A thermophilic bacterial strain B-60-8 for straw degradation and composting and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a thermophilic bacterial strain B-60-8 for straw degradation and composting, and its applications. The thermophilic strain B-60-8 is classified as *Thermobacterium jiuzhiense*, and was deposited on August 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35676. This invention aims to establish a straw composting strategy guided by H2O2 production capacity, and to verify the high efficiency of this bacterium in straw degradation under high-temperature conditions. The goal is to obtain a single-function strain that can adapt to the high-temperature period of straw composting and efficiently break down lignin using H2O2, thereby solving the problems of poor high-temperature adaptability and low lignin degradation efficiency of microbial agents. This provides a new microbial resource for the practical application of straw composting and bio-fertilizer preparation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a thermophilic strain B-60-8 used for the degradation and composting of straw and its applications. Background Technology

[0002] With the development of agricultural production, the treatment and resource utilization of crop straw has become an urgent environmental and agricultural issue. Returning straw to the field is an important way to achieve straw resource utilization, and composting is the key to its success. Microbial decomposition is widely recognized as the most promising straw treatment method due to its environmental friendliness and low cost. Among its key aspects, screening and obtaining functional bacterial strains for straw composting is the core foundation of this method.

[0003] The effective degradation of lignocellulose is the biggest challenge in straw composting. Hydrogen peroxide (H2O2), as an important reactive oxygen species, plays a crucial role in the biodegradation of lignin. It is not only an essential substrate for peroxidase-catalyzed reactions (such as lignin peroxidase and manganese peroxidase), but it can also generate hydroxyl radicals (·OH) through the non-enzymatic Fenton reaction, thereby attacking and cleaving the complex aromatic polymer structure of lignin, creating favorable conditions for the subsequent enzymatic hydrolysis of cellulose and hemicellulose. Therefore, microbial strains capable of stably producing H2O2 are considered an important resource for preparing straw composting inoculants.

[0004] Currently, some studies have reported on strains with H2O2 production capabilities used for biodegradation, such as certain types of Streptomyces (…). Streptomyces spp. ), white rot fungi ( Phanerochaete chrysosporium However, these existing strains still have significant drawbacks when applied to straw composting: they have poor environmental adaptability, low or unstable H2O2 production in the high-temperature environment of composting, and difficulty in continuously providing sufficient substrate for lignin-degrading enzyme systems, resulting in excessively long composting cycles and low efficiency. Summary of the Invention

[0005] To address the shortcomings of existing strains in current technologies, such as poor environmental adaptability, low or unstable H2O2 production in the high-temperature environment of composting, difficulty in continuously providing sufficient substrate for lignin-degrading enzyme systems, leading to excessively long composting cycles and low efficiency, this invention aims to establish a straw composting strategy guided by H2O2 production capacity. By verifying the high efficiency of this strain in straw degradation under high-temperature conditions, this invention seeks to obtain a novel straw composting functional strain with efficient and stable H2O2 production capacity, strong environmental adaptability, and multiple cellulose-degrading enzyme activities. This provides a new microbial resource for the practical application of straw composting and bio-fertilizer preparation. To achieve the above objectives, this invention adopts the following technical solution.

[0006] This invention provides a thermophilic bacterial strain B-60-8 for straw degradation and composting, wherein the thermophilic strain B-60-8 is classified and named *Thermobacterium jiuzhiense* (…). Caldifermentibacillus hisashii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 19, 2025, with accession number CGMCC No. 35676.

