Double-embedded particles and their preparation method, and their application in the decomposition of estrogen in wastewater

Abstract

This invention provides double-buried particles for wastewater treatment and their application in the decomposition of estrogen in wastewater. [Solution] Double-embedded particles are obtained by preparing artificial photosynthetic particles embedded with photocatalysts and white-rot fungi, and natural photosynthetic particles embedded with bacteria and green algae. The double-embedded particles have a good removal effect on bound estrogen and artificially synthesized free estrogen in wastewater. The double-embedded photosynthetic particles solve the problems of low decomposition efficiency of biological treatment and poor toxicity reduction effect of advanced oxidation treatment in existing technologies, and provide a new means of wastewater treatment.

Description

[Technical Field]

[0001] The present invention relates to the technical field of wastewater treatment, and more specifically, to double-embedded particles and a method for preparing them. This relates to the application of estrogen decomposition in wastewater. [Background technology]

[0002] The operation of wastewater treatment plants by local governments is facing new challenges and opportunities. Biochemical treatment processes are effective in reducing conventional pollution indicators and meeting basic environmental requirements. However, when faced with highly toxic, bio-resistant, and difficult-to-decompose contaminants, the solution is to deal with them. It becomes impossible to do so. For a long time, estrogen plays a special role in the body, so widely Although it has been studied and applied, its persistence and accumulation in the natural environment pose a significant ecological risk. This has gradually become clear. In the field of wastewater treatment, advanced oxidation technology is used to deeply purify estrogen, which is difficult to decompose. Biological enhancement technologies, as two cutting-edge strategies, are demonstrating their own unique advantages and limitations. First, high The oxidation technology has a double-edged sword: it has a fast reaction rate and excellent decomposition efficiency, but it also has the drawback of being able to decompose and generate The substance may have more serious toxicity problems than the original pollutant, and therefore it cannot be widely applied. The prospects are somewhat limited. Biological enhancement technologies reduce the toxicity of pollutants and the environment It is particularly excellent at contributing to restoration and ecosystem balance. However, it is also effective in producing persistent pollutants. Regarding the suppression of material degradation, the degradation efficiency of conventional biological enhancement techniques often fails to meet expectations. Furthermore, when used alone, it often fails to exert its intended effect, which diminishes its effectiveness. This is a significant limiting factor. [Overview of the project]

