CN106167771B - Bacillus megaterium D122, microbial inoculum thereof and preparation method of microbial inoculum - Google Patents
Bacillus megaterium D122, microbial inoculum thereof and preparation method of microbial inoculum Download PDFInfo
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Abstract
The invention relates to the technical field of microbial agents, and particularly relates to bacillus megaterium D122, a microbial agent thereof and a preparation method of the microbial agent, wherein the bacillus megaterium D122 is preserved in China center for type culture Collection with the preservation number of CCTCCM 2015751. The preparation method of the microbial inoculum comprises the following steps: (1) separating and screening; (2) purifying and storing; (3) culturing in a culture medium; (4) and D122 microbial inoculum is prepared to obtain the Bacillus megaterium D122 microbial inoculum. The bacillus megaterium D122 has the nitrogen-fixing enzyme activity and the function of secreting auxin indole acetic acid.
Description
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to bacillus megaterium D122, a microbial agent thereof and a preparation method of the microbial agent.
Background
Agricultural production relies on chemical fertilizers for a long time, so that a large amount of non-renewable resources are wasted, the problems of poor soil quality, reduced quality of agricultural products, environmental pollution and the like are caused, and the healthy survival of human beings is greatly influenced.
Bacillus megaterium (B.)Bacillus megaterium) The phosphorus-dissolving potassium-promoting nitrogen-fixing bacteria can well degrade phosphorus and potassium which cannot be utilized by plants in soil, improve soil fertility, fix nitrogen in air and promote crop yield increase. However, products produced in the microbial fertilizer industry in China show many problems, such as low effective viable count, high mixed bacteria rate, short effective period and the like, and the enthusiasm of farmers for use and the development of markets are seriously influenced. Domestic researches on the microbial fertilizer of the bacillus megaterium mainly focus on aspects of strain breeding, phosphate solubilizing effect, fermentation condition optimization, interaction with potassium solubilizing bacteria and the like, and researches on the protective agent of the bacillus megaterium are rare.
Disclosure of Invention
The present invention aims to overcome the above defects in the prior art and provide a bacillus megaterium D122, which has azotase activity and functions of secreting auxin indole acetic acid.
The invention also aims to overcome the defects in the prior art and provide the bacillus megaterium D122 microbial inoculum which has high nitrogenase activity, can secrete auxin, has a wide agricultural application range and high economic benefit.
The invention also aims to provide a preparation method of the bacillus megaterium D122 microbial inoculum, aiming at the defects in the prior art, the process is simple and mature, and the large-scale production can be realized.
The purpose of the invention is realized by the following technical scheme.
The Bacillus megaterium D122 is deposited in China Center for Type Collection, the address is university of Wuhan, and the Bacillus megaterium D122 is classified and named as Bacillus megaterium D122, the preservation date is 2015, 12 months and 15 days, and the preservation number is CCTCC NO: M2015751.
The bacillus megaterium D122 has a DNA sequence of a sequence table NO. 1.
The azotase activity of the bacillus megaterium D122 is 500-600 nmol/(mL-h), the indole acetic acid can be produced in the growth and metabolism process, and the secretion amount of the indole acetic acid is 100-200 mg/L.
Determination of nitrogenase Activity
1. Test method
The azotase activity of each strain was measured by acetylene reduction. Activating each preserved strain by using VM-Ethanol solid culture medium, picking 1-ring thallus by using an inoculating loop into a 1.5mL sterile centrifuge tube, diluting the thallus by using sterile water, inoculating the thallus into a 10 mL test tube filled with 5mL semi-solid culture medium according to the same inoculation amount, and sealing the test tube by using a reverse rubber plug. After culturing at 37 ℃ for 24 hours, 1/10 vol of 10% acetylene gas was injected, the culture was continued for 24 hours, and 0.5mL of the gas was extracted from the test tube and injected into a gas chromatograph (SP-2100, Beijing Tianpu Analyzer) to measure the acetylene and ethylene contents. The activity of the azotase was calculated according to the following formula (edited by the microorganism specialties of Beijing university of agriculture, 1986):
C=(h x×c×V)/(24.9×h s×t) (2.1)
wherein,h xis the peak area value of the sample;h sis standard C2H4Peak area value;cis standard C2H4Concentration (nmol/mL);
Vculture vessel volume (mL);tincubation time (h) for the sample;Cto produce C2H4Concentration [ nmol/(mL. h)]。
2. Test results
Combining the formula (2.1) and the azotase activity assay FIG. 4 shows that the azotase activity is 500-600 nmol/(mL. h). Therefore, the bacillus megaterium D122 can generate specific nitrogen fixing enzyme in the metabolic process and can spontaneously fix nitrogen in the atmosphere.
