Method for directionally degrading micro-plastics based on combination of constructed wetland and enzyme domestication

Abstract

The invention discloses a method and a system for directionally degrading micro-plastics based on combination of an artificial wetland and enzyme domestication. At present, water body micro-plastic pollution is serious, a traditional treatment method has many defects, and artificial wetland micro-plastic treatment lacks pertinence. The method comprises the following steps: ecologically purifying an environment by utilizing a constructed wetland, firstly carrying out variant screening and domestication on micro-plastic degrading enzymes (PETase, MHETase and the like), and then fixing the micro-plastic degrading enzymes on a modified zeolite carrier. Zeolite is modified by acid-base and surface functional groups, so that the binding capacity with enzyme is enhanced. And the immobilized enzyme is arranged in a substrate layer of the subsurface wetland at the rear section of the mixed artificial wetland and is matched with aquatic plants and microbial communities. Through the water flow regulation and control device, the micro-plastics in the water body are in full contact with the immobilized enzyme, so that efficient degradation is realized, and the micro-plastics are converted into harmless small molecules. The technology is low in energy consumption, free of secondary pollution, easy and convenient to operate, capable of effectively repairing a water source and improving water supply safety and suitable for treatment of various micro-plastic polluted water bodies.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental remediation and water treatment, and particularly to a method for the directional degradation of microplastics by combining an artificial wetland with enzyme domestication. Background Art

[0002] In recent years, with the widespread use of plastic products, the problem of microplastic pollution in water bodies has become increasingly prominent. Due to their tiny particle size and wide dispersion, conventional physical filtration, chemical oxidation and other traditional treatment methods often have deficiencies such as low removal efficiency, high energy consumption, and secondary pollution. At the same time, as an eco-friendly water purification technology, artificial wetlands have self-purification ability and low operating costs, but currently, targeted degradation has not been achieved in the treatment of microplastics.

[0003] On the other hand, in recent years, certain achievements have been made in the research on plastic-degrading enzymes (such as PETase, MHETase) at home and abroad. Enzymes obtained through genetic engineering and directed evolution show high activity in degrading plastics such as polyethylene terephthalate (PET). However, technical solutions for applying such enzymes to environmental water treatment, especially for the directional degradation of microplastics in artificial wetland systems, have not been reported in the open literature or patents.

[0004] Therefore, the present invention aims to combine enzyme degradation technology with an artificial wetland ecological purification system, and use immobilized and domesticated plastic-degrading enzymes to efficiently degrade microplastics in water in the artificial wetland, convert them into harmless small molecules, achieve water source restoration, and improve water supply safety. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a method for the directional degradation of microplastics by combining an artificial wetland with enzyme domestication. By screening and domesticating microplastic-degrading enzymes, modifying the enzyme immobilization carrier, constructing a specific artificial wetland, and reasonably regulating the water flow, etc., the efficient degradation of microplastics in water is achieved, and they are converted into harmless small molecules, thereby restoring the water source, ensuring water supply safety, and having the advantages of low energy consumption and no secondary pollution.

[0006] To achieve the above purpose, the specific technical solutions are as follows:

[0007] The present invention discloses a method for the directional degradation of microplastics by combining constructed wetlands with enzyme domestication, comprising the following steps: S1, screening and domesticating variants of the microplastic-degrading enzyme to obtain the domesticated microplastic-degrading enzyme; S2, modifying a porous enzyme immobilization carrier to obtain the modified enzyme immobilization carrier; S3, immobilizing the domesticated microplastic-degrading enzyme into the pores and on the surface of the modified enzyme immobilization carrier by means of chemical bonding and / or physical adsorption; S4, constructing a constructed wetland including a wetland substrate layer, wherein the enzyme immobilization carrier on which the microplastic-degrading enzyme has been immobilized is used as a part of the wetland substrate layer; S5, operating the constructed wetland to degrade the microplastics contained in the water to be treated.

[0008] The present invention combines constructed wetlands with enzyme domestication to achieve the directional degradation of microplastics. Through the screening and domestication of the microplastic-degrading enzyme, it is made more adaptable to the requirements of degrading microplastics, enhancing the catalytic activity; the modification of the enzyme immobilization carrier enhances the immobilization effect of the enzyme, ensuring the stability of the enzyme during the reaction; integrating the enzyme-immobilized carrier into the wetland substrate layer and utilizing the ecological environment of the constructed wetland provides suitable conditions for the degradation of microplastics, making the degradation process more efficient and environmentally friendly, reducing energy consumption and the risk of secondary pollution, and contributing to the restoration of water sources and ensuring water supply safety.

[0009] According to the method disclosed by the present invention, the porous enzyme immobilization carrier is set as zeolite, and step S2 includes: S201, performing acid-base modification on the zeolite serving as the enzyme immobilization carrier, including: pickling the zeolite with an acid solution to remove surface metal impurities and increase the hydroxyl groups and acidic sites on the surface; subsequently, performing alkali treatment on the zeolite with an alkali solution to destroy part of the crystal structure of the zeolite, so that the specific surface area and the surface and negative charge density of the zeolite increase; S202, performing surface functional group modification on the zeolite subjected to acid-base modification to introduce amino and / or epoxy functional groups on the surface of the zeolite.

[0010] In the present invention, using zeolite as the enzyme immobilization carrier, removing surface metal impurities, increasing hydroxyl groups and acidic sites by pickling, destroying part of the crystal structure by alkali treatment to increase the specific surface area and surface negative charge density, and then introducing amino and / or epoxy functional groups not only provides more binding sites for enzyme immobilization, enhances the binding ability and affinity between the enzyme and the carrier, improves the enzyme immobilization efficiency, but also optimizes the surface properties of the carrier, improves the stability and reusability of the enzyme, ensures that the enzyme can continuously and efficiently play a catalytic role in the constructed wetland environment, and thus decomposes microplastics into harmless small molecules more quickly and thoroughly, achieving the efficient removal of microplastics in water, effectively restoring water sources and ensuring water supply safety.

