A strain with high efficiency of degrading lignocellulose and application thereof

Abstract

The application discloses a strain for efficiently degrading lignocellulose and application thereof, and belongs to the technical field of microorganisms. The strain is Bacillus thuringiensis BT-L09 with the preservation number of CGMCC No. 39428. Enzyme activity determination results show that the strain has a complete lignocellulose degrading enzyme system, and the endoglucanase, exoglucanase, beta-glucosidase, cellulose comprehensive enzyme, xylanase, xylanase, laccase, lignin peroxidase and manganese peroxidase activities of the strain are excellent. In a corn stalk degradation test at 15 DEG C, the corn stalk degradation rate of the strain reaches 38.04% in 20 days. The strain not only helps to efficiently utilize biomass energy in lignocellulose, meets the demand of human beings for clean energy, but also can convert agricultural wastes into high value-added products, and improves the comprehensive utilization level of agricultural by-products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain that efficiently degrades lignocellulose and its applications. Background Technology

[0002] Lignocellulose is a major component of plant biomass, primarily composed of cellulose (30%–50%), hemicellulose (20%–35%), and lignin (15%–30%). Its highly complex structure is a core limiting factor for efficient conversion. Cellulose, as the main mechanical support framework, is a linear long-chain polymer formed by glucose monomers linked by β-1,4 glycosidic bonds. It exhibits high crystallinity and rigidity, and its molecules form a stable three-dimensional network structure through extensive hydrogen bonding interactions. Hemicellulose is a branched polysaccharide composed of various monosaccharide units such as xylose, arabinose, and glucose linked by α-1,4 glycosidic bonds. Its main function is to fill the gaps between cellulose microfibrils and provide adhesion. Lignin is a three-dimensional aromatic polymer compound formed by phenylpropane monomers covalently cross-linked to form a highly developed network structure. This network configuration encapsulates and anchors cellulose and hemicellulose, significantly enhancing the overall stability and biodegradability of the composite material.

[0003] Corn stalks, one of the world's most abundant agricultural byproducts, are primarily composed of lignocellulose and are a core component of the renewable biomass resource pool. This biomass not only provides a viable alternative to traditional petroleum-based fuels but also holds a strategic position in the field of biorefining. However, in Northeast my country, the long winters and low average annual temperatures present unique challenges to the resource utilization of corn stalks. Apart from a small amount used in biorefining and pulp and paper industries, the vast majority of corn stalks are still burned as low-value-added fuel for heating. This extensive method fails to fully unleash its biological potential, resulting in both low energy conversion efficiency and environmental burden.

[0004] Enzymatic hydrolysis technology has opened up new avenues for the efficient conversion of lignocellulose. Compared to traditional chemical treatments, this technology offers milder conditions and is particularly suitable for the naturally low-temperature environment of Northeast China. Achieving efficient degradation at low temperatures not only significantly reduces the energy costs and external heating infrastructure required to maintain the reaction temperature, but also utilizes the cold climate as a natural cold source, simplifying temperature control management during the enzymatic hydrolysis process and reducing the use of chemical additives. Targeted modification and regulation of cold-resistant microbial resources to optimize their specific functional expression has become a key scientific issue in promoting the utilization of plant biomass resources in cold regions and transforming the advantage of "cold" resources into an industrial advantage.

[0005] In recent years, the discovery of low-temperature cellulose-degrading bacteria has provided new ideas for the resource utilization of agricultural and forestry waste in cold regions. Li et al. isolated Bacillus sp. 3-2 from alpine meadow soil in the eastern Qilian Mountains. This bacterium can produce enzymes within a temperature range of 5-45℃ and pH 4.0-9.0, with an optimal enzyme production temperature of 20℃. Furthermore, it retains 60% of its relative enzyme activity at 5℃, demonstrating good cold adaptability. Kang Jingwei et al. isolated Acinetobacter sp. F16 from animal feces. At 15℃, it can simultaneously secrete CMCase (5.46 U / mL), xylanase (0.67 U / mL), and laccase (Lac, 1.48 U / mL). Its genome carries the cold shock protein genes cspG and capB, indicating potential for synergistic degradation of lignocellulose. The discovery of these low-temperature strains lays a germplasm foundation for straw return to the field and the development of bio-fertilizers in cold regions.