Qualitative content determination of auxin
(1) Selecting strains and inoculating respectively (OD 600=1.0, 0.5mL of bacterial suspension) into a 250mL Erlenmeyer flask containing 50mL of King liquid medium, with 3 replicates per group.
(2) After inoculation, the triangular flask is placed on a shaker, cultured for 3d at 28 ℃ and 125rpm and is to be tested.
(3) And (3) placing 50 muL of the bacterial suspension growing for 3d on the King liquid culture medium into a transparent centrifuge tube, and adding 50 muL of colorimetric solution.
(4) Setting a positive control and a negative control, adding 50 muL of plant growth hormone (IAA) with the concentration of 10mg/L into the positive control, and simultaneously adding 50 muL of colorimetric solution. And adding 50 mu LKING liquid culture medium in the negative control, and simultaneously adding 50 mu L colorimetric solution.
(5) Placing the positive control solution, the negative control solution and the determination solution on a white ceramic plate, placing the white ceramic plate at room temperature for 15min, observing the color change, wherein the color change is positive when the color change is red, which indicates that the IAA can be secreted, and the deeper the color is, the stronger the ability of secreting the IAA is; if the strain is not discolored, the strain is negative, which indicates that the strain cannot secrete IAA, and the strain is used as a judgment basis for judgment and analysis.
The culture medium and the reagent formula are as follows: king medium (1L): peptone 20g, K2HPO41.725g，MgSO4·7H2O1.5g, 15mL of glycerol, 0.1g of tryptophan and 1000mL of distilled water.
Quantitative determination of auxin
With minor modifications with reference to Riberio et al. The strain D122 is inoculated into LB liquid culture medium containing 1 g/L tryptophan, and is cultured for 48 hours at 30 ℃ and 180 r/min with shaking. The culture broth was centrifuged at 10000 r/min for 5min, 100mL of the supernatant was added to a 96-well plate, and mixed with 100mL of Salkowski's reagent (1 mL of 0.5 mol/L FeCl)3And 49 mL of 35% perchloric acid), standing at room temperature for 30 min, and measuring the absorbance at a wavelength of 530 nm by using a microplate reader. A standard curve was prepared using IAA standards of different concentrations, and the results are shown in Table 1.
TABLE 1 measurement results of Strain D122 IAA
| Bacterial strains | D141 | D52 | D87 | D122 |
| IAA concentration（mg/L） | 485.68 | 364.20 | 294.16 | 158.36 |
As can be seen from Table 1 and FIG. 5, the auxin concentration of Bacillus megaterium D122 is 158.36 mg/L, therefore, the Bacillus megaterium D122 can be judged to generate IAA in the growth and metabolism process and has the function of promoting the growth of crops.
A preparation method of a bacillus megaterium D122 microbial inoculum comprises the following steps:
(1) separating and screening
Selecting a soil sample containing the azotobacter colony of the bacillus megatherium D122, and culturing by using a separation screening culture medium to obtain the azotobacter colony;
(2) purifying and storing
Carrying out streak purification on the azotobacter colony obtained by separation and screening on a purification preservation culture medium, culturing and separating to obtain a single bacillus megaterium D122 colony, and preserving the single colony for later use; a single colony of B.megaterium D122 is shown in FIG. 1.
