Method for rapidly degrading refractory soluble organic matters in lake water body by using microbial agent

Abstract

The invention belongs to the technical field of water treatment, and particularly relates to a method for rapidly degrading refractory soluble organic matters in lake water by using a microbial agent. Through the synergistic effect of aerobic bacteria and anaerobic bacteria, the adsorption and catalysis functions of a catalyst carrier and mutual cooperation of biodegradation and catalytic oxidation, the metabolic limitation of traditional microorganisms on refractory organic matters such as benzene rings and long-chain hydrocarbons is broken through, efficient removal of the refractory organic matters in a water body is achieved, and the water quality is improved. The multi-stage porous structure rapidly adsorbs and enriches pollutants, the catalytic oxidation effect of the loaded metal oxide weakens the organic matter structure, meanwhile, the degradation effect of indigenous bacteria achieves thorough mineralization, the multi-step synergistic effect greatly improves the degradation efficiency, the concentration of refractory organics in a water body is remarkably reduced, and then the water quality is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of water treatment, and in particular relates to a method for rapidly degrading difficult-to-degrade soluble organic matter in lake water by utilizing microbial agents. Background Art

[0002] Refractory dissolved organic matter (RDOM) in natural waters primarily originates from plant degradation products, algal metabolites, and microbial secretions. These substances often contain complex aromatic structures and high-molecular-weight compounds, making them a significant component of organic matter in lake waters. With eutrophication and climate change, the content of RDOM in natural waters is often increasing, leading to problems such as decreased water transparency, changes in water color, and impaired ecosystem function. Currently, ecological restoration and control of RDOM in natural waters primarily utilize natural attenuation and dilution methods, ecological floating beds and aquatic plant restoration, and the enhancement of native microorganisms in natural waters.

[0003] Natural attenuation refers to the gradual degradation of organic matter through the water's own physical, chemical, and biological processes (such as photodegradation, adsorption, sedimentation, and natural microbial degradation). However, the structural characteristics of RDOM make it difficult to degrade through natural water purification processes. Natural attenuation is slow, requiring long treatment cycles, making it difficult to meet the demand for rapid remediation. Treatment effectiveness is significantly affected by environmental factors (such as temperature and light), resulting in poor stability. The removal efficiency of high-molecular-weight and complex RDOM is low, and dilution methods require large quantities of clean water, making them generally difficult to implement.

[0004] Using aquatic plants (such as reeds and cattails) to construct ecological floating beds or plant submerged plants for water remediation is a common ecological engineering approach. While these methods can improve water quality to some extent, their direct degradation capacity for RDOM is limited. Ecological floating beds primarily degrade dissolved organic matter through root adsorption and microbial attachment, limiting their direct degradation capacity for dissolved organic matter. They are highly seasonal, with their effectiveness significantly declining in winter. Regular harvesting and management are required, as plant litter can lead to secondary pollution. Large-scale application is limited by space constraints, making it difficult to implement in deep waters. There are no reports on their efficiency in degrading complex RDOM (such as aromatic substances).

[0005] Harnessing the inherent indigenous microbial communities in water bodies to degrade RDOM is an eco-friendly approach, such as enhancing local microbial activity through nutrient addition and increased aeration. However, relying on the natural degradation processes of indigenous microorganisms in natural water bodies cannot meet the requirements for rapid ecological restoration in terms of COD removal, aromatic structure degradation (UV254), and molecular weight reduction (E2:E3 ratio). These existing technologies primarily rely on the water's self-purification capacity or indirectly improve water quality through ecological engineering, and their ability to directly and efficiently degrade RDOM is limited.

[0006] In summary, existing natural water ecological restoration technologies have significant shortcomings in terms of treatment efficiency, thoroughness, and treatment cycle time. In particular, their direct degradation of RDOM is limited. Therefore, there is an urgent need to develop biotechnology that can rapidly and efficiently degrade RDOM in natural waters, thereby achieving rapid ecological restoration of water bodies. This present invention addresses these shortcomings of existing technologies by proposing a method for rapidly degrading recalcitrant soluble organic matter in lake waters using microbial agents, providing a new technical approach for water ecological restoration. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for rapidly degrading difficult-to-degrade soluble organic matter in lake water using microbial agents in response to existing problems.

[0008] The present invention is achieved through the following technical solutions: A method for rapidly degrading refractory soluble organic matter in lake water using a microbial agent comprises the following steps: S1. PLGA microspheres embedded with sodium nitrate and AQS were mixed with the catalyst carrier, vacuum adsorbed at 40°C for 1-2 hours, and then mixed with sodium carbonate-embedded calcium alginate / chitosan microspheres in a ratio of 2:1. Sterile water was added to fully moisten the microspheres. After stirring at room temperature for 4-5 hours, the microspheres were vacuum dried at 40-50°C for 10-12 hours. S2, immersing the catalyst support treated in step S1 in an anaerobic bacteria solution, vacuuming for 5 minutes, adding 5% CaCl2 and cross-linking for 20-30 minutes to obtain an anaerobic bacteria immobilized support; S3, immersing the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, and air-drying at 25°C for 30-40 minutes to obtain the immobilized bacteria agent; S4. Add the immobilized bacterial agent into the water to be treated.

