Biochar-laccase hybrid nanofilm based on interface bonding, preparation method and application in petroleum hydrocarbon pollution synergistic remediation and plant enhancement

Abstract

The invention belongs to the technical field of environmental engineering and pollution ecological restoration, and particularly relates to a biochar-laccase hybrid nanofilm based on interface bonding, a preparation method and application in petroleum hydrocarbon pollution synergistic restoration and plant enhancement. The biochar-laccase hybrid nanofilm comprises biochar and a laccase-inorganic hybrid nanoflower-shaped film, and laccase protein molecules in the laccase-inorganic hybrid nanoflower-shaped film and functional groups on the surface of the biochar are subjected to interface bonding to generate a composite film structure. The immobilized laccase has an ultrahigh specific surface area far exceeding a theoretical addition value, realizes efficient and stable immobilization of laccase, and shows excellent catalytic activity and environmental stability. When applied to petroleum hydrocarbon contaminated soil, the soil remediation agent can strengthen remediation plants such as oilseed rape to efficiently remove petroleum hydrocarbon through biological and non-biological mechanisms, obviously promote plant growth, realize the crossing from single pollution removal to pollution treatment and ecological restoration collaboration, and provide an innovative solution for green and sustainable soil remediation.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering and pollution ecological remediation technology, specifically relating to a biochar-laccase hybrid nanofilm based on interface bonding, its preparation method, and its application in the synergistic remediation and phytoremediation of petroleum hydrocarbon-contaminated soil. Background Technology

[0002] Petroleum hydrocarbons are chemically stable, highly hydrophobic, and difficult to biodegrade, making them highly susceptible to accumulation in soil environments. Excessive accumulation of petroleum hydrocarbons in soil not only undermines the ecological security of the soil environment but also endangers human health. Control and remediation technologies for petroleum hydrocarbon-contaminated soils primarily focus on adsorption, chemical oxidation, and bioremediation methods. However, adsorption methods suffer from drawbacks such as increased costs due to the addition of chemical reagents and the potential for secondary pollution. The stress caused by petroleum hydrocarbon pollution reduces the biodegradation efficiency of free microorganisms. Chemical oxidation methods suffer from demanding reaction conditions and the generation of byproducts. Therefore, finding new methods that can synergistically degrade petroleum hydrocarbons in soil through physical, chemical, and biological processes is crucial.

[0003] Enzyme catalysis is a promising bioremediation technology due to its low energy consumption, mild reaction conditions, environmental friendliness, and high selectivity. However, in the process of expanding its application in the remediation of polluted environments, free laccase still suffers from drawbacks such as high production costs, sensitivity to changes in environmental factors, and easy inactivation. Immobilization techniques such as physical adsorption, covalent cross-linking, and encapsulation to improve the catalytic activity and environmental tolerance of laccase also face significant challenges, including cumbersome immobilization processes, obstruction of enzyme active sites, and increased mass transfer resistance between the enzyme and substrate.

[0004] Organic-inorganic hybrid nanospheres represent a novel enzyme immobilization strategy that leverages the synergistic effects of enzyme molecules and metal ions to enhance mass transfer efficiency between enzymes and substrates, thereby improving enzyme catalytic activity and stability. While laccase-hybrid nanoflowers have demonstrated numerous advantages, several limitations remain in their widespread application. Their powdery structure makes them prone to aggregation and difficult to recycle during water purification processes, and the softness and poor rigidity of their petals limit their reusability. Existing research reports on remediation materials mostly focus on pollutant removal, often neglecting the simultaneous restoration of the ecological functions of contaminated soil. There is a lack of synergistic technologies capable of simultaneously and efficiently degrading pollutants while significantly promoting plant growth, achieving an integrated "remediation-restoration" process. Therefore, developing a novel composite material and its application methods that can significantly improve enzyme immobilization efficiency and stability while synergistically enhancing phytoremediation effects is of paramount importance for promoting the green and sustainable remediation of petroleum hydrocarbon-contaminated soils. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a biochar-laccase hybrid nanomembrane based on interfacial bonding, its preparation method, and its application in the synergistic remediation and phytoremediation of petroleum hydrocarbon-contaminated soil. This method combines laccase hybrid nanoflowers and carbon carriers based on interfacial bonding, resulting in a biochar-laccase hybrid nanomembrane composite material with a larger specific surface area, stronger pH stability, thermal stability, storage stability, and substrate affinity. It can also synergistically achieve efficient degradation of petroleum hydrocarbons in soil and promote plant (rapeseed) growth and biomass accumulation.

