阅读说明：本技术 一种水热预处理与高温混合酶协同降解植物生物质的方法 (Method for degrading plant biomass by cooperation of hydrothermal pretreatment and high-temperature mixed enzyme ) 是由 邱卫华 于 2020-06-23 设计创作，主要内容包括：本发明属于植物生物质原料高效降解技术领域。针对植物生物质原料水热预处理对木质素降解效果甚微,微生物预处理菌种种类稀缺、处理周期较长、糖分损耗较大等问题,提供一种水热预处理与高温混合酶协同降解植物生物质的方法。本发明包括以下步骤：1)植物生物质原料前处理；2)植物生物质原料水热预处理；3)嗜热毁丝霉的发酵培养；4)嗜热毁丝霉粗酶液的制备；5)水热预处理植物生物质原料的嗜热毁丝霉粗酶液降解。本发明所提供的技术方案具有能耗低、半纤维素和木质素组分降解效率高、纤维素组分回收率高、过程绿色环保等优点。(The invention belongs to the technical field of efficient degradation of plant biomass raw materials. Aiming at the problems that the hydrothermal pretreatment of plant biomass raw materials has little effect on lignin degradation, microbial pretreatment strains are scarce, the treatment period is long, the sugar loss is large and the like, the method for degrading the plant biomass by the cooperation of the hydrothermal pretreatment and high-temperature mixed enzyme is provided. The invention comprises the following steps: 1) pretreating a plant biomass raw material; 2) carrying out hydrothermal pretreatment on a plant biomass raw material; 3) fermenting and culturing myceliophthora thermophila; 4) preparing a myceliophthora thermophila crude enzyme solution; 5) carrying out hydrothermal pretreatment on the crude myceliophthora thermophila enzyme solution of the plant biomass raw material for degradation. The technical scheme provided by the invention has the advantages of low energy consumption, high degradation efficiency of hemicellulose and lignin components, high recovery rate of cellulose components, green and environment-friendly process and the like.)

1. The invention discloses a method for degrading plant biomass by combining hydrothermal pretreatment and high-temperature mixed enzyme, which is characterized by comprising the following steps of: 1) pretreatment of a biomass raw material; 2) carrying out hydrothermal pretreatment on a biomass raw material; 3) fermenting and culturing myceliophthora thermophila; 4) preparing a myceliophthora thermophila crude enzyme solution; 5) carrying out hydrothermal pretreatment on the crude myceliophthora thermophila enzyme solution of the plant biomass raw material for degradation.

2. The method of claim 1, wherein the plant biomass material comprises one or more of corn stover, wheat straw, cotton stalk, bagasse, corn cobs, wood chips, miscanthus, and peanut hulls.

3. The method for degrading the plant biomass by the cooperation of the hydrothermal pretreatment and the high-temperature mixed enzyme according to claim 1, wherein the pretreatment of the biomass raw material in the step 1) comprises cleaning, shearing and crushing to 40-60 meshes.

4. The method for the synergistic degradation of plant biomass through the hydrothermal pretreatment and the high-temperature enzyme mixing according to claim 1, wherein the parameters of the hydrothermal pretreatment of the biomass raw material in the step 2) are as follows: the hydrothermal pretreatment of the biomass raw material is carried out in a stainless steel high-pressure reaction kettle with a polytetrafluoroethylene lining. The hydrothermal pretreatment parameters are as follows: mixing and soaking the biomass raw material and distilled water according to a certain solid-liquid ratio, adding the mixture into a high-temperature high-pressure reaction kettle reaction tank, heating to a preset temperature, carrying out constant-temperature treatment for a certain time, and rapidly cooling to room temperature after the reaction is finished.

5. The method for the hydrothermal pretreatment and the high-temperature mixed enzyme synergistic degradation of the plant biomass according to claim 1, wherein the myceliophthora thermophila culture parameters in the step 3) are as follows: culturing myceliophthora thermophila by using a glucose potato juice culture medium under the conditions of 30-50 ℃ and 180-200 rpm shaking culture for 5-7 days.

6. The method for degrading plant biomass by combining hydrothermal pretreatment and high-temperature mixed enzyme in cooperation with the claim 1, wherein the preparation of the myceliophthora thermophila crude enzyme solution in the step 4) comprises the steps of centrifuging myceliophthora thermophila fermentation solution to respectively collect thalli and the fermentation solution, and centrifuging the thalli after the thalli are subjected to ultrasonic crushing to collect an extracting solution; mixing the fermentation liquor and the extracting solution, precipitating with 60-85% (w/w) ammonium sulfate, centrifuging, collecting protein, dissolving the protein precipitate with 0.2M PBS buffer solution with the pH value of 5.0-6.5, and dialyzing to remove the ammonium sulfate to obtain the crude myceliophthora thermophila enzyme solution.

