A preparation method of a remediation agent for groundwater pesticide pollution in karst topography

Abstract

The application discloses a preparation method of a reagent for repairing pesticide pollution of karst landform underground water, and relates to the technical field of underground water pollution repair. The reagent has the advantages of enhancing pesticide degradation effect, and by compounding hard water-resistant fungi, dehalogenation bacteria and denitrifying bacteria, the reagent can efficiently degrade pesticide pollutants in the complex environment of karst underground water, and better repair effect is achieved; the reagent can improve carrier stability and adsorption capacity, the biomimetic calcium phosphate nanocarrier has high specific surface area and excellent adsorption capacity, can effectively enhance the adsorption capacity of the reagent to pollutants, and improve repair effect; and the reagent has the advantages of high-efficiency repair mechanism, biological induction mineralization and physical adsorption strengthening of the bacteria-carrier complex are used to enhance the loading capacity of the bacterial group, and the intelligent coating technology is used to control the release rate of the bacterial group, so that precise repair is realized.

Description

Technical Field

[0001] This invention relates to the field of groundwater pollution remediation technology, specifically a method for preparing a pesticide pollution remediation agent for groundwater in karst landforms. Background Technology

[0002] Due to its high permeability, high calcium and sodium ion background, and rapid dynamic migration characteristics, karst landform groundwater is prone to widespread spread of pesticide pollutants, increasing the difficulty of pesticide pollution remediation.

[0003] Traditional remediation methods are often constrained by the complex environmental conditions of groundwater, making it difficult to effectively degrade or remove pesticide pollution.

[0004] Therefore, developing highly adaptable repair agents is an urgent problem to be solved.

[0005] The combination of biomimetic carriers and composite microbial agent systems, based on the degradation capabilities of natural microorganisms and by designing carriers with high specific surface area and adsorption performance, can effectively improve the degradation efficiency of pesticides, providing new ideas and technologies for the remediation of karst groundwater pollution. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a pesticide remediation agent for groundwater contaminated with pesticides in karst landforms, thereby addressing the high permeability and high calcium content of karst groundwater. 2+ / Mg 2+ Given the background and the characteristics of rapid dynamic migration, a "bionic carrier-composite microbial agent" drug system was constructed to solve the technical problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a pesticide remediation agent for groundwater contamination in karst landforms, comprising at least the following steps:

[0008] S1: Formulate a hard water tolerant fungi-denitrifying bacteria composite system to finally obtain the composite bacterial agent;

[0009] S2: Constructing a biomimetic calcium phosphate nanocarrier;

[0010] S3: Construct a bacterial-carrier complex;

[0011] S4: Complete the preparation of pesticide pollution remediation agents based on S1-S3.

[0012] Furthermore, S1 includes at least the following steps:

[0013] S1.1: Select strains and determine their morphology. The strains include white-rot fungi, dehalogenating bacteria, and denitrifying bacteria. The white-rot fungi include at least Trametes versicolor spore powder. The dehalogenating bacteria are in freeze-dried powder form. The denitrifying bacteria are in freeze-dried powder form.

[0014] S1.2: Pretreatment of the strain;

[0015] S1.3: Functional microbial community compounding, mixing activated spore powder of Ganoderma lucidum, freeze-dried powder of dehalogenated bacteria and freeze-dried powder of denitrifying bacteria in a ratio of 6:2:2, and then adding metabolic activators, including laccase activator, dehalogenation cofactor and denitrification promoter;

[0016] S1.4: Compound microbial agent forming: The mixed microbial community and metabolic activator in S1.3 are uniformly dispersed in a sterile protectant, and then freeze-dried under vacuum to obtain a compound microbial agent freeze-dried powder.

[0017] Furthermore, S1.2 includes at least the following steps:

[0018] Yunzhi spores were activated by inoculating spore powder into an acidic medium containing 1 mM CuSO4 and incubating with shaking at 25°C for 48 hours to induce calcium-binding protein expression. The acidic medium had a pH of 4.5 and contained 2% calcium carbonate.