[0006] The technical solution of the present invention is a biochar-laccase hybrid nanofilm based on interfacial bonding, comprising biochar and laccase-inorganic hybrid nanoflower-like film, wherein laccase protein molecules in the laccase-inorganic hybrid nanoflower-like film undergo interfacial bonding with functional groups on the surface of biochar to form a composite film structure.

[0007] The laccase-inorganic hybrid nanoflower-like film encapsulates laccase protein molecules with metal phosphates as crystal nuclei. The metal phosphates include, but are not limited to, copper phosphate, zinc phosphate, calcium phosphate, manganese phosphate, iron phosphate, or cobalt phosphate, with copper phosphate being preferred.

[0008] Biochar-laccase hybrid nanofilms based on interfacial bonding have a specific surface area greater than 200 m². 2 / g, preferably 300-350m 2 / g, further preferably 310-320m 2 / g.

[0009] The present invention also provides a method for preparing a biochar-laccase hybrid nanofilm based on interfacial bonding, the steps of which include: adding laccase-inorganic hybrid nanoflower material to a phosphate buffer solution containing biochar and reacting it to obtain an interfacially bonded biochar-laccase hybrid nanofilm.

[0010] The laccase-inorganic hybrid nanoflower material is in the form of microspheres, thin films, or sheets, preferably in the form of microspheres; laccase protein molecules are encapsulated with phosphate as the crystal nucleus, and the metal phosphates include, but are not limited to, copper phosphate, zinc phosphate, calcium phosphate, manganese phosphate, iron phosphate, or cobalt phosphate, preferably copper phosphate as the crystal nucleus for encapsulating laccase protein molecules; the specific surface area of ​​the laccase-inorganic hybrid nanoflower material is greater than 35 m² / g. 2 / g, preferably 40-45m 2 / g, further preferably 41-42m 2 / g.

[0011] The mass ratio of biochar to laccase-inorganic hybrid nanoflower material is 1:0.1-0.5, preferably 1:0.1; the concentration of biochar in phosphate buffer is 0.005-0.05 g / mL, preferably 0.01 g / mL.

[0012] The concentration of the phosphate buffer is 0.03-0.07 mol / L, preferably 0.05 mol / L; the pH is 4-6, preferably pH 5.5.

[0013] The biochar and laccase-inorganic hybrid nanoflower material are reacted under stirring for 4-8 hours, preferably 4 hours; the reaction temperature is 20-40℃.

[0014] The preparation of the laccase-inorganic hybrid nanoflower material includes: first, mixing the laccase solution with phosphate buffer, then adding the inorganic metal salt solution and mixing, and incubating at room temperature to obtain the laccase-inorganic hybrid nanoflower material.

[0015] The concentration of the laccase solution is 0.9-1.5 mg / mL, preferably 1 mg / mL; the concentration of the phosphate buffer is 0.008-0.05 mol / L, preferably 0.01 mol / L; the pH is 4.5-6.5, preferably pH 6.5; the volume ratio of the laccase solution to the phosphate buffer is 1:8-10, preferably 1:9.

[0016] The inorganic metal salts include, but are not limited to, copper, zinc, calcium, manganese, iron, or cobalt salts, such as copper sulfate, copper chloride, zinc chloride, zinc nitrate, calcium chloride, calcium nitrate, manganese chloride, manganese sulfate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, cobalt chloride, and cobalt nitrate, preferably copper salts, and more preferably copper sulfate or copper chloride. The volume ratio of the laccase solution to the inorganic metal salt solution is 1:0.1-0.2, preferably 1:0.1-0.15, and the concentration of the inorganic metal salt solution is 400-600 mmol / L.

[0017] The laccase-inorganic hybrid nanoflower material is in the form of microspheres, thin films, or sheets, preferably in the form of microspheres; laccase protein molecules are encapsulated with phosphate as the crystal nucleus, and the metal phosphates include, but are not limited to, copper phosphate, zinc phosphate, calcium phosphate, manganese phosphate, iron phosphate, or cobalt phosphate, preferably copper phosphate as the crystal nucleus for encapsulating laccase protein molecules; the specific surface area of ​​the laccase-inorganic hybrid nanoflower material is greater than 35 m² / g. 2 / g, preferably 40-45m 2 / g, further preferably 41-42m 2 / g.

