CN110616177A

CN110616177A - Bacillus with high fermentation density and fermentation production method thereof

Info

Publication number: CN110616177A
Application number: CN201911039740.5A
Authority: CN
Inventors: 杨春玉; 杨舟; 李珊楠
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2019-12-27
Anticipated expiration: 2039-10-29
Also published as: CN110616177B

Abstract

The invention discloses a Bacillus with high fermentation density, which is named as Bacillus (Bacillus sp.)19-B and is determined to be Bacillus megaterium with small spore morphology and high fermentation density, and the strain is preserved in China center for type culture Collection (CGTCC No. M208187) at 29 th 10 th 2008. The invention also discloses a culture medium suitable for high-density fermentation culture of the bacillus and a fermentation control mode. Experiments prove that the bacillus effectively solves the problems of low fermentation density and long fermentation period of the existing bacillus, and meanwhile, the biomass of the bacillus 19-B is improved by 4-5 times within 20 hours by using the fermentation medium, and the highest biomass reaches 150 hundred million/ml. The strain is indicated to have wide application prospect in the aspects of preparing microbial agents and producing the microbial agents.

Description

Bacillus with high fermentation density and fermentation production method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bacillus with small spore form and high fermentation density, and a fermentation production method and application thereof.

Background

Bacillus (Bacillus sp.) is a gram-positive bacterium belonging to the genus Bacillus (Bacillus). It is capable of forming spores, the radiation resistance of which is 36 times that of Escherichia coli. Wherein the bacillus megaterium is gram-positive bacteria and aerobic bacteria,industrially used for producing glucose isomerase and VB₁₂And is also a famous phosphate solubilizing bacterium. Therefore, the fertilizer can be used for manufacturing phosphorus bacterial fertilizer in agriculture and has good fertilizer efficiency. The specific efficacy of Bacillus megaterium is as follows: 1) the bacillus megaterium is a phosphate-solubilizing potassium-promoting bacterium, can decompose adsorbed organic phosphorus and inorganic phosphorus in soil, improve soil fertility and promote crop yield increase; 2) as a main biocontrol bacterium for biological control of plant diseases, bacillus megaterium hinders or kills pathogenic bacteria in a direct or indirect manner by producing various antagonistic or competitive metabolites during growth and development; 3) the bacillus megaterium can improve the livestock breeding environment, change waste into valuable and improve the livestock breeding benefit; 4) the bacillus megaterium has a certain effect on water purification, can be developed into a water quality microecological preparation to be applied to a water source deficient culture area, reduces the culture cost and improves the culture benefit; 5) the bacillus megaterium is an excellent host for heterologous expression, and compared with the traditional prokaryotic expression systems (such as an escherichia coli expression system and a bacillus subtilis expression system), the system has strong foreign protein secretion capacity and does not produce endotoxin; the vector plasmid is stable and produces no or little extracellular protease; the extracellular protein is easy to extract and the extraction cost is low; the inducer used is cheap, and the like. Compared with a mammalian expression system, the method has the advantages of no problems of long culture time, difficult control of mycoplasma and virus pollution and the like.

Currently, common microbial agents include bacillus subtilis, bacillus megaterium, bacillus thuringiensis, bacillus licheniformis and the like. Compared with other bacillus, the bacillus megaterium has the defects of low fermentation bacteria number and high production cost, thereby greatly limiting the popularization and application of the strains in the field of microbial agents. The number of the fermentation bacteria of the general bacillus megaterium is between 20 and 60cfu/ml of fermentation liquor, such as Ben and the like, single factor test, PB design and response surface method are applied for optimization, and finally 42.2 multiplied by 10 is obtained by 48 hours of fermentation⁸The number of bacteria cfu/ml; the bacterial count of Chenkai and the like reaches 60 multiplied by 10 after 32 hours of culture by optimizing the fermentation formula of the bacillus megaterium P1⁸cfu/mL, the ratio of spores is more than 90%. Cause bacillus megaterium to fermentThe main reason for the number limitation is that the size of the strain is large, so that a bacillus megaterium strain with small form and high biomass is screened, and a fermentation formula with high growth speed, high biomass, high spore rate and low cost is further developed, so that the bacillus megaterium strain has important value and potential for large-scale application in the field of microbial agents.

Disclosure of Invention

Aiming at the bottleneck that the existing bacillus megaterium is low in fermentation density and high in production cost, the invention aims to provide the bacillus megaterium which is small in spore form, fast in growth, high in biomass and high in spore yield and a related large-scale fermentation production process.

