A controllable electric field-enhanced enzyme degradation method and its application for in-situ remediation of organically contaminated soil

Abstract

The present invention discloses a controllable electric field-enhanced enzymatic degradation method and application for in-situ remediation of organic contaminated soil, belonging to the technical field of in-situ remediation of organic contaminated soil. The method comprises the following steps: setting electrode wells in the remediation area, the electrode wells being arranged in a rectangular shape to form a non-uniform symmetrical electric field; inserting electrodes into the electrode wells, fixing them, and connecting them to the corresponding output ends of a DC power supply; pumping an electrolyte containing immobilized enzymes into the anode wells and into the cathode wells; setting the potential gradient of the electric field for transporting the immobilized enzymes and starting the DC power supply; after completing the electrokinetic transport of the immobilized enzymes, adjusting the DC electric field potential gradient and starting electrokinetic enhanced degradation. The present invention achieves low-carbon, green in-situ remediation of organic contaminated soil based on enzyme bioelectrochemical degradation and microelectrode redox pathways.

Description

Technical Field

[0001] The present invention belongs to the technical field of in-situ remediation of organic contaminated soil, and in particular relates to a controllable electric field-enhanced enzyme degradation method and application for in-situ remediation of organic contaminated soil. Background Art

[0002] In recent years, my country's contaminated site remediation market has flourished, with the total project amount exceeding 10 billion for four consecutive years. Among them, organic contaminated sites account for about 43%, and the proportion of in-situ remediation technology applications is close to 50%, mainly thermal and chemical remediation. The remediation of contaminated sites is mostly for the purpose of land transfer and development. The pursuit of "short, flat and fast" in remediation projects has led to high energy consumption and high chemical input technologies becoming the mainstream of the market. The core technologies of 69 major domestic remediation engineering companies are concentrated in gas phase extraction, thermal and chemical remediation, etc. Based on the life cycle greenhouse gas emission coefficient of the State Grid's electricity (about 960g CO2 / kW·h), the CO2 emissions of gas phase extraction technology are about 500kg / t 土壤 , while the CO2 emissions of resistance heating (100℃) and electric conduction heating technology (500℃) are as high as 816kg / t 土壤 and 1224kg / t 土壤 In-situ chemical oxidation (ISCO) is often combined with other technologies such as multiphase extraction and thermal desorption, resulting in higher energy consumption and CO2 emissions. Under the "dual carbon" context, contaminated site remediation strategies will inevitably be adjusted, and the demand for green, low-energy in-situ remediation technologies for organically contaminated soils is growing.

[0003] Electrokinetic remediation (EK) refers to applying a direct current electric field to contaminated soil, and utilizing electrokinetic effects such as electrodialysis, electromigration, and electrophoresis to drive the directional migration of target substances. It has the advantages of not being affected by soil heterogeneity and low permeability. EK can effectively transport remediation functional agents and also stimulate microbial activity. Therefore, it is often coupled with bioremediation technology as an in situ remediation technology for the remediation of organic contaminated soil. However, in actual application, although a strong electric field (2.0V / cm) is beneficial to material transport, it is not conducive to stimulating bacterial strain activity to obtain higher organic pollutant degradation efficiency; a weak electric field (0.5-1.0V / cm) can effectively stimulate bacterial strain proliferation and degradation activity, but it will cause the strain to adhere and deposit in the soil. Summary of the Invention

[0004] To address the lack of green, low-energy in-situ remediation technologies for organically contaminated sites, the present invention proposes a controllable electric field-enhanced enzymatic degradation method and application for in-situ remediation of organically contaminated soil. This method addresses the high energy consumption and high chemical input issues of conventional in-situ remediation technologies. Biochar-immobilized laccase replaces degradation microorganisms. On the one hand, carrier immobilization ensures the activity and stability of laccase in harsh soil environments and reaction conditions; on the other hand, it resolves the technical conflict between the strain transport electric field and the biodegradation enhancement electric field during electrokinetic bioremediation. Simultaneously, the electron transfer acceleration and polarization effects of the DC electric field are utilized to achieve intramolecular power generation of the immobilized laccase and microelectropolarization of the carrier biochar, thereby realizing bioelectrochemical degradation and redox degradation of organic pollutants. The method of the present invention can address organic pollution problems in contaminated sites with different stratum properties and is particularly suitable for in-situ remediation of low-permeability contaminated strata.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A controllable electric field-enhanced enzymatic degradation method for in-situ remediation of organic contaminated soil comprises the following steps:

