Preparation method of sodium alginate mediated composite immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides

Abstract

The invention discloses a preparation method of sodium alginate mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading a trace amount of pyrethroid pesticides, which is characterized by comprising the following steps: preparing mesoporous molecular imprinting modified Fe3O4-NCs by a surface grafting method to obtain surface imprinting functionalized Fe3O4 nanostructure magnetic microspheres MIP-Fe3O4-NCs; the preparation method comprises the following steps: mixing MIP-Fe3O4-NCs activated by methanol with a sterilized sodium alginate solution, then adding a pyrethroid pesticide degrading bacterium solution, fully and uniformly stirring, then adding into cyclohexane, uniformly stirring again, dropwise adding a calcium chloride solution, and carrying out a cross-linking reaction at 4 DEG C for 2-6 hours to obtain the MIP-Fe3O4-NCs immobilized bacterium. The intercellular mass transfer resistance is reduced, the sensitivity of microorganisms to ultra-trace pyrethroid pesticides in the environment is greatly improved, and the degradation efficiency is improved.

Description

Technical Field

[0001] The invention relates to the degradation of pyrethroid pesticides, and in particular to a method for preparing sodium alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides. Background Art

[0002] Pyrethroid pesticides are insecticides with excellent biological activity and good environmental compatibility that are chemically synthesized by imitating natural pyrethroids. They are synthesized after organophosphorus and organochlorine, and are known as the third major milestone in the history of insecticides. Due to its high efficiency and wide range of applications, it is widely used in agricultural breeding, sanitary insecticides and other fields. However, pyrethroid insecticides are highly toxic. Traces of pyrethroid pesticides can cause stress and death to aquatic organisms, and pyrethroid pesticides are also highly toxic to mammals. Studies have shown that the large-scale use of pyrethroid pesticides poses a serious threat to human health. In order to protect human health and the balance of the ecosystem, exploring effective methods for degrading pyrethroid pesticides has become the focus of research attention of scientists at home and abroad.

[0003] At present, the degradation of pyrethroid pesticides mainly has the following pathways: First, photodegradation. Under the irradiation of sunlight, the covalent bonds between pyrethroid pesticide molecules are broken and the toxic effect is lost. Studies have shown that the photodegradation of pyrethroid pesticides is one of the important pathways for the migration and transformation of substances in the environment, and is also a common way for the degradation of such pesticide residues. The second is physical adsorption, which is also called van der Waals adsorption. This process uses van der Waals forces to adsorb pesticide molecules and separate them from the original environmental medium. Studies have shown that the adsorption of pyrethroid pesticides on glass surfaces and solid suspensions will accelerate the reduction of pesticide concentration and activity in water environments, and can be used as a low-cost natural adsorption medium to treat water environments contaminated by pyrethroid pesticides. Finally, there is biodegradation. Usually, physical and chemical degradation methods have the disadvantages of large side effects, easy to cause secondary pollution, high cost, and slow degradation efficiency. Biodegradation is a process that transforms pesticide molecules into smaller, simpler, and pollution-free molecules through biological oxidation or other biological transformation methods. Microbial degradation has low cost, high efficiency and no secondary pollution. It is considered to be a green engineering technology for efficiently degrading pesticide residues and is currently a hot topic in scientific research on removing pyrethroid pesticide residues.

[0004] The immobilized microorganism technology refers to immobilizing microorganisms on a certain carrier, which can not only maintain their high activity but also reproduce rapidly and in large quantities. It can not only increase the concentration of microorganisms but also resist the influence of adverse environments. In recent years, researchers at home and abroad have achieved remarkable results in using immobilized microorganisms to treat polluted water sources. Immobilized microorganisms have the advantages of rapid reproduction, accelerating reaction rates, easy operation, recyclability, and no environmental pollution. However, at the same time, there are many defects in using only the immobilization technology. In the degradation of pyrethroid pesticides at low concentrations, the mass transfer resistance between cells and the matrix is the most important defect, and the low concentration of pyrethroid pesticides will significantly affect the sensitivity of degrading bacteria, resulting in a further reduction in the degradation efficiency. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides, which has excellent enrichment ability, reduces the intercellular mass transfer resistance, greatly improves the sensitivity of microorganisms to trace pyrethroid pesticides in the environment, and improves the degradation efficiency.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides, comprising the following steps:

[0007] Step 1: Prepare mesoporous molecularly imprinted modified Fe3O4-NCs by surface grafting method to obtain surface imprinted functionalized Fe3O4 nanostructured magnetic microspheres MIP-Fe3O4-NCs;

[0008] Step 2: Mix the methanol-activated MIP-Fe3O4-NCs with the sterilized sodium alginate solution, then add the pyrethroid pesticide degrading bacteria liquid, stir well, add it to cyclohexane, stir well again, and then dropwise add calcium chloride solution, and carry out a cross-linking reaction at 4°C for 2-6 hours to obtain alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for degrading pyrethroid pesticides, where the ratio of MIP-Fe3O4-NCs, sodium alginate solution, Photobacterium phosphoreum seed solution, cyclohexane and calcium chloride solution is 10-20 mg: 20 mL: 1 mL: 20 mL: 5 mL.

