Method for synchronously treating feces and straws of livestock and poultry through double-layer immobilized enzyme microcapsules

Abstract

The invention provides a method for synchronously treating livestock manure and straw by using a double-layer immobilized enzyme microcapsule. The method comprises the following steps: preparing a temperature and pH sensitive double-layer immobilized enzyme microcapsule, and carrying out enzymolysis treatment on the livestock manure and straw. In the waste treatment process, along with the temperature rise, the thermal motion of a polymer chain is enhanced, and the outer network structure of the microcapsule becomes loose, so that the release of compound enzyme in the network is facilitated; acid is produced in the waste treatment process, so that the system is in an acid environment, carboxyl groups on hyaluronic acid molecular chains are protonized, electrostatic repulsive force between the molecular chains is weakened, an inner-layer gel network structure becomes loose, and release of embedded compound enzyme is promoted, and therefore, in the whole enzymolysis process, the enzyme activity is improved. The double-layer immobilized enzyme microcapsule continuously releases the compound enzyme, rapid proceeding of the enzymolysis reaction is promoted, the reaction time is 2-3 h, efficient resource reutilization of waste is achieved, and sustainable development of agriculture is assisted.

Description

Technical Field

[0001] The invention belongs to the technical field of solid waste treatment, and in particular relates to a method for synchronously treating livestock excrement and straw using double-layer immobilized enzyme microcapsules. Background Art

[0003] At present, the commonly used methods for treating urban and rural organic solid waste are aerobic composting and anaerobic digestion. Although composting requires little investment and is simple to operate, the odor and sewage generated during the treatment process have a greater impact on the surrounding environment; anaerobic digestion is a process in which organic matter is metabolized and decomposed by microorganisms in an oxygen-free environment, and finally a combustible mixed gas such as methane is formed. This method has a long fermentation cycle, high cost, low treatment efficiency, and the generated biogas residues and liquids need to be treated again. Therefore, in the process of urban and rural organic solid waste resource utilization, how to quickly and effectively achieve organic solid waste treatment has become a technical problem that the industry urgently needs to solve.

[0004] The use of enzyme technology in biotechnology to treat urban and rural organic solid waste is a new type of waste treatment technology that has emerged in China and abroad in recent years. Enzymes are a type of polymer biocatalyst that has the characteristics of specificity, high efficiency, and few by-products. They can usually catalyze reactions under natural conditions; however, the activity of free enzymes is easily affected by environmental factors (such as temperature and pH) and denatured or inactivated, and it is not easy to store for a long time, which hinders the industrial production and large-scale application of enzymes in the treatment of organic solid waste.

[0005] There are four main enzyme immobilization technologies currently under study: physical adsorption, embedding, covalent crosslinking and polymerization. Physical adsorption can cause weak adsorption, easy detachment of enzymes, and low immobilization effect; embedding can easily cause enzyme accumulation, reduce the contact area between reactants and enzymes, and thus affect the catalytic effect; covalent crosslinking of immobilized enzymes can cause functional groups to change the active center site of the enzyme, resulting in reduced enzyme activity. Therefore, it is necessary to study a method to optimize immobilized enzymes, improve the efficiency of enzyme treatment of organic solid waste, and promote the rapid resource utilization of organic solid waste. Summary of the invention

[0006] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for synchronously treating livestock manure and straw with double-layer immobilized enzyme microcapsules, including the preparation of temperature and pH sensitive double-layer immobilized enzyme microcapsules and enzymatic treatment of livestock manure and straw. As the temperature rises during waste treatment, the thermal motion of the polymer chain is enhanced, causing the outer network structure of the microcapsule to become loose, which is conducive to the release of the complex enzyme inside the network; acid production during waste treatment causes the system to be in an acidic environment, the carboxyl groups on the hyaluronic acid molecular chains are protonated, the electrostatic repulsion between the molecular chains is weakened, and the inner gel network structure becomes loose. This structural change will reduce the mechanical strength of the gel and the binding ability of the embedded material, thereby promoting the release of the embedded material-complex enzyme, so that during the entire enzymatic hydrolysis process, the double-layer immobilized enzyme microcapsules can continuously release the complex enzyme, promote the rapid progress of the enzymatic hydrolysis reaction, and improve the efficiency of organic solid waste treatment.