[0007] 1) Electrode wells are set up in the repair area and constructed using direct push drilling. The electrode wells are arranged in a rectangular shape to form a non-uniform symmetrical electric field;

[0008] 2) Insert the electrode into the electrode well, secure it, and connect it to the corresponding output terminal of the DC power supply;

[0009] 3) pumping the electrolyte containing the immobilized enzyme into the anode well and the electrolyte into the cathode well;

[0010] 4) Setting the potential gradient of the electric field for transporting the immobilized enzyme, starting the DC power supply, and starting to transport the immobilized enzyme into the repaired soil;

[0011] 5) After the electrokinetic transport of the immobilized enzyme is completed, the DC electric field potential gradient is adjusted to initiate electrokinetic enhanced degradation, thereby achieving bioelectrochemical degradation and microelectrode redox degradation of organic pollutants.

[0012] Furthermore, the electrode well has a pore size of 100-150 mm, the well wall is supported by a porous PVC screen plate, and the depth of the electrode well is consistent with the depth of soil contamination. The electrode well can be constructed using a Geoprobe or Powerprobe drilling rig.

[0013] Furthermore, the electrode is in a rod shape and can be made of graphite, stainless steel or titanium metal, with a diameter of 30-60 mm; the DC power supply has a constant voltage output and an operating voltage range of 0-200 V.

[0014] Furthermore, in the electrolyte containing the immobilized enzyme, the liquid-solid ratio of the electrolyte to the immobilized enzyme is 100 mL: (0.5-2) g.

[0015] Furthermore, the immobilized enzyme is immobilized using chitosan on nitrate-modified biochar as a carrier; the enzyme is laccase. The specific method for preparing the immobilized enzyme is as follows: chitosan (1% v / v acetic acid aqueous solution, i.e., acetic acid and water are prepared in a volume ratio of 1:100 to obtain a 1% v / v acetic acid aqueous solution; 0.5-1.5% w / v), laccase (100-500 U / L), glutaraldehyde (0.2-0.6% v / v), nitrate-modified biochar (20-40% w / v), and sodium tripolyphosphate (0.1-0.5% w / v) are added to an acetic acid-sodium acetate buffer in this order. Each addition of each material is shaken (150 rpm) for 10 minutes before the next material is added. After all materials are added, the mixture is shaken in a shaker at 25°C and 150 rpm for 24 hours to fully immobilize the laccase, and the immobilized enzyme is collected by centrifugation at 8000 rpm for 10 minutes. The preparation method of the nitric acid-modified biochar is as follows: concentrated nitric acid (content 68%) and biochar are mixed in a liquid-solid ratio of 100 mL: 10 g, and heated at 120° C. for 2 h.

[0016] Furthermore, the potential gradient in step 4) is 1.0-2 V / cm.

[0017] Furthermore, the electrokinetic transport in step 5) lasts for 10-100 hours.

[0018] Furthermore, in step 5), the potential gradient is 0.2-0.6 V / cm; and the electrokinetic enhanced degradation time is 5-15 days.

