Straw degrading bacterium and application thereof

Abstract

The invention provides a straw degrading bacterium and application of the straw degrading bacterium. The straw degrading bacterium is prepared from microbacterium sp. SDM28 (Microbacterium sp. SDM28) and bacillus (Bacillus sp. SDM29). Through mutual cooperation of microbacilli and bacillus, during straw degradation treatment, compound bacteria can synergistically generate high-activity cellulose degradation related enzymes, lignocellulose structures in straw can be efficiently decomposed, and especially the content of hemicellulose components can be remarkably reduced; the problems that existing straw degrading microorganisms are low in degrading efficiency and difficult to decompose complex lignocellulose structures are solved; meanwhile, the compound bacteria have mild requirements on environmental conditions, the application range focuses on the high-yield straw type of rice straw, large-scale application can be realized without complex regulation and control, resource waste and environmental pollution caused by slow natural decomposition after straw returning to the field are reduced, the resource utilization process of the straw can be promoted, and the economic benefit is increased. The requirements of large-scale and efficient straw treatment can be met.

Description

Technical Field

[0001] This invention relates to the field of agricultural straw waste treatment and microbial application technology, and in particular to a straw-degrading bacterium and its application. Background Technology

[0002] my country is the world's largest producer of crop straw, with an annual output of approximately 900 million tons, accounting for about one-fifth of the global total. Rice straw accounts for about 24% of this output. Rich in organic matter and nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium, rice is an important renewable biological resource. However, due to the short crop rotation period and the complex composition of straw, its natural decomposition after being returned to the field is slow, influenced by climatic conditions and soil environment. The large amount of waste generated in agricultural production, if not properly handled, can lead to resource waste and environmental pollution.

[0003] Currently, the main methods for straw treatment include physical methods (such as crushing and returning to the field), chemical methods (such as using chemical reagents for degradation), and biological methods. Among them, biological methods have received much attention due to their advantages such as environmental protection and low cost. However, straw has a complex composition, mainly composed of cellulose, hemicellulose, and lignin. These three main components form a lignocellulose structure that is difficult for microorganisms to decompose under natural conditions. Moreover, existing straw-degrading microorganisms have problems such as low degradation efficiency, narrow applicability, and stringent environmental requirements, making it difficult to meet the needs of large-scale and efficient straw treatment.

[0004] Therefore, it is necessary to provide a straw-degrading bacterium and its application to solve the above-mentioned technical problems. Summary of the Invention

[0005] This invention provides a straw-degrading bacterium and its application, which solves the problems of existing straw-degrading microorganisms having low degradation efficiency, narrow applicability, and demanding environmental conditions, making it difficult to efficiently decompose the complex lignocellulose structure of straw and meet the needs of large-scale, rapid processing of crop straw such as rice straw.

[0006] To solve the above-mentioned technical problems, the present invention provides a straw-degrading bacteria, comprising:

[0007] Microbacterium sp. SDM28 and Bacillus sp. SDM29.

[0008] The microbacterium is deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: October 13, 2025, address of deposit: Wuhan University, Wuhan, China, accession number: CCTCCNO: M20252205;

[0009] The Bacillus species is deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: October 13, 2025, address of deposit: Wuhan University, Wuhan, China, accession number: CCTCCNO: M202522056.

[0010] Preferably, the 16 rDNA nucleotide sequences of the microbacteria and Bacillus are as shown in SEQ ID NO. 1.

[0011] Preferably, after the microbes are cultured on carboxymethyl cellulose solid medium, the colonies exhibit a specific morphology, specifically being round, yellow, and opaque with neat edges and a diameter of 0.5-1.0 mm. Under an electron microscope, the bacterial cells are rod-shaped and have flagella. When the Bacillus is cultured on carboxymethyl cellulose solid medium, it can form colonies with distinct characteristics, being pale yellow, opaque, with a smooth surface, relatively neat edges, and a diameter between 1.0-1.5 mm. Under an electron microscope, the bacterial cells are short rod-shaped with spore structures, and the spores are located in the middle or off-center of the bacterial cell.

