A Method for Constructing and Applying Multi-Pond Artificial Wetland Systems Based on Rhizosphere Synergistic Symbiosis

Abstract

This invention provides a method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis and its application, relating to the field of ecological environment restoration technology. Specifically, it includes the following steps: preparing engineered algal strain A displaying laccase on its surface and engineered algal strain B displaying non-specific nucleases on its surface, and mixing them in a specific ratio to prepare a composite microalgae agent; introducing the prepared composite microalgae agent into a shallow pond at the front end of the wetland to construct a photocatalytic microalgae pretreatment unit; and planting submerged plants in the symbiotic ponds connected in series after the pretreatment unit. This invention, by employing a multi-pond artificial wetland system construction method based on rhizosphere synergistic symbiosis and utilizing synthetic biology surface display technology, anchors bacterial laccase and non-specific nucleases onto the cell surfaces of different Synechococcus algae, solving the problem of insufficient carbon source leading to easy saturation and short lifespan of the substrate adsorbed with phosphorus in existing artificial wetlands treating wastewater effluent, and compensating for the shortcomings of existing single ecological restoration technologies.

Description

Technical Field

[0001] This invention relates to the field of ecological environment restoration technology, specifically to a method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis and its application. Background Technology

[0002] With increasingly stringent requirements for water environment management, deep phosphorus removal and ecological security assurance of wastewater effluent have become crucial. Currently, constructed wetlands are widely used as the mainstream ecological restoration technology. Traditional wetland phosphorus removal mainly relies on the physicochemical adsorption and precipitation effects of substrates (such as zeolite, slag, gravel, etc.).

[0003] However, as operating time increases, the effective adsorption sites on the substrate surface are quickly filled and saturated, leading to an exponential decline in phosphorus removal capacity. Constructed wetlands face a significant "substrate saturation" bottleneck in terms of ultimate phosphorus removal. More seriously, when the internal environment of the wetland fluctuates (such as changes in pH or redox potential), the fixed phosphorus is easily chemically desorbed and released back into the water, causing a rebound in total phosphorus concentration in the effluent or even exceeding the standard, resulting in "secondary pollution." Furthermore, the frequent replacement and disposal of saturated substrate has always been a huge burden in operation and maintenance.

[0004] Furthermore, trace amounts of new pollutants (such as antibiotics) remaining in the wastewater are becoming a core factor threatening aquatic ecosystem security. Due to the nutrient-poor nature of the wastewater, the metabolic activity of native wetland microorganisms is low, making it difficult for them to secrete sufficient and highly active extracellular enzymes to effectively degrade antibiotics (such as tetracyclines and sulfonamides) with complex and stable chemical structures. Traditional biodegradation processes are slow and incomplete, and the biotoxicity of antibiotics, in turn, inhibits the function of wetland microbial communities, making it difficult to remove these persistent organic pollutants through mineralization.

[0005] A more insidious risk lies in the spread of resistance genes (ARGs). Existing wetland systems lack active blocking mechanisms against cell-free extracellular DNA (eDNA). The resistance-carrying eDNA released by damaged bacteria can easily adhere to the substrate or plant root surface and spread among environmental microorganisms through horizontal gene transfer, causing constructed wetlands to evolve into "reservoirs" of drug-resistant bacteria.

[0006] Therefore, given that existing single ecological restoration technologies are unable to overcome the shortcomings of easy saturation of substrate phosphorus removal and the lack of specific degradation mechanisms for antibiotics and resistance genes, there is an urgent need to develop a novel coupled purification system that integrates biological assimilation phosphorus removal with enzyme engineering-enhanced degradation. Summary of the Invention

[0007] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for constructing a multi-pond constructed wetland system based on rhizosphere synergistic symbiosis and its application. This method solves the problems of insufficient carbon sources in existing constructed wetlands when treating wastewater effluent, which leads to easy saturation and short lifespan of the substrate adsorbed with phosphorus, and the lack of active degradation mechanisms for new pollutants such as antibiotics and resistance genes, thus causing ecological risks.

