A method for purifying, propagating and processing bacillus for preparing bio-organic fertilizer

Abstract

The application relates to the technical field of bio-agriculture and microbial fertilizer preparation, and discloses a method for purifying, proliferating and treating bacillus for preparing bio-organic fertilizer, which comprises the following steps: step 1, bacillus strain preliminary screening: a soil sample or a compost sample containing target bacillus is taken, sterile physiological saline is added, and the mixture is subjected to oscillation culture at 28-30 DEG C and 120-150 r / min for 20-24 h to obtain a bacterial suspension; the bacterial suspension is subjected to heat shock treatment in a water bath at 80-85 DEG C for 10-15 min, and after cooling to room temperature, gradient dilution is performed to 10 ‑6 ‑10 ‑8 times. The application is characterized by the optimization design of gradient purification screening, graded proliferation culture and bacillus activation treatment, the culture medium formula and culture parameters are accurately matched with the growth requirements of the target bacillus, the survival rate of the bacillus agent bacillus obtained finally is greater than or equal to 95%, the effective viable bacterial count is greater than or equal to 2x10 11 CFU / g, and the attenuation rate of the effective viable bacterial count is less than or equal to 10% after 6 months of normal-temperature storage, so that the bacillus agent has the advantages of high purity, sufficient viable bacterial count and strong stability, and can efficiently meet the preparation requirements of bio-organic fertilizer.

Description

Technical Field

[0001] This invention relates to the field of bio-agriculture and microbial fertilizer preparation technology, specifically to a method for purifying, propagating, and treating Bacillus bacteria for preparing bio-organic fertilizer. Background Technology

[0002] In the field of bio-agriculture, bio-organic fertilizers are increasingly widely used due to their advantages in improving soil, enhancing crop quality, and being environmentally friendly and sustainable. Bacillus, as a core functional strain, directly determines the effectiveness of organic fertilizers based on its purity, viable cell count, and stability. Currently, the preparation of Bacillus generally suffers from insufficient strain purity, low propagation efficiency, and poor spore activation, resulting in difficulties in achieving the required number of effective viable cells in the inoculant. Furthermore, viable cells decay rapidly during room temperature storage, failing to meet the needs of large-scale production and long-term application. Simultaneously, existing culture medium formulations and parameters are poorly compatible, easily leading to problems such as strain growth imbalance and carrier aggregation, further restricting the production quality and application benefits of bio-organic fertilizers.

[0003] To address this issue, we propose a method for purifying, propagating, and treating Bacillus bacteria used in the preparation of bio-organic fertilizers. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for purifying, propagating, and treating Bacillus bacteria used in the preparation of bio-organic fertilizers, thus solving the problems mentioned in the background section.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for purifying, propagating, and treating Bacillus subtilis for preparing bio-organic fertilizer, comprising the following steps: Step 1: Initial screening of Bacillus strains: Take soil or compost samples containing the target Bacillus, add sterile physiological saline, and incubate with shaking at 28-30℃ and 120-150 r / min for 20-24 h to obtain a bacterial suspension; place the bacterial suspension in an 80-85℃ water bath for heat shock treatment for 10-15 min, cool to room temperature, and then serially dilute to 10⁻⁶. -6 -10 -8 times; Step 2: Gradient purification and screening: Spread the diluted bacterial suspension onto the first screening medium and incubate at 30-32℃ for 48-72 hours. Pick single colonies with consistent morphology. Inoculate the single colonies onto the second screening medium and incubate at 30-32℃ for 36-48 hours. Screen for strains with a clear zone diameter to colony diameter ratio ≥2.5. The first screening medium uses glucose as the carbon source and soybean meal as the nitrogen source, with 0.05-0.1% calcium carbonate and 0.02-0.03% Tween-80 added. The second screening medium uses sucrose as the carbon source and fish meal peptone as the nitrogen source, with 0.1-0.15% potassium dihydrogen phosphate and 0.03-0.05% magnesium sulfate added, and the pH is adjusted to 7.2-7.5. Step 3: Graded propagation culture: Inoculate the purified strain into seed culture medium and culture at 32-34℃ and 160-180 r / min for 18-20 h with shaking to obtain seed liquid; inoculate the seed liquid into propagation culture medium at an inoculation rate of 5-8%, and first culture at 34-36℃ and 180-200 r / min with shaking for 12-14 h, then adjust the temperature to 30-32℃ and the shaking speed to 150-160 r / min and continue to culture for 24-30 h; the propagation culture medium contains the following components by weight: 25-30 parts corn flour, 18-22 parts peanut meal powder, 5-8 parts yeast extract, 1.2-1.5 parts dipotassium hydrogen phosphate, 0.8-1.0 parts magnesium sulfate, 0.05-0.08 parts manganese sulfate, 2-3 parts betaine, 1000 parts water, and pH 7.0-7.3; Step 4: Spore activation and treatment: Centrifuge the propagated bacterial solution, collect the bacterial precipitate, resuspend it in a sterile buffer solution containing 0.5-0.8% trehalose and 0.3-0.5% proline, and activate it by standing at 25-28℃ for 6-8 hours; add the carrier to the activated bacterial solution, stir and mix evenly, and dry it at 45-50℃ and a vacuum degree of -0.06~-0.08MPa until the moisture content is ≤8% to obtain Bacillus inoculum; the carrier is a mixture of humic acid, diatomaceous earth and zeolite powder in a weight ratio of 3:2:1, and the amount of carrier added is 3-5 times the wet weight of the bacterial precipitate.

