CN106190887A - A kind of bacillus subtilis T400 and bacterial preparation process thereof - Google Patents
A kind of bacillus subtilis T400 and bacterial preparation process thereof Download PDFInfo
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Abstract
The present invention relates to microbial bacterial agent technical field, it is specifically related to a kind of bacillus subtilis T400 and bacterial preparation process thereof, described bacillus subtilis T400 is preserved in China typical culture collection center, preserving number is CCTCCM 2015755, this bacillus subtilis T400 can be by supplementing nitrogen required in process of crop growth from growing nitrogen-fixing, additionally it is possible to secretion auxin promotes plant growing.The bacterial preparation process of described bacillus subtilis T400 comprises the following steps: (1) separates, screens；(2) purification, preservation；(3) culture medium culturing: utilize fermentation medium fermentation culture list bacterium colony, obtain microbial inoculum；(4) thalline reclaims；(5) prepared by microbial inoculum.The technique of the present invention is the most ripe, is produced on a large scale, and the bacillus subtilis T400 prepared has higher nitrogenase activity and can secrete auxin, applied range, high financial profit.
Description
Technical field
The present invention relates to microbial bacterial agent technical field, be specifically related to a kind of bacillus subtilis T400 and be prepared by microbial inoculum
Method.
Background technology
Along with the continuous growth of world population, the demand of chemical nitrogen fertilizer is also being increased by Global Agriculture year by year.Chemical nitrogen fertilizer
Use the yield greatly improving agri-foodstuffs, but also to bring degradation under serious environmental pollution and soil fertility negative simultaneously
Impact.In nature, some prokaryotic micro-organisms passes through azotase at normal temperatures and pressures by the dinitrogen (N in air2) convert
For ammonia (NH3), this process is referred to as biological nitrogen fixation, and this quasi-microorganism is referred to as nitrogen-fixing microorganism.Add up according to FAO (Food and Agriculture Organization of the United Nation),
On the earth, combined state nitrogen total amount has 70% to derive from biological nitrogen fixation, and the nitrogen amount of annual whole world Microorganism incubation is up to 200,000,000 tons, about
Account for the 3/4 of world crops nitrogen requirement, be equivalent to more than 3 times of commercial production nitrogenous fertilizer.By biological nitrogen fixation be crops provide nitrogen source,
Improve yield, reduce fertilizer amount, reduce production cost, simultaneously facilitated agricultural sustainable development, be energy-saving and environmental protection,
Ecological friendly Nitrogen supplying mode.Therefore, profit carries out biological nitrogen fixation in various manners is the weight that the whole world is paid close attention at present
Want problem.
According to the marriage relation between nitrogen-fixing microorganism and host, nitrogen fixation can be divided into from growing nitrogen-fixing and symbiosis solid
Nitrogen, the former is such as azotobacter vinelandii, Klebsiella pneumoniae, bacillus megaterium etc., the latter such as root nodule bacteria and actinomycetes.Wherein, root
The research of tumor bacterium alreadys more than the history of 100 years, is the most clearly biological nitrogen fixation system studied so far, and at the U.S., bar
Western, Argentinian, Australian etc. it is widely used.But, root nodule bacteria have the harshest single-minded infectivity, are only limitted to pulse family and make
Thing production and application, can not realize symbiotic nitrogen fixation with crops such as grass family.Therefore, by being colonizated in rhizosphere, root table, or enter root
The spontaneous nitrogen-fixing microorganism of cortical tissue have highly important with gramineous crop association nitrogen fixations such as Oryza sativa L., Semen Maydis, Caulis Sacchari sinensis
Application prospect.
Spontaneous nitrogen-fixing microorganism is that typically in natural environment or culture medium and lives on one's own life, and can be by dividing in air
Sub-state nitrogen is fixed as a quasi-microorganism of ammonia.Spontaneous nitrogen-fixing microorganism need not and other biological associating just energy fixed nitrogen, has ready conditions
Wide, adapt to wide feature and some phytohormone can also be secreted, stimulate root system and the growth promoter of plant, azotobacter energy
Enough improve the Tiny ecosystem group of plant rhizosphere, play promotion plant growing, improve the effect of stress resistance of plant.But due to domestic
The most domestic outer research to azotobacter is started late, and greatly limit microbial bacterial agent in application agriculturally.
Summary of the invention
It is an object of the invention to for above-mentioned deficiency of the prior art, it is provided that a kind of bacillus subtilis T400, this merit
Can go back by supplementing nitrogen required in process of crop growth from growing nitrogen-fixing by microorganism fungus kind bacillus subtilis T400
Auxin can be secreted and promote plant growing.
It is another object of the present invention to for above-mentioned deficiency of the prior art, it is provided that a kind of bacillus subtilis T400's
Bacterial preparation process, technique is the most ripe, is produced on a large scale, and the bacillus subtilis T400 prepared has higher azotase
Activity and auxin, applied range, high financial profit can be secreted.
The purpose of the present invention is achieved through the following technical solutions.
A kind of bacillus subtilis T400, described bacillus subtilis Classification And Nomenclature: bacillus subtilis
T400Bacillus subtilis T400, is preserved in China typical culture collection center (China Center for Type
Collection), address: Wuhan, China Wuhan University, preservation date;On December 15th, 2015, deposit number: CCTCC
NO:M2015755.
The nitrogenase activity of described bacillus subtilis T400 is 600.000-800.000 nmol/ (mL h), and it is being given birth to
Can produce heteroauxing IAA in long metabolic process, heteroauxing concentration is 95-103 mg/L.Preferably, this bacillus subtilis
Bacterium T400 is the gram positive bacteria containing spore.
