Bacillus paralicheniformis CBJ-7 and application thereof

Abstract

The invention relates to bacillus paralicheniformis CBJ-7, and the preservation number of the bacillus paralicheniformis CBJ-7 is CGMCC (China General Microbiological Culture Collection Center) No.31960. The bacillus paralicheniformis CBJ-7 has the advantages that the preservation number is CGMCC No.31960; the bacillus paralicheniformis disclosed by the invention has the capacity of producing cellulase and amylase, and has an obvious antagonistic effect on fusarium oxysporum of cotton and bipolaris maydis. When applied to anaerobic fermentation of agricultural organic wastes, the biogas yield can be increased by more than 15%; when being used for composting the livestock and poultry manure, the organic fertilizer can effectively improve the fermentation temperature and shorten the decomposition time, and has important significance on promoting the recycling and energy utilization of organic wastes.

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 of the biogas projects in my country adopt medium-temperature fermentation. Biogas fermentation generally has problems such as low utilization rate and conversion rate of fermentation raw materials, which directly leads to poor comprehensive benefits of biogas production. In order to more effectively improve the efficiency of biogas fermentation, the main research at home and abroad is to improve the fermentation process and anaerobic digester, optimize fermentation process parameters, improve or increase inoculum, regulate fermentation pathways and add biogas fermentation promoters. Biogas fermentation is a process in which microorganisms of different metabolic types degrade complex organic matter and convert it into methane through synergistic metabolism. Therefore, adding functional microorganisms in the anaerobic fermentation process is an important measure to improve the decomposition and conversion rate of organic matter and biogas production.

[0003] At present, many strains of hydrolytic bacteria that can effectively improve the gas production rate of anaerobic fermentation have been selected, such as aerobic microorganisms: Trichoderma, Aspergillus, white rot fungi, etc., which are mainly used for the pretreatment of raw materials containing cellulose components such as straw. After pretreatment of rice straw with Aspergillus niger, Penicillium, Rhizopus, white rot fungi, etc., the biogas production can be increased by more than 30~60% compared with the control, and the effect of post-treatment with appropriate mixing of different strains is higher than that of a single bacterial agent. Applying facultative or anaerobic hydrolytic bacteria, hydrogen-producing acetogenic bacteria and methanogenic bacteria to the biogas fermentation system will not only significantly shorten the fermentation start-up time and improve the raw material conversion rate, but also significantly increase the biogas production. Therefore, one of the effective ways to solve this problem is to select and develop efficient biogas fermentation functional microorganisms to fundamentally improve the biogas production rate, methane content and fermentation efficiency. Summary of the invention

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

[0005] The present invention adopts the following technical solution: A bacillus paralicheniformis ( Bacillus paralicheniformis )CBJ-7, with the deposit number CGMCCNo.31960. The strain was deposited on September 13, 2024 at the General Microbiology Center of China Culture Collection Administration, located in Beijing, China.

[0006] Furthermore, the Bacillus paralicheniformis CBJ-7 grows on LB medium, and the colonies are irregular, milky white, moist, and opaque. The bacteria are rod-shaped, Gram-positive, form spores, and are facultatively anaerobic. It can liquefy gelatin, and the methyl red test is positive, gelatin is liquefied, starch is hydrolyzed, and biofilm is formed. The suitable growth temperature is 25-35° C. and the pH is 6.8-7.5.

[0007] Furthermore, the Bacillus paralicheniformis CBJ-7 has the ability to produce cellulase and amylase.

[0008] Furthermore, the Bacillus paralicheniformis CBJ-7 has the ability to degrade organic matter.

[0009] Furthermore, the Bacillus paralicheniformis CBJ-7 can antagonize cotton wilt pathogen and corn leaf blight pathogen.

[0010] An application of the above-mentioned Bacillus paralicheniformis CBJ-7 in the anaerobic fermentation process of agricultural organic waste.

[0011] An anaerobic fermentation promoter comprises the above-mentioned Bacillus paralicheniformis CBJ-7.

[0012] An application of the above-mentioned Bacillus paralicheniformis CBJ-7 in the aerobic composting process of agricultural organic waste.

[0013] A composting accelerator comprising the above-mentioned Bacillus paralicheniformis CBJ-7.

[0014] 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.

[0015] In each of the above applications, the inoculation amount of the Bacillus paralicheniformis CBJ-7 inoculant is 1-5% of the mass of the fermentation material, and the bacterial content of the Bacillus paralicheniformis CBJ-7 in the inoculant is not less than 10 8 Pieces / mL.

[0016] An application of the above-mentioned Bacillus paralicheniformis CBJ-7 in preventing and controlling cotton wilt pathogen and corn leaf blight pathogen.

[0017] A biological pesticide comprising the above-mentioned Bacillus paralicheniformis CBJ-7.

[0018] The beneficial effects of the present invention are: (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.

[0019] (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.

[0020] (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.

[0021] (4) The Bacillus paralicheniformis CBJ-7 of the present invention has a significant antagonistic effect on cotton wilt pathogen and corn leaf blight pathogen, and can be used in 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.