[0019] The dehalogenated bacteria were revived by placing the lyophilized dehalogenated bacteria powder in an oxygen-free bottle and incubating at 37°C for 24 hours. The oxygen-free bottle contained 0.1 mg / L of vitamin B. 12 10mM sodium acetate;

[0020] Denitrifying bacteria activation: The lyophilized powder of denitrifying bacteria was inoculated into nitrate medium and cultured at 30°C with aeration at 150 rpm until OD (dose-to-volume) was reached. 600 =1.0, the nitrate culture medium is 50 mg / L NO3. - The pH value is 7.2.

[0021] Furthermore, the laccase activator in S1.3 is 2,5-dimethoxybenzoic acid (1mM) added to the tragali activation medium;

[0022] The dehalogenation cofactor is cobalamin (0.05 mg / L) added to the dehalogenation bacteria resuscitation system;

[0023] The denitrification promoter is sodium molybdate (0.01 mM) supplemented into the denitrifying bacteria culture medium.

[0024] Furthermore, the defatted lactose-trehalose complex includes at least a defatted lactose-trehalose complex.

[0025] Furthermore, S2 includes at least the following steps:

[0026] S2.1: Raw material preparation, using calcium chloride (CaCl2) and disodium hydrogen phosphate (Na2HPO4) as raw materials, according to Ca... 2+ :PO4 3- Mix the components in a molar ratio of 1.67:1, adjust the pH of the solution to 9.0-10.0 to obtain NH3·H2O, and add sodium bicarbonate (NaHCO3) to make CO32-. 2- At a concentration of 20-50 mM, a precursor solution of hydroxyapatite (CHAP) is formed;

[0027] S2.2: Template-induced mineralization. 5-10 wt% of the amphiphilic block copolymer Pluronic F127 was added to the precursor solution as a mesoporous template, along with 0.1-0.5 wt% polyaspartic acid (PASP) to regulate crystal nucleation. The mixture was then hydrothermally reacted at 150°C for 24 hours to generate CO3. 2- Doped hydroxyapatite nuclei;

[0028] S2.3: Pore shaping and purification. The product was immersed in an ethanol-hydrochloric acid mixture (9:1, volume ratio, containing 0.1M HCl) and sonicated for 2 hours to remove the template. After centrifugation and washing, it was calcined at 400℃ under a nitrogen atmosphere for 2 hours to obtain a pore size of 20±2nm and a specific surface area >200m². 2 / g porous hydroxyapatite carrier;

[0029] S2.4: Surface functional modification. The calcined support was immersed in a 5 wt% polyaspartic acid (PASP) solution (pH 6.0), stirred at 60°C for 6 hours, washed with deionized water, and dried to form a functionalized support with a surface enriched with carboxylic acid groups. This functionalized support exhibits improved resistance to Pb. 2+ / Cd 2+ The adsorption capacity was increased to >200mg / g.

[0030] Furthermore, S3 includes at least the following steps:

[0031] The compound bacterial agent lyophilized powder was dispersed in sterile physiological saline containing 0.1% Tween 80 at a ratio of 1:10 (w / v) and allowed to stand at room temperature for 30 minutes.

[0032] Simultaneous dissolution of metabolic activators;

[0033] Biomimetic calcium phosphate nanocarriers with surface-modified polyaspartic acid (PASP) were immersed in a saturated CaCO3 solution (pH 8.0) and sonicated for 30 minutes to activate carboxylic acid groups.

[0034] The pretreated bacterial culture was mixed with activated carboxylic acid groups at a mass ratio of 1:5 and reacted in a constant temperature shaker (25℃, 100rpm) for 6 hours, maintaining the pH at 7.5-8.0 (with automatic addition of 0.1M NaHCO3 for adjustment).

[0035] Calcium-binding proteins secreted by bacteria bind to calcium on the carrier surface. 2+ The combination induces secondary mineralization of hydroxyapatite to form a bonding layer. The PASP carboxylic acid groups are cross-linked with the bacterial extracellular polysaccharide (EPS) through hydrogen bonding and coordination, with a loading rate of >90%.

