Special culture medium for culturing trichoderma reesei and producing cellulase for preparing lignocellulose biomass hydrolysis sugar and application of special culture medium

Abstract

The invention discloses a special culture medium for culturing trichoderma reesei and producing cellulase for preparing hydrolysis sugar of lignocellulose biomass (represented by corncobs or straws and the like) and application of the special culture medium. According to the culture medium, a mixture of industrial glucose with low price and a cereal processing byproduct bran with lower price is used for replacing microcrystalline cellulose and lactose or whey powder which are commonly used at present and are high in price, so that the material consumption cost of cellulase liquid submerged fermentation production is remarkably reduced; the production cost of the cellulase is reduced to the technical and economic indexes required by hydrolysis of (semi) cellulose components in lignocellulose biomass represented by corncobs or straws and the like to prepare hydrolyzed sugar, and the special requirements of fermentation production of cellulosic ethanol, other bulk bio-based products and the like are met.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and technology, and specifically relates to a special culture medium for culturing Trichoderma reesei and producing cellulase for preparing sugars from lignocellulosic biomass represented by corn cobs and straw, as well as a proprietary technical method for producing the cellulase using the culture medium. Background Art

[0002] The production of various energy and chemicals based on mineral resources such as oil, coal, and natural gas has greatly promoted economic and social development, particularly industrialization. However, these mineral resources are not only limited and non-renewable, but their development and utilization also emit large amounts of CO2, generating greenhouse gas emissions that have led to increasingly serious environmental problems. Therefore, the development and utilization of renewable and environmentally friendly alternative resources has become an urgent need for sustainable economic and social development.

[0003] Lignocellulosic biomass, synthesized through plant photosynthesis, is abundant and renewable. This is especially true for various agricultural and forestry wastes, such as corn cobs and straw. Biorefining technologies, which produce various bioenergy and bio-based chemicals, release CO₂ that can be absorbed by plant photosynthesis, without increasing the net CO₂ content in the atmosphere. This makes lignocellulosic biomass carbon neutral and environmentally friendly. Lignocellulosic biomass is widely recognized as a promising alternative to mineral resources such as oil, coal, and natural gas. Its development and utilization are crucial for sustainable economic and social development.

[0004] Cellulose, hemicellulose and lignin are the main components of lignocellulosic biomass, among which cellulose and hemicellulose are polysaccharides. After being hydrolyzed into monosaccharides, they can be used as basic raw materials for microbial fermentation to produce bioenergy and bio-based chemicals. However, the natural evolution process has caused cellulose, hemicellulose and lignin to be tightly entangled to form a complex. While providing support and protection for plant growth and development, it also increases the difficulty of degrading and utilizing lignocellulosic biomass, requiring appropriate pretreatment to achieve component separation. Although the cellulose and hemicellulose obtained after pretreatment can be hydrolyzed by acid or base catalysis, the chemical conversion technology route has prominent problems such as high acid and base consumption, harsh reaction conditions and many by-products. The enzymatic hydrolysis process based on cellulase has the advantages of being environmentally friendly, mild reaction conditions and few by-products, and is the main direction of the development of hydrolyzed sugar preparation technology for lignocellulosic biomass.

[0005] Cellulase is a complex enzyme. In addition to its main components, endo-, exo-, and β-glucosidases, it also contains xylanases, which hydrolyze hemicellulose, and various auxiliary proteins (enzymes) that promote hydrolysis. These enzymes and auxiliary proteins (enzymes) work synergistically to completely hydrolyze cellulose and hemicellulose: endo-, exo-, and β-glucosidases randomly hydrolyze cellulose chains to release reducing and non-reducing ends. Exo-, exo-, and β-glucosidases then hydrolyze cellobiose from these reducing and non-reducing ends. Finally, β-glucosidase hydrolyzes cellobiose to glucose (Trends in Biotechnology 2024, 42: 418-430). Trichoderma reesei is the microorganism with the highest cellulase production discovered to date, synthesizing and secreting cellulase under aerobic culture conditions (Microbial Cell Factory 2016, 15: 106). The development of biotechnology has provided efficient methods and tools for the transformation of Trichoderma reesei, and the enzyme production level has been continuously improved (Bioresource Technology 2023, 385: 129445). However, cellulase production needs to be achieved through liquid submerged fermentation. Culture medium is necessary for this process and is the main component of the cost of cellulase fermentation production.