[0019] The present invention also provides an application of the controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil in in-situ remediation of organic contaminated soil.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention adopts chitosan to strengthen physical adsorption + cross-linking fixation method to load laccase on biochar. Chitosan, as a biopolymer, can strengthen the protection of the enzyme, and ensure the activity of the immobilized enzyme and its green synthesis without affecting the conductivity of the material. The degradation strain is replaced by the immobilized enzyme, which ensures the activity and stability of the laccase in harsh soil environment and reaction conditions. The efficient electrodialysis effect of the strong electric field is used to achieve the directional migration (anode to cathode) of the immobilized enzyme in the repaired soil. After the electric transport of the immobilized enzyme is completed, the electric field strength is reduced, which can promote the adhesion and deposition of the immobilized enzyme while saving energy consumption, and realize the formation of the soil conductive network; the electron transfer acceleration and polarization effect of the direct current electric field are used to achieve the intramolecular power generation of laccase and the microelectropolarization of the carrier biochar, and then degrade the organic pollutants in the soil through bioelectrochemistry and redox pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0023] Figure 1 The activity of the biochar-immobilized laccase prepared by the present invention changes over time;

[0024] Figure 2 This is a laboratory verification simulation device for the controllable electric field-enhanced enzyme degradation method of the present invention; in the figure, 1-electric repair control system; 2-digital multimeter; 3-electrolyte recovery tank; 4-cathode well; 5-cathode; 6-porous sieve plate; 7-soil chamber; 8-anode well; 9-anode; 10-electrolyte (containing immobilized enzyme) storage tank; 11-peristaltic pump; 12-soil sampling point;

[0025] Figure 3 This is the laboratory verification result of the controllable electric field enhanced enzyme degradation method of the present invention;

[0026] Figure 4 This is a schematic diagram of a method specifically implemented when the present invention is actually applied on site;

[0027] Figure 5 The results of field application of the controllable electric field-enhanced enzyme degradation method of the present invention are shown in the figure. In the figure, (a) chloroform residue; (b) petroleum hydrocarbon residue. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The raw materials used in the following examples of the present invention are all commercially available.

[0034] Electric repair (EK) has low energy consumption when used as a means of material transportation, such as oxidizer and activator (268.61kW·h / t 土壤 ), surfactant (228.65kW·h / t 土壤 The transport rate of EK for electrically neutral substances is close to the electrodialysis flow rate (about 1.5 cm / h). It can effectively transport nano-scale (zero-valent iron, etc.) and micro-scale (biochar, etc.) repair functional materials, and can achieve the degradation of organic pollutants by relying on its own redox ability.

[0035] The bio-electrochemical system (BES) is considered to be a new in-situ environmental remediation technology with great development prospects. It is a process that uses microorganisms (such as bacteria) or biomolecules (such as enzymes) to efficiently catalyze electrode reactions. BES can provide continuous electrons to improve the biological oxidation and reduction efficiency of organic pollutants, overcoming the limitations of traditional in-situ bioremediation methods. At the same time, according to the microconductor theory of the soil electrokinetic remediation process, particles or membranes (microconductors) with electronic conductivity properties in the soil matrix are polarized by the electric field to become "microelectrodes", which can undergo redox reactions to degrade organic pollutants. Therefore, the present invention uses biocarbon-immobilized laccase to replace bacteria and couple with EK, which is not affected by the mass transfer electric field and can form a microreactor similar to BES in the soil, thereby achieving efficient removal of difficult-to-degrade organic pollutants.

[0036] The present invention provides a controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil, which mainly adopts a direct current electric field to achieve efficient transportation of biocarbon-immobilized laccase (immobilized enzyme) in the soil. When the immobilized enzyme migrates in the remediation soil through electrodialysis, a conductive network can be formed in the soil based on the conductive properties of the carbon carrier. The interaction between the electric field-soil conductive network-carbon-based carrier is used to stimulate the electron transfer pathway within the enzyme molecule, forming a large number of bioelectrochemical reaction systems with immobilized enzymes as the core, and realizing the redox of organic pollutants by direct electron transfer. In addition, the carbon carrier with electronic conductivity in the soil can also be polarized into a "microelectrode" by a direct current electric field, and redox reactions can occur to degrade organic pollutants. The present invention realizes low-carbon, green in-situ remediation of organic contaminated soil based on enzyme bioelectrochemical degradation and microelectrode redox.

