A method for rapidly degrading refractory dissolved organic matter in a lake water body by using a microbial agent

Abstract

The present application belongs to the technical field of water treatment, and particularly relates to a method for rapidly degrading refractory dissolved organic matter in lake water by using microbial agents. The present application breaks through the metabolic limitation of traditional microorganisms on refractory organic matter such as benzene ring and long-chain hydrocarbon by the synergistic effect of aerobic and anaerobic bacteria, the adsorption and catalytic function of catalyst carriers, the mutual cooperation of biodegradation and catalytic oxidation, realizes the efficient removal of refractory organic matter in water, rapidly adsorbs and enriches pollutants by the multi-stage pore structure, weakens the structure of organic matter by the catalytic oxidation of the loaded metal oxides, and realizes complete mineralization by the degradation of indigenous bacteria, so that the degradation efficiency is greatly improved, the concentration of refractory organic matter in water is significantly reduced, and the water quality is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of water treatment, and particularly relates to a method for rapidly degrading refractory dissolved organic matter in lake water by using microbial agents. BACKGROUND

[0002] Refractory dissolved organic matter (RDOM) in natural water bodies mainly comes from plant degradation products, algal metabolites and microbial secretions, etc. These substances usually contain complex aromatic structures and high molecular weight compounds, and are important components of organic matter in lake water. With water eutrophication and climate change, the content of RDOM in natural water bodies tends to increase, leading to problems such as decreased water transparency, water color change, and damaged ecosystem function. At present, the ecological restoration and treatment of RDOM in natural water bodies mainly adopts natural attenuation and dilution method, ecological floating bed and aquatic plant restoration method, and indigenous microorganism strengthening method in natural water bodies.

[0003] Natural attenuation refers to a method of relying on physical, chemical and biological processes (such as photodegradation, adsorption, sedimentation and natural microbial degradation) of the water body itself to gradually degrade organic matter. However, the structural characteristics of RDOM determine that this kind of substance is difficult to be degraded by natural water self-purification and other methods. The natural attenuation rate is slow, the treatment period is long, and it is difficult to meet the demand of rapid repair; the treatment effect is greatly affected by environmental factors (such as temperature, light, etc.), and the stability is poor; the removal efficiency of high molecular weight and complex structure RDOM is low; the dilution method needs a large amount of clean water source, which is usually difficult to implement.

[0004] Using aquatic plants (such as reed, cattail, etc.) to construct ecological floating bed or plant submerged plants for water remediation is a commonly used ecological engineering method. These methods can improve water quality to some extent, but have limited direct degradation ability of RDOM. Ecological floating bed mainly degrades dissolved organic matter through root adsorption and microbial attachment, and has limited direct degradation ability of dissolved organic matter; the effect is significantly decreased in winter due to strong seasonality; regular harvesting and management are required, otherwise plant littering will cause secondary pollution; large-area application is limited by space, and it is difficult to implement in deep water area; the degradation efficiency of complex structure RDOM (such as aromatic substances) has not been reported.

[0005] Using indigenous microorganism community in water body to degrade RDOM is an ecological friendly method, such as adding nutrients, increasing aeration and other measures to strengthen the activity of local microorganisms. However, the natural degradation process relying on indigenous microorganisms in natural water bodies cannot meet the demand of rapid ecological restoration of natural water bodies in terms of COD removal, aromatic structure degradation (UV254), molecular weight reduction (E2:E3 ratio), etc. These existing technologies mainly rely on the self-purification ability of water body or indirectly improve water quality through ecological engineering, and have limited ability to directly and efficiently degrade RDOM.

[0006] In summary, the existing natural water ecological restoration technology has obvious deficiencies in treatment efficiency, treatment completeness, treatment cycle and the like, and especially has limited direct degradation capacity for RDOM. Therefore, it is urgent to develop a biological technology capable of quickly and efficiently degrading RDOM in natural water to realize rapid ecological restoration of the water body. The present application proposes a method for quickly degrading refractory dissolved organic matter in lake water by using microbial agents, to provide a new technical path for water ecological restoration. SUMMARY

[0007] The present application aims at the existing problems, and provides a method for quickly degrading refractory dissolved organic matter in lake water by using microbial agents.

[0008] The present application is implemented by the following technical solutions:

[0009] A method for quickly degrading refractory dissolved organic matter in lake water by using microbial agents, comprising the following steps:

[0010] S1, the PLGA microspheres embedded with sodium nitrate and AQS are mixed with a catalyst carrier, vacuum adsorbed at 40 DEG C for 1-2h, mixed with sodium carbonate embedded calcium alginate / chitosan microspheres according to 2:1, sterile water is added to make the microspheres fully wet, stirred at room temperature for 4-5h, and vacuum dried at 40-50 DEG C for 10-12h;

[0011] S2, the catalyst carrier treated in step S1 is immersed in an anaerobic bacteria liquid, vacuum suctioned for 5min, 5% CaCl2 is added and crosslinked for 20-30min to obtain an anaerobic bacteria immobilized carrier;

[0012] S3, the anaerobic bacteria immobilized carrier is immersed in an aerobic bacteria glue liquid, air-dried at 25 DEG C for 30-40min to obtain an immobilized bacteria agent;

[0013] S4, the immobilized bacteria agent is put into the water to be treated.