[0006] Carbon source is the most important component in the culture medium of Trichoderma reesei and fermentation production of cellulase. Although the cellulose in the natural lignocellulose biomass is the natural carbon source of Trichoderma reesei culture and fermentation production of cellulase, solid cellulose requires bacterial strain to utilize the extremely low level of cellulase to hydrolyze into soluble sugars, which can then be utilized by cell growth, promote mycelial growth and induce synthesis and secretion of cellulase, and this liquid-solid heterogeneous enzyme catalysis reaction process rate is very slow (Journal of Biological Chemistry 1997, 272: 10169-10174). Li Hongbing et al. invented a method (CN105238704) for producing cellulase by solid-state fermentation using rice straw as main raw material. Although the solid-state fermentation process technology route is simple and the production equipment investment is low, the material heterogeneity causes process parameter control difficulty, and it is particularly difficult to amplify and realize large-scale production. In addition, the culture medium and equipment in the solid-state fermentation process are not sterilized thoroughly, and the contamination of foreign bacteria in the production process cannot be effectively prevented and controlled. The cellulase produced can generally only be used in industries with low requirements for hygiene indicators such as feed, and is not suitable for hydrolyzing cellulose and hemicellulose components in lignocellulosic biomass to prepare hydrolyzed sugars, as the basic raw material for the production of bioenergy and bio-based chemicals based on pure microbial culture liquid deep fermentation.

[0007] Liquid submerged fermentation is a good choice for producing cellulase that meets the needs of preparing sugars from lignocellulosic biomass hydrolysis, and a large amount of research has been carried out at home and abroad (Renewable and Sustainable Energy Reviews 2021, 151: 111622). In order to solve the prominent problem of the slow rate of Trichoderma reesei in utilizing natural lignocellulosic biomass such as straw, relatively easy-to-degrade microcrystalline cellulose has attracted attention, and a large amount of research has been carried out at home and abroad (Cellulose 2011, 18: 1527-1541). Feng Jiaxun et al. applied for a patented technology for liquid submerged fermentation of cellulase using microcrystalline cellulose as an inducer (CN201210276891). However, the high price of microcrystalline cellulose leads to the prominent problem of particularly high cost of producing cellulase by liquid submerged fermentation, which cannot meet the demand for low-cost preparation of sugars from lignocellulosic biomass represented by straw, etc. Moreover, microcrystalline cellulose is solid, and the problem of slow rate of absorption and utilization after degradation by the background low-dose cellulase of Trichoderma reesei strain still exists.

[0008] To address the high cost of microcrystalline cellulose and the slow degradation rate of the strain's native enzymes when using it as a carbon source, researchers developed soluble carbon sources and found that the most effective inducer was sophorose (J Bacteriol 1962, 83:400-408). However, sophorose resources are extremely scarce and expensive, making it unsuitable for industrial production. Further research has found that lactose can also be used as a carbon source and inducer for the production of cellulase by Trichoderma reesei (PLOS One 2013, 8:e62631; Appl Microbiol Biotechnol 1995, 44:106-111). Although the enzyme production effect is not as good as sophorose, lactose is relatively cheap. In addition, whey powder, a by-product of the dairy processing industry that contains lactose, is relatively inexpensive. Therefore, whey powder is currently a commonly used carbon source for the industrial production of cellulase in submerged liquid fermentation.

[0009] Compared with cellulase used in other industries, the production cost of cellulase used to prepare sugars from lignocellulosic biomass represented by corn cobs and straw must be further reduced before it has application value. However, the price of whey powder still cannot meet the needs of this cellulase fermentation production. Finding cheaper carbon sources to further reduce the cost of cellulase fermentation production is necessary to reduce the cost of preparing (hemi)cellulose hydrolyzed sugars. Summary of the Invention

[0010] The present invention aims to provide a low-cost culture medium for liquid fermentation of Trichoderma reesei to produce cellulase for preparing sugars from lignocellulosic biomass represented by straw and corn cobs, as well as a proprietary method for producing the cellulase using the culture medium.

[0011] In the first aspect of the present invention, 1. A method for preparing a special cellulase culture medium for fermenting Trichoderma reesei and producing (hemi)cellulose hydrolysis in lignocellulosic biomass is provided, comprising: S1. preparing a culture medium suitable for the growth and fermentation production of cellulase by Trichoderma reesei, the culture medium comprising a carbon source, a nitrogen source, and a salt; the carbon source is glucose and bran, and the weight ratio of glucose to bran is (1.5-2.5):1.

[0012] In one or more embodiments, a carbon source, a nitrogen source, an inorganic salt, etc.

[0013] In one or more embodiments, carbon sources, nitrogen sources, inorganic salts, corn steep liquor, etc.

[0014] In one or more embodiments, the weight ratio of glucose to bran is (1.6-2.4):1; preferably (1.8-2.2):1; more preferably 2:1.

[0015] In one or more embodiments, the culture medium is a culture medium suitable for the growth of Trichoderma reesei mycelium and the production of cellulase in a liquid submerged fermentation process; preferably, the culture medium comprises: glucose, bran, corn steep liquor, calcium chloride dihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, T1, and T2; preferably, T1 comprises: ferric citrate, zinc acetate dihydrate, and ethylenediaminetetraacetic acid; T2 comprises: boric acid, zinc sulfate heptahydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, ferrous sulfate heptahydrate, and manganese sulfate monohydrate.

