Bacillus thuringiensis and application thereof in lignocellulose degradation

Abstract

This invention discloses a strain of Bacillus thuringiensis and its application in the degradation of lignocellulose, belonging to the field of microbial technology. The microbial classification name is Bacillus thuringiensis, the strain name is LMU-81, and the accession number is CGMCC NO.36215. Strain LMU-81 was isolated from leaf litter soil and accumulated decaying wood in mangrove wetlands. It exhibits high lignocellulose enzyme activity, particularly lignin peroxidase (LiP), and can be used as a microbial inoculant in combination with other microorganisms to form a compound inoculant for the degradation of lignocellulose in sugarcane bagasse and other materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a strain of Bacillus thuringiensis and its application in the degradation of lignocellulose. Background Technology

[0002] Lignocellulosic biomass (LCB) is a promising renewable energy source rich in organic matter. Driven by the circular economy and bioeconomy, LCB is sustainably converted into bioenergy (such as biofuels, electricity, and heat) and high-value-added products (such as chemicals, biopolymers, and building materials) through biorefineries. As the most abundant biomass on Earth, LCB primarily originates from agricultural and forestry residues, energy crops, and municipal solid waste. Nearly 1.3 billion tons of LCB are produced globally each year. Of these biomass sources, sugarcane produces approximately 279 million tons of bagasse annually, with Brazil, India, China, and the United States being the four largest bagasse producers. LCB has the potential to mitigate global climate change as an alternative energy source; in the coming years, over 30% of fossil fuels may be replaced by biofuels, biochemicals, and biomaterials, potentially curbing humanity's over-reliance on fossil fuels. Currently, pretreatment, thermochemical methods, and biological pretreatment are used to depolymerize LCB to improve the utilization rate of lignocellulosic biomass. Chemical treatment is a widely used and efficient method that can selectively attack the complex structure of lignocellulose, but it has many limitations, including high cost and high energy and resource consumption. Furthermore, the byproducts generated by chemical reagents can lead to microbial inactivation and environmental pollution. Compared to chemical methods, biological treatment offers an environmentally friendly and economical solution. The goal of biomass fermentation is to utilize microorganisms to hydrolyze complex polysaccharides in biomass, further converting them into biofuels, materials, chemicals, or combustible gases. Therefore, obtaining enzyme-stable microorganisms with complete enzyme systems from the natural environment is a promising and sustainable strategy for efficiently depolymerizing LCBs.

[0003] Bacillus thuringiensis (Bt) Bacillus thuringiensis Bacillus thuringiensis (Bt) is a bacterium belonging to the family Bacillusaceae and the genus Bacillus. It is a group of aerobic or facultative anaerobic Gram-positive bacteria that produce parasporal crystals and spores, and is widely distributed in soil, water, air, and vegetation. Bt has been known for over 100 years. In 1956, T. Angus proved that the Cry protein produced by Bt has insecticidal activity. Since then, it has played a significant role in pest control and has been applied in various microbial insecticides. For example, it controls lepidopteran pests on crops such as vegetables, cotton, and corn, including pests such as the corn borer, cabbage white butterfly, and diamondback moth.

[0004] Previous studies have shown that most cellulases are derived from fungi, particularly Trichoderma and Aspergillus. Existing technologies document the use of Bacillus thuringiensis to decompose plant cellulose. The paper, "Identification of a Cellulase-Producing Strain and Optimization of Enzyme Production Conditions," (Journal of Shandong Agricultural University (Natural Science Edition), 2017, 48(4):556-561, in which the activity of hydroxymethyl cellulase (CMCase) produced by a cellulase-producing bacterium, X-2 Bacillus thuringiensis, reached 10.89 U / mL). Chinese Patent: Bacillus thuringiensis producing cellulase and inhibiting Vibrio splenita, and its application method; Application Publication No.: CN108841746A; Cellulase secreted by Bacillus thuringiensis XW008 can decompose crude fiber in feed; Chinese Patent: A strain of Bacillus thuringiensis and its application in treating vegetable waste and controlling nematodes; CN117701416A; Bacillus thuringiensis T1 has the function of degrading cellulose; Chinese Patent: A strain of Bacillus thuringiensis L16 and its application; Application Publication No.: CN120118805A; Bacillus thuringiensis L16 strain can produce protease, amylase, and cellulase. However, no reports have been found regarding Bacillus thuringiensis producing ligninase and degrading lignin. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus thuringiensis, which is classified and named as Bacillus thuringiensis. (Bacillus thuringiensis) The strain name is LMU-81, accession number: CGMCC NO.36215, depositary institution: China General Microbiological Culture Collection Center; deposit date: October 9, 2025. In addition to producing cellulase, strain LMU-81 can also produce ligninase, with high ligninase activity.