[0009] Further, step 1 is specifically as follows:

[0010] (1) Add 2 - 3 mL of Triton X-100 and 15 - 25 mL of cyclohexane into a round-bottom flask. After vigorous stirring, sequentially add 38.2 - 42.8 μL of 3-aminopropyltriethoxysilane, 50 - 70 μL of tetraethyl orthosilicate, and 200 μL of NH₃·H₂O. Stir for 8 - 12 h. After the reaction is completed, add 20 mL of acetone, shake for 15 min, then centrifuge to remove the supernatant. Wash the precipitate twice with anhydrous ethanol and ultrapure water respectively, and place it in a 60 °C vacuum drying oven in the dark to dry to obtain APTES-modified amino-functionalized silica microspheres;

[0011] (2) Sequentially add 400 μL of Fe₃O₄-NCs with a concentration of 1.25 - 2 mg / mL and 50 - 70 mg of amino-functionalized silica microspheres into a mixture composed of 1.5 mL of xylene, 10 - 15 mL of cyclohexane, and 30 - 40 μL of TEOS. Transfer it to a flask. After purging the oxygen in the flask with nitrogen, add 200 μL of NH₃·H₂O and stir for 2 - 4 h. Then add 100 - 200 μL of a pyrethroid pesticide solution with a concentration of 20 mg / mL, mix evenly, and stir in the dark for 6 - 8 h. Subsequently, add 11.6 - 14.8 μL of ethylene glycol dimethacrylate (EGDMA) and 15 mg of azobisisobutyronitrile (AIBN) into the flask, heat up to 60 °C, and continue to stir in the dark for 2 - 4 h. After the stirring is completed, add 10 - 15 mL of acetone for demulsification and sedimentation, then let it stand in a 35 °C water bath for 2 - 4 h, centrifuge and discard the supernatant. Resuspend the precipitate in ultrapure water, vortex for 1 - 2 min, centrifuge and discard the supernatant to remove unreacted TEOS, EGDMA, APTES, and MAA; then add 5 - 10 mL of a mixed solution composed of ethanol and acetonitrile with a volume ratio of 4:1 and sonicate for 5 min, then vortex for 30 - 50 min, centrifuge and remove the supernatant for the elution of pyrethroid pesticides, and repeat the elution 2 - 3 times; then add 10 - 20 mL of a mixed solution composed of methanol and acetic acid with a volume ratio of 9:1, shake for 15 min and then centrifuge to remove the precipitate, repeat the elution until the absorbance of the supernatant is close to 0 and the elution is completed to obtain MIP-Fe₃O₄-NCs.

[0012] Further, the preparation method of the Fe3O4-NCs described in step (2) is as follows: Weigh 5.4 - 8.1 g of FeCl3·6H2O and dissolve it in 150 mL of a mixed solvent composed of ultrapure water, ethanol, and n-hexane in a volume ratio of 3:5:7. Subsequently, add 6.1 - 9.2 g of sodium oleate, mix well by ultrasonic treatment, and then transfer it to a flask. Under magnetic stirring at 300 - 400 rpm, heat the solution to 70 - 80 °C and maintain for 4 - 6 h. After heating, perform phase separation to separate the upper layer of n-hexane. Transfer the remaining liquid to a flask containing 3 - 6 mL of a mixed solution composed of oleic acid and 1-octadecene in a volume ratio of 1:2, and add 10 - 15 mL of ethylene glycol. Heat it to 300 - 320 °C under a nitrogen atmosphere and evacuate for 20 - 40 min, maintaining the heating for 0.5 - 1.5 h. After heating is completed, cool it to room temperature, add 10 - 20 mL of a mixed solution composed of chloroform and ethanol in equal volume, shake it, and then centrifuge. Take the precipitate and wash it repeatedly with ultrapure water and ethanol 2 - 4 times, and dry it in vacuo at 60 °C to obtain square Fe3O4 nanoblocks Fe3O4-NCs.

[0013] Further, the solute in the pyrethroid pesticide solution described in step (2) is one or more of cyfluthrin, deltamethrin, and cypermethrin, and the solvent is a solvent composed of 3-aminopropyltriethoxysilane and methacrylic acid in a volume ratio of 2:1.

[0014] Further, the sodium alginate solution described in step 2 is a sodium alginate solution with a concentration of 25 g / L prepared using standard seawater with a salinity of 30‰, and the calcium chloride solution is a calcium chloride solution with a concentration of 20 g / L prepared using standard seawater with a salinity of 30‰.

[0015] Further, the pyrethroid pesticide-degrading bacterium bacterial solution described in step 2 is a Photobacterium ganghwense bacterial solution with an OD 600 = 2.0. The Photobacterium ganghwense is strain Nbuzhang1, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 2014580.

[0016] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a method for preparing alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides. This composite immobilized bacteria has specific adsorption ability for pesticides, enriches pesticides around the degrading bacteria, and improves the mass transfer ability of pesticides to the degrading bacteria. In addition, alginate in the composite not only acts as an outer shell, isolating the microorganisms from contact and interaction with the external harsh environment, maintaining the enzymes secreted by the bacterial cells and encapsulating them inside and on the surface of the microspheres; it also further mediates and protects the catalytic and adsorption abilities of the activated MIP-Fe3O4-NCs, covers the non-specific sites on the surface of MIP-Fe3O4-NCs, and reduces the non-specific adsorption ability of MIP-Fe3O4-NCs. And MIP-Fe3O4-NCs itself, as an excellent mesoporous adsorbent, attaches to the bacterial cells through the electrostatic force and hydrogen bonds of multiple groups, forming a good adsorption and degradation complex with the bacterial cells themselves, accelerating the ultra-trace adsorption and degradation of pyrethroid pesticides, that is, forming a cyclic degradation system of adsorption-degradation-re-adsorption-re-degradation.