[0012] To address the above problems, the present invention also provides a microbial preparation containing the aforementioned straw-degrading bacteria, wherein the microbial preparation is a mixture of microbacteria and Bacillus in a 1:1 ratio.

[0013] Preferably, the degrading bacteria or the microbial preparation is applied to crop straw.

[0014] Preferably, the degrading bacteria or the microbial preparation is used in the preparation of products that degrade crop straw.

[0015] Preferably, the crop straw is rice straw.

[0016] To address the above problems, the present invention also provides a product for degrading crop straw, wherein the product contains the straw-degrading bacteria or the microbial preparation.

[0017] To address the aforementioned problems, the present invention also provides a method for degrading crop straw, characterized in that the straw-degrading bacteria or the microbial preparation is applied to the crop straw.

[0018] Preferably, the crop straw is rice straw.

[0019] Compared with related technologies, the straw-degrading bacteria and their application provided by this invention have the following beneficial effects:

[0020] This invention provides a straw-degrading bacterium and its application. Through the synergistic interaction of Microbacterium and Bacillus, the composite bacteria can synergistically produce highly active cellulose-degrading enzymes during straw degradation treatment. This can efficiently decompose the lignocellulose structure in straw, especially significantly reducing the content of hemicellulose components. This avoids the problems of low degradation efficiency and difficulty in decomposing complex lignocellulose structures found in existing straw-degrading microorganisms. At the same time, this composite bacteria has mild environmental requirements and is applicable to rice straw, a high-yield straw type. It can be applied on a large scale without complex control, reducing resource waste and environmental pollution caused by slow natural decomposition of straw after returning it to the field, while promoting the resource utilization process of straw. This can meet the needs of large-scale and efficient straw treatment. Attached Figure Description

[0021] Figure 1 A schematic diagram of the morphology of the degrading bacteria Microbacterium sp. SDM28 and Bacillus sp. SDM29 provided by the present invention on carboxymethyl cellulose solid medium;

[0022] Figure 2 A schematic diagram of the phylogenetic tree of the degrading bacteria Microbacterium sp. SDM28 and Bacillus sp. SDM29 provided by the present invention;

[0023] Figure 3 A schematic diagram of the cellulase activity of the degrading bacteria Microbacterium sp. SDM28 and Bacillus sp. SDM29 provided by the present invention;

[0024] Figure 4 A schematic diagram of enzyme activity changes during straw degradation provided by this invention;

[0025] Figure 5 A schematic diagram of the degradation rate of rice straw provided by this invention;

[0026] Figure 6 A schematic diagram illustrating the changes in the component content of rice straw provided by this invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field; unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0028] Example 1

[0029] This invention involves culturing rice straw residues after 1 month and 4 months of degradation in an enrichment medium with shaking. Highly efficient rice straw decomposing strains are initially screened using a solid screening medium. The selected strains are then identified, their cellulase activity is measured, and they undergo Congo red staining experiments. The strains are then applied to the liquid degradation of rice straw, and the enzyme activity, straw component content, and straw degradation rate are measured during the degradation process. One strain of *Microbacterium* and one strain of *Bacillus* are obtained. The culture of *Microbacterium* is named *Microbacterium* sp. SDM28. This culture was received and registered by the China Center for Type Culture Collection on October 13, 2025, and will be preserved for thirty years from that date, with an extension of five years upon request for sample provision before the expiration date. The viability of this culture was tested by the China Center for Type Culture Collection on October 20, 2025, and the result was positive. The preservation number is CCTCC NO: M20252205.

[0030] The culture name and distinguishing characteristics of the Bacillus sp. SDM29 were received and registered by the China Center for Type Culture Collection on October 13, 2025, and will be preserved for thirty years from that date, with an extension of five years if a request for a sample is received before the expiration date. The viability of the culture was tested by the collection on October 20, 2025, and the result was positive. The accession number is CCTCC NO: M20252206.

