Bacillus paralicheniformis CBJ-7 and its application

Abstract

The present invention relates to a Bacillus paralicheniformis ( Bacillus paralicheniform ) CBJ-7, with a deposit number of CGMCC No. 31960. The Bacillus paralicheniformis strain of the present invention is capable of producing cellulase and amylase, and has a significant antagonistic effect against cotton wilt pathogen and corn leaf spot pathogen. When applied to anaerobic fermentation of agricultural organic waste, it can increase biogas production by more than 15%. When used in livestock and poultry manure composting, it can effectively increase fermentation temperature and shorten composting time, which is of great significance for promoting the resource and energy utilization of organic waste.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microbial fermentation, and specifically relates to a Bacillus paralicheniformis ( Bacillus paralicheniformis ) CBJ-7 and its applications. Background Art

[0002] Most biogas projects in my country utilize mesophilic fermentation, which is plagued by issues such as low raw material utilization and conversion rates, leading to poor overall biogas production efficiency. To more effectively improve biogas fermentation efficiency, research efforts at home and abroad have focused on improving fermentation processes and anaerobic digesters, optimizing fermentation process parameters, modifying or increasing the inoculum, manipulating the fermentation pathway, and adding biogas fermentation promoters. Biogas fermentation involves the coordinated metabolism of diverse microorganisms, which degrade complex organic matter and convert it into methane. Therefore, the addition of functional microorganisms to the anaerobic fermentation process is a crucial measure to increase both organic matter decomposition and conversion rates and biogas production.

[0003] Currently, several strains of hydrolytic bacteria have been bred that can effectively increase anaerobic fermentation gas production. These include aerobic microorganisms such as Trichoderma, Aspergillus, and white-rot fungi. These are primarily used for pretreatment of cellulose-containing feedstocks such as straw. Pretreatment of rice straw with Aspergillus niger, Penicillium, Rhizopus, and white-rot fungi can increase biogas production by 30–60% compared to the control, and the effect of post-treatment with appropriate mixtures of different strains is superior to that of a single agent. Incorporating facultative or anaerobic hydrolytic bacteria, hydrogen-producing and acetogenic bacteria, and methanogens into biogas fermentation systems not only significantly shortens fermentation startup time and improves feedstock conversion, but also significantly increases biogas production. Therefore, one effective approach to addressing this challenge is to select and develop highly efficient biogas fermentation microorganisms to fundamentally improve biogas production, methane content, and fermentation efficiency. Summary of the Invention

[0004] The present invention aims to provide a Bacillus paralicheniformis CBJ-7 having the ability to degrade cellulose and starch and can be used in the anaerobic fermentation or composting process of agricultural organic waste, especially carbon-rich raw materials, and its application.

[0005] The present invention adopts the following technical solutions:

[0006] A Bacillus paralicheniformis ( Bacillus paralicheniformis )CBJ-7, with the deposit number CGMCC No. 31960, was deposited on September 13, 2024 at the General Microbiology Center of China Culture Collection Administration, Beijing, China.

[0007] Furthermore, the Bacillus paralicheniformis CBJ-7 grows on LB medium, forming irregular, milky, moist, and opaque colonies. The bacteria are rod-shaped, Gram-positive, spore-forming, and facultatively anaerobic. It can liquefy gelatin, test positive in the methyl red test, liquefy gelatin, hydrolyze starch, and form biofilms. Its optimal growth temperature is 25-35°C and its pH is 6.8-7.5.

[0008] Furthermore, the Bacillus paralicheniformis CBJ-7 has the ability to produce cellulase and amylase.

[0009] Furthermore, the Bacillus paralicheniformis CBJ-7 has the ability to degrade organic matter.

[0010] Furthermore, the Bacillus paralicheniformis CBJ-7 can antagonize cotton wilt pathogen and corn leaf spot pathogen.

[0011] An application of the above-mentioned Bacillus paralicheniformis CBJ-7 in the anaerobic fermentation process of agricultural organic waste.

