Bacillus subtilis and application thereof

Abstract

The application relates to a Bacillus subtilis strain and an application thereof. The strain (Bacillus subtilis) XY01 is preserved in the China Center for Type Culture Collection on September 5, 2022, and the preservation number is CCTCC M20221379. The Bacillus subtilis XY01 degrades grass plants, is used for manufacturing straw pulp by a biological method, and is used for preparing humic acid by using straw pulp black liquor, realizes resource utilization of agricultural solid waste resources such as straw stalks, the black liquor generated in the pulping process is used for field application, and the production chain does not cause environmental pollution. The Bacillus subtilis is mild in the decomposition condition in the biological manufacturing of straw pulp, short in the production cycle, and friendly to the environment.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus subtilis and its applications, specifically to a strain of Bacillus subtilis and its application in the biological production of straw pulp and the preparation of humic acid from straw pulp black liquor. Background Technology

[0002] Pulp is a fibrous material made from plant fibers through various processing methods, and it is the main raw material for the papermaking industry. Based on the source of the raw materials, pulp can be divided into wood pulp, non-wood pulp, and waste paper pulp. Non-wood pulp is made from various non-wood fibers. With the advancement of solid agricultural waste resource utilization technology, the proportion and importance of straw pulp, made from grasses, reeds, bamboo, and cotton and hemp, are gradually increasing. Straw pulp typically contains a high amount of hemicellulose, and bleached straw pulp can be used to manufacture various medium and high-grade writing and printing papers.

[0003] Traditional straw pulp manufacturing processes mainly include chemical pulping, mechanical pulping, and chemimechanical pulping. Chemical pulping uses chemical methods to separate fibers from lignin, such as the caustic soda process, sulfate process, and sulfite process. Mechanical pulping includes disc milling mechanical pulping, thermomechanical pulping, and physical bursting pulping, or a combination of both. These methods suffer from drawbacks such as high pollution, high energy consumption, short fibers, and low pulp yield. In contrast, biopulping is an innovative and clean pulping technology that primarily uses microbial decomposition supplemented by physical decomposition. Biopulping produces long fibers, high pulp yield, strong folding endurance, low cost, and is environmentally friendly. However, biodecomposition requires stringent conditions, has a long decomposition cycle, and is susceptible to contamination by other microorganisms, leading to failure. Therefore, the industrial-scale implementation of biopulping has been slow both domestically and internationally. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Bacillus subtilis and its application. This Bacillus subtilis has mild decomposition conditions, short production cycle and is environmentally friendly in the biological production of straw pulp, and can be applied to straw pulp production.

[0005] A strain of Bacillus subtilis XY01 was deposited at the China Center for Type Culture Collection on September 5, 2022, with accession number CCTCC M20221379.

[0006] On the other hand, the application of a Bacillus subtilis strain XY01 in the biological production of straw pulp.

[0007] Furthermore, the application of Bacillus subtilis XY01 in the biological production of straw pulp is as follows: (1) Crushing and washing After removing the leaves and seeds from the grass, the grass stalks are obtained; they are then crushed and washed by a grass crusher or grass shredder to remove sand and dust, and squeezed to remove water, resulting in shredded grass. (2) Propagation of Bacillus subtilis XY01 Bacillus subtilis XY01 was propagated to a count >1×10⁻⁶ using either liquid or solid culture. 9 CFU / m; (3) Dilute and inoculate Dilute the propagated Bacillus subtilis XY01 to a count >1×10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation Ferment in a stacked environment at a temperature of 10℃~45℃, with a stack height of 1.5m~2m; ventilation and turning of the stack are combined to control the core temperature of the straw pile to <75℃, and compost for 5d~7d until the core temperature of the straw pile stabilizes at ≤55℃ and no longer rises, thus obtaining decomposed straw; (5) High-temperature alkaline hydrolysis The decaying straw was transferred to a high-temperature alkaline hydrolysis device, and the inorganic alkali to decaying straw mass-to-volume ratio was 5.5 kg / m³. 3 ~6.5Kg / m 3 An inorganic alkali, namely sodium hydroxide or potassium hydroxide, is added and the mixture is alkali-hydrolyzed at 140°C for 40 to 60 minutes. After the alkali hydrolysis is completed, the mixture is cooled, and the pH value of the solution after cooling is 7.5 to 8.5. The mixture is then extruded and separated to obtain straw fiber and black liquor. (6) Making straw pulp from grass fiber Grass fiber is pulped to obtain grass pulp; (7) Preparation of humic acid from black liquor After black liquor is concentrated or dried, humic acid is obtained.