[0017] In summary, the present invention uses alginate-mediated MIP-Fe3O4-NCs immobilized bacteria to have excellent enrichment ability for pyrethroid pesticides, reduce the intercellular mass transfer resistance, greatly improve the sensitive perception and response of microorganisms to trace pyrethroid pesticides in the environment, and simultaneously achieve the adsorption and degradation of trace pyrethroid pesticides in the environment.

[0018] The above-mentioned pyrethroid pesticide degrading bacteria is the Nbuzhang1 strain, classified and named as Photobacterium ganghwense Nbuzhang1, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2014580, and the deposit address is Wuhan University, Wuhan, China. Description of the Drawings

[0019] Figure 1 It is the scanning electron microscope images of different samples prepared in Specific Example 1, where A is Fe3O4-NCs and B is MIP-Fe3O4-NCs;

[0020] Figure 2 It is the scanning electron microscope images of different samples prepared in Specific Example 2, where A is Photobacterium ganghwense, B is sodium alginate beads, C is sodium alginate immobilized bacteria, D is activated carbon immobilized bacteria, E is MIP-Fe3O4-NCs immobilized bacteria, F is NIP-Fe3O4-NCs immobilized bacteria, G is the surface of MIP-Fe3O4-NCs immobilized bacteria, and H is the cross-section of MIP-Fe3O4-NCs immobilized bacteria;

[0021] Figure 3 It is the residual concentration of cyfluthrin in the culture medium after treatment in Specific Example 3, where A is the influence of the dosage of degrading bacteria on the pesticide residual concentration, and B is the influence of the dosage of MIP-Fe3O4-NCs on the pesticide residual concentration;

[0022] Figure 4 It is the selective test result of MIP-Fe3O4-NCs immobilized bacteria in Specific Example 4, where A is the pesticide residue amount in different experimental groups, B is the adsorption amount on the surface of microspheres in different experimental groups, and C is the total degradation amount in different experimental groups;

[0023] Figure 5 It is the analysis result of the degradation ability of MIP-Fe3O4-NCs immobilized bacteria on trace pyrethroid pesticide residues in Specific Example 5, where A is the pesticide residue amount at different initial concentrations, B is the adsorption amount on the surface of microspheres at different initial concentrations, and C is the total degradation amount at different initial concentrations;

[0024] Figure 6 It is the degradation analysis of MIP-Fe3O4-NCs immobilized bacteria on different concentrations of cypermethrin in actual seawater in Specific Example 6. A is the adsorption amount on the surface of microspheres at different initial concentrations, and B is the total degradation amount at different initial concentrations. Specific embodiments

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] Specific Example 1: Preparation of surface-imprinted functionalized Fe3O4 nanostructured magnetic microspheres (MIP-Fe3O4-NCs), the specific steps are as follows:

[0027] Step 1: Prepare square Fe3O4 nanoblocks (Fe3O4-NCs) by the high-efficiency thermal decomposition method.

[0028] First, weigh 5.4 - 8.1 g of FeCl3·6H2O and dissolve it in 150 mL of a mixed solvent (prepared by mixing ultrapure water, ethanol, and n - hexane in a volume ratio of 3:5:7). Subsequently, add 6.1 - 9.2 g of sodium oleate. After ultrasonic mixing, transfer the solution to a 250 mL flask. Under magnetic stirring at 300 - 400 rpm, heat the solution to 70 - 80 °C and maintain for 4 - 6 h. After heating, perform phase separation on the mixed solution, separate the upper - layer n - hexane, and transfer the remaining liquid to a three - necked flask. Add 3 - 6 mL of a mixed solution prepared by mixing oleic acid and octadecene in a volume ratio of 1:2, and add 10 - 15 mL of ethylene glycol. Heat the three - necked flask to 300 - 320 °C under a nitrogen atmosphere and evacuate for 30 min, and maintain heating for 1 h. After heating is completed, cool to room temperature, add 10 - 20 mL of a mixed solution prepared by mixing chloroform and ethanol in equal volumes, shake for 10 min, then centrifuge the solution at 9000 rpm for 20 min. Take the precipitate and wash it 3 times successively with 6 mL of ultrapure water and 6 mL of ethanol, and dry it in vacuo at 60 °C to obtain square Fe3O4 nanoblocks, which are dissolved in chloroform for standby;

[0029] Step 2: Prepare mesoporous molecularly imprinted modified Fe3O4 - NCs (MIP - Fe3O4 - NCs) by surface grafting method.

[0030] Add 2 - 3 mL of TritonX - 100 and 15 - 25 mL of cyclohexane to a round - bottom flask. After vigorous stirring for 15 min, sequentially add 38.2 - 42.8 μL of 3 - aminopropyltriethoxysilane (APTES), 50 - 70 μL of tetraethyl orthosilicate (TEOS), and 200 μL of NH3·H2O to the above - mentioned mixed solution and stir for 8 - 12 h. After the reaction is completed, add 20 mL of acetone to the reaction system, shake for 15 min, then centrifuge at 9000 rpm to remove the supernatant. Wash the precipitate 2 times with 10 - 20 mL of anhydrous ethanol and ultrapure water respectively, and dry it in a 60 °C vacuum drying oven in the dark to obtain APTES - modified amino - functionalized silica microspheres for standby;