[0037] The specific method includes the following steps:

[0038] (1) Electrode wells are set up in the repair area and constructed using direct push drilling for electrode installation; the electrode wells (electrodes) are arranged in a rectangular shape to form a non-uniform symmetrical electric field;

[0039] (2) Insert the electrode into the electrode well, fix it, and connect it to the corresponding output terminal of the DC power supply;

[0040] (3) pumping the electrolyte containing a certain amount of immobilized enzyme into the anode well and the electrolyte (without immobilized enzyme) into the cathode well;

[0041] (4) setting the potential gradient of the electric field for transporting the immobilized enzyme, starting the DC power supply, and starting to transport the immobilized enzyme into the repaired soil;

[0042] (5) After the electrokinetic transport of the immobilized enzyme is completed, the DC electric field potential gradient is adjusted to initiate electrokinetic enhanced degradation, thereby achieving bioelectrochemical degradation and microelectrode redox degradation of organic pollutants.

[0043] In the following preferred embodiments of the present invention, the electrode well can be constructed using a Geoprobe or Powerprobe drilling rig, with a diameter of 100-150 mm; the well wall is supported by a porous PVC screen; and the depth of the electrode well is consistent with the depth of soil contamination. The electrodes are rod-shaped and can be made of graphite, stainless steel, or titanium, with a diameter of 30-60 mm. The DC power supply has a constant voltage output range of 10-200 V, preferably 20 V.

[0044] The immobilized enzyme is immobilized by chitosan enhanced physical adsorption + cross-linking fixation method to achieve laccase immobilization. Chitosan (1% v / v acetic acid aqueous solution, 0.5-1.5% w / v, preferably 1% w / v), laccase (100-500 U / L, preferably 200 U / L), glutaraldehyde (0.2-0.6% v / v, preferably 0.6% v / v), nitric acid modified biochar (20-40% w / v, preferably 25% w / v), sodium tripolyphosphate (0.1-0.5% w / v, preferably 0.3% w / v) are added to acetic acid-sodium acetate buffer in order. Each time a material is added, it is shaken (150 rpm) and mixed for 10 minutes before the next material is added. After all the materials are added, they are placed in a shaker at 25°C and 150 rpm for 24 hours to fully immobilize the laccase, and the immobilized enzyme is collected by centrifugation at 8000 rpm for 10 minutes. The nitric acid-modified biochar is prepared using nitric acid-modified biochar as a carrier through a microwave hydrothermal method. The liquid-solid ratio of concentrated nitric acid (content 68%) to biochar is 100 mL:10 g, and the mixture is heated at 120° C. for 2 h.

[0045] In the following preferred embodiments of the present invention, the electrolyte in the anode well is an aqueous solution of sodium carbonate, sodium nitrate, or sodium sulfate, preferably an aqueous solution of sodium carbonate; the concentration is 50-200 mM, preferably 50-100 mM, more preferably 50 mM or 100 mM; the liquid-to-solid ratio of the electrolyte to the immobilized enzyme is 100 mL:(0.5-2) g, preferably 100 mL:2 g. The electrolyte in the cathode well is an aqueous solution of water, sodium carbonate, sodium nitrate, or sodium sulfate.

[0046] In step (4) of the following preferred embodiment of the present invention, the potential gradient of the electric field for transporting the immobilized enzyme is 1.0-2 V / cm, preferably 1 V / cm, 2 V / cm; the electrokinetic transport time of the immobilized enzyme is 10-100 h, preferably 100 h.

[0047] In step (5) of the following preferred embodiment of the present invention, the potential gradient of the electrokinetic enhanced degradation is 0.2-0.6 V / cm, preferably 0.2-0.5 V / cm, more preferably 0.2 V / cm, 0.5 V / cm; the electrokinetic enhanced degradation time is 5-15 d, preferably 15 d.

[0048] The following examples serve as further illustrations of the technical solutions of the present invention.