[0003] The present invention adopts the following technical solutions: The present invention provides double-embedded particles composed of artificial photosynthetic particles and natural photosynthetic particles, where the artificial photosynthetic particles consist of calcium alginate hydrogel, a photocatalyst embedded in the calcium alginate hydrogel, and white-rot fungi, and the photocatalyst is the MOF material NH2 -MIL-53(Fe), where the natural photosynthetic particles consist of calcium alginate hydrogel, green algae embedded in the calcium alginate hydrogel, and Bacillus subtilis, the particle diameter of the artificial photosynthetic particles is 2 - 5 mm, the particle diameter of the natural photosynthetic particles is 2 - 5 mm, and the number ratio of the artificial photosynthetic particles to the natural photosynthetic particles is (1 - 6):(1 - 6). In the present invention, the working principle of the artificial photosynthetic particles is that white-rot fungi have the characteristic of secreting laccase. In the presence of white-rot fungi, laccase (Lac) is generated in the system, and the photocatalyst captures visible light to generate electrons, and the electrons move to the T1 Cu(II) of laccase to initiate an enzymatic reaction. Since an organic covalent bond system is formed between the photocatalyst and laccase, the active site of laccase and the catalytic activity ability of laccase are enhanced, catalyzing the ring-opening and bond cleavage of estrogen, and easily removing mixed estrogen from pollutants. Green algae not only further remove mixed estrogen under photosynthesis and play a role in reducing the estrogen effect generated, but also generate oxygen for microbial respiration. Bacillus subtilis further decomposes the photocatalytic degradation estrogen intermediate, and generates various vitamins such as CO2 and vitamins B1 and B2 for microalgae absorption. As one aspect of the present invention, the preparation method of the photocatalyst is as follows: 1.08 - 2.02 ​​​​​​​Take g of FeCl3·6H2O and 0.73-0.91 g of NH2-H2BDC, and 50- Dissolve in 80 mL of organic solvent and perform hydrothermal synthesis at 150-170°C for 18-24 hours under sealed conditions. After cooling, wash with an organic solvent to remove the organic solvent, then remove the iron-based organometallic skeleton NH2-MIL-53(F e) is obtained and used as the photocatalyst, where the organic solvent is NN dimethylformamide. During the dissolution process, preferably, dissolution is promoted by ultrasonic or magnetic stirring. Alternatively, first dissolve 2-aminoterephthalic acid in an organic solvent, then add ferric chloride. The hydrothermal synthesis is preferably carried out in a polytetrafluoroethylene reaction vessel. During washing, Alternatively, the reaction product is cooled to room temperature (25°C). The washing is performed with DMF and anhydrous ethanol. The alternating washing and centrifugation can also be repeated three times. After the washing, preferably obtained The resulting product is dried. The drying temperature is preferably 70-90°C, and is 80°C. The drying time is preferably 6 hours. In another aspect of the present invention, the present invention provides a method for preparing the above-mentioned double-embedded particles, and the following steps Includes: S1, Preparation of artificial photosynthetic particles: 25-50 mL OD 600 Take a white rot fungal sap with a concentration of 0.8-1.0 and 5000-70 Centrifugation was performed at 00 rpm for 9-11 minutes to obtain white rot fungal cells, and the white rot fungal cells, 0 0.03-0.06 g of photocatalyst and 50-80 mL of sodium alginate with a mass concentration of 1-2% Mix the um solution and use a 2-5 mL syringe to dispense 50-100 mL with a mass concentration of 2-5%. Add to CaCl2 solution dropwise and allow to fix for 30-60 minutes to obtain artificial photosynthetic particles. Preparation of S2, natural photosynthetic particles: 25-50 mL OD 680Take a green algae solution with a pH of 0.8-1.0 and 4000-6000 rp Centrifuge at m for 10-15 minutes to collect the green algae, then add 25-50 mL of OD. 600 0. Take a Bacillus subtilis solution with a concentration of 8-1.0 and steep it in a well-ventilated chamber at 6000-8000 rpm for 10-15 minutes. After separating the heart and recovering the Bacillus subtilis cells, The green algae and Bacillus subtilis cells are suspended in 5-8 mL of ultrapure water, and the mass concentration is 50-80 mL. A fungal-algal mixture is obtained by uniformly mixing it with a 1-2% sodium alginate solution, and the fungal-algal mixture Using a 2-5 mL syringe, dispense 50-100 mL of a 2-5% CaCl2 solution. The mixture is added dropwise and allowed to react for 30-60 minutes to obtain natural photosynthetic particles. The sodium alginate solution and calcium chloride solution of S1 and S2 described above should be reduced before use. Sterilization is preferable, and the preferred sterilization method is treatment at 121°C for 30 minutes. The method for dispensing calcium chloride solution is to fill a 5 mL syringe with the solution to be dispensed. It is preferable. In one aspect of the present invention, the method for preparing the white rot fungal solution is as follows: white rot fungus The fungal cells were inoculated into a liquid potato complete medium, and the white rot fungal saturation OD600 was reduced to 0 at 28-32°C. Cultivate until the volume reaches 0.8-1.0 to obtain a white rot fungal solution, and add it to the aforementioned liquid potato medium. 1 liter of 0% potato juice, 20g glucose, 3g KH2PO4, MgSO4·7H2O It contains 1.5g and 1g of thiamine, and has a pH of 6. The method for preparing the aforementioned green algae solution is as follows: Green algae are inoculated into BG11 medium, and 24-26 Cultivate at °C and 2000 Lux for 1-2 weeks, then OD 680 This yields a green algae solution of 0.8-1.0, and The green alga mentioned is Chlorella. The method for preparing the Bacillus subtilis bacterial solution is as follows: Inoculate Bacillus subtilis into a nutrient-rich meat juice medium, and 28~ OD of Bacillus sublimate under conditions of 30℃ and 120-130 rpm 600 The value becomes 0.8 to 1.0. After culturing, add 5.0g of peptone, 3.0g of beef extract, and NaCl to the aforementioned nutrient broth medium. It contains 5.0g and 1 L of distilled water, with a pH of 7.0-7.2. In another aspect of the present invention, the present invention relates to the double-embedded particles or the particles prepared by the above preparation method. This further provides applications for the double-embedded particles in the decomposition of estrogen in wastewater. In one aspect of the present invention, the estrogen is conjugated estradiol and / or free-floating estrogen. It is mold release estradiol. Preferably, the conjugated estradiol is estradiol-3-sulfate, and the free The release agent is 17α-ethinylestradiol. The wastewater is preferably This is secondary wastewater from a sewage treatment plant. In one aspect of the present invention, the disassembly method is as follows: The double-embedded particles are added to estrogen-containing wastewater, and the concentration of the double-embedded particles in the wastewater reaches 60 The concentration is 0-800 particles / L, and after light irradiation treatment, the light irradiation intensity is 60-240 μmol·s. - 1 ·m -2 The temperature should be 20-25°C, and the decomposition should be completed after processing for 4-6 hours. It is possible. Preferably, in the present invention, the ratio of the number of artificial photosynthetic particles to natural photosynthetic particles is The ratio is 1-6:1-6, and it can also be (1-3):(1-3), or even 1:1. Good. The effective concentration of artificial photosynthetic particles in the double-embedded particles is preferably 100 to 600 particles / It is L, and may be 200 pieces / L, 300 pieces / L, 400 pieces / L, or 500 pieces / L. The effective concentration of natural photosynthetic particles is 100-600 particles / L, and also 200 particles / L and 300 particles / L. It may also be 400 / L or 500 / L. Specifically, it is decomposed using a photocatalytic batch-type bioreactor (PSBR), and the light irradiation intensity is preferably 120-220 μmol·s -1 ·m -2 and may also be 150 μmol·s -1 ·m -2 1[[ID=E14]] 80 μmol·s -1 ·m -2 or 200 μmol·s -1 ·m -2 or 220 μmol·s -1 ·m -2 It may also be. The temperature may be 21°C, 22°C, 23°C, or 24°C. During the operation of the photocatalytic batch-type bioreactor, the drainage ratio of each operation cycle is preferably 50%-55% and may also be 52% or 54%. The hydraulic retention time is preferably 4-6 h and may also be 5 h. The operation cycle includes sequential water intake, standing, aeration, sedimentation, and drainage. The water intake time is preferably 3-5 min and may also be 4 min. The standing time is preferably 5-10 min and may also be 6 min, 7 min, or 8 min. The aeration time is preferably 205-220 min and may also be 205 min, 210 min, 2 15 min, or 220 min. The aeration volume is preferably 0.5-1 L / min and more preferably 0.5 L / min. The sedimentation time is preferably 10-1 5 min and may also be 12 min, 13 min, or 14 min. The drainage time is preferably 3-5 min and may also be 4 min.