[0031] Subsequently, 400 μL of Fe3O4-NCs with a concentration of 1.25 - 2 mg / mL and 50 - 70 mg of amino-functionalized silica microspheres were successively added to a mixed solution composed of 1.5 mL of xylene, 10 - 15 mL of cyclohexane, and 30 - 40 μL of TEOS, transferred to a flask, and nitrogen was passed through for 1 h to remove the oxygen in the flask. Subsequently, 200 μL of NH3·H2O was added, and the mixture was stirred at 350 rpm for 2 - 4 h. Then, 100 - 200 μL of pyrethroid pesticides with a concentration of 20 mg / mL (the solute is one or more of cyfluthrin, deltamethrin, and cypermethrin, and the solvent is a solvent system composed of APTES and methacrylic acid (MAA) in a volume ratio of 2:1) was added and mixed evenly. After that, it was stirred in the dark for 6 - 8 h. Subsequently, 11.6 - 14.8 μL of ethylene glycol dimethacrylate (EGDMA) and 15 mg of azobisisobutyronitrile (AIBN) were added to the flask, the temperature was raised to 60 °C, and stirring was continued in the dark for 2 - 4 h. After the stirring was completed, 10 - 15 mL of acetone was added to the system for demulsification and sedimentation. Then, it was left standing in a 35 °C water bath for 2 - 4 h, centrifuged at 9000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 6 mL of ultrapure water, vortexed for 1 min, centrifuged at 9000 rpm for 20 min, and the supernatant was discarded to remove unreacted TEOS, EGDMA, APTES, and MAA. Then, 5 - 10 mL of a mixed solution composed of ethanol and acetonitrile in a volume ratio of 4:1 was added and sonicated for 5 min. Subsequently, it was vortexed for 40 min, centrifuged at 9000 rpm for 15 min, and the supernatant was removed for the elution of pyrethroid pesticides. The above elution steps were repeated 2 - 3 times. Subsequently, 10 - 20 mL of a mixed solution composed of methanol and acetic acid in a volume ratio of 9:1 was added, shaken for 15 min, and then centrifuged to remove the precipitate. This elution step was repeated several times, and the absorbance of the supernatant was measured until the absorbance of the supernatant was close to 0, and then the elution was completed to obtain MIP-Fe3O4-NCs, which was dried under vacuum at 30 °C and stored in a 4 °C refrigerator for later use.

[0032] Meanwhile, a control group of non-imprinted nanostructured magnetic microspheres (NIP-Fe3O4-NCs) without a template molecule was prepared. Except that the template molecule was replaced with an equal amount of xylene, the other steps were the same as above. Figure 1 Scanning electron microscope images of the prepared Fe3O4-NCs and MIP-Fe3O4-NCs.

[0033] As can be seen from Figure 1 A in, the prepared Fe3O4-NCs have a typical cubic structure with an edge length of about 15 - 20 nm, and the structure and size are basically uniform. As can be seen from Figure 1As can be seen from Figure B, the prepared MIP-Fe3O4-NCs are spherical and uniform-sized particles with a rough surface, indicating that a polymerization reaction occurred around the Fe3O4-NCs and a molecularly imprinted polymer was successfully grafted onto the surface of the Fe3O4-NCs.

[0034] Specific Example 2: Preparation of immobilized degrading bacteria using sodium alginate and MIP-Fe3O4-NCs. The specific steps are as follows:

[0035] Step 1: Preparation of degrading bacteria seed solution: The bacteria used in this experiment are highly efficient degrading strains of pyrethroid pesticides, namely strain Nbuzhang1, classified and named as Photobacterium ganghwense Nbuzhang1, deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2014580, and the Genbank accession number of the 16S rRNA of the strain is KR150790. The bacteria were inoculated into the first medium without pyrethroid pesticides and cultured at a temperature of 20 °C and a rotation speed of 180 rpm for 72 h. Subsequently, under the condition of 4500 rpm, centrifuged for 10 min, and the bacteria concentration was diluted to OD 600 = 2.0, and stored in a 4 °C refrigerator for later use. The preparation method of the first medium is as follows: Add 5 g of tryptone powder, 1 g of yeast extract, and 0.01 g of ferric phosphate into a beaker, dissolve with standard seawater with a salinity of 30‰, then make up the volume to 1 L, adjust the pH value to 5 with 0.1 mol / L hydrochloric acid, and sterilize by high-pressure steam for 40 min for later use.

[0036] Step 2: Preparation of sodium alginate-mediated MIP-Fe3O4-NCs immobilized bacteria

[0037] Take 20 - 50 mg of MIP-Fe3O4-NCs and dissolve them in 5 mL of methanol for activation, then transfer them to a beaker and air-dry in a fume hood. Prepare a 25 g / L sodium alginate solution and a 20 g / L calcium chloride solution using standard seawater with a salinity of 30‰, and add 10 - 20 mg of activated MIP-Fe3O4-NCs and 20 mL of the sodium alginate solution sterilized by high-pressure steam into the beaker, then add 1 mL of the Photobacterium ganghwense bacterial solution and stir well; Add the mixed solution into 20 mL of cyclohexane, stir evenly at a rotation speed of 200 rpm, and then dropwise add 5 mL of a 20 g / L calcium chloride solution. Then, store in a 4 °C refrigerator for crosslinking for 4 h to obtain sodium alginate-mediated MIP-Fe3O4-NCs immobilized bacteria, abbreviated as SA + B + MIPs, wash the immobilized bacteria 3 times with 30 mL of sterile standard seawater, and store in a 4 °C refrigerator for later use. At the same time, prepare the control group as follows:

[0038] Sodium alginate beads: A 20 g / L calcium chloride solution was added dropwise to a 25 g / L sodium alginate solution to form sodium alginate beads, abbreviated as SA;

[0039] Sodium alginate-immobilized bacteria: The preparation method was the same as in Step 2 above, except that MIP-Fe3O4-NCs were not added, abbreviated as SA+B;

[0040] Activated carbon-immobilized bacteria: The preparation method was the same as in Step 2 above, except that MIP-Fe3O4-NCs were replaced with activated carbon, abbreviated as SA+B+C;

[0041] NIP-Fe3O4-NCs-immobilized bacteria, the preparation method was the same as in Step 2 above, except that MIP-Fe3O4-NCs were replaced with NIP-Fe3O4-NCs, abbreviated as SA+B+NIPs;

[0042] Field emission scanning electron microscopy was used to scan Photobacterium glycinii and the prepared SA, SA+B+C, SA+B+MIPs, SA+B, SA+B+NIPs. Figure 2 These are the scanning electron microscope images of different samples.

[0043] As Figure 2 shown in A, Photobacterium glycinii is a typical rod-shaped bacterium with a diameter of about 1 μm. Figure 2 As shown in B, the single sodium alginate bead SA is porous and sponge-like. Figure 2 As shown in C, SA+B shows a structure similar to SA, and the immobilized bacteria are evenly distributed in the sodium alginate matrix. Figure 2 As shown in D, the activated carbon-supported immobilized bacteria show a dense texture structure, and compared with the SA material, its pore size and number are lower. Figure 2 E and Figure 2 F show the internal morphology of sodium alginate-mediated MIP-Fe3O4-NCs-immobilized bacteria and NIP-Fe3O4-NCs-immobilized bacteria. It can be seen that the degrading bacteria in the immobilized bacteria are evenly distributed in the sodium alginate matrix, and MIP-Fe3O4-NCs are concentrated around the degrading bacteria. Figure 2 G and Figure 2 H are the overall surface scanning image and overall cross-section scanning image of sodium alginate-mediated MIP-Fe3O4-NCs-immobilized bacteria beads. From the surface, it is relatively rough, which provides a larger specific surface area for the adsorption of pesticides and the transfer of media. In addition, from the cross-sectional view, it is found that the inside of the immobilized bacteria beads is hollow and porous, which provides a good environment for the adsorption and degradation of ultra-trace microorganisms.

[0044] Specific Example 3. Optimization of the conditions for MIP-Fe3O4-NCs-immobilized bacteria (taking cyhalothrin as an example)

[0045] 1. Method for determining pyrethroid pesticide residues: Add 0.5 g of sodium chloride to the culture solution to be tested, and then add a mixed solution composed of equal volumes of n - hexane and acetone with a volume twice that of the sample taken. Vortex for 5 min, centrifuge at 3000 rpm for 10 min, and take the supernatant. Then add a mixed solution composed of equal volumes of n - hexane and acetone with a volume equal to that of the sample taken for extraction again. Combine the two extraction solutions, dry them under nitrogen at 35 °C, and then dissolve them in a mixed solution composed of acetone and isooctane with a volume ratio of 1:9 and a volume equal to that of the sample taken. After vortexing for 3 min, filter through a 0.22 - μm filter membrane and detect and analyze by GC - ECD. The detection instrument is a GC - 2010 gas chromatograph (Shimadzu, Japan), an SPB - 5 quartz elastic capillary column (30 m × 0.25 mm × 0.25 μm); nitrogen is used as the carrier gas and the tail - blowing gas with a flow rate of 1.0 mL / min, the injection volume of the standard solution and the sample extract is 1.0 μL; the injection port temperature is 240 °C; the column temperature is maintained at 240 °C for 3 min, then increased to 290 °C at a rate of 5 °C / min and held for 5 min; the detector temperature is 320 °C.

[0046] 2. Optimization of the addition amount of degrading bacteria

[0047] Take the bacterial seed solution prepared in step 1 of specific example 2, and respectively subject 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL of the seed solution to the compound immobilization in step 2 of specific example 2, and add them to 20 mL of the second culture medium containing 1 mg / L of lambda - cyhalothrin pesticide. Each addition amount of degrading bacteria is subjected to 3 parallel experiments. The control group is to add the same amount of free bacteria to 20 mL of the second culture medium containing 1 mg / L of lambda - cyhalothrin pesticide, and each addition amount of degrading bacteria is subjected to 3 parallel experiments. After degrading for 48 h in a constant - temperature shaker at 20 °C and 180 r / min, take 2 mL of the culture solution to test the residual pesticide concentration and determine the optimal addition amount of the immobilized bacteria. The preparation method of the second culture medium is as follows: Weigh 0.01 g of ferric phosphate, make up the volume to 1 L with standard seawater with a salinity of 30‰, adjust the pH value to 5 with 0.1 mol / L hydrochloric acid, and sterilize by high - pressure steam for 40 min for standby.

[0048] The results are as Figure 3 shown in A below. As the amount of bacteria increases, the concentration of residual pesticide decreases, indicating that the degradation effect is increasing. However, when the amount of bacteria exceeds 1 mL, the change in the concentration of residual pesticide is not significant. This is because the degrading bacteria in the immobilized bacteria grow and reproduce during the process of degrading pesticides, the competition for territory by bacteria increases, the survival ability is limited, and the degradation ability decreases. Therefore, the optimal addition amount of bacteria is 1 mL, where the black line represents the control group and the red line represents the experimental group.