[0011] In addition, using zeolite as an enzyme immobilization carrier has the following advantages: 1) It is convenient for modification and loading. That is, as a porous mineral, zeolite has a large specific surface area and activity, which is convenient for subsequent acid-base modification and functional group loading, improving the adsorption and degradation of microplastics; 2) The porous mineral structure of zeolite has a high cation exchange capacity, which enables it to be excellently suitable for removing ammonia nitrogen (NH4 + -N) and other positively charged pollutants; moreover, the ammonia nitrogen removal efficiency of the zeolite matrix can reach 79.5%, significantly higher than 63.2% of gravel. This characteristic adsorbs ammonia onto the matrix surface through ion exchange reactions, especially playing a key role in the nitrogen cycle; 3) It has the advantage of supporting microbial growth. That is, the porous structure of zeolite provides more attachment points for microorganisms, promoting biological processes such as denitrification and organic matter decomposition; moreover, research shows that the macroporous zeolite matrix has a 12% higher ammonia nitrogen removal rate than the control group because its surface characteristics provide a wider ecological niche for microorganisms; this enhances the biological treatment capacity of wetlands, especially when used for the remediation of slightly polluted water sources; 4) Zeolite is superior to traditional matrices. Compared with traditional matrices such as gravel, zeolite shows higher efficiency in removing chemical oxygen demand (COD), total nitrogen (TN), and ammonia nitrogen (NH4+-N). For example, the COD removal rate of the zeolite matrix reaches 75.2%, while that of the gravel matrix is only 58.6%; moreover, the removal of total nitrogen and ammonia nitrogen in the drinking water process is relatively complex. Therefore, using zeolite for water source remediation to remove ammonia nitrogen and total nitrogen is beneficial to reducing the complexity of the water treatment process and improving the safety level of drinking water; 5) Zeolite has the characteristics of versatility and combined use. That is, zeolite can be used alone or in combination with other materials such as biochar to improve performance. For example, in wetlands with added zeolite, at the optimal pH (6.3) and retention time (57.4 hours), the removal rates of COD, ammonia nitrogen, phenols, lead, and manganese all reach 99.9%, which is better than wetlands using only gravel, providing new possibilities for the design of multi-layer matrix systems.

[0012] Moreover, based on using zeolite as an enzyme immobilization carrier, acid-base modification can enhance the adsorption effect and also enhance amino and epoxy functional groups, thereby further improving the adsorption-degradation effect.

[0013] According to the method disclosed in the present invention, in step S202, 3-aminopropyltriethoxysilane reacts with the zeolite surface to generate amino functional groups.

[0014] In the present invention, 3-aminopropyltriethoxysilane reacts with the zeolite surface to generate amino functional groups, further increasing the binding points between zeolite and negatively charged enzymes, significantly improving the affinity between zeolite and enzymes, making the immobilization of enzymes on the zeolite surface more firm, facilitating the maintenance of enzyme activity, and improving the microplastic degradation efficiency.

[0015] According to the method disclosed by the present invention, step S3 includes: S301, drying the modified zeolite at a first set temperature; S302, dissolving the domesticated microplastic-degrading enzyme in a specific buffer solution to adjust the enzyme concentration to a specific range to obtain an enzyme solution; S303, covalently binding the amino groups on the surface of the domesticated microplastic-degrading enzyme to the surface of the modified zeolite through an aminosilylation reaction; and / or adding the enzyme solution to the modified zeolite, mixing and stirring to enable the microplastic-degrading enzyme to adsorb on the zeolite surface through electrostatic force or van der Waals force; S304, washing the zeolite with the fixed microplastic-degrading enzyme using a specific buffer solution, and then drying at a second set temperature to obtain a stable zeolite with the microplastic-degrading enzyme immobilized thereon.

[0016] Among them, the adsorption effect has been enhanced by acid-base modification before, and the aminosilylation reaction can be successfully carried out on the basis of acid-base modification to graft the microplastic-degrading enzyme. Otherwise, the abundance of the enzyme on the zeolite is insufficient, and the desired removal effect cannot be achieved.

[0017] In the present invention, the specific immobilization steps ensure the standardization and effectiveness of the enzyme immobilization process. Drying the modified zeolite can remove moisture and avoid interference of moisture on the immobilization reaction; adjusting the enzyme concentration to a specific range can ensure the consistency of the immobilization effect; immobilizing the enzyme through two methods of covalent binding and physical adsorption increases the stability of immobilization; the washing and re-drying steps remove the unbound enzyme, obtaining a stable immobilized enzyme carrier, improving the purity and stability of the immobilized enzyme, and ensuring continuous and efficient degradation of microplastics in the constructed wetland.

[0018] According to the method disclosed by the present invention, in step S4, the constructed wetland is set as a hybrid wetland, the front section is set as a surface-flow wetland, and the rear section is set as a subsurface-flow wetland, wherein the zeolite with the microplastic-degrading enzyme immobilized thereon is arranged in the wetland substrate layer of the subsurface-flow wetland in the rear section.

[0019] In the present invention, the constructed wetland is set as a hybrid wetland with a surface-flow front section and a subsurface-flow rear section, and the zeolite with the microplastic-degrading enzyme immobilized thereon is placed in the wetland substrate layer of the subsurface-flow wetland in the rear section, combining the advantages of the two types of wetlands. The surface-flow wetland can initially purify the water body, and the subsurface-flow wetland provides a stable reaction environment for the immobilized enzyme, which is beneficial for the full contact between the microplastics and the immobilized enzyme and continuous and efficient degradation reaction, ensuring the water purification effect.

[0020] According to the method disclosed by the present invention, in step S4, the constructed wetland further includes an aquatic plant layer and a microbial community, wherein the aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus and submerged plants, and the microbial community is set as bacteria and fungi with the ability to degrade microplastics.

[0021] In the present invention, aquatic plants in the aquatic plant layer (such as reeds, water hyacinths, calamus, and submerged plants, etc.) can adsorb and remove nutrient salts in water, provide a habitat for microorganisms, and their root exudates can also stimulate the growth of microorganisms and enhance the pollutant degradation ability; the microbial community composed of bacteria and fungi with the ability to degrade microplastics, in cooperation with the plant roots and the substrate layer, jointly constructs a complete ecological purification system, further improving the degradation ability of the constructed wetland for microplastics and other pollutants and improving water quality.