[0007] The thermophilic strain B-60-8 (Thermobacterium jiuzhiense) provided by this invention can stably survive and function efficiently in high-temperature composting environments. This indicates strong environmental adaptability; the strain stably produces high levels of H2O2 in the high-temperature composting environment, thus continuously providing sufficient substrate for lignin-degrading enzyme systems, achieving a short composting cycle and high efficiency. Specifically, this strain was isolated from thermophilic fermentation compost. Its inherent thermophilic characteristics allow it to adapt well to and colonize the high-temperature composting period, effectively overcoming the technical bottleneck of suppressed activity or death of ordinary microorganisms during the heating stage, ensuring the continuous and efficient progress of straw composting. In terms of its mechanism of action, this strain produces high concentrations of hydrogen peroxide, utilizing reactive oxygen species-mediated non-enzymatic reactions to attack and break down the complex lignin structure in straw. This mechanism differs from the industry's conventional degradation pathways that rely on the direct secretion of cellulase or ligninase, providing a novel and efficient auxiliary decomposition channel for straw decomposition, thereby significantly improving the overall degradation efficiency of lignocellulose. Furthermore, this invention overcomes the dependence on complex complex microbial communities, achieving the above functions through the application of a single strain. This not only simplifies the preparation process of microbial agents, but also fundamentally ensures the stability of product functions and batch-to-batch consistency, laying the foundation for standardization and large-scale application.

[0008] Preferably, the nucleotide sequence of the 16S rDNA of the thermophilic strain B-60-8 is shown in SEQ ID NO.1:

[0009]

[0010] The present invention also provides the application of the thermophilic strain B-60-8 in the degradation of straw.

[0011] Preferably, the thermophilic strain B-60-8 can utilize H2O2 to degrade lignin, thereby promoting straw decomposition.

[0012] Preferably, the thermophilic strain B-60-8 is able to adapt to the high-temperature period of straw composting; wherein the temperature of the high-temperature period of straw composting is 50℃~60℃.

[0013] Preferably, the thermophilic strain B-60-8 is used to break down lignin under high temperature conditions of 50℃~60℃, thereby degrading straw and achieving straw decomposition.

[0014] Preferably, the thermophilic strain B-60-8 is used to prepare a straw composting inoculant.

[0015] Preferably, the straw composting microbial agent comprises live cells of the thermophilic strain B-60-8, fermentation broth, and / or spore preparation.

[0016] Preferably, the straw composting microbial agent is in the form of powder, granules, or liquid.

[0017] Preferably, when the straw composting microbial agent is in the form of powder or granules, the viable count of the straw composting microbial agent is 10. 8 CFU mL -1 ~10 9 CFU mL -1 When the straw composting microbial agent is in liquid form, the viable count of the straw composting microbial agent is 10. 8 CFU g -1 ~10 9 CFU g -1 .

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention provides a thermophilic bacterial strain B-60-8 for straw degradation and composting. This thermophilic strain B-60-8 can stably survive and function efficiently in high-temperature composting environments, demonstrating strong environmental adaptability. It stably produces high levels of H2O2 in the high-temperature composting environment, thus continuously providing sufficient substrate for lignin-degrading enzyme systems, achieving a short composting cycle and high efficiency. Specifically, this strain was isolated from thermophilic fermentation compost. Its inherent thermophilic characteristics allow it to adapt well to and colonize the high-temperature composting period, effectively overcoming the technical bottleneck of inhibited activity or death of ordinary microorganisms during the heating stage, ensuring the continuous and efficient progress of straw composting. In terms of its mechanism of action, this strain produces high concentrations of hydrogen peroxide, utilizing reactive oxygen species-mediated non-enzymatic reactions to attack and break down the complex lignin structure in straw. This mechanism differs from the industry's conventional degradation pathways that rely on the direct secretion of cellulase or ligninase, providing a novel and efficient auxiliary decomposition channel for straw decomposition, thereby significantly improving the overall degradation efficiency of lignocellulose. Furthermore, this invention breaks through the dependence on complex and complex bacterial communities, achieving the aforementioned functions through the application of a single strain. This not only simplifies the preparation process of the bacterial agent but also fundamentally ensures the stability of product functionality and batch-to-batch consistency, laying the foundation for standardized and large-scale application.

