Bacillus velezensis CA-06 and application thereof

Abstract

The invention belongs to the technical field of agriculture, and particularly relates to bacillus velezensis CA-06 and application thereof. The bacillus velezensis CA-06 is preserved in the China General Microbiological Culture Collection Center, and the preservation number of the bacillus velezensis CA-06 is CGMCC (China General Microbiological Culture Collection Center) No. 35570. The bacillus velezensis CA-06 provided by the invention has high cellulase activity, can significantly accelerate straw decomposition, realizes rapid resource utilization of straws, and improves the decomposition rate. The method has important significance in improving the straw utilization rate, improving the soil environment and promoting the sustainable high-quality development of agriculture.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, and in particular relates to a strain of Bacillus belye CA-06 and its applications. Background Technology

[0002] Straw, as the largest solid waste generated in agricultural production, accounts for hundreds of millions of tons of emissions globally each year. Its main components are cellulose, hemicellulose, and lignin, with cellulose accounting for 30% to 50%. It represents a highly promising renewable biomass resource. Traditional straw disposal methods primarily involve open burning and indiscriminate dumping, which not only results in a severe waste of biomass resources but also causes a series of environmental problems such as air pollution and soil degradation, hindering sustainable agricultural development. Therefore, achieving efficient resource utilization of straw (such as converting it into organic fertilizer, biofuel, and feed) has become a core requirement in the field of agricultural environmental protection and resource recycling.

[0003] Cellulose, the most abundant structural component of straw, has a dense crystalline structure formed by glucose residues linked by β-1,4 glycosidic bonds. It is difficult to directly decompose and utilize, and must rely on the synergistic action of cellulases (including endoglucanases, exoglucanases, and β-glucosidases) to degrade it into absorbable small-molecule sugars. Biodegradation has become the mainstream technology for straw cellulose degradation due to its advantages of being environmentally friendly, low-cost, and producing no secondary pollution. Screening for functional strains with high cellulase activity and stable degradation efficiency is a core prerequisite for the industrial application of this technology.

[0004] Currently, there are various strains available for straw degradation, but they generally suffer from low cellulase activity. To achieve the desired effect, it is necessary to increase the inoculation concentration or use them in combination with other strains, which increases the application cost and operational complexity, making it difficult to meet the needs of large-scale processing. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the purpose of this invention is to provide a strain of Bacillus belyssica CA-06 and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a strain of Bacillus belye ( Bacillus velezensis The strain CA-06 was deposited on August 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC); deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 35570.

[0007] The present invention also provides the application of the above-mentioned Bacillus belyssica CA-06 in the following aspects: (1) Degradation of cellulose; (2) Production of cellulase; (3) Decomposition of plant straw; (4) Degrading farmland residues.

[0008] Furthermore, the farmland residues include rice straw, wheat straw, or corn straw.

[0009] The present invention also provides a bacterial fermentation broth, the effective components of which include the above-mentioned Bacillus belye CA-06 or its fermentation products.

[0010] Furthermore, when the active ingredient is *Bacillus belyssica* CA-06, the effective viable count of *Bacillus belyssica* CA-06 in the fermentation broth is 2 × 10⁻⁶. 7-9 cfu / mL.

[0011] Furthermore, the fermentation product is the supernatant or whole fermentation liquid obtained from the fermentation of Bacillus belyssica CA-06.

[0012] The beneficial effects of this invention are as follows: This study is the first to isolate the red-necked longhorn beetle (Bauhinia purpurea) from the soil. Aromia bungii A functional strain of Bacillus belye, CA-06, with highly efficient cellulose degradation capabilities was isolated from the larval intestines. It exhibited excellent degradation efficiency on wheat straw, corn straw, and rice straw. Inoculation significantly accelerated cellulose decomposition and promoted straw degradation, facilitating direct application on insufficiently pretreated raw materials.

[0013] After inoculation at a 5% inoculum rate for 24 hours, the cellulase activity of strain CA-06 was detected using a Solarbio reagent kit. The activity was 64.64 U / mL after 1 day of fermentation, and the enzyme activity reached its maximum of 116.4 U / mL after 3 days of fermentation.