[0006] Table 1. Related enzyme activities in some reported cellulose-degrading strains

[0007] strains Temperature (°C) Endoglucanase (U / mL) Exoglucanase (U / mL) β-glucosidase (U / mL) Cellulose complex enzyme (U / mL) Xylanase (U / mL) Laccase (U / mL) Lignin peroxidase (U / mL) Manganese peroxidase (U / mL) Screening and Effects of Low-Temperature Resistant Straw Lignocellulose Degrading Fungal Straw Straw Straw Strains Alternaria + Alternaria / complex strain H 10 101.35 231.96 37.91 Screening of low-temperature cellulose-degrading composite bacterial strains MS15 (a complex bacterial strain) 15 2.2267 4.6925 Screening and Optimization of Enzyme Production Conditions for a Low-Temperature Cellulose-Degrading Bacterium YXS.3 9 130.21 Isolation, screening and identification of low-temperature degrading bacteria of corn stalks Penicillium / D5 15 31.5 29.6 52.7 Screening, identification and enzyme activity study of low-temperature cellulose-degrading fungi Isoponopontium globulus / ZY-50 16 128.34 2.31 2.43 7.89 Isolation and Identification of Low-Temperature Cellulose-Degrading Bacteria CF11 10 58.091 Screening, enzyme activity and whole genome analysis of Bacillus subtilis CH2 Bacillus CH2 28 40 32.18 Isolation and screening of cellulose-degrading bacteria and optimization of enzyme production conditions Bacillus licheniformis TB1 30 14.21 24.03 Screening of cellulose-degrading bacteria and study on their degradation characteristics Bacillus cereus YS 30 50.8 31.62

[0008] As shown in Table 1 above, although some degrading strains have been reported in previous studies, these strains generally suffer from key problems such as insufficient enzyme activity and incomplete enzyme system composition. Therefore, systematically screening for dominant strains with efficient lignocellulose degradation capabilities is particularly urgent. This work will not only provide important clues for a deeper understanding of microbial degradation mechanisms, but also lay a theoretical foundation and provide technical support for improving the utilization efficiency of lignocellulose biomass resources. Summary of the Invention

[0009] To address the challenges of existing technologies in the efficient degradation of lignocellulose, this invention identifies and develops a novel, highly efficient lignocellulose-degrading strain. This strain exhibits a superior complex expression system of cellulase, hemicellulase, and lignin-degrading enzymes, with enzyme activities significantly exceeding those of traditional strains. Most importantly, this strain demonstrates excellent lignocellulose degradation efficiency in corn straw substrates, achieving highly efficient synergistic depolymerization of lignin and cellulose components. The purpose of this invention is to provide a highly efficient lignocellulose-degrading strain and its applications.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention provides a strain that efficiently degrades lignocellulose, named *Bacillus thuringiensis* BT-L09. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39428, dated June 18, 2026. *Bacillus thuringiensis* BT-L09 exhibits cellulase, hemicellulase, and ligninase activities. The cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase, and cellulosic enzymes; the hemicellulase includes xylanase; and the ligninase includes one or more of laccase, lignin peroxidase, and manganese peroxidase.

[0012] This invention provides the application of the above-mentioned highly efficient lignocellulose-degrading strains in the fermentation production of cellulase and / or hemicellulase and / or ligninase. The cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase, and cellulosic complex enzymes; the hemicellulase includes xylanase; and the ligninase includes one or more of laccase, lignin peroxidase, and manganese peroxidase.

[0013] This invention also provides a microbial inoculant comprising the above-mentioned highly efficient lignocellulose-degrading strain and / or its fermentation products. The fermentation products comprise one or more of cellulase, hemicellulase, and ligninase. The cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase, and cellulosic enzymes; the hemicellulase includes xylanase; and the ligninase includes one or more of laccase, lignin peroxidase, and manganese peroxidase.

