Bacillus cereus BJ-2 for degrading straw and preparation method of Bacillus BJ-2 bacterial agent

Abstract

Bacillus cereus BJ-2 for degrading straw and a method for preparing a bacillus BJ-2 microbial agent, the present invention relates to a bacillus cereus for degrading straw and a method for preparing a microbial agent thereof. The deposit number of the bacillus cereus BJ-2 for degrading straw of the present invention is CCTCC NO: M 2023950. The microbial agent preparation method of Bacillus cereus BJ-2 is as follows: one, obtaining strains; two, obtaining seed liquid; three, adding the seed liquid to 1000g liquid culture medium, and adding the nutrient solution to the above-mentioned liquid culture medium, and culturing and fermenting in a shaking table. The bacillus cereus (Bacillus cereus) BJ-2 of the present invention effectively degrades straw residues in a short time, protects the ecological environment and human health, and is easy to use, low in cost, and the straw removal rate in the solution reaches more than 63%. The microbial agent prepared using Bacillus BJ-2 has the advantages of low production cost, ease of use, good removal effect, etc., is suitable for treating the pollution caused by straw in environments such as soil, and has very important theoretical and application value.

Description

Technical Field

[0001] The invention relates to a straw-degrading Bacillus cereus and a method for preparing a bacterial agent thereof. Background Art

[0002] Straw is the general term for the stems and leaves (ears) of mature crops, typically wheat, corn, sorghum, rice, bean stalks, and other crops (usually coarse grains) left after the seeds are harvested. Burning straw produces solid particulate matter, which contributes to PM2.5 concentrations in certain areas during specific periods, posing a serious threat to both urban and rural ecological environments and the physical and mental health of residents.

[0003] Lignin, a major component of straw, is a complex organic polymer that forms an important structural material in the supporting tissues of vascular plants and some algae. Lignin is particularly important in the formation of cell walls, especially in wood and bark, as it imparts rigidity and resists decay. Chemically, lignin is a cross-linked phenolic polymer.

[0004] Lignin, an amorphous polymer composed of phenylpropane units linked by carbon-carbon and ether bonds, is the second-largest biomass resource in the plant kingdom, second only to cellulose. As a typical biomass material, lignin is one of the few renewable aromatic compounds. Globally, lignin is the second-largest biomass resource in the plant kingdom after cellulose, with an annual global production of approximately 50 million tons. Of this, approximately 10% to 20% comes from agricultural residues, and 20% to 30% comes from forest biomass. These diverse sources and substantial production volumes are significant. Therefore, research and solutions to the problem of lignin residues are crucial for expanding its application in production and reducing pollution to subsequent crops and the environment.

[0005] In recent years, the theory of bioremediation has gradually become a focus of attention. Bioremediation technology, due to its ease of use, cost-effectiveness, and environmental friendliness, has become an effective method for treating organic pollutants in the environment. Numerous studies have demonstrated that microbial metabolic pathways can degrade straw, with the addition of highly efficient degrading strains being the most common and effective method of bioremediation. Microbial degradation of straw residues relies on natural forces and does not produce secondary pollution. By screening highly efficient straw-degrading strains, fermenting them into a microbial agent, and applying it to degrade straw residues, the goal is to eliminate straw residues in soil, water, and agricultural products. Summary of the Invention

[0006] The invention provides a Bacillus cereus BJ-2 for degrading straw and a preparation method of the Bacillus BJ-2 bacterial agent.

[0007] The straw-degrading Bacillus cereus BJ-2 of the present invention has a preservation number of CCTCC NO: M2023950, is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation date is June 5, 2023.

[0008] The preparation method of the above-mentioned Bacillus cereus BJ-2 bacterial agent is carried out according to the following steps:

[0009] First, Bacillus cereus BJ-2 was inoculated into LB liquid medium, cultured at 65°C and 180 rpm with shaking until the logarithmic growth phase, and washed with phosphate buffer solution with a pH of 7.0 to obtain the strain;

[0010] 2. Inoculate the strain obtained in step 1 into a seed bottle culture medium at an inoculum volume ratio of 10%, and culture with shaking at 65-70° C. and 150-200 rpm until the logarithmic growth phase to obtain a seed solution; wherein each 100 mL of the seed bottle culture medium is composed of 0.3-0.5% sucrose, 0.04-0.08% MgSO4·7H2O, 0.15-0.2% K2HPO4, 0.08-0.1% NaCl, 0.05-0.07% KH2PO4, 0.15-0.2% yeast extract and the balance water, and the pH is adjusted to 7.0;

