1100 microelectrode testing system in each embodiment. The comparison of the measurement data is as follows: Figure 3 As shown in the figure. A comparison reveals that after four months of rhizosphere regulation experiments, the ROL (Regenerative Oxygen Release) capacity of the artificial wetland plants in each embodiment was improved. The peak DO concentration measured in the matrix layer was 178.722 μmol / L in column A, 169.452 μmol / L in column B, 191.784 μmol / L in column C, and 186.782 μmol / L in column D. The main difference lies in the fact that after regulation, the DO concentration fluctuations were detectable in the root network group within a range of 28-80 mm below the matrix surface, with larger fluctuations and higher peak values ​​compared to the group without a root network, indicating a more vigorous and densely distributed reed root tissue structure in this area. The existing data are sufficient to prove that inducing redundant development of wetland plant roots is beneficial for promoting oxygen secretion from plant roots. Oxygen secretion from wetland plant roots can transport O2 from the leaf margin to the deep rhizosphere via aerenchyma, thereby increasing the DO concentration in the matrix layer and forming an O2 concentration gradient in the rhizosphere microenvironment. This will help the colonization of different types of rhizosphere microbial communities and the efficient degradation of pollutants. In addition, the root oxygen secretion capacity of the low C / N ratio influent group was slightly better than that of the high C / N ratio influent group.

[0078] Gas chromatography-mass spectrometry (GC-MS) was used to separate and qualitatively analyze the components of root exudates from constructed wetland plants in each example. The composition of root exudates measured in each group was summarized according to the category of organic compounds as follows: Figure 4 As shown. Among all root exudates measured in each set of examples, eight classes of organic compounds were detected: esters, alkanes, aldehydes, acids, phenols, ketones, alcohols, and amines. Among these, ethanol (C2H5OH), methyl acetate (C3H6O2), n-butyraldehyde (C4H8O), and ethyl propionate (C5H...) were also detected. 10O2), oxaloyl urea (C3H4N2O4), 2-ethylbutanal (C6H 12 Substances such as O were precisely and qualitatively detected in all RE groups. Ester compounds had the highest detection rate among all RE categories and are common components in REs, considered to be one of the key signaling molecules in the interaction between plant roots and rhizosphere microorganisms. The components and relative contents of root exudates detected in different examples varied considerably. Column C showed the most diverse range of components, including 27, with the highest relative content and the most diverse categories, covering all eight types of organic compounds measured. Column D detected 23 substances, and column B detected 21. In both examples, all compounds except amines were detected. Column A detected only 16 compounds, belonging to four categories: esters, aldehydes, alcohols, and acids, showing the fewest detected components. This suggests that applying vertical spatial stress to plant roots is beneficial for the release of root exudates in wetland plants. After redundant root development, more vigorous fibrous root tissue can enhance the carbon release function of plant roots, thereby influencing the modification of the plant rhizosphere microenvironment.

[0079] Figure 5 To measure the total TC concentration data of all effluents during the stable operation phase of the constructed wetlands in each embodiment, UHPLC-HRMS was used. Average values ​​were taken for inter-group comparison analysis, and the removal rate was calculated. Different lowercase letters in the figure indicate significant differences within the same antibiotic group (p<0.05). Figure 5 (a) As shown in the figure, during the stable operation phase of the experiment, the effluent concentrations of HCl-TC, HCl-CTC, HCl-DOX, and OTC in each embodiment were 6.71-20.84 μg / L, 15.14-29.64 μg / L, 1.08-7.84 μg / L, and 10.11-25.49 μg / L, respectively. Figure 5 (b) As shown in the figure, the removal rates reached 91.67%-97.32%, 88.14%-93.94%, 96.86%-99.57%, and 89.80%-95.96% respectively, indicating that the constructed wetland systems in this study all have high potential for TC removal. The average removal capacity for the four antibiotics, in descending order, was HCl-DOX > HCl-TC > OTC > HCl-CTC. The average TC removal rates of columns C and D after the root network were higher than those of columns A and B, respectively. This difference in removal rate indicates that plants and rhizosphere microbial communities play a certain role in TC removal. The root network group with optimal overall plant growth, richer rhizosphere microbial community, and more stable structure also had a higher TC removal rate.