[0016] In one or more embodiments, the fermentation medium further comprises a defoaming agent; preferably, it comprises 0.3 g / L of defoaming agent, or its dosage can be used according to conventional operations in the fermentation industry.

[0017] In one or more embodiments, the culture medium can meet the requirements of Trichoderma reesei for mycelial growth and cellulase production.

[0018] In another aspect of the present invention, a culture medium for culturing and fermenting Trichoderma reesei to produce a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass is provided; the culture medium comprises a carbon source, a nitrogen source, and a salt (which may also include inorganic salts, corn steep liquor, etc.); the carbon source is glucose and bran, and the weight ratio of glucose to bran is (1.5-2.5):1.

[0019] In one or more embodiments, the culture medium is a culture medium suitable for the growth of Trichoderma reesei mycelium and the production of cellulase in a liquid submerged fermentation process; preferably, the culture medium comprises: glucose, bran, corn steep liquor, calcium chloride dihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, T1, and T2; preferably, T1 comprises: ferric citrate, zinc acetate dihydrate, and ethylenediaminetetraacetic acid; T2 comprises: boric acid, zinc sulfate heptahydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, ferrous sulfate heptahydrate, and manganese sulfate monohydrate.

[0020] In another aspect of the present invention, there is provided a method for producing a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass, the method comprising:

[0021] (a) providing the aforementioned culture medium and culturing Trichoderma reesei;

[0022] (b) Cultivating Trichoderma reesei based on the culture medium of (a), and maintaining the reducing sugar concentration in the fermentation broth at 1-2 g / L by feeding, thereby inducing the biosynthesis of cellulase specifically for (hemi)cellulose hydrolysis in lignocellulosic biomass.

[0023] In one or more embodiments, in step (b), the feeding is performed with a syrup (mixed syrup) containing the inducer.

[0024] In one or more embodiments, the preparation method of the inducer-containing syrup includes: reverse catalyzing high-concentration glucose (syrup) with a β-glucosidase dosage of 5 to 12 U / g (glucose) at a temperature of 60 to 70°C and a pH of 4.5 to 5.0, thereby obtaining an inducer-containing syrup; the high-concentration glucose (syrup) contains 580 to 700 g / L of glucose; in the inducer-containing syrup, the inducers mainly include: sophorose, cellobiose and gentiobiose.

[0025] In one or more embodiments, during the fermentation culture process of step (b), a spore suspension or mycelium of Trichoderma reesei is inoculated into the culture medium, and when the reducing sugar concentration reaches 1 to 2 g / L in batch mode, the inducer-containing syrup (mixed syrup) is fed.

[0026] In one or more embodiments, the flow rate of the inducer-containing syrup is controlled to maintain the dissolved oxygen and reducing sugar concentrations in the fermentation broth at appropriate levels for enzyme production.

[0027] In one or more embodiments, in step (b): the mycelial growth temperature at the initial stage of fermentation is 30±1°C, preferably 30±0.5°C (more preferably 30±0.2°C).

[0028] In one or more embodiments, in step (b): after the feed stream is added with syrup and enters the enzyme production phase, the temperature is 28±1°C, preferably 28±0.5°C (more preferably 28±0.2°C).

[0029] In one or more embodiments, in step (b): the pH of the fermentation broth during mycelial growth and enzyme production phases is not lower than 4.5; for example, pH 4.5-5.0, preferably pH 4.5-4.8, more preferably pH 4.5-4.6.

[0030] In one or more embodiments, in step (b), the dissolved oxygen (DO) is not less than 15%; for example, the dissolved oxygen is 15-40%, preferably 20-35%, more preferably 20-30%.

[0031] In one or more embodiments, the culture is a batch culture.

[0032] In one or more embodiments, the fermentation time of step (b) is 120 to 160 hours, preferably 120 to 140 hours, and more preferably 120 to 130 hours.

[0033] In another aspect of the present invention, there is provided use of the culture medium in promoting the growth of Trichoderma reesei hyphae and biosynthesis of cellulase.

[0034] In another aspect of the present invention, there is provided a method for degrading cellulose or a material containing cellulose, the method comprising:

[0035] (i) producing a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass by culturing and fermenting Trichoderma reesei using the method described above;

[0036] (ii) using the cellulase produced in (i) to degrade cellulose or materials containing cellulose.

[0037] In one or more embodiments, the cellulose-containing material comprises lignocellulosic biomass, such as (but not limited to) corn cobs or straw.

