CN117904009A - Bacillus subtilis applicable to non-grain bio-based carbon source and fermentation production method thereof - Google Patents
Bacillus subtilis applicable to non-grain bio-based carbon source and fermentation production method thereof Download PDFInfo
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- CN117904009A CN117904009A CN202410312372.1A CN202410312372A CN117904009A CN 117904009 A CN117904009 A CN 117904009A CN 202410312372 A CN202410312372 A CN 202410312372A CN 117904009 A CN117904009 A CN 117904009A
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- bacillus subtilis
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a bacillus subtilis applicable to non-grain biological carbon sources and a fermentation production method thereof, which have the function of using the non-grain biological carbon sources mainly comprising ethanol, and the mixed carbon sources are used for replacing part of glucose and corn meal carbon sources, so that the adding time and the feeding speed of the mixed carbon sources in the culture process are adjusted, a brand-new fermentation process is obtained, and the viable count of the bacillus subtilis can be obviously improved; by providing the novel mixed carbon source as a fermentation medium to replace part of glucose and corn meal, the replacement of grain sources by non-grain biological carbon sources can be realized, the fermentation cost of bacillus subtilis can be greatly reduced, and the stability can be improved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis applicable to non-grain bio-based carbon sources and a fermentation production method thereof.
Background
Bacillus subtilis (Bacillus subtilis) is an aerobic bacillus gram-positive bacterium capable of producing spores, widely exists on the surfaces of soil and plants, has no pathogenicity, and can secrete enzymes such as protease, amylase, lipase, cellulase and the like. And can survive for more than 30 minutes under the high-temperature treatment of 100 ℃, and has the advantages of acid and alkali resistance, bile salt resistance and the like, so that the strain can keep higher activity in the production and application of agriculture and animal husbandry, and belongs to a high-efficiency microecological preparation strain.
Bacillus subtilis is regarded as one of the most widely used probiotics among many bacillus worldwide as a novel green probiotic species that is common and versatile in agricultural inoculants. The current high-density fermentation process of bacillus subtilis is a key core problem in the microbial inoculum production industry, and the important research content is to improve the viable count and reduce the production cost.
On the other hand, the main carbon sources in the fermentation medium commonly used by bacillus subtilis are glucose, corn meal and the like, fructose and sucrose are added to part of the fermentation medium, the carbon sources are all derived from grain resources and are expensive, competition with grain supply is increased by using the carbon sources as fermentation carbon sources, meanwhile, grain crops are usually required to be subjected to fine processing and purification to be used in the biological fermentation process, the processing steps increase the complexity and cost of production, and a series of chemicals and energy sources are possibly required to be used, so that the production cost is increased, and a certain influence is exerted on the environment and the sustainability. In recent years, with the aim of green bio-manufacturing, more and more biotechnology studies have been conducted to replace conventional grain-based carbon sources with non-grain bio-based carbon sources. Non-food bio-based carbon sources refer to those that do not rely on traditional food crops (e.g., corn, wheat, soybean, etc.) as raw materials. They may be carbon sources from other plants, microorganisms, marine organisms or other biological sources. These carbon sources can be used to produce renewable energy sources, bio-based chemicals, and other biological products. The development and utilization of non-grain bio-based carbon sources is to reduce the dependency on traditional grain crops, reduce competition for grain resources, improve sustainability, and reduce environmental impact associated with grain production. They can help address challenges in food and energy safety and promote sustainable development. Thus, finding a bacillus subtilis fermented with non-glucose and corn flour as main carbon sources is an effective means for green bio-production.
There are many types of non-grain bio-based carbon sources, if vegetable waste, plant straw, wood fiber, mono-or di-alkyd, etc. The applicant has long studied on electrochemical cascade synthesis biotechnology, found that industrial carbon exhaust carbon dioxide can prepare single products of monobasic and dibasic acids, alcohols and salts through an electrocatalytic technology or obtain mixed products with a certain proportion through controlling the types and current densities of catalysts, wherein the mixed products can greatly reduce the cost in the conversion process and improve the conversion efficiency, but the application of the mixed products is a difficult problem, and the monobasic and dibasic acids, alcohols and salts obtained through the electrocatalytic of carbon dioxide play a certain role in the culture and fermentation process of some strains with metabolic pathways, so the applicant considers that the carbon sources are applied to the biological fermentation process as non-grain biological-based carbon sources. According to the invention, the bacillus subtilis LTB-001 is screened from a potato planting farmland in Yi nationality county of Weining Yi nationality in Pitch, guizhou, and the fermentation medium is adjusted and optimized through research, so that the fermentation process of fermenting the bacillus subtilis by using a mixed product carbon source mainly comprising ethanol is adopted, the yield of the bacillus subtilis is improved, the limitation that only grain sources such as glucose, sucrose and fructose can be used for producing the bacillus subtilis is solved, the production cost is reduced, and the possibility of green production is provided.
Disclosure of Invention
The invention aims to provide a brand-new bacillus subtilis LTB-001 and a fermentation production method thereof, wherein a novel non-grain biological carbon source is adopted to replace a traditional grain carbon source in the fermentation process, so that the competition of grain resources is reduced, the sustainability is improved, the environmental influence related to grain production is reduced, and meanwhile, the fermentation process has high viable count.