[0049] 3. Optimization of the addition amount of MIP - Fe3O4 - NCs

[0050] 10 mg, 20 mg, 30 mg, 40 mg, and 50 mg of the MIP-Fe3O4-NCs prepared in step 2 of Specific Example 1 were respectively taken and subjected to the cell composite immobilization in step 2 of Specific Example 2, and added to 20 mL of a second culture medium containing 1 mg / L of lambda-cyhalothrin pesticide. The control group was the alginate-immobilized bacteria of 10 mg, 20 mg, 30 mg, 40 mg, and 50 mg without adding MIP-Fe3O4-NCs. After degrading for 48 h in a constant temperature shaker at 20 °C and 180 r / min, 2 mL of the culture solution was taken to test the residual pesticide concentration to determine the optimal addition amount of the adsorbent material.

[0051] The results are as Figure 3 shown in B of [reference], the pesticide degradation effect of SA + B is lower than that of SA + B + MIPs, indicating that loading MIP-Fe3O4-NCs can enhance the adsorption and degradation ability of trace pesticides. However, when the amount of MIP-Fe3O4-NCs exceeds 20 mg, the enhancement of the degradation effect is not obvious. To maximize the utilization of MIP-Fe3O4-NCs and avoid resource waste, the optimal addition amount of the system is 20.0 mg. B in the abscissa represents free bacteria.

[0052] Specific Example 4: Selectivity experiment of MIP-Fe3O4-NCs immobilized bacteria for degrading bacteria in mixed pesticides.

[0053] Method for measuring the amount of pesticide adsorbed on the microsphere surface: The microspheres used for degrading pesticides were taken out by filtering with a 0.22 μm filter membrane. The pesticides adsorbed on the surface of the microspheres were extracted with a mixed solution of n-hexane and acetone (v:v = 1:1) with twice the volume of the sample for 3 times, 15 min each time. The extraction solutions were combined and evaporated to dryness with a rotary evaporator, redissolved and fixed volume with acetone and isooctane (v:v = 1:9) with the volume of the sample, and filtered with a 0.22 μm filter membrane for GC-ECD detection and analysis.

[0054] Method for measuring the total degradation amount of different experimental groups: The specific calculation formula is as described in Formula 1. Formula 1: Total degradation amount of the experimental group = original pesticide concentration in the culture solution of the experimental group - residual amount in the culture solution - amount adsorbed on the microspheres.

[0055] Weigh 100 μg each of cyfluthrin, bifenthrin, deltamethrin, fenvalerate, and cypermethrin and add them together to 100 mL of the second culture medium as the basal medium. Prepare experimental groups G1 to G9 according to the requirements in Table 1. Place experimental groups G1 to G9 on a constant temperature shaker and degrade them for 48 h at 20 °C and 180 rpm. Take 10 mL of the culture solution, test the pesticide residue amount, and analyze the degradation effect. There are 3 parallel experiments for each experimental group. The results are as Figure 4 shown.

[0056] Table 1 Experimental group design

[0057]

[0058]

[0059] As Figure 4 shown in A, the residual amount of cyfluthrin in the culture solution after degradation in G6 is significantly lower than that of other structurally similar pyrethroid pesticides. And in seawater, except for the difference in the residual amount of cyfluthrin, the residual amounts of other pesticides are basically the same. From Figure 4 shown in B, the adsorption residual amounts of different pesticides on the surface of the immobilized degrading bacteria in different experimental groups are not very different, and the pesticide residual amounts are not much. Figure 4 Shown in C, among the degradation amounts of different pesticides, the degradation amounts of cyfluthrin in experimental groups G6 and G8 are significantly higher than those of other pesticides. These results indicate that the alginate-mediated MIP-Fe3O4-NCs immobilized bacteria have the strongest degradation ability for cyfluthrin in the mixed pesticides. This is because there are specific sites on MIP-Fe3O4-NCs in the MIP-Fe3O4-NCs immobilized bacteria that can specifically recognize cyfluthrin pesticides and will quickly recognize and adsorb cyfluthrin pesticides in the environment. However, there is no specific adsorption effect on other pyrethroid pesticides.

[0060] Specific Example 5. Analysis of the degradation ability of MIP-Fe3O4-NCs immobilized bacteria for trace pyrethroid pesticide residues.

[0061] Use standard seawater with a salinity of 30‰ to dilute and prepare 100 mL of cyfluthrin standard solutions with concentrations of 0.01 mg / L, 0.05 mg / L, and 0.1 mg / L respectively. Add free bacteria (B), alginate-immobilized bacteria (SA + B), and MIP-Fe3O4-NCs immobilized bacteria (SA + B + MIPs) into the above-mentioned cyfluthrin pesticide solutions with different concentrations respectively, so that the amount of bacteria is about 5.4×10 9were degraded for 48 h at 20 °C and 180 rpm. 10 mL of the sample was taken to test the degradation effect of each group. Each experimental group was run in triplicate. The results are as Figure 5 shown.