Specifically, the colony obtained by separation and screening is subjected to streak purification on a purification preservation culture medium, the single colony of the bacillus megaterium D122 is separated after being cultured for 36-48 hours at the constant temperature of 30 ℃, the single colony appearing on a plate is preserved in a test tube, and is cultured for 36-48 hours at the constant temperature of 30 ℃, and the single colony is preserved in a refrigerator at the temperature of 2-5 ℃, preferably a refrigerator at the temperature of 4 ℃.
(3) Culture medium culture
Inoculating the activated bacillus megaterium D122 slant into a sterilized liquid culture medium, and performing shaking culture to obtain a microorganism liquid fermentation broth;
(4) preparation of D122 microbial inoculum
And (3) taking a mixed solution of sodium chloride, sodium acetate and water as a liquid protective agent, adding the mixed solution into the cultured liquid fermentation liquor, uniformly mixing, standing for 24 hours, and determining that the initial bacteria number is 2.0-4.0 hundred million/mL to obtain the bacillus megaterium D122 liquid microbial inoculum.
Screening assay for liquid protectant
On the basis of experiments, sucrose, sodium chloride, molasses and sodium acetate are selected as screening objects of the liquid protective agent. Adding into the cultured liquid fermentation liquid according to a certain proportion, mixing well, adjusting water content, standing for 24h, and determining initial bacteria number. The cells were kept in the shade for 30 days, and the number of viable cells was measured again, using a treatment without the addition of a protective agent as a Control (CK). Let 7 treatments, repeat 3 times per treatment, see table 2.
TABLE 2 screening of protective Agents (unit: volume in fermentation broth/%)
| Sucrose | Sodium chloride | Molasses for health protection | Sodium acetate | |
| Process 1 | 10 | 7 | ||
| | 10 | 3 | ||
| Treatment 3 | 10 | 1.5 | ||
| Treatment 4 | 7 | 3 | ||
| Treatment 5 | 7 | 1.5 | ||
| Treatment 6 | 3 | 1.5 | ||
| CK | 0 | 0 | 0 | 0 |
The influence of the added protective agent on the effective viable count and the retention period of the microbial agent is as follows: adding the selected protective agent into the prepared microbial inoculum D122 according to a certain proportion, sealing and storing at room temperature. Standing for 24h to determine initial bacteria count, and determining viable bacteria count every 1-2 months, and treating without protectant as Control (CK).
And (3) screening results of the microbial inoculum protective agent: according to the influence result of adding different protective agents on the effective viable count and the storage life of the microbial agent, the optimal microbial agent protective agent formula is determined to be 7% of sodium chloride and 1.5% of sodium acetate.
In the step (4), the mass concentration of the sodium chloride is 6-8%, and the mass concentration of the sodium acetate is 1-2%.
The specific method for separating and screening in the step (1) comprises the following steps: selecting a soil sample, adding sterile water containing Tween 80, shaking, standing, centrifuging the supernatant, discarding the supernatant, retaining the precipitate, adding sterile water containing Tween 80 for suspension, centrifuging, removing the precipitate, centrifuging the supernatant, discarding the supernatant, retaining the precipitate, and suspending the precipitate with a phosphate buffer to obtain a sample solution; taking the sample liquid and a phosphate buffer solution for suspension mixing to obtain a diluted bacterium suspension; and heating the diluted bacterial suspension in a water bath, naturally cooling, sucking and coating the bacterial suspension on a nitrogen-free culture medium, and culturing to obtain a nitrogen-fixing bacterial colony.