[0007] Technical solution: A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules, comprising the following steps: S1. Mix and crush livestock manure and straw in a mass ratio of (5-7): (3-5), and steam explode for 3-5 minutes at a pressure of 1.5-2.5 MPa to obtain a pretreated product; S2. The pretreated product is added to a reaction tank, heated to 50-70°C, and the humidity is adjusted to 55-60% to obtain a substrate; S3. Add double-layer immobilized enzyme microcapsules to the substrate, mix evenly, perform enzymatic hydrolysis, cool, and obtain organic fertilizer.

[0008] Furthermore, the double-layer immobilized enzyme microcapsules in step S3 include inner-layer gel microspheres and outer-layer networks, and the specific preparation steps are as follows: Step 1: dissolving hyaluronic acid, dopamine and sucrose in water and stirring evenly to prepare a blended gel solution; Step 2: dissolving the complex enzyme in phosphate buffer to obtain a complex enzyme solution, then adding the complex enzyme solution to the blended gel solution, stirring evenly, and spray drying to obtain inner layer gel microspheres; Step 3: dissolving polysulfonate betaine and γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in a complex enzyme solution to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0009] Furthermore, in step 1, the mass ratio of hyaluronic acid, dopamine and sucrose is (10-15): (2-5): (1-2); and the mass fraction of the blended gel is 5-8%.

[0010] Furthermore, in step 2, the composite enzyme is a complex of protease, lipase and cellulase, and the mass ratio is (3-4): (1-3): (1-2).

[0011] Furthermore, the protease is any one of trypsin and papain; the lipase is any one of phosphatase, sterolase and carboxylesterase; the cellulase is any one of β-glucosidase, endoglucanase and exoglucanase.

[0012] Furthermore, in step 2, the mass volume ratio of the complex enzyme to the phosphate buffer is (15-20):1; and the volume ratio of the complex enzyme solution to the blended gel is 1:(5-10).

[0013] Furthermore, in step 3, the mass ratio of polysulfonated betaine to γ-aminobutyric acid is (1-2):1; and the mass fraction of the modified polysulfonated betaine solution is 3-5%.

[0014] Furthermore, in step 4, the mass volume ratio of the modified polysulfonated betaine to the complex enzyme solution is 1:(2-3).

[0015] Furthermore, in step 5, the mass volume ratio of the inner layer gel microspheres to the outer layer network solution is 1:(2-4).

[0016] Furthermore, in step S3, the mass ratio of the substrate to the double-layer immobilized enzyme microcapsule is (100-200):1; the temperature of the enzymatic hydrolysis reaction is 50-70°C, and the time is 2-3h.

