Bacillus altitudinis as well as complex microbial inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus altitudinis as well as a complex microbial inoculant and application thereof. The invention provides bacillus altitudinis, which is derived from intestinal tracts of earthworms, is named as DBN-EW28 and is preserved in China General Microbiological Culture Collection Center on February 14, 2025, the preservation number is CGMCC No.33530, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 yard, West Beichen Road, Chaoyang District, Beijing. The bacillus altitudinis DBN-EW28 strain disclosed by the invention has relatively high cellulase activity and antibacterial property, and is used for degrading crop straws when being combined with other strains, so that the contents of cellulose and hemicellulose in the crop straws are remarkably reduced, and the degradation effect of straw lignocellulose and the conversion rate of reducing sugar are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a highland bacillus, a composite bacterial agent and an application thereof. Background Art

[0002] At present, the comprehensive utilization of straw mainly covers three major directions: fertilizer, feed and energy. Among them, feed technology has attracted much attention because it can improve animal digestibility and nutrient utilization.

[0003] Traditional methods for processing straw into feed mainly include physical and chemical methods. Physical methods such as pulverization and steam explosion can improve the palatability of straw, but they cannot effectively break down the cell wall structure. Chemical methods such as alkalization and ammoniation improve degradation efficiency by destroying the lignin-cellulose complex. However, these methods are subject to problems such as high chemical dosage, high cost, and limited applicability. For example, ammoniation is only suitable for ruminant feed, while strong alkaline treatment can easily lead to soil salinization, making it difficult to meet the needs of modern green agriculture.

[0004] In recent years, biological methods have become a research hotspot due to their environmental friendliness. The enzyme system secreted by microorganisms is used to carry out targeted degradation of straw components. Although biological methods have the advantages of efficient degradation and no need for additional energy input, the degradation efficiency of a single strain or a simple compound bacterial agent is still not ideal. For example, in traditional cellulose decomposing bacteria (such as Trichoderma), their effect on cellulose wrapped in lignin is limited; and white rot fungi specifically for lignin degradation have slow metabolism and harsh enzyme production conditions. These existing technical solutions all have obvious limitations: either the degradation efficiency is insufficient, or the adaptability is not strong, or the synergistic mechanism is imperfect, and it is difficult to meet the needs of large-scale straw resource utilization.

[0005] In view of the above-mentioned technical bottlenecks, the development of composite microbial agents that have efficient lignocellulose degradation capabilities, strong environmental adaptability and optimized microbial community synergy mechanisms remains the key to breaking through straw resource utilization technology. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The present invention provides a Bacillus altitudinis strain DBN-EW28, which was deposited at the General Microbiology Center of the China Culture Collection Administration on February 14, 2025, and was named DBN-EW28. It was classified as Bacillus altitudinis, with a deposit number of CGMCC No. 33530, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the 16S rRNA sequence of the Bacillus subtilis is shown in Table SEQ ID No. 1.

[0009] The invention also provides a microecological preparation containing the Bacillus subtilis.

[0010] The present invention also provides the use of the Bacillus subtilis or the microecological preparation in degrading crop straw.

[0011] Preferably, the crop straw includes corn straw and oat straw.

[0012] The present invention also provides a composite bacterial agent containing the Bacillus subtilis.

[0013] Furthermore, the composite bacterial agent also includes Bacillus velezensis and Bacillus licheniformis.

[0014] Furthermore, the deposit number of the Bacillus velez is CICC 24433, and the deposit number of the Bacillus licheniformis is CGMCC No.7030.

[0015] The present invention also provides application of the composite bacterial agent in degrading crop straw.

[0016] Preferably, the crop straw includes corn straw and oat straw.