Specifically, the nitrogenase activity determination method of this bacillus subtilis T400 is as follows:
Test method:
Use acetylene reduction method that the nitrogenase activity of each bacterial strain is measured.The each bacterial strain VM-Ethanol solid that will preserve
Culture medium activates, and with inoculating loop picking 1 ring thalline in the sterile centrifugation tube of 1.5mL, dilutes with sterilized water, by identical inoculum concentration
Access equipped with in 10 mL test tubes of 5 mL semisolid culturemediums, by anti-mouth rubber stopper seal.After 37 DEG C are cultivated 24 h, inject 1/
10% acetylene gas of 10 volumes, continues to cultivate 24 h, extracts 0.5 mL gas injection gas chromatography instrument (sky, Beijing from test tube
General analytical tool factory SP-2100) in, measure acetylene, the content of ethylene.Nitrogenase activity size (Beijing is calculated by following equation
Agriculture university's microorganism specialty is compiled, and 1986):
C=(h x×c×V)/(24.9×h s×t) (2.1)
Wherein,h xFor sample peak area value；h sFor standard C2H4Peak area value；cFor standard C2H4Concentration (nmol/mL)；
VFor culture vessel volume (mL)；tFor sample culturing time (h)；CFor the C produced2H4Concentration [nmol/ (mL h)].
Result of the test:
Interpretation of result, as shown in Figure 4, can be calculated nitrogenase activity according to formula (2.1) is 600.000-800.000
nmol/(mL·h).It can be said that bright, bacillus subtilis T400 can produce specific azotase in metabolic process, can
With the nitrogen in spontaneous fixing air.
Specifically, the qualitative content assaying method of the heteroauxing of this bacillus subtilis T400 is:
The qualitative assay of auxin
(1) picking bacterial strain inoculate respectively (OD 600=1.0,0.5mL bacteria suspension) to the 250mL filling 50mL King fluid medium
In triangular flask, often 3 repetitions of group.
(2) after inoculation, triangular flask is placed on shaking table, 28 DEG C, 125rpm, cultivates 3d, to be measured.
(3) take above-mentioned bacteria suspension 50 L on King fluid medium after growth 3d to be placed in transparent centrifuge tube, simultaneously
Add 50 L color solutions.
(4) setting positive control and negative control, adding 50 L concentration in positive control is the auxin of 10mg/L
(IAA), add 50 L color solutions simultaneously.Negative control adds 50 LKing fluid mediums, adds 50 L color solutions simultaneously.
(5) positive control solution, cloudy shape comparison liquid and mensuration liquid are placed in white pottery porcelain plate juxtaposition 15min at room temperature, observe
Its color changes, and the color person of reddening be the positive, expression can producing IAA, and color more deeply feels and shows that the ability of producing IAA is the strongest；
If invariant color, be negative, represent this bacterial strain can not producing IAA, thus carry out discriminatory analysis as basis for estimation.Culture medium is
King culture medium (1L)；Agent prescription is peptone 20g, K2HPO41.725g, MgSO4·7H2O 1.5g, glycerol 15mL,
Tryptophan 0.1g, distilled water 1000mL.
Auxin quantitative determines
Method with reference to Riberio etc. is changed slightly.Bacterial strain T400 is inoculated into the LB fluid medium containing 1 g/L tryptophan
In, 30 DEG C, 180 r/min, shaken cultivation 48 h.Culture fluid 10000 r/min is centrifuged 5 min, takes 100 mL supernatant
It is added in 96 orifice plates, with 100mL Salkowski ' s reagent (1 mL 0.5 mol/L FeCl3With the 49 high chlorine of mL 35%
Acid) mixing, room temperature stands 30 min, measures absorbance by microplate reader under wavelength 530 nm.As it is shown in figure 5, it is dense by difference
The IAA standard substance of degree make standard curve, and measurement result is shown in Table 1.
Table 1 bacterial strain T400 IAA measurement result
| Bacterial strain | T400 | T665 | T808 | T188 |
| IAA concentration (mg/L) | 98.36 | 90.12 | 86.86 | 21.57 |
From Fig. 5 and Biao 1 it can be seen that the auxin concentration of bacillus subtilis T400 is 98.36 mg/L, therefore, it is possible to judge that
Bacillus subtilis T400 can produce IAA during growth metabolism, has the function promoting plant growth.
Described bacillus subtilis T400 has the DNA sequence of sequence table NO.1.
The bacterial preparation process of a kind of bacillus subtilis T400, comprises the following steps:
(1) separate, screen
Choose the soil sample of nitrogen-fixing bacteria bacterium colony containing bacillus subtilis T400, separated, screening, cultivate and obtain nitrogen-fixing bacteria bacterium
Fall.
(2) purification, preservation
In purification storage culture medium, the nitrogen-fixing bacteria bacterium colony separated, screening obtains being carried out purification of ruling, culture of isolated obtains withered
The grass mono-bacterium colony of bacillus cereus T400, preserves this list bacterium colony standby.Fig. 1 is that the mono-bacterium colony of bacillus subtilis T400 of isolated exists
The bacterium colony figure of 1000 times is amplified under microscope.
(3) culture medium culturing: utilize the fermentation medium fermentation culture mono-bacterium colony of bacillus subtilis T400, obtain microorganism
Bacterium solution；
Condition of culture is: the environmental factors affecting microbial growth has a lot, bigger fermentation impact is had liquid amount, connects
The amount of kind, cultivation temperature, rotating speed, ventilation, pH and fermentation time.
(4) thalline reclaims
The microbial inoculum produced by fermentation tank accesses aseptic disk centrifuge, centrifugal collection bacillus subtilis T400 bacterium
Body, quickly sloughs moisture, the pure mycopowder that preparation is dried, and measuring spore number is 500-800 hundred million/g.
(5) prepared by microbial inoculum
Being thoroughly mixed with matrix material Pulvis Talci and the peat composed of rotten mosses by dry pure mycopowder, carry out viable count detection, testing result shows this
Viable count scope is 4-20 hundred million/g, and product viable count complies fully with national standard " microbial bacterial agent " GB20287-2006 and requires 2
Hundred million/more than g, i.e. obtains bacillus subtilis T400 microbial inoculum.
