A strain B11 resistant to low temperature and saline-alkali and capable of degrading fiber lignin and its application

Abstract

The present invention discloses a strain B11 with cold and saline-alkali tolerance and the ability to degrade fiber lignin, which relates to the technical field of straw degradation. The strain B11 is Bacillus stratosphericus, and its taxonomic name is Bacillus stratosphericus, with the preservation number of CGMCC No. 30278. The present invention has strong cold tolerance (8 °C) and saline-alkali tolerance (9% NaCl, pH 13), and can be used for subsequent soil improvement and promoting crop growth.

Description

Technical Field

[0001] The present invention relates to the technical field of straw degradation, and more particularly to a strain B11 capable of resisting low temperature and salt and alkali and degrading fiber lignin, and its application. Background Art

[0002] Currently, soil salinization is widespread globally and is one of the main causes of soil degradation, a global environmental problem. According to incomplete statistics from the United Nations Educational, Scientific and Cultural Organization (UNESCO) and the Food and Agriculture Organization (FAO), saline-alkali land covers approximately 960 million hectares worldwide, accounting for 64.20% of total arable land. This area is increasing by 1.5 million hectares annually, severely damaging and weakening soil productivity. Against this backdrop, improving the quality of saline-alkali farmland has become a strategic option for addressing or mitigating national food security.

[0003] Inner Mongolia is my country's main grain-producing region, providing approximately 35 million tons of grain annually and playing an irreplaceable role in ensuring national food security. Effective improvement of saline-alkali soils has significantly increased the area of ​​arable land, which is of great significance to ensuring national food security.

[0004] Various measures, including deep tillage, straw incorporation, organic fertilizer application, and the use of beneficial microorganisms, can significantly improve the quality of saline-alkali soil. Compared to other salinization mitigation methods, straw incorporation is cost-effective and reduces environmental pollution, making it an effective measure for achieving green and sustainable agricultural development. In-situ straw incorporation can effectively improve soil salinization by inhibiting soil salt accumulation, increasing available nutrients, and regulating the structure of beneficial soil microorganisms, thereby alleviating salinity stress on crops. However, in the cold and arid regions of northern China (such as Inner Mongolia), low temperatures and droughts in autumn result in slow degradation of in-situ straw, severely impacting the sowing and emergence of crops the following year. The use of beneficial microorganisms can significantly accelerate straw degradation and simultaneously produce large amounts of extracellular polymeric substances (EPS, ACC, and IAA) to neutralize salt and alkali ions in the soil environment, alleviating soil salinization and promoting crop growth and development. Existing microbial resources for straw degradation are mostly room- or high-temperature-resistant, and screening for efficient low-temperature straw-degrading microorganisms is scarce. Furthermore, efficient degraders that tolerate saline-alkali conditions are even rarer.

[0005] As an important microbial resource, straw-degrading bacteria play a significant role in straw reuse and reducing soil salinization. They can accelerate straw degradation in low-temperature, saline-alkali environments, rapidly breaking straw down into simple compounds that can be used by crops. This improves soil's available nutrients, reduces soil salinity, and enhances crop resistance to biotic and abiotic stresses.

[0006] Therefore, for the saline-alkali land in the cold and arid areas of the north, it is an urgent problem that technicians in this field need to solve to screen out efficient straw degradation microorganisms suitable for the area, effectively improve the degradation efficiency of straw return to the field, reduce the degree of soil salinization, and provide technical guarantees for stable and increased crop yields. Summary of the Invention

[0007] In view of this, the present invention provides a low-temperature, salt-alkali-tolerant and cellulose lignin-degrading strain B11 and its application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A strain B11 that is resistant to low temperature, salt and alkali and can degrade cellulose lignin, the strain B11 is a stratosphere Bacillus, and is classified and named Bacillus stratosphericus It was deposited in the General Microbiology Center of China Culture Collection Administration on April 8, 2024, with the deposit number CGMCC No.30278, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The application of a low-temperature-resistant, salt-alkali-resistant and cellulose lignin-degrading strain B11 in the metabolic production of cellulase and ligninase.

[0011] Furthermore, the cellulase includes filter paper enzyme, endoglucanase, exoglucanase, β-glucosidase, xylanase, laccase, lignin peroxidase, and manganese peroxidase.

[0012] Application of a low-temperature-resistant, salt-alkali-resistant and cellulose lignin-degrading strain B11 in in-situ straw return to the field.

[0013] Furthermore, in saline-alkali soils of varying degrees, strain B11 significantly improved straw degradation efficiency and straw element release rate.

[0014] The application of the metabolites of a low-temperature-tolerant, salt-alkali-tolerant and cellulose lignin-degrading strain B11 in improving saline-alkali land and improving plant tolerance.

[0015] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:

[0016] This study screened cellulose-lignin-degrading bacteria from saline-alkali soils of varying degrees using CMC-Na (cellulose) and lignin (lignin). Strain B11 was identified as a stratospheric Bacillus based on its morphological, physiological, and biochemical properties, as well as 16S rDNA sequence analysis. The strain's filter paper enzyme activity was 35.26 U / mL, endoglucanase activity was 24.33 U / mL, exoglucanase activity was 31.25 U / mL, β-glucanidase activity was 24.26 U / mL, xylanase activity was 25.45 U / mL, laccase activity was 112.36 U / L, lignin peroxidase activity was 56.23 U / L, and manganese peroxidase activity was 49.26 U / L. Furthermore, the strain exhibited strong resistance to low temperatures (8°C) and salinity (9% NaCl, pH 13), making it suitable for subsequent soil improvement and crop growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0018] Figure 1 Distribution of locations where soil samples were collected;

[0019] Figure 2 Screening process;

[0020] Figure 3 Purified strain B11;

[0021] Figure 4 strain B11 in highly saline straw medium;

[0022] Figure 5 Straw degradation effect of strain B11;

[0023] Figure 6 Degradation effects of Congo red (left) and guaiacol (right) by the hydrolysis cycle of strain B11;

[0024] Figure 7 Results of the cold and salt-alkali resistance tests of strains B11 and B7;

