Application of a self-activated fenton-like composite repair material

Abstract

The application relates to a Fenton-like oxidation treatment method, in particular to application of a self-activation Fenton-like composite repair material, the composite repair material being CaO2@Fe3O4@BIP-BC, and taking protein imprinting biochar as a matrix, and magnetite and calcium peroxide being sequentially loaded on the surface. Compared with the prior art, the application has the advantages that 1) the biochar surface active groups and calcium peroxide improve the high efficiency and long-acting property of the Fenton-like, 2) the protein imprinting improves the selectivity of the target harmful bacteria and harmful bacteria gene removal, 3) the activation agent and the oxidant are simultaneously added, and the use cost and operation complexity are reduced. The method effectively solves the problems of the traditional Fenton-like oxidation treatment, such as poor specific removal effect on harmful bacteria and harmful bacteria genes, short treatment time, high required dosage, serious secondary pollution and the need of separate use of the activation agent and the oxidant, and realizes high efficiency and long-acting repair of soil and water bodies polluted by harmful bacteria and harmful bacteria genes through the Fenton-like reaction.

Description

TECHNICAL FIELD

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

[0002] With the large use and improper disposal of antibiotics and other antibacterial drugs, a large amount of harmful bacteria such as Antibiotics Resistance Bacterias (ARBs) and Antibiotics Resistance Genes (ARGs) and harmful bacteria genes are generated in the environment, which seriously endanger people's health. At the same time, the harmful bacteria and harmful bacteria genes are widely spread in the environment through sewage or other waste, which also threatens the ecological safety of soil and water. As a new type of pollutant, the harmful bacteria and harmful bacteria genes have the characteristics of persistence, easy spread and easy outbreak. The current common physical, biological and chemical treatment technologies cannot effectively control the spread and diffusion of harmful bacteria genes. Therefore, it is of great significance to develop new high-efficiency removal materials and technologies to realize the repair of soil and water contaminated by harmful bacteria and harmful bacteria genes and to protect the ecological safety of water and soil environment.

[0003] The Fenton-like oxidation treatment method is a mature soil and groundwater organic pollutant treatment technology, which is also gradually used for the removal of harmful bacteria and harmful bacteria genes in the water and soil environment. Common activators in the Fenton-like oxidation treatment include divalent iron, trivalent iron, zero-valent iron, magnetite and the like, and common oxidants include hydrogen peroxide, persulfate, periodate and the like. For example, a study uses pyrite (FeS2) to activate periodate (PI) to disinfect ARBs, and the results show that the FeS2 / PI system can disinfect 1x10 7 CFU mL -1 Kanamycin-resistant E. coli is disinfected to below the detection limit. However, the traditional Fenton-like oxidation treatment method often has problems such as poor reactivity, low utilization rate, Fe(III) / Fe(II) cycle limitation and the like in actual application. At the same time, the oxidant and the activator of the traditional Fenton-like method need to be added separately, which increases the operation complexity of the method, and a self-activated Fenton-like material needs to be developed to reduce the operation difficulty of the Fenton-like method. More importantly, the oxidation of the current Fenton-like material has broad spectrum, which not only removes harmful bacteria and harmful bacteria genes, but also causes inactivation of beneficial bacteria in the environment, and this problem can be solved by improving the specific removal effect of the material on harmful bacteria and harmful bacteria genes. In order to solve the above problems, it is necessary to develop a self-activated Fenton-like composite repair material which has high efficiency and long-term effect and can selectively remove harmful bacteria and harmful bacteria genes in the water and soil environment. SUMMARY

[0004] The present application aims to solve at least one of the above problems by providing an application of a self-activated Fenton-like composite repair material. The present application improves the efficiency and long-term effect of Fenton-like by (1) the surface active groups of biochar and calcium peroxide, (2) the protein imprinting improves the selectivity of removing target harmful bacteria and harmful bacteria genes, (3) biochar as a carrier simultaneously loads calcium peroxide and magnetite, realizes the simultaneous addition of activator and oxidant, reduces the use cost and operation complexity. By using the above technical innovation, the problems of poor selectivity of harmful bacteria and harmful gene removal, short treatment time, high required dose, serious secondary pollution, and the need to use activator and oxidant separately in the traditional Fenton-like oxidation treatment method are solved. The present application realizes efficient and long-term repair of soil and water bodies contaminated by target harmful bacteria and harmful bacteria genes through Fenton-like reaction. The biochar, iron oxide and calcium oxide and other substances in the material after the reaction will not have a great impact on the properties of soil and water even if they remain in the environment, and have good ecological and environmental safety.

