Bacillus subtilis WJL-2 and application thereof

Abstract

The invention discloses bacillus subtilis WJL-2 and application thereof, and relates to the technical field of microorganisms. The bacillus subtilis WJL-2 disclosed by the invention is preserved in Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC No: 65111, the preservation date is September 9, 2024, and the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. Results of the embodiment of the invention show that the strain WJL-2 has the capability of degrading cellulose, and when the strain WJL-2 is used for a liquid fermentation test, the degradation rate of the strain WJL-2 on sugarcane straws on the 12th day is 26.45%, and the degradation rate of the strain WJL-2 on straws on the 3rd day of culture reaches 26.37%. The highest hemicellulose degradation rate of the strain WJL-2 is 58.72%, and the highest cellulose degradation rate of the strain WJL-2 is 39.88%.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a strain of Bacillus subtilis WJL-2 and an application thereof. Background Art

[0002] my country has abundant straw resources, with about 1.8 billion mu of cultivated land. A large amount of cultivated land produces abundant straw resources, and crops produce about 820 million tons of straw each year. my country's crop straw resources are large in output, widely distributed, and diverse in variety. The region is vast and the technology for straw treatment is immature. Traditional straw treatment methods, such as burning and stacking, not only cause waste of resources, but may also cause serious environmental pollution and ecological damage. Therefore, it is particularly important to explore an environmentally friendly, efficient and economical straw treatment method. Microbial degradation, as an emerging straw treatment technology, has attracted much attention due to its environmental friendliness and sustainability.

[0003] Straw is mainly composed of cellulose, hemicellulose, lignin, ash, protein, fat and soluble sugar. Cellulose, as one of the main chemical components of straw, accounts for about 30%-50% of the dry weight of straw. Its efficient degradation is crucial for the overall utilization of straw. Currently, most of the reported cellulose-degrading strains are derived from soil, including Pseudomonas, Bacillus, Streptomyces, white rot fungi, etc., but there are few studies on strains from other sources. In addition, the strains also have problems such as low enzyme activity, poor stability and slow degradation rate, which significantly affect the degradation efficiency and application scope of straw.

[0004] Therefore, developing a bacterium that can safely and efficiently degrade straw is of great significance for the recycling of agricultural and sideline products. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a strain of Bacillus subtilis WJL-2. The technical problem to be solved by the present invention is to provide an application of the strain of Bacillus subtilis WJL-2 for safe and efficient degradation of straw.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A strain of Bacillus subtilis WJL-2, which is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No: 65111, a deposit date of September 9, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] Application of Bacillus subtilis WJL-2 in preparing products for straw degradation.

[0009] Application of Bacillus subtilis WJL-2 in the preparation of products for cellulose degradation.

[0010] Application of Bacillus subtilis WJL-2 in regulating cellulase activity.

[0011] The cellulase includes endoglucanase, β-glucanase and exoglucanase.

[0012] Application of Bacillus subtilis WJL-2 in regulating filter paper enzyme activity.

[0013] The product for straw degradation or the product for cellulose degradation is a degradation agent.

[0014] A degradation agent for straw degradation. The preparation method of the degradation agent comprises the following steps: inoculating a single colony of Bacillus subtilis WJL-2 into a 250mL conical flask filled with 100mL LB liquid culture medium, placing the flask in a shaking incubator at 28°C and 220r / min for culturing for 48h, centrifuging at 4000r / min to obtain bacterial cells, adjusting the bacterial liquid with physiological saline, and obtaining the degradation agent for straw degradation.

[0015] A degradation agent for cellulose degradation. The preparation method of the degradation agent comprises the following steps: inoculating a single colony of Bacillus subtilis WJL-2 into a 250mL conical flask filled with 100mL LB liquid culture medium, placing the flask in a shaking incubator at 28°C and 220r / min for 48h, centrifuging at 4000r / min to obtain bacterial cells, and adjusting the bacterial liquid with physiological saline to obtain the degradation agent for cellulose degradation.