[0025] Figure 8 Phylogenetic tree analysis of strain B11;

[0026] Figure 9 Analysis of temperature and humidity changes in soils with different degrees of salinity;

[0027] Figure 10Degradation efficiency of straw and its components by strain B11 in saline-alkali soils of different degrees;

[0028] Figure 11 The degradation of straw in saline-alkali soils with different degrees was observed by electron microscopy using strain B11;

[0029] Figure 12 Release rate of main elements of strain B11 in saline-alkali soils of different degrees;

[0030] Figure 13 The germination test of strain B11 was applied under saline-alkali stress;

[0031] Figure 14 Pot experiment of strain B11 under saline-alkali stress. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1

[0033] 1. Materials and Methods

[0034] 1.1 Soil sample collection

[0035] Conducted soil surveys in the four main banners (counties) of the Hetao Plain in Bayannur City, Inner Mongolia. Figure 1 A total of 24 soil samples were collected, and GPS location information and vegetation types were recorded (Table 1). Soil samples were collected using a three-point sampling method, where soil was collected at three points in an equilateral triangle with a side length of 5 meters. The average sampling depth was 0–20 cm. After obtaining the three soil samples, they were mixed and stored in a −80°C refrigerator for subsequent soil microbial screening.

[0036] Table 1 GPS information of soil samples and vegetation types

[0037] 1.2 Experimental reagents and equipment

[0038] Table 2 Summary of reagents and equipment

[0039] Table 3 Main instruments and equipment

[0040] 1.4 Test culture medium

[0041] (1) Cellulose culture medium: (NH3)2SO4 2.0 g, K2HPO4 1.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.2 g, MnSO4·7H2O 0.01 g, CMC-Na 10.0 g, agar 15-20 g, 1 L distilled water, pH around 7, sterilized at 121 ℃ for 30 min.

[0042] (2) Lignin culture medium: (NH3)2SO4 2.0 g, K2HPO4 1.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.2 g, MnSO4·7H2O 0.01 g, lig 1.0 g, agar 15-20 g, 1 L distilled water, sterilize at 121 ℃ for 30 min.

[0043] (3) LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 6-7. Sterilize at 121°C for 30 min. (If using solid medium, add 20 g agar.)

[0044] (4) Corn straw degradation medium: 2.0 g corn straw (1-2 cm stem), 50 mL soil extract, 8% NaCl, pH 9, sterilized at 121 °C for 30 min.

[0045] (5) Congo red-cellulose medium: KH2PO4 0.5 g, MgSO4·7H2O 0.25 g, gelatin 2.0 g, cellulose powder (Sigma) 1.88 g, Congo red 0.2 g, agar 14 g, 1 L distilled water, pH 8-8.5. Sterilize at 121°C for 30 min.

[0046] (6) Sodium carboxymethylcellulose medium: sodium carboxymethylcellulose (CMC-Na) 0.6 g, KH2PO4 0.15 g, MgSO4·7H2O 0.075 g, (NH3)2SO4 0.3 g, agar 16 g, 1 L distilled water, pH 8-8.5. Sterilize at 121 °C for 30 min.

[0047] (7) Culture medium for initial screening of salt-tolerant EPS-producing strains: 20 g sucrose, 0.2 g K2HPO4, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 3.0 g yeast extract, 20 g agar, soil extract to 1 L, pH 8-8.5. Sterilize at 121 °C for 30 min.

[0048] (8) Enzyme production medium: 0.6 g urea, 0.5 g peptone, 2.0 g (NH3)2SO4, 1.0 g K2HPO4, 0.05 g MgSO4·7H2O, 0.016 g MnSO4·7H2O, 0.017 g ZnSO4·7H2O, 0.02 g CaCl2, 0.2 g NaCl, 1 L distilled water, natural pH. Sterilize at 121°C for 30 min.

[0049] (9) Filter paper strip disintegration medium: (NH3)2SO4 3.00 g, KH2PO4 1.00 g, MgSO4·7H2O 0.40 g, Yeast extract 0.10 g, fixed volume 1 L, pH 7.0, sterilize at 121 ℃ for 30 min.

[0050] 1.4 Reagent Preparation

[0051] (1) DNS colorimetric reagent (3,5-dinitrosalicylic acid): Prepared according to the standard DNS reagent of the Ministry of Agriculture; citric acid buffer solution and glucose standard solution: Prepared according to QB 2583-2003 standard: Weigh 3,5-dinitrosalicylic acid (10 ± 0.1 g) and place it in approximately 600 mL of water. Gradually add 10 g of sodium hydroxide and dissolve it in a 50 °C water bath under magnetic stirring. Then, add 200 g of potassium sodium tartrate, 2 g of phenol (redistilled), and 5 g of anhydrous sodium sulfite in sequence. After all the solution is dissolved and clarified, cool to room temperature, dilute to 1000 mL with water, filter, store in a brown reagent bottle, and place in the dark for 7 days before use.

[0052] (2) Congo red staining solution: weigh 0.10 g of Congo red and dilute to 100 mL with deionized water;

[0053] (3) NaCl solution: Weigh 5.85 g of NaCl and dilute to 100 mL with deionized water.

[0054] (4) Whatman filter paper strips: Cut clean, starch-free Whatman filter paper into 1×6 cm strips.

[0055] (5) 1% glucose standard solution: Accurately weigh 0.1 g of dry glucose and dilute to 10 mL with deionized water. Prepare the solution immediately before use.

[0056] (6) 0.1 mol / L pH 4.5 citric acid buffer: Weigh 9.6 g of anhydrous citric acid and 2.7 g of sodium hydroxide and add them to 900 mL of distilled water while stirring. Finally, add distilled water to make up to 1 L. Test the pH value and adjust the solution to pH 4.5 with citric acid or sodium hydroxide dilution.

[0057] 2. Screening of low-temperature salt- and alkali-resistant degrading bacteria

[0058] 2.1 Enrichment culture

[0059] Weigh 5 g of soil sample and place it in a triangular flask containing 45 mL of sterile water and sterilized glass beads. Oscillate at 180 r / min for about 20 min to fully disperse the soil microorganisms.