[0038] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Schematic diagram of the Trichoderma reesei submerged fermentation process for cellulase production. 1. Air compressor; 2. Air control valve; 3. Air flow meter; 4. Gas distributor; 5. Fermenter; 6. Temperature sensor; 7. Temperature indicator and control unit; 8. Circulating cooling water control valve; 9. Circulating water pump; 10. Circulating water tank; 11. Cooling chamber; 12. Dissolved oxygen (DO) electrode; 13. DO indicator and control unit; 14. Stirring system; 15. pH electrode; 16. pH indicator and control unit; 17. Ammonia storage tank; 18. Peristaltic pump; 19. Mixed syrup storage tank; 20. Peristaltic pump; 21. Defoamer storage tank; 22. Peristaltic pump.

[0040] Figure 2 , using lactose (■) and glucose + bran (●) as carbon sources, Trichoderma reesei was used for submerged liquid fermentation to produce cellulase.

[0041] Figure 3 , device for hydrolyzing corn cob cellulose residue with cellulase (a) and enzymatic hydrolysis process to produce glucose (b). DETAILED DESCRIPTION

[0042] To address the high cost of using cellulosics, including microcrystalline cellulose, lactose, or whey powder, as carbon sources in existing submerged fermentation of Trichoderma reesei for cellulase production, the present invention provides a technical solution that significantly reduces costs. This solution utilizes relatively inexpensive industrial glucose and even less expensive bran, a byproduct of grain processing, as alternative carbon sources in a new culture medium for seed culture and fermentation production of cellulase at various stages.

[0043] In the present invention, the terms "containing," "having," or "include" encompass "comprising," "consisting primarily of," "consisting essentially of," and "consisting of." "Consisting primarily of," "consisting essentially of," and "consisting of" are subordinate concepts to "containing," "having," or "include." Unless otherwise specified, the components / ingredients listed after the term "containing," "having," or "include" are all active components / ingredients that exert the desired function / action.

[0044] In the present invention, the "inducer-containing syrup (mixed syrup)" mainly contains sophorose, cellobiose, gentiobiose and unconverted glucose, and they are prepared by a preferred method. Their content is unique and is beneficial to the growth of Trichoderma reesei and the production of cellulase as a carbon source and inducer.

[0045] In the present invention, the terms "enzymatic hydrolysis" and "hydrolysis" can be used interchangeably.

[0046] Unless otherwise specified, the "fermentation" and "cultivation" described in the claims and description of the present invention refer to the process for the purpose of growing mycelium of T. reesei strain and producing cellulase.

[0047] In the present invention, the "slant culture" and "seed culture" refer to cultures aimed at obtaining bacterial strains suitable for inoculation.

[0048] In the present invention, the "fermentation culture" is a fermentation for the purpose of growing mycelium of T. reesei strain and producing cellulase.

[0049] In the present invention, the scientific name of Trichoderma reesei "Trichoderma reesei" can be abbreviated as "T. reesei".

[0050] In the preferred embodiment of the present invention, a specific strain of T.reesei is used, which is the T.reesei SCB18 engineered strain (Biotechnol Biofuels 2017, 10: 272). However, as a general consensus in the art, in view of the commonalities of different strains of T.reesei, other types of T.reesei can also be applied to the technical solutions of the present invention. According to the technical solutions provided by the present invention, it is easy for those skilled in the art to operate and verify the growth and cellulase synthesis effects of the corresponding T.reesei. In addition, derivative strains of T.reesei strains (such as the strains in the embodiments) or recombinant strains after genetic engineering are also included in the present invention.

[0051] The culture medium of the present invention is a culture medium suitable for the growth of Trichoderma reesei and the production of cellulase, and the culture medium includes a carbon source, a nitrogen source, an inorganic salt, etc. Compared with the previous culture method, the technical solution of the present invention has been optimized and improved. In the culture system, expensive microcrystalline cellulose is no longer used, and lactose or whey powder is not used. Instead, low-cost glucose and bran are used as carbon sources. Preferably, according to the weight ratio, glucose: bran is (1.5-2.5):1. The culture medium of the present invention is particularly suitable for liquid submerged culture and fermentation of Trichoderma reesei (engineered) strains to produce special cellulase suitable for hydrolyzing cellulose and hemicellulose components in lignocellulosic biomass such as corn cobs and straw to produce sugar.

[0052] In a particularly preferred embodiment of the present invention, glucose and wheat bran replace the currently more expensive microcrystalline cellulose, lactose, or whey powder used as the carbon source in the culture medium. Glucose accounts for two-thirds of the total carbon source, and wheat bran accounts for one-third. This can meet the needs of Trichoderma reesei mycelial growth and cellulase production. Wheat bran is also inexpensive, costing only a few hundred yuan per ton.

[0053] As a preferred embodiment of the present invention, the culture medium includes: glucose, bran, corn steep liquor, calcium chloride dihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, T1, and T2. Preferably, T1 includes: ferric citrate, zinc acetate dihydrate, and ethylenediaminetetraacetic acid; and T2 includes: boric acid, zinc sulfate heptahydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, ferrous sulfate heptahydrate, and manganese sulfate monohydrate. It should be understood that the amounts of the components can be adjusted, regardless of the minor components, and can be replaced with substances of the same function, as long as they can provide sufficient nutrients for T. reesei growth and cellulase production.