[0062] Figure 5 shown in A. After 48 h of degradation by MIP-Fe3O4-NCs immobilized bacteria in a low pesticide residue environment, the pesticide residue in water was close to 0. After 48 h of degradation by free bacteria and sodium alginate immobilized bacteria, the pesticide residue in water was relatively high. According to the curve trend, it can be seen that the lower the concentration, the higher the amount of pesticide remaining in water relative to the initial addition amount, and the weaker the degradation ability. This indicates that trace amounts of pesticides can cause low sensitivity in the degradation bacteria and a decrease in degradation ability. From Figure 5 B, it can be seen that in a low pesticide residue environment, the amount of pesticide adsorbed on the surface of the microspheres in MIP-Fe3O4-NCs immobilized bacteria was lower than that in the microspheres of sodium alginate, indicating that the addition of the specific adsorption material enhanced the ability of the immobilized degradation bacteria to degrade pollutants. From Figure 5 C, it can be seen that the amount of pesticide degraded by MIP-Fe3O4-NCs immobilized bacteria was the highest.

[0063] The above results show that under the condition of low pesticide residues, the degradation abilities of free bacteria and immobilized bacteria are weak, while the MIP-Fe3O4-NCs immobilized bacteria show strong degradation ability. In an environment with low residual pollutants, the mass transfer resistance of pesticides from a large amount of liquid environment to the surface of bacteria is the key inhibitory factor for the degradation of low-concentration pesticides. It is inferred that the concentration of pesticides in the biodegradation system plays a key role. A low concentration of pesticides around the bacteria will reduce the degradation ability. The MIP-Fe3O4-NCs immobilized bacteria mediated by sodium alginate in the present invention have specific adsorption ability for pesticides. It enriches pesticides around the degrading bacteria and improves the mass transfer ability of pesticides to the degrading bacteria. In addition, the composite structure provides great help for the ultra-trace degradation ability of MIP-Fe3O4-NCs for pyrethroid pesticides. Sodium alginate in the composite not only acts as a shell, isolating the contact and interaction between microorganisms and the harsh external environment, maintaining the enzymes secreted by the bacteria and encapsulating them inside and on the surface of the microspheres; but also further mediates and protects the catalytic and adsorption abilities of MIP-Fe3O4-NCs after activation, covering the non-specific sites on the surface of MIP-Fe3O4-NCs and reducing the non-specific adsorption ability of MIP-Fe3O4-NCs. And MIP-Fe3O4-NCs itself, as an excellent mesoporous adsorbent, forms a good adsorption and degradation complex with the bacteria by electrostatic force and hydrogen bond of multiple groups, accelerating the ultra-trace adsorption and degradation of pyrethroid pesticides. In the seawater environment, the MIP-Fe3O4-NCs immobilized bacteria mediated by sodium alginate form a cyclic system of sodium alginate maintenance - MIP-Fe3O4-NCs adsorption - microbial degradation of pesticides, that is, a cyclic degradation system of adsorption - degradation - re-adsorption - re-degradation is formed.

[0064] Specific Example Six: Application of MIP-Fe3O4-NCs Immobilized Bacteria in the Degradation of Cyhalothrin Pesticide in Actual Seawater.

[0065] Step 1: Dilute and prepare 100 mL of a cyhalothrin standard solution with a concentration of 100 mg / L using standard seawater with a salinity of 30‰. Treat it according to the experimental group design in Table 1 of Specific Example Four. Degrade for 48 h under the conditions of 20 °C and 180 rpm, take 10 mL of samples to test the degradation effects of each experimental group, and conduct 3 parallel tests for each experimental group. The results are as Figure 6 shown.

[0066] From Figure 6As can be seen from A, as the initial concentration of the pesticide increases, the amount of pesticide adsorbed on the surface of the microspheres in the sodium alginate-immobilized bacteria increases. The amount of pesticide adsorbed on the surface of the sodium alginate-immobilized bacteria (SA+B) is less than that of the single sodium alginate microspheres (SA) because part of the pesticide adsorbed by the sodium alginate microspheres is degraded by the bacteria. The amount of pesticide adsorbed on the surface of the microspheres in the MIP-Fe3O4-NCs-immobilized bacteria is the lowest, which is due to the combined effect of the adsorption of MIP-Fe3O4-NCs and the biodegradation of microorganisms by sodium alginate as the loading medium. From Figure 6 As can be seen from B, the total degradation amount of experimental group G6 is much greater than that of other control groups, and the difference is more significant at low concentrations. This is mainly because the sodium alginate and MIP-Fe3O4-NCs present in G6 have good molecular selectivity, and the MIP-Fe3O4-NCs with high binding ability and fast binding kinetics are evenly dispersed in the MIP-Fe3O4-NCs-immobilized bacteria, avoiding the negative effects of the aggregation of MIP-Fe3O4-NCs in water and environmental polarity. The sodium alginate-immobilized microspheres with a porous structure, high surface area ratio and high biocompatibility provide an optimal environment for the growth and metabolism of bacteria. The enrichment of pesticides by the MIP-Fe3O4-NCs-immobilized bacteria increases the pesticide concentration around the degrading bacteria and enhances the degradation efficiency of the bacteria.