[0020] 2. This invention is the first to discover and utilize the unique characteristic of the thermophilic strain B-60-8, which produces high levels of H2O2. Through H2O2-mediated Fenton or Fenton-like reactions, it directly and efficiently attacks and breaks down the stubborn lignin structure in straw. Experimental data fully demonstrate that the straw degradation system incorporating this strain has a 4.4-fold higher lignin degradation rate than the control group, and the total weight loss rate of straw reaches 22.57% (compared to only 10.99% in the control group). This non-enzymatic degradation mechanism based on reactive oxygen species provides a novel technical approach to overcome the core bottleneck of "difficult lignin degradation" in straw composting, breaking through the limitations of traditional methods that rely solely on microbial secretion of cellulase / ligninase. This strain is a thermophilic bacterium, and its optimal growth and H2O2 production activity temperature is around 50℃, which highly matches the high-temperature period generated during straw composting. This characteristic effectively solves the technical problem of ordinary microbial agents being easily inactivated at high temperatures and unable to maintain their function throughout the entire cycle. It ensures that the strain can still stably colonize and efficiently perform its degradation function during the most critical heating and high-temperature periods of composting. The functional design and application scenarios of the strain in this invention closely revolve around the core objective of "promoting straw decomposition by degrading lignin through H2O2 production." Compared to existing technologies where similar strains are mainly used for producing proteases to degrade proteins, or only utilizing their broad-spectrum heat resistance to treat mixed organic waste, this invention is the first to clearly define the specific use and superior effect of *Bacillus jiuzhiense* in straw lignin degradation. This invention achieves efficient H2O2 enrichment and straw decomposition using only the single strain B-60-8, without relying on complex composite microbial communities. This fundamentally avoids problems such as inter-microbial antagonism, complex preparation processes, and poor batch stability that may exist when multiple microorganisms are combined, which is conducive to the standardized production, quality control, and large-scale application of the microbial agent, significantly reducing production costs and technical barriers.

[0021] 3. In this invention, strain B-60-8 (thermophilic strain B-60-8), which exhibits strong hydrogen peroxide production capacity and was obtained from samples screened during the high-temperature period of kitchen waste composting, was activated. 100 µL of the preserved bacterial culture was spread onto LB solid medium and incubated at 50°C for 24 hours. Single colonies were then streaked onto LB solid medium and incubated at 50°C for another 24 hours. Finally, single colonies were transferred to 1 mL of liquid LB medium and incubated at 50°C and 160 rpm. -1 After continuous incubation at a constant temperature on a shaker for 20 hours until turbidity is achieved, 1 mL of bacterial culture is added to 50 mL of LB liquid medium and incubated at 50℃ and 160 rpm. -1 The bacteria were continuously cultured at a constant temperature on a shaker for 12 hours until the mixture became turbid, thus preparing a high-concentration H2O2-producing bacterial solution.

[0022] Corn stalks were dried in a 105℃ oven until constant weight, passed through a 40-mesh sieve, and 2.3g of stalks were used to prepare 100mL of straw culture medium in a 250mL Erlenmeyer flask. The mouth of the flask was then sealed with sterile, breathable sealing film, and subsequently sterilized in an autoclave. The sterilized material was then allowed to stand at room temperature. The prepared high-concentration H2O2-producing bacterial solution was adjusted to 10... 9 CFU / mL was inoculated at a rate of 5% (v / v) into 100 mL of sterile straw culture medium and incubated at 50 °C and 160 rpm. -1 The samples were continuously incubated at a constant temperature on a shaker for 15 days. Every 3 days, 3 mL of the supernatant was collected to determine the viable bacterial count and hydrogen peroxide content for real-time monitoring, and the samples were saved for three-dimensional fluorescence spectroscopy analysis. After the straw culture was completed on the 15th day, the samples were filtered through filter paper, rinsed several times with distilled water, and dried at 65℃ to constant weight for determining the weight loss rate and lignocellulose content.

[0023] The culture medium used to cultivate the thermophilic strain B-60-8 described in this invention is:

[0024] (1) LB liquid medium: 5.0 g·L yeast extract -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 The solvent is water.

[0025] (2) LB solid medium: yeast extract 5.0 g·L -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 15.0 g / L agar -1 The solvent is water.