[0008] Furthermore, during the dilution in step (3), the propagated Bacillus subtilis XY01 was diluted to a bacterial count of 3 × 10⁻⁶. 6 CFU / mL ~ 4 × 10 6 CFU / mL.

[0009] Furthermore, the grass is rice straw or reed.

[0010] Furthermore, the liquid culture medium formula used for liquid culture is as follows: 32.0 g tryptone, 20.0 g yeast extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L, pH 7.0 ± 0.2.

[0011] Furthermore, the solid culture medium formula is as follows: 64.85% wheat bran, 20% corn flour, 15% soybean meal, 0.1% magnesium sulfate, and 0.05% manganese sulfate. Distilled water is added according to the mass ratio of feed (total mass of wheat bran, corn flour, soybean meal, magnesium sulfate, and manganese sulfate) to water of 1:1.2, with a pH of 7.0±0.2.

[0012] Furthermore, the high-temperature alkaline hydrolysis equipment is a tank-type high-temperature alkaline hydrolysis equipment or a continuous high-temperature alkaline hydrolysis equipment.

[0013] Beneficial effects of this invention: 1. Bacillus subtilis XY01 exhibits a short degradation cycle for grasses, requiring only 5–7 days, which is one-third the degradation cycle of white-rot fungi. It demonstrates strong resistance to contaminating microorganisms and rapidly colonizes to form a population suppression effect within 10 hours of inoculation. Degradation conditions are mild; Bacillus subtilis XY01 can efficiently decompose lignin and strip hemicellulose under pH 5–9 and temperatures 5℃–55℃, with particularly high biodegradation efficiency at pH 6.5–7.5 and temperatures 35℃–45℃. After enzymatic hydrolysis, the proportion of long fibers is high, the alkaline hydrolysis temperature is low, the alkali usage is one-quarter that of mechanical pulping, and the alkali residue is minimal.

[0014] 2. After drying, the black liquor contains >60% humic acid, of which >35% is fulvic acid. Its application to vegetables, farmland and fruit trees significantly increases yield and has a positive effect on alleviating soil salinization and reducing phosphorus and potassium leaching.

[0015] 3. By using Bacillus subtilis XY01 biological method to produce straw pulp and then using the black liquor from the straw pulp to prepare humic acid, the resource utilization of agricultural solid waste such as straw and stalks is realized. The black liquor generated during the pulping process is returned to the field, and the production chain does not cause environmental pollution.

[0016] Instruction manual illustrations Figure 1 This is a process flow diagram of the present invention for producing straw pulp using Bacillus subtilis XY01 biological method and preparing humic acid using straw pulp black liquor. Detailed Implementation

[0017] I. Obtaining Bacillus subtilis strain XY01 1. Culture medium Basic culture medium: NaNO3 2.5g, KH2PO4 1.0g, NaCl 0.5g, MgSO4·7H2O 0.5g, CaCl2 0.1g, trace element mixture 1mL, distilled water to a final volume of 1L, pH adjusted to 8.3.

[0018] Trace element mixture: FeCl3·6H2O 0.16g, ZnSO4·7H2O 1.5g, CoCl2·6H2O 0.16g, CuSO4·5H2O 0.15g, MnSO4·H2O 1.5g, H3BO3 0.3g, Na2MoO4·2H2O 0.1g, diluted with distilled water to 1L.

[0019] Primary screening medium for bacterial strains: basal medium components, with the addition of 1 mL / L of guaiacol.

[0020] Microbial strain rescreening medium: basic medium components, with 1 g / L of alkaline lignin added.

[0021] Nutrient agar medium: 10g peptone, 3g beef extract, 5g NaCl, 16g agar, 1000mL distilled water, pH 7.2.

[0022] Liquid culture medium: 10g peptone, 3g beef extract, 5g NaCl, 1000mL distilled water, pH 7.2.

[0023] Guaiacin-Nutrient Agar Medium: Nutrient agar medium with 0.4 mL / L of guaiacol added.

[0024] Aniline blue-nutrient agar medium: Nutrient agar medium with 0.1 g / L of aniline blue added.

[0025] 2. Isolation of strains (1) Sampling Microbial samples were collected from paddy fields in Yingkou City, Liaoning Province. The rice straw was harvested in autumn and observed after being piled outdoors for 30 days. The rice straw showed obvious decay or decomposition. 200g of the decomposed material was taken from this location, labeled with sample information, and stored at 4℃.