[0014] Further, the mass ratio of the PLGA microspheres to the catalyst carrier is 1:1;

[0015] The preparation method of the PLGA microspheres is as follows: sodium nitrate and anthraquinone-2-sulfonic acid sodium (AQS) are dissolved in ultrapure water according to a mass ratio of 10:1, and the mass-volume ratio of AQS to ultrapure water is 1g:50-60mL; then the solution is filtered and sterilized by using a 0.22μm filter membrane; poly (lactic-co-glycolic acid) (PLGA) is weighed according to a mass-volume ratio of 1g:20-30mL, and is dissolved in dichloromethane; the obtained sodium nitrate-AQS solution is added dropwise into the PLGA solution, and the volume ratio is 1:2; under ice bath conditions, the solution is emulsified by using a high-speed homogenizer at 10000rpm for 5-6min to form a stable W / O emulsion; the W / O emulsion is poured into an aqueous solution containing 2% PVA, and is continuously emulsified at 8000rpm for 8-10min to form a W / O / W multiple emulsion; the multiple emulsion is placed in a fume hood, and is magnetically stirred until dichloromethane is completely volatilized; the microspheres are collected by centrifugation at 3000-4000rpm for 10-20min at 4℃, and are washed with ultrapure water, and then are freeze-dried to obtain the PLGA microspheres.

[0016] Further, the preparation of the catalyst carrier comprises the following steps:

[0017] (1) After the natural attapulgite is crushed and sieved, it is uniformly mixed with a citric acid solution with a mass fraction of 6-8% according to a solid-liquid ratio of 1:5; after stirring treatment at 60-80℃ for 20-26h, the attapulgite is washed with deionized water until neutral, and is then dried at 90-100℃ for 20-30h; then the attapulgite is uniformly mixed with mullite hollow ceramic microbeads according to a mass ratio of 8-10:1; finally, the mixture is calcined in a muffle furnace at 700-750℃ for 4-6h to obtain a multi-level pore structure carrier;

[0018] (2) The multi-level pore structure carrier is dispersed in anhydrous ethanol according to a solid-liquid ratio of 1:10, and then 3-aminopropyltriethoxysilane (APTES) is added; after refluxing at 80-90℃ for 6-8h, the carrier is washed with ethanol for 3-4 times, and is then vacuum dried at 70-80℃ for 6-8h to obtain an amino-functionalized carrier;

[0019] (3) The amino-functionalized carrier is added into a core-shell catalyst suspension according to a solid-liquid ratio of 1:8, and 1% glutaraldehyde crosslinking agent is added; after stirring at 60-70℃ for 3-4h, the mixture is filtered and dried.

[0020] Further, the sieving in step (1) is sieving through a 200-mesh sieve;

[0021] The mullite hollow ceramic microbead has the following properties: particle size 30-60μm, wall thickness 2-5μm, density 0.6g / cm 3 , and temperature resistance >1200℃.

[0022] Further, the mass ratio of the 3-aminopropyltriethoxysilane to the support with hierarchical pore structure in step (2) is 0.1-0.15:1.

[0023] Further, the preparation method of the core-shell catalyst suspension in step (3) is as follows: a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 is prepared, the molar ratio of Fe 3+ to Fe 2+ is 2:1, the pH is adjusted to 10-11, and the mixture is stirred at 60-70°C for 1-2 h under N2 protection to generate Fe3O4 nanoparticles; the Fe3O4 nanoparticles are dispersed in a 0.05 mol / L KMnO4 solution, and a 0.1 mol / L MnSO4 solution is added dropwise, the molar ratio of Mn 7+ to Mn 2+ is 1:1, and the mixture is stirred at 40°C for 2-3 h.

[0024] Further, the preparation of the sodium carbonate-embedded calcium alginate / chitosan microspheres in step S1 comprises the following steps:

[0025] 1) Sodium carbonate is dissolved in ultrapure water at a mass / volume ratio of 1:10, and then sodium alginate with a mass of 1 / 5 of the mass of sodium carbonate is added to the sodium carbonate solution, which is stirred for 4-5 h and then filtered with a 0.45 μm filter membrane to remove bacteria;

[0026] 2) Chitosan is dissolved in a 1% acetic acid solution at a mass / volume ratio of 1 g / 100 mL, and after stirring for 20-26 h, the pH is adjusted to 5.5 with 1 M NaOH, and the solution is filtered with a 0.45 μm filter membrane to remove bacteria;

[0027] 3) The sodium carbonate / sodium alginate solution is loaded into a 5 mL syringe connected to a 22G needle, and the solution is dropped into a 0.05 g / mL calcium chloride crosslinking solution at a flow rate of 1 mL / min, and the crosslinking reaction is carried out at room temperature for 30 min to form calcium alginate microspheres, which are collected with tweezers, washed with ultrapure water for 3 times, and then transferred to the chitosan solution, and slowly stirred at room temperature for 20 min to allow the chitosan to deposit on the surface of the microspheres to form a coating, and then the microspheres are washed with ultrapure water for 3 times to remove unbound chitosan, and then the microspheres are laid flat on a sterile culture dish, and dried at room temperature under ventilation for 24 h, and then vacuum dried at 40°C for 8 h to constant weight.