[0006] Preferably, in step 2, the pH value of the first screening medium is 7.3-7.5, and intermittent aeration is used during the culture process. The aeration rate is 0.3-0.5 L / (L•min), each aeration lasts for 10-15 min, and aeration is performed once every 2 hours.

[0007] Preferably, the seed culture medium in step 3 comprises: 15-20 parts glucose, 10-12 parts peptone, 3-5 parts yeast extract, 0.8-1.0 parts potassium dihydrogen phosphate, 0.5-0.7 parts magnesium sulfate, and 1000 parts water, with a pH of 6.8-7.0; the seed culture is cultured to OD... 600When the value is 1.8-2.2, inoculate with the propagation culture medium.

[0008] Preferably, in step 3, during the propagation process, after the temperature is adjusted to 30-32℃, 0.3-0.5% glucose and 0.2-0.3% soybean meal hydrolysate are added to the propagation culture medium, with the amount added being 5-8% of the volume of the propagation culture medium.

[0009] Preferably, the sterile buffer solution in step 4 is a Tris-HCl buffer with a pH of 7.2-7.4, and the concentration of the bacterial suspension after resuspending is adjusted to 1×10⁻⁶. 10 -5×10 10 CFU / mL; during the drying process, a gradient temperature increase is used, first drying at 35-40℃ for 1-2 hours, and then increasing the temperature to 45-50℃ to continue drying for 3-4 hours.

[0010] Preferably, the target Bacillus is one or more of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens. When it is a mixture of multiple strains, each strain is mixed in a weight ratio of 1:1:1 after purification and then cultured for propagation.

[0011] Preferably, in step 4, the stirring speed is 80-100 r / min, the stirring time is 30-40 min, and the stirring is paused for 5 min every 10 min to avoid carrier agglomeration.

[0012] Preferably, the obtained Bacillus inoculum agent has a spore survival rate of ≥95% and an effective viable count of ≥2×10⁻⁶. 11 The effective viable count decreased by ≤10% after 6 months of storage at room temperature, with a CFU / g concentration.

[0013] (III) Beneficial Effects Compared with existing technologies, this invention provides a method for purifying, propagating, and treating Bacillus bacteria used in the preparation of bio-organic fertilizer, which has the following beneficial effects: This invention employs an optimized design for gradient purification and screening, graded propagation culture, and spore activation treatment. The culture medium formulation and parameters are precisely tailored to the growth requirements of the target Bacillus species, resulting in a final Bacillus agent with a spore survival rate ≥95% and an effective viable count ≥2×10⁻⁶. 11 With a CFU / g content and an effective viable count decay rate of ≤10% after 6 months of storage at room temperature, it possesses advantages such as high purity, sufficient viable count, and strong stability, and can efficiently meet the needs of bio-organic fertilizer preparation. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1 (Single strain: Bacillus subtilis) Initial screening of strains: Soil samples containing Bacillus subtilis were collected, sterile physiological saline was added, and the mixture was cultured at 29℃ and 130 rpm for 22 h with shaking to obtain a bacterial suspension; the suspension was then heat-shocked in a water bath at 82℃ for 12 min, cooled, and serially diluted to 10⁻⁶. -7 times.