Preferably, in described step (1), separate, screen method particularly includes: in soil sample, add sterilized water, concussion, quiet
Put, take supernatant centrifugal treating, abandon supernatant, retain precipitate, add sterilized water and suspend, centrifugal, go precipitation, take supernatant again
Centrifugal, abandon supernatant, retain precipitate, add phosphate buffer and suspend, obtain sample liquid；Sample liquid is added phosphate buffer
Suspend mixing, obtains the bacteria suspension of dilution；By the bacteria suspension heating in water bath of dilution, draw bacteria suspension after natural cooling and coat nothing
Nitrogen culture medium, cultivates and obtains nitrogen-fixing bacteria bacterium colony.
More specifically, the method for separation, screening is: take 500 g soil from farm, the yellow mud pool, Changping Town, Dongguan City, Guangdong Province
Sample, adds 3 L containing the sterilized water of 0.01% Tween 80, shakes 10min, stand half an hour, takes supernatant at 20 DEG C 8000
Rpm is centrifuged 20min.Abandon supernatant, retain precipitate, add the 30-50 mL sterilized water containing 0.01% Tween 80 and suspend, liquid in
20 DEG C of 5000 rpm is centrifuged 5 seconds, goes precipitation, and supernatant is poured in a dry sterile centrifugation tube 6000 rpm at 20 DEG C
Centrifugal 10min, abandons supernatant, retains precipitate, is suspended by the precipitate pH7.0 phosphate buffer of 10 mL, takes 1 mL above-mentioned
Sample liquid suspends with 9 mL phosphate buffers and mixes, and is 10 times of dilution bacteria suspensions.The bacteria suspension of dilution is placed in 75 DEG C of water-baths
Heat 15 min, draw 100 L after natural cooling and coat nitrogen-free agar, cultivate 36-48 hour for 30 DEG C and obtain the bud Han hay
The nitrogen-fixing bacteria bacterium colony of spore bacillus T400.
Preferably, purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony
In the 28-33 DEG C of constant temperature culture 36-48 hour isolated mono-bacterium colony of bacillus subtilis T400, the single bacterium colony that will occur on flat board
It is saved in test tube, preserves in 28-33 DEG C of constant temperature culture 36-48 hour is placed on 2-5 DEG C of refrigerator.
Preferably, the culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, logical
Tolerance, pH and fermentation time are further qualified, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 70-75% of tank body volume；Fermentation tank liquid amount will be according to the reality of fermentation tank
Size regulates and controls, and dress liquid can cause polluting too much, and liquid amount will also result in the situation of the wasting of resources very little.
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 5-10%；
Inoculum concentration refers to the seed liquor volume of subcultivation and the ratio of fermentating liquid volume.Suitable inoculum concentration scalable culture fluid viscosity and
Dissolved oxygen content, shorten growth peak time, make product synthesize in advance.The relation that fermentation time is bred with thalli growth
Can be drawn by growth curve, the suitableeest fermentation time enables to the metabolism amount of thalline and reaches maximum, and don't there will be
The phenomenon of thalline self-dissolving, it is ensured that the accuracy of fermentation results.
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 30-36 DEG C；
The growth of microorganism and the synthesis of product are required to carry out at its most most suitable temperature.Temperature is to ensure that what enzyme was lived
Essential condition, therefore must assure that the suitableeest temperature environment during the fermentation.
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 150-300rpm；
Shaking speed in sweat makes culture mix homogeneously, it is ensured that when each fluid space keeps identical growth
Phase, strengthening rotating speed can increase air contact area simultaneously, improves the concentration of dissolved oxygen in culture medium.
(3.5) ventilation: in terms of the research of fermentation technology, how based on aerobic fermentation, oxygen is primarily as respiratory chain
Terminal electron acceptor.Organic substance is converted into CO by aerobic respiration by aerobic microbiological2、H2O, and obtain energy.Hay spore
The ventilation of bacillus T400 is set according to different incubation times, after inoculation 0-12 hour, and ventilation controls at 0.9-
1.2vvm, it is preferable that ventilation controls after 1.04vvm, inoculation 13-42 hour, and ventilation controls at 1.15-1.6vvm, excellent
Selection of land, ventilation controls after 1.35vvm, inoculation 43-48 hour, and ventilation controls at 1.4-1.8vvm, it is preferable that set logical
Tolerance is 1.63vvm.
(3.6) pH:pH is the very important affecting parameters of growth of microorganism and Product formation, is to weigh metabolic activity
Aggregative indicator.The change of pH influences whether the structure that various enzyme is alive, bacterium utilizes speed and cell to substrate, thus affects bacterium
Growth and the synthesis of product.In bacillus subtilis T400 sweat, by mending the method regulation pH of acid or benefit alkali it is
7.1-7.4。
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 42-48 hour and is
Fermentation termination.
Preferably, in described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO3
1.0-1.4g, MgSO4·7H2O 0.6-1.2g, K2HPO41.0-2.0g, NaCl 0.1-0.4g, FeSO4·7H2O 0.001-
0.005g, NaMO4 2H2O 0.05-0.1g, sucrose 5-10g, agar 18-20g, distilled water 1000mL, the pH of described culture medium
For 7.1-7.4.Certainly, protection scope of the present invention is fallen within according to the mass ratio of above-mentioned quality.
Preferably, following steps are also included between described step (2) and step (3):
(S1) Gram’s staining: single bacterium colony after purification carries out Gram’s staining, screening obtains positive bacteria；
(S2) spore staining: positive bacteria carries out spore staining, screening obtains the gram positive bacteria list bacterium colony containing spore.
Gram’s staining and spore staining are the conventional methods of two kinds of Bacteria Identification, and dyeing can reduce qualification scope.Not
Dyed antibacterial is the least with surrounding index of refraction difference, the most extremely difficult observation.Antibacterial and ring after Gram’s staining
Border forms sharp contrast, it can clearly be observed that the form of antibacterial, arrangement and some strain belong to Gram-positive (G+) or
Person gram negative bacteria (G-), in order to taxonomic identification.Generally there is potential safety hazard in gram negative bacteria, at agricultural microbe application
During direct drop fabric feature after high temperature sterilize, exist may be pathogenic hidden danger.Spore staining is by endobacillary spore
Dye, can intuitively observe the features such as the size of spore, position, shape, reduce its qualification scope further.Bacillus cereus
There is long shelf-life, it is easy to the feature deposited, have a wide range of applications basis at agricultural microniological proudcts.
