Microbial complex microbial inoculant and method for degrading lignocellulose

Abstract

The invention relates to the field of microorganisms, and discloses a microbial composite inoculant and a method for degrading lignocellulose. The compound microbial agent comprises phanerochaete chrysosporium, aspergillus niger, trichoderma reesei, neurospora crassa and gloeophyllum trabeum. The phanerochaete chrysosporium is a white-rot fungus with strong performance, and can secrete a plurality of lignocelluloses at the same time. Aspergillus niger, trichoderma reesei, neurospora crassa and gloeophyllum trabeum are mainly used for secreting high-activity cellulase and hemicellulase. Destroying the lignocellulose structure needs the combined action of various lignocellulose enzyme systems, and after the five strains are mixed, the synergistic effect among the enzyme systems in the system can be remarkably enhanced, the lignocellulose structure is efficiently destroyed, and the degradation rate of the wood fiber raw material is increased.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a composite microbial agent and a method for degrading lignocellulose. Background Art

[0002] In recent years, with the increasingly serious global fossil energy crisis, scholars have paid increasing attention to wood fiber raw materials as the largest, most widely available and cheapest renewable resource on the earth. The main components of wood fiber raw materials are cellulose, including cellulose, hemicellulose and lignin, which account for about 70%-80% in total. However, the stability and difficulty of the dense structure of cellulose seriously hinder the large-scale resource utilization of wood fiber raw materials. Therefore, it is necessary to pretreat the wood fiber raw materials to destroy their cellulose structure.

[0003] At present, the methods commonly used to destroy the structure of lignocellulose are mainly divided into three categories: physical method, chemical method and biological method. However, due to the harsh reaction conditions and high energy consumption of physical methods, the high cost of chemical reagents and the easy secondary pollution, in contrast, the biological method has obvious advantages in low treatment cost, simple operation, low energy consumption and no secondary pollution. The biological method mainly uses various types of lignocellulose enzymes secreted by microorganisms to destroy the structure of lignocellulose and degrade lignocellulose. At present, the research on biodegradation of lignocellulose mainly focuses on a single microorganism. However, due to the small number of enzymes secreted by a single microorganism, its activity is not high, and the degree of damage to the structure of lignocellulose is also very limited, which limits the further resource application of biodegradation of lignocellulose.

[0004] Due to the insufficient efficiency of existing single microorganisms in degrading lignocellulose structures, there is an urgent need for a microbial composite agent that can efficiently degrade lignocellulose, which is of great significance for protecting the environment, saving energy, and promoting the resource utilization of lignocellulose raw materials. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of low degradation efficiency in the prior art of lignocellulose biodegradation technology, and to provide a microbial composite agent and a method for degrading lignocellulose. The synergistic effect between multiple microorganisms in the microbial composite agent is conducive to the complementary formation of enzyme systems secreted by them and can significantly improve the activity of lignocellulose enzymes, thereby increasing the degree of damage to the lignocellulose structure.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides a microbial composite agent, wherein the microbial composite agent comprises: Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora dactylifera.

[0007] A second aspect of the present invention provides a method for degrading lignocellulose, wherein the method comprises the following steps: pretreating the lignocellulosic raw material, and then inoculating the seed liquid of the microbial composite agent into the pretreated raw material for fermentation; wherein the microbial composite agent is the microbial composite agent provided by the present invention.

[0008] Through the above technical solution, the beneficial effects of the present invention are:

[0009] The microbial composite agent provided by the present invention includes Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala, and is used to degrade lignocellulose in agricultural waste. Compared with the traditional single biological degradation of lignocellulose, the microbial composite agent can utilize the synergistic effect between the enzyme systems of each strain to destroy the lignocellulose structure, thereby improving the biodegradability of components such as cellulose and hemicellulose.