[0060] 2.2 Initial screening

[0061] like Figure 2 As shown, 1 mL of soil sample dilution was taken and placed in a test tube containing 9 mL of sterile saline (0.9% NaCl solution) and mixed thoroughly. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 Soil dilution. Select 10 -4 ~10 -10 The soil dilution was spread on two selective media, carboxymethyl cellulose medium and alkaline lignin medium, respectively. The culture was carried out at 10°C for 7 days. The morphology and color of the colonies were observed and recorded. Specific colonies were screened and duplicate colonies were removed. Specific colonies were selected and streaked on LB medium for isolation and purification. The culture was carried out at 25°C for 1 to 7 days. The growth of the colonies was observed and recorded every day. Single colonies with different morphologies were streaked on new LB medium for isolation and purification until a pure strain was obtained ( Figure 3 ).

[0062] 2.3 Rescreening

[0063] like Figure 2 As shown in the results, 100 μL of the selected low-temperature and salt-alkali tolerant strains were added to straw degradation medium (low salinity pH 7, NaCl 2%; moderate salinity pH 9, NaCl 5%; high salinity pH 11, NaCl 8%) and cultured at 10°C for 28 days. The degradation of corn straw was observed and recorded. After 28 days, the corn straw was removed and dried at 80°C to constant weight. The weight loss rate was calculated, and strains with a straw weight loss rate of ≥30% were selected. The selected strains were numbered and each strain was liquid propagated. Specifically, each strain was selected and placed in 40 mL of sterile basal LB liquid medium and incubated at 25°C and 130 rpm. -1Culture at a constant temperature for 12–18 h, prepare glycerol stock at a bacterial suspension / glycerol (50% sterilized glycerol) (v / v) ratio of 1:1, place in a 2 mL cryovial, and store at −80°C until use.

[0064] The selected single strain B11, which is resistant to low temperature and salt and alkali, has a straw degradation rate of 34.51% in low salinity (pH 7, NaCl 2%) over 28 days, a maximum straw degradation rate of 35.36% in moderate salinity (pH 9, NaCl 5%) over 28 days, and a maximum straw degradation rate of 35.87% in high salinity (pH 11, NaCl 8%) over 28 days. Figure 4 , Figure 5 ).

[0065] 2.4 Determination of the degradation function of the strain

[0066] The degradation function of the purified strains was preliminarily identified, including the ability to degrade lignocellulose, starch, fat, and protein. The identification method is as follows:

[0067] (1) Cellulose-degrading bacteria: After staining the colony surface with 1 mg / mL Congo red solution for 1 hour, observe whether a transparent circle is produced on the agar medium surface and the size of the transparent circle to determine whether the strain has the ability to produce cellulase (see the results). Figure 6 Left).

[0068] (2) Lignin-degrading bacteria: Take 30 mL of 95% (v / v) ethanol solution, add 0.5 g of guaiacol, and drip it on the surface of the colony. If the titration area is reddish-brown, it means that the strain can produce laccase. Take 0.1% benzidine (v / v) and 0.4% hydrogen peroxide (v / v) in equal volumes and mix them, drip them on the edge of the colony. If the titration area is yellowish-brown, it means that the strain can produce peroxidase. The color depth can represent the ability of the strain to produce laccase and peroxidase (see the results). Figure 6 right).

[0069] (3) Starch (polysaccharide) degrading bacteria: Add 0.02 mol / L iodine solution to the surface of the colony. If a colorless transparent circle appears on the surface of the culture medium, the strain can degrade starch.

[0070] (4) Fat-degrading bacteria: Add 1.6% neutral red solution to the surface of the colony. If the surface of the culture medium turns dark blue, the strain can degrade fat.

[0071] (5) Protein-degrading bacteria: Use gelatin culture medium for identification. If the gelatin culture medium is liquefied, the strain can degrade protein.

[0072] Table 4 Straw degradation effect of strain B11 Note: “+” indicates that it has the corresponding degradation function, and “-” indicates that it does not have the corresponding degradation function.

[0073] 2.5 Determination of strains’ tolerance to low temperature and salt and alkali

[0074] (1) All the primary screening strains (glycerol bacteria preservation solution) were inoculated into basic sterilized LB liquid culture medium at a ratio of 1:1 (v / v) and activated in batches (35°C, 130 r·min -1 The activated strain was cultured at constant temperature for 12 hours. Streak the activated strain onto LB solid medium using a sterilized inoculating stick at 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, and 14°C (pH 8, 5% NaCl). The strains were cultured at constant temperature for 24–72 hours. Growth was observed and the results recorded. Colonies that grew within 72 hours were considered tolerant (+); those that did not grew were considered intolerant (-).

[0075] (2) Salt tolerance assessment: Prepare LB solid medium with NaCl content of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12% respectively, with a pH of 8.0. Sterilize the activated strains with sterilized inoculation sticks and culture them on plates. Incubate at 15°C for 24-72 hours to observe the growth of the strains and record the results to determine the salt tolerance of the strains. Colonies that grow within 72 hours are recorded as tolerant (+), while those that do not grow are intolerant (-).

[0076] (3) Identification of alkali resistance: After activation of all the primary screened strains, the pH values ​​of the culture medium were adjusted to 8, 9, 10, 11, 12, 13, 14, and 15, respectively, based on the highest salt concentration at which each strain could grow normally. The activated strains were streaked on plates using sterilized inoculation sticks and cultured at a constant temperature of 15°C for 24 to 72 hours. The growth of the strains was observed and the results were recorded to determine the salt tolerance of the strains. Colonies that grew within 72 hours were recorded as tolerant (+), while those that did not grew were recorded as intolerant (-).