Based on the above, the invention provides a bacillus subtilis applicable to non-grain bio-based carbon sources, which is characterized in that the bacillus subtilis is preserved in the microorganism strain collection in Guangdong province of China at the month 22 of 2023, and is named as LTB-001 strain, and the classification is named as: bacillus subtilis (Bacillus subtilis) with a preservation number of GDMCC No:64179 and a preservation address of 100 in martyrs of Guangzhou, guangdong province, academy of sciences of microbiology.
The 16s nucleotide sequence of the bacillus subtilis is shown as SEQ ID NO. 1.
The invention also provides the mixed carbon source for fermenting the bacillus subtilis, which comprises ethanol, formate and acetate, wherein the content of the ethanol in the fermentation broth is 10-40 g/L, the concentration of the formate is 5-10 g/L, and the concentration of the acetate in the fermentation broth is 5-15 g/L.
The mixed carbon source also comprises glucose and corn flour, wherein the concentration of the glucose in the fermentation liquor is 3-5g/L, and the concentration of the corn flour in the fermentation liquor is 20-30g/L.
The formate can be one or a mixture of sodium formate, potassium formate and ammonium formate.
The acetate can be one or a mixture of sodium acetate, potassium acetate and ammonium acetate.
The invention also provides a bacillus subtilis fermentation medium which comprises the mixed carbon source, 20-30 g/L of bean cake powder, 5-15 g/L of peptone, 0.1-0.3 g/L of manganese sulfate monohydrate, 1-3 g/L of sodium chloride, 0.5-1.5 g/L of magnesium sulfate heptahydrate, pH is adjusted to 7, and sterilization is performed for 30min at 121 ℃.
The invention also provides a bacillus subtilis fermentation production method, which comprises the following steps:
Inoculating 5-10% of seed liquid into a culture medium in a 15L fermentation tank, wherein the total volume of the culture medium is 10L, the fermentation temperature is 35-37 ℃, the aeration ratio is 0.4-1.0, the stirring rotation speed is 400-600 rpm/min, the fermentation time is 24-48 h, the pH is not controlled in the fermentation process, the feeding culture medium is started to be added when the thallus normally grows for 18h, and the feeding is completed at a constant speed of 1.39 mL/min for 12h until the fermentation is completed;
The fermentation medium is prepared from 3-5 g/L glucose, 20-30g/L bean cake powder, 5-15 g/L peptone, 0.1-0.3 g/L manganese sulfate monohydrate, 1-3 g/L sodium chloride and 0.5-1.5 g/L magnesium sulfate heptahydrate by preparing with deionized water, fixing the volume to 9L, sterilizing at 121 ℃ for 30 minutes, and cooling to normal temperature for use;
The feed medium is formed by directly mixing a feed medium 1 and a feed medium 2 in a ratio of 1:1, wherein the feed medium 1 and the feed medium 2 are as follows:
The feed supplement culture medium 1 is prepared from 200-800 g/L of ethanol by using sterile water, and the volume is fixed to 0.5L; the feed medium 2 is 100-200 g/L formate and 100-300 g/L acetate, the volume is fixed to 0.5L by deionized water, and the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to normal temperature for use.
The invention also provides a microbial agent, which comprises the bacillus subtilis LTB-001, and the viable count is more than or equal to 1000 hundred million cfu/g.
The invention also provides a microbial fertilizer, which comprises the bacillus subtilis LTB-001.
The invention also provides application of the bacillus subtilis LTB-001, the microbial agent or the microbial fertilizer in agricultural production, and particularly application to potato planting.
The invention has the beneficial effects that: 1. compared with the existing fermentation culture process of bacillus subtilis, the novel fermentation process of the invention has the function of using ethanol-based non-grain biological carbon sources, and the mixed carbon sources are used for replacing part of glucose and corn meal carbon sources, so that the adding time and the feeding speed of the mixed carbon sources in the culture process are adjusted, the novel fermentation process is obtained, and the viable count of the bacillus subtilis can be obviously improved; 2. by providing the novel mixed carbon source as a fermentation medium to replace part of glucose and corn meal, the replacement of grain sources by non-grain biological carbon sources can be realized, and the fermentation cost and stability of bacillus subtilis can be greatly reduced.
Drawings
Fig. 1: streaking a bacillus subtilis LTB-001 plate;
Fig. 2: bacillus subtilis LTB-001 spore staining pattern;
fig. 3: growth profile of bacillus subtilis LTB-001 in 15L tank.
Detailed Description
The applicant determines the growth level of the strain by using a conventional bacillus subtilis production culture medium taking glucose and corn meal as main carbon sources, and then, by comparison, the mixed carbon sources consisting of ethanol, formate and acetate are respectively used for replacing glucose and corn meal carbon sources with the same proportion on the basis of conventional fermentation for fermentation culture, and the fact that the ethanol concentration in the mixed carbon sources exceeds 40 g/L and the formate concentration exceeds 10 g/L has obvious inhibition effect on the growth of the strain, and the acetate concentration exceeds 15 g/L has obvious inhibition effect, because the high-concentration ethanol can damage the structure of certain proteins, so that part of proteins are denatured, and the proteins are important components of microorganisms, so that the denaturation of the proteins can lead to death of the bacillus subtilis. Meanwhile, formate and acetate with high concentration have an inhibiting effect on the growth of bacillus subtilis, and excessive acetate can cause acidification of the inside of cells to influence metabolic activity and growth speed, but when the concentration of ethanol is lower than 40 g/L, the concentration of formate is lower than 10 g/L and the concentration of acetate is lower than 15 g/L, the growth of the strain is promoted to a certain extent, and the promotion effect of a mixed carbon source of ethanol, formate and acetate is more obvious.