[0018] The mixture is incubated at room temperature for 24-72 hours to generate a precipitate. Free laccase is removed by washing with water, yielding the laccase-inorganic hybrid nanoflower material. Further, incubation at room temperature for 24-72 hours generates a blue precipitate, which is then washed with water to remove free laccase, yielding the laccase-copper phosphate hybrid nanoflower material.

[0019] The preparation of the biochar includes: calcining organic matter at high temperature in a nitrogen environment. The organic matter originates from animals, plants, microorganisms, etc., and can be plant roots, stems, leaves, flowers, fruits, seeds, epidermis, etc.; animal hooves, horns, hair, feathers, feces, etc.; or sludge or algae. Calcination is carried out at 400-700℃ for 2-5 hours.

[0020] Furthermore, it is prepared by high-temperature calcination of plant-derived organic matter in a nitrogen environment; further, it is prepared by heating straw or sawdust in a nitrogen environment at a heating rate of 5℃ / min to 400-700℃ and maintaining it for 2-5 hours, followed by natural cooling, to obtain biochar. Even further, it is selected from soybean straw and processed by crushing.

[0021] The laccase-inorganic hybrid nanoflower material provided by this invention can be used to degrade petroleum hydrocarbons, remediate petroleum hydrocarbon pollution, promote plant growth (especially root growth), and promote plant biomass accumulation. It can be used to prepare products for petroleum hydrocarbon degradation, petroleum hydrocarbon pollution remediation, promoting plant growth (especially root growth), and promoting plant biomass accumulation. The petroleum hydrocarbons include total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, or benzene compounds. The plants include plant seeds, seedlings, and plants, such as rapeseed seeds, rapeseed seedlings, and rapeseed plants. The products include, but are not limited to, reagents, fertilizers, nutrients, dispersants, degrading agents, and additives, and their forms can be solid, liquid, or semi-solid.

[0022] The biochar-laccase hybrid nanofilm based on interface bonding provided by this invention can be used to degrade petroleum hydrocarbons, remediate petroleum hydrocarbon pollution, promote plant growth (especially root growth), and promote plant biomass accumulation. It can be used to prepare products for petroleum hydrocarbon degradation, petroleum hydrocarbon pollution remediation, promoting plant growth (especially root growth), and promoting plant biomass accumulation. The petroleum hydrocarbons include total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, or benzene compounds. The plants include plant seeds, seedlings, and plants, such as rapeseed seeds, rapeseed seedlings, and rapeseed plants. The products include, but are not limited to, reagents, fertilizers, nutrients, dispersants, degrading agents, and additives, and their forms can be solid, liquid, or semi-solid.

[0023] The present invention also provides a product for petroleum hydrocarbon degradation, and / or petroleum hydrocarbon pollution remediation, and / or promoting plant growth, and / or promoting plant biomass accumulation, comprising the laccase-inorganic hybrid nanoflower material or the biochar-laccase hybrid nanofilm based on interface bonding provided by the present invention.

[0024] The petroleum hydrocarbons include total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, or benzene compounds, etc.

[0025] The plants mentioned include plant seeds, seedlings, and plants, such as rapeseed seeds, rapeseed seedlings, and rapeseed plants.

[0026] The products include, but are not limited to, reagents, fertilizers, nutrients, dispersants, degradants, additives, etc., and can be in solid, liquid, semi-solid, etc.

[0027] The present invention also provides a method for remediation of petroleum hydrocarbon pollution and / or phytoremediation, comprising the steps of adding the laccase-inorganic hybrid nanoflower material or the biochar-laccase hybrid nanofilm based on interface bonding provided above to the target material.

[0028] The targets include soil, rivers, lakes, oceans, wetlands, etc.

[0029] When the target material is soil, the mass ratio of biochar-laccase hybrid nanofilm based on interfacial bonding to soil is 0.1%-10%, and the soil moisture content is controlled at 30-50%.

[0030] The petroleum hydrocarbons include total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, or benzene compounds, etc.

[0031] The plants mentioned include plant seeds, seedlings, and plants, such as rapeseed seeds, rapeseed seedlings, and rapeseed plants.

