Application of self-activated Fenton-like composite repair material

Abstract

The invention relates to a Fenton-like oxidation treatment method, in particular to application of a self-activated Fenton-like composite repair material, the composite repair material is CaO2 (at) Fe3O4 (at) BIP-BC, western blot biochar is used as a matrix, and magnetite and calcium peroxide are sequentially loaded on the surface of the matrix. Compared with the prior art, the method has the advantages that the high efficiency and the long-term effect of Fenton-like are improved through biochar surface active groups and calcium peroxide, the selectivity of removing target harmful bacteria and harmful bacteria genes is improved through western blot, and the activating agent and the oxidizing agent are added at the same time, so that the use cost and the operation complexity are reduced. According to the method, the defects that traditional Fenton-like oxidation treatment is poor in harmful bacterium and harmful bacterium gene obligate removal effect, short in treatment time, high in required dosage and serious in secondary pollution, and an activating agent and an oxidizing agent need to be used separately are effectively overcome; the efficient and long-acting remediation of soil and water bodies polluted by harmful bacteria and harmful bacteria genes through the Fenton-like reaction is realized.

Description

Technical Field

[0001] The invention relates to a Fenton-like oxidation treatment method, and in particular to an application of a self-activated Fenton-like composite repair material. Background Art

[0002] With the extensive use and improper disposal of antibiotics and other antimicrobial drugs, a large number of harmful bacteria and harmful bacteria genes such as Antibiotics Resistance Bacterias (ARBs) and Antibiotics Resistance Genes (ARGs) have been produced in the environment, seriously endangering people's health. At the same time, harmful bacteria and harmful bacteria genes are widely spread in the environment through sewage or other wastes, posing a threat to the ecological safety of soil and water bodies. As a new type of pollutant, harmful bacteria and harmful bacteria genes are persistent, easy to spread, and easy to erupt. The current common physical, biochemical and chemical treatment technologies cannot effectively control the spread of harmful bacteria genes. Therefore, the development of new and efficient removal materials and technologies is of great significance to the restoration of soil and water bodies contaminated by harmful bacteria and harmful bacteria genes, and to ensure the ecological safety of the soil and water environment.

[0003] Fenton-like oxidation treatment is a mature technology for treating organic pollutants in soil and groundwater. It is now also gradually being used to remove harmful bacteria and harmful bacteria genes in soil and water environments. Commonly used activators in Fenton-like oxidation treatment include divalent iron, trivalent iron, zero-valent iron, magnetite, etc. Commonly used oxidants include hydrogen peroxide, persulfate, periodate, etc. For example, a study used pyrite (FeS2) to activate periodate (PI) to disinfect ARB. The results showed that the FeS2 / PI system can remove 1×10 7 CFU mL -1 Disinfection of kanamycin-resistant Escherichia coli to below the detection limit. However, traditional Fenton-like oxidation treatment methods often have problems such as poor reactivity, low utilization rate, and Fe(III) / Fe(II) cycle limitations in practical applications. At the same time, the oxidant and activator of the traditional Fenton-like method need to be added separately, which increases the operational complexity of the method. It is necessary to develop Fenton-like materials that can self-activate in water and soil environments to reduce the difficulty of Fenton-like operations. More importantly, the current oxidation effect of Fenton-like materials has a broad spectrum. While removing harmful bacteria and harmful bacteria genes, it will also inactivate beneficial bacteria in the environment. This problem can be solved by improving the material's specific removal effect on harmful bacteria and harmful bacteria genes. In order to solve the above problems, it is necessary to develop a self-activating Fenton-like composite remediation material that has high efficiency and long-term treatment, and can selectively remove harmful bacteria and harmful bacteria genes in water and soil environments. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a self-activated Fenton-like composite repair material in order to solve at least one of the above problems. This solution improves the efficiency and long-term effectiveness of Fenton-like by (1) biochar surface active groups and calcium peroxide, (2) protein imprinting improves the selectivity of removing target harmful bacteria and harmful bacteria genes, and (3) biochar is used as a carrier to load calcium peroxide and magnetite at the same time, so as to achieve the simultaneous addition of activators and oxidants, thereby reducing the use cost and operation complexity. By utilizing the above technical innovations, the problems of poor selective removal of harmful bacteria and harmful genes by traditional Fenton-like oxidation treatment methods, short treatment time, high required dosage, serious secondary pollution, and the need to use activators and oxidants separately are solved. The present invention realizes the efficient and long-term repair of soil and water bodies contaminated by target harmful bacteria and harmful bacteria genes through Fenton-like reactions. Even if the biochar, iron oxide, calcium oxide and other substances in the materials after the reaction remain in the environment, they will not have a significant impact on the properties of soil and water bodies, and have good ecological and environmental safety.

[0005] The inventors of this application have found in their research for many years that calcium peroxide is more stable than hydrogen peroxide, and is insoluble in water, has a certain sustained release effect, and is a good choice for a long-acting oxidant. Magnetite is relatively common in the natural environment and contains Fe 2+ , Fe 3+ , has strong oxidant activation performance, but it is easy to agglomerate when it exists alone, resulting in limited effect. Biochar has a good pore structure and is an excellent carrier. At the same time, the surface of biochar contains rich redox functional groups, which is beneficial to Fe 2+ / Fe 3+ The cyclic process promotes the efficiency of the Fenton reaction. Protein imprinting technology has good biocompatibility and adsorption selectivity for target bacteria. After protein imprinting modification of biochar, the selective adsorption of biochar to target harmful bacteria can be improved, which is conducive to improving the specific oxidation removal effect of harmful bacteria and the harmful bacterial genes contained in the self-activated Fenton-like material based on biochar carrier. Therefore, the preparation of a self-catalytic Fenton composite material with protein imprinted biochar as a carrier and calcium peroxide as a slow-release oxygen agent has strong feasibility and potential application value for achieving selective and efficient long-term removal of harmful bacterial genes in water and soil environments.