On the basis, a fermentation culture medium for replacing part of glucose and corn meal carbon sources with mixed carbon sources for optimizing the proportion of ethanol, formate and acetate is further designed, fermentation verification is carried out by utilizing a fermentation tank for multiple times, a feed supplement culture medium is designed, and a feeding mode is adjusted, so that an optimal fermentation method for fermenting bacillus subtilis by using the mixed carbon sources of ethanol, acetate and formate as main bodies is obtained.
Specifically, the invention provides a method for fermenting bacillus subtilis by using a mixed carbon source with ethanol as a main body, wherein the adopted carbon source comprises a mixed carbon source composed of ethanol, formate and acetate. The concentration of the ethanol in the fermentation liquor is preferably 10-40 g/L, the concentration of the formate in the fermentation liquor is preferably 5-10 g/L, and the concentration of the acetate in the fermentation liquor is preferably 5-15 g/L. Experiments prove that the bacillus subtilis can cause the cytoplasm to be highly concentrated and dehydrated at the later stage of the logarithmic phase of growth due to the lack of sufficient nutrient substances in the system, the cell membrane is invaginated to form a membrane to surround nuclear substances, and the membrane is surrounded by a multi-layer membrane to finally form mature spores to lose the growth and reproduction capability. In bacillus subtilis, phospholipids account for 10-15% of the dry weight of cytoplasmic membrane, which is the only membrane structure in cells, whereas ethanol at a certain concentration can inhibit the conversion of phospholipids and fatty acids into spores by changing their composition. When the concentration of ethanol is less than 10g/L, the composition of phospholipid and fatty acid cannot be changed, and further the formation of spores cannot be inhibited, and when the concentration of ethanol is more than 40 g/L, the structure of certain proteins is destroyed, so that partial proteins are denatured, and the death of thalli is directly caused. Meanwhile, acetate generates acetyl phosphate under the action of acetyl kinase, and can participate in synthesis of acetyl coenzyme A as an intermediate product, the acetyl coenzyme A directly participates in the central metabolic process of tricarboxylic acid cycle and the like to provide energy for strain growth, and if the concentration is too low, the basic carbon source requirement of strain growth cannot be met when the concentration is less than 5 g/L and the strain growth can be inhibited when the concentration is more than 15 g/L due to osmotic pressure and acetate tolerance. In addition, formate is converted by formate dehydrogenase, NAD+ is reduced to form NADH during the conversion, and NADH participates in a plurality of metabolic processes of the growth of Bacillus subtilis and provides reducing power, but if the concentration is too low, it is verified that the reducing power provided under the system is insufficient at less than 5 g/L, the optimal strain growth cannot be achieved, and the strain growth is inhibited due to osmotic pressure and formate tolerance at more than 10 g/L.
The ethanol, formate and acetate can be purchased and compounded commercially, or can be a mixture obtained by electrocatalytic, thermocatalytic or photocatalytic industrial tail gas carbon dioxide, such as a mixed carbon source obtained by a preparation method mentioned in patent application number CN202211251789.9, and according to the designed and verified addition amount of the ethanol, formate and acetate in the mixed carbon source, such as the mixed carbon source obtained by electrocatalytic carbon dioxide tail gas does not meet the concentration and proportion requirements, the corresponding ethanol, formate or acetate is supplemented to meet the bacillus subtilis fermentation culture requirement.
The formate can be one or a mixture of sodium formate, potassium formate and ammonium formate; the acetate can be one or more of sodium acetate, potassium acetate and ammonium acetate.
The carbon source also comprises glucose and corn flour, the concentration of the glucose in the fermentation liquid is 3-5 g/L, the concentration of the corn flour in the fermentation liquid is 20-30 g/L, the adding concentration of the glucose in the carbon source taking the glucose and the corn flour as main bodies in the traditional fermentation process is not less than 10 g/L, and the adding concentration of the corn flour is not less than 40 g/L. The invention aims to replace grain-based carbon sources glucose and corn meal with non-grain-based carbon sources, and a large amount of amylase is not secreted in the initial growth stage of bacillus subtilis, so that the carbon sources which can be directly utilized by thalli such as glucose are not separated, the amylase secreted by the bacillus subtilis begins to hydrolyze the corn meal to form reducing sugar for supplying the energy to the thalli when the quantity of the thalli is increased, if the concentration is too low, the initial growth requirement of the strain can not be met, the viable count of the bacillus subtilis is greatly reduced, if the concentration is too high, the cost is increased, and the viable count can not be further improved,
The fermentation temperature is preferably 35-37 ℃, and the pH value of the culture medium adopted by the fermentation is 6.0-9.0.