[0006] A strain of Bacillus thuringiensis, classified and named as: Bacillus thuringiensis (Bacillus thuringiensis The strain name is LMU-81, accession number: CGMCC NO.36215, depositary institution: China General Microbiological Culture Collection Center; deposit date: October 9, 2025.

[0007] The application of Bacillus thuringiensis in the preparation of a compound microbial agent that promotes the degradation of lignocellulose.

[0008] The Bacillus thuringiensis is used in the preparation of biological agents for the production of cellulase or ligninase; the cellulase includes one or more of exoglucosidase Exg, endoglucosidase Eng, β-glucosidase β-glucoside and filter paper enzyme FPA; the ligninase includes one or more of laccase Lac, lignin peroxidase LiP and manganese peroxidase MnP.

[0009] The beneficial effects of this invention are:

[0010] The strain LMU-81 described in this invention was collected from mangrove wetland soil in Fangchenggang City, Guangxi Province. Its microbial dominance stems from the unique, harsh, and variable growth environment of mangroves. The abundance of high-lignin, high-cellulose fallen leaves and branches in mangroves, coupled with high salinity and oxygen deficiency, provides a unique "training ground" for microorganisms, resulting in higher degradation activity and efficiency compared to terrestrial microorganisms. Secondly, mangrove soil is frequently oxygen-deficient due to periodic tidal inundation. Therefore, the strain must be capable of both aerobic respiration and anaerobic fermentation, which is particularly important for the traditionally believed requirement of strictly aerobic degradation of lignocellulose. In real-world industrial applications, the strain's sensitivity to oxygen levels is crucial to success, highlighting the significant research and application value of mangrove wetland microbial strains.

[0011] Based on its characteristics, it can be applied to the degradation of lignin, such as in composting and fermentation. It can also be combined with other microorganisms to form compound lignin-degrading agents, thus expanding the combination methods and selection range of lignin-degrading strains. Attached Figure Description

[0012] Figure 1 Electrophoresis image of PCR products from LMU-81; Figure 2 The colony morphology of LMU-81; Figure 3 Phylogenetic tree diagram of LMU-81; Figure 4 Bar chart of cellulase activity for LMU-81: Endoglucase Eng activity; Exoglucase Exg activity; β-glucosidase β-glu activity; Filter paper enzyme FPA activity; Figure 5 Bar chart showing ligninase activity of LMU-81: Lacase activity; Manganese peroxidase (MnP) activity; Lignin peroxidase (LiP) activity.

[0013] As shown in the uploaded microbial preservation certificate and microbial survival certificate, the strain preservation information is as follows: strain Bacillus thuringiensis (Bacillus thuringiensis)LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36215; and is referred to as strain LMU-81 in the following examples. Detailed Implementation

[0014] The reagent solutions and culture media used in the examples were prepared according to the following methods: Preparation of culture media and solutions used in the examples Culture medium: LB liquid culture medium (g·L) -1 ): NaCl 10.00 g, tryptone 10.00 g, yeast extract 5.00 g, adjust pH to approximately 7.0.

[0015] LB solid medium (g·L) -1 ): LB liquid medium with 20.00 g agar.

[0016] PDB medium (g·L) -1 ): 200.00 g of potatoes, cut into chunks and boil for about 30 minutes, collect the filtrate and add 20.00 g of glucose.

[0017] PDA medium (g·L) -1 ): PDB medium with 20.00 g agar.

[0018] CMC-Na liquid medium: Same as CMC-Na solid medium, but without agar powder.

[0019] Alkali lignin solid culture medium: 5 g alkali lignin, 2 g (NH4)2SO4, 0.50 g MgSO4·7H2O, 1 g K2HPO4, 0.50 g NaCl, 20 g agar powder, add water to 1000 mL, sterilize at 121 ℃ for 20 min and use.

[0020] Lignin enzyme production medium (g·L) -1 ): Alkali lignin 2.00 g, K2HPO4 1.00 g, MgSO4·7H2O 0.50 g, NaCl 0.50 g, (NH4)2SO4 2.00 g, CaCl2 0.10 g, MnSO4 0.02 g, FeSO4 0.05 g.

[0021] Reagent solution: 50% Glycerin: 50 g glycerin, 50 g H2O, sterilize by moist heat at 121 ℃ for 20 min.

[0022] 0.10 M tartaric acid buffer (pH 3.00): Mix 0.10 M tartaric acid solution and 0.10 M sodium tartrate solution until the pH is 3.00.