[0077] (4) Identification of salt and alkali tolerance: Based on the results of salt and alkali tolerance tests, the alkali tolerance of each strain was further identified. Based on the highest salt concentration at which each strain could grow normally, the NaCl content was set to 7%, 8%, 9%, 10%, and 11%, and the pH value of the culture medium was adjusted to 10, 11, 12, and 13 using NaOH (0.1 M, 1 M) and HCl (0.05 M, 1 M), respectively. The activated strains were streaked on plates using sterilized inoculation sticks and cultured at a constant temperature of 15°C for 24 to 72 hours. The growth of the strains was observed and the results were recorded to determine the salt tolerance of the strains. Colonies that grew within 72 hours were recorded as tolerant (+), while those that did not grew were recorded as intolerant (-).

[0078] Table 5 Identification of low temperature tolerance of strain B11 Note: “+” indicates that the strain can grow; “-” indicates that the strain cannot grow.

[0079] Table 6 Identification of salt tolerance of strain B11 Note: “+” indicates that the strain can grow; “-” indicates that the strain cannot grow.

[0080] Table 7 Identification of alkali resistance of strain B11 Note: “+” indicates that the strain can grow; “-” indicates that the strain cannot grow.

[0081] Table 8 Identification of salt and alkali tolerance of strain B11 Note: “+” indicates that the strain can grow; “-” indicates that the strain cannot grow.

[0082] Depend on Figure 7 As shown in Table 5-8, strain B11 can grow at 8°C, 9% NaCl, and pH 13. When subjected to both saline and alkali stress, it can grow at 9% NaCl and pH 12. The tolerance of the same strain B7 from the same batch of initial screening is slightly weaker than that of B11. Bacillus stratosphericus ) is a good low-temperature and salt-alkali resistant degradation strain.

[0083] 2.6 Molecular Biological Identification

[0084] Bacterial genomic DNA extraction method (Bacterial Genomic DNA Extraction Kit DP302).

[0085] (1) Take 1-5 mL of bacterial culture medium, centrifuge at 10,000 rpm for 1 min, and aspirate the supernatant as much as possible.

[0086] (2) Add 180 μL of buffer to the bacterial pellet. The buffer system (1 mL) is: Tris-HCl 20 μL; EDTA (pH 8.0) 4 μL; Trition 12 μL; Sterile water 964 μL; Lysozyme 0.02 g.

[0087] (3) After adding the buffer solution, incubate at 37°C for more than 30 min.

[0088] (4) Add 20 μL of Proteinase K solution to the centrifuge tube and mix well.

[0089] (5) Add 220 μL of buffer GB, shake for 15 seconds, and place at 70°C for 10 minutes (water bath). The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0090] (6) Add 220 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0091] (7) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column (the adsorption column is placed in a collection tube), leave it for 2 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid, and place the adsorption column in the collection tube.

[0092] (8) Add 500 μL of buffer GD to the adsorption column (please check whether anhydrous ethanol has been added before use), let it sit for 2 minutes, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and place the adsorption column in a collection tube.

[0093] (9) Add 600 μL of rinse solution PW to the adsorption column (check whether anhydrous ethanol has been added before use), let it sit for 2 minutes, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and place the adsorption column in a collection tube.

[0094] (10) Repeat step 8

[0095] (11) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid, place the adsorption column in a new centrifuge tube, and leave it at room temperature for several minutes (5 min) to completely dry the residual rinse liquid in the adsorption material.

[0096] (12) Add 50-200 μL (100 μL in this experiment) of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in a centrifuge tube.

[0097] The 16s rDNA of the isolated strain was amplified using universal primers as follows:

[0098] F: 5′-AGAGTTTGATCCTGGCTCA-3′, as shown in SEQ ID No. 1;

[0099] R: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID No. 2;

[0100] The PCR amplification reaction system (25 μL) consisted of 12.5 μL of Premix Taq™, 1 μL each of upstream and downstream primers (10 pmol / L), 3 μL of bacterial genomic DNA (1 μg / μL), and 7.5 μL of ddH₂O. The reaction conditions were: 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, 55°C for 45 s, and 72°C for 2 min, and finally 72°C for 10 min. After completion of the reaction, PCR products were detected by 1% agarose gel electrophoresis. The gel-recovered products were ligated with the pMD19-T vector and transformed into Trans-T1 competent cells. After blue-white screening and colony PCR identification, positive clones were sent to Nanjing GenScript Biotech Co., Ltd. for sequencing.

[0101] The results showed that the PCR band was single and clear, with a size of approximately 1500 bp, which was consistent with the expected size. The BLAST program on NCBI was used to compare the 16S rDNA sequences, and the phylogenetic tree was constructed using MEGA 7.0. The results showed that ( Figure 8 ), strain B11 and strain Bacillus stratosphericus The sequence similarity was the highest (99%). Based on the morphological characteristics, physiological and biochemical properties and 16S rDNA sequence analysis results of the strain B11 Stratosphere Bacillus ( Bacillus stratosphericus ), classified as Bacillus stratosphericus , deposited in the General Microbiology Center of China Culture Collection Administration on April 8, 2024, with the deposit number CGMCC No. 30278, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0102] Example 2

[0103] 3. Determination of Fiber Ligninase Activity

[0104] (1) Determination of total cellulase activity

[0105] Standard curve determination: Pipette 0-1.2 mL (0.0 / 0.2 / 0.4 / 0.6 / 0.8 / 1.0 / 1.2) of 1 g / L glucose standard solution, add deionized water to 2.00 mL after 0.2 mL intervals, add 3.0 mL of DNS reagent, mix well, boil in a boiling water bath for 5 minutes, cool with cold water, and dilute to 20.0 mL with deionized water. Measure the absorbance at 540 nm. Plot a standard curve with absorbance as the X-axis and glucose content as the Y-axis. The regression equation for this standard curve is: Y = 0.5952X + 0.01517, R 2=0.9923, indicating that the standard curve has good linearity and therefore meets the requirements and can be used as a standard curve for cellulase activity determination. The reaction solution was placed in a UV spectrophotometer to measure absorbance. The glucose content was determined according to the standard curve, and the cellulase activity was then calculated. Enzyme activity is calculated based on the substrate, sodium carboxymethyl cellulose, a pH of 6, and a constant temperature of 50°C in a water bath for 30 minutes. One unit of enzyme activity is defined as the amount of cellulase required to catalyze sodium carboxymethyl cellulose to produce 1 μg of glucose per 30 minutes.