When fermentation is carried out by adopting a fermentation tank, inoculating bacillus subtilis into a seed culture medium, carrying out aerobic fermentation culture for 6-8 hours, transferring the culture medium to the fermentation medium according to the inoculum size of 5-10%, wherein the fermentation time is 24-48 hours, the pH is not controlled in the fermentation process, and feeding the feed supplement culture medium is started in the 18 th hour, and the flow rate is 1.39 mL/min.
Obtaining seed liquid: streaking bacillus subtilis LTB-001 on an LB plate on an ultra-clean workbench by using an inoculating loop, and activating for 12-24 hours at 37 ℃ until a monoclonal strain grows;
Picking the monoclonal bacteria in an ultra-clean workbench by using a sterile inoculating loop, inoculating the monoclonal bacteria into a Ke-shi bottle, culturing for 24-48 hours at 37 ℃, pouring sterile water into the Ke-shi bottle after microscopic examination of spores is mature, scraping bacterial colonies on an inclined plane by using a sterilized bamboo stick, pouring a seed culture medium, and performing aerobic culture for 8 hours at 37 ℃ to obtain seed liquid.
Activation medium (LB medium): 1.5% -2% of agar is added to the peptone 10g/L, yeast powder 5 g/L and sodium chloride 10g/L to prepare a flat plate, and the flat plate is prepared by deionized water, sterilized at 121 ℃ for 30 minutes and then cooled to normal temperature for use.
Seed culture medium: glucose 5-10 g/L, corn flour 10-20 g/L, bean cake flour 10-20 g/L, peptone 5-15 g/L, manganese sulfate monohydrate 0.1-0.3 g/L, sodium chloride 1-3 g/L, magnesium sulfate heptahydrate 0.5-1.5 g/L, dipotassium hydrogen phosphate 1-2 g/L, and sterilizing by deionized water to 1L and 115 ℃ for 20min.
Fermentation tank medium: glucose 3-5 g/L, corn flour 20-30g/L, bean cake flour 20-30g/L, peptone 5-15 g/L, manganese sulfate monohydrate 0.1-0.3 g/L, sodium chloride 1-3 g/L, magnesium sulfate heptahydrate 0.5-1.5 g/L, and sterilizing at 115 deg.C for 20min by deionized water.
The feed medium is formed by directly mixing a feed medium 1 and a feed medium 2, wherein the feed medium 1 and the feed medium 2 are as follows:
The feed medium 1 is ethanol 200-800 g/L, and is prepared by sterile deionized water, and the volume is fixed to 0.5L; the feed medium 2 is 100-200 g/L formate, 100-300 g/L acetate, constant volume to 0.5L, sterilizing at 121deg.C for 30min, and cooling to normal temperature. Wherein the formate can be one or a mixture of sodium formate, potassium formate and ammonium formate; the acetate can be one or a mixture of sodium acetate, potassium acetate and ammonium acetate.
The following examples and drawings are used to describe embodiments of the present invention in detail, thereby solving the technical problems by applying the technical means to the present invention, and realizing the technical effects can be fully understood and implemented accordingly.
The strain used for fermentation is bacillus subtilis LTB-001, and is screened from a potato planting farmland in Miao county of Yi nationality of Pichia pastoris, and is classified and named as bacillus subtilis, and is preserved in the national institute of microbiology (microbiological analysis and detection center of Guangdong province) with a preservation number of GDMCC No.64179.
Experimental materials and reagents
1. Composition of culture medium for strain fermentation
Counting medium (NA medium): beef extract 3 g/L, peptone 10 g/L, sodium chloride 5 g/L, and agar 1.5-2.0%.
Activation medium (LB medium): 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride, and 1.5% -2% agar is added for preparing the flat plate.
Seed culture medium: glucose 5-10 g/L, corn flour 10-20 g/L, bean cake flour 10-20 g/L, peptone 5-15 g/L, manganese sulfate monohydrate 0.1-0.3 g/L, sodium chloride 1-3 g/L, magnesium sulfate heptahydrate 0.5-1.5 g/L, dipotassium hydrogen phosphate 1-2 g/L.
Fermentation tank medium: glucose 3-5 g/L, corn flour 20-30g/L, bean cake flour 20-30g/L, peptone 5-15 g/L, manganese sulfate monohydrate 0.1-0.3 g/L, sodium chloride 1-3 g/L, magnesium sulfate heptahydrate 0.5-1.5 g/L, and pH adjusted to 7.0.
The feed medium is formed by directly mixing a feed medium 1 and a feed medium 2 in a ratio of 1:1, wherein the feed medium 1 and the feed medium 2 are as follows:
the feed medium 1 is prepared from 200-800 g/L ethanol and sterile deionized water; the feed medium 2 is 100-200 g/L formate and 100-300 g/L acetate.
EXAMPLE 1 screening isolation and characterization of Bacillus subtilis LTB-001
1. Separation and screening of strains
(1) Under the aseptic condition, 50g of soil sample taken from a farmland planted by a potato in a county of Miao nationality of Ningyi of Pichia of Guizhou is added into an LB liquid culture medium for enrichment, and the culture is carried out at 37 ℃ and 180 r/min overnight. Placing the enriched bacterial liquid in a water bath at 80 ℃ for 20min, cooling, gradually diluting the bacterial liquid, coating the bacterial liquid on an LB solid plate, and culturing for 24h at 37 ℃.