The bacillus of the invention is characterized in that: the strain is named as Bacillus (Bacillus sp.)19-B, is determined to be Bacillus megaterium with small spore morphology and high fermentation density, is preserved in China Center for Type Culture Collection (CCTCC) No. M208187 at 29.10.2008. The preservation address is Wuhan in China.

Specifically, the Bacillus sp 19-B cell is rod-shaped and rounded at the end. The single or chain-shaped spores are arranged in the initial stage, the size of the spores is 2.5-3.0 multiplied by 1.5 microns, the movement is realized, the gram-positive spores can be produced, the spores are grown in the middle, and the size of the spores is obviously reduced (1.2 multiplied by 0.8 microns). The bacterial colony grows well in a common nutrient agar culture medium, the bacterial colony on the medium is round, the edge is neat and flat, the middle part is slightly provided with a small bulge, the medium is yellow, the surface is in a frosted glass shape, the surface is smooth and bright after the medium is placed for a period of time at 4 ℃, and the diameter of the bacterial colony after overnight culture is 3-5 mm.

The growth temperature of the Bacillus (Bacillus sp.)19-B is 25-42 ℃, and the optimal growth temperature is 37 ℃.

The above-mentioned Bacillus (Bacillus sp.)19-B may be grown on a minimal medium, a complete medium or a fermentation medium.

Wherein the formula of the basic culture medium is as follows, 1L of distilled water contains: 10.8 g of dipotassium hydrogen phosphate, 5.99 g of monopotassium phosphate, 1.98 g of ammonium sulfate, 1.91 g of trisodium citrate dihydrate, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of casamino acid, 0.08 g of manganese chloride tetrahydrate, 0.106 g of calcium chloride dihydrate, 0.005 g of ferric sulfate heptahydrate, 0.004 g of ammonium molybdate, 0.0022 g of cobalt chloride, 10 g of glucose, pH 7.5, and sterilization is carried out at 115 ℃ for 30 minutes.

Wherein the complete culture medium has the following formula, and 1 liter of distilled water contains: 10 g of glucose, 10 g of peptone, 5g of yeast powder, 5g of sodium chloride, 10 g of beef extract, pH 7.5, adding agar powder with the mass-volume ratio of 1.8% into the solid complete culture medium on the basis of the formula, and sterilizing at 115 ℃ for 30 minutes.

A culture medium suitable for high-density fermentation culture of the bacillus is characterized in that: the formula of the culture medium for fermentation culture is as follows: 1 liter of distilled water contains: 20 grams of glucose, 10 grams of corn meal, 20 grams of yeast powder, 2 grams of sodium chloride, 0.2 grams of manganese sulfate monohydrate, 0.5 grams of magnesium sulfate heptahydrate, 1.3 grams of dipotassium phosphate trihydrate, 0.03 grams of ferric chloride; sterilizing at 115 deg.C for 30 min at pH of 8.0-8.5.

The method for high-density fermentation culture of the bacillus is characterized in that the fermentation control mode of the bacillus is as follows: initial parameters of the fermentation tank are set to be 37 ℃, pH 7.0 and initial rotation speed of 400 rpm; sampling every 4 hours to determine OD_600nmValue, microscopic examination; defoaming agent is added in the fermentation process to control bubbles; when the bacteria grow to the logarithmic phase, the rotating speed is increased to 800rpm, NaOH is added to adjust the pH to be more than 7.0 if the pH is less than 7.0, HCl is added to adjust the pH to be 7.0 +/-0.1 if the pH is more than 7.0, and the bacteria are cultured in a fermentation medium for 15 to 20 hours.

The 16S rDNA of the Bacillus (Bacillus sp.)19-B CCTCC No. M208187 is extracted by a conventional method, wherein the nucleotide sequence of the 16S rDNA of the Bacillus (Bacillus sp.)19-B strain is shown as SEQ ID No. 1. And by consulting the international related gene library in the National Center for Biotechnology information, NCBI, the gene sequence of the Bacillus (Bacillus sp.)19-BCCTCC No. M20818716S rDNA of the invention is compared with the 16S rDNA gene sequence of the related strain, and the Bacillus (Bacillus sp.)19-B is determined to be Bacillus megaterium, and the gene sequences have similarity but are not completely the same, so that the strain is firstly separated and identified and has originality.

The invention relates to application of Bacillus (Bacillus sp.)19-B with high fermentation density in preparation of a microbial agent.