Advantages of the Invention

[0004] Compared with the prior art, the present invention has the following obvious advantages. (1) The present invention relates to the immobilization of a microbial strain having estrogen-degrading function, thereby improving wastewater Double embedding can significantly improve the survival capacity and decomposition efficiency of microorganisms in the environment. We propose a particle technology for the first time. Due to the stability of immobilized microbial particles, it can be used in complex aquatic environments. Even if microorganisms remain, they can continue to exert their decomposition action, thus reducing estradiol in wastewater. Estrogen such as 3-sulfate (E2-3S) and 17α-ethinylestradiol (EE2) It can effectively remove harmful substances. (2) By providing a relatively stable microenvironment for microorganisms, the dual-embedded particle technology provides these This effectively counteracts unfavorable factors and enables microorganisms to maintain efficient degradation activity under a wider range of environmental conditions. To make it possible to possess. (3) By using the double-embedded particle technology of the present invention, on the one hand, immobilized microbial particles The preparation and use of this product is relatively simple and does not require complex equipment or numerous chemicals. On the other hand, microbial decomposition can occur at room temperature without requiring additional energy support. It can be performed at normal pressure. (4) The present invention overcomes the shortcomings of current deep remediation processes for persistent organic pollutants, and To improve the low effectiveness and unstable operation of existing technologies for removing organic pollutants, and for secondary wastewater treatment plants. A gap between domestic and international technologies for the economical and efficient removal of persistent organic pollutants from wastewater. Fill in the blank. (5) The method provided by the present invention can be adjusted according to the characteristics and treatment requirements of different wastewater. It offers high flexibility and controllability, and can be applied to many different types of wastewater treatment. (6) The present invention involves embedding white rot fungi and NH2-MIL-53(Fe) to create artificial photosynthetic particles White rot fungi produce laccase, and NH2-MIL-53(Fe) solidifies the laccase. By functioning as a permanent carrier, the laccase (Lac) produced by white rot fungi The active site is covalently immobilized on the surface of NH2-MIL-53(Fe), and the catalytic activity of the system Not only is performance improved, but the accessibility distance to the active site is significantly shortened. (7) The present invention provides natural photosynthetic particles obtained by co-culturing green algae and Bacillus subtilis, which can be used in wastewater The CO2 produced is absorbed by microalgae, and at the same time, the oxygen released by the photosynthesis of the microalgae is also absorbed by microalgae. Metabolized by biological respiration, energy utilization rate improves to over 70%, and carbon emissions are reduced through aeration. The amount of emissions can be reduced by approximately 50%. Natural photosynthetic particles achieve self-sufficiency in oxygen and nutrients. Instead, the carbon, nitrogen, and phosphorus in wastewater are converted into high-value proteins and fats, and the carbon... By reducing waste, transforming it into valuable resources, and achieving near-zero urban wastewater discharge, we can achieve carbon neutrality. It can provide effective methods for achieving strategic goals. [Brief explanation of the drawing]

[0005] [Figure 1] This is a schematic diagram of the structural composition of the double-embedded particles. [Figure 2] This is a schematic diagram of the preparation process for double-embedded particles. [Figure 3] This is a scanning electron microscope image of a double-embedded particle. [Modes for carrying out the invention]