[0016] Application of Bacillus subtilis WJL-2 suspension in promoting straw degradation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The Bacillus subtilis WJL-2 disclosed in the present invention is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 65111, a deposit date of September 9, 2024, and a deposit address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The results of the embodiments of the present invention show that strain WJL-2 has the ability to degrade cellulose. In a liquid fermentation test using strain WJL-2, the degradation rate of sugarcane straw was 26.45% on the 12th day, and on the 3rd day of cultivation, the degradation rate of straw by strain WJL-2 reached 26.37%. The highest hemicellulose degradation rate of strain WJL-2 is 58.72% ( Fig.12 ), the cellulose degradation rate was the highest at 39.88%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a ventral view of the tested termite;

[0020] Figure 2 This is a diagram of the termite gut obtained by dissection;

[0021] Figure 3 This is a transparent image of strain WJL-2 obtained by staining with cellulose Congo red staining;

[0022] Figure 4 This is the phylogenetic tree of strain WJL-2 based on 16s rDNA sequence similarity;

[0023] Figure 5 This is the pH value change diagram of the straw liquid fermentation test of strain WJL-2;

[0024] Figure 6 is the glucose standard curve;

[0025] Figure 7 This is the change chart of filter paper enzyme activity in straw liquid fermentation test of strain WJL-2;

[0026] Figure 8 This is the change chart of β-glucanase activity in straw liquid fermentation test of strain WJL-2;

[0027] Fig. 9 This is the change diagram of endoglucanase activity in straw liquid fermentation test of strain WJL-2;

[0028] Fig.10 This is the exoglucanase activity change diagram of the straw liquid fermentation test of strain WJL-2;

[0029] Fig.11 This is the change diagram of straw degradation rate in straw liquid fermentation test of strain WJL-2;

[0030] Fig.12 This is the change diagram of hemicellulose degradation rate in straw liquid fermentation experiment of strain WJL-2;

[0031] Fig.13 This is a graph showing the changes in cellulose degradation rate in the straw liquid fermentation experiment of strain WJL-2. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0033] The capture site of the yellow-winged termite used in this application is Dayaoshan Nature Reserve, Jinxiu Yao Autonomous County, Laibin City, Guangxi ( Figure 1 ).

[0034] The formula of LB liquid / solid culture medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar powder (if it is liquid culture medium, agar powder does not need to be added), and 1 L deionized water.

[0035] The formula of sodium carboxymethyl cellulose (CMC-Na) culture medium is: sodium carboxymethyl cellulose (CMC-Na)

[0036] 10g, K2HPO4 1g, MgSO4·7H2O 0.5g, agar powder 13g, deionized water 1L.

[0037] Example 1

[0038] 1. Isolation and purification of Bacillus subtilis WJL-2 strain

[0039] Take 15 termites and soak them in 75% alcohol for 3 minutes for disinfection. Take out the termites with sterile tweezers, soak them in sterile water for 30 seconds, take them out and soak them in 75% alcohol for 30 seconds. Finally, rinse them repeatedly with sterile water several times to wash away the alcohol remaining on the surface of the termites.

[0040] Take two sterile tweezers to dissect the sterilized and washed termites. Use one pair of tweezers to hold the head and the other pair to hold the end of the tail and anus and pull it out slowly. Separate the termite intestine completely from the body, leaving the entire intestine ( Figure 2 ), and the rest was discarded. The above operations were all performed on a clean bench.

[0041] Place the termite intestines in a 250 mL conical flask filled with 100 mL sterile water, place 5 termite intestines in each flask, add a small amount of glass beads, repeat 3 times, and place in a constant temperature shaker at 28°C and 220 r / min for 24 hours to prepare the termite intestinal bacterial suspension. Dilute the bacterial suspension to 10 -1 , 10 -2 , 10 -3 , 10 -4 , use a pipette to draw 6 μl of the diluted termite intestinal bacterial suspension onto the sodium carboxymethyl cellulose medium, spread it with a spreading rod, and place the coated plate in a 28°C constant temperature incubator until the strain grows out. Continuously pick the strain to the carboxymethyl cellulose plate for purification and culture until a single colony is obtained.