[0010] The present invention has wide applicability to agricultural waste of wood fiber raw materials. The microbial composite agent provided by the present invention has a good degradation effect on wood fiber raw materials with a cellulose content of 35-45wt%, a hemicellulose content of 20-25wt%, and a lignin content of 2-6wt%, such as rice straw, and can effectively solve the defects of low straw utilization rate, low conversion rate, low economic benefit, and serious environmental pollution. The microbial composite agent provided by the present invention is simple to operate, low in cost, has no secondary pollution risks, and has both economic and environmental technical feasibility in degrading wood cellulose.

[0011] In a preferred embodiment of the present invention, by selecting the appropriate ratio of the number of live bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Pseudomonas fasciatus, the fermentation conditions when degrading lignocellulose, the specific types of the five bacteria, and selecting the appropriate culture medium, the ability of the microbial composite agent to degrade lignocellulose is further improved. After fermentation, the total solid degradation rate of rice straw is more than 70%, the hemicellulose degradation rate is more than 65%, the cellulose degradation rate is more than 50%, the lignin degradation rate is more than 15%, and the fermentation cycle is short, only about 7 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graph showing the degradation rate of each component of lignocellulose in lignocellulose raw material by the microbial composite agent in Example 1 of the present invention over time;

[0013] Figure 2 It is a curve chart of the changes in the activities of cellulase and hemicellulase during the fermentation process of the microbial composite agent in Example 1 of the present invention. DETAILED DESCRIPTION

[0014] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0015] A first aspect of the present invention provides a microbial composite agent, wherein the microbial composite agent comprises: Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Gloeophyllum trabeum.

[0016] Phanerochaete chrysosporium is a white rot fungus with strong performance, which can secrete multiple lignocellulases at the same time. Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala mainly secrete highly active cellulases and hemicellulases. The destruction of lignocellulose structure requires the joint action of multiple lignocellulase systems. The mixture of the above five strains can significantly enhance the synergistic effect between enzyme systems in the system, which is conducive to the complementary formation of the enzyme systems secreted by them and can significantly improve the activity of lignocellulase, thereby being able to efficiently destroy the lignocellulose structure and improve the degradation rate of lignocellulose raw materials.

[0017] According to the present invention, preferably, in the microbial composite agent, the ratio of the number of viable bacteria of the Phanerochaete chrysosporium, the Aspergillus niger, the Trichoderma reesei, the Neurospora crassa and the Chrysosporium is 1:(0.2-20):(0.2-20):(0.2-20):(0.1-10), preferably 1:(1-4):(1-4):(1-4):(0.5-2), more preferably 1:(1.5-2.5):(1.5-2.5):(1.5-2.5):(0.8-1.2).

[0018] According to the present invention, preferably, the strain number of Phanerochaete chrysosporium is CICC 40719 and / or the product number is B89537, and / or,

[0019] The strain number of the Aspergillus niger is CICC 40273 and / or ACCC 32589, and / or,

[0020] The strain number of the Trichoderma reesei is CICC 2626 and / or ACCC 30912, and / or,

[0021] The strain number of Neurospora crassa is CICC 40203 and / or ACCC 32257, and / or,

[0022] The strain number of the dense brown gill fungus is ACCC 51714.

[0023] Further, the strain number of the Phanerochaete chrysosporium is CICC 40719, and / or,

[0024] The strain number of the Aspergillus niger is CICC 40273 and / or ACCC 32589, and / or,

[0025] The strain number of the Trichoderma reesei is CICC 2626 and / or ACCC 30912, and / or,

[0026] The strain number of Neurospora crassa is CICC 40203 and / or ACCC 32257, and / or,

[0027] The strain number of the dense brown gill fungus is ACCC 51714.

[0028] Furthermore, the strain number of the Phanerochaete chrysosporium is CICC 40719.

[0029] The strain number of Aspergillus niger is CICC 40273,

[0030] The strain number of the Trichoderma reesei is CICC 2626,

[0031] The strain number of Neurospora crassa is CICC 40203,

[0032] The strain number of the dense brown gill fungus is ACCC 51714.