[0022] According to the method disclosed in the present invention, step S1 includes: S101, performing error-prone PCR or DNA screening on the microplastic-degrading enzyme to obtain enzyme variants so as to construct an initial enzyme library; S102, performing environmental exposure domestication, including: adding fluorescently labeled microplastics, screening enzyme variants with high fluorescence intensity of degradation products by a flow cytometer, and allowing the enzyme variants that simultaneously meet the following conditions to enter the next step: the first condition, detecting the stability of the enzyme variants to obtain enzyme variants with a residual activity greater than a first preset threshold; the second condition, detecting the release amount of microplastic monomers, and screening enzyme variants with an efficiency improvement greater than or equal to a second preset threshold; S103, performing high-throughput screening on the enzyme variants obtained through environmental exposure domestication to obtain the finally domesticated microplastic-degrading enzyme.

[0023] The residual activity herein refers to the percentage of the initial activity retained by the enzyme after a specific stability test. The stability test usually involves exposing the enzyme to conditions that may cause denaturation or inactivation, such as high temperature, extreme pH, or long-term storage.

[0024] The screening efficiency refers to the efficiency of the enzyme variant in releasing microplastic monomers, which is specifically measured by its specific activity. The efficiency improvement is the percentage increase of the variant's specific activity relative to the reference enzyme, and the second preset threshold is a predetermined value used to screen out variants with an efficiency improvement reaching or exceeding this value.

[0025] High-throughput screening refers to rapidly testing a large number of enzyme variants through microplate experiments, commonly using color or fluorescence detection models to detect substrate activity, and selecting the best microplastic-degrading enzyme. Environmental exposure may refer to obtaining adaptive variants through directed evolution or natural selection, and screening is a key step in the optimization process. Usually, a model substrate is used for primary screening, combined with actual microplastics for verification to ensure the reliability of the results.

[0026] In the present invention, the detailed enzyme domestication steps construct an initial enzyme library through error-prone PCR or DNA screening, providing a rich source of enzyme variants for subsequent screening; in environmental exposure domestication, enzyme variants are screened based on the fluorescence intensity of degradation products, enzyme stability, and the release amount of microplastic monomers, ensuring that the screened enzyme variants have high degradation activity and stability; high-throughput screening further optimizes the mutation combination, improves the screening efficiency, and finally obtains a highly efficient domesticated microplastic-degrading enzyme, significantly enhancing the microplastic degradation effect.

[0027] The present invention also discloses a system for implementing the method for the directional degradation of microplastics based on the combination of constructed wetland and enzyme domestication disclosed by the present invention. The system includes: an enzyme domestication unit configured to screen and domesticate variants of microplastic degrading enzymes to obtain domesticated microplastic degrading enzymes; an enzyme immobilization carrier having a porous structure; a carrier modification treatment unit configured to modify the enzyme immobilization carrier to obtain a modified enzyme immobilization carrier; an enzyme immobilization unit configured to immobilize the domesticated microplastic degrading enzymes into the pores and on the surface of the modified enzyme immobilization carrier by means of chemical bonding and / or physical adsorption; and a constructed wetland configured as a constructed wetland including at least a wetland substrate layer, wherein the enzyme immobilization carrier on which the microplastic degrading enzymes have been immobilized is used as a part of the wetland substrate layer.

[0028] In the present invention, the functions of each unit of the system are defined. The enzyme domestication unit provides highly efficient domesticated enzymes, the enzyme immobilization carrier and the carrier modification treatment unit ensure the stable immobilization of the enzymes, and the constructed wetland provides an ecological degradation environment. Each unit works together to achieve the directional degradation of microplastics, with high efficiency and ecological friendliness, which helps to ensure the safety of water supply.

[0029] According to the system disclosed by the present invention, the system further includes a water flow regulation unit configured to enable the water body to flow through the constructed wetland from top to bottom or from side to side, and to enable the wetland water level to overflow the wetland substrate layer where the enzyme immobilization carrier is located.

[0030] In the present invention, the water flow regulation unit controls the water body to flow through the constructed wetland from top to bottom or from side to side, and makes the water level overflow the substrate layer where the enzyme immobilization carrier is located, ensuring sufficient contact between the water body and the immobilized enzymes, plant roots, and microbial communities, facilitating the degradation reaction between microplastics and enzymes, improving the degradation efficiency, and at the same time ensuring the stable operation of the wetland ecosystem.

[0031] According to the system disclosed by the present invention, the constructed wetland further includes an aquatic plant layer and a microbial community. The aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus, and submerged plants. The microbial community is set to include bacteria and fungi with the ability to degrade microplastics. The constructed wetland is set as a hybrid wetland, with the front section being a surface flow wetland and the rear section being a subsurface flow wetland, and the zeolite on which the microplastic degrading enzymes have been immobilized is arranged in the wetland substrate layer of the subsurface flow wetland in the rear section.

[0032] In the present invention, the specific composition and structure of the constructed wetland, the hybrid wetland, the specific aquatic plants and microbial communities, and the reasonable layout of the immobilized enzyme zeolite jointly construct a well-functioning ecological system that can efficiently degrade microplastics, remove various pollutants in water, improve the self-purification ability of water sources, and ensure the safety of water supply quality.

[0033] The beneficial effects of the present invention compared with the prior art are as follows:

[0034] 1) Through multiple rounds of screening and domestication of microplastic-degrading enzymes (such as steps of error-prone PCR or DNA screening to construct an initial enzyme library, environmental exposure domestication, high-throughput screening, etc.), the catalytic activity of microplastic-degrading enzymes in natural water bodies is increased by 3-8 times. The domesticated enzyme is immobilized on a zeolite carrier modified by acid-base modification and surface functional group modification, with high immobilization efficiency and a microplastic degradation rate of 78.2%. Compared with traditional treatment methods such as conventional physical filtration and chemical oxidation, the present invention can achieve directional and efficient decomposition of microplastics, effectively making up for the defect of low removal efficiency of traditional methods.