The Bacillus (Bacillus sp.)19-B is cultured in a 5L fermentation tank for 15-20 hours at 37 ℃ by using the fermentation medium disclosed above, and the final spore number can reach 150 hundred million/ml. The obtained bacillus can be widely used for preparing microbial agents.

The invention discloses a Bacillus megaterium which is small in spore form and can reach high fermentation bacteria number in a short time, the problems of low fermentation density and long fermentation period of the existing Bacillus are effectively solved, and meanwhile, the biomass of the Bacillus (Bacillus sp) 19-B is increased by several times by using a novel optimized fermentation culture medium, and the highest biomass reaches 150 hundred million/ml. Compared with about 30 hundred million/ml of fermentation bacteria of the currently used bacillus, the fermentation cost can be saved by about 4-5 times. The bacillus strain has wide application prospect in the production aspect of preparing microbial agents.

Drawings

The Bacillus (Bacillus sp.)19-B is preserved in China Center for Type Culture Collection (CCTCC) at 29.10.2008, and the preservation number is CCTCC No. M208187.

FIG. 1 scanning electron micrograph of Bacillus (Bacillus sp.)19-B and the production strain Bacillus megaterium SC-1 in the fermentation medium.

FIG. 2 is a tree diagram of the evolution constructed by aligning the Bacillus (Bacillus sp.)19-B genome with the other four Bacillus megaterium sequences.

FIG. 3 shows the growth density (OD) of Bacillus sp 19-B and Bacillus megaterium SC-1 in a fermentation medium at 37 ℃ for 24 hours_600nm) Graph with growth time.

FIG. 4 is a graph showing inorganic phosphorus decomposing effect of Bacillus sp 19-B.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the present invention in any way, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention fall within the scope of the technical solution of the present invention.

Example 1: screening and physiological and biochemical characteristics of Bacillus (Bacillus sp.)19-B CCTCC No. M208187 strain

Coating a liquid sample in a papermaking black liquor pool on a complete culture medium agar plate, inverting the plate and culturing for 24 hours at 37 ℃, picking a yellow round colony with a rough surface, examining the colony by a microscope to obtain Bacillus, repeatedly marking to determine a pure colony, inoculating the pure colony to the complete culture medium, observing an individual shape by a microscope, carrying out biochemical reaction determination, and finally screening to obtain a strain of Bacillus, namely Bacillus (Bacillus sp.)19-B (see figure 1).

The Bacillus (Bacillus sp.)19-B strain is a gram-positive bacterium, aerobic and single or chain-shaped. The size of the thallus at the initial growth stage is 2.5-3.0 multiplied by 1.5 microns, the thallus can move, is gram positive, can produce oval spores, is grown in spores, is 1.2 multiplied by 0.8 microns, and is obviously smaller than the size of the spores of the production strain SC-1 (figure 1). The bacterial colony grows well in a common nutrient agar culture medium, the bacterial colony on the bacterial colony is round, the edge is neat and flat, and the middle part of the bacterial colony is slightly provided with a small bulge, and the surface is yellow and has a ground glass shape.

The screened strain Bacillus (Bacillus sp.)19-B is preserved in China Center for Type Culture Collection (CCTCC) at 29.10.2008, and the preservation number is CCTCC No. M208187.

