Preparation method of enzyme immobilization carrier for waste water of leaf mustard and application thereof

Abstract

This invention discloses a method for preparing an enzyme immobilization carrier for vegetable wastewater and its application, relating to the field of modified materials technology, including the following steps: (1) Adding iron oxide powder to a reactor containing water, dispersing it for 15-30 min, and then homogenizing it under inert gas protection; (2) Adding a modifier to the above system at 55°C under inert gas protection and reflux, then adding a surfactant and ethanol in sequence, and stirring for 30 min for homogenization; (3) Adding a mixed solution of styrene, crosslinking agent, epoxy monomer and pore-forming agent dropwise to the system, and stirring evenly; (4) Adding an initiator to the system, heating to 80°C, adding a metal salt solution, stirring continuously, and keeping warm at 80°C for 3.5 h; (5) Filtering to separate solid and liquid, obtaining open-structure eggshell-shaped magnetic microparticles, washing them, and drying them to constant weight; The above carrier can be used for enzyme immobilization and treatment of vegetable wastewater.

Description

Technical Field

[0001] This invention relates to the field of modified materials technology, and more specifically, to a method for preparing an enzyme immobilization carrier for vegetable wastewater and its application. Background Technology

[0002] With the improvement of people's living standards and the development of intensive vegetable cultivation, the output of vegetable waste (i.e., vegetable scraps) is increasing day by day. Fields, ditches, roadsides, vegetable distribution centers, and farmers' markets are the main sources of vegetable waste. Due to a lack of scientific and economical technologies for the treatment and utilization of vegetable waste, most of it is treated as waste or simply landfilled. Besides causing visual pollution, the high water content and easy rotting of vegetable waste lead to water, soil, and air pollution and the spread of pests and diseases during the decomposition process. Vegetable waste typically has a solid content of 3-19%, containing abundant organic matter such as sugars, hemicellulose, cellulose, and lignin. The residue after solid-liquid separation can be returned to the field as a usable resource after aerobic composting. However, there is a lack of targeted guidance methods for the treatment of vegetable wastewater.

[0003] Biological methods are the mainstream approach for wastewater treatment. Vegetable wastewater has a complex composition; even after solids are separated and recovered during the recycling process, it still contains a large amount of macromolecular organic matter. Conventional wastewater treatment processes struggle to degrade cellulose, hemicellulose, and lignin, which are abundant renewable organic matter in vegetable wastewater. Related enzymes can efficiently hydrolyze these substances into soluble small-molecule reducing sugars such as cellobiose and glucose, improving their utilization rate and offering considerable economic and environmental benefits. For the biodegradation of cellulose, hemicellulose, and lignin, enzyme immobilization technology has become a hot research topic to improve enzyme stability and resistance to inhibition.

[0004] The paper "Process Optimization of Cellulase Immobilization" published in *China Biogas* by Ma Yifang et al. reports a technique for preparing immobilized cellulase gel microspheres using sodium alginate and polyethylene glycol as raw materials and glutaraldehyde as a cross-linking agent. This achievement optimizes the preparation conditions of the immobilization carrier and effectively improves the enzymatic properties of the immobilized cellulase. However, further research is still lacking on the application of immobilized enzyme carriers in wastewater treatment and carrier recovery. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the first objective of the present invention is to provide a method for preparing eggshell-shaped magnetic microparticles, and the second objective is to provide the application of the above-mentioned eggshell-shaped magnetic microparticles in enzyme immobilization and treatment of vegetable wastewater.

[0006] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing eggshell-shaped magnetic microparticles, comprising the following steps:

[0007] (1) Add the iron oxide powder to a reactor containing water, disperse it for 15-30 minutes, and then homogenize it under inert gas protection;

[0008] (2) At 55°C, under inert gas protection and reflux, add the modifier to the above system, then add the surfactant and ethanol in sequence, and stir for 30 min to homogenize.

[0009] (3) Add a mixed solution of styrene, crosslinking agent, epoxy monomer and porogen to the system dropwise, and stir until homogeneous;

[0010] (4) Add an initiator to the system, heat to 80°C, add a metal salt solution, stir continuously, and keep warm at 80°C for 3.5 hours;

[0011] (5) Filtration was performed to separate solids and liquids, resulting in open-structure eggshell-shaped magnetic microparticles. These microparticles were then washed and dried to constant weight.

[0012] By adopting the above technical solution, metal salts are added during the emulsion polymerization process of preparing magnetic microparticles to break the emulsion, thereby rupturing the polymer droplets and preparing open-structure eggshell-shaped magnetic microparticles.

