CN113444674A - Culture method of bacillus mucilaginosus - Google Patents
Culture method of bacillus mucilaginosus Download PDFInfo
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Abstract
The invention discloses a method for culturing bacillus mucilaginosus, which comprises the following steps: the fermentation broth is treated by a strain activation-seed liquid culture-transfer fermentation medium-fermentation tank fermentation-viable count determination according to a conventional plate counting method or an absorbance method-post treatment spray drying method. Carbon and nitrogen are important elements that make up the cytoskeleton and are also important building blocks for bacterial metabolites, and are also energy sources for microbial growth. Therefore, different types and concentrations of the carbon source have different influences on the growth of the thalli and the formation of spores, and the carbon source selected from molasses is more favorable for generating the spores than other carbon sources. The invention selects 0.05 percent MgSO4 & 7H2O to be more beneficial to thallus culture. In thatThe addition of the surfactant TX10 with low concentration in the fermentation medium has weak promotion effect on colloid shake flask fermentation. The bacterial quantity of the strain fermentation liquor treated by the method can reach 700 multiplied by 108CFU/mL, obviously improved the bacterial load of product, have apparent economic benefits to reducing production cost.
Description
Technical Field
The invention relates to the technical field of microbial preparation, in particular to a culture method of bacillus mucilaginosus.
Background
Bacillus mucilaginosus (Bacillus mucosus) as one of important strains of Paenibacillus (Paenibacillus) can decompose silicate and other substances in potassium-containing minerals and release potassium ions, so that the Bacillus mucilaginosus is commonly called as potassium bacteria. Due to the functional diversity, the method is widely applied to many fields. In the agricultural field, bacillus mucilaginosus can convert molecular nitrogen in the environment into compound nitrogen which can be utilized by plants, and secrete substances such as auxin and the like to shorten the growth cycle of crops, so that the bacillus mucilaginosus is often added into crop fertilizers; in the aspect of industrial production, the bacillus mucilaginosus can decompose aluminosilicate minerals through organic acids formed outside cells, and is generally used in the fields of bioleaching, metallurgy and the like; in the aspect of environmental protection, the bacillus mucilaginosus has excellent flocculation activity, so the bacillus mucilaginosus has excellent application value in the fields of sewage treatment and the like.
At present, most of domestic bacillus mucilaginosus fertilizer production is still in an old fermentation process before continuation, firstly, the fermentation bacteria amount is low and is generally only about 5 hundred million/ml, and secondly, the production mode of direct adsorption or liquid filling of fermentation liquor after fermentation does not have a quick and effective post-treatment process, so that the bacteria amount of the bacillus mucilaginosus fertilizer product is low, the quality is not stable, and the sale and the application of the bacillus mucilaginosus fertilizer are seriously restricted.
Disclosure of Invention
The invention provides a culture method of bacillus mucilaginosus for making up the defects of the prior art.
The invention is realized by the following technical scheme: a method for culturing Bacillus mucilaginosus comprises the following steps:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank;
(5) measuring the number of viable bacteria in the fermentation liquor obtained in the step (3) or (4) by a conventional flat plate counting method or an absorbance method; pretreating the bacterial liquid at 80 ℃ for 20min before detecting the number of spores;
(6) treating the fermentation liquor by a post-treatment spray drying method;
further, the slant culture medium in the step (1) is 0.12 percent of potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Further, in the step (2), the seed culture medium is 1% of molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Further, a carbon source in the step (3); a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.0005% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; the pH value is 7.3-7.5.
Further, 0-0.1% of a surfactant TX10 is added into the fermentation medium in the step (3).
Compared with the prior art, the invention has the advantages that:
1. carbon and nitrogen are important elements that make up the cytoskeleton and are also important building blocks for bacterial metabolites, and are also energy sources for microbial growth. Therefore, different types and concentrations of the carbon source have different influences on the growth of the thalli and the formation of spores, and the carbon source selected from molasses is more favorable for generating the spores than other carbon sources. The addition of trace elements such as Mn, B, Zn, Cu, Mo and the like has certain influence on the growth of the bacillus mucilaginosus, has promotion effect at low concentration and inhibition effect at high concentration, and is more beneficial to thallus culture by selecting 0.05 percent of MgSO4 & 7H 2O. The addition of the surfactant TX10 with low concentration in the fermentation medium has weak promotion effect on colloid shake flask fermentation.