[0033] A second aspect of the present invention provides a method for degrading lignocellulose, wherein the method comprises the following steps: pretreating the lignocellulosic raw material, and then inoculating the seed liquid of the microbial composite agent into the pretreated raw material for fermentation; wherein the microbial composite agent is the microbial composite agent provided by the present invention.

[0034] According to the present invention, preferably, in the wood fiber raw material, the content of cellulose is 35-45wt%, the content of hemicellulose is 20-25wt%, and the content of lignin is 2-6wt%. The microbial composite agent provided by the present invention has wide applicability to agricultural waste of wood fiber raw materials, especially for wood fiber raw materials whose contents of cellulose, hemicellulose and lignin meet this range, such as rice straw, and has a good degradation effect.

[0035] According to the present invention, preferably, based on 100 g of the weight of the pretreated raw material, the inoculation amount of the seed solution is 0.1-10 mL, preferably 4.5-5.5 mL.

[0036] According to the present invention, preferably, the total concentration of live bacteria in the seed solution is 10 7 CFU / mL or more, preferably 10 7 -10 8 CFU / mL.

[0037] According to the present invention, preferably, the fermentation conditions include: temperature of 20-35°C, preferably 25-27°C; time of 90-240h, preferably 160-175h; stirring speed of 130-170rpm.

[0038] According to the present invention, preferably, the method for preparing the seed solution comprises: activating Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psoralea corylifolia independently in an activation medium, and then independently culture them in a culture medium.

[0039] According to the present invention, preferably, the activation medium and the expansion medium are both potato medium (PDB medium). The activation medium and the expansion medium each independently contain: potato slurry, glucose, potassium dihydrogen phosphate, magnesium sulfate and vitamin B1.

[0040] According to the present invention, preferably, based on the total volume of the activation medium or the expansion medium, the activation medium and the expansion medium each independently contain: 180-220 g / L potato slurry, 17-23 g / L glucose, 2.5-3.5 g / L potassium dihydrogen phosphate, 0.6-0.9 g / L magnesium sulfate and 6-10 mg / L vitamin B1. The potato slurry is obtained by cutting potatoes, boiling, melting and filtering.

[0041] In the present invention, the above-mentioned substances may exist in a form containing or not containing bound water when preparing the culture medium, and the magnesium sulfate may be magnesium sulfate heptahydrate, and its content in the culture medium is calculated in a form containing no bound water.

[0042] According to the present invention, preferably, the pH values ​​of the activation medium and the expansion medium are independently 5.8-6.5.

[0043] According to a specific embodiment of the present invention, the preparation method of the seed liquid comprises: picking single colonies of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala respectively and inoculating them into liquid PDB medium for activation, and then inoculating the activated Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala respectively into liquid PDB medium for expansion culture, and the five bacteria are respectively expanded to a viable bacterial concentration of 10 7 -10 8 CFU / mL, and then mixing the bacterial liquid of Phanerochaete chrysosporium, the bacterial liquid of Aspergillus niger, the bacterial liquid of Trichoderma reesei, the bacterial liquid of Neurospora crassa and the bacterial liquid of Psora crassa to obtain a seed liquid of a microbial composite agent; so that in the seed liquid of the microbial composite agent, the ratio of the number of viable bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora crassa is 1:(0.2-20):(0.2-20):(0.1-10), preferably 1:(1-4):(1-4):(1-4):(0.5-2), and more preferably 1:(1.5-2.5):(1.5-2.5):(1.5-2.5):(0.8-1.2).

[0044] Preferably, when the culture is expanded, based on the volume of the liquid PDB culture medium as 100 mL, the inoculation amount of the activated Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora dactylifera is independently 0.1-10 mL.

[0045] According to the present invention, preferably, the activation conditions include: temperature of 20-35°C, preferably 25-27°C; time of 1-5h, preferably 1.5-2.5h.