[0013] Furthermore, the mass ratio of the modifier to the iron oxide powder is 1:1.

[0014] Furthermore, the metal salt solution is one or both of sodium salt and calcium salt.

[0015] Furthermore, the metal salt solution is one or both of sodium chloride and calcium chloride.

[0016] Furthermore, the mass concentration of the metal salt solution is 2-30%, and the mass ratio of the metal salt solution to styrene is 1-5:10.

[0017] Furthermore, the mass ratio of styrene, crosslinking agent, epoxy monomer and porogen in the mixed solution is (70-80):(10-15):(10-15):(50-150).

[0018] Furthermore, the initiator is added at a mass of 1-10% of the mass of the mixed solution.

[0019] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0020] The above-mentioned application of eggshell-shaped magnetic microparticles as carriers in enzyme immobilization includes: adding the enzyme to a container filled with water, adding eggshell-shaped magnetic microparticles, and mixing to obtain a mixture; adding a modifier and immobilizing by shaking; filtering to obtain eggshell-shaped magnetic microparticles immobilized with the enzyme, and washing them until no free enzyme remains.

[0021] Furthermore, the enzyme is one or more of cellulase, hemicellulase, and ligninase.

[0022] Furthermore, the mass ratio of the eggshell-shaped magnetic microparticles to the enzyme is (1-10):1.

[0023] Further, the modifier is one or more selected from glutaraldehyde, polyethylene glycol, polyvinyl alcohol, tetraethyl orthosilicate, aminopropyltriethoxysilane, butyl propionate, and ethylenediamine.

[0024] Furthermore, the modifier has a mass fraction of 0.05-2.5%, and the amount of modifier added is 5-20% of the volume of the mixed liquid.

[0025] Furthermore, the oscillation immobilization conditions are as follows: immobilization temperature is 4-30℃, immobilization time is 2-10h, and oscillation frequency is 160rpm.

[0026] The application of the aforementioned immobilized enzyme eggshell-shaped magnetic microparticles in the treatment of vegetable wastewater includes: continuously operating the immobilized enzyme eggshell-shaped magnetic microparticles, inoculating anaerobic methanogenic activated sludge, and anaerobic fermenting the vegetable wastewater.

[0027] Furthermore, the eggshell-shaped magnetic microparticles that immobilize the enzyme are one or more of the following: eggshell-shaped magnetic microparticles that immobilize cellulase, eggshell-shaped magnetic microparticles that immobilize hemicellulase, and eggshell-shaped magnetic microparticles that immobilize ligninase.

[0028] In summary, the present invention has the following beneficial effects:

[0029] First, in the process of emulsion polymerization for preparing magnetic microparticles, the present invention adds a metal salt to break the emulsion, thereby causing the polymer droplets to rupture and prepare open-structure eggshell-shaped magnetic microparticles.

[0030] Secondly, in the application of the eggshell-shaped magnetic microparticles of the present invention for enzyme immobilization, the enzyme is surrounded on the inner side of the eggshell-shaped magnetic carrier to form a protective structure, which can reduce the friction and collision of the enzyme in the fluidized state, improve the stability and service life of the enzyme immobilization carrier, and also realize the recycling of enzyme by utilizing the magnetic characteristics of the magnetic microparticles.

[0031] Third, immobilized enzyme-like eggshell-shaped magnetic microparticles can be used for anaerobic fermentation treatment of vegetable wastewater, which can improve the degradation effect of cellulose substances in the wastewater. Attached Figure Description

[0032] Figure 1 These are microscopic images of the immobilized enzyme prepared in Example 1 of the present invention: (a): optical microscope image; (b): fluorescence microscope image.

[0033] Figure 2Here are optical microscope images of the immobilized enzymes prepared in Comparative Examples 2 and 3 of this invention, (a):

[0034] Comparative Example 2; (b): Comparative Example 3;

[0035] Figure 3 The following are the test results of anaerobic treatment of vegetable wastewater in the embodiments and comparative examples of the present invention: (a): COD detection results; (b): cumulative methane production detection results. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0037] Unless otherwise specified, all reagents used in the examples and comparative examples are commercially available.

[0038] In some specific embodiments, the modifier is selected from one or more of oleic acid, tartaric acid, γ-aminotriethoxysilane, γ-glycidoxypropyltrimethoxysilane, vinyltriethoxysilane, vinyltrichlorosilane, and γ-aminotrimethoxysilane.

[0039] In some specific embodiments, the surfactant is selected from one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, sodium laurate, and sodium oleate.

