Biological enzyme composition based on chitosan / lignin composite sponge carrier and preparation method thereof

Abstract

The invention discloses a preparation method of a biological enzyme composition based on a chitosan / lignin composite sponge carrier, and relates to the technical field of biological enzymes. Comprising the following steps: carrying out grafting reaction on chitosan colloid and palmitic acid under the action of a catalyst to obtain modified chitosan; mixing epoxidized lignin and modified chitosan, then adding a cross-linking agent, and carrying out a cross-linking reaction to obtain a chitosan / lignin composite sponge carrier; laccase and horse radish peroxidase are coated with chitosan gelatin layers, and enzyme microcapsules are obtained; and combining the chitosan / lignin composite sponge carrier with the enzyme micro-capsule to obtain the chitosan / lignin composite sponge. The prepared bio-enzyme composition based on the chitosan / lignin composite sponge carrier not only has a good adsorption effect on petroleum stains, but also can effectively decompose and remove the petroleum stains and thoroughly solve the problem of petroleum pollution, in addition, the chitosan / lignin composite sponge adsorption carrier used in the invention can be naturally degraded, and the bio-enzyme composition has a good application prospect. And no secondary pollution is caused to the soil.

Description

Technical Field

[0001] The present invention relates to the technical field of bioenzymes, and particularly to a bioenzyme composition based on a chitosan / lignin composite sponge carrier and a preparation method thereof. Background Art

[0002] At present, with the booming development of the petroleum industry, there is a risk of oil leakage at every link from oil extraction, transportation to storage. Once a leakage occurs, a large amount of oil flows into the environment such as the ocean and soil. Various complex organic compounds such as hydrocarbons and aromatics in the oil will cause serious damage to the ecological environment. Organisms such as fish and seabirds in the ocean will die due to contact with oil pollution, and the microbial community structure in the soil will also be changed, leading to the imbalance of the ecosystem. At the same time, oil pollution will also cause direct economic losses to industries such as tourism and fishery. Repairing the environment polluted by oil often requires huge human, material and time costs. Therefore, effectively treating the oil pollution caused by oil leakage has become a major issue of global concern.

[0003] Among many oil pollution treatment technologies, the adsorption method has become a relatively widely used method due to its advantages such as simple operation, relatively low cost, and less disturbance to the environment. Common adsorption materials include activated carbon, diatomite, natural fiber materials, etc. Activated carbon has a rich pore structure and a large specific surface area, and can adsorb oil; diatomite is widely sourced and inexpensive, and has certain adsorption properties; natural fiber materials such as cotton fiber and wood fiber are green and environmentally friendly, and can also adsorb oil to a certain extent. However, these traditional adsorption materials generally have a significant defect, that is, their adsorption effect on petroleum oil pollution is poor, and they can only adsorb the oil pollution on their own surface or inside the pores, only realizing the physical transfer of the oil pollution, and cannot completely decompose and remove the oil pollution. If the materials after adsorption saturation are not properly disposed of, it is easy to cause secondary pollution, and these materials are difficult to reuse, resulting in a large amount of solid waste. Summary of the Invention

[0004] The purpose of the present invention is to provide a bioenzyme composition based on a chitosan / lignin composite sponge carrier and a preparation method thereof, so as to solve the technical problems in the above background art that the traditional adsorption materials have poor adsorption effect on oil pollution, cannot decompose and remove oil pollution, and the adsorption materials are easy to cause secondary pollution to the soil. The bioenzyme composition based on the chitosan / lignin composite sponge carrier prepared by the present invention not only has a good adsorption effect on petroleum oil pollution, but also can effectively decompose and remove petroleum oil pollution, completely solving the problem of oil pollution. In addition, the chitosan / lignin composite sponge adsorption carrier used in the present invention can be naturally degraded and will not cause secondary pollution to the soil.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A preparation method of a bioenzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0007] S1. Dissolve chitosan in a citric acid solution and stir to form a chitosan colloid;

[0008] S2. Carry out a grafting reaction on the chitosan colloid and palmitic acid under the action of a catalyst to obtain modified chitosan;

[0009] S3. Pretreat lignin with an alkali solution, and then react it with epichlorohydrin to introduce epoxy functional groups to obtain epoxidized lignin;

[0010] S4. Mix the epoxidized lignin and the modified chitosan, then add a crosslinking agent and carry out a crosslinking reaction to obtain a chitosan / lignin composite sponge carrier;