[0009] Furthermore, the mass ratio of the PLGA microspheres to the catalyst carrier is 1:1; The preparation method of the PLGA microspheres is as follows: sodium nitrate and anthraquinone-2-sulfonate (AQS) are dissolved in ultrapure water at a mass ratio of 10:1, and the mass volume ratio of AQS to ultrapure water is 1g:50-60mL; then the microspheres are sterilized by filtration using a 0.22μm filter membrane; polylactic acid-glycolic acid copolymer (PLGA) is weighed and dissolved in dichloromethane at a mass volume ratio of 1g:20-30mL; the obtained sodium nitrate-AQS solution is added dropwise to the PLGA solution at a volume ratio of 1:2. Under ice bath conditions, emulsify with a high-speed homogenizer at 10,000 rpm for 5 to 6 minutes to form a stable W / O emulsion. Pour the W / O emulsion into an aqueous solution containing 2% PVA and continue emulsifying at 8,000 rpm for 8 to 10 minutes to form a W / O / W emulsion. Place the emulsion in a fume hood and stir magnetically until the dichloromethane is completely evaporated. Collect the microspheres by centrifugation at 3,000 to 4,000 rpm for 10 to 20 minutes at 4°C, wash with ultrapure water, and freeze-dry the PLGA microspheres.

[0010] Furthermore, the preparation of the catalyst support comprises the following steps: (1) After natural attapulgite is crushed and sieved, it is mixed evenly with a citric acid solution with a mass fraction of 6-8% at a solid-liquid ratio of 1:5, stirred at 60-80°C for 20-26 hours, washed with deionized water until neutral, and then dried at 90-100°C for 20-30 hours. It is then mixed evenly with mullite hollow ceramic microspheres at a mass ratio of 8-10:1, and finally calcined in a muffle furnace at 700-750°C for 4-6 hours to obtain a hierarchical pore structure carrier. (2) The multi-level pore structure carrier was dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then 3-aminopropyltriethoxysilane (APTES) was added. After refluxing at 80-90 °C for 6-8 h, it was washed with ethanol 3-4 times and vacuum dried at 70-80 °C for 6-8 h to obtain an amino-functionalized carrier. (3) Add the amino-functionalized carrier to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, add 1% glutaraldehyde crosslinker, stir at 60-70 °C for 3-4 h, and then filter and dry.

[0011] Furthermore, the sieving in step (1) is a 200 mesh sieve; The properties of the mullite hollow ceramic microspheres are: particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3 , temperature resistance>1200℃.

[0012] Furthermore, the mass ratio of 3-aminopropyltriethoxysilane to the multi-level pore structure carrier in step (2) is 0.1-0.15:1.

[0013] Furthermore, the preparation method of the core-shell catalyst suspension in step (3) is as follows: prepare a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 to control the Fe 3+ with Fe 2+ The molar ratio of 2:1 was adjusted to pH 10-11, and the mixture was stirred at 60-70 °C for 1-2 h under N2 protection to generate Fe3O4 nanoparticles. The Fe3O4 nanoparticles were dispersed in 0.05 mol / L KMnO4 solution, and 0.1 mol / L MnSO4 solution was added dropwise to control the Mn 7+ With Mn 2+ The molar ratio is 1:1, and the mixture is stirred at 40°C for 2~3h.

[0014] Furthermore, the preparation of the sodium carbonate embedded calcium alginate / chitosan microspheres described in step S1 comprises the following steps: 1) Dissolve sodium carbonate in ultrapure water at a mass-to-volume ratio of 1:10. Then weigh 1 / 5 the mass of sodium alginate and add it to the sodium carbonate solution. Stir for 4-5 hours and filter sterilize using a 0.45 μm filter membrane. 2) Dissolve chitosan in 1% acetic acid solution at a mass volume ratio of 1 g / 100 mL. Stir for 20-26 h, then adjust the pH to 5.5 with 1 M NaOH. Sterilize the solution by filtration through a 0.45 μm filter. 3) Pour the sodium carbonate / sodium alginate solution into a 5 mL syringe connected to a 22G needle. Add the solution dropwise into a 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min. Allow the cross-linking reaction to react at room temperature for 30 min to form calcium alginate microspheres. Collect the microspheres with tweezers and wash them three times with ultrapure water. Transfer the calcium alginate microspheres to the chitosan solution and stir slowly at room temperature for 20 min to allow chitosan to deposit on the surface of the microspheres to form a coating. Wash the microspheres three times with ultrapure water to remove unbound chitosan. Spread the microspheres flat in a sterile culture dish and air dry them at room temperature for 24 h. Then, vacuum dry them at 40°C for 8 h to constant weight.