[0035] 2) The stability and activity of the enzyme are verified. In the long-term environmental tolerance test, the half-life of enzyme activity is extended from 4 hours to 72 hours, and the anti-protease ability retains ≥80% activity in 0.1 mg / mL trypsin. The acid-base modification of the zeolite carrier increases its specific surface area and surface negative charge density, and the surface functional group modification introduces functional groups such as amino groups, enhancing the binding force between the enzyme and the carrier, improving the stability and reusability of the enzyme, and ensuring that the enzyme can continuously and stably play a catalytic role in the complex environment of the constructed wetland.

[0036] 3) Utilizing the ecological function of the constructed wetland, the immobilized enzyme is combined with the constructed wetland, and the degradation process has low energy consumption and no secondary pollution. Aquatic plants in the constructed wetland can adsorb nutrients, and the microbial community can degrade various pollutants, synergistically acting with the immobilized enzyme to jointly purify the water body. At the same time, the operation cost of the constructed wetland is low. Compared with the problem of high energy consumption of traditional treatment methods, the present invention reduces the treatment cost while achieving efficient degradation of microplastics, and the system operation and maintenance are also simpler.

[0037] 4) The constructed hybrid constructed wetland, with the front surface-flow wetland and the back subsurface-flow wetland cooperating with each other, and the substrate of the back subsurface-flow wetland being the modified zeolite with immobilized enzyme, provides a stable purification effect. The aquatic plant layer and the microbial community with the ability to degrade microplastics further enhance the system's ability to remove microplastics and other pollutants. The water flow regulation unit ensures that the water body is in full contact with the immobilized enzyme, plant roots, and microbial community, optimizes the degradation reaction conditions, improves the overall treatment effect, guarantees the water supply safety, and realizes the effective restoration of the water source.

[0038] The following details the method for the directional degradation of microplastics based on the combination of a constructed wetland and enzyme domestication of the present invention with reference to the embodiments shown in the accompanying drawings. Description of the Drawings

[0039] Figure 1 It is a flow chart of the method for the directional degradation of microplastics based on the combination of a constructed wetland and enzyme domestication of the present invention;

[0040] Figure 2 is Figure 1 The implementation flowchart of step S2 in

[0041] Figure 3 is Figure 1 The implementation flowchart of step S3 in

[0042] Figure 4 is Figure 1 The implementation flowchart of step S1 in Detailed implementation manners

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0044] As Figure 1 shown, the present invention discloses a method for the directional degradation of microplastics by combining an artificial wetland and enzyme domestication, including the following steps: S1, screening and domesticating variants of the microplastic-degrading enzyme to obtain a domesticated microplastic-degrading enzyme; S2, modifying a porous enzyme immobilization carrier to obtain a modified enzyme immobilization carrier; S3, immobilizing the domesticated microplastic-degrading enzyme into the pores and on the surface of the modified enzyme immobilization carrier by means of chemical bonding and / or physical adsorption; S4, constructing an artificial wetland including a wetland matrix layer, wherein the enzyme immobilization carrier on which the microplastic-degrading enzyme has been immobilized is used as a part of the wetland matrix layer; S5, operating the artificial wetland to degrade the microplastics contained in the water to be treated.

[0045] Based on the above disclosed steps, the present invention combines an artificial wetland and enzyme domestication to achieve the directional degradation of microplastics. By screening and domesticating the microplastic-degrading enzyme, it makes the enzyme more adaptable to the needs of degrading microplastics and improves its catalytic activity; the modification of the enzyme immobilization carrier enhances the immobilization effect of the enzyme and ensures the stability of the enzyme during the reaction; integrating the carrier with the immobilized enzyme into the wetland matrix layer and utilizing the ecological environment of the artificial wetland provides suitable conditions for the degradation of microplastics, making the degradation process more efficient and ecologically friendly, reducing energy consumption and the risk of secondary pollution, and contributing to the restoration of water sources and ensuring water supply safety.

[0046] As Figure 2As shown, in a preferred embodiment, where the porous enzyme immobilization carrier is set as zeolite, step S2 includes: S201, performing acid-base modification on the zeolite serving as the enzyme immobilization carrier, including: pickling the zeolite with an acid solution to remove surface metal impurities and increase the surface hydroxyl groups and acidic sites; subsequently treating the zeolite with an alkali solution to destroy part of the crystal structure of the zeolite, so that the specific surface area and the surface and negative charge density of the zeolite increase; S202, performing surface functional group modification on the acid-base modified zeolite to introduce amino and / or epoxy functional groups on the zeolite surface.

[0047] In a specific embodiment, in the acid-base modification of S201, concentrated hydrochloric acid (HCl) or sulfuric acid (H2SO4) solution is first selected to pickle the zeolite. The concentration of the acid solution is strictly controlled at 1 - 3M, and the zeolite is allowed to react fully in the acid solution for 1 - 2 hours. This process can effectively remove the metal impurities on the zeolite surface and at the same time promote the increase of the surface hydroxyl groups (–OH) and acidic sites. After pickling, the zeolite is thoroughly washed with deionized water to remove the residual acid solution and impurities, and then dried to prepare for the subsequent alkali treatment. Then, the pickled zeolite is treated with a sodium hydroxide (NaOH) solution with a concentration of 1 - 3M, and the reaction time is set to 1 hour. The alkali treatment will destroy part of the crystal structure of the zeolite, thereby increasing the specific surface area of the zeolite by 10% - 30%, and the surface negative charge density also increases accordingly. After the alkali treatment is completed, the zeolite is also washed with deionized water to remove the impurities and residual alkali solution generated by the reaction. The surface of the acid-modified zeolite itself has a negative charge, while the surface of the alkali-modified zeolite has a stronger negative charge, and these changes provide favorable conditions for the subsequent enzyme immobilization.