By the Gram’s staining result figure of Fig. 2 and the spore staining result figure of Fig. 3 it can be seen that bacillus subtilis T400 is
Gram positive bacteria, and containing spore.
Preferably, described step (S1) is Gram-stained method particularly includes:
(S1.1) smear: in aseptic operating platform, takes one piece of microscope slide, and on flame lamp, general plan is baked, and that removes on slide is miscellaneous
Matter；Dripping sterilized water in microscope slide central authorities, the single bacterium colony of picking, in water droplet, is smeared uniformly with the inoculating loop of calcination；Will
Specimen slide is crossed 3 times, back and forth with fixing cell above fire lamp；
(S1.2) just contaminate: dropping 2-5 drips ammonium oxalate crystal violet dye liquor, contaminates 1min, and incline dye liquor, and flowing water rinses to without purple；
(S1.3) mordant dyeing: first wash away back water with the iodine liquid (iodine 1.0g, potassium iodide 2.0g, distilled water 300.0mL) newly joined, then
Cover painting face 1min with iodine liquid, wash afterwards；
(S1.4) decolouring: remove after back water, drips 95% ethanol and carries out decolouring after the about 15-20 second, rinse with flowing water immediately；
(S1.5) redye: drip 1 sarranine dyeing liquor, contaminate 3-5min, blot with absorbent paper after washing；
(S1.6) microscopy: microscope slide is placed in optical microphotograph Microscopic observation coloration result.
Preferably, described step (S2) spore staining method particularly includes: in aseptic operating platform, take one piece of microscope slide,
On flame lamp, general plan is baked, and removes the impurity on slide.Sterilized water is dripped, picking single bacterium colony water droplet in microscope slide central authorities
In, smear uniformly with the inoculating loop of calcination.Specimen slide is crossed 3 times, back and forth with fixing cell above fire lamp.In coating
The region dropping 1-2 dropstone carbonic acid basic fuchsin dye liquor of thalline, dye 3min.Fall dye liquor with distilled water flushing, after air-drying, will carry
Slide is placed in optical microphotograph Microscopic observation.
Beneficial effects of the present invention:
(1) fixed nitrogen: bacillus subtilis Pseudomonas is widely present in the environment such as soil, plant rhizosphere, has strain safety, function many
The advantages such as sample, convenient for production, long shelf-life, are that current China microbial manure produces one of wide variety of strain, in micro-life
Thing fertilizer application field has the most considerable economic worth.The nitrogenase activity of described bacillus subtilis T400 is
600.000-800.000 nmol/ (mL h), the heteroauxing concentration that it produces during growth metabolism is 95-103 mg/
L。
(2) reduce nitrogen to put into: in maize field demonstration test, on the basis of fertilising cost is basically identical, test group
Joint nitrogen rate reaches 17.2%, and rate of growth reaches 29.24%, considerably improves the yield of Semen Maydis, illustrates that using T400 microbial inoculum is reducing
Nitrogen can bring bigger economic benefit while putting into.
(3) plant growth is promoted: in maize field demonstration test, it is right that the plant height of test group, stem leaf area thick, excellent is above
According to group, relative Ear height and lodging rate are below matched group, absolutely prove that T400 microbial inoculum has significantly rush to the growth of Semen Maydis
Enter effect.
Accompanying drawing explanation
Fig. 1 is the bacterium colony figure that the mono-bacterium colony of bacillus subtilis T400 of isolated amplifies 1000 times under the microscope.
Fig. 2 is bacillus subtilis T400 Gram’s staining result figure.
Fig. 3 is bacillus subtilis T400 spore staining result figure.
Fig. 4 is bacillus subtilis T400 nitrogenase activity determination result figure.
Fig. 5 is bacillus subtilis T400 IAA colorimetric design sketch.
Fig. 6 is the Semen Maydis pot experiment design sketch of bacillus subtilis T400 microbial inoculum.
Detailed description of the invention
The invention will be further described with the following Examples, as shown in Fig. 1-6.
Embodiment 1
The bacterial preparation process of a kind of bacillus subtilis T400 of the present embodiment, comprises the following steps:
(1) separate, screen
Choose the soil sample with bacillus subtilis T400 nitrogen-fixing bacteria bacterium colony, separated, screening, cultivate and obtain nitrogen-fixing bacteria bacterium colony；
(2) purification, preservation
In purification storage culture medium, the nitrogen-fixing bacteria bacterium colony separated, screening obtains being carried out purification of ruling, culture of isolated obtains withered
The grass mono-bacterium colony of bacillus cereus T400, preserves this list bacterium colony standby；
(3) culture medium culturing: utilize fermentation medium fermentation culture list bacterium colony, obtain microbial inoculum；
(4) thalline reclaims
Microbial inoculum is accessed aseptic disk centrifuge, centrifugal collection bacillus subtilis T400 thalline, quickly slough water
Point, the pure mycopowder that preparation is dried, measuring spore number is 50,000,000,000/g；
(5) prepared by microbial inoculum
Pure mycopowder will be dried mix with matrix material, carry out viable count detection, testing result show this viable count scope be 500,000,000/
G, i.e. obtains bacillus subtilis T400 microbial inoculum.
Described bacillus subtilis T400, is preserved in China typical culture collection center, and preserving number is CCTCCM
2015755.The nitrogenase activity of described bacillus subtilis T400 is 680.604 nmol/ (mL h), and it is in growth metabolism mistake
Can produce heteroauxing in journey, heteroauxing concentration is 98.36mg/L.Described bacillus subtilis T400 has sequence table
The DNA sequence of NO.1.
In described step (1), separate, screen method particularly includes: in soil sample, add sterilized water, concussion, stand, take
Clear liquid centrifugal treating, abandons supernatant, retains precipitate, adds sterilized water and suspends, centrifugal, goes precipitation, takes supernatant and be centrifuged, abandon
Supernatant, retains precipitate, adds phosphate buffer and suspends, obtains sample liquid；Sample liquid is added phosphate buffer and suspends mixed
Close, obtain the bacteria suspension of dilution；By the bacteria suspension heating in water bath of dilution, draw bacteria suspension after natural cooling and coat nitrogen-free cultivation
Base, cultivates and obtains nitrogen-fixing bacteria bacterium colony.
Purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 32 DEG C
38 hours isolated mono-bacterium colonies of bacillus subtilis T400 of constant temperature culture, are saved in test tube by the single bacterium colony occurred on flat board
In, preserve in 31 DEG C of constant temperature culture are placed on 4 DEG C of refrigerators for 42 hours.
The culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH
It is further qualified with fermentation time, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 75% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 5%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 32 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 200rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 1.04vvm, inoculation 13-42 hour, and ventilation controls after 1.35vvm, inoculation 43-48 hour, if
Determining ventilation is 1.63vvm；
(3.6) pH: in bacillus subtilis T400 sweat, is 7.2 by mending the method regulation pH of acid or benefit alkali；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 46 hours for fermentation eventually
Point.
In described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO31.0g,
MgSO4·7H2O 0.6g, K2HPO41.0g, NaCl 0.1g, FeSO4·7H2O 0.0015g, NaMO4 2H2O 0.05g, sugarcane
Sugar 5g, agar 18g, distilled water 1000mL, the pH of described culture medium are 7.1.
Embodiment 2
The present embodiment is with the difference of embodiment 1, also includes following steps between described step (2) and step (3):
(S1) Gram’s staining: single bacterium colony after purification carries out Gram’s staining, screening obtains positive bacteria；
(S2) spore staining: positive bacteria carries out spore staining, screening obtains the gram positive bacteria list bacterium colony containing spore.
Described step (S1) is Gram-stained method particularly includes:
(S1.1) smear: in aseptic operating platform, takes one piece of microscope slide, and on flame lamp, general plan is baked, and that removes on slide is miscellaneous
Matter；Dripping sterilized water in microscope slide central authorities, the single bacterium colony of picking, in water droplet, is smeared uniformly with the inoculating loop of calcination；Will
Specimen slide is crossed 3 times, back and forth with fixing cell above fire lamp；
(S1.2) just contaminating: drip 4 ammonium oxalate crystal violet dye liquors, contaminate 1min, incline dye liquor, and flowing water rinses to without purple；
(S1.3) mordant dyeing: first wash away back water with the iodine liquid newly joined, then cover painting face 1min with iodine liquid, wash afterwards；
(S1.4) decolouring: remove after back water, drips after 95% ethanol carries out decolouring about 18 seconds, rinses with flowing water immediately；
(S1.5) redye: drip 1 sarranine dyeing liquor, contaminate 4min, blot with absorbent paper after washing；
(S1.6) microscopy: microscope slide is placed in optical microphotograph Microscopic observation coloration result.
Described step (S2) spore staining method particularly includes: in aseptic operating platform, take one piece of microscope slide, at flame lamp
Upper general plan is baked, and removes the impurity on slide.Drip sterilized water in microscope slide central authorities, in picking single bacterium colony water droplet, use calcination
The inoculating loop crossed is smeared uniformly.Specimen slide is crossed 3 times, back and forth with fixing cell above fire lamp.District at coating thalline
Territory drips 2 dropstone carbonic acid basic fuchsin dye liquors, and dye 3min.
Remaining content of the present embodiment is same as in Example 1, repeats no more here.
Embodiment 3
The present embodiment is with the difference of embodiment 1 or 2, and in the preparation method of the present embodiment, step (4) thalline reclaims,
Measuring spore number is 60,000,000,000/g；Prepared by step (5) microbial inoculum, testing result shows that this viable count scope is 800,000,000/g.Described hay
The nitrogenase activity of bacillus cereus T400 is 692 nmol/ (mL h), and it can produce indole second during growth metabolism
Acid, heteroauxing concentration is 98.2mg/L.
Purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 28 DEG C
48 hours isolated mono-bacterium colonies of bacillus subtilis T400 of constant temperature culture, are saved in test tube by the single bacterium colony occurred on flat board
In, preserve in 28 DEG C of constant temperature culture are placed on 2 DEG C of refrigerators for 48 hours.
The culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH
It is further qualified with fermentation time, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 71% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 6%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 31 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 180rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 0.9vvm, inoculation 13-42 hour, and ventilation controls 1.15, after inoculation 43-48 hour, ventilation
Control at 1.4vvm；
(3.6) pH: in bacillus subtilis T400 sweat, is 7.2 by mending the method regulation pH of acid or benefit alkali；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 48 hours for fermentation eventually
Point.
In described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO31.2g,
MgSO4·7H2O 0.8g, K2HPO41.3g, NaCl 0.2g, FeSO4·7H2O 0.002g, NaMO4 2H2O 0.06g, sugarcane
Sugar 6g, agar 18g, distilled water 1000mL, the pH of described culture medium are 7.2.
Remaining content of the present embodiment is identical with embodiment 1 or 2, repeats no more here.
Embodiment 4
The present embodiment is with the difference of embodiment 1 or 2, and in the preparation method of the present embodiment, step (4) thalline reclaims,
Measuring spore number is 70,000,000,000/g；Prepared by step (5) microbial inoculum, testing result shows that this viable count scope is 1,000,000,000/g.Described hay
The nitrogenase activity of bacillus cereus T400 is 700 nmol/ (mL h), and it can produce indole second during growth metabolism
Acid, heteroauxing concentration is 101.45 mg/L.
Purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 30 DEG C
36 hours isolated mono-bacterium colonies of bacillus subtilis T400 of constant temperature culture, are saved in test tube by the single bacterium colony occurred on flat board
In, preserve in 30 DEG C of constant temperature culture are placed on 3 DEG C of refrigerators for 36 hours.
The culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH
It is further qualified with fermentation time, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 73% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 8%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 34 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 250rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 1.0vvm, inoculation 13-42 hour, and ventilation controls after 1.21vvm, inoculation 43-48 hour, logical
Tolerance controls at 1.6vvm；
(3.6) pH: in bacillus subtilis T400 sweat, is 7.3 by mending the method regulation pH of acid or benefit alkali；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 42 hours for fermentation eventually
Point.
