Heat-resistant corynebacterium nicotianae and application thereof in lignocellulose degradation

Abstract

The invention relates to the technical field of microorganisms, in particular to heat-resistant corynebacterium nicotianae and application of the heat-resistant corynebacterium nicotianae in lignocellulose degradation. The invention provides Glutamicobacter nicotianae WT08 which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Glutamicobacter nicotianae WT08 is CGMCC No.35741. The invention further provides a preparation method of the Glutamicobacter nicotianae WT08. The corynebacterium nicotianae WT08 can tolerate growth at a high temperature, can generate laccase, lignin peroxidase, cellulase and manganese peroxidase under a high-temperature condition, is a high-temperature-resistant lignocellulose degrading bacterium, can be used for composting treatment of raw materials such as agricultural and forestry wastes, can increase the quantity and activity of microorganisms in a high-temperature period of composting, and can be used for degrading the high-temperature-resistant lignocellulose degrading bacterium. The rapid degradation of the lignocellulose is promoted, and the method has a relatively good application value in the resource utilization of lignocellulose wastes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a heat-resistant Bacillus agri and its application in lignocellulose degradation. BACKGROUND

[0002] A large amount of agricultural and forestry wastes, including crop straw and fruit branch pruning (such as citrus pruning branches and apple pruning branches), are produced every year. These agricultural and forestry wastes not only contain a large amount of nitrogen, phosphorus, potassium and other nutrients, but also contain a large amount of lignin and crude fiber (Lyu S S, Lyu D G, Du G D, et al. Apple branch decomposition and nutrient turnover in the orchard soil [J]. Bioresources, 2017, 12(2):3108-3121). Lignin is a complex natural organic polymer compound composed of three monomers, coumaric alcohol, conifer alcohol and mustard alcohol, and is the basic framework of higher plants, providing rigidity and strength in plant tissues (JIN Z F, KATSUMATA K S, LAM T B, et al. Covalent linkages between cellulose and lignin in cell walls of coniferous and nonconiferous woods [J]. Biopolymers, 2006, 83(2):103-110). It mainly exists in plant cell walls, with a content of about 20%-35% in woody plants and about 15%-25% in herbaceous plants (Li J J. Preparation and curing mechanism of lignin-activated and modified phenolic resin [D]. Beijing Forestry University, 2019). Due to its complex properties, lignin is very slow to degrade naturally and has low effective utilization rate, resulting in great waste of biomass energy. In nature, lignin is mainly degraded by fungi, bacteria and other organisms and non-biological factors (Wang X X, Qi L H, Song J F, et al. Screening of cellulose and lignin degrading bacteria and their effects on composting of bacterial residue [J]. Henan Agricultural Science, 2024, 53(9):66-79. DOI:10.15933 / j.cnki.1004-3268.2024.09.007).

[0003] Composting is a common and effective method for treating agricultural and forestry wastes. Microorganisms that produce laccase, lignin peroxidase, manganese peroxidase and cellulase can degrade lignocellulose and realize its reuse, converting organic matter into humus and obtaining environmentally friendly and economically beneficial products.

[0004] A prolonged high-temperature phase exists during composting. High temperatures not only kill harmful microorganisms, weed seeds, and roundworm eggs in the raw materials, but are also crucial for the rapid degradation of lignin. Some scholars (AMT, BMV, A AH, et al. Biodegradation of lignin in a compost environment: a review[J]. Bioresource Technology, 2000, 72( 2):169-183.DOI:10.1016 / S0960-8524(99)00104-2.) believe that 40-50℃ is the optimal temperature for lignin degradation in compost. Controlling the compost temperature at 50℃ for 35 days can achieve a lignin degradation rate as high as 70%. However, when the temperature is no longer controlled, lignin degradation slows down. But the high-temperature phase also inhibits the population size and activity of microorganisms, thus affecting the lignin degradation efficiency. Therefore, screening heat-resistant lignin-degrading strains and inoculating them during composting increases the number and activity of microorganisms during the high-temperature period of composting, thereby promoting the rapid degradation of lignin and effectively improving the composting process and efficiency. Summary of the Invention

[0005] This invention provides a strain of Tobacco glutamate and its application in the degradation of lignocellulose.