[0046] Furthermore, the activation is carried out at a shaking table speed of 130-170 rpm.

[0047] According to the present invention, preferably, the conditions for the expansion culture include: a temperature of 20-35°C, preferably 25-27°C; and a time of 68-75h.

[0048] Furthermore, the expansion culture is carried out at a shaking table speed of 130-170 rpm.

[0049] According to the present invention, preferably, the pretreatment process includes: removing impurities, crushing and screening the wood fiber raw materials.

[0050] The present invention will be described in detail below through examples and comparative examples. In the following examples and comparative examples, unless otherwise specified, all are conventional methods; the reagents and materials used, unless otherwise specified, can be obtained from commercial channels. The determination methods and raw materials involved in each example and comparative example are as follows:

[0051] Determination of crude protein: Kjeldahl method GB / T 6432-2018;

[0052] Determination of total solids: Drying method HJ 613-2011;

[0053] Determination of the mass fraction of cellulose, hemicellulose and lignin: Paradigm washing method;

[0054] Determination of carboxymethyl cellulase (cellulase) and xylanase (hemicellulase) activities: Dinitrosalicylic acid (DNS) colorimetric method;

[0055] Total solid degradation rate formula: M1 is the mass of total solids in the system at the initial stage of fermentation, g; M2 is the mass of total solids in the system after the fermentation is completed, g;

[0056] The formula of lignocellulose degradation rate is: M begin Refers to the total mass of cellulose, hemicellulose or lignin in the system at the initial stage of fermentation, g; M end Refers to the total mass of cellulose, hemicellulose or lignin in the system after fermentation, g.

[0057] Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei and Neurospora crassa were purchased from China Industrial Culture Collection Center with numbers CICC 40719, CICC 40273, CICC 2626 and CICC 40203, respectively, and Folium sphaerocephalum was purchased from China Agricultural Culture Collection Center with number ACCC 51714.

[0058] Composition of liquid PDB culture medium: Based on the total volume of the liquid PDB culture medium, the liquid PDB culture medium contains: 200 g / L potato slurry (obtained by cutting, boiling, melting and filtering potatoes), 20 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 8 mg / L vitamin B1 and deionized water; pH value is 6; sterilize at 121°C for 20 min to obtain liquid PDB culture medium.

[0059] Wood fiber raw material: Rice straw was obtained from Siyang County, Jiangsu Province and stored in a -20°C refrigerator for future use. The composition and content are shown in Table 1.

[0060] Table 1

[0061] parameter <![CDATA[Total solids, % a > <![CDATA[Crude protein, % b > <![CDATA[Cellulose, % b > <![CDATA[Hemicellulose, % b > <![CDATA[Lignin, % b > Rice straw 91.51±0.81 4.60±0.13 41.61±0.41 22.34±0.30 4.63±0.08

[0062] Note: Values ​​are mean ± standard deviation; a means based on wet weight, b means based on dry weight.

[0063] The following examples are used to illustrate the method of using a microbial composite agent to degrade lignocellulose.

[0064] Example 1

[0065] The preparation method of the seed solution of the microbial composite agent is as follows:

[0066] Single colonies of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala were picked up and inoculated into shake flasks containing liquid PDB medium respectively, and activated for 2 h at a temperature of 26 ° C and a shaker speed of 150 rpm; then the activated Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala were inoculated into shake flasks containing liquid PDB medium respectively, with the volume of liquid PDB medium being 100 mL, and the inoculation amount of the activated Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala was 1 mL, and the culture was expanded for 72 h at a temperature of 26 ° C and a shaker speed of 150 rpm, and the five bacteria were expanded to a viable bacterial concentration of 10 7 CFU / mL, and then the culture solutions of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala obtained by expansion culture were mixed in a volume ratio of 1:2:2:2:1 to obtain the seed solution of the microbial composite inoculant, and the total concentration of live bacteria in the seed solution was 10 7 CFU / mL, the ratio of the number of live bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psoralea corylifolia in the seed solution was 1:2:2:2:1.