[0040] In some specific embodiments, the crosslinking agent is selected from one or more of divinylbenzene, N,N-methylenebisacrylamide, pentaerythritol triacrylate, ethylene glycol dimethacrylate, and pentaerythritol tetraacrylate.

[0041] In some specific embodiments, the epoxy monomer is selected from one or more of allyl glycidyl ether, glycidyl methacrylate, and glycidyl acrylate.

[0042] In some specific embodiments, the pore-forming agent is n-heptane.

[0043] In some specific embodiments, the initiator is selected from one or more of azobisisobutyronitrile, benzoyl peroxide, dodecyl peroxide, azobisisoheptanenitrile, potassium persulfate, and ammonium persulfate.

[0044] In some specific embodiments, the metal salt solution is one or both of sodium salt and calcium salt, preferably one or both of sodium chloride and calcium chloride.

[0045] Example 1

[0046] A method for preparing eggshell-shaped magnetic microparticles includes the following steps:

[0047] (1) Add 6g of iron oxide powder to a 250mL four-necked flask containing 180g of pure water, stir under ultrasonication for 30min, and then continue to homogenize under nitrogen protection by mechanical stirring for 10min.

[0048] (2) At 55°C, under nitrogen protection and reflux, 6g of oleic acid was added to the system and modified for 30min; 0.5g of sodium dodecyl sulfate was added; after 30min, 120mL of ethanol was added and the mixture was stirred for 30min to homogenize.

[0049] (3) Add a mixed solution of 28g styrene, 4g divinylbenzene, 4g glycidyl methacrylate and 30g n-heptane to the system dropwise, and stir until homogeneous;

[0050] (4) Add 1.5g of potassium persulfate to the system, heat to 80℃, add 10mL of 2% sodium chloride solution, stir continuously, and keep warm at 80℃ for 3.5h;

[0051] (5) Filtration was performed to separate solids and liquids, and eggshell-shaped magnetic particles were obtained. The particles were washed with pure water and ethanol and then dried under vacuum to constant weight.

[0052] Applications of enzyme immobilization using eggshell-shaped magnetic microparticles:

[0053] Add 100 mL of deionized water to an Erlenmeyer flask, add 2 g of cellulase, 2 g of hemicellulase, and 2 g of ligninase to the flask, add 10 g of eggshell-shaped magnetic microparticles, and shake to mix well to obtain a mixture; add 0.15% glutaraldehyde, and shake at 4 °C to immobilize; filter to obtain eggshell-shaped magnetic microparticles of immobilized enzyme, wash several times with pure water until no free enzyme is found in the washing solution.

[0054] Immobilized enzyme-containing eggshell-shaped magnetic microparticles for treating vegetable wastewater:

[0055] Take vegetable wastewater and adjust its COD to about 8000 mg / L. Add 150 mL of vegetable wastewater, 50 mL of anaerobic methanogenic activated sludge, and 1 g of eggshell-shaped magnetic microparticles with immobilized enzyme to an anaerobic fermentation bottle. Run the system continuously for 30 days, and measure the COD concentration and methane production of the system every 3 days.

[0056] Example 2

[0057] A method for preparing eggshell-shaped magnetic microparticles includes the following steps:

[0058] (1) Add 6g of iron oxide powder to a 250mL four-necked flask containing 180g of pure water, stir under ultrasonication for 30min, and then continue to homogenize under nitrogen protection by mechanical stirring for 10min.

[0059] (2) At 55°C, under nitrogen protection and reflux, 6g of tartaric acid was added to the system and modified for 30min; 1g of sodium dodecyl sulfonate was added; after 30min, 120mL of ethanol was added and the mixture was stirred for 30min to homogenize.

[0060] (3) Add dropwise a mixed solution of 26g styrene, 5g pentaerythritol triacrylate, 5g allyl glycidyl ether and 30g n-heptane to the system and stir until homogeneous;

[0061] (4) Add 1.5g of azobisisobutyronitrile to the system, heat to 80℃, add 10mL of 2% sodium chloride solution, stir continuously, and keep warm at 80℃ for 3.5h.

[0062] (5) Filtration was performed to separate solids and liquids, and eggshell-shaped magnetic particles were obtained. The particles were washed with pure water and ethanol and then dried under vacuum to constant weight.

[0063] Applications of enzyme immobilization using eggshell-shaped magnetic microparticles:

[0064] Add 100 mL of deionized water to an Erlenmeyer flask, add 6 g of cellulase to the flask, add 10 g of eggshell-shaped magnetic microparticles, and shake to mix well to obtain a mixture; add 0.1% polyethylene glycol, and shake at 4 °C to immobilize; filter to obtain eggshell-shaped magnetic microparticles with immobilized enzyme, wash several times with pure water until no free enzyme is found in the washing solution.