[0006] This invention isolated and screened a heat-resistant lignocellulose-degrading strain WT08 from high-temperature samples of crushed peach branches and sheep manure compost and the finished fertilizer product. Upon identification, this strain belongs to *Bacillus glutamate* (Tobacco glutamic acid bacteria). Glutamicibacter nicotianae This strain can tolerate high temperatures and still produce laccase, lignin peroxidase, cellulase, and manganese peroxidase at 50℃. The manganese peroxidase activity reached 16.05 U / mL on the 8th day of culture, providing an effective strain resource for the development of lignocellulose-degrading microbial agents for agricultural and forestry waste composting.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides *Bacillus nicotine* (Nicotiana glutamate) Glutamicibacter nicotianae WT08, this strain was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Bacillus nicotine*. Glutamicibacter nicotianae The accession number is CGMCC No. 35741.

[0009] Secondly, the present invention provides a microbial preparation comprising the *Tobacco glutamate* (Bacillus tobaccosifolius). Glutamicibacter nicotianae WT08.

[0010] The above-mentioned microbial preparations may be liquid or solid preparations, except that they contain the aforementioned *Tobacco glutamate* (…). Glutamicibacter nicotianae In addition to WT08, it may also contain excipients permitted in the field of microbial preparations.

[0011] In this invention, permitted excipients in the field of microbial preparations include carriers (adsorbents), protective agents / stabilizers, and nutritional supplements. The carrier can be an organic or inorganic carrier. Organic carriers include, but are not limited to, peat moss, humic acid, wheat bran, and rice husks; inorganic carriers include, but are not limited to, diatomaceous earth, zeolite powder, bentonite, kaolin, biochar, and vermiculite; protective agents / stabilizers can be sugars (e.g., trehalose, sucrose), alcohols (e.g., glycerol, mannitol), and amino acids (e.g., glutamic acid, proline); nutritional supplements can be nitrogen sources (e.g., yeast extract, soybean meal, ammonium sulfate), carbon sources (e.g., glucose, starch), and trace elements.

[0012] Thirdly, the present invention provides a lignocellulose-degrading microbial agent or a microbial agent for composting, said microbial agent comprising the *Bacillus glutamate* (…). Glutamicibacter nicotianae WT08.

[0013] The active microorganisms in the above-mentioned lignocellulose degrading microbial agents or microbial agents used for composting include *Bacillus glutamicum* (Tobacco glutamate). Glutamicibacter nicotianae In addition to WT08, it may also contain other microorganisms that have the function of degrading lignocellulose.

[0014] The aforementioned bacterial cells may be in liquid or solid form, and in addition to containing live microorganisms, may also contain excipients permitted in the field of microbial preparations.

[0015] Fourthly, the present invention provides a method for preparing the microbial preparation, the lignocellulose-degrading inoculant, or the inoculant for composting, the method comprising: culturing the *Bacillus glutamate* (…). Glutamicibacter nicotianae WT08.

[0016] Preferably, the culture temperature is 28-50℃. Tobacco glutamic acid bacteria (… Glutamicibacter nicotianae WT08 grows well in a temperature range of 28-50℃.

[0017] Fifthly, the present invention provides the *Tobacco glutamic acid bacteria* (… Glutamicibacter nicotianae The use of WT08 or the microbial preparations in the preparation of lignocellulose degradation products or products for composting.

[0018] Preferably, the product is a bacterial agent or a bacterial enzyme compound preparation.

[0019] Sixthly, the present invention provides the *Tobacco glutamate* (… Glutamicibacter nicotianae The use of WT08 or the microbial preparation or the lignocellulose degrading agent or composting agent in the preparation of laccase, lignin peroxidase, cellulase and / or manganese peroxidase.

[0020] Tobacco glutamic acid bacteria ( Glutamicibacter nicotianae WT08 can produce laccase, lignin peroxidase, cellulase and manganese peroxidase during the culture process. Among them, laccase, lignin peroxidase and manganese peroxidase can degrade lignin, and cellulase can degrade cellulose.

[0021] In a seventh aspect, the present invention provides the *Tobacco glutamate* (… Glutamicibacter nicotianae The use of WT08 or the microbial preparation or the lignocellulose degrading agent or the composting agent in the degradation of lignin, cellulose and / or lignocellulose.

[0022] Preferably, the degradation of lignin, cellulose and / or lignocellulose is carried out at 28-50°C.

[0023] Eighthly, the present invention provides the *Tobacco glutamate* (… Glutamicibacter nicotianae The application of WT08 or the microbial preparation or the lignocellulose degrading agent or the agent for composting in composting.

[0024] Preferably, the raw materials for the compost are those containing lignin, cellulose, and / or lignocellulose. The raw materials for the compost include, but are not limited to, agricultural and forestry waste, including crop straw, fruit shells, livestock and poultry manure, vegetable waste, agricultural product processing by-products, logging residues, processing residues, and afforestation pruning materials (dead branches, fallen leaves), etc.