[0017] Based on the above technical solution, the present invention has the following advantages and beneficial effects:

[0018] The present invention is the first to apply a composite bacterial agent of Bacillus altitudinis DBN-EW28, derived from the earthworm intestine, to the fermentation of straw. The Bacillus altitudinis DBN-EW28 strain has high cellulase activity and antibacterial properties, and is derived from the earthworm intestine, so it has a certain adaptability to the animal intestine. Moreover, after the present invention combines the Bacillus altitudinis DBN-EW28 strain with the Bacillus Velez CICC 24433 strain and the Bacillus licheniformis LC01 strain, the weight loss rate of the straw changes significantly, and the neutral detergent fiber of corn straw and the neutral detergent fiber, acid detergent lignin, and hemicellulose content of oat straw are all significantly reduced, greatly improving the degradation effect of straw lignocellulose and the reducing sugar conversion rate. In addition, by optimizing the composition and ratio of the fermentation agent, the straw prepared by the present invention has a higher nutritional value than the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the extraction process of earthworm intestinal microorganisms.

[0020] Figure 2 This is the colony characteristic diagram of Bacillus subtilis DBN-EW28.

[0021] Figure 3 These are the growth curves of Bacillus altitudinis DBN-EW28, Bacillus velezensis CICC 24433, and Bacillus licheniformis LC01.

[0022] Figure 4 The dynamic changes of reducing sugar content in straw degradation experiments, where (a) is corn straw and (b) is oat straw.

[0023] Figure 5 These are 500x scanning electron microscope photos of corn straw and oat grass after treatment.

[0024] Figure 6 This is the Fourier infrared spectrum of the treated straw. DETAILED DESCRIPTION

[0025] Specific operating steps and detailed explanations are given below for the above examples, but they do not constitute a limitation to the technical solution of the present invention.

[0026] Example 1 Isolation and screening of strains

[0027] The earthworms used in the present invention are obtained by laboratory cultivation. The breeding bed is made of rotten and well-rotted cow dung mixed with nutrient soil, which is placed in a cool and ventilated place. Watering is carried out regularly to ensure that the surface soil is moist. The earthworm species is Eisenia fetida.

[0028] 1. Isolation of cellulose-degrading bacteria from earthworm intestines

[0029] Sampling and processing: Select mature earthworms with distinct annuli, clean their bodies with neutral PBS buffer (pH 7.2-7.4), blot the surface liquid with sterile filter paper, and place on ice for 3 minutes to induce hypothermia. Pre-sterilized dissection instruments are disinfected with 75% ethanol and wiped with sterile filter paper. Fix the earthworms on a dissection board with their abdomen facing upwards. Figure 1 As shown, the four-stage nailing method was used to perform the dissection along the axial direction from the male orifice to the anus, and the abdominal incision was fixed section by section. The intestinal contents were collected into ice-cold centrifuge tubes and vortexed to mix.

[0030] Microbial isolation: The intestinal contents suspension was subjected to 10 -1 to 10 -7 Gradient dilution was performed, and 100 μL of each gradient solution was inoculated on the surface of the selective culture medium for cellulose-degrading bacteria. After uniform dispersion with a sterile glass applicator, the culture was cultured at 28°C for 3-5 days.

[0031] Formula of the selective culture medium for cellulose-degrading bacteria: 15 g / L CMC-Na, 1.0 g / L ammonium nitrate or 1.65 g / L ammonium sulfate, 1.0 g / L yeast extract, 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 20.0 g / L agar, natural pH, sterilization at 121°C for 20 min.

[0032] Strain purification: Select morphologically distinct colonies and subculture using the three-zone streak method in LB medium (37°C) and PDA medium (30°C). Repeat this process until a single, purified colony is obtained. Ultimately, 85 pure cultures were obtained and stored in glycerol tubes at -80°C.

[0033] 2. Screening of cellulose degradation ability of strains

[0034] Screening culture medium formula: same as the selective culture medium for cellulose-degrading bacteria.