(2) Selecting single bacteria with larger colony morphology, streaking, culturing at 37 ℃ for 24 hours, repeating the steps for 3 times to obtain pure bacterial strains, then culturing in an LB liquid medium added with 4% ethanol, culturing at 37 ℃ for 24 hours, and detecting the number of viable bacteria, wherein the total number of the bacterial strains is 79;
(3) Sequencing the strain with the highest viable count for 16s, wherein the sequencing result is shown as SEQ ID NO.1, and the classification is named as: bacillus subtilis (Bacillus subtilis), designated as Bacillus subtilis LTB-001, has been deposited at the university of Guangdong university institute of microbiology (center for analytical detection of microorganisms, guangdong province) under accession number GDMCC No.64179.
2. Physiological and biochemical characteristics
TABLE 1 physiological and biochemical characterization of Bacillus subtilis LTB-001
Note that: "+" indicates positive and "-" indicates negative
As shown in FIG. 2, the green cell body is the dyed spore, and the final spore rate of the bacillus subtilis LTB-001 is more than or equal to 95 percent. Analysis of the data in Table 1 shows that the bacillus subtilis LTB-001 can normally grow in a system containing 4% ethanol, can utilize carbon sources such as sodium formate and sodium acetate to perform physiological metabolism activities, and has the basis of fermentation by taking primary carbon and secondary carbon compounds as main carbon sources.
3. High temperature tolerance detection
The bacillus subtilis LTB-001 fermentation broth was continuously treated at room temperature (25 ℃), 80 ℃, 90 ℃ and 100 ℃ for 10 minutes, the room temperature treatment was used as a control group, each experimental group was subjected to dilution coating counting, and the survival rate was calculated after 24 hours of culture at 37 ℃, and the results were as follows:
TABLE 2 detection of Bacillus subtilis LTB-001 high temperature tolerance
As shown in Table 2, the survival rate of Bacillus subtilis LTB-001 after 10 minutes of treatment at 80℃was 99.61%, and the survival rate after 10 minutes of treatment at 100℃was as high as 90.30%. The results show that the strain has excellent heat resistance and is suitable for being applied to various fields such as agricultural bacterial agents.
4. Antibacterial property detection
The antibacterial activity of bacillus subtilis LTB-001 was tested using E.coli ATCC 25922 and Staphylococcus aureus ATCC 6538 as indicator bacteria. All strains used were from the university of Guangdong university institute of microorganisms. Specifically, after the LB solid culture medium is melted, the solid culture medium is cooled to about 45 ℃, 100 mu L of overnight cultured indicator bacteria culture solution is added into each 100mL of culture medium, and after being uniformly mixed, the mixture is poured into a sterile plate, and 15-20mL of indicator bacteria culture solution is poured into each plate. Placing 2 sterilized oxford cups after the solid culture medium in the plate is solidified, then adding 200 mu L of bacteria liquid to be tested into the oxford cups, repeating three groups of experiments, carefully transferring the plates to a 37 ℃ incubator for culturing for 24 hours after the plates are covered, and measuring the diameter of a bacteriostasis zone by using a vernier caliper, wherein the specific table is as follows:
TABLE 3 detection of antibacterial Properties of Bacillus subtilis LTB-001
Based on the results obtained in Table 3, it is known that the bacillus subtilis LTB-001 has good antibacterial capability, has excellent antibacterial performance on escherichia coli ATCC 25922 and staphylococcus aureus ATCC 6538, and is suitable for being applied to multiple fields such as agricultural bactericides.
EXAMPLE 2 fermentation of Bacillus subtilis LTB-001 in fermenter
Streaking bacillus subtilis LTB-001 on an LB plate on an ultra-clean workbench by using an inoculating loop, and activating for 12 hours at 37 ℃ until a monoclonal strain grows out;
picking the monoclonal thalli in an ultra-clean workbench by using a sterile inoculating loop, inoculating the monoclonal thalli into a Ke-shi bottle, culturing for 40 hours at 37 ℃, pouring sterile water into the Ke-shi bottle after microscopic examination of spores is mature, scraping bacterial colonies on an inclined plane by using a sterilized bamboo stick, pouring a seed culture medium, and performing aerobic culture for 8 hours at 37 ℃ to obtain a seed liquid.
Counting medium (NA medium): beef extract 3 g/L, peptone 10 g/L, sodium chloride 5 g/L, and agar 1.5-2.0%.
Activation medium (LB medium): 1.5% -2% of agar is added to the peptone 10g/L, yeast powder 5 g/L and sodium chloride 10g/L to prepare a flat plate, and the flat plate is prepared by deionized water and sterilized at 121 ℃ for 30min.
Seed culture medium: 10g/L glucose, 20 g/L corn meal, 20 g/L bean cake meal, 10g/L peptone, 0.2 g/L manganese sulfate monohydrate, 2 g/L sodium chloride, 1.0 g/L magnesium sulfate heptahydrate, and 1 g/L dipotassium hydrogen phosphate. Prepared by deionized water, fixed to 1L, sterilized at 115 ℃ for 20min.