[0011] S5. Add laccase and horseradish peroxidase to an acetic acid buffer solution to obtain an oxidase solution, and dissolve sodium alginate and gelatin in an acetic acid buffer solution to obtain a polymer solution;

[0012] S6. Use the oxidase solution as the core layer solution and the polymer solution as the shell layer solution, prepare microcapsules by coaxial electrospray method, and drop the microcapsules into a calcium chloride solution for crosslinking and curing to obtain enzyme microcapsules;

[0013] S7. Add the chitosan / lignin composite sponge carrier and the enzyme microcapsules to a phosphate buffer solution and carry out vacuum infiltration treatment to obtain the product.

[0014] In the technical solution of the present invention, by dissolving chitosan in a citric acid solution to form a colloid, and at the same time pretreating lignin with an alkali solution and then epoxidizing it, and then mixing and crosslinking the two, the prepared chitosan / lignin composite sponge carrier has a special three-dimensional porous structure. This three-dimensional structure can undergo moderate swelling when encountering oil. The internal porous structure is like a tiny storage space. After oil molecules enter the pores, it will cause the distance between chitosan and lignin molecular chains to increase, thereby triggering the swelling of the carrier, which helps to expand the adsorption area and improve the adsorption capacity. Compared with traditional adsorption materials such as activated carbon and diatomite, the adsorption capacity for oil pollution is improved. In addition, the chitosan / lignin composite sponge adsorption carrier used in the present invention can be naturally degraded and will not cause secondary pollution to the soil.

[0015] However, due to the strong hydrophilicity of chitosan itself, its affinity for hydrophobic petroleum oil pollution is limited, which has a certain impact on the adsorption of petroleum. To solve this problem, in the present invention, chitosan is grafted and modified with palmitic acid. The long carbon chain structure of palmitic acid can significantly improve the hydrophobicity of chitosan. After introducing palmitic acid, the surface properties of the modified chitosan change, making it easier to interact with petroleum hydrocarbon substances. On the other hand, the grafting reaction increases the length and steric hindrance of the chitosan molecular chain, which helps to construct a more open and ordered three-dimensional porous structure during the subsequent process of mixing and cross-linking with epoxidized lignin to form a composite sponge carrier. Through the above effects, the adsorption performance of the carrier for petroleum oil pollution is further improved.

[0016] In the present invention, laccase and horseradish peroxidase are loaded in the chitosan / lignin composite sponge carrier, and laccase and horseradish peroxidase are used to gradually oxidize and decompose hydrocarbon substances in petroleum into harmless substances such as carbon dioxide and water, fundamentally solving the problem of petroleum pollution. However, the research team of the present invention found in the experiment that directly loading laccase and horseradish peroxidase into the chitosan / lignin composite sponge carrier, the adsorption of the enzyme by soil particles, the competition of microorganisms, the interference of various chemical substances in the soil, and the disturbance of mechanical external forces seriously affect the activity and stability of the enzyme, thus affecting the decomposition ability of the enzyme for petroleum oil pollution. To further solve this problem, in the present invention, laccase and horseradish peroxidase are dissolved in acetic acid buffer as the core layer solution, and sodium alginate and gelatin are dissolved in acetic acid buffer as the shell layer solution, and microcapsules are prepared by coaxial electrospray method. This microcapsule structure provides a reliable protection barrier for the enzyme. The shell layer formed by sodium alginate and gelatin can not only isolate the erosion of various harmful substances in the soil on the enzyme, but also maintain the relative stability of the microenvironment where the enzyme is located, effectively resisting the influence of soil environment changes on the enzyme activity. In addition, sodium alginate and gelatin as the microcapsule shell layer materials have good film-forming properties and biocompatibility. The shell layer structure they form is relatively soft and has a certain elasticity, which can better fit the three-dimensional porous surface of the composite sponge carrier. In addition, the chemical properties of these two materials themselves make it easy for them to interact with the carrier surface, thereby improving the binding stability between the enzyme and the carrier and making the enzyme not easily detached from the carrier.

[0017] Preferably, in the step S2, the mass ratio of chitosan to palmitic acid is 1:0.1 - 0.4.