[0049] Example 1

[0050] Preparation of immobilized enzyme:

[0051] 1) Using commercially available biochar (200 mesh, 75 μm) as the raw material, nitric acid and biochar were mixed at a liquid-to-solid ratio of 1 mL:10 g, and heated at 120°C for 2 h using a microwave hydrothermal method to obtain nitric acid-modified biochar;

[0052] 2) To a 250 mL enzyme immobilization reaction system (containing 100 mL of acetic acid-sodium acetate buffer at pH 5), chitosan, laccase (200 U / L), glutaraldehyde, nitric acid-modified biochar, and sodium tripolyphosphate were added in the order specified (Table 1). Each addition was shaken (150 rpm) for 10 minutes before the next addition. After all materials were added, the mixture was shaken at 150 rpm at 25°C for 24 hours to fully immobilize the laccase. The immobilized laccase was then collected by centrifugation at 8000 rpm for 10 minutes.

[0053] Table 1 Optimization design of biochar-immobilized laccase synthesis

[0054]

[0055]

[0056] Performance determination of immobilized laccase:

[0057] Free laccase activity was determined using the ABTS assay: the amount of laccase required to convert 1 μmol of ABTS per minute. After mixing 500 μL of 1.5 mM ABTS solution (pH 4), 2.450 mL of 0.1 M citric acid-phosphate buffer (pH 3.5), and 50 μL of enzyme solution, absorbance was recorded at 420 nm using a UV spectrophotometer. The absorbance was measured every 10 seconds for at least 90 seconds. A standard curve was then plotted to calculate enzyme activity (Equation 1).

[0058] For the activity test of the immobilized enzyme, 0.03 g of sample was added to ABTS solution (3 mL, 1.5 mM, pH = 4), reacted at 25°C and 200 rpm for 30 min, and centrifuged at 8000 rpm for 10 min. The absorbance of the supernatant was measured, and the immobilized enzyme activity was expressed in U / g.

[0059]

[0060] Where: ΔA / Δt—the slope of the linear equation for the change in absorbance over time; V0—the volume of the reaction mixture during the test, mL; ε—the absorption constant, 36 / mol·cm; b—the thickness of the cuvette, cm; V—the volume of the test solution, mL; and df—the dilution factor of the test solution.

[0061] The initial laccase activity, residual laccase activity in the supernatant, and immobilized laccase activity were measured, and the immobilization rate and effective immobilization rate of biochar-immobilized laccase were calculated (Table 2). Comprehensive analysis showed that CL5 exhibited the best laccase immobilization effect, ensuring both a high effective immobilization rate and high immobilized laccase activity.

[0062] Table 2 Immobilization rate, effective immobilization rate and activity of laccase immobilized on biochar

[0063]

[0064] The free laccase and the best immobilized enzyme (CL5) were stored at 25℃ for 35 days, and samples were taken regularly to determine their activities. Figure 1 ), it can be seen that the immobilized enzyme still maintained approximately 48% of its relative activity after 35 days of storage, while the relative activity of the free enzyme was only about 12% of its initial value. After 14 days of storage, the immobilized enzyme's relative activity reached 87.6%, meeting the requirements for effective degradation activity during the electrokinetic enhanced degradation process.

[0065] Example 2

[0066] Based on the best immobilized enzyme obtained in Example 1, the laboratory effect verification of the controllable electric field enhanced enzyme degradation method was carried out. Figure 2 The electromechanical repair system consists of a DC power supply, relays, time-controlled switches, ammeter, voltmeter, and output terminals, a digital multimeter, electrode cables, electrodes, electrode wells, electrolyte storage tanks, peristaltic pumps, and electrolyte recovery tanks. The non-uniform and symmetrical electric field working electrodes (anode and cathode) are connected to the two power output ports of the electromechanical repair system.