[0012] An anaerobic fermentation promoter comprises the above-mentioned Bacillus paralicheniformis CBJ-7.

[0013] An application of the above-mentioned Bacillus paralicheniformis CBJ-7 in the aerobic composting process of agricultural organic waste.

[0014] A composting accelerator comprising the above-mentioned Bacillus paralicheniformis CBJ-7.

[0015] In the above applications, the agricultural organic waste includes but is not limited to livestock and poultry manure, crop straw or other fibrous organic solid waste.

[0016] In each of the above applications, the inoculation amount of the Bacillus paralicheniformis CBJ-7 agent is 1-5% of the mass of the fermentation material, and the bacterial content of the Bacillus paralicheniformis CBJ-7 in the agent is not less than 10 8 pieces / mL.

[0017] The invention relates to an application of the above-mentioned Bacillus paralicheniformis CBJ-7 in preventing and controlling cotton wilt pathogen and corn leaf spot pathogen.

[0018] A biopesticide comprising the above-mentioned Bacillus paralicheniformis CBJ-7.

[0019] The beneficial effects of the present invention are:

[0020] (1) The Bacillus paralicheniformis CBJ-7 of the present invention can increase the biogas production of agricultural organic waste such as livestock and poultry manure and straw by more than 15% under normal and medium temperature conditions.

[0021] (2) The Bacillus paralicheniformis CBJ-7 of the present invention can simultaneously produce cellulase and amylase, significantly improving the degradation efficiency of substrates containing multiple organic mixtures.

[0022] (3) In addition to being used for anaerobic fermentation of agricultural organic waste to produce biogas, the Bacillus paralicheniformis CBJ-7 of the present invention can also be used to improve the composting efficiency of organic waste.

[0023] (4) The Bacillus paralicheniformis CBJ-7 of the present invention has a significant antagonistic effect on cotton wilt pathogen and corn leaf spot pathogen, and can be used for the development of biocontrol agents. It can also be used in cotton fields or corn fields after anaerobic fermentation of organic waste into biogas manure or compost, which has a disease prevention effect and reduces the amount of pesticides used.

[0024] In summary, the present invention can not only effectively improve the anaerobic fermentation efficiency of livestock and poultry manure, but also has important significance for promoting the harmless, resource and energy utilization of organic waste, and has practical economic and social benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the phylogenetic tree of strain CBJ-7. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the embodiments and drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also fall within the protection scope of the present invention.

[0027] Example 1 Isolation and preservation of strain CBJ-7

[0028] Strain CBJ-7 was isolated from cow dung at a dairy farm in Shijiazhuang, Hebei Province, by enrichment culture, subculture, and gradient dilution methods.

[0029] The specific method is to collect fresh cow dung from a dairy farm, place a small amount of dung in a triangular tube filled with enrichment medium, and culture it at 35°C. After stable passage, isolate and screen the cells using a cellulase screening medium. Incubate at 35°C for 48–72 hours. Select the colony CBJ-7 with the largest ratio (D / d) of the clear zone diameter (D) to the colony diameter (d) in the screening medium. Inoculate the colony into LB medium and store at 4°C until ready for use.

[0030] Use culture medium:

[0031] (1) Enrichment medium: K2HPO4 1.0 g, MgSO4 0.5 g, (NH4)2SO4 2.0 g, NaCl 0.5 g, beef extract 0.5 g, peptone 1.0 g, sodium carboxymethyl cellulose (CMC-Na) 5.0 g, microcrystalline cellulose 5.0 g, straw powder 5.0 g, deionized water 1 L; pH 7.2-7.4.

[0032] (2) Cellulase screening culture medium: CMC-Na 5.0 g, K2HPO4 1.0 g, MgSO4 0.5 g, (NH4)2SO4 2.0 g, NaCl 0.5 g, beef extract 0.5 g, peptone 1.0 g, Congo red 1.5 g, agar 15 g, deionized water 1 L; pH 7.2-7.4.

[0033] (3) LB medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 15 g agar, 1 L deionized water; pH 7.0-7.4.