7. The method for degrading the plant biomass by the cooperation of the hydrothermal pretreatment and the high-temperature mixed enzyme according to claim 1, wherein the degradation of the plant biomass raw material in the step 5) is to add the crude myceliophthora thermophila enzyme solution obtained in the step 4) into the hydrothermal pretreatment plant biomass raw material obtained in the step 2), adjust the solid-to-liquid ratio to be 1: 5-1: 50, and degrade the biomass at 50 ℃ by heat preservation.

8. The plant biomass feedstock according to claim 2, wherein a plurality of plant biomass feedstocks are mixed in equal mass ratios for hydrothermal pretreatment.

9. The method for degrading the plant biomass by the cooperation of the hydrothermal pretreatment and the high-temperature mixed enzyme according to claim 3, wherein the solid-to-liquid ratio of the hydrothermal pretreatment biomass raw material to the distilled water in the step 2) is 1: 5-1: 20; the preset temperature is 140-220 ℃; the temperature maintaining time is 5-60 min.

10. The method for the hydrothermal pretreatment and the high-temperature enzyme mixing synergistic degradation of the plant biomass according to the claim 5, wherein the parameters of the ultrasonic disruption of the myceliophthora thermophila body in the step 4) are as follows: and opening the ice bath for 5-20 s, stopping for 5-20 s, and carrying out ultrasonic treatment for 20-30 min.

11. The method for the synergistic degradation of plant biomass through the hydrothermal pretreatment and the high-temperature enzyme mixing according to claim 6, wherein the concentration of the crude myceliophthora thermophila enzyme solution added in the step 5) is 50 mg-1 g of protein per 100g of biomass raw material.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of efficient utilization of biomass resources, and particularly relates to a method for degrading plant biomass by cooperation of hydrothermal pretreatment and high-temperature mixed enzyme.

[ background of the invention ]

The utilization of a novel industrial model to convert plant biomass raw materials into biological energy, materials and bulk chemicals with high added values has become a hot field of important development strategies and scientific researches in many countries. To convert a low-valued plant biomass feedstock into a feedstock for the production of a high-valued product, a high-concentration "sugar platform" needs to be constructed first. Therefore, breaking through natural barriers against biological and enzymatic attacks becomes a prerequisite and key to achieving a reasonable technical economy of plant biomass feedstocks.

The pretreatment technology can break the compact structure of the plant biomass raw material and increase the surface aperture and the surface area, thereby improving the cellulose enzymolysis rate of the raw material and the polysaccharide utilization rate of subsequent microorganisms. Among a plurality of pretreatment methods, the high-temperature hydrothermal pretreatment has the advantages of environmental protection, high efficiency, capability of removing a large amount of hemicellulose and partial lignin, low content of byproducts and the like because no chemical reagent is added, and is considered to be a low-cost green pretreatment method. However, hydrothermal pretreatment mostly hydrolyzes hemicellulose, and has little effect on lignin degradation in the feedstock. And the lignin is a key factor for restricting the construction of the subsequent enzymatic hydrolysis sugar platform of the plant biomass raw material. Thus, combined treatment of multiple pretreatment methods is the primary method to obtain a high concentration sugar platform. Biological pretreatment of plant biomass raw materials utilizes microorganisms and enzymes secreted by the microorganisms to achieve the purpose of degrading biomass. The pretreatment method has low cost, low energy consumption and environmental protection, and has low cost and energy consumption. The synergistic effect of the physical and chemical pretreatment method and the biological pretreatment method is beneficial to realizing the high-efficiency depolymerization of the plant biomass raw material. However, the existing plant biomass degradation strains mainly comprise room-temperature strains of brown rot fungi and white rot fungi, and have the problems of scarce species, long period, large sugar loss and the like. And the secreted enzyme preparation has poor thermostability. Myceliophthora thermophila (Myceliophthora thermophile) is a thermophilic fungus that produces cellulolytic enzymes with high thermal stability and is also an industrial producer of laccase. It secretes these enzyme preparations with high thermostability. The invention aims to provide a method for degrading plant biomass at high temperature by using a myceliophthora thermophila secretase preparation in cooperation with hydrothermal pretreatment.

[ summary of the invention ]

[ OBJECTS OF THE INVENTION ]

Aiming at the problems that the hydrothermal pretreatment has a very small effect on the degradation of lignin in plant biomass raw materials, and the microbial pretreatment has the defects of scarce strain types, long treatment period, large sugar loss and the like, the invention aims to provide the method for efficiently degrading the plant biomass raw materials by the cooperation of the hydrothermal pretreatment and high-temperature mixed enzyme.