[0042] 2. Qualitative screening of cellulose-degrading bacteria

[0043] The obtained single colony was inoculated into a shaking tube containing 5 mL of LB liquid medium and cultured in a shaking incubator at 28°C and 220 r / min for 12 hours to activate the strain. The activated strain was inoculated on sodium carboxymethyl cellulose medium with a toothpick, 3 spots were inoculated on each medium, repeated three times, and cultured at a constant temperature of 28°C until the strain grew out.

[0044] After the strain grows out, use a vernier caliper to measure the strain diameter (d), dye the culture medium with 1mg / mL Congo red dye for 25min, discard the dye, add 1mol / L NaCl solution for decolorization for 20min, and observe whether the colony produces a colorless transparent circle. Use the same method to measure the diameter of the transparent circle (D), record the diameter of the colony and the transparent circle, and calculate D / d.

[0045] The results are as follows Figure 3 As shown, a colorless transparent zone appeared around strain WJL-2 after staining with Congo red, and the ratio of the transparent zone diameter D to the colony diameter d (D / d) was 3.93 (Table 1), which proved that strain WJL-2 had the ability to degrade cellulose.

[0046] Table 1 Qualitative screening results of cellulose degrading bacteria

[0047] Strain number d: Average diameter of strain D (mm) D: Average diameter of hydrolysis circle (mm) D / d WJL-2 3.93 18.13 3.93±0.13

[0048] 3. Identification and preservation of Bacillus subtilis WJL-2 strain

[0049] The strain was inoculated into LB solid medium and the single colonies grown were observed. The colonies of strain WJL-2 were slightly yellow, opaque, and dry on the surface. After 7 days of culture, the edges spread outward and became irregular shapes. DNA was extracted according to the instructions of TIAN GEN Bacterial Genomic DNA Extraction Kit (centrifugal column type) (Beijing, China), and the kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Using bacterial universal primers 27F and 1492R, PCR amplification was performed with strain DNA as a template. The PCR product was detected by 1% agarose gel electrophoresis and sent to Shanghai Shenggong Biological Company for 16S rDNA sequencing. The 16SrDNA sequence of Bacillus subtilis WJL-2 is shown in SEQ ID NO.1. The sequencing results were submitted to the Gen Bank database of the National Center of Biotechnology Information (NCBI) of the United States for homology alignment search based on the basic local alignment search tool (BLAST). The 16S rRNA gene sequences of model strains with high homology were selected, and the neighbor-joining (NJ) method in MEGA v.7.0 software was used to construct a phylogenetic tree ( Figure 4). Combining the results of phylogenetic tree comparison analysis based on colony morphology and 16S rRNA gene sequence construction, strain WJL-2 was identified as Bacillus subtilis, which was deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No: 65111, deposit date: September 9, 2024, and deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0050] Example 2

[0051] The culture conditions in the initial culture system were optimized (the following are all single-factor experimental methods) to screen the optimal growth conditions of the strain, as follows:

[0052] The obtained single colony was inoculated into a 250 mL conical flask containing 100 mL LB liquid culture medium, placed in a shaking incubator at 28°C and 220 r / min for 48 h, and then inoculated into Hutcheson culture medium at a 9% inoculum.

[0053] 2g of sugarcane straw powder passed through a 20-mesh sieve was added to a 250mL conical flask containing 100mL of Hutcheson medium, and sterilized in a high-temperature autoclave at 121℃ for 15min. The treatment group was inoculated with 9% bacteria, and the control group was added with the same amount of physiological saline, with 3 replicates, and cultured in a shaker at 28℃ and 200r / min. Samples were taken on the 3rd, 7th, and 12th days of culture to measure pH, cellulase activity (including filter paper enzyme (FPA) activity, β-glucanase activity, endoglucanase (CMC) activity, exoglucanase (CBH) activity), straw degradation rate, hemicellulose content, and cellulose content.

[0054] 1. pH

[0055] The pH of the culture medium was measured with a handheld pH meter on days 3, 7, and 12.

[0056] The results are as follows Figure 5 As shown, the pH of the treatment group with strain WJL-2 increased with time and finally reached 7.68, while the pH of the control group without strain added fluctuated around 6.5.