[0034] Strain CBJ-7 grows on LB medium, producing irregular, milky, moist, and opaque colonies. The bacteria are rod-shaped, Gram-positive, spore-forming, and facultatively anaerobic. It can liquefy gelatin, test positive for methyl red, liquefy gelatin, hydrolyze starch, and form biofilms. Its optimal growth temperature is 25-35°C and its pH is 6.8-7.5.

[0035] 16S rDNA sequence analysis: bacterial culture in the logarithmic growth phase was selected, and genomic DNA of the strain was extracted using a universal bacterial genomic DNA extraction kit, which was used as a template for PCR amplification.

[0036] Amplification primers are universal bacterial primers:

[0037] The forward primer is Pf: 5′-AGAGTTTGACC TGGCTCAG-3′,

[0038] The reverse primer is Pr: 5′-ACGGCTACCTTGTTACGACT-3′.

[0039] PCR reaction program: pre-denaturation at 95°C for 4 min; 30 cycles of extension at 95°C for 1 min, 52°C for 1 min, and 72°C for 3 min; and 72°C for 10 min.

[0040] The PCR amplification product was subjected to agarose electrophoresis, and a band of about 1500 bp was detected. It was sent to Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to NCBI for homology comparison analysis with the existing 16S rDNA sequences in the database. The 16S rDNA gene sequences of closely related strains were selected from Genebank, and the phylogenetic tree was constructed using MEGA7.0. The results are as follows Figure 1 shown.

[0041] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain CBJ-7 was identified as Bacillus paralicheniformis ( Bacillus paralicheniformis Bacillus paralicheniformis CBJ-7 was deposited on September 13, 2024, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC No. 31960.

[0042] Example 2 Detection of amylase production by Bacillus paralicheniformis CBJ-7

[0043] The strain CBJ-7 was inoculated from the preserved slant onto starch detection medium and cultured in a constant temperature incubator at 30°C for 3 days. The growth and the presence of a transparent zone around the colony were observed. The diameter of the transparent zone (D) and the diameter of the colony (d) were measured, and the D / d ratio was calculated.

[0044] Starch detection medium: 5 g NaCl, 5 g beef extract, 10 g peptone, 10 g soluble starch, 15 g agar powder, appropriate amount of triphenyl blue, 1 L deionized water; pH 7.0-7.2.

[0045] The results showed that there was an obvious transparent zone around the strain CBJ-7, and the D / d value was greater than 2.0, indicating that the strain had the ability to produce amylase.

[0046] Example 3 Antagonism of Bacillus paralicheniformis CBJ-7 against plant pathogens

[0047] The plate standoff assay was used to determine whether strain CBJ-7 exhibited antagonistic effects against plant pathogens. A pathogen block was inoculated in the center of a PDA plate, and strain CBJ-7 was inoculated equidistantly on either side of the block. The plates were incubated at 25°C for 48 hours, with three replicates. Failure of pathogen growth near strain CBJ-7 indicated antagonistic activity. The results (Table 1) showed that strain CBJ-7 exhibited significant antagonistic effects against Fusarium wilt and Bipolaris davidii.

[0048] Table 1 Antagonistic activity of strain CBJ-7 against some pathogenic fungi

[0049] .

[0050] The growth rate method was used to determine the activity of CBJ-7 against cotton wilt pathogen ( Fusarium oxysporum ) and Bipolaris maydis ( Bipolaris maydis To investigate the antibacterial activity of CBJ-7, the researchers cultured the LB medium for 24 hours, centrifuged the supernatant, and sterilized it through a 0.22 μm sterile filter. A certain amount of the sterile filtrate was added to PDA medium at 40–45°C, mixed thoroughly, and then poured into 9 cm Petri dishes. A blank control was used without the sterile filtrate. After the culture solidified, colonies of Fusarium wilt and B. maydis were inoculated in the center of each plate. Three replicates were used for incubation at 28°C. Colony diameters were measured using the cross-hatch method, and inhibition rates were calculated. Results showed that CBJ-7 exhibited inhibition rates of 63.8% and 65.2% against Fusarium wilt and B. maydis, respectively.