[0016] Gradient purification and screening: Diluted bacterial suspensions were spread onto the first screening medium (pH 7.4, glucose as carbon source, soybean meal as nitrogen source, with 0.08% calcium carbonate and 0.025% Tween-80 added), and incubated at 31℃ for 60 h. Single colonies with consistent morphology were picked. The culture was then inoculated onto the second screening medium (sucrose as carbon source, fish meal peptone as nitrogen source, with 0.12% potassium dihydrogen phosphate and 0.04% magnesium sulfate added, pH 7.3), and incubated at 31℃ for 42 h. Strains with a clear zone diameter to colony diameter ratio ≥ 2.5 were screened. During the first screening medium incubation, intermittent aeration was performed at a rate of 0.4 L / (L•min), with each aeration lasting 12 min, and an interval of 2 h between aerations.

[0017] Graded propagation culture: The purified strain was inoculated into seed culture medium (18 parts glucose, 11 parts peptone, 4 parts yeast extract, 0.9 parts potassium dihydrogen phosphate, 0.6 parts magnesium sulfate, 1000 parts water, pH 6.9), and cultured at 33℃ with shaking at 170 rpm for 19 h until OD... 600 When the pH value is 2.0, inoculate the propagation medium (28 parts corn flour, 20 parts peanut meal powder, 6 parts yeast extract, 1.3 parts dipotassium hydrogen phosphate, 0.9 parts magnesium sulfate, 0.06 parts manganese sulfate, 2.5 parts betaine, 1000 parts water, pH 7.2) at a 6% inoculation rate. First, culture at 35℃ and 190 r / min with shaking for 13 h, then adjust the temperature to 31℃ and the shaking speed to 155 r / min, and add 0.4% glucose and 0.25% soybean meal hydrolysate (the amount added is 6% of the volume of the propagation medium), and continue to culture for 27 h.

[0018] Spore activation and treatment: The bacterial precipitate was collected by centrifugation of the propagated bacterial culture, resuspended in Tris-HCl buffer (pH 7.3) containing 0.6% trehalose and 0.4% proline, and the concentration was adjusted to 3 × 10⁻⁶. 10CFU / mL, activated at 26℃ for 7h; add carrier (humic acid: diatomaceous earth: zeolite powder = 3:2:1, the amount added is 4 times the wet weight of the bacterial precipitate), stir at 85r / min for 35min (pause for 5min every 10min), mix evenly, then gradually increase the temperature and dry (dry at 38℃ for 1.5h, then at 48℃ for 3.5h), vacuum degree -0.07MPa, dry until the moisture content is ≤8%, to obtain Bacillus inoculum.

[0019] Example 2 (Mixed strain: Bacillus subtilis + Bacillus licheniformis + Bacillus amyloliquefaciens) Initial screening of bacterial strains: Compost samples containing three different Bacillus species were taken, and sterile physiological saline was added to each. The samples were then cultured at 28°C and 120 rpm for 20 hours with shaking to obtain bacterial suspensions. These suspensions were then heat-shocked in an 80°C water bath for 10 minutes, cooled, and serially diluted to 10⁻⁶. -6 times.

[0020] Gradient purification and screening: Each bacterial suspension was spread onto the first screening medium (pH 7.3, same formulation as in Example 1) and incubated at 30°C for 48 h. Single colonies with consistent morphology were picked from each strain. These were then inoculated onto the second screening medium (pH 7.2, same formulation as in Example 1) and incubated at 30°C for 36 h. Strains with a clear zone diameter to colony diameter ratio ≥ 2.5 were selected. During the first screening medium incubation, intermittent aeration was performed at a rate of 0.3 L / (L•min) for 10 min at a time, with an interval of 2 h between each aeration.

[0021] Graded propagation culture: The three purified strains were inoculated separately into seed culture medium (15 parts glucose, 10 parts peptone, 3 parts yeast extract, 0.8 parts potassium dihydrogen phosphate, 0.5 parts magnesium sulfate, 1000 parts water, pH 6.8), and cultured at 32℃ with shaking at 160 rpm for 18 h until OD... 600 When the pH value is 1.8, mix the ingredients in a 1:1:1 weight ratio, and then inoculate them into the propagation medium (25 parts corn flour, 18 parts peanut meal powder, 5 parts yeast extract, 1.2 parts dipotassium hydrogen phosphate, 0.8 parts magnesium sulfate, 0.05 parts manganese sulfate, 2 parts betaine, 1000 parts water, pH 7.0) at a 5% inoculation rate. First, culture at 34℃ and 180 r / min with shaking for 12 h, then adjust the temperature to 30℃ and the shaking speed to 150 r / min, and add 0.3% glucose and 0.2% soybean meal hydrolysate (the amount added is 5% of the volume of the propagation medium), and continue to culture for 24 h.