[0006] Based on the above theoretical analysis, the purpose of the present invention is achieved through the following technical solutions:

[0007] Application of a self-activated Fenton-like composite repair material, which is CaO2@Fe3O4@BIP-BC, with protein-imprinted biochar as a matrix, and magnetite as an activator and calcium peroxide as a slow-release oxidant sequentially loaded on the surface, for treating harmful bacteria and harmful bacteria genes in the environment;

[0008] The protein imprinted biochar is obtained by combining the cell proteins of target harmful bacteria on the surface of biochar through protein recognition molecular imprinting technology, the magnetite is loaded on the surface of the protein imprinted biochar by coprecipitation, and the calcium peroxide is loaded on the surface of the magnetite by coprecipitation;

[0009] The composite repair material reacts with water in the environment through calcium peroxide, slowly releasing H2O2 and Fe 2+ / Fe 3+ A Fenton-like reaction occurs, achieving the inactivation of harmful bacteria and degradation of harmful bacteria genes in an aerobic environment.

[0010] The self-activated Fenton-like composite remediation material is used to treat harmful bacteria and harmful bacteria genes in the environment, and is particularly used to treat antibiotic-resistant bacteria and antibiotic-resistant genes in soil and groundwater.

[0011] The composite material firstly adsorbs the target harmful bacteria or harmful bacteria genes specifically on the surface of biochar through protein imprinting, and then the calcium peroxide in the material reacts with the water in the environment, and the slowly released H2O2 reacts with the Fe in the magnetite. 2+ / Fe 3+ A Fenton-like reaction occurs, thereby utilizing the generated active oxygen free radicals to inactivate the target harmful bacteria and degrade the harmful bacteria genes.

[0012] Preferably, in the CaO2@Fe3O4@BIP-BC, the mass ratio of Fe3O4 to BIP-BC is 12-1:6, and the particle size is 0.2-2.5 mm.

[0013] Preferably, the CaO2@Fe3O4@BIP-BC is prepared by the following steps:

[0014] S1: Preparation of biochar: The crushed biomass material is pyrolyzed by an oxygen-limited slow heating method, and after cooling, it is ground and sieved, and the sieved powder is soaked in an HCl solution, and then washed to obtain biochar;

[0015] S2: Preparation of protein-imprinted biochar: inoculate target harmful bacteria in LB medium containing protease, shake culture and centrifuge to obtain precipitate, redissolve the precipitate in lysis extraction buffer and heat, centrifuge again to obtain supernatant, add DNase and RNase to the supernatant and mix evenly, let stand in ice water to remove nucleic acid impurities, add acidified acetone-ethanol mixture to the supernatant and let stand in ice water to precipitate protein, collect the precipitate by centrifugation and wash, remove methanol by vacuum filtration and resuspend the precipitate in sample solubilization buffer, add biochar obtained in step S1 to the resuspended solution, mix and wash, and dry to obtain protein-imprinted biochar BIP-BC;

[0016] S3: Fe3O4@BIP-BC was prepared by coprecipitation method, and the specific steps were as follows: ferric chloride solution and ferrous chloride solution were prepared respectively, the protein imprinted biochar obtained in step S2 was dispersed in the ferric chloride solution, and after a certain contact time, the ferrous chloride solution was added thereto, and then an excess of ammonia solution was added and stirred continuously, the precipitate was separated and washed, and the Fe3O4@BIP-BC particles were obtained after freeze-drying;

[0017] S4: The preparation of CaO2@Fe3O4@BIP-BC is carried out by co-precipitation method, and the specific steps are: prepare calcium chloride solution, disperse the Fe3O4@BIP-BC particles obtained in step S3 in the calcium chloride solution, then add neutralizer and stabilizer and stir evenly, add hydrogen peroxide to the mixed solution, separate and wash the precipitate, and obtain the composite repair material after freeze-drying.

[0018] Preferably, in step S1:

[0019] The biomass material includes straw, sawdust, peel and other residues; the pyrolysis process is: preheating at 100°C for 1h in a nitrogen atmosphere, then heating to 300-700°C at 20°C / min for 2h; the sieving uses a 100-mesh screen; the concentration of the HCl solution is 10-15wt%; the soaking time is 12-24h;

[0020] In step S2:

[0021] The target harmful bacteria include but are not limited to Gram-negative bacteria such as Escherichia coli and Gram-positive bacteria such as Staphylococcus aureus; the LB medium containing protease is an LB medium with a trypsin concentration of 0.5-2.5%; the shaking culture is a continuous shaking culture at 37°C at a speed of 150-180rpm for 8-10 hours; the lysis and extraction buffer is pH=7, 20mM HEPES, 2-5wt% sodium dodecyl sulfate (SDS) and a mixture of protease inhibitors, the precipitate is redissolved in lysis extraction buffer at 10μL / mg wet cell weight; the heating is heated at 95°C for 3-5 minutes; the amount of DNase added is 1mg / mL, and the amount of RNase added is 0.5mg / mL; the concentration of the acidified acetone-ethanol mixture is 1.5mM; the sample solubilization buffer is a mixture of 8-10M urea, 2-4M thiazolinone, 2-4wt% CHAPS, 1-2wt% TCEP-HCl and 2-5% v / v carrier ampholytes; the biochar and the wet weight of the bacteria in the resuspension are added in equal weight;

[0022] In step S3:

[0023] Ferric chloride: protein imprinted biochar: ferrous chloride = 0.324g: 0.8g: 0.126g; the ammonia solution is added in excess; the contact time is 30-60min, and the continuous stirring time is 1h;

[0024] In step S4:

[0025] Calcium chloride: Fe3O4@BIP-BC particles: stabilizer: hydrogen peroxide = 1.078g: 0.3g: 5mL: 5mL; the neutralizer is ammonia water to adjust the reaction environment to be alkaline, and the stabilizer is PEG200.