[0032] The core of this invention lies in providing a biochar-laccase hybrid nanofilm based on interfacial bonding. This material consists of a biochar support rich in functional groups on its surface, and a laccase-inorganic metal salt hybrid nanofilm covering its surface through interfacial chemical bonding. The preparation method includes pretreating free laccase and inorganic metal salts to form laccase-inorganic hybrid nanoflowers through self-assembly, followed by addition to a biochar suspension. Through interfacial bonding-induced reaction between the biochar and the laccase-inorganic hybrid nanoflowers, a stable hybrid nanofilm structure is formed in situ. The material of this invention possesses an ultra-high specific surface area far exceeding the theoretical summation value, achieving efficient and stable immobilization of laccase, exhibiting excellent catalytic activity and environmental stability. Its application in petroleum hydrocarbon-contaminated soil can enhance the efficient removal of petroleum hydrocarbons by remediation plants such as rapeseed through biological and abiotic mechanisms, and significantly promote plant growth, achieving a leap from single pollution removal to synergistic pollution control and ecological restoration, providing an innovative solution for green and sustainable soil remediation. The advantages and positive effects of this invention are described below: (1) The functional groups on the surface of biochar form a stable interfacial bond structure with the protein molecules in the laccase-hybridized nanoflowers, effectively preventing the enzyme and nanoflowers from falling off and being lost during use, thus solving the core problems of easy inactivation of traditional immobilized enzymes and easy breakage and loss of nanoflower materials. Meanwhile, the specific surface area of ​​the composite material (314.478 m²) 2 / g) is much higher than the theoretical sum of biochar and nanoflower (116.958 m) 2 / g), providing abundant active reaction sites for pollutant adsorption and enzyme catalytic reactions.

[0033] (2) The unique hybrid nanomembrane structure retains the native conformation and active center of laccase to the greatest extent while immobilizing it, thus achieving high enzyme catalytic activity. The immobilized laccase has significantly enhanced tolerance to environmental factors (including pH, temperature changes, etc.), and its operational and storage stability is far superior to that of free laccase and traditional immobilized laccase, which greatly reduces the cost of practical applications and has key advantages for engineering applications.

[0034] (3) The biochar-laccase hybrid nanofilm composite material prepared in this invention, through the combined effect of "adsorption-enrichment-biodegradation," significantly improves the degradation efficiency of petroleum hydrocarbons in soil, with a degradation efficiency significantly higher than that of other single components. Furthermore, while degrading petroleum hydrocarbons, this composite material can significantly alleviate the stress of pollutants on plants and promote root growth and biomass accumulation in remediation plants (such as rapeseed). This achieves a synergistic win-win goal of pollution remediation efficiency and ecological restoration function, which is difficult to achieve with general remediation materials.

[0035] (4) The preparation process of this invention is green and mild. The entire preparation process is carried out at room temperature and pressure, with low energy consumption and no need for complex equipment or expensive reagents, which is in line with the principles of green chemistry. The raw materials are inexpensive and readily available. Biochar is derived from agricultural waste, realizing the resource utilization of waste and possessing great potential for large-scale production and application. Attached Figure Description

[0036] Figure 1 A flowchart illustrating the preparation method of biochar-laccase hybrid nanofilm composite material.

[0037] Figure 2 This is a SEM image of soybean straw biochar.

[0038] Figure 3 This is a SEM image of free laccase.

[0039] Figure 4 This is a SEM image of laccase-inorganic hybrid nanoflowers.

[0040] Figure 5 SEM image of biochar-laccase hybrid nanofilm.

[0041] Figure 6 This is the FTIR image of soybean straw biochar.

[0042] Figure 7 The image shows the FTIR spectrum of laccase-inorganic hybrid nanoflowers.

[0043] Figure 8 The image shows the FTIR spectrum of the biochar-laccase hybrid nanofilm.

[0044] Figure 9 A fully automated specific surface area (BET) analysis of soybean straw biochar.

[0045] Figure 10 A fully automated specific surface area (BET) analysis of laccase-inorganic hybrid nanoflowers.

[0046] Figure 11 A fully automated specific surface area (BET) analysis for biochar-laccase hybrid nanofilms.

[0047] Figure 12 The effect of pH on the relative activity of free laccase, laccase-inorganic hybrid nanoflowers, and biochar-laccase hybrid nanofilms.

[0048] Figure 13 The effect of temperature on the relative activity of free laccase, laccase-inorganic hybrid nanoflowers, and biochar-laccase hybrid nanofilms.

[0049] Figure 14 To assess the storage stability of free laccase, laccase-inorganic hybrid nanoflowers, and biochar-laccase hybrid nanofilms.