[0025] In a ninth aspect, the present invention provides a method for degrading lignin, cellulose, and / or lignocellulose, the method comprising: reacting the *Bacillus nicotine* (a type of bacteria) with... Glutamicibacter nicotianae WT08 or the microbial preparation or the lignocellulose degrading agent or the composting agent are mixed with raw materials containing lignin, cellulose and / or lignocellulose for treatment.

[0026] Preferably, the treatment is composting.

[0027] Preferably, the raw materials containing lignin, cellulose and / or lignocellulose include, but are not limited to, agricultural and forestry waste, wherein the agricultural and forestry waste includes crop straw, fruit shells, livestock and poultry manure, vegetable waste, agricultural product processing by-products, logging residues, processing residues, afforestation pruning materials (dead branches, fallen leaves), etc.

[0028] The beneficial effects of this invention include at least the following: The *Tobacco Glutamate Bacillus* WT08 provided by this invention can tolerate high temperature growth, growing well at 28-53℃, and can produce laccase, lignin peroxidase, cellulase and manganese peroxidase under high temperature conditions. It is a heat-resistant lignocellulose degrading bacterium that can be used for composting agricultural and forestry waste and other raw materials, increasing the number and activity of microorganisms during the high-temperature period of composting, promoting the rapid degradation of lignocellulose, effectively promoting the composting process and efficiency, and has good application value in the resource utilization of lignocellulose waste. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 The colony morphology (left) and Gram staining (right) of *Bacillus glutamate* WT08 in Example 1 of this invention are shown.

[0031] Figure 2 This is the phylogenetic tree of Tobacco Glutamate Bacillus WT08 in Example 1 of the present invention.

[0032] Figure 3 This is the growth curve of Tobacco Glutamate Bacillus WT08 in Example 1 of the present invention over 4 days at different temperatures.

[0033] Figure 4 This is the curve showing the change in laccase activity of Tobacco Glutamate Bacterium WT08 during 8 days of culture in Example 2 of the present invention.

[0034] Figure 5 This is the curve showing the change in lignin peroxidase activity of Tobacco Glutamate Bacterium WT08 during 8 days of culture in Example 2 of the present invention.

[0035] Figure 6 This is the curve showing the change in manganese peroxidase activity of Tobacco glutamate bacterium WT08 during 8 days of culture in Example 2 of the present invention.

[0036] Figure 7This is the curve showing the change in cellulase activity of *Bacillus glutamate* WT08 during 8 days of culture in Example 2 of the present invention.

[0037] Figure 8 This is a schematic diagram of the fermentation and composting process in Embodiment 3 of the present invention.

[0038] Figure 9 This is the fermentation temperature change curve under sterile conditions in Example 3 of the present invention.

[0039] Figure 10 This is the fermentation temperature change curve under the condition of having a microbial agent in Example 3 of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Example 1 Screening of heat-resistant lignocellulose-degrading strains 1. Experimental Methods: 1.1 Materials, Culture Media and Reagents 1.1.1 Source of Materials The test samples were taken from a composting plant in Pinggu District, Beijing. The samples were made from crushed peach branches and sheep manure composted at high temperature, as well as finished fertilizer products. Five samples were randomly selected, mixed, and stored.

[0042] 1.1.2 Culture medium LB basal medium: 5g yeast extract, 10g peptone, 10g NaCl, 15g agar, 1000mL deionized water, pH natural.

[0043] Lignin screening medium: 5g alkali lignin, 1g NaNO3, 1.2g Na2HPO4, 0.9g KH2PO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 15g agar, 1000mL distilled water, pH natural.

[0044] Cellulose screening medium: CMC-Na 5g, NaNO3 1g, Na2HPO4 1.2g, KH2PO4 0.9g, MgSO4·7H2O 0.5g, NaCl 0.5g, agar 15g, distilled water 1000mL, pH natural.

[0045] Fermentation medium: CMC-Na 5g, alkali lignin 2g, NaNO3 1g, Na2HPO4 1.2g, KH2PO4 0.9g, MgSO4·7H2O 0.5g, NaCl 0.5g, distilled water 1000mL, pH natural.

[0046] 0.4% guaiacol medium: 4 mL guaiacol, 1000 mL LB medium.

[0047] 0.1% Aniline Blue Medium: 1g Aniline Blue, 1000mL LB Medium.

[0048] Manganese chloride medium: MnCl2·4H2O 0.15 g, LB medium 1000 mL.

[0049] Brilliant Blue medium: 1g Brilliant Blue, 1000mL LB medium.