[0031] The isolation, screening, and identification process of the strains Microbacterium sp. SDM28 and Bacillus sp. SDM29 is as follows:

[0032] (1) Weigh 3g of rotten straw sample, cut the straw into 2mm pieces with sterile scissors, transfer it to a 100mL conical flask, add 50mL of enrichment medium, place it in a shaker at 28℃ and 180rpm for 3 days, take it out and shake it well, let it stand at room temperature for about 15min (after it has completely stood), and set the suspension aside for use.

[0033] (2) Initial screening: Under aseptic conditions, 450 μL of the enriched bacterial suspension was added to an Erlenmeyer flask containing 100 mL of screening medium and diluted to a 222× dilution. Each flask was shaken well, and 50 mL of the dilution was poured into a 90 cm culture dish. 160 μL of the liquid was transferred to each well of a 96-well cell culture plate using a pipette. Three cell culture plates were transferred from each flask. Three cell culture plates were transferred from the screening medium solution without the enriched bacterial suspension as a negative control. The culture plates were incubated at 28°C for 3-7 days. During the incubation period, the growth of microorganisms in the 96-well cell culture plates was observed. About 30% of the wells were retained as visibly turbid culture plates. 20 μL of the sample was transferred from each well of the retained culture plates to a 96-well PCR plate using a pipette and serially diluted to 10×. -4 10 -5 For each dilution, take 100 μL and spread it onto solid screening medium. Place the plate in a constant temperature incubator at 28°C and invert it for 3-7 days until a single colony grows.

[0034] (3) Secondary screening: After obvious colonies have grown on the solid screening medium plates, single colonies of different morphologies are picked, streaked and cultured, and continuously streaked in LB medium. The purification and isolation are repeated 3 times to obtain straw-degrading bacterial isolates. Figure 1 The strain was transferred to liquid LB medium and cultured at 28℃ and 180rpm for 1-2 days. 30% sterile glycerol was added, and the bacterial solution and sterile glycerol were mixed in a cryotube at a volume ratio of 2:1. The mixture was then stored in an ultra-low temperature freezer at -80℃ for later use.

[0035] (4) Screening of straw-degrading bacteria by Congo red staining test: Single strains isolated and purified were picked with an inoculation loop and inoculated onto CMC-Na solid medium. Each strain was repeated three times. The strains were incubated upside down in a constant temperature incubator at 28℃ for 2-3 days. After the colonies grew, they were stained with 1 mg / mL Congo red dye solution, so that the dye solution submerged the surface of the colonies. After standing for 30 min, the Congo red dye solution on the surface was discarded. The colonies were washed with 1 mol / L NaCl solution for 20 min to decolorize them, and the waste liquid was discarded. The diameter of the colony (d) and the diameter of the hydrolysis zone (D) around it were measured. The cellulose degradation ability of the strain was initially judged based on the D / d ratio.

[0036] (5) Results: Microbacterium sp. SDM28 and Bacillus sp. SDM29 showed clear zones on Congo red staining plates. The ratios of the diameter D of the clear zone to the diameter d of the colony were 5.61±0.92 and 8.09±2.92, respectively, indicating that they had straw degradation capabilities. Two strains were obtained through isolation and screening tests and named SDM28 and SDM29.

[0037] Congo red staining test results

[0038]

[0039] The culture media used in the above steps are as follows:

[0040] (1) Enrichment medium: 5g microcrystalline cellulose, 5g tryptone, 5g NaCl, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 1L deionized water, pH natural;

[0041] (2) Liquid screening medium: 5g sodium carboxymethyl cellulose (CMC-Na), 2g yeast extract, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 1L deionized water, pH natural;

[0042] (3) Solid screening medium: CMC-Na 5g, yeast extract 2g, K2PO4 0.5g, MgSO4·7H2O 0.5g, agar 20g, deionized water 1L, pH natural;

[0043] (4) LB (Luria-Bertani) medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1L deionized water (solid medium with 20g agar), pH natural;

