A method for synchronously treating livestock and poultry manure and straw by a double-layer immobilized enzyme microcapsule

Abstract

The present invention provides a method for synchronously treating livestock and poultry manure and straw by using a double-layer immobilized enzyme microcapsule, which includes the preparation of a temperature- and pH-sensitive double-layer immobilized enzyme microcapsule and the enzymatic hydrolysis treatment of livestock and poultry manure and straw. During the waste treatment process, as the temperature rises, the thermal motion of the polymer chains is enhanced, and the network structure of the outer layer of the microcapsule becomes loose, which is conducive to the release of the composite enzyme inside the network; the acid production during the waste treatment process makes the system in an acidic environment, the carboxyl groups on the hyaluronic acid molecular chain are protonated, and the electrostatic repulsion force between the molecular chains is weakened, and the inner layer gel network structure becomes loose, promoting the release of the entrapped composite enzyme. Therefore, during the entire enzymatic hydrolysis process, the double-layer immobilized enzyme microcapsule continuously releases the composite enzyme, promoting the rapid progress of the enzymatic hydrolysis reaction. The reaction time is 2-3 h, realizing the efficient resource recycling of waste and contributing to the sustainable development of agriculture.

Description

Technical Field

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

[0002] At present, the commonly used methods for treating urban and rural organic solid waste are aerobic composting and anaerobic digestion. Although composting has low investment and simple operation, the odor and sewage generated during the treatment process have a great impact on the surrounding environment. Anaerobic digestion is a process in which microorganisms metabolize and decompose organic matter in an anaerobic environment to finally form combustible mixed gases such as methane. This method has a long fermentation cycle, high cost, low treatment efficiency, and the biogas residues and biogas slurry generated need to be treated again. Therefore, in the process of resource utilization of urban and rural organic solid waste, how to quickly and effectively realize the treatment of organic solid waste has become a technical problem that the industry urgently needs to solve.

[0003] Using enzyme technology in biotechnology to treat urban and rural organic solid waste is a new type of waste treatment technology that has emerged at home and abroad in recent years. Enzymes are a class of high-molecular biological catalysts, which have the characteristics of specificity, high efficiency, and few by-products, and usually can carry out catalytic 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.

[0004] At present, there are mainly 4 ways of enzyme immobilization technology studied: physical adsorption, embedding, covalent cross-linking and polymerization. Among them, physical adsorption will cause problems such as weak adsorption force, easy detachment of enzymes, and low immobilization effect; the embedding method is easy to cause enzyme accumulation, reduce the contact area between reactants and enzymes, and thus affect the catalytic effect; covalent cross-linking of immobilized enzymes will cause the functional groups to change the active center site of the enzyme, resulting in a decrease in enzyme activity. Therefore, it is necessary to study an optimized method for immobilizing enzymes to improve the efficiency of enzyme treatment of organic solid waste and promote the rapid resource utilization of organic solid waste. Summary of the Invention

[0005] Technical problem to be solved: Aiming at the above technical problems, the object of the present invention is to provide a method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, including the preparation of temperature- and pH-sensitive double-layer immobilized enzyme microcapsules and the enzymatic hydrolysis treatment of livestock and poultry manure and straw. As the temperature rises during waste treatment, the thermal motion of the polymer chains enhances, causing the outer network structure of the microcapsules to become loose, which is conducive to the release of the composite enzyme inside the network; the acid production during the waste treatment process makes the system in an acidic environment, the carboxyl groups on the hyaluronic acid molecular chains are protonated, and the electrostatic repulsion between the molecular chains weakens, making the inner gel network structure loose. This structural change will reduce the mechanical strength of the gel and the binding ability to the embedded substances, thereby promoting the release of the embedded substance - the composite enzyme. Therefore, during the entire enzymatic hydrolysis process, the double-layer immobilized enzyme microcapsules can continuously release the composite enzyme, promoting the rapid progress of the enzymatic hydrolysis reaction and improving the treatment efficiency of organic solid waste.