[0050] Cellulose medium: After the colonies grow on the cellulose medium, they are stained with Congo red solution.

[0051] 1.1.3 Reagents Physiological saline (0.85%): NaCl 0.85g, deionized water 100mL.

[0052] 1 g / L Congo Red solution: 1 g Congo Red, 1000 mL deionized water.

[0053] 1 mol / L NaCl solution: 58.5 g NaCl, 1000 mL deionized water.

[0054] 1.2. Screening of heat-resistant lignocellulose-degrading strains 1.2.1 Initial screening of heat-resistant lignocellulose-degrading strains Take 10g of compost sample, add 90mL of 0.85% physiological saline, and incubate at 28℃ and 130r / min with shaking for 30min. Dilute the bacterial suspension to 10. -4 10 -6 10 -8 Each gradient was performed in triplicate. 100 μL of each gradient was spread onto a solid selection medium of cellulose or lignin and incubated at 28°C until a single colony could be picked. Single colonies of different morphologies were picked, purified three times, and inoculated onto slant agar plates for storage at 4°C.

[0055] Select morphologically distinct and vigorous strains from the isolated strains, inoculate them into cellulose or lignin selection media, and purify them three times. Retain the vigorous strains for later use.

[0056] 1.2.2 Secondary screening of high-temperature lignocellulose-degrading strains After activating the strains preserved from the initial screening, they were inoculated into CMC-Na medium, guaiacol medium, aniline blue medium, brilliant blue medium, and manganese chloride medium using the spot inoculation method. The inoculation diameter was about 5 mm. The cultures were incubated at 50°C, and the colony growth status and the color change of the medium were observed.

[0057] 1.3. Identification of Lignocellulose-Degrading Strains 1.3.1 Morphological identification of lignocellulose-degrading strains The strain WT08 obtained above was inoculated onto LB solid medium and placed in a constant temperature incubator at 50℃. The colony morphology was observed, Gram staining was performed, and the cell morphology was observed under an optical microscope.

[0058] 1.3.2 Molecular identification of lignocellulose-degrading strains Total DNA was extracted from strain WT08 using Tiangen Biotech's bacterial genomic DNA extraction kit. Using the extracted total DNA as a template, PCR amplification was performed using the universal 16S rDNA primers 27F (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.3: 5'-CTACGGCTACCTTGTTACGA-3'). The PCR reaction system and amplification program are shown in Tables 1 and 2, respectively.

[0059] Table 1 PCR reaction system PCR reaction system Volume Total volume 25 μL Template DNA 0.5-1 μL 2x power Taq PCR Master Mix 12.5 μL 27F 0.5 μL 1492R 0.5 μL ddH2O Supplement to 25 μL Table 2 Amplification Procedure

[0060] After amplification, 4 μL of the PCR product was taken and detected by electrophoresis on a 1% agarose gel. The remaining amplification product was sent to Beijing Biomed Gene Technology Co., Ltd. for sequencing. The sequencing results were compared and analyzed in NCBI. The phylogenetic tree of strain WT08 was constructed using the neighbor-joining method with the software Mega 7.0.

[0061] 1.4. Temperature tolerance and high-temperature growth of lignocellulose-degrading bacteria WT08 Strain strain WT08 was inoculated into LB liquid medium and spread onto LB plates. The minimum temperature was set at 28℃, the maximum at 78℃, with a temperature interval of 5℃, and each temperature was maintained for 48 hours. The growth of the strain was observed; the appearance of a single colony or turbidity of the bacterial solution indicated the corresponding temperature for its growth or tolerance. WT08 was continuously cultured in LB liquid medium for 4 days, with samples taken daily at each temperature, and the absorbance at λ=600 nm was measured.

[0062] 2. Experimental Results 2.1 Screening of lignocellulose-degrading strains Strain WT08 produced a 6-10 mm fading zone around the colony on aniline blue and brilliant blue solid media, but no obvious fading zone was observed on guaiacol, manganese sulfate, and CMC-Na solid media.

[0063] 2.2 Morphological identification of lignocellulose-degrading strains Figure 1 The morphological characteristics of strain WT08 are known: colony ( Figure 1 (Left) Pale yellow, opaque, smooth, moist, slightly raised, with smooth edges. Gram-positive bacteria ( Figure 1 (Right), the bacterial cells are short rod-shaped.

[0064] 2.3 Molecular identification of lignocellulose-degrading strains The 16S rDNA sequencing results of strain WT08 are shown in SEQ ID NO.1. Figure 2 It can be seen that strain WT08 is related to *Bacillus nicotine*. Glutamicibacter nicotianae (Gene Bank accession No. MK414827.1) clustered in the same branch with 99.93% homology, therefore strain WT08 was identified as *Bacillus nicotine*. Glutamicibacter nicotianae ).