More specifically, the separation and screening method comprises the following steps:
500 g of soil sample was collected from Chang Zheng Huang mud pond farm in Dongguan city, Guangdong province, 3L of sterile water containing 0.01% Tween 80 was added, shaking was carried out for 10min, standing was carried out for half an hour, and the supernatant was centrifuged at 4820rpm at 20 ℃ for 20 min. Discarding the supernatant, retaining the precipitate, adding 30-50 mL of sterile water containing 0.01% of Tween 80 for suspension, centrifuging the liquid at 20 ℃ and 5000 rpm for 5s, removing the precipitate, pouring the supernatant into a dry sterile centrifuge tube, centrifuging at 20 ℃ and 6000 rpm for 10min, discarding the supernatant, retaining the precipitate, and suspending the precipitate with 10 mL of phosphate buffer solution with pH 7.0 to obtain a sample solution; and (3) suspending and mixing 1 mL of the sample solution with 9 mL of phosphate buffer solution to obtain 10-fold diluted bacterial suspension. And (3) placing the diluted bacterial suspension in a water bath at 75 ℃ for heating for 15min, naturally cooling, sucking 100 mu L of the diluted bacterial suspension, coating the diluted bacterial suspension on a nitrogen-free culture medium, and culturing at 30 ℃ for 36-48 hours to obtain a nitrogen-fixing bacterial colony containing the D122 bacillus megaterium.
In the step (1), the separation and screening culture medium is prepared from the following raw materials in mass: CaCO31.0-1.4g，MgSO4·7H2O 0.6-1.2g，K2HPO41.0-2.0g，NaCl 0.1-0.4g，FeSO4·7H2O 0.001-0.005g，NaMO4·2H20.05-0.1 g of O, 5-10 g of sucrose, 18-20g of agar and 1000ml of distilled water, and the pH value of the separation and screening culture medium is 7.1-7.4. The invention relates to a separation and screening culture medium, belonging to an improved nitrogen-free culture medium.
The specific method for purifying and storing comprises the following steps: and (3) carrying out streak purification on the colonies obtained by separation and screening on a purification preservation culture medium, and carrying out constant-temperature culture at 30 ℃ for 36-48 hours to obtain a single bacillus subtilis D122 colony. The single colony appeared on the plate is preserved in a test tube, cultured at constant temperature of 30 ℃ for 36-48 hours, and preserved in a refrigerator at 4 ℃. The bacillus megaterium D122 is preserved in China center for type culture Collection with the preservation number: CCTCCM 2015751.
(2) The formula of the purified preservation culture medium is as follows: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 18.0 g of agar and 1000ml of distilled water, wherein the pH value of the purified preservation medium is 7.0-7.4.
In the culture of the culture medium in the step (3), the culture medium is prepared from the following raw materials by mass: CaCO31.0-1.4g，MgSO4·7H2O 0.6-1.2g，K2HPO41.0-2.0g，NaCl 0.1-0.4g，FeSO4·7H2O 0.001-0.005g，NaMO4·2H20.05-0.1 g of O, 5-10 g of sucrose, 18-20g of agar and 1000ml of distilled water, and the pH value of the culture medium is 7.1-7.4.
The method also comprises the following steps between the step (2) and the step (3):
(S1) gram stain: gram staining is carried out on the purified single colony, and positive bacteria are obtained through screening;
(S2) spore staining: and (3) carrying out spore staining on the positive bacteria, and screening to obtain gram-positive bacteria single colonies containing spores.
Spore staining and gram staining thereof
Gram staining and spore staining are two common methods of bacterial identification, and staining may narrow the scope of identification. The undyed bacteria have small refractive index difference with the surrounding environment and are extremely difficult to observe under a microscope. The gram-stained bacteria are in sharp contrast to the environment, and the morphology and arrangement of the bacteria and the gram-positive nature of certain species can be clearly observed (G)+) Or gram-negative bacteria (G)-) For classification and identification. Gram-negative bacteria generally have potential safety hazards, and structural characteristics are abandoned after direct high-temperature sterilization in the application process of agricultural microorganisms. The spore dyeing dyes the spores in the fungus body, so that the size, the position, the shape and other characteristics of the spores can be visually observed, and the identification range of the spores is further narrowed. The bacillus has the characteristics of long shelf life and easy storage, and has wide application basis in agricultural microbial products.
1. Gram stain
(1) Smearing: in a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking a single colony in the drop, and uniformly smearing the colony with a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells.
(2) Primary dyeing: dripping 2-5 drops of ammonium oxalate crystal violet dye solution, dyeing for 1min, pouring off the dye solution, and flushing with running water until no purple color is formed.