[0054] As a more preferred embodiment of the present invention, the culture medium comprises: 20 g / L glucose, 10 g / L bran, 23 g / L corn steep liquor, 0.6 g / L calcium chloride dihydrate, 5 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.9 g / L magnesium sulfate heptahydrate, 0.2 mL / L L1, 0.2 mL / L T2; wherein, T1 includes: 0.6% ferric citrate, 0.08% zinc acetate dihydrate and 0.08% ethylenediaminetetraacetic acid; T2 includes: 0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% ferrous sulfate heptahydrate and 1.6% manganese sulfate monohydrate; wherein, the amount of each component of the culture medium can fluctuate by 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 1-3% or 1-2%.

[0055] As a more preferred embodiment of the present invention, the culture medium further comprises: a defoaming agent; preferably, it comprises 0.3 g / L defoaming agent; the amount thereof can fluctuate by 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 1-3% or 1-2%.

[0056] In addition, those skilled in the art can adjust the composition of the fermentation medium based on common knowledge of T. reesei strain culture, such as appropriately increasing or decreasing certain inorganic salts to regulate mycelial growth, promote intracellular synthesis and extracellular secretion of cellulase, etc.

[0057] The present invention also includes a feeding step using a mixed syrup containing an inducer during the cultivation of Trichoderma reesei and the production of cellulase. The inducer-containing mixed syrup was prepared by the inventors after in-depth research and optimization; the syrup includes (primarily) sophorose, cellobiose, and gentiobiose. The inventors' testing has shown that the inducer-containing syrup contains a particularly high content of sophorose, which facilitates efficient induction.

[0058] As a preferred embodiment of the present invention, in the inducer-containing syrup (by weight ratio), the ratio of sophorose: cellobiose: gentiobiose is (100-130): (4-6): (35-55); preferably, the ratio of sophorose: cellobiose: gentiobiose is 114.4: 5.3: 43.7, and the ratio of each component can fluctuate by 30%, 20%, 10% or 5%.

[0059] The syrup containing the inducer is used to culture the engineered strain of Trichoderma reesei through controlled fed-batch culture. Unconverted glucose in the syrup preparation reaction process serves as a carbon source for mycelial growth. β-disaccharides such as sophorose, cellobiose and gentiobiose synthesized through the reverse catalytic reaction serve as inducers, thereby efficiently inducing the production of special cellulase for preparing sugars from lignocellulosic biomass by hydrolyzing sugars.

[0060] As a preferred embodiment of the present invention, the reverse catalytic reaction system is mixed evenly and reacted for 72±6h; preferably, the reaction is carried out for 72±4h (more preferably 72±2h). Preferably, at a temperature of 65-70°C and a pH of 4.5-5.0, a high concentration of glucose (syrup) is reversely catalyzed with a β-glucosidase dosage of 5-10U / g (glucose) to obtain a mixed syrup containing an inducer. Preferably, the high concentration of glucose (syrup) contains 580-700g / L of glucose, more preferably 600-650g / L. In the present invention, the reaction temperature of the β-glucosidase reverse catalytic synthesis reaction is preferably 65-70°C, which is completely different from the reaction temperature of 50°C for the hydrolysis of β-disaccharides such as cellobiose catalyzed by β-glucosidase.

[0061] As a particularly preferred embodiment of the present invention, spores or mycelium of the Trichoderma reesei strain are inoculated into the culture medium, and a syrup containing an inducer is added as an auxiliary flow to achieve enzyme production, specifically comprising: S1, culturing the Trichoderma reesei strain and producing enzymes as described above, inoculating a spore suspension or mycelium of the Trichoderma reesei strain into the culture medium according to a conventional proportion during the culture process, and batch culturing until the reducing sugar drops to a low level, and then starting to add the syrup containing the inducer; S2, controlling the flow rate of the syrup containing the inducer to maintain the dissolved oxygen and reducing sugar concentrations in the fermentation broth at appropriate levels for enzyme production.

[0062] As a preferred embodiment of the present invention, during the culture process, 5-10% by volume of T. reesei spore or mycelial suspension is inoculated and cultured at 30° C. After entering the enzyme production stage, the fermentation temperature is adjusted to 28° C.

[0063] As a preferred embodiment of the present invention, during the culture process, ammonia water is used to regulate the pH of the fermentation liquid to 4.5-5.0, and the dissolved oxygen in the fermentation liquid is controlled to be not less than 20% of the saturation value through ventilation and stirring.

[0064] As a preferred embodiment of the present invention, batch culture is carried out until the reducing sugar level drops to 1.0 g / L, and then feeding is started, and the syrup containing the inducer is fed.