[0065] Tobacco glutamic acid bacteria ( Glutamicibacter nicotianae WT08 was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Bacillus nicotine*. Glutamicibacter nicotianae The accession number is CGMCC No. 35741.

[0066] 2.4 Temperature tolerance and high-temperature growth of lignocellulose-degrading bacteria Based on the results of liquid culture medium and plate tests, strain WT08 can tolerate a maximum temperature of 53℃ for growth. Figure 3 As shown, strain WT08 grows well between 28-53℃, with an optimal growth temperature of 43℃. Growth gradually slows down after 53℃ and stops at 58℃.

[0067] Example 2: Determination of enzyme activities related to the heat-resistant lignocellulose-degrading strain WT08 1. Experimental Methods 1.1 Solutions and Buffers (1) Solution required for laccase (Lac) activity assay Sodium acetate buffer (pH=4.5): Weigh 9.8g of glacial acetic acid, add about 900mL of deionized water, adjust the pH to 4.5 with sodium hydroxide, bring the volume to 1000mL, and then accurately adjust the pH to 4.5 with sodium hydroxide.

[0068] ABTS solution (0.0005 mol / L): Weigh 0.2743 g ABTS and dilute to 1000 mL with deionized water.

[0069] (2) Solution required for lignin peroxidase (LiP) activity assay Azure B (0.16 mmol / L): Take 0.0489 g of Azure B and add deionized water to make up to 1000 mL.

[0070] Sodium tartrate buffer (50 mmol / L, pH=3.0): 7.5045 g tartaric acid, add 900 mL deionized water, adjust pH=3.0 with sodium hydroxide, bring the volume to 1000 mL with deionized water, and accurately adjust pH=3.0 with sodium hydroxide.

[0071] Hydrogen peroxide starter: 22.8 μL of 30% hydrogen peroxide was diluted with deionized water to a final volume of 250 mL.

[0072] (3) Solution required for manganese peroxidase (MnP) activity assay Malonic acid buffer (50 mmol / L): Add 1.0406 g malonic acid to 800 mL of deionized water, adjust the pH to 4.5 with sodium hydroxide, bring the volume to 1000 mL with deionized water, and then accurately adjust the pH to 4.5 with sodium hydroxide.

[0073] MnSO4 (10 mmol / L): Take 1.6902 g of MnSO4 and dilute to 1000 mL with deionized water.

[0074] 2,6-DMP (10 mmol / L): Take 1.5417 g of 2,6-DMP and dilute to 1000 mL with deionized water.

[0075] Hydrogen peroxide initiator: 100 μL of 30% hydrogen peroxide, diluted to 100 mL with deionized water.

[0076] (4) Solution required for cellulase activity assay Citrate buffer: Dissolve 0.966g of citric acid monohydrate in 150mL of deionized water, add 1.6g of trisodium citrate, and bring the volume to 200mL. Adjust the pH to 4.8.

[0077] Glucose standard solution (1g / L): Glucose was dried at 105℃ to constant weight, and 1g of dried glucose was taken and diluted to 1000mL.

[0078] CMC-Na buffer solution (5g / L): 5g CMC-Na was diluted to 1000mL with sodium citrate buffer and sterilized at 121℃ for 20min.

[0079] DNS solution (prepared using the Ghose method): Solution A: Dissolve 6.9g of crystalline phenol in 15.2mL of 10% NaOH solution, dilute with distilled water to 69mL, add 6.9g of sodium bisulfite to the solution and dissolve; Solution B: Dissolve 255g of potassium sodium tartrate in 300 mL of 10% NaOH solution, and then add 880 mL of 1% 3,5-dinitrosalicylic acid solution; Mix solutions A and B, store in a brown bottle for 7-10 days before use. Shelf life is 1 year when stored in a brown bottle.

[0080] 1.2 Preparation of crude enzyme solution and method for calculating enzyme activity *Bacillus glutamate* WT08 was inoculated into fermentation medium at a 1% inoculum and cultured at 50℃ and 130 rpm for 8 days. The culture broth was centrifuged at 4000 rpm for 15 min, and the supernatant was used as the crude enzyme solution. Three replicates were performed. The fermentation medium formula was as follows: CMC-Na 5g, alkali lignin 2g, NaNO3 1g, Na2HPO4 1.2g, KH2PO4 0.9g, MgSO4·7H2O 0.5g, NaCl 0.5g, distilled water 1000mL, pH natural.