[0017] Beneficial Effects

[0018] When treating livestock manure and straw waste, the present invention firstly performs pretreatment by steam explosion and hot alkali, wherein steam explosion can effectively destroy the complex organic structure and increase the surface area under high temperature and high pressure conditions; hot alkali pretreatment separates lignin and carbohydrates by destroying the lignin structure, degrading hemicellulose, increasing cellulose accessibility and reducing crystallinity, thereby significantly improving the efficiency of subsequent enzymatic hydrolysis treatment; The present invention immobilizes the complex enzyme, avoids the problem of reduced free enzyme activity due to environmental influences, prepares double-layer immobilized enzyme microcapsules, and continuously releases the complex enzyme, which is beneficial to improving the efficiency of enzyme treatment of organic solid waste and promoting resource utilization of organic solid waste; The double-layer immobilized enzyme microcapsule prepared by the invention comprises an inner pH-sensitive gel microsphere and an outer temperature-sensitive network, wherein the outer temperature-sensitive network comprises polysulfonated betaine and gamma-aminobutyric acid, the gamma-aminobutyric acid is grafted onto the polysulfonated betaine via an amino group, and the amino group on the lysine residue of the enzyme can be covalently bonded with the carboxyl group of the gamma-aminobutyric acid, thereby enhancing the binding force between the enzyme and the outer network and improving the immobilization effect; in addition, the polysulfonated betaine has a certain temperature responsiveness and is a network structure, and as the temperature rises during waste treatment, the thermal motion of the polymer chain on the polysulfonated betaine is enhanced, causing the network structure to become loose, which is conducive to the release of the complex enzyme inside the network; The inner pH-sensitive gel microspheres of the double-layer immobilized enzyme microcapsules prepared by the present invention include hyaluronic acid, dopamine and sucrose. The hyaluronic acid and dopamine are combined through electrostatic action to form spherical colloidal particles, which can achieve the embedding of complex enzymes. At the same time, the mechanical properties of hyaluronic acid are poor, and it is easy to deform or break, which affects the embedding effect. The modification of dopamine can improve its mechanical properties and enhance the embedding effect. In addition, due to the high viscosity of hyaluronic acid, it may cause serious and uneven agglomeration of particles during the embedding process, thereby reducing the embedding rate. The addition of sucrose can reduce the interaction between gel particles, reduce the agglomeration phenomenon, and can also combine with hyaluronic acid through hydrogen bonds to enhance the stability of the gel. The composite enzyme of the present invention can promote the growth and metabolism of microorganisms when treating waste. The microorganisms produce acid by decomposing organic matter such as cellulose, thereby reducing the pH of the system. In an acidic environment, the carboxyl groups on the hyaluronic acid molecular chains are protonated, the electrostatic repulsion between the molecular chains is weakened, and the gel network structure becomes loose. This structural change reduces the mechanical strength of the gel and the binding ability to the embedded material, achieving pH sensitivity, thereby promoting the release of the embedded material-the composite enzyme. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1

[0020] The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g hyaluronic acid, 3 g dopamine and 1 g sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:5, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:2, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0021] Example 2

[0022] The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:6, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:2, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0023] Example 3

[0024] The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:7, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2.5 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:3, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0025] Example 4

[0026] The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g hyaluronic acid, 3 g dopamine and 1 g sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:8, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:3 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:3, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0027] Example 5

[0028] The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g hyaluronic acid, 3 g dopamine and 1 g sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:10, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:3 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:4, stir and dry to obtain double-layer immobilized enzyme microcapsules.

[0029] Comparative Example 1

[0030] The difference between this comparative example and Example 3 is that only the inner layer gel microspheres are prepared, as follows: The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g hyaluronic acid, 3 g dopamine and 1 g sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: Take 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer to obtain a composite enzyme solution, then add it to the blended gel at a volume ratio of 1:7, stir evenly, spray dry, and obtain inner layer gel microspheres.

[0031] Comparative Example 2

[0032] The difference between this comparative example and Example 3 is that only the outer layer network is prepared, as follows: The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 2: Take 100 mg of complex enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer to obtain a complex enzyme solution. Step 3: Immerse the modified polysulfonated betaine in a complex enzyme solution at a mass volume ratio of 1:2.5, and dry to obtain an outer layer network.

[0033] Comparative Example 3

[0034] The difference between this comparative example and Example 3 is that γ-aminobutyric acid is not added to the outer network solution, specifically as follows: The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g hyaluronic acid, 3 g dopamine and 1 g sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:7, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 20 g of polysulfonate betaine in water, stirring evenly, and drying to obtain polysulfonate betaine with a mass fraction of 3%; Step 4: immersing polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2.5 to prepare an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:3, homogenize and dry to obtain double-layer immobilized enzyme microcapsules.

[0035] Comparative Example 4

[0036] The difference between this comparative example and Example 3 is that dopamine is not added to the inner layer of gel microspheres, as follows: The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 13 g of hyaluronic acid and 1 g of sucrose in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:7, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2.5 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:3, homogenize and dry to obtain double-layer immobilized enzyme microcapsules.