[0065] As a preferred embodiment of the present invention, controlling the flow rate of the syrup to maintain the reducing sugar at 1.0-2.0 g / L can better achieve cellulase production.

[0066] The cellulase of the present invention is used to hydrolyze cellulose and hemicellulose from lignocellulosic biomass, such as crop straw and forestry waste, to produce hydrolyzed sugars. In practical use, the cellulase exhibits excellent cellulose hydrolysis performance, and the resulting hydrolyzed sugars meet the technical and economic requirements for fermentation production of cellulosic ethanol and other bulk bio-based products.

[0067] In practical applications, parameters such as the cellulase dosage, enzymatic hydrolysis time, and sugar yield during the cellulose enzymatic hydrolysis process can be subjected to technical and economic analysis based on material conditions, cellulase costs, and production equipment, to achieve low-cost preparation of cellulose hydrolyzed sugars. The hydrolyzed sugars can be further used for microbial fermentation to produce bioenergy and bio-based chemicals.

[0068] The fermentation system of the present invention can be scaled up to accommodate industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on their general knowledge to facilitate the growth of the strain or the production of cellulase.

[0069] Based on the innovative disclosures of the present invention, the present invention also provides a strategy for degrading cellulose or cellulose-containing biomass, including the culture medium for fermenting Trichoderma reesei and producing cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass, and the method for preparing the inducer-containing syrup (mixed syrup).

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] 1. The culture medium formed by the present invention, which uses (industrial) glucose and bran as carbon sources, can significantly reduce the cost of cellulase fermentation production.

[0072] 2. The cellulose (crude) enzyme solution produced by the present invention is suitable for hydrolyzing cellulose and hemicellulose components in lignocellulosic biomass represented by corn cobs and straw to prepare hydrolyzed sugars.

[0073] The present invention is described in detail below in conjunction with the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, appropriate adjustments and improvements can be made without departing from the present invention, and these all fall within the scope of protection of the present invention.

[0074] Example 1: Batch fed-batch culture of T. reesei SCB18 for cellulase production

[0075] Equipment and process flow for producing cellulase by fed-batch culture of T. reesei SCB18 Figure 1 During operation, air from air compressor 1 is regulated by regulating valve 2 and measured by flowmeter 3, then enters fermenter 5 through air distributor 4 at a flow rate of 1 vvm (air volume per minute per fermenter volume). A temperature sensor 6 and indicator control unit 7, together with circulating water regulating valve 8, circulating water pump 9, circulating water tank 10, and fermenter bottom cooling system 11, form a temperature control system. A dissolved oxygen electrode (DO) 12 and indicator control unit 13 control the speed of agitator 14, maintaining the DO value in the fermentation broth at no less than 20% to meet the requirements of mycelial growth and cellulase synthesis. A pH electrode 15, indicator control unit 16, ammonia storage tank 17, and control peristaltic pump 18 form a pH control system, maintaining the pH at no less than 4.5 during the cultivation and fermentation process. A mixed syrup storage tank 19 is linked to a peristaltic pump 20 to control the addition of the mixed syrup by detecting the reducing sugar concentration in the fermentation broth. A defoamer storage tank 21 is linked to a peristaltic pump 22 to promptly eliminate foam generated during the cultivation and fermentation process.

[0076] The specific steps include:

[0077] 1. Slant culture and preparation of spore suspension

[0078] A small amount of T. reesei SCB18 spores were taken from the strain stored in a -80°C ultra-low temperature freezer and inoculated onto a solid medium containing 46 g / L potato dextrose agar (supplier: Qingdao Haibo Biotechnology Co., Ltd.). After culturing at 30°C for 7 days, the spores were washed with sterile water to prepare a suspension, which was then stored at 4°C for use.

[0079] 2. Shake flask seed culture

[0080] The spores washed in step 1 were inoculated into 250 mL Erlenmeyer flasks (8 shake flasks were used to meet the inoculation requirements of subsequent fermentation tank culture) with a liquid volume of 50 mL, and cultured at 30° C. and 220 rpm for 24 h to allow spore germination and prepare seeds. The culture medium consisted of: 20.0 g / L glucose monohydrate, 10.0 g / L corn steep liquor, 0.7 g / L calcium chloride dihydrate, 10.0 g / L potassium dihydrogen phosphate, 5.0 g / L ammonium sulfate, 1.0 g / L magnesium sulfate heptahydrate, 0.2 g / L T2 (0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% ferrous sulfate heptahydrate, and 1.6% manganese sulfate monohydrate), and 0.08 g / L defoamer.