[0026] (3) Straw culture medium: KH2PO4 1g, CaCl2 0.1g, MgSO4·7H2O 0.3g, NaCl 0.1g, FeCl3 0.01g, NaNO3 2.5g, corn straw 23g, diluted with distilled water to 1L, pH natural. Attached Figure Description

[0027] Figure 1 The colony and cell morphology of the thermophilic strain B-60-8 in this invention are shown; wherein:

[0028] A represents the colony morphology;

[0029] B represents the bacterial cell morphology;

[0030] The thermophilic strain B-60-8 is simply referred to as strain B-60-8.

[0031] Figure 2 This invention illustrates the effect of different temperatures on the growth of strain B-60-8.

[0032] Figure 3 This invention describes the production pattern of H2O2 by strain B-60-8 at 50℃.

[0033] Figure 4 This refers to the change in the viable count of strain B-60-8 in straw culture medium in this invention.

[0034] Figure 5 The dynamic changes and cumulative amount of H2O2 produced by strain B-60-8 in this invention are shown.

[0035] Figure 6 This refers to the difference in straw weight loss rate in this invention.

[0036] Figure 7 The difference in straw lignocellulose in this invention.

[0037] Figure 8 This shows the changes in the humification index (HIX) in this invention. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0039] Example 1: Obtaining strain B-60-8

[0040] 1. Strains Isolation and Purification

[0041] Take 10g of compost sample from the 60℃ high-temperature period and extract it in 100mL of sterile 0.85wt% NaCl solution to obtain the extract. Serial dilution was used to obtain a total of 68 strains of bacteria resistant to 60℃. The specific operation is as follows:

[0042] Take 100 μL of the extract and mix it with sterile water at a volume ratio of 1:9. Then, take another 100 μL of the mixed solution and mix it with sterile water at a volume ratio of 1:9. Repeat the above steps to dilute the bacterial solution to 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 and 10 -8 Take 0.1 mL of each of the following solutions, resulting in a dilution factor of 10: -5 10 -6 10 -7 and 10 -8The bacterial suspension was inoculated onto LB agar plates, with each dilution repeated three times, and incubated upside down in a 60°C incubator. Single colonies were picked up with sterilized toothpicks and repeatedly streaked for isolation and purification until no contaminants were observed under a microscope, resulting in 68 bacterial strains.

[0043] The extracellular hydrogen peroxide production capacity of 68 bacterial strains was identified, and the specific procedures are as follows:

[0044] Sixty-eight bacterial strains were activated in LB liquid medium and cultured at 60°C and 160 r / min to the logarithmic growth phase. The H2O2 production of the bacterial culture was measured using a kit. One strain was found to produce hydrogen peroxide at 60°C and was named B-60-8, i.e. strain B-60-8.

[0045] The reagent kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0038.

[0046] The 60℃ high-temperature compost sample was obtained by mixing kitchen waste from the canteen of the Institute of Organic Recycling, China Agricultural University (Suzhou) with commercially available wood chips at a mass ratio of 3:1 and a carbon-nitrogen ratio of 25:1. Water was added to bring the moisture content to 65%, and then 0.25wt% persulfate was added to provide an active oxygen environment for composting fermentation. The sample was collected after the compost temperature rose above 50℃.

[0047] Single colonies of the isolated strain B-60-8 were inoculated into LB liquid medium and cultured to obtain purified strain B-60-8.

[0048] The cultivation conditions were 60℃, shaking speed 160r / min, and cultivation time 16h.

[0049] LB liquid medium: yeast extract 5.0 g·L -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 The solvent is water.

[0050] Example 2: Identification of strain B-60-8

[0051] 1. Identification of strain B-60-8

[0052] The purified strain B-60-8 was identified and described using a multiphase identification method that included molecular biological identification and phenotypic characterization, proving that the strain belongs to *Heterobacter spp.* Caldifermentibacillus hisashii The details are as follows:

[0053] (1) Analysis of colony morphology characteristics

[0054] Strawberry strain B-60-8 was streaked in LB solid medium and incubated at 55°C for 24 hours for observation.