[0106] (2) Determination of filter paper enzyme activity (FPA):

[0107] Place a 1 cm × 5 cm filter paper strip (approximately 0.1 g) in a 25 mL stoppered colorimetric tube. Add 1.0 mL of 0.1 mol / L pH 4.5 citrate buffer to submerge the filter paper strip. Add 1.0 mL of crude enzyme solution (1.0 mL of inactivated crude enzyme solution for the blank control). Incubate at 50°C for 30 min. Add 3.0 mL of DNS reagent, mix thoroughly, boil in a boiling water bath for 5 min, cool with cold water, and dilute to 20.0 mL. Mix thoroughly and measure the absorbance at 540 nm. Repeat three times for each tube.

[0108] (3) Determination of EG or Cx enzyme (endo-β-1,4-glucosidase) activity:

[0109] The carboxymethyl cellulose (CMC) enzyme activity assay was used. To a 25-mL stoppered colorimetric tube, add 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution for the blank control) and 1.00 mL of 1% CMC-Na solution (0.1 mol / L citrate buffer, pH 4.5). The mixture was allowed to stand at 50°C for 30 min. Then, 3.00 mL of DNS reagent was added. The mixture was boiled in a boiling water bath for 5 min, cooled with cold water, and the volume was adjusted to 20.00 mL with deionized water. After mixing, the absorbance was measured at 540 nm. Repeat three times for each tube.

[0110] (4) Determination of CBH or C1 enzyme (exo-β-1,4-glucosidase) activity:

[0111] The microcrystalline cellulose (MCC) enzyme activity assay was performed. To a 25-mL stoppered colorimetric tube, add 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution for the blank control) and 1.00 mL of a 1% microcrystalline cellulose solution (0.1 mol / L pH 4.5 citrate buffer). The mixture was allowed to stand at 50°C for 30 min. Then, 3.00 mL of DNS reagent was added. The mixture was mixed and boiled in a boiling water bath for 5 min. Cooled with cold water, the volume was adjusted to 20.00 mL, and the absorbance was measured at 540 nm. Repeat three times for each tube.

[0112] (5) Determination of β-1,4-glucosidase activity:

[0113] To a 25 mL stoppered colorimetric tube, add 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution for the blank control) and 1.00 mL of 1% salicin solution (0.1 mol / L pH 4.5 citrate buffer). Incubate at 50°C for 30 min. Add 3.00 mL of DNS reagent, mix thoroughly, boil in a boiling water bath for 5 min, cool with cold water, and dilute to 20.00 mL. After mixing, measure the absorbance at 540 nm. Repeat three times for each tube.

[0114] (6) Determination of xylanase (XYA) activity:

[0115] Pipette 2 mL of enzyme solution, equilibrate at 37°C for 10 min, add to a graduated test tube, add 5 mL of DNS, shake for 3-5 seconds, add 2 mL of 100 mg / mL xylan solution, incubate at 37°C for 30 min, boil in a boiling water bath for 5 min, cool to room temperature, and dilute to 25 mL with distilled water. Measure the absorbance at 540 nm using a standard blank as a control, and record the value as AB. Pipette 2 mL of enzyme solution, equilibrate at 37°C for 10 min, add to a graduated test tube, add 2 mL of 100 mg / mL xylan solution, shake for 3-5 seconds, add 5 mL of DNS, incubate at 37°C for 30 min, boil in a boiling water bath for 5 min, cool to room temperature, and dilute to 25 mL with distilled water. Measure the absorbance at 540 nm using a standard blank as a control, and record the value as AE. Repeat three times.

[0116] Prepare a 10 mg / mL xylose solution using acetic acid-sodium acetate buffer (pH 5.5) after drying to constant weight. Pipette 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mL of the prepared xylose solution and dilute to 100 mL with acetic acid-sodium acetate buffer (pH 5.5) to prepare xylose standard solutions at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mg / mL. Pipette 2 mL of the xylose standard solution into a graduated test tube, add 2 mL of 0.1 mol / L acetic acid-sodium acetate buffer (pH 5.5) and 5 mL of DNS reagent, shake for 3-5 seconds, boil in a boiling water bath for 5 minutes, cool to room temperature, dilute to 25 mL with distilled water, adjust to zero with a standard blank solution, and measure absorbance at 540 nm. A standard curve was drawn with xylose concentration as the Y-axis and absorbance value (OD value) as the X-axis.

[0117] (7) Determination of laccase (Lac) activity:

[0118] ABTS method: Accurately pipette 10 mL each of 100 mM malonic acid-sodium malonate buffer (pH 4.5) and 0.6 mM ABTS solution into a test tube, mix thoroughly, and place in a 30°C water bath for 30 min. Place 1 mL of the mixed reagent in a 1.4 mL cuvette, zero the spectrophotometer at 420 nm, and accurately pipette 50 μL of the enzyme solution to be tested into the cuvette containing the mixed reagent. Immediately record the absorbance, recording every 30 seconds for a total of three times. Take the average value and convert it to OD per minute. 420 change.

[0119] (8) Determination of lignin peroxidase (Lip) activity:

[0120] Resveratrol method: Accurately pipette 0.5 mL of 200 mM tartaric acid buffer into a 1.4 mL cuvette, add 0.1 mL of 40 mM resveratrol, accurately add 50 μL of the enzyme solution to be tested, add 350 μL of distilled water, and after incubating at 30°C in a water bath, add 10 μL of 20 mM H2O2 solution to start the reaction. Quickly measure the absorbance at 310 nm, and measure again after 1 minute. Calculate the difference between the two, which is the OD per minute. 310 change.

[0121] (9) Determination of manganese peroxidase (Mnp) activity:

[0122] Accurately pipette 0.5 mL of 100 mM propionic acid-sodium malonate buffer into a 1.4 mL cuvette and add 0.1 mL of 10 mM manganese sulfate solution. Accurately add 50 μL of the enzyme solution to be tested and 350 μL of distilled water. Incubate in a 30°C water bath for 10 minutes. Add 0.01 mL of 10 mM hydrogen peroxide solution to initiate the reaction. Use a spectrophotometer to quickly measure the absorbance at 270 nm. Measure again 1 minute later and calculate the difference between the two, which is the absorbance change per minute (OD) 270 change).