(3) Mordant dyeing: washing with newly-prepared iodine solution (iodine 1.0g, potassium iodide 2.0g, and distilled water 300.0 mL), covering the coated surface with iodine solution for 1min, and washing with water.
(4) And (3) decoloring: after removing residual water, 95% alcohol was dropped thereto for decoloring for about 15 to 20 seconds, and then immediately washed with running water.
(5) Counterdyeing: dripping 1 drop of safranin staining solution, staining for 3-5min, washing with water, and blotting with absorbent paper.
(6) Microscopic examination: the slide glass was placed under an optical microscope to observe the staining results.
2. Spore staining
In a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking up a single colony water drop, and uniformly smearing the single colony water drop by using a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells. Dripping 1-2 drops of carbonate basic red dye solution into the zone coated with thallus, and dyeing for 3 min. The staining solution was rinsed off with distilled water, air dried, and the slides were placed under an optical microscope for observation.
As shown in FIGS. 2 and 3, it can be seen from the gram-stained and spore-stained results that Bacillus megaterium D122 is a gram-positive bacterium, rod-shaped, and contains spores.
The bacillus megaterium D122 microbial inoculum is prepared by the preparation method of the bacillus megaterium D122 microbial inoculum.
The invention has the beneficial effects that:
(1) according to the invention, the bacillus megaterium D122 is used as an original strain, and the azotobacter activity and the auxin secretion capacity of the bacillus megaterium are measured, so that the strain D122 has high-efficiency azotobacter activity and IAA secretion capacity. The azotase activity of the bacillus megaterium D122 is 500-600 nmol/(mL-h), the indole acetic acid can be produced in the growth and metabolism process, and the secretion amount of the indole acetic acid is 100-200 mg/L.
(2) In order to improve the spore survival rate of the bacillus megatherium D122 microbial inoculum, the special protective agent is added, so that the death rate of spores in the processing and storage processes is reduced, the product cost is effectively reduced, the economic benefit is improved, and guidance is provided for the production of microbial fertilizers in the future.
(3) According to the preparation method of the bacillus megaterium D122 microbial inoculum, the protective agent is added in a certain proportion, so that the survival rate of strains can be maintained at a higher level, and the storage stability of microbial fertilizer products and the shelf life of the products can be effectively prolonged.
Drawings
The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be derived on the basis of the following drawings without inventive effort.
FIG. 1 is a colony image of a single colony of isolated Bacillus megaterium D122 at a magnification of 10X 100 times under a microscope.
FIG. 2 is a graph showing the gram staining results of B.megaterium D122.
FIG. 3 is a graph showing the staining results of B.megaterium D122 spores.
FIG. 4 is a graph showing the results of the azotase activity assay of Bacillus megaterium D122.
FIG. 5 is a colorimetric effect graph of Bacillus megaterium D122 IAA.
Detailed Description
The invention is further described with reference to the following examples.
Example 1
The bacillus megaterium D122 is preserved in China center for type culture Collection with the preservation number of CCTCCM 2015751. The bacillus megaterium D122 has a DNA sequence of a sequence table NO. 1.
The azotase activity of the bacillus megaterium D122 is 500 nmol/(mL-h), the indole acetic acid can be produced in the growth and metabolism process, and the secretion amount of the indole acetic acid is 100 mg/L.
A preparation method of a bacillus megaterium D122 microbial inoculum comprises the following steps:
(1) separating and screening
Selecting a soil sample containing the azotobacter colony of the bacillus megatherium D122, and culturing by using a separation screening culture medium to obtain the azotobacter colony;
(2) purifying and storing
Purifying the azotobacter colonies obtained by separation and screening on a purification preservation culture medium, culturing and separating to obtain a single bacillus megaterium D122 colony, and preserving the single colony for later use;
(3) culture medium culture
Inoculating the activated bacillus megaterium D122 slant into a sterilized liquid culture medium, and performing shaking culture to obtain a microorganism liquid fermentation broth;
(4) preparation of D122 microbial inoculum
And (3) taking a mixed solution of sodium chloride, sodium acetate and water as a liquid protective agent, adding the liquid protective agent into the cultured liquid fermentation liquor according to a certain proportion, uniformly mixing, standing, and measuring the initial number of bacteria to be 2.0 hundred million/mL to obtain the bacillus megaterium D122 microbial inoculum.