[0037] Comparative Example 5

[0038] The difference between this comparative example and Example 3 is that no sucrose is added to the inner layer of gel microspheres, as follows: The preparation steps of double-layer immobilized enzyme microcapsules are as follows: Step 1: Dissolve 10 g of hyaluronic acid and 4 g of dopamine in water and stir evenly to prepare a blended gel with a mass fraction of 5%; Step 2: 100 mg of composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) is dissolved in 5 mL of phosphate buffer to obtain a composite enzyme solution, which is then added to the blended gel at a volume ratio of 1:7, stirred evenly, and spray-dried to obtain inner layer gel microspheres; Step 3: dissolving 10 g of polysulfonate betaine and 10 g of γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution with a mass fraction of 3%, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in the complex enzyme solution at a mass volume ratio of 1:2.5 to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution at a mass volume ratio of 1:3, homogenize and dry to obtain double-layer immobilized enzyme microcapsules.

[0039] Determination of complex enzyme embedding rate:

[0040] The double-layer immobilized enzyme microcapsules were added to acetic acid-sodium acetate buffer at pH 5.0, and the enzyme activity was determined after being broken by a homogenizer. The encapsulation rate was calculated as follows: Embedding rate (%) = (N0 / N) × 100% Wherein, N0 is the enzyme activity of the microcapsules formed by 1 mL of the mixed solution containing the complex enzyme, and N is the total enzyme activity of 1 mL of the mixed solution containing the complex enzyme.

[0041] Table 1 Composite enzyme embedding rate of immobilized enzyme microcapsules prepared in Examples 1-5 and Comparative Examples 1-5 Embedding rate (%) Embedding rate (%) Example 1 83.18 Comparative Example 1 50.23 Example 2 82.46 Comparative Example 2 — Example 3 88.21 Comparative Example 3 74.31 Example 4 85.73 Comparative Example 4 65.14 Example 5 80.55 Comparative Example 5 68.82 As shown in Table 1, the composite enzyme embedding rate of the double-layer immobilized enzyme microcapsules prepared in Examples 1-5 is 80.55-88.21%, while Comparative Example 1 only prepares the inner layer gel microspheres, and the embedding rate is only 50.23%; Comparative Example 2 only prepares the outer layer network, and embedding cannot be achieved; Comparative Example 3 does not add γ-aminobutyric acid, and the composite enzyme embedding rate is slightly reduced; Comparative Example 4 does not add dopamine, and the hyaluronic acid gel is easily deformed or broken, and the embedding rate drops significantly to 65.14%; Comparative Example 5 does not add sucrose, and the microcapsule embedding rate is 68.82%. It can be seen that the addition of sucrose can reduce the interaction between gel particles, reduce agglomeration, and improve the embedding effect of the composite enzyme. Therefore, the double-layer immobilized enzyme microcapsules prepared in Example 3 are selected for the subsequent treatment of livestock manure and straw waste.

[0042] Example 6

[0043] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 1.5MPa for 5min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 55°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 70° C. for 3 h, and cool to obtain organic fertilizer.

[0044] Example 7

[0045] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 2MPa for 3min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 60°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 2.5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 70° C. for 3 h, and cool to obtain organic fertilizer.

[0046] Example 8

[0047] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 1.5MPa for 5min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 55°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 50° C. for 3 h, and cool to obtain organic fertilizer.

[0048] Example 9

[0049] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 1.5MPa for 5min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 55°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 60° C. for 3 h, and cool to obtain organic fertilizer.

[0050] Example 10

[0051] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 1.5MPa for 5min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 55°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 70° C. for 2 h, and cool to obtain an organic fertilizer.

[0052] Embodiment 11

[0053] A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed, and steam exploded at a pressure of 1.5MPa for 5min to obtain a pretreated product; S2. The pretreated product was added to the reaction tank, heated to 55°C, and the humidity was adjusted to 55% to obtain a substrate; S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix well, perform enzymatic hydrolysis at 70° C. for 2.5 h, and cool to obtain an organic fertilizer.

[0054] Comparative Example 6

[0055] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 1 are used.