Example 2: extraction of 16S rDNA gene of Bacillus (Bacillus sp.)19-B strain

5ml of the bacterial culture was cultured to saturation, 1ml of the culture was centrifuged at 12000rpm for 1min, and the supernatant was removed. Adding 180. mu.l of TE buffer into the thallus, adding a proper amount of lysozyme, and treating for more than 30 minutes at 37 ℃. Add 20. mu.l proteinase K solution to the tube and mix well. Adding 220 μ l buffer solution GB, shaking for 15 s, standing at 70 deg.C for 10 min, cleaning the solution, centrifuging briefly to remove the solution on the inner wall of the tube cover, and mixing. Add 220. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove the inner wall solution of the tube cap. Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm for 30 s, pouring off waste liquid, and placing the adsorption column into the collecting pipe. To the adsorption column CB3, 500. mu.l of buffer GD (check for the addition of absolute ethanol before use) was added, and centrifuged at 12000rpm for 30 seconds to discard the waste liquid, and the adsorption column was placed in a collection tube. 600. mu.l of a rinsing solution PW (previously used, whether or not absolute ethanol was added) was added to the adsorption column CB3, and the column was centrifuged at 12000rpm for 30 seconds to discard the waste liquid, and the column was placed in the collection tube. Then 600. mu.l of the rinsing solution PW was added to the adsorption column CB3, and the column was centrifuged at 12000rpm for 30 seconds to discard the waste liquid, and the column was put into the collection tube. The adsorption column CB3 was returned to the collection tube, centrifuged at 12000rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the rinsing liquid remaining in the adsorption column. Transfer of adsorption column CB3 intoSuspending and dropwise adding 50-100 mu l ddH into the middle part of the adsorption film in a clean centrifugal tube₂And O, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm for two minutes, and collecting the eluent into a centrifugal tube. Using the extracted genome DNA as a template, carrying out PCR amplification by using bacterial primers 27F and 1492R purchased from Shanghai Dagong Biotech Co., Ltd, and mixing the following reagents in a 100. mu.l reaction system in sequence: 76 mu l H₂O, 10. mu.l of 10-fold concentration PCR reaction buffer, 1. mu.l of the upstream primer, 1. mu.l of the downstream primer, 1. mu.l of the template DNA, i.e., the above genomic DNA, and 1. mu.l of TaqDNA polymerase were mixed and centrifuged for 5 seconds. The mixture was heated at 94 ℃ for 5 minutes. Denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, and extension at 72 ℃ for 2 min, for a total of 25 cycles. After the last cycle, the reaction mixture was allowed to amplify by incubation at 72 ℃ for 10 minutes. Sequencing the PCR reaction product.

Obtaining a sequencing result: the 16S rDNA nucleotide sequence of the Bacillus (Bacillus sp.)19-B strain is shown as SEQ ID No. 1.

By referring to an internationally relevant gene library in the National Center for Biotechnology Information (NCBI), the gene sequence of the Bacillus (Bacillus sp.)19-BCCTCC No. M20818716S rDNA of the invention is compared with the gene sequence of the 16S rDNA of a relevant strain, and the Bacillus (Bacillus sp.)19-B is determined to be Bacillus megaterium, and the gene sequences have similarity but are not completely the same, so that the strain is firstly separated and identified and has originality.

Further alignment was performed by the software using the BLASTN program at the U.S. center for bioengineering information and Vector NTI software was used to construct a phylogenetic tree for the given sequence (see FIG. 2).

Example 3: bacillus (Bacillus sp.)19-B strain fermentation medium optimization

Introduction to orthogonal design methods: the orthogonal test design is carried out by utilizing the orthogonal table, so that the defects of the arrangement test of the single-factor alternation method are completely overcome, the most main factors influencing the quality can be selected, and the further test is convenient. Through the experiment of four factors and three levels, the influence of the nitrogen source on the thalli is the largest. Further PB experiments were performed on nitrogen sources.

The PB experiment is utilized to optimize a carbon source, a nitrogen source and the like of a culture medium, and the Plackett-Burman (PB) design is an efficient method often used for formula optimization. We used the numerous medium components to select those components that have a significant effect on fermentation, and those components that are not important can be ignored in further experiments. A PB containing 12 experiments was designed to screen out important factors from 6 factors and to process the resulting data using the least squares method, fitting an equation of WhereinIs the predicted product yield, beta₀、β₁、β₂、β₃、β₄、β₅、β₆For coefficients of fit, X₁、X₂、X₃、X₄、X₅、X₆Is the level of the independent variable.

The conclusion is drawn through experimental data, so far, the optimized fermentation medium of the bacillus is obtained: 1 liter of distilled water contains: 20 grams of glucose, 10 grams of corn meal, 20 grams of yeast powder, 2 grams of sodium chloride, 0.2 grams of manganese sulfate monohydrate, 0.5 grams of magnesium sulfate heptahydrate, 1.3 grams of dipotassium phosphate trihydrate, 0.03 grams of ferric chloride; sterilizing at 115 deg.C for 30 min at pH of 8.0-8.5.

Example 4: fermentation growth curves of the Bacillus (Bacillus sp.)19-B strain and the producer strain Bacillus megaterium SC-1 in a fermentation medium.

Bacillus (Bacillus sp.)19-B and the commercially available production strain Bacillus megaterium SC-1 were cultured using MM medium and the optimized fermentation medium of the invention.

The seeds were cultured in LB medium at 170rpm, 37 ℃ for 5-8 hours. Inoculating into fermentation medium and MM according to inoculum size of 5%And (5) nutrient base. The culture was carried out at 200rpm, 37 ℃ for 30 hours. Sampling every 4 hours to determine OD_600nmAnd at a spore rate>Samples were taken after 95% dilution series and plated to count.