[0018] In the technical solution of the present invention, as described above, chitosan is modified with palmitic acid. The long carbon chain structure of palmitic acid can significantly improve the hydrophobicity of chitosan, making it easier to interact with petroleum hydrocarbon substances and enhancing the adsorption capacity of the carrier for oil stains. Through experiments, it is found that when the mass ratio of chitosan to palmitic acid is less than 1:0.1, the carrier has strong adsorption performance for petroleum oil stains. As the amount of palmitic acid increases, the adsorption performance of the carrier for oil stains increases slowly. However, when the amount of palmitic acid increases to a mass ratio of chitosan to palmitic acid of 1:0.4, the present invention team finds that the decomposition performance of the bioenzyme composition for petroleum oil stains drops significantly. After research, it is found that this is because the binding of the enzyme microcapsule to the carrier depends on the interaction between their surfaces. The excessive long carbon chains of palmitic acid on the carrier surface will reduce its effective binding sites with the shell layer of the enzyme microcapsule (sodium alginate and gelatin), resulting in a decrease in the loading amount of the enzyme microcapsule on the carrier, and further leading to a decrease in the decomposition performance of the bioenzyme for petroleum oil stains. Therefore, the present invention strictly controls the mass ratio of chitosan to palmitic acid to be 1:0.1 - 0.4.

[0019] Preferably, in step S2, the grafting reaction temperature is 60 - 70 °C and the reaction time is 3 - 6 h.

[0020] Preferably, in step S3, the alkali solution is selected from one or more of sodium hydroxide and potassium hydroxide.

[0021] Preferably, in step S4, the mass ratio of epoxidized lignin to modified chitosan is 1:2 - 6.

[0022] Preferably, in step S4, the crosslinking agent is selected from one or more of glutaraldehyde and genipin.

[0023] Preferably, in step S5, the mass ratio of laccase to horseradish peroxidase is 1:0.5 - 0.8.

[0024] Preferably, in step S6, the mass concentration of the calcium chloride solution is 1.5%, and the crosslinking and curing time is controlled within 38 - 43 min.

[0025] Preferably, in step S7, the mass ratio of the chitosan / lignin composite sponge carrier to the enzyme microcapsule is 1:0.1 - 0.3.

[0026] A bioenzyme composition based on a chitosan / lignin composite sponge carrier is prepared by the above method.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. The composite sponge carrier prepared by mixing and crosslinking chitosan and lignin has a special three-dimensional porous structure. It expands moderately when encountering oil, increasing the adsorption area and enhancing the adsorption capacity. Compared with traditional adsorption materials such as activated carbon and diatomite, the adsorption capacity for oil pollution is significantly improved. Moreover, the chitosan / lignin composite sponge adsorption carrier can be naturally degraded without causing secondary pollution to the soil.

[0029] 2. Graft-modify chitosan with palmitic acid to improve its hydrophobicity, making it more likely to interact with petroleum hydrocarbons. At the same time, the grafting reaction increases the molecular chain length and steric hindrance of chitosan, which helps to construct a more open and ordered three-dimensional porous structure, further enhancing the adsorption performance of the carrier for oil pollution.

[0030] 3. Load laccase and horseradish peroxidase in the composite sponge carrier, which can oxidize and decompose petroleum hydrocarbons into harmless carbon dioxide and water.

[0031] 4. Prepare microcapsules by coaxial electrospray method, using sodium alginate and gelatin as the shell materials to provide a protection barrier for the enzyme, isolate the erosion of harmful substances in the soil, maintain the stability of the enzyme microenvironment, and resist the influence of soil environmental changes on the enzyme activity. Moreover, the shell materials have good film-forming properties and biocompatibility, can fit the porous surface of the carrier, interact with the carrier, improve the binding stability of the enzyme and the carrier, and prevent the enzyme from detaching from the carrier. Specific embodiments

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0033] Example 1

[0034] A preparation method of a bio-enzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0035] S1. Weigh 5 g of chitosan and add it to 300 mL of 0.1 mol / L citric acid solution, stir at 200 r / min for 1.5 h to form a chitosan colloid.

[0036] S2. Add palmitic acid to the chitosan colloid, with the mass ratio of chitosan to palmitic acid being 1:0.3. Then add 0.1 g of p-toluenesulfonic acid catalyst, and transfer the system to a 500 mL reaction kettle. React at 65 °C with stirring for 5 h to obtain modified chitosan.