2. The bacterial quantity of the strain fermentation liquor treated by the method can reach 700 multiplied by 108CFU/mL, obviously improved the bacterial load of product, have apparent economic benefits to reducing production cost.
Detailed Description
The detailed description is only a part of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Example 1
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; the pH value is 7.3-7.5.
The pH value can affect the permeability and stability of cell membranes and the dissolution and ionization of substances, and the factors can affect the absorption of nutrients by thalli and further affect the growth of the thalli.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; pretreating the bacterial liquid at 80 ℃ for 20min before detecting the number of spores;
(6) treating the fermentation liquor by a post-treatment spray drying method:
the bacillus mucilaginosus generated in the steps is used for generating steamed bread polysaccharide, so that the fermentation liquor is viscous, the phenomenon of spray drying viscous tower is caused, and the like, so that the spray drying fails.
The spray drying method comprises the following steps:
adding 0.5 wt% of Tween 80 and 5 wt% of light calcium carbonate according to the fermentation liquid amount, stirring uniformly, and heating to 40 deg.C for spray drying. The air inlet temperature of spray drying is 170-180 ℃, the air outlet temperature is 75-80 ℃, and the fermentation liquor can be sprayed and dried in journey, so that about 560 hundred million/g of bacterial powder is finally obtained.
The detection result in the step (5) shows that the bacillus mucilaginosus isAbsorbance average value (OD)600) Is 5.71.
Example 2
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; pH 7.3-7.5, 0.01% of surfactant TX 10.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; before detecting the number of spores, the bacterial liquid is pretreated for 20min at 80 ℃.
The detection result shows that the average value (OD) of the absorbance of the bacillus mucilaginosus is600) Is 5.72.
Example 3
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; pH 7.3-7.5, 0.05% of surfactant TX 10.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; before detecting the number of spores, the bacterial liquid is pretreated for 20min at 80 ℃.
The detection result shows that the average value (OD) of the absorbance of the bacillus mucilaginosus is600) It was 5.79.
Example 4
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; pH 7.3-7.5, 0.1% of surfactant TX 10.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; before detecting the number of spores, the bacterial liquid is pretreated for 20min at 80 ℃.
The detection result shows that the average value (OD) of the absorbance of the bacillus mucilaginosus is600) It was 5.89.
Example 5
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; pH 7.3-7.5, 0.2% of surfactant TX 10.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; before detecting the number of spores, the bacterial liquid is pretreated for 20min at 80 ℃.
The detection result shows that the average value (OD) of the absorbance of the bacillus mucilaginosus is600) Is 5.75.
Example 5
Preparing slant culture medium containing 0.12% potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% mgso4.7h2o; 2.0% agar; the pH was 7.5.
Preparing a seed culture medium, namely 1% molasses; 0.05% K2HPO 4; 0.05% mgso4.7h2o; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
Preparing a fermentation medium: a carbon source; a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% mgso4.7h2o; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; pH 7.3-7.5, 0.3% of surfactant TX 10.
Then, culturing strains, wherein the culture steps are as follows:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank. Specifically, the fermentation tank is pre-emptied for 1 hour, a fermentation culture medium is filled, the fermentation tank is sterilized again, the sterilization condition is 121-125 ℃, the sterilization time is 30-35 minutes, the fermentation culture medium is cooled to 30-35 ℃, and inoculation is carried out, wherein the inoculation amount is 1.7-1.9%; the fermentation medium amount in a 1 ton fermentation tank is 800-850L, the fermentation temperature is 37 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 1-5 cubic/min, and the fermentation is carried out for 20-24 hours to obtain fermentation liquor;
(5) the viable count is measured by a conventional flat plate counting method or an absorbance method; before detecting the number of spores, the bacterial liquid is pretreated for 20min at 80 ℃.
The detection result shows that the average value (OD) of the absorbance of the bacillus mucilaginosus is600) Is 1.4.
The invention belongs to the culture of microorganisms, the strain used in the embodiment is a Bacillus Subtilis 1 strain, the strain is obtained by breeding healthy pig intestines according to a conventional dilution plate separation method, and molasses is used as the carbon source. The bacterial quantity of the strain fermentation liquor treated by the method can reach 700 multiplied by 108CFU/mL, the bacterial load of the product is obviously improved.