[0005] The present application inventors found in years of research that calcium peroxide is more stable than hydrogen peroxide and is difficult to dissolve in water, has a certain slow-release effect, and is a good long-acting oxidant. Magnetite is common in natural environment and contains Fe 2+ , Fe 3+ , has strong oxidant activation performance, but is prone to agglomeration when alone, which limits the effect. Biochar has good pore structure and is an excellent carrier. The surface of biochar contains rich redox functional groups, which is beneficial to the Fe 2+ / Fe 3+ cycle process and promotes the efficiency of Fenton reaction. Protein imprinting technology has good biocompatibility and adsorption selectivity for target bacteria. After protein imprinting modification of biochar, the selectivity of biochar for target harmful bacteria can be improved, which is beneficial to improve the specific oxidation removal effect of self-activated Fenton-like material based on biochar carrier on harmful bacteria and harmful bacteria genes contained therein. Therefore, it has strong feasibility and potential application value to prepare a self-catalytic Fenton-like composite material using protein imprinting biochar as a carrier and calcium peroxide as a slow-release oxidant to realize selective and efficient long-term removal of harmful bacteria genes in water and soil environment.

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

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

[0008] The protein-imprinted biochar is obtained by imprinting the cell protein of the target harmful bacteria on the surface of the biochar through a protein-imprinting technology, the magnetite is loaded on the surface of the protein-imprinted biochar through a coprecipitation method, and the calcium peroxide is loaded on the surface of the magnetite through the coprecipitation method.

[0009] The composite repair material releases H2O2 and Fe 2+ / Fe 3+ to generate a Fenton-like reaction, so as to realize the inactivation of the harmful bacteria and the degradation of the harmful bacteria genes in an aerobic environment.

[0010] The self-activated Fenton-like composite repair material is used for treating the harmful bacteria and the harmful bacteria genes in the environment, and is especially used for treating the antibiotic-resistant bacteria and the antibiotic resistance genes in the soil and the underground water.

[0011] The composite material first specifically adsorbs the target harmful bacteria or harmful bacteria genes on the surface of the biochar through protein imprinting, and then slowly releases H2O2 and Fe 2+ / Fe 3+ to generate a Fenton-like reaction, so as to realize the inactivation of the target harmful bacteria and the degradation of the harmful bacteria genes by using the generated active oxygen free radicals.

[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 through the following steps:

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

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

[0016] S3: Preparation of Fe3O4@BIP-BC, which is performed by a co-precipitation method, and the specific steps are as follows: iron chloride solution and ferrous chloride solution are prepared respectively, the protein imprinting biochar obtained in step S2 is dispersed in the iron chloride solution, after a certain period of contact, the ferrous chloride solution is added, then an excess of ammonia water solution is added and continuous stirring is performed, the precipitate is separated and washed, and the Fe3O4@BIP-BC particles are obtained after freeze-drying;

[0017] S4: Preparation of CaO2@Fe3O4@BIP-BC, which is performed by a co-precipitation method, and the specific steps are as follows: calcium chloride solution is prepared, the Fe3O4@BIP-BC particles obtained in step S3 are dispersed in the calcium chloride solution, then a neutralizing agent and a stabilizer are added and stirred uniformly, hydrogen peroxide is added dropwise into the mixture, the precipitate is separated and washed, and the composite repair material is obtained after freeze-drying.

[0018] Preferably, in step S1:

[0019] The biomass material includes straw, sawdust, fruit peels, and other residues; the pyrolysis process is as follows: under a nitrogen atmosphere, preheating at 100℃ for 1h, then heating at 20℃ / min to 300-700℃ for constant temperature pyrolysis 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 culture medium containing protease is an LB culture medium with a trypsin concentration of 0.5-2.5%; the shaking culture is continuous shaking culture at 37℃ at a speed of 150-180rpm for 8-10 hours; the lysis extraction buffer is a mixture of pH=7, 20mM HEPES, 2-5wt% sodium dodecyl sulfate (SDS), and protease inhibitors, and the precipitate is resuspended in the lysis extraction buffer at a wet cell weight of 10μL / mg; the heating is at 95℃ for 3-5 minutes; the DNase is added at a concentration of 1mg / mL, and the RNase is added at a concentration of 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; the biochar and the bacteria in the resuspension are added in equal weight;

[0022] In step S3:

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

[0024] In step S4:

[0025] Calcium chloride: Fe3O4@BIP-BC particles: stabilizer: hydrogen peroxide = 1.078g: 0.3g: 5mL: 5mL; the neutralizing agent is ammonia water, the reaction environment is adjusted 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 jet method, including the following steps:

[0027] The grouting pipe is inserted into the deepest part of the contaminated soil, and the composite remediation material is uniformly mixed with water at a certain solid-liquid ratio, and then is injected into the contaminated soil along the rotary jet grouting pipe under the action of high-pressure grouting pump and compressed air. The grouting pipe is lifted upward at a certain interval to cover all the pollution depth, and the soil remediation treatment is carried out. The composite remediation material can fully contact with the contaminated soil due to the huge pressure and large jet range of high-pressure rotary jet, so as to achieve the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the soil.