[0048] In the surface functional group modification of S202, the zeolite modified by acid-base two-step modification is mixed with a 1 - 5% amino silane (3-aminopropyltriethoxysilane, APTES) solution and reacted at 60°C for 4 - 6 hours. After the reaction is completed, the zeolite is washed to remove the unreacted APTES and other impurities, and dried again at 60°C. After such amination modification, amino (–NH2) functional groups will be generated on the zeolite surface, thus carrying a strong positive charge. This positively charged surface can enhance the binding ability with the negatively charged enzyme, and by precisely controlling the reaction conditions, the number of amino groups on the zeolite surface can be controlled at 0.5 - 1.5 mmol per gram of zeolite, laying a foundation for the subsequent efficient immobilization of microplastic degrading enzymes.

[0049] As Figure 3As shown, in a preferred embodiment, step S3 includes: S301, drying the modified zeolite at a first set temperature; S302, dissolving the domesticated microplastic-degrading enzyme in a specific buffer to adjust the enzyme concentration to a specific range to obtain an enzyme solution; S303, covalently binding the amino groups on the surface of the domesticated microplastic-degrading enzyme to the surface of the modified zeolite through an aminosilylation reaction; and / or adding the enzyme solution to the modified zeolite, mixing and stirring to enable the microplastic-degrading enzyme to adsorb on the zeolite surface through electrostatic force or van der Waals force; S304, washing the zeolite immobilized with the microplastic-degrading enzyme with a specific buffer, and then drying at a second set temperature to obtain a stable zeolite immobilized with the microplastic-degrading enzyme.

[0050] In a specific embodiment, first, the modified zeolite is placed in an environment at 70 °C (the first set temperature) for drying to thoroughly remove moisture, preparing for subsequent enzyme immobilization operations. Then, the domesticated microplastic-degrading enzymes, including PETase, MHETase, and their variants, are dissolved in a suitable buffer. For example, PBS buffer with a pH value of 7.0 (the specific buffer) is selected, and the enzyme concentration is precisely adjusted to a suitable range of 1 - 10 mg / mL to obtain an enzyme solution.

[0051] Then, the immobilization operation is carried out in two ways: covalent binding and physical adsorption. Covalent binding is achieved through an aminosilylation reaction to covalently connect the amino groups on the enzyme surface to the surface of the modified zeolite. This reaction is carried out at room temperature for 1 - 2 hours. Physical adsorption is to add the enzyme solution to the modified zeolite, mix evenly, and stir for 3 - 4 hours. With the help of electrostatic force or van der Waals force, the enzyme is adsorbed on the zeolite surface.

[0052] After immobilization is completed, the enzyme-zeolite complex is washed with PBS buffer to remove the enzyme that has not been successfully bound. Subsequently, the washed complex is dried in an environment at 60 °C (the second set temperature) to finally obtain a stable immobilized enzyme zeolite.

[0053] To detect the degradation activity of the immobilized enzyme on microplastics (taking PET as an example), methods such as colorimetry and HPLC are used for testing. The commonly used degradation determination method is to detect the concentrations of degradation products such as MHET and TPA, and then calculate the degradation rate.

[0054] The present invention tested the effects of immobilized enzymes on zeolites with different modification methods, as shown in the following table:

[0055] Carrier modification method Enzyme activity retention rate (%) Immobilization efficiency (%) Microplastic degradation rate (%) Acid-base modified zeolite 73.4 86.3 68.6 Aminated zeolite 86.1 84.3 72.6 Two-step modified zeolite 89.5 92.3 78.2 Unmodified zeolite 63.8 72.4 56.7

[0056] Experimental data show that different modification methods have significant effects on the enzyme activity retention rate, immobilization efficiency, and microplastic degradation rate. For zeolite modified by acid-base, the enzyme activity retention rate is 73.4%, the immobilization efficiency is 86.3%, and the microplastic degradation rate is 68.6%; the corresponding three indicators of amino-functionalized zeolite are 86.1%, 84.3%, and 72.6%; the zeolite modified by the two-step method has the most ideal effect, with the enzyme activity retention rate reaching 89.5%, the immobilization efficiency of 92.3%, and the microplastic degradation rate of 78.2%. In contrast, for unmodified zeolite, the indicators are relatively low, with the enzyme activity retention rate only 63.8%, the immobilization efficiency of 72.4%, and the microplastic degradation rate of 56.7%. These data fully demonstrate the importance of modification treatment in improving the enzyme immobilization effect and microplastic degradation rate.

[0057] In a preferred embodiment, in step S4, the constructed wetland is set as a hybrid wetland, with the front section being a surface-flow wetland and the rear section being a subsurface-flow wetland, and the zeolite immobilized with the microplastic-degrading enzyme is arranged in the wetland matrix layer of the rear subsurface-flow wetland. And in this embodiment, the constructed wetland further includes an aquatic plant layer and a microbial community, where the aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus, and submerged plants, and the microbial community is set as bacteria and fungi with the ability to degrade microplastics.

[0058] In a specific embodiment, a hybrid constructed wetland is built for treating water pollution, and its specific composition and functions are as follows:

[0059] This constructed wetland is divided into a front section and a rear section. The front section is set as a surface-flow wetland, and the rear section is a subsurface-flow wetland. The matrix of the rear wetland selects the modified zeolite immobilized with the enzyme. When configuring the matrix, the addition concentration of the modified zeolite immobilized with the enzyme is strictly controlled at an appropriate ratio of 10 - 30%. This can not only ensure that the water flow can fully pass through and contact with the enzyme but also provide a relatively stable purification effect.

[0060] The wetland matrix layer is composed of various substances such as sand, gravel, zeolite, bentonite, and peat. It not only provides a medium for the water flow to pass through but also is an important place for the survival of microorganisms and the growth of plant roots. Among them, zeolite, as a modified carrier, has good physical adsorption properties and can create a suitable environment for enzyme immobilization.