[0015] Furthermore, the anaerobic bacterial solution described in step S2 is prepared by inoculating Desulfovibrio sp. and Paracoccus denitrificans into corresponding liquid culture media (Postgate C medium is used for Desulfovibrio sp., and inorganic salt medium containing nitrate is used for Paracoccus denitrificans), respectively, and culturing at 30°C and 150 rpm on a shaking table, and adjusting the concentration of the bacterial solution to 1×10 8CFU / mL, and then the two bacterial solutions were mixed in a volume ratio of 1:1 and added to a sodium alginate solution containing 0.1M CaCl2. The mass fraction of sodium alginate was 2%, and the volume ratio of the mixed bacterial solution to the sodium alginate solution containing 0.1M CaCl2 was 1:4.

[0016] Furthermore, the aerobic bacterial gel solution in step S3 is prepared by inoculating Bacillus subtilis into LB liquid culture medium, culturing at 37°C and 180 rpm in a shaking incubator for 18 to 24 hours until the logarithmic growth phase, measuring OD600 by spectrophotometry, and adjusting the concentration of the Bacillus subtilis bacterial solution to 2×10 8 CFU / mL, Bacillus subtilis culture liquid and 0.5% κ-carrageenan / 1% gelatin solution were mixed in a volume ratio of 1:3 and stirred thoroughly.

[0017] Compared with the prior art, the present invention has the following advantages: 1. The present invention achieves efficient removal of difficult-to-degrade organic matter in water bodies through the synergistic effect of aerobic and anaerobic bacteria, combined with the adsorption and catalytic functions of the catalyst carrier, and the mutual cooperation of biodegradation and catalytic oxidation. It breaks through the metabolic limitations of traditional microorganisms on difficult-to-degrade organic matter such as benzene rings and long-chain hydrocarbons, and realizes the high-efficiency removal of difficult-to-degrade organic matter in water bodies. The multi-level pore structure quickly adsorbs and enriches pollutants, the catalytic oxidation of the loaded metal oxides weakens the structure of organic matter, and the degradation of indigenous bacteria achieves thorough mineralization. The multi-step synergistic effect greatly improves the degradation efficiency and significantly reduces the concentration of difficult-to-degrade organic matter in water bodies, thereby improving water quality.

[0018] 2. The catalytic oxidation of the metal oxide nanoclusters of the present invention converts refractory organic matter into easily degradable small molecules, avoiding the formation of complex intermediates. The thorough decomposition by microorganisms completely mineralizes the small molecules into CO2 and H2O. The sodium alginate / gelatin coating is biodegradable, eliminating the risk of secondary pollution at multiple levels and ensuring the safety and environmental friendliness of water purification.

[0019] 3. The multi-level pore structure of the present invention provides a stable adsorption and reaction site. The gel network formed by the sodium alginate / gelatin coating enhances the material's stability, allowing it to maintain activity in water for a long time. The sustained release of the slow-release oxygen source and nutrients ensures the long-term metabolic activity of microorganisms, enabling this method to continuously and effectively remove refractory organic matter and achieve long-term water purification. DETAILED DESCRIPTION

[0020] In order to further explain the present invention, it is described below with reference to the following specific embodiments.

[0021] The strains of the present invention were purchased from the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) and Mingzhou Biotechnology, including Desulfovibrio sp. CGMCC 1.5190, Paracoccus denitrificans B98086, and Bacillus subtilis CGMCC 1.1086.

[0022] Desulfovibrio sulphuriformis culture medium (Postgate C medium, prepared according to standard recipe, required chemical reagents purchased from Sinopharm Chemical Reagent Co., Ltd.); Paracoccus denitrificans culture medium (nitrate inorganic salt medium, Sangon Biotech (Shanghai) Co., Ltd.); LB liquid medium (Sangon Biotech (Shanghai) Co., Ltd.).