[0026] Preferably, the CaO2@Fe3O4@BIP-BC is implemented by deep soil in-situ injection-high pressure rotary spraying method, comprising the following steps:

[0027] The grouting pipe is inserted into the deepest part of the contaminated soil, and the composite repair material is mixed with water at a certain solid-liquid ratio, and then sprayed into the contaminated soil along the rotary spraying grouting pipe under the action of a high-pressure grouting pump and compressed air. The grouting pipe is lifted upward at a certain interval to cover all the contaminated depths to repair the soil. Due to the huge pressure and large spraying range of the high-pressure rotary spraying, the composite repair material can fully contact with the contaminated soil, achieving the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the soil.

[0028] Preferably, the dosage of the composite repair material is 0.1-2.0% of the dry weight of the contaminated soil, the solid-liquid ratio of the composite repair material and water is 1:10-1:1000, the lifting spacing of the grouting pipe is 0.5-2.5m, the rotary spraying pressure is 0.5-2.5MPa, and the rotary spraying angle is 120-350°.

[0029] Preferably, the CaO2@Fe3O4@BIP-BC is implemented by an in-situ tillage method of the surface soil, comprising the following steps:

[0030] The composite repair material is evenly spread on the surface of the contaminated soil, and the soil is tilled to mix the composite repair material and the contaminated soil evenly to carry out soil repair treatment. During the repair process, the soil must have a certain moisture content to promote the release of hydrogen peroxide by contacting calcium peroxide with water. Through the self-activated Fenton-like effect and physical adsorption of the composite repair material, the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the surface soil is achieved.

[0031] Preferably, the amount of composite repair material applied is 0.05-2.5% of the dry weight of the contaminated soil, the tillage depth is 15-50 cm, the moisture content of the contaminated soil is 40-80% of the maximum field water holding capacity, and the treatment time is 10-20 days.

[0032] Preferably, the CaO2@Fe3O4@BIP-BC is implemented by gravity dosing and / or pressure dosing, comprising the following steps:

[0033] (a) Gravity addition method: The composite repair material is added into the water pump suction pipe or the water suction bell mouth of the suction well by gravity in a certain proportion, and the composite repair material and the polluted water are mixed evenly by the water pump impeller to repair the water body. The harmful bacteria and harmful bacteria genes in the water environment are removed through the adsorption effect of the composite repair material on harmful bacteria and harmful bacteria genes and the self-activated Fenton-like effect;

[0034] (b) Pressure addition method: After the composite repair material is mixed with water in a certain proportion, it is added into the water pipe or sewage pool passing through the polluted water body by a water pump or water ejector and mixed evenly to carry out water body repair treatment. The harmful bacteria and harmful bacteria genes in the water environment are removed through the adsorption effect of the composite repair material on harmful bacteria and harmful bacteria genes and the self-activated Fenton-like effect.

[0035] Preferably, the dosage of the composite repair material is 15-20 mg / L, and the hydraulic retention time is 2-3 h.

[0036] Preferably, the CaO2@Fe3O4@BIP-BC is mixed in water or water-containing soil, wherein the dosage of Fe3O4@BIP-BC is controlled to be 10-40 mg / L, and / or the concentration of CaO2 is 0.5-3 mM.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] In the common Fenton-like oxidation treatment method, the oxidant and activator need to be added to the water or soil environment separately, and there is no selectivity, and the bacteria in the environment are indiscriminately inactivated. There are also disadvantages such as poor contact between the activator and the oxidant, resulting in low reaction efficiency, easy to produce secondary pollution, short reaction time and difficulty in controlling the reaction effect.

[0039] The self-activated Fenton-like material prepared by the present invention is different from other Fenton-like oxidation treatment methods. The material can utilize the selective adsorption capacity of protein-imprinted biochar, the slow-release oxidation effect of calcium peroxide, and the activation effect of magnetite to innovatively achieve the purpose of self-catalytic slow release of active oxygen free radicals by the repair material, effectively improving the efficiency, long-term effectiveness and operability of Fenton-like advanced oxidation technology, and its selective adsorption of target bacteria reduces the impact on environmentally beneficial microorganisms during the repair process. It has the advantages of long action period, good treatment effect, stable structure, low cost, easy use and environmental friendliness:

[0040] 1) Biochar contains abundant oxygen-containing functional groups and π-π structures, which make it have a strong electron transfer effect and can accelerate the Fe 3+ / Fe 2+ The circulation rate is increased to improve the inactivation effect of the Fenton-like reaction on harmful bacteria and the oxidative degradation effect of harmful bacterial genes.

[0041] 2) The slow release of H2O2 by calcium peroxide gives the repair material a Fenton-like long-term effect, with a longer reaction time and no need for secondary addition.

[0042] 3) Use protein imprinting method to modify biochar so that it has selective adsorption effect on target harmful bacteria, improve its removal selectivity for target harmful bacteria and harmful bacteria genes in the water and soil environment, so as to reduce the killing effect of Fenton-like oxidation on beneficial bacteria in the water and soil environment.

[0043] 4) Calcium peroxide and magnetite were loaded on the protein-imprinted biochar in sequence, producing a self-activated Fenton-like reaction, so that the activator and oxidant could be added simultaneously when the repair material was used, solving the problem of the traditional Fenton-like reaction where the two agents had to be added separately, and reducing the cost of the material and the complexity of operation.

[0044] More specifically:

[0045] (1) The present invention uses biochar as a matrix to load magnetite as an activator, calcium peroxide as a H2O2 slow-release agent, and a two-step coprecipitation method to first load magnetite on the surface of biochar, and then add polyethylene glycol PEG200 as a protective agent, add ammonia water to create an alkaline environment to neutralize the HCl produced by the reaction, and load CaO2 on the material surface through coprecipitation to prepare a self-activated Fenton-like composite repair material, thereby innovatively achieving the purpose of self-catalytic generation of active free radicals by the repair material. It can solve the current problem that the oxidant and the activator need to be prepared and used separately, effectively improve the operability of Fenton-like advanced oxidation technology, and reduce the difficulty of actual repair work.