[0081] The methods for calculating the enzyme activities of laccase, lignin peroxidase, manganese peroxidase, and cellulase in the crude enzyme solution are as follows:

[0082] In the formula: n - the dilution factor of the enzyme solution during the determination; ε - the molar extinction coefficient; Vtotal - the total reaction volume; Venzyme - the volume of enzyme solution in the reaction system.

[0083] 1.3 Laccase activity assay Lac activity was determined using ABTS as a substrate. The reaction mixture (3 mL) consisted of 1 mL each of sodium acetate buffer, crude enzyme solution, and ABTS solution. The mixture was incubated at 37°C for 6 min, and the absorbance was measured at 420 nm. The molar extinction coefficient of the ABTS oxidation product was 36000 mol / L. -1 cm -1One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of ABTS per minute.

[0084] 1.4 Determination of manganese peroxidase activity MnP activity was determined using 2,6-DMP as a substrate. The reaction system consisted of 3.36 mL sodium malonate buffer (pH = 4.5), 0.2 mL MnSO4 solution, 0.2 mL enzyme solution, 0.2 mL 2,6-DMP, and 0.04 mL H2O2 to initiate the reaction. After incubating at 37°C for 6 min, the absorbance at 469 nm was measured. The molar extinction coefficient of the 2,6-DMP oxidation product at 469 nm was 55000 (mol / L). -1 cm -1 One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of substrate per minute.

[0085] 1.5 Determination of lignin peroxidase activity LiP activity was determined using Azure B as a substrate. 2.0 mL of tartrate buffer (pH=3.0), 0.5 mL of Azure B solution, and 1.0 mL of crude enzyme solution were added. 0.5 mL of H₂O₂ was added to initiate the reaction. After incubation at 37℃ for 20 min, the rate of decrease in absorbance at 651 nm was measured within 20 min. One unit of enzyme activity (U) was defined as a decrease of 0.1 OD units per milliliter of crude enzyme solution per minute.

[0086] 1.6 Cellulase Activity Assay Glucose standard curve: Take six test tubes and add 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of 1 g / L glucose standard solution, respectively, and then add water to a total volume of 2 mL. Add 2 mL of DNS solution to each tube, incubate in a boiling water bath for 5 min, cool to room temperature, and then take 1 mL of the solution from each tube and dilute to 5 mL with deionized water. Using the 0 mL test tube solution as the zero point, measure the OD value at 540 nm. Plot a standard curve with OD value on the ordinate and glucose concentration on the abscissa.

[0087] A unit of cellulase activity is defined as the amount of reducing sugar equivalent to 1 mg of glucose produced by hydrolyzing the corresponding substrate in 1 mL of enzyme solution within 30 min at 50℃. This is defined as 1 unit of enzyme activity, expressed as U / mL.

[0088] 2. Experimental Results 2.1 Lac activity The culture medium was incubated at 50℃ and 130 rpm for 8 consecutive days. Samples were taken every two days to test the laccase activity of strain WT08. The results are as follows: Figure 4As shown, this strain possesses laccase activity, which was detected at 0.109 U / mL on day 6.

[0089] 2.2 Lignin peroxidase activity The culture medium was incubated at 50℃ and 130 rpm for 8 consecutive days. Samples were taken every two days to detect the lignin peroxidase activity of strain WT08. The results are as follows: Figure 5 As shown, this strain still has lignin peroxidase activity at 50℃, and the lignin peroxidase activity was detected to be 0.024 U / mL on the 4th day.

[0090] 2.3 Manganese peroxidase activity The culture medium was incubated at 50℃ and 130 rpm for 8 consecutive days. Samples were taken every two days to detect the manganese peroxidase activity of strain WT08. The results are as follows: Figure 6 As shown, strain WT08 exhibits manganese peroxidase activity at 50°C, which rapidly increases to 16.05 U / mL on day 8.

[0091] 2.4 Cellulase Activity The culture medium was incubated at 50℃ and 130 rpm for 8 consecutive days. Samples were taken every two days to detect the cellulase activity of strain WT08. The results are as follows: Figure 7 As shown, strain WT08 was detected to have changes in cellulase activity at 50℃, with the highest value of 0.47 U / mL on day 4.