[0022] Spore activation and treatment: The bacterial precipitate was collected by centrifugation of the propagated bacterial culture, resuspended in Tris-HCl buffer (pH 7.2) containing 0.5% trehalose and 0.3% proline, and the concentration was adjusted to 1×10⁻⁶. 10CFU / mL, activated at 25℃ for 6h; added carrier (humic acid: diatomaceous earth: zeolite powder = 3:2:1, the amount added is 3 times the wet weight of the bacterial precipitate), stirred at 80r / min for 30min (pausing for 5min every 10min), mixed evenly, and then dried by gradient temperature increase (drying at 35℃ for 1h, then at 45℃ for 3h), vacuum degree -0.06MPa, dried until the moisture content is ≤8%, to obtain Bacillus inoculum.

[0023] Example 3 (Single strain: Bacillus amyloliquefaciens) Initial screening of strains: Soil samples containing Bacillus amyloliquefaciens were collected, sterile physiological saline was added, and the mixture was cultured at 30℃ and 150 rpm for 24 h with shaking to obtain a bacterial suspension; the suspension was then heat-shocked in an 85℃ water bath for 15 min, cooled, and serially diluted to 10⁻⁶. -8 times.

[0024] Gradient purification and screening: The diluted bacterial suspension was spread onto the first screening medium (pH 7.5, same formulation as in Example 1) and incubated at 32°C for 72 hours. Single colonies with consistent morphology were picked. The culture was then inoculated onto the second screening medium (pH 7.5, same formulation as in Example 1) and incubated at 32°C for 48 hours. Strains with a clear zone diameter to colony diameter ratio ≥ 2.5 were screened. During the first screening medium incubation, intermittent aeration was performed at a rate of 0.5 L / (L•min) for 15 minutes each time, with an interval of 2 hours between aerations.

[0025] Graded propagation culture: The purified strain was inoculated into seed culture medium (20 parts glucose, 12 parts peptone, 5 parts yeast extract, 1.0 part potassium dihydrogen phosphate, 0.7 parts magnesium sulfate, 1000 parts water, pH 7.0), and cultured at 34℃ with shaking at 180 rpm for 20 h until OD... 600 When the pH value is 2.2, inoculate the propagation medium (30 parts corn flour, 22 parts peanut meal powder, 8 parts yeast extract, 1.5 parts dipotassium hydrogen phosphate, 1.0 part magnesium sulfate, 0.08 parts manganese sulfate, 3 parts betaine, 1000 parts water, pH 7.3) at an 8% inoculation rate. First, culture at 36℃ and 200 r / min with shaking for 14 h, then adjust the temperature to 32℃ and the shaking speed to 160 r / min, and add 0.5% glucose and 0.3% soybean meal hydrolysate (the amount added is 8% of the volume of the propagation medium), and continue to culture for 30 h.

[0026] Spore activation and treatment: The bacterial precipitate was collected by centrifugation of the propagated bacterial culture, resuspended in Tris-HCl buffer (pH 7.4) containing 0.8% trehalose and 0.5% proline, and the concentration was adjusted to 5 × 10⁻⁶. 10CFU / mL, activated at 28℃ for 8 hours; add carrier (humic acid: diatomaceous earth: zeolite powder = 3:2:1, the amount added is 5 times the wet weight of the bacterial precipitate), stir at 100 r / min for 40 min (pause for 5 min every 10 min), mix evenly, then gradually increase the temperature and dry (dry at 40℃ for 2 h, then at 50℃ for 4 h), vacuum degree -0.08 MPa, dry until the moisture content is ≤8%, to obtain Bacillus inoculum.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for purifying, propagating, and treating Bacillus strains used in the preparation of bio-organic fertilizer, characterized in that, Includes the following steps: Step 1: Initial screening of Bacillus strains: Take soil or compost samples containing the target Bacillus, add sterile physiological saline, and incubate with shaking at 28-30℃ and 120-150 r / min for 20-24 h to obtain a bacterial suspension; place the bacterial suspension in an 80-85℃ water bath for heat shock treatment for 10-15 min, cool to room temperature, and then serially dilute to 10⁻⁶. -6 -10 -8 times; Step 2: Gradient purification and screening: Spread the diluted bacterial suspension onto the first screening medium and incubate at 30-32℃ for 48-72 hours. Pick single colonies with consistent morphology. Inoculate the single colonies onto the second screening medium and incubate at 30-32℃ for 36-48 hours. Screen for strains with a clear zone diameter to colony diameter ratio ≥2.