[0046] (2) Common methods for removing harmful bacteria and harmful genes lack selectivity and also pose a threat to beneficial bacteria in the environment, destroying the microbial diversity in the environment. Protein imprinting technology combines bacterial fragments with biochar to achieve selective adsorption of target harmful bacteria and reduce oxidative damage to beneficial microorganisms in the environment.

[0047] (3) Fenton-like oxidation treatment methods for removing harmful bacteria and harmful genes have been used, but are often limited by Fe 2+ / Fe 3+ Cycle step. In the present invention, biochar is used as a matrix to load magnetite for activating hydrogen peroxide, and the biochar not only activates hydrogen peroxide in the reaction, but also is beneficial to Fe 2+ / Fe3+ The method can be used to control and remove harmful bacteria and harmful genes in the environment, which has not been reported in previous research and development of pollution remediation materials. At the same time, the material preparation method is simple to operate, has low raw material cost, and no secondary pollution, and has strong practical application prospects.

[0048] (4) The current Fenton-like oxidation treatment method has a fast reaction rate. If harmful bacteria and harmful genes need to be effectively removed, the amount of oxidant added is large, while the reaction time is short, resulting in a high risk of resurrection of harmful bacteria. Calcium peroxide slowly reacts with water in the environment to produce hydrogen peroxide, thereby producing a continuous oxidizing environment, which can effectively remove harmful bacteria and reduce the risk of resurrection.

[0049] (5) The protein imprinting and two-step co-precipitation method used in the present invention is used to prepare the protein imprinting self-activated Fenton-like composite material. The overall preparation method is simple and feasible, and the raw materials used are cheap, easily available, environmentally friendly, and suitable for mass production. It has great application potential in the field of soil and groundwater remediation contaminated by harmful bacteria and harmful genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the adsorption isotherm of Fe3O4@BIP-BC and Fe3O4@BC in Example 2;

[0051] Figure 2 is the adsorption kinetics of Fe3O4@BIP-BC and Fe3O4@BC in Example 2;

[0052] Figure 3 is a competitive adsorption test in Example 2, wherein a) the mono-system competitive adsorption of Fe3O4@BIP-BC and Fe3O4@BC, and b) the binary system competitive adsorption of Fe3O4@BIP-BC;

[0053] Figure 4 Example 3 shows the inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria at different Fe3O4 to BIP-BC mass ratios;

[0054] Figure 5 The inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria at different CaO2 concentrations in Example 3;

[0055] Figure 6 The inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria at different Fe3O4@BIP-BC dosages in Example 3;

[0056] Figure 7 shows the inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria under different water matrix conditions in Example 4, including a) different HA concentrations, b) different pH, and c) different salinities. DETAILED DESCRIPTION

[0057] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] Unless otherwise specified in the following examples, the reagents used are conventional commercially available products, and the methods used are conventional means in the art.

[0059] All the contents in the following examples are percentage by mass or concentration by mass.

[0060] Example 1

[0061] A method for preparing a self-activated Fenton-like composite repair material for selectively removing harmful bacteria and harmful bacteria genes, the specific steps are as follows:

[0062] (I) Raw material pretreatment and preparation of protein-imprinted biochar

[0063] (1) Preparation and pretreatment of biochar:

[0064] (i) The collected corn stalk biomass material was washed, cut into small pieces of 3-5 cm, and dried at 75°C. The dried biomass material was crushed with a grinder, put into a crucible for compaction, covered and placed in a muffle furnace, and pyrolyzed by oxygen-limited slow heating method under nitrogen atmosphere. The pyrolysis process was preheated at 100°C for 1h, and then heated to 500°C for constant temperature pyrolysis for 2h. After cooling to room temperature, it was ground in a mortar, passed through a 100-mesh sieve, and placed in a brown wide-mouth bottle, sealed and stored for later use.

[0065] (ii) the initial corn biochar powder was soaked in HCl solution (10.0 wt%) for 12 h, the supernatant was discarded after standing, the powder was washed with deionized water, and then the sample was dried for later use;

[0066] (2) Preparation of protein-imprinted biochar: 100 mL of LB medium solution containing 0.5-2.5% trypsin was prepared according to the published ratio, inoculated with Escherichia coli HB101 bacteria, and 50 ug / mL of streptomycin was added to the medium, followed by overnight shaking culture at 37°C and 180 rpm. The cultured solution was centrifuged at 6000-8000 rpm for 5 min, the supernatant was discarded, and the precipitate was re-dissolved in lysis extraction buffer (pH = 7, 20 mM HEPES, 5 wt% sodium dodecyl sulfate (SDS) and a mixture of protease inhibitors) at a ratio of 10 μL / mg wet cell weight, and heated at 95°C for 5 minutes. Centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was separated from the cell material. DNase (1 mg / mL) and RNase (0.5 mg / mL) were added to the collected supernatant, mixed evenly with a vortex oscillator, and allowed to stand in ice water for 15 minutes to remove nucleic acid impurities. Then, 1.5 mM of acidified acetone-ethanol mixture was added to the supernatant and then allowed to stand in ice water for 30 minutes to precipitate the protein. The precipitate was collected by centrifugation at 10,000 rpm for 20 minutes at 4°C, washed three times with methanol, and the methanol was removed by rapid vacuum treatment. The precipitate was resuspended in a sample solubilization buffer (10 M urea, 4 M thiazolinone, 4 wt% CHAPS, 2 wt% TCEP-HCl, and 5% v / v carrier ampholyte 3-10 NL). The biochar obtained in step S1 was added to the resuspended solution, and then the mixture was mixed at 180 rpm for 16 hours and washed three times with physiological saline, and finally dried to obtain protein imprinted biochar BIP-BC for use.