[0028] Further, the anaerobic bacteria liquid in step S2 is that Desulfovibrio sp. and Paracoccus denitrificans are inoculated into corresponding liquid culture medium (Postgate C culture medium is used for Desulfovibrio sp., and inorganic salt culture medium containing nitrate is used for Paracoccus denitrificans), and the bacteria liquid is cultured under the condition of 30 DEG C and 150 rpm of a shaking table, the concentration of the bacteria liquid is adjusted to 1*10 8 CFU / mL, then the two bacteria liquids are mixed according to the volume ratio of 1:1 and added into a sodium alginate solution containing 0.1M CaCl2, the mass fraction of sodium alginate is 2%, and the volume ratio of the mixed bacteria liquid to the sodium alginate solution containing 0.1M CaCl2 is 1:4.

[0029] Further, the aerobic bacteria liquid in step S3 is that Bacillus subtilis is inoculated into LB liquid culture medium, and cultured under the condition of 37 DEG C and 180 rpm of a shaking table for 18-24h until the logarithmic growth phase, the OD600 is measured by using a spectrophotometer, the concentration of the Bacillus subtilis liquid is adjusted to 2*10 8 CFU / mL, and the Bacillus subtilis liquid is mixed with 0.5% kappa-carrageenan / 1% gelatin solution according to the volume ratio of 1:3, and fully stirred uniformly.

[0030] Compared with the prior art, the present application has the following advantages:

[0031] 1. The present application realizes efficient removal of refractory organic matter in water through the synergistic effect of aerobic and anaerobic bacteria, the adsorption and catalytic function of the catalyst carrier, the mutual cooperation of biodegradation and catalytic oxidation, and the breakthrough of the metabolic limitation of traditional microorganisms on benzene ring and long-chain hydrocarbon, etc.

[0032] 2. The catalytic oxidation of the metal oxide nanocluster in the present application converts refractory organic matter into small molecules which are easy to degrade, avoids the generation of complex intermediate products, and completely mineralizes the small molecules into CO2 and H2O through the complete decomposition of microorganisms.

[0033] 3、The multi-level pore structure of the present application provides stable adsorption and reaction sites, and the gel network formed by the sodium alginate / gelatin coating enhances the stability of the material, allowing it to maintain activity in water for a long time. The sustained release of oxygen source and nutrients ensures the long-term metabolic activity of microorganisms, enabling the method to continuously and effectively remove refractory organic matter and achieve long-term purification of water bodies. DETAILED DESCRIPTION

[0034] In order to further explain the present application, the following specific examples are described below.

[0035] The strains of the present application are purchased from the China General Microbiological Culture Collection Center (CGMCC) and Mingzhou Biology. Desulfovibrio sp. CGMCC 1.5190; Paracoccus denitrificans B98086; Bacillus subtilis CGMCC 1.1086.

[0036] Desulfovibrio culture medium (Postgate C medium, prepared according to the standard formula, and the required chemical reagents are purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd.); Paracoccus denitrificans culture medium (nitrate inorganic salt medium, Shenguo Bioengineering (Shanghai) Co., Ltd.); LB liquid medium (Shenguo Bioengineering (Shanghai) Co., Ltd.).

[0037] Example 1: A method for rapidly degrading refractory dissolved organic matter in lake water using microbial agents, comprising the following steps:

[0038] S1, the PLGA microspheres embedded with sodium nitrate and AQS are mixed with the catalyst carrier at a mass ratio of 1:1, then vacuum adsorbed at 40℃ for 1h, then mixed with the calcium alginate / chitosan microspheres embedded with sodium carbonate at a ratio of 2:1, sterile water is added to fully wet the microspheres, stirred at room temperature for 4h, and then vacuum dried at 40℃ for 10h;

[0039] 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 1 g:50 mL; then the bacteria are removed by filtering with a 0.22 μm filter membrane; PLGA is weighed and dissolved in dichloromethane at a mass-volume ratio of 1 g:20 mL, the obtained sodium nitrate-AQS solution is added to the PLGA solution at a volume ratio of 1:2, and under ice bath conditions, a stable W / O emulsion is formed by high-speed homogenizer 10000 rpm emulsification for 5 min, the W / O emulsion is poured into an aqueous solution containing 2% PVA, and the emulsion is continuously emulsified at 8000 rpm for 8 min to form a W / O / W multiple emulsion, the multiple emulsion is placed in a fume hood, and the dichloromethane is completely volatilized under magnetic stirring, the microspheres are collected by centrifugation at 3000 rpm for 10 min at 4 ℃, and the PLGA microspheres are washed with ultrapure water and freeze-dried;

[0040] The preparation of the catalyst carrier comprises the following steps:

[0041] (1) After the natural attapulgite is crushed through a 200-mesh sieve, it is uniformly mixed with a citric acid solution with a mass fraction of 6% at a solid-liquid ratio of 1:5, and then stirred and treated at 60 ℃ for 20 h, washed with deionized water until neutral, and dried at 90 ℃ for 20 h, and then uniformly mixed with mullite hollow ceramic microbeads (particle size 30-60 μm, wall thickness 2-5 μm, density 0.6 g / cm 3 , temperature resistance > 1200 ℃) at a mass ratio of 8:1, and finally calcined at 700 ℃ in a muffle furnace for 4-6 h to obtain a multi-level pore structure carrier;

[0042] (2) The multi-level pore structure carrier is dispersed in anhydrous ethanol at a solid-liquid ratio of 1:10, and then APTES is added, and the mass ratio of the APTES to the multi-level pore structure carrier is 0.1:1, and after refluxing at 80 ℃ for 6 h, the obtained product is washed with ethanol for 3 times and vacuum dried at 70 ℃ for 6 h to obtain an amino-functionalized carrier;

[0043] (3) The amino-functionalized carrier is added to a core-shell catalyst suspension at a solid-liquid ratio of 1:8, and 1% glutaraldehyde crosslinking agent is added, and then stirred at 60 ℃ for 3 h, and then dried by suction filtration;

[0044] The preparation method of the core-shell catalyst suspension is as follows: a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 is prepared, the molar ratio of Fe 3+ to Fe 2+ is controlled to be 2:1, the pH is adjusted to 10, Fe3O4 nanoparticles are generated by stirring at 60 ℃ for 1 h under N2 protection, the Fe3O4 nanoparticles are dispersed in a 0.05 mol / L KMnO4 solution, and a 0.1 mol / L MnSO4 solution is added dropwise, and the molar ratio of Mn7+ With Mn 2+ The molar ratio was 1:1, and the mixture was stirred at 40°C for 2 hours.

[0045] The preparation of sodium carbonate embedded calcium alginate / chitosan microspheres includes the following steps:

[0046] 1) Dissolve sodium carbonate in ultrapure water at a mass-to-volume ratio of 1:10, then weigh out sodium alginate (1 / 5 the mass of sodium carbonate) and add it to the sodium carbonate solution. Stir for 4 hours and then filter through a 0.45μm filter membrane for sterilization.

[0047] 2) Dissolve chitosan in 1% acetic acid solution at a mass-to-volume ratio of 1 g / 100 mL, stir for 20 h, adjust the pH to 5.5 with 1 M NaOH, and filter the solution through a 0.45 μm filter membrane for sterilization.

[0048] 3) Load the sodium carbonate / sodium alginate solution into a 5mL syringe, connect a 22G needle, and drop the solution into a 0.05g / mL calcium chloride crosslinking solution at a flow rate of 1mL / min. Perform the crosslinking reaction at room temperature for 30min to form calcium alginate microspheres. Collect the microspheres with tweezers, wash them three times with ultrapure water, and transfer the calcium alginate microspheres to a chitosan solution. Stir slowly at room temperature for 20min 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 evenly in a sterile petri dish and air dry at room temperature for 24h, then vacuum dry at 40℃ for 8h to constant weight.

[0049] S2. Immerse the catalyst support treated in step S1 into the anaerobic bacterial solution, vacuum suction for 5 min, add 5% CaCl2 for cross-linking for 20 min to obtain the anaerobic bacteria immobilized support.

[0050] The anaerobic bacterial solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans in a 1:1 ratio and adding them to a sodium alginate solution containing 0.1M CaCl2, wherein the mass fraction of sodium alginate is 2%.

[0051] S3. Immerse the anaerobic bacteria immobilization carrier in the aerobic bacteria gel solution and air dry at 25°C for 30 min to obtain the immobilized bacterial agent.

[0052] The aforementioned aerobic bacterial solution is prepared by adding Bacillus subtilis to a 0.5% κ-carrageenan / 1% gelatin solution;

[0053] S4. Simply add the immobilized bacterial agent into the water body to be treated.

[0054] Embodiment 2: A method for rapidly degrading refractory dissolved organic matter in a lake water body by using a microbial agent, comprising the following steps:

[0055] S1, the PLGA microspheres embedded with sodium nitrate and AQS are mixed with the catalyst carrier according to a mass ratio of 1:1, then vacuum adsorbed at 40°C for 1.5h, mixed with the sodium carbonate-embedded calcium alginate / chitosan microspheres according to a ratio of 2:1, added with sterile water to fully wet the microspheres, stirred at room temperature for 4.5h, and then vacuum dried at 45°C for 11h;