[0057] 2. Cellulase activity

[0058] ①Preparation of glucose standard curve

[0059] Take 6 clean test tubes and number them, add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of standard glucose solution respectively, then add 2, 1.8, 1.6, 1.4, 1.2, and 1 mL of distilled water to each test tube in the above order, add 1.5 mL of DNS reagent, place the test tubes in a boiling water bath for 5 min, take out and cool to room temperature, then add distilled water to make up to 10 mL, measure the OD value at 520 nm spectrophotometry, record the data and draw a glucose standard curve.

[0060] The results are as follows Figure 6 As shown, the equation of the glucose standard curve is: y = 0.8239x + 0.0397, and the correlation coefficient is: R 2 =0.9960, indicating that there is a good linear relationship between the glucose content and the absorbance value. The glucose content of the sample can be calculated using the absorbance value of the sample through the equation.

[0061] ②Preparation of crude enzyme solution

[0062] Take the relatively clear culture solution in the upper layer of the bottle and centrifuge it at 12000r / min for 2 minutes. The supernatant is the crude enzyme solution, which is diluted 2 times with deionized water and used for the next step of enzyme activity determination.

[0063] ③ Determination of four enzyme activities

[0064] The activities of four enzymes were measured respectively: filter paper enzyme (FPA) activity, endoglucanase (CMC) activity, β-glucanase activity, and exoglucanase (CBH) activity.

[0065] Filter paper enzyme activity: prepare 3 clean and dry test tubes, put 50mg filter paper strips in the test tubes, add 1.5mL citric acid buffer (0.05mol / L, pH4.5), put the test tubes in a 50℃ constant temperature water bath to preheat for 5min, take out and add 0.5mL enzyme solution and shake evenly, keep it in a water bath at 50℃ for 60min, take it out, add 1.5mL DNS reagent. Boil water bath for 10min, take it out and quickly cool it to room temperature, dilute it to 10mL with distilled water, take 200μL of reaction solution from each test tube to a clean 96-well plate, and measure the OD value at a wavelength of 520nm.

[0066] β-glucanase activity: prepare 3 clean and dry test tubes, add 1.5mL 0.5% salicin citrate buffer, put the test tubes into a 50℃ constant temperature water bath to preheat for 5min, take them out, add 0.5mL enzyme solution and shake evenly, keep them in a water bath at 50℃ for 30min, take them out, add 1.5mL DNS reagent. Boil water bath for 10min, take them out and quickly cool them to room temperature, dilute to 10mL with distilled water, take 200μL of the reaction solution from each test tube into a clean 96-well plate, and measure the OD value at a wavelength of 520nm.

[0067] Endoglucanase activity: prepare 3 clean and dry test tubes, add 1.5mL 1% CMC citric acid buffer, put the test tubes into a 50℃ constant temperature water bath to preheat for 5min, take them out, add 0.5mL enzyme solution and shake evenly, keep them in a water bath at 50℃ for 30min, take them out, add 1.5mL DNS reagent. Boil water bath for 10min, take them out and quickly cool them to room temperature, dilute to 10mL with distilled water, take 200μL of the reaction solution from each test tube into a clean 96-well plate, and measure the OD value at a wavelength of 520nm.

[0068] Exoglucanase activity: prepare 3 clean and dry test tubes, add 50mg of cotton wool and 1.5mL of citric acid buffer (0.05mol / L, pH5), preheat the test tubes in a constant temperature water bath at 45℃ for 5min, take them out and add 0.5mL of enzyme solution and shake evenly, keep them in a water bath at 45℃ for 24h, take them out and add 1.5mL of DNS reagent. Boil water bath for 10min, take them out and quickly cool them to room temperature, dilute them to 10mL with distilled water, take 200μL of the reaction solution from each test tube into a clean 96-well plate, and measure the OD value at a wavelength of 520nm.

[0069] Definition of enzyme activity unit (U / mL): Under the experimental conditions, the amount of enzyme required to hydrolyze the substrate to produce 1 μg of glucose per minute in 1 mL of enzyme solution is expressed as 1 enzyme activity unit.