[0123] The results are shown in Table 9. The cellulase and ligninase activities of the strains varied greatly. The filter paper enzyme activity of strain B11 was 35.26 U / mL, the endoglucanase activity was 24.33 U / mL, the exoglucanase activity was 31.25 U / mL, the β-glucosidase activity was 24.26 U / mL, the xylanase activity was 25.45 U / mL, the laccase activity was 112.36 U / L, the lignin peroxidase activity was 56.23 U / L, and the manganese peroxidase activity was 49.26 U / L.

[0124] Table 9 Fiber lignin degradation enzyme activity of strain B11

[0125] Example 3

[0126] 4. Straw degradation efficiency and straw element release rate of strain B11 in saline-alkali soils of varying degrees

[0127] 4.1 Test location

[0128] The experimental site was located in Wayao Village, Salaqi Town, Baotou City, Inner Mongolia. Three types of salinity levels were selected for straw return treatment: low, medium, and high. Baotou City, Inner Mongolia (41°13'-43°36'N, 113°26'-116°13'E) has a typical mid-temperate continental monsoon climate, an average altitude of 1.1 km, and a total area of ​​approximately 1.05×10 4 km 2 The region has little rainfall in summer, with an average annual rainfall of 185 mm, and low temperatures and drought in winter. The shallow groundwater level in Tumochuanping, Inner Mongolia, leads to solute enrichment and severe secondary salinization. The basic properties of saline-alkali soil are shown in Table 1. The B11 used in this study is Bacillus stratosphericus (CGMCC NO.30278), the bacteria were mixed with fillers (corn stalk husk powder, starch, and bran) in a ratio of 1:4 for fermentation, and then freeze-dried to prepare a dry powder inoculum with a viable bacterial count of 7.5×10 10 CFU / g.

[0129] Table 10 Chemical properties of saline-alkali soils of different degrees and content of corn straw components

[0130] 4.2 Experimental Design

[0131] Corn straw was dried at 70°C and cut into 1 cm × 1 cm squares. 20 g of corn straw was weighed and added to a 25 cm × 35 cm mesh bag with a pore size of 5 mm. 20 g of sieved soil was then added to increase the contact area between the straw and the soil. The composition of corn straw is shown in Table 10. This experiment was conducted on three saline-alkali soils with low (L), medium (M), and high (H) degrees of salinity. Bacillus stratosphericu After corn harvest, nylon bags were placed at a depth of 25 cm in the soil. Dry powdered microbial inoculant was then quantitatively applied to the surface of the nylon bags and the soil was filled. For the sterilization treatment, an equal amount of inactivated microbial inoculant was applied. Each treatment was repeated 7 times. Thermometers and hygrometers were placed to monitor the soil ( Figure 9 The total experimental time was 40 days. The straw was removed to determine the degradation rate of the straw and its components, and the soil around the straw was collected to determine the soil enzyme activity, chemical properties, and salt and alkaline ions.

[0132] 4.3 Measurement indicators

[0133] (1) Determination of corn straw degradation rate: The corn straw degradation rate was determined by the weight loss method. The calculation formula is: Straw degradation rate (%) = (W0-W1) / W0×100%, where W0 represents the weight of the straw in the culture medium before inoculation (g); W1 represents the weight of the degraded straw remaining after drying at the end of the culture (g). The results were repeated three times.

[0134] (2) Determination of corn straw composition: The dried corn straw was crushed and passed through a 1 mm sieve. The sieved straw was transferred to a special filter bag for neutralization and acid washing. Cellulose and hemicellulose were determined using a cellulose analyzer (ANKOM A200i). For lignin determination, the sample was washed with 72% concentrated sulfuric acid and ashed using a muffle furnace. The degradation efficiency of the straw components was calculated based on the loss of cellulose, hemicellulose, and lignin. The main elements in the straw were determined by the potassium dichromate volumetric method-external heating method, the total nitrogen content was determined by the H2SO4-H2O2 digestion-distillation method, the total phosphorus content was determined by the vanadium molybdenum yellow colorimetric method, and the total potassium content was determined by flame photometry.

[0135] Depend on Figure 10 It can be seen that the straw degradation rates of strain B11 in low, medium and high saline-alkali soils were 34.56%, 33.69% and 35.14% respectively; the cellulose degradation rates in low, medium and high saline-alkali soils were 25.91%, 26.29% and 28.17% respectively; the hemicellulose degradation rates in low, medium and high saline-alkali soils were 23.45%, 25.61% and 26.73% respectively; and the lignin degradation rates in low, medium and high saline-alkali soils were 28.15%, 30.27% and 32.56% respectively. The internal structure of the degraded straw in each treatment was observed by electron microscopy. Figure 11 Compared to the CK treatment, the internal structures of hemicellulose, cellulose, and lignin in the bacterial treatment were irregular, rough, and fragmented, with numerous cavities, significantly improving their degradation efficiency. In highly saline and alkaline environments, bacterial growth was greatest and structural damage was highest, consistent with the metabolic degradation patterns of bacterial enzymes.

[0136] Depend on Figure 12 It can be seen that the carbon release rates of strain B11 in low, medium and high saline-alkali soils were 10.23%, 12.78% and 15.64% respectively; the nitrogen release rates in low, medium and high saline-alkali soils were 12.45%, 13.69% and 15.16% respectively; the phosphorus release rates in low, medium and high saline-alkali soils were 8.36%, 10.45% and 11.23% respectively; and the potassium release rates in low, medium and high saline-alkali soils were 14.26%, 16.74% and 18.59% respectively.