[0022] 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

[0023] Figure 1 is the phylogenetic tree of strain CBJ-7. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with 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.

[0025] Example 1 Isolation and preservation of strain CBJ-7 The strain CBJ-7 was isolated from cow dung in a dairy farm in Shijiazhuang City, Hebei Province through enrichment culture, subculture and gradient dilution methods.

[0026] The specific method is: take fresh cow dung from the dairy farm, put a small amount of cow dung into a triangle containing enrichment medium, and enrich and culture at 35℃. After the passage is stable, use the cellulase primary screening medium for separation and screening, culture at 35℃ for 48~72 hours, select the colony CBJ-7 with a larger ratio of the transparent circle diameter (D) to the colony diameter (d) in the primary screening medium, inoculate it into LB medium, and store it at 4℃ for use.

[0027] Use culture medium: (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.

[0028] (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.

[0029] (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.

[0030] The strain CBJ-7 grows on LB medium, and the colonies are irregular, milky white, moist, and opaque. The bacteria are rod-shaped, Gram-positive, form spores, and are facultatively anaerobic. It can liquefy gelatin, and the methyl red test is positive, gelatin liquefies, starch hydrolyzes, and forms biofilm. The suitable growth temperature is 25~35℃ and the pH is 6.8~7.5.

[0031] 16S rDNA sequence analysis: Select bacterial solution in the logarithmic growth phase, use a universal bacterial genomic DNA extraction kit to extract the strain genomic DNA, and use it as a template for PCR amplification.

[0032] The amplification primers are universal bacterial primers: The forward primer is Pf: 5′-AGAGTTTGACC TGGCTCAG-3′, The reverse primer is Pr: 5′-ACGGCTACCTTGTTACGACT-3′.

[0033] PCR reaction program: pre-denaturation at 95°C for 4 min; 95°C for 1 min, 52°C for 1 min, 72°C for 3 min, 30 cycles of extension; 72°C for 10 min.

[0034] 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, and 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.

[0035] Based on the strain morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain CBJ-7 was identified as Bacillus paralicheniformis ( Bacillus paralicheniformis). Bacillus paralicheniformis CBJ-7 was sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation on September 13, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the preservation number: CGMCC No.31960.

[0036] Example 2 Detection of amylase production by Bacillus paralicheniformis CBJ-7 The slant from the strain CBJ-7 collection was inoculated on the starch detection medium. After culturing in a constant temperature incubator at 30℃ for 3 days, its growth and whether there was a transparent zone around the colony were observed, the diameter of the transparent zone (D) and the diameter of the colony (d) were measured respectively, and the D / d value was calculated.

[0037] Starch detection medium: NaCl 5 g, beef extract 5 g, peptone 10 g, soluble starch 10 g, agar powder 15 g, appropriate amount of triphenyl blue, deionized water 1 L; pH 7.0~7.2.

[0038] 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.

[0039] Example 3 Antagonism of Bacillus paralicheniformis CBJ-7 against plant pathogens The plate confrontation method was used to determine whether strain CBJ-7 had an antagonistic effect on plant pathogens: the pathogen block was inoculated in the center of the PDA plate, and the strain CBJ-7 was inoculated at equal distances on both sides of the block, and cultured at 25°C for 48 hours, repeated 3 times. The failure of pathogens to grow normally near strain CBJ-7 indicated an antagonistic effect. The results showed (see Table 1) that strain CBJ-7 had a significant antagonistic effect on cotton wilt pathogens and corn leaf spot pathogens.

[0040] Table 1 Antagonism of strain CBJ-7 against some pathogenic fungi .

[0041] The growth rate method was used to determine the activity of CBJ-7 against cotton wilt pathogen ( Fusarium oxysporum ) and Bipolaris maydis ( Bipolaris maydis) was tested for its antibacterial activity, that is, CBJ-7 was cultured in LB liquid medium for 24 h, centrifuged, and the supernatant was sterilized by 0.22 μm sterile filter membrane. A certain amount of sterile filtrate was added to PDA medium at 40-45 °C, mixed and poured into a 9 cm culture dish, and no sterile filtrate was added as a blank control. After the culture medium solidified, cotton wilt pathogen and corn leaf blight pathogen were inoculated in the center of each plate, repeated 3 times, and cultured at 28 °C. The colony diameter was measured by the cross method, and the inhibition rate was calculated. The results showed that the inhibition rates of CBJ-7 against cotton wilt pathogen and corn leaf blight pathogen were 63.8% and 65.2%, respectively.

[0042] Inhibition rate (%) = (colony diameter of control bacteria - colony diameter of treated bacteria) / (colony diameter of control bacteria - diameter of bacterial block) × 100.

[0043] Example 4 Anaerobic fermentation of cow dung 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.