[0067] The specific method of using microbial composite agents to degrade lignocellulose is as follows:

[0068] Take fresh rice straw, remove the debris in the straw, wash and crush it, pass it through a 60-mesh sieve, accurately weigh 300 g and place it in a 1L beaker, and sterilize it at 121°C for 20 min; then inoculate the seed liquid of the microbial composite agent into the crushed and sieved rice straw, with the weight of the crushed and sieved rice straw being 100 g, the inoculation amount of the seed liquid being 5 mL, and fermenting it in a fermenter at a temperature of 26°C and a stirring speed of 150 rpm for 168 h.

[0069] Depend on Figure 1 It can be seen that during the fermentation process, the degradation rates of cellulose, hemicellulose and lignin all showed an upward trend as the fermentation time increased. At 168h, the degradation rate of cellulose was 51.36%, the degradation rate of hemicellulose was 69.81%, and the degradation rate of lignin was 18.74%. This shows that the microbial composite agent of Example 1 has a good ability to degrade lignocellulose in rice straw.

[0070] Depend on Figure 2It can be seen that the microbial composite agent of Example 1 continuously secretes cellulase and hemicellulase during the fermentation process, and the enzyme activities continue to increase. At 168h, the cellulase activity is 30.14U / g, and the hemicellulase activity is 68.34U / g. The increase in the activity of the hydrolase in the system is beneficial to destroying the structure of lignocellulose and promoting the degradation of lignocellulose.

[0071] Example 2-16

[0072] The method of Example 1 is used to degrade lignocellulose, except that the ratio of the number of live bacteria of the five bacteria in the seed solution of the microbial composite agent is different or the fermentation conditions are different. The ratio of the number of live bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Pseudomonas fasciatus in the seed solution of the microbial composite agent, as well as the data of the fermentation temperature, fermentation time and seed solution inoculation amount are specifically shown in Table 2. Among them, the inoculation amount is the number of milliliters of seed solution inoculated based on the weight of the crushed and sieved rice straw as 100g.

[0073] Table 2

[0074]

[0075]

[0076] Embodiment 17

[0077] The lignocellulose was degraded according to the method of Example 1, except that Phanerochaete chrysosporium with a product number of B89537 purchased from Ningbo Mingzhou Biotechnology Co., Ltd. was used to replace Phanerochaete chrysosporium with a deposit number of CICC 40719, and Aspergillus niger with a deposit number of ACCC 32589 purchased from the China Agricultural Microbiological Culture Collection Administration Center was used to replace Aspergillus niger with a deposit number of CICC 40273; the total concentration of live bacteria in the seed solution was 10 7 CFU / mL, the ratio of the number of live bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psoralea corylifolia in the seed solution was 1:2:2:2:1.

[0078] Embodiment 18

[0079] The method of Example 1 was used to degrade lignocellulose, except that Trichoderma reesei with a deposit number of CICC 2626 was replaced by Trichoderma reesei purchased from the China Agricultural Microbial Culture Collection and with a deposit number of ACCC 30912, and Neurospora crassa with a deposit number of CICC 40203 was replaced by Neurospora crassa purchased from the China Agricultural Microbial Culture Collection and with a deposit number of ACCC 32257; the total concentration of live bacteria in the seed solution was 10 7CFU / mL, the ratio of the number of live bacteria of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psoralea corylifolia in the seed solution was 1:2:2:2:1.

[0080] Embodiment 19

[0081] The method of Example 1 was used to degrade lignocellulose, except that the lignocellulose raw material degraded by the microbial composite agent was different, and wheat straw (cellulose content was 35wt%, hemicellulose content was 30wt%, lignin content was 8wt%) was used instead of rice straw.