[0037] S3. Add 8 g of lignin to 200 mL of 1 mol / L sodium hydroxide solution, stir and pretreat at room temperature for 1 h, then add 10 mL of epichlorohydrin, heat to 65 °C, and stir and react for 2 h to obtain epoxidized lignin;

[0038] S4. Mix 2 g of epoxidized lignin with 10 g of modified chitosan, then add 1 mL of glutaraldehyde crosslinking agent with a mass concentration of 25%, and stir and react at 50 °C for 3 h to obtain a chitosan / lignin composite sponge carrier;

[0039] S5. Add 2 g of laccase and 1.5 g of horseradish peroxidase to 100 mL of acetate buffer (pH = 5.0, 0.1 mol / L), stir evenly to obtain an oxidase solution; add 3 g of sodium alginate and 2 g of gelatin to 150 mL of acetate buffer (pH = 5.0, 0.1 mol / L), heat to 50 °C, stir and dissolve to obtain a polymer solution;

[0040] S6. Load the oxidase solution and the polymer solution into the inner and outer nozzle liquid storage tanks of a coaxial electrospray device respectively, set the electrospray voltage to 15 kV, the propulsion speed to 0.5 mL / h for the inner nozzle and 1.0 mL / h for the outer nozzle, drop the electrosprayed microcapsules into a calcium chloride solution with a mass concentration of 1.5%, and carry out crosslinking and curing for 40 min to obtain enzyme microcapsules;

[0041] S7. Add 8 g of chitosan / lignin composite sponge carrier and 2.2 g of enzyme microcapsules to 200 mL of phosphate buffer (pH = 7.0, 0.05 mol / L), transfer the mixed solution to a vacuum infiltration device, and infiltrate at a vacuum degree of 10 - 3 Pa for 30 min to obtain a bioenzyme composition based on the chitosan / lignin composite sponge carrier.

[0042] Example 2

[0043] A preparation method of a bioenzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0044] S1. Weigh 5 g of chitosan and add it to 300 mL of 0.1 mol / L citric acid solution, stir at 200 r / min for 1.5 h to form a chitosan colloid;

[0045] S2. Add palmitic acid to the chitosan colloid, the mass ratio of chitosan to palmitic acid is 1:0.2, then add 0.1 g of p-toluenesulfonic acid catalyst, then transfer the system to a 500 mL reaction kettle, react at a temperature of 65 °C with stirring for 4 h to obtain modified chitosan;

[0046] S3. Add 8 g of lignin to 200 mL of 1 mol / L potassium hydroxide solution, stir and pretreat at room temperature for 1 h, then add 10 mL of epichlorohydrin, heat to 65 °C, and stir and react for 2 h to obtain epoxidized lignin;

[0047] S4. Mix 2 g of epoxidized lignin with 6 g of modified chitosan, then add 1 mL of 25% glutaraldehyde crosslinking agent, and stir and react at 50 °C for 3 h to obtain a chitosan / lignin composite sponge carrier;

[0048] S5. Add 2 g of laccase and 1.2 g of horseradish peroxidase to 100 mL of acetate buffer (pH = 5.0, 0.1 mol / L), stir evenly to obtain an oxidase solution; add 3 g of sodium alginate and 2 g of gelatin to 150 mL of acetate buffer (pH = 5.0, 0.1 mol / L), heat to 50 °C, stir and dissolve to obtain a polymer solution;

[0049] S6. Load the oxidase solution and the polymer solution into the inner and outer nozzle liquid storage tanks of a coaxial electrospray device respectively, set the electrospray voltage to 15 kV, the propulsion speed to 0.5 mL / h for the inner nozzle and 1.0 mL / h for the outer nozzle, drop the electrosprayed microcapsules into a 1.5% calcium chloride solution, and carry out crosslinking and curing for 39 min to obtain enzyme microcapsules;

[0050] S7. Add 8 g of chitosan / lignin composite sponge carrier and 1.0 g of enzyme microcapsules to 200 mL of phosphate buffer (pH = 7.0, 0.05 mol / L), transfer the mixed solution to a vacuum infiltration device, and infiltrate at a vacuum of 10 - 3 Pa for 30 min to obtain a bioenzyme composition based on the chitosan / lignin composite sponge carrier.