[0006] The following examples illustrate the double-embedded particles provided by the present invention, as well as the method for preparing them and wastewater The use in removing mixed estrogens within will be described in detail, but these are within the scope of protection of the present invention. It should not be understood as something that limits the scope. Example 1 In this example, artificial photosynthetic particles embedded with NH2-MIL-53(Fe) and white rot fungi were used. We label Particle 1, and Particle 2 refers to natural photosynthetic particles containing Bacillus subtilis and green algae, and Particle 1 and the grain Sub-sub This simulates secondary wastewater, containing estradiol-3-sulfate (E2-3S) and 17α-E It contains thinylestradiol (EE2). The specific preparation method and processing steps are as follows: The steps include: (1) Preparation of artificial photosynthetic particles: (I) Preparation of NH2-MIL-53(Fe): 1.08 g of FeCl3·6H2O, 0.73 Place g of NH2-H2BDC and 50 mL of DMF into a 250 mL conical flask. Ultrasonic treatment at 100W for 5 minutes was performed to promote dissolution, and the resulting solution was divided into 100 mL polytetraphthol. The mixture is sealed in a hydroethylene reaction vessel, subjected to a hydrothermal reaction at 170°C for 24 hours, cooled to room temperature, and then processed sequentially. Next, wash alternately with DMF and anhydrous ethanol, centrifuge three times, and centrifuge at 4000 rpm for 5 minutes. After centrifugation and material recovery, the material is dried at 80°C for 6 hours, and the photocatalyst NH2-MIL-53(Fe) is used. I got it. (II) Method for large-scale cultivation of white rot fungi: White rot fungi in slant test tubes in an ultra-clean bench Then, using an inoculation ring, take a certain amount of bacterial cells and add them to 100 mL of liquid potato at pH 6. Inoculate into a culture medium and osmote the bacterial suspension at 30°C. 600 The culture was continued until the concentration reached 0.8. (Liquid potato) The complete culture medium consists of 1 liter of 20% potato juice, 20 g of glucose, 3 g of KH2PO4, and MgS Contains 1.5g of O4·7H2O and 1g of thiamine. (III) Dissolution of embedding agent: Add 4g anhydrous CaCl2 to 100mL of ultrapure water and adjust the pH to 7. Prepare the solution to obtain a CaCl2 solution, and then vibrate 2 g of sodium alginate (SA) solid powder with an ultrasonic vibrator. Add 100 mL of water under low temperature, and help dissolve it in a 50°C water bath to adjust the pH to 7. Obtain a sodium alginate solution, and place the CaCl2 solution and the sodium alginate solution in a sterilization pot. In addition, the samples were sterilized at 121°C for 30 minutes. (IV) Particle embedding: Under room temperature conditions, weigh 0.05 g of photocatalyst NH2-MIL-53(Fe) and Prepared by taking 50 mL of white rot fungal solution and centrifuging it at 6000 rpm for 10 minutes. The bacterial cells were obtained and prepared. The weighed photocatalyst NH2-MIL-53(Fe) and the obtained bacterial cells were then combined. Mix with sodium alginate solution and use a 5 mL syringe to add 50 mL of CaCl2 solution. The mixture was dropped onto a substrate and allowed to react for 30 minutes to obtain approximately 300 artificial photosynthetic particles. (2) Preparation of natural photosynthetic particles: (I) Concentration culture of bacterial algae: Green algae (Chlorella) were inoculated into BG11 medium and cultured at 25°C for 2 Cultured at 000 Lux for 2 weeks, OD 680 A green algae solution of 1.0 was obtained, and 50 mL of the algae solution was taken. The green algae were collected by centrifuging at 4000 rpm for 10 minutes. The above procedure was performed in an ultra-clean environment. It took place inside the building. Bacillus subtilis is cultured in a nutrient medium containing beef juice (5.0g peptone, 3.0g beef extract, 5.0g NaCl). And inoculate with 1 L of distilled water (pH 7.0-7.2) and under conditions of 28°C and 120 rpm OD of bacterial solution 600 0.8 was cultured, 50 mL of bacterial suspension was taken, and under conditions of 6000 rpm 1 The bacterial cells were collected after centrifuging for 0 minutes. (II) The preparation of the embedding agent is the same as the dissolution of the embedding agent in step (III) of step (1). (III) The green algae and fungal cells collected in step (I) are suspended in 5 mL of ultrapure water, and 50 mL of a Mix uniformly with sodium luginate solution to obtain a fungal-algal mixture. 5 mL of the fungal-algal mixture was added to a packet. Using a needle-equipped injector, add the solution dropwise to 50 mL of CaCl2 solution and allow to fix for approximately 30 minutes. We obtained 300 natural photosynthetic particles. The white rot fungi, Chlorella, and Bacillus subtilis used in this invention are all commercially available products. (3) Method for treating wastewater containing double-embedded photosynthetic particles: (I) A 1L photodegradable batch biological reaction using artificial photosynthetic particles and natural photosynthetic particles in a 1:1 ratio. In addition to the container (PSBR), the effective concentration of artificial photosynthetic particles is approximately 300 particles / L, and natural photosynthesis The particle concentration is approximately 300 particles / L, and the light-illuminated culture conditions are: pH value of 7-8, and aeration intensity of 0. 5 mL / min, light irradiation cycle of 12 h / day, light irradiation intensity of 120 μmol·s -1 ·m -2 That was the case. (II) The photodegradation batch type biological reactor has an operating cycle of 4 hours, with each cycle consisting of 3 minutes of water intake and 5 minutes of water intake. The process includes min standing, 219 min aeration, 10 min sedimentation, and 3 min drainage. The height-to-diameter ratio of the bioreactor is 30:8, and the water intake, drainage, and aeration of the batch-type bioreactor are All of them are connected to the peristaltic pump, solenoid valve and aeration pump via an automatic controller. It operates dynamically, and the aeration system is located at the bottom of the batch-type bioreactor to supply sufficient oxygen. It was done. (III) The wastewater added to the photodegradation batch bioreactor is artificial simulated wastewater, where CO The D concentration is 200 mg / L, and ammonium nitrogen (NH4) + -N) Concentration 40 mg / L And phosphate (PO4 3- -P) The concentration is 3.5 mg / L, and estradiol-3- The sulfate (E2-3S) concentration is 2 μg / L, and 17α-ethinylestradiol (E The concentration of E2 is 2 μg / L. The E2-3S and EE2 content in the wastewater is ultrafast liquid. Quantitative detection is performed using chromatographic tandem mass spectrometry, and the specific steps are as follows: It is: 1. Pre-processing: Pre-process the sample, 2. Column connection: The column selected is BEH C18 1.7μm, 2.1×50mm. 3. Column cleaning: Before mounting the column in the instrument, clean it using the mobile phase and organic phase. The duration should be 5-10 minutes. 