[0023] 10 mM resveratrol: Weigh 1.682 g of resveratrol, dissolve it in distilled water, and bring the volume to 1 L.

[0024] 10 mM H2O2: Measure 1.02 mL of 30% H2O2 and add distilled water to bring the volume to 1 L.

[0025] 0.05 M Succinate Buffer (pH 4.50): Mix 0.05 M succinic acid solution and 0.05 M sodium succinate solution until the pH reaches 4.50. (0.05 M succinic acid solution: Weigh 0.59 g of succinic acid, dissolve in distilled water, and bring the volume to 100 mL; 0.05 M sodium succinate solution: Weigh 1.35 g of sodium succinate, dissolve in distilled water, and bring the volume to 100 mL).

[0026] 15 mM MnSO4: Weigh 2.535 g MnSO4, dissolve in distilled water, and bring the volume to 1 L.

[0027] 0.60 mM ABTS: Weigh 0.033 g ABTS, dissolve in distilled water, and bring the volume to 100 mL.

[0028] 0.05 M citric acid buffer solution (pH 5.00): Measure 20.50 mL of 0.10 M citric acid solution and 29.50 mL of 0.10 M sodium citrate solution, and add distilled water to a final volume of 100 mL.

[0029] Example 1

[0030] The screening steps for Bacillus thuringiensis sp. LMU-81 include: (1) Sample collection: Samples were collected from wetland soil, leaf litter soil and accumulated decaying wood in Pearl Bay Mangrove Nature Reserve, Shijiao Village, Fangchenggang City. (2) Enrichment: 10 g of each sample was placed in a sterile conical flask containing sterile water and glass beads, and incubated in a shaker at 28 ℃ and 180 rpm for 1 h. 5 mL of the suspension was then inoculated into 100 mL of CMC-Na liquid medium and alkali lignin liquid medium, and incubated at 28 ℃ and 130 rpm for 3-5 days to enrich the sample. (3) Separation and purification: Transfer 1 mL of the sample suspension to a 9 mL sterile water tube, then transfer 1 mL to another identical new test tube. Repeat this process stepwise to prepare 10 mL of the solution. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Sample solutions at different dilutions. Use a pipette to pipette 200 mL of 10... -4 10 -5 and 10 -6 Diluted solutions were spread onto CMC-Na and alkali lignin solid plates using a sterile spreader. The plates were allowed to stand at room temperature for 5–10 minutes to allow the bacterial suspension to immerse in the culture medium. The solid plates were then inverted and incubated at 28 °C for 5 days. Based on morphological analysis, bacteria and fungi were preliminarily distinguished, and the strains were isolated and purified on PDA and LB solid media, respectively.

[0031] Example 2

[0032] Screening of functional microorganisms: The isolated single strains were cultured on sodium carboxymethyl cellulose solid medium for 3-5 days, with three replicates for each strain. The plates were stained with 1 mg / mL Congo red solution for 30 min, then the stain was discarded, and the plates were destained with 1 M NaCl solution for 30 min. Plates showing a clear zone were selected, indicating that the strain may secrete cellulase; the size of this clear zone is considered an indicator for preliminary assessment of the microbial ability to secrete cellulase. Strain LMU-81; Diameter of clear zone (D / mm): 40.10±1.24; Colony diameter (d / mm): 7.13±0.19; D / d: 5.63±0.26; Taxonomy: Bacteria.

[0033] Example 3

[0034] molecular biological identification of strains Morphological and molecular identification of strain LMU-81: as shown in the appendix Figure 2 As shown, the colonies formed by strain LMU-81 on LB agar plates are round or oval with irregular edges, a characteristic consistent with the typical colony features of Bacillus thuringiensis. Under a microscope, the cells of LMU-81 are stained purple and rod-shaped, arranged in short chains, indicating that it is a Gram-positive bacterium.

[0035] After culturing strain LMU-81 in liquid medium, its genomic DNA was extracted and used as a template to amplify the strain's 16S rRNA gene using universal bacterial primers. The results of agarose gel electrophoresis of the PCR products are shown in the attached figure. Figure 3 As shown, specific bands of the expected size are displayed.

[0036] As attached Figure 3 As shown, strain LMU-81 is on the phylogenetic tree similar to Bacillus thuringiensis (Bt). Bacillus thuringiensis The most closely related species, LMU-81, is classified as Bacillus thuringiensis. Bacillus thuringiensis It belongs to the phylum Firmicutes, class Bacilli, order Bacillales, family Bacillaceae, and genus Bacillus. Bacillus ).