[0067] (II) Preparation of self-activated Fenton-like composite repair materials

[0068] (1) Preparation of Fe3O4@BIP-BC material by coprecipitation: Fe3O4@BIP-BC was prepared by coprecipitation. The specific operation method is as follows: 0.435 g of ferric chloride was dissolved in 20 mL of deionized water, and 1.2 g of protein-imprinted biochar was dispersed in the ferric chloride solution for 30 min. Then 0.126 g of ferrous chloride was weighed at a molar ratio of 2:1, dissolved in 20 mL of deionized water and added to the ferric chloride solution, and then 10 mL of ammonia solution was quickly added to the solution. After stirring for 1 h, the precipitate was washed with deionized water and anhydrous ethanol, and then dried using a freeze dryer to obtain Fe3O4@BIP-BC particles with a mass ratio of 1:4, which were stored at room temperature for use.

[0069] (2) Co-precipitation loading of CaO2@Fe3O4@BIP-BC material: CaO2@Fe3O4@BIP-BC was prepared by co-precipitation method. The specific operation method is as follows: 3.75g of CaCl2 particles were weighed and dissolved in deionized water, 1.5g of Fe3O4@BIP-BC particles were dispersed in the CaCl2 solution, and 15mL of ammonia water and 15mL of polyethylene glycol PEG200 were added after stirring for 15min. Ammonia water can neutralize the HCl produced by the reaction, and polyethylene glycol PEG200 is mainly used as a stabilizer. After mixing evenly, 15mL of H2O2 solution was slowly added dropwise. It can be observed that the solution gradually changed from black to black-gray, indicating that a co-precipitation reaction occurred and a CaO2@Fe3O4@BIP-BC composite material loaded with calcium peroxide was formed. The suspension was centrifuged and washed with anhydrous ethanol for multiple times. After being dried in a freeze dryer, a CaO2@Fe3O4@BIP-BC material with a mass ratio of 8:1:4 was obtained and stored at room temperature for use.

[0070] Example 2

[0071] The Fe3O4@BIP-BC particles (carrying protein imprinting) prepared in Example 1 and the separately prepared Fe3O4@BC particles (without protein imprinting) were used to test the adsorption performance of target harmful bacteria.

[0072] The preparation method of Fe3O4@BC particles is basically the same as that of Fe3O4@BIP-BC particles, except that no apparent protein blotting (i.e., sub-step (2) in step (1)) is performed during the preparation of Fe3O4@BC particles. CaO2 is not added to prevent the occurrence of a Fenton-like reaction that inactivates bacteria and affects the accuracy of the test results of adsorption performance.

[0073] (1) Adsorption isotherm

[0074] The Fe3O4@BIP-BC material and the Fe3O4@BC material were mixed with E. coli HB101 bacterial solution at a concentration of 0.2 mg / mL in a 200 mL system and mixed in an incubator for 2 hours. The initial OD600 of the bacterial suspension was 0.02, 0.04, 0.06, 0.08, 0.1 and 0.12. The bacteria-nanoparticle complex was separated under the action of a magnetic field, and the concentration of E. coli HB101 cells in the supernatant was detected.

[0075] (2) Adsorption kinetics

[0076] The Fe3O4@BIP-BC material and the Fe3O4@BC material were mixed with the E. coli HB101 solution with an initial OD600 of 0.1 in a 200 mL system at a concentration of 0.2 mg / mL and mixed in an incubator for 2 hours. Samples were taken from the mixed system at 0, 20, 40, 60, 80, 100, and 120 minutes, and the bacteria-nanoparticle complex was separated under the action of a magnetic field, and the concentration of E. coli HB101 cells in the supernatant was detected.

[0077] (3) Adsorption selectivity

[0078] In order to study the adsorption selectivity of Fe3O4@BIP-BC material and Fe3O4@BC material, Staphylococcus aureus, Shewanella CN32 and Escherichia coli DH5α were selected as competitive bacteria.

[0079] First, adsorption experiments of Escherichia coli HB101 bacteria and competing bacteria were carried out using a single bacterial solution to explore the adsorption selectivity of Fe3O4@BIP-BC material and Fe3O4@BC material; in short, 20 mg of Fe3O4@BIP-BC and Fe3O4@BC were added to two 100 mL bacterial solutions (OD600 = 0.1), respectively, and after mixing at 180 rpm for 2 hours at 25°C, the supernatant was taken to count the bacteria in the solution, and the amount of bacteria adsorbed by the material was calculated.

[0080] Staphylococcus aureus, Shewanella CN32 and Escherichia coli DH5α were selected as competitive cells to study the adsorption selectivity of Fe3O4@BIP-BC in a binary system. In short, 20 mg of Fe3O4@BIP-BC was added to a bacterial solution containing the same concentration of Escherichia coli HB101 and competitive bacteria (OD600 = 0.1). After the mixture was placed in a shaking incubator at 25°C and 180 rpm for 2 hours, the remaining bacteria in the supernatant were counted. The impact of competitive bacteria was evaluated by the displacement of target bacteria, and the calculation method is as follows:

[0081] Substitution rate = (HB101 adsorption amount - HB101 adsorption amount during competitive adsorption) / HB101 adsorption amount.

[0082] The adsorption test results are as follows Figure 1 -3 shows that the protein-imprinted biochar material has a higher adsorption capacity for the target bacteria than the original biochar. Combined with the results of the competitive adsorption experiment, it can be found that the adsorption capacity of the protein-imprinted biochar for the target bacteria is much higher than that of other bacteria, and when other bacteria are present, the adsorption of the material to the target bacteria is almost unaffected.

[0083] Example 3

[0084] 1) The effect of the CaO2@Fe3O4@BIP-BC material prepared in Example 1 on the treatment of harmful bacteria and harmful bacteria genes in water bodies using composite remediation materials under different activator ratios (Fe3O4:BIP-BC).