Fermentation tank medium: glucose 3 g/L, bean cake powder 30 g/L, peptone 10 g/L, manganese sulfate monohydrate 0.2 g/L, sodium chloride 2 g/L, magnesium sulfate heptahydrate 1.0 g/L, pH to 7.0, and sterilizing at 115 deg.C for 20min with deionized water.
The feed medium is formed by directly mixing a feed medium 1 and a feed medium 2 in a ratio of 1:1, wherein the feed medium 1 and the feed medium 2 are as follows:
the feed medium 1 is ethanol 600g/L and is prepared by sterile deionized water, and the volume is fixed to 0.5L; the feed medium 2 is formate 100 g/L, acetate 200 g/L, constant volume to 0.5L, sterilizing at 121deg.C for 30min, and cooling to normal temperature.
Preparing a fermentation medium and a feed medium according to the above formula.
Inoculating 10% of seed liquid into a culture medium in a 15L fermentation tank (the total volume of the culture medium is 10L), adjusting the fermentation temperature to 37 ℃, adjusting the aeration ratio to 0.4-1.0, stirring at a rotation speed of 400-600 rpm/min, controlling the fermentation time to 36h, controlling the pH in the early stage of fermentation, starting to add a feed supplement culture medium when the thalli normally grow for 18h, and feeding at a constant speed of 1.39 mL/min for 12h until the fermentation is finished, wherein the type of feed supplement acetate is sodium acetate and the type of formate is sodium formate.
The viable bacteria counting method comprises the following steps: 1mL of 1:10 sample homogenate is sucked by a 1mL sterile pipette or a micropipette, slowly injected into a sterile test tube filled with 9mL of physiological saline along the tube wall (note that the tip of the pipette does not touch the diluent), and the test tube is shaken or repeatedly blown by replacing 1 sterile pipette to be uniformly mixed to prepare 1:10 sample homogenate.
Taking 1mL sterile pipette or micropipette tip, and performing 10-fold increment of sample homogenization according to the operation sequence, wherein 1mL sterile pipette or tip is replaced for 1 time for each increment of dilution until the dilution is 10 8.
Samples with dilution factors of 10 7 and 10 8 were selected, 1mL of sample homogenate was drawn on NA plates for each dilution, spread evenly using a sterile spreading bar, and incubated at 37 ℃ for 24h, two plates for each dilution. Dilution from sample to plate coating was required to be completed within 15 min.
And selecting a plate with the colony count of 30-300 cfu and counting the total colony count of the plate without the growth of the spread colony. Plates below 30cfu record specific colony counts, and plates above 300cfu are recordable as more irreducible. The colony count per dilution should be the average of two plates.
Example 3
The same procedure as in example 2 was followed, except that the carbon source used was 5 g/L glucose +30 g/L okara +30 g/L ethanol +5 g/L sodium formate +10 g/L sodium acetate.
Comparative example 1
The same procedure as in example 2 was followed, except that 1g/L glucose +30 g/L okara +30 g/L ethanol +5 g/L sodium formate +10 g/L sodium acetate was used as the carbon source.
Comparative example 2
The same procedure as in example 2 was followed, except that 6 g/L glucose +30 g/L okara +30 g/L ethanol +5 g/L sodium formate +10 g/L sodium acetate was used as the carbon source.
Comparative example 3
The same procedure as in example 2 was followed, except that 10 g/L glucose +50 g/L okara powder was used as the carbon source.
TABLE 4 viable count of Bacillus subtilis LTB-001 with different glucose content
As is clear from the data in Table 4 and FIG. 3, the glucose of 3-5 g/L in examples 2 and 3 can be directly used in the initial stage of bacterial growth, and the amylase secreted by the bacterial cells increases after the number of the bacterial cells increases, so that the reducing sugar formed by hydrolysis of the corn meal in the system can be used for bacterial cell growth, and the final viable count is 220 hundred million cfu. In comparative example 1, since glucose is too little in the amount of a rapid carbon source that can be directly used by the cells, the amount of amylase secreted by the cells is insufficient to rapidly hydrolyze corn flour, resulting in slow growth of the cells in the early growth stage, thereby affecting the final viable count; in comparative example 2, when more glucose was added as a carbon source for growth, the viable count of the cells was almost the same as in examples 2 and 3, and the increase in viable count could not be significantly promoted by an excessive amount of glucose; in contrast, in comparative example 3, when only glucose and corn meal were used as carbon sources, the viable count was still lower than in examples 2 and 3, because the ethanol-based mixed carbon source had a higher nutrient enrichment than the single glucose carbon source, promoting the absorption and utilization of the carbon source by the strain, and facilitating the strain growth.
Comparative example 4
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+10 g/L ethanol+5 g/L sodium formate+10 g/L sodium acetate.
Comparative example 5
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+40 g/L ethanol+5 g/L sodium formate+10 g/L sodium acetate.
Comparative example 6
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+60 g/L ethanol+5 g/L sodium formate+10 g/L sodium acetate.