[0011] 3. Add 15-20 mL of the seed solution prepared in step 2 to 1000 g of liquid culture medium, and add 15-20 mL of nutrient solution to the above 1000 g of liquid culture medium, and culture and ferment in a shaker at 65-70° C. for 12-48 hours to obtain a Bacillus cereus BJ-2 preparation; wherein the liquid culture medium is prepared by adding 15 g of peptone, 4 g of yeast powder, 2.52 g of disodium hydrogen phosphate and 0.28 g of potassium dihydrogen phosphate to 1000 mL of distilled water, and adjusting the pH to 7.0; each 100 mL of nutrient solution is composed of 0.5-0.7% sucrose, 0.08-0.1% KH2PO4, 0.08-0.1% MgSO4·7H2O, 0.18-0.2% K2HPO4, 0.1-1.2% NaCl, 0.8-1.0% yeast powder and the remainder water.

[0012] The strain of the present invention is identified as Bacillus cereus by morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis. The strain grows rapidly on LB solid plate culture medium. The colonies are smooth, sticky, and large bacilli with blunt ends that rise above the surface. They can form chain-like arrangements and form oval spores within 6 hours of growth. The colonies are off-white, opaque, and have rough surfaces located in the center of the bacteria. The morphology is similar to wax, and the growth is uniform and turbid. The strain of the present invention is Gram-negative, can ferment glucose, sucrose, and mannitol, does not liquefy gelatin, does not produce indole, does not form H2S, can decompose and utilize citric acid, decompose urea, are negative for lysine ornithine, and are negative for ONPG, arginine, and oxidase. It is easy to grow on ordinary inorganic salt culture medium.

[0013] The Bacillus cereus BJ-2 of the present invention effectively degrades straw residues in a short period of time, protecting the ecological environment and human health. It is easy to use, low-cost, and achieves a straw removal rate of over 63% in the solution. The bacterial agent produced using Bacillus cereus BJ-2 has the advantages of low production cost, ease of use, and excellent removal efficiency. It is suitable for treating straw pollution in soil and other environments and has very important theoretical and practical value.

[0014] The straw-degrading Bacillus cereus BJ-2 of the present invention is deposited in the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China (zip code: 430072), with a deposit number of CCTCC NO: M2023950 and a deposit date of June 5, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the logarithmic growth phase of BJ-2;

[0016] Figure 2 The colony morphology is shown in the figure;

[0017] Figure 3 This is a 40x microscope photo;

[0018] Figure 4 This is a 100x microscope photo;

[0019] Figure 5 This is a BJ-2 electron microscope photo.

[0020] Figure 6 This is a photo of PCR electrophoresis bands;

[0021] Figure 7 The phylogenetic tree is constructed as shown. DETAILED DESCRIPTION

[0022] 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. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein may be combined with each other.

[0024] Specific embodiment 1: The Bacillus cereus BJ-2 for degrading straw in this embodiment has a preservation number of CCTCC NO: M 2023950, and is preserved in the China Center for Type Culture Collection (CCTCC), with a preservation date of June 5, 2023.

[0025] Isolation and identification of straw-degrading Bacillus cereus BJ-2 (degrading bacteria BJ-2) in this embodiment

[0026] 1. Isolation and screening of degradation bacteria BJ-2

[0027] Weigh 10.0 g of feces from cattle that have been eating straw for a long time (from the Experimental Station of the Northeast Geographic Institute, Gongzhuling City, Jilin Province on March 10, 2023) into a 250 mL conical flask, add 100 mL of inorganic salt culture medium, place the conical flask on a shaker (65°C, 180 rpm) and culture for 7 days, take 5 mL of culture solution, add 100 mL of separation culture medium, place the conical flask on a shaker (65°C, 180 rpm) and culture for 7 days, take 10 mL of culture solution and inoculate it into fresh separation culture medium, then culture the flask at 65°C for 7 days, and repeat the inoculation process for 5 times.

[0028] Before each inoculation, the turbid bacterial suspension in the isolation culture medium was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 5 serial dilutions, each dilution was separated by the coating method on solid separation and purification medium, cultured at 65 ° C for two days, single colonies were picked according to their different appearance and characteristics, repeatedly streaked and purified, and the same strains were merged according to the appearance and morphological characteristics of the colonies and the bacterial morphological characteristics observed by microscopic morphology, and the obtained strains were inoculated on the slant of solid separation and purification medium, stored and set aside.