[0036] The gradient adsorption process transfers the initially loaded complex to a vacuum adsorption tank (initial pressure -0.08MPa), and restores it to atmospheric pressure in stages (increasing by 0.02MPa every 0.5 hours), which promotes the embedding of bacteria into the carrier mesopores (pore size 20±2nm) to form a complex.

[0037] The complex was washed three times with a protective solution containing 5% trehalose (pH 7.0) to remove unbound cells.

[0038] Stabilization treatment was first performed by freeze fixation, in which the composite was immersed in a cryoprotectant (10% glycerol + 5% skim lactose) and pre-frozen at -80°C for 12 hours at a rate of 1°C / min. This was followed by vacuum freeze-drying (cold trap -50°C, vacuum 10Pa) for 48 hours to form porous bacteria-carrier composite particles (particle size 1-3 mm, porosity >40%). Next, intelligent coating was performed using fluidized bed spraying technology to coat the surface of the porous bacteria-carrier composite particles with a pH-sensitive ethyl cellulose membrane (thickness 50-100 μm). This pH-sensitive ethyl cellulose membrane slowly releases bacterial flora at pH 6.5-7.5 (release rate ≤5% / day) and rapidly disintegrates within 30 minutes at pH <6.0 (release rate >90%).

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] 1. This invention enhances the pesticide degradation effect by combining hard water-resistant fungi, dehalogenating bacteria and denitrifying bacteria, which can efficiently degrade pesticide pollutants in the complex environment of karst groundwater, achieving better remediation results.

[0041] 2. The agent of the present invention can improve the stability and adsorption capacity of the carrier. It adopts a biomimetic calcium phosphate nanocarrier, which has a high specific surface area and excellent adsorption capacity, and can effectively enhance the adsorption capacity of pollutants and improve the remediation effect.

[0042] 3. The agent of the present invention has a highly efficient repair mechanism. Through the bio-induced mineralization and physical adsorption enhancement of the bacterial-carrier complex, the loading capacity of the bacterial community is enhanced, and the release rate of the bacterial community is controlled by intelligent encapsulation technology to achieve precise repair.

[0043] 4. The reagent of the present invention has strong adaptability. The reagent system is designed for the high calcium background and dynamic migration characteristics of karst groundwater, and has strong adaptability. It can maintain high remediation efficiency under complex water quality conditions. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0045] A method for preparing a pesticide remediation agent for groundwater contamination in karst landforms, comprising at least the following steps:

[0046] S1: Formulate a hard water tolerant fungi-denitrifying bacteria composite system to finally obtain the composite bacterial agent;

[0047] S2: Constructing a biomimetic calcium phosphate nanocarrier;

[0048] S3: Construct a bacterial-carrier complex;

[0049] S4: Complete the preparation of pesticide pollution remediation agents based on S1-S3.

[0050] S1 includes at least the following steps:

[0051] S1.1: Select strains and determine their morphology. The strains include white-rot fungi, dehalogenating bacteria, and denitrifying bacteria. White-rot fungi include at least Coriolus versicolor spore powder. Dehalogenating bacteria are in freeze-dried powder form, and denitrifying bacteria are in freeze-dried powder form.

[0052] S1.2: Pretreatment of the strain;

[0053] In this embodiment, the strain source and morphology are as follows:

[0054] White-rot fungus: Trametes versicolor (CGMCC No. 5.776) spore powder, laccase activity ≥300U / g;

[0055] Dehalogenated bacteria: Dehalococcoides mccartyi strain 195 (ATCC BAA-2100) lyophilized powder;

[0056] Denitrifying bacteria: Pseudomonas stutzeriATCC 17588 freeze-dried powder.

[0057] S1.3: Functional microbial community compounding, mix activated spore powder of Ganoderma lucidum, freeze-dried powder of dehalogenated bacteria and freeze-dried powder of denitrifying bacteria in a ratio of 6:2:2, and then add metabolic activators, including laccase activator, dehalogenation cofactor and denitrification promoter.

[0058] S1.4: Compound microbial agent forming: The mixed microbial community and metabolic activator in S1.3 are uniformly dispersed in a sterile protectant, and then freeze-dried under vacuum to obtain a compound microbial agent freeze-dried powder.