[0023] Example 1: A method for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, comprising the following steps: S1. PLGA microspheres embedded with sodium nitrate and AQS were mixed with the catalyst carrier in a mass ratio of 1:1, vacuum adsorbed at 40°C for 1 hour, and then mixed with sodium carbonate embedded calcium alginate / chitosan microspheres in a ratio of 2:1. Sterile water was added to fully wet the microspheres. After stirring at room temperature for 4 hours, the mixture was vacuum dried at 40°C for 10 hours. The preparation method of the PLGA microspheres is as follows: sodium nitrate and AQS are dissolved in ultrapure water at a mass ratio of 10:1, and the mass volume ratio of AQS to ultrapure water is 1g:50mL; then, the mixture is sterilized by filtration using a 0.22μm filter membrane; PLGA is weighed and dissolved in dichloromethane at a mass volume ratio of 1g:20mL; the obtained sodium nitrate-AQS solution is added dropwise to the PLGA solution at a volume ratio of 1:2; emulsification is performed in an ice bath using a high-speed homogenizer at 10000rpm for 5min to form a stable W / O emulsion; the W / O emulsion is poured into an aqueous solution containing 2% PVA, and emulsification is continued at 8000rpm for 8min to form a W / O / W double emulsion; the double emulsion is placed in a fume hood and magnetically stirred until the dichloromethane is completely evaporated; the microspheres are collected by centrifugation at 3000rpm for 10min at 4°C, washed with ultrapure water, and freeze-dried. The preparation of the catalyst support comprises the following steps: (1) After natural attapulgite was crushed and passed through a 200-mesh sieve, it was mixed evenly with a 6% mass fraction of citric acid solution at a solid-liquid ratio of 1:5. After stirring at 60°C for 20 h, it was washed with deionized water until neutral and dried at 90°C for 20 h. Then, it was mixed with mullite hollow ceramic microspheres (particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3, temperature resistance>1200℃) were mixed in a mass ratio of 8:1, and finally calcined in a muffle furnace at 700℃ for 4~6h to obtain a multi-level pore structure carrier; (2) The multi-level pore structure carrier was dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then APTES was added. The mass ratio of APTES to the multi-level pore structure carrier was 0.1:1. After refluxing at 80°C for 6 hours, the carrier was washed with ethanol three times and vacuum dried at 70°C for 6 hours to obtain an amino-functionalized carrier. (3) Add the amino-functionalized support to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, add 1% glutaraldehyde crosslinker, stir at 60 °C for 3 h, and then filter and dry; The preparation method of the core-shell catalyst suspension is as follows: prepare a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 to control the Fe 3+ with Fe 2+ The molar ratio of 2:1 was adjusted to pH 10, and under N2 protection, the mixture was stirred at 60 °C for 1 h to generate Fe3O4 nanoparticles. The Fe3O4 nanoparticles were dispersed in 0.05 mol / L KMnO4 solution, and 0.1 mol / L MnSO4 solution was added dropwise to control the Mn 7+ With Mn 2+ The molar ratio was 1:1, and the mixture was stirred at 40 °C for 2 h; The preparation of the sodium carbonate embedded calcium alginate / chitosan microspheres comprises the following steps: 1) Dissolve sodium carbonate in ultrapure water at a mass-to-volume ratio of 1:10. Then weigh 1 / 5 of the mass of sodium carbonate into sodium alginate and add it to the sodium carbonate solution. Stir for 4 hours and filter sterilize using a 0.45 μm filter membrane. 2) Chitosan was dissolved in 1% acetic acid solution at a mass volume ratio of 1 g / 100 mL. After stirring for 20 h, the pH was adjusted to 5.5 with 1 M NaOH and the solution was sterilized by filtration through a 0.45 μm filter membrane. 3) Place the sodium carbonate / sodium alginate solution into a 5 mL syringe, connect a 22G needle, and drip the solution into a 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min. Allow the cross-linking reaction to proceed at room temperature for 30 min to form calcium alginate microspheres. Collect the microspheres with tweezers, wash them three times with ultrapure water, transfer the calcium alginate microspheres to the chitosan solution, and slowly stir them at room temperature for 20 min to allow chitosan to deposit on the surface of the microspheres to form a coating. Wash the microspheres three times with ultrapure water to remove unbound chitosan. Spread the microspheres flat on a sterile culture dish and air dry them at room temperature for 24 h. Then, vacuum dry them at 40°C for 8 h to constant weight. S2, immersing the catalyst support treated in step S1 in an anaerobic bacteria solution, vacuuming for 5 minutes, adding 5% CaCl2 for cross-linking for 20 minutes to obtain an anaerobic bacteria immobilized support; The anaerobic bacterial solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans in a ratio of 1:1 and adding the mixture to a sodium alginate solution containing 0.1M CaCl2, wherein the mass fraction of the sodium alginate is 2%. S3, immersing the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, and air-drying at 25°C for 30 minutes to obtain the immobilized bacteria agent; The aerobic bacterial gel solution is prepared by adding Bacillus subtilis to a 0.5% kappa-carrageenan / 1% gelatin solution; S4. Add the immobilized bacterial agent into the water to be treated.