TABLE 5 viable count of Bacillus subtilis LTB-001 fermented with different ethanol contents
As can be seen from the data in Table 5, the viable count is at a higher level when 10-40 g/L of ethanol is present in the system. This is because bacteria begin to sporulate by their self-protection mechanism when nutrients in the system are insufficient in the middle and late phases of the bacillus subtilis fermentation, and ethanol at a certain concentration can change the composition of phospholipids and fatty acids. In bacillus subtilis, phospholipids account for 10-15% of the dry weight of the cytoplasmic membrane, the only membrane structure in cells. Therefore, ethanol with a certain concentration can inhibit the formation of spores, and sodium formate and sodium acetate are added as carbon sources to provide bacterial growth, so that the bacteria can continuously reproduce and a higher viable count is obtained. In comparative example 6, an excessive amount of ethanol destroyed the cell membrane structure and function of the cells, resulting in a blocked metabolism of central carbon, outflow of intracellular substances or cell lysis, and the growth of the cells was significantly inhibited.
Comparative example 7
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+2 g/L sodium formate+10 g/L sodium acetate.
Comparative example 8
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+10 g/L sodium formate+10 g/L sodium acetate.
Comparative example 9
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+15 g/L sodium formate+10 g/L sodium acetate.
TABLE 6 viable count of Bacillus subtilis LTB-001 with different formate contents
As can be seen from the data in Table 6, the viable count of Bacillus subtilis was at a higher level when 5-10 g/L of sodium formate was present in the system. Sodium formate is finally converted by formate dehydrogenase, NAD+ is reduced to form NADH in the conversion process, and NADH participates in a plurality of metabolic processes of the growth of bacillus subtilis and provides reducing force to promote the growth of the strain. In comparative example 7, the formate was absent, so that the reduction power in the cells was insufficient, the metabolic capacity of the strain was lowered, and the number of viable bacteria was decreased. In comparative example 9, however, the excessive formate salt inhibited the growth of the strain due to its high osmotic pressure and formate tolerance, so that the selection of an appropriate formate salt concentration could promote the growth of the viable count of the strain.
Comparative example 10
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+5 g/L sodium formate+2 g/L sodium acetate.
Comparative example 11
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+5 g/L sodium formate+15 g/L sodium acetate.
Comparative example 12
The same procedure as in example 2 was followed, except that the carbon source used was 3 g/L glucose+30 g/L okara+30 g/L ethanol+5 g/L sodium formate+20 g/L sodium acetate.
TABLE 7 viable count of Bacillus subtilis LTB-001 with different acetate content
As can be seen from the data in Table 7, the viable count is at a higher level when an appropriate amount of sodium acetate is present in the system. Acetate acts on acetyl kinase to form acetyl phosphate, which can participate in the synthesis of acetyl-CoA as an intermediate product, and acetyl-CoA will directly participate in the isocenter metabolic process of tricarboxylic acid cycle, thereby providing energy for strain growth. In comparative example 10, when glucose and corn meal are consumed in the system, the energy substance required for the growth of the strain is lost due to the absence of acetate, and the added formate can provide reducing power for the growth of the strain but is difficult to directly provide the energy required for the growth of the strain, resulting in the failure of the strain to grow normally. In comparative example 12, excessive acetate inhibited the growth of the strain due to its high osmotic pressure and acetate tolerance, so that the selection of an appropriate acetate concentration could promote the growth of the viable count of the strain.
Comparative example 13
The same procedure as in example 2 was followed, except that the feeding time point was 16h after the start of fermentation.
Comparative example 14
The same procedure as in example 2 was followed, except that the feeding time point was 20h after the start of fermentation.
TABLE 8 viable count of Bacillus subtilis LTB-001 at different feed time points
As can be seen from the data in Table 8, when the feeding time point is earlier, the bacterial cells are in the late stage of logarithmic growth, and the addition of formate and acetate at high concentration in the feeding medium causes the pH in the system to rise, and the rise of pH promotes bacillus subtilis to form spores in advance to lose reproductive capacity, so that the number of viable bacteria is reduced. When the feeding time point is later, as nutrient substances in the system are depleted, partial thalli are converted into spore forms, and the feeding culture medium is added at the moment, the spores cannot be converted into the thallus forms to continue to grow and propagate, so that the number of viable bacteria is not high. Therefore, the feed medium is added at the time point when the bacterial body gradually changes into the spore, so that the formation of the spore can be inhibited, and meanwhile, nutrients are given to the bacterial body to enable the bacterial body to grow and reproduce normally, so that a higher viable count is obtained, and the time point is very critical.
Comparative example 15
The same procedure as in example 2 was followed, except that the feed rate was 1.19 mL/min.
Comparative example 16
The same procedure as in example 2 was followed, except that the feed rate was 1.67 mL/min.
TABLE 9 viable count of Bacillus subtilis LTB-001 at different feed rates
As can be seen from the data in Table 9, when the feed rate of the feed medium was slow, the amount of the mixed carbon source added was small, and a small amount of ethanol was insufficient to inhibit the transformation of bacterial cells into spores, while a small amount of formate and acetate did not satisfy the normal growth of bacterial cells, resulting in a low viable count. When the feeding speed of the feeding medium is high, excessive acetate and formate can lead to high pH of the culture system, leading thalli to form spores in advance and lose reproductive capacity, and leading the number of viable bacteria to be low.