[0080] Figure 6This figure compares the residual abundance of ARGs in the constructed wetland systems of various embodiments. In the figure, u represents the upper substrate sample, d represents the lower substrate sample, L represents the plant stem and leaf sample, and R represents the plant root sample (the same applies below); different lowercase letters indicate significant differences within the same ARG group (p<0.05). Figure 6 As shown in Figure (a), three target ARGs were detected in all collected matrix samples, with the highest concentration detected in the upper matrix of column D. intI1 , tet(A) and tet(M) All were the least abundant, with absolute abundances of 2.36 × 10⁻⁶. 7 copies / g, 7.83×10 6 copies / g and 2.07×10 5 The abundance of ARGs detected in the upper matrix of column C was also slightly lower than that in the group without root network, indicating that the application of vertical growth space stress in the rhizosphere is beneficial to accelerating the degradation and migration of ARGs in the upper root zone matrix layer and reducing residues. However, the effect of influent load regulation on the relative abundance of ARGs was not significant. In columns A, B, and C, the abundance levels of each target ARG detected in the upper and lower matrix layers were basically the same, with no significant difference. However, the detection of target ARGs in the lower matrix of column D was much higher than that in the upper layer, indicating that the microbial community in the lower layer of column D has a strong metabolic function for TCs. This can reasonably explain why the TCs concentration was the lowest and the removal rate was the highest in the effluent from the lower layer of column D. However, since there is no plant root growth in the deep lower layer of column D, more ARGs remained. This also indicates that plant roots have an important influence on the removal of ARGs in the constructed wetland matrix layer. Specifically, for different target ARGs, the absolute abundance of all samples was consistent, all being [missing information]. intI1 > tet (A) > tet(M) ,in intI1 and tet(A) The detected absolute abundance levels were roughly the same, all higher than tet(M) 2-3 orders of magnitude.

[0081] exist Figure 6 Figure (b) shows higher abundance in each plant sample. tet(A) and tet(M)The abundance of tetracycline ARGs was 1-2 orders of magnitude higher than that in the matrix samples. A cross-sectional comparison of the relative abundance of ARGs in the four experimental groups showed that the abundance of tetracycline ARGs in the root network group was significantly lower than that in the non-root network group in both the upper matrix and plant samples. Column D showed the lowest abundance of tetracycline ARGs in the upper matrix and plant stem and leaf samples, while column C showed the lowest abundance in the root samples. This indicates that the constructed wetland with root network exhibits a function of reducing ARG residues after the application of rhizosphere vertical spatial stress regulation. This difference is related to the differences in microbial community diversity and composition after regulation. The C / N ratio of the influent did not show a significant regularity in its effect on ARG environmental residues.

[0082] In summary, all the above embodiments can achieve simultaneous and efficient removal of the four types of TCs. The root network can regulate the rhizosphere microenvironment, allowing redundant root development in the constructed wetland, resulting in higher root oxygen secretion intensity and detection of more root exudates. At the same time, it can achieve a higher TC removal rate and less ARGs environmental residue. Adjusting the influent with a low C / N ratio can also optimize the plant rhizosphere microenvironment to a certain extent, but the regularity of its effect on TC removal rate and ARGs environmental residue is not significant.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics, characterized in that, include: The bottom layer of coarse gravel, and the water purification main matrix layer and fine sand layer sequentially filled on the surface of the bottom layer of coarse gravel; It also includes large fibrous-rooted wetland plants planted on the upper layer, with the rhizosphere of these plants located in the main substrate layer for water purification. The main substrate layer for water purification is a mixture of modified biochar and fine gravel. A double-layer root net is laid or not laid in the plant rhizosphere region 100 mm below the surface of the main substrate layer for water purification. Activated sludge from the mariculture tailwater pond is introduced into the plant rhizosphere region. The wet sludge density is 25 g / L. The mass ratio of C and N elements in the mariculture tailwater influent is controlled to be 3:1 or 6:

1. The volume ratio of the modified biochar to fine gravel is 1:(2.85-3.15).

2. The rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to claim 1, characterized in that, The modified biochar is sodium hydroxide-modified straw biochar with a particle size of 2-8 mm.

3. The rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to claim 1, characterized in that, The bottom coarse gravel is basalt crushed stone with a particle size of 10-20 mm.

4. The rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to claim 1, characterized in that, The fine gravel is slightly weathered basalt fragments with a particle size of 2-10 mm.

5. A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to claim 1, characterized in that, The fine sand layer has a particle size of 0.5-2 mm.

6. The rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to claim 1, characterized in that, The root mesh has a diameter of 1-2 mm.

7. A rhizosphere-regulated vertical flow constructed wetland system for removing tetracycline antibiotics according to any one of claims 1-6, characterized in that, The large, fibrous-rooted wetland plant is reed, with a plant height of 300-320 mm and a planting density of 200-250 plants / m². 2 .

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