[0067] Artificial simulated contaminated clay soil (naphthalene content 97.4 mg / kg) was loaded into the soil chamber using the wet filling method. The soil moisture content was adjusted to 30.0% (w / w), the filling height was 8.0 cm, and the soil density was 1.72 g / cm 3 To prevent soil from entering the electrode well through the holes, the outer wall of the electrode well in contact with the soil was covered with a 100-mesh nylon mesh. The electrode (graphite material, 120 mm long and 10 mm in diameter) was installed in the electrode well (50 mm long × 100 mm wide) and connected to the output terminal of the repair device power supply (DC power supply voltage was 20 V). A sodium carbonate solution (50 mM) containing 1.0% w / v immobilized enzyme was injected into the anode well as the electrolyte (liquid level 8.0 cm), and a sodium carbonate solution (50 mM) was pumped into the cathode well as the electrolyte (liquid level 8.0 cm). The electrokinetic transport of the immobilized enzyme was started, the potential gradient was set to 2 V / cm, and the transport time was 10 h. Afterwards, the potential gradient was adjusted to 0.5 V / cm, and the electrokinetic enhanced degradation was started, which lasted for 168 h.

[0068] After the operation, the content of naphthalene (15.1-17.8 mg / kg, average content 16.7 mg / kg) in the soil at different sampling points was ( Figure 3 ) are lower than the first category land screening value (25 mg / kg) in the "Soil Environmental Quality Construction Land Soil Pollution Risk Control Standard (Trial)" (GB36600-2018).

[0069] Example 3

[0070] The field application of the controlled electric field-enhanced enzymatic degradation method was carried out. A chemical plant relic site was selected. The production history primarily included pesticides and other chemical reagents. The soil in the test area was contaminated with a combination of chloroform (7.6 mg / kg) and petroleum hydrocarbons (2286.1 mg / kg) at a depth of 2 meters.

[0071] For specific implementation methods, see Figure 4 First, electrode wells (100 mm in diameter) were constructed within the selected test area using a Geoprobe drilling rig. The wells were spaced 1.5 m apart and supported by porous PVC screens. The wells were 2 m deep. Stainless steel (tube) electrodes (DN25, 33 mm in diameter) were inserted into the wells and secured, then connected to the power output of the electrokinetic remediation control system. Sodium carbonate (100 mM) electrolyte containing immobilized enzyme was pumped into the anode well at a liquid-to-solid ratio of 100 mL:2 g. Water was used as the electrolyte in the cathode well. The potential gradient of the electric field for transporting the immobilized enzyme was 1.0 V / cm (DC power supply voltage was 150 V) for 100 h. The potential gradient for electrokinetic degradation was 0.2 V / cm (DC power supply voltage was 30 V) for 15 days.

[0072] After the operation, the contents of chloroform (0.15-0.29 mg / kg, average content 0.23 mg / kg) and petroleum hydrocarbons (729-798 mg / kg, average content 747 mg / kg) in the soil of the remediation area were ( Figure 5 ) are lower than the first category land screening values ​​in the "Soil Environmental Quality Construction Land Soil Pollution Risk Control Standard (Trial)" (GB36600-2018) (chloroform: 0.3 mg / kg, petroleum hydrocarbons C10-C40: 826 mg / kg).

[0073] Comparative Example 1

[0074] The preparation of the immobilized enzyme was the same as in Example 1, except that laccase was replaced by horseradish peroxidase.

[0075] The application example is the same as Example 3. The results show that the chloroform content in the soil of the repair area is 1.09-1.42 mg / kg, with an average content of 1.31 mg / kg; the petroleum hydrocarbon content is 1153-1316 mg / kg, with an average content of 1228 mg / kg.

[0076] Comparative Example 2

[0077] The preparation of the immobilized enzyme was the same as in Example 1, except that commercially available biochar was used as the raw material to directly prepare the immobilized enzyme without modification.

[0078] The application example is the same as Example 3. The results show that the chloroform content in the soil of the repair area is 0.38-0.56 mg / kg, with an average content of 0.42 mg / kg; the petroleum hydrocarbon content is 957-1185 mg / kg, with an average content of 1048 mg / kg.

[0079] Comparative Example 3

[0080] The preparation method of the immobilized enzyme is the same as that in Example 1.