[0026] (2) Preliminary screening of bacterial strains Weigh 1g of the collected sample and add it to a 250mL Erlenmeyer flask containing 100mL of sterile distilled water. Recover the sample in a shaker at 200rpm and 37℃ for 1h. Let it stand for 10min. Take the supernatant and inoculate 50mL of it into a pre-sterilized 100mL primary screening medium at a volume ratio of 10%. Incubate the medium at 37℃ for 7d. Subculture the strains that show a color reaction (from colorless to purple) for 3 generations until each generation shows a color reaction and the time of the color reaction is stable. Inoculate the composite strains that show the above reaction into the secondary screening medium.

[0027] (3) Rescreening of bacterial strains The composite bacterial strains obtained in step (2) were statically cultured at 37°C for 10 days. The alkaline lignin decomposition rate in the system was measured at the end of the culture period, and the composite bacterial strains with the highest lignin decomposition rate were retained.

[0028] (4) Strain isolation After shaking and mixing the secondary screening culture obtained in step (3), take 1 mL of the liquid and serially dilute it with sterile water to 10⁻⁶. -5 Take 10 -3 10 -4 10 -5Spread 0.1 mL of each dilution onto nutrient agar plates and incubate at 37°C for 24 h. Pick a single colony and inoculate it into a slant tube of nutrient agar. Incubate at 37°C for 24 h and then store in a 4°C refrigerator for screening.

[0029] (5) Initial screening of degrading strains The pure culture strains obtained in step (4) were inoculated into the center of guaiacol-nutrient agar and aniline blue-nutrient agar media respectively using the spot inoculation method. After growing at 37℃ for 3 days, the diameter of the color development zone of the strains on guaiacol-nutrient agar media and the diameter of the decolorization zone of the strains on aniline blue-nutrient agar media were measured. The average value was taken after 3 replicates. Strains with large color development zones and obvious decolorization zones were screened, and 50% were eliminated. The strains were stored at 4℃ for later use.

[0030] (6) Rescreening of degrading strains The degradative screening strains were inoculated into sterilized liquid culture medium and cultured at 37°C and 250 rpm for 24 h using a shaker. Hemocytometer counting was performed, and the culture was diluted to 1 × 10⁻⁶. 5 CFU / mL was inoculated into rice straw shreds that had been crushed and washed using a straw crusher. Temperature and pH changes were observed and recorded at room temperature. Samples were weighed and taken daily, and the cellulose and lignin contents were determined according to the method in "NY / T 3494-2019 Determination of Cellulose, Hemicellulose and Lignin in Agricultural Biomass Raw Materials" until day 10.

[0031] The degradation data were statistically analyzed, and the decomposition ability of the strains was evaluated using weight loss rate, lignin loss rate, and cellulose loss rate as evaluation indicators. Three strains with high weight loss rate and (lignin loss rate / cellulose loss rate) > 10 were obtained and named XY01, XY02, and XY03 respectively according to their decomposition ability from high to low.

[0032] Table 1. Statistics on the ability of strains to decompose rice straw. Weight loss rate (%) 25.94 22.38 18.85 Lignin loss rate (%) 22.51 19.66 16.42 Cellulose loss rate (%) 1.42 1.85 1.16 Hemicellulose loss rate (%) 16.19 17.63 17.90 Lignin loss rate / cellulose loss rate 15.85 10.63 14.16 3. Strain identification Bacterial 16S rDNA sequence sequencing and GenBank comparison revealed that XY01 is Bacillus subtilis, which has wide applications in medicine, health, and food; XY02 is Pseudomonas fluorescens, which poses a risk of environmental pollution; and XY03 is Paenibacillus sp., whose pollution and pathogenicity are unknown.

[0033] Morphological characteristics of Bacillus subtilis XY01: (1) The colony surface is rough and opaque, dirty white, and wrinkles can form in liquid culture medium.

[0034] (2) Individual morphological characteristics of the bacteria: The cell size is (0.7μm~0.8μm)×(2.0μm~3.0μm), rod-shaped; the spore size is (0.6μm~0.9μm)×(1.0μm~1.5μm), elliptical, central or eccentric.

[0035] (3) Physiological and biochemical characteristics: Gram reaction positive, catalase positive, can undergo acetylmethyl methanol reaction, can hydrolyze starch, decompose tryptophan to form indole, culture temperature 5℃~55℃, optimal growth temperature 35℃~45℃, pH 5~9, optimal pH 6.5~7.5.