[0028] Preferably, the composite remediation material is added in an amount of 0.1-2.0% of the dry weight of the contaminated soil, the solid-liquid ratio of the composite remediation material and water is 1:10-1:1000, the lifting interval of the grouting pipe is 0.5-2.5m, the rotary jet pressure is 0.5-2.5MPa, and the rotary jet angle is 120-350°.

[0029] Preferably, the CaO2@Fe3O4@BIP-BC is implemented by surface soil in-situ plowing method, including the following steps:

[0030] The composite remediation material is uniformly spread on the surface of the contaminated soil, and plowing is carried out to uniformly mix the composite remediation material with the contaminated soil, so as to carry out soil remediation treatment. During the remediation process, the soil should have a certain water content to promote the contact between calcium peroxide and water to release hydrogen peroxide, so as to achieve the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the surface soil through the self-activation Fenton-like effect and physical adsorption effect of the composite remediation material.

[0031] Preferably, the composite remediation material is spread in an amount of 0.05-2.5% of the dry weight of the contaminated soil, the plowing depth is 15-50cm, the water content of the contaminated soil is 40-80% of the maximum field water holding capacity, and the treatment time is 10-20d.

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

[0033] (a) Gravity feeding method: the composite repair material is fed into the water suction pipe or the water suction horn of the water suction well in a certain proportion by gravity, the composite repair material is mixed uniformly with the contaminated water body by the water pump impeller, the water body is repaired and treated, and the harmful bacteria and harmful bacteria genes in the water environment are removed by the adsorption and self-activation Fenton effect of the composite repair material;

[0034] (b) Pressure feeding method: the composite repair material is mixed with water in a certain proportion, and then the composite repair material is fed into the water pipe or sewage tank through the contaminated water body by using a water pump or a water jet and is mixed uniformly, the water body is repaired and treated, and the harmful bacteria and harmful bacteria genes in the water environment are removed by the adsorption and self-activation Fenton effect of the composite repair material.

[0035] Preferably, the amount 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 addition amount 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 application has the following beneficial effects:

[0038] In the common Fenton-like oxidation treatment method, the oxidant and the activator need to be added into the water or soil environment respectively, and there is no selectivity, and the bacteria in the environment are inactivated without distinction, and there are the disadvantages of low reaction efficiency, easy secondary pollution, short reaction time, and difficult control of reaction effect caused by poor contact effect of the activator and the oxidant.

[0039] The self-activation Fenton material prepared in the present application is different from other Fenton oxidation treatment methods, and the material can utilize the selective adsorption capacity of the protein blotting biochar, the slow-release oxidation effect of calcium peroxide, and the activation effect of magnetite, and innovatively realize the purpose of self-catalytic slow release of active oxygen free radicals of the repair material, effectively improve the efficiency, long-acting and operability of the Fenton advanced oxidation technology, and the selective adsorption of the target bacteria reduces the influence of the beneficial microorganisms in the environment in the repair process, and has the advantages of long action period, good treatment effect, stable structure, low cost, convenient use, and environmental friendliness:

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

[0041] 2) The repair material has Fenton-like long-acting effect by the slow-release H2O2 effect of calcium peroxide, and the reaction time is longer, without the need for secondary addition.

[0042] 3) The biochar is modified by Western blotting method, so that it has selective adsorption effect on target harmful bacteria, improves the removal selectivity of target harmful bacteria and harmful bacteria genes in water and soil environment, and reduces the killing effect of Fenton-like oxidation on beneficial bacteria in water and soil environment.

[0043] 4) Calcium peroxide and magnetite are sequentially loaded on the Western blotting biochar to produce self-activated Fenton-like reaction, so that the activator and oxidant can be added at the same time when the repair material is used, the problem that the traditional Fenton-like reaction needs to add the two separately is solved, and the use cost and operation complexity of the material are reduced.

[0044] More specifically:

[0045] (1) The present application loads magnetite on biochar as an activator, and loads calcium peroxide as an H2O2 slow-release agent, and uses a two-step co-precipitation method to first load magnetite on the surface of biochar, and then adds polyethylene glycol PEG200 as a protective agent, and adds ammonia water to create an alkaline environment to neutralize the generated HCl, and co-precipitates CaO2 on the surface of the material to prepare a self-activated Fenton-like composite repair material, thereby innovatively realizing the purpose of self-catalyzing the production of active free radicals. The problem that the oxidant and the activator need to be prepared and used separately can be solved, the operability of Fenton-like advanced oxidation technology is effectively improved, and the difficulty of actual repair work is reduced.

[0046] (2) The common harmful bacteria and harmful gene removal method lacks selectivity, and also threatens the beneficial bacteria in the environment, destroys the microbial diversity in the environment, and the Western blotting technology realizes selective adsorption of target harmful bacteria by combining bacterial fragments with biochar, and reduces the oxidative damage to beneficial microorganisms in the environment.