[0006] Technical solution: A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0007] S1. Mix and crush livestock and poultry manure and straw according to a mass ratio of (5 - 7):(3 - 5), and perform steam explosion at a pressure of 1.5 - 2.5 MPa for 3 - 5 min to obtain a pretreatment product;

[0008] S2. Add the pretreatment product into a reaction tank, heat it to 50 - 70 °C, and adjust the humidity to 55 - 60% to obtain a substrate;

[0009] S3. Add double-layer immobilized enzyme microcapsules to the substrate, mix evenly, perform an enzymatic hydrolysis reaction, and cool to obtain organic fertilizer.

[0010] Further, the double-layer immobilized enzyme microcapsules in step S3 include an inner gel microsphere and an outer network, and the specific preparation steps are as follows:

[0011] Step 1: Dissolve hyaluronic acid, dopamine, and sucrose in water, stir evenly to obtain a co-coagulating gel solution;

[0012] Step 2: Take the composite enzyme and dissolve it in phosphate buffer solution to obtain a composite enzyme solution, then add it to the co-coagulating gel solution, stir evenly, and perform spray drying to obtain inner gel microspheres;

[0013] Step 3: Dissolve poly(sulfobetaine) and γ-aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution, and dry it to obtain modified poly(sulfobetaine);

[0014] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution to obtain an outer network solution;

[0015] Step 5: Place the inner layer of gel microspheres into the outer layer of network solution, stir and dry to obtain the double-layer immobilized enzyme microcapsules.

[0016] Furthermore, in the step 1, the mass ratio of hyaluronic acid, dopamine and sucrose is (10 - 15):(2 - 5):(1 - 2); the mass fraction of the co - coagulating gel is 5 - 8%.

[0017] Furthermore, in the step 2, the composite enzyme is a complex of protease, lipase and cellulase, and the mass ratio is (3 - 4):(1 - 3):(1 - 2).

[0018] 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.

[0019] Furthermore, in the step 2, the mass - to - volume ratio of the composite enzyme to the phosphate buffer solution is (15 - 20):1; the volume ratio of the composite enzyme solution to the co - coagulating gel is 1:(5 - 10).

[0020] Furthermore, in the step 3, the mass ratio of poly(sulfobetaine) and γ - aminobutyric acid is (1 - 2):1; the mass fraction of the modified poly(sulfobetaine) solution is 3 - 5%.

[0021] Furthermore, in the step 4, the mass - to - volume ratio of the modified poly(sulfobetaine) to the composite enzyme solution is 1:(2 - 3).

[0022] Furthermore, in the step 5, the mass - to - volume ratio of the inner layer of gel microspheres to the outer layer of network solution is 1:(2 - 4).

[0023] Furthermore, in the step S3, the mass ratio of the substrate to the double - layer immobilized enzyme microcapsules is (100 - 200):1; the temperature of the enzymatic hydrolysis reaction is 50 - 70 °C, and the time is 2 - 3 h.

[0024] Beneficial effects

[0025] When the present invention is used to treat livestock and poultry manure and straw waste, first, it is pretreated by steam explosion and hot alkali. Among them, steam explosion can effectively destroy the structure of complex organic substances 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 the accessibility of cellulose and reducing crystallinity, thus significantly improving the efficiency of subsequent enzymatic hydrolysis treatment;

[0026] The present invention immobilizes the composite enzyme, avoiding the problem of reduced activity of free enzyme caused by environmental impact, and prepares a double-layer immobilized enzyme microcapsule to continuously release the composite enzyme, which is beneficial to improving the efficiency of enzyme treatment of organic solid waste and promoting the resource utilization of organic solid waste;