4. Editing the instrument parameters: Mobile phase: ultrapure water, Organic phase: methanol, Injection flow rate: 0.25 m / s The flow rate is set to L / min, the injection volume to 20 μL, and the operating time to 6 min. 5. Analysis: Use either "Single Analysis" or "Fast Batch Processing" in the software to analyze the sample. Enter the bolt and bolt holder. 6. Data Analysis: Lab-solution Main Projector "Processing Tools", "Re-analysis" Open it to view the analysis results using ''. 7. Finally, wash the column with 100% methanol for 30 minutes. 8. Shutdown: Close the "LC" and "MC" buttons at the top of the software. The estrogen effect was measured using yeast cell bioluminescence, and the estrogen treatment step was The following applies: 1. Using an HLB solid-phase extraction column, add 3 ml of ethyl acetate, 3 ml of methanol and 6 Activate with mL of ultrapure water, and set the flow rate to 1-3 drops / s. 2. Add the sample dropwise to the activated HLB column, and set the flow rate to 1-2 drops / s. 3. After sampling, rinse the sample with 9 mL of ultrapure water to remove salt and impurities. Sushi, 4. Use a filter pump to dry the filler until it becomes a powder. 5. Elute the pear-shaped bolts using 6 mL each of ethyl acetate and methanol. 6. Increase the temperature of the nitrogen blowing device to 40°C, reduce the needle size, and extend the liquid level by 1 cm. Spray until wet, then use methanol to chromatograph a fixed volume of material. Then, a sample methanol extract was obtained. The testing method for estrogen effects is as follows: 1. Add 500 μl of yeast cells to 100 mL of leucine, uracil, and tryptophan-free solution. Inoculate into a yeast basal culture medium, 2. Culture under the conditions of 28°C and 200 rpm, OD 600 Shake until it reaches 0.6-0.8 Toushi, 3. Transfer 20 µl of estradiol (E2) standard material to an opaque 96-well plate. Used for detecting yeast estrogen, 4. Add 20 μl of methanol to three separate wells to use as a blank control. 5. Dilute the 20 μl sample methanol extract obtained above, and make the diluted solution opaque 96 In addition to well plates, 6. Add methanol and allow to evaporate until no visible wetting points remain. 200 μl of yeast The culture was added to an E2 96-well plate and used to detect the estrogen effect. 7. Cover the top with aluminum foil, set the culture temperature to 28°C and the time to 8 hours, and use an enzyme marker. The light intensity results were detected, and the enzyme marker conditions were set as follows: Bioluminescence was continuously detected for 8 hours at 28°C, with an accumulated time of 1 second / well every hour, and the following formula was used: The amount of bioluminescence was calculated according to 1). Bioluminescence = Minimum value + (Maximum value - Minimum value) / [1 + (Initial concentration / EC] 50 )] (1) Here, the minimum value refers to the minimum luminescence intensity of the detected yeast cells, and the maximum value refers to the maximum luminescence intensity of the detected yeast cells. This refers to the maximum luminescence intensity of the parent cell, and the initial concentration refers to the effective value of EE2 at 2 ug / L. EC50 refers to contaminants that cause estrogenic effects in 50% of the test yeast cell population. This refers to the concentration of EE2, i.e., the semi-effective concentration of EE2. Example 2 This embodiment provides a method for preparing double-embedded particles under different parameters, and includes the following steps: fruit: (1) Preparation of artificial photosynthetic particles: (I) Preparation of NH2-MIL-53(Fe): 2.02 g of FeCl3·6H2O, 0.9 Take 1 g of NH2-H2BDC and 80 mL of DMF and add it to a 250 mL conical flask. Place in a container, use 100W ultrasound for 5 minutes to promote dissolution, and then pour the resulting solution into a 100mL poly bottle. The mixture is sealed in a tetrafluoroethylene reaction vessel and subjected to a hydrothermal reaction at 150°C for 18 hours, then cooled to room temperature. Afterwards, the material is washed alternately with DMF and anhydrous ethanol, centrifuged three times, and then heated at 4000 rpm. After centrifuging for 5 minutes and recovering the material, dry it at 90°C for 6 hours to obtain the photocatalyst NH2-MIL-5. 3(Fe) was obtained. (II) Method for large-scale cultivation of white rot fungi: White rot fungi in slant test tubes in an ultra-clean bench Then, using an inoculation ring, a certain amount of bacterial cells is placed in 100 mL of pH 6 liquid potato culture medium. Seeds are planted and the fungal solution is OD at 28°C. 600 The culture was continued until the value reached 1.0. The liquid potato composite medium was 1L of 20% potato juice, 20g glucose, 3g KH2PO4, MgSO4·7H Contains 1.5g of 2O and 1g of thiamine. (III) Dissolution of embedding agent: Add 4g anhydrous CaCl2 to 100mL of ultrapure water and adjust the pH to 7. Prepare the solution to obtain a CaCl2 solution, and then vibrate 2 g of sodium alginate (SA) solid powder with an ultrasonic vibrator. Add 100 mL of water under low temperature, help dissolve in a 60°C water bath pot, adjust the pH to 7, and Obtain a sodium alginate solution, and place the CaCl2 solution and the sodium alginate solution into a sterilization pot. The samples were then sterilized at 121°C for 30 minutes. (IV) Particle embedding: Weigh 0.03 g of photocatalyst NH2-MIL-53(Fe) under room temperature conditions. I prepared the following: I took 80 mL of white rot fungal solution and centrifuged it at 7000 rpm for 11 minutes. The bacterial cells were separated and prepared. The weighed photocatalyst NH2-MIL-53(Fe) and the acquired Mix the bacterial cells with sodium alginate solution, and then use a 2 mL syringe to add 100 mL of CaC The particles were added dropwise to solution L2 and subjected to a fixation reaction for 60 minutes to obtain approximately 500 artificial photosynthetic particles. (2) Preparation of natural photosynthetic particles: (I) Concentration culture of bacterial algae: Green algae (Chlorella) were inoculated into BG11 medium and cultured at 26°C for 2 Cultured at 000 Lux for 1 week, OD 680 A 0.8 green algae solution was obtained, and 25 mL of the algae solution was 600 The green algae were collected by centrifuging at 0 rpm for 15 minutes. All of the above operations were performed in an ultra-clean bench. It took place in [location]. Bacillus subtilis is cultured in a nutrient medium containing beef juice (5.0g peptone, 3.0g beef extract, 5.0g NaCl). The bacteria were inoculated into 1 L of distilled water (pH 7.2) and the bacterial suspension was subjected to 30°C and 130 rpm conditions. D 600 Incubate until the concentration reaches 1.0, then incubate 25 mL of bacterial solution under 8000 rpm conditions for 15 minutes. The bacterial cells were recovered by centrifugation. (II) The preparation of the implantation material is the same as the dissolution of the implantation material in (III) of step (1). (III) The green algae and fungal cells collected in step (I) are suspended in 8 mL of ultrapure water, and 80 mL of a Mix uniformly with sodium luginate solution to obtain a fungal-algal mixture. 2 mL of the fungal-algal mixture was added. Using a syringe (with needle), add the solution dropwise to 100 mL of CaCl2 solution and allow it to fix for 60 minutes. Approximately 500 natural photosynthetic particles were obtained.