[0092] In summary, this invention isolated and screened a heat-resistant lignocellulose-degrading strain, WT08, which is a Gram-positive, short rod-shaped bacterium. 16S rDNA identification revealed that strain WT08 is related to *Bacillus tobacco glutamate* (…). Glutamicibacter nicotianae The strain (Gene Bank accession No. MK414827.1) showed 99.93% homology, therefore it was identified as *Bacillus tobaccomi*. Glutamicibacter nicotianae This *Glutamicinus tobaccoensis* strain was named WT08. *Glutamicinus tobaccoensis* WT08 grows well between 28-53℃, and can withstand temperatures up to 53℃. At 50℃, it still exhibits laccase, lignin peroxidase, cellulase, and manganese peroxidase activities. The manganese peroxidase activity reached 16.05 U / mL on day 8 of cultivation and showed a continuing upward trend. This strain provides an effective microbial resource for the development of high-temperature lignocellulose-degrading inoculants for agricultural and forestry waste composting.

[0093] Example 3 Composting fermentation experiment of heat-resistant lignocellulose degrading bacteria WT08 This embodiment utilizes *Bacillus nicotinic acid bacteria* (… Glutamicibacter nicotianaeComposting fermentation experiments were conducted using WT08. The original fermentation method in the Tuancheng Lake regulating pond utilized microorganisms from the natural environment. However, due to the difficulty in degrading lignocellulose, the composting cycle was long and the fermentation effect was poor. Natural fermentation experiments were conducted in 2019, requiring 1-3 years for complete decomposition. The decomposition standard referenced DB11 / T 840-2011, and the maturity assessment indicators are shown in Table 3. To address the above problems, a heat-resistant lignocellulose-degrading bacterium—*Bacillus tobaccomi* (…)—was used. Glutamicibacter nicotianae WT08 was used as a composting fermentation agent in a composting fermentation experiment to explore an efficient fermentation method. The specific methods and results are described below.

[0094] Table 3 Indicators for Determining Maturity Regarding the fermentation and composting treatment of green waste, the C / N ratio and moisture content are key influencing factors. After the green waste is crushed, the C / N ratio and moisture content are adjusted. An experiment using a C / N ratio of 50:1 was conducted. Before fermentation, the moisture content was adjusted to 50%-60%. When the temperature initially rose to 55℃-60℃, the mixture was stirred. While maintaining the temperature at 55℃-65℃, the moisture content was kept at 50%-60%. Subsequently, as the temperature decreased, stirring was performed every 7-12 days when the temperature fell below 55℃. Stirring was stopped when the temperature dropped below 35℃ and the temperature difference for two consecutive days did not exceed ±2℃. Studies by Chen Weiling, Yang Ning, and others have found that maintaining a fermentation temperature of 55℃ for more than 3 days or 50℃ for 5-7 days can ensure that pathogenic microorganisms in the compost are basically killed. This is an important condition for ensuring the harmlessness and maturity of compost (Chen Weiling, Li Shenghua. Effects of different composting measures on organic matter and biological indicators of tree branch compost [J]. Guangzhou Environmental Science, 2011, 26(1): 35-40; Yang Ning, Liu Hongquan, Sun Fangfang. Effects of different microbial agents on the composting effect of garden greening waste [J]. Forest Resources Cultivation, 2015, 29(4): 53-57.). In HJ 1266-2022, a higher standard requirement is given for the temperature of composting, as detailed in Table 4. In this experiment, this is used as the standard for whether the composting is complete.

[0095] Table 4 Temperature and Duration Requirements in the Primary Fermentation Unit for Composting For the specific process flow of fermentation and composting, please refer to Figure 8 Fermentation experiments were conducted with and without a microbial agent (containing *Bacillus glutamate* WT08). The temperature changes during the fermentation process are as follows: Figure 9 and Figure 10 As shown.

[0096] The composition of the greening compost obtained by composting with and without sterile agents was tested by PONY Testing. The test results are shown in Table 5 (sterile agent) and Table 6 (sterile agent), respectively.

[0097] Table 5

[0098] Table 6

[0099] Analysis of the above fermentation test data and test results shows that: 1. Without a sterile agent, fermentation time was 8 days above 55℃ and 0 days above 65℃, failing to fully meet the relevant standards of HJ1266-2022. With a sterile agent, fermentation time was 10 days above 55℃ and 4 days above 65℃, meeting the standard for complete decomposition. Furthermore, the compost temperature rose rapidly, significantly improving fermentation efficiency. Therefore, the degree of fermentation and decomposition with a sterile agent is significantly better than that without one.

[0100] 2. The compost produced by fermentation with and without microbial agents fully meets the national standards for organic substrates and is close to the standards for organic fertilizers (NY / T525-2021), making it an excellent organic nutrient substrate. However, the total nutrient content is higher with microbial agents than without. Furthermore, the seed germination index reaches 107.2 with microbial agents, significantly higher than that without. This indicates that the green waste is fully decomposed with microbial agents, far exceeding the degree of decomposition without them, and the compost has higher application value.