[0082] Embodiment 20

[0083] The method of Example 1 was used to degrade lignocellulose, except that the culture medium used for preparing the seed solution of the microbial composite agent was different, and the liquid PDB culture medium was replaced by SDA culture medium; the composition of the SDA culture medium was as follows: based on the total volume of the SDA culture medium, the SDA culture medium contained: 40 g / L glucose, 10 g / L peptone, 15 g / L agar, 200 mg / L chloramphenicol and deionized water, and was sterilized at 121° C. for 10 min before use.

[0084] Comparative Example 1

[0085] The method of Example 1 was used to degrade lignocellulose, except that the microbial composite agent did not contain Phanerochaete chrysosporium. The total concentration of live bacteria in the seed solution of the microbial composite agent was 10 7 CFU / mL, the ratio of the viable counts of Aspergillus niger, Trichoderma reesei, Neurospora crassa and Trichoderma spp. was 2:2:2:1.

[0086] Comparative Example 2

[0087] The method of Example 1 was used to degrade lignocellulose, except that the microbial composite agent did not contain Neurospora crassa. The total concentration of live bacteria in the seed solution of the microbial composite agent was 10 7 CFU / mL, the ratio of the viable counts of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei and Trichoderma spp. was 1:2:2:1.

[0088] Comparative Example 3

[0089] The method of Example 1 was used to degrade lignocellulose, except that the microbial composite agent did not contain dense brown gill fungus. The total concentration of live bacteria in the seed liquid of the microbial composite agent was 10 7 CFU / mL, the ratio of the viable counts of Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei and Neurospora crassa was 1:2:2:2.

[0090] Comparative Example 4

[0091] The method of Example 1 was followed to degrade lignocellulose, except that Pseudomonas aeruginosa purchased from the China Agricultural Microbial Culture Collection Center with a collection number of ACCC 60043 was used to replace the Pseudomonas aeruginosa with an equal number of viable cells.

[0092] Test Case

[0093] After the fermentation, the fermentation products of each embodiment and comparative example were dried and the changes in the contents of each component of the wood fiber raw material before and after fermentation were measured to analyze and investigate the degradation and utilization of the wood fiber raw material by the microbial composite agents of each embodiment and comparative example. The total solid degradation rate, cellulose degradation rate, hemicellulose degradation rate and lignin degradation rate of the wood fiber raw material, as well as the activities of cellulase and hemicellulase by the microbial composite agents of each embodiment and comparative example were measured respectively, and the results are shown in Table 3.

[0094] Table 3

[0095]

[0096]

[0097] From the results in Table 3, it can be seen that Examples 1-20 use the microbial composite agent of the present invention to degrade lignocellulose raw materials, and cellulose, hemicellulose and lignin have high degradation rates; the microbial composite agents of Comparative Examples 1-3 lack Phanerochaete chrysosporium, Neurospora crassa and Pseudomonas fasciatus, respectively, and Comparative Example 4 replaces Pseudomonas fasciatus with Pseudomonas aeruginosa, and the degradation rates of cellulose, hemicellulose and lignin are significantly reduced compared with Examples 1-20. This shows that the microbial composite agent of the present invention has a good ability to degrade lignocellulose.

[0098] In addition, Examples 2-10 all changed the ratio of the number of live bacteria of the five kinds of bacteria in the seed liquid of the microbial composite agent, Examples 11-16 changed the fermentation temperature, fermentation time and inoculation amount respectively, Examples 17 and 18 changed the source of bacteria, Example 19 used the microbial composite agent to degrade wheat straw, and Example 20 used different culture media. Compared with Example 1, the degradation rates of cellulose, hemicellulose and lignin were all reduced. This shows that the microbial composite agent of the present invention has a good degradation effect, especially for wood fiber raw materials whose contents of cellulose, hemicellulose and lignin meet a certain range, such as rice straw; when the five kinds of bacteria from a specific source are selected to match each other and the number of live bacteria of the five kinds of bacteria meets a certain ratio, the synergistic effect between the enzyme systems produced by the microbial composite agent can be further improved; in addition, the ability of the microbial composite agent to degrade lignocellulose can be further improved by selecting appropriate fermentation temperature, fermentation time, inoculation amount and culture medium.