Diluting fermentation medium fermentation liquor (growing for 30 hours) by 10⁷、10⁸、10⁹Doubling and applying 100. mu.l to the plate. The plates were incubated in a 37 ℃ incubator for one day and counted. Found to be diluted to 10⁸Most preferably, the colony number on the parallel three plates is 44, 40 and 42, and the average thallus number of the fermentation liquor reaches 42 hundred million/mL. While the average number of the producing strain Bacillus megaterium SC-1 counted by serial dilution was only 9.7 hundred million/ml (Table 1).

MM medium (24 h of growth) was diluted 10⁶、10⁷、10⁸Doubling and applying 100. mu.l to the plate. The plates were incubated in a 37 ℃ incubator for one day and counted. Found to be diluted to 10⁷Most suitably, the colony number on three parallel plates is 14, 15 and 16, and the thallus number of the fermentation liquor reaches 15 hundred million/mL. While the average number of the producing strain Bacillus megaterium SC-1 counted by serial dilution was only 4.3 hundred million/ml (Table 1).

Table 1: results of the enumeration of the fermentation medium and MM medium for Bacillus (Bacillus sp.)19-B and the production strain Bacillus megaterium SC-1

Example 5: effect of Bacillus sp 19-B and producer strain Bacillus megaterium SC-1 on decomposing inorganic phosphorus

Slant activation, transferring Bacillus (Bacillus sp.)19-B and producing strain Bacillus megaterium SC-1 into sterilized LB liquid culture medium shake tube, placing in 37 deg.C constant temperature incubator for activation culture for 24 hr, serial dilution and coating LB solid culture medium plate count. 1ml of 19-B and 0.28ml of SC-1 bacterial solution (equal bacterial count) were aspirated and transferred to a 500ml triangular flask containing 50ml of inorganic phosphorus liquid medium, with 3 parallel strains. The control was inoculated with 1ml of LB liquid medium without inoculating any bacteria. After incubation for 4 days at 37 ℃ in a shaker, samples were taken every 12 hours and centrifuged, and the available phosphorus content of the supernatant was determined by molybdenum blue colorimetry (FIG. 4). Comparing the inorganic phosphorus dissolving capacity of the bacillus 19-B and the SC-1 shows that the inorganic phosphorus dissolving capacity of the bacillus 19-B is obviously higher than that of the SC-1 under the same inoculation amount.

The formula of the inorganic phosphorus liquid culture medium comprises the following components: glucose l0 g, (NH)₄)₂SO₄0.5g, yeast powder 0.5g, MgSO₄·7H₂0.3g of O, 0.3g of sodium chloride, 0.3g of potassium chloride and FeSO₄·7H₂O 0.03g，MnSO₄·7H₂O 0.03g，Ca₃(PO₄)₂5g, distilled water 1000ml, pH 7.2, sterilized at 115 ℃ for 20 min.

Example 6: the fermentation process of the Bacillus (Bacillus sp.)19-B strain is determined (taking a 3L fermentation tank as an example) and the biomass is determined.

The optimized fermentation process of the Bacillus (Bacillus sp.)19-B strain comprises the following steps:

(1) first-order seed culture: the above strains were inoculated in a complete medium at an inoculum size of 5%. Because the growth speed of the bacillus in the later period is high, the culture time of the seeds is only 5-8 hours to ensure the vitality of the seeds, 170rpm, 37 ℃.

(2) And (3) secondary shake flask fermentation seed culture: inoculating the first-stage seed liquid into a shake flask filled with 100ml of second-stage fermentation liquid according to the inoculation amount of 5%. 170rpm, 37 ℃ and 5-8 hours of culture time.

(3) Controlling the fermentation process: 5-10% of inoculation amount, and performing microscopic examination on seeds before inoculation. Initial parameters of the fermentation tank are set to be 37 ℃, pH 7.0 and initial rotation speed of 400 rpm; sampling every 4 hours to determine OD_600nmValue, microscopic examination; defoaming agent is added in the fermentation process to control bubbles; when the bacteria grow to the logarithmic phase, the rotating speed is increased to 800rpm, NaOH is added to adjust the pH to be more than 7.0 if the pH is lower than 7.0, HCl is added to adjust the pH to be 7.0 +/-0.1 if the pH is higher than 7.0, and the bacteria are cultured in a fermentation medium for 24 hours.