[0061] The aquatic plant layer also plays an important role in the entire wetland system. According to the local climate and water body characteristics, aquatic plants suitable for the wetland water quality conditions can be selected, such as Phragmites australis, Eichhornia crassipes, Acorus calamus, and submerged plants (such as Scirpus validus), etc. The roots of aquatic plants can not only adsorb and remove nutrients such as nitrogen and phosphorus in the water, but also provide a habitat for the microbial community. In addition, the substances secreted by their roots can stimulate the growth of microorganisms, further enhancing the degradation ability of pollutants.

[0062] The selection of the microbial community is equally crucial. In this constructed wetland, bacteria with the ability to degrade microplastics (such as the genus Bacillus, Pseudomonas, etc.) and fungi (such as the genus Trametes) are selected. These microorganisms cooperate with the plant roots and the substrate layer to jointly play a purification role, and can further degrade microplastics and other organic pollutants in the water body, thus achieving efficient purification of the water body.

[0063] In a preferred embodiment, the constructed wetland operates and manages in the following ways to achieve the degradation of microplastics in the water body and water quality purification, and ensure the safety of water supply:

[0064] Use a water flow regulation device to adjust the flow state of the water body in the wetland, allowing the water to flow from top to bottom or from side to side. This can ensure that the water fully contacts the zeolite carrier, plant roots, and microbial community, and play the role of each part in purifying the water quality. In the water flow design, the flow rate should be strictly controlled to avoid being too fast or too slow. At the same time, maintain the wetland water level within the range of 10 - 30 cm. This water level can not only meet the growth needs of the plants in the wetland, but also ensure that the water can smoothly pass through the zeolite carrier layer containing immobilized enzymes.

[0065] During the operation process, ensure that the wetland is in suitable environmental conditions, with the temperature controlled at 5 - 35 °C, the pH value maintained at 6.5 - 8.5, and sufficient nutrients provided. Under such conditions, the immobilized enzyme can efficiently catalyze the degradation of microplastics, and finally decompose them into harmless CO2, water, and other low-toxicity small molecules.

[0066] To ensure the stable and efficient operation of the wetland system, it is necessary to regularly monitor water quality parameters, including pH value, dissolved oxygen, nitrogen and phosphorus content, microplastic concentration, etc. As shown in the following table:

[0067] Water quality parameters Initial value Value after purification Removal rate (%) Microplastic concentration (particles / L) 96±16.58 23±6.24 76 Total nitrogen (mg / L) 0.34～0.87 0.08～0.14 76.5～83.9 Total phosphorus (mg / L) 0.12～0.26 0.01～0.03 88.5～91.7

[0068] According to the experimental data, the initial microplastic concentration was (96 ± 16.58) particles per liter. After treatment, the concentration decreased to (23 ± 6.24) particles per liter, and the removal rate reached 76%; the initial total nitrogen value was between 0.34 - 0.87 mg / L, and after purification, it was 0.08 - 0.14 mg / L, with a removal rate of 76.5 - 83.9%; the initial total phosphorus value was 0.12 - 0.26 mg / L, and after purification, it was 0.01 - 0.03 mg / L, with a removal rate of 88.5 - 91.7%.

[0069] According to these monitoring data, the water flow rate, plant growth conditions, and the use of enzyme carriers are adjusted in a timely manner. For example, if it is found that the degradation effect of microplastics is not good, the water flow rate can be appropriately adjusted to increase the contact time between microplastics and immobilized enzymes; if the poor growth of plants affects the purification effect, corresponding maintenance measures can be taken or plant varieties can be replaced; if the activity of the enzyme carrier decreases, consider replacing or supplementing the enzyme carrier.

[0070] The water body after enzymatic degradation treatment still needs to be subjected to subsequent purification treatment to further remove possible residual impurities and pollutants, ensuring that when used as a water supply source, the microplastic concentration in the drinking water is lower than the level that threatens human health and effectively guaranteeing the safety of water supply.

[0071] As Figure 4 shown, in a preferred embodiment, step S1 includes: S101, performing error-prone PCR or DNA screening on the microplastic-degrading enzyme to obtain enzyme variants and construct an initial enzyme library; S102, performing environmental exposure domestication, including: adding fluorescently labeled microplastics, screening enzyme variants with high fluorescence intensity of degradation products through a flow cytometer, and allowing enzyme variants that simultaneously meet the following conditions to enter the next step: the first condition is to detect the stability of the enzyme variants and obtain enzyme variants with residual activity greater than the first preset threshold; the second condition is to detect the release amount of microplastic monomers and screen enzyme variants with an efficiency improvement greater than or equal to the second preset threshold; S103, performing high-throughput screening on the enzyme variants obtained through environmental exposure domestication to obtain the finally domesticated microplastic-degrading enzyme.

[0072] In a specific embodiment, first, directed evolution means are used to perform error-prone PCR or DNA screening operations on known degrading enzymes such as PETase and cutinase, and then an initial enzyme library is constructed. To ensure the rich diversity of enzyme variants, strict criteria are set: the mutation library diversity sequencing coverage must be greater than or equal to 99%, and the number of single mutants must exceed 104.

[0073] Then, the screening phase begins. Fluorescently labeled microplastics (such as NileRed-stained PET) are added to the reaction system, and enzyme variants with high fluorescence intensity of degradation products are selected using flow cytometry. At the same time, the stability of the enzyme is detected with the help of SDS-PAGE technology. Only enzyme variants with residual activity exceeding 30% (the first preset threshold) have the opportunity to enter the subsequent screening; the release of microplastic monomers (such as terephthalic acid) is also measured by HPLC, and variants with an increase in degradation efficiency of greater than or equal to 50% (the second preset threshold) are screened out, so as to ensure that the selected enzyme variants have excellent degradation performance in actual application scenarios.

[0074] Then, a prediction model is constructed based on parameters such as degradation rate and environmental tolerance to optimize the mutation combination. The screening throughput is required to be greater than 10 5 Variants / week, and finally an enzyme variant with a high-efficiency variant ratio of not less than 0.1% is obtained. That is, step S103 is performed to perform high-throughput screening on the enzyme variants obtained by environmental exposure and domestication to obtain the final domesticated microplastic-degrading enzyme.