[0008] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis, which specifically includes the following steps: Engineered algal strain A, which displays laccase on its surface, and engineered algal strain B, which displays non-specific nuclease on its surface, were prepared separately and then mixed in a certain proportion to prepare a composite microalgae inoculant. The prepared composite microalgae agent was inoculated into a shallow pond at the front end of the wetland to construct a photocatalytic microalgae pretreatment unit. Submerged plants are planted in the symbiotic ponds set up in series after the pretreatment unit. After the submerged plants are established, they are inoculated with a compound microalgae agent to form a biocatalytic film on the surface of the stems and leaves of the submerged plants, thus constructing a microalgae-submerged plant symbiotic unit. A sulfur-modified mixed substrate is laid at the bottom of a deep purification pond connected in series after the symbiotic pond, and emergent plants are planted on it to form a sulfur-containing substrate-emergent plant deep purification unit. A multi-pond artificial wetland system is constructed based on a photocatalytic microalgae pretreatment unit, a microalgae-submerged plant symbiotic unit, and a sulfur-containing matrix-emergent plant deep purification unit.

[0009] Furthermore, the preparation of the compound microalgae inoculant is as follows: Synechococcus was selected as the chassis cell; engineered algal strain A, which integrates the laccase gene (cotA) and the InP-N fusion expression cassette, and engineered algal strain B, which integrates the nuclease gene (nucA) and the InP-N fusion expression cassette, were constructed; algal strain A and algal strain B were mixed according to the cell number ratio, and the final optical density value of the bacterial solution was adjusted to obtain a composite microalgae inoculum.

[0010] Furthermore, the ratio of the number of engineered algae strain A to engineered algae strain B in the composite microalgae agent is 1:1 to 3:1, and the final optical density value of the bacterial solution is adjusted to 0.8-1.2.

[0011] Furthermore, the effective water depth of the photocatalytic microalgae pretreatment unit is 0.3-0.5m, and the hydraulic retention time is set to 24-48 hours.

[0012] Furthermore, the submerged plants are selected from a mixture of Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum, with a planting ratio of 2-3:3-5:1-3 and a planting density of 40-60 clumps / m². 2 .

[0013] Furthermore, submerged plants are planted in the symbiotic ponds connected in series after the pretreatment unit. After the submerged plants have established themselves, the specific procedure for inoculating with a compound microalgae agent is as follows: When the submerged plants have grown to 10-15cm below the water surface, dilute the compound microalgae agent to an OD730 of 0.4-0.6 and spray it evenly on the water surface.

[0014] Furthermore, the sulfur-modified mixed matrix is ​​prepared by mixing and laying the following raw materials in parts by weight: The composition includes 10-20 parts of natural pyrite particles, 15-25 parts of conductive modified biochar, 30-40 parts of zeolite, and 40-60 parts of gravel; the particle size of the pyrite particles is 3-5 mm, the particle size of the zeolite is 3-6 mm, and the particle size of the gravel is 8-16 mm.

[0015] Furthermore, the preparation method of the conductive modified biochar includes the following steps: Agricultural waste biomass is crushed and passed through a 100-mesh sieve; The screened biomass was immersed in a FeCl3 solution with a concentration of 1.0-1.5 mol / L for 12-24 hours with stirring. The impregnated biomass was dried and then pyrolyzed at 500-600℃ for 2-3 hours under nitrogen protection. After cooling, it was ground through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

[0016] An application of a multi-pond constructed wetland system based on rhizosphere synergistic symbiosis, wherein the multi-pond constructed wetland system is used for the treatment of wastewater effluent, and its operation and maintenance are as follows: The wastewater effluent flows sequentially through the pretreatment unit, symbiotic unit, and deep purification unit constructed above for purification. The accumulated phosphorus element is removed by periodically harvesting the above-ground parts of submerged and emergent plants.

[0017] Furthermore, the periodic harvesting specifically refers to harvesting 1 / 3 to 1 / 2 of the upper biomass of the submerged plants every 3-4 months for the microalgae-submerged plant symbiotic unit. For the sulfur-containing substrate-emergent plant deep purification unit, the above-ground withered parts of the emergent plants are harvested in late autumn and early winter each year, with a stubble height of 10-20cm.

[0018] (III) Beneficial Effects This invention provides a method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis and its application. Compared with existing technologies, it has the following advantages: This invention employs a rhizosphere-co-symbiotic multi-pond constructed wetland system construction method, utilizing synthetic biology surface display technology to anchor bacterial laccases and non-specific nucleases onto the cell surfaces of different Synechococcus species. This design not only avoids the problem of excessive metabolic burden caused by a single cell expressing multiple exogenous enzymes, ensuring the growth advantage of microalgae, but also endows the wetland system with dual purification functions. It solves the problems of insufficient carbon sources leading to easy saturation and short lifespan of the substrate for phosphorus adsorption in existing constructed wetlands treating wastewater effluent, as well as the lack of active degradation mechanisms for new pollutants such as antibiotics and resistance genes, thus addressing the shortcomings of single ecological restoration technologies. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0020] This application provides a method for constructing a multi-pond constructed wetland system based on rhizosphere synergistic symbiosis and its application. This method addresses the problems of insufficient carbon sources in existing constructed wetlands when treating wastewater effluent, which leads to easy saturation and short lifespan of the substrate adsorbed with phosphorus, as well as the lack of active degradation mechanisms for new pollutants such as antibiotics and resistance genes, thus causing ecological risks. This method also makes up for the shortcomings of single ecological restoration technologies.