[0014] In some embodiments, the microbial agent is a liquid agent, a solid agent, or a freeze-dried powder.

[0015] The present invention also provides the application of the above-mentioned highly efficient lignocellulose-degrading strains or the above-mentioned microbial agents in the preparation of lignocellulose-degrading enzyme preparations.

[0016] The present invention also provides a lignocellulose-degrading enzyme preparation, which comprises the above-mentioned strains that efficiently degrade lignocellulose or the above-mentioned microbial agents.

[0017] The present invention also provides the application of the above-mentioned strains, microbial agents or lignocellulose-degrading enzyme preparations that are highly efficient at degrading lignocellulose in the degradation of lignocellulose.

[0018] In some implementations, the lignocellulose is lignocellulose from corn stalks.

[0019] The present invention also provides a method for degrading lignocellulose, comprising the following steps: adding a lignocellulose-containing substance to a fermentation medium containing the above-mentioned highly efficient lignocellulose-degrading strain, and performing shaking culture to degrade lignocellulose; or adding a lignocellulose-containing substance to the fermentation product of the above-mentioned highly efficient lignocellulose-degrading strain to degrade lignocellulose.

[0020] In some embodiments, the lignocellulose-containing material is corn stalk; the shaking culture temperature is 10-32°C; and the fermentation product of the highly efficient lignocellulose-degrading strain is the fermentation product obtained by culturing the strain at a temperature of 10-32°C. Preferably, the temperature is 15°C.

[0021] The beneficial effects of this invention are as follows: The *Bacillus thuringiensis* BT-L09 strain of this invention, as determined by enzyme activity assays, exhibits the following activities: endoglucanase activity of 17.39 U / mL, exoglucanase activity of 6.51 U / mL, β-glucosidase activity of 30.91 U / mL, cellulase complex activity of 11.83 U / mL, xylanase activity of 16.24 U / mL, laccase activity of 18.25 U / mL, lignin peroxidase activity of 21.35 U / mL, and manganese peroxidase activity of 27.46 U / mL. This strain significantly outperforms existing strains in terms of lignocellulose-degrading enzyme activity, with the highest β-glucosidase activity among cellulase systems and the highest manganese peroxidase activity among lignin enzyme systems. This strain possesses a complete enzyme system and outstanding growth advantages, providing an ideal choice for developing compound degrading inoculants and demonstrating broad application prospects in the fermentation industry. Attached Figure Description

[0022] Figure 1 : Colony morphology characteristics of Bacillus thuringiensis BT-L09.

[0023] Figure 2 Figure: Results of Congo red staining experiment of Bacillus thuringiensis BT-L09 on carboxymethyl cellulose medium.

[0024] Figure 3 Figure: Comparison of Bacillus thuringiensis BT-L09 filter paper strip disintegration test results with the control group.

[0025] Figure 4 Enzyme activity diagrams related to cellulase and hemicellulase in Bacillus thuringiensis BT-L09.

[0026] Figure 5 : Ligninase-related enzyme activity diagram of Bacillus thuringiensis BT-L09.

[0027] Figure 6Gram staining image of Bacillus thuringiensis BT-L09.

[0028] Figure 7 Scanning electron microscope image of Bacillus thuringiensis BT-L09.

[0029] Figure 8 Electrophoresis image of 16S rDNA PCR products of Bacillus thuringiensis BT-L09.

[0030] Figure 9 Phylogenetic tree of Bacillus thuringiensis BT-L09 based on 16S rDNA sequence.

[0031] Figure 10 Line graphs showing the activities of endoglucanase and cellulosic enzyme in Bacillus thuringiensis BT-L09 at different temperatures.

[0032] Figure 11 Degradation rate of corn stalks by Bacillus thuringiensis BT-L09. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment is a screening embodiment of the strain described in this invention.