[0051] Example 3

[0052] A preparation method of a bioenzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0053] S1. Weigh 5 g of chitosan and add it to 300 mL of 0.1 mol / L citric acid solution, stir at 200 r / min for 1.5 h to form a chitosan colloid;

[0054] S2. Add palmitic acid to the chitosan colloid, the mass ratio of chitosan to palmitic acid is 1:0.25, then add 0.1 g of p-toluenesulfonic acid catalyst, then transfer the system to a 500 mL reaction kettle, and stir and react at a temperature of 65 °C for 4.5 h to obtain modified chitosan;

[0055] S3. Add 8 g of lignin to 200 mL of 1 mol / L sodium hydroxide solution, stir and pretreat at room temperature for 1 h, then add 10 mL of epichlorohydrin, heat to 65 °C, and stir and react for 2 h to obtain epoxidized lignin;

[0056] S4. Mix 2 g of epoxidized lignin with 8 g of modified chitosan, then add 1 mL of glutaraldehyde cross-linking agent with a mass concentration of 25%, and stir and react at 50 °C for 3 h to obtain a chitosan / lignin composite sponge carrier;

[0057] S5. Add 2 g of laccase and 1.3 g of horseradish peroxidase to 100 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), stir evenly to obtain an oxidase solution; add 3 g of sodium alginate and 2 g of gelatin to 150 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), heat to 50 °C, stir and dissolve to obtain a polymer solution;

[0058] S6. Fill the oxidase solution and the polymer solution into the inner and outer nozzle liquid storage tanks of a coaxial electrospray device respectively, set the electrospray voltage to 15 kV, the propulsion speed to 0.5 mL / h for the inner nozzle and 1.0 mL / h for the outer nozzle, drop the electrosprayed microcapsules into a calcium chloride solution with a mass concentration of 1.5%, and carry out cross-linking and curing for 40 min to obtain enzyme microcapsules;

[0059] S7. Add 8 g of chitosan / lignin composite sponge carrier and 1.5 g of enzyme microcapsules to 200 mL of phosphate buffer solution (pH = 7.0, 0.05 mol / L), transfer the mixed solution to a vacuum infiltration device, and infiltrate at a vacuum degree of 10 - 3 Pa for 30 min to obtain a bioenzyme composition based on the chitosan / lignin composite sponge carrier.

[0060] Example 4

[0061] A preparation method of a bioenzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0062] S1. Weigh 5 g of chitosan and add it to 300 mL of 0.1 mol / L citric acid solution, stir at 200 r / min for 1.5 h to form a chitosan colloid;

[0063] S2. Add palmitic acid to the chitosan colloid, the mass ratio of chitosan to palmitic acid is 1:0.4, then add 0.1 g of p-toluenesulfonic acid catalyst, then transfer the system to a 500 mL reaction kettle, react at a temperature of 70 °C and stir for 6 h to obtain modified chitosan;

[0064] S3. Add 8 g of lignin to 200 mL of 1 mol / L potassium hydroxide solution, stir and pretreat at room temperature for 1 h, then add 10 mL of epichlorohydrin, heat to 65 °C, and stir and react for 2 h to obtain epoxidized lignin;

[0065] S4. Mix 2 g of epoxidized lignin with 12 g of modified chitosan, then add 1 mL of glutaraldehyde crosslinking agent with a mass concentration of 25%, and carry out stirring reaction at 50 °C for 3 h to obtain a chitosan / lignin composite sponge carrier;

[0066] S5. Add 2 g of laccase and 1.6 g of horseradish peroxidase to 100 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), stir evenly to obtain an oxidase solution; add 3 g of sodium alginate and 2 g of gelatin to 150 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), heat to 50 °C, stir and dissolve to obtain a polymer solution;

[0067] S6. Load the oxidase solution and the polymer solution into the inner and outer nozzle liquid storage tanks of a coaxial electrospray device respectively, set the electrospray voltage to 15 kV, the propulsion speed to 0.5 mL / h for the inner nozzle and 1.0 mL / h for the outer nozzle, and drop the electrosprayed microcapsules into a calcium chloride solution with a mass concentration of 1.5% for crosslinking and curing for 43 min to obtain enzyme microcapsules;

[0068] S7. Add 8 g of chitosan / lignin composite sponge carrier and 2.4 g of enzyme microcapsules to 200 mL of phosphate buffer solution (pH = 7.0, 0.05 mol / L), transfer the mixture to a vacuum infiltration device, and carry out infiltration treatment at a vacuum degree of 10 - 3 Pa for 30 min to obtain a bioenzyme composition based on the chitosan / lignin composite sponge carrier.