[0021] To better understand the above technical solution, the following will provide a detailed explanation of the technical solution in conjunction with specific implementation methods.

[0022] The method for constructing multi-pond artificial wetland systems based on rhizosphere synergistic symbiosis specifically includes the following steps: S1. Preparation of engineered microalgae inoculants: Engineered algal strain A, which displays laccase on its surface, and engineered algal strain B, which displays non-specific nuclease on its surface, were prepared separately and then mixed in a certain proportion to prepare a composite microalgae inoculant. Among them, the cell ratio of engineered algae strain A to engineered algae strain B in the compound microalgae agent is 1:1 to 3:1, and the final optical density value of the bacterial solution is adjusted to 0.8-1.2. It should be noted that, specifically, in step S1, the chassis cells of both engineered algal strain A and engineered algal strain B are Synechococcus; the genome of engineered algal strain A integrates a fusion expression cassette composed of a strong promoter PcpcB, an ice nucleoprotein N-terminal domain gene InP-N, a flexible linker peptide gene (Gly4Ser)3, and a bacterial laccase gene cotA; the genome of engineered algal strain B integrates a fusion expression cassette composed of a strong promoter PcpcB, an ice nucleoprotein N-terminal domain gene InP-N, a flexible linker peptide gene (Gly4Ser)3, and a non-specific nuclease gene nucA.

[0023] The preparation of the compound microalgae inoculant is as follows: Synechococcus was selected as the chassis cell; engineered algal strain A, which integrates the laccase gene (cotA) and the InP-N fusion expression cassette, and engineered algal strain B, which integrates the nuclease gene (nucA) and the InP-N fusion expression cassette, were constructed; algal strain A and algal strain B were mixed according to the cell number ratio, and the final optical density value of the bacterial solution was adjusted to obtain a composite microalgae inoculum.

[0024] It should be noted that the engineered algal strain A was constructed, and its genome integrated a fusion expression cassette consisting of a strong promoter PcpcB, an ice nucleoprotein N-terminal domain gene InP-N, a flexible linker peptide gene (Gly4Ser)3, and a bacterial laccase gene cotA linked sequentially; the engineered algal strain B was constructed, and its genome integrated a fusion expression cassette consisting of a strong promoter PcpcB, an ice nucleoprotein N-terminal domain gene InP-N, a flexible linker peptide gene (Gly4Ser)3, and a non-specific nuclease gene nucA linked sequentially. After mixing algal strains A and B according to the cell number ratio and adjusting the final optical density value of the bacterial solution, a composite microalgae inoculum was prepared.

[0025] S2. Construct a photocatalytic microalgae pretreatment unit: The prepared composite microalgae agent was inoculated into a shallow pond at the front end of the wetland. The effective water depth of the photocatalytic microalgae pretreatment unit is 0.3-0.5m, and the hydraulic retention time (HRT) is set to 24-48 hours. S3. Constructing a microalgae-submerged plant symbiotic unit: Submerged plants are planted in the symbiotic ponds connected in series after the pretreatment unit. After the submerged plants are established, they are inoculated with the compound microalgae agent prepared in S2 to form a biocatalytic film on the surface of the stems and leaves of the submerged plants. Among them, the submerged plants selected are Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum, planted in a mixed ratio of 2-3:3-5:1-3, with a planting density of 40-60 clumps / m². 2 ; In step 3, the specific operation of inoculating with compound microalgae agent is as follows: when the submerged plants grow to 10-15cm below the water surface, dilute the compound microalgae agent to an OD730 of 0.4-0.6 and spray it evenly on the water surface. It should be noted that when inoculating the compound microalgae agent prepared in S2, spraying or immersion methods should be used.

[0026] S4. Construct a sulfur-containing matrix-emergent plant deep purification unit: A sulfur-modified mixed substrate was laid at the bottom of the deep purification pond set up in series after the symbiotic pond, and emergent plants were planted on it. Among them, emergent plants selected are reeds, cattails, and canna lilies, with a planting ratio of 1-3:2-4:3-5 and a planting density of 6-12 plants / m². 2 .