[0024] Example 2: A method for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, comprising the following steps: S1. PLGA microspheres embedded with sodium nitrate and AQS were mixed with the catalyst carrier in a mass ratio of 1:1, vacuum adsorbed at 40°C for 1.5 hours, and then mixed with sodium carbonate-embedded calcium alginate / chitosan microspheres in a ratio of 2:1. Sterile water was added to fully wet the microspheres. After stirring at room temperature for 4.5 hours, the mixture was vacuum dried at 45°C for 11 hours. The preparation method of the PLGA microspheres is as follows: sodium nitrate and AQS are dissolved in ultrapure water at a mass ratio of 10:1, and the mass volume ratio of AQS to ultrapure water is 1g:55mL; then, the mixture is sterilized by filtration using a 0.22μm filter membrane; PLGA is weighed and dissolved in dichloromethane at a mass volume ratio of 1g:25mL; the obtained sodium nitrate-AQS solution is added dropwise to the PLGA solution at a volume ratio of 1:2; emulsification is performed in an ice bath using a high-speed homogenizer at 10000rpm for 5.5min to form a stable W / O emulsion; the W / O emulsion is poured into an aqueous solution containing 2% PVA, and emulsification is continued at 8000rpm for 9min to form a W / O / W double emulsion; the double emulsion is placed in a fume hood and magnetically stirred until the dichloromethane is completely evaporated; the microspheres are collected by centrifugation at 3500rpm for 15min at 4°C, washed with ultrapure water, and freeze-dried. The preparation of the catalyst support comprises the following steps: (1) After natural attapulgite was crushed and passed through a 200-mesh sieve, it was mixed evenly with a 7% mass fraction of citric acid solution at a solid-liquid ratio of 1:5. After stirring at 70 °C for 23 h, it was washed with deionized water until neutral and dried at 95 °C for 25 h. It was then mixed with mullite hollow ceramic microspheres (particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3 , temperature resistance> 1200℃) were mixed in a mass ratio of 8~10:1, and finally calcined in a muffle furnace at 725℃ for 5h to obtain a multi-level pore structure carrier; (2) The multi-level pore structure carrier was dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then APTES was added. The mass ratio of APTES to the multi-level pore structure carrier was 0.12:1. After refluxing at 85°C for 7 hours, the carrier was washed three times with ethanol and vacuum dried at 75°C for 7 hours to obtain an amino-functionalized carrier. (3) Add the amino-functionalized support to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, add 1% glutaraldehyde crosslinker, stir at 65 °C for 3.5 h, and then filter and dry; The preparation method of the core-shell catalyst suspension is as follows: prepare a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 to control the Fe 3+ with Fe 2+ The molar ratio of 2:1 was adjusted to pH 10, and under N2 protection, the mixture was stirred at 65 °C for 1.5 h to generate Fe3O4 nanoparticles. The Fe3O4 nanoparticles were dispersed in 0.05 mol / L KMnO4 solution, and 0.1 mol / L MnSO4 solution was added dropwise to control the Mn 7+ With Mn 2+ The molar ratio was 1:1, and the mixture was stirred at 40 °C for 2.5 h; The preparation of the sodium carbonate embedded calcium alginate / chitosan microspheres comprises the following steps: 1) Dissolve sodium carbonate in ultrapure water at a mass-to-volume ratio of 1:10. Then weigh 1 / 5 of the mass of sodium carbonate into sodium alginate and add it to the sodium carbonate solution. Stir for 4.5 hours and filter sterilize using a 0.45 μm filter membrane. 2) Chitosan was dissolved in 1% acetic acid solution at a mass volume ratio of 1 g / 100 mL. After stirring for 23 h, the pH was adjusted to 5.5 with 1 M NaOH and the solution was sterilized by filtration through a 0.45 μm filter membrane. 3) Place the sodium carbonate / sodium alginate solution into a 5 mL syringe, connect a 22G needle, and drip the solution into a 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min. Allow the cross-linking reaction to proceed at room temperature for 30 min to form calcium alginate microspheres. Collect the microspheres with tweezers, wash them three times with ultrapure water, transfer the calcium alginate microspheres to the chitosan solution, and slowly stir them at room temperature for 20 min to allow chitosan to deposit on the surface of the microspheres to form a coating. Wash the microspheres three times with ultrapure water to remove unbound chitosan. Spread the microspheres flat on a sterile culture dish and air dry them at room temperature for 24 h. Then, vacuum dry them at 40°C for 8 h to constant weight. S2, immersing the catalyst support treated in step S1 in an anaerobic bacteria solution, vacuuming for 5 minutes, adding 5% CaCl2 for cross-linking for 25 minutes to obtain an anaerobic bacteria immobilized support; The anaerobic bacterial solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans in a ratio of 1:1 and adding the mixture to a sodium alginate solution containing 0.1M CaCl2, wherein the mass fraction of the sodium alginate is 2%. S3, immersing the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, and air-drying at 25°C for 35 minutes to obtain the immobilized bacteria agent; The aerobic bacterial gel solution is prepared by adding Bacillus subtilis to a 0.5% kappa-carrageenan / 1% gelatin solution; S4. Add the immobilized bacterial agent into the water to be treated.