[0035] Strain inoculation and culture: The isolated and purified strains were inoculated onto the primary screening culture medium and cultured in an incubator at 37°C (bacteria) or 28°C (fungi) for 4-6 days. The colonies were then stained with 1 mg / mL Congo red for 30 minutes, followed by decolorization with 1 mol / L NaCl solution for 30 minutes. After the NaCl solution was removed, the diameter of the hydrolysis zone around the colonies was measured. The zone diameter ratio (D / d) of the isolated strains is shown in Table 1:

[0036] Table 1 Strain circle diameter ratio

[0037]

[0038] Two strains with high carboxymethyl cellulose sodium degradation ability, DBN-EW28 and EW-29, were screened and their circle diameter ratios were 4.44±0.45 and 5.74±1.45, respectively.

[0039] Physiological, biochemical, and morphological characteristics of the strains are as follows: strain DBN-EW28 appears as a white, round, opaque object with a moist, shiny surface on LB medium, while strain EW-29 appears as a white, translucent object with a viscous, wrinkled texture on medium. Strain DBN-EW28 is Gram-positive and exhibits a mist-like, diffusive growth in semisolid medium. It is motile, catalase-positive, oxidase-positive, and positive in gelatin liquefaction and starch hydrolysis tests, as well as citrate utilization and malonate utilization. It is positive in the VP test and negative in the methyl red (MR) test. Strain EW-29 is Gram-positive and non-motile in semisolid medium. It is catalase-positive, oxidase-negative, and positive in gelatin liquefaction and starch hydrolysis tests, as well as citrate utilization and malonate utilization. It is positive in the VP test and negative in the methyl red (MR) test.

[0040] 3. Determination of antibacterial ability of strains - Oxford cup double-layer diffusion method

[0041] Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) were used as indicator bacteria, inoculated into 5 mL of LB liquid medium (1% inoculum) and cultured overnight at 37°C. A 3% water agar base medium (10 mL / plate) was prepared and, after solidification, placed in a sterile Oxford cup. The indicator bacteria were mixed with 40°C 0.8% agar LB semi-solid medium (final concentration 106 CFU / mL), and 10 mL / plate was spread as the top layer. After solidification, the Oxford cup was removed, the plate was marked, and 100 μL of the test solution was added to the wells. The plate was allowed to stand for 10-20 minutes, then incubated at 37°C for 18-24 hours. The diameter of the inhibition zone (including the Oxford cup aperture) was measured and recorded with a vernier caliper. The antibacterial activity was compared with that of the control group. The results showed that strain DBN-EW28 was significantly effective against Staphylococcus aureus ATCC25923, but had no inhibitory effect on Escherichia coli CICC10389. Strain EW-29 had no inhibitory effect on Staphylococcus aureus ATCC 25923 and Escherichia coli CICC10389. The results are shown in Table 2.

[0042] Table 2 Antibacterial test results comparison table

[0043]

[0044]

[0045] The present invention further performed PCR amplification on the 16s rRNA of the two strains using universal primers (forward primer 27F, reverse primer 1492R). The PCR amplification products were sent to Qingke Biotechnology Co., Ltd. for sequencing. The gene sequence obtained by 16s rRNA molecular identification was compared with the sequence in the NCBI library by BLAST, and the strain homology was compared. The strain phylogenetic tree (Neighbour-Joining) was constructed using MEGA 11 software. The sequence alignment results showed that DBN-EW28 had a homology of 100.00% with Bacillus altitudinis 41KF2Bt.26 and was identified as Bacillus altitudinis. EW-29 had a homology of 100.00% with Bacillus subtilis HSY21 and was identified as Bacillus subtilis.

[0046] The 16S rRNA sequence of DBN-EW28 is shown in Table SEQ ID No. 1:

[0047]

[0048] The isolated DBN-EW28 was deposited at the General Microbiology Center of the China Culture Collection Administration on February 14, 2025, and was classified and named: Bacillus altitudinis, with the deposit number CGMCC No. 33530. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0049] 4. Determination of enzyme activity of strains

[0050] The CMCase, FPA and β-glucosidase activities of the two screened strains DBN-EW28 and EW-29, Bacillus velezensis CICC 24433 and Bacillus licheniformis LC01 were determined.

[0051] Among them, the Bacillus licheniformis LC01 was deposited by the applicant on December 25, 2012 at the General Microbiology Center of the China Culture Collection Committee, abbreviated as CGMC C, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 7030. The strain has been published, see patent CN103039716B for details.