[0055] The colonies have irregular, flat or round edges, are yellow, transparent, and have a glossy surface. The bacteria are rod-shaped, with a width of 0.35 μm ± 0.05 μm and a length of 1.5 μm ± 0.5 μm. Figure 1 ).

[0056] LB solid medium: yeast extract 5.0 g·L⁻¹ -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 15.0 g / L agar -1 The solvent is water.

[0057] (2) Molecular biological identification results of strain B-60-8:

[0058] Single colonies of strain B-60-8 were picked and cultured overnight at 60°C in 100 mL LB broth. The bacterial culture was collected during the logarithmic growth phase. The culture was transferred to centrifuge tubes, centrifuged at high speed, and the precipitated bacterial cells were collected, discarding the supernatant. The collected bacterial cells were then rapidly frozen in liquid nitrogen, sealed with sealing film, and stored on dry ice. The samples were then sent to Guangdong Megagene Technology Co., Ltd. for single-strain whole-genome sequencing. Gene annotation, phylogenetic analysis, and genome similarity index calculation were performed on the sequencing data to determine the existence of new species.

[0059] The results are shown in Table 1.

[0060] Overnight stays refer to stays of 12 hours or more.

[0061] Table 1. Identification results of 16S rRNA and genome of strain B-60-8

[0062]

[0063] As shown in Table 1, the 16S rRNA identification results of strain B-60-8 were consistent with the genome identification results, both being *Heterobacter jiuzhi*. Therefore, strain B-60-8 was identified as *Heterobacter jiuzhi*. Furthermore, on August 19, 2025, it was deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35676.

[0064] The nucleotide sequence of the 16S rDNA of strain B-60-8 is shown in SEQ ID NO.1:

[0065]

[0066] (3) Physiological and biochemical characteristics

[0067] I. Gram staining:

[0068] Single colonies (samples) of strain B-60-8 were picked and smeared onto a glass slide containing water. After drying, the samples were fixed and pre-stained with crystal violet for 1 min. After rinsing, mordanting with iodine solution was performed, followed by rinsing with water. The samples were then destained with 95% (v / v) ethanol. The slide was agitated after adding ethanol, and the destaining time was controlled to 40 s depending on the thickness of the smear. After rinsing, excess water was gently absorbed with absorbent paper, followed by counterstaining with safranin dye for 1 min. After rinsing again, the samples were air-dried and observed using an oil immersion microscope. Gram-negative bacteria appeared red, and Gram-positive bacteria appeared purple.

[0069] II. Anaerobic growth assay:

[0070] Single colonies of strain B-60-8 were picked using the streak plate method and inoculated into GAM agar medium. The culture medium was then placed in AnaeroPac jars and incubated at 45°C for 48 hours. The colony growth was then observed and compared with that under aerobic conditions.

[0071] The GAM agar medium was purchased from Haibo Biotechnology Co., Ltd., catalog number HB8518-1.

[0072] III. Microbial motility testing:

[0073] Using a sterile inoculation loop, pick a single colony of strain B-60-8 and gently insert it into the center of the SIM medium to a depth of 0.8 cm, then gently rotate the inoculation loop to inoculate. After incubating the inoculated SIM medium at 60°C for 48 hours, observe whether there are any turbid diffusion areas in the medium.

[0074] The SIM culture medium was purchased from Hybio Biotechnology Co., Ltd., catalog number HB4112.

[0075] IV. Effects of different osmotic pressures, pH levels, and temperatures:

[0076] Strains B-60-8 were inoculated onto LB solid medium and cultured at 60°C for 48 h. The growth of strain B-60-8 was monitored under different NaCl concentrations (2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%, w / v).

[0077] Strains B-60-8 were inoculated onto LB solid medium and incubated at 60°C for 48 h. The growth of strain B-60-8 was monitored under different pH values ​​(5, 6, 7, 8, 9, and 10). The pH value was adjusted using sodium acetate and Tris buffer, respectively.