[0027] The double-layer immobilized enzyme microcapsule prepared by the present invention comprises an inner layer of pH-sensitive gel microspheres and an outer layer of temperature-sensitive network. The outer layer of temperature-sensitive network comprises poly(sulfobetaine) and γ-aminobutyric acid. γ-aminobutyric acid is grafted onto poly(sulfobetaine) through an amino group. The amino group on the lysine residue of the enzyme can covalently bind to the carboxyl group of γ-aminobutyric acid, thereby enhancing the binding force between the enzyme and the outer network and improving the immobilization effect; In addition, poly(sulfobetaine) has a certain temperature responsiveness and is a network structure. As the temperature increases during waste treatment, the thermal motion of the polymer chains on poly(sulfobetaine) is enhanced, resulting in the network structure becoming loose, which is beneficial to the release of the composite enzyme inside the network;

[0028] The inner layer of pH-sensitive gel microspheres of the double-layer immobilized enzyme microcapsule prepared by the present invention comprises hyaluronic acid, dopamine and sucrose. After hyaluronic acid and dopamine are combined through electrostatic interaction, spherical colloidal particles are formed, which can achieve the encapsulation of the composite enzyme; At the same time, the mechanical properties of hyaluronic acid are poor and it is easy to deform or break, affecting the encapsulation effect. The modification of dopamine can improve its mechanical properties and enhance the encapsulation effect; In addition, due to the high viscosity of hyaluronic acid, it may lead to serious and uneven particle aggregation during the encapsulation process, reducing the encapsulation rate. The addition of sucrose can reduce the interaction between gel particles, reduce the aggregation phenomenon, and can also bind to hyaluronic acid through hydrogen bonds to enhance the gel stability;

[0029] When the composite enzyme of the present invention treats waste, it can promote the growth and metabolism of microorganisms. The microorganisms produce acid by decomposing organic substances such as cellulose, thereby reducing the pH of the system. In an acidic environment, the carboxyl groups on the hyaluronic acid molecular chain are protonated, and the electrostatic repulsion force between the molecular chains is weakened, and the gel network structure becomes loose. This structural change will reduce the mechanical strength of the gel and the binding ability to the encapsulated substance, realizing pH sensitivity, thereby promoting the release of the encapsulated substance - the composite enzyme. Specific embodiments

[0030] The present invention will be further described below in conjunction with embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments:

[0031] Example 1

[0032] The preparation steps of the double-layer immobilized enzyme microcapsule are as follows:

[0033] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water, stir evenly to obtain a co - coagulating gel with a mass fraction of 5%;

[0034] 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 solution to obtain a composite enzyme solution, then add it to the co - coagulating gel with a volume ratio of 1:5, stir evenly, and spray - dry to obtain inner - layer gel microspheres;

[0035] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ - aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry it to obtain modified poly(sulfobetaine);

[0036] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass - to - volume ratio of 1:2 to obtain an outer - layer network solution;

[0037] Step 5: Place the inner - layer gel microspheres in the outer - layer network solution with a mass - to - volume ratio of 1:2, stir and dry to obtain double - layer immobilized enzyme microcapsules.

[0038] Example 2

[0039] The preparation steps of the double - layer immobilized enzyme microcapsules are as follows:

[0040] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water, stir evenly to obtain a co - coagulating gel with a mass fraction of 5%;

[0041] 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 solution to obtain a composite enzyme solution, then add it to the co - coagulating gel with a volume ratio of 1:6, stir evenly, and spray - dry to obtain inner - layer gel microspheres;

[0042] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ - aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry it to obtain modified poly(sulfobetaine);

[0043] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass - to - volume ratio of 1:2 to obtain an outer - layer network solution;

[0044] Step 5: Place the inner - layer gel microspheres in the outer - layer network solution with a mass - to - volume ratio of 1:2, stir and dry to obtain double - layer immobilized enzyme microcapsules.

[0045] Example 3

[0046] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0047] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine, and 1 g of sucrose in water, stir evenly, and prepare a coacervation gel with a mass fraction of 5%;

[0048] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase, and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the coacervation gel with a volume ratio of 1:7, stir evenly, and spray dry to obtain the inner-layer gel microspheres;

[0049] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly, prepare a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry to obtain the modified poly(sulfobetaine);

[0050] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:2.5 to obtain the outer-layer network solution;

[0051] Step 5: Place the inner-layer gel microspheres in the outer-layer network solution with a mass-to-volume ratio of 1:3, stir and dry to obtain the double-layer immobilized enzyme microcapsules.