Claims (5)
1. A method for culturing colloidal bacillus, which is characterized in that: the method comprises the following steps:
(1) strain activation
Inoculating Bacillus mucilaginosus to a slant culture medium for activation, controlling the temperature at 32 ℃, and culturing for 24-32 hours to obtain activated slant seeds;
(2) seed liquid culture
Inoculating the activated strain obtained in the step (1) into a 500 ml triangular flask filled with a seed culture medium by using an inoculating loop, and performing shake-flask culture at 31-33 ℃ and 200r/m in for 8-12 hours to obtain a seed solution for later use;
(3) transfer fermentation medium
The culture process parameters are as follows: the inoculation amount of the seed liquid is 7-9%, the liquid loading amount of a 250mL triangular flask is 30-60 mL, the initial pH is 7.0-7.5, the fermentation temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 40-48 h;
(4) fermentation in a fermentation tank: or inoculating the seed liquid obtained in the step (3) into a liquid fermentation culture medium, and fermenting by using a mechanical stirring type fermentation tank;
(5) measuring the number of viable bacteria in the fermentation liquor obtained in the step (3) or (4) by a conventional flat plate counting method or an absorbance method; pretreating the bacterial liquid at 80 ℃ for 20min before detecting the number of spores;
(6) and (4) processing the fermentation liquor by a post-processing spray drying method.
2. The method for culturing Bacillus mucilaginosus according to claim 1, wherein: the slant culture medium in the step (1) is 0.12 percent of potassium aluminum silicate; 0.5% sucrose; 0.2% Na2H PO 4; 0.0005% FeCl 3; 0.05% MgSO4 & 7H 2O; 2.0% agar; the pH was 7.5.
3. The method for culturing Bacillus mucilaginosus according to claim 1, wherein: in the step (2), the seed culture medium is 1 percent of molasses; 0.05% K2HPO 4; 0.05% MgSO4 & 7H 2O; 0.02% NaCl; 0.5% CaCO 3; the pH was 7.5.
4. The method for culturing Bacillus mucilaginosus according to claim 1, wherein: a carbon source in the step (3); a nitrogen source; 0.1-0.3% K2HPO 4; 0.02% NaCl 0.1% MgSO4 & 7H 2O; 0.05% FeCl 3; 0.1-0.7% of CaCO 3; wherein the carbon source is glucose, sucrose, soluble starch and molasses; the nitrogen source is bean tips, yeast extract and peptone; the pH value is 7.3-7.5.
5. The method for culturing Bacillus mucilaginosus according to claim 1, wherein: and (3) adding 0-0.1% of surfactant TX10 into the fermentation medium.
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| CN112941342A (en) * | 2021-01-29 | 2021-06-11 | 武汉科技大学 | Method for enriching vanadium by combining roasting, bioleaching and extraction of vanadium-containing shale |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001032587A1 (en) * | 1999-11-01 | 2001-05-10 | 'piksa Inter' Ltd | Biological addition to organic-mineral fertilizers |
| CN102260637A (en) * | 2010-05-28 | 2011-11-30 | 中国科学院过程工程研究所 | Low-density high-yield fermentation production method of colloid bacillus PM13 strain |
| CN103525728A (en) * | 2013-10-10 | 2014-01-22 | 合肥市钱鑫生物科技发展有限公司 | Submerged fermentation culture medium of bacillus mucilaginosus applied to microbial fertilizers |
-
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- 2021-08-24 CN CN202110974572.XA patent/CN113444674A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001032587A1 (en) * | 1999-11-01 | 2001-05-10 | 'piksa Inter' Ltd | Biological addition to organic-mineral fertilizers |
| CN102260637A (en) * | 2010-05-28 | 2011-11-30 | 中国科学院过程工程研究所 | Low-density high-yield fermentation production method of colloid bacillus PM13 strain |
| CN103525728A (en) * | 2013-10-10 | 2014-01-22 | 合肥市钱鑫生物科技发展有限公司 | Submerged fermentation culture medium of bacillus mucilaginosus applied to microbial fertilizers |
Non-Patent Citations (1)
| Title |
|---|
| 王雪 等: "胶质芽孢杆菌培养条件及发酵工艺的研究进展", 《过程工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941342A (en) * | 2021-01-29 | 2021-06-11 | 武汉科技大学 | Method for enriching vanadium by combining roasting, bioleaching and extraction of vanadium-containing shale |
| CN112941342B (en) * | 2021-01-29 | 2022-08-30 | 武汉科技大学 | Method for enriching vanadium by combining roasting, bioleaching and extraction of vanadium-containing shale |
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