[0029] According to the degradation effect, a strain numbered BJ-2 was finally obtained and named as the degrading bacteria BJ-2. Under pure culture conditions, this strain can degrade 63.2% of the initial concentration of 50.0 mg / L straw in 7 days.

[0030] HPLC assay conditions: Agilent 1260 high performance liquid chromatograph (Agilent, USA); detector: Agilent 1260 UV detector (Agilent, USA); chromatographic column: C18 reversed-phase column (Waters, 150 mm × 4.60 mm, 5 μm); mobile phase: methanol: water 60:40, V / V), flow rate 1.0 mL / min; column temperature 30°C; detection wavelength: 222 nm; injection volume: 10 μL.

[0031] The degradation rate is calculated as follows:

[0032]

[0033] The culture medium used in the above examples is as follows:

[0034] Mineral salt medium (MSM): NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, sucrose 1 g, dissolved in 1000 mL distilled water, pH = 7.0, autoclaved at 121°C for 30 min;

[0035] Isolation medium: NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, sucrose 1 g, different concentrations of straw and filter paper strips as nitrogen sources according to the experimental design requirements, make up to 1000 mL with distilled water, and adjust the pH to 7.0 (add 20 g agar to solid separation and purification medium (AMSM)).

[0036] The above culture media were sterilized in an autoclave at 121°C for 30 min. The culture medium was shaken at 30°C and 150 rpm until the logarithmic growth phase to obtain the strain.

[0037] 2. Identification of the degrading bacteria BJ-2

[0038] (1) Morphological identification of the degrading bacteria BJ-2

[0039] It will be in the logarithmic growth phase. Figure 1 As shown, the colony size is stable. The degrading bacteria BJ-2 isolated and purified above is described in single colony state. The colony morphology is shown in the figure Figure 2As shown, the degrading bacteria BJ-2 isolated and purified above grows very fast on LB solid plate medium. It is smooth and sticky, with blunt ends and large rods that can form chain-like arrangements and form oval spores within 6 hours of growth. The colonies are located in the center of the bacteria, grayish white, opaque, and have a rough surface. Its shape is waxy, with uniform turbid growth, sticky and easy to pick up. Electron microscopy observation shows that the surface is not smooth. Figure 3 This is a 40x microscope photo. Figure 4 Microscope 100x photo, Figure 5 This is a BJ-2 electron microscope photo.

[0040] (2) Analysis of physiological and biochemical characteristics

[0041] The physiological and biochemical characteristics of the degrading bacteria BJ-2 were determined with reference to Experimental Microbiology (Shen Ping, Fan Xiurong, Li Guangwu. Experimental Microbiology (3rd edition). Beijing: Higher Education Press, 1999.) and Manual of Identification of Common Bacteria (Dong Xiuzhu, Cai Miaoying. Manual of Identification of Common Bacteria. Beijing: Science Press, 2011.).

[0042] The test results showed that the strain was Gram-negative, could ferment glucose, sucrose, and mannitol, did not liquefy gelatin, did not produce indole, did not form H2S, could decompose and utilize citric acid, decompose urea, was negative for lysine ornithine, and was negative for ONPG, arginine, and oxidase.

[0043] (3) Homology analysis of 16S rDNA of degrading bacteria

[0044] The 16S rDNA gene was amplified using universal primers polymerase chain reaction (PCR) with primers 27F (5'-AGAGTTGATCCTGCTCAG-3') and 1492R (5'-GTTACCTTTACGACTT-3'). A 50 μL amplification system consisted of 25 μL of Premix Taq, 500 ng of template (Bacillus DNA), 10 μL each of 1492R and 27F, and sterile water to a final volume of 50 μL. PCR cycle parameters were: preheating at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1.5 minutes, and extension at 72°C for 10 minutes. Purification was performed using 1% agarose gel electrophoresis and EasyPure gel purification. The PCR electrophoresis bands are shown in the following images. Figure 6As shown. Extraction kit (Transgenic Biotechnology Co., Ltd., Beijing, China), and then sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, China). PCR products were purified and recovered using V-gene nucleic acid purification kit (TIANGEN), and the DNA was eluted into a collection tube and refrigerated at 4°C. The sequencing results were commissioned to Shanghai Sangon Engineering Co., Ltd. The sequencing results were compared for homology with the 16S rDNA sequences in Genbank using Blast software. The phylogenetic tree was constructed as shown in Figure 7 The 16S rDNA sequence of BJ-2 was obtained as shown in SEQ ID NO: 1. The 16S rDNA sequence of this strain has been submitted to the GenBank database (GenBank accession number: OR104997). It was found that the sequence has a 98% homology with the gene sequence of Bacillus cereus strain 8AQ and other strains.