[0036] Based on the results of bacterial 16S rDNA sequencing and GenBank alignment, this *Bacillus subtilis* XY01 has been named *Bacillus subtilis* XY01 and deposited on September 5, 2022, at the China Center for Type Culture Collection (CCTCC, located at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, accession number CCTCC M20221379). II. Application of Bacillus subtilis XY01 in biological straw pulp production and humic acid preparation from straw pulp black liquor (1) Crushing and washing After removing the leaves and seeds from the grass (rice straw or reeds), the grass stalks are obtained; they are then crushed and washed by a grass crusher or grass shredder to remove sand and dust, and squeezed to remove water, resulting in shredded grass. (2) Propagation of Bacillus subtilis XY01 Bacillus subtilis XY01 was propagated to a count >1×10⁻⁶ using either liquid or solid culture. 9 CFU / m; (3) Dilute and inoculate Dilute the propagated Bacillus subtilis XY01 to a count >1×10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation Ferment in a stacked environment at a temperature of 10℃~45℃, with a stack height of 1.5m~2m; ventilation and turning of the stack are combined to control the core temperature of the straw pile to <75℃, and compost for 5d~7d until the core temperature of the straw pile stabilizes at ≤55℃ and no longer rises, thus obtaining decomposed straw; (5) High-temperature alkaline hydrolysis The decomposed straw is transferred to a tank-type high-temperature alkaline hydrolysis device or a continuous high-temperature alkaline hydrolysis device, and the inorganic alkali to decomposed straw mass-to-volume ratio is 5.5–6.5 kg / m³.3 An inorganic alkali, namely sodium hydroxide or potassium hydroxide, is added and the mixture is alkali-hydrolyzed at 140°C for 40 to 60 minutes. After the alkali hydrolysis is completed, the mixture is cooled, and the pH value of the solution after cooling is 7.5 to 8.5. The mixture is then extruded and separated to obtain straw fiber and black liquor. (6) Making straw pulp from grass fiber Grass fiber is pulped to obtain grass pulp; (7) Preparation of humic acid from black liquor After black liquor is concentrated or dried, humic acid is obtained.

[0037] Example 1 (1) Crushing and washing After removing the leaves and seeds from rice, rice straw is obtained; it is then crushed and washed by a straw crusher to remove sand and dust, and squeezed to remove water, resulting in chopped straw. (2) Propagation of Bacillus subtilis XY01 Liquid culture medium formulation: 32.0 g tryptone, 20.0 g yeast extract, 5.0 g sodium chloride, distilled water to a final volume of 1 L, pH 7.0 ± 0.2; large-scale culture can be scaled up proportionally. Dispense 200 mL of liquid culture medium into 500 mL Erlenmeyer flasks, sterilize and cool, then inoculate. Incubate at 37°C and 350 rpm for 24 h on a shaker to obtain the liquid propagation strain *Bacillus subtilis* XY01. The *Bacillus subtilis* XY01 count is >1 × 10⁻⁶. 9 CFU / m; (3) Dilute and inoculate The propagated Bacillus subtilis XY01 was diluted to a bacterial count of 3 × 10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation Fermentation was carried out in a stacked environment at 30℃ with a stack height of 1.5m. Ventilation and turning of the stack were combined to control the core temperature of the straw pile to <75℃. The fermentation was completed after 5 days. The core temperature of the straw pile stabilized at ≤55℃ and no longer rose, thus obtaining decomposed straw. (5) High-temperature alkaline hydrolysis The decaying straw was transferred to a tank-type high-temperature alkaline hydrolysis device, and the inorganic alkali to decaying straw mass-to-volume ratio was 5.5 kg / m³. 3 Sodium hydroxide was added; alkaline hydrolysis was carried out at 140℃ for 40 min. After alkaline hydrolysis was completed, the solution was cooled. The pH value of the solution after cooling was 7.5. The straw fiber and black liquor were obtained by extrusion and separation. (6) Making straw pulp from grass fiber After refining, grass fibers are used to obtain grass pulp. The average fiber length is evaluated by wet weight method. Under the same pulping equipment conditions, the XY01 bio-pulp has a 7.33% higher average fiber length than mechanical pulp. After enzymatic hydrolysis, the proportion of long fibers is high and the alkaline hydrolysis temperature is low. (7) Preparation of humic acid from black liquor After drying the black liquor, humic acid was obtained, with a humic acid content of 63.28% and a fulvic acid content of 37.31%.