[0052] Example 4

[0053] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0054] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine, and 1 g of sucrose in water, stir evenly, and prepare a coacervation gel with a mass fraction of 5%;

[0055] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase, and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the coacervation gel with a volume ratio of 1:8, stir evenly, and spray dry to obtain the inner-layer gel microspheres;

[0056] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly, prepare a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry to obtain the modified poly(sulfobetaine);

[0057] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:3 to obtain the outer-layer network solution;

[0058] Step 5: Place the inner-layer gel microspheres in the outer-layer network solution with a mass-to-volume ratio of 1:3, stir and dry to obtain the double-layer immobilized enzyme microcapsules.

[0059] Example 5

[0060] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0061] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water, stir evenly to obtain a co-coagulating gel with a mass fraction of 5%;

[0062] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the co-coagulating gel, with a volume ratio of 1:10, stir evenly, and spray dry to obtain the inner layer gel microspheres;

[0063] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry to obtain the modified poly(sulfobetaine);

[0064] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution, with a mass-to-volume ratio of 1:3, to obtain the outer layer network solution;

[0065] Step 5: Place the inner layer gel microspheres in the outer layer network solution, with a mass-to-volume ratio of 1:4, stir and dry to obtain the double-layer immobilized enzyme microcapsules.

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 3 is that only the inner layer gel microspheres are prepared, and the specific steps are as follows:

[0068] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0069] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water, stir evenly to obtain a co-coagulating gel with a mass fraction of 5%;

[0070] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) and dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the co-coagulating gel, with a volume ratio of 1:7, stir evenly, and spray dry to obtain the inner layer gel microspheres.

[0071] Comparative Example 2

[0072] The difference between this comparative example and Example 3 is that only the outer layer network is prepared, and the specific steps are as follows:

[0073] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0074] Step 1: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry to obtain modified poly(sulfobetaine);

[0075] Step 2: Dissolve 100 mg of the composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1) in 5 mL of phosphate buffer to obtain a composite enzyme solution,

[0076] Step 3: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:2.5, and dry to prepare the outer network.

[0077] Comparative Example 3

[0078] The difference between this comparative example and Example 3 is that γ-aminobutyric acid is not added to the outer network solution, and the details are as follows:

[0079] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0080] Step 1: Dissolve 10 g of hyaluronic acid, 3 g of dopamine and 1 g of sucrose in water, stir evenly to obtain a coacervation gel with a mass fraction of 5%;

[0081] Step 2: Take 100 mg of the 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, and then add it to the coacervation gel with a volume ratio of 1:7, stir evenly, and spray dry to prepare the inner gel microspheres;

[0082] Step 3: Dissolve 20 g of poly(sulfobetaine) in water, stir evenly, and dry to obtain poly(sulfobetaine) with a mass fraction of 3%;

[0083] Step 4: Immerse the poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:2.5 to obtain the outer network solution;

[0084] Step 5: Place the inner gel microspheres in the outer network solution with a mass-to-volume ratio of 1:3, homogenize and dry to obtain the double-layer immobilized enzyme microcapsules.

[0085] Comparative Example 4

[0086] The difference between this comparative example and Example 3 is that dopamine is not added to the inner gel microspheres, and the details are as follows:

[0087] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0088] Step 1: Dissolve 13 g of hyaluronic acid and 1 g of sucrose in water, stir evenly to obtain a coacervation gel with a mass fraction of 5%;

[0089] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1), dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the coacervation gel with a volume ratio of 1:7. Stir evenly and perform spray drying to obtain inner layer gel microspheres;

[0090] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry it to obtain modified poly(sulfobetaine);

[0091] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:2.5 to obtain an outer layer network solution;

[0092] Step 5: Place the inner layer gel microspheres in the outer layer network solution with a mass-to-volume ratio of 1:3, homogenize and dry to obtain the double-layer immobilized enzyme microcapsules.