[0014] In indoor experiments, the bacterial suspension of Bacillus belyssica CA-06 of this invention (effective viable count of 2 × 10⁻⁶) was taken. 7 The spores (cfu / mL) were inoculated at a rate of 2% (v / v) into an inorganic salt medium containing 4.5 g / L wheat-rice-corn straw as the sole carbon source. After 15 days of fermentation, the degradation rates of wheat straw, rice straw, and corn straw reached 39.33%, 36.67%, and 32.67%, respectively.

[0015] In the field experiment, the bacterial suspension of Bacillus belyssica CA-06 of this invention (effective viable count of 2 × 10⁻⁶) was taken. 7The concentration of cfu / mL was added at a rate of 2% (V / m) to 15g of wheat straw, rice straw, or corn straw, respectively. After 15 days of fermentation, the degradation rates of wheat straw, rice straw, and corn straw reached 54%, 52%, and 44%, respectively. Attached Figure Description

[0016] Figure 1 This is a purified colony diagram of Bacillus belyssica CA-06.

[0017] Figure 2 This is a diagram of the hydrolysis zone produced by Bacillus belye CA-06 on a carboxymethyl cellulose agar plate.

[0018] Figure 3 Phylogenetic tree of Bacillus belyssica CA-06.

[0019] Figure 4 The absorbance standard curve of cellulase activity of Bacillus belyssica CA-06. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0021] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0022] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0023] 1. Separation and purification The larvae of the peach-necked longhorn beetle were disinfected by soaking in alcohol and then air-dried under sterile conditions. The abdominal cavity was cut open with a sterile scalpel to obtain the intestinal contents, which were placed in a mortar and pestle. 2 mL of physiological saline was added, and the mixture was ground evenly. The resulting suspension was diluted in three different gradients at 10⁻⁶. -6 10 -7 10 -8 The cultured bacteria were spread onto NA agar plates (5.0 g / L peptone, 5.0 g / L sodium chloride, 3.0 g / L beef extract, and 18.0 g / L agar powder) and incubated upside down at 25-30℃ for 3 days. The cultured bacteria were then purified to obtain a strain, which was named CA-06.

[0024] Identification Morphological identification like Figure 1 The image shows the colony morphology of purified CA-06 obtained on an agar plate.

[0025] (2) Determination of CA-06's ability to degrade cellulose Purified CA-06 was streaked onto sodium carboxymethyl cellulose medium (CMC-Na 10.0 g / L, peptone 5.0 g / L, yeast extract 0.5 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 1.5 g / L, NaCl 5.0 g / L, agar powder 15.0 g / L), and incubated upside down in a 30℃ incubator. For rapidly growing strains, sterilized pipette tips were used to collect the test strains, and the bacterial-containing tips were spot-inoculated once onto the sodium carboxymethyl cellulose medium, with three replicates. The plates were then incubated at 30℃ for 24 h. 5 mL of Congo red solution was used to stain the plates for 15 min, then the Congo red staining solution was discarded. 15 mL of NaCl solution was added for destaining, and after 15 min, the NaCl solution was discarded. This process was repeated three times, and the presence or absence of transparent hydrolysis zones around the colonies was observed.

[0026] like Figure 2 As shown, a transparent hydrolysis zone is visible around the colony.

[0027] (3) Molecular biological identification The above-mentioned strains were subjected to 16S rDNA sequencing and a phylogenetic tree was constructed. The sequencing results are shown in SEQ ID NO:1, and the phylogenetic tree is as follows: Figure 3 As shown. Based on the sequencing results and the above-mentioned microbiological characteristics and physiological and biochemical properties, this bacterium was identified as *Bacillus belye* (…). Bacillus velezensis ).