[0078] Meanwhile, strain B-60-8 was inoculated on LB solid medium, and the growth of strain B-60-8 was monitored under different temperature conditions (25°C, 30°C, 45°C, 50°C, 55°C, 60°C and 65°C).

[0079] V.API Biochemical Identification Kit:

[0080] The biochemical characteristics of strain B-60-8 were evaluated using the API ZYM and API 50CH kits at a culture temperature of 50°C.

[0081] The basic physiological and biochemical characteristics of strain B-60-8 are shown in Table 2.

[0082] Table 2. Basic physiological and biochemical characteristics of strain B-60-8

[0083]

[0084] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0085] Table 2 shows that strain B-60-8 is Gram-negative, can grow at temperatures ranging from 35℃ to 60℃, with an optimum temperature of 50℃, and can grow at NaCl concentrations ranging from 1% to 10%. It grows under pH conditions ranging from 5 to 10, with an optimum pH of 7. It lacks oxidase activity but possesses nitrate reductase activity, making it a facultative anaerobe with motility.

[0086] The physiological and biochemical results of strain B-60-8 obtained using API 50CH reagent strips are shown in Table 3.

[0087] Table 3. Physiological and biochemical results of strain B-60-8 using API 50CH reagent strips

[0088]

[0089] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0090] According to Table 3, the analysis of API 50CH carbohydrate metabolism results showed that the strain could utilize sugar alcohol substrates such as inositol and sorbitol, and could metabolize plant-derived glycosides such as amygdalin, arbutin, aesculin, and salicin. Further analysis revealed that strain B-60-8 lacked the ability to metabolize most common monosaccharides such as D-galactose, D-glucose, D-fructose, and D-mannose, as well as disaccharides such as D-cellobiose, D-maltose, D-lactose, and D-sucrose. The analysis also found that strain B-60-8 lacked the ability to utilize polysaccharides such as glycogen and inulin, as well as other sugar alcohols such as xylitol and arabinol.

[0091] The physiological and biochemical results of strain B-60-8 obtained using API ZYM reagent strips are shown in Table 4.

[0092] Table 4. Physiological and biochemical results of strain B-60-8 using API ZYM reagent strips

[0093]

[0094] Note: "+" indicates a positive reaction, "-" indicates a negative reaction, and "W" indicates a weak positive reaction.

[0095] As shown in Table 4 on the right, API ZYM enzymatic assays revealed that strain B-60-8 possessed esterase, acid phosphatase, and β-galactosidase activities, and exhibited weak α-glucosidase and β-glucosidase activities. This strain B-60-8 was completely lacking lipase, alkaline phosphatase, various other glycosidases (such as α-galactosidase, β-glucuronidase, and β-fructofuranosidase), and all tested proteolytic enzymes (such as leucine aminopeptidase and trypsin).

[0096] Example 2: Thermophilic H2O2 production characteristics of strain B-60-8

[0097] Strain strain B-60-8 was inoculated into LB liquid medium and incubated at 25℃, 35℃, 45℃, 50℃, 55℃, 60℃ and 65℃ for 160 r·min. -1 The OD was measured after continuous constant temperature incubation on a shaker for 48 hours. 600 Value. Result as follows Figure 2 As shown, strain B-60-8 can grow at temperatures ranging from 35℃ to 60℃, with the best growth at 50℃. Growth is poor below 35℃, and almost no growth at 25℃ and 65℃.

[0098] Therefore, strain B-60-8 was cultured at 50℃ for 160 r·min. -1 The mixture was continuously incubated at a constant temperature on a shaker for 48 hours, and the H2O2 production was measured every 4 hours. The results are as follows: Figure 3As shown, the strain reached its peak H2O2 production after 20 hours of cultivation. Therefore, considering the optimal growth temperature and H2O2 production time of strain B-60-8, subsequent cultivation was carried out at 50℃ and 160 rpm. -1 The bacterial solution formed after culturing the strain for 20 hours was used as a straw composting agent.