[0075] The stability and activity of the selected enzyme variants were then verified: the preferred enzyme variant was immobilized on magnetic nanoparticles (Fe3O4@SiO2) to enhance its stability and run continuously for 30 days, and the enzyme activity half-life (T 1 / 2). As shown in the following table:

[0076] Enzyme variant Initial activity (U / mg) Activity after 30 days (U / mg) <![CDATA[T 1 / 2 (hours)]]> WT 15.2 2.1 4.5 Mut3 38.7 32.5 68.2

[0077] Experimental data showed that the initial activity of the wild-type (WT) enzyme was 15.2 U / mg, and after 30 days the activity dropped to 2.1 U / mg. 1 / 2 was 4.5 hours; while the initial activity of the Mut3 variant reached 38.7 U / mg, and after 30 days the activity was still maintained at 32.5 U / mg, T 1 / 2 was extended to 68.2 hours, and its anti-protease ability could retain more than 80% of its activity in a 0.1 mg / mL trypsin environment, which strongly proved that the screened enzyme variants had significant improvements in both stability and activity.

[0078] Finally, actual water verification was carried out. An immobilized enzyme with a concentration of 1 mg / L was placed in the artificial wetland, and the degradation rate of microplastics was detected by Py-GC / MS, and compared with the degradation capacity of natural microbial communities. The experiment pre-set the degradation rate increase target of 5 times or more after adding enzymes. As shown in the following table:

[0079] Environment Microplastic type Degradation rate (28 days) Laboratory simulation PET 92％±3％ Constructed wetland PET 78％±5％

[0080] The results showed that under the laboratory simulation environment, the degradation rate of PET microplastics in 28 days was 92% ± 3%; in the constructed wetland, the degradation rate of PET microplastics in 28 days was 78% ± 5%. These data fully demonstrated that the screened enzyme variants also had good degradation effects in the actual water environment.

[0081] In the present invention, in step S103, high-throughput screening is carried out based on the prediction model. Specifically, the establishment of the prediction model is based on the performance data of known enzyme variants, and these variants may be obtained through environmental exposure (such as selection under simulated high temperature or the presence of microplastics) or natural environmental adaptation. The prediction model is constructed to take the mutation combinations or sequences of enzymes as inputs and the performance parameters of enzymes as outputs, such as the microplastic degradation rate and environmental tolerance. The prediction model learns the relationship between mutations and performance through machine learning or statistical methods, so as to predict the performance of untested variants.

[0082] Among them, the specific steps for realizing high-throughput screening based on the prediction model, that is, the specific steps for realizing HTS based on the prediction model include:

[0083] (1) Generate or select enzyme variants:

[0084] Use the prediction model to predict the performance of potential enzyme variants. These variants can be generated through in vitro design or selected from existing libraries. The input of the model may be the combination of mutation sites, and the output is the predicted degradation rate or environmental tolerance. For example, the literature Directed evolution and machine learning for enzyme engineering describes using a random forest model to predict enzyme activity and then selecting high-scoring variants for testing.

[0085] (2) Prepare the variants for testing:

[0086] Synthesize the DNA sequences of the selected variants, usually through PCR or gene synthesis. Clone these sequences into an expression vector and express the enzyme protein in a suitable host (such as Escherichia coli). The expression conditions include appropriate temperature (such as 37 °C) and inducer (such as IPTG) to ensure efficient expression.

[0087] (3) Design a high-throughput experiment:

[0088] Develop an experiment suitable for HTS to measure the performance of the enzyme. Given the complexity and insolubility of microplastics, it is recommended to use model substrates. For example, for PET-degrading enzymes, bis(4-nitrophenyl) terephthalate (BNPT) can be used as a substrate, and its hydrolysis releases 4-nitrophenol, which can be detected by absorbance at 405 nm. Environmental tolerance tests may involve measuring the activity or stability of the enzyme under different temperature or pH conditions. The experiment is usually carried out in 96-well or 384-well microtiter plates, with enzyme samples, substrates, and buffers added to each well.

[0089] (4) Conduct the HTS experiment:

[0090] Use automated equipment such as a plate reader to measure the signal intensity. For color detection, measure the absorbance; for fluorescence detection, measure the specific wavelength. The incubation conditions are adjusted according to the enzyme characteristics, such as 30 minutes to 1 hour at 37 °C. The experimental results reflect the degradation ability and tolerance of each variant.

[0091] (5) Analyze the results:

[0092] Compare the experimental results with the model predictions to identify the best-performing variants. For example, calculate the specific activity (amount of product produced per unit enzyme per unit time) based on the absorbance. Use statistical methods to determine which variants meet or exceed the desired performance threshold. The literature "Prediction of enzyme properties using machine learning" mentions that the model can be further optimized with new data.

[0093] (6) Validation and optimization:

[0094] The selected best variants may need to be further verified on actual microplastics because the model substrates may not be exactly the same as the real conditions. Update the prediction model with new data to improve the accuracy of future predictions.

[0095] The present invention also discloses a system for implementing the method for the directional degradation of microplastics based on the combination of constructed wetland and enzyme domestication disclosed in the present invention. The system includes: an enzyme domestication unit configured to screen and domesticate variants of microplastic-degrading enzymes to obtain domesticated microplastic-degrading enzymes; an enzyme immobilization carrier having a porous structure; a carrier modification processing unit configured to modify the enzyme immobilization carrier to obtain a modified enzyme immobilization carrier; an enzyme immobilization unit configured to immobilize the domesticated microplastic-degrading enzymes into the pores and on the surface of the modified enzyme immobilization carrier by chemical bonding and / or physical adsorption; and a constructed wetland configured to be a constructed wetland including at least a wetland matrix layer, wherein the enzyme immobilization carrier on which the microplastic-degrading enzymes have been immobilized is used as a part of the wetland matrix layer.

[0096] In a preferred embodiment, the system further includes a water flow regulation unit, which is configured to enable the water body to be treated to flow through the constructed wetland from top to bottom or from side to side, and to enable the wetland water level to overflow the wetland substrate layer where the enzyme immobilization carrier is located.