In described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO31.3g,
MgSO4·7H2O 1.0g, K2HPO41.6g, NaCl 0.3g, FeSO4·7H2O 0.003g, NaMO4 2H2O 0.08g, sugarcane
Sugar 8g, agar 19g, distilled water 1000mL, the pH of described culture medium are 7.3.
Remaining content of the present embodiment is identical with embodiment 1 or 2, repeats no more here.
Embodiment 5
The present embodiment is with the difference of embodiment 1 or 2, and in the preparation method of the present embodiment, step (4) thalline reclaims,
Measuring spore number is 75,000,000,000/g；Prepared by step (5) microbial inoculum, testing result shows that this viable count scope is 1,500,000,000/g.Described hay
The nitrogenase activity of bacillus cereus T400 is 750.346 nmol/ (mL h), and it can produce indole during growth metabolism
Acetic acid, heteroauxing concentration is 95.9 mg/L.
Purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 32 DEG C
40 hours isolated mono-bacterium colonies of bacillus subtilis T400 of constant temperature culture, are saved in test tube by the single bacterium colony occurred on flat board
In, preserve in 32 DEG C of constant temperature culture are placed on 3 DEG C of refrigerators for 40 hours.
The culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH
It is further qualified with fermentation time, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 74% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 9%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 34 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 285rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 1.1vvm, inoculation 13-42 hour, and ventilation controls after 1.45vvm, inoculation 43-48 hour, logical
Tolerance controls at 1.72vvm；
(3.6) pH: in bacillus subtilis T400 sweat, is 7.4 by mending the method regulation pH of acid or benefit alkali；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 48 hours for fermentation eventually
Point.
In described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO31.3g,
MgSO4·7H2O 1.1g, K2HPO41.8g, NaCl 0.3g, FeSO4·7H2O 0.004g, NaMO4 2H2O 0.09g, sugarcane
Sugar 9g, agar 20g, distilled water 1000mL, the pH of described culture medium are 7.4.
Remaining content of the present embodiment is identical with embodiment 1 or 2, repeats no more here.
Embodiment 6
The present embodiment is with the difference of embodiment 1 or 2, and in the preparation method of the present embodiment, step (4) thalline reclaims,
Measuring spore number is 80,000,000,000/g；Prepared by step (5) microbial inoculum, testing result shows that this viable count scope is 2,000,000,000/g.Described hay
The nitrogenase activity of bacillus cereus T400 is 786.375 nmol/ (mL h), and it can produce indole during growth metabolism
Acetic acid, heteroauxing concentration is 99.2mg/L.
Purification in described step (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 33 DEG C
36 hours isolated mono-bacterium colonies of bacillus subtilis T400 of constant temperature culture, are saved in test tube by the single bacterium colony occurred on flat board
In, preserve in 33 DEG C of constant temperature culture are placed on 5 DEG C of refrigerators for 36 hours.
The culture medium culturing of described step (3) is to fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH
It is further qualified with fermentation time, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 75% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 10%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 36 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 300rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 1.2vvm, inoculation 13-42 hour, and ventilation controls after 1.6vvm, inoculation 43-48 hour, ventilation
Amount controls at 1.8vvm；
(3.6) pH: in bacillus subtilis T400 sweat, is 7.4 by mending the method regulation pH of acid or benefit alkali；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, sets 42 hours for fermentation eventually
Point.
In described step (3) culture medium culturing, described culture medium is made up of the raw material of following quality: CaCO31.4g,
MgSO4·7H2O 1.2g, K2HPO42.0g, NaCl0.4g, FeSO4·7H2O 0.005g, NaMO4 2H2O 0.1g, sucrose
10g, agar 20g, distilled water 1000mL, the pH of described culture medium are 7.4.
Remaining content of the present embodiment is identical with embodiment 1 or 2, repeats no more here.
The 16S rDNA gene order strain identification of the bacillus subtilis T400 of the present invention
The individuality of antibacterial is small, and form is simple, and traditional method identifies that antibacterial is often according to they differential responses on Physiology and biochemistry
Main Basis as taxonomic identification.Since 20 century 70 later stages, the most general " formal " or " official " is thin
Bacterium sorting technique is as foundation with " primary Jie Shi determinative bacteriology handbook ".Physiology and biochemistry identify in, it will usually occur one or
The several physical signs of person does not meets the peculiar property that this strain is had, it is difficult to clearly identify this bacterial strain.At present, antibacterial
The physiological and biochemical index of bacterial strain is generally combined by the method identified with molecular biological characteristic, draws the most reliably conclusion.
Wherein to have become as at present antibacterial Bacillus subtilis the most in the world normal for the 16S rRNA gene evolution development system of DNA sequence analysis
Technological means (Kim et al, 2004；Prap et al, 1997).
Ribosome 16S rDNA gene order total length about 1550bp, is made up of conserved region alternately and variable region.Utilize
The universal primer of conservative region design, can amplify germy 16S rDNA fragment.16S rDNA gene sequence analysis
Ultimate principle be from micro-biological samples extraction 16S rDNA fragment, by clone, order-checking or enzyme action, probe hybridization obtain
The sequence information of 16S rDNA, then compare with sequence data or other data of 16S rDNA data base, determine its
Position in cladogram, thus identify microbe species that may be present in sample.16S rDNA fragment conservative region is utilized to set
The universal primer of meter, will not be complementary to abacterial DNA, and the difference of the 16S rDNA variable region of antibacterial can be used to distinguish not
Same bacterium.Therefore it is to be commonly recognized by certain bacterial strain 16S rDNA sequencing being obtained the way of final assay certificate
's.