[0007] Comparative Example 1 Compared to Example 1, the following differences apply: artificial photosynthetic particles are used instead of natural photosynthetic particles. 0.05g of MIL-53(Fe) material is added to a batch bioreactor (PSBR), and light is shone. Culture conditions: pH 7-8, aeration intensity 0.5 mL / min, light irradiation cycle 12 h / day, light irradiation Radiation intensity 120μmol·s -1 ·m -2 That's what I decided. Here is the preparation method for MIL-53(Fe): 0.83 g of terephthalic acid in 28 mL of NN It was added to a beaker containing dimethylformamide (DMF) and completely dissolved under magnetic stirring. Then, an equimolar amount of FeCl3·6H2O (1.35g) was added. The mixture was left at room temperature for 30 minutes. After stirring, transfer the solution to a 100 mL polytetrafluoroethylene-lined autoclave. The mixture was transferred and reacted at 170°C for 24 hours. Finally, the autoclave was allowed to cool naturally to room temperature. Afterwards, the material was washed alternately with anhydrous ethanol and NN dimethylformamide and centrifuged three times. After recovery, the material was dried at 80°C for 6 hours to obtain the photocatalytic MIL-53(Fe) material. Comparative Example 2 Compared to Example 1, it differs in the following respects, with artificial photosynthetic particles and natural photosynthetic particles being used instead. 0.05g of NH2-MIL-53(Fe) is added to a 1L batch bioreactor (PSBR). In addition, the conditions for light-illuminated culture were: pH value 7-8, aeration intensity 0.5 mL / min, and light irradiation cycle 12 hours. / day, light irradiation intensity 120μmol·s -1 ·m -2 That's what I decided. Comparative Example 3 Compared to Example 1, the following points differ: the addition of artificial photosynthetic particles was omitted, and approximately 300 particles were used. Only natural photosynthetic particles were added to a 1L batch bioreactor (PSBR), and the conditions for light-illuminated culture were applied. pH value 7-8, aeration intensity 0.5 mL / min, light irradiation cycle 12 h / day, light irradiation intensity 12 0 μmol·s -1 ·m -2 That's what I decided. The results of the decomposition of mixed estrogen in wastewater from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were examined. Output and analysis: 100 mL water sample filtered through a 0.45 μm glass fiber filter. Filter with a Blenn (aqueous system), store in a refrigerator at 4°C after filtration, and then proceed with the solid-phase extraction procedure and This was then used for nitrogen spraying, and finally, estrogen concentration and effect were measured. Each experiment was repeated three times. The analysis was performed using the mean ± standard deviation. The E2-3S removal rate was determined according to the following formula (2). The EE2 removal rate was calculated. E2-3S removal rate or EE2 removal rate = (1-C / C0) × 100% (2) Here, C0 is the initial concentration of E2-3S or EE2, and C is the E2-3S after the reaction. The values ​​represent the concentration of EE2, and the results are shown in Table 1. Table 1: E2-3S / EE2 removal rate and estrogen effect reduction efficiency in different modes As can be seen from Table 1 of TIFF0007854582000001.tif38115, the double-embedded particles of Example 1 removed E2-3S and EE2. The decongestive effect was observed in NH2-MIL-53(Fe) (Comparative Example 2) and natural photosynthetic particles (Comparative Example 3). The percentages were also high, at 13.9%, 5.8%, 9.2%, and 4.4%, respectively. Using cell luminescence, E2 was used as a positive standard substance for the estrogen mimic test, with a concentration range of 1 0 -13 M~10 -7 We obtained a reduction in the estrogen effect by exposing M to E2. Regarding the effect reduction effect, the double-embedded particles are made of NH2-MIL-53(Fe)MOF material and natural materials. These figures were significantly higher than those of naturally photosynthetic particles, at 98.2% and 89.3%, respectively. As can be seen from the results, the NH2-MIL-53(Fe)MOF material and fungal-algal symbiotic bond The processing method for double-embedded particles is for E2-3S and EE2, in particular bound estrogen (E2- Although it has a high removal effect against 3S, NH2-MIL-53(Fe)MOF material is es The ability to reduce the trogenic effect was not strong. As can be seen from the analysis of the results, the present invention The law aims to effectively remove bound and free estrogens from wastewater. This can be achieved, and at the same time, reduce the concentration of estrogen-active substances in wastewater and the ecological risk level. This can be done. As can be seen from the results, artificial photosynthetic particles and natural photosynthetic particles in wastewater It breaks down conjugated estrogens (E2-3S) and synthetic free estrogens (EE2). They work together to fulfill their roles, as natural photosynthetic particles alone have a significantly lower efficiency in removing estrogen. To lower. Comparative Example 4 Compared to Example 1, it differs in the following respects, and in the green algae in the natural photosynthetic particles in step (2). Instead of using the term "group," I used cyanobacteria (Anabaena). Comparative Example 5 Compared to Example 1, the following differences exist: the withered grass in the natural photosynthetic particles in step (2) Instead of bacteria, brown, round-shaped nitrogen-fixing bacteria were used. The aforementioned brown, round-shaped nitrogen-fixing bacteria were cultured in nitrogen-fixing culture medium. It uses a base, and its composition is 0.5g yeast extract, 20.0g mannitol, and KH2PO4 0.2g, K2HPO40.8g, MgSO4.7H2O 0.2g, CaSO4·2 H2O 0.1g, FeCl3 trace amount 0.036g, Na2MoO4·2H2O 0.08 Contains 4g and 1.0 L of distilled water. The decomposition effects of Comparative Examples 4 and 5 were detected and analyzed according to the method in Example 1, and the results are shown in Table 2. It will be done. Table 2: E2-3S / EE2 removal rate and estrogen effect reduction efficiency in different modes TIFF0007854582000002.tif39118 As can be seen from Table 2, Anabaena was used instead of microalgae, and brown round nitrates were used instead of bacteria. When immobilized bacteria are used, the removal rate of E2-3S and EE2 is obtained from Chlorella and Bacillus subtilis. The effect was significantly lower than that of naturally