[0027] In step 4, the sulfur-modified mixed matrix is ​​prepared by mixing and laying the following raw materials in parts by weight: 10-20 parts of natural pyrite particles, 15-25 parts of conductive modified biochar, 30-40 parts of zeolite, and 40-60 parts of gravel; the particle size of the pyrite particles is 3-5 mm, the particle size of the zeolite is 3-6 mm, and the particle size of the gravel is 8-16 mm. The method for preparing the conductive modified biochar includes the following steps: S41, crush agricultural and forestry waste biomass (such as straw, waste wood, waste bamboo) and pass it through a 100-mesh sieve; S42, the screened biomass is immersed in a FeCl3 solution with a concentration of 1.0-1.5 mol / L and stirred for 12-24 hours; S43 involves drying the impregnated biomass, pyrolyzing it at 500-600℃ for 2-3 hours under nitrogen protection, cooling it, and then grinding it through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

[0028] S5. Operation and Maintenance: The wastewater effluent flows sequentially through the pretreatment unit, symbiotic unit, and deep purification unit constructed above for purification. The enriched phosphorus element is removed by periodically harvesting the above-ground parts of submerged and emergent plants. The specific strategy for periodic harvesting is as follows: For the microalgae-submerged plant symbiotic unit, harvest 1 / 3 to 1 / 2 of the upper biomass of the submerged plant every 3-4 months; for the sulfur-containing substrate-emergent plant deep purification unit, harvest the above-ground withered parts of the emergent plant in late autumn and early winter each year, leaving a stubble height of 10-20cm.

[0029] This scheme constructs a multi-pond artificial wetland system with rhizosphere synergistic symbiosis. The specific construction steps and parameters are as follows: Shallow ponds, symbiotic ponds, and deep purification ponds are sequentially set up at the front end of the wetland. Based on the above construction method, a photocatalytic microalgae pretreatment unit, a microalgae-submerged plant symbiotic unit, and a sulfur-containing substrate-emergent plant deep purification unit are constructed respectively. The construction steps and parameters in the construction scheme are shown in the following embodiments: Example 1 Firstly, the preparation of conductive modified biochar and microalgae inoculants is detailed below: (1) Preparation of conductive modified biochar: (a) Agricultural waste is crushed by a pulverizer and then sieved through a 100-mesh screen; (b) Take the sieved biomass powder and immerse it in a 1.0 mol / L FeCl3 solution. Stir and immerse for 12 hours at room temperature to allow iron ions to fully enter the biomass pores. (c) The impregnated biomass is dried at 80°C, placed in a tube furnace, heated to 500°C under a nitrogen protective atmosphere, and pyrolyzed and carbonized for 2 hours. (d) After cooling to room temperature, remove the material, grind it, and pass it through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

[0030] (2) Preparation of engineered microalgae inoculants: Synechococcus UTEX 2973 was selected as the chassis cell. Two engineered algal strains, A and B, were constructed, one integrating the laccase gene (cotA) and the InP-N fusion expression cassette. Algal strains A and B were mixed at a 1:1 cell ratio, and the final optical density (OD730) of the bacterial culture was adjusted to 0.8 using BG11 medium to obtain the composite microalgae inoculum.

[0031] Secondly: The setup of the Duotang artificial wetland system is as follows: (3) Construction of photocatalytic microalgae pretreatment unit: A shallow pond was set up at the very front of the wetland, with an effective water depth of 0.3m. The compound microalgae agent prepared in step (2) was inoculated, and the hydraulic retention time was set to 24 hours.

[0032] (4) Construction of microalgae-submerged plant symbiotic units: A symbiotic pond is connected in series after the pretreatment unit. Submerged plants are mixed and planted, with a planting ratio (plant number ratio) of Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum of 2:3:1, and a planting density of 40 clumps / m². 2 Once the submerged plants have grown stably and reached a height of 15cm below the water surface, dilute the compound microalgae agent from step (2) to an OD730 of 0.4 and spray it evenly onto the water surface.

[0033] (5) Construction of a sulfur-containing substrate-emergent plant deep purification unit: A deep purification pond is connected in series after the symbiotic pond. The bottom is laid with a sulfur-modified mixed substrate, composed of the following raw materials in parts by weight: 10 parts natural pyrite particles (3mm diameter), 15 parts conductive modified biochar (obtained in step 1), 30 parts zeolite (3mm diameter), and 40 parts gravel (8mm diameter). Emergent plants are mixed and planted on the substrate layer, with reeds, cattails, and thaliana planted in a ratio of 1:2:3 at a density of 6 plants / m². 2 .