[0085] The composite repair material was prepared substantially according to the steps of Example 1.

[0086] The initial concentration was set to 10 6 100mL of Escherichia coli HB101 solution with a CFU / mL was placed in 5 250mL glass conical bottles, and composite materials were prepared in sequence according to the Fe3O4:BIP-BC mass ratio of 2:1, 1:1, 1:2, 1:4, and 1:6. The dosage of Fe3O4@BIP-BC was controlled to 30mg / L, and the CaO2 concentration in the 200mL reaction system was 1mM. After the repair material and the contaminated water sample were fully contacted and mixed evenly, the mixed system was placed in a shaking incubator and shaken at 25℃ and 180rpm for 3h. Water samples were taken at fixed time points to calculate the number of residual bacteria in the water body and the removal rate. The results are as follows Figure 4 As shown. Figure 4 It can be seen that different proportions of activator materials have different removal rates for Escherichia coli HB101, among which the best effects are achieved when the ratio of Fe3O4:BIP-BC is 1:4 and 1:6. In actual engineering use, the optimal ratio should be determined in combination with the material preparation and application costs.

[0087] 2) The effect of the CaO2@Fe3O4@BIP-BC material prepared in Example 1 on the effect of the composite repair material at different CaO2 concentrations in treating harmful bacteria and harmful bacteria genes in water bodies.

[0088] The composite repair material was prepared substantially according to the steps of Example 1.

[0089] The initial concentration was set to 10 6 100mL of Escherichia coli HB101 solution with CFU / mL was placed in 5 250mL glass conical bottles, and the composite materials were prepared in sequence according to the Fe3O4:BIP-BC mass ratio of 1:4, where the dosage of Fe3O4@BIP-BC was controlled at 30mg / L, and the CaO2 concentration in the 200mL reaction system was 0.5, 1, 2, and 3mM, respectively. After the repair material and the contaminated water sample were fully contacted and mixed evenly, the mixed system was placed in a shaking incubator and shaken at 25℃ and 180rpm for 3h. Water samples were taken at fixed time points to calculate the number of residual bacteria in the water body and the removal rate. The results are as follows Figure 5 As shown. Figure 5It can be seen that different CaO2 concentrations have different removal rates for Escherichia coli HB101, among which the best effect is achieved when the CaO2 concentration is 1mM. In actual engineering use, the optimal ratio should be determined in combination with material preparation and application costs.

[0090] 3) The effect of the CaO2@Fe3O4@BIP-BC material prepared in Example 1 on the effect of the composite repair material on treating harmful bacteria and harmful bacteria genes in water bodies at different Fe3O4@BIP-BC dosages.

[0091] The composite repair material was prepared substantially according to the steps of Example 1.

[0092] The initial concentration was set to 10 6 100mL of Escherichia coli HB101 solution with CFU / mL was placed in 5 250mL glass conical bottles, and composite materials were prepared in sequence according to the Fe3O4:BIP-BC mass ratio of 1:4. The dosage of Fe3O4@BIP-BC was controlled to 10, 20, 30, and 40mg / L, respectively, so that the CaO2 concentration in the 200mL reaction system was 1mM. After the repair material and the contaminated water sample were fully contacted and mixed evenly, the mixed system was placed in an oscillating incubator at 25℃ and 180rpm for 3h. Water samples were taken at fixed time points to calculate the number of residual bacteria in the water body and the removal rate. The results are as follows Figure 6 As shown. Figure 6 It can be seen that different Fe3O4@BIP-BC dosages have different removal rates for Escherichia coli HB101, among which the best effect is achieved when the Fe3O4@BIP-BC dosage is 30 mg / L and 40 mg / L. In actual engineering use, the optimal ratio should be determined in combination with material preparation and application costs.

[0093] Example 4

[0094] The inactivation performance of the CaO2@Fe3O4@BIP-BC material prepared in Example 1 against harmful bacteria under different water matrix conditions was investigated, and the bacterial solution configuration was referenced to Example 1.

[0095] In the experiment considering the effect of solution pH, harmful bacteria inactivation experiments were conducted by adding HCl or NaOH to adjust the initial solution pH to 3, 5, 7, and 9;

[0096] In the experiment considering the effect of solution salinity, the solution ionic strength was adjusted by adding different amounts of NaCl suspension into the reaction system, and harmful bacteria inactivation experiments were carried out under ionic concentrations of 0mM, 15mM, 75mM, 150mM, and 300mM.

[0097] To determine the effect of symbiotic humic acid (HA), HA was added to the disinfection system, and harmful bacteria removal experiments were carried out under the conditions of final HA concentrations of 0, 0.1, 1, and 10 mg / L.

[0098] The test results are shown in Figure 7. The results show that pH has little effect on the bacterial inactivation effect. The inactivation rate is slow when the pH is high, which may be because the reaction of CaO2 and water is inhibited when the pH is high; HA concentration has a great influence on the bacterial inactivation effect. When the HA concentration is high, it competes with bacteria and the bacterial inactivation effect is poor; salinity has little effect on bacterial inactivation. The bacterial inactivation rate is different under different salinity conditions, but the bacteria are eventually inactivated.

[0099] Application Example 1

[0100] Deep soil in-situ injection - high pressure rotary jet method:

[0101] The grouting pipe is inserted into the deepest part of the contaminated soil layer, and the self-activated Fenton-like composite repair material of Example 1 is mixed with water at a certain solid-liquid ratio and then sprayed into the contaminated deep soil along the rotary spraying grouting pipe under the action of a high-pressure grouting pump and compressed air. At the same time, the grouting pipe with a nozzle is gradually lifted upward at a certain interval to ensure that the material can cover all the contaminated depths. Due to the huge pressure and large spraying range of the high-pressure rotary spraying, the composite repair material can fully contact with the contaminated soil, achieving the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the soil.