[0099] Example 3: Straw colonization and H2O2 production capacity of strain B-60-8

[0100] Strain strain B-60-8 was inoculated into LB liquid medium and incubated at 50°C and 160 rpm. -1 After culturing strain B-60-8 for 20 hours under certain conditions, a bacterial culture was formed, and the culture was adjusted to 10... 9 After CFU / mL, it was added to 100 mL of sterile straw culture medium at a ratio of 5% (v / v) and incubated at 50 °C and 160 rpm. -1 The cells were continuously incubated at a constant temperature on a shaker for 15 days, and the supernatant was collected every 3 days for the determination of H2O2 and viable bacteria count. H2O2 was determined using an H2O2 detection kit, and viable bacteria count was determined using the plate count method.

[0101] The straw culture medium is made from materials with the following final concentrations:

[0102] 1g KH2PO4, 0.1g CaCl2, 0.3g MgSO4·7H2O, 0.1g NaCl, 0.01g FeCl3, 2.5g NaNO3, and 23g corn stalks, add distilled water to 1L, pH to natural.

[0103] The results of changes in viable bacterial count and dynamic changes and accumulation of H2O2 produced by strain B-60-8 in straw culture medium are as follows: Figure 4 and Figure 5 As shown, strain B-60-8 increased the H2O2 content in the straw culture medium environment by 474.4%, and the viable bacterial count in the straw culture medium was maintained at 10. 10 CFU / mL.

[0104] Example 4: Straw degradation and decomposition promotion effects of strain B-60-8

[0105] A sterile straw culture medium without added microbial agents was set as the control (CK). The experimental group was treated with strain B-60-8 (experimental group: strain B-60-8 was inoculated into LB liquid medium and incubated at 50℃ and 160 rpm). -1 After culturing the strain for 20 hours under certain conditions, a bacterial culture was formed, and the culture was adjusted to 10... 9 After CFU / mL, it was added at a ratio of 5% to 100 mL of sterile straw culture medium, with three replicates per treatment group.

[0106] Corn stalks were dried in a 105℃ oven to constant weight, passed through a 40-mesh sieve, and 2.3g of stalks were placed in a 250mL Erlenmeyer flask containing 100mL of straw culture medium. The flask mouth was then sealed with sterile, breathable sealing film and placed in an autoclave for sterilization. The sterilized material was then allowed to stand at room temperature. The bacterial culture prepared according to the method in Example 2 was adjusted to 10... 9 CFU / ml was inoculated at a rate of 5% (v / v) into 100 mL of sterile straw culture medium and incubated at 50 °C and 160 rpm. -1 The straw was continuously cultured at a constant temperature on a shaker for 15 days. After the 15th day of culture, the straw was collected, filtered through filter paper, rinsed several times with distilled water, and dried at 65℃ to constant weight for determination of weight loss rate and lignocellulose content.

[0107] The weightlessness rate is calculated using the following formula:

[0108] Weight loss rate = (original straw mass - dried sample mass) / original straw mass × 100%;

[0109] In the above formula, the unit of weight loss rate is %;

[0110] The contents of lignin, cellulose, and hemicellulose were determined using kits. These kits were purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog numbers BC4200, BC4280, and BC4440.

[0111] DOM extraction and humification index (HIX) determination: 2g of air-dried sample was weighed and mixed with ultrapure water at a ratio of 1:10 (g:mL), shaken for 24h (temperature 25°C, rotation speed 200r / min), then centrifuged at 10000r / min for 20min. The supernatant was filtered through a 0.45μm filter membrane, and the DOC content in the filtrate was determined using the potassium dichromate oxidation method. The remaining filtrate was used for three-dimensional fluorescence spectroscopy analysis to obtain the HIX results. The air-dried sample refers to straw collected on day 15 after cultivation, filtered through filter paper, rinsed multiple times with distilled water, and then dried at 65℃ to constant weight.