[0070] Enzyme activity = (G×n×1000) / V×T.

[0071] In the enzyme activity formula, G: the glucose content corresponding to the absorbance value of the sample in the standard curve, in mg; V: the volume of crude enzyme solution added to the sample, in ml; T: the reaction time of the sample, in min; n: the dilution factor of the crude enzyme solution; 1000: the conversion value between mg and μg.

[0072] The results are as follows Figure 7-10 As shown in Figure 2, the filter paper enzyme activity and β-glucosidase activity of strain WJL-2 did not change much, and the highest filter paper enzyme activity was 61.76 mg / L ( Figure 7); the highest β-glucosidase activity was 20.08 mg / L ( Figure 8 ); The endoglucanase activity of strain WJL-2 decreased over time and finally reached 19.47 mg / L ( Fig. 9 ); the exoglucanase activity of strain WJL-2 was the highest at 1.60 mg / L ( Fig.10 ).

[0073] 3. Straw degradation rate

[0074] The liquid culture solution was filtered out, and the straw was repeatedly washed with sterile water for 3 times to remove the soluble substances on the surface. After the filtration, the straw was placed in a forced air drying oven at 70°C to dry to a constant weight and weighed.

[0075] Straw degradation rate = (weight of straw before treatment - weight of straw after treatment) / weight of straw before treatment × 100%.

[0076] The results are as follows Fig.11 As shown, in the liquid fermentation test using strain WJL-2, the degradation rate of sugarcane straw was 26.45% on the 12th day, while on the 3rd day of cultivation, the degradation rate of straw by strain WJL-2 reached 26.37%.

[0077] 4. Hemicellulose and cellulose content

[0078] The hemicellulose and cellulose contents of straw were determined using commercial kits purchased from Suzhou Keming Biotechnology Co., Ltd. (Suzhou, China) according to the instructions for determination of hemicellulose and cellulose contents of Suzhou Keming.

[0079] The results are as follows Fig.12 , 13 As shown, the hemicellulose degradation rate of strain WJL-2 was the highest at 58.72% ( Fig.12 ), the highest cellulose degradation rate was 39.88% ( Fig.13 ).

[0080] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis WJL-2, which is deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number: GDMCCNo: 65111, the deposit date: September 9, 2024, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Bacillus subtilis WJL-2 according to claim 1 in preparing products for straw degradation.

3. Use of the Bacillus subtilis WJL-2 according to claim 1 in the preparation of products for cellulose degradation.

4. Use of the Bacillus subtilis WJL-2 according to claim 1 in regulating cellulase activity.

5. The use according to claim 4, characterized in that: The cellulase includes endoglucanase, β-glucanase and exoglucanase.

6. Use of the Bacillus subtilis WJL-2 according to claim 1 in regulating the activity of filter paper enzymes.

7. The use according to claim 2 or 3, characterized in that: The product for straw degradation or the product for cellulose degradation is a degradation agent.

8. A degradation agent for straw degradation, characterized in that: The preparation method of the degradation agent comprises the following steps: inoculating a single colony of Bacillus subtilis WJL-2 into a 250 mL conical flask filled with 100 mL LB liquid culture medium, placing the flask in a shaking incubator at 28°C and 220 r / min for 48 hours, centrifuging at 4000 r / min to obtain bacterial cells, adjusting the bacterial liquid with physiological saline, and obtaining the degradation agent for straw degradation.

9. A degradation agent for cellulose degradation, characterized in that: The preparation method of the degradation agent comprises the following steps: inoculating a single colony of Bacillus subtilis WJL-2 into a 250 mL conical flask filled with 100 mL LB liquid culture medium, placing the flask in a shaking incubator at 28°C and 220 r / min for 48 hours, centrifuging at 4000 r / min to obtain bacterial cells, adjusting the bacterial liquid with physiological saline, and obtaining a degradation agent for cellulose degradation.

10. Use of the bacterial suspension of Bacillus subtilis WJL-2 according to claim 1 in promoting straw degradation.

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