[0038] Example 2 (1) Crushing and washing After removing the leaves and seeds from the reeds, the reed stalks are obtained; they are then crushed and washed by a grass powdering machine to remove sand and dust, and squeezed to remove water, resulting in crushed reed stalks. (2) Propagation of Bacillus subtilis XY01 Solid culture medium formulation: wheat bran 64.85%, corn flour 20%, soybean meal 15%, magnesium sulfate 0.1%, manganese sulfate 0.05%, material-to-water ratio 1:1.2, pH 7.0±0.2; after sterilization and cooling, the solid culture medium was inoculated with liquid propagation culture at an inoculation rate of 0.1%; the culture temperature was 37℃, and the culture time was 48 hours, yielding Bacillus subtilis XY01 propagated in solid culture, with a Bacillus subtilis XY01 count > 1×10⁻⁶. 9 CFU / m; (3) Dilute and inoculate The propagated Bacillus subtilis XY01 was diluted to a bacterial count of 4 × 10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation Fermentation was carried out in a stacked environment at 10℃ with a stack height of 1.8m. Ventilation and turning of the stack were combined to control the core temperature of the straw pile to <75℃. The fermentation was completed after 7 days. The core temperature of the straw pile stabilized at ≤55℃ and no longer rose, thus obtaining decomposed straw. (5) High-temperature alkaline hydrolysis The decaying straw was transferred to a tank-type high-temperature alkaline hydrolysis device, and the inorganic alkali to decaying straw mass-to-volume ratio was 6.0 kg / m³. 3 Add potassium hydroxide; perform alkaline hydrolysis at 140℃ for 1 hour; after alkaline hydrolysis, cool the solution. The pH of the solution after cooling is 8.2. Separate by pressing to obtain straw fiber and black liquor. (6) Making straw pulp from grass fiber After refining, straw fibers are used to obtain straw pulp. The average fiber length is evaluated by wet weight method. Under the same pulping equipment conditions, XY01 biological pulp is 4.37% longer than chemical pulp (sodium hydroxide hydrolysis). The proportion of long fibers is higher after enzymatic hydrolysis, and the hydrolysis temperature is lower. (7) Preparation of humic acid from black liquor After drying the black liquor, humic acid was obtained, which contained 60.42% humic acid and 35.59% fulvic acid.

[0039] Example 3 (1) Crushing and washing After removing the leaves and seeds from wheat, wheat straw is obtained; it is then crushed and washed by a straw crusher to remove sand and dust, and squeezed to remove water, resulting in chopped straw. (2) Propagation of Bacillus subtilis XY01 Liquid culture medium formulation: 32.0 g tryptone, 20.0 g yeast extract, 5.0 g sodium chloride, distilled water to a final volume of 1 L, pH 7.0 ± 0.2; large-scale culture can be scaled up proportionally. Dispense 200 mL of liquid culture medium into 500 mL Erlenmeyer flasks, sterilize and cool, then inoculate. Incubate at 37°C and 350 rpm for 24 h on a shaker to obtain the liquid propagation strain *Bacillus subtilis* XY01. The *Bacillus subtilis* XY01 count is >1 × 10⁻⁶. 9 CFU / m; (3) Dilute and inoculate The propagated Bacillus subtilis XY01 was diluted to a bacterial count of 3 × 10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation Fermentation was carried out in a stacked environment at 20℃ with a stack height of 2m. Ventilation was combined with turning the stack to control the core temperature of the straw pile to <75℃. The fermentation was completed after 6 days of composting. The core temperature of the straw pile stabilized at ≤55℃ and no longer rose, thus obtaining decomposed straw. (5) High-temperature alkaline hydrolysis The decomposed straw is transferred to a tank-type high-temperature alkaline hydrolysis device or a continuous high-temperature alkaline hydrolysis device, and the inorganic alkali to decomposed straw mass-to-volume ratio is 6.5 kg / m³. 3 Sodium hydroxide was added and the mixture was alkali-hydrolyzed at 140℃ for 40 min. After the alkali hydrolysis was completed, the solution was cooled. The pH value of the solution after cooling was 8.0. The straw fiber and black liquor were obtained by extrusion and separation. (6) Making straw pulp from grass fiber After refining, grass fibers are used to obtain grass pulp. The average fiber length is evaluated by wet weight method. Under the same equipment conditions, XY01 bio-pulp increases the length by 6.85% compared with mechanical pulp. After enzymatic hydrolysis, the proportion of long fibers is high and the alkaline hydrolysis temperature is low. (7) Preparation of humic acid from black liquor After drying the black liquor, humic acid was obtained, which contained 65.12% humic acid and 38.66% fulvic acid.