, authentication method
(1) PCR reaction system (25 μ L):
10×PCR Buffer 2.5μL
dNTP(2.5mM) m 2.0μL
Primer 2 7F(10 μM) 0.5 μ L
Primer 1492R(10 μM) 0.5 μ L
DNA profiling 100ng
Taq enzyme (5U/ μ L) 0.5 μ L
ddH2O 19μL
(2) PCR reaction condition: 95 DEG C of denaturation 5 min, 95 DEG C of degeneration 30 s, 58 DEG C of annealing 30 s, 72 DEG C of extensions
80 s, 35 circulations, 72 DEG C extend 10 min.ABI 3730 xl DNA analysis instrument (Applied Biosystems, Inc.) is used to enter
Row DNA sequencing.
, sequencing result
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg
atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaca
3, homology analysis
Identify that this antibacterial isBacillus subtilis, bacillus subtilis.
Application examples: the bacillus subtilis T400 microbial inoculum of the present invention is carried out Semen Maydis and Oryza sativa L. pot experiment
1, nursery
By soaked overnight in Semen Maydis and rice paddy seed warm water, it is seeded in the plastic cup equipped with soil, natural conditions nursery 12-14
My god, during plant about 10cm, select the uniform Semen Maydis of growing way and rice transplanting in flowerpot.
2, soil pretreatment
After soil air-dries, cross 1 mm sieve.Each process arranges 3 repetitions.Each flowerpot is respectively provided with soil 4.0 kg, carbamide
407 mg, calcium superphosphate 162 mg and potassium sulfate 418 mg..
3, test is arranged
Select and have from growing nitrogen-fixing and promote that the bacillus subtilis T400 microbial inoculum of plant growth function carries out pot test effect contrast
Test, every basin adds microbial inoculum 1g.Arranging bentonite and be added to control experiment, every basin adds bentonite 1g.Water every day in right amount, often
The water yield of basin is identical, sow uniformly, do not make water flow out at the bottom of basin in case fertilizer loss and produce error.
4, result of the test
After transplanting seedlings 45 days, measure rice tillering number, plant height and chlorophyll content.Data acquisition SPSS 17.0 carries out statistical analysis.
After 64th day, measure Semen Maydis plant height, the number of blade and single-strain fresh weight thereof.Can show that inoculation T400 bacillus subtilis microbial agent is permissible
It is obviously promoted Semen Maydis and paddy growth, is shown in Table 2, table 3 and Fig. 6.
Table 2 microbial inoculum T400 Oryza sativa L. results from pot experiment test
Note: the same column shoulder different lowercase alphabet of note show significant difference (P<0.05).
Table 3 microbial inoculum T400 Semen Maydis results from pot experiment test
| Bacterial strain | Plant height (cm) | The number of blade (sheet) | Single-strain fresh weight (g) |
| T400 | 106.0 | 10.5 | 42.2 |
| CK | 77.6 | 8.8 | 29.2 |
From table 2, table 3 and Fig. 6 is it can be seen that utilize the bacillus subtilis T400 microbial inoculum of the present invention to carry out Oryza sativa L. and Semen Maydis is potted plant
Test, microbial inoculum T400 considerably improves the tiller number of Oryza sativa L., plant height and chlorophyll content, and the increment rate of tiller number reaches
70.67%, the increment rate of plant height is 19.34%, and the increment rate of chlorophyll content is 49.34%.The plant height of milpa is at comparison
1.36 times of the plant plant height of reason, the number of blade is 1.19 times of the number of blade of control treatment, and individual plant plant fresh weight is control treatment
1.44 times.
The present invention by Guangdong Province's introduction innovation start-up group funded projects research and develop, prepare bacillus subtilis T400 and
Microbial inoculum has wide market prospect, high financial profit.
Last it should be noted that, above example is only in order to illustrate technical scheme, rather than the present invention is protected
Protecting the restriction of scope, although having made to explain to the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent, without deviating from the reality of technical solution of the present invention
Matter and scope.
SEQUENCE LISTING
<110>Dongguan Baode Bio-engineering Co., Ltd.
<120>a kind of bacillus subtilis T400 and bacterial preparation process thereof
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>959
<212>DNA
<213>the 16s rDNA gene order of bacillus subtilis (Bacillus subtilis) T400
<400>1
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaca 959
Claims (10)
1. a bacillus subtilis T400, it is characterised in that: described bacillus subtilis T400 is preserved in Chinese Typical Representative and cultivates
Thing preservation center, preserving number is CCTCCM 2015755.
A kind of bacillus subtilis T400 the most according to claim 1, it is characterised in that: described bacillus subtilis T400
Nitrogenase activity be 600.000-800.000nmol/ (mL h), its heteroauxing produced during growth metabolism is dense
Degree is 95-103mg/L.
A kind of bacillus subtilis T400 the most according to claim 1, it is characterised in that: described bacillus subtilis T400
There is the DNA sequence of sequence table NO.1.