[0028] 1. Experimental Methods 100 mL of LB liquid culture medium was placed in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 30 min. When the temperature dropped below 45 °C, 5% of *Bacillus belyssica* CA-06 was inoculated and cultured at 30 °C and 180 rpm on a rotating shaker for 24 h. The *Bacillus belyssica* culture was centrifuged at 6000 rpm for 20 min, the supernatant was discarded, and 1 mL of extraction buffer was added for every 5 million bacteria. The bacteria were disrupted by sonication on ice (200 W, 3 seconds of sonication, 10-second interval, repeated 30 times). The mixture was then centrifuged at 8000 g at 4 °C for 10 min, the supernatant was collected, and placed on ice for analysis.

[0029] Preheat the microplate reader for at least 30 minutes and adjust the wavelength to 540nm.

[0030] The standard (10 mg anhydrous glucose, loss on drying <0.2%) was diluted with distilled water to 1, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg / mL.

[0031] Cellulase activity was measured using a Solarbio reagent kit (BC2545, 100T / 48S). 50 μL of reagent one, 200 μL of reagent two, and 50 μL of distilled water were added sequentially to the control and assay tubes, respectively. The assay tubes were then filled with 50 μL of sample (the supernatant from the initial ultrasonic disruption and centrifugation), while the control tubes were filled with 50 μL of boiled sample (inactivated sample after a 5-minute boiling water bath). All samples were mixed thoroughly and incubated at 40°C for 30 minutes. Immediately after incubation, the tubes were boiled in boiling water for 15 minutes (sealing with plastic wrap to prevent bursting) to obtain the saccharified solution.

[0032] Add 15 μL of saccharification solution to the control tube and the test tube, respectively. Add 15 μL of standard solution to the standard tube. Then add 35 μL of reagent three to each of the control tube, test tube, and standard tube. Mix well and develop the color in a boiling water bath for 15 min (cover with sealing film to prevent bursting). Cool. Add 250 μL of distilled water to each tube. Mix well, and transfer 200 μL to a 96-well plate to measure the absorbance value A. Calculate ΔA = Atest tube - Acontrol tube, and ΔAstandard = Astandard - A (0 mg / mL). Construct a standard curve.

[0033] 2. Experimental Results Based on the concentration (y, mg / mL) and absorbance ΔA standard of the standard tube ( x (△A standard), establish a standard curve ( Figure 3 ), to determine △A ( x The sample concentration (y, mg / mL) was calculated by substituting the values ​​of ΔA (as determined by the formula). The final result showed that the cellulase activity of strain CA-06 was 64.64 U / mL after 1 day of fermentation, reaching a maximum of 116.4 U / mL after 3 days, and then decreasing and stabilizing after 5 days. In the formula: 1000: unit conversion factor, 1 mg / mL = 1000 ug / L; Vreaction total: total volume of the reaction system, 0.35 mL; Vsample: volume of sample added, 0.05 mL; Vsample total: volume of extract added, 1 mL; T: reaction time, 30 min; Cpr: sample protein concentration, mg / mL; W: sample mass, g; 500: total bacterial or cell count, 5 million.

[0034] 1. Experimental Methods The CA-06 after double screening was made into fermentation broth. 100 mL of LB liquid culture medium was placed in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 30 min. When the temperature dropped to 45 °C, a single colony of CA-06 was picked up using an inoculation loop and inoculated into the LB liquid culture medium. The culture was then incubated at 30 °C on a rotating shaker at 180 rpm for 24 h to obtain the fermentation broth of CA-06. The viable cell count was determined to be 2.0 × 10⁻⁶. 9 CFU / mL. 1 mL of fermentation broth was inoculated into 50 mL of liquid JGJJ medium (KH₂PO₄ 1.0 g, NaCl 0.1 g, MgSO₄·7H₂O 0.3 g, NaNO₃ 2.5 g, CaCl₂ 0.1 g, FeCl₃ 0.01 g, wheat straw, rice straw, and corn straw 1.5 g each, diluted with double-distilled water to 1000 mL) and incubated at 37 ℃. After 15 days, the precipitate and bacterial cells on the straw were removed sequentially by gauze filtration, distilled water rinsing, and dilute hydrochloric acid rinsing. The treated straw was dried in an 80 ℃ oven to constant weight, and the straw degradation rate was calculated using the weight loss method. The degradation efficiency on rice straw was analyzed based on the degradation rate. A treatment group using distilled water instead of the bacterial strain served as a control, and each treatment group had three replicates.