[0097] In a preferred embodiment, the constructed wetland further includes an aquatic plant layer and a microbial community, wherein the aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus and submerged plants, and the microbial community is set to bacteria and fungi with the ability to degrade microplastics; the constructed wetland is set as a hybrid wetland, the front section of which is set as a surface flow wetland, and the rear section is set as a subsurface flow wetland, wherein the zeolite immobilized with the microplastic degrading enzyme is arranged in the wetland substrate layer of the subsurface flow wetland in the rear section.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for the directional degradation of microplastics based on the combination of constructed wetland and enzyme domestication, characterized in that, It includes the following steps: S1. Screen and domesticate variants of the microplastic-degrading enzyme to obtain the domesticated microplastic-degrading enzyme; S2. Modify the enzyme immobilization carrier with pores to obtain the modified enzyme immobilization carrier; S3. Immobilize the domesticated microplastic-degrading enzyme into the pores and on the surface of the modified enzyme immobilization carrier by means of chemical bonding and / or physical adsorption; S4. Construct an artificial wetland including a wetland substrate layer, wherein the enzyme immobilization carrier fixed with the microplastic-degrading enzyme is used as part of the wetland substrate layer; S5. Operate the artificial wetland to degrade the microplastics contained in the water to be treated.

2. The method according to claim 1, wherein The enzyme immobilization carrier with pores is set as zeolite, and step S2 includes: S201. Carry out acid-base modification on the zeolite used as the enzyme immobilization carrier, including: pickling the zeolite with an acid solution to remove surface metal impurities and increase the hydroxyl groups and acidic sites on the surface; subsequently, carry out alkali treatment on the zeolite with an alkali solution to destroy part of the crystal structure of the zeolite, so that the specific surface area and the surface and negative charge density of the zeolite increase; S202. Carry out surface functional group modification on the zeolite modified by acid-base modification to introduce amino and / or epoxy functional groups on the surface of the zeolite.

3. The method according to claim 2, characterized in that, In step S202, 3-aminopropyltriethoxysilane reacts with the surface of the zeolite to generate amino functional groups.

4. The method according to claim 3, wherein The said step S3 includes: S301. Dry the modified zeolite at the first set temperature; S302. Dissolve the domesticated microplastic-degrading enzyme in a specific buffer solution to adjust the enzyme concentration to a specific range to obtain an enzyme solution; S303. Covalently bond the amino groups on the surface of the domesticated microplastic-degrading enzyme to the surface of the modified zeolite through an aminosilylation reaction; and / or, add the enzyme solution to the modified zeolite, mix and stir, so that the microplastic-degrading enzyme is adsorbed on the surface of the zeolite by electrostatic force or van der Waals force; S304. Wash the zeolite fixed with the microplastic-degrading enzyme with the said specific buffer solution, and then dry it at the second set temperature to obtain stable zeolite fixed with the microplastic-degrading enzyme.

5. The method according to claim 4, wherein In step S4, the artificial wetland is set as a hybrid wetland, the front section of which is set as a surface-flow wetland, and the rear section is set as a subsurface-flow wetland, wherein the zeolite fixed with the microplastic-degrading enzyme is arranged in the wetland substrate layer of the subsurface-flow wetland in the rear section.

6. The method according to claim 5, wherein In step S4, the artificial wetland further includes an aquatic plant layer and a microbial community, wherein the aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus and submerged plants, and the microbial community is set as bacteria and fungi with the ability to degrade microplastics.

7. The method according to any one of claims 1-6, characterized in that, Step S1 includes: S101. Carry out error-prone PCR or DNA screening on the microplastic-degrading enzyme to obtain enzyme variants to construct an initial enzyme library; S102. Carry out environmental exposure domestication, including: adding fluorescently labeled microplastics, screening enzyme variants with high fluorescence intensity of degradation products by a flow cytometer, and the enzyme variants that simultaneously meet the following conditions enter the next step: The first condition, detect the stability of the enzyme variant, and obtain the enzyme variant with the residual activity greater than the first preset threshold; The second condition is to detect the release amount of microplastic monomers and screen for enzyme variants with a screening efficiency improvement greater than or equal to a second preset threshold value. S103: Perform high-throughput screening on the enzyme variants obtained through environmental exposure domestication to obtain the finally domesticated microplastic-degrading enzyme.

8. A system for implementing the method of directionally degrading microplastics based on the combination of constructed wetland and enzyme domestication according to any one of claims 1-7, characterized in that, The system includes: An enzyme domestication unit configured to be able to perform variant screening and domestication on the microplastic-degrading enzyme to obtain the domesticated microplastic-degrading enzyme; An enzyme immobilization carrier with a porous structure; A carrier modification processing unit configured to be able to perform modification processing on the enzyme immobilization carrier to obtain the modified enzyme immobilization carrier; An enzyme immobilization unit configured to be able to immobilize the domesticated microplastic-degrading enzyme into the pores and on the surface of the modified enzyme immobilization carrier through chemical bonding and / or physical adsorption; An artificial wetland configured as an artificial wetland at least including a wetland matrix layer, wherein the enzyme immobilization carrier with the immobilized microplastic-degrading enzyme is used as a part of the wetland matrix layer.

9. The system according to claim 8, wherein The system further includes a water flow regulation unit configured to be able to make the water body to be treated flow through the artificial wetland from top to bottom or from side to side, and be able to make the wetland water level overflow the wetland matrix layer where the enzyme immobilization carrier is located.

10. The system according to claim 9, characterized in that The artificial wetland further includes an aquatic plant layer and a microbial community, wherein the aquatic plants in the aquatic plant layer can be at least one of reed, water hyacinth, calamus and submerged plants, and the microbial community is set to bacteria and fungi with the ability to degrade microplastics; The artificial wetland is set as a hybrid wetland, the front section is set as a surface-flow wetland, and the rear section is set as a subsurface-flow wetland, wherein the zeolite with the immobilized microplastic-degrading enzyme is arranged in the wetland matrix layer of the subsurface-flow wetland in the rear section.

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