[0093] Comparative Example 5

[0094] The difference between this comparative example and Example 3 is that sucrose is not added to the inner layer gel microspheres, specifically as follows:

[0095] The preparation steps of the double-layer immobilized enzyme microcapsules are as follows:

[0096] Step 1: Dissolve 10 g of hyaluronic acid and 4 g of dopamine in water, stir evenly to obtain a coacervation gel with a mass fraction of 5%;

[0097] Step 2: Take 100 mg of the composite enzyme (the mass ratio of papain, phosphatase and β-glucosidase is 3:2:1), dissolve it in 5 mL of phosphate buffer solution to obtain a composite enzyme solution, and then add it to the coacervation gel with a volume ratio of 1:7. Stir evenly and perform spray drying to obtain inner layer gel microspheres;

[0098] Step 3: Dissolve 10 g of poly(sulfobetaine) and 10 g of γ-aminobutyric acid in water, stir evenly to obtain a modified poly(sulfobetaine) solution with a mass fraction of 3%, and dry it to obtain modified poly(sulfobetaine);

[0099] Step 4: Immerse the modified poly(sulfobetaine) in the composite enzyme solution with a mass-to-volume ratio of 1:2.5 to obtain an outer layer network solution;

[0100] Step 5: Place the inner layer gel microspheres in the outer layer network solution with a mass-to-volume ratio of 1:3, homogenize and dry to obtain the double-layer immobilized enzyme microcapsules.

[0101] Determination of the encapsulation efficiency of the composite enzyme:

[0102] The double-layer immobilized enzyme microcapsules were added to an acetic acid-sodium acetate buffer solution with a pH of 5.0. After being broken by a homogenizer, the enzyme activity was measured. The calculation formula for the encapsulation rate is as follows:

[0103] Encapsulation rate (%) = (N0 / N) × 100%

[0104] In the formula, N0 is the enzyme activity of the microcapsules formed by 1 mL of the mixed solution containing the composite enzyme, and N is the total enzyme activity of 1 mL of the mixed solution containing the composite enzyme.

[0105] Table 1 Encapsulation rate of the composite enzyme in the immobilized enzyme microcapsules prepared in Examples 1-5 and Comparative Examples 1-5

[0106] Entrapment efficiency (%) Entrapment efficiency (%) 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

[0107] As can be seen from Table 1, the encapsulation rate of the composite enzyme in the double-layer immobilized enzyme microcapsules prepared in Examples 1-5 was 80.55 - 88.21%. In Comparative Example 1, only the inner layer gel microspheres were prepared, and the encapsulation rate was only 50.23%. In Comparative Example 2, only the outer layer network was prepared, and encapsulation could not be achieved. In Comparative Example 3, γ-aminobutyric acid was not added, and the encapsulation rate of the composite enzyme decreased slightly. In Comparative Example 4, dopamine was not added, and the hyaluronic acid gel was prone to deformation or fragmentation, and the encapsulation rate decreased significantly to 65.14%. In Comparative Example 5, sucrose was not added, and the encapsulation rate of the microcapsules was 68.82%. It can be seen that the addition of sucrose can reduce the interaction between gel particles, reduce the aggregation phenomenon, and improve the encapsulation effect of the composite enzyme. Therefore, the double-layer immobilized enzyme microcapsules prepared in Example 3 were selected for subsequent treatment of livestock and poultry manure and straw waste.

[0108] Example 6

[0109] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0110] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, crush them, and perform steam explosion at a pressure of 1.5 MPa for 5 min to obtain a pretreatment product;

[0111] S2. Add the pretreatment product to a reaction tank, heat it to 55 °C, and adjust the humidity to 55% to obtain a substrate;

[0112] S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, and carry out an enzymatic hydrolysis reaction at 70 °C for 3 h, then cool to obtain organic fertilizer.