[0035] The relative degradation rate (RDR) of straw was calculated using the formula RDR = [(M1-M2) / M1] × 100%, where M1 and M2 are the masses (g) of the residue in the control group and the residue in the experimental group, respectively.

[0036] 2. Experimental Results Table 1. Effects of CA-06 on wheat straw degradation

[0037] Table 2. Effects of CA-06 on the degradation of rice straw

[0038] Table 3. Effects of CA-06 on the degradation of corn straw

[0039] Material source and pretreatment Wheat straw, rice straw, and corn straw harvested from the same season and the same experimental base were collected, and soil and impurities were removed. After air drying, the straw was cut into small pieces of about 3-5 cm, mixed well, and set aside. The straw was then dried at 60℃ until constant weight, weighed, and the data was recorded.

[0040] Test methods Sample preparation: Each type of straw was tested separately. 15.0 g of dried straw was placed into each mesh bag, and each treatment was replicated 3 times.

[0041] Group processing: Control (CK): No CA-06 fermentation broth was added to the mesh bag; only an equal volume of sterile distilled water was added to adjust the substrate moisture content to 55%-65% of the maximum water holding capacity. Treatment (T): CA-06 fermentation broth (bacterial concentration consistent with Example 3) was added to the mesh bag, with the same volume as the sterile distilled water in the control group. Finally, the substrate moisture content was adjusted to 55%-65% (consistent with the control group).

[0042] Cultivation conditions: Place the mesh bag containing straw into a cultivation pot containing a small amount of sterilized soil, and keep it ventilated; place it in a constant temperature incubator for cultivation, with temperature and humidity conditions as described in the previous example.

[0043] Sampling and measurement: Samples were taken at 15 days and 30 days of cultivation. The corresponding mesh bags were removed, the soil attached to the surface was gently brushed off, and the straw was dried at 60-70 ℃ to constant weight. The dry weight of the straw was then recorded.

[0044] The relative degradation rate (RDR) of straw was calculated using the formula RDR = [(M1﹣ M2 ) / M1 ]×100%, where M1 and M2 are the masses (g) of the residue in the control group and the residue in the experimental group, respectively.

[0045] Test results Table 4. Effects of CA-06 on field degradation of wheat straw

[0046] Table 5. Effects of CA-06 on field degradation of rice straw.

[0047] Table 6. Effects of CA-06 on field degradation of corn straw

[0048] Numerical data show that, compared with the control, the degradation rate of all three types of straw increased after the application of CA-06, indicating that CA-06 can significantly improve the field decomposition rate of straw and promote nutrient return.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Bacillus velezensis CA-06, accession number: CGMCC No.35570.

2. The application of Bacillus belyssica CA-06 as described in claim 1 in the degradation of cellulose.

3. The use of Bacillus belyssica CA-06 as described in claim 1 in the production of cellulase.

4. The application of Bacillus belyssica CA-06 as described in claim 1 in the decomposition of plant straw.

5. The application of Bacillus belyssica CA-06 as described in claim 1 in the degradation of farmland residues.

6. The application according to claim 5, characterized in that, The farmland residues include rice straw, wheat straw, or corn straw.

7. A bacterial fermentation broth, characterized in that, The active ingredient includes Bacillus belyssica CA-06 or its fermentation product as described in claim 1.

8. The bacterial fermentation broth according to claim 7, characterized in that, When the active ingredient is Bacillus belyssica CA-06, the effective viable count of Bacillus belyssica CA-06 in the fermentation broth is 2 × 10⁻⁶. 7-9 cfu / mL.

9. The bacterial fermentation broth according to claim 7, characterized in that, The fermentation product is the supernatant or whole fermentation liquid obtained from the fermentation of Bacillus belyssica CA-06.

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