[0044] Using 250 mL anaerobic bottles, cow dung was used as raw material, and the medium temperature fully mixed anaerobic fermentation process was set up with 2 treatments. Treatment 1: blank control. Treatment 2: adding 1.5% of the fermentation liquid of Bacillus paralicheniformis CBJ-7. The organic load TS was 12% (TS is also called dry matter concentration, which refers to the percentage of dry matter in the total weight when a certain amount of fermentation liquid is placed in an oven at 100~105℃ and dried to constant weight). The slurry in the normal fermentation biogas tank was used as the inoculum, the inoculation amount was 25% (volume ratio), the fermentation temperature was medium temperature 35±1℃, the fermentation time was 30 days, and each treatment was paralleled 5 times. The results are shown in Table 2.

[0045] Table 2 Biogas production .

[0046] As can be seen from Table 2, when anaerobic fermentation was carried out with cow dung as the raw material 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 was significantly higher than the control, indicating that the addition of the bacterial agent CBJ-7 can significantly improve the methane production efficiency of anaerobic fermentation of cow dung.

[0047] Example 5 Anaerobic fermentation of corn straw 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.

[0048] A 250 mL anaerobic bottle was used, corn stalks were used as raw materials, and a medium-temperature fully mixed anaerobic fermentation process was used, with two treatments. Treatment 1: blank control. Treatment 2: adding 2.0% of the fermentation bacterial liquid of Bacillus paralicheniformis CBJ-7 of the present invention. The organic load TS was 13%, the slurry in the normal fermentation biogas digester was used as the inoculum, the inoculation amount was 25% (volume ratio), the fermentation temperature was medium temperature 35±1℃, the fermentation time was 50 days, and each treatment was paralleled 5 times. The results are shown in Table 3.

[0049] Table 3 Biogas production .

[0050] 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 was 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.

[0051] Example 6 Application of Bacillus paralicheniformis CBJ-7 in anaerobic fermentation of cow dung + straw 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.

[0052] A 250 mL anaerobic bottle was used, with 50% cow dung + 50% corn straw powder as raw materials, and a medium temperature fully mixed anaerobic fermentation process was used. Two treatments were set up: treatment 1: blank control; treatment 2: adding 5% mass fraction of Bacillus paralicheniformis CBJ-7 fermentation liquid. The organic load TS was 12% (TS is also called dry matter concentration, which refers to the percentage of dry matter in the total weight when a certain amount of fermentation liquid is placed in an oven at 100-105 ℃ and dried to constant weight). The biogas slurry in the normal fermentation biogas digester was used as the inoculum, the inoculum amount was 25% (volume ratio), the fermentation temperature was medium temperature 35±2℃, the fermentation time was 30 days, and each treatment was paralleled 5 times.

[0053] The specific results are shown in Table 4. When agricultural waste corn stalks and livestock and poultry manure were used as raw materials for anaerobic fermentation, after adding Bacillus paralicheniformis CBJ-7, the total volatile acid (VFA) increased by 19.7% compared with the control, and the biogas production rate increased by 21.1% compared with the control, which was significantly higher than the control, indicating that the addition of the 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.

[0054] Table 4 Biogas production .

[0055] Example 7-9 Application of Bacillus paralicheniformis CBJ-7 in mixed composting of cow dung and corn straw 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.

[0056] The composting test was conducted in a foam box with an effective volume of 10 L. A ventilation pipe was set at the bottom of the box, and a 5-10 mm gravel cushion was laid on top, on which the composting test was conducted. The size of the compost pile was: length × width × height = 0.6 m × 0.5 m × 0.4 m. The indoor temperature was controlled at 25-28 °C.

[0057] The composting materials are cow dung and corn stalks, which are mixed evenly at different mass ratios. The moisture content in the pile is controlled to be 50%~60%. A control group and a microbial agent group (2% added according to the dry matter mass ratio) are set up respectively for aerobic composting for 45 days. The compost and environmental temperature are measured twice a day, and the average value is recorded. In the first week of composting, the pile is manually turned regularly according to the actual situation of the pile (when the temperature reaches above 55°C), and then it is manually turned every 3 days. When the germination index GI value is >80%, the compost is mature. Samples are collected after each turning. The sampling method uses the "five-point method", and then the five collected samples are mixed to ensure the representativeness of the samples and stored in a -20°C refrigerator for subsequent determination.

[0058] Table 5 Parameter changes during composting .

[0059] 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 3~5d, and the decomposition time can be shortened by 4~7d. 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 progress.

[0060] The present invention has been described in detail according to the above-mentioned embodiments. It should be noted that the above embodiments are only for the purpose of illustrating the invention. Without departing from the spirit and essence of the present invention, those skilled in the art can design various alternatives and improvements of the present invention, which should all be understood to be within the scope of protection of the present invention.

Claims

1. A Bacillus paralicheniformis ( Bacillus paralicheniformis ) CBJ-7, characterized in that, 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 as claimed in claim 1 in the anaerobic fermentation process of agricultural organic waste.

5. An anaerobic fermentation promoter, characterized in that It comprises the Bacillus paralicheniformis CBJ-7 as claimed in claim 1.

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

7. A composting accelerator, characterized in that: It comprises the Bacillus paralicheniformis CBJ-7 as claimed in 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: It comprises the Bacillus paralicheniformis CBJ-7 as claimed in claim 1.

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