[0034] Thirdly: Operations and maintenance, as detailed below: (6) Operation and maintenance: For the microalgae-submerged plant symbiotic unit, one-third of the biomass of the submerged plant is harvested every three months; for the deep purification unit, the withered parts of the emergent plant are harvested at the end of November each year, leaving a stubble height of 10cm.

[0035] Example 2 This scheme constructs a multi-pond artificial wetland system with rhizosphere symbiosis. Compared with Example 1, the specific adjusted parameters for Example 2 are as follows: (1) Preparation of conductive modified biochar: (a) Agricultural waste is crushed by a pulverizer and then sieved through a 100-mesh screen; (b) Take the sieved biomass powder and immerse it in a 1.2 mol / L FeCl3 solution, stirring and immersing for 18 hours at room temperature; (c) The impregnated biomass is dried at 85°C, placed in a tube furnace, and heated to 550°C under a nitrogen protective atmosphere for pyrolysis and carbonization for 2.5 hours. (d) After cooling to room temperature, remove the material, grind it, and pass it through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

[0036] (2) Preparation of engineered microalgae inoculants: The construction of the chassis cells and genes is the same as in Example 1. Algal strain A and algal strain B are mixed at a cell ratio of 2:1, and the final optical density value (OD730) of the bacterial solution is adjusted to 1.0 using BG11 medium to obtain a compound microalgae inoculum.

[0037] (3) Construction of photocatalytic microalgae pretreatment unit: A shallow pond was set up at the very front of the wetland, with an effective water depth of 0.4m. The compound microalgae agent prepared in step (2) was inoculated, and the hydraulic retention time was set to 36 hours.

[0038] (4) Construction of microalgae-submerged plant symbiotic units: Symbiotic ponds were connected in series after the pretreatment units. Submerged plants were mixed and planted, with the planting ratio of Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum being 2.5:4:2, and the planting density being 50 clumps / m².2 After the submerged plants have established themselves, dilute the compound microalgae agent in step (2) to an OD730 of 0.5 and spray it evenly onto the water surface.

[0039] (5) Construction of a sulfur-containing substrate-emergent plant deep purification unit: A deep purification pond is connected in series after the symbiotic pond. The bottom is laid with a sulfur-modified mixed substrate, composed of the following raw materials in parts by weight: 15 parts natural pyrite particles (4mm diameter), 20 parts conductive modified biochar (obtained in step 1), 35 parts zeolite (5mm diameter), and 50 parts gravel (12mm diameter). Emergent plants are mixed and planted on the substrate layer, with reeds, cattails, and thaliana in a planting ratio of 2:3:4, at a planting density of 9 plants / m². 2 .

[0040] (6) Operation and maintenance: For the microalgae-submerged plant symbiotic unit, half of the biomass of the submerged plant is harvested every 3.5 months; for the deep purification unit, the withered parts of the emergent plant are harvested in mid-November each year, leaving a stubble height of 15cm.

[0041] Example 3 This scheme constructs a multi-pond artificial wetland system with rhizosphere symbiosis. Compared with Examples 1 and 2, the specific adjusted parameters for Example 3 are as follows: (1) Preparation of conductive modified biochar: (a) Agricultural waste is crushed by a pulverizer and then sieved through a 100-mesh screen; (b) Take the sieved biomass powder and immerse it in a 1.5 mol / L FeCl3 solution, stirring and immersing for 24 hours at room temperature; (c) The impregnated biomass is dried at 90°C, placed in a tube furnace, and heated to 600°C under a nitrogen protective atmosphere for pyrolysis and carbonization for 3 hours. (d) After cooling to room temperature, remove the material, grind it, and pass it through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

[0042] (2) Preparation of engineered microalgae inoculants: The construction of the chassis cells and genes is the same as in Example 1. Algal strain A and algal strain B are mixed at a cell ratio of 3:1, and the final optical density value (OD730) of the bacterial solution is adjusted to 1.2 using BG11 medium to obtain a composite microalgae inoculum.

[0043] (3) Construction of photocatalytic microalgae pretreatment unit: A shallow pond was set up at the very front of the wetland, with an effective water depth of 0.5m. The compound microalgae agent prepared in step (2) was inoculated, and the hydraulic retention time was set to 48 hours.

[0044] (4) Construction of microalgae-submerged plant symbiotic units: A symbiotic pond is connected in series after the pretreatment unit. Submerged plants are mixed and planted, with *Vallisneria natans*, *Elodea nuttallii*, and *Ceratophyllum demersum* planted in a ratio of 3:5:3, at a density of 60 clumps / m². 2 After the submerged plants have established themselves, dilute the compound microalgae agent in step (2) to an OD730 of 0.6 and spray it evenly onto the water surface.