[0044] (5) CMC-Na solid culture medium: CMC-Na 2g, (NH4)2SO4 2g, KH2PO4 1g, MgSO4·7H2O 0.5g, agar 20g, distilled water 1L, pH natural;

[0045] Single colonies of the two selected bacterial strains were picked and cultured in 50 mL LB broth with shaking for 12 h. 2 mL of each colony was transferred to a 10 mL centrifuge tube and sent to Anhui General Biotechnology Co., Ltd. for DNA extraction. The DNA was amplified and sequenced using the universal primers 27F / 1492R for bacterial 16S rRNA gene. The 16S rRNA sequence information of the two strains and other strains was searched in NCBI, and sequence alignment was performed. Mega11.0 software was used for analysis and phylogenetic tree construction. SDM28 was grouped with *Microbacterium*; SDM29 was grouped with *Bacillus*. Figure 2 ).

[0046] Example 2

[0047] Assay of cellulose-degrading enzyme activity of straw-degrading bacteria:

[0048] Single colonies of each strain were picked and cultured in 50 mL LB liquid medium at 28 °C for 24 h in a shaker to prepare the enzyme solution for testing. Each treatment was performed in triplicate, with one control. The β-glucosidase activity (β-GC), filter paper cellulase activity (FPA), and carboxymethyl cellulase activity (CL) of each single strain were determined using an enzyme activity kit (purchased from Suzhou Keming Biotechnology Co., Ltd.). Enzyme activity assays were performed according to the kit instructions. Figure 3 );

[0049] Experimental results: Both Microbacterium sp. SDM28 and Bacillus sp. SDM29 have strong cellulose degradation capabilities. The β-glucosidase, filter paper cellulase, and carboxymethyl cellulase activities of Microbacterium sp. SDM28 were 5.75 nmol / min / mL, 0.26 U / mL, and 23.76 ug / min / mL, respectively; while the three enzyme activities of Bacillus sp. SDM29 were 8.43 nmol / min / mL, 0.16 U / mL, and 25.15 ug / min / mL, respectively.

[0050] Example 3

[0051] Application of straw-degrading bacteria on rice straw:

[0052] Cut the dried straw into 2-3 cm pieces, soak in 1% NaOH for 24 hours, wash with water until the pH is neutral, and then dry for later use. Weigh 3 g of rice straw into a 250 mL Erlenmeyer flask, add 100 mL of sterile propagation medium, and inoculate with 5% bacterial solution. The OD600nm of the straw-degrading bacteria in the bacterial solution is approximately 0.6. Use 5% sterile water instead of the bacterial solution as a blank control (S0). Incubate at 28℃ for 30 days.

[0053] (1) The culture medium used in the above experiments:

[0054] Propagation culture medium: 2g yeast powder, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 1L distilled water.

[0055] (2) Determination of enzyme activity during straw degradation:

[0056] At 6, 15, and 30 days during the degradation process, 2 mL of fermentation broth was collected into centrifuge tubes, and the activities of β-glucosidase (β-GC) and β-xylosidase (β-GC) were measured using a kit. Figure 4 Perform enzyme activity assays according to the kit instructions.

[0057] After 30 days of cultivation, the straw residue was removed, dried at 60℃ to constant weight, and weighed. The degradation rate was calculated based on the mass of the straw before and after degradation. Figure 5 The degradation rate is calculated using the following formula:

[0058] Straw degradation rate = (Original rice straw dry weight (g) - Decomposed rice straw dry weight (g)) / Original rice straw dry weight (g) × 100%

[0059] (3) Determination of component content after straw degradation:

[0060] The dried straw was ground into a fine powder, and the contents of cellulose, hemicellulose, and lignin in the straw were determined using a modified Van Soest washing method (differential gravity method). Figure 6 ).