[0081] 3. Fermentation to produce cellulase

[0082] (1) Fermentation

[0083] Preparation of inducer-containing syrup (mixed syrup): A 600 g / L glucose solution was prepared, and 10 IU of commercial β-glucosidase (purchased from Ningxia Xiasheng Industrial Group Co., Ltd.) was added per gram of glucose. The solution was placed in a reactor at pH 4.8 and 65°C. The reaction system was stirred for 72 hours to achieve uniform mixing. This yielded an inducer-containing syrup (mixed syrup), which was then analyzed for its main components. The mixed syrup was determined to contain 488.5 g / L of unconverted glucose, 114.4 g / L of sophorose, 5.3 g / L of cellobiose, and 43.7 g / L of gentiobiose.

[0084] The seed liquid is inoculated at a 10% inoculum rate into a fermentation tank containing 4L of fermentation medium. The fermentation temperature is controlled at 30°C, and the pH is automatically controlled to 4.5 by feeding ammonia water. By coupling the fermentation tank stirring speed and the dissolved oxygen (DO) value, the DO is kept at no less than 20% (DO can be reduced to about 5% under industrial production conditions). The reducing sugar concentration in the fermentation broth in the fermentation tank is sampled every 4 hours. When the reducing sugar concentration in the fermentation broth is lower than 1.0g / L, the fermentation temperature is adjusted to 28°C, and the syrup containing the inducer (mixed syrup) is started. The syrup flow acceleration is adjusted according to the reducing sugar concentration to keep the reducing sugar concentration in the fermentation broth at 1.0-2.0g / L.

[0085] Wherein, the fermentation medium formula is: 20.0g / L glucose, 10.0g / L bran, 23.0g / L corn steep liquor, 0.6g / L calcium chloride dihydrate, 5.0g / L potassium dihydrogen phosphate, 4.8g / L ammonium sulfate, 0.9g / L magnesium sulfate heptahydrate, 0.2mL / L T1 (0.6% ferric citrate, 0.08% zinc acetate dihydrate and 0.08% ethylenediaminetetraacetic acid), 0.2mL / L T2 (0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% cupric sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% ferrous sulfate heptahydrate and 1.6% manganese sulfate monohydrate), and 0.3g / L defoamer.

[0086] At the same time, fermentation with lactose as the carbon source was used as a comparison, that is, in the above fermentation medium, other components remained unchanged, and "20.0 g / L glucose, 10.0 g / L bran" was replaced by "30.0 g / L lactose".

[0087] (2) Result detection

[0088] The cellulase activity (filter paper enzyme activity FPU) was determined using the standard method promulgated by the International Union of Pure and Applied Chemistry (IUPAC) (Pure and Applied Chemistry 1987, 59(2): 257-268).

[0089] Cellulase production results Figure 2 As shown in the figure, the cellulase activity in the fermentation broth reached 73.0 FPU / mL after 110 h of culture. If the fermentation time is continued to be extended to 120 h, the enzyme activity in the fermentation broth slowly increased to 76.7 FPU / mL. At this time, the fermentation and enzyme production can be stopped to avoid the degradation of the synthesized cellulase by the by-product protease secreted by the bacteria. The enzyme production level in the culture medium after 103 h of fermentation with lactose as the carbon source is 63.2 FPU / mL. If the fermentation time is continued to be extended to 120 h, the enzyme activity in the fermentation broth increases to 69.4 FPU / mL.

[0090] According to the above, the use of cheap glucose and bran as carbon sources in the culture medium does not affect the constant production of cellulase. After about 80 hours of fermentation, the cellulase production using glucose and bran as carbon sources increased by about 10.5% compared with lactose as the carbon source, which is a significant increase.

[0091] Example 2: Hydrolysis of corncob cellulose residue with self-produced cellulase

[0092] Taking the hydrolysis of cellulose residue remaining after the hemicellulose hydrolysis component xylose is extracted from corn cobs of Jinan Shengquan Group Co., Ltd. to produce furfural and other products as an example, the hydrolysis performance of the cellulase prepared in Example 1 is evaluated, which specifically includes the following steps:

[0093] 1. Provide experimental materials. The measured moisture content of cellulose residue is 67.5% (dry matter content 32.5%) and the cellulose content is 65.0%.

[0094] 2. In Figure 3 (a) The enzymatic hydrolysis was carried out in a reactor with a total volume of 5 L and a working volume of 3 L. The enzymatic hydrolysis temperature was controlled at 50° by electric heating, the pH was adjusted to 5.0 using 20% NaOH solution, and appropriate stirring was performed to ensure uniform suspension of the solid material.

[0095] 3. The dosage of cellulase is 10FPU per gram of dry material, which is added to the enzymatic hydrolysis reactor along with the material; the initial material dry matter concentration is 10-12%, and the material is fed as the viscosity decreases until the dry matter concentration reaches 25%. The glucose release during the enzymatic hydrolysis process is as follows: Figure 3 (b) shown.