[0050] Figure 15 The effects of biochar (BC), free laccase, laccase-inorganic hybrid nanoflowers (NFs), and biochar-laccase hybrid nanofilms (BCNFs) on the remediation of petroleum hydrocarbons in soil were investigated. Detailed Implementation

[0051] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a further detailed account of the present invention: a biochar-laccase hybrid nanofilm based on interfacial bonding, its preparation method, and its application in the synergistic remediation and phytoremediation of petroleum hydrocarbon-contaminated soils. The advantages and features of the present invention will become clearer from the following description and claims. Example 1: The preparation method of biochar-laccase hybrid nanofilm based on interfacial bonding in this embodiment includes the following steps: (1) Preparation of laccase-copper phosphate hybrid nanoflowers Measure 5 mL of 1 mg / mL laccase solution into a 100 mL beaker, add 45 mL of 0.01 mol / L pH 6.5 phosphate buffer solution, and add 600 μL of 500 mmol / L CuSO4 solution. Stir well and incubate at room temperature for 2 days. A blue precipitate will form. Separate the obtained blue precipitate and wash it with water 3 times to remove unreacted free laccase, and you can get laccase-copper phosphate hybrid nanoflowers.

[0052] (2) Preparation of soybean straw biochar Remove unwanted leaves from the collected soybean straw, wash it with deionized water, air dry it naturally, cut it into small pieces of about 1 cm, grind it with a pulverizer, and pass it through a 100-mesh sieve. Weigh a certain amount of the pre-treated soybean straw biomass powder into an alumina pot, cover it, and place it in a tubular furnace. Purge with nitrogen, set the heating rate to 5℃ / min, heat it from room temperature to 500℃ and maintain it for 2 hours. After the reaction is complete, allow it to cool naturally. The resulting black solid powder is biochar. (3) Preparation of biochar-laccase hybrid nanofilm composite material First, disperse 1g of soybean straw biochar in 100mL of 0.05mol / L pH 5.5 phosphate buffer solution and sonicate for 30min to ensure homogeneity. Then, add 0.1mg of laccase-copper phosphate hybrid nanoflowers and magnetically stir at 500rpm for 4h. After the reaction is complete, separate the solid sample and wash it three times with water to obtain the biochar-laccase hybrid nanofilm composite material. The preparation process is described in [link to preparation procedure]. Figure 1 .

[0053] Example 2: Biochar, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms obtained in Example 1 were characterized and tested: The laccase, biochar, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms from Example 1 were analyzed by field emission scanning electron microscopy (SEM). Figure 2 Soybean straw biochar, Figure 3 It is free laccase. Figure 4 For laccase-copper phosphate hybrid nanoflowers, Figure 5 It is a biochar-laccase hybrid nanofilm composite material. (The text abruptly ends here, so the translation also ends here.) Figure 2 It can be seen that soybean straw biochar has a porous carbonaceous framework structure with irregular pore structure and relatively thick pore walls. Free laccase ( Figure 3 It has an irregular three-dimensional structure with a relatively smooth surface but covered with curved wrinkles. The blue laccase-copper phosphate hybrid nanoflower precipitate formed through self-assembly ( Figure 4 They exhibit a relatively regular microsphere structure, which facilitates the generation of a larger specific surface area, promotes substrate-enzyme contact, and enhances enzyme catalytic activity. Figure 5 It can be observed that the overall structure of laccase-copper phosphate nanoflora is destroyed, while the surface structure remains unchanged, forming a thin film covering the surface of biochar.

[0054] Fourier transform infrared (FTIR) spectroscopy was performed on the biochar, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilm from Example 1. Figure 6 Soybean straw biochar, Figure 7 For laccase-copper phosphate hybrid nanoflowers, Figure 8 This is a biochar-laccase hybrid nanofilm composite material. The biochar surface possesses abundant oxygen-containing functional groups such as hydroxyl, carboxyl, and ether groups, providing it with numerous reactive sites and strong biocompatibility as a carrier material. Laccase protein molecules were successfully encapsulated in laccase-copper phosphate hybrid nanoflowers using Cu3(PO4)2 as the crystal nucleus. When the biochar carrier matrix is ​​combined with the laccase nanoflowers, [the following is observed] at 3300-3400 cm⁻¹. -1 Absorption peaks within the range and at 1600 cm⁻¹ -1 The absorption peaks at the point disappeared because the functional groups on the surface of biochar interacted with the protein molecules in the laccase-hybridized nanoflowers during the reaction, forming a stable composite film structure.