[0061] Accurately weigh 1g (W1) of rice straw powder into a 100mL Erlenmeyer flask, add 50mL of neutral detergent, heat, and maintain a gentle boil for 1 hour. Filter and wash the sample until neutral, ensuring no neutral detergent residue remains. Wash 2-3 times with acetone, and dry in a 105℃ oven to constant weight, weighing (W2). Add 50mL of acidic detergent to the dried residue, following the same method, and dry and weigh to obtain W3. Then place the dried residue in a beaker and add 5mL of 72% concentrated sulfuric acid that has been chilled in the refrigerator beforehand. Hydrolyze at room temperature for 3 hours, then add 50mL of distilled water and let stand overnight at room temperature. The next day, filter using a pre-weighed sintered crucible (W0), wash with distilled water until neutral, and dry at 105℃ to constant weight, weighing (W0+W4). The straw residue was placed in a muffle furnace along with a sand core crucible and heated at 550°C until the residue ash was obtained, then cooled to constant weight in a desiccator and weighed (W0+W5). The straw component content was calculated using the following formula.

[0062]

[0063]

[0064]

[0065] In the formula, W1 is the original dry weight of straw; W2 is the dry weight after neutral washing; W3 is the dry weight after acid washing; W4 is the dry weight after hydrolysis with 72% sulfuric acid; and W5 is the dry weight after residue restoration.

[0066] The experimental results showed that after treating rice straw with a compound microbial culture of Microbacterium sp. SDM28 and Bacillus sp. SDM29 for 30 days, the β-glucosidase activity of the bacterial solution was 5.56, 4.08, and 6.16 nmol / min / mL on days 6, 15, and 30, respectively; the β-xylosidase activity was 0.68, 1.56, and 2.51 nmol / min / mL, respectively. These enzyme activities were significantly higher than those of the control (CK) without bacterial treatment. After 30 days of treatment with the compound microbial culture, the straw degradation rate was 24.59%, significantly higher than that of the control (12.74%). The compound microbial treatment mainly reduced the hemicellulose content of the straw.

[0067] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A straw-degrading bacterium, characterized in that, include: Microbacterium sp. SDM28 and Bacillus sp. SDM29. The microbacterium is deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: October 13, 2025, address of deposit: Wuhan University, Wuhan, China, accession number: CCTCCNO: M20252205; The Bacillus species is deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: October 13, 2025, address of deposit: Wuhan University, Wuhan, China, accession number: CCTCCNO: M20252206.

2. The straw-degrading bacteria according to claim 1, characterized in that, The 16 rDNA nucleotide sequences of the microbacteria and Bacillus are shown in SEQ ID NO.

1.

3. The straw-degrading bacteria according to claim 1, characterized in that, When cultured on carboxymethyl cellulose solid medium, the *Microbacterium* species exhibits a specific morphology, specifically being round, yellow, and opaque with neat edges and a diameter of 0.5-1.0 mm. Under an electron microscope, the bacterial cells are rod-shaped and flagellated. When cultured on carboxymethyl cellulose solid medium, the *Bacillus* species form colonies with distinct characteristics: pale yellow, opaque, smooth, with relatively neat edges, and a diameter between 1.0-1.5 mm. Under an electron microscope, the bacterial cells are short rod-shaped with spore structures, and the spores are located in the middle or off-center of the bacterial cell.

4. A microbial preparation containing straw-degrading bacteria as described in any one of claims 1-3, wherein the microbial preparation is a mixture of microbacteria and Bacillus in a 1:1 ratio.

5. The application of the microbial preparation according to claim 3 on crop straw.

6. The application of the microbial preparation according to claim 3 in the preparation of products that degrade crop straw.

7. The application according to claim 5 or 6, characterized in that, The crop straw mentioned is rice straw.

8. A product for degrading crop straw, characterized in that, The product contains the straw-degrading bacteria of claim 1 or the microbial preparation of claim 4.

9. A method for degrading crop straw, characterized in that, The straw-degrading bacteria of claim 1 or the microbial preparation of claim 4 are applied to crop straw.

10. The method according to claim 9, characterized in that, The crop straw mentioned is rice straw.

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