[0056] The preparation method of the PLGA microspheres is as follows: sodium nitrate and AQS are dissolved in ultrapure water according to a mass ratio of 10:1, and the mass-volume ratio of AQS to ultrapure water is 1g:55mL; then filtered and sterilized by using a 0.22μm filter membrane; PLGA is weighed according to a mass-volume ratio of 1g:25mL, dissolved in dichloromethane, and the obtained sodium nitrate-AQS solution is added dropwise into the PLGA solution according to a volume ratio of 1:2, emulsified in an ice bath at a high-speed homogenizer of 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 emulsified at 8000rpm for 9min to form a W / O / W multiple emulsion, the multiple emulsion is placed in a fume hood and magnetically stirred until the dichloromethane is completely volatilized, the microspheres are collected by centrifugation at 3500rpm for 15min at 4°C, and the PLGA microspheres are washed with ultrapure water and freeze-dried;

[0057] The preparation of the catalyst carrier comprises the following steps:

[0058] (1) The natural attapulgite is crushed through a 200-mesh sieve, mixed uniformly with a citric acid solution with a mass fraction of 7% according to a solid-liquid ratio of 1:5, stirred at 70°C for 23h, washed with deionized water until neutral, dried at 95°C for 25h, then mixed uniformly with mullite hollow ceramic microbeads (particle size 30-60μm, wall thickness 2-5μm, density 0.6g / cm 3 , temperature resistance >1200°C) according to a mass ratio of 8-10:1, and finally calcined at 725°C in a muffle furnace for 5h to obtain a multi-level pore structure carrier;

[0059] (2) The multi-level pore structure carrier is dispersed in anhydrous ethanol according to a solid-liquid ratio of 1:10, then APTES is added, the mass ratio of APTES to the multi-level pore structure carrier is 0.12:1, refluxed at 85°C for 7h, washed with ethanol for 3 times, and vacuum dried at 75°C for 7h to obtain an amino-functionalized carrier;

[0060] (3) The amino-functionalized carrier is added into a core-shell catalyst suspension according to a solid-liquid ratio of 1:8, 1% glutaraldehyde crosslinking agent is added, stirred at 65°C for 3.5h, then filtered and dried.

[0061] The preparation method of the core-shell catalyst suspension is as follows: a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 is prepared, the molar ratio of Fe 3+ to Fe 2+ is 2:1, the pH is adjusted to 10, and the mixture is stirred at 65°C for 1.5 h under N2 protection to generate Fe3O4 nanoparticles; the Fe3O4 nanoparticles are dispersed in a 0.05 mol / L KMnO4 solution, a 0.1 mol / L MnSO4 solution is added dropwise, the molar ratio of Mn 7+ to Mn 2+ is 1:1, and the mixture is stirred at 40°C for 2.5 h;

[0062] The preparation of the sodium carbonate-embedded calcium alginate / chitosan microspheres comprises the following steps:

[0063] 1) Sodium carbonate is dissolved in ultrapure water at a mass-volume ratio of 1:10, then sodium alginate with a mass of 1 / 5 of the mass of sodium carbonate is added to the sodium carbonate solution, the mixture is stirred for 4.5 h, and then filtered with a 0.45 μm filter membrane to remove bacteria;

[0064] 2) Chitosan is dissolved in a 1% acetic acid solution at a mass-volume ratio of 1 g / 100 mL, the mixture is stirred for 23 h, the pH is adjusted to 5.5 with 1M NaOH, and the solution is filtered with a 0.45 μm filter membrane to remove bacteria;

[0065] 3) The sodium carbonate / sodium alginate solution is loaded into a 5 mL syringe, connected to a 22G needle, and dropped into a 0.05 g / mL calcium chloride crosslinking solution at a flow rate of 1 mL / min, the crosslinking reaction is carried out at room temperature for 30 min, the calcium alginate microspheres are collected with tweezers, washed with ultrapure water for 3 times, the calcium alginate microspheres are transferred to the chitosan solution, slowly stirred at room temperature for 20 min to allow the chitosan to deposit on the surface of the microspheres to form a coating, the microspheres are washed with ultrapure water for 3 times to remove unbound chitosan, the microspheres are laid flat on a sterile culture dish, dried at room temperature under ventilation for 24 h, and dried at 40°C under vacuum for 8 h to a constant weight;

[0066] S2, the catalyst carrier treated in step S1 is immersed in an anaerobic bacteria solution, vacuum suctioned for 5 min, and 5% CaCl2 is added for crosslinking for 25 min to obtain an anaerobic bacteria immobilized carrier;

[0067] The anaerobic bacteria solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans at a ratio of 1:1 in a sodium alginate solution containing 0.1M CaCl2, and the mass fraction of sodium alginate is 2%;

[0068] S3, immerse the anaerobic bacteria immobilized carrier into the aerobic bacteria glue solution, dry at 25℃ for 35min to obtain the immobilized bacteria agent;

[0069] The aerobic bacteria glue solution is prepared by adding Bacillus subtilis into a 0.5% K-carrageenan / 1% gelatin solution;

[0070] S4, put the immobilized bacteria agent into the water body to be treated.