[0025] Example 3: A method for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, comprising the following steps: S1. PLGA microspheres embedded with sodium nitrate and AQS were mixed with the catalyst carrier in a mass ratio of 1:1, vacuum adsorbed at 40°C for 2 h, and then mixed with sodium carbonate embedded calcium alginate / chitosan microspheres in a ratio of 2:1. Sterile water was added to fully wet the microspheres. After stirring at room temperature for 5 h, the mixture was vacuum dried at 50°C for 12 h. The preparation method of the PLGA microspheres is as follows: sodium nitrate and AQS are dissolved in ultrapure water at a mass ratio of 10:1, and the mass volume ratio of AQS to ultrapure water is 1g:60mL; then, the mixture is sterilized by filtration using a 0.22μm filter membrane; PLGA is weighed and dissolved in dichloromethane at a mass volume ratio of 1g:30mL; the obtained sodium nitrate-AQS solution is added dropwise to the PLGA solution at a volume ratio of 1:2; emulsification is performed at 10000rpm for 6min in an ice bath using a high-speed homogenizer to form a stable W / O emulsion; the W / O emulsion is poured into an aqueous solution containing 2% PVA, and emulsification is continued at 8000rpm for 10min to form a W / O / W double emulsion; the double emulsion is placed in a fume hood and magnetically stirred until the dichloromethane is completely evaporated; the microspheres are collected by centrifugation at 4000rpm for 20min at 4°C, washed with ultrapure water, and freeze-dried. The preparation of the catalyst support comprises the following steps: (1) After natural attapulgite was crushed and passed through a 200-mesh sieve, it was mixed evenly with 8% citric acid solution at a solid-liquid ratio of 1:5. After stirring at 80°C for 26 h, it was washed with deionized water until neutral and dried at 100°C for 30 h. Then, it was mixed with mullite hollow ceramic microspheres (particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3 , temperature resistance>1200℃) were mixed in a mass ratio of 8~10:1, and finally calcined in a muffle furnace at 750℃ for 6h to obtain a multi-level pore structure carrier; (2) The multi-level pore structure carrier was dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then APTES was added. The mass ratio of APTES to the multi-level pore structure carrier was 0.15:1. After refluxing at 90°C for 8 hours, the carrier was washed with ethanol four times and vacuum dried at 80°C for 8 hours to obtain an amino-functionalized carrier. (3) Add the amino-functionalized support to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, add 1% glutaraldehyde crosslinker, stir at 70 °C for 4 h, and then filter and dry; The preparation method of the core-shell catalyst suspension is as follows: prepare a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 to control the Fe 3+ with Fe 2+ The molar ratio of 2:1 was adjusted to pH 11, and under N2 protection, the mixture was stirred at 70 °C for 2 h to generate Fe3O4 nanoparticles. The Fe3O4 nanoparticles were dispersed in 0.05 mol / L KMnO4 solution, and 0.1 mol / L MnSO4 solution was added dropwise to control the Mn 7+ With Mn 2+ The molar ratio was 1:1, and the mixture was stirred at 40 °C for 3 h; The preparation of the sodium carbonate embedded calcium alginate / chitosan microspheres comprises the following steps: 1) Dissolve sodium carbonate in ultrapure water at a mass-to-volume ratio of 1:10. Then weigh 1 / 5 of the mass of sodium carbonate into sodium alginate and add it to the sodium carbonate solution. Stir for 5 hours and filter sterilize using a 0.45 μm filter membrane. 2) Chitosan was dissolved in 1% acetic acid solution at a mass volume ratio of 1 g / 100 mL. After stirring for 26 h, the pH was adjusted to 5.5 with 1 M NaOH and the solution was sterilized by filtration through a 0.45 μm filter membrane. 3) Place the sodium carbonate / sodium alginate solution into a 5 mL syringe, connect a 22G needle, and drip the solution into a 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min. Allow the cross-linking reaction to proceed at room temperature for 30 min to form calcium alginate microspheres. Collect the microspheres with tweezers, wash them three times with ultrapure water, transfer the calcium alginate microspheres to the chitosan solution, and slowly stir them at room temperature for 20 min to allow chitosan to deposit on the surface of the microspheres to form a coating. Wash the microspheres three times with ultrapure water to remove unbound chitosan. Spread the microspheres flat on a sterile culture dish and air dry them at room temperature for 24 h. Then, vacuum dry them at 40°C for 8 h to constant weight. S2, immersing the catalyst support treated in step S1 in an anaerobic bacteria solution, vacuuming for 5 minutes, adding 5% CaCl2 for cross-linking for 30 minutes to obtain an anaerobic bacteria immobilized support; The anaerobic bacterial solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans in a ratio of 1:1 and adding the mixture to a sodium alginate solution containing 0.1M CaCl2, wherein the mass fraction of the sodium alginate is 2%. S3, immersing the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, and air-drying at 25°C for 40 minutes to obtain the immobilized bacteria agent; The aerobic bacterial gel solution is prepared by adding Bacillus subtilis to a 0.5% kappa-carrageenan / 1% gelatin solution; S4. Add the immobilized bacterial agent into the water to be treated.