[0065] Immobilized enzyme-containing eggshell-shaped magnetic microparticles for treating vegetable wastewater:

[0066] Take vegetable wastewater and adjust its COD to about 8000 mg / L. Add 150 mL of vegetable wastewater, 50 mL of anaerobic methanogenic activated sludge, and 0.5 g of eggshell-shaped magnetic microparticles with immobilized enzyme to an anaerobic fermentation bottle. Run the system continuously for 30 days and measure the COD concentration and methane production of the system every 3 days.

[0067] Example 3

[0068] A method for preparing eggshell-shaped magnetic microparticles includes the following steps:

[0069] (1) Add 6g of iron oxide powder to a 250mL four-necked flask containing 180g of pure water, stir under ultrasonication for 30min, and then continue to homogenize under nitrogen protection by mechanical stirring for 10min.

[0070] (2) At 55°C, under nitrogen protection and reflux, 6g of γ-aminotriethoxysilane was added to the system and modified for 30min; 1g of sodium oleate was added; after 30min, 120mL of ethanol was added and stirred for 30min to homogenize.

[0071] (3) Add a mixed solution of 26g styrene, 5g ethylene glycol dimethacrylate, 5g glycidyl acrylate and 30g n-heptane to the system and stir until homogeneous;

[0072] (4) Add 1.5g of ammonium persulfate to the system, heat to 80℃, add 10mL of 2% sodium chloride solution, stir continuously, and keep warm at 80℃ for 3.5h;

[0073] (5) Filtration was performed to separate solids and liquids, and eggshell-shaped magnetic particles were obtained. The particles were washed with pure water and ethanol and then dried under vacuum to constant weight.

[0074] Applications of enzyme immobilization using eggshell-shaped magnetic microparticles:

[0075] Add 100 mL of deionized water to an Erlenmeyer flask, add 6 g of hemicellulase to the flask, add 10 g of eggshell-shaped magnetic microparticles, and shake to mix well to obtain a mixture; add 0.15% ethylenediamine, and shake at 4 °C to immobilize; filter to obtain eggshell-shaped magnetic microparticles of immobilized enzyme, wash several times with pure water until no free enzyme is found in the washing solution.

[0076] Immobilized enzyme-containing eggshell-shaped magnetic microparticles for treating vegetable wastewater:

[0077] Take vegetable wastewater and adjust its COD to about 8000 mg / L. Add 150 mL of vegetable wastewater, 50 mL of anaerobic methanogenic activated sludge, and 1 g of eggshell-shaped magnetic microparticles with immobilized enzyme to an anaerobic fermentation bottle. Run the system continuously for 30 days, and measure the COD concentration and methane production of the system every 3 days.

[0078] Comparative Example 1

[0079] Take vegetable wastewater and adjust its COD to about 8000 mg / L. Add 150 mL of vegetable wastewater and 50 mL of inoculated anaerobic methanogenic activated sludge to an anaerobic fermentation bottle. Run the system continuously for 30 days, and measure the COD concentration and methane production of the system every 3 days.

[0080] Comparative Example 2

[0081] The method for preparing eggshell-shaped magnetic particles differs from that in Example 1 in that the sodium chloride solution concentration is 0.5%, while the remaining steps and reagent usage are the same as in Example 1.

[0082] Comparative Example 3

[0083] The method for preparing eggshell-shaped magnetic particles differs from that in Example 1 in that the sodium chloride solution concentration is 30%, while the remaining steps and reagent usage are the same as in Example 1.

[0084] Performance testing and results

[0085] The eggshell-shaped magnetic microparticles of the immobilized enzyme prepared in Example 1 were observed using optical and fluorescence microscopy, and the results are as follows: Figure 1 As shown.

[0086] COD concentration detection of anaerobic treatment of vegetable wastewater in each embodiment and comparative example: The supernatant was taken and the COD was measured using the corresponding reagent kit and 5B-3B(V11) multi-parameter water quality analyzer from Lianhua Technology Co., Ltd.

[0087] Methane production was measured in the anaerobic treatment of vegetable wastewater in the various embodiments and comparative examples: the gas production of anaerobic fermentation was determined by the drainage method, and the methane production was determined by gas chromatography (GC-7860Plus, Nuoxi Instruments, China).