[0047] (3) The method for removing harmful bacteria and harmful genes by Fenton-like oxidation treatment has been applied, but it is often limited by the Fe 2+ / Fe 3+ cycling step. The present application loads magnetite on biochar to activate hydrogen peroxide, and the biochar not only has an activating effect on hydrogen peroxide, but also is beneficial to the cycling of Fe 2+ / Fe3+ The Fenton reaction efficiency is improved by circulation, and the method is used for control and removal of harmful bacteria and harmful genes in the environment, which has not been reported in the previous pollution remediation material research and development work. Meanwhile, the material preparation method is simple to operate, the raw material cost is low, there is no secondary pollution, and has strong practical application prospect.

[0048] (4) The current Fenton-like oxidation treatment method has a fast reaction rate, if the harmful bacteria and harmful genes need to be effectively removed, the oxidant dosage is large, and the reaction time is short, which leads to a high risk of revival and regeneration of harmful bacteria, and the calcium peroxide slowly reacts to generate hydrogen peroxide after contacting with water in the environment, thereby generating a sustained oxidation environment, which can effectively remove harmful bacteria and reduce the risk of revival and regeneration.

[0049] (5) The protein blot and two-step co-precipitation method are used to prepare the protein blot self-activation Fenton-like composite material, the overall preparation method is simple and feasible, the raw materials are cheap and easy to obtain, environment-friendly, and suitable for batch production, and has great application potential in the field of harmful bacteria and harmful gene contaminated soil and groundwater remediation. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Adsorption isotherms of Fe3O4@BIP-BC and Fe3O4@BC in Example 2;

[0051] Figure 2 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) is a competitive adsorption of a unary system of Fe3O4@BIP-BC and Fe3O4@BC, and b) is a competitive adsorption of a binary system of Fe3O4@BIP-BC;

[0053] Figure 4 In Example 3, the inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria under different mass ratios of Fe3O4 and BIP-BC;

[0054] Figure 5 In Example 3, the inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria under different CaO2 concentrations;

[0055] Figure 6 In Example 3, the inactivation effect of CaO2@Fe3O4@BIP-BC on harmful bacteria under different Fe3O4@BIP-BC dosages;

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

[0057] The present application will be described in detail below with reference to the accompanying drawings and specific examples.

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

[0059] In the following examples, the mass percentage or mass concentration is used.

[0060] Example 1

[0061] A preparation method of 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 blotting biochar

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

[0064] (i) The collected corn straw biomass material was washed, cut into small pieces of 3-5 cm, dried at 75°C, and then ground into powder using a grinder. The dried biomass material was then loaded into a crucible, compacted, covered, and placed in a muffle furnace under a nitrogen atmosphere. The slow oxygen-limited heating method was used for pyrolysis treatment. The pyrolysis process was first preheated at 100°C for 1 h, then heated to 500°C for 2 h. After cooling to room temperature, the material was ground in a mortar and sieved through a 100-mesh screen. The sieved material was then sealed in a brown jar and stored for later use.

[0065] (ii) The initial corn biochar powder obtained was first soaked in HCl solution (10.0 wt%) for 12 h. After standing, the supernatant was discarded, and the powder was washed with deionized water. The sample was then dried and ready for use.

[0066] (2) Preparation of protein blotting biochar: 100 mL LB medium solution containing trypsin at a concentration of 0.5-2.5% was prepared according to the published proportion, E. coli HB101 bacteria was inoculated, 50 ug / mL of streptomycin was added to the medium, and then it was placed in a 37°C, 180 rpm condition for overnight shaking culture. After the culture, the solution was centrifuged at 6000-8000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended in lysis extraction buffer (pH = 7, 20 mM HEPES, 5 wt% sodium dodecyl sulfate (SDS) and a mixture of protease inhibitors) at a proportion of 10 μL / mg wet cell weight, and heated at 95°C for 5 min. Centrifuge at 4000 rpm for 15 min at 4°C, separate the supernatant from the cell material. After adding DNase (1 mg / mL) and RNase (0.5 mg / mL) to the collected supernatant, mix well with a vortex mixer, and stand in ice water for 15 min to remove nucleic acid impurities. Then add 1.5 mM acidified acetone-ethanol mixture to the supernatant and stand in ice water for 30 min to precipitate the protein, centrifuge at 10000 rpm for 20 min at 4°C to collect the precipitate, wash with methanol three times, remove the methanol by rapid vacuum treatment, and resuspend the precipitate in 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). Add the biochar obtained in step S1 to the resuspension, then mix the mixture at 180 rpm for 16 h, wash with normal saline three times, and finally dry to obtain protein blotting biochar BIP-BC for standby.