[0071] Embodiment 3: a method for rapidly degrading refractory dissolved organic matter in a lake water body by using a microbial agent, comprising the following steps:

[0072] S1, mix the PLGA microspheres embedded with sodium nitrate and AQS with the catalyst carrier according to a mass ratio of 1:1, vacuum adsorb at 40℃ for 2h, then mix with the calcium alginate / chitosan microspheres embedded with sodium carbonate according to a mass ratio of 2:1, add sterile water to fully wet the microspheres, stir at room temperature for 5h, and then vacuum dry at 50℃ for 12h;

[0073] The preparation method of the PLGA microspheres is as follows: dissolve sodium nitrate and AQS in ultrapure water according to a mass ratio of 10:1, and the mass / volume ratio of AQS to ultrapure water is 1g:60mL; then filter and sterilize with a 0.22μm filter membrane; weigh PLGA according to a mass / volume ratio of 1g:30mL, dissolve in dichloromethane, and then add the obtained sodium nitrate-AQS solution to the PLGA solution according to a volume ratio of 1:2, emulsify in a high-speed homogenizer at 10000rpm for 6min under ice bath conditions to form a stable W / O emulsion, continue to emulsify at 8000rpm for 10min in an aqueous solution containing 2% PVA to form a W / O / W multiple emulsion, place the multiple emulsion in a fume hood, magnetically stir until the dichloromethane is completely volatilized, centrifuge at 4000rpm for 20min at 4℃ to collect the microspheres, wash with ultrapure water, and then freeze-dry the PLGA microspheres;

[0074] The preparation of the catalyst carrier comprises the following steps:

[0075] (1) crush the natural attapulgite to pass through a 200-mesh sieve, mix with a citric acid solution with a mass fraction of 8% according to a solid-liquid ratio of 1:5, stir and treat at 80℃ for 26h, then wash with deionized water until neutral, dry at 100℃ for 30h, then mix with mullite hollow ceramic microbeads (particle size 30~60μm, wall thickness 2~5μm, density 0.6g / cm 3 , temperature resistance >1200℃) according to a mass ratio of 8~10:1, and finally calcine in a muffle furnace at 750℃ for 6h to obtain a multi-level pore structure carrier;

[0076] (2) The multi-level pore structure carrier is dispersed in anhydrous ethanol according to a solid-liquid ratio of 1:10, then APTES is added, the mass ratio of the APTES to the multi-level pore structure carrier is 0.15:1, after refluxing at 90°C for 8h, the obtained product is washed with ethanol for 4 times, and then vacuum dried at 80°C for 8h to obtain an amino-functionalized carrier;

[0077] (3) The amino-functionalized carrier is added into a core-shell catalyst suspension according to a solid-liquid ratio of 1:8, 1% glutaraldehyde crosslinking agent is added, stirring is carried out at 70°C for 4h, then the product is dried by suction filtration to obtain a product;

[0078] The preparation method of the core-shell catalyst suspension is as follows: a mixed solution of 0.2mol / L FeCl3 and 0.1mol / L FeSO4 is prepared, the molar ratio of Fe 3+ to Fe 2+ is 2:1, the pH value is adjusted to 11, Fe3O4 nanoparticles are generated by stirring at 70°C for 2h under N2 protection; the Fe3O4 nanoparticles are dispersed in a 0.05mol / L KMnO4 solution, a 0.1mol / L MnSO4 solution is added dropwise, the molar ratio of Mn 7+ to Mn 2+ is 1:1, and stirring is carried out at 40°C for 3h;

[0079] The preparation of the sodium carbonate-embedded calcium alginate / chitosan microsphere includes the following steps:

[0080] 1) Sodium carbonate is dissolved in ultrapure water according to a mass-volume ratio of 1:10, then sodium alginate with a mass of 1 / 5 of the mass of sodium carbonate is added into the sodium carbonate solution, stirring is carried out for 5h, and then the solution is filtered with a 0.45μm filter membrane to remove bacteria;

[0081] 2) Chitosan is dissolved in a 1% acetic acid solution according to a mass-volume ratio of 1g / 100mL, stirring is carried out for 26h, then the pH value is adjusted to 5.5 by using 1M NaOH, and the solution is filtered with a 0.45μm filter membrane to remove bacteria;

[0082] 3) The sodium carbonate / sodium alginate solution is loaded into a 5mL syringe, a 22G needle is connected, the solution is dropped into a 0.05g / mL calcium chloride crosslinking solution at a flow rate of 1mL / min, crosslinking reaction is carried out at room temperature for 30min, calcium alginate microspheres are collected by using tweezers, the microspheres are washed with ultrapure water for 3 times, the calcium alginate microspheres are transferred into the chitosan solution, slow stirring is carried out at room temperature for 20min, chitosan is deposited on the surface of the microspheres to form a coating, the microspheres are washed with ultrapure water for 3 times to remove unbound chitosan, the microspheres are laid flat on a sterile culture dish, and then the dish is dried at room temperature under ventilation for 24h, and then vacuum dried at 40°C for 8h to a constant weight;

[0083] S2, immerse the catalyst carrier treated in step S1 into an anaerobic bacteria solution, vacuum suction for 5 min, add 5% CaCl2 for cross-linking for 30 min to obtain an anaerobic bacteria immobilized carrier;

[0084] The anaerobic bacteria solution is prepared by mixing Desulfovibrio sp. and Paracoccus denitrificans at a ratio of 1:1 in a 0.1M CaCl2-containing sodium alginate solution, and the mass fraction of sodium alginate is 2%;

[0085] S3, immerse the anaerobic bacteria immobilized carrier into an aerobic bacteria glue solution, and dry at 25℃ for 40 min to obtain an immobilized bacteria agent;

[0086] The aerobic bacteria glue solution is prepared by adding Bacillus subtilis to a 0.5% kappa-carrageenan / 1% gelatin solution;

[0087] S4, the immobilized bacteria agent is put into the water body to be treated.