4. the bacterial preparation process of a kind of bacillus subtilis T400 described in claim 1-3 any one, it is characterised in that:
Comprise the following steps:
Separate, screen
Choose the soil sample of nitrogen-fixing bacteria bacterium colony containing bacillus subtilis T400, separated, screening, cultivate and obtain nitrogen-fixing bacteria bacterium
Fall；
Purification, preservation
Being purified by the nitrogen-fixing bacteria bacterium colony separated, screening obtains in culture medium, culture of isolated obtains bacillus subtilis
The mono-bacterium colony of T400, preserves this list bacterium colony standby；
(3) culture medium culturing: utilize the culture medium fermentation culture mono-bacterium colony of bacillus subtilis T400, obtain microbial inoculum；
Thalline reclaims
Collect bacillus subtilis T400 thalline by centrifugal for microbial inoculum, quickly slough moisture, the pure mycopowder that preparation is dried, survey
Normal bud spore number is 500-800 hundred million/g；
(5) prepared by microbial inoculum
To be dried pure mycopowder to mix with matrix material, and carry out viable count detection, testing result shows that this viable count scope is 4-20
Hundred million/g, i.e. obtains bacillus subtilis T400 microbial inoculum.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 4, it is characterised in that: described step
Suddenly in (1), separate, screen method particularly includes: in soil sample, add sterilized water, concussion, stand, take supernatant centrifugal treating,
Abandon supernatant, retain precipitate, add sterilized water and suspend, centrifugal, go precipitation, take supernatant and be centrifuged again, abandon supernatant, it is heavy to retain
Shallow lake thing, adds phosphate buffer and suspends, obtain sample liquid；Sample liquid is added phosphate buffer suspension mixing, obtains dilution
Bacteria suspension；By the bacteria suspension heating in water bath of dilution, draw bacteria suspension after natural cooling and coat nitrogen-free agar, cultivate and consolidated
Nitrogen bacterium bacterium colony.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 4, it is characterised in that: described step
Suddenly purification in (2), preservation method particularly includes: step (1) is cultivated obtain nitrogen-fixing bacteria bacterium colony in 28-33 DEG C of constant temperature culture 36-
48 hours, the mono-bacterium colony of isolated bacillus subtilis T400, list bacterium colony is saved in test tube, in 28-33 DEG C of constant temperature culture
Within 36-48 hour, it is placed in 2-5 DEG C of refrigerator preservation.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 4, it is characterised in that: described step
Suddenly the culture medium culturing of (3) fermentation tank liquid amount, inoculum concentration, cultivation temperature, rotating speed, ventilation, pH and fermentation time are made into
One step limits, particularly as follows:
(3.1) liquid amount: fermentation tank liquid amount is the 70-75% of tank body volume；
(3.2) inoculum concentration: the inoculum concentration of bacillus subtilis T400 is 5-10%；
(3.3) cultivation temperature: the shake-flask culture temperature of bacillus subtilis T400 is 30-36 DEG C；
(3.4) rotating speed: in sweat, the shaking speed of bacillus subtilis T400 is 150-300rpm；
(3.5) ventilation: the ventilation of bacillus subtilis T400 is set according to different incubation times, and after inoculation, 0-12 is little
Time, ventilation controls after 0.9-1.2vvm, inoculation 13-42 hour, and ventilation controls 43-48 after 1.15-1.6vvm, inoculation
Hour, ventilation controls at 1.4-1.8vvm；
(3.6) pH: in bacillus subtilis T400 sweat, remains by mending the method regulation pH of acid or benefit alkali
7.1-7.4；
(3.7) fermentation time: according to bacillus subtilis T400 growing state in shaking flask, set its fermentation time as 42-
48 hours.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 4, it is characterised in that: described step
Suddenly, in (3) culture medium culturing, described culture medium is made up of the raw material of following quality: 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, NaMO4·2H2O
0.05-0.1g, sucrose 5-10g, agar 18-20g, distilled water 1000mL, the pH of described culture medium are 7.1-7.4.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 4, it is characterised in that: described step
Suddenly further comprising the steps of between (2) and step (3):
(S1) Gram’s staining: single bacterium colony after purification carries out Gram’s staining, screening obtains positive bacteria；
(S2) spore staining: positive bacteria carries out spore staining, screening obtains the gram positive bacteria list bacterium colony containing spore.
The bacterial preparation process of a kind of bacillus subtilis T400 the most according to claim 9, it is characterised in that: described
Step (S1) is Gram-stained method particularly includes:
(S1.1) smear: in aseptic operating platform, takes one piece of microscope slide, and on flame lamp, general plan is baked, and that removes on microscope slide is miscellaneous
Matter；Dripping sterilized water in microscope slide central authorities, the picking single bacillus subtilis mono-bacterium colony of T400 is in water droplet, with calcination
Inoculating loop is smeared uniformly；Specimen slide is crossed 3 times, back and forth with fixing cell above fire lamp；
(S1.2) just contaminate: dropping 2-5 drips ammonium oxalate crystal violet dye liquor, contaminates 1min, and incline dye liquor, and flowing water rinses to without purple；
(S1.3) mordant dyeing: first wash away back water with the iodine liquid newly joined, then wash after iodine liquid covering painting face 1min；
(S1.4) decolouring: remove after back water, drips 95% ethanol and carries out decolouring after the about 15-20 second, rinse with flowing water immediately；
(S1.5) redye: drip 1 sarranine dyeing liquor, contaminate 3-5min, blot with absorbent paper after washing；
(S1.6) microscopy: microscope slide is placed in optical microphotograph Microscopic observation coloration result.
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| CN115895936A (en) * | 2022-08-08 | 2023-04-04 | 河北农业大学 | Bacillus subtilis 2-22 and application thereof |
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| CN103275895A (en) * | 2013-05-24 | 2013-09-04 | 南京农业大学 | Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof |
| CN104560789A (en) * | 2014-12-13 | 2015-04-29 | 郑州市污水净化有限公司 | Peanut growth promoting rhizobacteria HS2 and application thereof |
| CN104611252A (en) * | 2014-12-13 | 2015-05-13 | 苏新宏 | Tobacco plant growth promoting bacterium TC6 and application thereof |
| CN104630090A (en) * | 2014-12-13 | 2015-05-20 | 河南农业大学 | Corn rhizosphere growth promoting bacteria YM3 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103275895A (en) * | 2013-05-24 | 2013-09-04 | 南京农业大学 | Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof |
| CN104560789A (en) * | 2014-12-13 | 2015-04-29 | 郑州市污水净化有限公司 | Peanut growth promoting rhizobacteria HS2 and application thereof |
| CN104611252A (en) * | 2014-12-13 | 2015-05-13 | 苏新宏 | Tobacco plant growth promoting bacterium TC6 and application thereof |
| CN104630090A (en) * | 2014-12-13 | 2015-05-20 | 河南农业大学 | Corn rhizosphere growth promoting bacteria YM3 and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112831437A (en) * | 2021-01-20 | 2021-05-25 | 河北冀微生物技术有限公司 | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate |
| CN112831437B (en) * | 2021-01-20 | 2021-11-23 | 河北冀微生物技术有限公司 | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate |
| CN115895936A (en) * | 2022-08-08 | 2023-04-04 | 河北农业大学 | Bacillus subtilis 2-22 and application thereof |
| CN115895936B (en) * | 2022-08-08 | 2023-06-16 | 河北农业大学 | Bacillus subtilis 2-22 and application thereof |
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