[0059] S1.2 includes at least the following steps:

[0060] Yunzhi spores were activated by inoculating spore powder into an acidic medium containing 1 mM CuSO4 and incubating with shaking at 25°C for 48 hours to induce calcium-binding protein expression. The acidic medium had a pH of 4.5 and contained 2% calcium carbonate.

[0061] The dehalogenated bacteria were revived by placing the dehalogenated lyophilized powder in an oxygen-free bottle and incubating at 37°C for 24 hours. The oxygen-free bottle contained 0.1 mg / L of vitamin B. 12 10mM sodium acetate;

[0062] Denitrifying bacteria activation: The lyophilized powder of denitrifying bacteria was inoculated into nitrate medium and cultured at 30°C with aeration at 150 rpm until OD (dose-to-volume) was reached. 600 =1.0, nitrate medium is 50 mg / L NO3 - The pH value is 7.2.

[0063] The laccase activator in S1.3 is 2,5-dimethoxybenzoic acid (1 mM) added to the activated medium of Trametes versicolor.

[0064] The dehalogenation cofactor was cobalamin (0.05 mg / L) added to the dehalogenation bacteria resuscitation system;

[0065] The denitrification promoter is sodium molybdate (0.01 mM) added to the denitrifying bacteria culture medium.

[0066] The skimmed lactose-trehalose complex includes at least the skimmed lactose-trehalose complex.

[0067] S2 includes at least the following steps:

[0068] S2.1: Raw material preparation, using calcium chloride (CaCl2) and disodium hydrogen phosphate (Na2HPO4) as raw materials, according to Ca... 2+ :PO4 3- Mix the components in a molar ratio of 1.67:1, adjust the pH of the solution to 9.0-10.0 to obtain NH3·H2O, and add sodium bicarbonate (NaHCO3) to make CO32-. 2- At a concentration of 20-50 mM, a precursor solution of hydroxyapatite (CHAP) is formed;

[0069] S2.2: Template-induced mineralization. 5-10 wt% of the amphiphilic block copolymer Pluronic F127 was added to the precursor solution as a mesoporous template, along with 0.1-0.5 wt% polyaspartic acid (PASP) to regulate crystal nucleation. The mixture was then hydrothermally reacted at 150°C for 24 hours to generate CO3. 2- Doped hydroxyapatite nuclei;

[0070] S2.3: Pore shaping and purification. The product was immersed in an ethanol-hydrochloric acid mixture (9:1, volume ratio, containing 0.1M HCl) and sonicated for 2 hours to remove the template. After centrifugation and washing, it was calcined at 400℃ under a nitrogen atmosphere for 2 hours to obtain a pore size of 20±2nm and a specific surface area >200m². 2 / g porous hydroxyapatite carrier;

[0071] S2.4: Surface functional modification. The calcined support was immersed in a 5 wt% polyaspartic acid (PASP) solution (pH 6.0), stirred at 60°C for 6 hours, washed with deionized water, and dried to form a functionalized support with a surface enriched with carboxylic acid groups. This functionalized support exhibits improved resistance to Pb. 2 + / Cd 2 The adsorption capacity of + is increased to >200mg / g;

[0072] Performance characterization was performed using XRD patterns (JCPDS 09-0432) and EDS analysis (Ca / P = 1.67) to verify the composition. BET analysis showed uniformity of the mesoporous structure (pore size distribution ±2 nm). XPS spectroscopy confirmed successful PASP grafting (N1s peak binding energy 399.8 eV). The support in Ca... 2+ =200mg / L simulated groundwater immersion for 30 days without structural collapse (Ca 2+ Adsorption capacity <5 mg / g), and for Pb 2+ The Langmuir adsorption capacity reached 235 mg / g.

[0073] S3 includes at least the following steps:

[0074] The compound bacterial agent lyophilized powder was dispersed in sterile physiological saline containing 0.1% Tween 80 at a ratio of 1:10 (w / v) and allowed to stand at room temperature for 30 minutes.