[0088] Comparative Example 1

[0089] Comparative Example 1 and Example 2 are compared in the preparation of the catalyst carrier. Steps (1) and (2) are omitted, and the natural attapulgite is directly added to the core-shell catalyst suspension at a solid-liquid ratio of 1:8, 1% glutaraldehyde cross-linking agent is added, stirring at 65℃ for 4.5h, then filtering and drying, and the other steps are the same as Example 2.

[0090] Comparative Example 2

[0091] Comparative Example 2 and Example 2 are compared. The catalyst carrier is replaced by an amino-functionalized carrier, and the other steps are the same as Example 2.

[0092] Comparative Example 3

[0093] Step S1 omits the operation of "mixing with sodium carbonate embedded calcium alginate / chitosan microspheres at a ratio of 2:1", and the other steps are the same as Example 2.

[0094] Prepare a simulated natural water body containing 50mg / L phenol, adjust the pH to 7.2, control the temperature at 25±1℃, and then use the methods of Examples 1-3 and Comparative Examples 1-3 to treat, respectively, and set 3 parallel experiments for each group.

[0095] 1. Degradation efficiency monitoring

[0096] Sample at 0h, 6h, 12h, 24h, 48h, 72h, respectively, determine the concentration of phenol in the water body by high performance liquid chromatograph, and calculate the removal rate at different time points.

[0097] The test results are shown in Table 1 below.

[0098] Table 1

[0099] ;

[0100] 2. COD removal rate determination

[0101] (1) Sampling: water samples at 0h, 6h, 12h, 24h, 48h and 72h were taken from the experimental group and each comparative example, respectively, and the water samples were thoroughly mixed before sampling, and 3 parallel samples were taken at each time point.

[0102] (2) Digestion: 20mL of water sample (if the COD value is high, the sample needs to be diluted) was accurately taken into a 250mL ground reflux conical flask, 10mL of potassium dichromate standard solution and a few small glass beads or zeolite were added, a ground reflux condenser tube was connected, 30mL of sulfuric acid-silver sulfate solution was slowly added from the top of the condenser tube, the conical flask was gently shaken to mix the solution, and heated for 2h (the time was counted from the beginning of boiling).

[0103] (3) Titration: after cooling, the condenser tube wall was washed with 90mL of water, and the conical flask was removed. The total volume of the solution should not be less than 140mL, otherwise the end point of titration is not obvious due to too much acidity. 3 drops of nitrilotriacetic acid indicator solution were added, and the solution was titrated with ferrous ammonium sulfate standard solution. The color of the solution changed from yellow to blue-green to red-brown, which was the end point. The amount of ferrous ammonium sulfate standard solution used was recorded.

[0104] (4) Blank test: distilled water was used instead of water sample, and the determination was carried out according to the same procedure. The amount of ferrous ammonium sulfate standard solution used in the blank test was recorded.

[0105] The test results are shown in Table 2 below.

[0106] Table 2

[0107] ;

[0108] As can be seen from Tables 1 and 2 above, the phenol removal rate is 93.5% and the COD removal rate is 91.6% at 72h, and the values of the two are close and are at a high level. This shows that the present application can quickly adsorb phenol through multi-level pores, catalyze its oxidative cracking into small molecules by metal oxide nanoclusters, and completely mineralize the intermediate products by microorganisms, realizing the whole process efficient treatment of pollutants from adsorption, degradation to mineralization.