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

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

[0069] (2) Coprecipitation loading of CaO2@Fe3O4@BIP-BC: CaO2@Fe3O4@BIP-BC was prepared by coprecipitation method. The specific operation method is as follows: 3.75 g of CaCl2particles were weighed and dissolved in deionized water, 1.5 g of Fe3O4@BIP-BC particles were dispersed in the CaCl2solution, after stirring for 15 min, 15 mL of ammonia water and 15 mL of polyethylene glycol PEG200 were added, the ammonia water can neutralize the HCl produced in the reaction, and the polyethylene glycol PEG200 is mainly used as a stabilizer. After mixing evenly, slowly add 15 mL of H2O2solution, it can be observed that the solution gradually changes from black to black gray, indicating that the coprecipitation reaction has occurred, forming a CaO2@Fe3O4@BIP-BC composite material loaded with calcium peroxide. The suspension was centrifuged and washed with anhydrous ethanol several times, and then dried in a freeze dryer to obtain CaO2@Fe3O4@BIP-BC material with a mass ratio of 8:1:4, which was stored at room temperature for use.

[0070] Example 2

[0071] The Fe3O4@BIP-BC particles (carrying protein imprint) prepared in Example 1 and the Fe3O4@BC particles (without protein imprint) prepared 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, the difference is that the apparent protein imprint in the preparation process of Fe3O4@BC particles is not performed (i.e. sub-step (2) in step (1)). CaO2 is not added to prevent the occurrence of Fenton-like reaction to inactivate the bacteria and affect the accuracy of the test results of the adsorption performance.

[0073] (1) Adsorption isotherm

[0074] Fe3O4@BIP-BC material and 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 h. 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] Fe3O4@BIP-BC material and Fe3O4@BC material were mixed with E. coli HB101 bacterial solution with initial OD600 of 0.1 at a concentration of 0.2 mg / mL in a 200 mL system, and mixed in an incubator for 2 h. At 0, 20, 40, 60, 80, 100, 120 min, samples were taken from the mixed system, and the bacterial-nanoparticle complex was separated under the action of a magnetic field. The concentration of E. coli HB101 cells in the supernatant was detected.

[0077] (3) Adsorption selectivity

[0078] To study the adsorption selectivity of Fe3O4@BIP-BC material and Fe3O4@BC material, Staphylococcus aureus, Shewanella CN32 and E. coli DH5a were selected as competitive bacteria.

[0079] First, a single bacterial solution was used to perform adsorption experiments of E. coli HB101 bacteria and competitive bacteria to explore the adsorption selectivity of Fe3O4@BIP-BC material and Fe3O4@BC material. Briefly, 20 mg of Fe3O4@BIP-BC and Fe3O4@BC were added to two bacterial solutions (OD600 = 0.1) of 100 mL, mixed at 25°C and 180 rpm for 2 hours, and then the supernatant was taken to count the bacteria in the solution and calculate the amount of bacteria adsorbed by the material.

[0080] Staphylococcus aureus, Shewanella CN32 and E. coli DH5a were selected as competitive cells to study the adsorption selectivity of Fe3O4@BIP-BC in a binary system. Briefly, 20 mg of Fe3O4@BIP-BC was added to a bacterial solution containing E. coli HB101 and competitive bacteria (OD600 = 0.1) at the same concentration. The mixture was placed in a shaking incubator at 25°C and 180 rpm for 2 hours, and then the remaining bacteria in the supernatant were counted. The effect of competitive bacteria was evaluated by displacement of target bacteria, and the calculation method was as follows:

[0081] Replacement rate = (HB101 adsorption amount - HB101 adsorption amount when competitive adsorption) / HB101 adsorption amount.

[0082] The results of the adsorption experiment are shown in Table Figure 1 -3, which shows that the protein-imprinted biochar material has a higher adsorption amount of target bacteria than the original biochar. Combined with the results of the competitive adsorption experiment, it can be found that the protein-imprinted biochar has a much higher adsorption amount of target bacteria than other bacteria, and the adsorption of target bacteria by the material is almost not affected in the presence of other bacteria.

[0083] Example 3

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

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

[0086] 100 mL of E. coli HB101 bacterial solution with an initial concentration of 10 6 CFU / mL was respectively placed in 5 250 mL glass conical flasks, and the composite material was prepared according to the Fe3O4:BIP-BC mass ratio of 2:1, 1:1, 1:2, 1:4, and 1:6. The Fe3O4@BIP-BC dosage was controlled at 30 mg / L, and the CaO2 concentration in the 200 mL reaction system was 1 mM. After the repair material and the contaminated water sample were fully contacted and uniformly mixed, the mixed system was placed in a shaking incubator at 25°C and 180 rpm for 3 h. The residual bacteria in the water body were calculated at the fixed time point, and the removal rate was calculated. The results are shown in Figure 4 As can be seen from Figure 4 , different ratios of activator materials have different removal rates for E. coli HB101, and the effect is best when the Fe3O4:BIP-BC ratio is 1:4 and 1:6. In actual engineering use, the best ratio should be determined by considering the material preparation and application cost.