[0052] The Bacillus velezensis CICC 24433 is from the China Industrial Microorganism Culture Collection Center.

[0053] Cellulase activity assay:

[0054] Formula of cellulase production culture medium: KH2PO4 2g / L, (NH4)2SO4 1.4g / L, MgSO4·7H2O0.3g / L, CaCl2 0.3g / L, FeSO4·7H2O 0.005g / L, MnSO4 0.0016g / L, ZnSO4·7H2O0.0014g / L, CoCl20.002g / L, CMC-Na 5g / L, peptone 5g / L, pH 7.0, sterilization at 121℃ for 20min.

[0055] Strain Cultivation and Crude Enzyme Preparation: Four strains were inoculated into LB liquid medium and cultured at 37°C, 180 rpm, and shaken until the exponential growth phase. After adjusting the OD600 of the culture to 1.0 with sterile water, the culture was transferred to a 150 mL Erlenmeyer flask containing 50 mL of enzyme production medium at a 5% inoculum rate. The culture was cultured at 37°C, 160 rpm, for 1-2 days. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes to obtain the crude enzyme solution.

[0056] Definition of enzyme activity unit (U / mL): The amount of cellulase required to catalyze the hydrolysis of substrate to produce 1 μg of glucose in 1 mL of enzyme solution for 1 min is one unit of enzyme activity.

[0057] Establishment of standard curve: prepare glucose gradient solution, take 0-1.4mL of 1mg / mL glucose standard solution, add distilled water to 2.0mL, add 2.0mL DNS reagent, boil in water bath for 10min, and then dilute to 25mL. Measure the absorbance at 540nm to establish the standard curve.

[0058] Endoglucanase (CMCase) Assay: Prepare four test tubes containing 2.0 mL of 1% CMC-Na. Add 1.0 mL of crude enzyme solution to three sample tubes. Incubate at 50°C for 30 minutes, then add 2.0 mL of DNS reagent. Add 1.0 mL of inactivated enzyme solution to a blank tube. Boil in a water bath for 10 minutes, adjust the volume to 25 mL, measure absorbance at 540 nm, and calculate enzyme activity based on the standard curve.

[0059] Filter Paper Enzyme (FPA) Assay: Four test tubes containing 2.0 mL of pH 4.8 buffer and 0.5 g of filter paper strips were preheated at 50°C for 5 minutes. Three sample tubes were then added with 1.0 mL of crude enzyme solution and incubated for 60 minutes. A blank tube was supplemented with inactivated enzyme solution and 2.0 mL of DNS reagent. The mixture was then brought to volume in a boiling water bath for 10 minutes. The absorbance was then measured and the enzyme activity was calculated.

[0060] β-glucosidase (β-gase) assay: replace CMC-Na with 1% salicin and follow the CMCase assay procedure.

[0061] Here are the results:

[0062] strain DBN-EW28 had CMCase of 8.56 U / mL, FPA of 1.66 U / mL, and β-gase of 1.90 U / mL;

[0063] The CMCase, FPA, and β-gase contents of strain EW-29 were 12.71 U / mL, 2.00 U / mL, and 0.36 U / mL, respectively;

[0064] strain LC01 had CMCase of 3.89 U / mL, FPA of 1.58 U / mL, and β-gase of 1.76 U / mL;

[0065] The CMCase, FPA and β-gase contents of strain CICC 24433 were 23.07 U / mL, 2.58 U / mL and 1.67 U / mL, respectively.

[0066] Example 2 Screening of composite bacterial agents

[0067] 1. Inter-strain antagonism test

[0068] The four strains were streaked onto LB medium at intersections, and then incubated at 37°C for 1-2 days to observe whether the growth of the indicator bacteria was inhibited (e.g., broken lines, sparse colonies). The observations showed no significant mutual inhibition between the four strains.