[0088] like Figure 1 As shown, the open-structure eggshell-shaped magnetic microparticles prepared by this invention differ from conventional circular closed-structure magnetic microparticles in that they form an uneven internal containment space. Combining the same perspective of bright-field and fluorescence fields, it can be observed that there are obvious strongly fluorescent particles inside the magnetic microparticles. This is because enzymes, as proteins, have stronger fluorescence intensity than magnetic microparticles, indicating that the enzyme has successfully attached itself. The eggshell-shaped magnetic microparticles encapsulate the enzyme on the inside, forming a protective structure and buffer space, which can reduce friction and collision of the enzyme in fluidized state during wastewater treatment, improving the stability and lifespan of the immobilized enzyme carrier. However, when the concentration of the salt solution used as a demulsifier is too low or too high, the desired structure cannot be achieved, such as... Figure 2 As shown, when the salt solution concentration is too low, demulsification fails, resulting in a conventional spherical closed structure (a); when the salt solution concentration is too high, complete demulsification occurs, resulting in a disordered morphology (b), and an eggshell-like structure for enzyme immobilization cannot be obtained. Figure 3 It can be seen that the COD degradation amount in the examples and comparative examples increases with time. The eggshell-shaped magnetic microparticles of the immobilized enzyme can completely or nearly completely degrade COD within 30 days, while the COD degradation amount of conventional degradation methods, such as Comparative Example 1, is only 1 / 2 after 30 days. Combined with the cumulative methane production test results, the cumulative methane production of the examples tends to stabilize at 25-30 days, while the cumulative methane production of the comparative example is still less than 1 / 2 of that of the examples at 30 days. This shows that the immobilized enzyme carrier of the present invention has excellent effect in treating vegetable wastewater, can shorten the degradation time, and improve the degradation rate of cellulose substances in vegetable wastewater.

[0089] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing eggshell-shaped magnetic microparticles, characterized in that, Includes the following steps: (1) Add the iron oxide powder to a reactor containing water, disperse it for 15-30 minutes, and then homogenize it under inert gas protection; (2) At 55°C, under inert gas protection and reflux, a modifier is added to the above system, followed by the addition of a surfactant and ethanol, and the mixture is stirred for 30 min to homogenize. The modifier is selected from one or more of oleic acid, tartaric acid, γ-aminotriethoxysilane, γ-glycidoxypropyltrimethoxysilane, vinyltriethoxysilane, vinyltrichlorosilane, and γ-aminotrimethoxysilane. (3) Add a mixed solution of styrene, crosslinking agent, epoxy monomer and porogen to the system dropwise, and stir until uniform. The epoxy monomer is selected from one or more of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate. (4) Add an initiator to the system, heat to 80°C, add a metal salt solution, stir continuously, and keep warm at 80°C for 3.5 hours. The metal salt solution is one or two of sodium salt and calcium salt. (5) Filter to separate solid and liquid, and obtain open-structure eggshell-shaped magnetic particles. Wash them and dry them to constant weight.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the modifier to the iron oxide powder is 1:

1.

3. The preparation method according to claim 1, characterized in that, The metal salt solution is one or both of sodium chloride and calcium chloride.

4. The preparation method according to claim 1, characterized in that, The mass concentration of the metal salt solution is 2-30%, and the mass ratio of the metal salt solution to styrene is 1-5:

10.

5. The preparation method according to claim 1, characterized in that, The mass ratio of styrene, crosslinking agent, epoxy monomer and porogen in the mixed solution is (70-80): (10-15): (10-15): (50-150).

6. The application of the eggshell-shaped magnetic microparticles according to any one of claims 1-5 as a carrier for enzyme immobilization, characterized in that, The enzyme is added to a container filled with water, followed by eggshell-shaped magnetic microparticles, and mixed thoroughly to obtain a mixture. A modifier is added, and the mixture is shaken to immobilize it. The mixture is then filtered to obtain eggshell-shaped magnetic microparticles containing the immobilized enzyme, and washed until no free enzyme remains. The modifier is one or more of the following: glutaraldehyde, polyethylene glycol, polyvinyl alcohol, tetraethyl orthosilicate, aminopropyltriethoxysilane, butyl propionate, and ethylenediamine.

7. The application according to claim 6, characterized in that, The mass ratio of eggshell-shaped magnetic microparticles to enzymes is (1-10):

1.

8. The application of the eggshell-shaped magnetic microparticles with immobilized enzymes according to claim 7 in the treatment of vegetable wastewater, characterized in that, The process involves continuously operating eggshell-shaped magnetic microparticles containing immobilized enzymes, inoculating anaerobic methanogenic activated sludge, and anaerobic fermenting vegetable wastewater.

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