In the step (4), the mass concentration of the sodium chloride is 6% and the mass concentration of the sodium acetate is 1%.
The specific method for separating and screening in the step (1) comprises the following steps: selecting a soil sample, adding sterile water containing Tween 80, shaking, standing, centrifuging the supernatant, discarding the supernatant, retaining the precipitate, adding sterile water containing Tween 80 for suspension, centrifuging, removing the precipitate, centrifuging the supernatant, discarding the supernatant, retaining the precipitate, and suspending the precipitate with a phosphate buffer to obtain a sample solution; taking the sample liquid and a phosphate buffer solution for suspension mixing to obtain a diluted bacterium suspension; and heating the diluted bacterial suspension in a water bath, naturally cooling, sucking and coating the bacterial suspension on a nitrogen-free culture medium, and culturing to obtain a nitrogen-fixing bacterial colony.
In the step (1), the separation and screening culture medium is prepared from the following raw materials in mass: CaCO31.0g，MgSO4·7H2O 0.6g，K2HPO41.0g，NaCl 0.1g，FeSO4·7H2O 0.001g，NaMO4·2H20.05 g of O, 5g of cane sugar, 18g of agar and 1000ml of distilled water, and the pH value of the separation and screening culture medium is 7.1.
The bacillus megaterium D122 microbial inoculum is prepared by the preparation method of the bacillus megaterium D122 microbial inoculum.
Example 2
The present embodiment is different from embodiment 1 in that the following steps are further included between step (2) and step (3) of the present embodiment:
(S1) gram stain: gram staining is carried out on the purified single colony, and positive bacteria are obtained through screening;
(S2) spore staining: and (3) carrying out spore staining on the positive bacteria, and screening to obtain gram-positive bacteria single colonies containing spores.
Specifically, the gram staining method is as follows:
(1) smearing: in a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking a single colony in the drop, and uniformly smearing the colony with a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells.
(2) Primary dyeing: dripping 2-5 drops of ammonium oxalate crystal violet dye solution, dyeing for 1min, pouring off the dye solution, and flushing with running water until no purple color is formed.
(3) Mordant dyeing: washing with newly-prepared iodine solution (iodine 1.0g, potassium iodide 2.0g, and distilled water 300.0 mL), covering the coated surface with iodine solution for 1min, and washing with water.
(4) And (3) decoloring: after removing residual water, 95% alcohol was dropped thereto for decoloring for about 15 to 20 seconds, and then immediately washed with running water.
(5) Counterdyeing: dripping 1 drop of safranin staining solution, staining for 3-5min, washing with water, and blotting with absorbent paper.
(6) Microscopic examination: the slide glass was placed under an optical microscope to observe the staining results.
Specifically, the spore staining method comprises the following steps:
in a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking up a single colony water drop, and uniformly smearing the single colony water drop by using a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells. Dripping 1-2 drops of carbonate basic red dye solution into the zone coated with thallus, and dyeing for 3 min.
The rest of this embodiment is the same as embodiment 1, and is not described herein again.
Example 3
This example differs from example 1 or 2 in that the azotase activity of B.megaterium D122 in this example is 520 nmol/(mL. h), which can produce indole acetic acid during the growth metabolism, and the amount of indole acetic acid secreted is 120 mg/L.
The initial number of the bacillus megaterium D122 microbial inoculum is 2.5 hundred million/mL.
In the step (4), the mass concentration of the sodium chloride is 6.5%, and the mass concentration of the sodium acetate is 1.5%.
In the step (1), the separation and screening culture medium is prepared from the following raw materials in mass: CaCO31.2g，MgSO4·7H2O 0.8g，K2HPO41.5g，NaCl 0.2g，FeSO4·7H2O 0.002g，NaMO4·2H20.06 g of O, 6g of cane sugar, 18g of agar and 1000ml of distilled water, and the pH value of the separation and screening culture medium is 7.2.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Example 4
This example differs from example 1 or 2 in that the azotase activity of B.megaterium D122 in this example is 550 nmol/(mL. h), which can produce indole acetic acid during the growth metabolism, and the amount of indole acetic acid secreted is 150 mg/L.