[0096] 4. After 72 hours of enzymatic hydrolysis, the glucose concentration was 146.0 g / L; after 96 hours of enzymatic hydrolysis, the glucose concentration was 156.0 g / L; after 120 hours of enzymatic hydrolysis, the glucose concentration was 164.0 g / L; the calculated enzymatic hydrolysis yield of cellulose was 80.4%.

[0097] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person of ordinary skill in the art may make various adjustments and improvements without departing from the scope of the present invention, and these modifications fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims. All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually.

Claims

1. A method for preparing a culture medium for fermenting Trichoderma reesei and producing a cellulase-specific culture medium for hydrolyzing (hemi)cellulose in lignocellulosic biomass, characterized in that: include: S1. Prepare a culture medium suitable for the growth of Trichoderma reesei and fermentation to produce cellulase, wherein the culture medium includes a carbon source, a nitrogen source, and a salt; the carbon source is glucose and bran, and the weight ratio of glucose to bran is (1.5-2.5):

1.

2. The method according to claim 1, wherein The culture medium is a culture medium suitable for the growth of Trichoderma reesei mycelium and the production of cellulase in a liquid submerged fermentation process; preferably, the culture medium comprises: glucose, bran, corn steep liquor, calcium chloride dihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, T1, and T2; preferably, T1 comprises: ferric citrate, zinc acetate dihydrate, and ethylenediaminetetraacetic acid; and T2 comprises: boric acid, zinc sulfate heptahydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, ferrous sulfate heptahydrate, and manganese sulfate monohydrate.

3. A culture medium for culturing and fermenting Trichoderma reesei to produce a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass; the culture medium comprises a carbon source, a nitrogen source, and a salt; the carbon source is glucose and bran, and the weight ratio of glucose to bran is (1.5-2.5):

1.

4. The culture medium for culturing and fermenting Trichoderma reesei to produce a cellulase specifically for (hemi)cellulose hydrolysis in lignocellulosic biomass according to claim 3, wherein: The culture medium is a culture medium suitable for the growth of Trichoderma reesei mycelium and the production of cellulase in a liquid submerged fermentation process; preferably, the culture medium comprises: glucose, bran, corn steep liquor, calcium chloride dihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, T1, and T2; preferably, T1 comprises: ferric citrate, zinc acetate dihydrate, and ethylenediaminetetraacetic acid; and T2 comprises: boric acid, zinc sulfate heptahydrate, copper sulfate pentahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, ferrous sulfate heptahydrate, and manganese sulfate monohydrate.

5. A method for producing a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass, characterized in that: The method comprises: (a) providing the culture medium according to claim 3 or 4, and culturing Trichoderma reesei; (b) Cultivating Trichoderma reesei based on the culture medium of (a), and maintaining the reducing sugar concentration in the fermentation broth at 1-2 g / L by feeding, thereby inducing the biosynthesis of cellulase specifically for (hemi)cellulose hydrolysis in lignocellulosic biomass.

6. The method according to claim 5, wherein In step (b), the feed is carried out using a syrup containing an inducer; The preparation method of the inducer-containing syrup includes: at a temperature of 60-70°C and a pH of 4.5-5.0, using a β-glucosidase dosage of 5-12 U / g (glucose) to reversely catalyze high-concentration glucose to synthesize the inducer-containing syrup; the high-concentration glucose is 580-700 g / L; in the inducer-containing syrup, the inducers mainly include: sophorose, cellobiose and gentiobiose.

7. The method according to claim 6, wherein During the fermentation culture process of step (b), the spore suspension or mycelium of Trichoderma reesei is inoculated into the culture medium, and the culture is carried out in batch mode until the reducing sugar concentration reaches 1-2 g / L, and then the inducer-containing syrup is fed as feed.

8. The method according to claim 5, wherein In step (b): The mycelial growth temperature at the initial stage of fermentation is 30±1°C, preferably 30±0.5°C. After the feed stream is added with syrup and enters the enzyme production stage, the temperature is 28±1°C, preferably 28±0.5°C. The pH of the fermentation broth during mycelial growth and enzyme production stages is not lower than 4.5; for example, pH 4.5 to 5.0, preferably pH 4.5 to 4.8, more preferably pH 4.5 to 4.

6. The dissolved oxygen is not less than 15%; for example, the dissolved oxygen is 15 to 40%, preferably 20 to 35%, more preferably 20 to 30%.

9. Use of the culture medium according to claim 3 or 4 in promoting the growth of Trichoderma reesei hyphae and the synthesis of cellulase.

10. A method for degrading cellulose or a material containing cellulose, characterized in that: The method comprises: (i) producing a cellulase specifically for hydrolyzing (hemi)cellulose in lignocellulosic biomass by culturing and fermenting Trichoderma reesei using the method according to any one of claims 5 to 7; (ii) using the cellulase produced in (i) to degrade cellulose or materials containing cellulose.

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