[0055] The specific surface area of ​​the biochar, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms from Example 1 was analyzed using a fully automated method. Figure 9 Soybean straw biochar, Figure 10 For laccase-copper phosphate hybrid nanoflowers, Figure 11 The composite material is a biochar-laccase hybrid nanofilm. The BET specific surface areas of soybean straw biochar, laccase-copper phosphate hybrid nanoflowers, and the biochar-laccase hybrid nanofilm composite are 75.708 m². 2 / g, 41.250m 2 / g and 314.478m 2 / g. The specific surface area of ​​the biochar-laccase hybrid nanofilm was 4.15 times and 7.62 times that of soybean straw biochar and laccase-copper phosphate hybrid nanoflowers, respectively. The laccase-copper phosphate hybrid nanoflowers, in the form of a thin film, can effectively increase the specific surface area of ​​the composite material and increase the effective active adsorption sites of the composite material.

[0056] Example 3: The stability of the free laccase, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms described in this invention was tested, including pH stability, temperature stability, and storage stability. The steps and results are as follows: (1) pH stability The pH stability of free and immobilized laccase was evaluated by incubating them in phosphate buffer solutions under a range of different pH conditions. The pH range was set from 3.0 to 8.0, and the incubation time was 12 h. Using ABTS as a substrate, the activity of free and immobilized laccase was determined spectrophotometrically, with maximum enzyme activity defined as 100%.

[0057] like Figure 12 As shown, free laccase, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms exhibited the highest activity at pH values ​​of 3, 2, and 3, respectively. Due to the presence of electrostatic interactions, OH...- and H + The uneven concentration distribution of free and immobilized laccase in their microenvironments and reaction media leads to a shift in their optimal pH activities. Compared to free laccase, laccase-copper phosphate hybrid nanoflowers are more stable under stronger acidic conditions. This is because the alkaline microenvironment in the solution creates a balanced microenvironment for the carrier and protons, which is more conducive to maintaining the conformational stability of the enzyme. Under alkaline conditions (pH = 8), the relative residual enzyme activities of free laccase, laccase-copper phosphate hybrid nanoflowers, and biochar-laccase hybrid nanofilms are 8.75%, 18.48%, and 28.66%, respectively, indicating that immobilized laccase has stronger pH stability than free laccase. This behavior is attributed to the existence of multiple binding sites between the enzyme and the carrier matrix, which effectively improves the stability of the conjugated system.

[0058] (2) Temperature stability The thermal stability of free and immobilized laccase was investigated by measuring the remaining enzyme activity after incubation at different temperatures (25℃, 35℃, 45℃, 55℃, 65℃, 75℃, 85℃) for 1 h. Enzyme activity at 25℃ was defined as 100%.

[0059] like Figure 13 As shown, free laccase exhibits the highest relative activity at 55℃. Laccase-copper phosphate hybrid nanoflowers and biochar-laccase hybrid nanofilms show the highest relative enzyme activity at 65℃. Compared to free laccase, laccase immobilized in the carrier material offers some protection against temperature changes, resulting in higher thermal stability. When the temperature increases to 85℃, the remaining relative enzyme activity of free laccase is only 4.35%, while the relative enzyme activities of laccase-copper phosphate hybrid nanoflowers and biochar-laccase hybrid nanofilms remain at 26.71% and 38.44%, respectively. Immobilized laccase allows for stronger interactions between the enzyme and the carrier, protecting the protease from folding and denaturation. This not only provides a suitable microenvironment for laccase but also enhances its thermal stability.

[0060] (3) Storage stability To further investigate storage stability, free and immobilized laccase were stored at room temperature for 50 days, and their remaining activity was measured every 10 days at the optimal pH and temperature. Initial enzyme activity was defined as 100%.

[0061] like Figure 14As shown, after 50 days of storage, the relative residual enzyme activity of free laccase was 40.35%, while the relative enzyme activity of laccase-copper phosphate hybrid nanoflowers and biochar-laccase hybrid nanofilms remained at around 60%. The storage stability of immobilized laccase was significantly higher than that of free laccase. On the one hand, the nanoflower structure formed by the chelation of laccase with copper ions is more conducive to the stability of the protease's spatial structure. On the other hand, the excellent surface properties and chemical stability of biochar, through its immobilization effect, confine the laccase to a specific spatial region, preventing changes in the enzyme's conformational structure and thus conferring good storage stability.