[0069] Example 5

[0070] A preparation method of a bioenzyme composition based on a chitosan / lignin composite sponge carrier, comprising the following steps:

[0071] S1. Weigh 5 g of chitosan and add it to 300 mL of 0.1 mol / L citric acid solution, stir at 200 r / min for 1.5 h to form a chitosan colloid;

[0072] S2. Add palmitic acid to the chitosan colloid, the mass ratio of chitosan to palmitic acid is 1:0.1, then add 0.1 g of p-toluenesulfonic acid catalyst, then transfer the system to a 500 mL reaction kettle, react at a temperature of 60 °C with stirring for 3 h to obtain modified chitosan;

[0073] S3. Add 8 g of lignin to 200 mL of 1 mol / L sodium hydroxide solution, stir and pretreat at room temperature for 1 h, then add 10 mL of epichlorohydrin, heat to 65 °C, and stir and react for 2 h to obtain epoxidized lignin;

[0074] S4. Mix 2 g of epoxidized lignin with 4 g of modified chitosan, then add 1 mL of glutaraldehyde cross-linking agent with a mass concentration of 25%, and carry out stirring reaction at 50 °C for 3 h to obtain a chitosan / lignin composite sponge carrier;

[0075] S5. Add 2 g of laccase and 1 g of horseradish peroxidase to 100 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), stir evenly to obtain an oxidase solution; add 3 g of sodium alginate and 2 g of gelatin to 150 mL of acetate buffer solution (pH = 5.0, 0.1 mol / L), heat to 50 °C, stir and dissolve to obtain a polymer solution;

[0076] S6. Load the oxidase solution and the polymer solution into the inner and outer nozzle liquid storage tanks of a coaxial electrospray device respectively, set the electrospray voltage to 15 kV, the propulsion speed to 0.5 mL / h for the inner nozzle and 1.0 mL / h for the outer nozzle, drop the electrosprayed microcapsules into a calcium chloride solution with a mass concentration of 1.5%, and carry out cross-linking and curing for 38 min to obtain enzyme microcapsules;

[0077] S7. Add 8 g of chitosan / lignin composite sponge carrier and 0.8 g of enzyme microcapsules to 200 mL of phosphate buffer solution (pH = 7.0, 0.05 mol / L), transfer the mixture to a vacuum infiltration device, and infiltrate at a vacuum degree of 10 - 3 Pa for 30 min to obtain a bio-enzyme composition based on the chitosan / lignin composite sponge carrier.

[0078] Comparative Example 1

[0079] The difference between Comparative Example 1 and Example 1 is that the chitosan / lignin composite sponge carrier is replaced with activated carbon, and the remaining operation steps are the same as those in Example 1.

[0080] Comparative Example 2

[0081] The difference between Comparative Example 2 and Example 1 is that the chitosan / lignin composite sponge carrier is replaced with diatomite, and the remaining operation steps are the same as those in Example 1.

[0082] Comparative Example 3

[0083] The difference between Comparative Example 3 and Example 1 is that in the preparation process of the chitosan / lignin composite sponge carrier, chitosan is not modified with palmitic acid, and the remaining operation steps are the same as those in Example 1.

[0084] Comparative Example 4

[0085] The difference between Comparative Example 1 and Example 1 is that the outer layer of laccase and horseradish peroxidase is not coated with sodium alginate / gelatin layer by electrospray technology, and the remaining operation steps are the same as those in Example 1.

[0086] Comparative Example 5

[0087] The difference between Comparative Example 5 and Example 4 is that the mass ratio of chitosan to palmitic acid is 1:0.5, and the remaining operation steps are the same as those in Example 4.

[0088] Comparative Example 6

[0089] The difference between Comparative Example 5 and Example 4 is that the mass ratio of chitosan to palmitic acid is 1:0.6, and the remaining operation steps are the same as those in Example 4.

[0090] Performance test:

[0091] 1. Crude oil adsorption effect test:

[0092] Accurately measure 5 g of the samples of the examples and comparative examples, and then prepare a solution of crude oil at 1000 mg / L with an organic solvent such as n-hexane. Take 100 mL of this solution and place it in multiple identical conical flasks. Put the prepared samples into them respectively, seal them, and oscillate in a constant temperature oscillator at 25 °C and 150 r / min for 24 hours to allow the samples to fully adsorb petroleum. After the oscillation, separate the samples and the solution by filtration or centrifugation, and use an ultraviolet-visible spectrophotometer to measure the remaining petroleum concentration in the supernatant. According to the change in concentration before and after adsorption, calculate the crude oil adsorption amount according to the formula adsorption amount (mg / g) = (initial petroleum concentration - remaining petroleum concentration) × solution volume ÷ sample mass.