[0075] Simultaneous dissolution of metabolic activators;

[0076] Biomimetic calcium phosphate nanocarriers with surface-modified polyaspartic acid (PASP) were immersed in a saturated CaCO3 solution (pH 8.0) and sonicated for 30 minutes to activate carboxylic acid groups.

[0077] The pretreated bacterial culture was mixed with activated carboxylic acid groups at a mass ratio of 1:5 and reacted in a constant temperature shaker (25℃, 100rpm) for 6 hours, maintaining the pH at 7.5-8.0 (with automatic addition of 0.1M NaHCO3 for adjustment).

[0078] Calcium-binding proteins secreted by bacteria bind to calcium on the carrier surface. 2+ The combination induces secondary mineralization of hydroxyapatite to form a bonding layer. The PASP carboxylic acid groups are cross-linked with the bacterial extracellular polysaccharide (EPS) through hydrogen bonding and coordination, with a loading rate of >90%.

[0079] The gradient adsorption process transfers the initially loaded complex to a vacuum adsorption tank (initial pressure -0.08MPa), and restores it to atmospheric pressure in stages (increasing by 0.02MPa every 0.5 hours), which promotes the embedding of bacteria into the carrier mesopores (pore size 20±2nm) to form a complex.

[0080] The complex was washed three times with a protective solution containing 5% trehalose (pH 7.0) to remove unbound cells.

[0081] Stabilization treatment was first performed by freeze fixation, in which the composite was immersed in a cryoprotectant (10% glycerol + 5% skim lactose) and pre-frozen at -80°C for 12 hours at a rate of 1°C / min. This was followed by vacuum freeze-drying (cold trap -50°C, vacuum 10Pa) for 48 hours to form porous bacteria-carrier composite particles (particle size 1-3 mm, porosity >40%). Next, intelligent coating was performed using fluidized bed spraying technology to coat the surface of the porous bacteria-carrier composite particles with a pH-sensitive ethyl cellulose membrane (thickness 50-100 μm). The pH-sensitive ethyl cellulose membrane slowly releases the bacterial flora at pH 6.5-7.5 (release rate ≤5% / day) and rapidly disintegrates within 30 minutes at pH <6.0 (release rate >90%).

[0082] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a pesticide remediation agent for groundwater contamination in karst landforms, characterized in that: At least the following steps are included: S1: Formulate a hard water tolerant fungi-denitrifying bacteria composite system to finally obtain the composite bacterial agent; S1 includes at least the following steps: S1.1: Select strains and determine their morphology. The strains include white-rot fungi, dehalogenating bacteria, and denitrifying bacteria. The white-rot fungi include at least Trametes versicolor spore powder. The dehalogenating bacteria are in freeze-dried powder form. The denitrifying bacteria are in freeze-dried powder form. S1.2: Pretreatment of the bacterial strain; S1.3: Functional microbial community compounding, mixing activated spore powder of Ganoderma lucidum, freeze-dried powder of dehalogenated bacteria and freeze-dried powder of denitrifying bacteria in a ratio of 6:2:2, and then adding metabolic activators, including laccase activator, dehalogenation cofactor and denitrification promoter; S1.4: Compound microbial agent forming: The mixed microbial community and metabolic activator in S1.3 are uniformly dispersed in a sterile preservative, and then freeze-dried under vacuum to obtain a lyophilized compound microbial agent powder. S2: Constructing a biomimetic calcium phosphate nanocarrier; S2 includes at least the following steps: S2.1: Raw material preparation, using calcium chloride and disodium hydrogen phosphate as raw materials, according to... Mix the components in a specific molar ratio, adjust the pH of the solution to 9.0-10.0, and obtain... And add sodium bicarbonate to make At a concentration of 20-50 mM, a hydroxyapatite carbonate precursor solution is formed. S2.2: Template-induced mineralization. 5-10 wt% of the amphiphilic block copolymer Pluronic F127 was added to the precursor solution as a mesoporous template, along with 0.1-0.5 wt% polyaspartic acid to regulate crystal nucleation. The mixture was then hydrothermally reacted at 150℃ for 24 hours to generate… Doped hydroxyapatite nuclei; S2.3: Pore formation and purification. The product was immersed in an ethanol-hydrochloric acid mixture and sonicated for 2 hours to remove the template. After centrifugation and washing, it was calcined at 400℃ in a nitrogen atmosphere for 2 hours to obtain a porous hydroxyapatite support with a pore size of 20±2nm and a specific surface area of ​​>200m² / g. S2.4: Surface functional modification. The calcined support is immersed in a 5wt% polyaspartic acid solution, stirred at 60℃ for 6 hours, washed with deionized water, and dried to form a functionalized support with a surface enriched with carboxylic acid groups. The adsorption capacity was increased to >200 mg / g S3: Construct a bacterial-carrier complex; The S3 includes at least the following steps: Disperse the compound bacterial agent lyophilized powder in sterile physiological saline containing 0.1% Tween 80 at a ratio of 1:10 and let it stand at room temperature for 30 minutes. Simultaneous dissolution of metabolic activators; Biomimetic calcium phosphate nanocarriers with surface-modified polyaspartic acid were immersed in saturated water. The solution was sonicated for 30 minutes to activate the carboxylic acid groups. The pretreated bacterial solution was mixed with activated active carboxylic acid groups at a mass ratio of 1:5 and reacted in a constant temperature shaker for 6 hours, maintaining the pH at 7.5-8.