[0036] The preparation method of the culture medium used in this embodiment is as follows:

[0037] Sodium carboxymethyl cellulose medium: 10.0 g sodium carboxymethyl cellulose, 1.0 g peptone, 1.0 g yeast extract powder, 6.0 g NaNO3, 1.0 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g KH2PO4, 15.0 g agar powder, 1000 mL distilled water, pH 7.0 ± 0.2, sterilized by moist heat at 121℃ for 20 min (add 20 g / L agar to solid medium, do not add agar to liquid medium).

[0038] Tryptone-Soybean Agar Medium (TSA): 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g NaCl, 14 g agar powder, 1000 mL distilled water, pH 7.2 ± 0.1, sterilized by moist heat at 121℃ for 20 min.

[0039] Hutchison's liquid medium: KH2PO4 1.0 g, MgSO4·7H2O 0.3 g, NaCl 0.1 g, NaNO3 2.5 g, FeCl3 0.01 g, CaCl2 0.1 g, distilled water 1000 mL, pH 7.2 ± 0.1, sterilized by moist heat at 121℃ for 20 min.

[0040] Liquid fermentation medium: K2HPO4·3H2O 13.3 g, KH2PO4 4.0 g, MgSO4·7H2O 0.2 g, (NH4)2SO4 0.5 g, corn straw powder (passed through 50 mesh) 5 g, distilled water 1000 mL, pH 7.0 ± 0.2, sterilized before use.

[0041] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride (NaCl), 1000 mL distilled water, sterilized at 121°C for 20 min by moist heat.

[0042] Sampling and extraction: Soil samples under decaying straw were collected from different cornfields in Longtan District, Jilin City, Jilin Province in March 2023. 2.5 g of each sample was placed in a 100 mL sterile water conical flask, shaken at 15℃ and 220 r / min for 1 h, and the bacterial solution was collected. The precipitate was discarded by centrifugation, and the supernatant was kept for later use.

[0043] Serial dilution and plating culture: The supernatant was serially diluted to 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 The solution was prepared by taking 100 μL of 10... -4 10 -5 10 -6 The diluted solution was spread on TSA plates and incubated at 15°C for 5 days.

[0044] Isolation and purification: Based on preliminary differentiation according to colony morphology, single colonies of different morphologies were picked and inoculated onto TSA solid medium for isolation. Single colonies cultured for 5 days were selected and streaked repeatedly on their respective media for purification six times until pure cultures were obtained. The colony morphology characteristics of Bacillus thuringiensis BT-L09 screened in this invention are shown in the figure below. Figure 1 As shown, it is a grayish-white and opaque colony with obvious irregular radial wrinkles on the surface and a depression in the middle.

[0045] Preliminary screening: (1) The purified single strain was inoculated onto sodium carboxymethyl cellulose solid medium and cultured at 15°C for 5 days. It was then stained with 1 mg / mL Congo red solution for 30 min, the dye was discarded, and the strain was repeatedly washed with 1 mol / L NaCl solution until the eluent was nearly colorless. The transparent zone was observed; the presence of a transparent zone on the surface of the medium indicated that the strain might produce cellulase. The colony diagram of Bacillus thuringiensis BT-L09 obtained in this invention under this screening is shown below. Figure 2 As shown in the figure, A is the BT-L09 bacterial zone, and B is the transparent hydrolysis zone formed around the BT-L09 strain. (2) The purified single strain was inoculated into LB liquid medium and cultured at 15℃ and 220 r / min for 1 day to prepare seed liquid. 1 mL of seed liquid was inoculated into Hutchinson's liquid medium containing filter paper strips and cultured at 15℃ and 150 r / min for 15 days. The disintegration of the filter paper strips was observed. The disintegration of the filter paper indicates that the strain has the ability to produce cellulase complex. The results of the Bacillus thuringiensis BT-L09 obtained by screening in this invention under this screening are as follows. Figure 3 As shown, the left image is Hutchinson's liquid medium containing filter paper strips without inoculated strain seed solution, and B is Hutchinson's liquid medium containing filter paper strips inoculated with BT-L09 seeds. It can be seen that the filter paper strips in the BT-L09 inoculated medium show obvious disintegration. The strains obtained through the above two screening methods are the primary screening strains.