[0045] (5) Construction of a sulfur-containing substrate-emergent plant deep purification unit: A deep purification pond is connected in series after the symbiotic pond. The bottom is laid with a sulfur-modified mixed substrate, composed of the following raw materials in parts by weight: 20 parts natural pyrite particles (5mm diameter), 25 parts conductive modified biochar, 40 parts zeolite (6mm diameter), and 60 parts gravel (16mm diameter). Emergent plants are mixed and planted on the substrate layer, with reeds, cattails, and thaliana planted in a ratio of 3:4:5, at a planting density of 12 plants / m². 2 .

[0046] (6) Operation and maintenance: For the microalgae-submerged plant symbiotic unit, half of the biomass of the submerged plant is harvested every 4 months; for the deep purification unit, the withered parts of the emergent plant are harvested in early December each year, leaving a stubble height of 20cm.

[0047] After 90 days of operation, the water quality of the control group (Class A standard effluent from a municipal wastewater treatment plant) and the effluent treated by the system in Examples 1-3 were tested. The experimental methods are as follows: (1) Configuration of experimental instruments Total phosphorus testing instruments: UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan); autoclave.

[0048] Tetracycline detection instruments: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS, Agilent 1290 Infinity II LC / 6470 Triple Quad MS, Agilent Technologies, USA); fully automated solid phase extraction (SPE); twelve-well water bath nitrogen blowing system.

[0049] Resistance gene detection instruments: Real-Time qPCR instrument (ABI 7500, Applied Biosystems, USA); NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA); high-speed refrigerated centrifuge.

[0050] (2) Specific testing methods for each indicator 1) Method for determining total phosphorus (TP) The national standard method "Determination of Total Phosphorus in Water - Ammonium Molybdate Spectrophotometric Method" (GB 11893-89) was adopted. Specific steps: A suitable amount of homogeneous water sample was placed in a colorimetric tube, potassium persulfate solution was added, and the sample was digested in a 120°C autoclave for 30 minutes. After cooling, ascorbic acid and molybdate colorimetric reagent were added sequentially, and the sample was allowed to stand at room temperature for 15 minutes for color development. The absorbance was measured at 700 nm using a UV-Vis spectrophotometer, and the total phosphorus concentration was calculated using the standard curve.

[0051] 2) Methods for determining tetracycline (TC) Since the tetracycline concentration in the effluent was at a trace level (ng / L), solid-phase extraction (SPE) enrichment combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used for quantitative analysis.

[0052] Enrichment and purification: 500 mL of water sample was filtered through a 0.45 μm glass fiber membrane. An Oasis HLB solid-phase extraction column (500 mg / 6 mL) was activated sequentially with methanol and ultrapure water. The filtered water sample was enriched on the extraction column at a flow rate of 5 mL / min, followed by rinsing with ultrapure water and vacuum drying. Finally, 10 mL of methanol was used as elution, and the eluent was concentrated to near dryness at 40°C using a nitrogen blowdown apparatus. The eluent was then diluted to volume with 1 mL of initial mobile phase, filtered through a 0.22 μm organic filter membrane, and ready for analysis.

[0053] Instrumental analysis: HPLC-MS / MS was used for detection, with a C18 reversed-phase column. Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile, with gradient elution. Mass spectrometry was performed in electrospray ionization (ESI+) mode and multiple reaction monitoring (MRM) mode, with accurate quantification using the internal standard method.

[0054] (3) Method for determining the abundance of tetracycline resistance gene (tetA) The absolute abundance of the tetA gene in water was determined using real-time quantitative PCR (qPCR).

[0055] Nucleic acid extraction: Take 500 mL of water sample and filter it through a 0.22 μm polycarbonate microporous membrane to retain microorganisms and extracellular DNA in the water. Cut the filter membrane into pieces and place them in centrifuge tubes. Extract total genomic DNA using a commercial water DNA extraction kit (such as the FastDNASPIN Kit for Soil / Water). Determine the DNA concentration and purity using a NanoDrop 2000 (OD260 / 280 should be between 1.8 and 2.0).

[0056] qPCR amplification: Specific primers were designed for the tetA gene. The PCR reaction system (20 μL) included: 10 μL of SYBR Green fluorescent dye premix, 0.4 μL each of forward and reverse primers, 2 μL of DNA template, and sterile water to a final volume of 20 μL.