0. Calcium-binding proteins secreted by bacteria and the carrier surface The combination induces secondary mineralization of hydroxyapatite to form a bonding layer. The PASP carboxylic acid groups are cross-linked with the extracellular polysaccharides of the bacteria through hydrogen bonding and coordination, with a loading rate of >90%. The gradient adsorption process transfers the initially loaded complex to a vacuum adsorption tank and gradually restores it to atmospheric pressure, which promotes the embedding of bacteria into the carrier mesopores to form a complex. Clean and purify by rinsing the complex three times with a protective solution containing 5% trehalose to remove unbound cells; The stabilization process begins with freeze-fixation, where the composite is immersed in a cryoprotectant and pre-frozen at -80°C for 12 hours at a rate of 1°C / min. This is followed by vacuum freeze-drying for 48 hours to form porous bacteria-carrier composite particles. Next, intelligent coating is applied using fluidized bed spraying technology to coat the surface of the porous bacteria-carrier composite particles with a pH-sensitive ethyl cellulose membrane. This pH-sensitive ethyl cellulose membrane slowly releases the bacterial community at pH 6.5-7.5 and rapidly disintegrates within 30 minutes at pH < 6.

0. S4: Complete the preparation of pesticide pollution remediation agents based on S1-S3.

2. The preparation method of a pesticide remediation agent for groundwater pollution in karst landforms according to claim 1, characterized in that: S1.2 includes at least the following steps: Yunzhi spore activation, inoculating spore powder into a solution containing... The expression of calcium-binding protein was induced by incubation in an acidic medium at 25°C with shaking for 48 hours. The acidic medium had a pH of 4.5 and contained 2% calcium carbonate. The dehalogenated bacteria were revived by placing the lyophilized dehalogenated bacteria powder in an anaerobic flask and incubating at 37°C for 24 hours. The anaerobic flask contained 0.1 mg / L vitamin C. 10 mM sodium acetate; Denitrifying bacteria were activated by inoculating lyophilized denitrifying bacteria powder into nitrate medium and incubating at 30°C and 150 rpm with aeration. The nitrate culture medium is The pH value is 7.

2.

3. The preparation method of a pesticide remediation agent for groundwater pollution in karst landforms according to claim 1, characterized in that: The laccase activator in S1.3 is 2,5-dimethoxybenzoic acid added to the activated medium of Trametes versicolor. The dehalogenation cofactor is cobalamin added to the dehalogenation bacteria resuscitation system; The denitrification promoter is sodium molybdate supplemented into the denitrifying bacteria culture medium.

4. The preparation method of a pesticide remediation agent for groundwater pollution in karst landforms according to claim 1, characterized in that: The defatted lactose-trehalose complex includes at least a defatted lactose-trehalose complex.