[0046] Enzyme activity rescreening:

[0047] A. Preparation of crude enzyme solution: The initially screened strains were inoculated into TSA medium and cultured at 15℃ for 2 days. The cultured strains were then inoculated into LB liquid medium and cultured in a constant temperature shaker at 15℃ and 150 r / min for 1 day to prepare the seed culture. 1 mL of the seed culture was inoculated into 50 mL of liquid fermentation medium and cultured in a shaker at 15℃ and 220 r / min for 7 days. After the culture was completed, the bacterial culture was centrifuged at 4℃ and 8000 r / min for 15 min, and the supernatant was the crude enzyme solution.

[0048] B. Determination of the glucose / xylose standard curve: Accurately weigh 0.1 g of glucose / xylose, dissolve in an appropriate amount of distilled water, transfer to a 100 mL volumetric flask and dilute to volume to obtain the standard stock solution. Take 6 test tubes and add different volumes of distilled water and standard stock solution to prepare a concentration gradient. Add 3 mL of DNS reagent to each test tube, mix well, boil in a water bath for 10 min, cool, and dilute to 25 mL. Measure the absorbance at a wavelength of 540 nm.

[0049] C. Endoglucanase activity: Mix 1.5 mL of 1% sodium carboxymethyl cellulose solution with 0.5 mL of crude enzyme solution (the control group is inactivated crude enzyme solution), incubate at 50°C for 30 min, add 3.0 mL of DNS reagent, incubate at boiling water for 10 min, cool, and measure the absorbance at 540 nm. Enzyme activity unit definition (U / mL): The amount of enzyme required to catalyze the production of 1 μg of glucose from 1 mL of substrate per minute.

[0050] D. Cellulase Total Activity (FPA) Assay: Take 50 mg of filter paper, 1.5 mL of pH 4.8 sodium citrate buffer, and 0.5 mL of crude enzyme solution (the control is inactivated crude enzyme solution). Incubate at 50°C for 60 min, add 3.0 mL of DNS reagent, incubate at boiling water for 10 min, and measure the absorbance at 540 nm after cooling. The definition of enzyme activity unit is the same as above.

[0051] E. Determination of exoglucanase activity: Use 50mg of defatted cotton instead of filter paper, and follow the same steps as FPA determination.

[0052] F. Determination of β-glucosidase activity: Take 1.5 mL of 1% salicin (prepared with pH 4.5 sodium citrate buffer) and 0.5 mL of crude enzyme solution (the control is inactivated crude enzyme solution), incubate at 37℃ for 30 min, add 3.0 mL of DNS reagent, incubate in boiling water for 10 min, cool, and then measure the absorbance at 540 nm. The definition of enzyme activity unit is the same as above.

[0053] G. Determination of xylanase activity: Take 1.5 mL of 1% xylan (prepared with pH 4.6 acetate buffer) and 0.5 mL of crude enzyme solution (the control is inactivated crude enzyme solution), incubate at 50℃ for 30 min, add 3.0 mL of DNS reagent, incubate at boiling water for 10 min, cool, and then measure the absorbance at 540 nm. Enzyme activity unit definition (U / mL): The amount of enzyme required to catalyze the production of 1 μg of xylose from 1 mL of substrate per minute.

[0054] H. Laccase Activity Assay: Using 0.5 mL of 0.5 mol / L ABTS as substrate, add 2 mL of pH 4.5 citrate-sodium citrate buffer and 1 mL of crude enzyme solution. Initiate the reaction at 30℃ and measure the absorbance change within the first minute of the reaction at 420 nm. Enzyme activity unit definition (U / mL): 1 μmol of ABTS produced per minute. + • The amount of enzyme required for free radicals. The molar absorptivity of the ABTS oxidation product at 420 nm is 36000 / mol·L / cm.

[0055] M. Assay for lignin peroxidase activity: 1.5 mL of 0.1 mol / L tartaric acid buffer (pH 3.0), 1.0 mL of 10 mmol / L resveratrol, and 0.4 mL of crude enzyme solution were added sequentially. 0.1 mL of 10 mmol / L H₂O₂ was added to initiate the reaction. The absorbance change was measured at 310 nm at 30°C during the first 3 minutes of the reaction. Enzyme activity unit definition (U / mL): The amount of enzyme required to catalyze the conversion of 1 μmol of resveratrol per minute. The molar absorptivity of the resveratrol oxidation product at 310 nm is 9300 / mol·L / cm.