[0056] Comparative Example 7

[0057] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 2 are used.

[0058] Comparative Example 8

[0059] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 3 are used.

[0060] Comparative Example 9

[0061] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 4 are used.

[0062] Comparative Example 10

[0063] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 5 are used.

[0064] Comparative Example 11

[0065] The difference between this comparative example and Example 6 is that no pretreatment is performed, as follows: A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules comprises the following steps: S1. 300kg of livestock manure and 200kg of straw were mixed and crushed to obtain a substrate; S2. Add the substrate to the reaction tank, adjust the substrate humidity to 55%, add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3, mix well, perform enzymatic hydrolysis at 70 ° C for 3 h, cool, and obtain organic fertilizer.

[0066] Performance Test:

[0067] (1) Waste degradation rate The degradation rates of protein, fat, lignin and cellulose of livestock manure and straw in Examples 6-11 and Comparative Examples 6-11 were determined. Protein was determined by the fully automatic Kjeldahl method, fat content was determined by the Soxhlet extraction method, lignin was determined by the nitric acid-ethanol method, and cellulose was determined by the 72% concentrated sulfuric acid hydrolysis method.

[0068] Table 2 Degradation rates of protein, fat, lignin and cellulose in wastes in Examples 6-11 and Comparative Examples 6-11

[0069] As shown in Table 2, in Examples 6, 8, and 9, only the enzymolysis temperature was changed. As the enzymolysis temperature increased, the degradation rates of protein, fat, lignin, and cellulose increased, indicating that the double-layer immobilized enzyme microcapsules prepared by the present invention had a certain temperature sensitivity. In Examples 6, 10, and 11, only the enzymolysis time was changed. As the enzymolysis time increased, the degradation rates of microorganisms in the waste increased. Decomposition of organic matter such as cellulose produces acid, causing the pH of the system to gradually decrease, the carboxyl groups on the hyaluronic acid molecular chains to be protonated, the electrostatic repulsion between the molecular chains to be weakened, and the gel network structure to become loose. This structural change will reduce the mechanical strength of the gel and the binding ability of the embedded substance, thereby achieving pH sensitivity, thereby promoting the release of the embedded complex enzyme; Comparative Example 6 uses a complex enzyme embedded in only the inner layer gel network, Comparative Example 7 uses a complex enzyme immobilized in only the outer layer network, Comparative Example 8 uses microcapsules without adding γ-aminobutyric acid, Comparative Example 9 uses microcapsules without adding dopamine, and Comparative Example 10 uses microcapsules without adding sucrose. The degradation rates of protein, fat, lignin and cellulose in the waste are all reduced. It can be seen that the double-layer immobilized enzyme microcapsules prepared by the present invention are used to achieve efficient enzymatic hydrolysis of waste; Comparative Example 11 does not pretreat the waste, and the degradation rate is also reduced.

[0070] (2) Organic fertilizer performance According to NY / T 525-2021 "Organic Fertilizer", the performance indicators of the organic fertilizers produced in Example 6 and Comparative Examples 6-11 were measured, including the mass fraction of organic matter, the mass fraction of total nutrients (nitrogen, phosphorus and potassium), the mass fraction of water, pH, the number of fecal coliform bacteria, and the total arsenic and total cadmium in heavy metals.