[0113] Example 7

[0114] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0115] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, pulverize them, and subject them to steam explosion at a pressure of 2 MPa for 3 min to obtain a pretreatment product;

[0116] S2. Add the pretreatment product to a reaction tank, heat it to 60 °C, and adjust the humidity to 55% to obtain a substrate;

[0117] S3. Add 2.5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, carry out an enzymatic hydrolysis reaction at 70 °C for 3 h, and cool to obtain organic fertilizer.

[0118] Example 8

[0119] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0120] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, pulverize them, and subject them to steam explosion at a pressure of 1.5 MPa for 5 min to obtain a pretreatment product;

[0121] S2. Add the pretreatment product to a reaction tank, heat it to 55 °C, and adjust the humidity to 55% to obtain a substrate;

[0122] S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, carry out an enzymatic hydrolysis reaction at 50 °C for 3 h, and cool to obtain organic fertilizer.

[0123] Example 9

[0124] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0125] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, pulverize them, and subject them to steam explosion at a pressure of 1.5 MPa for 5 min to obtain a pretreatment product;

[0126] S2. Add the pretreatment product to a reaction tank, heat it to 55 °C, and adjust the humidity to 55% to obtain a substrate;

[0127] S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, carry out an enzymatic hydrolysis reaction at 60 °C for 3 h, and cool to obtain organic fertilizer.

[0128] Example 10

[0129] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0130] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, pulverize them, and perform steam explosion at a pressure of 1.5 MPa for 5 min to obtain a pretreatment product;

[0131] S2. Add the pretreatment product to a reaction tank, heat it to 55 °C, and adjust the humidity to 55% to prepare a substrate;

[0132] S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, carry out an enzymatic hydrolysis reaction at 70 °C for 2 h, and cool to obtain organic fertilizer.

[0133] Example 11

[0134] A method for synchronously treating livestock and poultry manure and straw with double-layer immobilized enzyme microcapsules, comprising the following steps:

[0135] S1. Mix 300 kg of livestock and poultry manure and 200 kg of straw, pulverize them, and perform steam explosion at a pressure of 1.5 MPa for 5 min to obtain a pretreatment product;

[0136] S2. Add the pretreatment product to a reaction tank, heat it to 55 °C, and adjust the humidity to 55% to prepare a substrate;

[0137] S3. Add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3 to the substrate, mix evenly, carry out an enzymatic hydrolysis reaction at 70 °C for 2.5 h, and cool to obtain organic fertilizer.

[0138] Comparative Example 6

[0139] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 1 are used.

[0140] Comparative Example 7

[0141] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 2 are used.

[0142] Comparative Example 8

[0143] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 3 are used.

[0144] Comparative Example 9

[0145] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 4 are used.

[0146] Comparative Example 10

[0147] The difference between this comparative example and Example 6 is that the microcapsules prepared in Comparative Example 5 are used.

[0148] Comparative Example 11

[0149] The difference between this comparative example and Example 6 lies in that no pretreatment was carried out, specifically as follows:

[0150] A method for synchronously treating livestock and poultry manure and straw with a double-layer immobilized enzyme microcapsule, comprising the following steps:

[0151] S1. Mix and crush 300 kg of livestock and poultry manure and 200 kg of straw to obtain a substrate;

[0152] S2. Add the substrate to a reaction tank, adjust the humidity of the substrate to 55%, add 5 kg of the double-layer immobilized enzyme microcapsules prepared in Example 3, mix evenly, carry out an enzymatic hydrolysis reaction at 70 °C for 3 h, and cool to obtain organic fertilizer.

[0153] Performance test:

[0154] (1) Degradation rate of waste

[0155] Determine the degradation rates of proteins, fats, lignins, and celluloses in livestock and poultry manure and straw in Examples 6-11 and Comparative Examples 6-11. Among them, the protein is determined by the automatic Kjeldahl method, the fat content is determined by the Soxhlet extraction method, the lignin is determined by the nitric acid-ethanol method, and the cellulose is determined by the 72% concentrated sulfuric acid hydrolysis method.