[0137] Example 4

[0138] 5. Metabolites of strain B11

[0139] (1) Determination of indoleacetic acid (IAA) content: The IAA production capacity of the strain was determined by referring to the Salkowskis colorimetric method. The method is as follows: the activated strain (1 mL) was inoculated into a solution containing L-tryptophan (200 mg·L -1 ) in DF liquid medium (40 mL) (with the non-inoculated treatment as the control group), 30°C, 180 r·min -1 After 48 h of constant temperature culture, OD was measured. 600 After keeping the absorbance values ​​of each strain consistent, the color was measured at 10000 r·min -1 Centrifuge for 3 minutes, take 1 mL of the supernatant and add 4 mL of alkowski colorimetric solution and mix well (the same operation as the control group), stand at room temperature in the dark for 20 minutes, and measure the OD 530 The IAA content (mg·L -1 ).

[0140] The standard curve was made based on the OD value of the 3-indoleacetic acid standard sample. The brief method is as follows: prepare the 3-indoleacetic acid standard solution, add 4 mL of Salkowski colorimetric solution, mix well, and stand at room temperature in the dark for 20 minutes. Measure the OD 530 A standard curve was prepared based on the concentration of the 3-indoleacetic acid standard solution and its corresponding absorbance value.

[0141] (2) Detection of deaminase (ACC) content: 7.5 mL of activated strain culture medium was taken and incubated at 30 °C and 200 r·min -1 After culturing at constant temperature for 24 h, the -1 Centrifuge for 10 min (4°C) and collect the precipitate; then resuspend the cells in 5 mL of DF nitrogen-free medium (without (NH4)2SO4) and incubate at 30°C, 200 rpm. -1 Incubate at constant temperature for 24 h, continue centrifugation under the above conditions, collect the precipitate, and then add Tris-HCl buffer (5 mL, 0.1 mol·L -1 , pH 7.6) to resuspend and wash the cells, repeat twice; resuspend the cells again in 7.5 mL DF nitrogen-free medium, and add 45 μL sterile ACC solution (0.5 mol·L -1 ), placed in a shaker, 30℃, 200 r·min -1 Culture at constant temperature for 24 h (this process is to induce the strain to produce ACC deaminase); then the culture solution was heated at 5000 r·min -1 Centrifuge for 20 min, collect the precipitate, add Tris-HCl solution (5 mL, 0.1 mol·L -1, pH 7.6) and resuspend and wash the cells, repeat twice, collect the precipitate; take 100 μL of cell disruption solution (dissolved by shaking with toluene) and store at 4 °C for use in protein content determination.

[0142] Take 200 μL of the remaining cell lysis solution and place it in a 1.5 mL centrifuge tube. Add ACC solution (20 μL, 0.5 mol·L -1 ) and mixed them evenly. The other one was not added with ACC solution and reacted at 30°C for 15 min. Then 1 mL of HCl solution (0.56 mol·L) was added to each -1 ) and mix well, 11000 r·min -1 Centrifuge for 10 min (4°C), take the supernatant (800 μL each) and place it in a 5 mL centrifuge tube for later use; add HCl solution (800 μL, 0.56 mol·L -1 ), 2,4-dinitrophenylhydrazine reaction solution (300 μL), react at 30 °C for 30 min, and then add NaOH solution (22 mL, 2 mol·L -1 ) and mix well, and measure OD after color development 540 The absorbance value of the sample was substituted into the regression equation of the standard curve to obtain the content of α-ketobutyric acid, and the amount of α-ketobutyric acid (μmol) was calculated. The total protein content (mg) in the cell lysate was then determined according to the Bradford method. Finally, the amount of α-ketobutyric acid produced by bacterial cells per unit protein content per unit time was defined as ACC deaminase activity, and the ACC deaminase activity was calculated according to the formula (U·mg -1 ), calculated as follows. Three replicates were set for each strain.

[0143] (3) Determination of phosphate solubility: The phosphate solubility of the strain was determined by referring to the molybdenum antimony colorimetric method. The method is as follows: the activated strain (1 mL) was inoculated into PKO medium (50 mL), and the control group was inoculated with an equal amount of sterile water. The culture medium was placed at 30°C and 150 rpm. -1 Cultured at constant temperature for 7 days; then at 11000 r·min -1 Centrifuge for 5 minutes and collect the supernatant. Add 1-2 drops of dinitrophenol indicator to the supernatant (30 mL) and adjust with a small amount of NaOH solution (1 M, 10 M) or HCl solution (1 M, 5 M) until the solution just turns slightly yellow. Then accurately add 5 mL of molybdenum antimony mixed color development solution, shake thoroughly, and dilute to 50 mL with deionized water. After reacting at room temperature above 15°C for 30 minutes (within 8 hours), measure and record the OD 700 The phosphorus content (mg·L) was calculated based on the standard curve. -1 ).

[0144] Standard curve drawing: Accurately absorb 5 mg·L -1 0, 2, 4, 6, 8, and 10 mL of K2HPO4 standard solution were placed in a 50 mL volumetric flask. A blank solution of equal volume to the sample solution used for colorimetric determination was added. 1 to 2 drops of dinitrophenol indicator were added, and NaOH solution (1M, 10M) and HCl solution (1M, 5M) were added dropwise until the solution just turned slightly yellow. 5 mL of molybdenum antimony mixed colorimetric solution was then accurately added. The solution was shaken thoroughly and made up to 50 mL with deionized water. The solution was reacted at room temperature above 15°C for 30 minutes. The OD value corresponding to each standard solution was determined. 700 A standard curve is prepared based on the concentration of the standard solution and its corresponding absorbance value.

[0145] (4) Determination of iron carrier synthesis capacity: The activated strain (0.5 mL) was inoculated into MKB liquid culture medium (5 mL). At the same time, an equal amount of MKB liquid culture medium without the inoculated strain (1 mL) was added for the determination of the reference value (Ar). The mixture was incubated at 30°C and 150 rpm. -1 After 48 hours of culture, take 1 mL of culture medium and mix it with CAS detection solution at a ratio of 1:1 (v / v). After 1 hour at room temperature, measure the OD 630 The experimental group is labeled A, and the reference group is labeled Ar. The measurement is adjusted to zero with distilled water. The A / Ar ratio represents the relative siderophore content in the sample. A lower value indicates a greater ability of the strain to produce siderophores. (Note: During the experiment, if the strain produces siderophores, the reaction system will appear orange; if it does not, the reaction system will remain blue.)