[0026] Comparative Example 1 Compared with Example 2, in the preparation of the catalyst carrier, this comparative example 1 omitted steps (1) and (2), and the natural attapulgite was directly added to the core-shell catalyst suspension at a solid-liquid ratio of 1:8. 1% glutaraldehyde cross-linking agent was added, and the mixture was stirred at 65°C for 4.5 hours, and then filtered and dried. The other steps were the same as those in Example 2.

[0027] Comparative Example 2 Compared with Example 2, this comparative example 2 is that the catalyst support is replaced with an amino-functionalized support, and the other steps are the same as those in Example 2.

[0028] Comparative Example 3 In step S1, the operation of "embedding calcium alginate / chitosan microspheres with sodium carbonate and mixing them in a ratio of 2:1" was omitted, and the other steps were the same as those in Example 2.

[0029] Simulated natural water containing 50 mg / L phenol was prepared, the pH was adjusted to 7.2, and the temperature was controlled at 25±1° C., and then treated using the methods of Examples 1 to 3 and Comparative Examples 1 to 3, respectively, with three parallel experiments set up for each group.

[0030] 1. Degradation efficiency monitoring Samples were taken at 0h, 6h, 12h, 24h, 48h and 72h, and the concentration of phenol in the water was determined by high performance liquid chromatography, and the removal rate at different time points was calculated.

[0031] The test results are shown in Table 1 below.

[0032] Table 1 ; 2. COD removal rate determination (1) Sampling: Take water samples from the experimental group and each control group at 0h, 6h, 12h, 24h, 48h, and 72h respectively. Ensure that the water samples are fully mixed before sampling. Take three parallel samples at each time point.

[0033] (2) Digestion: Accurately draw 20 mL of water sample (if the COD value is high, dilute it before sampling) into a 250 mL ground-mouth reflux conical flask, add 10 mL of potassium dichromate standard solution and several small glass beads or zeolites, connect a ground-mouth reflux condenser, slowly add 30 mL of sulfuric acid-silver sulfate solution from the top of the condenser, gently shake the conical flask to mix the solution, and heat to reflux for 2 h (time starts from the beginning of boiling). (3) Titration: After cooling, rinse the condenser tube with 90 mL of water and remove the conical flask. The total volume of the solution must not be less than 140 mL, otherwise the titration endpoint will not be obvious due to the high acidity. Add 3 drops of ferrochlore indicator solution and titrate with ammonium ferrous sulfate standard solution. The endpoint is when the color of the solution changes from yellow to blue-green to reddish-brown. Record the amount of ammonium ferrous sulfate standard solution used.

[0034] (4) Blank test: Replace the water sample with distilled water and perform the test according to the same steps. Record the amount of ammonium ferrous sulfate standard solution used in the blank test.

[0035] The test results are shown in Table 2 below.

[0036] Table 2 ; As shown in Tables 1 and 2 above, the 72-hour phenol removal rate reached 93.5%, and the COD removal rate reached 91.6%, both of which are close and high. This demonstrates that the present invention achieves efficient treatment of pollutants throughout the entire process, from adsorption and degradation to mineralization, by rapidly adsorbing phenol through multi-stage pores, catalyzing its oxidative decomposition into small molecules through metal oxide nanoclusters, and then thoroughly mineralizing the intermediate products through microorganisms.

[0037] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for rapidly degrading refractory soluble organic matter in lake water using microbial agents, characterized in that: The steps include: S1. PLGA microspheres embedded with sodium nitrate and AQS were mixed with the catalyst carrier, vacuum adsorbed at 40°C for 1-2 hours, and then mixed with sodium carbonate embedded calcium alginate / chitosan microspheres in a ratio of 2:

1. Sterile water was added to fully moisten the microspheres. After stirring at room temperature for 4-5 hours, the microspheres were vacuum dried at 40-50°C for 10-12 hours. S2, immersing the catalyst support treated in step S1 in an anaerobic bacteria solution, vacuuming for 5 minutes, adding 5% CaCl2 and cross-linking for 20 to 30 minutes to obtain an anaerobic bacteria immobilized support; S3, immersing the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, and air-drying at 25°C for 30-40 minutes to obtain the immobilized bacteria agent; S4. Add the immobilized bacterial agent into the water to be treated.