[0112] The results of the difference in straw weight loss rate after adding strain B-60-8 are as follows: Figure 6 As shown, at the end of the composting process, the straw weight loss rate of the experimental group inoculated with strain B-60-8 of this invention was 22.57%, significantly higher than that of the control group (CK) at 10.99%, with a relative improvement rate of 105%. This result indicates that strain B-60-8 provided by this invention can greatly promote the decomposition of straw organic matter and effectively shorten the composting cycle.

[0113] The results of the difference in straw lignocellulose after adding strain B-60-8 are as follows: Figure 7As shown, the degradation rates of lignin, cellulose, and hemicellulose in straw reached 19.68%, 17.57%, and 6.07%, respectively, significantly higher than those in the control group. Among these, the degradation rate of lignin showed the most significant increase, being 4.4 times that of the control group. This indicates that strain B-60-8 can effectively promote lignin degradation.

[0114] The results of the change in humification index after adding strain B-60-8 are as follows: Figure 8 As shown, during the composting process, the humification index (HIX) of the experimental group inoculated with strain B-60-8 of this invention was significantly higher than that of the control group (CK). The HIX value of the experimental group reached a peak of 0.653 on day 12 and remained at a high level throughout the middle and late stages of composting. This indicates that strain B-60-8 of this invention can not only accelerate straw degradation but also significantly promote the synthesis and accumulation of humus, achieving efficient straw composting and obtaining compost products with more thorough composting and more stable quality. This provides an effective solution to the problem of unstable effects of existing straw composting technologies.

[0115] In summary, the strain B-60-8 provided by this invention increased the H2O2 content in a 50℃ high-temperature straw culture medium environment by 474.4%, and the viable bacterial count in the straw culture medium was maintained at 10. 10 The concentration of CFU / mL was increased, and the degradation rates of lignin, cellulose and hemicellulose in straw reached 19.68%, 17.57% and 6.07% respectively under a high temperature of 50℃, which effectively promoted the decomposition of straw.

[0116] The thermophilic bacterial strain B-60-8 provided by this invention can be used to prepare straw composting inoculant. The straw composting inoculant contains live cells of the thermophilic strain B-60-8 and / or fermentation broth. The straw composting inoculant is available in powder, granule, or liquid form.

[0117] When the straw composting inoculant is in powder or granule form, the viable count of the straw composting inoculant is 10. 8 CFU mL -1 When the straw composting microbial agent is in liquid form, the viable count of the straw composting microbial agent is 10. 9 CFU g -1 .

[0118] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The application of a thermophilic bacterial strain B-60-8 for straw degradation and composting in the degradation of straw, characterized in that, The thermophilic strain B-60-8 is classified as *Heterobacter jiuzhi* (…). Caldifermentibacillus hisashii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 19, 2025, with accession number CGMCC No. 35676; The thermophilic strain B-60-8 can utilize H2O2 to degrade lignin, thereby promoting straw decomposition; The thermophilic strain B-60-8 is able to adapt to the high-temperature period of straw composting; wherein, the high-temperature period of straw composting is 50℃~60℃; The thermophilic strain B-60-8 is used to break down lignin under high temperature conditions of 50℃~60℃, thereby degrading straw and achieving straw decomposition.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the thermophilic strain B-60-8 is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The thermophilic strain B-60-8 is used to prepare straw composting inoculant.

4. The application according to claim 3, characterized in that, The straw composting microbial agent contains live cells of the thermophilic strain B-60-8 and / or fermentation broth.

5. The application according to claim 4, characterized in that, The straw composting microbial agent is available in powder, granule, or liquid form.

6. The application according to claim 5, characterized in that, When the straw composting microbial agent is in the form of powder or granules, the viable count of the straw composting microbial agent is 10. 8 CFU mL -1 ~10 9 CFU mL -1 When the straw composting microbial agent is in liquid form, the viable count of the straw composting microbial agent is 10. 8 CFU g -1 ~10 9 CFU g -1 .

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