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

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

[0089] 100 mL of E. coli HB101 bacterial solution with an initial concentration of 10 6 CFU / mL was respectively placed in 5 250 mL glass conical flasks, and the composite material was prepared according to the Fe3O4:BIP-BC mass ratio of 2:1, 1:1, 1:2, 1:4, and 1:6. The Fe3O4@BIP-BC dosage was controlled at 30 mg / L, and the CaO2 concentration in the 200 mL reaction system was 1 mM. After the repair material and the contaminated water sample were fully contacted and uniformly mixed, the mixed system was placed in a shaking incubator at 25°C and 180 rpm for 3 h. The residual bacteria in the water body were calculated at the fixed time point, and the removal rate was calculated. The results are shown in Figure 5 As can be seen from Figure 5It is known that different CaO2 concentrations have different removal rates for Escherichia coli HB101, with the best effect achieved when the CaO2 concentration is 1 mM. In actual engineering applications, the optimal ratio should be determined by considering the material preparation and application costs.

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

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

[0092] Each was configured with an initial concentration of 10 6 100 mL of CFU / mL *E. coli* HB101 bacterial suspension was divided into five 250 mL Erlenmeyer flasks. Composite materials were prepared sequentially using a Fe3O4:BIP-BC mass ratio of 1:4, with Fe3O4@BIP-BC dosages successively controlled at 10, 20, 30, and 40 mg / L, ensuring a CaO2 concentration of 1 mM in the 200 mL reaction system. After thorough mixing of the remediation material with the contaminated water sample, the mixture was placed in a shaking incubator at 25°C and 180 rpm for 3 hours. Water samples were taken at fixed time points to calculate the number of residual bacteria and the removal rate. The results are as follows: Figure 6 As shown. By Figure 6 It can be seen that different dosages of Fe3O4@BIP-BC have different removal rates for Escherichia coli HB101. The best results were achieved when the dosages of Fe3O4@BIP-BC were 30 mg / L and 40 mg / L. In actual engineering applications, the optimal ratio should be determined by considering the 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 aqueous substrate conditions was investigated. The bacterial solution preparation was as described in Example 1.

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

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

[0097] To determine the effect of the symbiotic humic acid (HA), the HA was added into the disinfection system, and the harmful bacteria removal experiment was carried out under the condition that the final concentration of the HA was 0, 0.1, 1 and 10 mg / L respectively.

[0098] The test results are shown in Figure 7, and the results show that the pH has little effect on the bacteria inactivation effect, the inactivation speed is slow when the pH is high, and the reaction of CaO2 with water is inhibited when the pH is high; the HA concentration has a great effect on the bacteria inactivation effect, the bacteria inactivation effect is poor when the HA concentration is high because the bacteria compete with each other; the salinity has little effect on the bacteria inactivation effect, the bacteria inactivation rate is different under different salinity conditions, but the bacteria are inactivated finally.

[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 remediation material of Example 1 is mixed with water at a certain solid-liquid ratio, and then is sprayed into the contaminated deep soil under the action of the high-pressure grouting pump and compressed air along the rotary jet grouting pipe, and the grouting pipe with nozzles is gradually lifted at a certain interval to ensure that the material can cover all the pollution depth. The composite remediation material can fully contact with the contaminated soil due to the high pressure and large jet range of the high-pressure rotary jet, so as to achieve the purpose of in-situ removal of harmful bacteria and harmful bacteria genes in the soil.

[0102] In the application example, the composite remediation material is added in an amount of 1.0% of the dry weight of the contaminated soil, the solid-liquid ratio of the material and water in the injection slurry is controlled to be 1:100, the initial insertion depth of the grouting pipe is consistent with the maximum depth of the contaminated soil, the lifting interval of the grouting pipe can be 1.0 m, the rotary jet pressure can be 2.0 MPa, and the rotary jet angle can be 270°.

[0103] In the application example, the composite remediation material is added in an amount of 1.0% of the dry weight of the contaminated soil, the solid-liquid ratio of the material and water in the injection slurry is controlled to be 1:100, the initial insertion depth of the grouting pipe is consistent with the maximum depth of the contaminated soil, the lifting interval of the grouting pipe can be 1.0 m, the rotary jet pressure can be 2.0 MPa, and the rotary jet angle can be 270°.

[0104] Application Example 2

[0105] Surface soil in-situ plowing method:

[0106] The self-activated Fenton-like composite repair material of Example 1 is uniformly applied on the surface of the contaminated soil, and then the soil is plowed to mix the repair material with the soil uniformly. During the repair process, the soil is ensured to have a certain moisture content to promote the contact of calcium peroxide with water to release hydrogen peroxide, so as to achieve the purpose of removing harmful bacteria and harmful bacteria genes in the surface soil in situ through the self-activated Fenton-like effect and physical adsorption effect of the material.