[0067] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A preparation method of sodium alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides, which is characterized in that It includes the following steps: Step 1: Prepare mesoporous molecularly imprinted modified Fe3O4-NCs by surface grafting method to obtain surface imprinted functionalized Fe3O4 nanostructured magnetic microspheres MIP-Fe3O4-NCs; Step 2: Mix the methanol-activated MIP-Fe3O4-NCs with the sterilized sodium alginate solution, then add the pyrethroid pesticide-degrading bacteria solution. After stirring well, add it to cyclohexane. After stirring evenly again, dropwise add calcium chloride solution, and carry out cross-linking reaction at 4°C for 2-6 h to obtain sodium alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for degrading pyrethroid pesticides, where the ratio of MIP-Fe3O4-NCs, sodium alginate solution, Pseudomonas fluorescens seed solution, cyclohexane and calcium chloride solution is 10-20 mg: 20 mL: 1 mL: 20 mL: 5 mL.

2. The preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides according to claim 1, characterized in that Step 1 is specifically as follows: (1) Add 2-3 mL of Triton X-100 and 15-25 mL of cyclohexane to a round-bottom flask. After vigorous stirring, sequentially add 38.2-42.8 μL of 3-aminopropyltriethoxysilane, 50-70 μL of tetraethyl orthosilicate and 200 μL of NH3·H2O, stir for 8-12 h. After the reaction is completed, add 20 mL of acetone, shake for 15 min, then centrifuge to remove the supernatant, wash the precipitate twice with absolute ethanol and ultrapure water respectively, and place it in a 60°C vacuum drying oven in the dark for drying to obtain APTES-modified amino-functionalized silica microspheres; (2) Add 400 μL of Fe3O4-NCs with a concentration of 1.25 - 2 mg / mL and 50 - 70 mg of amino-functionalized silica microspheres successively into a mixed solution composed of 1.5 mL of xylene, 10 - 15 mL of cyclohexane, and 30 - 40 μL of TEOS, transfer it to a flask, purge the oxygen in the flask with nitrogen, then add 200 μL of NH3·H2O and stir for 2 - 4 h. After that, add 100 - 200 μL of pyrethroid pesticide solution with a concentration of 20 mg / mL, mix well, and stir in the dark for 6 - 8 h. Subsequently, add 11.6 - 14.8 μL of ethylene glycol dimethacrylate (EGDMA) and 15 mg of azobisisobutyronitrile (AIBN) into the flask, heat up to 60 °C, and continue to stir in the dark for 2 - 4 h. After the stirring is completed, add 10 - 15 mL of acetone for demulsification and sedimentation, then let it stand in a water bath at 35 °C for 2 - 4 h, centrifuge and discard the supernatant. The precipitate is resuspended in ultrapure water, vortexed for 1 - 2 min, centrifuged again, and the supernatant is discarded to remove unreacted TEOS, EGDMA, APTES, and MAA. Then add 5 - 10 mL of a mixed solution composed of ethanol and acetonitrile in a volume ratio of 4:1 and sonicate for 5 min, then vortex for 30 - 50 min, centrifuge and remove the supernatant for the elution of pyrethroid pesticides, and repeat the elution 2 - 3 times. Subsequently, add 10 - 20 mL of a mixed solution composed of methanol and acetic acid in a volume ratio of 9:1, shake for 15 min and then centrifuge to remove the precipitate, and repeat the elution until the absorbance of the supernatant is close to 0, and then the elution is completed to obtain MIP-Fe3O4-NCs.

3. The preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides according to claim 2, characterized in that The preparation method of Fe3O4-NCs described in step (2) is as follows: Weigh 5.4 - 8.1 g of FeCl3·6H2O and dissolve it in 150 mL of a mixed solvent composed of ultrapure water, ethanol, and n-hexane in a volume ratio of 3:5:

7. Then add 6.1 - 9.2 g of sodium oleate, mix well by ultrasonic, and transfer it to a flask. Under magnetic stirring at 300 - 400 rpm, heat the solution to 70 - 80 °C and maintain it for 4 - 6 h. After heating, perform phase separation, separate the upper layer of n-hexane, transfer the remaining liquid to a flask containing 3 - 6 mL of a mixed solution composed of oleic acid and octadecene in a volume ratio of 1:2, and add 10 - 15 mL of ethylene glycol. Heat it to 300 - 320 °C under a nitrogen atmosphere and evacuate for 20 - 40 min, maintaining the heating for 0.5 - 1.5 h. After heating is completed, cool it to room temperature, add 10 - 20 mL of a mixed solution composed of chloroform and ethanol in equal volume, shake and then centrifuge. Take the precipitate and wash it repeatedly with ultrapure water and ethanol 2 - 4 times, and dry it in a vacuum at 60 °C to obtain square Fe3O4 nanoblocks Fe3O4-NCs.

4. The preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides according to claim 2, characterized in that The solute in the pyrethroid pesticide solution described in step (2) is one or more of cyfluthrin, deltamethrin, and cypermethrin, and the solvent is a solvent composed of 3-aminopropyltriethoxysilane and methacrylic acid in a volume ratio of 2:

1.

5. The preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides according to claim 1, characterized in that: The sodium alginate solution described in Step 2 is a sodium alginate solution with a concentration of 25 g / L prepared using standard seawater with a salinity of 30‰, and the calcium chloride solution is a calcium chloride solution with a concentration of 20 g / L prepared using standard seawater with a salinity of 30‰.

6. The preparation method of alginate-mediated MIP-Fe3O4-NCs immobilized bacteria for adsorbing and degrading trace pyrethroid pesticides according to claim 1, characterized in that: The pyrethroid pesticide-degrading bacterial liquid described in Step 2 is a Photobacterium ganghwense bacterial liquid with an OD 600 = 2.

0. The Photobacterium ganghwense is strain Nbuzhang1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2014580.

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