[0057] Reaction procedure and quantification: Amplification was performed on a real-time PCR instrument using the following program: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 15 seconds; 60°C annealing extension for 34 seconds, for a total of 40 cycles. Each amplification included a plasmid containing a known copy number of the tetA gene fragment as a standard. A standard curve was plotted to calculate the absolute copy number of the tetA gene in the sample (unit: copies / mL).

[0058] The test results are shown in Table 1: Table 1. Water body detection results of Examples 1-3 As shown in Table 1, the multi-pond constructed wetland system of microalgae-plant-microbe rhizosphere synergistic symbiosis constructed in this invention can effectively remove phosphorus and new pollutants from the effluent. Compared with the control group, the total phosphorus in the effluent of the three embodiments consistently met the Class III surface water standard (≤0.2 mg / L), and the removal effects on antibiotics and resistance genes were all 1-2 orders of magnitude higher. This system not only achieves deep phosphorus removal but also demonstrates excellent performance in reducing biotoxicity risks. The comprehensive purification effect of Example 2 is significantly better than that of other embodiments, and its core advantage stems from the precise synergy of the three-in-one system of "engineered microalgae-conductive matrix-plant rhizosphere".

[0059] Regarding antibiotic removal, the conductive modified biochar (pyrolyzed at 550℃) prepared in Example 2, combined with pyrite, constructed a superior electron transport channel, significantly accelerating the oxidative degradation of antibiotics by laccases on the microalgae surface. In terms of resistance gene removal, the appropriate plant planting density and the compound microalgae inoculant formed a highly active biocatalytic membrane, and the nucleases on the surface efficiently cleaved extracellular resistance genes. Furthermore, the plant configuration in Example 2 (the ratio of Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum, and the ratio of Reed, Typha, and Thalia dealbata) achieved an optimal balance between root oxygen secretion and biomass growth, ensuring efficient phosphorus assimilation and providing a guarantee for the formation of an alternating aerobic / anaerobic environment in the rhizosphere, demonstrating the crucial role of process parameter optimization in improving system performance.

[0060] In summary, compared with existing technologies, it has the following beneficial effects: 1. This invention utilizes a multi-pond artificial wetland system construction method based on rhizosphere synergistic symbiosis, employing a "functional division of labor" microbial community combination strategy. Using synthetic biology surface display technology, bacterial laccase and non-specific nucleases are anchored onto the cell surfaces of different Synechococcus species. This design not only avoids the problem of excessive metabolic load caused by a single cell expressing multiple exogenous enzymes, ensuring the growth advantage of microalgae, but also endows the wetland system with dual purification functions: laccase can effectively oxidize and degrade refractory antibiotics such as tetracycline and sulfonamides, while nucleases can actively cleave extracellular resistance genes (eDNA) in the water, blocking the horizontal spread of drug resistance at the source and solving the problem of high ecological risks from new pollutants in effluent. 2. This invention constructs a microalgae-submerged plant symbiotic system, in which engineered microalgae attach to the surface of plant leaves to form a highly active biocatalytic film. The plant provides a huge specific surface area for the microalgae to attach, preventing the microalgae from being lost with the water flow; at the same time, the in-situ dissolved oxygen produced by the photosynthesis of the microalgae directly supplies the laccase (which uses oxygen as an electron acceptor) on the surface, maintaining the enzyme's highly efficient oxidation activity and solving the problem of decreased enzyme activity due to hypoxia in the bottom layer of traditional wetlands and inside biofilms; 3. This invention introduces conductive modified biochar and pyrite into the matrix, constructing a highly efficient electron transport chain of "microalgae (enzyme source / electron donor) - conductive matrix (electron mediator) - pollutants (electron acceptor)". The conductive matrix acts as an electron shuttle, significantly accelerating the electron transfer rate in the laccase-catalyzed oxidation of antibiotics. Furthermore, pyrite can also act as an electron donor to drive the sulfur autotrophic denitrification process, achieving deep denitrification under conditions of low carbon-to-nitrogen ratio in the effluent (lack of organic carbon source) without the need for an external carbon source. 4. This invention proposes a long-term phosphorus removal mechanism based on biological assimilation. Unlike traditional matrix adsorption methods (which are easily saturated and desorbed), this system utilizes the efficient absorption of phosphorus by submerged plants and attached microalgae during their growth, converting dissolved phosphorus in the water into biomass phosphorus in the plants and algae. By periodically harvesting the upper parts of submerged plants and the aboveground parts of emergent plants, phosphorus is periodically removed from the water system, thus overcoming the bottleneck of matrix adsorption saturation and achieving persistent and sustainable purification of phosphorus-containing wastewater.