[0156] Table 2 Degradation rates of proteins, fats, lignins, and celluloses of waste in Examples 6-11 and Comparative Examples 6-11

[0157]

[0158] As can be seen from Table 2, in Examples 6, 8, and 9, only the enzymatic hydrolysis temperature was changed. As the enzymatic hydrolysis temperature increased, the degradation rates of proteins, fats, lignins, and celluloses all increased, indicating that the double-layer immobilized enzyme microcapsules prepared by the present invention have a certain temperature sensitivity; in Examples 6, 10, and 11, only the enzymatic hydrolysis time was changed. As the enzymatic hydrolysis time extended, the microorganisms in the waste passed through

[0159] The decomposition of organic substances such as cellulose produces acids, causing the pH of the system to gradually decrease. The carboxyl groups on the hyaluronic acid molecular chain are protonated, the electrostatic repulsion between molecular chains weakens, and the gel network structure becomes loose. This structural change reduces the mechanical strength of the gel and the binding ability to the embedded substances, achieving pH sensitivity, thereby promoting the release of the embedded composite enzyme; Comparative Example 6 uses a composite enzyme embedded only in the inner gel network, Comparative Example 7 uses a composite enzyme immobilized only in the outer network, Comparative Example 8 does not add γ-aminobutyric acid to the microcapsules, Comparative Example 9 does not add dopamine to the microcapsules, and Comparative Example 10 does not add sucrose to the microcapsules. The degradation rates of proteins, fats, lignin, and cellulose in the waste have all decreased. It can be seen that the double-layer immobilized enzyme microcapsules prepared in the present invention are used to achieve efficient enzymatic hydrolysis of waste; Comparative Example 11 does not pre-treat the waste, and the degradation rate also decreases.

[0160] (2)Organic fertilizer performance

[0161] 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 moisture, the pH value, the number of fecal coliform bacteria, and the total arsenic and total cadmium in heavy metals.

[0162] Table 3 Performance indicators of the organic fertilizers produced in Example 6 and Comparative Examples 6-11

[0163] 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 Moisture mass fraction (%) 22.54 29.82 29.15 27.56 27.17 27.39 28.59 pH value 5.7 7.5 7.2 6.7 6.5 6.4 7.1 Fecal coliform count (per 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

[0164] As can be seen from Table 3, the performance indicators of the organic fertilizers produced in Example 6 and Comparative Examples 6-11 all meet the regulations in NY / T 525-2021 "Organic Fertilizer". Comparative Example 6 uses a composite enzyme embedded only in the inner gel network, and Comparative Example 7 uses a composite enzyme immobilized only in the outer network for enzymatic hydrolysis of waste. The performance indicators of the organic fertilizers produced by both are significantly lower than those of Example 6. Comparative Example 8 does not add γ-aminobutyric acid to the microcapsules, Comparative Example 9 does not add dopamine to the microcapsules, Comparative Example 10 does not add sucrose to the microcapsules, and Comparative Example 11 does not perform pretreatment. The performance indicators of the organic fertilizers produced by enzymatic hydrolysis of these four are slightly lower than those of Example 6. Therefore, it can be seen that the double-layer immobilized enzyme microcapsules prepared in the present invention can achieve a higher degree of enzymatic hydrolysis and the production of organic fertilizers, promoting the resource recycling of livestock manure and straw.

[0165] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall 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. The livestock manure and straw were mixed and crushed in a ratio of (5-7): (3-5), and steam exploded at a pressure of 1.5-2.5MPa for 3-5min 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. Adding double-layer immobilized enzyme microcapsules to the substrate, mixing evenly, performing enzymatic hydrolysis, cooling, and obtaining organic fertilizer; 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.

2. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, 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%.

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

4. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 3, 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.

5. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, 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).

6. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, 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%.

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

8. The method for simultaneously treating livestock manure and straw using double-layer immobilized enzyme microcapsules according to claim 1, 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).

9. 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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