The initial number of the bacillus megaterium D122 microbial inoculum is 3 hundred million/mL.
In the step (4), the mass concentration of the sodium chloride is 7% and the mass concentration of the sodium acetate is 1.5%.
In the step (1), the separation and screening culture medium is prepared from the following raw materials in mass: CaCO31.3g，MgSO4·7H2O 1.0g，K2HPO41.8g，NaCl 0.3g，FeSO4·7H2O 0.004g，NaMO4·2H20.09 g of O, 8g of sucrose, 19g of agar and 1000ml of distilled water, and the pH value of the separation and screening medium is 7.3.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Example 5
This example differs from example 1 or 2 in that the azotase activity of Bacillus megaterium D122 in this example is 580 nmol/(mL. h), which can produce indole acetic acid during the growth metabolism, and the amount of indole acetic acid secreted is 180 mg/L.
The initial number of the bacillus megaterium D122 microbial inoculum is 4.0 hundred million/mL.
In the step (4), the mass concentration of the sodium chloride is 7.5%, and the mass concentration of the sodium acetate is 1.5%.
In the step (1), the separation and screening culture medium is prepared from the following raw materials in mass: CaCO31.4g，MgSO4·7H2O 1.2g，K2HPO42.0g，NaCl 0.4g，FeSO4·7H2O 0.005g，NaMO4·2H20.1g of O, 10g of cane sugar, 20g of agar and 1000ml of distilled water, and the pH value of the separation and screening culture medium is 7.4.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Identification of 16SrDNA sequence strain of Bacillus megaterium D122
The bacteria are tiny in individuals and simple in shape, and the traditional method for identifying the bacteria is often used as a main basis for classification and identification according to different physiological and biochemical reactions of the bacteria. Since the late 70 s in the 20 th century, the international general "official" or "official" classification of bacteria was based on Bergey's Manual of bacteriology of identification. In physiological and biochemical identification, one or more physiological indexes do not conform to the unique properties of the strain, and the strain is difficult to be clearly identified. Currently, methods for identifying bacteria usually combine physiological and biochemical indicators of strains with molecular biological characteristics to draw more reliable conclusions. Wherein the 16S rRNA gene evolutionary development system of DNA sequence analysis has become a common technical means for the heterogeneous classification and identification of bacteria in the international (Kim et al, 2004; Prap et al, 1997).
The ribosome 16S rDNA gene sequence has a total length of about 1550bp and consists of alternative conserved region and variable region. The 16S rDNA fragments of all bacteria can be amplified by using a universal primer designed by a conserved region. The 16S rDNA sequence analysis technology is based on the basic principle that 16S rDNA fragments are extracted from a microorganism sample, 16S rDNA sequence information is obtained through cloning, sequencing or enzyme digestion and probe hybridization, and then the sequence information is compared with sequence data or other data of a 16S rDNA database to determine the position of the sequence information in an evolutionary tree, so that the possible microorganism species in the sample can be identified. The universal primer designed by using the conserved region of the 16S rDNA fragment can not be complementary to non-bacterial DNA, and the difference of the 16S rDNA variable region of bacteria can be used for distinguishing different bacteria. It is therefore generally accepted that the final identification is obtained by sequencing the 16S rDNA of a strain.
1. The method comprises the following steps:
(1) PCR reaction (25. mu.L):
10×PCR Buffer 2.5μL
dNTP(2.5mM) m 2.0μL
primer 27F (10. mu.M) 0.5. mu.L
Primer 1492R (10. mu.M) 0.5. mu.L
DNA template 100ng
Taq enzyme (5U/. mu.L) 0.5. mu.L
ddH2O 19μL
(2) And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 80 s, 35 cycles, and extension at 72 ℃ for 10 min. DNA sequencing was performed using an ABI 3730 xl DNA Analyzer (applied biosystems).