[0056] N, Manganese Peroxidase Activity Assay: 2.0 mL of 0.05 mol / L succinate buffer (pH 4.5), 0.5 mL of 15 mmol / L MnSO4, and 0.4 mL of crude enzyme solution were added sequentially. 0.1 mL of 10 mmol / L H2O2 was added to initiate the reaction. The absorbance change was measured at 240 nm wavelength within the first 3 minutes of the reaction at 37℃. Enzyme activity unit definition (U / mL): The amount of enzyme required to catalyze the conversion of 1 μmol MnSO4 per minute. 3+ The molar absorptivity at 240 nm is 49600 / mol·L / cm.

[0057] Through the above-mentioned secondary screening, a strain with excellent enzyme activities was obtained and named BT-L09. The results of the enzyme activities of BT L09 are as follows: Figure 4 , 5 As shown, its endoglucanase activity was 17.39 U / mL, exoglucanase activity was 6.51 U / mL, β-glucosidase activity was 30.91 U / mL, cellulosic enzyme activity was 11.83 U / mL, xylanase activity was 16.24 U / mL, laccase activity was 18.25 U / mL, lignin peroxidase activity was 21.35 U / mL, and manganese peroxidase activity was 27.46 U / mL.

[0058] Example 2

[0059] This embodiment is an example of the identification of the strain described in this invention.

[0060] A. Gram staining: Prepare smears of fresh bacterial cells from TSA plates, stain with crystal violet for 1 min, wash with water; mordant with iodine solution for 1 min, wash with water; decolorize with 95% ethanol until colorless or pale purple, wash with water; counterstain with safranin for 1 min, wash with water; observe and photograph under an oil immersion microscope after drying. The Gram staining image of strain BT-L09 is shown below. Figure 6 As shown, it is a Gram-positive bacterium.

[0061] B. Scanning electron microscopy (SEM) observation: Glutaraldehyde was added to the surface of the colonies for fixation. The colonies were gently scraped, and the bacterial suspension was collected. After centrifugation, the suspension was fixed overnight with 2.5% glutaraldehyde (prepared in PBS). The colonies were washed three times with PBS, each time incubated for 30 min. The colonies were then dehydrated sequentially with 30%, 50%, 70%, 85%, 90%, and 100% ethanol, for 15 min each time. After centrifugation at 10,000 rpm, the colonies were replaced with 100% tert-butanol two or three times. The colonies were then frozen at 80°C for 12 h, freeze-dried for 12 h, and observed under a 15,000x electron microscope. The SEM image of strain BT-L09 is shown below. Figure 7 As shown, the bacterial cells are shrunken and concave rod-shaped, uniform in size, about 0.8 × 3 μm, with wavy peritrichous flagella and no exposed spores.

[0062] C. Molecular Identification: Bacterial strain BT L09 was inoculated into LB liquid medium and cultured with shaking for 12 h. 1 mL of bacterial culture was collected by centrifugation, and 400 μL of Buffer Digestion was added. The culture was incubated at 65°C for 1 h until complete lysis. 200 μL of Buffer PB was added, and the culture was incubated at 20°C for 5 min. After centrifugation, the supernatant was collected, and an equal volume of isopropanol was added. The culture was incubated at room temperature for 2 min, centrifuged, and the supernatant was discarded. The cells were washed twice with 75% ethanol, dried, and then dissolved in 50 μL of TE Buffer. PCR amplification and sequencing of 16S rDNA were performed. The sequencing results were compared using BLAST in the NCBI database. Sequences of strains with high similarity were selected, and multiple alignments were performed using MAFFT software. A phylogenetic tree was constructed using software such as FastTree / iqtree / raxml. The gel electrophoresis verification image of PCR amplification of BT-L09 16S rDNA is shown below. Figure 8 As shown, the amplification length is approximately 1500 bp, consistent with the expected experimental results. Phylogenetic tree identification results ( Figure 9 The results indicate that strain BT-L09 is Bacillus thuringiensis. Strain BT-L09 was deposited on June 18, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Bacillus thuringiensis, with accession number CGMCC No. 39428.