[0071] Table 3 Performance indicators of organic fertilizers produced in Example 6 and Comparative Examples 6-11 Example 6 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Comparative Example 10 Comparative Example 11 Organic matter mass fraction (%) 71.45 52.61 55.13 66.42 67.63 68.02 62.12 Total nutrient mass fraction (%) 6.22 4.13 4.34 5.79 5.82 5.87 4.68 Water content (%) 22.54 29.82 29.15 27.56 27.17 27.39 28.59 pH 5.7 7.5 7.2 6.7 6.5 6.4 7.1 Fecal coliform count (pcs / g) 56 84 81 67 66 65 70 Total arsenic (mg / kg) 3.22 5.78 5.91 4.22 3.97 4.06 5.21 Total cadmium (mg / kg) 0.54 1.15 1.27 0.84 0.89 0.81 1.17 It can be seen from Table 3 that the performance indicators of the organic fertilizers produced by Example 6 and Comparative Examples 6-11 all meet the requirements of NY / T 525-2021 "Organic Fertilizer". Comparative Example 6 uses a composite enzyme embedded in only the inner layer gel network, and Comparative Example 7 uses a composite enzyme immobilized in only the outer layer network for enzymolysis of waste. The performance indicators of the organic fertilizers produced by both are significantly lower than those in Example 6. The microcapsules used in Comparative Example 8 do not add γ-aminobutyric acid, the microcapsules used in Comparative Example 9 do not add dopamine, and the microcapsules used in Comparative Example 10 do not add sucrose. Comparative Example 11 is not pretreated. The performance indicators of the organic fertilizers produced by enzymolysis of these four are slightly lower than those in Example 6. Therefore, it can be seen that the double-layer immobilized enzyme microcapsules prepared by the present invention can achieve a higher degree of enzymolysis and production of organic fertilizers, and promote the resource recycling of poultry and livestock manure and straw.

[0072] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules, characterized in that: The following steps are involved: S1. Mix and crush livestock manure and straw in a mass ratio of (5-7): (3-5), and steam explode for 3-5 minutes at a pressure of 1.5-2.5 MPa to obtain a pretreated product; S2. The pretreated product is added to a reaction tank, heated to 50-70°C, and the humidity is adjusted to 55-60% to obtain a substrate; S3. Add double-layer immobilized enzyme microcapsules to the substrate, mix evenly, perform enzymatic hydrolysis, cool, and obtain organic fertilizer.

2. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, characterized in that: The double-layer immobilized enzyme microcapsules in step S3 include inner-layer gel microspheres and outer-layer networks, and the specific preparation steps are as follows: Step 1: dissolving hyaluronic acid, dopamine and sucrose in water and stirring evenly to prepare a blended gel solution; Step 2: dissolving the complex enzyme in phosphate buffer to obtain a complex enzyme solution, then adding the complex enzyme solution to the blended gel solution, stirring evenly, and spray drying to obtain inner layer gel microspheres; Step 3: dissolving polysulfonate betaine and γ-aminobutyric acid in water, stirring evenly to obtain a modified polysulfonate betaine solution, and drying to obtain modified polysulfonate betaine; Step 4: immersing the modified polysulfonated betaine in a complex enzyme solution to obtain an outer network solution; Step 5: Place the inner layer gel microspheres in the outer layer network solution, stir and dry to obtain double-layer immobilized enzyme microcapsules.

3. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: In step 1, the mass ratio of hyaluronic acid, dopamine and sucrose is (10-15): (2-5): (1-2); the mass fraction of the blended gel is 5-8%.

4. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: The complex enzyme in step 2 is a complex of protease, lipase and cellulase, and the mass ratio is (3-4): (1-3): (1-2).

5. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 4, characterized in that: The protease is any one of trypsin and papain; the lipase is any one of phosphatase, sterolase and carboxylesterase; the cellulase is any one of β-glucosidase, endoglucanase and exoglucanase.

6. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: In step 2, the mass volume ratio of the complex enzyme to the phosphate buffer is (15-20):1; the volume ratio of the complex enzyme solution to the blended gel is 1:(5-10).

7. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: In step 3, the mass ratio of polysulfonate betaine to γ-aminobutyric acid is (1-2):1; the mass fraction of the modified polysulfonate betaine solution is 3-5%.

8. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: In step 4, the mass volume ratio of the modified polysulfonated betaine to the complex enzyme solution is 1:(2-3).

9. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 2, characterized in that: In step 5, the mass volume ratio of the inner layer gel microspheres to the outer layer network solution is 1:(2-4).

10. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, characterized in that: In step S3, the mass ratio of the substrate to the double-layer immobilized enzyme microcapsule is (100-200):1; the temperature of the enzymatic hydrolysis reaction is 50-70° C., and the time is 2-3 hours.

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