2. Sequencing results
tcgagcgaac tgattagaag cttgcttcta tgacgttagc ggcggacggg tgagtaacac
gtgggcaacc tgcctgtaag actgggataa cttcgggaaa ccgaagctaa taccggatag
gatcttctcc ttcatgggag atgattgaaa gatggtttcg gctatcactt acagatgggc
ccgcggtgca ttagctagtt ggtgaggtaa cggctcaccaaggcaacgat gcatagccga
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg
aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtacgaga gtaactgctc
gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa
tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct
taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tggggaactt
gagtgcagaa gagaaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag
gaacaccagt ggcgaaggcg gctttttggt ctgtaactga cgctgaggcg cgaaagcgtg
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt
tagagggttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta
cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt
ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaactcta
gagatagagc gttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca
gcatttagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa
agggctgcaa gaccgcgagg tcaagccaat cccataaaac cattctcagt tcggattgta
ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc
gaagtcggtg gagtaaccgt aag
3. Homology analysis
Identifying the bacterium asBacillus megateriumBacillus megaterium.
The invention is funded and researched by introducing an innovative entrepreneurial team project in Guangdong province, and the prepared bacillus megaterium D122 and the microbial inoculum thereof have wide market prospect and high economic benefit.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
<0001>
SEQUENCE LISTING
<110> Dongguan City, Purcha bioengineering Co., Ltd
<120> bacillus megaterium D122, microbial inoculum thereof and preparation method of microbial inoculum
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>1403
<212>DNA
<213> 16s rDNA Gene sequence of Bacillus megaterium D122
<400>1
tcgagcgaac tgattagaag cttgcttcta tgacgttagc ggcggacggg tgagtaacac 60
gtgggcaacc tgcctgtaag actgggataa cttcgggaaa ccgaagctaa taccggatag 120
gatcttctcc ttcatgggag atgattgaaa gatggtttcg gctatcactt acagatgggc 180
ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcatagccga 240
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360
aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtacgaga gtaactgctc 420
gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480
tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct 540
taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tggggaactt 600
gagtgcagaa gagaaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660
gaacaccagt ggcgaaggcg gctttttggt ctgtaactga cgctgaggcg cgaaagcgtg 720
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780
tagagggttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta 840
cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900
ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaactcta 960
gagatagagc gttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080
gcatttagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa 1200
agggctgcaa gaccgcgagg tcaagccaat cccataaaac cattctcagt tcggattgta 1260
ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380
gaagtcggtg gagtaaccgt aag 1403
Claims (3)
1. A Bacillus megaterium D122, wherein: the bacillus megaterium D122 is preserved in China center for type culture Collection with the preservation number of CCTCCM 2015751.
2. The bacillus megaterium D122 according to claim 1, wherein: the bacillus megaterium D122 has a DNA sequence of a sequence table NO. 1.
3. The bacillus megaterium D122 according to claim 1, wherein: the azotase activity of the bacillus megaterium D122 is 500-600 nmol/(mL-h), the indole acetic acid can be produced in the growth and metabolism process, and the secretion amount of the indole acetic acid is 100-200 mg/L.
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|---|---|---|---|---|
| CN101914468A (en) * | 2010-07-15 | 2010-12-15 | 广西大学 | Nitrogen-fixing bacillus megaterium strain DL7 and application thereof |
| CN102747018A (en) * | 2012-07-16 | 2012-10-24 | 南京农业大学 | Bacillus megaterium and application thereof |
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2016
- 2016-05-20 CN CN201610336831.5A patent/CN106167771B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914468A (en) * | 2010-07-15 | 2010-12-15 | 广西大学 | Nitrogen-fixing bacillus megaterium strain DL7 and application thereof |
| CN102747018A (en) * | 2012-07-16 | 2012-10-24 | 南京农业大学 | Bacillus megaterium and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 烟草根际固氮菌的筛选、鉴定及优化培养;刘晓璐;《中国烟草学报》;20150228(第01期);全文 * |
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