[0037] Example 4

[0038] Cellulase activity assay: (1) Preparation of crude enzyme solution The selected strains were cultured into a bacterial suspension and inoculated into the enzyme-producing fermentation medium at a ratio of 5%. The suspension was cultured at 28 ℃ and 180 rpm for 5 days with shaking. 5 mL of the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant obtained after centrifugation was the crude enzyme solution.

[0039] (2) Plot the glucose standard curve Using a pipette, pipette 0.50 mL of glucose standard series solutions into 25 mL test tubes. Then, add 1.50 mL of 0.05 M pH 5.00 citrate buffer to each tube. For the control group, add 2 mL of the same concentration of citrate buffer. Finally, add 3 mL of DNS reagent to all tubes and mix thoroughly. Perform triplicate for each sample and control tube. Place the tubes in a boiling water bath for 10 min, then immediately remove and cool. Make up to 25 mL with distilled water and mix again. Using a blank tube as a control, measure its absorbance at 540 nm. Plot a standard curve with glucose concentration as the X-axis and absorbance as the Y-axis, and calculate the linear regression equation. The glucose standard curve equation is y = 0.7446x - 0.0422, R0. 2 = 0.9984, indicating that the curve has a good linear relationship.

[0040] (3) Cellulase activity assay Eng Activity Assay: The amount of reducing sugar released during hydrolysis was determined by the DNS colorimetric method, and the cellulase activity was calculated. 1 mL of 1% carboxymethyl cellulose solution was used as the substrate, and 0.50 mL of enzyme solution was added. The mixture was vortexed and incubated at 50 °C for 30 min to complete the enzyme reaction. After the reaction was complete, 3 mL of DNS colorimetric reagent was immediately added to the mixture to stop the enzyme reaction. The treated sample was heated in a boiling water bath for 10 min and then cooled in water to maintain color stability. Next, an inactivated enzyme solution was used as a blank control, and the absorbance was measured at 540 nm using a UV spectrophotometer. Each experimental sample was performed in triplicate. Cellulase activity is defined as the amount of enzyme required to release 1 µmol of glucose by hydrolyzing the corresponding substrate in 1 min at 50 °C, expressed as U / mL. Enzyme activity is calculated using the following formula: Where G is the glucose content (mg) determined by the standard curve; V is the total reaction volume (mL); T is the reaction time (min); and v is the enzyme volume (mL).

[0041] Exg activity assay: Using 1 mL of 1% microcrystalline cellulose as substrate, add 0.50 mL of enzyme solution, vortex to mix thoroughly, incubate the mixture at 50 ℃ for 30 min, remove the test tube, and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, cool in water to maintain color stability, use inactivated enzyme solution as a blank, and measure the absorbance at 540 nm using a UV spectrophotometer. Each experimental sample is performed in triplicate. Enzyme activity is calculated using the enzyme activity formula.

[0042] β-Glu activity assay: Using 1 mL of 1% salicin as substrate, add 0.50 mL of enzyme solution, vortex to mix thoroughly, incubate the mixture at 50 ℃ for 30 min, remove the test tube, and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, cool in water to maintain color stability, use inactivated enzyme solution as a blank, and measure the absorbance at 540 nm using a UV spectrophotometer. Each experimental sample was performed in triplicate. Enzyme activity was calculated using the enzyme activity formula.

[0043] Filter paper activity (FPA) assay: Using 50 mg of starch-free filter paper as substrate, add 0.50 mL of enzyme solution and 1 mL of 0.05 M pH 5.00 citrate buffer, vortex to mix thoroughly, and incubate the mixture at 50 ℃ for 30 min. Remove the test tube and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, then cool in water to maintain color stability. Use inactivated enzyme solution as a blank. Measure the absorbance at 540 nm using a UV spectrophotometer. Perform three replicates for each sample. Calculate enzyme activity using the enzyme activity formula.

[0044] The levels of exoglucanase Exg were 12.42 U / mL, endoglucanase Eng was 3.52 U / mL, β-glucosidase b-glu was 1.41 U / mL, and filter paper enzyme FPA was 8.07 U / mL, as determined by the assay.

[0045] (4) Ligninase activity assay LiP activity assay: 1.50 mL of 0.10 M tartaric acid buffer, 1 mL of 10 mM resveratrol, 0.40 mL of crude enzyme solution were added sequentially, followed by 0.10 mL of 10 mM H₂O₂. The reaction was initiated at 30 °C. The absorbance change of the reaction solution at 310 nm was measured within the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to oxidize resveratrol to produce 1 mmol of veratraldehyde per minute.