[0063] Example 3

[0064] A. The activities of endoglucanase and cellulosic enzyme in the screened strain BT-L09 were measured at different temperatures (this study focuses on endoglucanase and cellulosic enzyme. The former randomly hydrolyzes β-1,4-glycosidic bonds within the cellulose molecular chain to open the chain, while the latter characterizes the overall efficiency of multi-component synergistic degradation of natural substrates; both are sufficient to anchor the optimal temperature of the whole enzyme system). The experimental temperatures were 10℃, 15℃, 20℃, 25℃, and 30℃, and the specific experimental methods were the same as those in the enzyme activity rescreening in Example 1. Figure 10 The graph shows the activities of endoglucanase and cellulosic enzyme in strain BT-L09 at different temperatures. It can be seen that the activities of endoglucanase and cellulosic enzyme in this strain are better at 15℃ than at other temperatures, with activities of 17.00 U / mL and 12.30 U / mL, respectively.

[0065] B. Dry the corn stalks at 105℃ to constant weight. Add 5g of corn stalks to a 50mL / 250mL liquid fermentation medium, inoculate with 1mL of Bacillus thuringiensis BT-L09 seed culture, and culture with shaking at 15℃ / 28℃ / 32℃ and 150r / min for 20 days. Every 5 days, take the culture medium, centrifuge at 4℃ and 3500r / min for 10min, discard the supernatant, rinse repeatedly with deionized water to remove bacterial cells, centrifuge again, dry at 105℃ to constant weight, measure the weight of the stalks before and after fermentation, and calculate the degradation rate of the corn stalks.

[0066] Corn stalk degradation rate = (mass of stalks before fermentation − mass of stalks after fermentation) / mass of stalks before fermentation × 100%.

[0067] like Figure 11 The results showed that the strain had the best degradation effect on corn stalks at 15℃, and the degradation rate reached 38.04% after 20 days of shaking culture.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain that degrades lignocellulose, characterized in that: The strain is Bacillus thuringiensis BT-L09, with accession number CGMCC No.39428.

2. The use of the lignocellulose-degrading strain according to claim 1 in the production of cellulase, hemicellulase and / or ligninase.

3. The application according to claim 2, characterized in that: The cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase, and cellulosic enzyme; the hemicellulase is xylanase; and the ligninase includes one or more of laccase, lignin peroxidase, and manganese peroxidase.

4. A microbial inoculant, characterized in that: It includes the strain that degrades lignocellulose as described in claim 1 and / or its fermentation products.

5. The use of the lignocellulose-degrading strain of claim 1 or the microbial agent of claim 4 in the preparation of lignocellulose-degrading enzyme preparations.

6. A lignocellulose-degrading enzyme preparation, characterized in that: The strain comprising the lignocellulose-degrading strain of claim 1 or the microbial agent of claim 4.

7. The application of the lignocellulose-degrading strain of claim 1, the microbial agent of claim 4, or the lignocellulose-degrading enzyme preparation of claim 6 in the degradation of lignocellulose.

8. The application according to claim 7, characterized in that: The lignocellulose is the lignocellulose found in corn stalks.

9. A method for degrading lignocellulose, characterized in that, Includes the following steps: Add the lignocellulose-containing substance to the fermentation medium containing the lignocellulose-degrading strain of claim 1, and perform shaking culture to degrade lignocellulose; Alternatively, a substance containing lignocellulose may be added to the fermentation product of the lignocellulose-degrading strain described in claim 1 to degrade lignocellulose.

10. The method for degrading lignocellulose according to claim 9, characterized in that: The shaking culture temperature is 10-32℃; the fermentation product of the lignocellulose-degrading strain is the fermentation product of the strain cultured at 10-32℃.

Images