Example 4 field experiments with Bacillus subtilis LTB-001
The bacillus subtilis LTB-001 bacterial obtained in the example 2 is prepared into a microbial agricultural microbial inoculum by conventional compounding and is applied to a potato planting field in Zhanjiang. The experiment uses agricultural microbial agents prepared from bacillus subtilis LTB-001 as a raw material as an experiment group, and uses the agricultural microbial agents of the bacillus curdlan and the bacillus jungdeh potato which are conventionally purchased in the market as a control group 1 and a control group 2. The experiment period is 60 days, the same farm administrator manages the planting and raising, each treatment of the experiment is divided into 3 repetitions of each treatment of 30 mu of land, and each repetition of each 10 mu of land is used for guaranteeing the accuracy of the experiment.
Table 10 application of bacillus subtilis LTB-001 in potato planting
As can be seen from the results in table 10, the agricultural microbial inoculum treatment made from the bacillus subtilis LTB-001 as a raw material has a significant effect on the weight of the lower monolith, which is significantly increased by 34.5% compared to control group 1, by 31.1% compared to control group 2, and other indexes have no significant difference between treatment and control (p < 0.05). The results show that the bacillus subtilis LTB-001 has excellent potential for preparing the agricultural microbial inoculum.
By combining the physiological and biochemical characteristics, metabolic pathway analysis and experimental data of the bacillus subtilis LTB-001, the bacillus subtilis LTB-001 strain can obtain higher viable count through the fermentation process in a mixed carbon source mainly comprising ethanol, formate and acetate. The fermentation process using the mixed carbon source mainly comprising ethanol, formate and acetate has high efficiency and innovation in reducing the carbon source of grains and fermenting bacillus subtilis.
All of the above-described primary implementations of this intellectual property are not intended to limit other forms of implementing this new product and/or new method. Those skilled in the art will utilize this important information and the above modifications to achieve a similar implementation. But all modifications or adaptations belong to the reserved rights based on the new products of the invention.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
The 16s nucleotide sequence of the bacillus subtilis is shown in SEQ ID NO. 1:
AGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG.
Claims (9)
1. A bacillus subtilis suitable for use as a non-grain bio-based carbon source, characterized in that: the bacillus subtilis is named as LTB-001, and the preservation number is GDMCC No:64179.
2. The bacillus subtilis suitable for use with non-grain biobased carbon sources of claim 1, wherein: the nucleotide sequence of the bacillus subtilis is shown as SEQ ID NO. 1.
3. Use of a non-grain bio-based carbon source in a bacillus subtilis fermentation production process according to claim 1 or 2, characterized in that: the carbon source comprises ethanol, formate and acetate, wherein the content of the ethanol in the fermentation broth is 10-40 g/L, the concentration of the formate is 5-10 g/L, and the concentration of the acetate in the fermentation broth is 5-15 g/L.
4. A use according to claim 3, wherein: the carbon source also comprises glucose and corn flour, wherein the concentration of the glucose in the fermentation liquor is 3-5g/L, and the concentration of the corn flour in the fermentation liquor is 20-30g/L.
5. Fermentation medium of bacillus subtilis for use with non-grain bio-based carbon sources according to claim 1 or 2, characterized in that: comprising the non-grain bio-based carbon source according to claim 3 or 4, 20-30 g/L of bean cake powder, 5-15 g/L of peptone, 0.1-0.3 g/L of manganese sulfate monohydrate, 1-3 g/L of sodium chloride, 0.5-1.5 g/L of magnesium sulfate heptahydrate, adjusting the pH to 7, and sterilizing at 121 ℃ for 30min.
6. The fermentation production method of bacillus subtilis applicable to non-grain bio-based carbon sources according to claim 1 or 2, characterized by comprising:
inoculating the seed liquid into a culture medium in a fermentation tank according to 5-10%, wherein the fermentation temperature is 35-37 ℃, the fermentation time is 24-48 h, and feeding a feed supplement culture medium when the thalli grow normally for 18h until the fermentation is finished;
the culture medium in the fermentation tank is glucose 3-5 g/L, bean cake powder 20-30g/L, peptone 5-15 g/L, manganese sulfate monohydrate 0.1-0.3 g/L, sodium chloride 1-3 g/L, magnesium sulfate heptahydrate 0.5-1.5 g/L, which are prepared by deionized water, the volume is fixed to 9L, and the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to normal temperature for use;
The feed medium is formed by directly mixing a feed medium 1 and a feed medium 2 in a ratio of 1:1, wherein the feed medium 1 and the feed medium 2 are as follows:
The feed supplement culture medium 1 is prepared from 200-800 g/L of ethanol by using sterile water, and the volume is fixed to 0.5L; the feed medium 2 is 100-200 g/L formate and 100-300 g/L acetate, the volume is fixed to 0.5L by deionized water, and the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to normal temperature for use.
7. A microbial agent is characterized in that: the bacillus subtilis applicable to the non-grain bio-based carbon source according to claim 1 or 2, wherein the viable count is more than or equal to 1000 hundred million cfu/g.
8. A microbial fertilizer, which is characterized in that: a bacillus subtilis comprising the suitable non-grain biobased carbon source according to claim 1 or 2.
9. Use of the bacillus subtilis according to claim 1 or 2, the microbial agent according to claim 7, or the microbial fertilizer according to claim 8 in agricultural production.
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