[0093] 2. Crude oil decomposition effect test:

[0094] For the selection of components of the simulated crude oil, n-heptane (representing saturated alkanes), toluene (representing aromatic hydrocarbons), and hexadecane (representing long-chain alkanes) were chosen and mixed in a volume ratio of 3:2:5. Using n-hexane as the solvent, 150 mL of n-heptane, 100 mL of toluene, and 250 mL of hexadecane were accurately measured into a container, and then n-hexane was added to make the volume up to 1 L and stirred well to prepare 1 L of a simulated crude oil solution with a concentration of 5000 mg / L. Then, a clean and well-sealed 250 mL stoppered conical flask was selected as the reaction vessel. 1 g of enzyme composition samples for the examples and comparative examples were prepared, and 3 parallel samples were set for each group. 200 mL of the simulated crude oil solution was added to each conical flask, and the respective samples were added to the corresponding flasks. Subsequently, the conical flasks were placed in a constant temperature shaking incubator, with the temperature set at 30 °C and the shaking speed at 100 r / min. Every 6 hours, 5 mL of the reaction solution was taken from each conical flask with a pipette and analyzed using a gas chromatograph equipped with a flame ionization detector (FID) and a DB-5 capillary column (30 m × 0.25 mm × 0.25 μm). The inlet temperature was 250 °C, the detector temperature was 300 °C, the initial column temperature was maintained at 50 °C for 2 min, then heated to 300 °C at a rate of 10 °C / min and maintained for 5 min. The carrier gas was nitrogen, with a flow rate of 1 mL / min and a split ratio of 20:1, to measure the content of the remaining petroleum simulation components. Finally, according to the formula: decomposition rate (%) = (initial crude oil hydrocarbon content - remaining crude oil hydrocarbon content) ÷ initial crude oil hydrocarbon content × 100%, using the decomposition rate data after 72 hours as the final evaluation index, the average value of the 3 parallel samples of each group of samples was calculated to evaluate the decomposition effect of the enzyme on the crude oil.

[0095]

[0096] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier, characterized in that: The following steps are involved: S1, dissolving chitosan in citric acid solution and stirring to form chitosan colloid; S2, grafting chitosan colloid with palmitic acid under the action of a catalyst to obtain modified chitosan; S3, pre-treating the lignin with alkali solution, and then reacting it with epichlorohydrin to introduce epoxy functional groups to obtain epoxidized lignin; S4, mixing the epoxidized lignin and the modified chitosan, and then adding a cross-linking agent to carry out a cross-linking reaction to obtain a chitosan / lignin composite sponge carrier; S5, adding laccase and horseradish peroxidase to an acetate buffer to obtain an oxidase solution, dissolving sodium alginate and gelatin in the acetate buffer to obtain a polymer solution; S6, using the oxidase solution as the core solution and the polymer solution as the shell solution, preparing microcapsules by coaxial electrospraying, dropping the microcapsules into a calcium chloride solution for cross-linking and curing, and obtaining enzyme microcapsules; S7. Add the chitosan / lignin composite sponge carrier and the enzyme microcapsule into phosphate buffer and perform vacuum infiltration treatment to obtain the product.

2. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In the step S2, the mass ratio of chitosan to palmitic acid is 1:0.1-0.

4.

3. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In the step S2, the grafting reaction temperature is 60-70° C., and the reaction time is 3-6 hours.

4. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S3, the alkali solution is selected from one or more of sodium hydroxide and potassium hydroxide.

5. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S4, the mass ratio of epoxidized lignin to modified chitosan is 1:2-6.

6. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S4, the cross-linking agent is selected from one or more of glutaraldehyde and genipin.

7. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S5, the mass ratio of laccase to horseradish peroxidase is 1:0.5-0.

8.

8. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S6, the mass concentration of the calcium chloride solution is 1.5%, and the cross-linking curing time is controlled at 38-43 minutes.

9. The method for preparing a bio-enzyme composition based on a chitosan / lignin composite sponge carrier according to claim 1, characterized in that: In step S7, the mass ratio of the chitosan / lignin composite sponge carrier to the enzyme microcapsule is 1:0.1-0.

3.

10. A bio-enzyme composition based on a chitosan / lignin composite sponge carrier, characterized in that: The method is prepared by any one of claims 1 to 9.

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