[0062] Example 4: Remediation experiment of petroleum hydrocarbon contaminated soil: 1g of petroleum hydrocarbon contaminated soil sample (concentration 5.4g / kg) was accurately weighed into a reaction flask. Different catalysts with a mass concentration of 0.05g / g were added, and an appropriate amount of water was added to the reaction flask to control the moisture content at approximately 40%. After the reaction system was thoroughly mixed, the catalytic reaction was carried out. The removal rate of petroleum hydrocarbons in the soil was measured after 1, 2, 3, 4, and 5 days of reaction. A blank control group was also set up, and each sample was subjected to three parallel treatments.

[0063] Figure 15 As shown in the figure, after 5 days of treatment, the removal rate of petroleum hydrocarbons in the blank control group without any remediation materials was 22.01%, while the removal rate in the soybean straw biochar treatment group was 40.79%, and the removal rate of petroleum hydrocarbons by free laccase was 43.96%. As can be seen from the figure, the removal rate of petroleum hydrocarbons by free laccase initially increased rapidly and then stabilized. This is because free laccase applied to the soil undergoes conformational transformation or inactivation due to changes in soil environmental factors, resulting in a decrease in its catalytic activity. Furthermore, the poor oxidation efficiency of laccase and the steric hindrance between laccase and petroleum hydrocarbon macromolecules are also major reasons for the incomplete degradation of pollutants. Compared with free laccase, the removal rate of petroleum hydrocarbons by laccase-copper phosphate hybrid nanoflowers was improved, reaching 55.95%. This indicates that laccase immobilization can effectively improve enzyme stability and catalytic activity. The biochar-laccase hybrid nanofilm composite material achieved a petroleum hydrocarbon removal rate of 69.88%, significantly outperforming other treatment groups. This is likely due to the synergistic effect of biochar adsorption and enzyme-catalyzed degradation. Specifically, the adsorption process promotes the transfer of petroleum hydrocarbons from the soil to the active sites of enzymes on the biochar surface, enhancing electron transfer between the enzymes and pollutants, thereby improving the petroleum hydrocarbon removal efficiency.

[0064] Example 5: Application of biochar-laccase hybrid nanomembranes in the synergistic remediation and phytoremediation of petroleum hydrocarbon-contaminated soil: A pot experiment was conducted on rapeseed with three treatment groups: 1) rapeseed cultivated in clean soil (T1), 2) rapeseed cultivated in petroleum hydrocarbon-contaminated soil (T2), and 3) rapeseed cultivated in petroleum hydrocarbon-contaminated soil treated with biochar-laccase hybrid nanomembrane composite material (T3). The petroleum hydrocarbon concentration was 11.44 g / kg, and the amount of biochar-laccase hybrid nanomembrane composite material added was 5% (w / w) of the soil mass. Ten rapeseed seedlings were transplanted into each pot and managed routinely for 30 days. During the experiment, water management was kept consistent across all treatment groups. After the experiment, five rapeseed plants from each treatment were selected for subsequent physiological and biochemical index measurements, and the removal rate of petroleum hydrocarbons in the soil was determined. The results are shown in Table 1.

[0065] Table 1. Degradation rate of petroleum hydrocarbons and physiological and biochemical indicators of rapeseed in soil under different treatment groups.

[0066] Without the addition of remediation materials, the removal rate of petroleum hydrocarbons by rapeseed was only 25.39% after 30 days of remediation. With the application of biochar-laccase hybrid nanomembrane composite material, the removal rate of petroleum hydrocarbons reached 85.29%, significantly reducing the residual amount of petroleum hydrocarbons in the soil. This alleviated the toxic effects of high-concentration petroleum hydrocarbon pollution stress on remediated plants and further promoted the metabolic capacity of plant roots and rhizosphere microorganisms for petroleum hydrocarbons. Rapeseed cultivated in clean soil was unaffected by pollution stress and exhibited good growth, with a biomass of 0.2353 g. Rapeseed plants grown in petroleum hydrocarbon-contaminated soil were stunted, with a biomass of 0.1004 g. After treatment with biochar-laccase hybrid nanomembrane composite material, the biomass of rapeseed reached 0.2846 g, showing a significant promoting effect compared to the clean soil group. This result indicates that biochar-laccase hybrid nanomembrane composite material can act as a biochar-based synergistic fertilizer to promote the absorption and transformation of nutrients in rapeseed, thereby enhancing plant growth and increasing biomass.