[0102] Among them, the dosage of composite repair material is controlled at 0.1-2.0% of the dry weight of contaminated soil, the solid-liquid ratio of material and water in the injection slurry is controlled at 1:10-1:1000, the initial insertion depth of the grouting pipe is consistent with the maximum depth of the contaminated soil, the lifting spacing of the grouting pipe can be 0.5-2.5m, the rotary spraying pressure can be 0.5-2.5MPa, and the rotary spraying angle can be 120-350°.

[0103] In this application example, the dosage of composite repair material is controlled at 1.0% of the dry weight of the contaminated soil, the solid-liquid ratio of material and water in the injection slurry is controlled at 1:100, the initial insertion depth of the grouting pipe is consistent with the maximum depth of the contaminated soil, the lifting spacing of the grouting pipe can be 1.0m, the rotary spraying pressure can be 2.0MPa, and the rotary spraying angle can be 270°.

[0104] Application Example 2

[0105] In-situ tillage of topsoil:

[0106] The self-activated Fenton-like composite remediation material of Example 1 is evenly spread on the surface of the contaminated soil, and then the soil is plowed to evenly mix the remediation material and the soil. During the remediation process, the soil must have a certain moisture content to promote the release of hydrogen peroxide by contact between calcium peroxide and water. The purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the surface soil is achieved through the self-activated Fenton-like effect and physical adsorption effect of the material.

[0107] The amount of composite repair material applied is controlled to be 0.05-2.5% of the dry weight of the contaminated soil, the tillage depth of the soil is generally 15-50 cm, the soil moisture content is adjusted to 40-80% of the maximum field water holding capacity after the composite repair material is applied, and the treatment time of the repair material is preferably 10-20 days.

[0108] In this application example, the amount of composite repair material applied is controlled to 1.5% of the dry weight of the contaminated soil, the soil plowing depth is about 20-30 cm, and the soil moisture content is adjusted to about 60% of the maximum field water holding capacity after the composite repair material is applied. The treatment time of the repair material is preferably 15 days.

[0109] Application Example 3

[0110] Gravity dosing method:

[0111] The self-activated Fenton-like material of Example 1 is added into the water suction pipe of a water pump or at the water suction bell of a water suction well in a certain proportion by gravity, and the agent and the polluted water are mixed evenly by the water pump impeller. The harmful bacteria and harmful bacteria genes in the water environment are removed through the adsorption effect of the material on harmful bacteria and harmful bacteria genes and the self-activated Fenton-like effect.

[0112] Among them, the dosage of the agent is 15-20 mg / L, and the hydraulic retention time is 2-3h.

[0113] In this application example, the dosage of the agent is 18 mg / L and the hydraulic retention time is 2.5 h.

[0114] Application Example 4

[0115] Pressure dosing method:

[0116] After the self-activated Fenton-like agent of Example 1 is mixed with water in a certain proportion, the agent is added into a water pipe or a sewage pool using a water pump or a water ejector. The agent is then evenly mixed with the polluted water in the water pipe or the pool, and the harmful bacteria and harmful bacteria genes in the water environment are removed through the adsorption effect of the material on harmful bacteria and harmful bacteria genes and the self-activated Fenton-like effect.

[0117] Among them, the dosage of the agent is 15-20 mg / L, and the hydraulic retention time is 2-3h.

[0118] In this application example, the dosage of the agent is 16 mg / L and the hydraulic retention time is 3 h.

[0119] In summary, the present invention uses biochar material with good adsorption capacity as the matrix, combines protein imprinting technology with co-precipitation method to prepare a self-activated Fenton-like composite remediation material with magnetite as the activator and calcium peroxide as the slow-release oxidant, which is used to remove harmful bacteria and harmful genes in water / soil environments:

[0120] Calcium peroxide reacts slowly with the water in the environment, slowly releasing H2O2, and maintaining an oxidative environment for a long time to completely inactivate harmful bacteria and degrade harmful genes.

[0121] Simultaneously loading calcium peroxide and magnetite on biochar enables the one-time addition of the activator and oxidant, simplifies the operating steps of traditional Fenton-like treatment, improves application efficiency, and reduces raw material and labor costs.

[0122] The protein-imprinted biochar can selectively adsorb target harmful bacteria in the environment and promote Fe 3+ Reduction to improve the efficiency of the Fenton reaction.

[0123] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An application of a self-activated Fenton-like composite repair material, characterized in that: The composite repair material is CaO2@Fe3O4@BIP-BC, which uses protein-imprinted biochar as a matrix, and magnetite as an activator and calcium peroxide as a slow-release oxidant are sequentially loaded on the surface to treat harmful bacteria and harmful bacteria genes in the environment. The protein imprinted biochar is obtained by combining the cell proteins of target harmful bacteria on the surface of biochar through protein recognition molecular imprinting technology, the magnetite is loaded on the surface of the protein imprinted biochar by coprecipitation, and the calcium peroxide is loaded on the surface of the magnetite by coprecipitation; The composite repair material reacts with water in the environment through calcium peroxide, slowly releasing H2O2 and Fe 2+ / Fe 3+ A Fenton-like reaction occurs, which inactivates harmful bacteria and degrades their genes.

2. The use of a self-activated Fenton-like composite repair material according to claim 1, characterized in that: In the CaO2@Fe3O4@BIP-BC, the mass ratio of Fe3O4 to BIP-BC is 12-1:6, and the particle size is 0.2-2.5 mm.