[0061] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis, characterized in that, Engineered algal strain A, which displays laccase on its surface, and engineered algal strain B, which displays non-specific nuclease on its surface, were prepared separately and then mixed in a certain proportion to prepare a composite microalgae inoculant. The prepared composite microalgae agent was inoculated into a shallow pond at the front end of the wetland to construct a photocatalytic microalgae pretreatment unit. Submerged plants are planted in the symbiotic ponds set up in series after the pretreatment unit. After the submerged plants are established, they are inoculated with a compound microalgae agent to form a biocatalytic film on the surface of the stems and leaves of the submerged plants, thus constructing a microalgae-submerged plant symbiotic unit. A sulfur-modified mixed substrate is laid at the bottom of a deep purification pond connected in series after the symbiotic pond, and emergent plants are planted on it to form a sulfur-containing substrate-emergent plant deep purification unit. A multi-pond artificial wetland system is constructed based on a photocatalytic microalgae pretreatment unit, a microalgae-submerged plant symbiotic unit, and a sulfur-containing matrix-emergent plant deep purification unit.

2. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The preparation of the compound microalgae inoculant is as follows: Synechococcus was selected as the chassis cell; Construct an engineered algal strain A that integrates the laccase gene (cotA) with the InP-N fusion expression cassette, and an engineered algal strain B that integrates the nuclease gene (nucA) with the InP-N fusion expression cassette; Algal strain A and algal strain B were mixed according to the cell number ratio, and the final optical density value of the bacterial solution was adjusted to obtain a composite microalgae inoculum.

3. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The ratio of the number of cells of engineered algae strain A to engineered algae strain B in the composite microalgae agent is 1:1 to 3:1, and the final optical density value of the bacterial solution is adjusted to 0.8-1.

2.

4. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The effective water depth of the photocatalytic microalgae pretreatment unit is 0.3-0.5m, and the hydraulic retention time is set to 24-48 hours.

5. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The submerged plants used are a mixture of Vallisneria natans, Elodea nuttallii, and Ceratophyllum demersum, planted in a ratio of 2-3:3-5:1-3, with a planting density of 40-60 clumps / m². 2 .

6. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, Submerged plants are planted in the symbiotic ponds connected in series after the pretreatment unit. After the submerged plants have established themselves, the specific procedure for inoculating with the compound microalgae agent is as follows: When the submerged plants have grown to 10-15cm below the water surface, dilute the compound microalgae agent to an OD730 of 0.4-0.6 and spray it evenly on the water surface.

7. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The sulfur-modified mixed matrix is ​​prepared by mixing and laying the following raw materials in parts by weight: 10-20 parts of natural pyrite particles, 15-25 parts of conductive modified biochar, 30-40 parts of zeolite, and 40-60 parts of gravel. The pyrite particles have a diameter of 3-5 mm, the zeolite particles have a diameter of 3-6 mm, and the gravel particles have a diameter of 8-16 mm.

8. The method for constructing a multi-pond artificial wetland system based on rhizosphere synergistic symbiosis as described in claim 1, characterized in that, The method for preparing the conductive modified biochar includes the following steps: Agricultural waste biomass is crushed and passed through a 100-mesh sieve; The screened biomass was immersed in a FeCl3 solution with a concentration of 1.0-1.5 mol / L for 12-24 hours with stirring. The impregnated biomass was dried and then pyrolyzed at 500-600℃ for 2-3 hours under nitrogen protection. After cooling, it was ground through a 50-mesh sieve to obtain iron-loaded conductive modified biochar.

9. An application of a multi-pond constructed wetland system based on rhizosphere synergistic symbiosis, characterized in that, The Duotang constructed wetland system is constructed using the construction method described in any one of claims 1-8; The aforementioned multi-pond constructed wetland system is used for the treatment of wastewater effluent, and its operation and maintenance are as follows: Wastewater effluent flows sequentially through the constructed pretreatment unit, symbiotic unit, and deep purification unit for purification. The accumulated phosphorus element is removed by periodically harvesting the above-ground parts of submerged and emergent plants.

10. The application of a multi-pond constructed wetland system based on rhizosphere synergistic symbiosis as described in claim 9, characterized in that, The periodic harvesting specifically refers to harvesting 1 / 3 to 1 / 2 of the upper biomass of the submerged plants every 3-4 months for the microalgae-submerged plant symbiotic unit. For the sulfur-containing substrate-emergent plant deep purification unit, the above-ground withered parts of the emergent plants are harvested in late autumn and early winter each year, with a stubble height of 10-20cm.

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