[0067] In summary, the biochar-laccase hybrid nanofilm composite material provided by this invention significantly improves the degradation efficiency of petroleum hydrocarbons in soil. Furthermore, while degrading petroleum hydrocarbons, this composite material can significantly alleviate the stress of pollutants on plants and promote the accumulation of remediation plant biomass. This achieves a synergistic win-win goal of pollution remediation efficiency and ecological restoration function, which is difficult to achieve with conventional remediation materials.

[0068] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they shall still fall within the protection scope of the present invention.

Claims

1. A biochar-laccase hybrid nanofilm based on interfacial bonding, characterized in that, The membrane comprises biochar and laccase-inorganic hybrid nanoflower-like film. The laccase-inorganic hybrid nanoflower-like film encapsulates laccase protein molecules with metal phosphate as the crystal nucleus. The laccase protein molecules in the laccase-inorganic hybrid nanoflower-like film undergo interfacial bonding with the functional groups on the surface of biochar to form a composite membrane structure.

2. A method for preparing biochar-laccase hybrid nanofilms based on interfacial bonding, characterized in that the steps include... include: Laccase-inorganic hybrid nanoflower material was added to a phosphate buffer solution containing biochar and reacted to obtain an interfacially bonded biochar-laccase hybrid nanofilm.

3. The preparation method according to claim 2, characterized in that, The mass ratio of biochar to laccase-inorganic hybrid nanoflower material is 1:0.1-0.5, the concentration of biochar in phosphate buffer is 0.005-0.05 g / mL, the concentration of phosphate buffer is 0.03-0.07 mol / L, and the pH is 4-6.

4. The preparation method according to claim 2, characterized in that, The preparation of the laccase-inorganic hybrid nanoflower material includes: first, mixing the laccase solution with phosphate buffer, then adding the inorganic metal salt solution and mixing, and incubating at room temperature to obtain the laccase-inorganic hybrid nanoflower material.

5. The preparation method according to claim 4, characterized in that, The concentration of the laccase solution is 0.9-1.5 mg / mL; the concentration of the phosphate buffer is 0.008-0.05 mol / L, and the pH is 4.5-6.5; the volume ratio of the laccase solution to the phosphate buffer is 1:8-10; the inorganic metal salt is selected from any one or any combination of copper, zinc, calcium, manganese, iron, or cobalt salts.

6. The laccase-inorganic hybrid nanoflower material prepared by the preparation method of claim 4, or the biochar-laccase hybrid nanofilm based on interface bonding as described in claim 1, or the biochar-laccase hybrid nanofilm based on interface bonding prepared by the preparation methods of claims 2-5, may be used for petroleum hydrocarbon degradation, and / or petroleum hydrocarbon pollution remediation, and / or promoting plant growth, and / or promoting plant biomass accumulation, or for preparing products for petroleum hydrocarbon degradation, and / or petroleum hydrocarbon pollution remediation, and / or promoting plant growth, and / or promoting plant biomass accumulation.

7. A product for the degradation of petroleum hydrocarbons, and / or the remediation of petroleum hydrocarbon pollution, and / or the promotion of plant growth, and / or the promotion of plant biomass accumulation, characterized in that, The material contains laccase-inorganic hybrid nanoflower material prepared by the preparation method of claim 4, or a biochar-laccase hybrid nanofilm based on interface bonding as described in claim 1, or a biochar-laccase hybrid nanofilm based on interface bonding prepared by the preparation methods of claims 2-5.

8. The product according to claim 7, characterized in that, The product includes any one of the following: reagent, fertilizer, nutrient, dispersant, degrader, and additive.

9. A method for remediation of petroleum hydrocarbon pollution and / or phytofortification, characterized in that the steps include... include: Add to the target material the laccase-inorganic hybrid nanoflower material prepared by the preparation method of claim 4, or the biochar-laccase hybrid nanofilm based on interface bonding as described in claim 1, or the biochar-laccase hybrid nanofilm based on interface bonding prepared by the preparation methods of claims 2-5.

10. The method according to claim 9, characterized in that, Petroleum hydrocarbons include any one or any combination of total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, or benzene series compounds.

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