[0051] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial block diameter) × 100.

[0052] Example 4 Anaerobic fermentation of cow dung

[0053] The strain CBJ-7 was cultured in LB liquid medium at 30°C and 180 rpm for 48 h. The bacterial concentration was detected and adjusted to 1.0×10 8 CFU / mL.

[0054] Using 250 mL anaerobic bottles, a mesophilic, fully mixed anaerobic fermentation process was conducted using cow dung as the feedstock. Two treatments were set up. Treatment 1 consisted of a blank control. Treatment 2 included the addition of 1.5% (by mass) of a fermentation broth of Bacillus paralicheniformis CBJ-7. The organic loading (TS) was 12% (TS, also known as dry matter concentration, refers to the percentage of dry matter in the total weight of a fermentation broth dried to a constant weight in an oven at 100-105°C). Biogas slurry from a normal fermentation digester was used as the inoculum at a volume ratio of 25%. Fermentation temperature was mesophilic at 35 ± 1°C, and fermentation time was 30 days. Five replicates were run for each treatment. Results are shown in Table 2.

[0055] Table 2 Biogas production

[0056] .

[0057] As shown in Table 2, when anaerobic fermentation of cow dung was carried out and the Bacillus paralicheniformis of the present invention was added, the biogas production was increased by 19.1% compared with the control, and the total volatile acid (VFA) was increased by 15.8% compared with the control, which were significantly higher than the control. This shows that the addition of the bacterial agent CBJ-7 can significantly improve the methane production efficiency of anaerobic fermentation of cow dung.

[0058] Example 5 Anaerobic fermentation of corn straw

[0059] The strain CBJ-7 was cultured in LB liquid medium at 30°C and 180 rpm for 48 h. The bacterial concentration was detected and adjusted to 1.0×10 8 CFU / mL.

[0060] Corn straw was used as the raw material in 250 mL anaerobic flasks under a mesophilic, fully mixed anaerobic fermentation process with two treatments. Treatment 1 consisted of a blank control. Treatment 2 included the addition of a 2.0% (mass fraction) fermentation broth of the present invention's Bacillus paralicheniformis CBJ-7. The inoculum was 25% (volume ratio) of biogas slurry from a normal fermentation digester, with a 13% organic load (TS). The fermentation temperature was mesophilic at 35 ± 1°C, and the fermentation time was 50 days. Five replicates were run for each treatment. The results are shown in Table 3.

[0061] Table 3 Biogas production

[0062] .

[0063] As shown in Table 3, when corn straw was used as raw material for anaerobic fermentation and Bacillus paralicheniformis was added, the biogas production increased by 27.4% compared with the control, and the total volatile acid (VFA) increased by 23.5% compared with the control, which were significantly higher than the control. This shows that the addition of the bacterial agent CBJ-7 can significantly improve the methane production efficiency of anaerobic fermentation of corn straw.

[0064] Example 6 Application of Bacillus paralicheniformis CBJ-7 in anaerobic fermentation of cow dung and straw

[0065] The strain CBJ-7 was cultured in LB liquid medium at 30°C and 180 rpm for 48 h. The bacterial concentration was detected and adjusted to 1.0×10 8 CFU / mL.

[0066] A mesophilic, fully mixed anaerobic fermentation process was conducted using 250 mL anaerobic flasks with 50% cow dung and 50% corn straw meal as the raw materials. Two treatments were set up: Treatment 1: a blank control; Treatment 2: a fermentation solution supplemented with 5% by mass of Bacillus paralicheniformis CBJ-7. The organic loading (TS) was 12% (TS refers to the percentage of dry matter content of the fermentation solution after drying to a constant weight in an oven at 100-105°C). Biogas slurry from a normal fermentation digester was used as the inoculum at a volume ratio of 25%. Fermentation temperature was mesophilic at 35 ± 2°C, and fermentation time was 30 days. Five replicates were run for each treatment.