[0046] MnP activity assay: MnP can convert Mn 2+ Oxidized to Mn 3+ Add 2 mL of 0.05 M succinate buffer, 0.50 mL of 15 mM MnSO4, 0.4 mL of crude enzyme solution, and finally 0.10 mL of 10 mM H2O2. ， The reaction was initiated at 30 °C. The absorbance change of the reaction solution at 240 nm wavelength was measured during the first 3 minutes. One unit of enzyme activity was defined as the oxidation of 1 mmol Mn per minute. 2+ The required amount of enzyme.

[0047] Lac activity assay: 0.50 mL of 0.60 mM ABTS, 2 mL of 0.05 M citrate buffer, and finally 1 mL of crude enzyme solution were added sequentially. The reaction was started at 25 °C, and the absorbance change of the reaction solution at 420 nm wavelength was detected within the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to catalyze 1 mmol of ABTS per minute.

[0048] The formula for calculating enzyme activity is as follows: Where ΔA is the absorbance change; ε is the molar absorptivity (mol) -1 ·L·cm -1 ); d is the optical path length of the cuvette (cm); V is the total reaction volume (mL); v is the crude enzyme solution volume (mL); T is the reaction time (min).

[0049] As attached Figure 5 As shown, the highest Lac enzyme activity of strain LMU-81 was 11.59 U / mL; the highest LiP enzyme activity was 22.65 U / mL; and the highest manganese peroxidase (MnP) enzyme activity was 6.62 U / mL.

[0050] Example 5

[0051] Filter paper strip disintegration experiment: The main component of filter paper strips is cellulose. Using filter paper strips as the sole carbon source in the culture medium effectively induces the secretion of cellulase by the strains, reflecting the overall level of cellulase production. Strains initially screened using the Congo red staining method were further subjected to filter paper strip disintegration experiments to evaluate their activity in cellulose degradation.

[0052] LMU-81 can degrade filter paper strips into a flocculent state. After the filter paper disintegration experiment, the fermentation broth in the culture medium was repeatedly rinsed with distilled water to remove the bacterial cells attached to the filter paper strips. The culture was then centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was repeatedly washed with a small amount of distilled water to remove the remaining filter paper strips several times until the effluent was clear. The remaining filter paper strips were then dried at 80 ℃ for 24 h, taken out and weighed at room temperature, and analyzed by: Filter paper weight loss (%) = [(Filter paper weight - Residual filter paper weight) / Filter paper weight] × 100% The calculated weight loss rate of the filter paper was 47.7%.

[0053] Example 6

[0054] Determination of lignocellulose degradation rate: The microorganisms were inoculated at a rate of 10% into 100 mL of lignocellulose degradation liquid culture medium and cultured in a constant temperature shaker at 180 rpm and 28 ℃. After the culture was completed, a mixture of dilute hydrochloric acid and dilute nitric acid was added to the fermentation broth to remove residual cells. The broth was then repeatedly rinsed with distilled water. The culture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed several times with a small amount of distilled water to remove degradation residues until the effluent was clear. The effluent was then dried in a drying oven at 105 ℃ to constant weight. The changes in cellulose, hemicellulose, and lignin content were calculated by measuring the contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and ash in the degradation residues during the washing process. Cellulose content was calculated as the difference between ADF and ADL, hemicellulose content as the difference between NDF and ADF, and lignin content as the difference between ADL and Ash. A lignocellulose degradation liquid culture medium without added microorganisms was used as a control. The relative degradation rate of lignocellulose was determined, and the lignocellulose degradation rate was calculated using the following formula: Lignocellulose degradation rate (%) = [(bagasse weight - residual bagasse weight) / bagasse weight] × 100% The calculated lignocellulose degradation rate of strain LMU-81 was 44.1%.

Claims

1. A strain of Bacillus thuringiensis, characterized in that, The strain was classified and named as: Bacillus thuringiensis (Bacillus thuringiensis The strain name is LMU-81, accession number: CGMCC NO.36215, depositary institution: China General Microbiological Culture Collection Center; deposit date: October 9, 2025.

2. The application of Bacillus thuringiensis as described in claim 1 in the preparation of a compound microbial agent that promotes the degradation of lignocellulose.

3. The use of Bacillus thuringiensis as described in claim 1 in the preparation of biological agents for the production of cellulase or ligninase; the cellulase includes one or more of exoglucosidase Exg, endoglucosidase Eng, β-glucosidase β-glucoside and filter paper enzyme FPA; the ligninase includes one or more of laccase Lac, lignin peroxidase LiP and manganese peroxidase MnP.

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