[0045] 3. Growth characteristics analysis

[0046] The growth experiment of the strain with the optimum temperature, optimum pH and substrate concentration was carried out.

[0047] In the temperature experiment, straw was added to 100 mL of inorganic salt culture medium to make its concentration reach 50 mg / L, the pH was adjusted to 7, 5% of BJ-2 bacterial suspension was inoculated, and the culture temperature was adjusted to 30℃, 45℃, 50℃, 65℃, and 70℃, respectively, and cultured in a shaking incubator at 180 rpm.

[0048] In the pH value experiment, the pH values ​​of 100 mL of inorganic salt culture medium were adjusted to 5, 7, and 9, respectively, lignin was added to make its concentration reach 50 mg / L, 5% volume percentage of BJ-2 bacterial suspension was inoculated, and cultured in a shaking incubator at 65°C and 180 rpm.

[0049] The substrate concentration experiment was carried out by adding straw debris to the inorganic salt culture medium to make the final concentrations of 10mg / L, 20mg / L, 50mg / L, 80mg / L, and 100mg / L, respectively. The BJ-2 bacterial suspension was inoculated with a volume percentage of 5%, and the culture was shaken at 65℃ and 180rpm.

[0050] Samples were collected at 3, 5, 7, 9, and 11 days after each treatment to determine the growth of the straw-degrading bacteria BJ-2 and straw weight. Each treatment was repeated three times, with an uninoculated control used to culture, observe, and record the growth of the strain.

[0051] The results showed that the optimal growth temperature of the degrading bacterium BJ-2 was 20-45°C, and the optimal growth pH was alkaline 7.2±0.2. Based on the morphological, physiological and biochemical characteristics of the above strain BJ-2 and the results of 16S rDNA sequence analysis, the degrading bacterium BJ-2 was identified as Bacillus cereus BJ-2.

[0052] Specific embodiment 2: The preparation method of the bacterial agent of Bacillus cereus BJ-2 described in specific embodiment 1 is carried out according to the following steps:

[0053] First, Bacillus cereus BJ-2 was inoculated into LB liquid medium, cultured at 65°C and 180 rpm with shaking until the logarithmic growth phase, and washed with phosphate buffer solution with a pH of 7.0 to obtain the strain;

[0054] 2. Inoculate the strain obtained in step 1 into a seed bottle culture medium at an inoculum volume ratio of 10%, and culture with shaking at 65-70° C. and 150-200 rpm until the logarithmic growth phase to obtain a seed solution; wherein each 100 mL of the seed bottle culture medium is composed of 0.3-0.5% sucrose, 0.04-0.08% MgSO4·7H2O, 0.15-0.2% K2HPO4, 0.08-0.1% NaCl, 0.05-0.07% KH2PO4, 0.15-0.2% yeast extract and the balance water, and the pH is adjusted to 7.0;

[0055] 3. Add 15-20 mL of the seed solution prepared in step 2 to 1000 g of liquid culture medium, and add 15-20 mL of nutrient solution to the above 1000 g of liquid culture medium, and culture and ferment in a shaker at 65-70° C. for 12-48 hours to obtain a Bacillus cereus BJ-2 preparation; wherein the liquid culture medium is prepared by adding 15 g of peptone, 4 g of yeast powder, 2.52 g of disodium hydrogen phosphate and 0.28 g of potassium dihydrogen phosphate to 1000 mL of distilled water, and adjusting the pH to 7.0; each 100 mL of nutrient solution is composed of 0.5-0.7% sucrose, 0.08-0.1% KH2PO4, 0.08-0.1% MgSO4·7H2O, 0.18-0.2% K2HPO4, 0.1-1.2% NaCl, 0.8-1.0% yeast powder and the remainder water.

[0056] The pH value of the nutrient solution in step three of this embodiment is natural.

[0057] Specific embodiment 3: This embodiment differs from specific embodiment 2 in that in step 1, LB medium is prepared by adding 10g NaCl, 5g yeast extract powder, and 10g tryptone to 1000mL distilled water, and the pH is adjusted to 7.0. Other steps and parameters are the same as those in specific embodiment 1.