[0069] 2. The four strains were compounded in different combinations of 2, 3 and 4 strains. The strain seed solution after 1 day of culture was adjusted to OD 600 After the concentration reaches about 1.0, the bacterial suspension of each strain is mixed and inoculated into the fermentation medium at a volume ratio of 1:1:1:1 with a 5% inoculum volume, based on the principle of keeping the total inoculation volume unchanged. The composite bacterial agent composition is as follows:

[0070] Table 3 Combination of compound bacterial agents

[0071]

[0072] Fermentation medium formula: corn straw powder 15 g / L, (NH4)2SO4 4 g / L, KH2PO4 1.0 g / L, NaCl 0.1 g / L, MgSO4·7H2O 0.3 g / L, CaCl2 0.1 g / L, FeCl3 0.01 g / L, pH 7.0-7.2, sterilization at 121°C for 20 min.

[0073] After 1-2 days of fermentation, the supernatant was obtained by centrifugation and the cellulase and hemicellulase activities of each bacterial combination were determined by DNS method. The cellulase and hemicellulase activities of four single strains under the same conditions were also determined.

[0074] Definition of hemicellulase activity unit (U / mL): The amount of xylanase or mannanase required to catalyze the hydrolysis of substrate to produce 1 μmol of xylose or mannose in 1 mL of enzyme solution for 1 min is one unit of enzyme activity.

[0075] Hemicellulase activity determination: 1% xylan (source leaf, beech) and 0.5% carob bean gum were used as substrates, and 0.1 mL of crude enzyme solution was added to 0.9 mL of substrate solution (inactivated crude enzyme solution was used as control). After shaking, the solution was placed in a 50°C water bath for 30 minutes. After that, 1 mL of DNS reagent was added. After boiling in a water bath for 10 minutes, the solution was taken out and quickly cooled to room temperature. The volume was made up to 25 mL, and the OD was measured after shaking.540 At the same time, a standard curve was drawn and the hemicellulase activity was calculated based on the standard curve.

[0076] Since the hemicellulose component of straw is relatively easy to decompose, and most cellulolytic enzymes such as β-1,4-endoglucanase (EG), cellulose biohydrolases (CBHs), and β-glucosidase (β-gase) can simultaneously act on the hemicellulose backbone and cleave β-1,4-glycosidic bonds, hemicellulase is only used as an auxiliary screening indicator in this embodiment, and the main indicator is cellulase activity. The cellulase and hemicellulase activities of each strain and combination using corn straw powder as the enzyme substrate are shown in Table 4, thereby screening the combination T7 with higher cellulase activity. The CMCase activity of this bacterial agent combination is 22.05 U / mL, the FPA is 2.94 U / mL, the β-gase activity is 2.97 U / mL, the xylanase activity is 2.74 U / mL, and the mannanase activity is 0.95 U / mL. The activities of CMCase, FPA, β-gase, xylanase, and mannanase in this combination were all higher than those of the four single strains and other combination treatments, indicating that strains LC01, CICC 24433, and DBN-EW28 had significant synergistic effects and enhanced enzyme production performance compared with single strains.

[0077] Table 4 Cellulase and hemicellulase activities of strains / combinations

[0078]

[0079]

[0080] Example 3 Preparation of composite bacterial agent

[0081] 1. Fermentation of Bacillus altitudinis DBN-EW28, Bacillus velezensis CICC 24433, and Bacillus licheniformis LC01:

[0082] (1) Preparation of fermentation medium: cornmeal 0.5%, soybean meal 1%, sucrose 0.4%, fish meal 0.6%, KH2PO4 0.1%, FeSO4·7H2O 0.025%, MgSO4·7H2O 0.05%, MnSO4 0.024%, CaCO3 0.1%, defoamer 0.05%, pH 7.2.

[0083] (2) Fermentation tank culture: sterilize at 121°C for 30 min, cool to 37°C, inoculate the seed solution of each strain with a cell age of 12 h, and inoculate at a volume of 0.1%. During the fermentation period, the rotation speed was maintained at 220 rpm, the ventilation volume was 1:0.3 vvm, and the tank pressure was 0.05 MPa. The fermentation was terminated after 24 h, and the number of viable bacteria in the fermentation was ≥1.0 × 10 11 CFU / mL.