3. The use of a self-activated Fenton-like composite repair material according to claim 1, characterized in that: The CaO2@Fe3O4@BIP-BC is prepared by the following steps: S1: Preparation of biochar: The biomass material crushed to 100-120 mesh was pyrolyzed by oxygen-limited slow heating method, and then ground and sieved after cooling. The sieved powder was immersed in HCl solution, filtered by using a 0.45 μm filter membrane, and the powder trapped on the filter paper was washed with deionized water for 3-5 times to obtain washed biochar; S2: Preparation of protein-imprinted biochar: inoculate target harmful bacteria in LB medium containing protease, shake culture and centrifuge to obtain precipitate, redissolve the precipitate in lysis extraction buffer and heat, centrifuge again to obtain supernatant, add DNase and RNase to the supernatant and mix evenly, let stand in ice water to remove nucleic acid impurities, add acidified acetone-ethanol mixture to the supernatant and let stand in ice water to precipitate protein, collect the precipitate by centrifugation and wash, remove methanol by vacuum filtration and resuspend the precipitate in sample solubilization buffer, add biochar obtained in step S1 to the resuspended solution, mix and wash, and dry to obtain protein-imprinted biochar BIP-BC; S3: Fe3O4@BIP-BC was prepared by coprecipitation method, and the specific steps were as follows: ferric chloride solution and ferrous chloride solution were prepared respectively, the protein imprinted biochar obtained in step S2 was dispersed in the ferric chloride solution, and after a certain contact time, the ferrous chloride solution was added thereto, and then an excess of ammonia solution was added and stirred continuously, the precipitate was separated and washed, and the Fe3O4@BIP-BC particles were obtained after freeze-drying; S4: The preparation of CaO2@Fe3O4@BIP-BC is carried out by co-precipitation method, and the specific steps are: prepare calcium chloride solution, disperse the Fe3O4@BIP-BC particles obtained in step S3 in the calcium chloride solution, then add neutralizer and stabilizer and stir evenly, add hydrogen peroxide to the mixed solution, separate and wash the precipitate, and obtain the composite repair material after freeze-drying.

4. The use of a self-activated Fenton-like composite repair material according to claim 3, characterized in that: In step S1: The pyrolysis process is as follows: preheating at 100°C for 1 hour in a nitrogen atmosphere, and then heating to 300-700°C for constant temperature pyrolysis for 2 hours; the sieving is performed using a 100-mesh screen; the concentration of the HCl solution is 10-15wt%; the soaking time is 12-24 hours; In step S2: The target harmful bacteria include Gram-negative bacteria and Gram-positive bacteria; the LB medium containing protease is an LB medium with a trypsin concentration of 0.5-2.5%; the shaking culture is a continuous shaking culture at 37°C at a speed of 150-180rpm for 8-10 hours; the lysis and extraction buffer is pH=7, 20mM HEPES, 2-5wt% sodium dodecyl sulfate and a mixture of protease inhibitors, the precipitate is redissolved in a lysis extraction buffer at 10μL / mg wet cell weight; the heating is heated at 95°C for 3-5 minutes; the amount of DNase added is 1mg / mL, and the amount of RNase added is 0.5mg / mL; the concentration of the acidified acetone-ethanol mixture is 1.5mM; the sample solubilization buffer is a mixture of 8-10M urea, 2-4M thiazolinone, 2-4wt% CHAPS, 1-2wt% TCEP-HCl and 2-5% v / v carrier ampholyte 3-10NL; the biochar and the wet weight of the bacteria in the resuspension are added in equal weight; In step S3: Ferric chloride: protein imprinted biochar: ferrous chloride = 0.324g: 0.8g: 0.126g; the contact time is 30-60min, and the continuous stirring time is 1h; In step S4: Calcium chloride: Fe3O4@BIP-BC particles: stabilizer: hydrogen peroxide = 1.078 g: 0.3 g: 5 mL: 5 mL; the neutralizer is ammonia water, and the stabilizer is PEG200.

5. The use of a self-activated Fenton-like composite repair material according to claim 1, characterized in that: The CaO2@Fe3O4@BIP-BC is implemented by deep soil in-situ injection-high pressure rotary spraying method, which includes the following steps: Insert the grouting pipe into the deepest part of the contaminated soil, mix the composite repair material and water in a certain solid-liquid ratio and then spray it into the contaminated soil. Lift the grouting pipe upward at a certain interval to cover all contaminated depths and carry out soil repair.

6. The use of a self-activating Fenton-like composite repair material according to claim 5, characterized in that: The dosage of composite repair material is 0.1-2.0% of the dry weight of contaminated soil, the solid-liquid ratio of composite repair material and water is 1:10-1:1000, the lifting spacing of grouting pipe is 0.5-2.5m, the rotary spraying pressure is 0.5-2.5MPa, and the rotary spraying angle is 120-350°.

7. The use of a self-activated Fenton-like composite repair material according to claim 1, characterized in that: The CaO2@Fe3O4@BIP-BC is implemented by in-situ tillage of the surface soil, including the following steps: The composite repair material is evenly spread on the surface of the contaminated soil, and the soil is tilled to make the composite repair material and the contaminated soil evenly mixed to carry out soil repair treatment.

8. The use of a self-activated Fenton-like composite repair material according to claim 7, characterized in that: The application rate of composite repair material is 0.05-2.5% of the dry weight of contaminated soil, the tillage depth is 15-50cm, the moisture content of contaminated soil is 40-80% of the maximum field water holding capacity, and the treatment time is 10-20d.

9. The use of a self-activated Fenton-like composite repair material according to claim 1, characterized in that: The CaO2@Fe3O4@BIP-BC is implemented by gravity dosing and / or pressure dosing, and includes the following steps: (a) Gravity addition method: Use gravity to add the composite repair material in a certain proportion into the water pump suction pipe or the water suction bell mouth of the suction well, and use the water pump impeller to mix the composite repair material and the polluted water evenly to repair the water body; (b) Pressure addition method: After the composite repair material is mixed with water in a certain proportion, a water pump or water ejector is used to add the composite repair material into a water pipe or sewage pool passing through the polluted water body and mix it evenly to carry out water body repair treatment.

10. The use of a self-activated Fenton-like composite repair material according to claim 9, characterized in that: The dosage of composite repair material is 15-20 mg / L, and the hydraulic retention time is 2-3 h.

Images