[0067] Specific results are shown in Table 4. When agricultural waste corn straw and livestock and poultry manure were used as raw materials for anaerobic fermentation, the addition of Bacillus paralicheniformis CBJ-7 increased the total volatile acid (VFA) by 19.7% compared with the control, and the biogas production rate increased by 21.1% compared with the control, which were significantly higher than the control. This indicates that the addition of strain CBJ-7 can significantly promote the hydrolysis efficiency of the raw materials and improve the methane production efficiency of the anaerobic fermentation of the raw materials.

[0068] Table 4 Biogas production

[0069] .

[0070] Examples 7-9 Application of Bacillus paralicheniformis CBJ-7 in mixed composting of cow dung and corn straw

[0071] The strain CBJ-7 was cultured in LB liquid medium at 30°C and 180 rpm for 48 h. The bacterial concentration was detected and adjusted to 1.0×10 8 CFU / mL.

[0072] Composting experiments were conducted in a foam box with an effective volume of 10 L. A ventilation tube was installed at the bottom of the box, and a 5-10 mm gravel bedding layer was laid on top. The compost pile dimensions were: length × width × height = 0.6 m × 0.5 m × 0.4 m. The indoor temperature was maintained at 25-28°C.

[0073] Compost was made from cow dung and corn stalks, mixed uniformly at varying mass ratios. The moisture content of the compost pile was controlled at 50% to 60%. A control group and a group supplemented with a microbial agent (2% by dry matter weight) were set up for aerobic composting for 45 days. Compost and ambient temperatures were measured twice daily, and the average values ​​were recorded. During the first week of composting, the compost was manually turned regularly based on the actual composting conditions (when the temperature reached above 55°C), and then every three days thereafter. The compost was considered mature when the germination index (GI) value exceeded 80%. Samples were collected after each turning using the "five-point" sampling method. The five samples were then mixed to ensure representativeness and stored at -20°C for subsequent analysis.

[0074] Table 5 Parameter changes during composting

[0075] .

[0076] As shown in Table 5, the addition of Bacillus paralicheniformis to the mixed aerobic composting of cow dung and corn straw can effectively increase the maximum temperature reached by fermentation. Compared with the control, the maximum temperature can be increased by 9~12℃, the high temperature duration can be extended by 3~5 days, and the decomposition time can be shortened by 4~7 days. This shows that the addition of Bacillus paralicheniformis CBJ-7 to the mixed aerobic composting of cow dung and corn straw can effectively promote the composting effect and process.

[0077] The present invention has been described in detail based on the above embodiments. It should be noted that the above embodiments are merely for the purpose of illustrating the invention. Without departing from the spirit and substance of the present invention, those skilled in the art may devise various alternatives and improvements of the present invention, all of which should be understood to be within the scope of protection of the present invention.

Claims

1. A Bacillus paralicheniformis ( Bacillus paralicheniformis ) CBJ-7, characterized by, The deposit number is CGMCC No.31960.

2. The Bacillus paralicheniformis CBJ-7 according to claim 1, characterized in that It has the ability to produce cellulase and amylase.

3. The Bacillus paralicheniformis CBJ-7 according to claim 1, characterized in that It can antagonize cotton wilt fungus and corn leaf spot fungus.

4. Use of the Bacillus paralicheniformis CBJ-7 according to claim 1 in the anaerobic fermentation process of agricultural organic waste.

5. An anaerobic fermentation promoter, characterized in that The method comprises the Bacillus paralicheniformis CBJ-7 according to claim 1 .

6. Use of the Bacillus paralicheniformis CBJ-7 according to claim 1 in aerobic composting of agricultural organic waste.

7. A composting accelerator, characterized in that: The method comprises the Bacillus paralicheniformis CBJ-7 according to claim 1 .

8. Use of the Bacillus paralicheniformis CBJ-7 according to claim 1 in controlling cotton wilt pathogen and corn leaf blight pathogen.

9. A biological pesticide, characterized in that: The method comprises the Bacillus paralicheniformis CBJ-7 according to claim 1 .

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