[0101] 3. After adding the microbial agent, the fermentation process can be completed in 15 days. After 30 days in the aging box, it can be put into use. After using the microbial agent, the fermentation cycle of the traditional fermentation method is shortened from 1 year to 45 days, which greatly improves the fermentation efficiency and accelerates the resource conversion of green waste.

[0102] Fermented and decomposed materials play important roles: First, the bio-organic substrate produced by fermentation can replace chemical fertilizers in green spaces, fundamentally avoiding the risk of water pollution from chemical fertilizers; furthermore, the high-temperature fermentation process kills pathogens in green waste, preventing their survival and spread, and significantly reducing pesticide use. Second, bio-organic substrates improve soil, increasing soil permeability and organic matter content, achieving carbon sequestration and reducing carbon dioxide emissions. Third, decomposed substrates can be used to cultivate mushrooms, enriching the understory ecological community. Fourth, conducting ecological substrate seedling experiments explores new methods of "soilless cultivation," expands new ideas for plant propagation, and saves on maintenance costs.

[0103] The utilization of *Bacillus glutamate* WT08 for the resource utilization of landscaping waste has yielded significant results. Nearly 300 tons of garden waste were processed using high-temperature fermentation technology, producing 260 tons of bio-organic substrate, replacing approximately 1200 kg of chemical fertilizer annually. Simultaneously, it saves approximately 80,000 yuan annually in transportation and disposal costs. Furthermore, it reduces the risk of algal toxin accumulation and lowers drinking water source treatment costs, demonstrating significant economic benefits. In addition, it enables the efficient recycling of garden waste, achieving carbon sequestration and reduction, avoiding environmental pollution caused by traditional treatment methods, eliminating safety hazards, stabilizing soil and protecting slopes, enriching biodiversity, and protecting a healthy aquatic ecosystem. Moreover, the fermented material can replace chemical fertilizers to provide nutrients for plants, reduce the risk of water pollution, protect water quality, and create a superior aquatic environment, demonstrating excellent ecological benefits.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Tobacco glutamic acid bacteria ( Glutamicibacter nicotianae WT08, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35741.

2. A microbial preparation, characterized in that, The microbial preparation comprises *Bacillus nicotine* as described in claim 1. Glutamicibacter nicotianae WT08.

3. A lignocellulose-degrading microbial agent or a microbial agent for composting, characterized in that, The microbial agent comprises *Bacillus nicotine* as described in claim 1. Glutamicibacter nicotianae WT08.

4. The method for preparing the microbial preparation according to claim 2 or the lignocellulose-degrading microbial agent according to claim 3 or the microbial agent for composting, characterized in that, The method includes: culturing the *Tobacco glutamate* bacteria as described in claim 1 (… Glutamicibacter nicotianae WT08; Preferably, the culture temperature is 28-50℃.

5. The *Tobacco glutamate* strain described in claim 1 (… Glutamicibacter nicotianae The use of the microbial preparation described in WT08 or claim 2 in the preparation of lignocellulose degradation products or products for composting.

6. The *Tobacco glutamate* strain described in claim 1 (… Glutamicibacter nicotianae The use of the microbial preparation of WT08 or claim 2 or the lignocellulose degrading agent of claim 3 or the composting agent in the preparation of laccase, lignin peroxidase, cellulase and / or manganese peroxidase.

7. The *Tobacco glutamate* strain described in claim 1 (… Glutamicibacter nicotianae The application of the microbial preparation of claim WT08 or claim 2 or the lignocellulose-degrading microbial agent of claim 3 or the microbial agent for composting in the degradation of lignin, cellulose and / or lignocellulose.

8. The Tobacco Glutamate Bacillus (Tobacco Glutamate Bacillus) according to claim 1 Glutamicibacter nicotianae The application of the microbial preparation of WT08 or claim 2 or the lignocellulose degrading agent of claim 3 or the microbial agent for composting in composting.

9. A method for degrading lignin, cellulose, and / or lignocellulose, characterized in that, The method includes: administering the *Tobacco glutamate* (as described in claim 1) Glutamicibacter nicotianae The microbial preparation described in WT08 or claim 2, or the lignocellulose-degrading microbial agent described in claim 3, or the microbial agent for composting, is mixed with raw materials containing lignin, cellulose, and / or lignocellulose for treatment.

10. The method according to claim 9, characterized in that, The treatment is composting.

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