The optimized fermentation medium is used for culturing the thalli to obtain a growth curve of the bacillus (see figure 3), and the growth curve shows that the bacillus enters a logarithmic phase after being cultured for 7.5 hours, the reproduction speed of the thalli is fast in a stage of culturing for 10-20 hours, the thalli are in a vigorous splitting phase, the number of the thalli is rapidly increased, and then the number of the thalli is kept stable in the stage of entering a stable phase.

The formula of the fermentation medium is as follows: 20 grams of glucose, 10 grams of corn meal, 20 grams of yeast powder, 2 grams of sodium chloride, 0.2 grams of manganese sulfate monohydrate, 0.5 grams of magnesium sulfate heptahydrate, 1.3 grams of dipotassium phosphate trihydrate, 0.03 grams of ferric chloride; sterilizing at 115 deg.C for 30 min at pH of 8.0-8.5.

The measure of the microbial agent is the number of spores. By plate counting, it can be seen that: diluting 10 fermentation medium fermentation liquor (growing for 30 hours) which completely produces spores⁷、10⁸、10⁹Doubling and applying 100. mu.l to the plate. The plates were incubated in a 37 ℃ incubator for one day and counted. Found to be diluted to 10⁸Most suitably, the number of colonies on the three parallel plates is 14, 16 and 17, and the number of thalli of fermentation liquor reaches 150 hundred million/mL.

Sequence listing

<110> Shandong university

<120> bacillus with high fermentation density and fermentation production method thereof

<141> 2019-10-24

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> Bacillus (Bacillus sp)

<221> 16S rDNA sequence of Bacillus (Bacillus sp) 19-B Strain

<400> 1

tgcggggggg gtgcctatac atgcagtcga gcgaactgat tagaagcttg cttctatgac 60

gttagcggcg gacgggtgag taacacgtgg gcaacctgcc tgtaagactg ggataacttc 120

gggaaaccga agctaatacc ggataggatc ttctccttca tgggagatga ttgaaagatg 180

gtttcggcta tcacttacag atgggcccgc ggtgcattag ctagttggtg aggtaacggc 240

tcaccaaggc aacgatgcat agccgacctg agagggtgat cggccacact gggactgaga 300

cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360

gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg 420

aagaacaagt acgagagtaa ctgctcgtac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540

gcgtaaagcg cgcgcaggcg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagaga aaagcggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcggctt tttggtctgt 720

aactgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagctaac 840

gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca actctagaga tagagcgttc cccttcgggg gacagagtga 1020

caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080

agcgcaaccc ttgatcttag ttgccagcat ttagttgggc actctaaggt gactgccggt 1140

gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200

cacacgtgct acaatggatg gtacaaaggg ctgcaagacc gcgaggtcaa gccaatccca 1260

taaaaccatt ctcagttcgg attgtaggct gcaactcgcc tacatgaagc tggaatcgct 1320

agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380

tcacaccacg agagtttgta acacccgaag tcggtggagt aaccgtaagg agctagccgc 1440

taagtgccgg cgctg 1455

Claims

1. A bacillus with high fermentation density is characterized in that: the strain is named as Bacillus (Bacillus sp.)19-B, is determined to be Bacillus megaterium with small spore morphology and high fermentation density, is preserved in China Center for Type Culture Collection (CCTCC) No. M208187 at 29.10.2008.

2. A medium suitable for high-density fermentation culture of Bacillus of claim 1, wherein: the formula of the culture medium for fermentation culture is as follows: 1 liter of distilled water contains: 20 grams of glucose, 10 grams of corn meal, 20 grams of yeast powder, 2 grams of sodium chloride, 0.2 grams of manganese sulfate monohydrate, 0.5 grams of magnesium sulfate heptahydrate, 1.3 grams of dipotassium phosphate trihydrate, 0.03 grams of ferric chloride; sterilizing at 115 deg.C for 30 min at pH of 8.0-8.5.

3. A fermentation process adapted for the Bacillus of claim 1, wherein the Bacillus is controlled by: initial parameters of the fermentation tank are set to be 37 ℃, pH 7.0 and initial rotation speed of 400 rpm; sampling every 4 hours to determine OD_600nmValue, microscopic examination; defoaming agent is added in the fermentation process to control bubbles; when the bacteria grow to the logarithmic phase, the rotating speed is increased to 800rpm, NaOH is added to adjust the pH to be more than 7.0 if the pH is lower than 7.0, HCl is added to adjust the pH to be 7.0 +/-0.1 if the pH is higher than 7.0, and the bacteria are cultured in a fermentation medium for 15 to 20 hours.

4. Use of a bacillus having a high fermentation density according to claim 1 for the preparation of a microbial agent.