[ technical solution ] A

The invention provides a method for degrading plant biomass by the cooperation of hydrothermal pretreatment and high-temperature mixed enzyme, which comprises the following steps: 1) pretreatment of a biomass raw material; 2) carrying out hydrothermal pretreatment on a biomass raw material; 3) culturing myceliophthora thermophila; 4) preparing a myceliophthora thermophila crude enzyme solution; 5) carrying out hydrothermal pretreatment on the crude myceliophthora thermophila enzyme solution of the plant biomass raw material for degradation.

The plant biomass raw material comprises one or more of corn straw, wheat straw, cotton stalk, bagasse, corncob, wood chip, miscanthus and peanut shell.

The pretreatment of the plant biomass raw material comprises cleaning, shearing and crushing to 40-60 meshes.

The plant biomass raw material is characterized in that when various biomass raw materials are subjected to hydrothermal pretreatment, the raw materials are mixed according to equal mass.

The biomass raw material hydrothermal pretreatment parameters in the step 2) of the invention are as follows: the hydrothermal pretreatment of the biomass raw material is carried out in a stainless steel high-pressure reaction kettle with a polytetrafluoroethylene lining. The hydrothermal pretreatment parameters are as follows: mixing and soaking the biomass raw material and distilled water according to a certain solid-liquid ratio, adding the mixture into a high-temperature high-pressure reaction kettle reaction tank, heating to a preset temperature, carrying out constant-temperature treatment for a certain time, and rapidly cooling to room temperature after the reaction is finished.

The myceliophthora thermophila culture parameters in the step 3) of the invention are as follows: culturing myceliophthora thermophila by using a glucose potato juice culture medium under the conditions of 30-50 ℃ and 180-200 rpm shaking culture for 5-7 days.

The preparation of the myceliophthora thermophila crude enzyme solution in the step 4) of the invention means that myceliophthora thermophila fermentation liquor is centrifuged to respectively collect thalli and the fermentation liquor, and the thalli are centrifuged to collect extracting solution after being crushed by ultrasonic waves; mixing the fermentation liquor and the extracting solution, precipitating with 60-85% (w/w) ammonium sulfate, centrifuging, collecting protein, dissolving the protein precipitate with 0.2M PBS buffer solution with the pH value of 5.0-6.5, and dialyzing to remove the ammonium sulfate to obtain the crude myceliophthora thermophila enzyme solution.

The degradation of the plant biomass raw material in the step 5) refers to adding the myceliophthora thermophila crude enzyme liquid obtained in the step 4) into the hydrothermal pretreatment plant biomass raw material obtained in the step 2), adjusting the solid-liquid ratio to be 1: 5-1: 50, and preserving heat at 50 ℃ to degrade biomass.

The solid-to-liquid ratio of the hydrothermal pretreatment biomass raw material to the distilled water in the step 2) is 1: 5-1: 20; the preset temperature is 140-220 ℃; the temperature maintaining time is 5-60 min.

The ultrasonic crushing parameters of the myceliophthora thermophila in the step 4) of the invention are as follows: and opening the ice bath for 5-20 s, stopping for 5-20 s, and carrying out ultrasonic treatment for 20-30 min.

The crude myceliophthora thermophila enzyme solution in the step 5) of the invention is added with the concentration of 50 mg-1 g of protein per 100g of biomass raw material.

[ PROBLEMS ] the present invention

The hydrothermal pretreatment has the advantages of environmental protection, low cost and the like for breaking the biological degradation-resistant barrier of the plant biomass, but has little effect on the degradation of lignin in the plant biomass raw material, and the degradation efficiency of hemicellulose is low when the treatment temperature is low (less than or equal to 150 ℃); most of microbial pretreatment strains of plant biomass raw materials are normal-temperature microbial agents, and the problems of long treatment period, large sugar loss and the like exist. Myceliophthora thermophila can grow at a high temperature of 50-60 ℃, and can secrete enzymes for degrading cellulose and hemicellulose and various lignin degrading enzymes.

The invention synergistically degrades plant biomass by mixing hydrothermal pretreatment and myceliophthora thermophila with the high-temperature enzyme preparation, and has the following beneficial effects:

1) the intensity of hydrothermal pretreatment can be reduced, and the purpose of saving energy is achieved;

2) after the pretreatment intensity is reduced, the concentration of an inhibitor in the hydrothermal pretreatment biomass raw material can be reduced, so that the inhibition on the enzyme activity is reduced, and the subsequent biological pretreatment effect of a high-temperature enzyme system is improved;

3) the intracellular enzyme and the extracellular enzyme secreted by the myceliophthora thermophila have stronger heat resistance, and can carry out biological pretreatment at higher temperature (50-60 ℃), thereby reducing the cooling energy consumption of hydrothermal pretreatment products, and can be linked with subsequent cellulose hydrolysis (50 ℃), so that the overall energy consumption of the plant biomass pretreatment and enzymolysis processes is reduced.

[ detailed description ] embodiments

The technical solution of the present invention is further illustrated by the following examples. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.