[0146] (5) Determination of extracellular polymeric substances (EPS): The EPS content was determined by referring to the Congo red agar method. The method is as follows: the activated strain (1 mL) was inoculated into LB liquid medium (40 mL) containing 5% salinity, and the mixture was incubated at 37°C and 150 rpm. -1 The cells were cultured overnight at a constant temperature for 72 h; then the cells were rotated at 8000 r·min -1 Centrifuge for 10 minutes to remove bacterial cells. Mix the supernatant with anhydrous ethanol at a 1:2 (v / v) ratio and incubate overnight at 4°C. Separate the precipitate by centrifugation to obtain EPS. Dry the EPS in hot air at 40°C for 2–3 days to obtain the dry weight, which is the EPS content.

[0147] The results showed that strain B11 could secrete 61.91 g / L of extracellular polymers, 65.37% of siderophores, and 46.37 mg / L of phosphate solubilization to improve saline-alkali environment, as well as 5.34 mg / L of indoleacetic acid and 0.24 U / mg of dehydrogenase to promote plant growth under adverse conditions.

[0148] 6. Strain B11 promotes corn seedling growth under saline-alkali stress

[0149] The experimental soil was collected from saline-alkali farmland in western Bayannur City, Inner Mongolia, China (40°13'-42°28'N, 105°12'-109°53'E). The top 0-25 cm of uncontaminated saline-alkali soil was collected. The homogenized soil was air-dried and passed through a 2.0 mm sieve for subsequent potting experiments. The maize variety used in this experiment was Yudika 159. Before germination, the maize seeds were surface-sterilized with 10% (v / v) hydrogen peroxide (H2O2) and rinsed with deionized water. The seeds were then evenly distributed on damp filter paper and germinated at 25°C for 48-72 hours until radicles emerged.

[0150] Germination test: The test corn variety was Dika 159. Corn germination effect: Select corn seeds of uniform size and fullness for surface disinfection (soak in 10% sodium hypochlorite solution for 10 minutes, rinse with sterile water for more than three times until odorless), and germinate for 12 to 24 hours on a sterilized surface dish (with gauze soaked in sterile water inside). Then select seeds of uniform size and successfully germinated and soak them in the bacterial suspension for 2 to 3 hours. For the non-inoculated treatment, soak the seeds in an equal amount of sterile distilled water at the same time. The bacterial suspension was prepared as follows: the strain was inoculated into a sterilized basic LB liquid medium (40 mL) and cultured (35°C, 130 r·min -1 Constant temperature culture) 12-16h, 10000 r·min -1 Centrifuge for 10 minutes, take the bacterial precipitate and dilute it with sterile distilled water to OD 600 The saline solution was set to pH 9 NaCl 5%, and the seeds pre-treated with soaking seeds (10 seeds were evenly sown in each culture dish) were sown in culture dishes containing vermiculite (50 g). After that, the bacterial suspension was regularly replenished every other day according to the amount of water loss, and an equal amount of sterile water was added for the non-inoculated treatment. The light incubator was kept at 28°C and the humidity was maintained at around 70%. The growth was observed by 12h / 12h light / dark culture. After 7 days of culture, the germination rate, bud length, root length, dry weight and fresh weight were counted. Three replicates were set for each treatment.

[0151] A greenhouse pot experiment was conducted in a greenhouse at Inner Mongolia Agricultural University (Inner Mongolia, China), with two treatments: one treated with B11 and one untreated (CK). Plants were grown under natural light for 40 days, with six replicates per treatment. Each pot (30 cm diameter × 20 cm base diameter × 25 cm height) was filled with 6 kg of soil. 100 g of dry powdered microbial inoculum was applied to the soil surface, while the untreated treatment received 100 g of inactivated dry powdered microbial inoculum, providing a similar microbial flora. Ten pre-germinated maize seeds were sown evenly in each pot and thinned to five plants after 14 days. During plant growth, daytime temperatures ranged from 20-30°C and nighttime temperatures from 10-20°C. Deionized water was added regularly to maintain soil water holding capacity at approximately 60%.

[0152] In the germination test ( Figure 13 ) After the strain B11 was applied, the germination rate was 85.75%, the shoot length was 16.18 cm, the root length was 23.45 cm, the fresh weight was 2.87 g, and the dry weight was 0.43 g; the germination rate increased by 12.35 percentage points compared with the control, the shoot length increased by 5.89 cm, the root length increased by 12.78 cm, the fresh weight increased by 1.12 times, and the dry weight increased by 0.19 g compared with the control. In the potted experiment ( Figure 14 ), exogenously applied strain B11 resulted in plant height of 49.61 cm, stem diameter of 7.50 cm, SPAD of 46.31, fresh weight of 18.06 g, and dry weight of 7.47 g. Compared with the control, plant height increased by 16.89 cm, stem diameter increased by 3.75 cm, SPAD increased by 21.45, fresh weight increased by 9.74 g, and dry weight increased by 4.12 g. These results indicate that strain B11 significantly improves the salt-alkali tolerance of maize seedlings and promotes their growth under saline-alkali stress.

[0153] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain B11 that is resistant to low temperature, saline-alkali, and can degrade fiber lignin, characterized in that, The strain B11 is Bacillus stratosphericus, and its taxonomic name is Bacillus stratosphericus , and its preservation number is CGMCC No. 30278.

2. Use of the strain B11 as claimed in claim 1 in the production of cellulase and ligninase.

3. The application according to claim 2, wherein The cellulase includes filter paper enzyme, endoglucanase, exoglucanase, β-glucosidase, xylanase; The ligninase includes lignin peroxidase and manganese peroxidase.

4. Use of the strain B11 as claimed in claim 1 in the in-situ straw returning to the field.

5. The application according to claim 4, wherein, In saline-alkali soils with different degrees, the strain B11 significantly improves the straw degradation efficiency and the straw element release rate.

6. Use of the strain B11 as claimed in claim 1 in improving the saline-alkali tolerance of plants.

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