occurring photosynthetic particles, with values ​​of 6.3% and 3.0%, respectively. At the same time, there was a significant difference in the reduction of the effect, and when the microalgae was Anabaena, The effect is about 58 ng / L higher than that of Chlorella. This is because cyanobacteria are affected by estrogen. It is thought that this is to release algal toxins. If the bacteria are brown round nitrogen-fixing bacteria, then their effect The concentration is about 3 ng / L higher than that of Bacillus subtilis, which is because brown, round-shaped nitrogen-fixing bacteria are denitrifying bacteria. This is thought to be the reason. As can be seen from the analysis of the results, the microalgae defined in this invention are The combination of Lorera and Bacillus subtilis has a good wastewater treatment effect. Example 3 Compared to Example 1, the following differences exist, with the photoirradiation cycle in step (3) being 18 hours / day. That is the case. Example 4 Compared to Example 1, the following differences exist, and the light irradiation cycle in step (3) is 6 hours / day. be. The decomposition effects of Examples 3 and 4 were detected and analyzed according to the method described in Example 1, and the results are shown in Table 3. It is shown here. Table 3: E2-3S / EE2 removal rate and estrogen effect reduction efficiency in different modes As can be seen from Table 3 of TIFF0007854582000003.tif38115, when the light irradiation period is 18h / day, E2-3S and EE2 In contrast, they have high removal rates, of 98.8% and 88.1%, respectively, for total estrogen. The reduction effect is only 4.6 ng / L. When the light irradiation period is 6 hours, E2-3S is approximately The removal rates of EE2 decreased slightly, to 94.3% and 78.6%, respectively, and the total est The LOGEN reduction effect was only 29.8 ng / L. As can be seen from the analysis of the results, light irradiation The longer the period, the more advantageous it is for estrogen removal; therefore, the light irradiation period set in this invention is Setting this to 6-18 hours / day is appropriate. Example 5 Compared to Example 1, the following differences exist: the light irradiation intensity in step (3) is 240 μm ol m -2 s -1 That is the case. Example 6 Compared to Example 1, the following differences exist, and the light irradiation intensity during step (3) is 60 μMo lm -2 s -1 That is the case. The decomposition effect of Examples 5 and 6 was detected and analyzed according to the method described in Example 1, and the results were This is shown in Table 4. Table 4: E2-3S / EE2 removal rate and estrogen effect reduction efficiency in different modes As can be seen from Table 4 of TIFF0007854582000004.tif39115, the light irradiation intensity is 240 μmol m -2 s -1 If so, E2 -3S and EE2 showed strong removal rates and effect reduction, with removal rates of 99.0% and 87% respectively. The concentration is 0.4%, and the effective value is 3.9 ng / L. The light irradiation intensity is 60 μmol m -2 s -1 In this case, the removal rates of E2-3S and EE2 decrease slightly, and the total reduction in effect increases. The removal rates were 90.2% and 66.4%, respectively, and the effective value was 19.7 ng / L. Light irradiation intensity of 60 μmol m -2 s -1 From 240 μmol m -2 s -1 Up to As a result, the removal rate of E2-3S and the reduction of estrogen effects remain essentially unchanged, EE 2 is greatly affected by light illumination. As can be seen from the analysis of the results, the light illumination set in this invention The injection intensity range is 60 μmol m -2 s -1 ~240 μmol m -2 s -1 Set to It is reasonable to do so. Example 7 Compared to Example 1, the following differences exist, and the concentration of artificial photosynthetic particles in Step 3 is approximately 6 The concentration is 00 particles / L, and the concentration of natural photosynthetic particles is 600 particles / L. Example 8 Compared to Example 1, the following differences exist, and the concentration of artificial photosynthetic particles in step 3 is approximately 1 The concentration is 00 particles / L, and the concentration of natural photosynthetic particles is 300 particles / L. Example 9 Compared to Example 1, the following differences exist: the concentration of artificial photosynthetic particles in step 3 is approximately 3 The concentration is 00 particles / L, while the concentration of natural photosynthetic particles is 100 particles / L. The decomposition effects of Example 7 and Comparative Examples 6 and 7 were detected and analyzed according to the method described in Example 1. The results are shown in Table 5. Table 5: E2-3S / EE2 removal rate and estrogen effect reduction efficiency in different modes As can be seen from Table 5 of TIFF0007854582000005.tif43120, the ratio of the number of artificial photosynthetic particles to natural photosynthetic particles is 1:1, and the work After reducing the concentration to 1x, the removal rate and effectiveness reduction of E2-3S and EE2 increased, and the removal rate The results were 99.5% and 89.1%, respectively, with an effect value of 0.9 ng / L. Artificial photocombination When the ratio of mature particles to naturally occurring photosynthetic particles is 1:3, the removal rate and effectiveness of E2-3S are reduced. The rate did not change significantly, but the removal rate of EE2 was greatly affected, and the removal rate of E2-3S was 94.32%. The percentage is 68.2% for EE2 removal rate and 10.3 ng / L for overall effect reduction. When the ratio of artificial photosynthetic particles to natural photosynthetic particles is 3:1, E2-3S and EE2 are removed. The rate was hardly affected, with removal rates of 95.4% and 86.6% respectively, resulting in a low overall effect. The reduced value is 23.8 ng / L. As can be seen from the analysis of the results, the artificial set in this invention It is reasonable to set the ratio of photosynthetic particles to naturally occurring photosynthetic particles to (1-6):(1-6). ru. The above are merely preferred embodiments of the present invention, and those skilled in the art will not deviate from the principles of the present invention. Without doing so, several improvements and modifications can be made, and these improvements and modifications are All of the above are considered to fall within the scope of protection of this invention.