5. The first screening medium uses glucose as the carbon source and soybean meal as the nitrogen source, with 0.05-0.1% calcium carbonate and 0.02-0.03% Tween-80 added. The second screening medium uses sucrose as the carbon source and fish meal peptone as the nitrogen source, with 0.1-0.15% potassium dihydrogen phosphate and 0.03-0.05% magnesium sulfate added, and the pH is adjusted to 7.2-7.

5. Step 3: Graded propagation culture: Inoculate the purified strain into seed culture medium and culture at 32-34℃ and 160-180r / min for 18-20h to obtain seed liquid; Inoculate the seed culture solution into the propagation medium at an inoculation rate of 5-8%, and first culture at 34-36℃ and 180-200 r / min with shaking for 12-14 h. Then adjust the temperature to 30-32℃ and the shaking speed to 150-160 r / min and continue culturing for 24-30 h. The propagation medium contains the following components by weight: 25-30 parts corn flour, 18-22 parts peanut meal, 5-8 parts yeast extract, 1.2-1.5 parts dipotassium hydrogen phosphate, 0.8-1.0 parts magnesium sulfate, 0.05-0.08 parts manganese sulfate, 2-3 parts betaine, 1000 parts water, and a pH of 7.0-7.

3. Step 4: Spore activation and treatment: Centrifuge the propagated bacterial solution, collect the bacterial precipitate, resuspend it in a sterile buffer solution containing 0.5-0.8% trehalose and 0.3-0.5% proline, and activate it by standing at 25-28℃ for 6-8 hours; add the carrier to the activated bacterial solution, stir and mix evenly, and dry it at 45-50℃ and a vacuum degree of -0.06~-0.08MPa until the moisture content is ≤8% to obtain Bacillus inoculum; the carrier is a mixture of humic acid, diatomaceous earth and zeolite powder in a weight ratio of 3:2:1, and the amount of carrier added is 3-5 times the wet weight of the bacterial precipitate.

2. The method for purifying, propagating, and treating Bacillus spores for preparing bio-organic fertilizer according to claim 1, characterized in that, In step 2, the pH value of the first screening medium is 7.3-7.

5. During the culture process, intermittent aeration is used, with an aeration rate of 0.3-0.5 L / (L•min), each aeration lasting 10-15 min, and aeration is performed once every 2 hours.

3. The method for purifying, propagating, and treating Bacillus spores for preparing bio-organic fertilizer according to claim 1, characterized in that, The seed culture medium in step 3 comprises: 15-20 parts glucose, 10-12 parts peptone, 3-5 parts yeast extract, 0.8-1.0 parts potassium dihydrogen phosphate, 0.5-0.7 parts magnesium sulfate, and 1000 parts water, with a pH of 6.8-7.0; the seed culture is then cultured to OD... 600 When the value is 1.8-2.2, inoculate with the propagation culture medium.

4. The method for purifying, propagating, and treating Bacillus strains for preparing bio-organic fertilizer according to claim 1, characterized in that, In step 3, during the propagation process, after adjusting the temperature to 30-32℃, add 0.3-0.5% glucose and 0.2-0.3% soybean meal hydrolysate to the propagation medium. The amount added is 5-8% of the volume of the propagation medium.

5. The method for purifying, propagating, and treating Bacillus spores for preparing bio-organic fertilizer according to claim 1, characterized in that, The sterile buffer solution mentioned in step 4 is a Tris-HCl buffer with a pH of 7.2-7.

4. The concentration of the bacterial culture after resuspending is adjusted to 1×10⁻⁶. 10 -5×10 10 CFU / mL; during the drying process, a gradient temperature increase is used, first drying at 35-40℃ for 1-2 hours, and then increasing the temperature to 45-50℃ to continue drying for 3-4 hours.

6. The method for purifying, propagating, and treating Bacillus spores for preparing bio-organic fertilizer according to claim 1, characterized in that, The target Bacillus is one or more of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens. When it is a mixture of multiple strains, each strain is mixed in a weight ratio of 1:1:1 after purification and then cultured for propagation.

7. The method for purifying, propagating, and treating Bacillus strains for preparing bio-organic fertilizer according to claim 1, characterized in that, In step 4, the stirring speed is 80-100 r / min, the stirring time is 30-40 min, and the stirring is paused for 5 min every 10 min to avoid carrier agglomeration.

8. The method for purifying, propagating, and treating Bacillus spores for preparing bio-organic fertilizer according to claim 1, characterized in that, The obtained Bacillus inoculum agent had a spore survival rate of ≥95% and an effective viable count of ≥2×10⁻⁶. 11 The effective viable count decreased by ≤10% after 6 months of storage at room temperature, with a CFU / g concentration.