[0084] 2. Preparation of composite bacterial agent:

[0085] The fermentation broths of Bacillus altitudinis DBN-EW28, Bacillus velezensis CICC 24433, and Bacillus licheniformis LC01 were centrifuged to enrich the cells, and the concentrations of the three bacterial broths were adjusted to 2.0 × 10 8 CFU / mL, the bacterial solutions of the three strains were mixed in a volume ratio of 1:1:1 to obtain a straw degradation composite bacterial agent.

[0086] Example 4 Application of microbial agent in straw degradation

[0087] The composite bacterial agent obtained in Example 3 was used to conduct an application test on straw cellulose degradation.

[0088] Corn straw and oat straw were cut into 2-3 cm segments and dried in an oven at 80°C to constant weight. The initial lignocellulose content of the straw is shown in Table 5.

[0089] Table 5 Initial lignocellulose content of straw

[0090]

[0091] Each type of straw was treated with two treatments (blank control and treatment with added microbial agent), with a total of four treatments (two types of straw × two treatments). The inoculated microbial agent treatment group was prepared by adding 20 g of straw, 400 mL of liquid fermentation nutrient solution, and a suspension of cellulose-degrading composite microorganisms (OD 600 The mixture was inoculated with 3% of the culture medium and cultured at 37°C and 150 rpm for 9 days. 5 mL of the bacterial liquid was sampled on the 1st, 2nd, 3rd, 5th, 7th, and 9th days and stored in a -20°C refrigerator for the determination of reducing sugars. At the end of the fermentation on the 9th day, a solid straw sample was taken, repeatedly rinsed with dilute acid, then rinsed with distilled water, and dried at 75°C to constant weight. The weight loss rate of the straw before and after the experiment was calculated, and the acid detergent fiber, neutral detergent fiber, acid detergent lignin, crude fiber, and hemicellulose contents in the straw were determined by the Van Soest washing method. The crude ash content was determined by the muffle furnace combustion method. The straw samples were also subjected to electron microscopy scanning and Fourier transform infrared spectroscopy analysis.

[0092] Fermentation nutrient solution formula: peptone 2 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1.0 g / L, NaCl 0.5 g / L, CaCl2 0.2 g / L, yeast extract 0.5 g / L, pH natural, autoclave at 121°C for 20 min.

[0093] Figure 4 The results showed that the addition of cellulose-degrading composite bacterial agents during the fermentation and degradation of corn straw and oat straw could increase the reducing sugar content in the fermentation liquid at the initial stage of fermentation, and the reducing sugar content showed a trend of first increasing and then decreasing during the fermentation process. This was because the microorganisms in the bacterial agent in the initial stage degraded the straw lignocellulose, causing the macromolecular cellulose or hemicellulose to decompose into reducing sugars such as glucose that are easily absorbed by animals, and the further growth and reproduction of the microorganisms consumed part of the reducing sugar, so that the reducing sugar content showed a downward trend in the later stage, and the straw itself dissolved part of the reducing sugar, resulting in the reducing sugar content in the inoculated bacterial agent group at the end of fermentation being lower than that in the blank control group.

[0094] Figure 5 The results showed that the addition of bacterial agents caused some damage to the surface structure of the straw. The surface structure of the blank control straw was smooth and dense, while the inoculation of composite bacterial agents caused multiple breaks in the straw, and the structure became loose and irregular.