Claims

1. A method for preparing double-embedded particles, The double-embedded particles consist of artificial photocatalytic particles and natural photosynthetic particles. The artificial photocatalytic particles consist of calcium alginate hydrogel and the calcium alginate The photocatalyst and white rot fungus are embedded in a muhydrogel, and the photocatalyst is an iron-based organogold Genus skeleton NH ２ -MIL-53(Fe), The aforementioned natural photosynthetic particles consist of calcium alginate hydrogel and the calcium alginate hydrogel. It contains green algae and Bacillus subtilis embedded in the gel, The particle diameter of the artificial photocatalytic particles is 2 to 5 mm, and the particle diameter of the natural photosynthetic particles is 2 The size is ~5 mm, and the ratio of the number of artificial photocatalytic particles to natural photosynthetic particles is (1-6):(1 ~6) The method for preparing the double-embedded particles includes the following steps: S1. Preparation of artificial photocatalytic particles: Collect 25-50 mL of white rot fungal fluid with an OD600 of 0.8-1.0, and then 5000-7 The white rot fungal cells were obtained by centrifuging at 000 rpm for 9 to 11 minutes, and the white rot fungal cells were obtained. Dissolve 0.03-0.06 g of photocatalyst in 50-80 mL of sodium alginate solution at a mass concentration of 1-2%. Mix with the um solution and use a 2-5 mL syringe to dispense 50-100 mL of 2-5% C solution by mass. Add to aCl2 solution dropwise and allow to fixate for 30-60 min to obtain artificial photocatalytic particles. S2. Preparation of natural photosynthetic particles: Take 25-50 mL of green algae liquid with an OD680 of 0.8-1.0 and ferment at 4000-6000 rpm. Centrifugation was performed for 10-15 minutes at m to collect the green algae, and 25-50 mL of OD600 was added. Take a Bacillus subtilis solution with a concentration of 8-1.0 and disperse it for 10-15 minutes under conditions of 6000-8000 rpm. After separating the heart and recovering the Bacillus subtilis cells, The green algae and Bacillus subtilis cells are suspended in 5-8 mL of ultrapure water, and a mass concentration of 1 is added to 50-80 mL. Mix uniformly with a 2% sodium alginate solution to obtain a fungal-algal mixture, and the fungal-algal mixture Dispense the solution into 50-100 mL of a 2-5% CaCl2 solution using a 2-5 mL syringe. After grinding, a fixation reaction is carried out for 30-60 minutes to obtain natural photosynthetic particles. The method for preparing the white rot fungal solution is as follows: white rot fungal cells are transferred to a liquid jar. Inoculate into a complete culture medium for sweet potatoes, and at 28-32°C, the OD600 of the white rot fungus saturates to 0.8-1.

0. The mixture is cultured until it reaches a certain stage, and a white rot fungal solution is obtained. The liquid potato mixture is 20% potato 1 liter of broth, 20 g of glucose, 3 g of KH2PO4, 1.5 g of MgSO4・7H2O It contains 1 g of thiamine and has a pH of 6. The method for preparing the aforementioned green algae solution is as follows: Green algae are inoculated into BG11 medium, and 24-26 Cultivate at °C and 2000 Lux for 1-2 weeks to obtain a green algae solution with an OD680 of 0.8-1.

0. The green alga mentioned is Chlorella. The method for preparing the Bacillus subtilis bacterial suspension is as follows: Inoculate Bacillus subtilis into a nutrient-rich meat juice medium, and 28- Under conditions of 30°C and 120-130 rpm, the OD600 of the Bacillus subtilis bacterial solution becomes 0.8-1.

0. The culture was continued until the nutrient broth medium consisted of 5.0 g peptone, 3.0 g beef extract, and NaCl. It contains 5.0 g and 1 L of distilled water, and the pH is 7.0 to 7.

2. A method for preparing double-embedded particles, characterized by the features described above.

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