[0040] Example 4 The biodegradation temperature in this embodiment is 45°C, and other conditions are the same as in Example 3; Bacillus subtilis XY01 can decompose lignin and strip hemicellulose at temperatures ranging from 5℃ to 55℃, especially at 35℃ to 45℃, where it can efficiently decompose lignin and strip hemicellulose. Therefore, the bacteria can grow normally and decompose straw better at 45℃.

[0041] The above are merely specific embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of Bacillus subtilis XY01 in the biological production of straw pulp, characterized by: Bacillus subtilis ( Bacillus subtilis XY01 was deposited at the China Center for Type Culture Collection on September 5, 2022, with accession number CCTCCM20221379.

2. The application of Bacillus subtilis XY01 according to claim 1 in the biological production of straw pulp, characterized in that: Specific process as follows: (1) Crushing and washing: After removing the leaves and seeds from the grass, grass stalks are obtained. The grass is either rice straw or reed. The grass is crushed and washed by a grass crusher or grass shredder to remove sand and dust, and then squeezed to remove water, resulting in shredded grass. (2) Propagation of Bacillus subtilis XY01: Bacillus subtilis XY01 was propagated to a count >1×10⁻⁶ using liquid or solid culture. 9 CFU / mL; (3) Dilute and inoculate: Dilute the propagated Bacillus subtilis XY01 to a count > 1 × 10⁻⁶. 6 CFU / mL, inoculated onto chopped straw, with the mass ratio of chopped straw to diluted Bacillus subtilis XY01 suspension being 1:0.5, and stirred thoroughly; (4) Biodegradation: fermentation in a pile at a temperature of 10℃~45℃, with a pile height of 1.5m~2m; ventilation and turning of the pile are combined to control the core temperature of the straw pile to <75℃, and compost for 5d~7d until the core temperature of the straw pile stabilizes to ≤55℃ and no longer rises, thus obtaining decomposed straw. (5) High-temperature alkaline hydrolysis: Transfer the decomposed straw to the high-temperature alkaline hydrolysis equipment, and apply the inorganic alkali to the decomposed straw at a mass-to-volume ratio of 5.5 kg / m³. 3 ~6.5Kg / m 3 An inorganic alkali, namely sodium hydroxide or potassium hydroxide, is added and the mixture is alkali-hydrolyzed at 140°C for 40 to 60 minutes. After the alkali hydrolysis is completed, the mixture is cooled, and the pH value of the solution after cooling is 7.5 to 8.

5. The mixture is then extruded and separated to obtain straw fiber and black liquor. (6) Grass fiber is used to make grass pulp. Grass fiber is ground into pulp to obtain grass pulp; (7) Preparation of humic acid from black liquor: Humic acid is obtained by concentrating or drying black liquor.

3. The application of Bacillus subtilis XY01 according to claim 2 in the biological production of straw pulp, characterized in that: In step (3), the propagated Bacillus subtilis XY01 was diluted to a bacterial count of 3 × 10⁻⁶. 6 CFU / mL ~ 4 × 10 6 CFU / mL.

4. The application of Bacillus subtilis XY01 according to claim 2 in the biological production of straw pulp, characterized in that: The liquid culture medium formula used for liquid culture is as follows: 32.0 g tryptone, 20.0 g yeast extract, 5.0 g sodium chloride, and distilled water to a final volume of 1 L, pH=7.0±0.

2.

5. The application of Bacillus subtilis XY01 according to claim 2 in the biological production of straw pulp, characterized in that: solid... Culture medium formula: 64.85% wheat bran, 20% corn flour, 15% soybean meal, 0.1% magnesium sulfate, 0.05% manganese sulfate, and distilled water, with a material-to-water mass ratio of 1:1.2 and pH=7.0±0.

2.

6. The application of Bacillus subtilis XY01 according to claim 2 in the biological production of straw pulp, characterized in that: The high-temperature alkaline hydrolysis equipment is either a tank-type high-temperature alkaline hydrolysis equipment or a continuous high-temperature alkaline hydrolysis equipment.

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