[0099] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A microbial composite agent, characterized in that: The microbial composite bacterial agent comprises: Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala.

2. The microbial composite agent according to claim 1, characterized in that: In the microbial composite agent, the ratio of the number of live bacteria of the Phanerochaete chrysosporium, the Aspergillus niger, the Trichoderma reesei, the Neurospora crassa and the Pseudomonas syringae is 1:(0.2-20):(0.2-20):(0.2-20):(0.1-10), preferably 1:(1-4):(1-4):(1-4):(0.5-2), more preferably 1:(1.5-2.5):(1.5-2.5):(1.5-2.5):(0.8-1.2); Preferably, the strain number of Phanerochaete chrysosporium is CICC 40719, and / or, The strain number of the Aspergillus niger is CICC 40273 and / or ACCC 32589, and / or, The strain number of the Trichoderma reesei is CICC 2626 and / or ACCC 30912, and / or, The strain number of Neurospora crassa is CICC 40203 and / or ACCC 32257, and / or, The strain number of the dense brown gill fungus is ACCC 51714; Preferably, the strain number of Phanerochaete chrysosporium is CICC 40719, The strain number of Aspergillus niger is CICC 40273, The strain number of the Trichoderma reesei is CICC 2626, The strain number of Neurospora crassa is CICC 40203, The strain number of the dense brown gill fungus is ACCC 51714.

3. A method for degrading lignocellulose, characterized in that: The method comprises the following steps: pre-treating the wood fiber raw material, and then inoculating the seed liquid of the microbial composite agent into the pre-treated raw material for fermentation; wherein the microbial composite agent is the microbial composite agent according to claim 1 or 2; Preferably, in the wood fiber raw material, the cellulose content is 35-45wt%, the hemicellulose content is 20-25wt%, and the lignin content is 2-6wt%.

4. The method according to claim 3, characterized in that Based on the weight of the pretreated raw material as 100 g, the inoculation amount of the seed solution is 0.1-10 mL, preferably 4.5-5.5 mL; Preferably, the total concentration of live bacteria in the seed solution is 10 7 CFU / mL and above.

5. The method according to claim 3 or 4, characterized in that: The fermentation conditions include: a temperature of 20-35°C, preferably 25-27°C; a fermentation time of 90-240h, preferably 160-175h.

6. The method according to any one of claims 3 to 5, characterized in that: The method for preparing the seed solution comprises: activating Phanerochaete chrysosporium, Aspergillus niger, Trichoderma reesei, Neurospora crassa and Psora sphaerocephala independently in an activation medium, and then independently culture them in a culture medium.

7. The method according to claim 6, characterized in that The activation medium and the expansion medium each independently contain: potato slurry, glucose, potassium dihydrogen phosphate, magnesium sulfate and vitamin B1; Preferably, based on the total volume of the activation medium or the expansion medium, the activation medium and the expansion medium each independently contain: 180-220 g / L potato slurry, 17-23 g / L glucose, 2.5-3.5 g / L potassium dihydrogen phosphate, 0.6-0.9 g / L magnesium sulfate and 6-10 mg / L vitamin B1; Preferably, the pH values ​​of the activation medium and the expansion medium are independently 5.8-6.

5.

8. The method according to claim 6 or 7, characterized in that: The activation conditions include: temperature of 20-35°C, preferably 25-27°C; time of 1-5h, preferably 1.5-2.5h.

9. The method according to any one of claims 6 to 8, characterized in that: The conditions for the expansion culture include: a temperature of 20-35°C, preferably 25-27°C; and a time of 68-75h.

10. The method according to any one of claims 3 to 9, characterized in that: The pretreatment process includes: removing impurities, crushing and screening the wood fiber raw materials.

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