2. The method of claim 1, wherein the method comprises: The mass ratio of the PLGA microspheres to the catalyst carrier is 1:1; The preparation method of the PLGA microspheres is as follows: sodium nitrate and AQS are dissolved in ultrapure water at a mass ratio of 10:1, and then filtered and sterilized; PLGA is weighed and dissolved in dichloromethane at a mass volume ratio of 1 g:20-30 mL; the obtained sodium nitrate-AQS solution is added dropwise to the PLGA solution at a volume ratio of 1:2; emulsification is performed at 10,000 rpm for 5-6 minutes under ice bath conditions to form a stable W / O emulsion; the W / O emulsion is poured into an aqueous solution containing 2% PVA, and emulsification is performed at 8,000 rpm for 8-10 minutes to form a W / O / W double emulsion; the double emulsion is placed in a fume hood and magnetically stirred until the dichloromethane is completely volatilized; the microspheres are collected by centrifugation at 3,000-4,000 rpm at 4°C, washed with ultrapure water, and freeze-dried to obtain the PLGA microspheres.

3. The method for rapidly degrading refractory soluble organic matter in lake water using a microbial agent according to claim 1, characterized in that: The preparation of the catalyst support comprises the following steps: (1) After natural attapulgite is crushed and sieved, it is mixed evenly with a citric acid solution with a mass fraction of 6-8% at a solid-liquid ratio of 1:5, stirred at 60-80°C for 20-26 hours, washed with deionized water until neutral, and then dried at 90-100°C for 20-30 hours. Then, it is mixed evenly with mullite hollow ceramic microspheres at a mass ratio of 8-10:1, and finally calcined in a muffle furnace at 700-750°C for 4-6 hours to obtain a hierarchical pore structure carrier; (2) The multi-level pore structure carrier was dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then APTES was added. After refluxing at 80-90°C for 6-8 hours, it was washed with ethanol 3-4 times and vacuum dried at 70-80°C for 6-8 hours to obtain an amino-functionalized carrier. (3) The amino-functionalized support was added to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, 1% glutaraldehyde crosslinker was added, and the mixture was stirred at 60-70°C for 3-4 hours, and then filtered and dried.

4. The method of claim 3 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The sieving in step (1) is a 200 mesh sieve; The properties of the mullite hollow ceramic microspheres are: particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3 , temperature resistance>1200℃.

5. The method of claim 3 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The mass ratio of 3-aminopropyltriethoxysilane to the multi-level pore structure carrier described in step (2) is 0.1 to 0.15:

1.

6. The method of claim 3 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The preparation method of the core-shell catalyst suspension in step (3) is as follows: prepare a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 to control the Fe 3+ with Fe 2+ The molar ratio of 2:1 was adjusted to pH 10-11, and under N2 protection, the mixture was stirred at 60-70℃ for 1-2h to generate Fe3O4 nanoparticles. The Fe3O4 nanoparticles were dispersed in 0.05mol / L KMnO4 solution, and 0.1mol / L MnSO4 solution was added dropwise to control the Mn 7+ With Mn 2+ The molar ratio is 1:1, and the mixture is stirred at 40°C for 2 to 3 hours.

7. The method of claim 1 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The preparation of sodium carbonate embedded calcium alginate / chitosan microspheres described in step S1 comprises the following steps: 1) Sodium carbonate was dissolved in ultrapure water at a mass-to-volume ratio of 1:10, and 0.2 times the mass of sodium carbonate as sodium alginate was added. The mixture was stirred for 4-5 hours and then filtered and sterilized to obtain a sodium carbonate / sodium alginate solution; 2) Dissolve chitosan in 1% acetic acid solution at a mass volume ratio of 1 g:100 mL, stir for 20-26 h, adjust the pH to 5.5 with 1 M NaOH, and filter sterilize to obtain a chitosan solution; 3) Use a syringe to drop the sodium carbonate / sodium alginate solution into the 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min. The cross-linking reaction is carried out at room temperature for 30 minutes to form calcium alginate microspheres. The microspheres are collected and washed three times with ultrapure water. The calcium alginate microspheres are transferred to the above chitosan solution and stirred at room temperature for 20 to 30 minutes. The microspheres are washed three times with ultrapure water. The microspheres are spread flat in a sterile culture dish and dried under ventilation at room temperature for 24 hours. The microspheres are vacuum dried at 40°C for 8 hours to constant weight to obtain sodium carbonate embedded in calcium alginate / chitosan microspheres.

8. The method of claim 1 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The anaerobic bacterial solution in step S2 is prepared by mixing Desulfovibrio sulphuri and Paracoccus denitrificans in a ratio of 1:1 and adding the mixture to a sodium alginate solution containing 0.1M CaCl2, wherein the mass fraction of the sodium alginate is 2%.

9. The method of claim 1 for rapidly degrading refractory soluble organic matter in lake water using a microbial agent, characterized in that: The aerobic bacterial gel solution in step S3 is prepared by adding Bacillus subtilis to a 0.5% κ-carrageenan / 1% gelatin solution.