[0107] In the application example, the application amount of the composite repair material is controlled to be 1.5% of the dry weight of the contaminated soil, the plowing depth of the soil is about 20-30 cm, the moisture content of the soil after the application of the composite repair material is adjusted to about 60% of the maximum field water holding capacity, and the treatment time of the repair material is preferably 15 days.

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

[0109] Application Example 3

[0110] Gravity addition method:

[0111] The self-activated Fenton-like material of Example 1 is added into the water suction pipe of the water pump or the water suction horn of the water suction well by gravity in a certain proportion. The impeller of the water pump is used to mix the reagent with the contaminated water uniformly. Through the adsorption effect and the self-activated Fenton-like effect of the material on harmful bacteria and harmful bacteria genes, the harmful bacteria and harmful bacteria genes in the water environment are removed.

[0112] In the application example, the dosage of the reagent is 18 mg / L, and the hydraulic retention time is 2.5 h.

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

[0114] Application Example 4

[0115] Pressure addition method:

[0116] The self-activated Fenton-like reagent of Example 1 is mixed with water in a certain proportion, and then the reagent is added into the water pipe or sewage pool by using a water pump or a water jet. Subsequently, the reagent is mixed with the contaminated water uniformly in the water pipe or the sewage pool. Through the adsorption effect and the self-activated Fenton-like effect of the material on harmful bacteria and harmful bacteria genes, the harmful bacteria and harmful bacteria genes in the water environment are removed.

[0117] In the application example, the dosage of the reagent is 18 mg / L, and the hydraulic retention time is 2.5 h.

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

[0119] In summary, the self-activated Fenton composite repair material prepared by using the biochar material with good adsorption capacity as the matrix, combining the Western blotting technology and the co-precipitation method, and taking the magnetite as the activator and the calcium peroxide as the slow-release oxidant, is used for removing the harmful bacteria and harmful genes in the water / soil environment.

[0120] The calcium peroxide slowly reacts with the moisture in the environment to slowly release H2O2, and the oxidation environment is maintained for a long time to completely inactivate the harmful bacteria and degrade the harmful genes.

[0121] The calcium peroxide and the magnetite are simultaneously loaded on the biochar to realize one-time addition of the activator and the oxidant, simplify the operation steps of the traditional Fenton treatment, improve the application efficiency, and reduce the raw material and labor costs.

[0122] The biochar modified by the Western blotting serves as the matrix material, can selectively adsorb the target harmful bacteria in the environment, and promotes the reduction of Fe 3+ to improve the Fenton reaction efficiency.

[0123] The above description of the embodiments is for facilitating the understanding and use of the application by the ordinary skilled in the art. The person skilled in the art can obviously easily make various modifications to the embodiments, and apply the general principles described herein to other embodiments without the need for creative labor. Therefore, the application is not limited to the above embodiments, and the improvements and modifications made by the person skilled in the art according to the disclosure of the application without departing from the scope of the application should be within the protection scope of the application.

Claims

1. Application of a self-activated Fenton-like composite repair material, characterized in that, The composite repair material is CaO2@Fe3O4@BIP-BC, which is prepared by loading magnetite as an activator and calcium peroxide as a slow-release oxidant on the surface of protein imprinting biochar as a substrate, and is used for treating harmful bacteria and harmful bacteria genes in the environment. The protein imprinting biochar is obtained by combining the cell protein of target harmful bacteria on the surface of biochar through a protein recognition molecular imprinting technology, the magnetite is loaded on the surface of the protein imprinting biochar through a coprecipitation method, and the calcium peroxide is loaded on the surface of the magnetite through a coprecipitation method; wherein, the biochar is prepared by the following method: biomass material crushed to 100-120 mesh is pyrolyzed by a limited oxygen slow heating method, and after cooling, grinding and sieving, the sieved powder is soaked in an HCl solution, 0.45 μm filter membrane is used for suction filtration, and the powder intercepted on the filter paper is washed with deionized water for 3-5 times to obtain the washed biochar; the protein imprinting biochar is prepared by the following method: target harmful bacteria are inoculated in an LB medium containing protease, and after oscillation culture, the precipitate is obtained by centrifugation, the precipitate is resuspended in a lysis extraction buffer and heated, and the supernatant is obtained by centrifugation again, DNase and RNase are added to the supernatant and mixed uniformly, and nucleic acid impurities are removed by standing in ice water, acidified acetone-ethanol mixed solution is added to the supernatant to precipitate proteins in ice water, the precipitate is collected by centrifugation and washed, methanol is removed by vacuum suction filtration, and the precipitate is resuspended in a sample solubilization buffer, biochar obtained in step S1 is added to the resuspension and mixed and washed, and the protein imprinting biochar BIP-BC is obtained by drying. The composite repair material releases H2O2 and Fe 2+ / Fe 3+ to realize inactivation of harmful bacteria and degradation of harmful bacteria genes through Fenton-like reaction.