[0058] Example 1 Preparation of a bacterial agent for degrading Bacillus cereus BJ-2 (straw soil bioremediation bacterial agent)

[0059] 1) Bacillus cereus BJ-2 was inoculated into LB liquid medium and cultured at 65°C and 180 rpm with shaking until the logarithmic growth phase to obtain a strain; the LB medium was prepared by adding 10 g of NaCl, 5 g of yeast extract powder, and 5 g of tryptone to 1000 mL of distilled water, and the pH was adjusted to 7.0;

[0060] 2) Inoculate the cultured bacteria into a seed flask at a 10% inoculum volume ratio and incubate at 65°C, 180 rpm, and shake at the logarithmic growth phase to obtain a seed solution. The medium composition per 1000 mL seed flask is: NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.4 g, sucrose 3.0 g, yeast extract 0.2 g. Make up to 1000 mL with water and adjust the pH to 7.0.

[0061] 3) 20 mL of the obtained seed solution (OD 600 =2) adding 1000 g of fermentation medium and 20 mL of nutrient solution to 1000 g of liquid culture medium, and culturing and fermenting in a shaker at 28° C. for 48 h to obtain a soil bioremediation agent of the degradation bacteria BJ-2 strain.

[0062] Example 2 Effect of Bacillus cereus BJ-2BJ-2 bacterial agent on soil remediation

[0063] First, 1000 g of soil was weighed, an aqueous solution made from straw was added, and a soil bioremediation agent containing the BJ-2 strain (Bacillus cereus BJ-2BJ-2 agent obtained in Example 1) was applied to the soil at a weight percentage of 10%. The soil was thoroughly mixed and incubated at a constant temperature of 65°C in an incubator. Samples were taken regularly to determine the residual lignin in the liquid.

[0064] The results showed that the straw degradation rate was significantly improved by using soil bioremediation agents containing the BJ-2 strain. The lignin degradation rate reached 63.2% after adding the BJ-2 agent to the soil for 7 days, while the degradation rate was only 11.1% without adding the agent.

Claims

1. Bacillus cereus BJ-2 for degrading straw, characterized by The straw-degrading Bacillus cereus BJ-2 has a deposit number of CCTCC NO: M 2023950 and is deposited in the China Center for Type Culture Collection (CCTCC). The deposit date is June 5, 2023.

2. The method for preparing a microbial agent of Bacillus cereus BJ-2 as claimed in claim 1, wherein The preparation method of the bacterial agent of Bacillus cereus BJ-2 is carried out according to the following steps:

1. Inoculate Bacillus cereus BJ-2 into LB liquid medium, culture at 65°C and 180 rpm with shaking until the logarithmic growth phase, and wash with phosphate buffer solution with a pH of 7.0 to obtain a strain; 2. Inoculate the strain obtained in step 1 into a seed bottle culture medium at an inoculum volume ratio of 10%, and culture with shaking at 65-70° C. and 150-200 rpm until the logarithmic growth phase to obtain a seed solution; wherein each 100 mL of the seed bottle culture medium is composed of 0.3-0.5% sucrose, 0.04-0.08% MgSO4·7H2O, 0.15-0.2% K2HPO4, 0.08-0.1% NaCl, 0.05-0.07% KH2PO4, 0.15-0.2% yeast extract and the balance water, and the pH is adjusted to 7.0; 3. Add 15-20 mL of the seed solution prepared in step 2 to 1000 g of liquid culture medium, and add 15-20 mL of nutrient solution to the above 1000 g of liquid culture medium, and culture and ferment in a shaker at 65-70° C. for 12-48 hours to obtain a Bacillus cereus BJ-2 preparation; wherein the liquid culture medium is prepared by adding 15 g of peptone, 4 g of yeast powder, 2.52 g of disodium hydrogen phosphate and 0.28 g of potassium dihydrogen phosphate to 1000 mL of distilled water, and adjusting the pH to 7.0; each 100 mL of nutrient solution is composed of 0.5-0.7% sucrose, 0.08-0.1% KH2PO4, 0.08-0.1% MgSO4·7H2O, 0.18-0.2% K2HPO4, 0.1-1.2% NaCl, 0.8-1.0% yeast powder and the remainder water.

3. The method for preparing a microbial agent of Bacillus cereus BJ-2 according to claim 2, wherein In step 1, LB medium was prepared by adding 10 g of NaCl, 5 g of yeast extract powder, and 10 g of tryptone to 1000 mL of distilled water, and adjusting the pH to 7.0.

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