[0081] The application example was the same as Example 3, except that the liquid-to-solid ratio of electrolyte to immobilized enzyme was 100 mL:0.4 g. The results showed that the chloroform content in the soil of the remediation area was 0.51-0.77 mg / kg, with an average of 0.61 mg / kg; the petroleum hydrocarbon content was 1206-1441 mg / kg, with an average of 1354 mg / kg.

[0082] Comparative Example 4

[0083] The preparation method of the immobilized enzyme is the same as that in Example 1.

[0084] The application example is the same as Example 3, except that the potential gradient of the electric field for transporting the immobilized enzyme is 2.0 V / cm (voltage 300 V). The results showed that the chloroform content in the soil of the remediation area was 0.43-0.65 mg / kg, with an average content of 0.53 mg / kg; the petroleum hydrocarbon content was 1038-1176 mg / kg, with an average content of 1095 mg / kg.

[0085] Comparative Example 5

[0086] The preparation method of the immobilized enzyme is the same as that in Example 1.

[0087] The application example is the same as Example 3, except that the potential gradient for electrokinetic degradation is 1.0 V / cm. The results showed that the chloroform content in the soil of the remediation area was 0.33-0.46 mg / kg, with an average content of 0.39 mg / kg; the petroleum hydrocarbon content was 875-984 mg / kg, with an average content of 906 mg / kg.

[0088] The above are merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil, characterized in that: The following steps are involved: 1) Electrode wells are set up in the repair area, and the electrode wells are arranged in a rectangular shape to form a non-uniform symmetrical electric field; 2) Insert the electrode into the electrode well, secure it, and connect it to the corresponding output terminal of the DC power supply; 3) pumping the electrolyte containing the immobilized enzyme into the anode well and the electrolyte into the cathode well; 4) Setting the potential gradient of the electric field for transporting the immobilized enzyme and starting the DC power supply; 5) After the electrokinetic transport of the immobilized enzyme is completed, the DC electric field potential gradient is adjusted to initiate electrokinetic enhanced degradation; The immobilized enzyme uses nitrate-modified biochar as a carrier and is immobilized using chitosan; the enzyme is laccase; the preparation method of the immobilized enzyme is as follows: 0.5-1.5% w / v chitosan, 100-500 U / L laccase, 0.2-0.6% v / v glutaraldehyde, 15-35% w / v nitric acid-modified biochar, and 0.1-0.5% w / v sodium tripolyphosphate are added to an acetic acid-sodium acetate buffer in this order; each time a material is added, it is shaken and mixed at a speed of 150 r / min for 10 minutes before the next material is added; after all the materials are added, it is placed in a shaker at 25° C. and 150 r / min and shaken for 24 hours to fully immobilize the laccase, and the immobilized enzyme is collected by centrifugation at 8000 r / min for 10 minutes; the preparation method of the nitric acid-modified biochar is as follows: concentrated nitric acid with a content of 68% and biochar are mixed at a liquid-solid ratio of 100 mL:10 g, and heated at 120° C. for 2 hours.

2. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: The electrode well has a pore size of 100-150 mm, the well wall is supported by a porous PVC sieve plate, and the depth of the electrode well is consistent with the depth of soil pollution.

3. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: The electrodes are made of graphite, stainless steel or titanium, with a diameter of 30-60 mm; the DC power supply has a constant voltage output, and the operating voltage range is 10-200 V.

4. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: In the electrolyte containing the immobilized enzyme, the liquid-solid ratio of the electrolyte to the immobilized enzyme is 100 mL: (0.5-2) g.

5. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: The potential gradient in step 4) is 1.0-2 V / cm.

6. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: The electrokinetic transport in step 5) lasts for 10-100 hours.

7. The controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to claim 1, characterized in that: In step 5), the potential gradient is 0.2-0.6 V / cm; and the electrokinetic enhanced degradation time is 5-15 days.

8. Use of the controllable electric field enhanced enzyme degradation method for in-situ remediation of organic contaminated soil according to any one of claims 1 to 7 in in-situ remediation of organic contaminated soil.

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