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: biomass material crushed to 100-120 mesh is pyrolyzed by a limited oxygen slow heating method, and after cooling, grinding and sieving, the sieved powder is soaked in an HCl solution, 0.45 μm filter membrane is used for suction filtration, and the powder intercepted on the filter paper is washed with deionized water for 3-5 times to obtain the washed biochar; S2: preparation of protein imprinting biochar: target harmful bacteria are inoculated in an LB medium containing protease, and after oscillation culture, the precipitate is obtained by centrifugation, the precipitate is resuspended in a lysis extraction buffer and heated, and the supernatant is obtained by centrifugation again, DNase and RNase are added to the supernatant and mixed uniformly, and nucleic acid impurities are removed by standing in ice water, acidified acetone-ethanol mixed solution is added to the supernatant to precipitate proteins in ice water, the precipitate is collected by centrifugation and washed, methanol is removed by vacuum suction filtration, and the precipitate is resuspended in a sample solubilization buffer, biochar obtained in step S1 is added to the resuspension and mixed and washed, and the protein imprinting biochar BIP-BC is obtained by drying. S3: Preparation of Fe3O4@BIP-BC, which is carried out by a co-precipitation method, and the specific steps are as follows: iron chloride solution and ferrous chloride solution are respectively configured, the protein imprinting biochar obtained in step S2 is dispersed in the iron chloride solution, after a certain period of contact, the ferrous chloride solution is added, then an excess of ammonia water solution is added and continuous stirring is carried out, the precipitate is separated and washed, and the Fe3O4@BIP-BC particles are obtained after freeze-drying; S4: Preparation of CaO2@Fe3O4@BIP-BC, which is carried out by a co-precipitation method, and the specific steps are as follows: a calcium chloride solution is configured, the Fe3O4@BIP-BC particles obtained in step S3 are dispersed in the calcium chloride solution, then a neutralizing agent and a stabilizer are added and stirred uniformly, hydrogen peroxide is added dropwise into the mixture, the precipitate is separated and washed, and the composite repair material is obtained 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: under a nitrogen atmosphere, preheat at 100℃ for 1h, then heat to 300-700℃ for constant temperature pyrolysis for 2h; the sieving uses a 100 mesh screen; the concentration of the HCl solution is 10-15wt%; the soaking time is 12-24h; In step S2: The target harmful bacteria include gram-negative bacteria and gram-positive bacteria; the LB culture medium containing protease is an LB culture medium with a trypsin concentration of 0.5-2.5%; the shaking culture is continuous shaking culture at 37℃ at a speed of 150-180 rpm for 8-10 hours; the lysis extraction buffer is a mixture of pH=7, 20 mM HEPES, 2-5 wt% sodium dodecyl sulfate and protease inhibitors, and the precipitate is resuspended in the lysis extraction buffer at 10 μL / mg wet cell weight; the heating is heating at 95℃ for 3-5 minutes; the addition amount of DNase is 1 mg / mL, and the addition amount of RNase is 0.5 mg / mL; the concentration of the acidified acetone-ethanol mixture is 1.5 mM; the sample solubilization buffer is a mixture of 8-10 M urea, 2-4 M thiazolinone, 2-4 wt% CHAPS, 1-2 wt% TCEP-HCl and 2-5 % v / v carrier ampholyte 3-10 NL; the biochar and the bacteria wet weight in the resuspension are added in equal weight; In step S3: Iron chloride: protein imprinting 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 = 1.078g: 0.3g: 5mL: 5mL; the neutralizing agent 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 jet method, including the following steps: The grouting pipe is inserted into the deepest part of the contaminated soil, the composite remediation material is mixed with water at a certain solid-liquid ratio, and then is sprayed into the contaminated soil, the grouting pipe is lifted upward at a certain interval to cover all the pollution depth, and the soil is remediated.

6. The use of a self-activated Fenton-like composite repair material according to claim 5, characterized in that, The dosage of the composite remediation material is 0.1-2.0% of the dry weight of the contaminated soil, the solid-liquid ratio of the composite remediation material and water is 1:10-1:1000, the lifting interval of the grouting pipe is 0.5-2.5 m, the spraying pressure is 0.5-2.5 MPa, and the 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 the surface soil in-situ plowing method, and includes the following steps: The composite remediation material is uniformly applied on the surface of the contaminated soil, and is plowed to mix the composite remediation material with the contaminated soil, and the soil is remediated.

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

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 the gravity dosing method or the pressure dosing method, and includes the following steps: (a) Gravity dosing method: the composite remediation material is dosed in the water pump suction pipe or the water suction horn mouth of the water suction well by gravity, the composite remediation material is mixed with the contaminated water by the water pump impeller, and the water body is remediated; (b) Pressure dosing method: the composite remediation material is mixed with water at a certain ratio, and then is dosed into the water pipe or sewage tank through the contaminated water body by the water pump or water jet, and is mixed uniformly, and the water body is remediated.

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

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