[0095] like Figure 6 (Left) shows the Fourier infrared spectrum of corn stalks at 3414 cm -1 A broad absorption peak appears at 2919 cm, which is related to the -OH stretching vibration of cellulose, hemicellulose and lignin molecules. After treatment, the absorption peak of hydroxyl group moves to the direction of larger wave number, indicating the exposure of cellulose, hemicellulose and lignin. -1 The absorption peak observed at 1513 cm comes from the vibration of the C=C skeleton of the benzene ring, indicating that the benzene ring skeleton has been affected to a certain extent during the treatment process; -1 The absorption peak at 1327 cm comes from the vibration of the aromatic ring skeleton; -1 The absorption peaks at 1374, 1247, 1162, and 1053 cm are characteristic peaks of the condensation of the syringyl (S) ring and the guaiacyl (G) ring of lignin; -1 The absorption peaks at are caused by cellulose and hemicellulose -CH2 swing and COO- stretching, COC asymmetric stretching, COC antisymmetric stretching vibration, and CH deformation. These characteristic absorption peaks of cellulose and hemicellulose have moved to a certain extent after treatment, indicating that the addition of microbial agents has also affected the cellulose and hemicellulose structures in corn straw. Figure 6 As shown on the right, oat grass has the following wavelengths: 3414, 2919, 1513, 1374, 1247, 1162, 1053 cm-1 Absorption peaks also appeared at , indicating that the structure of cellulose, hemicellulose and lignin of oat straw was affected to a certain extent after inoculation with the fungus.

[0096] The weight loss rate of straw before and after degradation is shown in Table 6.

[0097] Table 6 Straw weight loss rate

[0098]

[0099] By adding the composite bacterial agent, the weight loss rate of corn straw increased from 12.55% to 20.96%, and the weight loss rate of oat straw increased from 33.35% to 45.09%. In addition, the weight loss rate of the straw inoculated with the composite bacterial agent was improved to a certain extent compared with the inoculation of the three single strains alone, indicating that the strains played a synergistic role and promoted the degradation of straw.

[0100] The lignocellulose content of straw is shown in Tables 7 and 8.

[0101] Table 7 Effect of adding composite microbial agent on corn straw degradation

[0102]

[0103] Table 8 Effect of adding composite microbial agents on the degradation of oat grass

[0104]

[0105] Note: The number of samples in the table is n=4, and the values ​​in the table are mean ± standard deviation. Significant differences of p<0.05 are represented by different letters, * represents significant differences at the 5% significance level (p<0.05), ** represents significant differences at the 1% significance level (p<0.01), *** represents extremely significant (p<0.001), and NS represents not significant differences.

[0106] The addition of the composite inoculant significantly reduced the neutral detergent fiber content of corn straw, while also reducing crude fiber, crude ash, acid detergent fiber, acid detergent lignin, and hemicellulose to some extent. The neutral detergent fiber, acid detergent lignin, hemicellulose, and crude ash content of oat straw were significantly reduced, with some reductions in crude fiber and acid detergent fiber. This suggests that the composite inoculant significantly degrades cellulose, hemicellulose, and lignin in straw.

[0107] The above is a general description and detailed implementation scheme of the present invention, but the protection scope of the present invention is not limited thereto. Any solution that is replaced or modified based on the ideas or implementation schemes of the present invention falls within the protection scope of the present invention.

Claims

1. A strain of Bacillus altitudinis, characterized in that: Its deposit number is CGMCC No.33530.

2. The Bacillus sp. according to claim 1, characterized in that Its 16S rRNA sequence is shown in Table SEQ ID No.

1.

3. A microecological preparation containing the Bacillus subtilis according to claim 1 or 2.

4. Use of the Bacillus subtilis according to claim 1 or 2 or the probiotic preparation according to claim 3 in degrading crop straw.

5. The use according to claim 4, characterized in that The crop straws include corn straws and oat straws.

6. A composite bacterial agent, characterized in that: Contains the Bacillus sp. according to claim 1.

7. The composite bacterial agent according to claim 6, characterized in that The composite bacterial agent also includes Bacillus velezensis and Bacillus licheniformis.

8. The composite bacterial agent according to claim 7, characterized in that The deposit number of the Bacillus Velez is CICC 24433, and the deposit number of the Bacillus licheniformis is CGMCC No.7030.

9. Use of the composite bacterial agent according to any one of claims 6 to 8 in degrading crop straw.

10. The use according to claim 9, characterized in that The crop straws include corn straws and oat straws.

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