[0109] The above description is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent substitutions or changes within the technical scope disclosed by the present application and according to the technical solution and inventive concept of the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for rapidly degrading refractory dissolved organic matter in a lake water body using a microbial inoculum, characterized in that, It comprises the following steps: S1, the PLGA microspheres embedded with sodium nitrate and AQS are mixed with a catalyst carrier, vacuum adsorbed at 40 DEG C for 1-2h, mixed with sodium carbonate embedded calcium alginate / chitosan microspheres according to 2:1, sterile water is added to make the microspheres fully wet, stirred at room temperature for 4-5h, vacuum dried at 40-50 DEG C for 10-12h; The preparation of the catalyst carrier comprises the following steps: (1) the natural attapulgite is crushed and sieved, then mixed with a citric acid solution with a mass fraction of 6-8% according to a solid-liquid ratio of 1:5, stirred and treated at 60-80 DEG C for 20-26h, then washed with deionized water until neutral, dried at 90-100 DEG C for 20-30h, then mixed with mullite hollow ceramic microbeads according to a mass ratio of 8-10:1, finally calcined in a muffle furnace at 700-750 DEG C for 4-6h to obtain a multi-level pore structure carrier; (2) the multi-level pore structure carrier is dispersed in anhydrous ethanol according to a solid-liquid ratio of 1:10, then 3-aminopropyl triethoxysilane is added, refluxed at 80-90 DEG C for 6-8h, washed with ethanol for 3-4 times, then vacuum dried at 70-80 DEG C for 6-8h to obtain an amino-functionalized carrier; (3) the amino-functionalized carrier is added to a core-shell catalyst suspension according to a solid-liquid ratio of 1:8, 1% glutaraldehyde crosslinking agent is added, stirred at 60-70 DEG C for 3-4h, then filtered and dried; The preparation method of the core-shell catalyst suspension is as follows: a mixed solution of 0.2 mol / L FeCl3 and 0.1 mol / L FeSO4 is prepared, the molar ratio of Fe 3+ to Fe 2+ is 2:1, the pH is adjusted to 10-11, and stirring is carried out at 60-70 DEG C for 1-2 h under N2 protection to generate Fe3O4 nanoparticles; the Fe3O4 nanoparticles are dispersed in a 0.05 mol / L KMnO4 solution, a 0.1 mol / L MnSO4 solution is added dropwise, the molar ratio of Mn 7+ to Mn 2+ is 1:1, and stirring is carried out at 40 DEG C for 2-3 h. S2, the catalyst carrier treated in step S1 is immersed in an anaerobic bacteria liquid, vacuum suctioned for 5min, 5% CaCl2 is added and crosslinked for 20-30min to obtain an anaerobic bacteria immobilized carrier; S3, the anaerobic bacteria immobilized carrier is immersed in an aerobic bacteria glue liquid, air dried at 25 DEG C for 30-40min to obtain an immobilized bacteria agent; S4, the immobilized bacteria agent is put into the water body to be treated.

2. The method for rapidly degrading refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, 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 according to a mass ratio of 10:1, filtered and sterilized; PLGA is weighed, dissolved in dichloromethane according to a mass-volume ratio of 1g:20-30mL, the obtained sodium nitrate-AQS solution is added to the PLGA solution, the volume ratio is 1:2, emulsified at 10000rpm under ice bath conditions for 5-6min to form a stable W / O emulsion, the W / O emulsion is poured into an aqueous solution containing 2% PVA, emulsified at 8000rpm for 8-10min to form a W / O / W multiple emulsion, the multiple emulsion is placed in a fume hood, magnetically stirred until dichloromethane is completely volatilized, the microspheres are collected by centrifugation at 3000-4000rpm at 4 DEG C, washed with ultrapure water, then freeze-dried to obtain the PLGA microspheres.

3. The method for rapidly degrading the refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, In step (1), the sieving is 200 mesh sieving; The properties of the hollow ceramic microbeads are: particle size 30-60 mu m, wall thickness 2-5 mu m, density 0.6 g / cm 3 , temperature resistance > 1200 DEG C.

4. The method for rapidly degrading refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, In step (2), the mass ratio of 3-aminopropyl triethoxysilane to the multi-level pore structure carrier is 0.1-0.15:

1.

5. The method for rapidly degrading the refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, The preparation of the sodium carbonate embedded calcium alginate / chitosan microspheres in step S1 comprises the following steps: 1) Dissolve sodium carbonate in ultrapure water with a mass-volume ratio of 1:10, add 0.2 times the mass of sodium alginate, stir for 4-5 h, filter and sterilize to obtain a sodium carbonate / sodium alginate solution; 2) Dissolve chitosan in 1% acetic acid solution with a mass-volume ratio of 1 g:100 mL, stir for 20-26 h, adjust the pH to 5.5 with 1M NaOH, filter and sterilize to obtain a chitosan solution; 3) Use a syringe to drop the sodium carbonate / sodium alginate solution into a 0.05 g / mL calcium chloride cross-linking solution at a flow rate of 1 mL / min, cross-link at room temperature for 30 min to form calcium alginate microspheres, collect the microspheres, wash with ultrapure water 3 times, transfer the calcium alginate microspheres to the chitosan solution described above, stir at room temperature for 20-30 min, wash the microspheres with ultrapure water 3 times, spread the microspheres on a sterile culture dish, dry in a ventilated oven at room temperature for 24 h, and vacuum dry at 40°C for 8 h to a constant weight to obtain sodium carbonate-embedded calcium alginate / chitosan microspheres.

6. The method for rapidly degrading refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, The anaerobic bacteria solution in step S2 is a mixture of Desulfovibrio and Paracoccus denitrificans in a 1:1 ratio added to a 0.1M CaCl2 sodium alginate solution, with a mass fraction of 2% sodium alginate.

7. The method for rapidly degrading refractory dissolved organic matter in a lake water body by using microbial inoculants according to claim 1, characterized in that, The aerobic bacteria solution in step S3 is Bacillus subtilis added to a 0.5% κ-carrageenan / 1% gelatin solution.